Accuracy of malaria diagnosis by microscopy, rapid diagnostic test, and PCR methods and evidence of antimalarial overprescription in non-severe febrile patients in two Tanzanian hospitals.

PubMed

Nicastri, Emanuele; Bevilacqua, Nazario; Sañé Schepisi, Monica; Paglia, Maria G; Meschi, Silvia; Ame, Shaali M; Mohamed, Jape A; Mangi, Sabina; Fumakule, Robert; Di Caro, Antonino; Capobianchi, Maria R; Kitua, Andrew; Molteni, Fabrizio; Racalbuto, Vincenzo; Ippolito, Giuseppe

2009-05-01

The study was aimed to evaluate the malaria over/underdiagnosis and over/underprescription of antimalarial drugs. Between February and March 2007 blood samples were collected from 336 non-severe febrile outpatients attended in two peripheral Tanzanian hospitals. Microscopy and a rapid diagnostic test (RDT) were done locally and the accuracy evaluated by qualitative polymerase chain reaction (PCR) for Plasmodium spp. The testing was performed at National Institute for Infectious Diseases Lazzaro Spallanzani (INMI), Rome, Italy. As a result of PCR, we identified 26 malaria cases out of 336 (7.7%) patients. Microscopy and RDT accuracies were 93.5% and 97.6%, respectively. Overprescription and underdiagnosis rates were 29.3% and 30.8%, respectively. On-field training, clinical management of febrile illness, and malaria microscopy in remote settings should be considered.

Quantitative analysis of diet structure by real-time PCR, reveals different feeding patterns by two dominant grasshopper species

PubMed Central

Huang, Xunbing; Wu, Huihui; McNeill, Mark Richard; Qin, Xinghu; Ma, Jingchuan; Tu, Xiongbing; Cao, Guangchun; Wang, Guangjun; Nong, Xiangqun; Zhang, Zehua

2016-01-01

Studies on grasshopper diets have historically employed a range of methodologies, each with certain advantages and disadvantages. For example, some methodologies are qualitative instead of quantitative. Others require long experimental periods or examine population-level effects, only. In this study, we used real-time PCR to examine diets of individual grasshoppers. The method has the advantage of being both fast and quantitative. Using two grasshopper species, Oedaleus asiaticus and Dasyhippus barbipes, we designed ITS primer sequences for their three main host plants, Stipa krylovii, Leymus chinensis and Cleistogenes squarrosa and used real-time PCR method to test diet structure both qualitatively and quantitatively. The lowest detection efficiency of the three grass species was ~80% with a strong correlation between actual and PCR-measured food intake. We found that Oedaleus asiaticus maintained an unchanged diet structure across grasslands with different grass communities. By comparison, Dasyhippus barbipes changed its diet structure. These results revealed why O. asiaticus distribution is mainly confined to Stipa-dominated grassland, and D. barbipes is more widely distributed across Inner Mongolia. Overall, real-time PCR was shown to be a useful tool for investigating grasshopper diets, which in turn offers some insight into grasshopper distributions and improved pest management. PMID:27562455

Evaluation of blood collection filter papers for HIV-1 DNA PCR.

PubMed

Masciotra, Silvina; Khamadi, Samoel; Bilé, Ebi; Puren, Adrian; Fonjungo, Peter; Nguyen, Shon; Girma, Mulu; Downing, Robert; Ramos, Artur; Subbarao, Shambavi; Ellenberger, Dennis

2012-10-01

The collection of dried blood spots (DBS) on Whatman 903 cards has facilitated for years the detection of HIV-1 in infants by DNA PCR as early as 4-6 weeks after birth in resource-limited settings (RLS), but alternate blood collection devices are proving to be necessary. The qualitative detection of HIV-1 DNA by PCR from DBS prepared on three commercially available blood collection cards was evaluated at the Centers for Disease Control and Prevention (CDC) and in four laboratories in Africa. DBS were prepared on Ahlstrom grade 226, Munktell TFN and Whatman 903, and stored under a variety of conditions. DBS were stored at ambient temperature (RT), 37°C with high humidity, and -20°C for varying lengths of time. The presence of HIV-1 DNA was tested using Roche Amplicor HIV-1 DNA (v 1.5) weekly for 4 weeks and at weeks 8 and 12 (RT and 37°C), at weeks 4, 8, and 18 (-20°C) of storage. DBS specimens were also tested after international shipment at RT. In addition, after nearly 3 years storage at -20°C, DBS were also evaluated independently using the COBAS Ampliprep/TaqMan HIV-1 Qual and Abbott RealTime HIV-1 Qualitative tests. HIV-1 DNA was detected equally well on the three blood collection cards regardless of storage conditions and PCR assay. Ahlstrom 226 and Munktell TFN papers were comparable to Whatman 903 for HIV-1 DNA detection and may be considered as optional blood collection devices in resource-limited countries. Published by Elsevier B.V.

Diagnosis of ocular toxoplasmosis by two polymerase chain reaction (PCR) examinations: qualitative multiplex and quantitative real-time.

PubMed

Sugita, Sunao; Ogawa, Manabu; Inoue, Shizu; Shimizu, Norio; Mochizuki, Manabu

2011-09-01

To establish a two-step polymerase chain reaction (PCR) diagnostic system for ocular toxoplasmosis. A total of 13 ocular fluid samples (11 aqueous humor and 2 vitreous fluid) were collected from 13 patients with clinically suspected ocular toxoplasmosis. Ten ocular samples from other uveitis patients and 20 samples from subjects without ocular inflammation were used as controls. Two polymerase chain reaction (PCR) methods, i.e., qualitative multiplex PCR and quantitative real-time PCR, were used to measure the toxoplasma genome (T. gondii B1 gene). Qualitative multiplex PCR detected T. gondii B1 gene in the ocular fluids of 11 out of 13 patients with clinically suspected ocular toxoplasmosis. In real-time PCR, we detected high copy numbers of T. gondii DNA (5.1 × 10(2)-2.1 × 10(6) copies/mL) in a total of 10 patients (10/13, 77%). Only ocular toxoplasmosis scar lesions were observed in the three real-time PCR-negative patients. PCR assay results for the samples from the two control groups were all negative. The two-step PCR examination to detect toxoplasma DNA is a useful tool for diagnosing ocular toxoplasmosis.

Development and in-house validation of the event-specific qualitative and quantitative PCR detection methods for genetically modified cotton MON15985.

PubMed

Jiang, Lingxi; Yang, Litao; Rao, Jun; Guo, Jinchao; Wang, Shu; Liu, Jia; Lee, Seonghun; Zhang, Dabing

2010-02-01

To implement genetically modified organism (GMO) labeling regulations, an event-specific analysis method based on the junction sequence between exogenous integration and host genomic DNA has become the preferential approach for GMO identification and quantification. In this study, specific primers and TaqMan probes based on the revealed 5'-end junction sequence of GM cotton MON15985 were designed, and qualitative and quantitative polymerase chain reaction (PCR) assays were established employing the designed primers and probes. In the qualitative PCR assay, the limit of detection (LOD) was 0.5 g kg(-1) in 100 ng total cotton genomic DNA, corresponding to about 17 copies of haploid cotton genomic DNA, and the LOD and limit of quantification (LOQ) for quantitative PCR assay were 10 and 17 copies of haploid cotton genomic DNA, respectively. Furthermore, the developed quantitative PCR assays were validated in-house by five different researchers. Also, five practical samples with known GM contents were quantified using the developed PCR assay in in-house validation, and the bias between the true and quantification values ranged from 2.06% to 12.59%. This study shows that the developed qualitative and quantitative PCR methods are applicable for the identification and quantification of GM cotton MON15985 and its derivates.

Automation of laboratory testing for infectious diseases using the polymerase chain reaction-- our past, our present, our future.

PubMed

Jungkind, D

2001-01-01

While it is an extremely powerful and versatile assay method, polymerase chain reaction (PCR) can be a labor-intensive process. Since the advent of commercial test kits from Roche and the semi-automated microwell Amplicor system, PCR has become an increasingly useful and widespread clinical tool. However, more widespread acceptance of molecular testing will depend upon automation that allows molecular assays to enter the routine clinical laboratory. The forces driving the need for automated PCR are the requirements for diagnosis and treatment of chronic viral diseases, economic pressures to develop more automated and less expensive test procedures similar to those in the clinical chemistry laboratories, and a shortage in many areas of qualified laboratory personnel trained in the types of manual procedures used in past decades. The automated Roche COBAS AMPLICOR system has automated the amplification and detection process. Specimen preparation remains the most labor-intensive part of the PCR testing process, accounting for the majority of the hands-on-time in most of the assays. A new automated specimen preparation system, the COBAS AmpliPrep, was evaluated. The system automatically releases the target nucleic acid, captures the target with specific oligonucleotide probes, which become attached to magnetic beads via a biotin-streptavidin binding reaction. Once attached to the beads, the target is purified and concentrated automatically. Results of 298 qualitative and 57 quantitative samples representing a wide range of virus concentrations analyzed after the COBAS AmpliPrep and manual specimen preparation methods, showed that there was no significant difference in qualitative or quantitative hepatitis C virus (HCV) assay performance, respectively. The AmpliPrep instrument decreased the time required to prepare serum or plasma samples for HCV PCR to under 1 min per sample. This was a decrease of 76% compared to the manual specimen preparation method. Systems that can analyze more samples with higher throughput and that can answer more questions about the nature of the microbes that we can presently only detect and quantitate will be needed in the future.

Quantification of fungal abundance on cultural heritage using real time PCR targeting the β-actin gene

PubMed Central

Ettenauer, Jörg; Piñar, Guadalupe; Tafer, Hakim; Sterflinger, Katja

2014-01-01

The traditional methodology used for the identification of microbes colonizing our cultural heritage was the application of cultivation methods and/or microscopy. This approach has many advantages, as living microorganisms may be obtained for physiological investigations. In addition, these techniques allow the quantitative and qualitative assessment of the investigated environment. Quantitative analyses are done by plate count and the determination of abundance by the colony forming unit (CFU). Nevertheless, these techniques have many drawbacks that lead to an underestimation of the cell numbers and do not provide a comprehensive overview of the composition of the inhabiting microbiota. In the last decades, several molecular techniques have been developed enabling many advantages over the cultivation approach. Mainly PCR-based, fingerprinting techniques allow a qualitative detection and identification of the microbiota. In this study, we developed a real time PCR method as a simple, rapid and reliable tool to detect and quantify fungal abundance using the β-actin gene, which is known to appear as a single-copy gene in fungi. To this end, five different indoor thermal insulation materials applied for historical buildings that were previously tested for their bio-susceptibility against various fungi were subjected to qPCR analyses. The obtained results were compared with those obtained from a previous study investigating the bio-susceptibility of the insulation materials using classical cultivation experiments. Both results correlated well, revealing that Perlite plaster was the most suitable insulation material, showing the lowest fungal CFU and qPCR values. In contrast, insulations made of wood showed to be not recommendable from the microbiological point of view. In addition, the potential of qPCR was tested in other materials of cultural heritage, as old parchments, showing to be a suitable method for measuring fungal abundance in these delicate materials. PMID:24904567

Qualitative PCR method for Roundup Ready soybean: interlaboratory study.

PubMed

Kodama, Takashi; Kasahara, Masaki; Minegishi, Yasutaka; Futo, Satoshi; Sawada, Chihiro; Watai, Masatoshi; Akiyama, Hiroshi; Teshima, Reiko; Kurosawa, Yasunori; Furui, Satoshi; Hino, Akihiro; Kitta, Kazumi

2011-01-01

Quantitative and qualitative methods based on PCR have been developed for genetically modified organisms (GMO). Interlaboratory studies were previously conducted for GMO quantitative methods; in this study, an interlaboratory study was conducted for a qualitative method for a GM soybean, Roundup Ready soy (RR soy), with primer pairs designed for the quantitative method of RR soy studied previously. Fourteen laboratories in Japan participated. Each participant extracted DNA from 1.0 g each of the soy samples containing 0, 0.05, and 0.10% of RR soy, and performed PCR with primer pairs for an internal control gene (Le1) and RR soy followed by agarose gel electrophoresis. The PCR product amplified in this PCR system for Le1 was detected from all samples. The sensitivity, specificity, and false-negative and false-positive rates of the method were obtained from the results of RR soy detection. False-negative rates at the level of 0.05 and 0.10% of the RR soy samples were 6.0 and 2.3%, respectively, revealing that the LOD of the method was somewhat below 0.10%. The current study demonstrated that the qualitative method would be practical for monitoring the labeling system of GM soy in kernel lots.

Evaluation of four endogenous reference genes and their real-time PCR assays for common wheat quantification in GMOs detection.

PubMed

Huang, Huali; Cheng, Fang; Wang, Ruoan; Zhang, Dabing; Yang, Litao

2013-01-01

Proper selection of endogenous reference genes and their real-time PCR assays is quite important in genetically modified organisms (GMOs) detection. To find a suitable endogenous reference gene and its real-time PCR assay for common wheat (Triticum aestivum L.) DNA content or copy number quantification, four previously reported wheat endogenous reference genes and their real-time PCR assays were comprehensively evaluated for the target gene sequence variation and their real-time PCR performance among 37 common wheat lines. Three SNPs were observed in the PKABA1 and ALMT1 genes, and these SNPs significantly decreased the efficiency of real-time PCR amplification. GeNorm analysis of the real-time PCR performance of each gene among common wheat lines showed that the Waxy-D1 assay had the lowest M values with the best stability among all tested lines. All results indicated that the Waxy-D1 gene and its real-time PCR assay were most suitable to be used as an endogenous reference gene for common wheat DNA content quantification. The validated Waxy-D1 gene assay will be useful in establishing accurate and creditable qualitative and quantitative PCR analysis of GM wheat.

Evaluation of Four Endogenous Reference Genes and Their Real-Time PCR Assays for Common Wheat Quantification in GMOs Detection

PubMed Central

Huang, Huali; Cheng, Fang; Wang, Ruoan; Zhang, Dabing; Yang, Litao

2013-01-01

Proper selection of endogenous reference genes and their real-time PCR assays is quite important in genetically modified organisms (GMOs) detection. To find a suitable endogenous reference gene and its real-time PCR assay for common wheat (Triticum aestivum L.) DNA content or copy number quantification, four previously reported wheat endogenous reference genes and their real-time PCR assays were comprehensively evaluated for the target gene sequence variation and their real-time PCR performance among 37 common wheat lines. Three SNPs were observed in the PKABA1 and ALMT1 genes, and these SNPs significantly decreased the efficiency of real-time PCR amplification. GeNorm analysis of the real-time PCR performance of each gene among common wheat lines showed that the Waxy-D1 assay had the lowest M values with the best stability among all tested lines. All results indicated that the Waxy-D1 gene and its real-time PCR assay were most suitable to be used as an endogenous reference gene for common wheat DNA content quantification. The validated Waxy-D1 gene assay will be useful in establishing accurate and creditable qualitative and quantitative PCR analysis of GM wheat. PMID:24098735

Short communication: comparing real-time PCR and bacteriological cultures for Streptococcus agalactiae and Staphylococcus aureus in bulk-tank milk samples.

PubMed

Zanardi, G; Caminiti, A; Delle Donne, G; Moroni, P; Santi, A; Galletti, G; Tamba, M; Bolzoni, G; Bertocchi, L

2014-09-01

For more than 30 yr, a control plan for Streptococcus agalactiae and Staphylococcus aureus has been carried out in more than 1,500 dairy herds of the province of Brescia (northern Italy). From 2010 to 2011, the apparent prevalence of Strep. agalactiae has been relatively stable around 10%, but the apparent prevalence of Staph. aureus has been greater than 40% with an increasing trend. The aim of this paper was to estimate and compare the diagnostic accuracy of 3 assays for the detection of Strep. agalactiae and Staph. aureus in bulk-tank milk samples (BTMS) in field conditions. The assays were a qualitative and a quantitative bacteriological culture (BC) for each pathogen and a homemade multiplex real-time PCR (rt-PCR). Because a gold standard was not available, the sensitivities (Se) and specificities (Sp) were evaluated using a Bayesian latent class approach. In 2012 we collected one BTMS from 165 dairy herds that were found positive for Strep. agalactiae in the previous 2-yr campaigns of eradication plan. In most cases, BTMS collected in these herds were positive for Staph. aureus as well, confirming the wide spread of this pathogen. At the same time we also collected composite milk samples from all the 8,624 lactating cows to evaluate the within-herd prevalence of Strep. agalactiae. Streptococcus agalactiae samples were cultured using a selective medium Tallium Kristalviolette Tossin, whereas for Staph. aureus, we used Baird Parker modified medium with added Rabbit Plasma Fibrinogen ISO-Formulation. In parallel, BTMS were tested using the rt-PCR. Regarding Strep. agalactiae, the posterior median of Se and Sp of the 2 BC was similar [qualitative BC: Se=98%, posterior credible interval (95%PCI): 94-100%, and Sp=99%, 95%PCI: 96-100%; quantitative BC: Se=99%, 95%PCI: 96-100%, and Sp=99%, 95%PCI: 95-100%] and higher than those of the rt-PCR (at 40 cycle threshold, Se=92%, 95%PCI: 85-97%; Sp=94%, 95%PCI: 88-98%). Also in case of Staph. aureus, the posterior medians of BC were generally higher than those of rt-PCR. In fact, although the Se of BC was slightly lower (rt-PCR at 40 cycle threshold, median Se=99%, 95%PCI: 97-100%, and qualitative BC, median Se=94%, 95%PCI: 87-99%), the Sp was much higher (rt-PCR at 40 cycle threshold, median Sp=67%, 95%PCI: 38-97%; qualitative BC, median Sp=95%; 95%PCI: 76-100%). Our study confirms that BC and rt-PCR are reliable diagnostic tools to detect Strep. agalactiae and Staph. aureus, and rt-PCR results should be confirmed by BC carried out on BTMS and possibly on composite milk samples. Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Active CMV and EBV infections in renal transplant recipients with unexplained fever and elevated serum creatinine.

PubMed

Hasannia, Tahereh; Moosavi Movahed, Seyed Majid; Vakili, Rosita; Rafatpanah, Houshang; Hekmat, Reza; Valizadeh, Narges; Rezaee, Seyed Abdolrahim

2016-10-01

Proper identification of active cytomegalovirus (CMV) and Epstein-Barr virus (EBV) infections are helpful for monitoring antiviral treatment in transplant recipients. Qualitative and quantitative CMV, EBV DNA PCR techniques in the context of serological tests are performed for early detection and differentiation of active and latent CMV and EBV infections in renal transplantation. Basically, 129 renal transplanted recipients monitored carefully and hospitalized for unexplained elevated creatinine levels or high fever and 21 of their donors were studied. CMV DNA was detected in 63.5% of the febrile episodes following transplantation and in 46.42% of readmitted patients using qualitative PCR method. In the first group, 15% of the patients and in the second group 42.85% of the patients had copy numbers more than cutoff point (900 copies/mL). Cutoff point had 100% sensitivity and 82.5% specificity for active and symptomatic CMV infection. Only 15.5% of the subjects were positive for EBV infection by qualitative PCR method. Among them 5% had >2000 copies/mL and were symptomatic. One subject with a history of three times hospitalization had higher EBV viral load and developed post-transplant lymphoproliferative disorder. CMV load was significantly correlated with elevated creatinine levels (OR = 3.1, p = 0.006), abnormal heart sounds (OR = 4.7; p = 0.02) and hypertension (OR = 3.6; p = 0.03). Only qRT-PCR could differentiate between latent and active infections and might be clinically useful for monitoring symptomatic CMV and EBV infections and initiation of the antiviral therapy. Elevated creatinine levels, hypertension, and abnormal heart sounds could be considered as main manifestations of HCMV infection in kidney recipients.

Identification of fecal contamination sources in water using host-associated markers.

PubMed

Krentz, Corinne A; Prystajecky, Natalie; Isaac-Renton, Judith

2013-03-01

In British Columbia, Canada, drinking water is tested for total coliforms and Escherichia coli, but there is currently no routine follow-up testing to investigate fecal contamination sources in samples that test positive for indicator bacteria. Reliable microbial source tracking (MST) tools to rapidly test water samples for multiple fecal contamination markers simultaneously are currently lacking. The objectives of this study were (i) to develop a qualitative MST tool to identify fecal contamination from different host groups, and (ii) to evaluate the MST tool using water samples with evidence of fecal contamination. Singleplex and multiplex polymerase chain reaction (PCR) were used to test (i) water from polluted sites and (ii) raw and drinking water samples for presence of bacterial genetic markers associated with feces from humans, cattle, seagulls, pigs, chickens, and geese. The multiplex MST assay correctly identified suspected contamination sources in contaminated waterways, demonstrating that this test may have utility for heavily contaminated sites. Most raw and drinking water samples analyzed using singleplex PCR contained at least one host-associated marker. Singleplex PCR was capable of detecting host-associated markers in small sample volumes and is therefore a promising tool to further analyze water samples submitted for routine testing and provide information useful for water quality management.

[Retrospective study of the implementation of the qualitative PCR technique in biological samples for monitoring toxoplasmosis in pediatric patients receiving hematopoietic stem cell transplantation].

PubMed

Nigro, Mónica G; Figueroa, Carlos; Ledesma, Bibiana A

2014-01-01

Toxoplasmosis is an opportunistic infection caused by the parasite Toxoplasma gondii. The infection is severe and difficult to diagnose in patients receiving allogeneic hematopoietic stem cell transplantation (HSCT). Twelve patients receiving HSCT were monitored post-transplant, by qualitative PCR at the Children's Hospital S.A.M.I.C. "Prof. Dr. Juan P. Garrahan". The monitoring of these patients was defined by a history of positive serology for toxoplasmosis in the donor or recipient and because their hematologic condition did not allow the use of trimethoprim-sulfamethoxazole for prophylaxis. During the patients' monitoring, two of them with positive PCR results showed signs of illness by T. gondii and were treated with pyrimethamine-clindamycin. In two other patients, toxoplasmosis was the cause of death and an autopsy finding, showing negative PCR results. Four patients without clinical manifestations received treatment for toxoplasmosis because of positive PCR detection. In four patients there were no signs of toxoplasmosis disease and negative PCR results during follow-up. The qualitative PCR technique proved useful for the detection of toxoplasmosis reactivation in HSCT recipients, but has limitations in monitoring and making clinical decisions due to the persistence of positive PCR over time and manifestations of toxicity caused by the treatment. Copyright © 2014 Asociación Argentina de Microbiología. Publicado por Elsevier España. All rights reserved.

Characterization of the exogenous insert and development of event-specific PCR detection methods for genetically modified Huanong No. 1 papaya.

PubMed

Guo, Jinchao; Yang, Litao; Liu, Xin; Guan, Xiaoyan; Jiang, Lingxi; Zhang, Dabing

2009-08-26

Genetically modified (GM) papaya (Carica papaya L.), Huanong No. 1, was approved for commercialization in Guangdong province, China in 2006, and the development of the Huanong No. 1 papaya detection method is necessary for implementing genetically modified organism (GMO) labeling regulations. In this study, we reported the characterization of the exogenous integration of GM Huanong No. 1 papaya by means of conventional polymerase chain reaction (PCR) and thermal asymmetric interlaced (TAIL)-PCR strategies. The results suggested that one intact copy of the initial construction was integrated in the papaya genome and which probably resulted in one deletion (38 bp in size) of the host genomic DNA. Also, one unintended insertion of a 92 bp truncated NptII fragment was observed at the 5' end of the exogenous insert. Furthermore, we revealed its 5' and 3' flanking sequences between the insert DNA and the papaya genomic DNA, and developed the event-specific qualitative and quantitative PCR assays for GM Huanong No. 1 papaya based on the 5' integration flanking sequence. The relative limit of detection (LOD) of the qualitative PCR assay was about 0.01% in 100 ng of total papaya genomic DNA, corresponding to about 25 copies of papaya haploid genome. In the quantitative PCR, the limits of detection and quantification (LOD and LOQ) were as low as 12.5 and 25 copies of papaya haploid genome, respectively. In practical sample quantification, the quantified biases between the test and true values of three samples ranged from 0.44% to 4.41%. Collectively, we proposed that all of these results are useful for the identification and quantification of Huanong No. 1 papaya and its derivates.

Single Fluorescence Channel-based Multiplex Detection of Avian Influenza Virus by Quantitative PCR with Intercalating Dye

PubMed Central

Ahberg, Christian D.; Manz, Andreas; Neuzil, Pavel

2015-01-01

Since its invention in 1985 the polymerase chain reaction (PCR) has become a well-established method for amplification and detection of segments of double-stranded DNA. Incorporation of fluorogenic probe or DNA intercalating dyes (such as SYBR Green) into the PCR mixture allowed real-time reaction monitoring and extraction of quantitative information (qPCR). Probes with different excitation spectra enable multiplex qPCR of several DNA segments using multi-channel optical detection systems. Here we show multiplex qPCR using an economical EvaGreen-based system with single optical channel detection. Previously reported non quantitative multiplex real-time PCR techniques based on intercalating dyes were conducted once the PCR is completed by performing melting curve analysis (MCA). The technique presented in this paper is both qualitative and quantitative as it provides information about the presence of multiple DNA strands as well as the number of starting copies in the tested sample. Besides important internal control, multiplex qPCR also allows detecting concentrations of more than one DNA strand within the same sample. Detection of the avian influenza virus H7N9 by PCR is a well established method. Multiplex qPCR greatly enhances its specificity as it is capable of distinguishing both haemagglutinin (HA) and neuraminidase (NA) genes as well as their ratio. PMID:26088868

Event specific qualitative and quantitative polymerase chain reaction detection of genetically modified MON863 maize based on the 5'-transgene integration sequence.

PubMed

Yang, Litao; Xu, Songci; Pan, Aihu; Yin, Changsong; Zhang, Kewei; Wang, Zhenying; Zhou, Zhigang; Zhang, Dabing

2005-11-30

Because of the genetically modified organisms (GMOs) labeling policies issued in many countries and areas, polymerase chain reaction (PCR) methods were developed for the execution of GMO labeling policies, such as screening, gene specific, construct specific, and event specific PCR detection methods, which have become a mainstay of GMOs detection. The event specific PCR detection method is the primary trend in GMOs detection because of its high specificity based on the flanking sequence of the exogenous integrant. This genetically modified maize, MON863, contains a Cry3Bb1 coding sequence that produces a protein with enhanced insecticidal activity against the coleopteran pest, corn rootworm. In this study, the 5'-integration junction sequence between the host plant DNA and the integrated gene construct of the genetically modified maize MON863 was revealed by means of thermal asymmetric interlaced-PCR, and the specific PCR primers and TaqMan probe were designed based upon the revealed 5'-integration junction sequence; the conventional qualitative PCR and quantitative TaqMan real-time PCR detection methods employing these primers and probes were successfully developed. In conventional qualitative PCR assay, the limit of detection (LOD) was 0.1% for MON863 in 100 ng of maize genomic DNA for one reaction. In the quantitative TaqMan real-time PCR assay, the LOD and the limit of quantification were eight and 80 haploid genome copies, respectively. In addition, three mixed maize samples with known MON863 contents were detected using the established real-time PCR systems, and the ideal results indicated that the established event specific real-time PCR detection systems were reliable, sensitive, and accurate.

[Method validation according to ISO 15189 and SH GTA 04: application for the detection of KRAS mutations using PCR TaqMan assay].

PubMed

Harlé, Alexandre; Dubois, Cindy; Rouyer, Marie; Merlin, Jean-Louis

2013-01-01

Since January 16(th) 2010, the French legislation requires that the medical laboratories must be accredited according to ISO 15189 standards. Thus, all medical laboratories in France must be accredited for at least part of their biological tests before the end of October 2013. Molecular biology tests are also concerned by the accreditation. Validation of molecular biology methods is made difficult, for reasons related to the methods, but also by the type of analytes that are basically rare. This article describes the validation of the qualitative detection of KRAS mutations in metastatic colorectal cancer using TaqMan PCR according to ISO 15189 and to the technical guide for accreditation in Human Health, SH-GTA-04, edited by the COFRAC.

Bottlenecks and opportunities for delivering integrated pediatric HIV services in Nepal

PubMed Central

Diese, Mulamba; Shrestha, Lexman; Pradhan, Birendra; Singh, Dipendra; Raaijmakers, Hendrikus; Kisesa, Annefrida; Chamla, Dick; Ntambue, Mukengeshayi Abel

2016-01-01

Background In children, integration of HIV in MNCH services has been shown to incr. ease uptake of early infant diagnosis. This article examines bottlenecks and opportunities for scaling up integrated pediatric HIV services in Nepal. Methods This is a descriptive study using both mixed qualitative and quantitative methods, conducted in January 2015 in 19 facilities in five regions of Nepal most affected by HIV epidemic. The qualitative methods comprised in-depth structured interviews with key informants (leadership of The National Center for AIDS and STD Control and National Public Health Laboratory, district management teams, medical officers in charge of health facilities and HIV clinics, frontline staff at antenatal care and HIV clinics and laboratory). The quantitative methods were used to abstract data of HIV-infected pregnant women seen between January and December 2014, HIV-exposed infants aged less than 12 months, and HIV infected children aged less than 15 years who were initiated HIV treatment from 2010 to 2014. Structured tools were used to collect data which were analysed using IBM SPSS. Results Of the 19 facilities assessed, 18(98%), 18(98%), 14(75%), and 11(58%) provided prevention of mother-to-child transmission (PMTCT), Expanded Program on Immunization (EPI), pediatric ART and nutrition rehabilitation services, respectively. However, only 1(5%) facility collected onsite dried blood spots (DBS) for PCR HIV testing and 6(32%) facilities provided counselling and referral for DBS. In 2014, of the 121 HIV-exposed infants recorded, only 21(17%) received PCR test. The median turnaround time of the PCR test results was 54 days. Of the 21 records with PCR test, 11(52.5%) were from PMTCT clinics, 7(33%) from Nutritional rehabilitation clinics, and 3(14.5%) from pediatric outpatient clinic. Conversely, 934 children were initiated ART between 2010 and 2014, of which 5% were infants and 29% aged between 1 and 5 years. 298(32%) had comorbidities of which 64% had malnutrition. A total of 534(57%) had tuberculosis (TB) status assessed of which 58(11%) had active TB. Infants had lowest retention (63%), high mortality (17.4%), and loss to follow-up (10.9%). Conclusion Few facilities collect DBS and few children receive PCR tests with limited linkage to ART. This has led to late ART initiation, comorbidities, including TB coinfections and poor outcomes. The results indicate that there are opportunities for improving HIV case finding among HIV-exposed infants in PMTCT, EPI, TB, and nutrition services if provider initiated testing and counselling at the point of service delivery is institutionalized in these settings. PMID:26945139

Development and Validation of Broad-Range Qualitative and Clade-Specific Quantitative Molecular Probes for Assessing Mercury Methylation in the Environment.

PubMed

Christensen, Geoff A; Wymore, Ann M; King, Andrew J; Podar, Mircea; Hurt, Richard A; Santillan, Eugenio U; Soren, Ally; Brandt, Craig C; Brown, Steven D; Palumbo, Anthony V; Wall, Judy D; Gilmour, Cynthia C; Elias, Dwayne A

2016-10-01

Two genes, hgcA and hgcB, are essential for microbial mercury (Hg) methylation. Detection and estimation of their abundance, in conjunction with Hg concentration, bioavailability, and biogeochemistry, are critical in determining potential hot spots of methylmercury (MeHg) generation in at-risk environments. We developed broad-range degenerate PCR primers spanning known hgcAB genes to determine the presence of both genes in diverse environments. These primers were tested against an extensive set of pure cultures with published genomes, including 13 Deltaproteobacteria, nine Firmicutes, and nine methanogenic Archaea genomes. A distinct PCR product at the expected size was confirmed for all hgcAB(+) strains tested via Sanger sequencing. Additionally, we developed clade-specific degenerate quantitative PCR (qPCR) primers that targeted hgcA for each of the three dominant Hg-methylating clades. The clade-specific qPCR primers amplified hgcA from 64%, 88%, and 86% of tested pure cultures of Deltaproteobacteria, Firmicutes, and Archaea, respectively, and were highly specific for each clade. Amplification efficiencies and detection limits were quantified for each organism. Primer sensitivity varied among species based on sequence conservation. Finally, to begin to evaluate the utility of our primer sets in nature, we tested hgcA and hgcAB recovery from pure cultures spiked into sand and soil. These novel quantitative molecular tools designed in this study will allow for more accurate identification and quantification of the individual Hg-methylating groups of microorganisms in the environment. The resulting data will be essential in developing accurate and robust predictive models of Hg methylation potential, ideally integrating the geochemistry of Hg methylation to the microbiology and genetics of hgcAB IMPORTANCE: The neurotoxin methylmercury (MeHg) poses a serious risk to human health. MeHg production in nature is associated with anaerobic microorganisms. The recent discovery of the Hg-methylating gene pair, hgcA and hgcB, has allowed us to design and optimize molecular probes against these genes within the genomic DNA for microorganisms known to methylate Hg. The protocols designed in this study allow for both qualitative and quantitative assessments of pure-culture or environmental samples. With these protocols in hand, we can begin to study the distribution of Hg-methylating organisms in nature via a cultivation-independent strategy. Copyright © 2016 Christensen et al.

Using multiple PCR and CE with chemiluminescence detection for simultaneous qualitative and quantitative analysis of genetically modified organism.

PubMed

Guo, Longhua; Qiu, Bin; Chi, Yuwu; Chen, Guonan

2008-09-01

In this paper, an ultrasensitive CE-CL detection system coupled with a novel double-on-column coaxial flow detection interface was developed for the detection of PCR products. A reliable procedure based on this system had been demonstrated for qualitative and quantitative analysis of genetically modified organism-the detection of Roundup Ready Soy (RRS) samples was presented as an example. The promoter, terminator, function and two reference genes of RRS were amplified with multiplex PCR simultaneously. After that, the multiplex PCR products were labeled with acridinium ester at the 5'-terminal through an amino modification and then analyzed by the proposed CE-CL system. Reproducibility of analysis times and peak heights for the CE-CL analysis were determined to be better than 0.91 and 3.07% (RSD, n=15), respectively, for three consecutive days. It was shown that this method could accurately and qualitatively detect RRS standards and the simulative samples. The evaluation in terms of quantitative analysis of RRS provided by this new method was confirmed by comparing our assay results with those of the standard real-time quantitative PCR (RT-QPCR) using SYBR Green I dyes. The results showed a good coherence between the two methods. This approach demonstrated the possibility for accurate qualitative and quantitative detection of GM plants in a single run.

A preamplification approach to GMO detection in processed foods.

PubMed

Del Gaudio, S; Cirillo, A; Di Bernardo, G; Galderisi, U; Cipollaro, M

2010-03-01

DNA is widely used as a target for GMO analysis because of its stability and high detectability. Real-time PCR is the method routinely used in most analytical laboratories due to its quantitative performance and great sensitivity. Accurate DNA detection and quantification is dependent on the specificity and sensitivity of the amplification protocol as well as on the quality and quantity of the DNA used in the PCR reaction. In order to enhance the sensitivity of real-time PCR and consequently expand the number of analyzable target genes, we applied a preamplification technique to processed foods where DNA can be present in low amounts and/or in degraded forms thereby affecting the reliability of qualitative and quantitative results. The preamplification procedure utilizes a pool of primers targeting genes of interest and is followed by real-time PCR reactions specific for each gene. An improvement of Ct values was found comparing preamplified vs. non-preamplified DNA. The strategy reported in the present study will be also applicable to other fields requiring quantitative DNA testing by real-time PCR.

Characterization and application of a quantitative DNA marker that discriminates sex in Chinook salmon (Oncorhynchus tshawytscha)

USGS Publications Warehouse

Clifton, D.R.; Rodriguez, R.J.

1997-01-01

A qualitative male-specific DNA marker (OT-24) was amplified by spPCR (single-primer polymerase chain reaction) from chinook salmon (Oncorhynchus tshawytscha) DNA along with several non-sex-linked products. The termini of the male-specific product were sequenced, and a pair of PeR primers were constructed for marker-specific PCR amplification. Dual primer PCR (dpPCR), with the marker-specific primers, amplified a product from both nudes and females. The amount of dpPCR product amplified from males was at least 100-fold greater than that from females. The quantitative difference between males and females was consistent among geographically distinct populations from western U.S. rivers. In addition, DNA sequence analysis indicated that OT-24 was highly conserved among geographically distinct salmon populations. The qualitative spPCR product segregated through several genetic crosses indicating equal sex ratios among progeny. Identification of the male and female juveniles by dpPCR was consistent with the spPCR analysis. There was no tissue specificity observed by spPCR or dpPCR analysis of this marker. A rapid DNA extraction method and dpPCR analysis were used to nonlethally determine sex ratios in wild spring chinook salmon adults, withheld for genetic and behavioral studies, prior to their development of gross sexual differences in their external morphology.

Characterization and application of a quantitative DNA marker that discriminates sex in chinook salmon (Oncorhynchus tshawytscha)

USGS Publications Warehouse

Clifton, D.R.; Rodriguez, R.J.

1997-01-01

A qualitative male-specific DNA marker (OT-24) was amplified by spPCR (single-primer polymerase chain reaction) from chinook salmon (Oncorhynchus tshawytscha) DNA along with several non-sex-linked products. The termini of the male-specific product were sequenced, and a pair of PeR primers were constructed for marker-specific PCR amplification. Dual primer PCR (dpPCR), with the marker-specific primers, amplified a product from both nudes and females. The amount of dpPCR product amplified from males was at least 100-fold greater than that from females. The quantitative difference between males and females was consistent among geographically distinct populations from western U.S. rivers. In addition, DNA sequence analysis indicated that OT-24 was highly conserved among geographically distinct salmon populations. The qualitative spPCR product segregated through several genetic crosses indicating equal sex ratios among progeny. Identification of the male and female juveniles by dpPCR was consistent with the spPCR analysis. There was no tissue specificity observed by spPCR or dpPCR analysis of this marker. A rapid DNA extraction method and dpPCR analysis were used to nonlethally determine sex ratios in wild spring chinook salmon adults, withheld for genetic and behavioral studies, prior to their development of gross sexual differences in their external morphology.

Comparison of oral fluid collection methods for the molecular detection of hepatitis B virus.

PubMed

Portilho, M M; Mendonça, Acf; Marques, V A; Nabuco, L C; Villela-Nogueira, C A; Ivantes, Cap; Lewis-Ximenez, L L; Lampe, E; Villar, L M

2017-11-01

This study aims to compare the efficiency of four oral fluid collection methods (Salivette, FTA Card, spitting and DNA-Sal) to detect HBV DNA by qualitative PCR. Seventy-four individuals (32 HBV reactive and 42 with no HBV markers) donated serum and oral fluid. In-house qualitative PCR to detect HBV was used for both samples and commercial quantitative PCR for serum. HBV DNA was detected in all serum samples from HBV-infected individuals, and it was not detected in control group. HBV DNA from HBV group was detected in 17 samples collected with Salivette device, 16 samples collected by FTA Card device, 16 samples collected from spitting and 13 samples collected by DNA-Sal device. Samples that corresponded to a higher viral load in their paired serum sample could be detected using all oral fluid collection methods, but Salivette collection device yielded the largest numbers of positive samples and had a wide range of viral load that was detected. It was possible to detect HBV DNA using all devices tested, but higher number of positive samples was observed when samples were collected using Salivette device, which shows high concordance to viral load observed in the paired serum samples. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd. All rights reserved.

Occurrence of a Sequence in Marine Cyanophages Similar to That of T4 g20 and Its Application to PCR-Based Detection and Quantification Techniques†

PubMed Central

Fuller, Nicholas J.; Wilson, William H.; Joint, Ian R.; Mann, Nicholas H.

1998-01-01

Viruses are ubiquitous components of marine ecosystems and are known to infect unicellular phycoerythrin-containing cyanobacteria belonging to the genus Synechococcus. A conserved region from the cyanophage genome was identified in three genetically distinct cyanomyoviruses, and a sequence analysis revealed that this region exhibited significant similarity to a gene encoding a capsid assembly protein (gp20) from the enteric coliphage T4. The results of a comparison of gene 20 sequences from three cyanomyoviruses and T4 allowed us to design two degenerate PCR primers, CPS1 and CPS2, which specifically amplified a 165-bp region from the majority of cyanomyoviruses tested. A competitive PCR (cPCR) analysis revealed that cyanomyovirus strains could be accurately enumerated, and it was demonstrated that quantification was log-linear over ca. 3 orders of magnitude. Different calibration curves were obtained for each of the three cyanomyovirus strains tested; consequently, cPCR performed with primers CPS1 and CPS2 could lead to substantial inaccuracies in estimates of phage abundance in natural assemblages. Further sequence analysis of cyanomyovirus gene 20 homologs would be necessary in order to design primers which do not exhibit phage-to-phage variability in priming efficiency. It was demonstrated that PCR products of the correct size could be amplified from seawater samples following 100× concentration and even directly without any prior concentration. Hence, the use of degenerate primers in PCR analyses of cyanophage populations should provide valuable data on the diversity of cyanophages in natural assemblages. Further optimization of procedures may ultimately lead to a sensitive assay which can be used to analyze natural cyanophage populations both quantitatively (by cPCR) and qualitatively following phylogenetic analysis of amplified products. PMID:9603813

Rapid diagnosis of common deletional α-thalassemia in the Chinese population by qPCR based on identical primer homologous fragments.

PubMed

Long, Ju

2016-05-01

In China, -(SEA), -α(3.7) and -α(4.2) are common deletional α-thalassemia alleles. Gap-PCR is the currently used detection method for these alleles, whose disadvantages include time-consuming procedure and increased potential for PCR product contamination. Therefore, this detection method needs to be improved. Based on identical-primer homologous fragments, a qPCR system was developed for deletional α-thalassemia genotyping, which was composed of a group of quantitatively-related primers and their corresponding probes plus two groups of qualitatively-related primers and their corresponding probes. In order to verify the accuracy of the qPCR system, known genotype samples and random samples are employed. The standard curve result demonstrated that designed primers and probes all yielded good amplification efficiency. In the tests of known genotype samples and random samples, sample detection results were consistent with verification results. In detecting αα, -(SEA), -α(3.7) and -α(4.2) alleles, deletional α-thalassemia alleles are accurately detected by this method. In addition, this method is provided with a wider detection range, greater speed and reduced PCR product contamination risk when compared with current common gap-PCR detection reagents. Copyright © 2016 Elsevier B.V. All rights reserved.

PCR und ELISA - Alternativen zum Maustest für die Analyse des Botulismus-Neurotoxin-C1 Giftbildungspotentiales in Umweltproben? [PCR and ELISA - in vitro alternatives to the mouse-bioassay for assessing the botulinum-neurotoxin-C1 production potential in environmental samples?

USGS Publications Warehouse

Zechmeister, T.C.; Farnleitner, A.H.; Rocke, T.E.; Pittner, F.; Rosengarten, R.; Mach, R.L.; Herzig, A.; Kirschner, A.K.T.

2002-01-01

Botulism is one of the most important bird diseases world-wide and is caused by the intoxication with Botulinum-Neurotoxin-C1 (BoNt-C1), which is produced by toxigenic clostridia under appropriate conditions. Avian botulism leads regularly to large losses among the migrating bird populations breeding and resting at the saltwater pools of the Austrian national park Neusiedler See-Seewinkel. Despite of its ethical dubiousness and its high technical expense the mouse-bioassay is still used as the routine standard method for the detection of BoNt-C1. According to the 3R-concept, in vitro alternative methods for the qualitative detection of BoNt-C1 (immunostick-ELISA) and a corresponding BoNt-C1 gene fragment (nested-PCR) were established. In order to estimate the BoNt-C1 production potential the methods were tested with sediment samples from different saltwater pools subjected to cultivation conditions appropriate for in vitro BoNt-C1-production. With the mouse-bioassay, 52 out of 77 samples were found to have a positive toxin production potential. The immunostick-ELISA showed a similar sensitivity as the mouse-bioassay and exhibited a highly significant positive correlation (r=0.94; p<0.001) with the mouse-bioassay in detecting BoNt-C1. The nested-PCR approach revealed higher numbers of positive BoNt-C1 gene fragment detections as compared to the direct toxin analysis approaches. A weak correlation (r=0.21; p=0.07) with the mouse-bioassay was discernible, no correlation was found with the immunostick-ELISA (r=0.09; p=0.46). Obviously, the PCR approach detected the BoNt-C1 gene fragment in some of the samples where no toxin expression has occurred. Thus it is suggested that the qualitative immunostick-ELISA represents a potential in vitro alternative to the mouse-bioassay for assessing the BoNt-C1 production potential in environmental samples. In contrast, qualitative BoNt-C1 gene fragment detection via PCR led to an overestimation of the actual toxin production potential.

[Examination of processed vegetable foods for the presence of common DNA sequences of genetically modified tomatoes].

PubMed

Kitagawa, Mamiko; Nakamura, Kosuke; Kondo, Kazunari; Ubukata, Shoji; Akiyama, Hiroshi

2014-01-01

The contamination of processed vegetable foods with genetically modified tomatoes was investigated by the use of qualitative PCR methods to detect the cauliflower mosaic virus 35S promoter (P35S) and the kanamycin resistance gene (NPTII). DNA fragments of P35S and NPTII were detected in vegetable juice samples, possibly due to contamination with the genomes of cauliflower mosaic virus infecting juice ingredients of Brassica species and soil bacteria, respectively. Therefore, to detect the transformation construct sequences of GM tomatoes, primer pairs were designed for qualitative PCR to specifically detect the border region between P35S and NPTII, and the border region between nopaline synthase gene promoter and NPTII. No amplification of the targeted sequences was observed using genomic DNA purified from the juice ingredients. The developed qualitative PCR method is considered to be a reliable tool to check contamination of products with GM tomatoes.

Event-specific qualitative and quantitative PCR detection of the GMO carnation (Dianthus caryophyllus) variety Moonlite based upon the 5'-transgene integration sequence.

PubMed

Li, P; Jia, J W; Jiang, L X; Zhu, H; Bai, L; Wang, J B; Tang, X M; Pan, A H

2012-04-27

To ensure the implementation of genetically modified organism (GMO)-labeling regulations, an event-specific detection method was developed based on the junction sequence of an exogenous integrant in the transgenic carnation variety Moonlite. The 5'-transgene integration sequence was isolated by thermal asymmetric interlaced PCR. Based upon the 5'-transgene integration sequence, the event-specific primers and TaqMan probe were designed to amplify the fragments, which spanned the exogenous DNA and carnation genomic DNA. Qualitative and quantitative PCR assays were developed employing the designed primers and probe. The detection limit of the qualitative PCR assay was 0.05% for Moonlite in 100 ng total carnation genomic DNA, corresponding to about 79 copies of the carnation haploid genome; the limit of detection and quantification of the quantitative PCR assay were estimated to be 38 and 190 copies of haploid carnation genomic DNA, respectively. Carnation samples with different contents of genetically modified components were quantified and the bias between the observed and true values of three samples were lower than the acceptance criterion (<25%) of the GMO detection method. These results indicated that these event-specific methods would be useful for the identification and quantification of the GMO carnation Moonlite.

Limitations of Rapid Diagnostic Testing in Patients with Suspected Malaria: A Diagnostic Accuracy Evaluation from Swaziland, a Low-Endemicity Country Aiming for Malaria Elimination

PubMed Central

Ranadive, Nikhil; Kunene, Simon; Darteh, Sarah; Ntshalintshali, Nyasatu; Nhlabathi, Nomcebo; Dlamini, Nomcebo; Chitundu, Stanley; Saini, Manik; Murphy, Maxwell; Soble, Adam; Schwartz, Alanna; Greenhouse, Bryan

2017-01-01

Abstract Background. The performance of Plasmodium falciparum–specific histidine-rich protein 2–based rapid diagnostic tests (RDTs) to evaluate suspected malaria in low-endemicity settings has not been well characterized. Methods. Using dried blood spot samples from patients with suspected malaria at 37 health facilities from 2012 to 2014 in the low-endemicity country of Swaziland, we investigated the diagnostic accuracy of histidine-rich protein 2–based RDTs using qualitative polymerase chain reaction (PCR) (nested PCR targeting the cytochrome b gene) and quantitative PCR as reference standards. To explore reasons for false-negative and/or false-positive results, we used pfhrp2/3-specific PCR and logistic regression analyses of potentially associated epidemiological factors. Results. From 1353 patients, 93.0% of RDT-positive (n = 185) and 31.2% of RDT-negative samples (n = 340) were available and selected for testing. Compared with nested PCR, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of RDTs were 51.7%, 94.1%, 67.3%, and 89.1%, respectively. After exclusion of samples with parasite densities <100/μL, which accounted for 75.7% of false-negative results and 33.3% of PCR-detectable infections, the sensitivity, specificity, PPV, and NPV were 78.8%, 93.7%, 62.3%, and 97.1%. Deletions of pfhrp2 were not detected. False-positivity was more likely during the second year and was not associated with demographics, recent malaria, health facility testing characteristics, or potential DNA degradation. Conclusions. In the low-transmission setting of Swaziland, we demonstrated low sensitivity of RDT for malaria diagnosis, owing to an unexpectedly high proportion of low-density infection among symptomatic subjects. The PPV was also low, requiring further investigation. A more accurate point-of-care diagnostic may be needed to support malaria elimination efforts. PMID:28369268

Limitations of Rapid Diagnostic Testing in Patients with Suspected Malaria: A Diagnostic Accuracy Evaluation from Swaziland, a Low-Endemicity Country Aiming for Malaria Elimination.

PubMed

Ranadive, Nikhil; Kunene, Simon; Darteh, Sarah; Ntshalintshali, Nyasatu; Nhlabathi, Nomcebo; Dlamini, Nomcebo; Chitundu, Stanley; Saini, Manik; Murphy, Maxwell; Soble, Adam; Schwartz, Alanna; Greenhouse, Bryan; Hsiang, Michelle S

2017-05-01

The performance of Plasmodium falciparum-specific histidine-rich protein 2-based rapid diagnostic tests (RDTs) to evaluate suspected malaria in low-endemicity settings has not been well characterized. Using dried blood spot samples from patients with suspected malaria at 37 health facilities from 2012 to 2014 in the low-endemicity country of Swaziland, we investigated the diagnostic accuracy of histidine-rich protein 2-based RDTs using qualitative polymerase chain reaction (PCR) (nested PCR targeting the cytochrome b gene) and quantitative PCR as reference standards. To explore reasons for false-negative and/or false-positive results, we used pfhrp2/3-specific PCR and logistic regression analyses of potentially associated epidemiological factors. From 1353 patients, 93.0% of RDT-positive (n = 185) and 31.2% of RDT-negative samples (n = 340) were available and selected for testing. Compared with nested PCR, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of RDTs were 51.7%, 94.1%, 67.3%, and 89.1%, respectively. After exclusion of samples with parasite densities <100/μL, which accounted for 75.7% of false-negative results and 33.3% of PCR-detectable infections, the sensitivity, specificity, PPV, and NPV were 78.8%, 93.7%, 62.3%, and 97.1%. Deletions of pfhrp2 were not detected. False-positivity was more likely during the second year and was not associated with demographics, recent malaria, health facility testing characteristics, or potential DNA degradation. In the low-transmission setting of Swaziland, we demonstrated low sensitivity of RDT for malaria diagnosis, owing to an unexpectedly high proportion of low-density infection among symptomatic subjects. The PPV was also low, requiring further investigation. A more accurate point-of-care diagnostic may be needed to support malaria elimination efforts. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America.

Development and in-house validation of the event-specific polymerase chain reaction detection methods for genetically modified soybean MON89788 based on the cloned integration flanking sequence.

PubMed

Liu, Jia; Guo, Jinchao; Zhang, Haibo; Li, Ning; Yang, Litao; Zhang, Dabing

2009-11-25

Various polymerase chain reaction (PCR) methods were developed for the execution of genetically modified organism (GMO) labeling policies, of which an event-specific PCR detection method based on the flanking sequence of exogenous integration is the primary trend in GMO detection due to its high specificity. In this study, the 5' and 3' flanking sequences of the exogenous integration of MON89788 soybean were revealed by thermal asymmetric interlaced PCR. The event-specific PCR primers and TaqMan probe were designed based upon the revealed 5' flanking sequence, and the qualitative and quantitative PCR assays were established employing these designed primers and probes. In qualitative PCR, the limit of detection (LOD) was about 0.01 ng of genomic DNA corresponding to 10 copies of haploid soybean genomic DNA. In the quantitative PCR assay, the LOD was as low as two haploid genome copies, and the limit of quantification was five haploid genome copies. Furthermore, the developed PCR methods were in-house validated by five researchers, and the validated results indicated that the developed event-specific PCR methods can be used for identification and quantification of MON89788 soybean and its derivates.

Kinetics of Poliovirus Shedding following Oral Vaccination as Measured by Quantitative Reverse Transcription-PCR versus Culture

PubMed Central

Begum, Sharmin; Uddin, Md Jashim; Platts-Mills, James A.; Liu, Jie; Kirkpatrick, Beth D.; Chowdhury, Anwarul H.; Jamil, Khondoker M.; Haque, Rashidul; Petri, William A.; Houpt, Eric R.

2014-01-01

Amid polio eradication efforts, detection of oral polio vaccine (OPV) virus in stool samples can provide information about rates of mucosal immunity and allow estimation of the poliovirus reservoir. We developed a multiplex one-step quantitative reverse transcription-PCR (qRT-PCR) assay for detection of OPV Sabin strains 1, 2, and 3 directly in stool samples with an external control to normalize samples for viral quantity and compared its performance with that of viral culture. We applied the assay to samples from infants in Dhaka, Bangladesh, after the administration of trivalent OPV (tOPV) at weeks 14 and 52 of life (on days 0 [pre-OPV], +4, +11, +18, and +25 relative to vaccination). When 1,350 stool samples were tested, the sensitivity and specificity of the quantitative PCR (qPCR) assay were 89 and 91% compared with culture. A quantitative relationship between culture+/qPCR+ and culture−/qPCR+ stool samples was observed. The kinetics of shedding revealed by qPCR and culture were similar. qPCR quantitative cutoffs based on the day +11 or +18 stool samples could be used to identify the culture-positive shedders, as well as the long-duration or high-frequency shedders. Interestingly, qPCR revealed that a small minority (7%) of infants contributed the vast majority (93 to 100%) of the total estimated viral excretion across all subtypes at each time point. This qPCR assay for OPV can simply and quantitatively detect all three Sabin strains directly in stool samples to approximate shedding both qualitatively and quantitatively. PMID:25378579

Value of Different Assays for Detection of Human Cytomegalovirus (HCMV) in Predicting the Development of HCMV Disease in Human Immunodeficiency Virus-Infected Patients

PubMed Central

Blank, Brian S. N.; Meenhorst, Pieter L.; Mulder, Jan Willem; Weverling, Gerrit Jan; Putter, Hein; Pauw, Wouter; van Dijk, Willemien C.; Smits, Paul; Lie-A-Ling, Sonja; Reiss, Peter; Lange, Joep M. A.

2000-01-01

In the present prospective study, five blood tests for detection of human cytomegalovirus (HCMV), nucleic acid sequence-based amplification (NASBA) for detection of early (immediate-early antigen) and late (pp67) mRNA, PCR for detection of HCMV DNA (DNA PCR), culture, and pp65 antigenemia assay, and culture and DNA PCR of urine and throat swab specimens were compared for their abilities to predict the development of disease caused by HCMV (HCMV disease). Of 101 human immunodeficiency virus (HIV)-infected patients with ≤100 CD4+ lymphocytes per mm3, 25 patients developed HCMV disease. The pp65 antigenemia assay (sensitivity, 50%; specificity, 89%) and DNA PCR of blood (sensitivity, 69%; specificity, 75%) were most accurate in predicting the development of HCMV disease within the next 12 months. Both blood culture and late pp67 mRNA NASBA had high specificities (91 and 90%, respectively) but low sensitivities (25 and 13%, respectively). The sensitivities of urine culture, DNA PCR, throat swab specimen culture, DNA PCR, and NASBA of blood for detection of the immediate-early antigen were 73, 87, 53, 67, and 63%, respectively, and the specificities were 58, 46, 76, 60, and 72%, respectively. The positive predictive values of all tests however, were low and did not exceed 50%. In conclusion, virological screening by these qualitative assays for detection of HCMV is of limited value for prediction of the development of HCMV disease in HIV-infected patients. PMID:10655346

Multilaboratory comparison of hepatitis C virus viral load assays.

PubMed

Caliendo, A M; Valsamakis, A; Zhou, Y; Yen-Lieberman, B; Andersen, J; Young, S; Ferreira-Gonzalez, A; Tsongalis, G J; Pyles, R; Bremer, J W; Lurain, N S

2006-05-01

We report a multilaboratory evaluation of hepatitis C virus (HCV) viral load assays to determine their linear range, reproducibility, subtype detection, and agreement. A panel of HCV RNA samples ranging in nominal concentration from 1.0 to 7.0 log10 IU/ml was constructed by diluting a clinical specimen (genotype 1b). Replicates of the panel were tested in multiple laboratories using the Abbott TaqMan analyte-specific reagent (Abbott reverse transcription-PCR [RT-PCR]), Roche TaqMan RUO (Roche RT-PCR), Roche Amplicor Monitor HCV 2.0 (Roche Monitor), and Bayer VERSANT HCV RNA 3.0 (Bayer bDNA) assays. Bayer bDNA-negative specimens were tested reflexively using the Bayer VERSANT HCV RNA qualitative assay (Bayer TMA). Abbott RT-PCR and Roche RT-PCR detected all 28 replicates with a concentration of 1.0 log10 IU/ml and were linear to 7.0 log10 IU/ml. Roche Monitor and Bayer bDNA detected 27 out of 28 and 13 out of 28 replicates, respectively, of 3.0 log10 IU/ml. Bayer TMA detected all seven replicates with 1.0 log10 IU/ml. Bayer bDNA was the most reproducible of the four assays. The mean viral load values for panel members in the linear ranges of the assays were within 0.5 log10 for the different tests. Eighty-nine clinical specimens of various genotypes (1 through 4) were tested in the Bayer bDNA, Abbott RT-PCR, and Roche RT-PCR assays. For Abbott RT-PCR, mean viral load values were 0.61 to 0.96 log10 greater than the values for Bayer bDNA assay for samples with genotype 1, 2, or 3 samples and 0.08 log10 greater for genotype 4 specimens. The Roche RT-PCR assay gave mean viral load values that were 0.28 to 0.82 log10 greater than those obtained with the Bayer bDNA assay for genotype 1, 2, and 3 samples. However, for genotype 4 samples the mean viral load value obtained with the Roche RT-PCR assay was, on average, 0.15 log10 lower than that of the Bayer bDNA. Based on these data, we conclude that the sensitivity and linear range of the Abbott and Roche RT-PCR assays enable them to be used for HCV diagnostics and therapeutic monitoring. However, the differences in the viral load values obtained with the different assays underscore the importance of using one assay when monitoring response to therapy.

Development and validation of duplex, triplex, and pentaplex real-time PCR screening assays for the detection of genetically modified organisms in food and feed.

PubMed

Huber, Ingrid; Block, Annette; Sebah, Daniela; Debode, Frédéric; Morisset, Dany; Grohmann, Lutz; Berben, Gilbert; Stebih, Dejan; Milavec, Mojca; Zel, Jana; Busch, Ulrich

2013-10-30

Worldwide, qualitative methods based on PCR are most commonly used as screening tools for genetically modified material in food and feed. However, the increasing number and diversity of genetically modified organisms (GMO) require effective methods for simultaneously detecting several genetic elements marking the presence of transgenic events. Herein we describe the development and validation of a pentaplex, as well as complementary triplex and duplex real-time PCR assays, for the detection of the most common screening elements found in commercialized GMOs: P-35S, T-nos, ctp2-cp4-epsps, bar, and pat. The use of these screening assays allows the coverage of many GMO events globally approved for commercialization. Each multiplex real-time PCR assay shows high specificity and sensitivity with an absolute limit of detection below 20 copies for the targeted sequences. We demonstrate by intra- and interlaboratory tests that the assays are robust as well as cost- and time-effective for GMO screening if applied in routine GMO analysis.

The use of digital PCR to improve the application of quantitative molecular diagnostic methods for tuberculosis.

PubMed

Devonshire, Alison S; O'Sullivan, Denise M; Honeyborne, Isobella; Jones, Gerwyn; Karczmarczyk, Maria; Pavšič, Jernej; Gutteridge, Alice; Milavec, Mojca; Mendoza, Pablo; Schimmel, Heinz; Van Heuverswyn, Fran; Gorton, Rebecca; Cirillo, Daniela Maria; Borroni, Emanuele; Harris, Kathryn; Barnard, Marinus; Heydenrych, Anthenette; Ndusilo, Norah; Wallis, Carole L; Pillay, Keshree; Barry, Thomas; Reddington, Kate; Richter, Elvira; Mozioğlu, Erkan; Akyürek, Sema; Yalçınkaya, Burhanettin; Akgoz, Muslum; Žel, Jana; Foy, Carole A; McHugh, Timothy D; Huggett, Jim F

2016-08-03

Real-time PCR (qPCR) based methods, such as the Xpert MTB/RIF, are increasingly being used to diagnose tuberculosis (TB). While qualitative methods are adequate for diagnosis, the therapeutic monitoring of TB patients requires quantitative methods currently performed using smear microscopy. The potential use of quantitative molecular measurements for therapeutic monitoring has been investigated but findings have been variable and inconclusive. The lack of an adequate reference method and reference materials is a barrier to understanding the source of such disagreement. Digital PCR (dPCR) offers the potential for an accurate method for quantification of specific DNA sequences in reference materials which can be used to evaluate quantitative molecular methods for TB treatment monitoring. To assess a novel approach for the development of quality assurance materials we used dPCR to quantify specific DNA sequences in a range of prototype reference materials and evaluated accuracy between different laboratories and instruments. The materials were then also used to evaluate the quantitative performance of qPCR and Xpert MTB/RIF in eight clinical testing laboratories. dPCR was found to provide results in good agreement with the other methods tested and to be highly reproducible between laboratories without calibration even when using different instruments. When the reference materials were analysed with qPCR and Xpert MTB/RIF by clinical laboratories, all laboratories were able to correctly rank the reference materials according to concentration, however there was a marked difference in the measured magnitude. TB is a disease where the quantification of the pathogen could lead to better patient management and qPCR methods offer the potential to rapidly perform such analysis. However, our findings suggest that when precisely characterised materials are used to evaluate qPCR methods, the measurement result variation is too high to determine whether molecular quantification of Mycobacterium tuberculosis would provide a clinically useful readout. The methods described in this study provide a means by which the technical performance of quantitative molecular methods can be evaluated independently of clinical variability to improve accuracy of measurement results. These will assist in ultimately increasing the likelihood that such approaches could be used to improve patient management of TB.

Analysis of a quantitative PCR assay for CMV infection in liver transplant recipients: an intent to find the optimal cut-off value.

PubMed

Martín-Dávila, P; Fortún, J; Gutiérrez, C; Martí-Belda, P; Candelas, A; Honrubia, A; Barcena, R; Martínez, A; Puente, A; de Vicente, E; Moreno, S

2005-06-01

Preemptive therapy required highly predictive tests for CMV disease. CMV antigenemia assay (pp65 Ag) has been commonly used for rapid diagnosis of CMV infection. Amplification methods for early detection of CMV DNA are under analysis. To compare two diagnostic methods for CMV infection and disease in this population: quantitative PCR (qPCR) performed in two different samples, plasma and leukocytes (PMNs) and using a commercial diagnostic test (COBAS Amplicor Monitor Test) versus pp65 Ag. Prospective study conducted in liver transplant recipients from February 2000 to February 2001. Analyses were performed on 164 samples collected weekly during early post-transplant period from 33 patients. Agreements higher than 78% were observed between the three assays. Optimal qPCR cut-off values were calculated using ROC curves for two specific antigenemia values. For antigenemia >or=10 positive cells, the optimal cut-off value for qPCR in plasma was 1330 copies/ml, with a sensitivity (S) of 58% and a specificity (E) of 98% and the optimal cut-off value for qPCR-cells was 713 copies/5x10(6) cells (S:91.7% and E:86%). Using a threshold of antigenemia >or=20 positive cells, the optimal cut-off values were 1330 copies/ml for qPCR-plasma (S 87%; E 98%) and 4755 copies/5x10(6) cells for qPCR-cells (S 87.5%; E 98%). Prediction values for the three assays were calculated in patients with CMV disease (9 pts; 27%). Considering the assays in a qualitative way, the most sensitive was CMV PCR in cells (S: 100%, E: 54%, PPV: 40%; NPV: 100%). Using specific cut-off values for disease detection the sensitivity, specificity, PPV and NPV for antigenemia >or=10 positive cells were: 89%; 83%; 67%; 95%, respectively. For qPCR-cells >or=713 copies/5x10(6) cells: 100%; 54%; 33% and 100% and for plasma-qPCR>or=1330 copies/ml: 78%, 77%, 47%, 89% respectively. Optimal cut-off for viral load performed in plasma and cells can be obtained for the breakpoint antigenemia value recommended for initiating preemptive therapy with high specificities and sensitivities. Diagnostic assays like CMV pp65 Ag and quantitative PCR for CMV have similar efficiency and could be recommended as methods of choice for diagnosis and monitoring of active CMV infection after transplantation.

Establishment and application of event-specific polymerase chain reaction methods for two genetically modified soybean events, A2704-12 and A5547-127.

PubMed

Li, Xiang; Pan, Liangwen; Li, Junyi; Zhang, Qigang; Zhang, Shuya; Lv, Rong; Yang, Litao

2011-12-28

For implementation of the issued regulations and labeling policies for genetically modified organism (GMO) supervision, the polymerase chain reaction (PCR) method has been widely used due to its high specificity and sensitivity. In particular, use of the event-specific PCR method based on the flanking sequence of transgenes has become the primary trend. In this study, both qualitative and quantitative PCR methods were established on the basis of the 5' flanking sequence of transgenic soybean A2704-12 and the 3' flanking sequence of transgenic soybean A5547-127, respectively. In qualitative PCR assays, the limits of detection (LODs) were 10 copies of haploid soybean genomic DNA for both A2704-12 and A5547-127. In quantitative real-time PCR assays, the LODs were 5 copies of haploid soybean genomic DNA for both A2704-12 and A5547-127, and the limits of quantification (LOQs) were 10 copies for both. Low bias and acceptable SD and RSD values were also achieved in quantification of four blind samples using the developed real-time PCR assays. In addition, the developed PCR assays for the two transgenic soybean events were used for routine analysis of soybean samples imported to Shanghai in a 6 month period from October 2010 to March 2011. A total of 27 lots of soybean from the United States and Argentina were analyzed: 8 lots from the Unites States were found to have the GM soybean A2704-12 event, and the GM contents were <1.5% in all eight analyzed lots. On the contrary, no GM soybean A5547-127 content was found in any of the eight lots. These results demonstrated that the established event-specific qualitative and quantitative PCR methods could be used effectively in routine identification and quantification of GM soybeans A2704-12 and A5547-127 and their derived products.

A Short Interspersed Nuclear Element (SINE)-Based Real-Time PCR Approach to Detect and Quantify Porcine Component in Meat Products.

PubMed

Zhang, Chi; Fang, Xin; Qiu, Haopu; Li, Ning

2015-01-01

Real-time PCR amplification of mitochondria gene could not be used for DNA quantification, and that of single copy DNA did not allow an ideal sensitivity. Moreover, cross-reactions among similar species were commonly observed in the published methods amplifying repetitive sequence, which hindered their further application. The purpose of this study was to establish a short interspersed nuclear element (SINE)-based real-time PCR approach having high specificity for species detection that could be used in DNA quantification. After massive screening of candidate Sus scrofa SINEs, one optimal combination of primers and probe was selected, which had no cross-reaction with other common meat species. LOD of the method was 44 fg DNA/reaction. Further, quantification tests showed this approach was practical in DNA estimation without tissue variance. Thus, this study provided a new tool for qualitative detection of porcine component, which could be promising in the QC of meat products.

Development and validation of broad-range qualitative and clade-specific quantitative molecular probes for assessing mercury methylation in the environment

DOE Office of Scientific and Technical Information (OSTI.GOV)

Christensen, Geoff A.; Wymore, Ann M.; King, Andrew J.

Two genes, hgcA and hgcB, are essential for microbial mercury (Hg)-methylation. Detection and estimation of their abundance, in conjunction with Hg concentration, bioavailability and biogeochemistry is critical in determining potential hot spots of methylmercury (MeHg) generation in at-risk environments. We developed broad-range degenerate PCR primers spanning known hgcAB genes to determine the presence of both genes in diverse environments. These primers were tested against an extensive set of pure cultures with published genomes, including 13 Deltaproteobacteria, nine Firmicutes, and nine methanogenic Archaea. A distinct PCR product at the expected size was confirmed for all hgcAB+ strains tested via Sanger sequencing.more » Additionally, we developed clade-specific degenerate quantitative primers (qPCR) that targeted hgcA for each of the three dominant Hg-methylating clades. The clade-specific qPCR primers amplified hgcA from 64%, 88% and 86% of tested pure cultures of Deltaproteobacteria, Firmicutes and Archaea, respectively, and were highly specific for each clade. Amplification efficiencies and detection limits were quantified for each organism. Primer sensitivity varied among species based on sequence conservation. Finally, to begin to evaluate the utility of our primer sets in nature, we tested hgcA and hgcAB recovery from pure cultures spiked into sand and soil. These novel quantitative molecular tools designed in this study will allow for more accurate identification and quantification of the individual Hg-methylating groups of microorganisms in the environment. Here, the resulting data will be essential in developing accurate and robust predictive models of Hg-methylation potential, ideally integrating the geochemistry of Hg methylation to the microbiology and genetics of hgcAB.« less

Development and validation of broad-range qualitative and clade-specific quantitative molecular probes for assessing mercury methylation in the environment

DOE PAGES

Christensen, Geoff A.; Wymore, Ann M.; King, Andrew J.; ...

2016-07-15

Two genes, hgcA and hgcB, are essential for microbial mercury (Hg)-methylation. Detection and estimation of their abundance, in conjunction with Hg concentration, bioavailability and biogeochemistry is critical in determining potential hot spots of methylmercury (MeHg) generation in at-risk environments. We developed broad-range degenerate PCR primers spanning known hgcAB genes to determine the presence of both genes in diverse environments. These primers were tested against an extensive set of pure cultures with published genomes, including 13 Deltaproteobacteria, nine Firmicutes, and nine methanogenic Archaea. A distinct PCR product at the expected size was confirmed for all hgcAB+ strains tested via Sanger sequencing.more » Additionally, we developed clade-specific degenerate quantitative primers (qPCR) that targeted hgcA for each of the three dominant Hg-methylating clades. The clade-specific qPCR primers amplified hgcA from 64%, 88% and 86% of tested pure cultures of Deltaproteobacteria, Firmicutes and Archaea, respectively, and were highly specific for each clade. Amplification efficiencies and detection limits were quantified for each organism. Primer sensitivity varied among species based on sequence conservation. Finally, to begin to evaluate the utility of our primer sets in nature, we tested hgcA and hgcAB recovery from pure cultures spiked into sand and soil. These novel quantitative molecular tools designed in this study will allow for more accurate identification and quantification of the individual Hg-methylating groups of microorganisms in the environment. Here, the resulting data will be essential in developing accurate and robust predictive models of Hg-methylation potential, ideally integrating the geochemistry of Hg methylation to the microbiology and genetics of hgcAB.« less

Establishing diagnostic cut-off criteria for the COBAS AmpliPrep/COBAS TaqMan HIV-1 Qualitative test through validation against the Amplicor DNA test v1.5 for infant diagnosis using dried blood spots.

PubMed

Maritz, Jean; Preiser, Wolfgang; van Zyl, Gert U

2012-02-01

As antibody testing cannot confirm HIV-1 infection in children less than 18 months of age, diagnosis in these children depends on nucleic acid testing. The COBAS(®) AmpliPrep/COBAS(®) TaqMan(®) (CAP/CTM, Roche(®) Molecular Systems, Inc., Branchburg, NJ) HIV-1 Qualitative test is a total nucleic acid real-time PCR assay utilising whole EDTA blood or dried blood spots (DBS), which recently replaced the Roche(®) AMPLICOR(®) DNA test v1.5 (Amplicor) as the diagnostic HIV PCR assay in many South African laboratories. For the Amplicor assay, stringent diagnostic criteria were previously formulated for the local population, and a comparison reported the CAP/CTM's sensitivity at 99.7% and specificity at 100% for both sample types compared to these Amplicor criteria. To validate the assay prior to introduction in our laboratory and to define stringent diagnostic cut-off criteria. Whole EDTA blood samples from patients younger than 18 months sent for routine HIV-1 diagnosis were tested by Amplicor, and positive results were confirmed from DBS. CAP/CTM assays were subsequently performed from DBS. The CAP/CTM had a sensitivity of 98.8% and a specificity of 97.1%, but a positive predictive value (PPV) of only 78.7% compared to the Amplicor assay. Samples positive by CAP/CTM but negative by Amplicor displayed poor amplification curves compared to concordant positive samples. Upon re-testing those with sufficient material available by CAP/CTM, all showed negative results. The decreased PPV may either be due to false positive CAP/CTM results, or increased sensitivity compared to the Amplicor assay. Criteria were formulated for defining presumed false-positive results. Copyright © 2011 Elsevier B.V. All rights reserved.

Have you tried spermine? A rapid and cost-effective method to eliminate dextran sodium sulfate inhibition of PCR and RT-PCR.

PubMed

Krych, Łukasz; Kot, Witold; Bendtsen, Katja M B; Hansen, Axel K; Vogensen, Finn K; Nielsen, Dennis S

2018-01-01

The Dextran Sulfate Sodium (DSS) induced colitis mouse model is commonly used to investigate human inflammatory bowel disease (IBD). Nucleic acid extracts originating from these animals are often contaminated with DSS, which is a strong inhibitor of many enzymatic based molecular biology reactions including PCR and reverse-transcription (RT). Methods for removing DSS from nucleic acids extracts exist for RNA, but no effective protocol for DNA or cDNA is currently available. However, spermine has previously been shown to be an effective agent for counteracting DSS inhibition of polynucleotide kinase, which led to the hypothesis, that spermine could be used to counteract DSS inhibition of PCR and RT. We investigated the means of adding spermine in an adequate concentration to PCR based protocols (including qPCR, two-step RT-qPCR, and amplicon sequencing library preparation) to remove DSS inhibition. Within the range up to 0.01g/L, spermine can be added to PCR/qPCR or RT prophylactically without a significant reduction of reaction efficiency. Addition of spermine at the concentration of 0.08g/L can be used to recover qualitative PCR signal inhibited by DSS in concentrations up to 0.32g/L. For optimal quantitative analysis, the concentration of spermine requires fine adjustment. Hence, we present here a simple fluorometric based method for adjusting the concentration of spermine ensuring an optimal efficiency of the reaction exposed to an unknown concentration of DSS. In conclusion, we demonstrate a cost effective and easy method to counteract DSS inhibition in PCR and two-step RT-qPCR. Fixed or fine-tuned concentrations of spermine can be administered depending on the qualitative or quantitative character of the analysis. Copyright © 2017 Elsevier B.V. All rights reserved.

Development of real-time PCR method for the detection and the quantification of a new endogenous reference gene in sugar beet "Beta vulgaris L.": GMO application.

PubMed

Chaouachi, Maher; Alaya, Akram; Ali, Imen Ben Haj; Hafsa, Ahmed Ben; Nabi, Nesrine; Bérard, Aurélie; Romaniuk, Marcel; Skhiri, Fethia; Saïd, Khaled

2013-01-01

KEY MESSAGE : Here, we describe a new developed quantitative real-time PCR method for the detection and quantification of a new specific endogenous reference gene used in GMO analysis. The key requirement of this study was the identification of a new reference gene used for the differentiation of the four genomic sections of the sugar beet (Beta vulgaris L.) (Beta, Corrollinae, Nanae and Procumbentes) suitable for quantification of genetically modified sugar beet. A specific qualitative polymerase chain reaction (PCR) assay was designed to detect the sugar beet amplifying a region of the adenylate transporter (ant) gene only from the species of the genomic section I of the genus Beta (cultivated and wild relatives) and showing negative PCR results for 7 species of the 3 other sections, 8 related species and 20 non-sugar beet plants. The sensitivity of the assay was 15 haploid genome copies (HGC). A quantitative real-time polymerase chain reaction (QRT-PCR) assay was also performed, having high linearity (R (2) > 0.994) over sugar beet standard concentrations ranging from 20,000 to 10 HGC of the sugar beet DNA per PCR. The QRT-PCR assay described in this study was specific and more sensitive for sugar beet quantification compared to the validated test previously reported in the European Reference Laboratory. This assay is suitable for GMO quantification in routine analysis from a wide variety of matrices.

Evaluation of a new automated Abbott RealTime MTB RIF/INH assay for qualitative detection of rifampicin/isoniazid resistance in pulmonary and extra-pulmonary clinical samples of Mycobacterium tuberculosis.

PubMed

Ruiz, Pilar; Causse, Manuel; Vaquero, Manuel; Gutierrez, Juan Bautista; Casal, Manuel

2017-01-01

A new automated real-time PCR assay for the detection of rifampicin (RIF) and isoniazid (INH) resistance in Mycobacterium tuberculosis (MTB) was evaluated. A total of 163 clinical samples (128 pulmonary and 35 extra-pulmonary) were processed using four PCR assay kits: Abbott RealTime MTB RIF/INH, Genotype MTBDRplus, Xpert/MTB RIF, and Anyplex MTB/MDR. The results of phenotypic drug-susceptibility testing using BACTECMGIT 960 were used as reference. The sensitivity and specificity of the new Abbott RealTime MTB RIF/INH assay in comparison with phenotypic testing was 96.3% (95%CI 87.32%-100%) for RIF and 100% (95%CI 99.3%-100%) for INH; the sensitivity was 78.8% (95%CI 66.8%-90.9%) and the specificity was 100% (95%CI 98.9%-100%). The Abbott RealTime MTB RIF/INH test could be a valid method for detecting the most common mutations in strains resistant to RIF and INH.

A Rare Case of Transfusion Transmission of Hepatitis A Virus to Two Patients with Haematological Disease

PubMed Central

da Silva, Suely Gonçalves Cordeiro; Leon, Luciane Almeida Amado; Alves, Gilda; Brito, Selma Magalhães; Sandes, Valcieny de Souza; Lima, Magda Maria Adorno Ferreira; Nogueira, Marta Colares; Tavares, Rita de Cássia Barbosa da Silva; Dobbin, Jane; Apa, Alexandre; de Paula, Vanessa Salete; Oliveira, Jaqueline Mendes de Oliveira; Pinto, Marcelo Alves; Ferreira Jr, Orlando da Costa; Motta, Iara de Jesus Ferreira

2016-01-01

Summary Background This paper describes the transmission of hepatitis A virus (HAV) to two blood recipients from a healthy donor that later presented to the blood bank with jaundice. Methods The RNA of HAV was detected by qualitative nested reverse transcription polymerase chain reaction (nested RT-PCR) and quantified by real-time RT-PCR. HAV RNA samples were genotyped by direct sequencing of PCR products. A sequence from a fragment of 168 bp from the VP1/2A HAV region was used to construct a phylogenetic tree. Case Report A 31-year-old male donor accepted for donation of a whole blood unit returned to the blood bank with clinical jaundice 20 days after donation. His serological and NAT tests were negative for HBV and HCV. Serological tests for HAV IgM and IgG were negative on donation sample but positive on follow-up sample, confirming donor's HAV acute infection. Both recipients of red blood cells (R1) and platelet concentrate (R2) from the same implicated donation were HAV IgM-negative and IgG-positive. Qualitative PCR was positive on samples from all three individuals and phylogenetic analysis of viruses proved HAV transmission to the two recipients of blood products. HAV viral load on donor follow-up sample and the platelet recipient was 1.3 and 1.5 × 103 IU/ml, respectively. The RBC recipient, also infected by HCV, was undergoing bone marrow transplantation and died from fulminant hepatitis, 26 days after the implicated HAV transfusion. Conclusion The blood donor, a garbage collector, spontaneously returned to the blood bank when developing jaundice. This highlights the importance of donor education to immediately report to blood banks of any signs and symptoms related to infectious disease developed after blood donation. The fact that one immunocompromised patient with HCV infection died from fulminant hepatitis after receiving a HAV-contaminated platelet transfusion underpins the importance of a HAV vaccination program for these group of patients. PMID:27226795

Prevalence of hepatitis-C virus genotypes and potential transmission risks in Malakand Khyber Pakhtunkhwa, Pakistan.

PubMed

Nazir, Nausheen; Jan, Muhammad Rasul; Ali, Amjad; Asif, Muhammad; Idrees, Muhammad; Nisar, Mohammad; Zahoor, Muhammad; Abd El-Salam, Naser M

2017-08-22

Hepatitis C virus (HCV) is a leading cause of chronic liver disease and frequently progresses towards liver cirrhosis and Hepatocellular Carcinoma (HCC). This study aimed to determine the prevalence of HCV genotypes and their association with possible transmission risks in the general population of Malakand Division. Sum of 570 serum samples were collected during March 2011 to January 2012 from suspected patients visited to different hospitals of Malakand. The suspected sera were tested using qualitative PCR and were then subjected to molecular genotype specific assay. Quantitative PCR was also performed for determination of pre-treatment viral load in confirmed positive patients. Out of 570 serum samples 316 sera were seen positive while 254 sera were found negative using qualitative PCR. The positive samples were then subjected to genotyping assay out of 316, type-specific PCR fragments were seen in 271 sera while 45 samples were found untypable genotypes. Genotype 3a was seen as a predominant genotype (63.3%) with a standard error of ±2.7%. Cramer's V statistic and Liklihood-Ratio statistical procedures are used to measure the strength and to test the association, respectively, between the dependent variable, genotype, and explanatory variables (e.g. gender, risk, age and area/districts). The dependent variable, genotype, is observed statistically significant association with variable risk factors. This implies that the genotype is highly dependent on how the patient was infected. In contrast, the other covariates, for example, gender, age, and district (area) no statistical significant association are observed. The association between gender-age indicates that the mean age of female was older by 10.5 ± 2.3 years with 95% confidence level using t-statistic. It was concluded from the present study that the predominant genotype was 3a in the infected population of Malakand. This study also highlights the high prevalence rate of untypable genotypes which an important issue of health care setup in Malakand and create complications in therapy of infected patients. Major mode of HCV transmission is multiple uses and re-uses of needles/injections. ISRCTN ISRCTN73824458. Registered: 28 September 2014.

Brief Report: High Sensitivity and Specificity of the Cepheid Xpert HIV-1 Qualitative Point-of-Care Test Among Newborns in Botswana.

PubMed

Ibrahim, Maryanne; Moyo, Sikhulile; Mohammed, Terence; Mupfumi, Lucy; Gaseitsiwe, Simani; Maswabi, Kenneth; Ajibola, Gbolahan; Gelman, Rebecca; Batlang, Oganne; Sakoi, Maureen; Auletta-Young, Chloe; Makhema, Joseph; Lockman, Shahin; Shapiro, Roger L

2017-08-15

HIV point-of-care (POC) testing allows for early infant HIV diagnosis and treatment, but POC accuracy at birth and in the setting of antiretroviral prophylaxis for the prevention of mother-to-child HIV transmission is unknown. We evaluated the Cepheid Xpert HIV-1 Qual POC test against the Roche Taqman HIV-1 DNA polymerase chain reaction (PCR) platform using dried blood spots from 15 HIV-infected and 75 HIV-exposed uninfected newborns. These infants were screened for HIV at <96 hours of life at 5 hospital maternity wards in Botswana; all infants received postexposure antiretroviral prophylaxis with single-dose nevirapine and zidovudine, and most mothers received 3-drug antiretroviral therapy in pregnancy and at delivery. Fourteen of the 15 PCR positive samples tested positive by Cepheid POC, yielding a sensitivity of 93.3% (95% confidence interval: 68.1 to 99.8). Baseline viral load among positive infants ranged from <40 to >10,000,000 copies/mL, with a median of 2403 copies/mL. The HIV RNA for the infant with false-negative POC testing was 1661 copies/mL. Of note, 2 infants with low HIV RNA (<40 and 272 copies/mL) were correctly identified as HIV positive by Cepheid POC. All the 75 PCR-negative samples tested negative by Cepheid POC, yielding a specificity of 100% (95% confidence interval: 96.1 to 100). Our study demonstrates high sensitivity and specificity for the Cepheid POC assay in the first week of life despite early infection and antiretroviral prophylaxis. This platform may be a useful approach for adding early infant HIV diagnosis to current testing programs.

Qualitative and quantitative distribution of PCV2 in wild boars and domestic pigs in Germany.

PubMed

Reiner, Gerald; Bronnert, Bastian; Hohloch, Corinna; Fresen, Christina; Haack, Ingo; Willems, Hermann; Reinacher, Manfred

2010-09-28

Porcine circovirus 2 (PCV2), the causative agent of postweaning multisystemic wasting syndrome (PMWS), has been detected in North American and European wild boars at prevalences arguing for high circulation rates among populations. Systematic data on the qualitative distribution of PCV2 infections and on PCVD (PCV2 diseases) in wild boars are rare, however, and quantitative data about viral loads are missing. To be able to judge the PCV2/PCVD situation in wild boars, evaluation of the nationwide qualitative and quantitative distribution of PCV2 and PCVD in Germany was the objective of the present study. Wild boar samples were compared with domestic pig samples of the same greater areas, including tonsils, lungs, spleen, Lnn. bronchiales and Lnn. mesenterici of 349 wild boars and 348 domestic pigs. All of the wild boars and 308 of the domestic pigs have been apparently free of PCVD, 40 of the domestic pigs had been rejected from slaughter due to health problems (i.e. wasting). Tissues were examined by pathohistology, immunohistology (IHC), nested PCR (nPCR and quantitative PCR (qPCR). One wild boar (0.3%) and 8.7% of the domestic pigs were classified as PCVD-affected, based on pathohistology and IHC. PCV2 DNA was detected in 63.1% and 45.4% of the wild boars by nPCR and qPCR, respectively, and in 100% and 98.8% of the domestic pigs. PCV2 loads differed significantly between wild boars (average: 10(2.8) PCV2 genomes/microg extracted sample DNA) and domestic pigs (average: 10(4.2) PCV2 genomes/microg of sample DNA). The qualitative detection of PCV2 DNA in tissues of wild boars and domestic pigs was abundant and not of any pathological relevance. The overall load of PCV2 in domestic pigs was relatively high and borderline with respect to PCVD, and there was no difference between apparently healthy pigs and pigs rejected from slaughter in this respect. Most of the wild boars were infected with PCV2 at loads less relevant for PCVD. (c) 2010 Elsevier B.V. All rights reserved.

Identification and quantification of three genetically modified insect resistant cotton lines using conventional and TaqMan real-time polymerase chain reaction methods.

PubMed

Yang, Litao; Pan, Aihu; Zhang, Kewei; Guo, Jinchao; Yin, Changsong; Chen, Jianxiu; Huang, Cheng; Zhang, Dabing

2005-08-10

As the genetically modified organisms (GMOs) labeling policies are issued in many countries, qualitative and quantitative polymerase chain reaction (PCR) techniques are increasingly used for the detection of genetically modified (GM) crops in foods. Qualitative PCR and TaqMan real-time quantitative PCR methods to detect and identify three varieties of insect resistant cotton, i.e., Mon531 cotton (Monsanto Co.) and GK19 and SGK321 cottons (Chinese Academy of Agricultural Sciences), which were approved for commercialization in China, were developed in this paper. Primer pairs specific to inserted DNAs, such as Cowpea trypsin inhibitor (CpTI) gene of SGK321 cotton and the specific junction DNA sequences containing partial Cry1A(c) gene and NOS terminator of Mon531, GK19, and SGK321 cotton varieties were designed to conduct the identified PCR assays. In conventional specific identified PCR assays, the limit of detection (LOD) was 0.05% for Mon531, GK19, or SGK321 in 100 ng of cotton genomic DNA for one reaction. Also, the multiplex PCR method for screening the three GM cottons was also established, which could save time and cost in practical detection. Furthermore, a real-time quantitative PCR assay based on TaqMan chemistry for detection of insect resistant gene, Cry1A(c), was developed. This assay also featured the use of a standard plasmid as a reference molecule, which contained both a specific region of the transgene Cry1A(c) and an endogenous stearoyl-acyl carrier protein desaturase (Sad1) gene of the cotton. In quantitative PCR assay, the quantification range was from 0.01 to 100% in 100 ng of the genome DNA template, and in the detection of 1.0, 3.0, and 5.0% levels of three insect resistant cotton lines, respectively, all of the relative standard deviations (RSDs) were less than 8.2% except for the GM cotton samples with 1.0% Mon531 or GK19, which meant that our real-time PCR assays involving the use of reference molecule were reliable and practical for GM insect resistant cottons quantification. All of these results indicated that our established conventional and TaqMan real-time PCR assays were applicable to detect the three insect resistant cottons qualitatively and quantitatively.

Denman Island disease in Washington State, USA: distribution and prevalence in Pacific and Olympia oysters.

PubMed

Elston, Ralph; Friedman, Carolyn; Gustafson, Lori; Meyer, Gary; Rogers, Russell

2015-05-21

We sampled over 2400 wild, feral, and cultured Pacific oysters Crassostrea gigas and Olympia oysters Ostrea lurida in Washington State, USA, from 2002 to 2006 to estimate the prevalence of infection with Mikrocytos mackini, the causative agent of Denman Island disease. Both histology and qualitative PCR methods were used. Estimates of true prevalence of M. mackini infection in C. gigas, after accounting for imperfect test sensitivity, ranged from mean values of 0 to 10.0% by histology and 0 to 8.4% based on pooled PCR samples. M. mackini was not detected in any of the O. lurida samples. Results suggest a lower prevalence of the pathogen and severity of this oyster disease in Washington than that indicated in previous reports from British Columbia, Canada, potentially attributable to higher seawater temperatures in the Washington sample locations.

Head-to-head comparison of the diagnostic accuracies of BD Veritor™ System RSV and Quidel® Sofia® RSV FIA systems for respiratory syncytial virus (RSV) diagnosis.

PubMed

Kanwar, Neena; Hassan, Ferdaus; Nguyen, Ashley; Selvarangan, Rangaraj

2015-04-01

Respiratory syncytial virus (RSV) is one of the most common causes of severe lower respiratory tract disease among infants and young children. BD Veritor™ System RSV (BD) and Quidel(®) Sofia(®) RSV FIA (QD) are the new generation lateral flow digital immunoassay (DIA) tests with an instrumented read for the qualitative detection of RSV viral antigens. To compare the diagnostic accuracies of BD and QD for RSV detection using fresh nasopharyngeal aspirates and nasopharyngeal swab specimens collected in universal transport media during 2013-2014 respiratory season. The two DIA tests were performed simultaneously on randomly selected specimens on a weekly basis during the RSV season until 200 fresh remnant specimens were enrolled. Real-time RT-PCR assay results were used to compare and evaluate the performance of both RSV DIA assays. Among 200 specimens tested, RSV real-time RT-PCR assay detected RSV in 104 samples, while QD detected 84 samples and BD detected 74 samples as positive. The overall sensitivity for detection of RSV in comparison to PCR was 71.15% (61.3-79.4) for BD and 80.77% (71.6-87.6) for QD system (P=0.36). The specificity was 100% (95.2-100) for both systems. The work flow analysis revealed that the overall specimen processing time was significantly lower for BD as compared with the QD assay. In comparison with the real-time PCR, the QD system showed a higher sensitivity than that of the BD system, but the difference did not reach statistical significance (P=0.36). Both BD and QD systems were found comparable in terms of specificity. Copyright © 2015 Elsevier B.V. All rights reserved.

Comparative Evaluation of Four Real-Time PCR Methods for the Quantitative Detection of Epstein-Barr Virus from Whole Blood Specimens.

PubMed

Buelow, Daelynn; Sun, Yilun; Tang, Li; Gu, Zhengming; Pounds, Stanley; Hayden, Randall

2016-07-01

Monitoring of Epstein-Barr virus (EBV) load in immunocompromised patients has become integral to their care. An increasing number of reagents are available for quantitative detection of EBV; however, there are little published comparative data. Four real-time PCR systems (one using laboratory-developed reagents and three using analyte-specific reagents) were compared with one another for detection of EBV from whole blood. Whole blood specimens seeded with EBV were used to determine quantitative linearity, analytical measurement range, lower limit of detection, and CV for each assay. Retrospective testing of 198 clinical samples was performed in parallel with all methods; results were compared to determine relative quantitative and qualitative performance. All assays showed similar performance. No significant difference was found in limit of detection (3.12-3.49 log10 copies/mL; P = 0.37). A strong qualitative correlation was seen with all assays that used clinical samples (positive detection rates of 89.5%-95.8%). Quantitative correlation of clinical samples across assays was also seen in pairwise regression analysis, with R(2) ranging from 0.83 to 0.95. Normalizing clinical sample results to IU/mL did not alter the quantitative correlation between assays. Quantitative EBV detection by real-time PCR can be performed over a wide linear dynamic range, using three different commercially available reagents and laboratory-developed methods. EBV was detected with comparable sensitivity and quantitative correlation for all assays. Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Evaluation of Altona Diagnostics RealStar Zika Virus Reverse Transcription-PCR Test Kit for Zika Virus PCR Testing

PubMed Central

Lombos, Ernesto; Tang, Elaine; Perusini, Stephen; Eshaghi, Alireza; Nagra, Sandeep; Frantz, Christine; Olsha, Romy; Kristjanson, Erik; Dimitrova, Kristina; Safronetz, David; Drebot, Mike

2017-01-01

ABSTRACT With the emerging Zika virus (ZIKV) epidemic, accessible real-time reverse transcription-PCR (rRT-PCR) assays are needed to streamline testing. The commercial Altona Diagnostics RealStar ZIKV rRT-PCR test kit (Altona PCR) has been approved for emergency use authorization by the U.S. FDA. Our aim was to verify the Altona PCR by comparing it to the CDC-designed dual-target ZIKV rRT-PCR reference assay (reference PCR) and describe the demographics of patients tested for ZIKV by rRT-PCR in Ontario, Canada. A large set of clinical specimens was tested for ZIKV by the Altona PCR and the reference PCR. Positive or equivocal specimens underwent PCR and Sanger sequencing targeting the ZIKV NS5 gene. A total of 671 serum specimens were tested by the reference PCR: 58 (8.6%) were positive, 193 (28.8%) were equivocal, and 420 (62.6%) were negative. Ninety percent of the reference PCR-positive patients were tested in the first 5 days after symptom onset. The Altona PCR was performed on 284/671 specimens tested by the reference PCR. The Altona PCR was positive for 53/58 (91%) reference PCR-positive specimens and 16/193 (8%) reference PCR-equivocal specimens; the ZIKV NS5 PCR was positive for all 68 Altona PCR-positive specimens and negative for all 181 Altona PCR-negative specimens that underwent the NS5 PCR. The Altona PCR has very good sensitivity (91%) and specificity (97%) compared to the reference PCR. The Altona PCR can be used for ZIKV diagnostic testing and has less extensive verification requirements than a laboratory-developed test. PMID:28298448

Comprehensive GMO detection using real-time PCR array: single-laboratory validation.

PubMed

Mano, Junichi; Harada, Mioko; Takabatake, Reona; Furui, Satoshi; Kitta, Kazumi; Nakamura, Kosuke; Akiyama, Hiroshi; Teshima, Reiko; Noritake, Hiromichi; Hatano, Shuko; Futo, Satoshi; Minegishi, Yasutaka; Iizuka, Tayoshi

2012-01-01

We have developed a real-time PCR array method to comprehensively detect genetically modified (GM) organisms. In the method, genomic DNA extracted from an agricultural product is analyzed using various qualitative real-time PCR assays on a 96-well PCR plate, targeting for individual GM events, recombinant DNA (r-DNA) segments, taxon-specific DNAs, and donor organisms of the respective r-DNAs. In this article, we report the single-laboratory validation of both DNA extraction methods and component PCR assays constituting the real-time PCR array. We selected some DNA extraction methods for specified plant matrixes, i.e., maize flour, soybean flour, and ground canola seeds, then evaluated the DNA quantity, DNA fragmentation, and PCR inhibition of the resultant DNA extracts. For the component PCR assays, we evaluated the specificity and LOD. All DNA extraction methods and component PCR assays satisfied the criteria set on the basis of previous reports.

Molecular-level evaluation of selected periodontal pathogens from subgingival regions in canines and humans with periodontal disease

PubMed Central

Polkowska, Izabela; Bartoszcze-Tomaszewska, Małgorzata; Sobczyńska-Rak, Aleksandra; Matuszewski, Łukasz

2017-01-01

Dogs commonly serve as a model for various human conditions, including periodontal diseases. The aim of this study was to identify the anaerobic bacteria that colonize the subgingival areas in dogs and humans by using rapid real-time polymerase chain reaction (RT-PCR)-based tests and to compare the results obtained in each species. Bacterial microflora evaluations, both quantitative and qualitative, were performed by applying ready-made tests on twelve dogs and twelve humans. Five samples were collected from each subject's deepest gingival pockets and joined to form a collective sample. The results of the study revealed interspecies similarities in the prevalences of Porphyromonas (P.) gingivalis, Treponema denticola, Tannerella forsythia, and Fusobacterium nucleatum. Red complex bacteria comprised the largest portion of the studied bacterial complexes in all study groups, with P. gingivalis being the most commonly isolated bacterium. The results show similarities in the prevalence of bacterial microflora in dogs and humans. Microbiological analysis of gingival pockets by using rapid real-time PCR-based tests in clinical practice, both veterinary and human, can facilitate the choice of appropriate pharmacological treatment and can provide a basis for subsequent verification of the treatment's effectiveness. PMID:27297417

Molecular-level evaluation of selected periodontal pathogens from subgingival regions in canines and humans with periodontal disease.

PubMed

Gołyńska, Magdalena; Polkowska, Izabela; Bartoszcze-Tomaszewska, Małgorzata; Sobczyńska-Rak, Aleksandra; Matuszewski, Łukasz

2017-03-30

Dogs commonly serve as a model for various human conditions, including periodontal diseases. The aim of this study was to identify the anaerobic bacteria that colonize the subgingival areas in dogs and humans by using rapid real-time polymerase chain reaction (RT-PCR)-based tests and to compare the results obtained in each species. Bacterial microflora evaluations, both quantitative and qualitative, were performed by applying ready-made tests on twelve dogs and twelve humans. Five samples were collected from each subject's deepest gingival pockets and joined to form a collective sample. The results of the study revealed interspecies similarities in the prevalences of Porphyromonas ( P .) gingivalis, Treponema denticola, Tannerella forsythia , and Fusobacterium nucleatum . Red complex bacteria comprised the largest portion of the studied bacterial complexes in all study groups, with P. gingivalis being the most commonly isolated bacterium. The results show similarities in the prevalence of bacterial microflora in dogs and humans. Microbiological analysis of gingival pockets by using rapid real-time PCR-based tests in clinical practice, both veterinary and human, can facilitate the choice of appropriate pharmacological treatment and can provide a basis for subsequent verification of the treatment's effectiveness.

Clinical evaluation of the Abbott RealTime MTB Assay for direct detection of Mycobacterium tuberculosis-complex from respiratory and non-respiratory samples.

PubMed

Hinić, Vladimira; Feuz, Kinga; Turan, Selda; Berini, Andrea; Frei, Reno; Pfeifer, Karin; Goldenberger, Daniel

2017-05-01

Rapid and reliable diagnosis is crucial for correct management of tuberculosis. The Abbott RealTime MTB Assay represents a novel qualitative real-time PCR assay for direct detection of M. tuberculosis-complex (MTB) DNA from respiratory samples. The test targets two highly conserved sequences, the multi-copy insertion element IS6110 and the protein antigen B (PAB) gene of MTB, allowing even the detection of IS6610-deficient strains. We evaluated this commercial diagnostic test by analyzing 200 respiratory and, for the first time, 87 non-respiratory clinical specimens from our tertiary care institution and compared its results to our IS6110-based in-house real-time PCR for MTB as well as MTB culture. Overall sensitivity for Abbott RealTime MTB was 100% (19/19) in smear positive and 87.5% (7/8) in smear negative specimens, while the specificity of the assay was 100% (260/260). For both non-respiratory smear positive and smear negative specimens Abbott RealTime MTB tests showed 100% (8/8) sensitivity and 100% (8/8) specificity. Cycle threshold (Ct) value analysis of 16 MTB positive samples showed a slightly higher Ct value of the Abbott RealTime MTB test compared to our in-house MTB assay (mean delta Ct = 2.55). In conclusion, the performance of the new Abbott RealTime MTB Assay was highly similar to culture and in-house MTB PCR. We document successful analysis of 87 non-respiratory samples with the highly automated Abbott RealTime MTB test with no inhibition observed. Copyright © 2017 Elsevier Ltd. All rights reserved.

Synthesis of O-serogroup specific positive controls and real-time PCR standards for nine clinically relevant non-O157 STECs.

PubMed

Conrad, Cheyenne C; Gilroyed, Brandon H; McAllister, Tim A; Reuter, Tim

2012-10-01

Non-O157 Shiga toxin producing Escherichia coli (STEC) are gaining recognition as human pathogens, but no standardized method exists to identify them. Sequence analysis revealed that STEC can be classified on the base of variable O antigen regions into different O serotypes. Polymerase chain reaction is a powerful technique for thorough screening and complex diagnosis for these pathogens, but requires a positive control to verify qualitative and/or quantitative DNA-fragment amplification. Due to the pathogenic nature of STEC, controls are not readily available and cell culturing of STEC reference strains requires biosafety conditions of level 2 or higher. In order to bypass this limitation, controls of stacked O-type specific DNA-fragments coding for primer recognition sites were designed to screen for nine STEC serotypes frequently associated with human infection. The synthetic controls were amplified by PCR, cloned into a plasmid vector and transferred into bacteria host cells. Plasmids amplified by bacterial expression were purified, serially diluted and tested as standards for real-time PCR using SYBR Green and TaqMan assays. Utility of synthetic DNA controls was demonstrated in conventional and real-time PCR assays and validated with DNA from natural STEC strains. Copyright © 2012 Elsevier B.V. All rights reserved.

Development of a qualitative real-time PCR method to detect 19 targets for identification of genetically modified organisms.

PubMed

Peng, Cheng; Wang, Pengfei; Xu, Xiaoli; Wang, Xiaofu; Wei, Wei; Chen, Xiaoyun; Xu, Junfeng

2016-01-01

As the amount of commercially available genetically modified organisms (GMOs) grows recent years, the diversity of target sequences for molecular detection techniques are eagerly needed. Considered as the gold standard for GMO analysis, the real-time PCR technology was optimized to produce a high-throughput GMO screening method. With this method we can detect 19 transgenic targets. The specificity of the assays was demonstrated to be 100 % by the specific amplification of DNA derived from reference material from 20 genetically modified crops and 4 non modified crops. Furthermore, most assays showed a very sensitive detection, reaching the limit of ten copies. The 19 assays are the most frequently used genetic elements present in GM crops and theoretically enable the screening of the known GMO described in Chinese markets. Easy to use, fast and cost efficient, this method approach fits the purpose of GMO testing laboratories.

Data processing of qualitative results from an interlaboratory comparison for the detection of “Flavescence dorée” phytoplasma: How the use of statistics can improve the reliability of the method validation process in plant pathology

PubMed Central

Renaudin, Isabelle; Poliakoff, Françoise

2017-01-01

A working group established in the framework of the EUPHRESCO European collaborative project aimed to compare and validate diagnostic protocols for the detection of “Flavescence dorée” (FD) phytoplasma in grapevines. Seven molecular protocols were compared in an interlaboratory test performance study where each laboratory had to analyze the same panel of samples consisting of DNA extracts prepared by the organizing laboratory. The tested molecular methods consisted of universal and group-specific real-time and end-point nested PCR tests. Different statistical approaches were applied to this collaborative study. Firstly, there was the standard statistical approach consisting in analyzing samples which are known to be positive and samples which are known to be negative and reporting the proportion of false-positive and false-negative results to respectively calculate diagnostic specificity and sensitivity. This approach was supplemented by the calculation of repeatability and reproducibility for qualitative methods based on the notions of accordance and concordance. Other new approaches were also implemented, based, on the one hand, on the probability of detection model, and, on the other hand, on Bayes’ theorem. These various statistical approaches are complementary and give consistent results. Their combination, and in particular, the introduction of new statistical approaches give overall information on the performance and limitations of the different methods, and are particularly useful for selecting the most appropriate detection scheme with regards to the prevalence of the pathogen. Three real-time PCR protocols (methods M4, M5 and M6 respectively developed by Hren (2007), Pelletier (2009) and under patent oligonucleotides) achieved the highest levels of performance for FD phytoplasma detection. This paper also addresses the issue of indeterminate results and the identification of outlier results. The statistical tools presented in this paper and their combination can be applied to many other studies concerning plant pathogens and other disciplines that use qualitative detection methods. PMID:28384335

Data processing of qualitative results from an interlaboratory comparison for the detection of "Flavescence dorée" phytoplasma: How the use of statistics can improve the reliability of the method validation process in plant pathology.

PubMed

Chabirand, Aude; Loiseau, Marianne; Renaudin, Isabelle; Poliakoff, Françoise

2017-01-01

A working group established in the framework of the EUPHRESCO European collaborative project aimed to compare and validate diagnostic protocols for the detection of "Flavescence dorée" (FD) phytoplasma in grapevines. Seven molecular protocols were compared in an interlaboratory test performance study where each laboratory had to analyze the same panel of samples consisting of DNA extracts prepared by the organizing laboratory. The tested molecular methods consisted of universal and group-specific real-time and end-point nested PCR tests. Different statistical approaches were applied to this collaborative study. Firstly, there was the standard statistical approach consisting in analyzing samples which are known to be positive and samples which are known to be negative and reporting the proportion of false-positive and false-negative results to respectively calculate diagnostic specificity and sensitivity. This approach was supplemented by the calculation of repeatability and reproducibility for qualitative methods based on the notions of accordance and concordance. Other new approaches were also implemented, based, on the one hand, on the probability of detection model, and, on the other hand, on Bayes' theorem. These various statistical approaches are complementary and give consistent results. Their combination, and in particular, the introduction of new statistical approaches give overall information on the performance and limitations of the different methods, and are particularly useful for selecting the most appropriate detection scheme with regards to the prevalence of the pathogen. Three real-time PCR protocols (methods M4, M5 and M6 respectively developed by Hren (2007), Pelletier (2009) and under patent oligonucleotides) achieved the highest levels of performance for FD phytoplasma detection. This paper also addresses the issue of indeterminate results and the identification of outlier results. The statistical tools presented in this paper and their combination can be applied to many other studies concerning plant pathogens and other disciplines that use qualitative detection methods.

Novel multiplex qualitative detection using universal primer-multiplex-PCR combined with pyrosequencing.

PubMed

Shang, Ying; Xu, Wentao; Wang, Yong; Xu, Yuancong; Huang, Kunlun

2017-12-15

This study described a novel multiplex qualitative detection method using pyrosequencing. Based on the principle of the universal primer-multiplex-PCR, only one sequencing primer was employed to realize the detection of the multiple targets. Samples containing three genetically modified (GM) crops in different proportions were used to validate the method. The dNTP dispensing order was designed based on the product sequences. Only 12 rounds (ATCTGATCGACT) of dNTPs addition and, often, as few as three rounds (CAT) under ideal conditions, were required to detect the GM events qualitatively, and sensitivity was as low as 1% of a mixture. However, when considering a mixture, calculating signal values allowed the proportion of each GM to be estimated. Based on these results, we concluded that our novel method not only realized detection but also allowed semi-quantitative detection of individual events. Copyright © 2017. Published by Elsevier Ltd.

The use of comparative duplex PCR in monitoring of patients with non-Hodgkin's lymphoma and chronic lymphocytic leukaemia.

PubMed

Slavícková, A; Forsterová, K; Ivánek, R; Cerný, J; Klener, P

2005-01-01

Various quantitative PCR approaches have been utilized during the last years to provide information about the treatment efficacy and the risk of recurrent disease in haematological malignancies. Apart from the frequently used real-time PCR, cost-saving modified standard PCR methods may be applied as well. This report evaluates the utility of the end-point comparative duplex PCR. We have used this method for monitoring of 35 patients with either NHL or CLL and observed a good correlation between quantitative molecular results and clinical outcome. There was also an agreement between comparative duplex PCR and real-time PCR in patients who were monitored by both methods. We therefore believe that use of this technique should be strongly considered instead of simple qualitative detection in monitoring of therapeutic outcome in NHL or CLL patients.

Real-time PCR using SYBR Green for the detection of Shigella spp. in food and stool samples.

PubMed

Mokhtari, W; Nsaibia, S; Gharbi, A; Aouni, M

2013-02-01

Shigella spp are exquisitely fastidious Gram negative organisms that frequently get missed in the detection by traditional culture methods. For this reason, this work has adapted a classical PCR for detection of Shigella in food and stool specimens to real-time PCR using the SYBR Green format. This method follows a melting curve analysis to be more rapid and provide both qualitative and quantitative data about the targeted pathogen. A total of 117 stool samples with diarrhea and 102 food samples were analyzed in Public Health Regional Laboratory of Nabeul by traditional culture methods and real-time PCR. To validate the real-time PCR assay, an experiment was conducted with both spiked and naturally contaminated stool samples. All Shigella strains tested were ipaH positive and all non-Shigella strains yielded no amplification products. The melting temperature (T(m) = 81.5 ± 0.5 °C) was consistently specific for the amplicon. Correlation coefficients of standard curves constructed using the quantification cycle (C(q)) versus copy numbers of Shigella showed good linearity (R² = 0.995; slope = 2.952) and the minimum level of detection was 1.5 × 10³ CFU/g feces. All food samples analyzed were negative for Shigella by standard culture methods, whereas ipaH was detected in 8.8% culture negative food products. Moreover, the ipaH specific PCR system increased the detection rate over that by culture alone from 1.7% to 11.1% among patients with diarrhea. The data presented here shows that the SYBR Green I was suitable for use in the real-time PCR assay, which provided a specific, sensitive and efficient method for the detection and quantification of Shigella spp in food and stool samples. Copyright © 2012 Elsevier Ltd. All rights reserved.

Development and Validation of a Rotor-Gene Real-Time PCR Assay for Detection, Identification, and Quantification of Chlamydia trachomatis in a Single Reaction

PubMed Central

Jalal, Hamid; Stephen, Hannah; Curran, Martin D.; Burton, Janet; Bradley, Michelle; Carne, Christopher

2006-01-01

A multitarget real-time PCR (MRT-PCR) for detection of Chlamydia trachomatis DNA was developed and validated. There were three targets for amplification in a single reaction: the cryptic plasmid (CP), the major outer membrane protein (MOMP) gene, and an internal control. The assay had the following characteristics: (i) detection and confirmation of the presence of C. trachomatis DNA in a single reaction, (ii) detection of all genovars of C. trachomatis without any cross-reactivity with pathogenic bacteria or commensal organisms of the oropharynx and genital tract, (iii) a 95% probability of detection with three copies of MOMP and one copy of CP per reaction mixture, (iv) identification of the inhibition of amplification, (v) a quantitative dynamic range of 25 to 250,000 genome copies per reaction mixture, (vi) high intra- and interassay reproducibilities, and (vii) correct identification of all samples in the validation panel. There were 146 COBAS Amplicor PCR (Amplicor PCR)-positive samples and 122 Amplicor PCR-negative samples in the panel. MRT-PCR detected CP DNA alone in 6 (4%) Amplicor PCR-positive samples and both CP and MOMP DNAs in 140 (96%) of 146 Amplicor PCR-positive samples. The quantity of MOMP DNA in 95 (68%) of 140 samples was within the dynamic range of the assay. The median C. trachomatis load in these samples was 321 genome copies per reaction mixture (range, 26 to 40,137 genome copies per reaction mixture). Due to the inclusion of two different C. trachomatis-specific targets, the assay confirmed 259 (97%) of 268 results in a single reaction. This assay could be used in the qualitative format for the routine detection of C. trachomatis and in the quantitative format for study of the pathogenesis of C. trachomatis-associated diseases. The assay demonstrated the potential to eliminate the need for confirmatory testing in almost all samples, thus reducing the turnaround time and the workload. PMID:16390971

Principles and applications of polymerase chain reaction in medical diagnostic fields: a review

PubMed Central

Valones, Marcela Agne Alves; Guimarães, Rafael Lima; Brandão, Lucas André Cavalcanti; de Souza, Paulo Roberto Eleutério; de Albuquerque Tavares Carvalho, Alessandra; Crovela, Sergio

2009-01-01

Recent developments in molecular methods have revolutionized the detection and characterization of microorganisms in a broad range of medical diagnostic fields, including virology, mycology, parasitology, microbiology and dentistry. Among these methods, Polymerase Chain Reaction (PCR) has generated great benefits and allowed scientific advancements. PCR is an excellent technique for the rapid detection of pathogens, including those difficult to culture. Along with conventional PCR techniques, Real-Time PCR has emerged as a technological innovation and is playing an ever-increasing role in clinical diagnostics and research laboratories. Due to its capacity to generate both qualitative and quantitative results, Real-Time PCR is considered a fast and accurate platform. The aim of the present literature review is to explore the clinical usefulness and potential of both conventional PCR and Real-Time PCR assays in diverse medical fields, addressing its main uses and advances. PMID:24031310

Simplex and duplex event-specific analytical methods for functional biotech maize.

PubMed

Lee, Seong-Hun; Kim, Su-Jeong; Yi, Bu-Young

2009-08-26

Analytical methods are very important in the control of genetically modified organism (GMO) labeling systems or living modified organism (LMO) management for biotech crops. Event-specific primers and probes were developed for qualitative and quantitative analysis for biotech maize event 3272 and LY 038 on the basis of the 3' flanking regions, respectively. The qualitative primers confirmed the specificity by a single PCR product and sensitivity to 0.05% as a limit of detection (LOD). Simplex and duplex quantitative methods were also developed using TaqMan real-time PCR. One synthetic plasmid was constructed from two taxon-specific DNA sequences of maize and two event-specific 3' flanking DNA sequences of event 3272 and LY 038 as reference molecules. In-house validation of the quantitative methods was performed using six levels of mixing samples, from 0.1 to 10.0%. As a result, the biases from the true value and the relative deviations were all within the range of +/-30%. Limits of quantitation (LOQs) of the quantitative methods were all 0.1% for simplex real-time PCRs of event 3272 and LY 038 and 0.5% for duplex real-time PCR of LY 038. This study reports that event-specific analytical methods were applicable for qualitative and quantitative analysis for biotech maize event 3272 and LY 038.

A Targeted Q-PCR-Based Method for Point Mutation Testing by Analyzing Circulating DNA for Cancer Management Care.

PubMed

Thierry, Alain R

2016-01-01

Circulating cell-free DNA (cfDNA) is a valuable source of tumor material available with a simple blood sampling enabling a noninvasive quantitative and qualitative analysis of the tumor genome. cfDNA is released by tumor cells and exhibits the genetic and epigenetic alterations of the tumor of origin. Circulating cell-free DNA (cfDNA) analysis constitutes a hopeful approach to provide a noninvasive tumor molecular test for cancer patients. Based upon basic research on the origin and structure of cfDNA, new information on circulating cell-free DNA (cfDNA) structure, and specific determination of cfDNA fragmentation and size, we revisited Q-PCR-based method and recently developed a the allele-specific-Q-PCR-based method with blocker (termed as Intplex) which is the first multiplexed test for cfDNA. This technique, named Intplex(®) and based on a refined Q-PCR method, derived from critical observations made on the specific structure and size of cfDNA. It enables the simultaneous determination of five parameters: the cfDNA total concentration, the presence of a previously known point mutation, the mutant (tumor) cfDNA concentration (ctDNA), the proportion of mutant cfDNA, and the cfDNA fragmentation index. Intplex(®) has enabled the first clinical validation of ctDNA analysis in oncology by detecting KRAS and BRAF point mutations in mCRC patients and has demonstrated that a blood test could replace tumor section analysis for the detection of KRAS and BRAF mutations. The Intplex(®) test can be adapted to all mutations, genes, or cancers and enables rapid, highly sensitive, cost-effective, and repetitive analysis. As regards to the determination of mutations on cfDNA Intplex(®) is limited to the mutational status of known hotspot mutation; it is a "targeted approach." However, it offers the opportunity in detecting quantitatively and dynamically mutation and could constitute a noninvasive attractive tool potentially allowing diagnosis, prognosis, theranostics, therapeutic monitoring, and follow-up of cancer patients expanding the scope of personalized cancer medicine.

Clinical Impact of Hepatitis C Infection in Military Active Duty Women

DTIC Science & Technology

1996-07-01

presence of viral RNA (HCV RNA) by qualitative and quantitative assays: RT PCR (primers 5’ UTR) bDNA (Chiron Laboratories, Emeryville, CA) Amplicor...PCR) bDNA Eq/mL x 10’ Amplicor Copies/mL 1 Positive 27.11 (Positive) 340,135 (Positive) 2 Negative < 3.5 (Negative) (Negative) 3 Negative < 3.5...the advantage of measuring titer of RNA in serum, however the sensitivity is low. 8 TABLE 3 Comparison of the Sensitivity of HCV RNA by RT-PCR, bDNA

A new LAMP-based assay for the molecular diagnosis of toxoplasmosis: comparison with a proficient PCR assay.

PubMed

Varlet-Marie, Emmanuelle; Sterkers, Yvon; Perrotte, Marina; Bastien, Patrick

2018-05-01

Toxoplasmosis is generally a benign infection caused by the protozoan parasite Toxoplasma gondii but can have severe consequences in fetuses of mothers infected during pregnancy (congenital toxoplasmosis) and immunocompromised individuals. PCR-based diagnostic tests have become crucial for its diagnosis. However, this molecular diagnosis essentially relies upon laboratory-developed methods and suffers from a lack of standardization, leading to great variation in methods and performance among laboratories. With the need for accreditation of clinical microbiological laboratories, the use of commercial PCR kits has become an attractive alternative; but thorough evaluation of newly commercialized kits by proficient groups is necessary before any recommendation can be made to parasitology laboratories by health authorities or learned societies. Here, we compared the performance of an original commercial method, the Iam TOXO Q-LAMP (DiaSorin®), using Loop-mediated isothermal amplification (LAMP) technology, with our reference laboratory-developed method using real-time PCR. The kit was first tested using amniotic fluid (AF) and plasma samples (either negative or spiked with live T. gondii tachyzoites at different concentrations (from 7 to 10 5  tachyzoites/mL)). It was then assessed using a cohort of 11 AF, five placental and 32 blood clinical samples preserved at -20 °C. For the processing of placental/blood samples, a pretreatment step was used, which did not strictly follow the manufacturer's recommendations. The practical ease of use and compliance with good laboratory practices were also evaluated. Although the LAMP assay was less sensitive than the laboratory-developed method at very low parasite concentrations (0.1 T. gondii genome equivalents/mL), the two methods yielded identical results qualitatively and, in some instances, quantitatively, particularly for AF samples. Copyright © 2018. Published by Elsevier Ltd.

New PCR Test That Recognizes All Human Prototypes of Enterovirus: Application for Clinical Diagnosis

PubMed Central

Bourlet, Thomas; Caro, Valerie; Minjolle, Sophie; Jusselin, Isabelle; Pozzetto, Bruno; Crainic, Radu; Colimon, Ronald

2003-01-01

We describe a new PCR test (Penter RT-PCR) that recognizes all 64 prototypes of enterovirus. Sixty clinical samples were analyzed in parallel with this Penter RT-PCR and previously described PCR tests: 34 and 32 samples tested positive, respectively. This assay is suitable for use in clinical diagnosis, and its ability to amplify all known serotypes makes it more useful than other consensus PCR tests. PMID:12682177

Measurement issues associated with quantitative molecular biology analysis of complex food matrices for the detection of food fraud.

PubMed

Burns, Malcolm; Wiseman, Gordon; Knight, Angus; Bramley, Peter; Foster, Lucy; Rollinson, Sophie; Damant, Andrew; Primrose, Sandy

2016-01-07

Following a report on a significant amount of horse DNA being detected in a beef burger product on sale to the public at a UK supermarket in early 2013, the Elliott report was published in 2014 and contained a list of recommendations for helping ensure food integrity. One of the recommendations included improving laboratory testing capacity and capability to ensure a harmonised approach for testing for food authenticity. Molecular biologists have developed exquisitely sensitive methods based on the polymerase chain reaction (PCR) or mass spectrometry for detecting the presence of particular nucleic acid or peptide/protein sequences. These methods have been shown to be specific and sensitive in terms of lower limits of applicability, but they are largely qualitative in nature. Historically, the conversion of these qualitative techniques into reliable quantitative methods has been beset with problems even when used on relatively simple sample matrices. When the methods are applied to complex sample matrices, as found in many foods, the problems are magnified resulting in a high measurement uncertainty associated with the result which may mean that the assay is not fit for purpose. However, recent advances in the technology and the understanding of molecular biology approaches have further given rise to the re-assessment of these methods for their quantitative potential. This review focuses on important issues for consideration when validating a molecular biology assay and the various factors that can impact on the measurement uncertainty of a result associated with molecular biology approaches used in detection of food fraud, with a particular focus on quantitative PCR-based and proteomics assays.

Development and evaluation of event-specific quantitative PCR method for genetically modified soybean A2704-12.

PubMed

Takabatake, Reona; Akiyama, Hiroshi; Sakata, Kozue; Onishi, Mari; Koiwa, Tomohiro; Futo, Satoshi; Minegishi, Yasutaka; Teshima, Reiko; Mano, Junichi; Furui, Satoshi; Kitta, Kazumi

2011-01-01

A novel real-time PCR-based analytical method was developed for the event-specific quantification of a genetically modified (GM) soybean event; A2704-12. During the plant transformation, DNA fragments derived from pUC19 plasmid were integrated in A2704-12, and the region was found to be A2704-12 specific. The pUC19-derived DNA sequences were used as primers for the specific detection of A2704-12. We first tried to construct a standard plasmid for A2704-12 quantification using pUC19. However, non-specific signals appeared with both qualitative and quantitative PCR analyses using the specific primers with pUC19 as a template, and we then constructed a plasmid using pBR322. The conversion factor (C(f)), which is required to calculate the amount of the genetically modified organism (GMO), was experimentally determined with two real-time PCR instruments, the Applied Biosystems 7900HT and the Applied Biosystems 7500. The determined C(f) values were both 0.98. The quantitative method was evaluated by means of blind tests in multi-laboratory trials using the two real-time PCR instruments. The limit of quantitation for the method was estimated to be 0.1%. The trueness and precision were evaluated as the bias and reproducibility of relative standard deviation (RSD(R)), and the determined bias and RSD(R) values for the method were each less than 20%. These results suggest that the developed method would be suitable for practical analyses for the detection and quantification of A2704-12.

Laboratory Evaluation of the Alere q Point-of-Care System for Early Infant HIV Diagnosis.

PubMed

Hsiao, Nei-yuan; Dunning, Lorna; Kroon, Max; Myer, Landon

2016-01-01

Early infant diagnosis (EID) and prompt linkage to care are critical to minimise the high morbidity and mortality associated with infant HIV infection. Attrition in the "EID cascade" is common; however, point-of-care (POC) EID assays with same-day result could facilitate prompt linkage of HIV-infected infant to treatment. Despite a number of POC EID assays in development, few have been independently evaluated and data on new technologies are urgently needed to inform policy. We compared Alere q 1/2 Detect POC system laboratory test characteristics with the local standard of care (SOC), Roche CAP/CTM HIV-1 qualitative PCR in an independent laboratory-based evaluation in Cape Town, South Africa. Routinely EID samples collected between November 2013 and September 2014 were each tested by both SOC and POC systems. Repeat testing was done to troubleshoot any discrepancy between POC and SOC results. Overall, 1098 children with a median age of 47 days (IQR, 42-117) were included. Birth PCR (age <7 days) comprised of 8% (n = 92) tests while 56% (n = 620) of children tested as part of routine EID (ages 6-14 weeks). In the overall direct comparison, Alere q Detect achieved sensitivity of 95.5% (95% CI, 91.7-97.9%) and a specificity of 99.8% (95% CI, 99.1-100%). Following repeat testing of discordant samples and exclusion of any inconclusive results, the POC assay sensitivity and specificity were 96.9% (95% CI 93.4-98.9%) and 100% (lower 95% CI 98%) respectively. Among birth PCR tests the POC assay had slightly lower sensitivity (93.3% vs 96.5% in routine EID) and higher assay error rate (10% vs 5% in samples of older children, p = 0.04). Our results indicate this POC assay performs well for EID in the laboratory. The high specificity and thus high positive predictive value would suggest a positive POC result may be adequate for immediate infant ART initiation. While POC testing for EID may have particular utility for birth testing at delivery facilities, the lower sensitivity and error rate requires further attention, as does field implementation of POC EID technologies in other clinical care settings.

Development and application of a multi-targeting reference plasmid as calibrator for analysis of five genetically modified soybean events.

PubMed

Pi, Liqun; Li, Xiang; Cao, Yiwei; Wang, Canhua; Pan, Liangwen; Yang, Litao

2015-04-01

Reference materials are important in accurate analysis of genetically modified organism (GMO) contents in food/feeds, and development of novel reference plasmid is a new trend in the research of GMO reference materials. Herein, we constructed a novel multi-targeting plasmid, pSOY, which contained seven event-specific sequences of five GM soybeans (MON89788-5', A2704-12-3', A5547-127-3', DP356043-5', DP305423-3', A2704-12-5', and A5547-127-5') and sequence of soybean endogenous reference gene Lectin. We evaluated the specificity, limit of detection and quantification, and applicability of pSOY in both qualitative and quantitative PCR analyses. The limit of detection (LOD) was as low as 20 copies in qualitative PCR, and the limit of quantification (LOQ) in quantitative PCR was 10 copies. In quantitative real-time PCR analysis, the PCR efficiencies of all event-specific and Lectin assays were higher than 90%, and the squared regression coefficients (R(2)) were more than 0.999. The quantification bias varied from 0.21% to 19.29%, and the relative standard deviations were from 1.08% to 9.84% in simulated samples analysis. All the results demonstrated that the developed multi-targeting plasmid, pSOY, was a credible substitute of matrix reference materials, and could be used as a reliable reference calibrator in the identification and quantification of multiple GM soybean events.

Nine year trends of dengue virus infection in Mumbai, Western India.

PubMed

Shastri, Jayanthi; Williamson, Manita; Vaidya, Nilima; Agrawal, Sachee; Shrivastav, Om

2017-01-01

Dengue virus (DENV) causes a wide range of diseases in humans, from acute febrile illness Dengue fever (DF) to life-threatening Dengue hemorrhagic fever (DHF) or Dengue shock syndrome (DSS). Factors believed to be responsible for spread of Dengue virus infection include explosive population growth, unplanned urban overpopulation with inadequate public health systems, poor standing water and vector control, climate changes and increased international recreational, business, military travel to endemic areas. All of these factors must be addressed to control the spread of Dengue and other mosquito-borne infections. The detection of Dengue virus RNA by reverse transcriptase PCR (RT-PCR) in human serum or plasma samples is highly indicative of acute Dengue fever. Moreover, the method is able to identify the Dengue virus serotype by demonstrating defined sequence homologies in the viral genomic RNA. During the nine year period of this study analysis, 6767 strongly suspected cases were tested by RT-PCR. 1685 (24.9%) were Dengue PCR positive and confirmed as Dengue cases. Observations on the seasonality were based on the nine year's data as the intensity of sampling was at its maximum during monsoon season. Dengue typing was done on 100 positive samples after storage of Dengue RNA at - 80°C. Dengue serotypes were detected in 69 samples of which Dengue 2 was most predominant. 576 samples were processed for NS1 antigen and PCR simultaneously. 19/576 were positive (3.3 %) for NS1 as well as by PCR. 23/576 samples were negative for NS1 antigen, but were positive by RT-PCR. The remaining 534 samples which were negative for NS1 antigen were also negative by Dengue RT-PCR. In this study we sought to standardize rapid, sensitive, and specific fluorogenic probe-based RT-PCR assay to screen and serotype a representative range of Dengue viruses that are found in and around Mumbai. Qualitative Dengue virus TaqMan assays could have tremendous utility for the epidemiological investigation of Dengue illness and especially for the study of the viremic response with candidate live-attenuated dengue virus vaccines.

Wheat-specific gene, ribosomal protein l21, used as the endogenous reference gene for qualitative and real-time quantitative polymerase chain reaction detection of transgenes.

PubMed

Liu, Yi-Ke; Li, He-Ping; Huang, Tao; Cheng, Wei; Gao, Chun-Sheng; Zuo, Dong-Yun; Zhao, Zheng-Xi; Liao, Yu-Cai

2014-10-29

Wheat-specific ribosomal protein L21 (RPL21) is an endogenous reference gene suitable for genetically modified (GM) wheat identification. This taxon-specific RPL21 sequence displayed high homogeneity in different wheat varieties. Southern blots revealed 1 or 3 copies, and sequence analyses showed one amplicon in common wheat. Combined analyses with sequences from common wheat (AABBDD) and three diploid ancestral species, Triticum urartu (AA), Aegilops speltoides (BB), and Aegilops tauschii (DD), demonstrated the presence of this amplicon in the AA genome. Using conventional qualitative polymerase chain reaction (PCR), the limit of detection was 2 copies of wheat haploid genome per reaction. In the quantitative real-time PCR assay, limits of detection and quantification were about 2 and 8 haploid genome copies, respectively, the latter of which is 2.5-4-fold lower than other reported wheat endogenous reference genes. Construct-specific PCR assays were developed using RPL21 as an endogenous reference gene, and as little as 0.5% of GM wheat contents containing Arabidopsis NPR1 were properly quantified.

Impact of Enteroviral Polymerase Chain Reaction Testing on Length of Stay for Infants 60 Days Old or Younger.

PubMed

Aronson, Paul L; Lyons, Todd W; Cruz, Andrea T; Freedman, Stephen B; Okada, Pamela J; Fleming, Alesia H; Arms, Joseph L; Thompson, Amy D; Schmidt, Suzanne M; Louie, Jeffrey; Alfonzo, Michael J; Monuteaux, Michael C; Nigrovic, Lise E

2017-10-01

To determine the impact of a cerebrospinal fluid enterovirus polymerase chain reaction (PCR) test performance on hospital length of stay (LOS) in a large multicenter cohort of infants undergoing evaluation for central nervous system infection. We performed a planned secondary analysis of a retrospective cohort of hospitalized infants ≤60 days of age who had a cerebrospinal fluid culture obtained at 1 of 18 participating centers (2005-2013). After adjustment for patient age and study year as well as clustering by hospital center, we compared LOS for infants who had an enterovirus PCR test performed vs not performed and among those tested, for infants with a positive vs negative test result. Of 19 953 hospitalized infants, 4444 (22.3%) had an enterovirus PCR test performed and 945 (21.3% of tested infants) had positive test results. Hospital LOS was similar for infants who had an enterovirus PCR test performed compared with infants who did not (incident rate ratio 0.98 hours; 95% CI 0.89-1.06). However, infants PCR positive for enterovirus had a 38% shorter LOS than infants PCR negative for enterovirus (incident rate ratio 0.62 hours; 95% CI 0.57-0.68). No infant with a positive enterovirus PCR test had bacterial meningitis (0%; 95% CI 0-0.4). Although enterovirus PCR testing was not associated with a reduction in LOS, infants with a positive enterovirus PCR test had a one-third shorter LOS compared with infants with a negative enterovirus PCR test. Focused enterovirus PCR test use could increase the impact on LOS for infants undergoing cerebrospinal fluid evaluation. Copyright © 2017 Elsevier Inc. All rights reserved.

Chronic myelogenous leukemia: laboratory diagnosis and monitoring.

PubMed

Wang, Y L; Bagg, A; Pear, W; Nowell, P C; Hess, J L

2001-10-01

Rapid developments have occurred both in laboratory medicine and in therapeutic interventions for the management of patients with chronic myelogenous leukemia (CML). With a wide array of laboratory tests available, selecting the appropriate test for a specific diagnostic or therapeutic setting has become increasingly difficult. In this review, we first discuss, from the point of view of laboratory medicine, the advantages and disadvantages of several commonly used laboratory assays, including cytogenetics, fluorescence in situ hybridization (FISH), and qualitative and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). We then discuss, from the point of view of clinical care, the test(s) of choice for the most common clinical scenarios, including diagnosis and monitoring of the therapeutic response and minimal residual disease in patients treated with different therapies. The purpose of this review is to help clinicians and laboratory physicians select appropriate tests for the diagnosis and monitoring of CML, with the ultimate goal of improving the cost-effective usage of clinical laboratories and improving patient care. Copyright 2001 Wiley-Liss, Inc.

Collaborative trial validation studies of real-time PCR-based GMO screening methods for detection of the bar gene and the ctp2-cp4epsps construct.

PubMed

Grohmann, Lutz; Brünen-Nieweler, Claudia; Nemeth, Anne; Waiblinger, Hans-Ulrich

2009-10-14

Polymerase Chain Reaction (PCR)-based screening methods targeting genetic elements commonly used in genetically modified (GM) plants are important tools for the detection of GM materials in food, feed, and seed samples. To expand and harmonize the screening capability of enforcement laboratories, the German Federal Office of Consumer Protection and Food Safety conducted collaborative trials for interlaboratory validation of real-time PCR methods for detection of the phosphinothricin acetyltransferase (bar) gene from Streptomyces hygroscopicus and a construct containing the 5-enolpyruvylshikimate-3-phosphate synthase gene from Agrobacterium tumefaciens sp. strain CP4 (ctp2-cp4epsps), respectively. To assess the limit of detection, precision, and accuracy of the methods, laboratories had to analyze two sets of 18 coded genomic DNA samples of events LLRice62 and MS8 with the bar method and NK603 and GT73 with the ctp2-cp4epsps method at analyte levels of 0, 0.02, and 0.1% GM content, respectively. In addition, standard DNAs were provided to the laboratories to generate calibration curves for copy number quantification of the bar and ctp2-cp4epsps target sequences present in the test samples. The study design and the results obtained are discussed with respect to the difficult issue of developing general guidelines and concepts for the collaborative trial validation of qualitative PCR screening methods.

A Complementary Isothermal Amplification Method to the U.S. EPA Quantitative Polymerase Chain Reaction Approach for the Detection of Enterococci in Environmental Waters

PubMed Central

2017-01-01

We report a novel molecular assay, based on helicase-dependent amplification (HDA), for the detection of enterococci as markers for fecal pollution in water. This isothermal assay targets the same Enterococcus 23S rRNA gene region as the existing quantitative polymerase chain reaction (qPCR) assays of U.S. Environmental Protection Agency Methods 1611 and 1609 but can be entirely performed on a simple heating block. The developed Enterococcus HDA assay successfully discriminated 15 enterococcal from 15 non-enterococcal reference strains and reliably detected 48 environmental isolates of enterococci. The limit of detection was 25 target copies per reaction, only 3 times higher than that of qPCR. The applicability of the assay was tested on 30 environmental water sample DNA extracts, simulating a gradient of fecal pollution. Despite the isothermal nature of the reaction, the HDA results were consistent with those of the qPCR reference. Given this performance, we conclude that the developed Enterococcus HDA assay has great potential as a qualitative molecular screening method for resource-limited settings when combined with compatible up- and downstream processes. This amplification strategy can pave the way for developing a new generation of rapid, low-cost, and field-deployable molecular diagnostic tools for water quality monitoring. PMID:28541661

A simplified and accurate detection of the genetically modified wheat MON71800 with one calibrator plasmid.

PubMed

Kim, Jae-Hwan; Park, Saet-Byul; Roh, Hyo-Jeong; Park, Sunghoon; Shin, Min-Ki; Moon, Gui Im; Hong, Jin-Hwan; Kim, Hae-Yeong

2015-06-01

With the increasing number of genetically modified (GM) events, unauthorized GMO releases into the food market have increased dramatically, and many countries have developed detection tools for them. This study described the qualitative and quantitative detection methods of unauthorized the GM wheat MON71800 with a reference plasmid (pGEM-M71800). The wheat acetyl-CoA carboxylase (acc) gene was used as the endogenous gene. The plasmid pGEM-M71800, which contains both the acc gene and the event-specific target MON71800, was constructed as a positive control for the qualitative and quantitative analyses. The limit of detection in the qualitative PCR assay was approximately 10 copies. In the quantitative PCR assay, the standard deviation and relative standard deviation repeatability values ranged from 0.06 to 0.25 and from 0.23% to 1.12%, respectively. This study supplies a powerful and very simple but accurate detection strategy for unauthorized GM wheat MON71800 that utilizes a single calibrator plasmid. Copyright © 2014 Elsevier Ltd. All rights reserved.

Event-specific qualitative and quantitative detection of five genetically modified rice events using a single standard reference molecule.

PubMed

Kim, Jae-Hwan; Park, Saet-Byul; Roh, Hyo-Jeong; Shin, Min-Ki; Moon, Gui-Im; Hong, Jin-Hwan; Kim, Hae-Yeong

2017-07-01

One novel standard reference plasmid, namely pUC-RICE5, was constructed as a positive control and calibrator for event-specific qualitative and quantitative detection of genetically modified (GM) rice (Bt63, Kemingdao1, Kefeng6, Kefeng8, and LLRice62). pUC-RICE5 contained fragments of a rice-specific endogenous reference gene (sucrose phosphate synthase) as well as the five GM rice events. An existing qualitative PCR assay approach was modified using pUC-RICE5 to create a quantitative method with limits of detection correlating to approximately 1-10 copies of rice haploid genomes. In this quantitative PCR assay, the square regression coefficients ranged from 0.993 to 1.000. The standard deviation and relative standard deviation values for repeatability ranged from 0.02 to 0.22 and 0.10% to 0.67%, respectively. The Ministry of Food and Drug Safety (Korea) validated the method and the results suggest it could be used routinely to identify five GM rice events. Copyright © 2017 Elsevier Ltd. All rights reserved.

Association Mapping of the High-Grade Myopia MYP3 Locus Reveals Novel Candidates UHRF1BP1L, PTPRR, and PPFIA2

PubMed Central

Hawthorne, Felicia; Feng, Sheng; Metlapally, Ravikanth; Li, Yi-Ju; Tran-Viet, Khanh-Nhat; Guggenheim, Jeremy A.; Malecaze, Francois; Calvas, Patrick; Rosenberg, Thomas; Mackey, David A.; Venturini, Cristina; Hysi, Pirro G.; Hammond, Christopher J.; Young, Terri L.

2013-01-01

Purpose. Myopia, or nearsightedness, is a common ocular genetic disease for which over 20 candidate genomic loci have been identified. The high-grade myopia locus, MYP3, has been reported on chromosome 12q21–23 by four independent linkage studies. Methods. We performed a genetic association study of the MYP3 locus in a family-based high-grade myopia cohort (n = 82) by genotyping 768 single-nucleotide polymorphisms (SNPs) within the linkage region. Qualitative testing for high-grade myopia (sphere ≤ −5 D affected, > −0.5 D unaffected) and quantitative testing on the average dioptric sphere were performed. Results. Several genetic markers were nominally significantly associated with high-grade myopia in qualitative testing, including rs3803036, a missense mutation in PTPRR (P = 9.1 × 10−4) and rs4764971, an intronic SNP in UHRF1BP1L (P = 6.1 × 10−4). Quantitative testing determined statistically significant SNPs rs4764971, also found by qualitative testing (P = 3.1 × 10−6); rs7134216, in the 3′ untranslated region (UTR) of DEPDC4 (P = 5.4 × 10−7); and rs17306116, an intronic SNP within PPFIA2 (P < 9 × 10−4). Independently conducted whole genome expression array analyses identified protein tyrosine phosphatase genes PTPRR and PPFIA2, which are in the same gene family, as differentially expressed in normal rapidly growing fetal relative to normal adult ocular tissue (confirmed by RT-qPCR). Conclusions. In an independent high-grade myopia cohort, an intronic SNP in UHRF1BP1L, rs4764971, was validated for quantitative association, and SNPs within PTPRR (quantitative) and PPFIA2 (qualitative and quantitative) approached significance. Three genes identified by our association study and supported by ocular expression and/or replication, UHRF1BP1L, PTPRR, and PPFIA2, are novel candidates for myopic development within the MYP3 locus that should be further studied. PMID:23422819

Development and clinical validation of the Genedrive point-of-care test for qualitative detection of hepatitis C virus.

PubMed

Llibre, Alba; Shimakawa, Yusuke; Mottez, Estelle; Ainsworth, Shaun; Buivan, Tan-Phuc; Firth, Rick; Harrison, Elliott; Rosenberg, Arielle R; Meritet, Jean-François; Fontanet, Arnaud; Castan, Pablo; Madejón, Antonio; Laverick, Mark; Glass, Allison; Viana, Raquel; Pol, Stanislas; McClure, C Patrick; Irving, William Lucien; Miele, Gino; Albert, Matthew L; Duffy, Darragh

2018-04-03

Recently approved direct acting antivirals provide transformative therapies for chronic hepatitis C virus (HCV) infection. The major clinical challenge remains to identify the undiagnosed patients worldwide, many of whom live in low-income and middle-income countries, where access to nucleic acid testing remains limited. The aim of this study was to develop and validate a point-of-care (PoC) assay for the qualitative detection of HCV RNA. We developed a PoC assay for the qualitative detection of HCV RNA on the PCR Genedrive instrument. We validated the Genedrive HCV assay through a case-control study comparing results with those obtained with the Abbott RealTi m e HCV test. The PoC assay identified all major HCV genotypes, with a limit of detection of 2362 IU/mL (95% CI 1966 to 2788). Using 422 patients chronically infected with HCV and 503 controls negative for anti-HCV and HCV RNA, the Genedrive HCV assay showed 98.6% sensitivity (95% CI 96.9% to 99.5%) and 100% specificity (95% CI 99.3% to 100%) to detect HCV. In addition, melting peak ratiometric analysis demonstrated proof-of-principle for semiquantification of HCV. The test was further validated in a real clinical setting in a resource-limited country. We report a rapid, simple, portable and accurate PoC molecular test for HCV, with sensitivity and specificity that fulfils the recent FIND/WHO Target Product Profile for HCV decentralised testing in low-income and middle-income countries. This Genedrive HCV assay may positively impact the continuum of HCV care from screening to cure by supporting real-time treatment decisions. NCT02992184. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Absolute quantification of olive oil DNA by droplet digital-PCR (ddPCR): Comparison of isolation and amplification methodologies.

PubMed

Scollo, Francesco; Egea, Leticia A; Gentile, Alessandra; La Malfa, Stefano; Dorado, Gabriel; Hernandez, Pilar

2016-12-15

Olive oil is considered a premium product for its nutritional value and health benefits, and the ability to define its origin and varietal composition is a key step towards ensuring the traceability of the product. However, isolating the DNA from such a matrix is a difficult task. In this study, the quality and quantity of olive oil DNA, isolated using four different DNA isolation protocols, was evaluated using the qRT-PCR and ddPCR techniques. The results indicate that CTAB-based extraction methods were the best for unfiltered oil, while Nucleo Spin-based extraction protocols showed greater overall reproducibility. The use of both qRT-PCR and ddPCR led to the absolute quantification of the DNA copy number. The results clearly demonstrate the importance of the choice of DNA-isolation protocol, which should take into consideration the qualitative aspects of DNA and the evaluation of the amplified DNA copy number. Copyright © 2016 Elsevier Ltd. All rights reserved.

[Detection of rubella virus RNA in clinical material by real time polymerase chain reaction method].

PubMed

Domonova, É A; Shipulina, O Iu; Kuevda, D A; Larichev, V F; Safonova, A P; Burchik, M A; Butenko, A M; Shipulin, G A

2012-01-01

Development of a reagent kit for detection of rubella virus RNA in clinical material by PCR-RT. During development and determination of analytical specificity and sensitivity DNA and RNA of 33 different microorganisms including 4 rubella strains were used. Comparison of analytical sensitivity of virological and molecular-biological methods was performed by using rubella virus strains Wistar RA 27/3, M-33, "Orlov", Judith. Evaluation of diagnostic informativity of rubella virus RNAisolation in various clinical material by PCR-RT method was performed in comparison with determination of virus specific serum antibodies by enzyme immunoassay. A reagent kit for the detection of rubella virus RNA in clinical material by PCR-RT was developed. Analytical specificity was 100%, analytical sensitivity - 400 virus RNA copies per ml. Analytical sensitivity of the developed technique exceeds analytical sensitivity of the Vero E6 cell culture infection method in studies of rubella virus strains Wistar RA 27/3 and "Orlov" by 11g and 31g, and for M-33 and Judith strains is analogous. Diagnostic specificity is 100%. Diagnostic specificity for testing samples obtained within 5 days of rash onset: for peripheral blood sera - 20.9%, saliva - 92.5%, nasopharyngeal swabs - 70.1%, saliva and nasopharyngeal swabs - 97%. Positive and negative predictive values of the results were shown depending on the type of clinical material tested. Application of reagent kit will allow to increase rubella diagnostics effectiveness at the early stages of infectious process development, timely and qualitatively perform differential diagnostics of exanthema diseases, support tactics of anti-epidemic regime.

Overdiagnosis of Clostridium difficile Infection in the Molecular Test Era.

PubMed

Polage, Christopher R; Gyorke, Clare E; Kennedy, Michael A; Leslie, Jhansi L; Chin, David L; Wang, Susan; Nguyen, Hien H; Huang, Bin; Tang, Yi-Wei; Lee, Lenora W; Kim, Kyoungmi; Taylor, Sandra; Romano, Patrick S; Panacek, Edward A; Goodell, Parker B; Solnick, Jay V; Cohen, Stuart H

2015-11-01

Clostridium difficile is a major cause of health care-associated infection, but disagreement between diagnostic tests is an ongoing barrier to clinical decision making and public health reporting. Molecular tests are increasingly used to diagnose C difficile infection (CDI), but many molecular test-positive patients lack toxins that historically defined disease, making it unclear if they need treatment. To determine the natural history and need for treatment of patients who are toxin immunoassay negative and polymerase chain reaction (PCR) positive (Tox-/PCR+) for CDI. Prospective observational cohort study at a single academic medical center among 1416 hospitalized adults tested for C difficile toxins 72 hours or longer after admission between December 1, 2010, and October 20, 2012. The analysis was conducted in stages with revisions from April 27, 2013, to January 13, 2015. Patients undergoing C difficile testing were grouped by US Food and Drug Administration-approved toxin and PCR tests as Tox+/PCR+, Tox-/PCR+, or Tox-/PCR-. Toxin results were reported clinically. Polymerase chain reaction results were not reported. The main study outcomes were duration of diarrhea during up to 14 days of treatment, rate of CDI-related complications (ie, colectomy, megacolon, or intensive care unit care) and CDI-related death within 30 days. Twenty-one percent (293 of 1416) of hospitalized adults tested for C difficile were positive by PCR, but 44.7% (131 of 293) had toxins detected by the clinical toxin test. At baseline, Tox-/PCR+ patients had lower C difficile bacterial load and less antibiotic exposure, fecal inflammation, and diarrhea than Tox+/PCR+ patients (P < .001 for all). The median duration of diarrhea was shorter in Tox-/PCR+ patients (2 days; interquartile range, 1-4 days) than in Tox+/PCR+ patients (3 days; interquartile range, 1-6 days) (P = .003) and was similar to that in Tox-/PCR- patients (2 days; interquartile range, 1-3 days), despite minimal empirical treatment of Tox-/PCR+ patients. No CDI-related complications occurred in Tox-/PCR+ patients vs 10 complications in Tox+/PCR+ patients (0% vs 7.6%, P < .001). One Tox-/PCR+ patient had recurrent CDI as a contributing factor to death within 30 days vs 11 CDI-related deaths in Tox+/PCR+ patients (0.6% vs 8.4%, P = .001). Among hospitalized adults with suspected CDI, virtually all CDI-related complications and deaths occurred in patients with positive toxin immunoassay test results. Patients with a positive molecular test result and a negative toxin immunoassay test result had outcomes that were comparable to patients without C difficile by either method. Exclusive reliance on molecular tests for CDI diagnosis without tests for toxins or host response is likely to result in overdiagnosis, overtreatment, and increased health care costs.

Use of polymerase chain reaction technique to confirm VecTest screening results in Plasmodium falciparum and Plasmodium vivax VK 210 laboratory-infected Anopheles stephensi mosquitoes.

PubMed

Santos-Ciminera, Patricia D; Acheé, Nicole L; Quinnan, Gerald V; Roberts, Donald R

2004-09-01

We evaluated polymerase chain reaction (PCR) to confirm immunoassays for malaria parasites in mosquito pools after a failure to detect malaria with PCR during an outbreak in which pools tested positive using VecTest and enzyme-linked immunosorbent assay (ELISA). We combined VecTest, ELISA, and PCR to detect Plasmodium falciparum and Plasmodium vivax VK 210. Each mosquito pool, prepared in triplicate, consisted of 1 exposed Anopheles stephensi and up to 9 unfed mosquitoes. The results of VecTest and ELISA were concordant. DNA from a subset of the pools, 1 representative of each ratio of infected to uninfected mosquitoes, was extracted and used as template in PCR. All P. vivax pools were PCR positive but some needed additional processing for removal of apparent inhibitors before positive results were obtained. One of the pools selected for P. falciparum was negative by PCR, probably because of losses or contamination during DNA extraction; 2 remaining pools at this ratio were PCR positive. Testing pools by VecTest, ELISA, and PCR is feasible, and PCR is useful for confirmation of immunoassays. An additional step might be needed to remove potential inhibitors from pools prior to PCR.

Evaluation of a shortened QIAsymphony DNA extraction protocol for stool samples using a multiplex real-time PCR for the detection of enteric pathogens.

PubMed

van Zanten, E; Wisselink, G J; Stoll, S; Alvarez, R; Kooistra-Smid, A M D

2011-02-01

A shortened DNA extraction protocol for the QIAsymphony SP was evaluated by quantitative and qualitative comparison of real-time PCR results of 150 co-extracted stool samples. The average ∆Cycle threshold value for positive pathogenic targets was 0.28 Ct. A consensus of 96.91%, with a correlation coefficient of 0.9880 was recorded. Copyright © 2010 Elsevier B.V. All rights reserved.

Cytomegalovirus Urinary Shedding in HIV-infected Pregnant Women and Congenital Cytomegalovirus Infection.

PubMed

Adachi, Kristina; Xu, Jiahong; Ank, Bonnie; Watts, D Heather; Mofenson, Lynne M; Pilotto, Jose Henrique; Joao, Esau; Santos, Breno; Fonseca, Rosana; Kreitchmann, Regis; Pinto, Jorge; Mussi-Pinhata, Marisa M; Gray, Glenda; Theron, Gerhard; Morgado, Mariza G; Bryson, Yvonne J; Veloso, Valdilea G; Klausner, Jeffrey D; Moye, Jack; Nielsen-Saines, Karin

2017-08-01

Cytomegalovirus (CMV) urinary shedding in pregnant women infected with human immunodeficiency virus (HIV) was evaluated to determine whether it poses an increased risk for congenital CMV infection (cCMV). A subset of mother-infant pairs enrolled in the perinatal NICHD HPTN 040 study (distinguished by no antiretroviral use before labor) was evaluated. Maternal and infant urines were tested by qualitative real-time polymerase chain reaction (RT-PCR) for CMV DNA with quantitative RT-PCR performed on positive specimens. Urine specimens were available for 260 women with 85.4% from the Americas and 14.6% from South Africa. Twenty-four women (9.2%) had detectable CMV viruria by qualitative PCR. Maternal CMV viruria was not associated with mean CD4 cell counts or HIV viral load but was associated with younger maternal age (P = .02). Overall, 10 of 260 infants (3.8%) had cCMV. Women with detectable peripartum CMV viruria were more likely to have infants with cCMV than those without: 20.8% (5/24) versus 2.1% (5/236), (P = .0001). Women with CMV viruria had significantly higher rates of HIV perinatal transmission (29.2% vs. 8.1%, P = .002). They were 5 times (adjusted odds ratio [aOR] = 5.6, 95% confidence interval [CI] 1.9-16.8) and nearly 30 times (aOR, 29.7; 95% CI, 5.4-164.2) more likely to transmit HIV and CMV to their infants, respectively. Maternal gonorrhea (aOR, 19.5; 95% CI, 2.5-151.3) and higher maternal HIV log10 viral load (OR, 2.8; 95% CI, 1.3-6.3) were also significant risk factors for cCMV. In this cohort of HIV-infected pregnant women not on antiretrovirals, urinary CMV shedding was a significant risk factor for CMV and HIV transmission to infants. NCT00099359. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com

Agreement Rate of Rapid Urease Test, Conventional PCR, and Scorpion Real-Time PCR in Detecting Helicobacter Pylori from Tonsillar Samples of Patients with Chronic Tonsillitis

PubMed Central

Najafipour, Reza; Farivar, Taghi Naserpour; Pahlevan, Ali Akbar; Johari, Pouran; Safdarian, Farshid; Asefzadeh, Mina

2012-01-01

Background: Helicobacter pylori is capable of inducing systemic inflammatory reactions through immunological processes. There are several methods to identify the presence of H. pylori in clinical samples including rapid urease test (RUT), conventional polymerase chain reaction (PCR), and the Scorpion real-time PCR. Aim: The aim of the present study is to compare the agreement rate of these tests in identifying H. pylori in tonsillar biopsy specimens collected from patients with chronic tonsillitis. Materials and Methods: A total of 103 tonsil biopsy samples from patients with clinical signs of chronic tonsillitis were examined with RUT, PCR, and Scorpion real-time PCR. The degree of agreement between the three tests was later calculated. Results: There was a poor degree of agreement between RUT and PCR and also RUT and Scorpion real-time PCR (Kappa=0.269 and 0.249, respectively). In contrast with RUT, there was a strong degree of agreement between PCR and Scorpion real-time PCR (Kappa=0.970). Conclusion: The presence of a strong agreement between the Scorpion real-time PCR and PCR as well as its technical advantage over the conventional PCR assay, made the Scorpion real-time PCR an appropriate laboratory test to investigate the presence of H. pylori in tonsillar biopsy specimens in patients suffering from chronic tonsillitis. PMID:22754245

Duration of Group A Streptococcus PCR positivity following antibiotic treatment of pharyngitis.

PubMed

Homme, Jason H; Greenwood, Corryn S; Cronk, Lisa B; Nyre, Lisa M; Uhl, James R; Weaver, Amy L; Patel, Robin

2018-02-01

Polymerase chain reaction (PCR) has high sensitivity and specificity for detection of group A streptococcus (GAS) in throat swabs and is routinely used for GAS pharyngitis diagnosis at our institution. Herein we defined the natural history of throat swab GAS PCR and culture positivity during and following treatment of GAS pharyngitis. Fifty children with a PCR positive GAS throat swab were recruited for participation. Four additional throat swabs were collected over 2 weeks following the initial positive PCR result (during and following a standard course of antibiotic therapy) and tested for GAS using rapid real-time PCR and culture. After the initial positive swab, 45% had a positive PCR 2-4 days, 20% 5-7 days, 18% 8-10 days, 25% 11-13days, and 20% 14-18days later. The median time to a negative PCR was 4 days with the nadir in positive PCR results approximating the end of a typical 10-day treatment interval. Seven subjects remained persistently PCR positive. Culture results remained positive at a stable rate for each time interval, ranging from 5-10%. If a patient presents with symptoms of GAS pharyngitis after previous positive GAS PCR testing and treatment with appropriate antibiotics, it is reasonable to use PCR testing for GAS pharyngitis testing beginning one week after initial testing. Further studies are warranted to determine if this time frame can be applied to PCR testing used to detect other infections. Copyright © 2017 Elsevier Inc. All rights reserved.

Use of Selected Essential Oils to Control Aflatoxin Contaminated Stored Cashew and Detection of Aflatoxin Biosynthesis Gene

PubMed Central

Abd El-Aziz, Abeer R. M.; Mahmoud, Mohamed A.; Al-Othman, Monira R.; Al-Gahtani, Munirah F.

2015-01-01

Aspergillus spp. associated with cashew from the regions of Riyadh, Dammam, and Abha were isolated and three different culture media were used to qualitatively measure aflatoxin production by Aspergillus via UV light (365 nm), which was expressed as positive or negative. The obtained data showed that six isolates of A. flavus and four isolates of A. parasiticus were positive for aflatoxin production, while all isolates of A. niger were negative. Five commercially essential oils (thyme, garlic, cinnamon, mint, and rosemary) were tested to determine their influence on growth and aflatoxin production in A. flavus and A. parasiticus by performing high-performance liquid chromatography (HPLC). The results showed that the tested essential oils caused highly significant inhibition of fungal growth and aflatoxin production in A. flavus and A. parasiticus. The extent of the inhibition of fungal growth and aflatoxin production was dependent on the type and concentration of essential oils applied. The results indicate that cinnamon and thyme oils show strong antimicrobial potential. PCR was used with four sets of primer pairs for nor-1, omt-1, ver-1, and aflR genes, enclosed in the aflatoxin biosynthetic pathway. The interpretation of the results revealed that PCR is a rapid and sensitive method. PMID:25705718

Use of selected essential oils to control aflatoxin contaminated stored cashew and detection of aflatoxin biosynthesis gene.

PubMed

Abd El-Aziz, Abeer R M; Mahmoud, Mohamed A; Al-Othman, Monira R; Al-Gahtani, Munirah F

2015-01-01

Aspergillus spp. associated with cashew from the regions of Riyadh, Dammam, and Abha were isolated and three different culture media were used to qualitatively measure aflatoxin production by Aspergillus via UV light (365 nm), which was expressed as positive or negative. The obtained data showed that six isolates of A. flavus and four isolates of A. parasiticus were positive for aflatoxin production, while all isolates of A. niger were negative. Five commercially essential oils (thyme, garlic, cinnamon, mint, and rosemary) were tested to determine their influence on growth and aflatoxin production in A. flavus and A. parasiticus by performing high-performance liquid chromatography (HPLC). The results showed that the tested essential oils caused highly significant inhibition of fungal growth and aflatoxin production in A. flavus and A. parasiticus. The extent of the inhibition of fungal growth and aflatoxin production was dependent on the type and concentration of essential oils applied. The results indicate that cinnamon and thyme oils show strong antimicrobial potential. PCR was used with four sets of primer pairs for nor-1, omt-1, ver-1, and aflR genes, enclosed in the aflatoxin biosynthetic pathway. The interpretation of the results revealed that PCR is a rapid and sensitive method.

Efficacy of Recombinase Polymerase Amplification to Diagnose Trypanosoma cruzi Infection in Dogs with Cardiac Alterations from an Endemic Area of Mexico.

PubMed

Jimenez-Coello, Matilde; Shelite, Thomas; Castellanos-Gonzalez, Alejandro; Saldarriaga, Omar; Rivero, Rocio; Ortega-Pacheco, Antonio; Acevedo-Arcique, Carlos; Amaya-Guardia, Karla; Garg, Nisha; Melby, Peter; Travi, Bruno L

2018-05-16

Chagas disease is a lingering Public Health problem in Latin America with ∼5.7 million people infected with Trypanosoma cruzi. Transmission is still taking place in most countries of the Americas, including the United States. Dogs are frequently infected with T. cruzi and its high infection prevalence is associated with increased risk of Chagas disease in humans. The city of Mérida in the Yucatan peninsula is endemic for Chagas disease and canines are frequently infected with T. cruzi. The objective of this study was to evaluate the performance of a qualitative point of care (POC) molecular test (RPA-LF, recombinase polymerase amplification-lateral flow) developed in our laboratory for identifying infected dogs. We used retrospective samples of dogs that came for consultation because of cardiac alterations and proved to be infected with T. cruzi as determined by enzyme-linked immunosorbent assay (ELISA), Western blot, and quantitative PCR (qPCR). The analytical sensitivity indicated that RPA-LF amplified T. cruzi DNA in samples containing almost equal to one to two parasites per reaction. Serial twofold dilutions of T. cruzi epimastigotes showed that the test had 95% (19/20) repeatability at concentrations of two parasites per reaction. The test showed no cross reactivity with human DNA or other protozoan parasites (Trypanosoma rangeli, Leishmania spp., and Plasmodium spp.). RPA-LF had the capacity to amplify all discrete typing units (DTUs I-VI) of T. cruzi that circulate in domestic or extradomestic environments. The RPA-LF had 93.2% (95% confidence interval 87.2-98.1) sensitivity and excellent agreement with qPCR used as gold standard (Cohen's Kappa test = 0.963). ELISA was positive in 96.6% (85/88) of dogs, which together with the molecular tests confirmed the frequent contact with infected triatomine bugs in the city of Mérida. These preliminary results on the diagnostic efficacy of the RPA-LF deserve further large-scale field testing of this POC test for T. cruzi infection in endemic areas.

Comparison of the clinical performances of the AdvanSure HPV Screening Real-Time PCR, the Abbott Real-Time High-Risk HPV Test, and the Hybrid Capture High-Risk HPV DNA Test for Cervical Cancer Screening.

PubMed

Chung, Hae-Sun; Hahm, Chorong; Lee, Miae

2014-09-01

The clinical performance of three human papillomavirus (HPV) DNA commercial assays for cervical cancer screening was evaluated; the AdvanSure HPV Screening Real-Time PCR (AdvanSure PCR; LG Life Sciences) that was developed recently for the detection of both high-risk and low-risk genotypes, the Abbott RealTime High-Risk HPV Test (Abbott PCR; Abbott Molecular) and the Hybrid Capture High-Risk HPV DNA test (HC2; Qiagen). The three different HPV DNA tests were compared using cytology samples obtained from 619 women who underwent routine cervical cancer screening. The gold-standard assay was histopathological confirmation of cervical intraepithelial neoplasia of grade 2 or worse. The clinical sensitivities of the AdvanSure PCR, the Abbott PCR and the HC2 for the detection of cervical intraepithelial neoplasia of grade 2 or worse were 95.5%, 95.5% and 100%, respectively, while the clinical specificities were 61.6%, 86.4% and 83.3%, respectively. There were no significant differences in the clinical sensitivities of the Abbott PCR and the AdvanSure PCR compared to the HC2. The clinical specificities of the Abbott PCR and the AdvanSure PCR for the detection of HPV types 16/18 were 97.8% and 98.5%, respectively. For cervical cancer screening, all three tests showed relatively good clinical sensitivities, but the AdvanSure PCR had lower clinical specificity than the Abbott PCR and the HC2. The AdvanSure PCR and the Abbott PCR assays have the advantage of being automated and the ability to distinguish between HPV types 16/18 and other HPV types. The two real-time PCR assays could be useful tools in HPV testing for cervical cancer screening. Copyright © 2014 Elsevier B.V. All rights reserved.

Comparison of the AdvanSure human papillomavirus screening real-time PCR, the Abbott RealTime High Risk human papillomavirus test, and the Hybrid Capture human papillomavirus DNA test for the detection of human papillomavirus.

PubMed

Hwang, Yusun; Lee, Miae

2012-05-01

We evaluated the performance of various commercial assays for the molecular detection of human papillomavirus (HPV); the recently developed AdvanSure HPV Screening real-time PCR assay (AdvanSure PCR) and the Abbott RealTime High Risk HPV PCR assay (Abbott PCR) were compared with the Hybrid Capture 2 HPV DNA Test (HC2). All 3 tests were performed on 177 samples, and any sample that showed a discrepancy in any of the 3 tests was genotyped using INNO-LiPA HPV genotyping and/or sequencing. On the basis of these results, we obtained a consensus HPV result, and the performance of each test was evaluated. We also evaluated high-risk HPV 16/18 detection by using the 2 real-time PCR assays. Among the 177 samples, 65 were negative and 75 were positive in all 3 assays; however, the results of the 3 assays with 37 samples were discrepant. Compared with the consensus HPV result, the sensitivities and specificities of HC2, AdvanSure PCR, and Abbott PCR were 97.6%, 91.7%, and 86.9% and 83.9%, 98.8%, and 100.0%, respectively. For HPV type 16/18 detection, the concordance rate between the AdvanSure PCR and Abbott PCR assays was 98.3%; however, 3 samples were discrepant (positive in AdvanSure PCR and negative in Abbott PCR) and were confirmed as HPV type 16 by INNO-LiPA genotyping and/or sequencing. For HPV detection, the AdvanSure HPV Screening real-time PCR assay and the Abbott PCR assay are less sensitive but more specific than the HC2 assay, but can simultaneously differentiate type 16/18 HPV from other types.

Diagnostic validation of three test methods for detection of cyprinid herpesvirus 3 (CyHV-3).

PubMed

Clouthier, Sharon C; McClure, Carol; Schroeder, Tamara; Desai, Megan; Hawley, Laura; Khatkar, Sunita; Lindsay, Melissa; Lowe, Geoff; Richard, Jon; Anderson, Eric D

2017-03-06

Cyprinid herpesvirus 3 (CyHV-3) is the aetiological agent of koi herpesvirus disease in koi and common carp. The disease is notifiable to the World Organisation for Animal Health. Three tests-quantitative polymerase chain reaction (qPCR), conventional PCR (cPCR) and virus isolation by cell culture (VI)-were validated to assess their fitness as diagnostic tools for detection of CyHV-3. Test performance metrics of diagnostic accuracy were sensitivity (DSe) and specificity (DSp). Repeatability and reproducibility were measured to assess diagnostic precision. Estimates of test accuracy, in the absence of a gold standard reference test, were generated using latent class models. Test samples originated from wild common carp naturally exposed to CyHV-3 or domesticated koi either virus free or experimentally infected with the virus. Three laboratories in Canada participated in the precision study. Moderate to high repeatability (81 to 99%) and reproducibility (72 to 97%) were observed for the qPCR and cPCR tests. The lack of agreement observed between some of the PCR test pair results was attributed to cross-contamination of samples with CyHV-3 nucleic acid. Accuracy estimates for the PCR tests were 99% for DSe and 93% for DSp. Poor precision was observed for the VI test (4 to 95%). Accuracy estimates for VI/qPCR were 90% for DSe and 88% for DSp. Collectively, the results show that the CyHV-3 qPCR test is a suitable tool for surveillance, presumptive diagnosis and certification of individuals or populations as CyHV-3 free.

Virus excretion and antibody dynamics in goats inoculated with a field isolate of peste des petits ruminants virus.

PubMed

Liu, W; Wu, X; Wang, Z; Bao, J; Li, L; Zhao, Y; Li, J

2013-11-01

A field isolate of peste des petits ruminants virus (PPRV) from an outbreak in Tibet, China, was inoculated into goats to investigate the dynamics of virus excretion and antibody production. Further, animals received PPRV vaccine strain Nigeria 75/1. Ocular, nasal and oral samples were tested for the presence of virus antigen by one-step real-time qualitative RT-PCR (qRT-PCR); competitive ELISA (c-ELISA) was used for the measurement of specific antibodies against PPRV. Virus particles could be detected as early as day 3 post-inoculation (pi) and virus excretion lasted for up to day 26 pi. All four goats inoculated with the PPRV field isolate were seropositive as early as day 10 pi. In animals inoculated with the vaccine strain, antibody was detected at day 14 pi, and levels of neutralizing antibodies remained above the protection threshold level (1 : 8) for 8 months. Both virus particles and neutralizing antibodies were detected earlier in goats challenged with the field isolate than in those receiving the vaccine strain. © 2013 Blackwell Verlag GmbH.

The IRIDICA PCR/Electrospray Ionization-Mass Spectrometry Assay on Bronchoalveolar Lavage for Bacterial Etiology in Mechanically Ventilated Patients with Suspected Pneumonia.

PubMed

Strålin, Kristoffer; Ehn, Fredrik; Giske, Christian G; Ullberg, Måns; Hedlund, Jonas; Petersson, Johan; Spindler, Carl; Özenci, Volkan

2016-01-01

We studied the diagnostic performance of the IRIDICA PCR/electrospray ionization-mass spectrometry (PCR/ESI-MS) assay applied on bronchoalveolar lavage (BAL) samples, from 51 mechanically ventilated patients with suspected pneumonia, in a prospective study. In 32 patients with X-ray verified pneumonia, PCR/ESI-MS was positive in 66% and BAL culture was positive in 38% (p = 0.045), and either of the methods was positive in 69%. The following BAL result combinations were noted: PCR/ESI-MS+/culture+, 34%; PCR/ESI-MS+/culture-, 31%; PCR/ESI-MS-/culture+, 3.1%; PCR/ESI-MS-/culture-, 31%; kappa 0.36 (95% confidence interval (CI), 0.10-0.63). In pneumonia patients without prior antibiotic treatment, optimal agreement was noted with 88% PCR/ESI-MS+/culture+ and 12% PCR/ESI-MS-/culture- (kappa 1.0). However, in patients with prior antibiotic treatment, the test agreement was poor (kappa 0.16; 95% CI, -0.10-0.44), as 10 patients were PCR/ESI-MS+/culture-. In 8/10 patients the pathogens detected by PCR/ESI-MS could be detected by other conventional tests or PCR tests on BAL. Compared with BAL culture, PCR/ESI-MS showed specificities and negative predictive values of ≥87% for all individual pathogens, an overall sensitivity of 77% and positive predictive value (PPV) of 42%. When other conventional tests and PCR tests were added to the reference standard, the overall PPV increased to 87%. The PCR/ESI-MS semi-quantitative level tended to be higher for PCR/ESI-MS positive cases with pneumonia compared with cases without pneumonia (p = 0.074). In conclusion, PCR/ESI-MS applied on BAL showed a promising performance and has potential to be clinically useful in mechanically ventilated patients with suspected pneumonia. The usefulness of the method for establishment of pneumonia etiology and selection of antibiotic therapy should be further studied.

O-5S quantitative real-time PCR: a new diagnostic tool for laboratory confirmation of human onchocerciasis.

PubMed

Mekonnen, Solomon A; Beissner, Marcus; Saar, Malkin; Ali, Solomon; Zeynudin, Ahmed; Tesfaye, Kassahun; Adbaru, Mulatu G; Battke, Florian; Poppert, Sven; Hoelscher, Michael; Löscher, Thomas; Bretzel, Gisela; Herbinger, Karl-Heinz

2017-10-02

Onchocerciasis is a parasitic disease caused by the filarial nematode Onchocerca volvulus. In endemic areas, the diagnosis is commonly confirmed by microscopic examination of skin snip samples, though this technique is considered to have low sensitivity. The available melting-curve based quantitative real-time PCR (qPCR) using degenerated primers targeting the O-150 repeat of O. volvulus was considered insufficient for confirming the individual diagnosis, especially in elimination studies. This study aimed to improve detection of O. volvulus DNA in clinical samples through the development of a highly sensitive qPCR assay. A novel hydrolysis probe based qPCR assay was designed targeting the specific sequence of the O. volvulus O-5S rRNA gene. A total of 200 clinically suspected onchocerciasis cases were included from Goma district in South-west Ethiopia, from October 2012 through May 2013. Skin snip samples were collected and subjected to microscopy, O-150 qPCR, and the novel O-5S qPCR. Among the 200 individuals, 133 patients tested positive (positivity rate of 66.5%) and 67 negative by O-5S qPCR, 74 tested positive by microscopy (37.0%) and 78 tested positive by O-150 qPCR (39.0%). Among the 133 O-5S qPCR positive individuals, microscopy and O-150 qPCR detected 55.6 and 59.4% patients, respectively, implying a higher sensitivity of O-5S qPCR than microscopy and O-150 qPCR. None of the 67 individuals who tested negative by O-5S qPCR tested positive by microscopy or O-150 qPCR, implying 100% specificity of the newly designed O-5S qPCR assay. The novel O-5S qPCR assay is more sensitive than both microscopic examination and the existing O-150 qPCR for the detection of O. volvulus from skin snip samples. The newly designed assay is an important step towards appropriate individual diagnosis and control of onchocerciasis.

The IRIDICA PCR/Electrospray Ionization–Mass Spectrometry Assay on Bronchoalveolar Lavage for Bacterial Etiology in Mechanically Ventilated Patients with Suspected Pneumonia

PubMed Central

Ehn, Fredrik; Giske, Christian G.; Ullberg, Måns; Hedlund, Jonas; Petersson, Johan; Spindler, Carl; Özenci, Volkan

2016-01-01

We studied the diagnostic performance of the IRIDICA PCR/electrospray ionization–mass spectrometry (PCR/ESI-MS) assay applied on bronchoalveolar lavage (BAL) samples, from 51 mechanically ventilated patients with suspected pneumonia, in a prospective study. In 32 patients with X-ray verified pneumonia, PCR/ESI-MS was positive in 66% and BAL culture was positive in 38% (p = 0.045), and either of the methods was positive in 69%. The following BAL result combinations were noted: PCR/ESI-MS+/culture+, 34%; PCR/ESI-MS+/culture-, 31%; PCR/ESI-MS-/culture+, 3.1%; PCR/ESI-MS-/culture-, 31%; kappa 0.36 (95% confidence interval (CI), 0.10–0.63). In pneumonia patients without prior antibiotic treatment, optimal agreement was noted with 88% PCR/ESI-MS+/culture+ and 12% PCR/ESI-MS-/culture- (kappa 1.0). However, in patients with prior antibiotic treatment, the test agreement was poor (kappa 0.16; 95% CI, -0.10–0.44), as 10 patients were PCR/ESI-MS+/culture-. In 8/10 patients the pathogens detected by PCR/ESI-MS could be detected by other conventional tests or PCR tests on BAL. Compared with BAL culture, PCR/ESI-MS showed specificities and negative predictive values of ≥87% for all individual pathogens, an overall sensitivity of 77% and positive predictive value (PPV) of 42%. When other conventional tests and PCR tests were added to the reference standard, the overall PPV increased to 87%. The PCR/ESI-MS semi-quantitative level tended to be higher for PCR/ESI-MS positive cases with pneumonia compared with cases without pneumonia (p = 0.074). In conclusion, PCR/ESI-MS applied on BAL showed a promising performance and has potential to be clinically useful in mechanically ventilated patients with suspected pneumonia. The usefulness of the method for establishment of pneumonia etiology and selection of antibiotic therapy should be further studied. PMID:27463099

A naked-eye colorimetric "PCR developer"

NASA Astrophysics Data System (ADS)

Valentini, Paola; Pompa, Pier Paolo

2016-04-01

Despite several advances in molecular biology and diagnostics, Polymerase Chain Reaction (PCR) is currently the gold standard for nucleic acids amplification and detection, due to its versatility, low-cost and universality, with estimated <10 billion reactions per year and a worldwide market of several billion dollars/year. Nevertheless, PCR still relies on the laborious, time-consuming, and multi-step gel electrophoresis-based detection, which includes gel casting, electrophoretic run, gel staining, and gel visualization. In this work, we propose a "PCR developer", namely a universal one-step, one-tube method, based on controlled aggregation of gold nanoparticles (AuNPs), to detect PCR products by naked eye in few minutes, with no need for any instrumentation. We demonstrated the specificity and sensitivity of the PCR developer on different model targets, suitable for a qualitative detection in real-world diagnostics (i.e., gene rearrangements, genetically modified organisms, and pathogens). The PCR developer proved to be highly specific and ultra-sensitive, discriminating down to few copies of HIV viral DNA, diluted in an excess of interfering human genomic DNA, which is a clinically relevant viral load. Hence, it could be a valuable tool for both academic research and clinical applications.

Development, validation and testing costs of an in-house real-time PCR assay for the detection of Chlamydia trachomatis.

PubMed

Santos, Camila Gurgel Dos; Sabidó, Meritxell; Leturiondo, André Luiz; Ferreira, Cynthia de Oliveira; da Cruz, Thielle Pereira; Benzaken, Adele Schwartz

2017-03-01

To improve the screening of Chlamydia trachomatis(C. trachomatis) in Brazil, an accurate and affordable method is needed. The objective of this study was to develop and assess the performance and costs of a new in-house real-time PCR (qPCR) assay for the diagnosis of C. trachomatis infection. Asymptomatic women aged 14-25 years who attended primary health services in Manaus, Brazil, were screened for C. trachomatis using the Digene Hybrid Capture II CT-ID (HCII CT-ID) DNA test. A subset of cervical specimens were tested using an in-house qPCR and a commercial qPCR, ArtusC. trachomatis Plus RG PCR 96 CE (Artus qPCR) kit, as a reference test. A primer/probe based on the sequence of cryptic plasmid (CP) was designed. An economic evaluation was conducted from the provider's perspective. The primers were considered specific for C. trachomatis because they did not amplify any product from non-sexually transmitted bacterial species tested. Overall, 292 specimens were tested by both the commercial kit (Artus qPCR) and the in-house qPCR. Of those, one resulted in no amplification and was excluded from the analysis. The sensitivity, specificity, and positive and negative predictive values of the in-house qPCR were 99.5 % [95 % confidence interval (CI): 97.1-100], 95.1 % (95 % CI: 89-98.4), 97.4 % (95 % CI: 94-99.1) and 99.0 % (95 % CI: 94.5-100), respectively. The cost per case of C. trachomatis was £0.44 ($0.55) for HCII CT-ID, £1.16 ($1.45) for Artus qPCR and £1.06 ($1.33) for in-house qPCR. We have standardized an in-house qPCR to detect cervical C. trachomatis targeting CP. The in-house qPCR showed excellent accuracy and was more affordable than the commercial qPCR kit.

Accurate PCR Detection of Influenza A/B and Respiratory Syncytial Viruses by Use of Cepheid Xpert Flu+RSV Xpress Assay in Point-of-Care Settings: Comparison to Prodesse ProFlu.

PubMed

Cohen, Daniel M; Kline, Jennifer; May, Larissa S; Harnett, Glenn Eric; Gibson, Jane; Liang, Stephen Y; Rafique, Zubaid; Rodriguez, Carina A; McGann, Kevin M; Gaydos, Charlotte A; Mayne, Donna; Phillips, David; Cohen, Jason

2018-02-01

The Xpert Flu+RSV Xpress Assay is a fast, automated in vitro diagnostic test for qualitative detection and differentiation of influenza A and B viruses and respiratory syncytial virus (RSV) performed on the Cepheid GeneXpert Xpress System. The objective of this study was to establish performance characteristics of the Xpert Flu+RSV Xpress Assay compared to those of the Prodesse ProFlu+ real-time reverse transcription-PCR (RT-PCR) assay (ProFlu+) for the detection of influenza A and B viruses as well as RSV in a Clinical Laboratory Improvement Amendments (CLIA)-waived (CW) setting. Overall, the assay, using fresh and frozen nasopharyngeal (NP) swabs, demonstrated high concordance with results of the ProFlu+ assay in the combined CW and non-CW settings with positive percent agreements (PPA) (100%, 100%, and 97.1%) and negative percent agreements (NPA) (95.2%, 99.5%, and 99.6%) for influenza A and B viruses and RSV, respectively. In conclusion, this multicenter study using the Cepheid Xpert Flu+RSV Xpress Assay demonstrated high sensitivities and specificities for influenza A and B viruses and RSV in ∼60 min for use at the point-of-care in the CW setting. Copyright © 2018 American Society for Microbiology.

Cross-sectional, descriptive study of Chagas disease among citizens of Bolivian origin living in Munich, Germany

PubMed Central

Navarro, Miriam; Berens-Riha, Nicole; Hohnerlein, Stefan; Seiringer, Peter; von Saldern, Charlotte; Garcia, Sarah; Blasco-Hernández, Teresa; Navaza, Bárbara; Shock, Jonathan; Bretzel, Gisela; Hoelscher, Michael; Löscher, Thomas; Albajar-Viñas, Pedro; Pritsch, Michael

2017-01-01

Purpose Chagas disease (CD) has become a global health issue mainly due to migration. Germany lacks surveillance data and is home to a large Latin American immigrant population. Recognising that Bolivia is the country with the highest CD prevalence in Latin America, this cross-sectional, descriptive pilot study investigated CD and associated factors among citizens of Bolivian origin living in Munich, Germany. Methods Participants completed a questionnaire in order to collect socioeconomic and health-related data. In addition, serology was performed. In case of positive serological tests, PCR diagnostic and clinical staging together with disease management was initiated. Qualitative research was conducted to identify personal and community barriers as well as strategies to increase CD awareness among the population at risk. Results Between June 2013 and June 2014, 43 people from Bolivia (or descendants) were enrolled. A total of 9.3% (4/43), of whom two women were of childbearing age, tested seropositive (ELISA and IFAT), and one also by PCR. For 2/4 positive participants, clinical evaluation was performed and the indeterminate form of CD was diagnosed. Knowledge about CD symptoms and ways of transmission were completely absent among 55.8% (24/43, 2/4 with CD) and 30.2% (13/43, 1/4 with CD) of participants, respectively. A total of 27.9% (12/43, 0/4 with CD) of participants had donated blood prior to the study, whereas 62.8% (27/43, 3/4 with CD) were motivated to donate blood in the future. The qualitative research identified lack of knowledge as well as stigma and fears related to CD. Conclusions Despite the small number of participants, the prevalence of CD as well as the potential risk of non-vectorial transmission was alarming. Campaigns adapted for Latin American migrants as well as control strategies should be developed and put in place in order to prevent non-vectorial transmission and actively detect cases of CD in Germany. PMID:28093440

Comparison of Performance Characteristics of Aspergillus PCR in Testing a Range of Blood-Based Samples in Accordance with International Methodological Recommendations.

PubMed

Springer, Jan; White, P Lewis; Hamilton, Shanna; Michel, Denise; Barnes, Rosemary A; Einsele, Hermann; Löffler, Juergen

2016-03-01

Standardized methodologies for the molecular detection of invasive aspergillosis (IA) have been established by the European Aspergillus PCR Initiative for the testing of whole blood, serum, and plasma. While some comparison of the performance of Aspergillus PCR when testing these different sample types has been performed, no single study has evaluated all three using the recommended protocols. Standardized Aspergillus PCR was performed on 423 whole-blood pellets (WBP), 583 plasma samples, and 419 serum samples obtained from hematology patients according to the recommendations. This analysis formed a bicenter retrospective anonymous case-control study, with diagnosis according to the revised European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) consensus definitions (11 probable cases and 36 controls). Values for clinical performance using individual and combined samples were calculated. For all samples, PCR positivity was significantly associated with cases of IA (for plasma, P = 0.0019; for serum, P = 0.0049; and for WBP, P = 0.0089). Plasma PCR generated the highest sensitivity (91%); the sensitivities for serum and WBP PCR were 80% and 55%, respectively. The highest specificity was achieved when testing WBP (96%), which was significantly superior to the specificities achieved when testing serum (69%, P = 0.0238) and plasma (53%, P = 0.0002). No cases were PCR negative in all specimen types, and no controls were PCR positive in all specimens. This study confirms that Aspergillus PCR testing of plasma provides robust performance while utilizing commercial automated DNA extraction processes. Combining PCR testing of different blood fractions allows IA to be both confidently diagnosed and excluded. A requirement for multiple PCR-positive plasma samples provides similar diagnostic utility and is technically less demanding. Time to diagnosis may be enhanced by testing multiple contemporaneously obtained sample types. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

pcr: an R package for quality assessment, analysis and testing of qPCR data

PubMed Central

Ahmed, Mahmoud

2018-01-01

Background Real-time quantitative PCR (qPCR) is a broadly used technique in the biomedical research. Currently, few different analysis models are used to determine the quality of data and to quantify the mRNA level across the experimental conditions. Methods We developed an R package to implement methods for quality assessment, analysis and testing qPCR data for statistical significance. Double Delta CT and standard curve models were implemented to quantify the relative expression of target genes from CT in standard qPCR control-group experiments. In addition, calculation of amplification efficiency and curves from serial dilution qPCR experiments are used to assess the quality of the data. Finally, two-group testing and linear models were used to test for significance of the difference in expression control groups and conditions of interest. Results Using two datasets from qPCR experiments, we applied different quality assessment, analysis and statistical testing in the pcr package and compared the results to the original published articles. The final relative expression values from the different models, as well as the intermediary outputs, were checked against the expected results in the original papers and were found to be accurate and reliable. Conclusion The pcr package provides an intuitive and unified interface for its main functions to allow biologist to perform all necessary steps of qPCR analysis and produce graphs in a uniform way. PMID:29576953

Clinical utility of RT-PCR in assessing HER 2 gene expression versus traditional IHC and FISH in breast cancer patients.

PubMed

Suryavanshi, Moushumi; Mehta, Anurag; Jaipuria, Jiten; Kumar, Dushyant; Vishwakarma, Gayatri; Panigrahi, Manoj Kumar; Verma, Haristuti; Saifi, Mumtaz; Sharma, Sanjeev; Tandon, Simran; Doval, D C; Das, Bhudev C

2018-02-09

IHC and FISH are used for categorizing HER 2 status in breast cancer at the protein and DNA level, respectively. HER 2 expression at the RNA level is quantitative, cheaper, easier to standardize and free from interobserver variation. 115 consecutive patients were tested by IHC, FISH and RT-PCR (test cohort). Assuming FISH result to be the response variable, ROC curves for RT-PCR ratio were analyzed to label HER 2 negative, equivocal and positive cases as RT-PCR score 1, 2 and 3, respectively. Inter-relationships between RT-PCR, IHC and FISH were defined. 'Clinical benefit' of a test was defined as proportion of patients labeled unequivocally as HER 2 positive or negative. Population for 1 year was simulated constraint to previous reports of HER 2 positivity and IHC category distribution by a meta-analysis of previous studies that evaluated concordance between IHC and FISH to determine HER 2 status (simulation cohort). Four diagnostic pathways in the simulation cohort were defined-(1) initial IHC, followed by FISH (conventional pathway); (2) initial RT-PCR, followed by FISH; (3) initial IHC, followed by RT-PCR and then by FISH; (4) initial RT-PCR, followed by IHC and then by FISH. The clinical benefit of IHC and RT-PCR in the four pathways was analyzed and sensitivity analysis for incremental cost-effectiveness ratio and cost-benefit comapring RT-PCR against IHC, both as first-line tests and among those with IHC score 2 as a reflex second-line test was performed by the Monte Carlo technique. 115 patients comprised the study population. While none with IHC score of 0 or 1 was FISH positive for HER 2, all cases with IHC score of 3 were FISH positive. 43 cases were assigned IHC score of 2. Thus, 72 patients benefited from the initial IHC testing [clinical benefit 62.6%], with the overall concordance between IHC and FISH being 100% for those with IHC score of 0, 1 and 3 (conclusive IHC categories). For RT-PCR with 100% concordance, 15.7% (115-97 = 18) patients would have benefited from RT-PCR testing if it was used as a first-line test. If RT-PCR would have been used as a second-line test among those with IHC score 2 (n = 43), then only 6 patients would have been assigned a conclusive RT-PCR category (category 1 or 3) translating to a clinical benefit of 14% (6/43) as a second-line test. As a second-line test it had 51% probability to prove more cost-effective than the conventional pathway, provided the cost of RT-PCR was 0.4 times the cost of IHC. Also in a three-step pathway, RT-PCR upfront would have 56% probability of higher cost-benefit provided the cost of RT-PCR was 0.1 times the cost of IHC. RT-PCR results were found to be suboptimal to IHC in terms of discriminative ability and clinical benefit; thus, it is unlikely to replace IHC as a first-line test in the near future.

Effectiveness of Quantitative Real Time PCR in Long-Term Follow-up of Chronic Myeloid Leukemia Patients.

PubMed

Savasoglu, Kaan; Payzin, Kadriye Bahriye; Ozdemirkiran, Fusun; Berber, Belgin

2015-08-01

To determine the use of the Quantitative Real Time PCR (RQ-PCR) assay follow-up with Chronic Myeloid Leukemia (CML) patients. Cross-sectional observational. Izmir Ataturk Education and Research Hospital, Izmir, Turkey, from 2009 to 2013. Cytogenetic, FISH, RQ-PCR test results from 177 CMLpatients' materials selected between 2009 - 2013 years was set up for comparison analysis. Statistical analysis was performed to compare between FISH, karyotype and RQ-PCR results of the patients. Karyotyping and FISH specificity and sensitivity rates determined by ROC analysis compared with RQ-PCR results. Chi-square test was used to compare test failure rates. Sensitivity and specificity values were determined for karyotyping 17.6 - 98% (p=0.118, p > 0.05) and for FISH 22.5 - 96% (p=0.064, p > 0.05) respectively. FISH sensitivity was slightly higher than karyotyping but there was calculated a strong correlation between them (p < 0.001). RQ-PCR test failure rate did not correlate with other two tests (p > 0.05); however, karyotyping and FISH test failure rate was statistically significant (p < 0.001). Besides, the situation needed for karyotype analysis, RQ-PCR assay can be used alone in the follow-up of CMLdisease.

Faster and economical screening for vancomycin-resistant enterococci by sequential use of chromogenic agar and real-time polymerase chain reaction.

PubMed

Tan, Thean Yen; Jiang, Boran; Ng, Lily Siew Yong

2017-08-01

Screening for vancomycin-resistant enterococci (VRE) by culture takes days to generate results, while polymerase chain reaction (PCR) testing directly from clinical specimens lacks specificity. The aims of this study were to develop a real-time PCR to detect and identify Enterococcus faecium, Enterococcus faecalis, and vanA and vanB genes, and to evaluate the impact of this PCR on test-reporting times when performing it directly from suspect VRE isolates present on screening chromogenic media. The tetraplex PCR primers were designed to amplify E. faecium, E. faecalis, and vanA and vanB genes, with melt-curve analysis of PCR products. Following analytical and clinical validation of the molecular assay, PCR testing was performed for target colonies present on VRE chromogenic media. PCR results were evaluated against conventional phenotypic identification and susceptibility testing, with the time to result being monitored for both modalities. A total of 519 colonies from clinical specimens were tested concurrently by real-time PCR and phenotypic methods. In all, 223 isolates were identified with phenotypic vancomycin resistance (vanA, n = 108; vanB, n = 105; non-vanA/vanB = 10), with complete agreement between PCR and phenotypic testing for vancomycin-resistant E. faecium and E. faecalis. The majority (88.6%) of PCR results were reported, on average, 24.8 hours earlier than those of phenotypic testing, with 68% reduction in total costs. The use of culture on selective media, followed by direct colony PCR confirmation allows faster and economical VRE screening. Copyright © 2015. Published by Elsevier B.V.

Comparative Evaluation of Three Nucleic Acid-Based Assays for BK Virus Quantification

PubMed Central

Descamps, Veronique; Martin, Elodie; Morel, Virginie; François, Catherine; Helle, François; Duverlie, Gilles; Castelain, Sandrine

2015-01-01

With the growing importance of BK virus (BKV), effective and efficient screening for BKV replication in plasma and urine samples is very important for monitoring renal transplant and hematopoietic stem cell transplant recipients, who are at increased risk of BKV-associated diseases. However, recent assays proposed by many manufacturers have not been tested, and the available tests have not been standardized. The aim of the present study was to evaluate and compare the performances of three commercially available kits, R-gene, GeneProof, and RealStar, on plasma and urine specimens from patients infected with various genotypes and to determine the correlations with the results from a reference laboratory. A qualitatively excellent global agreement (96.8%) was obtained. RealStar PCR tended to give a higher sensitivity, especially for subtype Ib1 samples. Comparison of 30 plasma samples and 53 urine samples showed a good agreement between the three assays, with Spearman's Rho correlation coefficient values falling between 0.92 and 0.98 (P < 0.001). Moreover, a perfect correlation was obtained for comparison of the assay performances with the AcroMetrix BKV panel (P < 0.001 for all comparisons). According to Bland-Altman analysis, more than 95% (240/249 comparisons) of sample comparisons were situated in the range of the mean ± 2 standard deviations (SD). The greatest variability between assays was observed for 10.2% of subtype Ib2 samples, with differences of >1 log10 copies/ml. In conclusion, this study demonstrated the reliable and comparable performances of the R-gene, GeneProof, and RealStar real-time PCR systems for quantification of BKV in urine and plasma samples. All three real-time PCR assays are appropriate for screening of BKV replication in patients. PMID:26424842

Development of a qualitative, multiplex real-time PCR kit for screening of genetically modified organisms (GMOs).

PubMed

Dörries, Hans-Henno; Remus, Ivonne; Grönewald, Astrid; Grönewald, Cordt; Berghof-Jäger, Kornelia

2010-03-01

The number of commercially available genetically modified organisms (GMOs) and therefore the diversity of possible target sequences for molecular detection techniques are constantly increasing. As a result, GMO laboratories and the food production industry currently are forced to apply many different methods to reliably test raw material and complex processed food products. Screening methods have become more and more relevant to minimize the analytical effort and to make a preselection for further analysis (e.g., specific identification or quantification of the GMO). A multiplex real-time PCR kit was developed to detect the 35S promoter of the cauliflower mosaic virus, the terminator of the nopaline synthase gene of Agrobacterium tumefaciens, the 35S promoter from the figwort mosaic virus, and the bar gene of the soil bacterium Streptomyces hygroscopicus as the most widely used sequences in GMOs. The kit contains a second assay for the detection of plant-derived DNA to control the quality of the often processed and refined sample material. Additionally, the plant-specific assay comprises a homologous internal amplification control for inhibition control. The determined limits of detection for the five assays were 10 target copies/reaction. No amplification products were observed with DNAs of 26 bacterial species, 25 yeasts, 13 molds, and 41 not genetically modified plants. The specificity of the assays was further demonstrated to be 100% by the specific amplification of DNA derived from reference material from 22 genetically modified crops. The applicability of the kit in routine laboratory use was verified by testing of 50 spiked and unspiked food products. The herein described kit represents a simple and sensitive GMO screening method for the reliable detection of multiple GMO-specific target sequences in a multiplex real-time PCR reaction.

Multiplex PCR Tests for Detection of Pathogens Associated with Gastroenteritis

PubMed Central

Zhang, Hongwei; Morrison, Scott; Tang, Yi-Wei

2016-01-01

Synopsis A wide range of enteric pathogens can cause infectious gastroenteritis. Conventional diagnostic algorithms including culture, biochemical identification, immunoassay and microscopic examination are time consuming and often lack sensitivity and specificity. Advances in molecular technology have as allowed its use as clinical diagnostic tools. Multiplex PCR based testing has made its way to gastroenterology diagnostic arena in recent years. In this article we present a review of recent laboratory developed multiplex PCR tests and current commercial multiplex gastrointestinal pathogen tests. We will focus on two FDA cleared commercial syndromic multiplex tests: Luminex xTAG GPP and Biofire FimArray GI test. These multiplex tests can detect and identify multiple enteric pathogens in one test and provide results within hours. Multiplex PCR tests have shown superior sensitivity to conventional methods for detection of most pathogens. The high negative predictive value of these multiplex tests has led to the suggestion that they be used as screening tools especially in outbreaks. Although the clinical utility and benefit of multiplex PCR test are to be further investigated, implementing these multiplex PCR tests in gastroenterology diagnostic algorithm has the potential to improve diagnosis of infectious gastroenteritis. PMID:26004652

Detection of Echinococcus multilocularis by MC-PCR: evaluation of diagnostic sensitivity and specificity without gold standard.

PubMed

Wahlström, Helene; Comin, Arianna; Isaksson, Mats; Deplazes, Peter

2016-01-01

A semi-automated magnetic capture probe-based DNA extraction and real-time PCR method (MC-PCR), allowing for a more efficient large-scale surveillance of Echinococcus multilocularis occurrence, has been developed. The test sensitivity has previously been evaluated using the sedimentation and counting technique (SCT) as a gold standard. However, as the sensitivity of the SCT is not 1, test characteristics of the MC-PCR was also evaluated using latent class analysis, a methodology not requiring a gold standard. Test results, MC-PCR and SCT, from a previous evaluation of the MC-PCR using 177 foxes shot in the spring (n=108) and autumn 2012 (n=69) in high prevalence areas in Switzerland were used. Latent class analysis was used to estimate the test characteristics of the MC-PCR. Although it is not the primary aim of this study, estimates of the test characteristics of the SCT were also obtained. This study showed that the sensitivity of the MC-PCR was 0.88 [95% posterior credible interval (PCI) 0.80-0.93], which was not significantly different than the SCT, 0.83 (95% PCI 0.76-0.88), which is currently considered as the gold standard. The specificity of both tests was high, 0.98 (95% PCI 0.94-0.99) for the MC-PCR and 0.99 (95% PCI 0.99-1) for the SCT. In a previous study, using fox scats from a low prevalence area, the specificity of the MC-PCR was higher, 0.999% (95% PCI 0.997-1). One reason for the lower estimate of the specificity in this study could be that the MC-PCR detects DNA from infected but non-infectious rodents eaten by foxes. When using MC-PCR in low prevalence areas or areas free from the parasite, a positive result in the MC-PCR should be regarded as a true positive. The sensitivity of the MC-PCR (0.88) was comparable to the sensitivity of SCT (0.83).

Evaluation of a Turbidimetric β-d-Glucan Test for Detection of Pneumocystis jirovecii Pneumonia.

PubMed

Dichtl, Karl; Seybold, Ulrich; Wagener, Johannes

2018-07-01

Currently, diagnosis of Pneumocystis jirovecii pneumonia (PJP) relies on analysis of lower respiratory specimens, either by microscopy or quantitative real-time PCR (qPCR). Thus, bronchoscopy is required, which is associated with increased risk of respiratory failure. We assessed the value of noninvasive serologic β-d-glucan (BDG) testing for laboratory diagnosis of PJP using a newly available turbidimetric assay. We identified 73 cases of PJP with positive qPCR results from lower respiratory specimens for Pneumocystis and serology samples dating from 1 week before to 4 weeks after qPCR. In addition, 25 sera from controls with suspected PJP but specimens negative for Pneumocystis by qPCR were identified. Sera were tested with a turbidimetric BDG assay (Fujifilm Wako Chemicals Europe GmbH, Neuss, Germany), using an 11-pg/ml cutoff. Sensitivity and specificity were calculated based on qPCR test results as a reference. The turbidimetric BDG assay identified 63/73 patients with positive or slightly positive qPCR tests for an overall sensitivity of 86%; after exclusion of cases with only slightly positive qPCR results, sensitivity was 91%. No correlation between serum BDG levels and respiratory specimen DNA levels was found. Serologic BDG testing was negative in 25/25 controls with negative qPCR for a specificity of 100% using the predefined cutoff. In 22/25 samples (88%), no BDG was detected. Serologic BDG testing using the turbidimetric assay showed high sensitivity and specificity compared to qPCR of lower respiratory specimens for the diagnosis of PJP. Both turnover time and test performance will allow clinicians to delay or in some cases forego bronchoscopy. Copyright © 2018 American Society for Microbiology.

[Real-time PCR detection of Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae DNA in clinical specimens].

PubMed

Vacková, Z; Lžičařová, D; Stock, N K; Kozáková, J

2015-10-01

The study aim was to implement a molecular real-time polymerase chain reaction (PCR) assay recommended by the CDC (Centers for Disease Control and Prevention) for the detection of Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae in clinical (culture negative) specimens from patients with suspected invasive bacterial disease. Clinical specimens are referred to the National Reference Laboratory (NRL) for Meningococcal Infections, Unit for Airborne Bacterial Infections, Centre for Epidemiology and Microbiology, National Institute of Public Health from various regions of the Czech Republic. Clinical specimens are, in particular, cerebrospinal fluid, anti-coagulated blood or serum and, exceptionally, post-mortem specimens. The NRL has implemented molecular diagnosis of these bacterial pathogens involved in meningitis and sepsis from clinical specimens since 1999. The first diagnostic method was semi-nested PCR followed by electrophoretic analysis. In 2014, a molecular qualitative real-time PCR assay was implemented.

Real-Time PCR in Clinical Microbiology: Applications for Routine Laboratory Testing

PubMed Central

Espy, M. J.; Uhl, J. R.; Sloan, L. M.; Buckwalter, S. P.; Jones, M. F.; Vetter, E. A.; Yao, J. D. C.; Wengenack, N. L.; Rosenblatt, J. E.; Cockerill, F. R.; Smith, T. F.

2006-01-01

Real-time PCR has revolutionized the way clinical microbiology laboratories diagnose many human microbial infections. This testing method combines PCR chemistry with fluorescent probe detection of amplified product in the same reaction vessel. In general, both PCR and amplified product detection are completed in an hour or less, which is considerably faster than conventional PCR detection methods. Real-time PCR assays provide sensitivity and specificity equivalent to that of conventional PCR combined with Southern blot analysis, and since amplification and detection steps are performed in the same closed vessel, the risk of releasing amplified nucleic acids into the environment is negligible. The combination of excellent sensitivity and specificity, low contamination risk, and speed has made real-time PCR technology an appealing alternative to culture- or immunoassay-based testing methods for diagnosing many infectious diseases. This review focuses on the application of real-time PCR in the clinical microbiology laboratory. PMID:16418529

Clinical antibacterial effectiveness of the self-adjusting file system.

PubMed

Neves, M A S; Rôças, I N; Siqueira, J F

2014-04-01

To evaluate in vivo the antibacterial effectiveness of the self-adjusting file (SAF) using molecular methods. Root canals from single-rooted teeth with apical periodontitis were instrumented using the SAF system under continuous irrigation with 2.5% NaOCl. DNA extracts from samples taken before and after instrumentation were subjected to quantitative analysis of total bacteria counts and levels of streptococci by quantitative real-time polymerase chain reaction (qPCR). The reverse-capture checkerboard assay was also used to identify 28 bacterial taxa before (S1) and after (S2) SAF instrumentation. SAF was also compared with a conventional hand nickel-titanium instrumentation technique for total bacterial reduction. Data from qPCR were analysed statistically within groups using the Wilcoxon matched pairs test and between groups using the Mann-Whitney U-test and the Fisher's exact test, with significance level set at P < 0.05. Self-adjusting file significantly reduced the total bacterial counts from a mean number of 1.96 × 10(7) cells to 1.34 × 10(4) cells (P < 0.001). Quantitatively, the 99.9% reduction in total bacterial counts associated with the SAF system was significantly superior to the 95.1% reduction obtained by hand instrumentation (P < 0.001). Qualitatively, SAF resulted in significantly more cases with negative PCR results for bacteria (54.5%) than hand instrumentation (4.5%) (P < 0.001). The SAF system succeeded in significantly reducing the streptococcal levels, but four cases still harboured these bacteria in S2. Checkerboard analysis revealed that not only streptococci but also some anaerobic and even as-yet-uncultivated bacteria may resist the effects of chemomechanical procedures. The SAF instrumentation system was highly effective in reducing bacterial populations from infected root canals and performed significantly better than hand instrumentation. However, because half of the samples still had detectable bacteria after preparation with SAF, supplementary disinfection is still required to maximize bacterial elimination. © 2013 International Endodontic Journal. Published by John Wiley & Sons Ltd.

A new multiplex real-time PCR test for HSV1/2 and syphilis: an evaluation of its impact in the laboratory and clinical setting.

PubMed

Scott, Laura Jane; Gunson, Rory N; Carman, William F; Winter, Andrew J

2010-12-01

To develop, evaluate and implement a new multiplex real-time PCR test for the detection of herpes simplex virus (HSV)1, HSV2 and syphilis in a single sample using a single test. A multiplex real-time PCR test detecting HSV1, HSV2 and Treponema pallidum was designed, validated and evaluated for a period of 6 months on patients attending the Sandyford Initiative (a series of genitourinary medicine clinics in and around Glasgow). A total of 692 samples were tested, and T pallidum PCR positives were confirmed by a second PCR at the Scottish Reference Laboratory (SBSTIRL). All PCR results were aligned with dark ground microscopy findings and serological results where available and compared. The laboratory validation of the multiplex assay showed the test to be sensitive, specific and robust. Of the 692 samples, 139 were positive for HSV1, 136 for HSV2, 15 for syphilis, one for both syphilis and HSV1, and 401 were negative; the reference laboratory confirmed all T pallidum PCR-positive samples. The PCR test was more sensitive than both dark ground microscopy and serological testing for the diagnosis of primary syphilis. The introduction of this new test has led to a better turnaround time for the diagnosis of genital ulcer disease, better detection of primary syphilis infection, and the detection of unexpected cases of syphilis where the aetiological agent suspected was HSV.

[Application of droplet digital PCR technology for genetic testing and prenatal diagnosis of spinal muscular atrophy].

PubMed

Zou, Yang; Xu, Peiwen; Li, Jie; Huang, Sexin; Gao, Ming; Kang, Ranran; Gao, Xuan; Gao, Yuan

2016-10-01

To explore the clinical application of droplet digital PCR (ddPCR) for genetic testing and prenatal diagnosis of spinal muscular atrophy (SMA) with deletion of SMN1 gene exon 7. A total of 138 clinical samples, including 121 peripheral blood, 13 amniotic fluid, 2 umbilical cord blood and 2 chorionic villi from 56 SMA families, were tested by both ddPCR and multiplex ligation-dependent probe amplification (MLPA). Results of the two approaches were analyzed with commercial software QuantaSoft (ddPCR) and Coffalyser (MLPA), respectively. Among the 138 cases, 25 had two copies, 84 had one copy, and 29 had null copy of exon 7 of the SMN1 gene. The results of ddPCR and MLPA were completely consistent. As a rapid, precise and economically efficient method, ddPCR will provide a new choice for genetic testing of SMA.

Inter-laboratory analysis of selected genetically modified plant reference materials with digital PCR.

PubMed

Dobnik, David; Demšar, Tina; Huber, Ingrid; Gerdes, Lars; Broeders, Sylvia; Roosens, Nancy; Debode, Frederic; Berben, Gilbert; Žel, Jana

2018-01-01

Digital PCR (dPCR), as a new technology in the field of genetically modified (GM) organism (GMO) testing, enables determination of absolute target copy numbers. The purpose of our study was to test the transferability of methods designed for quantitative PCR (qPCR) to dPCR and to carry out an inter-laboratory comparison of the performance of two different dPCR platforms when determining the absolute GM copy numbers and GM copy number ratio in reference materials certified for GM content in mass fraction. Overall results in terms of measured GM% were within acceptable variation limits for both tested dPCR systems. However, the determined absolute copy numbers for individual genes or events showed higher variability between laboratories in one third of the cases, most possibly due to variability in the technical work, droplet size variability, and analysis of the raw data. GMO quantification with dPCR and qPCR was comparable. As methods originally designed for qPCR performed well in dPCR systems, already validated qPCR assays can most generally be used for dPCR technology with the purpose of GMO detection. Graphical abstract The output of three different PCR-based platforms was assessed in an inter-laboratory comparison.

Performance of a Highly Sensitive Mycobacterium tuberculosis Complex Real-Time PCR Assay for Diagnosis of Pulmonary Tuberculosis in a Low-Prevalence Setting: a Prospective Intervention Study.

PubMed

Vinuesa, Víctor; Borrás, Rafael; Briones, María Luisa; Clari, María Ángeles; Cresencio, Vicenta; Giménez, Estela; Muñoz, Carmen; Oltra, Rosa; Servera, Emilio; Scheelje, Talia; Tornero, Carlos; Navarro, David

2018-05-01

The potential impact of routine real-time PCR testing of respiratory specimens from patients with presumptive tuberculosis in terms of diagnostic accuracy and time to tuberculosis treatment inception in low-prevalence settings remains largely unexplored. We conducted a prospective intervention cohort study. Respiratory specimens from 1,020 patients were examined by acid-fast bacillus smear microscopy, tested by a real-time Mycobacterium tuberculosis complex PCR assay (Abbott RealTi me MTB PCR), and cultured in mycobacterial media. Seventeen patients tested positive by PCR (5 were acid-fast bacillus smear positive and 12 acid-fast bacillus smear negative), and Mycobacterium tuberculosis was recovered from cultures for 12 of them. Patients testing positive by PCR and negative by culture ( n = 5) were treated and deemed to have responded to antituberculosis therapy. There were no PCR-negative/culture-positive cases, and none of the patients testing positive for nontuberculous mycobacteria ( n = 20) yielded a positive PCR result. The data indicated that routine testing of respiratory specimens from patients with presumptive tuberculosis by the RealTi me MTB PCR assay improves the tuberculosis diagnostic yield and may reduce the time to antituberculosis treatment initiation. On the basis of our data, we propose a novel mycobacterial laboratory algorithm for tuberculosis diagnosis. Copyright © 2018 American Society for Microbiology.

Economic evaluation of laboratory testing strategies for hospital-associated Clostridium difficile infection.

PubMed

Schroeder, Lee F; Robilotti, Elizabeth; Peterson, Lance R; Banaei, Niaz; Dowdy, David W

2014-02-01

Clostridium difficile infection (CDI) is the most common cause of infectious diarrhea in health care settings, and for patients presumed to have CDI, their isolation while awaiting laboratory results is costly. Newer rapid tests for CDI may reduce this burden, but the economic consequences of different testing algorithms remain unexplored. We used decision analysis from the hospital perspective to compare multiple CDI testing algorithms for adult inpatients with suspected CDI, assuming patient management according to laboratory results. CDI testing strategies included combinations of on-demand PCR (odPCR), batch PCR, lateral-flow diagnostics, plate-reader enzyme immunoassay, and direct tissue culture cytotoxicity. In the reference scenario, algorithms incorporating rapid testing were cost-effective relative to nonrapid algorithms. For every 10,000 symptomatic adults, relative to a strategy of treating nobody, lateral-flow glutamate dehydrogenase (GDH)/odPCR generated 831 true-positive results and cost $1,600 per additional true-positive case treated. Stand-alone odPCR was more effective and more expensive, identifying 174 additional true-positive cases at $6,900 per additional case treated. All other testing strategies were dominated by (i.e., more costly and less effective than) stand-alone odPCR or odPCR preceded by lateral-flow screening. A cost-benefit analysis (including estimated costs of missed cases) favored stand-alone odPCR in most settings but favored odPCR preceded by lateral-flow testing if a missed CDI case resulted in less than $5,000 of extended hospital stay costs and <2 transmissions, if lateral-flow GDH diagnostic sensitivity was >93%, or if the symptomatic carrier proportion among the toxigenic culture-positive cases was >80%. These results can aid guideline developers and laboratory directors who are considering rapid testing algorithms for diagnosing CDI.

Economic Evaluation of Laboratory Testing Strategies for Hospital-Associated Clostridium difficile Infection

PubMed Central

Robilotti, Elizabeth; Peterson, Lance R.; Banaei, Niaz; Dowdy, David W.

2014-01-01

Clostridium difficile infection (CDI) is the most common cause of infectious diarrhea in health care settings, and for patients presumed to have CDI, their isolation while awaiting laboratory results is costly. Newer rapid tests for CDI may reduce this burden, but the economic consequences of different testing algorithms remain unexplored. We used decision analysis from the hospital perspective to compare multiple CDI testing algorithms for adult inpatients with suspected CDI, assuming patient management according to laboratory results. CDI testing strategies included combinations of on-demand PCR (odPCR), batch PCR, lateral-flow diagnostics, plate-reader enzyme immunoassay, and direct tissue culture cytotoxicity. In the reference scenario, algorithms incorporating rapid testing were cost-effective relative to nonrapid algorithms. For every 10,000 symptomatic adults, relative to a strategy of treating nobody, lateral-flow glutamate dehydrogenase (GDH)/odPCR generated 831 true-positive results and cost $1,600 per additional true-positive case treated. Stand-alone odPCR was more effective and more expensive, identifying 174 additional true-positive cases at $6,900 per additional case treated. All other testing strategies were dominated by (i.e., more costly and less effective than) stand-alone odPCR or odPCR preceded by lateral-flow screening. A cost-benefit analysis (including estimated costs of missed cases) favored stand-alone odPCR in most settings but favored odPCR preceded by lateral-flow testing if a missed CDI case resulted in less than $5,000 of extended hospital stay costs and <2 transmissions, if lateral-flow GDH diagnostic sensitivity was >93%, or if the symptomatic carrier proportion among the toxigenic culture-positive cases was >80%. These results can aid guideline developers and laboratory directors who are considering rapid testing algorithms for diagnosing CDI. PMID:24478478

Bayesian estimation of test characteristics of real-time PCR, bacteriological culture and California mastitis test for diagnosis of intramammary infections with Staphylococcus aureus in dairy cattle at routine milk recordings.

PubMed

Mahmmod, Yasser S; Toft, Nils; Katholm, Jørgen; Grønbæk, Carsten; Klaas, Ilka C

2013-11-01

Danish farmers can order a real-time PCR mastitis diagnostic test on routinely taken cow-level samples from milk recordings. Validation of its performance in comparison to conventional mastitis diagnostics under field conditions is essential for efficient control of intramammary infections (IMI) with Staphylococcus aureus (S. aureus). Therefore, the objective of this study was to estimate the sensitivity (Se) and specificity (Sp) of real-time PCR, bacterial culture (BC) and California mastitis test (CMT) for the diagnosis of the naturally occurring IMI with S. aureus in routinely collected milk samples using latent class analysis (LCA) to avoid the assumption of a perfect reference test. Using systematic random sampling, a total of 609 lactating dairy cows were selected from 6 dairy herds with bulk tank milk PCR cycle threshold (Ct) value ≤39 for S. aureus. At routine milk recordings, automatically obtained cow-level (composite) milk samples were analyzed by PCR and at the same milking, 2436 quarter milk samples were collected aseptically for BC and CMT. Results showed that 140 cows (23%) were positive for S. aureus IMI by BC while 170 cows (28%) were positive by PCR. Estimates of Se and Sp for PCR were higher than test estimates of BC and CMT. SeCMT was higher than SeBC however, SpBC was higher than SpCMT. SePCR was 91%, while SeBC was 53%, and SeCMT was 61%. SpPCR was 99%, while SpBC was 89%, and SpCMT was 65%. In conclusion, PCR has a higher performance than the conventional diagnostic tests (BC and CMT) suggesting its usefulness as a routine test for accurate diagnosis of S. aureus IMI from dairy cows at routine milk recordings. The use of LCA provided estimates of the test characteristics for two currently diagnostic tests (BC, CMT) and a novel technique (real-time PCR) for diagnosing S. aureus IMI under field conditions at routine milk recordings in Denmark. Copyright © 2013 Elsevier B.V. All rights reserved.

Bayesian estimation of true prevalence, sensitivity and specificity of three diagnostic tests for detection of Escherichia coli O157 in cattle feces.

PubMed

Ekong, Pius S; Sanderson, Michael W; Bello, Nora M; Noll, Lance W; Cernicchiaro, Natalia; Renter, David G; Bai, Jianfa; Nagaraja, T G

2017-12-01

Cattle are a reservoir for Escherichia coli O157 and they shed the pathogen in their feces. Fecal contaminants on the hides can be transferred onto carcasses during processing at slaughter plants, thereby serving as a source of foodborne infection in humans. The detection of E. coli O157 in cattle feces is based on culture, immunological, and molecular methods We evaluated the diagnostic sensitivity and specificity of one culture- and two PCR-based tests for the detection of E. coli O157 in cattle feces, and its true prevalence using a Bayesian implementation of latent class models. A total of 576 fecal samples were collected from the floor of pens of finishing feedlot cattle in the central United States during summer 2013. Samples were enriched and subjected to detection of E. coli O157 by culture (immunomagnetic separation, plating on a selective medium, latex agglutination, and indole testing), conventional PCR (cPCR), and multiplex quantitative PCR (mqPCR). The statistical models assumed conditional dependence of the PCR tests and high specificity for culture (mode=99%; 5th percentile=97%). Prior estimates of test parameters were elicited from three experts. Estimated posterior sensitivity (posterior median and 95% highest posterior density intervals) of culture, cPCR, and mqPCR was 49.1% (44.8-53.4%), 59.7% (55.3-63.9%), and 97.3% (95.1-99.0%), respectively. Estimated posterior specificity of culture, cPCR, and mqPCR were 98.7% (96.8-99.8%), 94.1% (87.4-99.1%), and 94.8% (84.1-99.9%), respectively. True prevalence was estimated at 91.3% (88.1-94.2%). There was evidence of a weak conditional dependence between cPCR and mqPCR amongst test positive samples, but no evidence of conditional dependence amongst test negative samples. Sensitivity analyses showed that overall our posterior inference was rather robust to the choice of priors, except for inference on specificity of mqPCR, which was estimated with considerable uncertainty. Our study evaluates performance of three diagnostic tests for detection of E. coli O157 in feces of feedlot cattle which is important for quantifying true fecal prevalence and adjusting for test error in risk modeling. Copyright © 2017 Elsevier B.V. All rights reserved.

PCR Testing of IVC Filter Tops as a Method for Detecting Murine Pinworms and Fur Mites.

PubMed

Gerwin, Philip M; Ricart Arbona, Rodolfo J; Riedel, Elyn R; Henderson, Kenneth S; Lipman, Neil S

2017-11-01

We evaluated PCR testing of filter tops from cages maintained on an IVC system through which exhaust air is filtered at the cage level as a method for detecting parasite-infected and -infested cages. Cages containing 4 naïve Swiss Webster mice received 360 mL of uncontaminated aspen chip or α-cellulose bedding (n = 18 cages each) and 60 mL of the same type of bedding weekly from each of the following 4 groups of cages housing mice infected or infested with Syphacia obvelata (SO), Aspiculuris tetraptera (AT), Myocoptes musculinus (MC), or Myobia musculi (MB) and Radfordia affinis (RA; 240 mL bedding total). Detection rates were compared at 30, 60, and 90 d after initiating bedding exposure, by using PCR analysis of filter tops (media extract and swabs) and testing of mouse samples (fur swab [direct] PCR testing, fecal flotation, anal tape test, direct examination of intestinal contents, and skin scrape). PCR testing of filter media extract detected 100% of all parasites at 30 d (both bedding types) except for AT (α-cellulose bedding, 67% detection rate); identified more cages with fur mites (MB and MC) than direct PCR when cellulose bedding was used; and was better at detecting parasites than all nonmolecular methods evaluated. PCR analysis of filter media extract was superior to swab and direct PCR for all parasites cumulatively for each bedding type. Direct PCR more effectively detected MC and all parasites combined for aspen chip compared with cellulose bedding. PCR analysis of filter media extract for IVC systems in which exhaust air is filtered at the cage level was shown to be a highly effective environmental testing method.

PCR Testing of IVC Filter Tops as a Method for Detecting Murine Pinworms and Fur Mites

PubMed Central

Gerwin, Philip M; Arbona, Rodolfo J Ricart; Riedel, Elyn R; Henderson, Kenneth S; Lipman, Neil S

2017-01-01

We evaluated PCR testing of filter tops from cages maintained on an IVC system through which exhaust air is filtered at the cage level as a method for detecting parasite- infected and -infested cages. Cages containing 4 naïve Swiss Webster mice received 360 mL of uncontaminated aspen chip or α-cellulose bedding (n = 18 cages each) and 60 mL of the same type of bedding weekly from each of the following 4 groups of cages housing mice infected or infested with Syphacia obvelata (SO), Aspiculuris tetraptera (AT), Myocoptes musculinus (MC), or Myobia musculi (MB) and Radfordia affinis (RA; 240 mL bedding total). Detection rates were compared at 30, 60, and 90 d after initiating bedding exposure, by using PCR analysis of filter tops (media extract and swabs) and testing of mouse samples (fur swab [direct] PCR testing, fecal flotation, anal tape test, direct examination of intestinal contents, and skin scrape). PCR testing of filter media extract detected 100% of all parasites at 30 d (both bedding types) except for AT (α-cellulose bedding, 67% detection rate); identified more cages with fur mites (MB and MC) than direct PCR when cellulose bedding was used; and was better at detecting parasites than all nonmolecular methods evaluated. PCR analysis of filter media extract was superior to swab and direct PCR for all parasites cumulatively for each bedding type. Direct PCR more effectively detected MC and all parasites combined for aspen chip compared with cellulose bedding. PCR analysis of filter media extract for IVC systems in which exhaust air is filtered at the cage level was shown to be a highly effective environmental testing method. PMID:29256370

[Prevalence of hepatitis C virus in the blood bank at Centro Médico Nacional La Raza].

PubMed

Benítez-Arvizu, Gamaliel; Cortez-Gómez, Rudyard; Novelo-Garza, Bárbara Alicia; Malagón-Martínez, Araceli; Guerra-Márquez, Angel; Alvarado-Maldonado, María Del Consuelo; Rodríguez-Bartolo, Mireya; Argüelles-Pimentel, Rosa María; Sánchez-Barrera, Reyna Gabriela

2006-01-01

There are no records on the prevalence of infection by HCV in Mexican population. The central area of Mexico is a highly dense demographic zone and is the influence area of the second blood bank in Latin America in terms of affluence. We prospectively studied the prevalence and genotypes of HCV infection in 5105 individuals attending the Central Blood Bank of Centro Médico Nacional La Raza regardless if they were accepted or rejected as donors. We applied a quimiolumiscence assay as a screening test. A recombinant immunoassay (RIBA) and a qualitative polymerase chain reaction (PCR) were performed as confirmatory tests and to detect viremia, respectively. Virus genotype was identified by means of a Line Immuno Probe Assay in PCR positive samples. The overall prevalence of HCV infection was 0.195% (10/5105). Viremia was detected in 90% of the subjects. The prevalence of accepted donors (0.087%) was significantly lower (p = 0.017) than that of the rejected ones (0.421%). Among viremic subjects, 60 % were infected with genotype 2 and 40% with a subtype combination (a/b) of genotype 1. The prevalence of HCV infection in our population was significantly lower than the world mean prevalence estimated in 3 %. A higher prevalence of genotype 2 in asymptomatic individuals contrasts with previous studies with a selected population where genotype 1 prevailed.

PERFORMANCE EVALUATION OF A PROTOTYPE ARCHITECT ANTIBODY ASSAY FOR BABESIA MICROTI.

PubMed

Cheng, Kevin; Coller, Kelly E; Marohnic, Christopher C; Pfeiffer, Zachary A; Fino, James R; Elsing, Randee R; Bergsma, Janet; Marcinkus, Marilee A; Kar, Alak K; Gumbs, Orlando H; Otis, Kathy S; Fishpaugh, Jeffrey; Schultz, Phillip W; Pope, Mark R; Narvaez, Alfredo R; Wong, Susan J; Madison-Antenucci, Susan; Leary, Thomas P; Dawson, George J

2018-05-09

The tick-borne protozoan Babesia microti is responsible for more than 200 cases of transfusion-transmitted babesiosis (TTB) infection in the United States over the last 30 years. Measures to mitigate the risk of TTB include nucleic acid testing (NAT) and B. microti antibody testing. A fully automated prototype B. microti antibody test was developed on the ARCHITECT instrument. The specificity was determined to be 99.98% in volunteer blood donors (n=28,740) from areas considered as low endemic for B. microti The sensitivity of the prototype test was studied in experimentally-infected macaques; a total of 128 samples were detected compared to 125 with the indirect fluorescent antibody test (IFA), additionally, 83 (89.2%) of the PCR positive samples were detected compared to 81 (87.1%) using the IFA test. All PCR positive samples that tested negative in the prototype antibody test were pre-seroconversion period samples. Following seroconversion, periods of intermittent parasitemia occurred; 17 PCR negative samples drawn in-between PCR positive bleed dates, tested positive both by the prototype test (robust reactivity) and IFA (marginal reactivity) prior to the administration of therapeutic drugs, indicating that the PCR test failed to detect samples from persistently infected macaques. The prototype assay detected 56 of 58 (96.6%) human subjects diagnosed with clinical babesiosis by both PCR and IFA testing. Overall, the prototype anti-babesia assay provides a highly sensitive and specific test for the diagnosis of B. microti infection. While PCR is preferred for detection of window period parasitemia, antibody tests detect infected subjects during periods of low level parasitemia. Copyright © 2018 Cheng et al.

[Evaluation of cytomegalovirus quantification in blood by the R-gene real-time PCR test].

PubMed

Marque-Juillet, S; Touzard, A; Monnier, S; Fernand-Laurent, C; Therby, A; Rigaudeau, S; Harzic, M

2010-04-01

Diagnosing the presence of cytomegalovirus (CMV) in the blood of immunodepressed patients is often done by quantitative polymerase chain reaction (Q-PCR) even though the reference method remains the antigenemia pp65 (Ag-pp65) test. To define the predictive value of the Q-PCR in the diagnosis of CMV disease and assess treatment efficacy using the CMV R-gene test. To compare the Q-PCR results and feasibility with those of the Ag-pp65 test. The Q-PCR was performed in 34 whole blood samples (frozen at -80 degrees C until use) from five patients diagnosed with CMV disease, defined as the presence of clinical signs and Ag-pp65 in the nuclei of more than two cells. After extraction, viral DNA was quantified in each sample using the Q-PCR CMV R-gene kit according to the manufacturer's instructions. Immediately after blood was drawn, the Ag-pp65 test had been performed in 32 samples using CINAkit (Argene). The 16 samples positive by the Ag-pp65 test were also positive by PCR; six samples negative by the Ag-pp65 test were positive by PCR; and the remaining 10 samples were negative by both techniques. During treatment, the two markers' kinetics were similar. The CMV R-gene test has a predictive value as good as that of the Ag-pp65 test but is fast and easier to use. A prospective study with a greater number of patients is needed to define the prediction threshold for CMV disease. Copyright 2009 Elsevier Masson SAS. All rights reserved.

Sustained high-throughput polymerase chain reaction diagnostics during the European epidemic of Bluetongue virus serotype 8.

PubMed

van Rijn, Piet A; Heutink, René G; Boonstra, Jan; Kramps, Hans A; van Gennip, René G P

2012-05-01

A real-time reverse transcription polymerase chain reaction assay (PCR test) based on genome segment 10 of Bluetongue virus (BTV) was developed. The PCR test consists of robotized viral RNA isolation from blood samples and an all-in-one method including initial denaturation of genomic double-stranded RNA, reverse transcription polymerase chain reaction (RT-PCR), and real-time detection and analysis. Reference strains of the 24 recognized BTV serotypes, isolates from different years, and geographic origins were detected. Other orbiviruses such as African horse sickness virus, Epizootic hemorrhagic disease virus, and Equine encephalosis virus were not detected. Experimentally infected animals were PCR positive from 2 days postinoculation, which was earlier than fever, other clinical signs, or seroconversion. The diagnostic sensitivity and specificity were very close to or even 100%. The PCR test played a key role in the detection of BTV serotype 8 in August 2006 in The Netherlands. The outbreak in a completely naive ruminant population allowed for further evaluation of the PCR test with field samples. In 2006, the correlation between enzyme-linked immunosorbent assay and PCR results was estimated to be 95%. In the following years, the PCR test was used for diagnosis of diseased animals, for testing of healthy animals for trade purposes, and for detection of BTV RNA in different species of the insect vector, Culicoides. In the autumn of 2008, BTV serotype 6 unexpectedly emerged in northwest Europe and was also detected with the PCR test developed in the current study. The performance in routine use over 5 years has been recorded and evaluated.

Development of a test for bovine tuberculosis in cattle based on measurement of gamma interferon mRNA by real-time PCR.

PubMed

Gan, W; Zhou, X; Yang, H; Chen, H; Qiao, J; Khan, S H; Yang, L; Yin, X; Zhao, D

2013-08-03

The infection status of cattle for bovine tuberculosis (bTB) was determined by real-time PCR, comparing the levels of IFN-γ mRNA in blood cultures stimulated with either bovine or avian tuberculin with non-stimulated control (phosphate buffer saline, PBS) blood culture. Totally, 137 cattle were tested to validate the assay, in which 54 were IFN-γ real-time quantitative PCR (RT-qPCR) positive, while the remaining 83 were found negative. Meanwhile, the IFN-γ ELISA test was carried out using the Bovigam IFN-γ detection ELISA kit and these results were used as a standard. The results of the single intradermal tuberculin tests (SIDT) and IFN-γ RT-qPCR tests were compared and revealed that the RT-qPCR correlated better with the ELISA and its accuracy was higher than SIDT. This indicates the RT-qPCR is a useful diagnostic method for bTB in cattle. However, several limitations remain for our approach, such as lack of a TB lesions or postmortem test results as a gold standard. Further improvements should be made in the future to increase accuracy of diagnosis of bTB in cattle.

Relative Accuracy of Nucleic Acid Amplification Tests and Culture in Detecting Chlamydia in Asymptomatic Men

PubMed Central

Cheng, Hong; Macaluso, Maurizio; Vermund, Sten H.; Hook, Edward W.

2001-01-01

Published estimates of the sensitivity and specificity of PCR and ligase chain reaction (LCR) for detecting Chlamydia trachomatis are potentially biased because of study design limitations (confirmation of test results was limited to subjects who were PCR or LCR positive but culture negative). Relative measures of test accuracy are less prone to bias in incomplete study designs. We estimated the relative sensitivity (RSN) and relative false-positive rate (RFP) for PCR and LCR versus cell culture among 1,138 asymptomatic men and evaluated the potential bias of RSN and RFP estimates. PCR and LCR testing in urine were compared to culture of urethral specimens. Discordant results (PCR or LCR positive, but culture negative) were confirmed by using a sequence including the other DNA amplification test, direct fluorescent antibody testing, and a DNA amplification test to detect chlamydial major outer membrane protein. The RSN estimates for PCR and LCR were 1.45 (95% confidence interval [CI] = 1.3 to 1.7) and 1.49 (95% CI = 1.3 to 1.7), respectively, indicating that both methods are more sensitive than culture. Very few false-positive results were found, indicating that the specificity levels of PCR, LCR, and culture are high. The potential bias in RSN and RFP estimates were <5 and <20%, respectively. The estimation of bias is based on the most likely and probably conservative parameter settings. If the sensitivity of culture is between 60 and 65%, then the true sensitivity of PCR and LCR is between 90 and 97%. Our findings indicate that PCR and LCR are significantly more sensitive than culture, while the three tests have similar specificities. PMID:11682509

Analytical Comparison of In Vitro-Spiked Human Serum and Plasma for PCR-Based Detection of Aspergillus fumigatus DNA: a Study by the European Aspergillus PCR Initiative

PubMed Central

Mengoli, Carlo; Springer, Jan; Bretagne, Stéphane; Cuenca-Estrella, Manuel; Klingspor, Lena; Lagrou, Katrien; Melchers, Willem J. G.; Morton, C. Oliver; Barnes, Rosemary A.; Donnelly, J. Peter; White, P. Lewis

2015-01-01

The use of serum or plasma for Aspergillus PCR testing facilitates automated and standardized technology. Recommendations for serum testing are available, and while serum and plasma are regularly considered interchangeable for use in fungal diagnostics, differences in galactomannan enzyme immunoassay (GM-EIA) performance have been reported and are attributed to clot formation. Therefore, it is important to assess plasma PCR testing to determine if previous recommendations for serum are applicable and also to compare analytical performance with that of serum PCR. Molecular methods testing serum and plasma were compared through multicenter distribution of quality control panels, with additional studies to investigate the effect of clot formation and blood fractionation on DNA availability. Analytical sensitivity and time to positivity (TTP) were compared, and a regression analysis was performed to identify variables that enhanced plasma PCR performance. When testing plasma, sample volume, preextraction-to-postextraction volume ratio, PCR volume, duplicate testing, and the use of an internal control for PCR were positively associated with performance. When whole-blood samples were spiked and then fractionated, the analytical sensitivity and TTP were superior when testing plasma. Centrifugation had no effect on DNA availability, whereas the presence of clot material significantly lowered the concentration (P = 0.028). Technically, there are no major differences in the molecular processing of serum and plasma, but the formation of clot material potentially reduces available DNA in serum. During disease, Aspergillus DNA burdens in blood are often at the limits of PCR performance. Using plasma might improve performance while maintaining the methodological simplicity of serum testing. PMID:26085614

Analytical Comparison of In Vitro-Spiked Human Serum and Plasma for PCR-Based Detection of Aspergillus fumigatus DNA: a Study by the European Aspergillus PCR Initiative.

PubMed

Loeffler, Juergen; Mengoli, Carlo; Springer, Jan; Bretagne, Stéphane; Cuenca-Estrella, Manuel; Klingspor, Lena; Lagrou, Katrien; Melchers, Willem J G; Morton, C Oliver; Barnes, Rosemary A; Donnelly, J Peter; White, P Lewis

2015-09-01

The use of serum or plasma for Aspergillus PCR testing facilitates automated and standardized technology. Recommendations for serum testing are available, and while serum and plasma are regularly considered interchangeable for use in fungal diagnostics, differences in galactomannan enzyme immunoassay (GM-EIA) performance have been reported and are attributed to clot formation. Therefore, it is important to assess plasma PCR testing to determine if previous recommendations for serum are applicable and also to compare analytical performance with that of serum PCR. Molecular methods testing serum and plasma were compared through multicenter distribution of quality control panels, with additional studies to investigate the effect of clot formation and blood fractionation on DNA availability. Analytical sensitivity and time to positivity (TTP) were compared, and a regression analysis was performed to identify variables that enhanced plasma PCR performance. When testing plasma, sample volume, preextraction-to-postextraction volume ratio, PCR volume, duplicate testing, and the use of an internal control for PCR were positively associated with performance. When whole-blood samples were spiked and then fractionated, the analytical sensitivity and TTP were superior when testing plasma. Centrifugation had no effect on DNA availability, whereas the presence of clot material significantly lowered the concentration (P = 0.028). Technically, there are no major differences in the molecular processing of serum and plasma, but the formation of clot material potentially reduces available DNA in serum. During disease, Aspergillus DNA burdens in blood are often at the limits of PCR performance. Using plasma might improve performance while maintaining the methodological simplicity of serum testing. Copyright © 2015 Loeffler et al.

Prevalence of PCR detectable malaria infection among febrile patients with a negative Plasmodium falciparum specific rapid diagnostic test in Zanzibar.

PubMed

Baltzell, Kimberly A; Shakely, Deler; Hsiang, Michelle; Kemere, Jordan; Ali, Abdullah Suleiman; Björkman, Anders; Mårtensson, Andreas; Omar, Rahila; Elfving, Kristina; Msellem, Mwinyi; Aydin-Schmidt, Berit; Rosenthal, Philip J; Greenhouse, Bryan

2013-02-01

We screened for malaria in 594 blood samples from febrile patients who tested negative by a Plasmodium falciparum-specific histidine-rich protein-2-based rapid diagnostic test at 12 health facilities in Zanzibar districts North A and Micheweni, from May to August 2010. Screening was with microscopy, polymerase chain reaction (PCR) targeting the cytochrome b gene (cytbPCR) of the four major human malaria species, and quantitative PCR (qPCR). The prevalence of cytbPCR-detectable malaria infection was 2% (12 of 594), including 8 P. falciparum, 3 Plasmodium malariae, and 1 Plasmodium vivax infections. Microscopy identified 4 of 8 P. falciparum infections. Parasite density as estimated by microscopy or qPCR was > 4,000 parasites/μL in 5 of 8 cytbPCR-detectable P. falciparum infections. The infections that were missed by the rapid diagnostic test represent a particular challenge in malaria elimination settings and highlight the need for more sensitive point-of-care diagnostic tools to improve case detection of all human malaria species in febrile patients.

Reduced-cost Chlamydia trachomatis-specific multiplex real-time PCR diagnostic assay evaluated for ocular swabs and use by trachoma research programmes.

PubMed

Butcher, Robert; Houghton, Jo; Derrick, Tamsyn; Ramadhani, Athumani; Herrera, Beatriz; Last, Anna R; Massae, Patrick A; Burton, Matthew J; Holland, Martin J; Roberts, Chrissy H

2017-08-01

Trachoma, caused by the intracellular bacterium Chlamydia trachomatis (Ct), is the leading infectious cause of preventable blindness. Many commercial platforms are available that provide highly sensitive and specific detection of Ct DNA. However, the majority of these commercial platforms are inaccessible for population-level surveys in resource-limited settings typical to trachoma control programmes. We developed two low-cost quantitative PCR (qPCR) tests for Ct using readily available reagents on standard real-time thermocyclers. Each multiplex qPCR test targets one genomic and one plasmid Ct target in addition to an endogenous positive control for Homo sapiens DNA. The quantitative performance of the qPCR assays in clinical samples was determined by comparison to a previously evaluated droplet digital PCR (ddPCR) test. The diagnostic performance of the qPCR assays were evaluated against a commercial assay (artus C. trachomatis Plus RG PCR, Qiagen) using molecular diagnostics quality control standards and clinical samples. We examined the yield of Ct DNA prepared from five different DNA extraction kits and a cold chain-free dry-sample preservation method using swabs spiked with fixed concentrations of human and Ct DNA. The qPCR assay was highly reproducible (Ct plasmid and genomic targets mean total coefficients of variance 41.5% and 48.3%, respectively). The assay detected 8/8 core specimens upon testing of a quality control panel and performed well in comparison to commercially marketed comparator test (sensitivity and specificity>90%). Optimal extraction and sample preservation methods for research applications were identified. We describe a pipeline from collection to diagnosis providing the most efficient sample preservation and extraction with significant per test cost savings over a commercial qPCR diagnostic assay. The assay and its evaluation should allow control programs wishing to conduct independent research within the context of trachoma control, access to an affordable test with defined performance characteristics. Copyright © 2017. Published by Elsevier B.V.

Detection of Echinococcus multilocularis by MC-PCR: evaluation of diagnostic sensitivity and specificity without gold standard

PubMed Central

Wahlström, Helene; Comin, Arianna; Isaksson, Mats; Deplazes, Peter

2016-01-01

Introduction A semi-automated magnetic capture probe-based DNA extraction and real-time PCR method (MC-PCR), allowing for a more efficient large-scale surveillance of Echinococcus multilocularis occurrence, has been developed. The test sensitivity has previously been evaluated using the sedimentation and counting technique (SCT) as a gold standard. However, as the sensitivity of the SCT is not 1, test characteristics of the MC-PCR was also evaluated using latent class analysis, a methodology not requiring a gold standard. Materials and methods Test results, MC-PCR and SCT, from a previous evaluation of the MC-PCR using 177 foxes shot in the spring (n=108) and autumn 2012 (n=69) in high prevalence areas in Switzerland were used. Latent class analysis was used to estimate the test characteristics of the MC-PCR. Although it is not the primary aim of this study, estimates of the test characteristics of the SCT were also obtained. Results and discussion This study showed that the sensitivity of the MC-PCR was 0.88 [95% posterior credible interval (PCI) 0.80–0.93], which was not significantly different than the SCT, 0.83 (95% PCI 0.76–0.88), which is currently considered as the gold standard. The specificity of both tests was high, 0.98 (95% PCI 0.94–0.99) for the MC-PCR and 0.99 (95% PCI 0.99–1) for the SCT. In a previous study, using fox scats from a low prevalence area, the specificity of the MC-PCR was higher, 0.999% (95% PCI 0.997–1). One reason for the lower estimate of the specificity in this study could be that the MC-PCR detects DNA from infected but non-infectious rodents eaten by foxes. When using MC-PCR in low prevalence areas or areas free from the parasite, a positive result in the MC-PCR should be regarded as a true positive. Conclusion The sensitivity of the MC-PCR (0.88) was comparable to the sensitivity of SCT (0.83). PMID:26968153

PCR-based study of conserved and variable DNA sequences of Tritrichomonas foetus isolates from Saskatchewan, Canada.

PubMed Central

Riley, D E; Wagner, B; Polley, L; Krieger, J N

1995-01-01

The protozoan parasite Tritrichomonas foetus causes infertility and spontaneous abortion in cattle. In Saskatchewan, Canada, the culture prevalence of trichomonads was 65 of 1,048 (6%) among 1,048 bulls tested within a 1-year period ending in April 1994. Saskatchewan was previously thought to be free of the parasite. To confirm the culture results, possible T. foetus DNA presence was determined by the PCR. All of the 16 culture-positive isolates tested were PCR positive by a single-band test, but one PCR product was weak. DNA fingerprinting by both T17 PCR and randomly amplified polymorphic DNA PCR revealed genetic variation or polymorphism among the T. foetus isolates. T17 PCR also revealed conserved loci that distinguished these T. foetus isolates from Trichomonas vaginalis, from a variety of other protozoa, and from prokaryotes. TCO-1 PCR, a PCR test designed to sample DNA sequence homologous to the 5' flank of a highly conserved cell division control gene, detected genetic polymorphism at low stringency and a conserved, single locus at higher stringency. These findings suggested that T. foetus isolates exhibit both conserved genetic loci and polymorphic loci detectable by independent PCR methods. Both conserved and polymorphic genetic loci may prove useful for improved clinical diagnosis of T. foetus. The polymorphic loci detected by PCR suggested either a long history of infection or multiple lines of T. foetus infection in Saskatchewan. Polymorphic loci detected by PCR may provide data for epidemiologic studies of T. foetus. PMID:7615746

Species Identification of Fox-, Mink-, Dog-, and Rabbit-Derived Ingredients by Multiplex PCR and Real-Time PCR Assay.

PubMed

Wu, Qingqing; Xiang, Shengnan; Wang, Wenjun; Zhao, Jinyan; Xia, Jinhua; Zhen, Yueran; Liu, Bang

2018-05-01

Various detection methods have been developed to date for identification of animal species. New techniques based on PCR approach have raised the hope of developing better identification methods, which can overcome the limitations of the existing methods. PCR-based methods used the mitochondrial DNA (mtDNA) as well as nuclear DNA sequences. In this study, by targeting nuclear DNA, multiplex PCR and real-time PCR methods were developed to assist with qualitative and quantitative analysis. The multiplex PCR was found to simultaneously and effectively distinguish four species (fox, dog, mink, and rabbit) ingredients by the different sizes of electrophoretic bands: 480, 317, 220, and 209 bp. Real-time fluorescent PCR's amplification profiles and standard curves showed good quantitative measurement responses and linearity, as indicated by good repeatability and coefficient of determination R 2  > 0.99. The quantitative results of quaternary DNA mixtures including mink, fox, dog, and rabbit DNA are in line with our expectations: R.D. (relative deviation) varied between 1.98 and 12.23% and R.S.D. (relative standard deviation) varied between 3.06 and 11.51%, both of which are well within the acceptance criterion of ≤ 25%. Combining the two methods is suitable for the rapid identification and accurate quantification of fox-, dog-, mink-, and rabbit-derived ingredients in the animal products.

Usefulness of multiplex PCR methods and respiratory viruses’ distribution in children below 15 years old according to age, seasons and clinical units in France: A 3 years retrospective study

PubMed Central

Collin, Gilles; Ichou, Houria; Charpentier, Charlotte; Bendhafer, Samia; Dumitrescu, Madalina; Allal, Lahcene; Cojocaru, Bogdan; Desfrère, Luc; Descamps, Diane; Mandelbrot, Laurent; Houhou-Fidouh, Nadhira

2017-01-01

Background To date, only influenza and RSV testing are recommended for respiratory viruses’ detection in paediatric units. In this study, we described, according to seasons, ages and clinical units, the results obtained in children (<15 years old) by multiplex-PCR (mPCR) tests allowing a quick and wide range detection of all respiratory viruses. These results were also compared with RSV specific detection. Methods All nasopharyngeal mPCR and RSV tests requested by clinicians in our French teaching hospitals group between 2011 and 2014 were retrospectively included. All repeated samples for the same children in the same month were discarded. Results Of the 381 mPCR tests (344 children) performed, 51.4% were positive. Positivity and viral co-infection rates were higher in the 6–36 months old strata (81% and 25%, p<0.0001 and p = 0.04, respectively). Viral distribution showed strong variations across ages. During specific influenza epidemic periods, only 1/39 (2.5%) mPCR tests were positive for influenza and 19/39 (48.7%) for other viruses. During specific RSV epidemic periods, only 8/46 (17.4%) mPCR tests were positive for RSV and 14/46 (30.4%) for other viruses. 477/1529 (31.2%) of RSV immunochromatography-tests were positive. Among the negatives immunochromatography-test also explored by mPCR, 28/62 (31%) were positive for other respiratory viruses. Conclusion This study provides a wide description of respiratory viruses’ distribution among children in hospital settings using mPCR over 3 years. It emphasizes the number of undiagnosed respiratory viruses according to the current diagnosis practice in France and gives a better picture of respiratory viruses identified in hospital settings by mPCR all over the year in France. PMID:28235002

Assessment of real-time PCR method for detection of EGFR mutation using both supernatant and cell pellet of malignant pleural effusion samples from non-small-cell lung cancer patients.

PubMed

Shin, Saeam; Kim, Juwon; Kim, Yoonjung; Cho, Sun-Mi; Lee, Kyung-A

2017-10-26

EGFR mutation is an emerging biomarker for treatment selection in non-small-cell lung cancer (NSCLC) patients. However, optimal mutation detection is hindered by complications associated with the biopsy procedure, tumor heterogeneity and limited sensitivity of test methodology. In this study, we evaluated the diagnostic utility of real-time PCR using malignant pleural effusion samples. A total of 77 pleural fluid samples from 77 NSCLC patients were tested using the cobas EGFR mutation test (Roche Molecular Systems). Pleural fluid was centrifuged, and separated cell pellets and supernatants were tested in parallel. Results were compared with Sanger sequencing and/or peptide nucleic acid (PNA)-mediated PCR clamping of matched tumor tissue or pleural fluid samples. All samples showed valid real-time PCR results in one or more DNA samples extracted from cell pellets and supernatants. Compared with other molecular methods, the sensitivity of real-time PCR method was 100%. Concordance rate of real-time PCR and Sanger sequencing plus PNA-mediated PCR clamping was 98.7%. We have confirmed that real-time PCR using pleural fluid had a high concordance rate compared to conventional methods, with no failed samples. Our data demonstrated that the parallel real-time PCR testing using supernatant and cell pellet could offer reliable and robust surrogate strategy when tissue is not available.

Spatiotemporal behavior and nonlinear dynamics in a phase conjugate resonator

NASA Technical Reports Server (NTRS)

Liu, Siuying Raymond

1993-01-01

The work described can be divided into two parts. The first part is an investigation of the transient behavior and stability property of a phase conjugate resonator (PCR) below threshold. The second part is an experimental and theoretical study of the PCR's spatiotemporal dynamics above threshold. The time-dependent coupled wave equations for four-wave mixing (FWM) in a photorefractive crystal, with two distinct interaction regions caused by feedback from an ordinary mirror, was used to model the transient dynamics of a PCR below threshold. The conditions for self-oscillation were determined and the solutions were used to define the PCR's transfer function and analyze its stability. Experimental results for the buildup and decay times confirmed qualitatively the predicted behavior. Experiments were carried out above threshold to study the spatiotemporal dynamics of the PCR as a function of Pragg detuning and the resonator's Fresnel number. The existence of optical vortices in the wavefront were identified by optical interferometry. It was possible to describe the transverse dynamics and the spatiotemporal instabilities by modeling the three-dimensional-coupled wave equations in photorefractive FWM using a truncated modal expansion approach.

Quantitative competitive (QC) PCR for quantification of porcine DNA.

PubMed

Wolf, C; Lüthy, J

2001-02-01

Many meat products nowadays may contain several species in different proportions. To protect consumers from fraud and misdeclarations, not only a qualitative but also a quantitative monitoring of ingredients of complex food products is necessary. DNA based techniques like the polymerase chain reaction (PCR) are widely used for identification of species but no answer to the proportional amount of a certain species could be given using current techniques. In this study we report the development and evaluation of a quantitative competitive polymerase chain reaction (QC-PCR) for detection and quantification of porcine DNA using a new porcine specific PCR system based on the growth hormone gene of sus scrofa. A DNA competitor differing by 30 bp in length from the porcine target sequence was constructed and used for PCR together with the target DNA. Specificity of the new primers was evaluated with DNA from cattle, sheep, chicken and turkey. The competitor concentration was adjusted to porcine DNA contents of 2 or 20% by coamplification of mixtures containing porcine and corresponding amounts of bovine DNA in defined ratios.

PCR detection of thermophilic spore-forming bacteria involved in canned food spoilage.

PubMed

Prevost, S; Andre, S; Remize, F

2010-12-01

Thermophilic bacteria that form highly heat-resistant spores constitute an important group of spoilage bacteria of low-acid canned food. A PCR assay was developed in order to rapidly trace these bacteria. Three PCR primer pairs were designed from rRNA gene sequences. These primers were evaluated for the specificity and the sensitivity of detection. Two primer pairs allowed detection at the species level of Geobacillus stearothermophilus and Moorella thermoacetica/thermoautrophica. The other pair allowed group-specific detection of anaerobic thermophilic bacteria of the genera Thermoanaerobacterium, Thermoanaerobacter, Caldanerobium and Caldanaerobacter. After a single enrichment step, these PCR assays allowed the detection of 28 thermophiles from 34 cans of spoiled low-acid food. In addition, 13 ingredients were screened for the presence of these bacteria. This PCR assay serves as a detection method for strains able to spoil low-acid canned food treated at 55°C. It will lead to better reactivity in the canning industry. Raw materials and ingredients might be qualified not only for quantitative spore contamination, but also for qualitative contamination by highly heat-resistant spores.

[Development of pseudoviral competitive internal controls for RT-PCR detection of dengue virus].

PubMed

Hang, Xiao-Tong; Li, Jian-Dong; Zhang, Quan-Fu; Li, Chuan; Zhang, Shuo; Liang, Mi-Fang; Li, De-Xin

2010-02-01

Development of pseudoviral competitive internal controls for RT-PCR laboratory detection of dengue virus. The internal controls target gene were obtained by insertion of a 180 bp non-related DNA fragment into RT-PCR detection target of dengue virus between the forward and reverse PCR primer binding regions. A yellow florescence protein reporter gene was induced at downstream of internal controls target gene via internal ribosome entry site gene. HEK 293T cells were transfected with plasmid containing this whole cassette and lentiviral packaging support plasmid. Pseudoviral particle was recovered from the supernatant and analyzed quantitatively and qualitatively in simulated samples at the same tube under different experimental conditions. The established pseudoviral competitive internal controls can be used in the RT-PCR detection of different serotype dengue virus and the whole detection process can be monitored. The obtained fragment is easy to be differentiated in agarose electrophoresis. The pseudoviral competitive internal controls could be used for the quality control of the laboratory diagnosis process, simple to prepare, stable for storage, easy to be transformed into internal controls for other RNA virus.

Development of a One-Step Duplex RT-PCR Method for the Simultaneous Detection of VP3/VP1 and VP1/P2B Regions of the Hepatitis A Virus.

PubMed

Kim, Mi-Ju; Lee, Shin-Young; Kim, Hyun-Joong; Lee, Jeong Su; Joo, In Sun; Kwak, Hyo Sun; Kim, Hae-Yeong

2016-08-28

The simultaneous detection and accurate identification of hepatitis A virus (HAV) is critical in food safety and epidemiological studies to prevent the spread of HAV outbreaks. Towards this goal, a one-step duplex reverse-transcription (RT)-PCR method was developed targeting the VP1/P2B and VP3/VP1 regions of the HAV genome for the qualitative detection of HAV. An HAV RT-qPCR standard curve was produced for the quantification of HAV RNA. The detection limit of the duplex RT-PCR method was 2.8 × 10(1) copies of HAV. The PCR products enabled HAV genotyping analysis through DNA sequencing, which can be applied for epidemiological investigations. The ability of this duplex RT-PCR method to detect HAV was evaluated with HAV-spiked samples of fresh lettuce, frozen strawberries, and oysters. The limit of detection of the one-step duplex RT-PCR for each food model was 9.4 × 10(2) copies/20 g fresh lettuce, 9.7 × 10(3) copies/20 g frozen strawberries, and 4.1 × 10(3) copies/1.5 g oysters. Use of a one-step duplex RT-PCR method has advantages such as shorter time, decreased cost, and decreased labor owing to the single amplification reaction instead of four amplifications necessary for nested RT-PCR.

Clinical and epidemiological use of nested PCR targeting the repetitive element IS1111 associated with the transposase gene from Coxiella burnetii.

PubMed

Mares-Guia, Maria Angélica M M; Guterres, Alexandro; Rozental, Tatiana; Ferreira, Michelle Dos Santos; Lemos, Elba R S

Q fever is a worldwide zoonosis caused by Coxiella burnetii-a small obligate intracellular Gram-negative bacterium found in a variety of animals. It is transmitted to humans by inhalation of contaminated aerosols from urine, feces, milk, amniotic fluid, placenta, abortion products, wool, and rarely by ingestion of raw milk from infected animals. Nested PCR can improve the sensitivity and specificity of testing while offering a suitable amplicon size for sequencing. Serial dilutions were performed tenfold to test the limit of detection, and the result was 10× detection of C. burnetti DNA with internal nested PCR primers relative to trans-PCR. Different biological samples were tested and identified only in nested PCR. This demonstrates the efficiency and effectiveness of the primers. Of the 19 samples, which amplify the partial sequence of C. burnetii, 12 were positive by conventional PCR and nested PCR. Seven samples-five spleen tissue samples from rodents and two tick samples-were only positive in nested PCR. With these new internal primers for trans-PCR, we demonstrate that our nested PCR assay for C. burnetii can achieve better results than conventional PCR. Published by Elsevier Editora Ltda.

Performance of a new gelled nested PCR test for the diagnosis of imported malaria: comparison with microscopy, rapid diagnostic test, and real-time PCR.

PubMed

Iglesias, Nuria; Subirats, Mercedes; Trevisi, Patricia; Ramírez-Olivencia, Germán; Castán, Pablo; Puente, Sabino; Toro, Carlos

2014-07-01

Microscopy and rapid diagnostic tests (RDTs) are the techniques commonly used for malaria diagnosis but they are usually insensitive at very low levels of parasitemia. Nested PCR is commonly used as a reference technique in the diagnosis of malaria due to its high sensitivity and specificity. However, it is a cumbersome assay only available in reference centers. We evaluated a new nested PCR-based assay, BIOMALAR kit (Biotools B&M Labs, Madrid, Spain) which employs ready-to-use gelled reagents and allows the identification of the main four species of Plasmodium. Blood samples were obtained from patients with clinical suspicion of malaria. A total of 94 subjects were studied. Fifty-two (55.3%) of them were malaria-infected subjects corresponding to 48 cases of Plasmodium falciparum, 1 Plasmodium malariae, 2 Plasmodium vivax, and 1 Plasmodium ovale. The performance of the BIOMALAR test was compared with microscopy, rapid diagnostic test (RDT) (BinaxNOW® Malaria) and real-time quantitative PCR (qPCR). The BIOMALAR test showed a sensitivity of 98.1% (95% confidence interval [CI], 89.7-100), superior to microscopy (82.7% [95% CI, 69.7-91.8]) and RDT (94.2% [95% CI, 84.1-98.8]) and similar to qPCR (100% [95% CI, 93.2-100]). In terms of specificity, the BIOMALAR assay showed the same value as microscopy and qPCR (100% [95% CI, 93.2-100]). Nine subjects were submicroscopic carriers of malaria. The BIOMALAR test identified almost all of them (8/9) in comparison with RDT (6/9) and microscopy (0/9). In conclusion, the BIOMALAR is a PCR-based assay easy to use with an excellent performance and especially useful for diagnosis submicroscopic malaria.

Prevalence of Anaplasma phagocytophilum in small rodents in France.

PubMed

Chastagner, A; Moinet, M; Perez, G; Roy, E; McCoy, K D; Plantard, O; Agoulon, A; Bastian, S; Butet, A; Rantier, Y; Verheyden, H; Cèbe, N; Leblond, A; Vourc'h, G

2016-07-01

Anaplasma phagocytophilum is an emerging zoonotic tick-borne pathogen affecting a wide range of mammals. Rodents are suspected to be natural reservoirs for this bacterium, but their role in the epidemiologic cycles affecting domestic animals and wild ungulates has not been demonstrated. This study aimed to improve our knowledge on A. phagocytophilum prevalence in Apodemus sylvaticus, A. flavicollis and Myodes glareolus using data collected in 2010 in one area in eastern France and in 2012-2013 in two others areas in western France. Rodents were captured in each site and infection was tested using qualitative real-time PCR assays on either blood or spleen samples. Prevalence showed high variability among sites. The highest prevalence was observed in the most eastern site (with an average infection rate of 22.8% across all species), whereas no rodent was found to be PCR positive in the south-west site and only 6.6% were positive in the north-west of France. Finally, a significant increase in prevalence was observed in autumn samples compared to spring samples in the north-west, but no change was found in the other two sites. Copyright © 2016 Elsevier GmbH. All rights reserved.

Development and validation of a multiplex quantitative polymerase chain reaction assay for the detection of Mollicutes impurities in human cells, cultured under good manufacturing practice conditions, and following European Pharmacopoeia requirements and the International Conference on Harmonization guidelines.

PubMed

Vanni, Irene; Ugolotti, Elisabetta; Raso, Alessandro; Di Marco, Eddi; Melioli, Giovanni; Biassoni, Roberto

2012-07-01

The clinical applications of in vitro manipulated cultured cells and their precursors are often made use of in therapeutic trials. However, tissue cultures can be easily contaminated by the ubiquitous Mollicutes micro-organisms, which can cause various and severe alterations in cellular function. Thus methods able to detect and trace Mollicutes impurities contaminating cell cultures are required before starting any attempt to grow cells under good manufacturing practice (GMP) conditions. We developed a multiplex quantitative polymerase chain reaction (qPCR) assay specific for the 16S-23S rRNA intergenic spacer regions, for the Tuf and P1 cytoadhesin genes, able to detect contaminant Mollicutes species in a single tube reaction. The system was validated by analyzing different cell lines and the positive samples were confirmed by 16S and P1 cytoadhesin gene dideoxy sequencing. Our multiplex qPCR detection system was able to reach a sensitivity, specificity and robustness comparable with the culture and the indicator cell culture method, as required by the European Pharmacopoeia guidelines. We have developed a multiplex qPCR method, validated following International Conference on Harmonization (ICH) guidelines, as a qualitative limit test for impurities, assessing the validation characteristics of limit of detection and specificity. It also follows the European Pharmacopoeia guidelines and Food and Drug Administration (FDA) requirements.

A Novel High-Throughput Method for Molecular Detection of Human Pathogenic Viruses Using a Nanofluidic Real-Time PCR System

PubMed Central

Coudray-Meunier, Coralie; Fraisse, Audrey; Martin-Latil, Sandra; Delannoy, Sabine; Fach, Patrick; Perelle, Sylvie

2016-01-01

Human enteric viruses are recognized as the main causes of food- and waterborne diseases worldwide. Sensitive and quantitative detection of human enteric viruses is typically achieved through quantitative RT-PCR (RT-qPCR). A nanofluidic real-time PCR system was used to develop novel high-throughput methods for qualitative molecular detection (RT-qPCR array) and quantification of human pathogenic viruses by digital RT-PCR (RT-dPCR). The performance of high-throughput PCR methods was investigated for detecting 19 human pathogenic viruses and two main process controls used in food virology. The conventional real-time PCR system was compared to the RT-dPCR and RT-qPCR array. Based on the number of genome copies calculated by spectrophotometry, sensitivity was found to be slightly better with RT-qPCR than with RT-dPCR for 14 viruses by a factor range of from 0.3 to 1.6 log10. Conversely, sensitivity was better with RT-dPCR than with RT-qPCR for seven viruses by a factor range of from 0.10 to 1.40 log10. Interestingly, the number of genome copies determined by RT-dPCR was always from 1 to 2 log10 lower than the expected copy number calculated by RT-qPCR standard curve. The sensitivity of the RT-qPCR and RT-qPCR array assays was found to be similar for two viruses, and better with RT-qPCR than with RT-qPCR array for eighteen viruses by a factor range of from 0.7 to 3.0 log10. Conversely, sensitivity was only 0.30 log10 better with the RT-qPCR array than with conventional RT-qPCR assays for norovirus GIV detection. Finally, the RT-qPCR array and RT-dPCR assays were successfully used together to screen clinical samples and quantify pathogenic viruses. Additionally, this method made it possible to identify co-infection in clinical samples. In conclusion, given the rapidity and potential for large numbers of viral targets, this nanofluidic RT-qPCR assay should have a major impact on human pathogenic virus surveillance and outbreak investigations and is likely to be of benefit to public health. PMID:26824897

Comparison between two commercially available serological tests and polymerase chain reaction in the diagnosis of Cryptosporidium in animals and diarrhoeic children.

PubMed

Helmy, Yosra A; Krücken, Jürgen; Nöckler, Karsten; von Samson-Himmelstjerna, Georg; Zessin, Karl-H

2014-01-01

For the detection of Cryptosporidium species in 804 animals and 165 diarrhoeic children (<10 years) in Egypt, two copro-antigen tests, the RIDASCREEN® Cryptosporidium test [enzyme immunoassay (EIA)] and the RIDA®QUICK Cryptosporidium/Giardia Combi [immuno-chromatographic test (ICT)] as well as polymerase chain reaction (PCR) were used. Prevalence of Cryptosporidium was 15.0, 19.5 and 32.3% in animals and 2.4, 6.7 and 49.1% in children using EIA, ICT and PCR, respectively.Using PCR as reference method, animal samples sensitivity (Se) of the EIA was 46.5% when questionable samples were considered positive, whereas specificity (Sp) was 100%. Se of the ICT was 60.4% while Sp was 100%. Positive predictive values (PPVs) for both EIA and ICT test were 100%, and negative predictive values (NPVs) for EIA were 79.7 and 84.1% for ICT. For the children samples, the Se of EIA was 5%, Sp was 100%, PPV was 100% and NPV was 52.2%, while the Se of ICT was 13.6%, Sp was 100%, PPV was 100% and NPV was 54.6%.The Kappa score of agreement between PCR and ICT was 67.4%, 54.1% between PCR and EIA and 84.4% between ICT and EIA. Until the second serial dilution of the EIA and ICT test, 9 × 10(3) oocysts/μl of Cryptosporidia was detected, whereas in PCR, they were detected until the sixth serial dilution. Copro-antigen tests were easy to perform and less time-consuming but less sensitive compared to PCR. They obviously are best applicable for screening and epidemiological studies of large numbers of subjects, for batch specimen processing and in isolated or rural areas where reliable tests like PCR are unfeasible. When in children, a single stool sample is used for the diagnosis of clinical cases; better results can be obtained when non-standardized PCR due low specificity is coupled with copro-antigen tests.

Comparative evaluation of molecular diagnostic tests for Nucleospora salmonis and prevalence in migrating juvenile salmonids from the Snake River, USA

USGS Publications Warehouse

Badil, Samantha; Elliott, Diane G.; Kurobe, Tomofumi; Hedrick, Ronald P.; Clemens, Kathy; Blair, Marilyn; Purcell, Maureen K.

2011-01-01

Nucleospora salmonis is an intranuclear microsporidian that primarily infects lymphoblast cells and contributes to chronic lymphoblastosis and a leukemia-like condition in a range of salmonid species. The primary goal of this study was to evaluate the prevalence of N. salmonis in out-migrating juvenile hatchery and wild Chinook salmon Oncorhynchus tshawytscha and steelhead O. mykiss from the Snake River in the U.S. Pacific Northwest. To achieve this goal, we first addressed the following concerns about current molecular diagnostic tests for N. salmonis: (1) nonspecific amplification patterns by the published nested polymerase chain reaction (nPCR) test, (2) incomplete validation of the published quantitative PCR (qPCR) test, and (3) whether N. salmonis can be detected reliably from nonlethal samples. Here, we present an optimized nPCR protocol that eliminates nonspecific amplification. During validation of the published qPCR test, our laboratory developed a second qPCR test that targeted a different gene sequence and used different probe chemistry for comparison purposes. We simultaneously evaluated the two different qPCR tests for N. salmonis and found that both assays were highly specific, sensitive, and repeatable. The nPCR and qPCR tests had good overall concordance when DNA samples derived from both apparently healthy and clinically diseased hatchery rainbow trout were tested. Finally, we demonstrated that gill snips were a suitable tissue for nonlethal detection of N. salmonis DNA in juvenile salmonids. Monitoring of juvenile salmonid fish in the Snake River over a 3-year period revealed low prevalence of N. salmonis in hatchery and wild Chinook salmon and wild steelhead but significantly higher prevalence in hatchery-derived steelhead. Routine monitoring of N. salmonis is not performed for all hatchery steelhead populations. At present, the possible contribution of this pathogen to delayed mortality of steelhead has not been determined.

Retrospective Review of Treponema pallidum PCR and Serology Results: Are Both Tests Necessary?

PubMed

Brischetto, Anna; Gassiep, Ian; Whiley, David; Norton, Robert

2018-05-01

There has been a resurgence of syphilis diagnoses in Australia. We investigated whether our Treponema pallidum PCR test provides any additional diagnostic information over syphilis serology (chemiluminescence immunoassay [CMIA], Treponema pallidum particle agglutination [TPPA] assay, and the rapid plasma reagin [RPR] flocculation test). A retrospective audit of all T. pallidum PCR requests that came through our laboratory from January 2010 to June 2017 was conducted; data collected included age, gender, site of swab, and results from T. pallidum PCR, syphilis serology, and herpes simplex virus 1 (HSV-1) and HSV-2 PCRs. A total of 441 T. pallidum PCR tests were performed; on average, 3 T. pallidum PCRs per month were requested in 2011, and this rate increased to 17.2 requests per month in 2017. A total of 323 patients had both T. pallidum PCR and syphilis serology performed, with 67% of swabs taken from the genitals. T. pallidum PCR gave positive results for 61/323 (19%) patients; of these 61 patients, 59 (97%) also had positive syphilis serology results ( T. pallidum PCR sensitivity, 68%; specificity, 99%; positive predictive value, 97%; negative predictive value, 89%). Syphilis serology was positive for 91/323 patients (28%); of these 91 patients, 61 (66%) were also T. pallidum PCR positive (syphilis serology sensitivity, 97%; specificity, 88%; positive predictive value, 60%; negative predictive value, 99%). The Cohen's kappa value was 0.74, indicating substantial agreement between the two tests. Our results show that most patients with positive T. pallidum PCR results also had positive syphilis serology. Therefore, T. pallidum PCR adds little clinical value over serology for the diagnosis of syphilis in certain clinical settings. Copyright © 2018 American Society for Microbiology.

A Journal-Neutral Ratio for Marketing Faculty Scholarship Assessment

ERIC Educational Resources Information Center

Elbeck, Matt; Baruca, Arne

2015-01-01

This article proposes a journal-neutral Publication to Citation Ratio (PCR) to complement qualitative methods to evaluate a marketing educator's scholarship for reappointment, promotion, tenure, and post-tenure review (RPTP) decisions. We empirically establish a minimum time period to evaluate scholarship data, then benchmark publication and…

On-chip quantitative detection of pathogen genes by autonomous microfluidic PCR platform.

PubMed

Tachibana, Hiroaki; Saito, Masato; Shibuya, Shogo; Tsuji, Koji; Miyagawa, Nobuyuki; Yamanaka, Keiichiro; Tamiya, Eiichi

2015-12-15

Polymerase chain reaction (PCR)-based genetic testing has become a routine part of clinical diagnoses and food testing. In these fields, rapid, easy-to-use, and cost-efficient PCR chips are expected to be appeared for providing such testing on-site. In this study, a new autonomous disposable plastic microfluidic PCR chip was created, and was utilized for quantitative detection of pathogenic microorganisms. To control the capillary flow of the following solution in the PCR microchannel, a driving microchannel was newly designed behind the PCR microchannel. This allowed the effective PCR by simply dropping the PCR solution onto the inlet without any external pumps. In order to achieve disposability, injection-molded cyclo-olefin polymer (COP) of a cost-competitive plastic was used for the PCR chip. We discovered that coating the microchannel walls with non-ionic surfactant produced a suitable hydrophilic surface for driving the capillary flow through the 1250-mm long microchannel. As a result, quantitative real-time PCR with the lowest initial concentration of human, Escherichia coli (E. coli), and pathogenic E. coli O157 genomic DNA of 4, 0.0019, 0.031 pg/μl, respectively, was successfully achieved in less than 18 min. Our results indicate that the platform presented in this study provided a rapid, easy-to-use, and low-cost real-time PCR system that could be potentially used for on-site gene testing. Copyright © 2015 Elsevier B.V. All rights reserved.

PCR testing for diagnosis of Ichthyophonus hoferi: reply to LaPatra & Kocan (2013).

PubMed

Hamazaki, Toshihide; Kahler, Eryn; Borba, Bonnie M; Burton, Tamara

2013-11-06

LaPatra & Kocan (2013) critiqued our paper Hamazaki et al. (2013; Dis Aquat Org 105:21-25) for data not supporting the conclusions of 'PCR testing is as accurate as culture…', but they neither pointed out what part of our data did not support our conclusion, nor did they provide any contrary scientific evidence supporting their argument that PCR testing is less accurate than culture. In the absence of any contradictory data, we stand by our data and our conclusion: PCR test is as suitable as culture as a diagnostic and field surveillance tool.

Evaluation of clinical sensitivity and specificity of hepatitis B virus (HBV), hepatitis C virus, and human immunodeficiency Virus-1 by cobas MPX: Detection of occult HBV infection in an HBV-endemic area.

PubMed

Ha, Jihye; Park, Younhee; Kim, Hyon-Suk

2017-11-01

Transfusion-transmitted infectious diseases remain a major concern for blood safety, particularly with hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus (HIV). Nucleic acid testing (NAT) in donor screening shortens the serologically negative window period and reduces virus transmission. The cobas MPX (Roche Molecular Systems, Inc., Branchburg, New Jersey) is a recently developed multiplex qualitative PCR system that enables the simultaneous detection of HBV, HCV, and HIV with improved sensitivity and throughput using cobas 6800 and 8800 instruments. The aim of this study was to conduct an evaluation of the clinical sensitivity and specificity of cobas MPX detection of HBV, HCV, and HIV in clinical specimens. Among samples referred for HBV, HCV, and HIV-1 quantification at Severance Hospital, Yonsei University College of Medicine, positive samples were selected to evaluate sensitivity. A total of 843 samples was tested using both cobas MPX and COBAS AmpliPrep/COBAS TaqMan Tests for HBV, HCV, and HIV-1 using the cobas 8800 system and a COBAS TaqMan 96 analyzer, respectively. Samples that showed discrepancies were confirmed by nested PCR. The cobas MPX achieved excellent sensitivity and specificity for the detection of HBV, HCV, and HIV-1 in clinical samples. We found that the lower limit of detection (LOD) of blood screening by NAT actually improves clinical sensitivity, and occult HBV infection prevalence among healthy employees of the hospital was rather high. Copyright © 2017 Elsevier B.V. All rights reserved.

Soft fruit traceability in food matrices using real-time PCR.

PubMed

Palmieri, Luisa; Bozza, Elisa; Giongo, Lara

2009-02-01

Food product authentication provides a means of monitoring and identifying products for consumer protection and regulatory compliance. There is a scarcity of analytical methods for confirming the identity of fruit pulp in products containing Soft Fruit. In the present work we have developed a very sensible qualitative and quantitative method to determine the presence of berry DNAs in different food matrices. To our knowledge, this is the first study that shows the applicability, to Soft Fruit traceability, of melting curve analysis and multiplexed fluorescent probes, in a Real-Time PCR platform. This methodology aims to protect the consumer from label misrepresentation.

Comparison of nested PCR and qPCR for the detection and quantitation of BoHV6 DNA.

PubMed

Kubiś, Piotr; Materniak, Magdalena; Kuźmak, Jacek

2013-12-01

Nested PCR and qPCR (quantitative PCR) tests based on glycoprotein B (gB) gene were designed for detecting Bovine herpesvirus 6 (BoHV6) in bovine whole blood samples and wild ruminant blood clots (deer and roe-deer). This virus, commonly known as BLHV (bovine lymphotropic herpesvirus) belongs to the Herpesviridae family, subfamily Gammaherpesvirinae and Macavirus genus. DNA isolated from 92 dairy cow blood samples and 69 wild ruminant clots were examined for the presence of BoHV6 using nested PCR and qPCR tests. Viral DNA was detected by using nested PCR in 59 out of 92 bovine blood samples (64.1%), and by qPCR in 68 out of 92 bovine blood samples (73.9%), but none out of 69 DNA samples isolated from wild ruminant blood clots, was positive in both assays. The specificity of nested PCR and qPCR was confirmed by using BoHV1, BoHV4, BoHV6, BFV, BIV, and BLV DNA. The sensitivity of nested PCR and qPCR was determined using a serially 10-fold diluted vector pCR2.1HgB (2 × 10(0)-2 × 10(6)copies/reaction). In this testing, qPCR was more sensitive than the nested PCR, detecting two copies of BoHV6 whilst the limit of detection for nested PCR was 20 copies. In all qPCR assays, the coefficients of determination (R(2)) ranged between 0.990 and 0.999, and the calculated amplification efficiencies (Eff%) within the range of 89.7-106.9. The intra- and inter-assay CV (coefficient of variation) values did not exceed 4%. Copyright © 2013 Elsevier B.V. All rights reserved.

Evaluation of Aspergillus PCR protocols for testing serum specimens.

PubMed

White, P Lewis; Mengoli, Carlo; Bretagne, Stéphane; Cuenca-Estrella, Manuel; Finnstrom, Niklas; Klingspor, Lena; Melchers, Willem J G; McCulloch, Elaine; Barnes, Rosemary A; Donnelly, J Peter; Loeffler, Juergen

2011-11-01

A panel of human serum samples spiked with various amounts of Aspergillus fumigatus genomic DNA was distributed to 23 centers within the European Aspergillus PCR Initiative to determine analytical performance of PCR. Information regarding specific methodological components and PCR performance was requested. The information provided was made anonymous, and meta-regression analysis was performed to determine any procedural factors that significantly altered PCR performance. Ninety-seven percent of protocols were able to detect a threshold of 10 genomes/ml on at least one occasion, with 83% of protocols reproducibly detecting this concentration. Sensitivity and specificity were 86.1% and 93.6%, respectively. Positive associations between sensitivity and the use of larger sample volumes, an internal control PCR, and PCR targeting the internal transcribed spacer (ITS) region were shown. Negative associations between sensitivity and the use of larger elution volumes (≥100 μl) and PCR targeting the mitochondrial genes were demonstrated. Most Aspergillus PCR protocols used to test serum generate satisfactory analytical performance. Testing serum requires less standardization, and the specific recommendations shown in this article will only improve performance.

Evaluation of Aspergillus PCR Protocols for Testing Serum Specimens▿†

PubMed Central

White, P. Lewis; Mengoli, Carlo; Bretagne, Stéphane; Cuenca-Estrella, Manuel; Finnstrom, Niklas; Klingspor, Lena; Melchers, Willem J. G.; McCulloch, Elaine; Barnes, Rosemary A.; Donnelly, J. Peter; Loeffler, Juergen

2011-01-01

A panel of human serum samples spiked with various amounts of Aspergillus fumigatus genomic DNA was distributed to 23 centers within the European Aspergillus PCR Initiative to determine analytical performance of PCR. Information regarding specific methodological components and PCR performance was requested. The information provided was made anonymous, and meta-regression analysis was performed to determine any procedural factors that significantly altered PCR performance. Ninety-seven percent of protocols were able to detect a threshold of 10 genomes/ml on at least one occasion, with 83% of protocols reproducibly detecting this concentration. Sensitivity and specificity were 86.1% and 93.6%, respectively. Positive associations between sensitivity and the use of larger sample volumes, an internal control PCR, and PCR targeting the internal transcribed spacer (ITS) region were shown. Negative associations between sensitivity and the use of larger elution volumes (≥100 μl) and PCR targeting the mitochondrial genes were demonstrated. Most Aspergillus PCR protocols used to test serum generate satisfactory analytical performance. Testing serum requires less standardization, and the specific recommendations shown in this article will only improve performance. PMID:21940479

Clinical Performance of Aspergillus PCR for Testing Serum and Plasma: a Study by the European Aspergillus PCR Initiative.

PubMed

White, P Lewis; Barnes, Rosemary A; Springer, Jan; Klingspor, Lena; Cuenca-Estrella, Manuel; Morton, C Oliver; Lagrou, Katrien; Bretagne, Stéphane; Melchers, Willem J G; Mengoli, Carlo; Donnelly, J Peter; Heinz, Werner J; Loeffler, Juergen

2015-09-01

Aspergillus PCR testing of serum provides technical simplicity but with potentially reduced sensitivity compared to whole-blood testing. With diseases for which screening to exclude disease represents an optimal strategy, sensitivity is paramount. The associated analytical study confirmed that DNA concentrations were greater in plasma than those in serum. The aim of the current investigation was to confirm analytical findings by comparing the performance of Aspergillus PCR testing of plasma and serum in the clinical setting. Standardized Aspergillus PCR was performed on plasma and serum samples concurrently obtained from hematology patients in a multicenter retrospective anonymous case-control study, with cases diagnosed according to European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) consensus definitions (19 proven/probable cases and 42 controls). Clinical performance and clinical utility (time to positivity) were calculated for both kinds of samples. The sensitivity and specificity for Aspergillus PCR when testing serum were 68.4% and 76.2%, respectively, and for plasma, they were 94.7% and 83.3%, respectively. Eighty-five percent of serum and plasma PCR results were concordant. On average, plasma PCR was positive 16.8 days before diagnosis and was the earliest indicator of infection in 13 cases, combined with other biomarkers in five cases. On average, serum PCR was positive 10.8 days before diagnosis and was the earliest indicator of infection in six cases, combined with other biomarkers in three cases. These results confirm the analytical finding that the sensitivity of Aspergillus PCR using plasma is superior to that using serum. PCR positivity occurs earlier when testing plasma and provides sufficient sensitivity for the screening of invasive aspergillosis while maintaining methodological simplicity. Copyright © 2015 White et al.

Polymerase chain reaction assay for the detection of Helicobacter pylori in gastric biopsy specimens: comparison with culture, rapid urease test, and histopathological tests.

PubMed Central

Fabre, R; Sobhani, I; Laurent-Puig, P; Hedef, N; Yazigi, N; Vissuzaine, C; Rodde, I; Potet, F; Mignon, M; Etienne, J P

1994-01-01

Ulcer recurrence is probably related to residual Helicobacter pylori (H pylori). Histological examination and culture are considered to be the most specific tests. CLO test is a rapid but less specific test, which is usually used as an alternative test to culture. The aim of this study was to investigate the efficiency of a simplified polymerase chain reaction (PCR) assay as a procedure for the diagnosis of gastric H pylori infection of patients. Biopsy specimens were obtained from antral mucosa of 58 patients at endoscopy and submitted to four tests for detection of H pylori. The bacteria were found in 53%, 43%, 48%, and 50% of patients according to the results of PCR, CLO test, culture, and histological examination. Twenty three patients had both negative histology and negative culture and PCR was negative in all of these. Thirteen patients were not classified because only histology or culture was positive and 10 of these had a positive PCR test. When the diagnosis of H pylori was established by agreement with both histology and culture or three positive tests out of four, 29 patients were H pylori positive (28 having had three positive tests and one displaying positive histology and culture), and 26 were negative, and three undetermined. PCR proved the most sensitive and specific test. These results suggest the simplified PCR assay may be a valuable test for the detection of H pylori. Images p906-a PMID:8063217

Testing for Genetically Modified Foods Using PCR

ERIC Educational Resources Information Center

Taylor, Ann; Sajan, Samin

2005-01-01

The polymerase chain reaction (PCR) is a Nobel Prize-winning technique that amplifies a specific segment of DNA and is commonly used to test for the presence of genetic modifications. Students use PCR to test corn meal and corn-muffin mixes for the presence of a promoter commonly used in genetically modified foods, the cauliflower mosaic virus 35S…

9 CFR 147.30 - Laboratory procedure recommended for the polymerase chain reaction (PCR) test for Mycoplasma...

Code of Federal Regulations, 2010 CFR

2010-01-01

... the polymerase chain reaction (PCR) test for Mycoplasma gallisepticum and M. synoviae. 147.30 Section... Examination Procedures § 147.30 Laboratory procedure recommended for the polymerase chain reaction (PCR) test... should consist of the following sequences: ER12JA07.005 (c) Polymerase chain reaction. (1) Treat each...

9 CFR 147.30 - Laboratory procedure recommended for the polymerase chain reaction (PCR) test for Mycoplasma...

Code of Federal Regulations, 2011 CFR

2011-01-01

... the polymerase chain reaction (PCR) test for Mycoplasma gallisepticum and M. synoviae. 147.30 Section... Examination Procedures § 147.30 Laboratory procedure recommended for the polymerase chain reaction (PCR) test... should consist of the following sequences: ER12JA07.005 (c) Polymerase chain reaction. (1) Treat each...

ELISA versus PCR for diagnosis of chronic Chagas disease: systematic review and meta-analysis

PubMed Central

2010-01-01

Background Most current guidelines recommend two serological tests to diagnose chronic Chagas disease. When serological tests are persistently inconclusive, some guidelines recommend molecular tests. The aim of this investigation was to review chronic Chagas disease diagnosis literature and to summarize results of ELISA and PCR performance. Methods A systematic review was conducted searching remote databases (MEDLINE, LILACS, EMBASE, SCOPUS and ISIWeb) and full texts bibliography for relevant abstracts. In addition, manufacturers of commercial tests were contacted. Original investigations were eligible if they estimated sensitivity and specificity, or reliability -or if their calculation was possible - of ELISA or PCR tests, for chronic Chagas disease. Results Heterogeneity was high within each test (ELISA and PCR) and threshold effect was detected only in a particular subgroup. Reference standard blinding partially explained heterogeneity in ELISA studies, and pooled sensitivity and specificity were 97.7% [96.7%-98.5%] and 96.3% [94.6%-97.6%] respectively. Commercial ELISA with recombinant antigens studied in phase three investigations partially explained heterogeneity, and pooled sensitivity and specificity were 99.3% [97.9%-99.9%] and 97.5% [88.5%-99.5%] respectively. ELISA's reliability was seldom studied but was considered acceptable. PCR heterogeneity was not explained, but a threshold effect was detected in three groups created by using guanidine and boiling the sample before DNA extraction. PCR sensitivity is likely to be between 50% and 90%, while its specificity is close to 100%. PCR reliability was never studied. Conclusions Both conventional and recombinant based ELISA give useful information, however there are commercial tests without technical reports and therefore were not included in this review. Physicians need to have access to technical reports to understand if these serological tests are similar to those included in this review and therefore correctly order and interpret test results. Currently, PCR should not be used in clinical practice for chronic Chagas disease diagnosis and there is no PCR test commercially available for this purpose. Tests limitations and directions for future research are discussed. PMID:21108793

Real-time PCR detection of Plasmodium directly from whole blood and filter paper samples

PubMed Central

2011-01-01

Background Real-time PCR is a sensitive and specific method for the analysis of Plasmodium DNA. However, prior purification of genomic DNA from blood is necessary since PCR inhibitors and quenching of fluorophores from blood prevent efficient amplification and detection of PCR products. Methods Reagents designed to specifically overcome PCR inhibition and quenching of fluorescence were evaluated for real-time PCR amplification of Plasmodium DNA directly from blood. Whole blood from clinical samples and dried blood spots collected in the field in Colombia were tested. Results Amplification and fluorescence detection by real-time PCR were optimal with 40× SYBR® Green dye and 5% blood volume in the PCR reaction. Plasmodium DNA was detected directly from both whole blood and dried blood spots from clinical samples. The sensitivity and specificity ranged from 93-100% compared with PCR performed on purified Plasmodium DNA. Conclusions The methodology described facilitates high-throughput testing of blood samples collected in the field by fluorescence-based real-time PCR. This method can be applied to a broad range of clinical studies with the advantages of immediate sample testing, lower experimental costs and time-savings. PMID:21851640

Non-radioactive detection of trinucleotide repeat size variability.

PubMed

Tomé, Stéphanie; Nicole, Annie; Gomes-Pereira, Mario; Gourdon, Genevieve

2014-03-06

Many human diseases are associated with the abnormal expansion of unstable trinucleotide repeat sequences. The mechanisms of trinucleotide repeat size mutation have not been fully dissected, and their understanding must be grounded on the detailed analysis of repeat size distributions in human tissues and animal models. Small-pool PCR (SP-PCR) is a robust, highly sensitive and efficient PCR-based approach to assess the levels of repeat size variation, providing both quantitative and qualitative data. The method relies on the amplification of a very low number of DNA molecules, through sucessive dilution of a stock genomic DNA solution. Radioactive Southern blot hybridization is sensitive enough to detect SP-PCR products derived from single template molecules, separated by agarose gel electrophoresis and transferred onto DNA membranes. We describe a variation of the detection method that uses digoxigenin-labelled locked nucleic acid probes. This protocol keeps the sensitivity of the original method, while eliminating the health risks associated with the manipulation of radiolabelled probes, and the burden associated with their regulation, manipulation and waste disposal.

Deep Diversity: Novel Approach to Overcoming the PCR Bias Encountered During Environmental Analysis of Microbial Populations for Alpha-Diversity

NASA Technical Reports Server (NTRS)

Ramirez, Gustavo A; Vaishampayan, Parag A.

2011-01-01

Alpha-diversity studies are of crucial importance to environmental microbiologists. The polymerase chain reaction (PCR) method has been paramount for studies interrogating microbial environmental samples for taxon richness. Phylogenetic studies using this technique are based on the amplification and comparison of the 16S rRNA coding regions. PCR, due disproportionate distribution of microbial species in the environment, increasingly favors the amplification of the most predominant phylotypes with every subsequent reaction cycle. The genetic and chemical complexity of environmental samples are intrinsic factors that exacerbate an inherit bias in PCR-based quantitative and qualitative studies of microbial communities. We report that treatment of a genetically complex total genomic environmental DNA extract with Propidium Monoazide (PMA), a DNA intercalating molecule capable of forming a covalent cross-linkage to organic moieties upon light exposure, disproportionally inactivates predominant phylotypes and results in the exponential amplification of previously shadowed microbial ?-diversity quantified as a 19.5% increase in OUTs reported via phylogenetic screening using PhyloChip.

Comparison of the performance of three PCR assays for the detection and differentiation of Theileria orientalis genotypes.

PubMed

Perera, Piyumali K; Gasser, Robin B; Pulford, David J; Stevenson, Mark A; Firestone, Simon M; McFadden, Andrew M J; Jabbar, Abdul

2015-03-31

Oriental theileriosis is a tick-borne disease of bovines caused by the members of the Theileria orientalis complex. Recently, we developed a multiplexed tandem (MT) PCR to detect, differentiate and quantitate four genotypes (i.e., buffeli, chitose, ikeda and type 5) of T. orientalis. In this study, we used MT PCR to assess the prevalence and infection intensity of four T. orientalis genotypes in selected cattle herds that experienced oriental theileriosis outbreaks in New Zealand, and compared the sensitivities and specificities of MT PCR, PCR-high resolution melting (PCR-HRM) and a TaqMan qPCR. MT PCR, PCR-HRM analysis for T. orientalis and a TaqMan qPCR assay for ikeda genotype were employed to test 154 and 88 cattle blood samples from North (where oriental theileriosis outbreaks had occurred; designated as Group 1) and South (where no outbreaks had been reported; Group 2) Islands of New Zealand, respectively. Quantitative data from MT PCR assay were analyzed using generalized linear model and paired-sample t-test. The diagnostic specificity and sensitivity of the assays were estimated using a Bayesian latent class modeling approach. In Group 1, 99.4% (153/154) of cattle were test-positive for T. orientalis in both the MT PCR and PCR-HRM assays. The apparent prevalences of genotype ikeda in Group 1 were 87.6% (134/153) and 87.7% (135/154) using the MT PCR and Ikeda TaqMan qPCR assays, respectively. Using the MT PCR test, all four genotypes of T. orientalis were detected. The infection intensity estimated for genotype ikeda was significantly higher (P = 0.009) in severely anaemic cattle than in those without anaemia, and this intensity was significantly higher than that of buffeli (P < 0.001) in the former cattle. Bayesian latent class analysis showed that the diagnostic sensitivities (97.1-98.9%) and specificities (96.5-98.9%) of the three PCR assays were very comparable. The present findings show the advantages of using the MT PCR assay as a useful tool for in-depth epidemiological and transmission studies of T. orientalis worldwide.

Diagnosis of Caprine Arthritis Encephalitis Virus infection in dairy goats by ELISA, PCR and Viral Culture.

PubMed

Panneum, S; Rukkwamsuk, T

2017-03-01

For preventive and control strategies of Caprine Arthritis Encephalitis Virus (CAEV) infection in dairy goats, performance of the available diagnostic tests was described as one of the most important and necessary aspects. The study aimed at evaluating the diagnostic test performance, including PCR, ELISA and viral culture, for CAEV infection in dairy goats in Thailand. Blood samples of 29 dairy goats from five low- to medium-prevalence herds and one very low-prevalence herd were collected for PCR and ELISA methods. The performance of these two diagnostic methods was evaluated by comparing with cytopathic effects (CPE) in the co-cultivation of CAEV and primary synovial cells. Results indicated that sensitivity, specificity were, respectively, 69.6%, 100%, for PCR; and 95.7%, 83.3% for ELISA. The PCR assay tended to have lower sensitivity and higher specificity than ELISA. When multiple tests were applied, parallel testing provided sensitivity and specificity of 98.7% and 83.3%, while series testing showed sensitivity and specificity of 66.6% and 100% respectively. These results indicated that combination of ELISA and PCR provided some advantages and possibly offered optimal methods to detect CAEV-infected goats. Kappa value of the agreement between PCR and ELISA test was 0.34, indicating fair agreement. Regarding the possibility of antigenic variation between CAEV strains used in both PCR and ELISA assays, the actual circulating CAEV strain should be reviewed in order to develop and enhance the diagnostic tests using the CAE viral antigens derived from specific local strains of Thailand.

Development of a general method for detection and quantification of the P35S promoter based on assessment of existing methods

PubMed Central

Wu, Yuhua; Wang, Yulei; Li, Jun; Li, Wei; Zhang, Li; Li, Yunjing; Li, Xiaofei; Li, Jun; Zhu, Li; Wu, Gang

2014-01-01

The Cauliflower mosaic virus (CaMV) 35S promoter (P35S) is a commonly used target for detection of genetically modified organisms (GMOs). There are currently 24 reported detection methods, targeting different regions of the P35S promoter. Initial assessment revealed that due to the absence of primer binding sites in the P35S sequence, 19 of the 24 reported methods failed to detect P35S in MON88913 cotton, and the other two methods could only be applied to certain GMOs. The rest three reported methods were not suitable for measurement of P35S in some testing events, because SNPs in binding sites of the primer/probe would result in abnormal amplification plots and poor linear regression parameters. In this study, we discovered a conserved region in the P35S sequence through sequencing of P35S promoters from multiple transgenic events, and developed new qualitative and quantitative detection systems targeting this conserved region. The qualitative PCR could detect the P35S promoter in 23 unique GMO events with high specificity and sensitivity. The quantitative method was suitable for measurement of P35S promoter, exhibiting good agreement between the amount of template and Ct values for each testing event. This study provides a general P35S screening method, with greater coverage than existing methods. PMID:25483893

Sensitivity of a real-time PCR method for the detection of transgenes in a mixture of transgenic and non-transgenic seeds of papaya (Carica papaya L.).

PubMed

Nageswara-Rao, Madhugiri; Kwit, Charles; Agarwal, Sujata; Patton, Mariah T; Skeen, Jordan A; Yuan, Joshua S; Manshardt, Richard M; Stewart, C Neal

2013-09-01

Genetically engineered (GE) ringspot virus-resistant papaya cultivars 'Rainbow' and 'SunUp' have been grown in Hawai'i for over 10 years. In Hawai'i, the introduction of GE papayas into regions where non-GE cultivars are grown and where feral non-GE papayas exist have been accompanied with concerns associated with transgene flow. Of particular concern is the possibility of transgenic seeds being found in non-GE papaya fruits via cross-pollination. Development of high-throughput methods to reliably detect the adventitious presence of such transgenic material would benefit both the scientific and regulatory communities. We assessed the accuracy of using conventional qualitative polymerase chain reaction (PCR) as well as real-time PCR-based assays to quantify the presence of transgenic DNA from bulk samples of non-GE papaya seeds. In this study, an optimized method of extracting high quality DNA from dry seeds of papaya was standardized. A reliable, sensitive real-time PCR method for detecting and quantifying viral coat protein (cp) transgenes in bulk seed samples utilizing the endogenous papain gene is presented. Quantification range was from 0.01 to 100 ng/μl of GE-papaya DNA template with a detection limit as low as 0.01% (10 pg). To test this system, we simulated transgene flow using known quantities of GE and non-GE DNA and determined that 0.038% (38 pg) GE papaya DNA could be detected using real-time PCR. We also validated this system by extracting DNA from known ratios of GE seeds to non-GE seeds of papaya followed by real-time PCR detection and observed a reliable detection limit of 0.4%. This method for the quick and sensitive detection of transgenes in bulked papaya seed lots using conventional as well as real-time PCR-based methods will benefit numerous stakeholders. In particular, this method could be utilized to screen selected fruits from maternal non-GE papaya trees in Hawai'i for the presence of transgenic seed at typical regulatory threshold levels. Incorporation of subtle differences in primers and probes for variations in cp worldwide should allow this method to be utilized elsewhere when and if deregulation of transgenic papaya occurs.

Sensitivity of a real-time PCR method for the detection of transgenes in a mixture of transgenic and non-transgenic seeds of papaya (Carica papaya L.)

PubMed Central

2013-01-01

Background Genetically engineered (GE) ringspot virus-resistant papaya cultivars ‘Rainbow’ and ‘SunUp’ have been grown in Hawai’i for over 10 years. In Hawai’i, the introduction of GE papayas into regions where non-GE cultivars are grown and where feral non-GE papayas exist have been accompanied with concerns associated with transgene flow. Of particular concern is the possibility of transgenic seeds being found in non-GE papaya fruits via cross-pollination. Development of high-throughput methods to reliably detect the adventitious presence of such transgenic material would benefit both the scientific and regulatory communities. Results We assessed the accuracy of using conventional qualitative polymerase chain reaction (PCR) as well as real-time PCR-based assays to quantify the presence of transgenic DNA from bulk samples of non-GE papaya seeds. In this study, an optimized method of extracting high quality DNA from dry seeds of papaya was standardized. A reliable, sensitive real-time PCR method for detecting and quantifying viral coat protein (cp) transgenes in bulk seed samples utilizing the endogenous papain gene is presented. Quantification range was from 0.01 to 100 ng/μl of GE-papaya DNA template with a detection limit as low as 0.01% (10 pg). To test this system, we simulated transgene flow using known quantities of GE and non-GE DNA and determined that 0.038% (38 pg) GE papaya DNA could be detected using real-time PCR. We also validated this system by extracting DNA from known ratios of GE seeds to non-GE seeds of papaya followed by real-time PCR detection and observed a reliable detection limit of 0.4%. Conclusions This method for the quick and sensitive detection of transgenes in bulked papaya seed lots using conventional as well as real-time PCR-based methods will benefit numerous stakeholders. In particular, this method could be utilized to screen selected fruits from maternal non-GE papaya trees in Hawai’i for the presence of transgenic seed at typical regulatory threshold levels. Incorporation of subtle differences in primers and probes for variations in cp worldwide should allow this method to be utilized elsewhere when and if deregulation of transgenic papaya occurs. PMID:24004548

Detection and Differentiation of Leishmania spp. in Clinical Specimens by Use of a SYBR Green-Based Real-Time PCR Assay.

PubMed

de Almeida, Marcos E; Koru, Ozgur; Steurer, Francis; Herwaldt, Barbara L; da Silva, Alexandre J

2017-01-01

Leishmaniasis in humans is caused by Leishmania spp. in the subgenera Leishmania and Viannia Species identification often has clinical relevance. Until recently, our laboratory relied on conventional PCR amplification of the internal transcribed spacer 2 (ITS2) region (ITS2-PCR) followed by sequencing analysis of the PCR product to differentiate Leishmania spp. Here we describe a novel real-time quantitative PCR (qPCR) approach based on the SYBR green technology (LSG-qPCR), which uses genus-specific primers that target the ITS1 region and amplify DNA from at least 10 Leishmania spp., followed by analysis of the melting temperature (T m ) of the amplicons on qPCR platforms (the Mx3000P qPCR system [Stratagene-Agilent] and the 7500 real-time PCR system [ABI Life Technologies]). We initially evaluated the assay by testing reference Leishmania isolates and comparing the results with those from the conventional ITS2-PCR approach. Then we compared the results from the real-time and conventional molecular approaches for clinical specimens from 1,051 patients submitted to the reference laboratory of the Centers for Disease Control and Prevention for Leishmania diagnostic testing. Specimens from 477 patients tested positive for Leishmania spp. with the LSG-qPCR assay, specimens from 465 of these 477 patients also tested positive with the conventional ITS2-PCR approach, and specimens from 10 of these 465 patients had positive results because of retesting prompted by LSG-qPCR positivity. On the basis of the T m values of the LSG-qPCR amplicons from reference and clinical specimens, we were able to differentiate four groups of Leishmania parasites: the Viannia subgenus in aggregate; the Leishmania (Leishmania) donovani complex in aggregate; the species L (L) tropica; and the species L (L) mexicana, L (L) amazonensis, L (L) major, and L (L) aethiopica in aggregate. Copyright © 2016 American Society for Microbiology.

Quality assurance in RT-PCR-based BCR/ABL diagnostics--results of an interlaboratory test and a standardization approach.

PubMed

Burmeister, T; Maurer, J; Aivado, M; Elmaagacli, A H; Grünebach, F; Held, K R; Hess, G; Hochhaus, A; Höppner, W; Lentes, K U; Lübbert, M; Schäfer, K L; Schafhausen, P; Schmidt, C A; Schüler, F; Seeger, K; Seelig, R; Thiede, C; Viehmann, S; Weber, C; Wilhelm, S; Christmann, A; Clement, J H; Ebener, U; Enczmann, J; Leo, R; Schleuning, M; Schoch, R; Thiel, E

2000-10-01

Here we describe the results of an interlaboratory test for RT-PCR-based BCR/ABL analysis. The test was organized in two parts. The number of participating laboratories in the first and second part was 27 and 20, respectively. In the first part samples containing various concentrations of plasmids with the ela2, b2a2 or b3a2 BCR/ABL transcripts were analyzed by PCR. In the second part of the test, cell samples containing various concentrations of BCR/ABL-positive cells were analyzed by RT-PCR. Overall PCR sensitivity was sufficient in approximately 90% of the tests, but a significant number of false positive results were obtained. There were significant differences in sensitivity in the cell-based analysis between the various participants. The results are discussed, and proposals are made regarding the choice of primers, controls, conditions for RNA extraction and reverse transcription.

A comparative study of digital PCR and real-time qPCR for the detection and quantification of HPV mRNA in sentinel lymph nodes of cervical cancer patients.

PubMed

Carow, Katrin; Read, Christina; Häfner, Norman; Runnebaum, Ingo B; Corner, Adam; Dürst, Matthias

2017-10-30

Qualitative analyses showed that the presence of HPV mRNA in sentinel lymph nodes of cervical cancer patients with pN0 status is associated with significantly decreased recurrence free survival. To further address the clinical potential of the strategy and to define prognostic threshold levels it is necessary to use a quantitative assay. Here, we compare two methods of quantification: digital PCR and standard quantitative PCR. Serial dilutions of 5 ng-5 pg RNA (≙ 500-0.5 cells) of the cervical cancer cell line SiHa were prepared in 5 µg RNA of the HPV-negative human keratinocyte cell line HaCaT. Clinical samples consisted of 10 sentinel lymph nodes with varying HPV transcript levels. Reverse transcription of total RNA (5 µg RNA each) was performed in 100 µl and cDNA aliquots were analyzed by qPCR and dPCR. Digital PCR was run in the RainDrop ® Digital PCR system (RainDance Technologies) using a probe-based detection of HPV E6/E7 cDNA PCR products with 11 µl template. qPCR was done using a Rotor Gene Q 5plex HRM (Qiagen) amplifying HPV E6/E7 cDNA in a SYBR Green format with 1 µl template. For the analysis of both, clinical samples and serial dilution samples, dPCR and qPCR showed comparable sensitivity. With regard to reproducibility, both methods differed considerably, especially for low template samples. Here, we found with qPCR a mean variation coefficient of 126% whereas dPCR enabled a significantly lower mean variation coefficient of 40% (p = 0.01). Generally, we saw with dPCR a substantial reduction of subsampling errors, which most likely reflects the large cDNA amounts available for analysis. Compared to real-time PCR, dPCR shows higher reliability. Thus, our HPV mRNA dPCR assay holds promise for the clinical evaluation of occult tumor cells in histologically tumor-free lymph nodes in future studies.

A proof of concept study to assess the potential of PCR testing to detect natural Mycobacterium bovis infection in South American camelids.

PubMed

Crawshaw, Timothy R; Chanter, Jeremy I; McGoldrick, Adrian; Line, Kirsty

2014-02-07

Cases of Mycobacterium bovis infection South American camelids have been increasing in Great Britain. Current antemortem immunological tests have some limitations. Cases at post mortem examination frequently show extensive pathology. The feasibility of detecting Mycobacterium bovis DNA in clinical samples was investigated. A sensitive extraction methodology was developed and used on nasal swabs and faeces taken post-mortem to assess the potential for a PCR test to detect Mycobacterium bovis in clinical samples. The gross pathology of the studied South American camelids was scored and a significantly greater proportion of South American camelids with more severe pathology were positive in both the nasal swab and faecal PCR tests. A combination of the nasal swab and faecal PCR tests detected 63.9% of all the South American camelids with pathology that were tested. The results suggest that antemortem diagnosis of Mycobacterium bovis in South American camelids may be possible using a PCR test on clinical samples, however more work is required to determine sensitivity and specificity, and the practicalities of applying the test in the field.

Performance of a RT-PCR Assay in Comparison to FISH and Immunohistochemistry for the Detection of ALK in Non-Small Cell Lung Cancer.

PubMed

Hout, David R; Schweitzer, Brock L; Lawrence, Kasey; Morris, Stephan W; Tucker, Tracy; Mazzola, Rosetta; Skelton, Rachel; McMahon, Frank; Handshoe, John; Lesperance, Mary; Karsan, Aly; Saltman, David L

2017-08-01

Patients with lung cancers harboring an activating anaplastic lymphoma kinase ( ALK ) rearrangement respond favorably to ALK inhibitor therapy. Fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) are validated and widely used screening tests for ALK rearrangements but both methods have limitations. The ALK RGQ RT-PCR Kit (RT-PCR) is a single tube quantitative real-time PCR assay for high throughput and automated interpretation of ALK expression. In this study, we performed a direct comparison of formalin-fixed paraffin-embedded (FFPE) lung cancer specimens using all three ALK detection methods. The RT-PCR test (diagnostic cut-off Δ C t of ≤8) was shown to be highly sensitive (100%) when compared to FISH and IHC. Sequencing of RNA detected full-length ALK transcripts or EML4-ALK and KIF5B-ALK fusion variants in discordant cases in which ALK expression was detected by the ALK RT-PCR test but negative by FISH and IHC. The overall specificity of the RT-PCR test for the detection of ALK in cases without full-length ALK expression was 94% in comparison to FISH and sequencing. These data support the ALK RT-PCR test as a highly efficient and reliable diagnostic screening approach to identify patients with non-small cell lung cancer whose tumors are driven by oncogenic ALK.

Performance of a RT-PCR Assay in Comparison to FISH and Immunohistochemistry for the Detection of ALK in Non-Small Cell Lung Cancer

PubMed Central

Hout, David R.; Lawrence, Kasey; Morris, Stephan W.; Tucker, Tracy; Mazzola, Rosetta; Skelton, Rachel; McMahon, Frank; Handshoe, John; Lesperance, Mary; Karsan, Aly

2017-01-01

Patients with lung cancers harboring an activating anaplastic lymphoma kinase (ALK) rearrangement respond favorably to ALK inhibitor therapy. Fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) are validated and widely used screening tests for ALK rearrangements but both methods have limitations. The ALK RGQ RT-PCR Kit (RT-PCR) is a single tube quantitative real-time PCR assay for high throughput and automated interpretation of ALK expression. In this study, we performed a direct comparison of formalin-fixed paraffin-embedded (FFPE) lung cancer specimens using all three ALK detection methods. The RT-PCR test (diagnostic cut-off ΔCt of ≤8) was shown to be highly sensitive (100%) when compared to FISH and IHC. Sequencing of RNA detected full-length ALK transcripts or EML4-ALK and KIF5B-ALK fusion variants in discordant cases in which ALK expression was detected by the ALK RT-PCR test but negative by FISH and IHC. The overall specificity of the RT-PCR test for the detection of ALK in cases without full-length ALK expression was 94% in comparison to FISH and sequencing. These data support the ALK RT-PCR test as a highly efficient and reliable diagnostic screening approach to identify patients with non-small cell lung cancer whose tumors are driven by oncogenic ALK. PMID:28763012

Quantification of hepatitis C virus RNA in serum by branched DNA-based signal amplification assays.

PubMed

Pawlotsky, J M; Martinot-Peignoux, M; Poveda, J D; Bastie, A; Le Breton, V; Darthuy, F; Rémiré, J; Erlinger, S; Dhumeaux, D; Marcellin, P

1999-05-01

The objective of the study was to compare the clinical sensitivity and specificity of versions 1.0 and 2.0 of the branched DNA (bDNA)-based hepatitis C virus (HCV) RNA quantification assay, and also to compare the values yielded by the two versions according to the HCV genotype. Serum samples from 268 patients tested routinely by a non-quantitative HCV RNA PCR assay (group A) were tested with version 2.0 of the bDNA assay. Samples from 342 HCV PCR-positive patients with chronic hepatitis C eligible for interferon treatment (group B) were tested with both version 1.0 and version 2.0 of the bDNA assay. Version 2.0 had a clinical sensitivity of 92% (95% confidence interval (CI): 87-97%) in group A and 89% (86-92%) in group B. In group B, the gain in sensitivity with bDNA 2.0 was 16% relative to bDNA 1.0 (P < 0.001). The log values of the two assays correlated with samples positive by both assays (r = 0.83, P < 0.0001), but the distribution of values was larger in samples containing HCV genotypes 2 and 3. The mean ratio of assay 2.0/assay 1.0 values was 1.69 +/- 1.44 (range: 0.33-13.43). The mean ratio was close to 1 with samples containing genotype 1 or 4, but ranged from 0.33 to more than 5. The mean ratio was close to 3 with samples containing genotype 2 or 3, and ranged from 0.5 to more than 13. HCV RNA levels were significantly lower in samples containing genotype 4 than in those containing other genotypes. Sera from 200 anti-HCV-negative, HCV RNA PCR-negative blood donors (group C), and from 164 anti-HCV-negative patients with symptoms of chronic liver disease (group D) were used to assess the clinical specificity of bDNA 2.0. In addition, samples with an HCV RNA titer between 0.2 (assay cutoff) and 0.5 MEq/ml from a group of 546 patients tested routinely for HCV RNA load by bDNA 2.0 (group E) were retested by bDNA 2.0 and by qualitative PCR. The specificity of bDNA 2.0 was 100% (98-100%) in group C and 99% (97-100%) in group D. Among the 41 samples from group E, 38 were positive by bDNA 2.0 retesting (36 were PCR-positive) and three were negative by bDNA 2.0 retesting (all were PCR-positive). It is concluded that version 2.0 of the bDNA assay is markedly more sensitive than version 1.0 and has a good specificity. In contrast with version 1.0, version 2.0 is not influenced by the HCV genotype. The relationship between values obtained with assays 1.0 and 2.0 on clinical specimens is not linear, indicating that HCV RNA titers cannot reliably be calculated from the results of version 1.0.

[The establishment of a novel method of nano-immunomagnetic separation and Real-time PCR for detecting Vibrio cholerae from seafood].

PubMed

Cheng, Jinxia; Zeng, Jing; Liu, Li; Wei, Haiyan; Zhao, Xiaojuan; Zhang, Ximeng; Zhang, Lei; Zhang, Haiyu

2014-02-01

A novel method of Nano-Immunomagnetic Separation (Nano-IMS) plus Real-time PCR was established for detecting Vibrio cholerae. The Nano-Immunomagnetic Beads were created by using the monoclonal antibody of Vibrio cholerae, which was named Nano-IMB-Vc. Nano-IMB-Vc has specific adsorption of Vibrio cholerae, combined with Real-time PCR technology, a method for rapid detection of Vibrio cholerae was established. The capture specificity of Nano-IMB-Vc was tested by using 15 bacteria strains. The specificity of Real-time PCR method was tested by using 102 targets and 101 non-targets bacteria strains. The sensitivity of Nano-IMS plus Real-time PCR were tested in pure culture and in artificial samples and compared with NMKL No.156. The capture ratio of Nano-IMB-Vc was reached 70.2% at the level of 10(3) CFU/ml. In pure culture, the sensitivity of Nano-IMS plus Real-time PCR was reached at 5.4×10(2) CFU/ml. The specific of Real-time PCR method was tested by using 102 targets and 101 non-targets bacteria. The results showed that 102 strains of Vibrio cholerae test results were all positive, and the rest of the 101 strains of non-target bacteria test results were negative. No cross-reaction was founded. Add 1 CFU vibrio cholerae per 25 g sample, it could be detect with Nano-IMS plus Real-time PCR method after 8 hours enrichment. The Nano-IMS plus Real-time PCR method of Vibrio cholerae established in this study has good specificity and sensitivity, which could be applied to the rapid detection of Vibrio cholerae.

Quantification of integrated HIV DNA by repetitive-sampling Alu-HIV PCR on the basis of poisson statistics.

PubMed

De Spiegelaere, Ward; Malatinkova, Eva; Lynch, Lindsay; Van Nieuwerburgh, Filip; Messiaen, Peter; O'Doherty, Una; Vandekerckhove, Linos

2014-06-01

Quantification of integrated proviral HIV DNA by repetitive-sampling Alu-HIV PCR is a candidate virological tool to monitor the HIV reservoir in patients. However, the experimental procedures and data analysis of the assay are complex and hinder its widespread use. Here, we provide an improved and simplified data analysis method by adopting binomial and Poisson statistics. A modified analysis method on the basis of Poisson statistics was used to analyze the binomial data of positive and negative reactions from a 42-replicate Alu-HIV PCR by use of dilutions of an integration standard and on samples of 57 HIV-infected patients. Results were compared with the quantitative output of the previously described Alu-HIV PCR method. Poisson-based quantification of the Alu-HIV PCR was linearly correlated with the standard dilution series, indicating that absolute quantification with the Poisson method is a valid alternative for data analysis of repetitive-sampling Alu-HIV PCR data. Quantitative outputs of patient samples assessed by the Poisson method correlated with the previously described Alu-HIV PCR analysis, indicating that this method is a valid alternative for quantifying integrated HIV DNA. Poisson-based analysis of the Alu-HIV PCR data enables absolute quantification without the need of a standard dilution curve. Implementation of the CI estimation permits improved qualitative analysis of the data and provides a statistical basis for the required minimal number of technical replicates. © 2014 The American Association for Clinical Chemistry.

Detection and Species Determination of Malaria Parasites by PCR: Comparison with Microscopy and with ParaSight-F and ICT Malaria Pf Tests in a Clinical Environment

PubMed Central

Tham, Jill M.; Lee, Szu Hee; Tan, Theresa M. C.; Ting, Robert C. Y.; Kara, Ursula A. K.

1999-01-01

A rapid procedure for the diagnosis of malaria infections directly from dried blood spots by PCR amplification was evaluated with samples from 52 patients. Plasmodium infections were identified with a genus-specific primer set, and species differentiation between Plasmodium falciparum and Plasmodium vivax was analyzed by multiplex PCR. The PCR test with any of the three primer sets was able to detect as few as four parasites per microliter by gel electrophoresis or by nonisotopic paper hybridization chromatography. The diagnoses obtained by PCR correlated closely with those obtained by Giemsa staining except for two samples observed to have mixed P. falciparum-P. vivax infections. These were initially missed by microscopic analysis. In comparison with antigen-capture assays for P. falciparum, the PCR assays were able to detect three infections that were missed by the ParaSight-F test. The PCR test was negative for nine ParaSight-F-positive samples and one ICT Malaria Pf-positive sample, and these were confirmed to be false-positive results. The PCR thus gave no false-negative or false-positive results. Patients undergoing antimalarial therapy were also monitored by the PCR assay. Four of seven patients who were PCR positive for P. vivax at the time of discharge were later readmitted to the hospital with a recurrence of P. vivax infection. We would like to propose that PCR is a sensitive and easy method that can serve as a useful addition to microscopy for the diagnosis and the clinical monitoring of treatment of malaria. PMID:10203469

Development of an Internal Positive Control for Rapid Diagnosis of Avian Influenza Virus Infections by Real-Time Reverse Transcription-PCR with Lyophilized Reagents

PubMed Central

Das, Amaresh; Spackman, Erica; Senne, Dennis; Pedersen, Jan; Suarez, David L.

2006-01-01

We developed an internal positive control (IPC) RNA to help ensure the accuracy of the detection of avian influenza virus (AIV) RNA by reverse transcription (RT)-PCR and real-time RT-PCR (RRT-PCR). The IPC was designed to have the same binding sites for the forward and reverse primers of the AIV matrix gene as the target amplicon, but it had a unique internal sequence used for the probe site. The amplification of the viral RNA and the IPC by RRT-PCR were monitored with two different fluorescent probes in a multiplex format, one specific for the AIV matrix gene and the other for the IPC. The RRT-PCR test was further simplified with the use of lyophilized bead reagents for the detection of AIV RNA. The RRT-PCR with the bead reagents was more sensitive than the conventional wet reagents for the detection of AIV RNA. The IPC-based RRT-PCR detected inhibitors in blood, kidney, lungs, spleen, intestine, and cloacal swabs, but not allantoic fluid, serum, or tracheal swabs The accuracy of RRT-PCR test results with the lyophilized beads was tested on cloacal and tracheal swabs from experimental birds inoculated with AIV and compared with virus isolation (VI) on embryonating chicken eggs. There was 97 to 100% agreement of the RRT-PCR test results with VI for tracheal swabs and 81% agreement with VI for cloacal swabs, indicating a high level of accuracy of the RRT-PCR assay. The same IPC in the form of armored RNA was also used to monitor the extraction of viral RNA and subsequent detection by RRT-PCR. PMID:16954228

Usefulness of Two Aspergillus PCR Assays and Aspergillus Galactomannan and β-d-Glucan Testing of Bronchoalveolar Lavage Fluid for Diagnosis of Chronic Pulmonary Aspergillosis

PubMed Central

Urabe, Naohisa; Sano, Go; Suzuki, Junko; Hebisawa, Akira; Nakamura, Yasuhiko; Koyama, Kazuya; Ishii, Yoshikazu; Tateda, Kazuhiro; Homma, Sakae

2017-01-01

ABSTRACT We evaluated the usefulness of an Aspergillus galactomannan (GM) test, a β-d-glucan (βDG) test, and two different Aspergillus PCR assays of bronchoalveolar lavage fluid (BALF) samples for the diagnosis of chronic pulmonary aspergillosis (CPA). BALF samples from 30 patients with and 120 patients without CPA were collected. We calculated the sensitivity, specificity, positive predictive value, negative predictive value, positive likelihood ratio, negative likelihood ratio, and diagnostic odds ratio for each test individually and in combination with other tests. The optical density index values, as determined by receiver operating characteristic analysis, for the diagnosis of CPA were 0.5 and 100 for GM and βDG testing of BALF, respectively. The sensitivity and specificity of the GM test, βDG test, and PCR assays 1 and 2 were 77.8% and 90.0%, 77.8% and 72.5%, 86.7% and 84.2%, and 66.7% and 94.2%, respectively. A comparison of the PCR assays showed that PCR assay 1 had a better sensitivity, a better negative predictive value, and a better negative likelihood ratio and PCR assay 2 had a better specificity, a better positive predictive value, and a better positive likelihood ratio. The combination of the GM and βDG tests had the highest diagnostic odds ratio. The combination of the GM and βDG tests on BALF was more useful than any single test for diagnosing CPA. PMID:28330887

A multiplex PCR method of detecting recombinant DNAs from five lines of genetically modified maize.

PubMed

Matsuoka, T; Kuribara, H; Akiyama, H; Miura, H; Goda, Y; Kusakabe, Y; Isshiki, K; Toyoda, M; Hino, A

2001-02-01

Seven lines of genetically modified (GM) maize have been authorized in Japan as foods and feeds imported from the USA. We improved a multiplex PCR method described in the previous report in order to distinguish the five lines of GM maize. Genomic DNA was extracted from GM maize with a silica spin column kit, which could reduce experimental time and improve safety in the laboratory and potentially in the environment. We sequenced recombinant DNA (r-DNA) introduced into GM maize, and re-designed new primer pairs to increase the specificity of PCR to distinguish five lines of GM maize by multiplex PCR. A primer pair for the maize intrinsic zein gene (Ze1) was also designed to confirm the presence of amplifiable maize DNA. The lengths of PCR products using these six primer pairs were different. The Ze1 and the r-DNAs from the five lines of GM maize were qualitatively detected in one tube. The specific PCR bands were distinguishable from each other on the basis of the expected length. The r-DNA could be detected from maize samples containing 0.5% of each of the five lines of GM maize. The sensitivity would be acceptable to secure the verification of non-GMO materials and to monitor the reliability of the labeling system.

Archaea in the foregut of macropod marsupials: PCR and amplicon sequence-based observations.

PubMed

Klieve, A V; Ouwerkerk, D; Maguire, A J

2012-11-01

To investigate, using culture-independent techniques, the presence and diversity of methanogenic archaea in the foregut of kangaroos. DNA was extracted from forestomach contents of 42 kangaroos (three species), three sheep and three cattle. Four qualitative and quantitative PCR assays targeting the archaeal domain (16S rRNA gene) or the functional methanogenesis gene, mcrA, were used to determine the presence and population density of archaea in kangaroos and whether they were likely to be methanogens. All ruminal samples were positive for archaea, produced PCR product of expected size, contained high numbers of archaea and high numbers of cells with mcrA genes. Kangaroos were much more diverse and contradictory. Fourteen kangaroos had detectable archaea with numbers 10- to 1000-fold fewer than sheep and cattle. Many kangaroos that did not possess archaea were positive for the mcrA gene and had detectable numbers of cells with this gene and vice versa. DNA sequence analysis of kangaroos' archaeal 16S rRNA gene clones show that many methanogens were related to Methanosphaera stadmanae. Other sequences were related to non-methanogenic archaea (Thermoplasma sp.), and a number of kangaroos had mcrA gene sequences related to methane oxidising archaea (ANME). Discrepancies between qualitative and quantitative PCR assays for archaea and the mcrA gene suggest that the archaeal communities are very diverse and it is possible that novel species exist. Archaea (in general) were below detectable limits in many kangaroos, especially Red kangaroos; when present they are in lower numbers than in ruminants, and the archaea are not necessarily methanogenic. The determination of why this is the case in the kangaroo foregut could assist in reducing emissions from other ecosystems in the future. © 2012 The Authors Journal of Applied Microbiology © 2012 The Society for Applied Microbiology.

Utility of PCR, Culture, and Antigen Detection Methods for Diagnosis of Legionellosis.

PubMed

Chen, Derrick J; Procop, Gary W; Vogel, Sherilynn; Yen-Lieberman, Belinda; Richter, Sandra S

2015-11-01

The goal of this retrospective study was to evaluate the performance of different diagnostic tests for Legionnaires' disease in a clinical setting where Legionella pneumophila PCR had been introduced. Electronic medical records at the Cleveland Clinic were searched for Legionella urinary antigen (UAG), culture, and PCR tests ordered from March 2010 through December 2013. For cases where two or more test methods were performed and at least one was positive, the medical record was reviewed for relevant clinical and epidemiologic factors. Excluding repeat testing on a given patient, 19,912 tests were ordered (12,569 UAG, 3,747 cultures, and 3,596 PCR) with 378 positive results. The positivity rate for each method was 0.4% for culture, 0.8% for PCR, and 2.7% for UAG. For 37 patients, at least two test methods were performed with at least one positive result: 10 (27%) cases were positive by all three methods, 16 (43%) were positive by two methods, and 11 (30%) were positive by one method only. For the 32 patients with medical records available, clinical presentation was consistent with proven or probable Legionella infection in 84% of the cases. For those cases, the sensitivities of culture, PCR, and UAG were 50%, 92%, and 96%, respectively. The specificities were 100% for culture and 99.9% for PCR and UAG. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Utility of PCR, Culture, and Antigen Detection Methods for Diagnosis of Legionellosis

PubMed Central

Chen, Derrick J.; Procop, Gary W.; Vogel, Sherilynn; Yen-Lieberman, Belinda

2015-01-01

The goal of this retrospective study was to evaluate the performance of different diagnostic tests for Legionnaires' disease in a clinical setting where Legionella pneumophila PCR had been introduced. Electronic medical records at the Cleveland Clinic were searched for Legionella urinary antigen (UAG), culture, and PCR tests ordered from March 2010 through December 2013. For cases where two or more test methods were performed and at least one was positive, the medical record was reviewed for relevant clinical and epidemiologic factors. Excluding repeat testing on a given patient, 19,912 tests were ordered (12,569 UAG, 3,747 cultures, and 3,596 PCR) with 378 positive results. The positivity rate for each method was 0.4% for culture, 0.8% for PCR, and 2.7% for UAG. For 37 patients, at least two test methods were performed with at least one positive result: 10 (27%) cases were positive by all three methods, 16 (43%) were positive by two methods, and 11 (30%) were positive by one method only. For the 32 patients with medical records available, clinical presentation was consistent with proven or probable Legionella infection in 84% of the cases. For those cases, the sensitivities of culture, PCR, and UAG were 50%, 92%, and 96%, respectively. The specificities were 100% for culture and 99.9% for PCR and UAG. PMID:26292304

Latent class analysis of the diagnostic characteristics of PCR and conventional bacteriological culture in diagnosing intramammary infections caused by Staphylococcus aureus in dairy cows at dry off

PubMed Central

2012-01-01

Background Staphylococcus aureus is one of the most common causes of intramammary infections in dairy cows at dry off. Reliable identification is important for disease management on herd level and for antimicrobial treatment of infected animals. Our objective was to evaluate the test characteristics of PathoProof ™ Mastitis PCR Assay and bacteriological culture (BC) in diagnosing bovine intramammary infections caused by S. aureus at dry off at different PCR cycle threshold (Ct)-value cut-offs. Methods Sterile quarter samples and non-sterile composite samples from 140 animals in seven herds were collected in connection with the dairy herd improvement (DHI) milk recording. All quarter samples were analyzed using BC whereas all composite samples were analyzed with PathoProof ™ Mastitis PCR Assay. Latent class analysis was used to estimate test properties for PCR and BC in the absence of a perfect reference test. The population was divided into two geographically divided subpopulations and the Hui-Walter 2-test 2-populations model applied to estimate Se, Sp for the two tests, and prevalence for the two subpopulations. Results The Se for PCR increased with increasing Ct-value cut-off, accompanied by a small decrease in Sp. For BC the Se decreased and Sp increased with increasing Ct-value cut-off. Most optimal test estimates for the real-time PCR assay were at a Ct-value cut-off of 37; 0.93 [95% posterior probability interval (PPI) 0.60-0.99] for Se and 0.95 [95% PPI 0.95-0.99] for Sp. At the same Ct-value cut-off, Se and Sp for BC were 0.83 [95% PPI 0.66-0.99] and 0.97 [95% PPI 0.91-0.99] respectively. Depending on the chosen PCR Ct-value cut-off, the prevalence in the subpopulations varied; the prevalence increased with increasing PCR Ct-value cut-offs. Conclusion Neither BC nor real-time PCR is a perfect test in detecting IMI in dairy cows at dry off. The changes in sensitivity and prevalence at different Ct-value cut-offs for both PCR and BC may indicate a change in the underlying disease definition. At low PCR Ct-value cut-offs the underlying disease definition may be a truly/heavily infected cow, whereas at higher PCR Ct-value cut-offs the disease definition may be a S. aureus positive cow. PMID:23164432

Simultaneous detection of Fusarium culmorum and F. graminearum in plant material by duplex PCR with melting curve analysis.

PubMed

Brandfass, Christoph; Karlovsky, Petr

2006-01-23

Fusarium head blight (FHB) is a disease of cereal crops, which has a severe impact on wheat and barley production worldwide. Apart from reducing the yield and impairing grain quality, FHB leads to contamination of grain with toxic secondary metabolites (mycotoxins), which pose a health risk to humans and livestock. The Fusarium species primarily involved in FHB are F. graminearum and F. culmorum. A key prerequisite for a reduction in the incidence of FHB is an understanding of its epidemiology. We describe a duplex-PCR-based method for the simultaneous detection of F. culmorum and F. graminearum in plant material. Species-specific PCR products are identified by melting curve analysis performed in a real-time thermocycler in the presence of the fluorescent dye SYBR Green I. In contrast to multiplex real-time PCR assays, the method does not use doubly labeled hybridization probes. PCR with product differentiation by melting curve analysis offers a cost-effective means of qualitative analysis for the presence of F. culmorum and F. graminearum in plant material. This method is particularly suitable for epidemiological studies involving a large number of samples.

Accuracy of real-time PCR, Gram stain and culture for Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae meningitis diagnosis.

PubMed

Wu, Henry M; Cordeiro, Soraia M; Harcourt, Brian H; Carvalho, Mariadaglorias; Azevedo, Jailton; Oliveira, Tainara Q; Leite, Mariela C; Salgado, Katia; Reis, Mitermayer G; Plikaytis, Brian D; Clark, Thomas A; Mayer, Leonard W; Ko, Albert I; Martin, Stacey W; Reis, Joice N

2013-01-22

Although cerebrospinal fluid (CSF) culture is the diagnostic reference standard for bacterial meningitis, its sensitivity is limited, particularly when antibiotics were previously administered. CSF Gram staining and real-time PCR are theoretically less affected by antibiotics; however, it is difficult to evaluate these tests with an imperfect reference standard. CSF from patients with suspected meningitis from Salvador, Brazil were tested with culture, Gram stain, and real-time PCR using S. pneumoniae, N. meningitidis, and H. influenzae specific primers and probes. An antibiotic detection disk bioassay was used to test for the presence of antibiotic activity in CSF. The diagnostic accuracy of tests were evaluated using multiple methods, including direct evaluation of Gram stain and real-time PCR against CSF culture, evaluation of real-time PCR against a composite reference standard, and latent class analysis modeling to evaluate all three tests simultaneously. Among 451 CSF specimens, 80 (17.7%) had culture isolation of one of the three pathogens (40 S. pneumoniae, 36 N. meningitidis, and 4 H. influenzae), and 113 (25.1%) were real-time PCR positive (51 S. pneumoniae, 57 N. meningitidis, and 5 H. influenzae). Compared to culture, real-time PCR sensitivity and specificity were 95.0% and 90.0%, respectively. In a latent class analysis model, the sensitivity and specificity estimates were: culture, 81.3% and 99.7%; Gram stain, 98.2% and 98.7%; and real-time PCR, 95.7% and 94.3%, respectively. Gram stain and real-time PCR sensitivity did not change significantly when there was antibiotic activity in the CSF. Real-time PCR and Gram stain were highly accurate in diagnosing meningitis caused by S. pneumoniae, N. meningitidis, and H. influenzae, though there were few cases of H. influenzae. Furthermore, real-time PCR and Gram staining were less affected by antibiotic presence and might be useful when antibiotics were previously administered. Gram staining, which is inexpensive and commonly available, should be encouraged in all clinical settings.

Direct urine polymerase chain reaction for chlamydia and gonorrhoea: a simple means of bringing high-throughput rapid testing to remote settings?

PubMed

Rahimi, Frashta; Goire, Namraj; Guy, Rebecca; Kaldor, John M; Ward, James; Nissen, Michael D; Sloots, Theo P; Whiley, David M

2013-08-01

Background Rapid point-of-care tests (POCTs) for chlamydia (Chlamydia trachomatis) and gonorrhoea (Neisseria gonorrhoeae) have the potential to confer health benefits in certain populations even at moderate sensitivities; however, suitable POCTs for these organisms are currently lacking. In this study, we investigated the use of direct urine polymerase chain reaction (PCR), with the view of implementing a simplified PCR strategy for high-throughput chlamydia and gonorrhoea screening in remote settings. Briefly, a simple dilution of the urine was performed before adding it directly to a real-time PCR reaction. The method was evaluated using 134 stored urine specimens that had been submitted for chlamydia and gonorrhoea testing and had been tested using a commercial C. trachomatis and N. gonorrhoeae PCR method. These included samples that were PCR-positive for chlamydia (n=87), gonorrhoea (n=16) or both (n=2). Direct urine testing was conducted using previously described in-house real-time PCR methods for C. trachomatis and N. gonorrhoeae as well as for recognised N.gonorrhoeae antimicrobial resistance mechanisms. The overall sensitivities and specificities of the direct urine PCR were 78% and 100% for chlamydia, and 83% and 100% for gonorrhoea. N.gonorrhoeae penicillin and quinolone resistance mechanisms were characterised in 14 of the 18 N. gonorrhoeae-positive samples. The results of this study show that the simplified PCR strategy may be a feasible approach for rapid screening and improving chlamydia and gonorrhoea treatment in remote settings.

Cross-sectional, descriptive study of Chagas disease among citizens of Bolivian origin living in Munich, Germany.

PubMed

Navarro, Miriam; Berens-Riha, Nicole; Hohnerlein, Stefan; Seiringer, Peter; von Saldern, Charlotte; Garcia, Sarah; Blasco-Hernández, Teresa; Navaza, Bárbara; Shock, Jonathan; Bretzel, Gisela; Hoelscher, Michael; Löscher, Thomas; Albajar-Viñas, Pedro; Pritsch, Michael

2017-01-16

Chagas disease (CD) has become a global health issue mainly due to migration. Germany lacks surveillance data and is home to a large Latin American immigrant population. Recognising that Bolivia is the country with the highest CD prevalence in Latin America, this cross-sectional, descriptive pilot study investigated CD and associated factors among citizens of Bolivian origin living in Munich, Germany. Participants completed a questionnaire in order to collect socioeconomic and health-related data. In addition, serology was performed. In case of positive serological tests, PCR diagnostic and clinical staging together with disease management was initiated. Qualitative research was conducted to identify personal and community barriers as well as strategies to increase CD awareness among the population at risk. Between June 2013 and June 2014, 43 people from Bolivia (or descendants) were enrolled. A total of 9.3% (4/43), of whom two women were of childbearing age, tested seropositive (ELISA and IFAT), and one also by PCR. For 2/4 positive participants, clinical evaluation was performed and the indeterminate form of CD was diagnosed. Knowledge about CD symptoms and ways of transmission were completely absent among 55.8% (24/43, 2/4 with CD) and 30.2% (13/43, 1/4 with CD) of participants, respectively. A total of 27.9% (12/43, 0/4 with CD) of participants had donated blood prior to the study, whereas 62.8% (27/43, 3/4 with CD) were motivated to donate blood in the future. The qualitative research identified lack of knowledge as well as stigma and fears related to CD. Despite the small number of participants, the prevalence of CD as well as the potential risk of non-vectorial transmission was alarming. Campaigns adapted for Latin American migrants as well as control strategies should be developed and put in place in order to prevent non-vectorial transmission and actively detect cases of CD in Germany. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

Monitoring food pathogens: Novel instrumentation for cassette PCR testing

PubMed Central

Hunt, Darin; Figley, Curtis; Lauzon, Jana; Figley, Rachel; Pilarski, Linda M.; McMullen, Lynn M.; Pilarski, Patrick M.

2018-01-01

In this manuscript, we report the design and development of a fast, reliable instrument to run gel-based cassette polymerase chain reactions (PCR). Here termed the GelCycler Mark II, our instrument is a miniaturized molecular testing system that is fast, low cost and sensitive. Cassette PCR utilizes capillary reaction units that carry all reagents needed for PCR, including primers and Taq polymerase, except the sample, which is loaded at the time of testing. Cassette PCR carries out real time quantitative PCR followed by melt curve analysis (MCA) to verify amplicon identity at the expected melt temperature (Tm). The cassette PCR technology is well developed, particularly for detecting pathogens, and has been rigorously validated for detecting pathogenic Escherichia coli in meat samples. However, the work has been hindered by the lack of a robust and stable instrument to carry out the PCR, which requires fast and accurate temperature regulation, improved light delivery and fluorescent recording, and faster PCR reactions that maintain a high sensitivity of detection. Here, we report design and testing of a new instrument to address these shortcomings and to enable standardized testing by cassette PCR and commercial manufacture of a robust and accurate instrument that can be mass produced to deliver consistent performance. As a corollary to our new instrument development, we also report the use of an improved design approach using a machined aluminum cassette to meet the new instrument standards, prevent any light bleed across different trenches in each cassette, and allow testing of a larger number of samples for more targets in a single run. The GelCycler Mark II can detect and report E. coli contamination in 41 minutes. Sample positives are defined in as having a melt curve comparable to the internal positive control, with peak height exceeding that of the internal negative control. In a fractional analysis, as little as 1 bacterium per capillary reaction unit is directly detectable, with no enrichment step, in 35 cycles of PCR/MCA, in a total time of 53 minutes, making this instrument and technology among the very best for speed and sensitivity in screening food for pathogenic contamination. PMID:29746561

Identification of Streptococcus pyogenes - Phenotypic Tests vs Molecular Assay (spy1258PCR): A Comparative Study.

PubMed

Abraham, Tintu; Sistla, Sujatha

2016-07-01

Traditionally Group A Streptococcus pyogenes (GAS) is differentiated from other beta haemolytic streptococci (BHS) by certain presumptive tests such as bacitracin sensitivity and production of Pyrollidonyl Aryl Sulfatase (PYR). The phenotypic and genotypic confirmatory tests are Lancefield grouping for cell wall carbohydrate antigen and PCR for spy1258 gene respectively. Reliance on presumptive tests alone may lead to misidentification of isolates. To compare the predictive values of routine phenotypic tests with spy1258 PCR for the identification of Streptococcus pyogenes. This comparative analytical study was carried out in the Department of Microbiology, JIPMER, Puducherry, over a period of 18 months (1(st) November 2013 to 30(th) April 2015). Two hundred and six consecutive BHS isolates from various clinical samples were subjected to phenotypic tests such as bacitracin sensitivity, PYR test and Lancefield grouping. The results were compared with spy1258 PCR which was considered 95 the confirmatory test for identification. The sensitivity and specificity of phenotypic tests were as follows; Susceptibility to bacitracin - 95.42%, 70.96%, PYR test - 95.42%, 77.41%, Lancefield grouping- 97.71%, 80.64%. Clinical laboratories should not depend on bacitracin sensitivity as a single presumptive test for the routine identification of GAS but should use supplemental tests such as PYR test or latex agglutination test and for best results use spy1258 PCR.

Performance of automated multiplex PCR using sonication fluid for diagnosis of periprosthetic joint infection: a prospective cohort.

PubMed

Renz, Nora; Feihl, Susanne; Cabric, Sabrina; Trampuz, Andrej

2017-12-01

Sonication of explanted prostheses improved the microbiological diagnosis of periprosthetic joint infections (PJI). We evaluated the performance of automated multiplex polymerase chain reaction (PCR) using sonication fluid for the microbiological diagnosis of PJI. In a prospective cohort using uniform definition criteria for PJI, explanted joint prostheses were investigated by sonication and the resulting sonication fluid was analyzed by culture and multiplex PCR. McNemar's Chi-squared test was used to compare the performance of diagnostic tests. Among 111 patients, PJI was diagnosed in 78 (70%) and aseptic failure in 33 (30%). For the diagnosis of PJI, the sensitivity and specificity of periprosthetic tissue culture was 51 and 100%, of sonication fluid culture 58 and 100%, and of sonication fluid PCR 51 and 94%, respectively. Among 70 microorganisms, periprosthetic tissue culture grew 52 (74%), sonication fluid culture grew 50 (71%) and sonication fluid PCR detected 37 pathogens (53%). If only organisms are considered, for which primers are included in the test panel, PCR detected 37 of 58 pathogens (64%). The sonication fluid PCR missed 19 pathogens (predominantly oral streptococci and anaerobes), whereas 7 additional microorganisms were detected only by PCR (including Cutibacterium spp. and coagulase-negative staphylococci). The performance of multiplex PCR using sonication fluid is comparable to culture of periprosthetic tissue or sonication fluid. The advantages of PCR are short processing time (< 5 h) and fully automated procedure. However, culture technique is still needed due to the low sensitivity and the need of comprehensive susceptibility testing. Modification of primers or inclusion of additional ones may improve the performance of PCR, especially of low-virulent organisms.

The detectability half-life in arthropod predator-prey research: what it is, why we need it, how to measure it, and how to use it.

PubMed

Greenstone, Matthew H; Payton, Mark E; Weber, Donald C; Simmons, Alvin M

2014-08-01

Molecular gut-content analysis enables detection of arthropod predation with minimal disruption of ecosystem processes. Most assays produce only qualitative results, with each predator testing either positive or negative for target prey remains. Nevertheless, they have yielded important insights into community processes. For example, they have confirmed the long-hypothesized role of generalist predators in retarding early-season build-up of pest populations prior to the arrival of more specialized predators and parasitoids and documented the ubiquity of secondary and intraguild predation. However, raw qualitative gut-content data cannot be used to assess the relative impact of different predator taxa on prey population dynamics: they must first be weighted by the relative detectability periods for molecular prey remains for each predator-prey combination. If this is not carried out, interpretations of predator impact will be biased towards those with the longest detectabilities. We review the challenges in determining detectability half-lives, including unstated assumptions that have often been ignored in the performance of feeding trials. We also show how detectability half-lives can be used to properly weight assay data to rank predators by their importance in prey population suppression, and how sets of half-lives can be used to test hypotheses concerning predator ecology and physiology. We use data from 32 publications, comprising 97 half-lives, to generate and test hypotheses on taxonomic differences in detectability half-lives and discuss the possible role of the detectability half-life in interpreting qPCR and next-generation sequencing data. © 2013 John Wiley & Sons Ltd.

Impact of rapid methicillin-resistant Staphylococcus aureus polymerase chain reaction testing on mortality and cost effectiveness in hospitalized patients with bacteraemia: a decision model.

PubMed

Brown, Jack; Paladino, Joseph A

2010-01-01

Patients hospitalized with Staphylococcus aureus bacteraemia have an unacceptably high mortality rate. Literature available to date has shown that timely selection of the most appropriate antibacterial may reduce mortality. One tool that may help with this selection is a polymerase chain reaction (PCR) assay that distinguishes methicillin (meticillin)-resistant S. aureus (MRSA) from methicillin-susceptible S. aureus (MSSA) in less than 1 hour. To date, no information is available evaluating the impact of this PCR technique on clinical or economic outcomes. To evaluate the effect of a rapid PCR assay on mortality and economics compared with traditional empiric therapy, using a literature-derived model. A literature search for peer-reviewed European (EU) and US publications regarding treatment regimens, outcomes and costs was conducted. Information detailing the rates of infection, as well as the specificity and sensitivity of a rapid PCR assay (Xpert MRSA/SA Blood Culture PCR) were obtained from the peer-reviewed literature. Sensitivity analysis varied the prevalence rate of MRSA from 5% to 80%, while threshold analysis was applied to the cost of the PCR test. Hospital and testing resource consumption were valued with direct medical costs, adjusted to year 2009 values. Adjusted life-years were determined using US and WHO life tables. The cost-effectiveness ratio was defined as the cost per life-year saved. Incremental cost-effectiveness ratios (ICERs) were calculated to determine the additional cost necessary to produce additional effectiveness. All analyses were performed using TreeAge Software (2008). The mean mortality rates were 23% for patients receiving empiric vancomycin subsequently switched to semi-synthetic penicillin (SSP) for MSSA, 36% for patients receiving empiric vancomycin treatment for MRSA, 59% for patients receiving empiric SSP subsequently switched to vancomycin for MRSA and 12% for patients receiving empiric SSP for MSSA. Furthermore, with an MRSA prevalence of 30%, the numbers of patients needed to test in order to save one life were 14 and 16 compared with empiric vancomycin and SSP, respectively. The absolute mortality difference for MRSA prevalence rates of 80% and 5% favoured the PCR testing group at 2% and 10%, respectively, compared with empiric vancomycin and 18% and 1%, respectively, compared with empiric SSP. In the EU, the cost-effectiveness ratios for empiric vancomycin- and SSP-treated patients were Euro 695 and Euro 687 per life-year saved, respectively, compared with Euro 636 per life-year saved for rapid PCR testing. In the US, the cost-effectiveness ratio was $US 898 per life-year saved for empiric vancomycin and $US 820 per life-year saved for rapid PCR testing. ICERs demonstrated dominance of the PCR test in all instances. Threshold analysis revealed that PCR testing would be less costly overall, even at greatly inflated assay prices. Rapid PCR testing for MRSA appears to have the potential to reduce mortality rates while being less costly than empiric therapy in the EU and US, across a wide range of MRSA prevalence rates and PCR test costs.

qPCR-High resolution melt analysis for drug susceptibility testing of Mycobacterium leprae directly from clinical specimens of leprosy patients.

PubMed

Araujo, Sergio; Goulart, Luiz Ricardo; Truman, Richard W; Goulart, Isabela Maria B; Vissa, Varalakshmi; Li, Wei; Matsuoka, Masanori; Suffys, Philip; Fontes, Amanda B; Rosa, Patricia S; Scollard, David M; Williams, Diana L

2017-06-01

Real-Time PCR-High Resolution Melting (qPCR-HRM) analysis has been recently described for rapid drug susceptibility testing (DST) of Mycobacterium leprae. The purpose of the current study was to further evaluate the validity, reliability, and accuracy of this assay for M. leprae DST in clinical specimens. The specificity and sensitivity for determining the presence and susceptibility of M. leprae to dapsone based on the folP1 drug resistance determining region (DRDR), rifampin (rpoB DRDR) and ofloxacin (gyrA DRDR) was evaluated using 211 clinical specimens from leprosy patients, including 156 multibacillary (MB) and 55 paucibacillary (PB) cases. When comparing the results of qPCR-HRM DST and PCR/direct DNA sequencing, 100% concordance was obtained. The effects of in-house phenol/chloroform extraction versus column-based DNA purification protocols, and that of storage and fixation protocols of specimens for qPCR-HRM DST, were also evaluated. qPCR-HRM results for all DRDR gene assays (folP1, rpoB, and gyrA) were obtained from both MB (154/156; 98.7%) and PB (35/55; 63.3%) patients. All PCR negative specimens were from patients with low numbers of bacilli enumerated by an M. leprae-specific qPCR. We observed that frozen and formalin-fixed paraffin embedded (FFPE) tissues or archival Fite's stained slides were suitable for HRM analysis. Among 20 mycobacterial and other skin bacterial species tested, only M. lepromatosis, highly related to M. leprae, generated amplicons in the qPCR-HRM DST assay for folP1 and rpoB DRDR targets. Both DNA purification protocols tested were efficient in recovering DNA suitable for HRM analysis. However, 3% of clinical specimens purified using the phenol/chloroform DNA purification protocol gave false drug resistant data. DNA obtained from freshly frozen (n = 172), formalin-fixed paraffin embedded (FFPE) tissues (n = 36) or archival Fite's stained slides (n = 3) were suitable for qPCR-HRM DST analysis. The HRM-based assay was also able to identify mixed infections of susceptible and resistant M. leprae. However, to avoid false positives we recommend that clinical specimens be tested for the presence of the M. leprae using the qPCR-RLEP assay prior to being tested in the qPCR-HRM DST and that all specimens demonstrating drug resistant profiles in this assay be subjected to DNA sequencing. Taken together these results further demonstrate the utility of qPCR-HRM DST as an inexpensive screening tool for large-scale drug resistance surveillance in leprosy.

A proof of concept study to assess the potential of PCR testing to detect natural Mycobacterium bovis infection in South American camelids

PubMed Central

2014-01-01

Background Cases of Mycobacterium bovis infection South American camelids have been increasing in Great Britain. Current antemortem immunological tests have some limitations. Cases at post mortem examination frequently show extensive pathology. The feasibility of detecting Mycobacterium bovis DNA in clinical samples was investigated. Findings A sensitive extraction methodology was developed and used on nasal swabs and faeces taken post-mortem to assess the potential for a PCR test to detect Mycobacterium bovis in clinical samples. The gross pathology of the studied South American camelids was scored and a significantly greater proportion of South American camelids with more severe pathology were positive in both the nasal swab and faecal PCR tests. A combination of the nasal swab and faecal PCR tests detected 63.9% of all the South American camelids with pathology that were tested. Conclusions The results suggest that antemortem diagnosis of Mycobacterium bovis in South American camelids may be possible using a PCR test on clinical samples, however more work is required to determine sensitivity and specificity, and the practicalities of applying the test in the field. PMID:24507471

Comparison of Test Results for Zika Virus RNA in Urine, Serum, and Saliva Specimens from Persons with Travel-Associated Zika Virus Disease - Florida, 2016.

PubMed

Bingham, Andrea M; Cone, Marshall; Mock, Valerie; Heberlein-Larson, Lea; Stanek, Danielle; Blackmore, Carina; Likos, Anna

2016-05-13

In May 2015, Zika virus was reported to be circulating in Brazil. This was the first identified introduction of the virus in the Region of the Americas. Since that time, Zika virus has rapidly spread throughout the region. As of April 20, 2016, the Florida Department of Health Bureau of Public Health Laboratories (BPHL) has tested specimens from 913 persons who met state criteria for Zika virus testing. Among these 913 persons, 91 met confirmed or probable Zika virus disease case criteria and all cases were travel-associated (1). On the basis of previous small case studies reporting real time reverse-transcription polymerase chain reaction (RT-PCR) detection of Zika virus RNA in urine, saliva, and semen (2-6), the Florida Department of Health collected multiple specimen types from persons with suspected Zika virus disease. Test results were evaluated by specimen type and number of days after symptom onset to determine the most sensitive and efficient testing algorithm for acute Zika virus disease. Urine specimens were collected from 70 patients with suspected Zika virus disease from zero to 20 days after symptom onset. Of these, 65 (93%) tested positive for Zika virus RNA by RT-PCR. Results for 95% (52/55) of urine specimens collected from persons within 5 days of symptom onset tested positive by RT-PCR; only 56% (31/55) of serum specimens collected on the same date tested positive by RT-PCR. Results for 82% (9/11) of urine specimens collected >5 days after symptom onset tested positive by RT-PCR; none of the RT-PCR tests for serum specimens were positive. No cases had results that were exclusively positive by RT-PCR testing of saliva. BPHL testing results suggest urine might be the preferred specimen type to identify acute Zika virus disease.

Usefulness of Two Aspergillus PCR Assays and Aspergillus Galactomannan and β-d-Glucan Testing of Bronchoalveolar Lavage Fluid for Diagnosis of Chronic Pulmonary Aspergillosis.

PubMed

Urabe, Naohisa; Sakamoto, Susumu; Sano, Go; Suzuki, Junko; Hebisawa, Akira; Nakamura, Yasuhiko; Koyama, Kazuya; Ishii, Yoshikazu; Tateda, Kazuhiro; Homma, Sakae

2017-06-01

We evaluated the usefulness of an Aspergillus galactomannan (GM) test, a β-d-glucan (βDG) test, and two different Aspergillus PCR assays of bronchoalveolar lavage fluid (BALF) samples for the diagnosis of chronic pulmonary aspergillosis (CPA). BALF samples from 30 patients with and 120 patients without CPA were collected. We calculated the sensitivity, specificity, positive predictive value, negative predictive value, positive likelihood ratio, negative likelihood ratio, and diagnostic odds ratio for each test individually and in combination with other tests. The optical density index values, as determined by receiver operating characteristic analysis, for the diagnosis of CPA were 0.5 and 100 for GM and βDG testing of BALF, respectively. The sensitivity and specificity of the GM test, βDG test, and PCR assays 1 and 2 were 77.8% and 90.0%, 77.8% and 72.5%, 86.7% and 84.2%, and 66.7% and 94.2%, respectively. A comparison of the PCR assays showed that PCR assay 1 had a better sensitivity, a better negative predictive value, and a better negative likelihood ratio and PCR assay 2 had a better specificity, a better positive predictive value, and a better positive likelihood ratio. The combination of the GM and βDG tests had the highest diagnostic odds ratio. The combination of the GM and βDG tests on BALF was more useful than any single test for diagnosing CPA. Copyright © 2017 American Society for Microbiology.

Soft Fruit Traceability in Food Matrices using Real-Time PCR

PubMed Central

Palmieri, Luisa; Bozza, Elisa; Giongo, Lara

2009-01-01

Food product authentication provides a means of monitoring and identifying products for consumer protection and regulatory compliance. There is a scarcity of analytical methods for confirming the identity of fruit pulp in products containing Soft Fruit. In the present work we have developed a very sensible qualitative and quantitative method to determine the presence of berry DNAs in different food matrices. To our knowledge, this is the first study that shows the applicability, to Soft Fruit traceability, of melting curve analysis and multiplexed fluorescent probes, in a Real-Time PCR platform. This methodology aims to protect the consumer from label misrepresentation. PMID:22253987

Establishment of a nested-ASP-PCR method to determine the clarithromycin resistance of Helicobacter pylori.

PubMed

Luo, Xiao-Feng; Jiao, Jian-Hua; Zhang, Wen-Yue; Pu, Han-Ming; Qu, Bao-Jin; Yang, Bing-Ya; Hou, Min; Ji, Min-Jun

2016-07-07

To investigate clarithromycin resistance positions 2142, 2143 and 2144 of the 23SrRNA gene in Helicobacter pylori (H. pylori) by nested-allele specific primer-polymerase chain reaction (nested-ASP-PCR). The gastric tissue and saliva samples from 99 patients with positive results of the rapid urease test (RUT) were collected. The nested-ASP-PCR method was carried out with the external primers and inner allele-specific primers corresponding to the reference strain and clinical strains. Thirty gastric tissue and saliva samples were tested to determine the sensitivity of nested-ASP-PCR and ASP-PCR methods. Then, clarithromycin resistance was detected for 99 clinical samples by using different methods, including nested-ASP-PCR, bacterial culture and disk diffusion. The nested-ASP-PCR method was successfully established to test the resistance mutation points 2142, 2143 and 2144 of the 23SrRNA gene of H. pylori. Among 30 samples of gastric tissue and saliva, the H. pylori detection rate of nested-ASP-PCR was 90% and 83.33%, while the detection rate of ASP-PCR was just 63% and 56.67%. Especially in the saliva samples, nested-ASP-PCR showed much higher sensitivity in H. pylori detection and resistance mutation rates than ASP-PCR. In the 99 RUT-positive gastric tissue and saliva samples, the H. pylori-positive detection rate by nested-ASP-PCR was 87 (87.88%) and 67 (67.68%), in which there were 30 wild-type and 57 mutated strains in gastric tissue and 22 wild-type and 45 mutated strains in saliva. Genotype analysis showed that three-points mixed mutations were quite common, but different resistant strains were present in gastric mucosa and saliva. Compared to the high sensitivity shown by nested-ASP-PCR, the positive detection of bacterial culture with gastric tissue samples was 50 cases, in which only 26 drug-resistant strains were found through analyzing minimum inhibitory zone of clarithromycin. The nested-ASP-PCR assay showed higher detection sensitivity than ASP-PCR and drug sensitivity testing, which could be performed to evaluate clarithromycin resistance of H. pylori.

Establishment of a nested-ASP-PCR method to determine the clarithromycin resistance of Helicobacter pylori

PubMed Central

Luo, Xiao-Feng; Jiao, Jian-Hua; Zhang, Wen-Yue; Pu, Han-Ming; Qu, Bao-Jin; Yang, Bing-Ya; Hou, Min; Ji, Min-Jun

2016-01-01

AIM: To investigate clarithromycin resistance positions 2142, 2143 and 2144 of the 23SrRNA gene in Helicobacter pylori (H. pylori) by nested-allele specific primer-polymerase chain reaction (nested-ASP-PCR). METHODS: The gastric tissue and saliva samples from 99 patients with positive results of the rapid urease test (RUT) were collected. The nested-ASP-PCR method was carried out with the external primers and inner allele-specific primers corresponding to the reference strain and clinical strains. Thirty gastric tissue and saliva samples were tested to determine the sensitivity of nested-ASP-PCR and ASP-PCR methods. Then, clarithromycin resistance was detected for 99 clinical samples by using different methods, including nested-ASP-PCR, bacterial culture and disk diffusion. RESULTS: The nested-ASP-PCR method was successfully established to test the resistance mutation points 2142, 2143 and 2144 of the 23SrRNA gene of H. pylori. Among 30 samples of gastric tissue and saliva, the H. pylori detection rate of nested-ASP-PCR was 90% and 83.33%, while the detection rate of ASP-PCR was just 63% and 56.67%. Especially in the saliva samples, nested-ASP-PCR showed much higher sensitivity in H. pylori detection and resistance mutation rates than ASP-PCR. In the 99 RUT-positive gastric tissue and saliva samples, the H. pylori-positive detection rate by nested-ASP-PCR was 87 (87.88%) and 67 (67.68%), in which there were 30 wild-type and 57 mutated strains in gastric tissue and 22 wild-type and 45 mutated strains in saliva. Genotype analysis showed that three-points mixed mutations were quite common, but different resistant strains were present in gastric mucosa and saliva. Compared to the high sensitivity shown by nested-ASP-PCR, the positive detection of bacterial culture with gastric tissue samples was 50 cases, in which only 26 drug-resistant strains were found through analyzing minimum inhibitory zone of clarithromycin. CONCLUSION: The nested-ASP-PCR assay showed higher detection sensitivity than ASP-PCR and drug sensitivity testing, which could be performed to evaluate clarithromycin resistance of H. pylori. PMID:27433095

Detection of Genetically Modified Maize in Processed Foods Sold Commercially in Iran by Qualitative PCR

PubMed Central

Rabiei, Maryam; Mehdizadeh, Mehrangiz; Rastegar, Hossein; Vahidi, Hossein; Alebouyeh, Mahmoud

2013-01-01

Detection of genetically modified organisms (GMOs) in food is an important issue for all the subjects involved in food control and customer’s right. Due to the increasing number of GMOs imported to Iran during the past few years, it has become necessary to screen the products in order to determine the identity of the consumed daily foodstuffs. In this study, following the extraction of genomic DNA from processed foods sold commercially in Iran, qualitative PCR was performed to detect genetically modified maize. The recombinant DNA target sequences were detected with primers highly specific for each investigated transgene such as CaMV35s gene, Bt-11, MON810 and Bt-176 separately. Based on the gel electrophoresis results, Bt- 11 and MON810 events were detected in some maize samples, while, in none of them Bt- 176 modified gene was detected. For the first time, the results demonstrate the presence of genetically modified maize in Iranian food products, reinforcing the need for the development of labeling system and valid quantitative methods in routine analyses. PMID:24250568

Detection of genetically modified maize in processed foods sold commercially in iran by qualitative PCR.

PubMed

Rabiei, Maryam; Mehdizadeh, Mehrangiz; Rastegar, Hossein; Vahidi, Hossein; Alebouyeh, Mahmoud

2013-01-01

Detection of genetically modified organisms (GMOs) in food is an important issue for all the subjects involved in food control and customer's right. Due to the increasing number of GMOs imported to Iran during the past few years, it has become necessary to screen the products in order to determine the identity of the consumed daily foodstuffs. In this study, following the extraction of genomic DNA from processed foods sold commercially in Iran, qualitative PCR was performed to detect genetically modified maize. The recombinant DNA target sequences were detected with primers highly specific for each investigated transgene such as CaMV35s gene, Bt-11, MON810 and Bt-176 separately. Based on the gel electrophoresis results, Bt- 11 and MON810 events were detected in some maize samples, while, in none of them Bt- 176 modified gene was detected. For the first time, the results demonstrate the presence of genetically modified maize in Iranian food products, reinforcing the need for the development of labeling system and valid quantitative methods in routine analyses.

Factors influencing polymerase chain reaction outcomes in patients with clinically suspected ocular tuberculosis.

PubMed

Balne, Praveen Kumar; Modi, Rohit Ramesh; Choudhury, Nuzhat; Mohan, Neha; Barik, Manas Ranjan; Padhi, Tapas Ranjan; Sharma, Savitri; Panigrahi, Satya Ranjan; Basu, Soumyava

2014-03-25

Polymerase chain reaction (PCR) assay can be a useful method for definitive diagnosis in paucibacillary infections such as ocular tuberculosis (TB). In this study, we have evaluated factors affecting PCR outcomes in patients with clinically suspected ocular TB. Patients with clinically suspected ocular TB were investigated by PCR of aqueous or vitreous samples. Three control groups were also tested: group 1 included culture-proven non-tuberculous endophthalmitis, group 2 culture-negative non-tuberculous endophthalmitis, and group 3 patients undergoing surgery for uncomplicated cataract. PCR targeted one or more of following targets: IS6110, MPB64, and protein b genes of Mycobacterium tuberculosis complex. Multiple regression analysis (5% level of significance) was done to evaluate the associations between positive PCR outcome and laterality of disease, tuberculin skin test (TST)/interferon-gamma release assay (IGRA), chest radiography, and type of sample (aqueous or vitreous). The main outcome measures were positive PCR by one or more gene targets, and factors influencing positive PCR outcomes. All 114 samples were tested for MPB64, 110 for protein b, and 88 for IS6110. MPB64 was positive in 70.2% (n = 80) of tested samples, protein b in 40.0% (n = 44), and IS6110 in only 9.1% (n = 8). DNA sequencing of amplicons from four randomly chosen PCR reactions showed homology for M. tuberculosis complex. Of the 80 PCR-positive patients, 71 completed a full course of antitubercular therapy, of which 65 patients (91.5%) had complete resolution of inflammation at final follow-up. Among controls, 12.5% (3 out of 24) in group 1 and 18.7% (6 out of 32) in group 2 also tested positive by PCR. No PCR-positive outcome was observed in control group 3 (n = 25). Multiple regression analysis revealed significant association of positive PCR outcome with bilateral presentation, but not with a positive TST/IGRA, chest radiography, or type of sample (aqueous/vitreous) used. Careful selection of gene targets can yield high PCR positivity in clinically suspected ocular TB. Bilateral disease presentation but not any evidence of latent systemic TB influences PCR outcomes. False-positive results may be seen in ocular inflammation unrelated to ocular TB.

PCR examination of bronchoalveolar lavage samples is a useful tool in pre-clinical diagnosis of ovine pulmonary adenocarcinoma (Jaagsiekte).

PubMed

Voigt, K; Brügmann, M; Huber, K; Dewar, P; Cousens, C; Hall, M; Sharp, J M; Ganter, M

2007-12-01

Ovine pulmonary adenocarcinoma (OPA) is a contagious lung tumour of sheep caused by Jaagsiekte sheep retrovirus (JSRV). The disease is a particular problem in flocks in many parts of the world. The aim of the study was to assess screening methods for individual animals as a prelude to future eradication trials. Results of histological examination were used as the standard to evaluate the relative sensitivity and specificity of an established heminested polymerase chain reaction (PCR) test for JSRV proviral DNA from blood and bronchoalveolar lavage (BAL) samples. PCR results from tissue samples are included as control data. PCR testing of blood samples was found to have an estimated sensitivity of only 10% (95% confidence interval (CI) 3-20) while the sensitivity of the PCR test on BAL samples was 89% (CI 79-96) in comparison to the results of histological examination. We conclude that PCR testing of BAL samples is an effective confirmatory test for sheep with suspected clinical OPA. It is also a useful tool for the pre-clinical identification of individual infected sheep within an infected flock and therefore may prove beneficial in future control or eradication programmes.

Prevalence of Trichomonas vaginalis in Women Visiting 2 Obstetrics and Gynecology Clinics in Daegu, South Korea.

PubMed

Goo, Youn-Kyoung; Shin, Won-Sik; Yang, Hye-Won; Joo, So-Young; Song, Su-Min; Ryu, Jae-Sook; Lee, Won-Myung; Kong, Hyun-Hee; Lee, Won-Ki; Lee, Sang-Eun; Lee, Won-Ja; Chung, Dong-Il; Hong, Yeonchul

2016-02-01

This study explored epidemiological trends in trichomoniasis in Daegu, South Korea. Wet mount microscopy, PCR, and multiplex PCR were used to test for Trichomonas vaginalis in vaginal swab samples obtained from 621 women visiting 2 clinics in Daegu. Of the 621 women tested, microscopy detected T. vaginalis in 4 (0.6%) patients, PCR detected T. vaginalis in 19 (3.0%) patients, and multiplex PCR detected T. vaginalis in 12 (1.9%) patients. Testing via PCR demonstrated high sensitivity and high negative predictive value for T. vaginalis. Among the 19 women who tested positive for T. vaginalis according to PCR, 94.7% (18/19) reported vaginal signs and symptoms. Notably, more than 50% of T. vaginalis infections occurred in females younger than 30 years old, and 58% were unmarried. Multiplex PCR, which simultaneously detects pathogens from various sexually transmitted infections, revealed that 91.7% (11/12) of patients were infected with 2 or more pathogens. Mycoplasma hominis was the most prevalent co-infection pathogen with T. vaginalis, followed by Ureaplasma urealyticum and Chlamydia trachomatis. Our results indicate that PCR and multiplex PCR are the most sensitive tools for T. vaginalis diagnosis, rather than microscopy which has been routinely used to detect T. vaginalis infections in South Korea. Therefore, clinicians should take note of the high prevalence of T. vaginalis infections among adolescent and young women in order to prevent persistent infection and transmission of this disease.

Pneumocystis PCR: It Is Time to Make PCR the Test of Choice.

PubMed

Doyle, Laura; Vogel, Sherilynn; Procop, Gary W

2017-01-01

The testing strategy for Pneumocystis at the Cleveland Clinic changed from toluidine blue staining to polymerase chain reaction (PCR). We studied the differences in positivity rates for these assays and compared each with the detection of Pneumocystis in companion specimens by cytology and surgical pathology. We reviewed the results of all Pneumocystis test orders 1 year before and 1 year after the implementation of a Pneumocystis -specific PCR. We also reviewed the corresponding cytology and surgical pathology results, if performed. Finally, we reviewed the medical records of patients with rare Pneumocystis detected by PCR in an effort to differentiate colonization vs true disease. Toluidine blue staining and surgical pathology had similar sensitivities and negative predictive values, both of which were superior to cytology. There was a >4-fold increase in the annual detection of Pneumocystis by PCR compared with toluidine blue staining (toluidine blue staining: 11/1583 [0.69%] vs PCR: 44/1457 [3.0%]; chi-square P < .001). PCR detected 1 more case than surgical pathology and was far more sensitive than cytology. Chart review demonstrated that the vast majority of patients with rare Pneumocystis detected were immunosuppressed, had radiologic findings supportive of this infection, had no other pathogens detected, and were treated for pneumocystosis by the clinical team. PCR was the most sensitive method for the detection of Pneumocystis and should be considered the diagnostic test of choice. Correlation with clinical and radiologic findings affords discrimination of early true disease from the far rarer instances of colonization.

Sources of psychrophilic and psychrotolerant clostridia causing spoilage of vacuum-packed chilled meats, as determined by PCR amplification procedure.

PubMed

Broda, D M; Boerema, J A; Brightwell, G

2009-07-01

To determine possible preslaughter and processing sources of psychrophilic and psychrotolerant clostridia causing spoilage of vacuum-packed chilled meats. Molecular methods based on the polymerase chain reaction (PCR) amplification of specific 16S rDNA fragments were used to detect the presence of Clostridium gasigenes, Clostridium estertheticum, Clostridium algidicarnis and Clostridium putrefaciens in a total of 357 samples collected from ten slaughter stock supply farms, slaughter stock, two lamb-processing plants, their environments, dressed carcasses and final vacuum-packed meat stored at -0.5 degrees C for 5(1/2) weeks. Clostridium gasigenes, C. estertheticum and C. algidicarnis/C. putrefaciens were commonly detected in farm, faeces, fleece and processing environmental samples collected at the slaughter floor operations prior to fleece removal, but all these micro-organisms were detected in only 4 out of 26 cooling floor and chiller environmental samples. One out of 42 boning room environmental samples tested positive for the presence of C. gasigenes and C. estertheticum, but 25 out of 42 of these samples were positive for C. algidicarnis/C. putrefaciens. Nearly all of the 31 faecal samples tested positive for the presence of C. gasigenes and C. estertheticum; however, only two of these samples were positive for C. algidicarnis and/or C. putrefaciens. Clostridial species that were subject to this investigation were frequently detected on chilled dressed carcasses. The major qualitative and quantitative differences between the results of PCR detection obtained with the primers specific for 'blown pack' -causing clostridia (C. gasigenes and C. estertheticum) and those obtained with primers specific for C. algidicarnis and C. putrefaciens suggest that the control of meat spoilage caused by different groups of meat clostridia is best approached individually for each group. This paper provides information significant for controlling meat spoilage-causing clostridia in the meat-processing plants.

Development and Evaluation of a Rapid and Sensitive EBOV-RPA Test for Rapid Diagnosis of Ebola Virus Disease.

PubMed

Yang, Mingjuan; Ke, Yuehua; Wang, Xuesong; Ren, Hang; Liu, Wei; Lu, Huijun; Zhang, Wenyi; Liu, Shiwei; Chang, Guohui; Tian, Shuguang; Wang, Lihua; Huang, Liuyu; Liu, Chao; Yang, Ruifu; Chen, Zeliang

2016-06-01

Confirming Ebola virus disease (EVD), a deadly infectious disease, requires real-time RT-PCR, which takes up to a few hours to yield results. Therefore, a rapid diagnostic assay is imperative for EVD diagnosis. A rapid nucleic acid test based on recombinase polymerase amplification (EBOV-RPA) was developed to specifically detect the 2014 outbreak strains. The EBOV-RPA assay was evaluated by testing samples from suspected EVD patients in parallel with RT-PCR. An EBOV-RPA, which could be completed in 20 min, was successfully developed. Of 271 patients who tested positive for Ebola virus by RT-PCR, 264 (sensitivity: 97%, 95% CI: 95.5-99.3%) were positive by EBOV-RPA; 101 of 104 patients (specificity: 97%, 95% CI: 93.9-100%) who tested negative by RT-PCR were also negative by EBOV-RPA. The sensitivity values for samples with a Ct value of <34, which accounted for 95.59% of the samples, was 100%. Discordant samples positive by RT-PCR but negative by EBOV-RPA had significantly high Ct values. Results of external quality assessment samples with EBOV-RPA were 100%, consistent with those of RT-PCR. The EBOV-RPA assay showed 97% sensitivity and 97% specificity for all EVD samples tested, making it a rapid and sensitive test for EVD diagnosis.

A multiplex PCR method for detection of Aspergillus spp. and Mycobacterium tuberculosis in BAL specimens.

PubMed

Amini, F; Kachuei, R; Noorbakhsh, F; Imani Fooladi, A A

2015-06-01

The aim of this study was the detection of Aspergillus species and Mycobacterium tuberculosis together in bronchoalveolar lavage (BAL) using of multiplex PCR. In this study, from September 2012 until June 2013, 100 bronchoalveolar lavage (BAL) specimens were collected from patients suspected of tuberculosis (TB). After the direct and culture test, multiplex PCR were utilized in order to diagnose Aspergillus species and M. tuberculosis. Phenol-chloroform manual method was used in order to extract DNA from these microorganisms. Aspergillus specific primers, M. tuberculosis designed primers and beta actin primers were used for multiplex PCR. In this study, by multiplex PCR method, Aspergillus species were identified in 12 samples (12%), positive samples in direct and culture test were respectively 11% and 10%. Sensitivity and specificity of this method in comparison to direct test were respectively 100% and 98.8%, also sensitivity and specificity of this method in comparison to culture test were respectively 100% and 97.7%. In this assay, M. tuberculosis was identified in 8 samples (8%). Mycobacterium-positive samples in molecular method, direct and culture test were respectively 6%, 5% and 7%. Sensitivity and specificity of PCR method in comparison to direct test were 80% and 97.8% also sensitivity and specificity of this method in comparison to culture test was 71.4% and 98.9%. In the present study, multiplex PCR method had higher sensitivity than direct and culture test in order to identify and detect Aspergillus, also this method had lower sensitivity for identification of M. tuberculosis, suggesting that the method of DNA extraction was not suitable. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Toxoplasma gondii infections in chickens - performance of various antibody detection techniques in serum and meat juice relative to bioassay and DNA detection methods.

PubMed

Schares, G; Koethe, M; Bangoura, B; Geuthner, A-C; Randau, F; Ludewig, M; Maksimov, P; Sens, M; Bärwald, A; Conraths, F J; Villena, I; Aubert, D; Opsteegh, M; Van der Giessen, J

2018-05-19

Chickens, especially if free-range, are frequently exposed to Toxoplasma gondii, and may represent an important reservoir for T. gondii. Poultry products may pose a risk to humans, when consumed undercooked. In addition, chickens are regarded as sensitive indicators for environmental contamination with T. gondii oocysts and have been used as sentinels. The aim of the present study was to determine the suitability of commonly used antibody detection methods, i.e. the modified agglutination test (MAT), IFAT and ELISA to detect T. gondii-infected chickens. Samples of experimentally and naturally infected chickens were used. The infection state of all chickens was determined by Magnetic-Capture (MC-) real-time PCR (RT PCR). Naturally exposed chickens were additionally examined by mouse bioassay and conventional RT PCR on acidic pepsin digests (PD-RT PCR). Blood serum and meat juice of various sources were tested for antibodies to T. gondii. In naturally infected chickens, there was substantial agreement between the mouse bioassay and MC-RT PCR or the mouse bioassay and conventional PD-RT PCR. PD-RT PCR was slightly more sensitive than MC-RT PCR, as all (26/26) bioassay-positive chickens also tested positive in at least one of the tissues tested (heart, drumstick). By MC-RT PCR, 92.3% (24/26) of the naturally infected bioassay-positive chickens were positive. The diagnostic sensitivity of MC-RT PCR was clearly related to the organ examined. Based on a quantitative assessment of the MC-RT PCR results in experimentally infected chickens, brain and heart tissues harbored an at least 100 times higher parasite concentration than breast, thigh or drumstick musculature. In naturally infected chickens, only three out of 24 birds, which were MC-RT PCR-positive in heart samples, also tested positive in drumstick musculature. Under experimental conditions, the agreement between MC-RT PCR and the serological techniques revealed 100% diagnostic sensitivity and specificity. Under field conditions, examinations of sera by ELISA, IFAT and MAT showed good performance in identifying chickens that were positive in either a mouse bioassay, MC-RT PCR, or PD-RT PCR as illustrated by diagnostic sensitivities of 87.5%, 87.5% and 65.2%, respectively, and diagnostic specificities of 86.2%, 82.8% and 100%, respectively. The examination of meat juice samples from breast, drumstick or heart musculature revealed similar or even better results in the ELISA. The results in the MAT with meat juice from breast musculature were less consistent than those of ELISA and IFAT because a number of negative chickens tested false-positive in the MAT. The MAT performed similar to ELISA and IFAT when applied to test meat juice samples collected from heart, thigh or drumstick musculature. Copyright © 2018 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.

See what you eat--broad GMO screening with microarrays.

PubMed

von Götz, Franz

2010-03-01

Despite the controversy of whether genetically modified organisms (GMOs) are beneficial or harmful for humans, animals, and/or ecosystems, the number of cultivated GMOs is increasing every year. Many countries and federations have implemented safety and surveillance systems for GMOs. Potent testing technologies need to be developed and implemented to monitor the increasing number of GMOs. First, these GMO tests need to be comprehensive, i.e., should detect all, or at least the most important, GMOs on the market. This type of GMO screening requires a high degree of parallel tests or multiplexing. To date, DNA microarrays have the highest number of multiplexing capabilities when nucleic acids are analyzed. This trend article focuses on the evolution of DNA microarrays for GMO testing. Over the last 7 years, combinations of multiplex PCR detection and microarray detection have been developed to qualitatively assess the presence of GMOs. One example is the commercially available DualChip GMO (Eppendorf, Germany; http://www.eppendorf-biochip.com), which is the only GMO screening system successfully validated in a multicenter study. With use of innovative amplification techniques, promising steps have recently been taken to make GMO detection with microarrays quantitative.

Development and validation of a duplex real-time PCR assay for the diagnosis of equine piroplasmosis.

PubMed

Lobanov, Vladislav A; Peckle, Maristela; Massard, Carlos L; Brad Scandrett, W; Gajadhar, Alvin A

2018-03-02

Equine piroplasmosis (EP) is an economically significant infection of horses and other equine species caused by the tick-borne protozoa Theileria equi and Babesia caballi. The long-term carrier state in infected animals makes importation of such subclinical cases a major risk factor for the introduction of EP into non-enzootic areas. Regulatory testing for EP relies on screening of equines by serological methods. The definitive diagnosis of EP infection in individual animals will benefit from the availability of sensitive direct detection methods, for example, when used as confirmatory assays for non-negative serological test results. The objectives of this study were to develop a real-time quantitative polymerase chain reaction (qPCR) assay for simultaneous detection of both agents of EP, perform comprehensive evaluation of its performance and assess the assay's utility for regulatory testing. We developed a duplex qPCR targeting the ema-1 gene of T. equi and the 18S rRNA gene of B. caballi and demonstrated that the assay has high analytical sensitivities for both piroplasm species. Validation of the duplex qPCR on samples from 362 competitive enzyme-linked immunosorbent assay (cELISA)-negative horses from Canada and the United States yielded no false-positive reactions. The assay's performance was further evaluated using samples collected from 430 horses of unknown EP status from a highly endemic area in Brazil. This set of samples was also tested by a single-target 18S rRNA qPCR for T. equi developed at the OIE reference laboratory for EP in Japan, and a previously published single-target 18S rRNA qPCR for B. caballi whose oligonucleotides we adopted for use in the duplex qPCR. Matching serum samples were tested for antibodies to these parasites using cELISA. By the duplex qPCR, T. equi-specific 18S rRNA qPCR and cELISA, infections with T. equi were detected in 87.9% (95% confidence interval, CI: 84.5-90.7%), 90.5% (95% CI: 87.3-92.3%) and 87.4% (95% CI: 84.0-90.2%) of the horses, respectively. The B. caballi prevalence estimates were 9.3% (95% CI: 6.9-12.4%) by the duplex qPCR and 7.9% (95% CI: 5.7-10.9%) by the respective single-target qPCR assay. These values were markedly lower compared to the seroprevalence of 58.6% (95% CI: 53.9-63.2%) obtained by B. caballi-specific cELISA. The relative diagnostic sensitivity of the duplex qPCR for T. equi was 95.5%, as 359 of the 376 horses with exposure to T. equi confirmed by cELISA had parasitemia levels above the detection limit of the molecular assay. In contrast, only 39 (15.5%) of the 252 horses with detectable B. caballi-specific antibodies were positive for this piroplasm species by the duplex qPCR. The duplex qPCR described here performed comparably to the existing single-target qPCR assays for T. equi and B. caballi and will be more cost-effective in terms of results turnaround time and reagent costs when both pathogens are being targeted for disease control and epidemiological investigations. These validation data also support the reliability of the ema-1 gene-specific oligonucleotides developed in this study for confirmatory testing of non-negative serological test results for T. equi by qPCR. However, the B. caballi-specific qPCR cannot be similarly recommended as a confirmatory assay for routine regulatory testing due to the low level of agreement with serological test results demonstrated in this study. Further studies are needed to determine the transmission risk posed by PCR-negative equines with detectable antibodies to B. caballi.

Diagnostic testing for pandemic influenza in Singapore: a novel dual-gene quantitative real-time RT-PCR for the detection of influenza A/H1N1/2009.

PubMed

Lee, Hong Kai; Lee, Chun Kiat; Loh, Tze Ping; Tang, Julian Wei-Tze; Chiu, Lily; Tambyah, Paul A; Sethi, Sunil K; Koay, Evelyn Siew-Chuan

2010-09-01

With the relative global lack of immunity to the pandemic influenza A/H1N1/2009 virus that emerged in April 2009 as well as the sustained susceptibility to infection, rapid and accurate diagnostic assays are essential to detect this novel influenza A variant. Among the molecular diagnostic methods that have been developed to date, most are in tandem monoplex assays targeting either different regions of a single viral gene segment or different viral gene segments. We describe a dual-gene (duplex) quantitative real-time RT-PCR method selectively targeting pandemic influenza A/H1N1/2009. The assay design includes a primer-probe set specific to only the hemagglutinin (HA) gene of this novel influenza A variant and a second set capable of detecting the nucleoprotein (NP) gene of all swine-origin influenza A virus. In silico analysis of the specific HA oligonucleotide sequence used in the assay showed that it targeted only the swine-origin pandemic strain; there was also no cross-reactivity against a wide spectrum of noninfluenza respiratory viruses. The assay has a diagnostic sensitivity and specificity of 97.7% and 100%, respectively, a lower detection limit of 50 viral gene copies/PCR, and can be adapted to either a qualitative or quantitative mode. It was first applied to 3512 patients with influenza-like illnesses at a tertiary hospital in Singapore, during the containment phase of the pandemic (May to July 2009).

Molecular diagnosis of Legionella infections--Clinical utility of front-line screening as part of a pneumonia diagnostic algorithm.

PubMed

Gadsby, Naomi J; Helgason, Kristjan O; Dickson, Elizabeth M; Mills, Jonathan M; Lindsay, Diane S J; Edwards, Giles F; Hanson, Mary F; Templeton, Kate E

2016-02-01

Urinary antigen testing for Legionella pneumophila serogroup 1 is the leading rapid diagnostic test for Legionnaires' Disease (LD); however other Legionella species and serogroups can also cause LD. The aim was to determine the utility of front-line L. pneumophila and Legionella species PCR in a severe respiratory infection algorithm. L. pneumophila and Legionella species duplex real-time PCR was carried out on 1944 specimens from hospitalised patients over a 4 year period in Edinburgh, UK. L. pneumophila was detected by PCR in 49 (2.7%) specimens from 36 patients. During a LD outbreak, combined L. pneumophila respiratory PCR and urinary antigen testing had optimal sensitivity and specificity (92.6% and 98.3% respectively) for the detection of confirmed cases. Legionella species was detected by PCR in 16 (0.9%) specimens from 10 patients. The 5 confirmed and 1 probable cases of Legionella longbeachae LD were both PCR and antibody positive. Front-line L. pneumophila and Legionella species PCR is a valuable addition to urinary antigen testing as part of a well-defined algorithm. Cases of LD due to L. longbeachae might be considered laboratory-confirmed when there is a positive Legionella species PCR result and detection of L. longbeachae specific antibody response. Copyright © 2015 The British Infection Association. Published by Elsevier Ltd. All rights reserved.

A novel comprehensive set of fungal Real time PCR assays (fuPCR) for the detection of fungi in immunocompromised haematological patients-A pilot study.

PubMed

Rahn, Sebastian; Schuck, Anna; Kondakci, Mustafa; Haas, Rainer; Neuhausen, Nicole; Pfeffer, Klaus; Henrich, Birgit

2016-12-01

Fungal infections are recognized in an increasing number of patients with immunological deficits and are associated with high rates of mortality (Brown et al., 2012a). In this pilot-study, a rapid Real time PCR (fuPCR) was designed for the detection and differentiation of fungal pathogens in clinical specimens of haematological patients. The fuPCR, targeting the internal transcribed spacer region 2 (ITS2) of rDNA region, is comprised of seven multiplex reactions, which were shown to be specific and sensitive for a comprehensive spectrum of clinically relevant fungal species. This was validated by testing respective fungal DNAs in each fuPCR reaction and 28 respiratory samples of fungal pneumonia-proven patients. Clinical sample sets of throat swab, EDTA-blood and blood sera from 50 patients with severe haematological malignancies, including haematopoietic stem cell transfer (HSCT), and samples from 30 healthy individuals were then analysed. In a first step, 198 samples of immunosuppressed patients were solely examined by fuPCR; and 50.8% (33/65) respiratory swabs, 4.8% (3/63) EDTA blood samples and 1.4% (1/70) blood serum samples were tested positive. In a second step, 56 respiratory samples of immunosuppressed patients and 30 of healthy individuals were simultaneously analysed by fuPCR and standard cultivation techniques. By both methods 30.4% (17/56) swabs of the immunocompromised patients were tested positive, 37.5% (21/56) were tested negative and 32.1% (18/56) were tested fuPCR positive and culture negative. In analysing the blood samples of the immunocompromised patients 5.4% (3/56) EDTA blood samples and 16.1% (9/56) sera samples were tested fuPCR-positive, whereas all samples of 30 healthy individuals with no signs of immunological deficits were tested negative by fuPCR. 38.9% (14/36) of the fungi detected in respiratory samples of the immunosuppressed patients, belonged to Candida spp., 47.2% (17/36) to Saccharomyces spp., 5.6% (2/36) to Cladosporium spp. and 8.3% (3/36) to Alternaria spp., whereas cultivation only identified Candida spp. (10/17) and Saccharomyces spp. (7/17). In this pilot study a novel fuPCR assay was developed and validated for the simultaneous and comprehensive detection of fungal pathogens in clinical respiratory specimens of haematological patients. Future work will focus on the validation of the blood-stream detected fungi in pathogenicity of these patients. Copyright © 2016 Elsevier GmbH. All rights reserved.

The intellectual developmental disorders Mexico study: situational diagnosis, burden, genomics and intervention proposal.

PubMed

Lazcano-Ponce, Eduardo; Katz, Gregorio; Rodríguez-Valentín, Rocío; Castro, Filipa de; Allen-Leigh, Betania; Márquez-Caraveo, María Elena; Ramírez-García, Miguel Ángel; Arroyo-García, Eduardo; Medina-Mora, María Elena; Ángeles, Gustavo; Urquieta-Salomón, José Edmundo; Salvador-Carulla, Luis

2016-01-01

This study aims to generate evidence on intellectual development disorders (IDD) in Mexico. IDD disease burden will be estimated with a probabilistic model, using population-based surveys. Direct and indirect costs of catastrophic expenses of families with a member with an IDD will be evaluated. Genomic characterization of IDD will include: sequencing participant exomes and performing bioinformatics analyses to identify de novo or inherited variants through trio analysis; identifying genetic variants associated with IDD, and validating randomly selected variants by polymerase chain reaction (PCR) and sequencing or real-time quantitative PCR (qPCR). Delphi surveys will be done on best practices for IDD diagnosis and management. An external evaluation will employ qualitative case studies of two social and labor inclusion programs for people with IDD. The results will constitute scientific evidence for the design, promotion and evaluation of public policies, which are currently absent on IDD.

Quantification by real-time PCR of Trypanosoma cruzi DNA in samples of Triatoma infestans used in xenodiagnosis of chronic Chagas disease patients.

PubMed

Saavedra, Miguel; Zulantay, Inés; Apt, Werner; Castillo, Juan; Araya, Eduardo; Martínez, Gabriela; Rodríguez, Jorge

2016-07-04

Trypanosoma cruzi multiplies and differentiates in the digestive tract of triatomine insects. Xenodiagnosis (XD) is a parasitological tool in which the insect vectors acts as a biological culture medium to amplify and detect T. cruzi infection in mammals. The sensitivity of XD has been overcome by the application of PCR in fecal samples (FS) of XD (PCR-XD). In this study, T. cruzi amplified in Triatoma infestans fed by XD on individuals with chronic Chagas disease (CChD) is quantified by real-time PCR (qPCR-XD). Under informed consent, 100 individuals were evaluated. In 21 of them XD, PCR-XD and qPCR-XD were positive. For the contrary, 79 were negative XD. In 58 (73.4 %) and 66 cases (83.5 %) of them, PCR-XD (Fisher's exact test P = 0.005) and qPCR-XD (Fisher's exact test: P = 0.037) respectively, were positive. In cases with positive XD, qPCR-XD allowed to establish that in 9/21 cases (42.9 %) the parasite burden fluctuated between 100 and 1,000 par. eq./ml. Otherwise, in 32/79 (40.5 %) cases with negative XD, a parasite burden between 1 and 10 par. eq./ml was determined. All samples showed amplification of exogenous internal control (X12, Ct average: 31.8), so problems in the DNA extraction (excess or loss of genetic material), unspecific amplification and/or inhibition in qPCR-XD reactions were ruled out. Additionally, in all the patients qPCR in blood (qPCR-B) was performed. In the cases with positive XD, the concordance between the positivity of qPCR-XD and qPCR-B was 100 %, nevertheless, the parasite burden in blood was lower and different than XD (Chi-square test: χ (2)  = 91.82, df = 5, P = 0.0001). In the cases with negative XD the ranges of qPCR-XD and qPCR-B were similar (Chi-square test: χ (2)  = 6.71, df = 5, P = 0.1520). This study allowed the detection and quantification of T. cruzi by qPCR-XD in FS of Tr. infestans fed on patients with CChD. The highest parasite burden was observed in positive XD cases. qPCR-XD could be used in different studies related with the complex T. cruzi-vector-host interactions.

Detection of Yersinia Enterocolitica Species in Pig Tonsils and Raw Pork Meat by the Real-Time Pcr and Culture Methods.

PubMed

Stachelska, M A

2017-09-26

The aim of the present study was to establish a rapid and accurate real-time PCR method to detect pathogenic Yersinia enterocolitica in pork. Yersinia enterocolitica is considered to be a crucial zoonosis, which can provoke diseases both in humans and animals. The classical culture methods designated to detect Y. enterocolitica species in food matrices are often very time-consuming. The chromosomal locus _tag CH49_3099 gene, that appears in pathogenic Y. enterocolitica strains, was applied as DNA target for the 5' nuclease PCR protocol. The probe was labelled at the 5' end with the fluorescent reporter dye (FAM) and at the 3' end with the quencher dye (TAMRA). The real-time PCR cycling parameters included 41 cycles. A Ct value which reached a value higher than 40 constituted a negative result. The developed for the needs of this study qualitative real-time PCR method appeared to give very specific and reliable results. The detection rate of locus _tag CH49_3099 - positive Y. enterocolitica in 150 pig tonsils was 85 % and 32 % with PCR and culture methods, respectively. Both the Real-time PCR results and culture method results were obtained from material that was enriched during overnight incubation. The subject of the study were also raw pork meat samples. Among 80 samples examined, 7 ones were positive when real-time PCR was applied, and 6 ones were positive when classical culture method was applied. The application of molecular techniques based on the analysis of DNA sequences such as the Real-time PCR enables to detect this pathogenic bacteria very rapidly and with higher specificity, sensitivity and reliability in comparison to classical culture methods.

Quantification of Campylobacter spp. in pig feces by direct real-time PCR with an internal control of extraction and amplification.

PubMed

Leblanc-Maridor, Mily; Garénaux, Amélie; Beaudeau, François; Chidaine, Bérangère; Seegers, Henri; Denis, Martine; Belloc, Catherine

2011-04-01

The rapid and direct quantification of Campylobacter spp. in complex substrates like feces or environmental samples is crucial to facilitate epidemiological studies on Campylobacter in pig production systems. We developed a real-time PCR assay for detecting and quantifying Campylobacter spp. directly in pig feces with the use of an internal control. Campylobacter spp. and Yersinia ruckeri primers-probes sets were designed and checked for specificity with diverse Campylobacter, related organisms, and other bacterial pathogens before being used in field samples. The quantification of Campylobacter spp. by the real-time PCR then was realized on 531 fecal samples obtained from experimentally and naturally infected pigs; the numeration of Campylobacter on Karmali plate was done in parallel. Yersinia ruckeri, used as bacterial internal control, was added to the samples before DNA extraction to control DNA-extraction and PCR-amplification. The sensitivity of the PCR assay was 10 genome copies. The established Campylobacter real-time PCR assay showed a 7-log-wide linear dynamic range of quantification (R²=0.99) with a detection limit of 200 Colony Forming Units of Campylobacter per gram of feces. A high correlation was found between the results obtained by real-time PCR and those by culture at both qualitative and quantitative levels. Moreover, DNA extraction followed by real-time PCR reduced the time needed for analysis to a few hours (within a working day). In conclusion, the real-time PCR developed in this study provides new tools for further epidemiological surveys to investigate the carriage and excretion of Campylobacter by pigs. Copyright © 2011 Elsevier B.V. All rights reserved.

[Clinical importance of semi-quantitative monitoring of lymphomas using the comparative polymerase chain reaction].

PubMed

Slavícková, A; Ivánek, R; Cerný, J; Sálková, J; Trnĕný, M

2002-11-22

PCR techniques detecting interchromosomal translocation and clonal immunoglobulin gene rearrangement (IgH) as disease markers in non-Hodgkin's lymphomas (NHL) has been utilised past ten years. However, qualitative PCR detection of persisted minimal residual disease cannot provide clinically useful prognostic information and presently, quantitative approaches are required to predict patient outcome and assess response to the treatment. In some cases, "end-point" quantifying techniques, such as comparative PCR, are applicable and the relative estimation of differences in target quantity may serve in disease monitoring rather than absolute number of target copies. Our method of comparative PCR employs co-amplification of sequences of interest (clonal CDR3, bcl2/Jh) and the segment of Hras 1 gene(ras) as an internal standard. Serial dilutions of stored diagnostic DNAs from blood and bone marrow are examined in the same PCR and, after gel densitometry, the amount of initial target is assessed by comparing exponential products of co-amplification. The comparative PCR assay was utilized in monitoring of NHL patients cured either with conventional therapy, or with high-dose regimens and transplantation with stem cells, or with chimaeric anti-CD20 monoclonal antibody (Rituximab). Results from 50 monitored intervals obtained during several months up to several years were supplemented with clinical statements retrospectively. Some of patients became PCR-negative, reappearance of PCR-positivity was observed as well. The decrease or increase of disease marker corresponded to clinical observations. Results obtained from bone marrow were in agreement with those obtained from blood. End-point quantifying PCR comparative assay may provide an information on the increased risk of relapse and impact of the therapy. The predictive value of these methods depends on the frequency of sample taking and on the sensitivity of the method, which should be monitored in negative cases.

Comparison of electron microscopy, ELISA, real time RT-PCR and insulated isothermal RT-PCR for the detection of Rotavirus group A (RVA) in feces of different animal species.

PubMed

Soltan, Mohamed A; Tsai, Yun-Long; Lee, Pei-Yu A; Tsai, Chuan-Fu; Chang, Hsiao-Fen G; Wang, Hwa-Tang T; Wilkes, Rebecca P

2016-09-01

There is no gold standard for detection of Rotavirus Group A (RVA), one of the main causes of diarrhea in neonatal animals. Sensitive and specific real-time RT-PCR (rtRT-PCR) assays are available for RVA but require submission of the clinical samples to diagnostic laboratories. Patient-side immunoassays for RVA protein detection have shown variable results, particularly with samples from unintended species. A sensitive and specific test for detection of RVA on the farm would facilitate rapid management decisions. The insulated isothermal RT-PCR (RT-iiPCR) assay works in a portable machine to allow sensitive and specific on-site testing. The aim of this investigation was to evaluate a commercially available RT-iiPCR assay for RVA detection in feces from different animal species. This assay was compared to an in-house rtRT-PCR assay and a commercially available rtRT-PCR kit, as well as an ELISA and EM for RVA detection. All three PCR assays targeted the well-conserved NSP5 gene. Clinical fecal samples from 108 diarrheic animals (mainly cattle and horses) were tested. The percentage of positive samples by ELISA, EM, in-house rtRT-PCR, commercial rtRT-PCR, and RT-iiPCR was 29.4%, 31%, 36.7%, 51.4%, 56.9%, respectively. The agreement between different assays was high (81.3-100%) in samples containing high viral loads. The sensitivity of the RT-iiPCR assay appeared to be higher than the commercially available rtRT-PCR assay, with a limit of detection (95% confidence index) of 3-4 copies of in vitro transcribed dsRNA. In conclusion, the user-friendly, field-deployable RT-iiPCR system holds substantial promise for on-site detection of RVA. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

The Role of PCR in the Diagnosis of Candida Vulvovaginitis-a New Gold Standard?

PubMed

Sobel, J D; Akins, Robert A

2015-06-01

PCR is recognized as a reliable technique for detection of all types of microorganisms. Being highly objective and reproducible also sensitive and specific, PCR is now widely used for sexually transmitted infection (STI) diagnosis. Potential, however, exists for detecting non-pathogens, and not identifying a pathogenic state decreases specificity or clinical significance. PCR Candida tests of vaginal specimens are now widely available and frequently used offering a modest to moderate increase in sensitivity and are likely to replace traditional culture and DNA homology testing. Nevertheless, there remain considerable gaps in our knowledge regarding the usefulness and applications of these expensive tests.

Development and first evaluation of a novel multiplex real-time PCR on whole blood samples for rapid pathogen identification in critically ill patients with sepsis.

PubMed

van de Groep, Kirsten; Bos, Martine P; Savelkoul, Paul H M; Rubenjan, Anna; Gazenbeek, Christel; Melchers, Willem J G; van der Poll, Tom; Juffermans, Nicole P; Ong, David S Y; Bonten, Marc J M; Cremer, Olaf L

2018-04-26

Molecular tests may enable early adjustment of antimicrobial therapy and be complementary to blood culture (BC) which has imperfect sensitivity in critically ill patients. We evaluated a novel multiplex real-time PCR assay to diagnose bloodstream pathogens directly in whole blood samples (BSI-PCR). BSI-PCR included 11 species- and four genus-specific PCRs, a molecular Gram-stain PCR, and two antibiotic resistance markers. We collected 5 mL blood from critically ill patients simultaneously with clinically indicated BC. Microbial DNA was isolated using the Polaris method followed by automated DNA extraction. Sensitivity and specificity were calculated using BC as reference. BSI-PCR was evaluated in 347 BC-positive samples (representing up to 50 instances of each pathogen covered by the test) and 200 BC-negative samples. Bacterial species-specific PCR sensitivities ranged from 65 to 100%. Sensitivity was 26% for the Gram-positive PCR, 32% for the Gram-negative PCR, and ranged 0 to 7% for yeast PCRs. Yeast detection was improved to 40% in a smaller set-up. There was no overall association between BSI-PCR sensitivity and time-to-positivity of BC (which was highly variable), yet Ct-values were lower for true-positive versus false-positive PCR results. False-positive results were observed in 84 (4%) of the 2200 species-specific PCRs in 200 culture-negative samples, and ranged from 0 to 6% for generic PCRs. Sensitivity of BSI-PCR was promising for individual bacterial pathogens, but still insufficient for yeasts and generic PCRs. Further development of BSI-PCR will focus on improving sensitivity by increasing input volumes and on subsequent implementation as a bedside test.

Direct Fluorescence Detection of Allele-Specific PCR Products Using Novel Energy-Transfer Labeled Primers.

PubMed

Winn-Deen

1998-12-01

Background: Currently analysis of point mutations can be done by allele-specific polymerase chain reaction (PCR) followed by gel analysis or by gene-specific PCR followed by hybridization with an allele-specific probe. Both of these mutation detection methods require post-PCR laboratory time and run the risk of contaminating subsequent experiments with the PCR product liberated during the detection step. The author has combined the PCR amplification and detection steps into a single procedure suitable for closed-tube analysis. Methods and Results: Allele-specific PCR primers were designed as Sunrise energy-transfer primers and contained a 3' terminal mismatch to distinguish between normal and mutant DNA. Cloned normal (W64) and mutant (R64) templates of the beta3-adrenergic receptor gene were tested to verify amplification specificity and yield. A no-target negative control was also run with each reaction. After PCR, each reaction was tested for fluorescence yield by measuring fluorescence on a spectrofluorimeter or fluorescent microtitreplate reader. The cloned controls and 24 patient samples were tested for the W64R mutation by two methods. The direct fluorescence results with the Sunrise allele-specific PCR method gave comparable genotypes to those obtained with the PCR/ restriction digest/gel electrophoresis control method. No PCR artifacts were observed in the negative controls or in the PCR reactions run with the mismatched target. Conclusions: The results of this pilot study indicate good PCR product and fluorescence yield from allele-specific energy-transfer labeled primers, and the capability of distinguishing between normal and mutant alleles based on fluorescence alone, without the need for restriction digestion, gel electrophoresis, or hybridization with an allele-specific probe.

Development of an updated PCR assay for detection of African swine fever virus.

PubMed

Luo, Yuzi; Atim, Stella A; Shao, Lina; Ayebazibwe, Chrisostom; Sun, Yuan; Liu, Yan; Ji, Shengwei; Meng, Xing-Yu; Li, Su; Li, Yongfeng; Masembe, Charles; Ståhl, Karl; Widén, Frederik; Liu, Lihong; Qiu, Hua-Ji

2017-01-01

Due to the current unavailability of vaccines or treatments for African swine fever (ASF), which is caused by African swine fever virus (ASFV), rapid and reliable detection of the virus is essential for timely implementation of emergency control measures and differentiation of ASF from other swine diseases with similar clinical presentations. Here, an improved PCR assay was developed and evaluated for sensitive and universal detection of ASFV. Primers specific for ASFV were designed based on the highly conserved region of the vp72 gene sequences of all ASFV strains available in GenBank, and the PCR assay was established and compared with two OIE-validated PCR tests. The analytic detection limit of the PCR assay was 60 DNA copies per reaction. No amplification signal was observed for several other porcine viruses. The novel PCR assay was more sensitive than two OIE-validated PCR assays when testing 14 strains of ASFV representing four genotypes (I, V, VIII and IX) from diverse geographical areas. A total of 62 clinical swine blood samples collected from Uganda were examined by the novel PCR, giving a high agreement (59/62) with a superior sensitive universal probe library-based real-time PCR. Eight out of 62 samples tested positive, and three samples with higher Ct values (39.15, 38.39 and 37.41) in the real-time PCR were negative for ASFV in the novel PCR. In contrast, one (with a Ct value of 29.75 by the real-time PCR) and two (with Ct values of 29.75 and 33.12) ASFV-positive samples were not identified by the two OIE-validated PCR assays, respectively. Taken together, these data show that the novel PCR assay is specific, sensitive, and applicable for molecular diagnosis and surveillance of ASF.

Comparison of quantitative cytomegalovirus (CMV) PCR in plasma and CMV antigenemia assay: clinical utility of the prototype AMPLICOR CMV MONITOR test in transplant recipients.

PubMed

Caliendo, A M; St George, K; Kao, S Y; Allega, J; Tan, B H; LaFontaine, R; Bui, L; Rinaldo, C R

2000-06-01

The correlation between the prototype AMPLICOR CMV MONITOR test (Roche Molecular Systems), a quantitative PCR assay, and the cytomegalovirus (CMV) pp65 antigenemia assay was evaluated in transplant recipients. Sequential blood specimens were collected on 29 patients (491 specimens), the leukocyte fraction was tested by CMV antigenemia, and quantitative PCR was performed on plasma specimens. None of the 15 patients (242 specimens) who were antigenemia negative were positive for CMV DNA by PCR, and none of these patients developed active CMV disease. There were 14 antigenemia-positive patients, 8 of whom developed active CMV disease. In all patients, there was a good association between the antigenemia and PCR assays. Ganciclovir-resistant virus was isolated from three patients with active CMV disease. These three patients had persistently elevated levels of antigenemia and CMV DNA by PCR when resistance to ganciclovir developed. This standardized, quantitative CMV PCR assay on plasma has clinical utility for the diagnosis of active disease and in monitoring the response to antiviral therapy in transplant recipients.

Comparison of Quantitative Cytomegalovirus (CMV) PCR in Plasma and CMV Antigenemia Assay: Clinical Utility of the Prototype AMPLICOR CMV MONITOR Test in Transplant Recipients

PubMed Central

Caliendo, Angela M.; St. George, Kirsten; Kao, Shaw-Yi; Allega, Jessica; Tan, Ban-Hock; LaFontaine, Robert; Bui, Larry; Rinaldo, Charles R.

2000-01-01

The correlation between the prototype AMPLICOR CMV MONITOR test (Roche Molecular Systems), a quantitative PCR assay, and the cytomegalovirus (CMV) pp65 antigenemia assay was evaluated in transplant recipients. Sequential blood specimens were collected on 29 patients (491 specimens), the leukocyte fraction was tested by CMV antigenemia, and quantitative PCR was performed on plasma specimens. None of the 15 patients (242 specimens) who were antigenemia negative were positive for CMV DNA by PCR, and none of these patients developed active CMV disease. There were 14 antigenemia-positive patients, 8 of whom developed active CMV disease. In all patients, there was a good association between the antigenemia and PCR assays. Ganciclovir-resistant virus was isolated from three patients with active CMV disease. These three patients had persistently elevated levels of antigenemia and CMV DNA by PCR when resistance to ganciclovir developed. This standardized, quantitative CMV PCR assay on plasma has clinical utility for the diagnosis of active disease and in monitoring the response to antiviral therapy in transplant recipients. PMID:10834964

Detection methods for biotech cotton MON 15985 and MON 88913 by PCR.

PubMed

Lee, Seong-Hun; Kim, Jin-Kug; Yi, Bu-Young

2007-05-02

Plants derived through agricultural biotechnology, or genetically modified organisms (GMOs), may affect human health and ecological environment. A living GMO is also called a living modified organism (LMO). Biotech cotton is a GMO in food or feed and also an LMO in the environment. Recently, two varieties of biotech cotton, MON 15985 and MON 88913, were developed by Monsanto Co. The detection method is an essential element for the GMO labeling system or LMO management of biotech plants. In this paper, two primer pairs and probes were designed for specific amplification of 116 and 120 bp PCR products from MON 15985 and MON 88913, respectively, with no amplification from any other biotech cotton. Limits of detection of the qualitative method were all 0.05% for MON 15985 and MON 88913. The quantitative method was developed using a TaqMan real-time PCR. A synthetic plasmid, as a reference molecule, was constructed from a taxon-specific DNA sequence of cotton and two construct-specific DNA sequences of MON 15985 and MON 88913. The quantitative method was validated using six samples that contained levels of biotech cotton mixed with conventional cotton ranging from 0.1 to 10.0%. As a result, the biases from the true value and the relative deviations were all within the range of +/-20%. Limits of quantitation of the quantitative method were all 0.1%. Consequently, it is reported that the proposed detection methods were applicable for qualitative and quantitative analyses for biotech cotton MON 15985 and MON 88913.

PCR-Restriction Fragment Length Polymorphism for Rapid, Low-Cost Identification of Isoniazid-Resistant Mycobacterium tuberculosis▿

PubMed Central

Caws, Maxine; Tho, Dau Quang; Duy, Phan Minh; Lan, Nguyen Thi Ngoc; Hoa, Dai Viet; Torok, Mili Estee; Chau, Tran Thi Hong; Van Vinh Chau, Nguyen; Chinh, Nguyen Tran; Farrar, Jeremy

2007-01-01

PCR-restriction fragment length poymorphism (PCR-RFLP) is a simple, robust technique for the rapid identification of isoniazid-resistant Mycobacterium tuberculosis. One hundred consecutive isolates from a Vietnamese tuberculosis hospital were tested by MspA1I PCR-RFLP for the detection of isoniazid-resistant katG_315 mutants. The test had a sensitivity of 80% and a specificity of 100% against conventional phenotypic drug susceptibility testing. The positive and negative predictive values were 1 and 0.86, respectively. None of the discrepant isolates had mutant katG_315 codons by sequencing. The test is cheap (less than $1.50 per test), specific, and suitable for the rapid identification of isoniazid resistance in regions with a high prevalence of katG_315 mutants among isoniazid-resistant M. tuberculosis isolates. PMID:17428939

Leishmania Infection: Laboratory Diagnosing in the Absence of a “Gold Standard”

PubMed Central

Rodríguez-Cortés, Alhelí; Ojeda, Ana; Francino, Olga; López-Fuertes, Laura; Timón, Marcos; Alberola, Jordi

2010-01-01

There is no gold standard for diagnosing leishmaniases. Our aim was to assess the operative validity of tests used in detecting Leishmania infection using samples from experimental infections, a reliable equivalent to the classic definition of gold standard. Without statistical differences, the highest sensitivity was achieved by protein A (ProtA), immunoglobulin (Ig)G2, indirect fluorescenece antibody test (IFAT), lymphocyte proliferation assay, quantitative real-time polymerase chain reaction of bone marrow (qPCR-BM), qPCR-Blood, and IgG; and the highest specificity by IgG1, IgM, IgA, qPCR-Blood, IgG, IgG2, and qPCR-BM. Maximum positive predictive value was obtained simultaneously by IgG2, qPCR-Blood, and IgG; and maximum negative predictive value by qPCR-BM. Best positive and negative likelihood ratios were obtained by IgG2. The test having the greatest, statistically significant, area under the receiver operating characteristics curve was IgG2 enzyme-linked immunosorbent assay (ELISA). Thus, according to the gold standard used, IFAT and qPCR are far from fulfilling the requirements to be considered gold standards, and the test showing the highest potential to detect Leishmania infection is Leishmania-specific ELISA IgG2. PMID:20134001

Leishmania infection: laboratory diagnosing in the absence of a "gold standard".

PubMed

Rodríguez-Cortés, Alhelí; Ojeda, Ana; Francino, Olga; López-Fuertes, Laura; Timón, Marcos; Alberola, Jordi

2010-02-01

There is no gold standard for diagnosing leishmaniases. Our aim was to assess the operative validity of tests used in detecting Leishmania infection using samples from experimental infections, a reliable equivalent to the classic definition of gold standard. Without statistical differences, the highest sensitivity was achieved by protein A (ProtA), immunoglobulin (Ig)G2, indirect fluorescenece antibody test (IFAT), lymphocyte proliferation assay, quantitative real-time polymerase chain reaction of bone marrow (qPCR-BM), qPCR-Blood, and IgG; and the highest specificity by IgG1, IgM, IgA, qPCR-Blood, IgG, IgG2, and qPCR-BM. Maximum positive predictive value was obtained simultaneously by IgG2, qPCR-Blood, and IgG; and maximum negative predictive value by qPCR-BM. Best positive and negative likelihood ratios were obtained by IgG2. The test having the greatest, statistically significant, area under the receiver operating characteristics curve was IgG2 enzyme-linked immunosorbent assay (ELISA). Thus, according to the gold standard used, IFAT and qPCR are far from fulfilling the requirements to be considered gold standards, and the test showing the highest potential to detect Leishmania infection is Leishmania-specific ELISA IgG2.

Clostridium difficile PCR ribotypes 001 and 176 - the common denominator of C. difficile infection epidemiology in the Czech Republic, 2014.

PubMed

Krutova, Marcela; Matejkova, Jana; Kuijper, Ed J; Drevinek, Pavel; Nyc, Otakar

2016-07-21

In 2014, 18 hospitals in the Czech Republic participated in a survey of the incidence of Clostridium difficile infections (CDI) in the country. The mean CDI incidence was 6.1 (standard deviation (SD):7.2) cases per 10,000 patient bed-days and 37.8 cases (SD: 41.4) per 10,000 admissions. The mean CDI testing frequency was 39.5 tests (SD: 25.4) per 10,000 patient bed-days and 255.8 tests (SD: 164.0) per 10,000 admissions. A total of 774 C. difficile isolates were investigated, of which 225 (29%) belonged to PCR ribotype 176, and 184 isolates (24%) belonged to PCR ribotype 001. Multilocus variable-number tandem repeat analysis (MLVA) revealed 27 clonal complexes formed by 84% (190/225) of PCR ribotype 176 isolates, and 14 clonal complexes formed by 77% (141/184) of PCR ribotype 001 isolates. Clonal clusters of PCR ribotypes 176 and 001 were observed in 11 and 7 hospitals, respectively. Our data demonstrate the spread of two C. difficile PCR ribotypes within 18 hospitals in the Czech Republic, stressing the importance of standardising CDI testing protocols and implementing mandatory CDI surveillance in the country. This article is copyright of The Authors, 2016.

Evaluation of Line Immunoassay to Detect HTLV-1 Infection in an Endemic Area, Southwestern Japan; Comparison with Polymerase Chain Reaction and Western Blot.

PubMed

Umeki, Kazumi; Umekita, Kunihiko; Hashikura, Yuuki; Yamamoto, Ikuo; Kubo, Kazuyoshi; Nagatomo, Yasuhiro; Okayama, Akihiko

2017-02-01

Human T-lymphotropic virus type 1 (HTLV-1) has been recognized as a cause of adult T-cell leukemia/lymphoma, HTLV-1-associated myelopathy/tropical spastic paraparesis, and HTLV-1-associated uveitis. HTLV-1 infection is normally detected by screening for HTLV-1 antibodies, and positive samples are confirmed by Western blot (WB). However, WB fails to confirm some samples that were positive for HTLV-1 antibodies on screening. Line immunoassay (LIA) is commonly used in Europe and Brazil, but not in Japan. Therefore, we evaluated the performance of LIA as a method of confirming HTLV-1 antibodies using samples in Japan. LIA was compared with polymerase chain reaction (PCR) and WB using 50 negative and 70 positive samples tested by chemiluminescent enzyme immunoassay (CLEIA) in Miyazaki, Japan, an HTLV-1 endemic area. LIA (INNO-LIA HTLVI/II Score) and WB (Problot HTLV-I) were performed according to the manufacturer's instructions. Real-time PCR for HTLV-1 pX region was performed using DNA derived from white blood cells. The samples that tested negative by real-time PCR were further tested by nested PCR. All 50 CLEIA negative samples were determined to be negative by LIA and PCR. Of the 70 positive samples, 66 tested positive by both of LIA and PCR. Three samples tested negative by LIA and PCR, and the remaining sample (PCR negative) showed non-specific staining in LIA and WB. WB showed more indeterminate results than LIA. Gp21 antibody in LIA demonstrated a high ability to discriminate between positive and negative PCR results. Furthermore, the degree of gp21 antibody reaction by LIA showed correlation with HTLV-1 proviral loads (PVLs). Our results indicate that LIA performs well in confirming HTLV-1 seropositivity by showing a low incidence of indeterminate results and good agreement with PCR using samples in Japan, although the number of samples tested was small. In addition, semi-quantitative antibody titer to gp21 correlated well with HTLV-1 PVLs. Further study including larger samples is necessary to determine the positioning of LIA for HTLV-1 detection in Japan.

Development of a PCR Assay to Detect Low Level Trypanosoma cruzi in Blood Specimens Collected with PAXgene Blood DNA Tubes for Clinical Trials Treating Chagas Disease.

PubMed

Wei, Bo; Chen, Lei; Kibukawa, Miho; Kang, John; Waskin, Hetty; Marton, Matthew

2016-12-01

Chagas disease is caused by the parasitic infection of Trypanosoma cruzi (T. cruzi). The STOP CHAGAS clinical trial was initiated in 2011 to evaluate posaconazole in treating Chagas disease, with treatment success defined as negative qualitative PCR results of detecting the parasites in blood specimens collected post-treatment. PAXgene Blood DNA tubes were utilized as a simple procedure to collect and process blood specimens. However, the PAXgene blood specimens challenged published T. cruzi PCR methods, resulting in poor sensitivity and reproducibility. To accurately evaluate the treatment efficacy of the clinical study, we developed and validated a robust PCR assay for detecting low level T. cruzi in PAXgene blood specimens. The assay combines a new DNA extraction method with a custom designed qPCR assay, resulting in limit of detection of 0.005 and 0.01 fg/μl for K98 and CL Brener, two representative strains of two of T. cruzi's discrete typing units. Reliable qPCR standard curves were established for both strains to measure parasite loads, with amplification efficiency ≥ 90% and the lower limit of linearity ≥ 0.05 fg/μl. The assay successfully analyzed the samples collected from the STOP CHAGAS study and may prove useful for future global clinical trials evaluating new therapies for asymptomatic chronic Chagas disease.

Highly sensitive and quantitative evaluation of the EGFR T790M mutation by nanofluidic digital PCR.

PubMed

Iwama, Eiji; Takayama, Koichi; Harada, Taishi; Okamoto, Isamu; Ookubo, Fumihiko; Kishimoto, Junji; Baba, Eishi; Oda, Yoshinao; Nakanishi, Yoichi

2015-08-21

The mutation of T790M in EGFR is a major mechanism of resistance to treatment with EGFR-TKIs. Only qualitative detection (presence or absence) of T790M has been described to date, however. Digital PCR (dPCR) analysis has recently been applied to the quantitative detection of target molecules in cancer with high sensitivity. In the present study, 25 tumor samples (13 obtained before and 12 after EGFR-TKI treatment) from 18 NSCLC patients with activating EGFR mutations were evaluated for T790M with dPCR. The ratio of the number of T790M alleles to that of activating mutation alleles (T/A) was determined. dPCR detected T790M in all 25 samples. Although T790M was present in all pre-TKI samples from 13 patients, 10 of these patients had a low T/A ratio and manifested substantial tumor shrinkage during treatment with EGFR-TKIs. In six of seven patients for whom both pre- and post-TKI samples were available, the T/A ratio increased markedly during EGFR-TKI treatment. Highly sensitive dPCR thus detected T790M in all NSCLC patients harboring activating EGFR mutations whether or not they had received EGFR-TKI treatment. Not only highly sensitive but also quantitative detection of T790M is important for evaluation of the contribution of T790M to EGFR-TKI resistance.

Canine Skin and Conjunctival Swab Samples for the Detection and Quantification of Leishmania infantum DNA in an Endemic Urban Area in Brazil

PubMed Central

de Almeida Ferreira, Sidney; Leite, Rodrigo Souza; Ituassu, Leonardo Trindade; Almeida, Gregório Guilherme; Souza, Daniel Menezes; Fujiwara, Ricardo Toshio; de Andrade, Antero Silva Ribeiro; Melo, Maria Norma

2012-01-01

Background We evaluated kDNA PCR/hybridization and quantitative real-time PCR (qPCR) targeting the gene of DNA polymerase of Leishmania infantum for CVL diagnosis and assessment of parasite load in clinical samples obtained invasively and non-invasively. Methodology/Principal Findings Eighty naturally infected dogs from an endemic urban area in Brazil were used. Animals were divided into two groups based on the presence or absence of CVL clinical sings. Skin biopsies, bone marrow, blood and conjunctival swabs samples were collected and submitted to L. infantum DNA detection. In addition, anti-Leishmania antibody titers were measured by Immunofluorescence antibody test. The symptomatic dogs had increased titers compared to asymptomatic dogs (P = 0.025). The frequencies of positive results obtained by kDNA PCR/hybridization for asymptomatic and symptomatic dogs, respectively, were as follows: right conjunctiva, 77.5% and 95.0%; left conjunctiva, 75.0% and 87.5%; skin, 45.0% and 75.0%; bone marrow, 50.0% and 77.5%; and blood, 27.5% and 22.5%. In both groups, the parasite load in the skin samples was the highest (P<0.0001). The parasite loads in the conjunctival swab and bone marrow samples were statistically equivalent within each group. The parasite burden in conjunctival swabs was higher in the dogs with clinical signs than in asymptomatic dogs (P = 0.028). This same relationship was also observed in the bone marrow samples (P = 0.002). No differences in amastigotes load in the skin were detected between the groups. Conclusions The conjunctival swab is a suitable clinical sample for qualitative molecular diagnosis of CVL. The highest parasite burdens were detected in skin regardless of the presence of VL-associated clinical signs. The qPCR results emphasized the role of dogs, particularly asymptomatic dogs, as reservoirs for CVL because of the high cutaneous parasite loads. These results may help to explain the maintenance of high transmission rates and numbers of CVL cases in endemic urban regions. PMID:22506084

Comprehensive Panel of Real-Time TaqMan™ Polymerase Chain Reaction Assays for Detection and Absolute Quantification of Filoviruses, Arenaviruses, and New World Hantaviruses

PubMed Central

Trombley, Adrienne R.; Wachter, Leslie; Garrison, Jeffrey; Buckley-Beason, Valerie A.; Jahrling, Jordan; Hensley, Lisa E.; Schoepp, Randal J.; Norwood, David A.; Goba, Augustine; Fair, Joseph N.; Kulesh, David A.

2010-01-01

Viral hemorrhagic fever is caused by a diverse group of single-stranded, negative-sense or positive-sense RNA viruses belonging to the families Filoviridae (Ebola and Marburg), Arenaviridae (Lassa, Junin, Machupo, Sabia, and Guanarito), and Bunyaviridae (hantavirus). Disease characteristics in these families mark each with the potential to be used as a biological threat agent. Because other diseases have similar clinical symptoms, specific laboratory diagnostic tests are necessary to provide the differential diagnosis during outbreaks and for instituting acceptable quarantine procedures. We designed 48 TaqMan™-based polymerase chain reaction (PCR) assays for specific and absolute quantitative detection of multiple hemorrhagic fever viruses. Forty-six assays were determined to be virus-specific, and two were designated as pan assays for Marburg virus. The limit of detection for the assays ranged from 10 to 0.001 plaque-forming units (PFU)/PCR. Although these real-time hemorrhagic fever virus assays are qualitative (presence of target), they are also quantitative (measure a single DNA/RNA target sequence in an unknown sample and express the final results as an absolute value (e.g., viral load, PFUs, or copies/mL) on the basis of concentration of standard samples and can be used in viral load, vaccine, and antiviral drug studies. PMID:20439981

Microarrays--new possibilities for detecting biological factors hazardous for humans and animals, and for use in environmental protection.

PubMed

Mirski, Tomasz; Bartoszcze, Michał; Bielawska-Drózd, Agata; Gryko, Romuald; Kocik, Janusz; Niemcewicz, Marcin; Chomiczewski, Krzysztof

2016-01-01

Both the known biological agents that cause infectious diseases, as well as modified (ABF-Advanced Biological Factors) or new, emerging agents pose a significant diagnostic problem using previously applied methods, both classical, as well as based on molecular biology methods. The latter, such as PCR and real-time PCR, have significant limitations, both quantitative (low capacity), and qualitative (limited number of targets). The article discusses the results of studies on using the microarray method for the identification of viruses (e.g. Orthopoxvirus group, noroviruses, influenza A and B viruses, rhino- and enteroviruses responsible for the FRI (Febrile Respiratory Illness), European bunyaviruses, and SARS-causing viruses), and bacteria (Mycobacterium spp., Yersinia spp., Campylobacter spp., Streptococcus pneumoniae, Salmonella typhi, Salmonella enterica, Staphylococcus aureus, Neisseria meningitidis, Clostridium difficile , Helicobacter pylori), including multiple antibiotic-resistant strains. The method allows for the serotyping and genotyping of bacteria, and is useful in the diagnosis of genetically modified agents. It allows the testing of thousands of genes in one experiment. In addition to diagnosis, it is applicable for gene expression studies, analysis of the function of genes, microorganisms virulence, and allows the detection of even single mutations. The possibility of its operational application in epidemiological surveillance, and in the detection of disease outbreak agents is demonstrated.

Addition of allochthonous fungi to a historically contaminated soil affects both remediation efficiency and bacterial diversity.

PubMed

Federici, Ermanno; Leonardi, Vanessa; Giubilei, Maria A; Quaratino, Daniele; Spaccapelo, Roberta; D'Annibale, Alessandro; Petruccioli, Maurizio

2007-11-01

Botryosphaeria rhodina DABAC P82 and Pleurotus pulmonarius CBS 664.97 were tested for their ability to grow and to degrade aromatic hydrocarbons in an aged contaminated soil. To evaluate the impact of indigenous microflora on the overall process, incubations were performed on both fumigated and nonfumigated soils. Fungal colonization by B. rhodina was unexpectedly lower in the fumigated than in the nonfumigated soil while the growth of P. pulmonarius showed an opposite response. Degradation performances and detoxification by both fungi in the nonfumigated soil were markedly higher than those observed in the fumigated one. Heterotrophic bacterial counts in nonfumigated soil augmented with either B. rhodina or P. pulmonarius were significantly higher than those of the corresponding incubation control (6.7 +/- 0.3 x 10(8) and 8.35 +/- 0.6 x 10(8), respectively, vs 9.2 +/- 0.3 x 10(7)). Bacterial communities of both incubation controls and fungal-augmented soil were compared by numerical analysis of denaturing gradient gel electrophoresis profiles of polymerase chain reaction (PCR)-amplified 16S ribosomal RNA (rRNA) genes and cloning and sequencing of PCR-amplified 16S rRNA genes. Besides increasing overall diversity, fungal augmentation led to considerable qualitative differences with respect to the pristine soil.

Marzipan: polymerase chain reaction-driven methods for authenticity control.

PubMed

Brüning, Philipp; Haase, Ilka; Matissek, Reinhard; Fischer, Markus

2011-11-23

According to German food guidelines, almonds are the only oilseed ingredient allowed for the production of marzipan. Persipan is a marzipan surrogate in which the almonds are replaced by apricot or peach kernels. Cross-contamination of marzipan products with persipan may occur if both products are produced using the same production line. Adulterations or dilutions, respectively, of marzipan with other plant-derived products, for example, lupine or pea, have also been found. Almond and apricot plants are closely related. Consequently, classical analytical methods for the identification/differentiation often fail or are not sensitive enough to quantify apricot concentrations below 1%. Polymerase chain reaction (PCR)-based methods have been shown to enable the differentiation of closely related plant species in the past. These methods are characterized by high specificity and low detection limits. Isolation methods were developed and evaluated especially with respect to the matrix marzipan in terms of yield, purity, integrity, and amplificability of the isolated DNA. For the reliable detection of apricot, peach, pea, bean, lupine, soy, cashew, pistachio, and chickpea, qualitative standard and duplex PCR methods were developed and established. The applicability of these methods was tested by cross-reaction studies and analysis of spiked raw pastes. Contaminations at the level of 0.1% could be detected.

Comparison of a PfHRP2-based rapid diagnostic test and PCR for malaria in a low prevalence setting in rural southern Zambia: implications for elimination.

PubMed

Laban, Natasha M; Kobayashi, Tamaki; Hamapumbu, Harry; Sullivan, David; Mharakurwa, Sungano; Thuma, Philip E; Shiff, Clive J; Moss, William J

2015-01-28

Rapid diagnostic tests (RDTs) detecting histidine-rich protein 2 (PfHRP2) antigen are used to identify individuals with Plasmodium falciparum infection even in low transmission settings seeking to achieve elimination. However, these RDTs lack sensitivity to detect low-density infections, produce false negatives for P. falciparum strains lacking pfhrp2 gene and do not detect species other than P. falciparum. Results of a PfHRP2-based RDT and Plasmodium nested PCR were compared in a region of declining malaria transmission in southern Zambia using samples from community-based, cross-sectional surveys from 2008 to 2012. Participants were tested with a PfHRP2-based RDT and a finger prick blood sample was spotted onto filter paper for PCR analysis and used to prepare blood smears for microscopy. Species-specific, real-time, quantitative PCR (q-PCR) was performed on samples that tested positive either by microscopy, RDT or nested PCR. Of 3,292 total participants enrolled, 12 (0.4%) tested positive by microscopy and 42 (1.3%) by RDT. Of 3,213 (98%) samples tested by nested PCR, 57 (1.8%) were positive, resulting in 87 participants positive by at least one of the three tests. Of these, 61 tested positive for P. falciparum by q-PCR with copy numbers ≤ 2 x 10(3) copies/μL, 5 were positive for both P. falciparum and Plasmodium malariae and 2 were positive for P. malariae alone. RDT detected 32 (53%) of P. falciparum positives, failing to detect three of the dual infections with P. malariae. Among 2,975 participants enrolled during a low transmission period between 2009 and 2012, sensitivity of the PfHRP2-based RDT compared to nested PCR was only 17%, with specificity of >99%. The pfhrp gene was detected in 80% of P. falciparum positives; however, comparison of copy number between RDT negative and RDT positive samples suggested that RDT negatives resulted from low parasitaemia and not pfhrp2 gene deletion. Low-density P. falciparum infections not identified by currently used PfHRP2-based RDTs and the inability to detect non-falciparum malaria will hinder progress to further reduce malaria in low transmission settings of Zambia. More sensitive and specific diagnostic tests will likely be necessary to identify parasite reservoirs and achieve malaria elimination.

Diagnostic Accuracy of Molecular Amplification Tests for Human African Trypanosomiasis—Systematic Review

PubMed Central

Boer, Kimberly R.; Dyserinck, Heleen C.; Büscher, Philippe; Schallig, Henk D. H. F.; Leeflang, Mariska M. G.

2012-01-01

Background A range of molecular amplification techniques have been developed for the diagnosis of Human African Trypanosomiasis (HAT); however, careful evaluation of these tests must precede implementation to ensure their high clinical accuracy. Here, we investigated the diagnostic accuracy of molecular amplification tests for HAT, the quality of articles and reasons for variation in accuracy. Methodology Data from studies assessing diagnostic molecular amplification tests were extracted and pooled to calculate accuracy. Articles were included if they reported sensitivity and specificity or data whereby values could be calculated. Study quality was assessed using QUADAS and selected studies were analysed using the bivariate random effects model. Results 16 articles evaluating molecular amplification tests fulfilled the inclusion criteria: PCR (n = 12), NASBA (n = 2), LAMP (n = 1) and a study comparing PCR and NASBA (n = 1). Fourteen articles, including 19 different studies were included in the meta-analysis. Summary sensitivity for PCR on blood was 99.0% (95% CI 92.8 to 99.9) and the specificity was 97.7% (95% CI 93.0 to 99.3). Differences in study design and readout method did not significantly change estimates although use of satellite DNA as a target significantly lowers specificity. Sensitivity and specificity of PCR on CSF for staging varied from 87.6% to 100%, and 55.6% to 82.9% respectively. Conclusion Here, PCR seems to have sufficient accuracy to replace microscopy where facilities allow, although this conclusion is based on multiple reference standards and a patient population that was not always representative. Future studies should, therefore, include patients for which PCR may become the test of choice and consider well designed diagnostic accuracy studies to provide extra evidence on the value of PCR in practice. Another use of PCR for control of disease could be to screen samples collected from rural areas and test in reference laboratories, to spot epidemics quickly and direct resources appropriately. PMID:22253934

Diagnostic accuracy of molecular amplification tests for human African trypanosomiasis--systematic review.

PubMed

Mugasa, Claire M; Adams, Emily R; Boer, Kimberly R; Dyserinck, Heleen C; Büscher, Philippe; Schallig, Henk D H F; Leeflang, Mariska M G

2012-01-01

A range of molecular amplification techniques have been developed for the diagnosis of Human African Trypanosomiasis (HAT); however, careful evaluation of these tests must precede implementation to ensure their high clinical accuracy. Here, we investigated the diagnostic accuracy of molecular amplification tests for HAT, the quality of articles and reasons for variation in accuracy. Data from studies assessing diagnostic molecular amplification tests were extracted and pooled to calculate accuracy. Articles were included if they reported sensitivity and specificity or data whereby values could be calculated. Study quality was assessed using QUADAS and selected studies were analysed using the bivariate random effects model. 16 articles evaluating molecular amplification tests fulfilled the inclusion criteria: PCR (n = 12), NASBA (n = 2), LAMP (n = 1) and a study comparing PCR and NASBA (n = 1). Fourteen articles, including 19 different studies were included in the meta-analysis. Summary sensitivity for PCR on blood was 99.0% (95% CI 92.8 to 99.9) and the specificity was 97.7% (95% CI 93.0 to 99.3). Differences in study design and readout method did not significantly change estimates although use of satellite DNA as a target significantly lowers specificity. Sensitivity and specificity of PCR on CSF for staging varied from 87.6% to 100%, and 55.6% to 82.9% respectively. Here, PCR seems to have sufficient accuracy to replace microscopy where facilities allow, although this conclusion is based on multiple reference standards and a patient population that was not always representative. Future studies should, therefore, include patients for which PCR may become the test of choice and consider well designed diagnostic accuracy studies to provide extra evidence on the value of PCR in practice. Another use of PCR for control of disease could be to screen samples collected from rural areas and test in reference laboratories, to spot epidemics quickly and direct resources appropriately.

Pneumocystis PCR: It Is Time to Make PCR the Test of Choice

PubMed Central

Doyle, Laura; Vogel, Sherilynn

2017-01-01

Abstract Background The testing strategy for Pneumocystis at the Cleveland Clinic changed from toluidine blue staining to polymerase chain reaction (PCR). We studied the differences in positivity rates for these assays and compared each with the detection of Pneumocystis in companion specimens by cytology and surgical pathology. Methods We reviewed the results of all Pneumocystis test orders 1 year before and 1 year after the implementation of a Pneumocystis-specific PCR. We also reviewed the corresponding cytology and surgical pathology results, if performed. Finally, we reviewed the medical records of patients with rare Pneumocystis detected by PCR in an effort to differentiate colonization vs true disease. Results Toluidine blue staining and surgical pathology had similar sensitivities and negative predictive values, both of which were superior to cytology. There was a >4-fold increase in the annual detection of Pneumocystis by PCR compared with toluidine blue staining (toluidine blue staining: 11/1583 [0.69%] vs PCR: 44/1457 [3.0%]; chi-square P < .001). PCR detected 1 more case than surgical pathology and was far more sensitive than cytology. Chart review demonstrated that the vast majority of patients with rare Pneumocystis detected were immunosuppressed, had radiologic findings supportive of this infection, had no other pathogens detected, and were treated for pneumocystosis by the clinical team. Conclusion PCR was the most sensitive method for the detection of Pneumocystis and should be considered the diagnostic test of choice. Correlation with clinical and radiologic findings affords discrimination of early true disease from the far rarer instances of colonization. PMID:29062861

Utility of Urinary Protein-Creatinine Ratio and Protein Content in a 24-Hour Urine Collection in Systemic Lupus Erythematosus: A Systematic Review and Meta-Analysis.

PubMed

Medina-Rosas, Jorge; Yap, Kristy S; Anderson, Melanie; Su, Jiandong; Touma, Zahi

2016-09-01

To systematically review literature on the utility of spot urinary protein-creatinine ratio (PCR) as a screening test for proteinuria and its ability to accurately measure proteinuria compared with 24-hour urine collection (24H-P) in patients with systemic lupus erythematosus (SLE). We conducted a literature search (1900-2015) for articles comparing PCR and 24H-P in SLE patients in the databases Medline, Web of Science, and Embase. Included studies and their results were critically appraised and analyzed. Thirteen studies (1,001 patients; 84.01% women) were included. Ten studies reported on Pearson's correlation (range 0.67-0.94), and 3 studies reported on Spearman's correlation (range 0.78-1.00). The meta-analysis of studies with Pearson's correlation showed a high overall correlation of 0.80 between 24H-P and PCR, yet with high heterogeneity (I(2)  = 97.2%). Correlation analysis is not sufficient to evaluate the utility of a new test against the gold standard test, and analysis on agreement is required. Seven studies reported on agreement: 3 studies analyzed concordance correlation coefficient (0.48-0.94), 3 analyzed intraclass correlation coefficient (0.66-0.95), and 1 analyzed kappa coefficient (0.58). These results confirmed that the agreement between 24H-P and PCR was inappropriate. Three studies included Bland-Altman plots, and the results also demonstrated poor agreement between both tests. The PCR has a utility as a screening test for proteinuria in SLE patients. The studies' results of 24H-P and PCR showed poor agreement between both tests, signifying that PCR should not be a substitute for the gold standard test (24H-P) to accurately measure proteinuria. © 2016, American College of Rheumatology.

qPCR-High resolution melt analysis for drug susceptibility testing of Mycobacterium leprae directly from clinical specimens of leprosy patients

PubMed Central

Goulart, Luiz Ricardo; Truman, Richard W.; Goulart, Isabela Maria B.; Vissa, Varalakshmi; Li, Wei; Matsuoka, Masanori; Suffys, Philip; Fontes, Amanda B.; Rosa, Patricia S.; Scollard, David M.; Williams, Diana L.

2017-01-01

Background Real-Time PCR-High Resolution Melting (qPCR-HRM) analysis has been recently described for rapid drug susceptibility testing (DST) of Mycobacterium leprae. The purpose of the current study was to further evaluate the validity, reliability, and accuracy of this assay for M. leprae DST in clinical specimens. Methodology/Principal findings The specificity and sensitivity for determining the presence and susceptibility of M. leprae to dapsone based on the folP1 drug resistance determining region (DRDR), rifampin (rpoB DRDR) and ofloxacin (gyrA DRDR) was evaluated using 211 clinical specimens from leprosy patients, including 156 multibacillary (MB) and 55 paucibacillary (PB) cases. When comparing the results of qPCR-HRM DST and PCR/direct DNA sequencing, 100% concordance was obtained. The effects of in-house phenol/chloroform extraction versus column-based DNA purification protocols, and that of storage and fixation protocols of specimens for qPCR-HRM DST, were also evaluated. qPCR-HRM results for all DRDR gene assays (folP1, rpoB, and gyrA) were obtained from both MB (154/156; 98.7%) and PB (35/55; 63.3%) patients. All PCR negative specimens were from patients with low numbers of bacilli enumerated by an M. leprae-specific qPCR. We observed that frozen and formalin-fixed paraffin embedded (FFPE) tissues or archival Fite’s stained slides were suitable for HRM analysis. Among 20 mycobacterial and other skin bacterial species tested, only M. lepromatosis, highly related to M. leprae, generated amplicons in the qPCR-HRM DST assay for folP1 and rpoB DRDR targets. Both DNA purification protocols tested were efficient in recovering DNA suitable for HRM analysis. However, 3% of clinical specimens purified using the phenol/chloroform DNA purification protocol gave false drug resistant data. DNA obtained from freshly frozen (n = 172), formalin-fixed paraffin embedded (FFPE) tissues (n = 36) or archival Fite’s stained slides (n = 3) were suitable for qPCR-HRM DST analysis. The HRM-based assay was also able to identify mixed infections of susceptible and resistant M. leprae. However, to avoid false positives we recommend that clinical specimens be tested for the presence of the M. leprae using the qPCR-RLEP assay prior to being tested in the qPCR-HRM DST and that all specimens demonstrating drug resistant profiles in this assay be subjected to DNA sequencing. Conclusion/Significance Taken together these results further demonstrate the utility of qPCR-HRM DST as an inexpensive screening tool for large-scale drug resistance surveillance in leprosy. PMID:28570560

Effects of canine parvovirus strain variations on diagnostic test results and clinical management of enteritis in dogs.

PubMed

Markovich, Jessica E; Stucker, Karla M; Carr, Alaina H; Harbison, Carole E; Scarlett, Janet M; Parrish, Colin R

2012-07-01

To estimate the prevalence of canine parvovirus (CPV) strains among dogs with enteritis admitted to a referral hospital in the southwestern United States during an 11-month period and to compare diagnostic test results, disease severity, and patient outcome among CPV strains. Prospective observational study. 72 dogs with histories and clinical signs of parvoviral enteritis. For each dog, a fecal sample or rectal swab specimen was evaluated for CPV antigen via an ELISA. Subsequently, fecal samples (n = 42 dogs) and pharyngeal swab specimens (16) were obtained and tested for CPV antigen via an ELISA and CPV DNA via a PCR assay. For specimens with CPV-positive results via PCR assay, genetic sequencing was performed to identify the CPV strain. 56 dogs tested positive for CPV via ELISA or PCR assay. For 42 fecal samples tested via both ELISA and PCR assay, 27 had positive results via both assays, whereas 6 had positive PCR assay results only. Ten pharyngeal swab specimens yielded positive PCR assay results. Genetic sequencing was performed on 34 fecal or pharyngeal swab specimens that had CPV-positive PCR assay results; 25 (73.5%) were identified as containing CPV type-2c, and 9 (26.5%) were identified as containing CPV type-2b. No association was found between CPV strain and disease severity or clinical outcome. CPV type-2b and CPV type-2c posed similar health risks for dogs; therefore, genetic sequencing of CPV does not appear necessary for clinical management of infected patients. The diagnostic tests used could detect CPV type-2c.

Differential Diagnosis of Malaria on Truelab Uno®, a Portable, Real-Time, MicroPCR Device for Point-Of-Care Applications.

PubMed

Nair, Chandrasekhar Bhaskaran; Manjula, Jagannath; Subramani, Pradeep Annamalai; Nagendrappa, Prakash B; Manoj, Mulakkapurath Narayanan; Malpani, Sukriti; Pullela, Phani Kumar; Subbarao, Pillarisetti Venkata; Ramamoorthy, Siva; Ghosh, Susanta K

2016-01-01

Sensitive and specific detection of malarial parasites is crucial in controlling the significant malaria burden in the developing world. Also important is being able to identify life threatening Plasmodium falciparum malaria quickly and accurately to reduce malaria related mortality. Existing methods such as microscopy and rapid diagnostic tests (RDTs) have major shortcomings. Here, we describe a new real-time PCR-based diagnostic test device at point-of-care service for resource-limited settings. Truenat® Malaria, a chip-based microPCR test, was developed by bigtec Labs, Bangalore, India, for differential identification of Plasmodium falciparum and Plasmodium vivax parasites. The Truenat Malaria tests runs on bigtec's Truelab Uno® microPCR device, a handheld, battery operated, and easy-to-use real-time microPCR device. The performance of Truenat® Malaria was evaluated versus the WHO nested PCR protocol. The Truenat® Malaria was further evaluated in a triple-blinded study design using a sample panel of 281 specimens created from the clinical samples characterized by expert microscopy and a rapid diagnostic test kit by the National Institute of Malaria Research (NIMR). A comparative evaluation was done on the Truelab Uno® and a commercial real-time PCR system. The limit of detection of the Truenat Malaria assay was found to be <5 parasites/μl for both P. falciparum and P. vivax. The Truenat® Malaria test was found to have sensitivity and specificity of 100% each, compared to the WHO nested PCR protocol based on the evaluation of 100 samples. The sensitivity using expert microscopy as the reference standard was determined to be around 99.3% (95% CI: 95.5-99.9) at the species level. Mixed infections were identified more accurately by Truenat Malaria (32 samples identified as mixed) versus expert microscopy and RDTs which detected 4 and 5 mixed samples, respectively. The Truenat® Malaria microPCR test is a valuable diagnostic tool and implementation should be considered not only for malaria diagnosis but also for active surveillance and epidemiological intervention.

Differential Diagnosis of Malaria on Truelab Uno®, a Portable, Real-Time, MicroPCR Device for Point-Of-Care Applications

PubMed Central

Nair, Chandrasekhar Bhaskaran; Manjula, Jagannath; Subramani, Pradeep Annamalai; Nagendrappa, Prakash B.; Manoj, Mulakkapurath Narayanan; Malpani, Sukriti; Pullela, Phani Kumar; Subbarao, Pillarisetti Venkata; Ramamoorthy, Siva; Ghosh, Susanta K.

2016-01-01

Background Sensitive and specific detection of malarial parasites is crucial in controlling the significant malaria burden in the developing world. Also important is being able to identify life threatening Plasmodium falciparum malaria quickly and accurately to reduce malaria related mortality. Existing methods such as microscopy and rapid diagnostic tests (RDTs) have major shortcomings. Here, we describe a new real-time PCR-based diagnostic test device at point-of-care service for resource-limited settings. Methods Truenat® Malaria, a chip-based microPCR test, was developed by bigtec Labs, Bangalore, India, for differential identification of Plasmodium falciparum and Plasmodium vivax parasites. The Truenat Malaria tests runs on bigtec’s Truelab Uno® microPCR device, a handheld, battery operated, and easy-to-use real-time microPCR device. The performance of Truenat® Malaria was evaluated versus the WHO nested PCR protocol. The Truenat® Malaria was further evaluated in a triple-blinded study design using a sample panel of 281 specimens created from the clinical samples characterized by expert microscopy and a rapid diagnostic test kit by the National Institute of Malaria Research (NIMR). A comparative evaluation was done on the Truelab Uno® and a commercial real-time PCR system. Results The limit of detection of the Truenat Malaria assay was found to be <5 parasites/μl for both P. falciparum and P. vivax. The Truenat® Malaria test was found to have sensitivity and specificity of 100% each, compared to the WHO nested PCR protocol based on the evaluation of 100 samples. The sensitivity using expert microscopy as the reference standard was determined to be around 99.3% (95% CI: 95.5–99.9) at the species level. Mixed infections were identified more accurately by Truenat Malaria (32 samples identified as mixed) versus expert microscopy and RDTs which detected 4 and 5 mixed samples, respectively. Conclusion The Truenat® Malaria microPCR test is a valuable diagnostic tool and implementation should be considered not only for malaria diagnosis but also for active surveillance and epidemiological intervention. PMID:26784111

Shedding and seroprevalence of pathogenic Leptospira spp. in sheep and cattle at a New Zealand Abattoir.

PubMed

Fang, F; Collins-Emerson, J M; Cullum, A; Heuer, C; Wilson, P R; Benschop, J

2015-06-01

A cross-sectional study was carried out on sheep and cattle slaughtered at a New Zealand abattoir from September to November 2010 to investigate the supplier-specific shedding rate, renal carriage rate and seroprevalence of leptospires. In the 2008/2009 season, this abattoir experienced three human leptospirosis cases from 20 staff, of which two were hospitalized. Urine, kidney and blood samples were collected from carcasses of 399 sheep (six suppliers, 17 slaughter lines) and 146 cattle (three suppliers, 22 slaughter lines). The urine and kidney samples were tested by quantitative real-time PCR (qPCR), while serum samples (from coagulated blood samples) were tested by microscopic agglutination test (MAT). In total, 27% (73/274; 95% CI: 18-37) of urine samples tested positive by qPCR. Species-specific shedding rates (prevalence of positive urine qPCR) were 31% (95% CI: 17-48) for sheep and 21% (95% CI: 14-30) for cattle. For 545 kidney samples tested, 145 were qPCR positive (27%; 95% CI: 17-39). The average prevalence of kidney qPCR positivity was 29% (95% CI: 17-45) for sheep and 21% (95% CI: 15-28) for cattle. Three hundred and thirty of 542 sampled sheep and cattle had antibodies against Leptospira borgpetersenii serovar Hardjobovis (Hardjobovis) and/or Leptospira interrogans serovar Pomona (Pomona), based on reciprocal MAT titre ≥1 : 48 (overall seroprevalence of 61%; 95% CI: 48-73). Seroprevalence was 57% (95% CI: 40-72) for sheep and 73% (95% CI: 59-83) for cattle. Among the seropositive animals, 41% (70/170; 95% CI: 30-54) were shedding (tested positive by urine qPCR) and 42% (137/330; 95% CI: 30-54) had renal carriage (tested positive by kidney qPCR). Some risk management options for abattoirs or farms to prevent human leptospirosis infections include vaccination of maintenance hosts, the use of personal protective equipment, and the application of urine qPCR to detect shedding status of stock as surveillance and as an alert. © 2014 Blackwell Verlag GmbH.

Droplet Digital PCR for BCR/ABL(P210) Detecting of CML: A High Sensitive Method of the Minimal Residual Disease& Disease Progression.

PubMed

Wang, Wen-Jun; Zheng, Chao-Feng; Liu, Zhuang; Tan, Yan-Hong; Chen, Xiu-Hua; Zhao, Bin-Liang; Li, Guo-Xia; Xu, Zhi-Fang; Ren, Fang-Gang; Zhang, Yao-Fang; Chang, Jian-Mei; Wang, Hong-Wei

2018-04-25

The present study intended to establish a droplet digital PCR (dd-PCR) for monitoring minimal residual disease (MRD) in patients with BCR/ABL (P210)-positive CML, thereby achieving deep-level monitoring of tumor load and determining the efficacy for guided clinically individualized treatment. Using dd-PCR and RT-qPCR, two cell suspensions were obtained from K562 cells and normal peripheral blood mononuclear cells by gradient dilution and were measured at the cellular level. At peripheral blood(PB) level, 61 cases with CML-chronic phase (CML-CP) were obtained after tyrosine kinase inhibitors (TKIs) treatment and regular follow-ups. By RT-qPCR, BCR/ABL (P210) fusion gene was undetectable in PB after three successive analyses, which were performed once every three months. At the same time, dd-PCR was performed simultaneously with the last equal amount of cDNA. Ten CML patients with MR4.5 were followed up by the two methods. At the cellular level, consistency of results of dd-PCR and RT-qPCR reached R 2 ≥0.99, with conversion equation of Y=33.148X 1.222 (Y: dd-PCR results; X: RT-qPCR results). In the dd-PCR test, 11 of the 61 CML patients (18.03%) tested positive and showed statistically significant difference (P<0.01). In the follow-up of 10 CML patients who were in MR4.5, 10 patients loss of MR4.0, and 4 were tested positive by dd-PCR 3 months earlier than by RT-qPCR. In contrast with RT-qPCR, dd-PCR is more sensitive, thus enabling accurate conversion of dd-PCR results into internationally standard RT-qPCR results by conversion equation, to achieve a deeper molecular biology-based stratification of BCR/ABL(P210) MRD. It has some reference value to monitor disease progression in clinic. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Pre-Clinical Testing of Real-Time PCR Assays for Diarrheal Disease Agents of Genera Escherichia and Shigella

DTIC Science & Technology

2014-05-16

FOR DIARRHEAL DISEASE AGENTS OF GENERA ESCHERICHIA AND SHIGELLA May 16, 2014 Reporting Period: October 1, 2010 to September 30, 2013...10-2010 - 30-09-2013 PRE-CLINICAL TESTING OF REAL-TIME PCR ASSAYS FOR DIARRHEAL DISEASE AGENTS OF GENERA ESCHERICHIA AND SHIGELLA ...Texas (MOA 2007 - 2013. Agreement No.: DODI 4000.19; AFI 25-201). Pre-clinical test results qualify ETEC and Shigella real-time PCR assays as lead

Latent class analysis of real time qPCR and bacteriological culturing for the diagnosis of Streptococcus agalactiae in cow composite milk samples.

PubMed

Holmøy, Ingrid H; Toft, Nils; Jørgensen, Hannah J; Mørk, Tormod; Sølverød, Liv; Nødtvedt, Ane

2018-06-01

Streptococcus agalactiae (S. agalactiae) has re-emerged as a mastitis pathogen among Norwegian dairy cows. The Norwegian cattle health services recommend that infected herds implement measures to eradicate S. agalactiae, this includes a screening of milk samples from all lactating cows. The performance of the qPCR-test currently in use for this purpose has not been evaluated under field conditions. The objective of this study was to estimate the sensitivity and specificity of the real-time qPCR assay in use in Norway (Mastitis 4 qPCR, DNA Diagnostics A/S, Risskov, Denmark) and compare it to conventional bacteriological culturing for detection of S. agalactiae in milk samples. Because none of these tests are considered a perfect reference test, the evaluation was performed using latent class models in a Bayesian analysis. Aseptically collected cow-composite milk samples from 578 cows belonging to 6 herds were cultured and tested by qPCR. While 37 (6.4%) samples were positive for S. agalactiae by bacteriological culture, 66 (11.4%) samples were positive by qPCR. The within-herd prevalence in the six herds, as estimated by the latent class models ranged from 7.7 to 50.8%. At the recommended cut-off (cycle threshold 37), the sensitivity of the qPCR was significantly higher at 95.3 (95% posterior probability interval [PPI] [84.2; 99.6]) than that of bacteriological culture at 58.2 (95% PPI [43.8; 74.4]). However, bacterial culture had a higher specificity of 99.7 (95% PPI [98.5; 100.0]) compared to the qPCR at 98.5 (95% PPI [94.6; 99.9]). The median estimated negative predictive values of qPCR was consistently higher than those of the BC at all estimated prevalences, and the superiority of the qPCR increased with increasing within-herd prevalence. The median positive predictive values of BC was in general higher than the estimates for the qPCR, however, at the highest prevalence the predictive ability of both tests were similar. Copyright © 2018 Elsevier B.V. All rights reserved.

Cost-effectiveness of a modified two-step algorithm using a combined glutamate dehydrogenase/toxin enzyme immunoassay and real-time PCR for the diagnosis of Clostridium difficile infection.

PubMed

Vasoo, Shawn; Stevens, Jane; Portillo, Lena; Barza, Ruby; Schejbal, Debra; Wu, May May; Chancey, Christina; Singh, Kamaljit

2014-02-01

The analytical performance and cost-effectiveness of the Wampole Toxin A/B EIA, the C. Diff. Quik Chek Complete (CdQCC) (a combined glutamate dehydrogenase antigen/toxin enzyme immunoassay), two RT-PCR assays (Progastro Cd and BD GeneOhm) and a modified two-step algorithm using the CdQCC reflexed to RT-PCR for indeterminate results were compared. The sensitivity of the Wampole Toxin A/B EIA, CdQCC (GDH antigen), BD GeneOhm and Progastro Cd RT-PCR were 85.4%, 95.8%, 100% and 93.8%, respectively. The algorithm provided rapid results for 86% of specimens and the remaining indeterminate results were resolved by RT-PCR, offering the best balance of sensitivity and cost savings per test (algorithm ∼US$13.50/test versus upfront RT-PCR ∼US$26.00/test). Copyright © 2012. Published by Elsevier B.V.

A cost effective real-time PCR for the detection of adenovirus from viral swabs

PubMed Central

2013-01-01

Compared to traditional testing strategies, nucleic acid amplification tests such as real-time PCR offer many advantages for the detection of human adenoviruses. However, commercial assays are expensive and cost prohibitive for many clinical laboratories. To overcome fiscal challenges, a cost effective strategy was developed using a combination of homogenization and heat treatment with an “in-house” real-time PCR. In 196 swabs submitted for adenovirus detection, this crude extraction method showed performance characteristics equivalent to viral DNA obtained from a commercial nucleic acid extraction. In addition, the in-house real-time PCR outperformed traditional testing strategies using virus culture, with sensitivities of 100% and 69.2%, respectively. Overall, the combination of homogenization and heat treatment with a sensitive in-house real-time PCR provides accurate results at a cost comparable to viral culture. PMID:23758993

Rapid diagnostic tests for malaria at sites of varying transmission intensity in Uganda.

PubMed

Hopkins, Heidi; Bebell, Lisa; Kambale, Wilson; Dokomajilar, Christian; Rosenthal, Philip J; Dorsey, Grant

2008-02-15

In Africa, fever is often treated presumptively as malaria, resulting in misdiagnosis and the overuse of antimalarial drugs. Rapid diagnostic tests (RDTs) for malaria may allow improved fever management. We compared RDTs based on histidine-rich protein 2 (HRP2) and RDTs based on Plasmodium lactate dehydrogenase (pLDH) with expert microscopy and PCR-corrected microscopy for 7000 patients at sites of varying malaria transmission intensity across Uganda. When all sites were considered, the sensitivity of the HRP2-based test was 97% when compared with microscopy and 98% when corrected by PCR; the sensitivity of the pLDH-based test was 88% when compared with microscopy and 77% when corrected by PCR. The specificity of the HRP2-based test was 71% when compared with microscopy and 88% when corrected by PCR; the specificity of the pLDH-based test was 92% when compared with microscopy and >98% when corrected by PCR. Based on Plasmodium falciparum PCR-corrected microscopy, the positive predictive value (PPV) of the HRP2-based test was high (93%) at all but the site with the lowest transmission rate; the pLDH-based test and expert microscopy offered excellent PPVs (98%) for all sites. The negative predictive value (NPV) of the HRP2-based test was consistently high (>97%); in contrast, the NPV for the pLDH-based test dropped significantly (from 98% to 66%) as transmission intensity increased, and the NPV for expert microscopy decreased significantly (99% to 54%) because of increasing failure to detect subpatent parasitemia. Based on the high PPV and NPV, HRP2-based RDTs are likely to be the best diagnostic choice for areas with medium-to-high malaria transmission rates in Africa.

Patients with diffuse uveitis and inactive toxoplasmic retinitis lesions test PCR positive for Toxoplasma gondii in their vitreous and blood.

PubMed

Novais, Eduardo A; Commodaro, Alessandra G; Santos, Fábio; Muccioli, Cristina; Maia, André; Nascimento, Heloisa; Moeller, Cecilia T A; Rizzo, Luiz V; Grigg, Michael E; Belfort, Rubens

2014-07-01

To determine if patients with inactive chorioretinitis lesions who experience chronic toxoplasmic uveitis test PCR positive for Toxoplasma in their ocular fluids. Two patients undergoing long-term anti-toxoplasmic treatment developed chronic uveitis and vitritis. They underwent therapeutic and diagnostic pars plana vitrectomy. Patient specimens were tested for toxoplasmosis by real-time PCR and nested PCR. Patient specimens were also tested for the presence of Toxoplasma antibodies that recognise allelic peptide motifs to determine parasite serotype. Patients tested positive for Toxoplasma by real-time PCR at the B1 gene in the vitreous and aqueous humours of patient 1, but only the vitreous of patient 2. Patients were not parasitemic by real-time PCR in plasma and blood. During surgery, only old hyperpigmented toxoplasmic scars were observed; there was no sign of active retinitis. Multilocus PCR-DNA sequence genotyping at B1, NTS2 and SAG1 loci established that two different non-archetypal Toxoplasma strains had infected patients 1 and 2. A peptide-based serotyping ELISA confirmed the molecular findings. No active lesions were observed, but both patients possessed sufficient parasite DNA in their vitreous to permit genotyping. Several hypotheses to explain the persistence of the vitritis and anterior uveitis in the absence of active retinitis are discussed. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Universal reverse-transcriptase real-time PCR for infectious hematopoietic necrosis virus (IHNV)

USGS Publications Warehouse

Purcell, Maureen K.; Thompson, Rachel L.; Garver, Kyle A.; Hawley, Laura M.; Batts, William N.; Sprague, Laura; Sampson, Corie; Winton, James R.

2013-01-01

Infectious hematopoietic necrosis virus (IHNV) is an acute pathogen of salmonid fishes in North America, Europe and Asia and is reportable to the World Organization for Animal Health (OIE). Phylogenetic analysis has identified 5 major virus genogroups of IHNV worldwide, designated U, M, L, E and J; multiple subtypes also exist within those genogroups. Here, we report the development and validation of a universal IHNV reverse-transcriptase real-time PCR (RT-rPCR) assay targeting the IHNV nucleocapsid (N) gene. Properties of diagnostic sensitivity (DSe) and specificity (DSp) were defined using laboratory-challenged steelhead trout Oncorhynchus mykiss, and the new assay was compared to the OIE-accepted conventional PCR test and virus isolation in cell culture. The IHNV N gene RT-rPCR had 100% DSp and DSe and a higher estimated diagnostic odds ratio (DOR) than virus culture or conventional PCR. The RT-rPCR assay was highly repeatable within a laboratory and highly reproducible between laboratories. Field testing of the assay was conducted on a random sample of juvenile steelhead collected from a hatchery raceway experiencing an IHN epizootic. The RT-rPCR detected a greater number of positive samples than cell culture and there was 40% agreement between the 2 tests. Overall, the RT-rPCR assay was highly sensitive, specific, repeatable and reproducible and is suitable for use in a diagnostic setting.

Evaluation of Legionella real-time PCR against traditional culture for routine and public health testing of water samples.

PubMed

Collins, S; Stevenson, D; Walker, J; Bennett, A

2017-06-01

To evaluate the usefulness of Legionella qPCR alongside traditional culture for enumeration of Legionella from water samples as part of both routine and public health investigation testing. Routine water samples (n = 2002) and samples from public health investigations (n = 215) were analysed by culture and qPCR for Legionella spp., Legionella pneumophila and L. pneumophila sg-1. A negative qPCR result was highly predictive of a negative culture result for all water systems (negative predictive values, NPV from 97·4 to 100%). Positive predictive values (PPV) were lower (0-50%). Results for qPCR were generally larger than culture with average log 10 differences of 1·1 for Legionella spp. and 1·2 for L. pneumophila. Alert and action levels of 1000 and 10 000 GU per litre, respectively, are proposed for Legionella qPCR for hot and cold water systems (HCWS). The use of qPCR significantly reduced the time to results for public health investigations by rapidly identifying potential sources and ruling out others, thus enabling a more rapid and efficient response. The high NPV of qPCR supports its use to rapidly screen out negative samples without culture. Alert and action levels for Legionella qPCR for HCWS are proposed. Quantitative PCR will be a valuable tool for both routine and public health testing. This study generated comparative data of >2000 water samples by qPCR and culture. Action and alert levels have been recommended that could enable duty holders to interpret qPCR results to facilitate timely Legionella control and public health protection. © 2017 Crown copyright. Journal of Applied Microbiology © 2017 The Society for Applied Microbiology.

9 CFR 147.6 - Procedure for determining the status of flocks reacting to tests for Mycoplasma gallisepticum...

Code of Federal Regulations, 2010 CFR

2010-01-01

... Agency may examine reactors by the in vivo bio-assay, PCR-based procedures, and/or culture procedures before final determination of the flock status is made. (13) If the in vivo bio-assay, PCR-based... classification for which it was tested. (14) If the in vivo bio-assay, PCR-based procedures, or culture...

Bayesian modelling to estimate the test characteristics of coprology, coproantigen ELISA and a novel real-time PCR for the diagnosis of taeniasis.

PubMed

Praet, Nicolas; Verweij, Jaco J; Mwape, Kabemba E; Phiri, Isaac K; Muma, John B; Zulu, Gideon; van Lieshout, Lisette; Rodriguez-Hidalgo, Richar; Benitez-Ortiz, Washington; Dorny, Pierre; Gabriël, Sarah

2013-05-01

To estimate and compare the performances of coprology, copro-Ag ELISA and real-time polymerase chain reaction assay (copro-PCR) for detection of Taenia solium tapeworm carriers. The three diagnostic tests were applied on 817 stool samples collected in two Zambian communities where taeniasis is endemic. A Bayesian approach was used to allow estimation of the test characteristics. Two (0.2%; 95% Confidence Interval (CI): 0-0.8), 67 (8.2%; 95% CI: 6.4-10.3) and 10 (1.2%; 95% CI: 0.5-2.2) samples were positive using coprology, copro-Ag ELISA and copro-PCR, respectively. Specificities of 99.9%, 92.0% and 99.0% were determined for coprology, copro-Ag ELISA and copro-PCR, respectively. Sensitivities of 52.5%, 84.5% and 82.7% were determined for coprology, copro-Ag ELISA and copro-PCR, respectively. We urge for additional studies exploring possible cross-reactions of the copro-Ag ELISA and for the use of more sensitive tests, such as copro-PCR, for the detection of tapeworm carriers, which is a key factor in controlling the parasite in endemic areas. © 2013 Blackwell Publishing Ltd.

[Correlation between results of PCR and specific serological tests in diagnosis of Epstein-Barr virus in patients with mononucleosis syndrome].

PubMed

Banko, A V; Lazarević, I B; Cupić, M D; Knezević, A M; Stevanović, G D; Krejović-Trivić, S B; Jovanović, T P

2009-01-01

Routine laboratory diagnosis of infectious mononucleosis is based on EBV serological testing, but due to problems in interpretation of results, molecular methods, especially PCR, are often necessary. The aim of the present study was to investigate correlation between results of PCR and specific serological tests in diagnosis of Epstein-Barr virus in patients with mononucleosis syndrome. The study comprised 68 patients with mononucleosis syndrome. Their blood samples were tested using ELISA for detection of 4 EBV specific antibodies (anti-VCA IgM and IgG, anti-EA-D IgG and anti-EBNA-1 IgG) and PCR for detection of EBV DNA. According to results of serology 42 patients had acute primary infection, 2 reactivation, 1 chronic active infection, 19 past infection, and 4 have been EBV seronegative. EBV DNA was detected in 17 patients (25%) and all of them were serologically defined as acutely infected. PCR was useful for resolving unclear serology results. Specific serology is the first step in diagnosis of IM, but PCR may serve as a useful additional diagnostic tool for clarifying serological dilemmas, reaching final diagnosis and defining status of the infection.

Comparison of nine different real-time PCR chemistries for qualitative and quantitative applications in GMO detection.

PubMed

Buh Gasparic, Meti; Tengs, Torstein; La Paz, Jose Luis; Holst-Jensen, Arne; Pla, Maria; Esteve, Teresa; Zel, Jana; Gruden, Kristina

2010-03-01

Several techniques have been developed for detection and quantification of genetically modified organisms, but quantitative real-time PCR is by far the most popular approach. Among the most commonly used real-time PCR chemistries are TaqMan probes and SYBR green, but many other detection chemistries have also been developed. Because their performance has never been compared systematically, here we present an extensive evaluation of some promising chemistries: sequence-unspecific DNA labeling dyes (SYBR green), primer-based technologies (AmpliFluor, Plexor, Lux primers), and techniques involving double-labeled probes, comprising hybridization (molecular beacon) and hydrolysis (TaqMan, CPT, LNA, and MGB) probes, based on recently published experimental data. For each of the detection chemistries assays were included targeting selected loci. Real-time PCR chemistries were subsequently compared for their efficiency in PCR amplification and limits of detection and quantification. The overall applicability of the chemistries was evaluated, adding practicability and cost issues to the performance characteristics. None of the chemistries seemed to be significantly better than any other, but certain features favor LNA and MGB technology as good alternatives to TaqMan in quantification assays. SYBR green and molecular beacon assays can perform equally well but may need more optimization prior to use.

An international inter-laboratory ring trial to evaluate a real-time PCR assay for the detection of bovine herpesvirus 1 in extended bovine semen.

PubMed

Wang, Jianning; O'Keefe, Joseph; Orr, Della; Loth, Leo; Banks, Malcolm; Wakeley, Philip; West, Donna; Card, Roderick; Ibata, Georgina; Van Maanen, Kees; Thoren, Peter; Isaksson, Mats; Kerkhofs, Pierre

2008-01-01

Six laboratories participated in a ring trial to evaluate the reliability of a real-time PCR assay for the detection of bovine herpesvirus 1 (BoHV-1) from extended bovine semen. Sets of coded samples were prepared and distributed to each of the laboratories. The sample panel contained semen from naturally and artificially infected bulls, serial dilutions of positive semen with negative semen, semen from uninfected seronegative bulls, negative semen spiked with virus, as well as serial dilutions of reference virus. The samples were tested using a previously validated real-time PCR assay for the detection of BoHV-1 in each participating laboratory. The PCR tests were conducted with four different real-time PCR amplification platforms, including RotorGene 3000, Stratagene MX 3000/4000, ABI 7900, and Roche LightCycler 2.0. Virus isolation using one set of samples was performed in one laboratory. The results of the laboratories were compared with one another, and with those of virus isolation. It was found that the sensitivity and specificity of the real-time PCR test was greater than those of virus isolation (82.7% versus 53.6% and 93.6% versus 84.6%, respectively). A high level of agreement on PCR testing results between the laboratories was achieved (kappa value 0.59-0.95). The results of this study indicate that the real-time PCR assay is suitable for the detection of BoHV-1 in extended semen, and would be a good substitute for the slow and laborious virus isolation, for the screening testing at artificial insemination centres and for international trade.

Improved detection of canine Angiostrongylus vasorum infection using real-time PCR and indirect ELISA.

PubMed

Jefferies, Ryan; Morgan, Eric R; Helm, Jenny; Robinson, Matthew; Shaw, Susan E

2011-12-01

This study reports the development of a real-time PCR assay and an indirect ELISA to improve on current detection of canine Angiostrongylus vasorum infection. A highly specific fluorescent probe-based, real-time PCR assay was developed to target the A. vasorum second internal transcribed spacer region and detected DNA in EDTA blood, lung tissue, broncho-alveolar larvage fluid, endotracheal mucus, pharyngeal swabs and faecal samples. PCR was fast (∼1 h), highly efficient when using EDTA blood samples, consistently detected a single molecule of parasite DNA and did not amplify DNA from other parasitic nematodes or definitive host species. An indirect ELISA was also developed using the soluble protein fraction from adult A. vasorum worms. Some cross-reactive antigen recognition was observed when tested against sera from dogs infected with Crenosoma vulpis (n = 8), Toxocara canis (n = 5) and Dirofilaria immitis (n = 5). This was largely overcome by setting the cut-off for a positive result at an appropriately high level. Field evaluation of the real-time PCR and ELISA was conducted by testing sera and EDTA blood from dogs with suspected A. vasorum infection (n = 148) and compared with the Baermann's larval migration test in faeces. Thirty-one dogs were positive by at least one test. Of these, 20 (65%) were detected by the Baermann method, 18 (58%) by blood PCR, 24 (77%) by ELISA and 28 (90%) by blood PCR and ELISA together. Combined testing using real-time PCR and ELISA therefore improved the detection rate of A. vasorum infection and holds promise for improved clinical diagnosis and epidemiological investigation.

Mycobacterium paratuberculosis detection in cow's milk in Argentina by immunomagnetic separation-PCR.

PubMed

Gilardoni, Liliana Rosa; Fernández, Bárbara; Morsella, Claudia; Mendez, Laura; Jar, Ana María; Paolicchi, Fernando Alberto; Mundo, Silvia Leonor

2016-01-01

The aim of this study was to standardize a diagnosis procedure to detect Mycobacterium avium subsp. paratuberculosis (Map) DNA in raw cow milk samples under field conditions. A procedure that combines both immunomagnetic separation and IS900-PCR detection (IMS-IS1 PCR) was employed on milk samples from 265 lactating Holstein cows from Map infected and uninfected herds in Argentina. IMS-IS1 PCR results were analyzed and compared with those obtained from milk and fecal culture and serum ELISA. The extent of agreement between both tests was determined by the Kappa test. IMS-IS1 PCR showed a detection limit of 10(1) CFU of Map/mL of milk, when 50:50 mix of monoclonal and polyclonal antibodies were used to coat magnetic beads. All of the 118 samples from the Map uninfected herds were negative for the set of the tests. In Map infected herds, 80 out of 147 cows tested positive by milk IMS-IS1 PCR (55%), of which 2 (1.4%) were also positive by milk culture, 15 (10%) by fecal culture, and 20 (14%) by serum ELISA. Kappa statistics (95% CI) showed a slight agreement between the different tests (<0.20), and the proportions of agreement were ≤0.55. The IMS-IS1 PCR method detected Map in milk of the cows that were not positive in other techniques. This is the first report dealing with the application of IMS-IS1 PCR in the detection of Map in raw milk samples under field conditions in Argentina. Copyright © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

Prevalence of Vector-Borne Pathogens in Southern California Dogs With Clinical and Laboratory Abnormalities Consistent With Immune-Mediated Disease.

PubMed

Kidd, L; Qurollo, B; Lappin, M; Richter, K; Hart, J R; Hill, S; Osmond, C; Breitschwerdt, E B

2017-07-01

Studies investigating the prevalence of vector-borne pathogens in southern California dogs are limited. Occult infections might be misdiagnosed as idiopathic immune-mediated disease. (1) To determine the prevalence of vector-borne pathogens in southern California dogs with compatible clinical findings using PCR and serologic panels and (2) to determine whether testing convalescent samples and repeating PCR on acute samples using the same and different gene targets enhance detection. Forty-two client-owned dogs with clinical signs of vector-borne disease presenting to specialty practices in San Diego County. Combined prospective and retrospective observational study. Forty-two acute and 27 convalescent samples were collected. Acute samples were prospectively tested for antibodies to Rickettsia, Ehrlichia, Bartonella, Babesia, Borrelia, and Anaplasma species. PCR targeting Ehrlichia, Babesia, Anaplasma, hemotropic Mycoplasma, and Bartonella species was also performed. Retrospectively, convalescent samples were tested for the same organisms using serology, and for Ehrlichia, Babesia, Anaplasma, and Bartonella species using PCR. Acute samples were retested using PCR targeting Ehrlichia and Babesia species. Evidence of exposure to or infection with a vector-borne pathogen was detected in 33% (14/42) of dogs. Ehrlichia and Babesia species were most common; each was identified in 5 dogs. Convalescent serologic testing, repeating PCR, and using novel PCR gene targets increased detection by 30%. Repeated testing using serology and PCR enhances detection of infection by vector-borne pathogens in dogs with clinical signs of immune-mediated disease. Larger prevalence studies of emerging vector-borne pathogens in southern California dogs are warranted. Copyright © 2017 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.

Comparison of culture and biochemical tests with PCR for detection of Brachyspira hyodysenteriae and Brachyspira pilosicoli.

PubMed

Råsbäck, T; Fellström, C; Gunnarsson, A; Aspán, A

2006-08-01

Traditional culture and biochemical tests (CBT) were compared with PCR for sensitivity and detection of Brachyspira hyodysenteriae and Brachyspira pilosicoli in seeded faeces and clinical samples from diarrhoeic pigs. A duplex PCR system was developed based on primers detecting the tlyA-gene of B. hyodysenteriae and the 16S rRNA-gene of B. pilosicoli. Sensitivities for the PCR system were determined on seeded faeces, using DNA that had been recovered from primary cultures or extracted directly from faeces. Compared to CBT, PCR applied to DNA extracted directly from faeces lowered the sensitivity by a factor of 1000 to 10,000. B. hyodysenteriae and B. pilosicoli detection was compared for CBT and PCR using 200 clinical samples. CBT detected more B. hyodysenteriae isolates in the clinical samples than PCR, but fewer B. pilosicoli positive samples. An atypical strongly haemolytic isolate was detected only by CBT.

PCR detection and quantitation of predominant anaerobic bacteria in human and animal fecal samples.

PubMed Central

Wang, R F; Cao, W W; Cerniglia, C E

1996-01-01

PCR procedures based on 16S rRNA gene sequences specific for 12 anaerobic bacteria that predominate in the human intestinal tract were developed and used for quantitative detection of these species in human (adult and baby) feces and animal (rat, mouse, cat, dog, monkey, and rabbit) feces. Fusobacterium prausnitzii, Peptostreptococcus productus, and Clostridium clostridiiforme had high PCR titers (the maximum dilutions for positive PCR results ranged from 10(-3) to 10(-8)) in all of the human and animal fecal samples tested. Bacteroides thetaiotaomicron, Bacteroides vulgatus, and Eubacterium limosum also showed higher PCR titers (10(-2) to 10(-6)) in adult human feces. The other bacteria tested, including Escherichia coli, Bifidobacterium adolescentis, Bifidobacterium longum, Lactobacillus acidophilus, Eubacterium biforme, and Bacteroides distasonis, were either at low PCR titers (less than 10(-2)) or not detected by PCR. The reported PCR procedure including the fecal sample preparation method is simplified and rapid and eliminates the DNA isolation steps. PMID:8919784

Comparison of the performance of laboratory tests in the diagnosis of feline infectious peritonitis.

PubMed

Stranieri, Angelica; Giordano, Alessia; Paltrinieri, Saverio; Giudice, Chiara; Cannito, Valentina; Lauzi, Stefania

2018-05-01

We compared the performance of clinicopathologic and molecular tests used in the antemortem diagnosis of feline infectious peritonitis (FIP). From 16 FIP and 14 non-FIP cats, we evaluated retrospectively the sensitivity, specificity, and likelihood ratios (LRs) of serum protein electrophoresis, α 1 -acid glycoprotein (AGP) on peripheral blood, screening reverse-transcription nested PCR (RT-nPCR) on the 3'-untranslated region (3'-UTR), and spike (S) gene sequencing on peripheral blood, body cavity effusions, and tissue, as well as body cavity cytology and delta total nucleated cell count (ΔTNC). Any of these tests on blood, and especially the molecular tests, may support or confirm a clinical diagnosis of FIP. A negative result does not exclude the disease except for AGP. Cytology, 3'-UTR PCR, and ΔTNC may confirm a clinical diagnosis on effusions; cytology or 3'-UTR PCR may exclude FIP. Conversely, S gene sequencing is not recommended based on the LRs. On tissues, S gene sequencing is preferable when histology is highly consistent with FIP, and 3'-UTR PCR when FIP is unlikely. Combining one test with high LR+ with one with low LR- (e.g., molecular tests and AGP on blood, ΔTNC and cytology in effusions) may improve the diagnostic power of the most used laboratory tests.

Nested polymerase chain reaction on blood clots for gene encoding 56 kDa antigen and serology for the diagnosis of scrub typhus.

PubMed

Prakash, J A J; Kavitha, M L; Mathai, E

2011-01-01

Scrub typhus is a zoonotic illness endemic in the Asia-Pacific region. Early diagnosis and appropriate management contribute significantly to preventing adverse outcomes including mortality. Serology is widely used for diagnosing scrub typhus. Recent reports suggest that polymerase chain reaction (PCR) could be a rapid and reliable alternative. This study assessed the utility of these tests for scrub typhus diagnosis. Nested PCR to detect the 56 kDa antigen gene of O. tsutsugamushi was performed on blood clots from 87 individuals with clinically suspected scrub typhus. Weil-Felix test and scrub typhus IgM ELISA were performed on serum samples from the same patients. As a gold standard reference test was not available, latent class analysis (LCA) was used to assess the performance of the three tests. The LCA analysis showed the sensitivity of Weil-Felix test, IgM ELISA and PCR to be 59%, 100% and 58% respectively. The specificity of ELISA was only 73%, whereas those of the Weil-Felix test and PCR were 94% and 100% respectively. Nested PCR using blood clots while specific, lacked sensitivity as compared to IgM ELISA. In resource-poor settings Weil-Felix test still remains valuable despite its moderate sensitivity.

An insulated isothermal PCR method on a field-deployable device for rapid and sensitive detection of canine parvovirus type 2 at points of need.

PubMed

Wilkes, Rebecca P; Lee, Pei-Yu A; Tsai, Yun-Long; Tsai, Chuan-Fu; Chang, Hsiu-Hui; Chang, Hsiao-Fen G; Wang, Hwa-Tang T

2015-08-01

Canine parvovirus type 2 (CPV-2), including subtypes 2a, 2b and 2c, causes an acute enteric disease in both domestic and wild animals. Rapid and sensitive diagnosis aids effective disease management at points of need (PON). A commercially available, field-deployable and user-friendly system, designed with insulated isothermal PCR (iiPCR) technology, displays excellent sensitivity and specificity for nucleic acid detection. An iiPCR method was developed for on-site detection of all circulating CPV-2 strains. Limit of detection was determined using plasmid DNA. CPV-2a, 2b and 2c strains, a feline panleukopenia virus (FPV) strain, and nine canine pathogens were tested to evaluate assay specificity. Reaction sensitivity and performance were compared with an in-house real-time PCR using serial dilutions of a CPV-2b strain and 100 canine fecal clinical samples collected from 2010 to 2014, respectively. The 95% limit of detection of the iiPCR method was 13 copies of standard DNA and detection limits for CPV-2b DNA were equivalent for iiPCR and real-time PCR. The iiPCR reaction detected CPV-2a, 2b and 2c and FPV. Non-targeted pathogens were not detected. Test results of real-time PCR and iiPCR from 99 fecal samples agreed with each other, while one real-time PCR-positive sample tested negative by iiPCR. Therefore, excellent agreement (k = 0.98) with sensitivity of 98.41% and specificity of 100% in detecting CPV-2 in feces was found between the two methods. In conclusion, the iiPCR system has potential to serve as a useful tool for rapid and accurate PON, molecular detection of CPV-2. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Nasal Methicillin-Resistant Staphylococcus aureus (MRSA) PCR Testing Reduces the Duration of MRSA-Targeted Therapy in Patients with Suspected MRSA Pneumonia.

PubMed

Baby, Nidhu; Faust, Andrew C; Smith, Terri; Sheperd, Lyndsay A; Knoll, Laura; Goodman, Edward L

2017-04-01

The objective of this study was to evaluate the impact of pharmacist-ordered methicillin-resistant Staphylococcus aureus (MRSA) PCR testing on the duration of empirical MRSA-targeted antibiotic therapy in patients with suspected pneumonia. This is a retrospective analysis of patients who received vancomycin or linezolid for suspected pneumonia before and after the implementation of a pharmacist-driven protocol for nasal MRSA PCR testing. Patients were included if they were adults of >18 years of age and initiated on vancomycin or linezolid for suspected MRSA pneumonia. The primary endpoint was the duration of vancomycin or linezolid therapy. After screening 368 patients, 57 patients met inclusion criteria (27 pre-PCR and 30 post-PCR). Baseline characteristics were similar between the two groups, with the majority of patients classified as having health care-associated pneumonia (68.4%). The use of the nasal MRSA PCR test reduced the mean duration of MRSA-targeted therapy by 46.6 h (74.0 ± 48.9 h versus 27.4 ± 18.7 h; 95% confidence interval [CI], 27.3 to 65.8 h; P < 0.0001). Fewer patients in the post-PCR group required vancomycin serum levels and dose adjustment (48.1% versus 16.7%; P = 0.02). There were no significant differences between the pre- and post-PCR groups regarding days to clinical improvement (1.78 ± 2.52 versus 2.27 ± 3.34; P = 0.54), length of hospital stay (11.04 ± 9.5 versus 8.2 ± 7.8; P = 0.22), or hospital mortality (14.8% versus 6.7%; P = 0.41). The use of nasal MRSA PCR testing in patients with suspected MRSA pneumonia reduced the duration of empirical MRSA-targeted therapy by approximately 2 days without increasing adverse clinical outcomes. Copyright © 2017 American Society for Microbiology.

Direct detection of Mycobacterium tuberculosis complex in bovine and bubaline tissues through nested-PCR.

PubMed

Araújo, Cristina P; Osório, Ana Luiza A R; Jorge, Klaudia S G; Ramos, Carlos A N; Souza Filho, Antonio F; Vidal, Carlos E S; Vargas, Agueda P C; Roxo, Eliana; Rocha, Adalgiza S; Suffys, Philip N; Fonseca, Antônio A; Silva, Marcio R; Barbosa Neto, José D; Cerqueira, Valíria D; Araújo, Flábio R

2014-01-01

Post-mortem bacterial culture and specific biochemical tests are currently performed to characterize the etiologic agent of bovine tuberculosis. Cultures take up to 90 days to develop. A diagnosis by molecular tests such as PCR can provide fast and reliable results while significantly decreasing the time of confirmation. In the present study, a nested-PCR system, targeting rv2807, with conventional PCR followed by real-time PCR, was developed to detect Mycobacterium tuberculosis complex (MTC) organisms directly from bovine and bubaline tissue homogenates. The sensitivity and specificity of the reactions were assessed with DNA samples extracted from tuberculous and non-tuberculous mycobacteria, as well as other Actinomycetales species and DNA samples extracted directly from bovine and bubaline tissue homogenates. Regarding the analytical sensitivity, DNA of the M. bovis AN5 strain was detected up to 1.5 pg by nested-PCR, whereas DNA of M. tuberculosis H37Rv strain was detected up to 6.1 pg. The nested-PCR system showed 100% analytical specificity for MTC when tested with DNA of reference strains of non-tuberculous mycobacteria and closely-related Actinomycetales. A clinical sensitivity level of 76.7% was detected with tissues samples positive for MTC by means of the culture and conventional PCR. A clinical specificity of 100% was detected with DNA from tissue samples of cattle with negative results in the comparative intradermal tuberculin test. These cattle exhibited no visible lesions and were negative in the culture for MTC. The use of the nested-PCR assay to detect M. tuberculosis complex in tissue homogenates provided a rapid diagnosis of bovine and bubaline tuberculosis.

Direct detection of Mycobacterium tuberculosis complex in bovine and bubaline tissues through nested-PCR

PubMed Central

Araújo, Cristina P.; Osório, Ana Luiza A.R.; Jorge, Klaudia S.G.; Ramos, Carlos A.N.; Souza Filho, Antonio F.; Vidal, Carlos E.S.; Vargas, Agueda P.C.; Roxo, Eliana; Rocha, Adalgiza S.; Suffys, Philip N.; Fonseca, Antônio A.; Silva, Marcio R.; Barbosa Neto, José D.; Cerqueira, Valíria D.; Araújo, Flábio R.

2014-01-01

Post-mortem bacterial culture and specific biochemical tests are currently performed to characterize the etiologic agent of bovine tuberculosis. Cultures take up to 90 days to develop. A diagnosis by molecular tests such as PCR can provide fast and reliable results while significantly decreasing the time of confirmation. In the present study, a nested-PCR system, targeting rv2807, with conventional PCR followed by real-time PCR, was developed to detect Mycobacterium tuberculosis complex (MTC) organisms directly from bovine and bubaline tissue homogenates. The sensitivity and specificity of the reactions were assessed with DNA samples extracted from tuberculous and non-tuberculous mycobacteria, as well as other Actinomycetales species and DNA samples extracted directly from bovine and bubaline tissue homogenates. Regarding the analytical sensitivity, DNA of the M. bovis AN5 strain was detected up to 1.5 pg by nested-PCR, whereas DNA of M. tuberculosis H37Rv strain was detected up to 6.1 pg. The nested-PCR system showed 100% analytical specificity for MTC when tested with DNA of reference strains of non-tuberculous mycobacteria and closely-related Actinomycetales. A clinical sensitivity level of 76.7% was detected with tissues samples positive for MTC by means of the culture and conventional PCR. A clinical specificity of 100% was detected with DNA from tissue samples of cattle with negative results in the comparative intradermal tuberculin test. These cattle exhibited no visible lesions and were negative in the culture for MTC. The use of the nested-PCR assay to detect M. tuberculosis complex in tissue homogenates provided a rapid diagnosis of bovine and bubaline tuberculosis. PMID:25242951

Characterization of Chinese Haemophilus parasuis Isolates by Traditional Serotyping and Molecular Serotyping Methods

PubMed Central

Ma, Lina; Wang, Liyan; Chu, Yuefeng; Li, Xuerui; Cui, Yujun; Chen, Shengli; Zhou, Jianhua; Li, Chunling; Lu, Zhongxin; Liu, Jixing; Liu, Yongsheng

2016-01-01

Haemophilus parasuis is classified mainly through serotyping, but traditional serotyping always yields non-typable (NT) strains and unreliable results via cross-reactions. Here, we surveyed the serotype prevalence of Chinese H. parasuis isolates using traditional serotyping (gel immuno-diffusion test, GID) and molecular serotyping (multiplex PCR, mPCR). We also investigated why discrepant results between these methods were obtained, and investigated mPCR failure through whole-genome sequencing. Of the 100 isolate tested, 73 (73%) and 93 (93%) were serotyped by the GID test and mPCR, respectively, with a concordance rate of 66% (66/100). Additionally, mPCR reduced the number of NT isolates from 27 (27%) for the GID testing, to seven (7%). Eleven isolates were sequenced, including nine serotype-discrepant isolates from mPCR and GID typing (excluding strains that were NT by GID only) and two NT isolates from both methods, and their in silico serotypes were obtained from genome sequencing based on their capsule loci. The mPCR results were supported by the in silico serotyping of the seven serotype-discrepant isolates. The discrepant results and NT isolates determined by mPCR were attributed to deletions and unknown sequences in the serotype-specific region of each capsule locus. Compared with previous investigations, this study found a similar predominant serotype profile, but a different prevalence frequency for H. parasuis, and the five most prevalent serotypes or strain groups were serotypes 5, 4, NT, 7 and 13 for mPCR, and serotypes 5, NT, 4, 7 and 13/10/14 for GID. Additionally, serotype 7 was recognized as a principal serotype in this work. PMID:28005999

Simultaneous detection of bovine and porcine DNA in pharmaceutical gelatin capsules by duplex PCR assay for Halal authentication.

PubMed

Nikzad, Jafar; Shahhosseini, Soraya; Tabarzad, Maryam; Nafissi-Varcheh, Nastaran; Torshabi, Maryam

2017-02-14

In the pharmaceutical industry, hard- and soft-shelled capsules are typically made from gelatin, commonly derived from bovine and porcine sources. To ensure that pharmaceutical products comply with halal regulations in Muslim countries (no porcine products allowed), development of a valid, reliable, quick, and most importantly, cost-effective tests are of utmost importance. We developed a species-specific duplex polymerase chain reaction (PCR) assay targeting 149 bp porcine and 271 bp bovine mitochondrial DNA (mtDNA) to simultaneously detect both porcine and bovine DNA (in one reaction at the same time) in gelatin. Some additional simplex PCR tests (targeting 126 bp bovine and 212 bp porcine mtDNA) and real-time PCR using a commercially available kit (for identification of porcine DNA) were used to verify the selectivity and sensitivity of our duplex PCR. After optimization of DNA extraction and PCR methods, hard/soft pharmaceutical gelatin capsules (containing drug) were tested for the presence of porcine and/or bovine DNA. Duplex PCR detected the presence of as little as 0.1% porcine DNA, which was more accurate than the commercially available kit. Of all gelatin capsules tested (n = 24), 50% contained porcine DNA (pure porcine gelatin alone or in combination with bovine gelatin). Duplex PCR presents an easy-to-follow, quick, low-cost and reliable method to simultaneously detect porcine and bovine DNAs (>100 bp) in minute amounts in highly processed gelatin-containing pharmaceutical products (with a 0.1% sensitivity for porcine DNA) which may be used for halal authentication. Simultaneous detection of porcine and bovine DNA in gelatin capsules by duplex PCR.

Value of PCR in sonication fluid for the diagnosis of orthopedic hardware-associated infections: Has the molecular era arrived?

PubMed

Renz, Nora; Cabric, Sabrina; Morgenstern, Christian; Schuetz, Michael A; Trampuz, Andrej

2018-04-01

Bone healing disturbance following fracture fixation represents a continuing challenge. We evaluated a novel fully automated polymerase chain reaction (PCR) assay using sonication fluid from retrieved orthopedic hardware to diagnose infection. In this prospective diagnostic cohort study, explanted orthopedic hardware materials from consecutive patients were investigated by sonication and the resulting sonication fluid was analyzed by culture (standard procedure) and multiplex PCR (investigational procedure). Hardware-associated infection was defined as visible purulence, presence of a sinus tract, implant on view, inflammation in peri-implant tissue or positive culture. McNemar's chi-squared test was used to compare the performance of diagnostic tests. For the clinical performance all pathogens were considered, whereas for analytical performance only microorganisms were considered for which primers are included in the PCR assay. Among 51 patients, hardware-associated infection was diagnosed in 38 cases (75%) and non-infectious causes in 13 patients (25%). The sensitivity for diagnosing infection was 66% for peri-implant tissue culture, 84% for sonication fluid culture, 71% (clinical performance) and 77% (analytical performance) for sonication fluid PCR, the specificity of all tests was >90%. The analytical sensitivity of PCR was higher for gram-negative bacilli (100%), coagulase-negative staphylococci (89%) and Staphylococcus aureus (75%) than for Cutibacterium (formerly Propionibacterium) acnes (57%), enterococci (50%) and Candida spp. (25%). The performance of sonication fluid PCR for diagnosis of orthopedic hardware-associated infection was comparable to culture tests. The additional advantage of PCR was short processing time (<5 h) and fully automated procedure. With further improvement of the performance, PCR has the potential to complement conventional cultures. Copyright © 2018 Elsevier Ltd. All rights reserved.

Comparison of FilmArray and Quantitative Real-Time Reverse Transcriptase PCR for Detection of Zaire Ebolavirus from Contrived and Clinical Specimens

PubMed Central

Southern, Timothy R.; Racsa, Lori D.; Albariño, César G.; Fey, Paul D.; Hinrichs, Steven H.; Murphy, Caitlin N.; Herrera, Vicki L.; Sambol, Anthony R.; Hill, Charles E.; Ryan, Emily L.; Kraft, Colleen S.; Campbell, Shelley; Sealy, Tara K.; Schuh, Amy; Ritchie, James C.; Lyon, G. Marshall; Mehta, Aneesh K.; Varkey, Jay B.; Ribner, Bruce S.; Brantly, Kent P.; Ströher, Ute; Iwen, Peter C.

2015-01-01

Rapid, reliable, and easy-to-use diagnostic assays for detection of Zaire ebolavirus (ZEBOV) are urgently needed. The goal of this study was to examine the agreement among emergency use authorization (EUA) tests for the detection of ZEBOV nucleic acids, including the BioFire FilmArray BioThreat (BT) panel, the FilmArray BT-E panel, and the NP2 and VP40 quantitative real-time reverse transcriptase (qRT) PCR assays from the Centers for Disease Control and Prevention (CDC). Specimens used in this study included whole blood spiked with inactivated ZEBOV at known titers and whole-blood, plasma, and urine clinical specimens collected from persons diagnosed with Ebola virus disease (EVD). The agreement for FilmArray and qRT-PCR results using contrived whole-blood specimens was 100% (6/6 specimens) for each ZEBOV dilution from 4 × 107 to 4 × 102 50% tissue culture infective dose (TCID50)/ml, as well as the no-virus negative-control sample. The limit of detection for FilmArray and qRT-PCR assays with inactivated ZEBOV, based on duplicate positive results, was determined to be 4 × 102 TCID50/ml. Rates of agreement between FilmArray and qRT-PCR results for clinical specimens from patients with EVD were 85% (23/27 specimens) for whole-blood specimens, 90% (18/20 specimens) for whole-blood specimens tested by FilmArray testing and matched plasma specimens tested by qRT-PCR testing, and 85% (11/13 specimens) for urine specimens. Among 60 specimens, eight discordant results were noted, with ZEBOV nucleic acids being detected only by FilmArray testing in four specimens and only by qRT-PCR testing in the remaining four specimens. These findings demonstrate that the rapid and easy-to-use FilmArray panels are effective tests for evaluating patients with EVD. PMID:26157148

Bat white-nose syndrome: a real-time TaqMan polymerase chain reaction test targeting the intergenic spacer region of Geomyces destructanstructans.

USGS Publications Warehouse

Muller, Laura K.; Lorch, Jeffrey M.; Lindner, Daniel L.; O'Connor, Michael; Gargas, Andrea; Blehert, David S.

2013-01-01

The fungus Geomyces destructans is the causative agent of white-nose syndrome (WNS), a disease that has killed millions of North American hibernating bats. We describe a real-time TaqMan PCR test that detects DNA from G. destructans by targeting a portion of the multicopy intergenic spacer region of the rRNA gene complex. The test is highly sensitive, consistently detecting as little as 3.3 fg of genomic DNA from G. destructans. The real-time PCR test specifically amplified genomic DNA from G. destructans but did not amplify target sequence from 54 closely related fungal isolates (including 43 Geomyces spp. isolates) associated with bats. The test was further qualified by analyzing DNA extracted from 91 bat wing skin samples, and PCR results matched histopathology findings. These data indicate the real-time TaqMan PCR method described herein is a sensitive, specific, and rapid test to detect DNA from G. destructans and provides a valuable tool for WNS diagnostics and research.

Reduction of the use of antimicrobial drugs following the rapid detection of Streptococcus agalactiae in the vagina at delivery by real-time PCR assay.

PubMed

Poncelet-Jasserand, E; Forges, F; Varlet, M-N; Chauleur, C; Seffert, P; Siani, C; Pozzetto, B; Ros, A

2013-08-01

To assess whether the determination of the presence of group B streptococci (GBS) in the vagina using a rapid polymerase chain reaction (PCR) assay at delivery was able to spare useless antimicrobial treatments, as compared with conventional culture at 34-38 weeks of gestation. Practical evaluation and prospective cost-effectiveness analysis. A university hospital in France. A cohort of 225 women in labour at the University-Hospital of Saint-Etienne. Each woman had a conventional culture performed at 34-38 weeks of gestation. At the beginning of labour, two vaginal swabs were sampled for rapid PCR testing and culture. The decision to prescribe a prophylactic antimicrobial treatment or not was taken according to the result of the PCR test. A comparative cost-effectiveness analysis of the two diagnostic strategies was carried out. Number of women receiving inadequate prophylactic antimicrobial drugs following each testing strategy, costs of PCR testing and culture, frequency of vaginal GBS, and diagnostic performance of the PCR test at delivery. The percentage of unnecessarily treated women was significantly reduced using the rapid test versus conventional culture (4.5 and 13.6%, respectively; P < 0.001). The rate of vaginal GBS at delivery was 12.5%. The incremental cost-effectiveness ratio (ICER) for each inadequate management avoided was €36 and €173 from the point of view of the healthcare system and hospital, respectively. The PCR assay reduced the number of inadequate antimicrobial treatments aimed to prevent the early onset of GBS disease. However, this strategy generates extra costs that must be put into balance with its clinical benefits. © 2013 The Authors BJOG An International Journal of Obstetrics and Gynaecology © 2013 RCOG.

Rapid and sensitive insulated isothermal PCR for point-of-need feline leukaemia virus detection.

PubMed

Wilkes, Rebecca P; Anis, Eman; Dunbar, Dawn; Lee, Pei-Yu A; Tsai, Yun-Long; Lee, Fu-Chun; Chang, Hsiao-Fen G; Wang, Hwa-Tang T; Graham, Elizabeth M

2018-04-01

Objectives Feline leukaemia virus (FeLV), a gamma retrovirus, causes diseases of the feline haematopoietic system that are invariably fatal. Rapid and accurate testing at the point-of-need (PON) supports prevention of virus spread and management of clinical disease. This study evaluated the performance of an insulated isothermal PCR (iiPCR) that detects proviral DNA, and a reverse transcription (RT)-iiPCR that detects both viral RNA and proviral DNA, for FeLV detection at the PON. Methods Mycoplasma haemofelis, feline coronavirus, feline herpesvirus, feline calicivirus and feline immunodeficiency virus were used to test analytical specificity. In vitro transcribed RNA, artificial plasmid, FeLV strain American Type Culture Collection VR-719 and a clinical FeLV isolate were used in the analytical sensitivity assays. A retrospective study including 116 clinical plasma and serum samples that had been tested with virus isolation, real-time PCR and ELISA, and a prospective study including 150 clinical plasma and serum samples were implemented to evaluate the clinical performances of the iiPCR-based methods for FeLV detection. Results Ninety-five percent assay limit of detection was calculated to be 16 RNA and five DNA copies for the RT-iiPCR, and six DNA copies for the iiPCR. Both reactions had analytical sensitivity comparable to a reference real-time PCR (qPCR) and did not detect five non-target feline pathogens. The clinical performance of the RT-iiPCR and iiPCR had 98.82% agreement (kappa[κ] = 0.97) and 100% agreement (κ = 1.0), respectively, with the qPCR (n = 85). The agreement between an automatic nucleic extraction/RT-iiPCR system and virus isolation to detect FeLV in plasma or serum was 95.69% (κ = 0.95) and 98.67% (κ = 0.85) in a retrospective (n = 116) and a prospective (n = 150) study, respectively. Conclusions and relevance These results suggested that both RT-iiPCR and iiPCR assays can serve as reliable tools for PON FeLV detection.

Review: Diagnostic accuracy of PCR-based detection tests for Helicobacter Pylori in stool samples.

PubMed

Khadangi, Fatemeh; Yassi, Maryam; Kerachian, Mohammad Amin

2017-12-01

Although different methods have been established to detect Helicobacter pylori (H. pylori) infection, identifying infected patients is an ongoing challenge. The aim of this meta-analysis was to provide pooled diagnostic accuracy measures for stool PCR test in the diagnosis of H. pylori infection. In this study, a systematic review and meta-analysis were carried out on various sources, including MEDLINE, Web of Sciences, and the Cochrane Library from April 1, 1999, to May 1, 2016. This meta-analysis adheres to the guidelines provided by the Preferred Reporting Items for Systematic Reviews and Meta-Analyses report (PRISMA Statement). The clinical value of DNA stool PCR test was based on the pooled false positive, false negative, true positive, and true negative of different genes. Twenty-six of 328 studies identified met the eligibility criteria. Stool PCR test had a performance of 71% (95% CI: 68-73) sensitivity, 96% (95% CI: 94-97) specificity, and 65.6 (95% CI: 30.2-142.5) diagnostic odds ratio (DOR) in diagnosis of H. pylori. The DOR of genes which showed the highest performance of stool PCR tests was as follows: 23S rRNA 152.5 (95% CI: 55.5-418.9), 16S rRNA 67.9 (95%CI: 6.4-714.3), and glmM 68.1 (95%CI: 20.1-231.7). The sensitivity and specificity of stool PCR test are relatively in the same spectrum of other diagnostic methods for the detection of H. pylori infection. In descending order of significance, the most diagnostic candidate genes using PCR detection were 23S rRNA, 16S rRNA, and glmM. PCR for 23S rRNA gene which has the highest performance could be applicable to detect H. pylori infection. © 2017 John Wiley & Sons Ltd.

Development of reverse transcription-PCR assays specific for detection of equine encephalitis viruses.

PubMed

Linssen, B; Kinney, R M; Aguilar, P; Russell, K L; Watts, D M; Kaaden, O R; Pfeffer, M

2000-04-01

Specific and sensitive reverse transcription-PCR (RT-PCR) assays were developed for the detection of eastern, western, and Venezuelan equine encephalitis viruses (EEE, WEE, and VEE, respectively). Tests for specificity included all known alphavirus species. The EEE-specific RT-PCR amplified a 464-bp region of the E2 gene exclusively from 10 different EEE strains from South and North America with a sensitivity of about 3,000 RNA molecules. In a subsequent nested PCR, the specificity was confirmed by the amplification of a 262-bp fragment, increasing the sensitivity of this assay to approximately 30 RNA molecules. The RT-PCR for WEE amplified a fragment of 354 bp from as few as 2,000 RNA molecules. Babanki virus, as well as Mucambo and Pixuna viruses (VEE subtypes IIIA and IV), were also amplified. However, the latter viruses showed slightly smaller fragments of about 290 and 310 bp, respectively. A subsequent seminested PCR amplified a 195-bp fragment only from the 10 tested strains of WEE from North and South America, rendering this assay virus specific and increasing its sensitivity to approximately 20 RNA molecules. Because the 12 VEE subtypes showed too much divergence in their 26S RNA nucleotide sequences to detect all of them by the use of nondegenerate primers, this assay was confined to the medically important and closely related VEE subtypes IAB, IC, ID, IE, and II. The RT-PCR-seminested PCR combination specifically amplified 342- and 194-bp fragments of the region covering the 6K gene in VEE. The sensitivity was 20 RNA molecules for subtype IAB virus and 70 RNA molecules for subtype IE virus. In addition to the subtypes mentioned above, three of the enzootic VEE (subtypes IIIB, IIIC, and IV) showed the specific amplicon in the seminested PCR. The practicability of the latter assay was tested with human sera gathered as part of the febrile illness surveillance in the Amazon River Basin of Peru near the city of Iquitos. All of the nine tested VEE-positive sera showed the expected 194-bp amplicon of the VEE-specific RT-PCR-seminested PCR.

Nested-PCR real time as alternative molecular tool for detection of Borrelia burgdorferi compared to the classical serological diagnosis of the blood.

PubMed

Sroka-Oleksiak, Agnieszka; Ufir, Krzysztof; Salamon, Dominika; Bulanda, Malgorzata; Gosiewski, Tomasz

Lyme disease, caused by Borrelia burgdorferi, is a multisystem disease that often makes difficulties to recognize caused by their genetic heterogenity. Currently, the gold standard for the detection of Lyme disease (LD) is serologic diagnostics based mainly on tests: ELISA and Western blot (WB). These methods, however, are subject to consider- able defect, especially in the initial phase of infection due to the occurrence of so-called serological window period and low specificity. For this reason, they might be replaced by molecular methods, for example polymerase chain reaction (PCR), which should be more sensitivity and specificity. In the present study we attempt to optimize the PCR reaction conditions and enhance existing test sensitivity by applying the equivalent of real time PCR - nested PCR for detection B. burgdorferi DNA in the patient's blood. The study involved 94 blood samples of patients with suspected LD. From each sample, 1.5 ml of blood was used for the isolation of bacterial DNA and PCR real time am- plification and its equivalent, in nested version. The remaining part earmarked for serologi- cal testing. Optimization of the reaction conditions made experimentally, using gradient of the temperature and gradient of the magnesium ions concentration for reaction real time in nested-PCR and PCR version. The results show that the nested-PCR real time, has a much higher sensitivity 45 (47.8%) of positive results for the detection of B. burgdorferi compared to the single- variety, without a preceding pre-amplification 2 (2.1%). Serological methods allowed the detection of infection in 41 (43.6%) samples. These results support of the nested PCR method as a better molecular tool for the detection of B. burgdorferi infection than classical PCR real time reaction. The nested-PCR real time method may be considered as a complement to ELISA and WB mainly in the early stages of infection, when in the blood circulating B. burgdorferi cells. By contrast, the results of serological and molecular tests should always be carried out tak- ing into account the patient's clinical status.

Virological Diagnosis of Central Nervous System Infections by Use of PCR Coupled with Mass Spectrometry Analysis of Cerebrospinal Fluid Samples

PubMed Central

Lévêque, Nicolas; Legoff, Jérôme; Mengelle, Catherine; Mercier-Delarue, Séverine; N'guyen, Yohan; Renois, Fanny; Tissier, Fabien; Simon, François; Izopet, Jacques

2014-01-01

Viruses are the leading cause of central nervous system (CNS) infections, ahead of bacteria, parasites, and fungal agents. A rapid and comprehensive virologic diagnostic testing method is needed to improve the therapeutic management of hospitalized pediatric or adult patients. In this study, we assessed the clinical performance of PCR amplification coupled with electrospray ionization-time of flight mass spectrometry analysis (PCR-MS) for the diagnosis of viral CNS infections. Three hundred twenty-seven cerebrospinal fluid (CSF) samples prospectively tested by routine PCR assays between 2004 and 2012 in two university hospital centers (Toulouse and Reims, France) were retrospectively analyzed by PCR-MS analysis using primers targeted to adenovirus, human herpesviruses 1 to 8 (HHV-1 to -8), polyomaviruses BK and JC, parvovirus B19, and enteroviruses (EV). PCR-MS detected single or multiple virus infections in 190 (83%) of the 229 samples that tested positive by routine PCR analysis and in 10 (10.2%) of the 98 samples that tested negative. The PCR-MS results correlated well with herpes simplex virus 1 (HSV-1), varicella-zoster virus (VZV), and EV detection by routine PCR assays (kappa values [95% confidence intervals], 0.80 [0.69 to 0.92], 0.85 [0.71 to 0.98], and 0.84 [0.78 to 0.90], respectively), whereas a weak correlation was observed with Epstein-Barr virus (EBV) (0.34 [0.10 to 0.58]). Twenty-six coinfections and 16 instances of uncommon neurotropic viruses (HHV-7 [n = 13], parvovirus B19 [n = 2], and adenovirus [n = 1]) were identified by the PCR-MS analysis, whereas only 4 coinfections had been prospectively evidenced using routine PCR assays (P < 0.01). In conclusion, our results demonstrated that PCR-MS analysis is a valuable tool to identify common neurotropic viruses in CSF (with, however, limitations that were identified regarding EBV and EV detection) and may be of major interest in better understanding the clinical impact of multiple or neglected viral neurological infections. PMID:24197874

Systematic review and meta-analysis: rapid diagnostic tests versus placental histology, microscopy and PCR for malaria in pregnant women.

PubMed

Kattenberg, Johanna H; Ochodo, Eleanor A; Boer, Kimberly R; Schallig, Henk Dfh; Mens, Petra F; Leeflang, Mariska Mg

2011-10-28

During pregnancy, malaria infection with Plasmodium falciparum or Plasmodium vivax is related to adverse maternal health and poor birth outcomes. Diagnosis of malaria, during pregnancy, is complicated by the absence or low parasite densities in peripheral blood. Diagnostic methods, other than microscopy, are needed for detection of placental malaria. Therefore, the diagnostic accuracy of rapid diagnostic tests (RDTs), detecting antigen, and molecular techniques (PCR), detecting DNA, for the diagnosis of Plasmodium infections in pregnancy was systematically reviewed. MEDLINE, EMBASE and Web of Science were searched for studies assessing the diagnostic accuracy of RDTs, PCR, microscopy of peripheral and placental blood and placental histology for the detection of malaria infection (all species) in pregnant women. The results of 49 studies were analysed in metandi (Stata), of which the majority described P. falciparum infections. Although both placental and peripheral blood microscopy cannot reliably replace histology as a reference standard for placental P. falciparum infection, many studies compared RDTs and PCR to these tests. The proportion of microscopy positives in placental blood (sensitivity) detected by peripheral blood microscopy, RDTs and PCR are respectively 72% [95% CI 62-80], 81% [95% CI 55-93] and 94% [95% CI 86-98]. The proportion of placental blood microscopy negative women that were negative in peripheral blood microscopy, RDTs and PCR (specificity) are 98% [95% CI 95-99], 94% [95% CI 76-99] and 77% [95% CI 71-82]. Based on the current data, it was not possible to determine if the false positives in RDTs and PCR are caused by sequestered parasites in the placenta that are not detected by placental microscopy. The findings suggest that RDTs and PCR may have good performance characteristics to serve as alternatives for the diagnosis of malaria in pregnancy, besides any other limitations and practical considerations concerning the use of these tests. Nevertheless, more studies with placental histology as reference test are urgently required to reliably determine the accuracy of RDTs and PCR for the diagnosis of placental malaria. P. vivax-infections have been neglected in diagnostic test accuracy studies of malaria in pregnancy.

Systematic review and meta-analysis: rapid diagnostic tests versus placental histology, microscopy and PCR for malaria in pregnant women

PubMed Central

2011-01-01

Background During pregnancy, malaria infection with Plasmodium falciparum or Plasmodium vivax is related to adverse maternal health and poor birth outcomes. Diagnosis of malaria, during pregnancy, is complicated by the absence or low parasite densities in peripheral blood. Diagnostic methods, other than microscopy, are needed for detection of placental malaria. Therefore, the diagnostic accuracy of rapid diagnostic tests (RDTs), detecting antigen, and molecular techniques (PCR), detecting DNA, for the diagnosis of Plasmodium infections in pregnancy was systematically reviewed. Methods MEDLINE, EMBASE and Web of Science were searched for studies assessing the diagnostic accuracy of RDTs, PCR, microscopy of peripheral and placental blood and placental histology for the detection of malaria infection (all species) in pregnant women. Results The results of 49 studies were analysed in metandi (Stata), of which the majority described P. falciparum infections. Although both placental and peripheral blood microscopy cannot reliably replace histology as a reference standard for placental P. falciparum infection, many studies compared RDTs and PCR to these tests. The proportion of microscopy positives in placental blood (sensitivity) detected by peripheral blood microscopy, RDTs and PCR are respectively 72% [95% CI 62-80], 81% [95% CI 55-93] and 94% [95% CI 86-98]. The proportion of placental blood microscopy negative women that were negative in peripheral blood microscopy, RDTs and PCR (specificity) are 98% [95% CI 95-99], 94% [95% CI 76-99] and 77% [95% CI 71-82]. Based on the current data, it was not possible to determine if the false positives in RDTs and PCR are caused by sequestered parasites in the placenta that are not detected by placental microscopy. Conclusion The findings suggest that RDTs and PCR may have good performance characteristics to serve as alternatives for the diagnosis of malaria in pregnancy, besides any other limitations and practical considerations concerning the use of these tests. Nevertheless, more studies with placental histology as reference test are urgently required to reliably determine the accuracy of RDTs and PCR for the diagnosis of placental malaria. P. vivax-infections have been neglected in diagnostic test accuracy studies of malaria in pregnancy. PMID:22035448

[Comparison of the CMV antigenemia test and CMV-DNA PCR results in solid organ transplant recipients].

PubMed

Özkarata, Emre; Özkarataş, Emre; Özbek, Ö Alpay; Avkan Oğuz, Vildan; Sayıner, A Arzu

2016-01-01

Cytomegalovirus (CMV) infection is among the most common important viral infections in solid organ transplant (SOT) recipients. Diagnostic tests for detecting CMV replication are widely used for this group of patients, however there is no clear agreement on the cut-off levels for interpretation of clinical decisions especially when the low level of viral load is detected. In this study, CMV pp65 antigenemia test results were compared with plasma CMV-DNA levels detected by quantitative real-time polymerase chain reaction (qPCR) in samples of kidney and liver transplant recipients in the Central Laboratory of Dokuz Eylul University Hospital between 2011 and 2013, and the correlation between these two tests and viral load equivalent to antigenemia positivity were determined. In the study, pp65 antigenemia and CMV-DNA qPCR results were evaluated retrospectively. The samples from the same patients were included if the time between antigenemia and CMV-DNA qPCR tests were less than 48 hours. SPSS v15.0 was used for correlation, regression and ROC curve analysis. The results of the 217 samples collected from 100 patients (59 male, 41 female; age range: 16-71, mean age: 46 ± 13 years), 36 liver and 64 kidney recipients were evaluated in the study. Of the patients 80% were CMV IgM negative, IgG positive; 1% was CMV IgG and IgM positive; 2% were CMV IgM and IgG negative, while for 17 patients serological results could not be reached. CMV pp65 antigenemia and CMV-DNA were both negative in 102 (47%) samples, while both were positive in 37 (17%) samples. The single sample from a case with CMV IgM and IgG positivity yielded negative results for both antigenemia and CMV-DNA tests. In 78 samples antigenemia were negative and CMV-DNA qPCR were positive, while there were no samples with antigenemia positive and qPCR negative. Mean values of antigenemia and qPCR tests were 23 positive cells/200.000 leukocytes (range: 1 to 230 positive cells) and 12.595 copies/ml (range: 180 to 106.311 copies/ml), respectively. There was a significant correlation between antigenemia and qPCR results among the samples that were positive by both assays (r= 0.785). ROC curve analysis showed that CMV viral load of 205 copies/ml in plasma corresponds to ≥ 1 pp65 antigen positive cells per 200.000 leukocytes (sensitivity: 91.7%, specificity: 90.3%). Higher analytical sensitivity of qPCR test can be explained by the results of CMV-DNA PCR positive and antigenemia negative samples. Non-existence of samples with antigen positive and PCR negative results supported this finding. ROC analysis showed that any sample with CMV-DNA qPCR result less than 205 copies/ml, could be accepted as pp65 antigenemia negative. This viral load value is valid only for the studied patient group and assays, therefore could be changed according to study population and tests.

Loop-Mediated Isothermal Amplification for Laboratory Confirmation of Buruli Ulcer Disease—Towards a Point-of-Care Test

PubMed Central

Beissner, Marcus; Phillips, Richard Odame; Battke, Florian; Bauer, Malkin; Badziklou, Kossi; Sarfo, Fred Stephen; Maman, Issaka; Rhomberg, Agata; Piten, Ebekalisai; Frimpong, Michael; Huber, Kristina Lydia; Symank, Dominik; Jansson, Moritz; Wiedemann, Franz Xaver; Banla Kere, Abiba; Herbinger, Karl-Heinz; Löscher, Thomas; Bretzel, Gisela

2015-01-01

Background As the major burden of Buruli ulcer disease (BUD) occurs in remote rural areas, development of point-of-care (POC) tests is considered a research priority to bring diagnostic services closer to the patients. Loop-mediated isothermal amplification (LAMP), a simple, robust and cost-effective technology, has been selected as a promising POC test candidate. Three BUD-specific LAMP assays are available to date, but various technical challenges still hamper decentralized application. To overcome the requirement of cold-chains for transport and storage of reagents, the aim of this study was to establish a dry-reagent-based LAMP assay (DRB-LAMP) employing lyophilized reagents. Methodology/Principal Findings Following the design of an IS2404 based conventional LAMP (cLAMP) assay suitable to apply lyophilized reagents, a lyophylization protocol for the DRB-LAMP format was developed. Clinical performance of cLAMP was validated through testing of 140 clinical samples from 91 suspected BUD cases by routine assays, i.e. IS2404 dry-reagent-based (DRB) PCR, conventional IS2404 PCR (cPCR), IS2404 qPCR, compared to cLAMP. Whereas qPCR rendered an additional 10% of confirmed cases and samples respectively, case confirmation and positivity rates of DRB-PCR or cPCR (64.84% and 56.43%; 100% concordant results in both assays) and cLAMP (62.64% and 52.86%) were comparable and there was no significant difference between the sensitivity of the assays (DRB PCR and cPCR, 86.76%; cLAMP, 83.82%). Likewise, sensitivity of cLAMP (95.83%) and DRB-LAMP (91.67%) were comparable as determined on a set of 24 samples tested positive in all routine assays. Conclusions/Significance Both LAMP formats constitute equivalent alternatives to conventional PCR techniques. Provided the envisaged availability of field friendly DNA extraction formats, both assays are suitable for decentralized laboratory confirmation of BUD, whereby DRB-LAMP scores with the additional advantage of not requiring cold-chains. As validation of the assays was conducted in a third-level laboratory environment, field based evaluation trials are necessary to determine the clinical performance at peripheral health care level. PMID:26566026

Molecular diagnosis of canine visceral leishmaniasis: a comparative study of three methods using skin and spleen from dogs with natural Leishmania infantum infection.

PubMed

Reis, Levi Eduardo Soares; Coura-Vital, Wendel; Roatt, Bruno Mendes; Bouillet, Leoneide Érica Maduro; Ker, Henrique Gama; Fortes de Brito, Rory Cristiane; Resende, Daniela de Melo; Carneiro, Mariângela; Giunchetti, Rodolfo Cordeiro; Marques, Marcos José; Carneiro, Cláudia Martins; Reis, Alexandre Barbosa

2013-11-08

Polymerase chain reaction (PCR) and its variations represent highly sensitive and specific methods for Leishmania DNA detection and subsequent canine visceral leishmaniasis (CVL) diagnosis. The aim of this work was to compare three different molecular diagnosis techniques (conventional PCR [cPCR], seminested PCR [snPCR], and quantitative PCR [qPCR]) in samples of skin and spleen from 60 seropositive dogs by immunofluorescence antibody test and enzyme-linked immunosorbent assay. Parasitological analysis was conducted by culture of bone marrow aspirate and optical microscopic assessment of ear skin and spleen samples stained with Giemsa, the standard tests for CVL diagnosis. The primers L150/L152 and LINR4/LIN17/LIN19 were used to amplify the conserved region of the Leishmania kDNA minicircle in the cPCR, and snPCR and qPCR were performed using the DNA polymerase gene (DNA pol α) primers from Leishmania infantum. The parasitological analysis revealed parasites in 61.7% of the samples. Sensitivities were 89.2%, 86.5%, and 97.3% in the skin and 81.1%, 94.6%, and 100.0% in spleen samples used for cPCR, snPCR, and qPCR, respectively. We demonstrated that the qPCR method was the best technique to detect L. infantum in both skin and spleen samples. However, we recommend the use of skin due to the high sensitivity and sampling being less invasive. Copyright © 2013 Elsevier B.V. All rights reserved.

Recent sequence variation in probe binding site affected detection of respiratory syncytial virus group B by real-time RT-PCR.

PubMed

Kamau, Everlyn; Agoti, Charles N; Lewa, Clement S; Oketch, John; Owor, Betty E; Otieno, Grieven P; Bett, Anne; Cane, Patricia A; Nokes, D James

2017-03-01

Direct immuno-fluorescence test (IFAT) and multiplex real-time RT-PCR have been central to RSV diagnosis in Kilifi, Kenya. Recently, these two methods showed discrepancies with an increasing number of PCR undetectable RSV-B viruses. Establish if mismatches in the primer and probe binding sites could have reduced real-time RT-PCR sensitivity. Nucleoprotein (N) and glycoprotein (G) genes were sequenced for real-time RT-PCR positive and negative samples. Primer and probe binding regions in N gene were checked for mismatches and phylogenetic analyses done to determine molecular epidemiology of these viruses. New primers and probe were designed and tested on the previously real-time RT-PCR negative samples. N gene sequences revealed 3 different mismatches in the probe target site of PCR negative, IFAT positive viruses. The primers target sites had no mismatches. Phylogenetic analysis of N and G genes showed that real-time RT-PCR positive and negative samples fell into distinct clades. Newly designed primers-probe pair improved detection and recovered previous PCR undetectable viruses. An emerging RSV-B variant is undetectable by a quite widely used real-time RT-PCR assay due to polymorphisms that influence probe hybridization affecting PCR accuracy. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Development of a reference material of a single DNA molecule for the quality control of PCR testing.

PubMed

Mano, Junichi; Hatano, Shuko; Futo, Satoshi; Yoshii, Junji; Nakae, Hiroki; Naito, Shigehiro; Takabatake, Reona; Kitta, Kazumi

2014-09-02

We developed a reference material of a single DNA molecule with a specific nucleotide sequence. The double-strand linear DNA which has PCR target sequences at the both ends was prepared as a reference DNA molecule, and we named the PCR targets on each side as confirmation sequence and standard sequence. The highly diluted solution of the reference molecule was dispensed into 96 wells of a plastic PCR plate to make the average number of molecules in a well below one. Subsequently, the presence or absence of the reference molecule in each well was checked by real-time PCR targeting for the confirmation sequence. After an enzymatic treatment of the reaction mixture in the positive wells for the digestion of PCR products, the resultant solution was used as the reference material of a single DNA molecule with the standard sequence. PCR analyses revealed that the prepared samples included only one reference molecule with high probability. The single-molecule reference material developed in this study will be useful for the absolute evaluation of a detection limit of PCR-based testing methods, the quality control of PCR analyses, performance evaluations of PCR reagents and instruments, and the preparation of an accurate calibration curve for real-time PCR quantitation.

Droplet digital PCR (ddPCR) vs quantitative real-time PCR (qPCR) approach for detection and quantification of Merkel cell polyomavirus (MCPyV) DNA in formalin fixed paraffin embedded (FFPE) cutaneous biopsies.

PubMed

Arvia, Rosaria; Sollai, Mauro; Pierucci, Federica; Urso, Carmelo; Massi, Daniela; Zakrzewska, Krystyna

2017-08-01

Merkel cell polyomavirus (MCPyV) is associated with Merkel cell carcinoma and high viral load in the skin was proposed as a risk factor for the occurrence of this tumour. MCPyV DNA was detected, with lower frequency, in different skin cancers but since the viral load was usually low, the real prevalence of viral DNA could be underestimated. To evaluate the performance of two assays (qPCR and ddPCR) for MCPyV detection and quantification in formalin fixed paraffin embedded (FFPE) tissue samples. Both assays were designed to simultaneous detection and quantification of both MCPyV as well as house-keeping DNA in clinical samples. The performance of MCPyV quantification was investigated using serial dilutions of cloned target DNA. We also evaluated the applicability of both tests for the analysis of 76 FFPE cutaneous biopsies. The two approaches resulted equivalent with regard to the reproducibility and repeatability and showed a high degree of linearity in the dynamic range tested in the present study. Moreover, qPCR was able to quantify ≥10 5 copies per reaction, while the upper limit of ddPCR was 10 4 copies. There was not significant difference between viral load measured by the two methods The detection limit of both tests was 0,15 copies per reaction, however, the number of positive samples obtained by ddPCR was higher than that obtained by qPCR (45% and 37% respectively). The ddPCR represents a better method for detection of MCPyV in FFPE biopsies, mostly these containing low copies number of viral genome. Copyright © 2017 Elsevier B.V. All rights reserved.

Comparison of Direct Sequencing, Real-Time PCR-High Resolution Melt (PCR-HRM) and PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) Analysis for Genotyping of Common Thiopurine Intolerant Variant Alleles NUDT15 c.415C>T and TPMT c.719A>G (TPMT*3C).

PubMed

Fong, Wai-Ying; Ho, Chi-Chun; Poon, Wing-Tat

2017-05-12

Thiopurine intolerance and treatment-related toxicity, such as fatal myelosuppression, is related to non-function genetic variants encoding thiopurine S-methyltransferase (TPMT) and Nudix hydrolase 15 (NUDT15). Genetic testing of the common variants NUDT15:NM_018283.2:c.415C>T (Arg139Cys, dbSNP rs116855232 T allele) and TPMT: NM_000367.4:c.719A>G (TPMT*3C, dbSNP rs1142345 G allele) in East Asians including Chinese can potentially prevent treatment-related complications. Two complementary genotyping approaches, real-time PCR-high resolution melt (PCR-HRM) and PCR-restriction fragment length morphism (PCR-RFLP) analysis were evaluated using conventional PCR and Sanger sequencing genotyping as the gold standard. Sixty patient samples were tested, revealing seven patients (11.7%) heterozygous for NUDT15 c.415C>T, one patient homozygous for the variant and one patient heterozygous for the TPMT*3C non-function allele. No patient was found to harbor both variants. In total, nine out of 60 (15%) patients tested had genotypic evidence of thiopurine intolerance, which may require dosage adjustment or alternative medication should they be started on azathioprine, mercaptopurine or thioguanine. The two newly developed assays were more efficient and showed complete concordance (60/60, 100%) compared to the Sanger sequencing results. Accurate and cost-effective genotyping assays by real-time PCR-HRM and PCR-RFLP for NUDT15 c.415C>T and TPMT*3C were successfully developed. Further studies may establish their roles in genotype-informed clinical decision-making in the prevention of morbidity and mortality due to thiopurine intolerance.

Evaluation of a Commercial Multiplex PCR for Rapid Detection of Multi Drug Resistant Gram Negative Infections

PubMed Central

Chavada, Ruchir; Maley, Michael

2015-01-01

Introduction: Community and healthcare associated infections caused by multi-drug resistant gram negative organisms (MDR GN) represent a worldwide threat. Nucleic Acid Detection tests are becoming more common for their detection; however they can be expensive requiring specialised equipment and local expertise. This study was done to evaluate the utility of a commercial multiplex tandem (MT) PCR for detection of MDR GN. Methods: The study was done on stored laboratory MDR GN isolates from sterile and non-sterile specimens (n=126, out of stored 567 organisms). Laboratory validation of the MT PCR was done to evaluate sensitivity, specificity and agreement with the current phenotypic methods used in the laboratory. Amplicon sequencing was also done on selected isolates for assessing performance characteristics. Workflow and cost implications of the MT PCR were evaluated. Results: The sensitivity and specificity of the MT PCR were calculated to be 95% and 96.7% respectively. Agreement with the phenotypic methods was 80%. Major lack of agreement was seen in detection of AmpC beta lactamase in enterobacteriaceae and carbapenemase in non-fermenters. Agreement of the MT PCR with another multiplex PCR was found to be 87%. Amplicon sequencing confirmed the genotype detected by MT PCR in 94.2 % of cases tested. Time to result was faster for the MT PCR but cost per test was higher. Conclusion: This study shows that with carefully chosen targets for detection of resistance genes in MDR GN, rapid and efficient identification is possible. MT PCR was sensitive and specific and likely more accurate than phenotypic methods. PMID:26464612

Development, validation and implementation of a quadruplex real-time PCR assay for identification of potentially toxigenic corynebacteria.

PubMed

De Zoysa, Aruni; Efstratiou, Androulla; Mann, Ginder; Harrison, Timothy G; Fry, Norman K

2016-12-01

Toxigenic corynebacteria are uncommon in the UK; however, laboratory confirmation by the national reference laboratory can inform public health action according to national guidelines. Standard phenotypic tests for identification and toxin expression of isolates can take from ≥24 to ≥48 h from receipt. To decrease the time to result, a real-time PCR (qPCR) assay was developed for confirmation of both identification of Corynebacterium diphtheriae and Corynebacterium ulcerans/Corynebacterium pseudotuberculosis and detection of the diphtheria toxin gene. Target genes were the RNA polymerase β-subunit-encoding gene (rpoB) and A-subunit of the diphtheria toxin gene (tox). Green fluorescent protein DNA (gfp) was used as an internal process control. qPCR results were obtained within 3 to 4 h after receipt of isolate. The assay was validated according to published guidelines and demonstrated high diagnostic sensitivity (100 %), high specificity (98-100 %) and positive and negative predictive values of 91 to 100 % and 100 %, respectively, compared to both block-based PCR and the Elek test, together with a greatly reduced time from isolate receipt to reporting. Limitations of the qPCR assay were the inability to distinguish between C. ulcerans and C. pseudotuberculosis and that the presence of the toxin gene as demonstrated by qPCR may not always predict toxin expression. Thus, confirmation of expression of diphtheria toxin is always sought using the phenotypic Elek test. The new qPCR assay was formally introduced as the front-line test for putative toxigenic corynebacteria to inform public health action in England and Wales on 1 April 2014.

Use of PCR and Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis Techniques for Differentiation of Prevotella intermedia Sensu Stricto and Prevotella nigrescens

PubMed Central

Premaraj, Thyagaseely; Kato, Naoki; Fukui, Katsuhito; Kato, Haru; Watanabe, Kunitomo

1999-01-01

Primers were designed from 16S rRNA sequences of Prevotella intermedia sensu stricto and Prevotella nigrescens and were used to discriminate these two species by PCR. The results were compared with those from the PCR technique using primers designed from arbitrarily primed PCR products by Guillot and Mouton (E. Guillot and C. Mouton, J. Clin. Microbiol. 35:1876–1882, 1997). The specificities of both assays were studied by using P. intermedia ATCC 25611, P. nigrescens ATCC 33563, 174 clinical isolates of P. intermedia sensu lato, and 59 reference strains and 58 clinical isolates of other Prevotella species and/or common oral flora. In addition, the usefulness and reliability of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the differentiation of the two species were examined by comparing the results with those from PCR assays. The controversial lipase test for distinguishing these species was also carried out. Unambiguous differentiation was made by both PCR assays, and the results matched each other. The SDS-PAGE assay was found to misidentify a few strains tested, compared with the results of PCR assays. The lipase test was positive for both species, including the reference strains of P. intermedia and P. nigrescens. We conclude that both PCR assays are simple, rapid, reliable, and specific methods which could be used in clinical studies and that the lipase test is not valuable in the differentiation. The reliable discrimination of the two species by SDS-PAGE is questionable. PMID:10074526

Quantitative and Qualitative Changes in V-J α Rearrangements During Mouse Thymocytes Differentiation

PubMed Central

Pasqual, Nicolas; Gallagher, Maighréad; Aude-Garcia, Catherine; Loiodice, Mélanie; Thuderoz, Florence; Demongeot, Jacques; Ceredig, Rod; Marche, Patrice Noël; Jouvin-Marche, Evelyne

2002-01-01

Knowledge of the complete nucleotide sequence of the mouse TCRAD locus allows an accurate determination V-J rearrangement status. Using multiplex genomic PCR assays and real time PCR analysis, we report a comprehensive and systematic analysis of the V-J recombination of TCR α chain in normal mouse thymocytes during development. These respective qualitative and quantitative approaches give rise to four major points describing the control of gene rearrangements. (a) The V-J recombination pattern is not random during ontogeny and generates a limited TCR α repertoire; (b) V-J rearrangement control is intrinsic to the thymus; (c) each V gene rearranges to a set of contiguous J segments with a gaussian-like frequency; (d) there are more rearrangements involving V genes at the 3′ side than 5′ end of V region. Taken together, this reflects a preferential association of V and J gene segments according to their respective positions in the locus, indicating that accessibility of both V and J regions is coordinately regulated, but in different ways. These results provide a new insight into TCR α repertoire size and suggest a scenario for V usage during differentiation. PMID:12417627

Effect of Genital Sampling Site on the Detection and Quantification of Ureaplasma Species with Quantitative Polymerase Chain Reaction during Pregnancy.

PubMed

Faron, Gilles; Vancutsem, Ellen; Naessens, Anne; Buyl, Ronald; Gucciardo, Leonardo; Foulon, Walter

2017-01-01

Objective . This study aimed to compare the qualitative and quantitative reproducibility of quantitative PCR (qPCR) for Ureaplasma species (Ureaplasma spp.) throughout pregnancy and according to the genital sampling site. Study Design . Between 5 and 14 weeks of gestation (T1), vaginal, fornix, and two cervical samples were taken. Sampling was repeated during the 2nd (T2) and 3rd (T3) trimester in randomly selected T1 positive and negative women. Qualitative and quantitative reproducibility were evaluated using, respectively, Cohen's kappa ( κ ) and interclass correlation coefficients (ICC) and repeated measures ANOVA on the log-transformed mean number of DNA copies for each sampling site. Results . During T1, 51/127 women were positive for U. parvum and 8 for U. urealyticum (4 patients for both). Sampling was repeated for 44/55 women at T2 and/or T3; 43 (97.7%) remained positive at the three timepoints. κ ranged between 0.83 and 0.95 and the ICC for cervical samples was 0.86. Conclusions . Colonization by Ureaplasma spp. seems to be very constant during pregnancy and vaginal samples have the highest detection rate.

Cytomegalovirus infection in inflammatory bowel disease is not associated with worsening of intestinal inflammatory activity.

PubMed

do Carmo, Alexandre Medeiros; Santos, Fabiana Maria; Ortiz-Agostinho, Carmen Lucia; Nishitokukado, Iêda; Frota, Cintia S; Gomes, Flavia Ubeda; Leite, André Zonetti de Arruda; Pannuti, Claudio Sérgio; Boas, Lucy Santos Vilas; Teixeira, Magaly Gemio; Sipahi, Aytan Miranda

2014-01-01

Cytomegalovirus is highly prevalent virus and usually occurs in immunocompromised patients. The pathophysiology and treatment of inflammatory bowel disease often induce a state of immunosuppression. Because this, there are still doubts and controversies about the relationship between inflammatory bowel disease and cytomegalovirus. Evaluate the frequency of cytomegalovirus in patients with inflammatory bowel disease and identify correlations. Patients with inflammatory bowel disease underwent an interview, review of records and collection of blood and fecal samples. The search for cytomegalovirus was performed by IgG and IgM blood serology, by real-time PCR in the blood and by qualitative PCR in feces. Results were correlated with red blood cell levels, C-reactive protein levels, erythrocyte sedimentation rates and fecal calprotectin levels for each patient. Among the 400 eligible patients, 249 had Crohn's disease, and 151 had ulcerative colitis. In the group of Crohn's disease, 67 of the patients had moderate or severe disease, but 126 patients presented with active disease, based on the evaluation of the fecal calprotectin. In patients with ulcerative colitis, only 21 patients had moderate disease, but 76 patients presented with active disease, based on the evaluation of the fecal calprotectin. A large majority of patients had positive CMV IgG. Overall, 10 patients had positive CMV IgM, and 9 patients had a positive qualitative detection of CMV DNA by PCR in the feces. All 400 patients returned negative results after the quantitative detection of CMV DNA in blood by real-time PCR. Analyzing the 19 patients with active infections, we only found that such an association occurred with the use of combined therapy (anti-TNF-alpha + azathioprine). The findings show that latent cytomegalovirus infections are frequent and active cytomegalovirus infection is rare. We did not find any association between an active infection of CMV and inflammatory bowel disease activity.

Roche/BIOTECON Diagnostics LightCycler foodproof L. monocytogenes detection kit in combination with ShortPrep foodproof II Kit. Performance-Tested Method 070401.

PubMed

Junge, Benjamin; Berghof-Jäger, Kornelia

2006-01-01

A method was developed for the detection of L. monocytogenes in food based on real-time polymerase chain reaction (PCR). This advanced PCR method was designed to reduce the time needed to achieve results from PCR reactions and to enable the user to monitor the amplification of the PCR product simultaneously, in real-time. After DNA isolation using the Roche/BIOTECON Diagnostics ShortPrep foodproof II Kit (formerly called Listeria ShortPrep Kit) designed for the rapid preparation of L. monocytogenes DNA for direct use in PCR, the real-time detection of L. monocytogenes DNA is performed by using the Roche/BIOTECON Diagnostics LightCycler foodproof L. monocytogenes Detection Kit. This kit provides primers and hybridization probes for sequence-specific detection, convenient premixed reagents, and different controls for reliable interpretation of results. For repeatability studies, 20 different foods, covering the 15 food groups recommended from the AOAC Research Institute (AOAC RI) for L. monocytogenes detection were analyzed: raw meats, fresh produce/vegetables, processed meats, seafood, egg and egg products, dairy (cultured/noncultured), spices, dry foods, fruit/juices, uncooked pasta, nuts, confectionery, pet food, food dyes and colorings, and miscellaneous. From each food 20, samples were inoculated with a low level (1-10 colony-forming units (CFU)/25 g) and 20 samples with a high level (10-50 CFU/25 g) of L. monocytogenes. Additionally, 5 uninoculated samples were prepared from each food. The food samples were examined with the test kits and in correlation with the cultural methods according to U.S. Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) or U.S. Department of Agriculture (USDA)/Food Safety and Inspection Service (FSIS) Microbiology Laboratory Guidebook. After 48 h of incubation, the PCR method in all cases showed equal or better results than the reference cultural FDA/BAM or USDA/FSIS methods. Fifteen out of 20 tested food types gave exactly the same amount of positive samples for both methods in both inoculation levels. For 5 out of 20 foodstuffs, the PCR method resulted in more positives than the reference method after 48 h of incubation. Following AOAC RI definition, these were false positives because they were not confirmed by the reference method (false-positive rate for low inoculated foodstuffs: 5.4%; for high inoculated foodstuffs: 7.1%). Without calculating these unconfirmed positives, the PCR method showed equal sensitivity results compared to the alternative method. With the unconfirmed PCR-positives included into the calculations, the alternative PCR method showed a higher sensitivity than the microbiological methods (low inoculation level: 100 vs 98.0%; sensitivity rate: 1; high inoculation level: 99.7 vs 97.7%; sensitivity rate, 1). All in-house and independently tested uninoculated food samples were negative for L. monocytogenes. The ruggedness testing of both ShortPrep foodproof II Kit and Roche/BIOTECON LightCycler foodproof L. monocytogenes Detection Kit showed no noteworthy influences to any variation of the parameters component concentration, apparatus comparison, tester comparison, and sample volumes. In total, 102 L. monocytogenes isolates (cultures and pure DNA) were tested and detected for the inclusivity study, including all isolates claimed by the AOAC RI. The exclusivity study included 60 non-L. monocytogenes bacteria. None of the tested isolates gave a false-positive result; specificity was 100%. Three different lots were tested in the lot-to-lot study. All 3 lots gave equal results. The stability study was subdivided into 3 parts: long-term study, stress test, and freeze-defrost test. Three lots were tested in 4 time intervals within a period of 13 months. They all gave comparable results for all test intervals. For the stress test, LightCycler L. monocytogenes detection mixes were stored at different temperatures and tested at different time points during 1 month. Stable results were produced at all storage temperatures. The freeze-defrost analysis showed no noteworthy aggravation of test results. The independent validation study examined by Campden and Chorleywood Food Research Association Group (CCFRA) demonstrated again that the LightCycler L. monocytogenes detection system shows a comparable sensitivity to reference methods. With both the LightCycler PCR and BAM methods, 19 out of 20 inoculated food samples were detected. The 24 h PCR results generated by the LightCycler system corresponded directly with the FDA/BAM culture results. However, the 48 h PCR results did not relate exactly to the FDA/BAM results, as one sample found to be positive by the 48 h PCR could not be culturally confirmed and another sample which was negative by the 48 h PCR was culturally positive.

Performance of nested RT-PCR on CSF for tuberculous meningitis diagnosis in HIV-infected patients.

PubMed

Gualberto, F A S; Gonçalves, M G; Fukasawa, L O; Santos, A M Ramos Dos; Sacchi, C T; Harrison, L H; Boulware, D R; Vidal, J E

2017-10-01

Timely diagnosis of tuberculous meningitis (TBM) in patients with human immunodeficiency virus (HIV) infection remains a challenge. Despite the current scale-up of the Xpert® MTB/RIF assay, other molecular diagnostic tools are necessary, particularly in referral centres in low- and middle-income countries without Xpert testing. To determine the diagnostic performance of nested real-time polymerase chain reaction (nRT-PCR) in HIV-infected TBM patients categorised according to standardised clinical case definitions. Based on clinical, laboratory and imaging data, HIV-infected patients with suspected TBM were prospectively categorised as 'definite TBM', 'probable TBM', 'possible TBM' or 'not TBM'. We evaluated nRT-PCR sensitivity and specificity in diagnosing TBM among definite TBM cases, and among definite + probable TBM cases. Ninety-two participants were enrolled in the study. nRT-PCR sensitivity for definite TBM (n = 8) was 100% (95%CI 67-100) and 86% (95%CI 60-96) for both definite and probable TBM (n = 6). Assuming that 'not TBM' patients (n = 74) were true-negatives, nRT-PCR specificity was 100% (95%CI 95-100). The possible TBM group (n = 4) had no nRT-PCR positives. The nRT-PCR is a useful rule-in test for HIV-infected patients with TBM according to international consensus case definitions. As nRT-PCR cannot exclude TBM, studies comparing and combining nRT-PCR with other assays are necessary for a rule-out test.

Simplified PCR for detection of Haemophilus ducreyi and diagnosis of chancroid.

PubMed Central

West, B; Wilson, S M; Changalucha, J; Patel, S; Mayaud, P; Ballard, R C; Mabey, D

1995-01-01

A simplified PCR was developed for detection of Haemophilus ducreyi in samples from chancroid patients. The strategy included a straightforward chloroform extraction sample preparation method, a one-tube nested PCR to minimize contamination risks, and a colorimetric method for detection of products. Primers were designed from published nucleotide sequences of the 16S rRNA gene of H. ducreyi, with longer outer primers for annealing at a higher temperature and shorter inner primers labelled with biotin and digoxigenin for binding with avidin and colorimetric detection. The PCR technique detected all 35 strains of H. ducreyi tested, from four different geographical regions, and was negative for other, related strains of bacteria and for the common contaminating bacteria tested. Of 25 samples from H. ducreyi culture-positive chancroid patients, 24 were PCR positive and 1 produced a weak reaction. Of 83 samples from clinical cases of chancroid in the Republic of South Africa, 69 were PCR positive. The sensitivity of PCR compared with that of clinical diagnosis was 83%. All 50 negative control samples were negative. Encouraging results were also obtained with a consecutive series of 25 genital ulcer patients in Tanzania, of whom 9 were PCR positive. The adaptations of this simplified PCR strategy, at the sensitivity and specificity levels obtained, mean it will be useful for detection of H. ducreyi in areas where the organism is endemic, particularly where testing by culture is difficult or impossible. PMID:7540625

Hybrid capture-II and LCR-E7 PCR assays for HPV typing in cervical cytologic samples.

PubMed

Yamazaki, H; Sasagawa, T; Basha, W; Segawa, T; Inoue, M

2001-10-15

As part of an ongoing cohort study in the Hokuriku region of Japan, cervical cell samples from histologically confirmed normal (n = 114) or abnormal (n = 286) women were examined for the presence of HPV DNA using a second-generation hybrid capture assay (HCA-II) and LCR-E7 PCR. HCA-II detected low-risk (HPV-6, -11, -42, 43 and -44) and high-risk (HPV-16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, -59 and -68) HPV types, while LCR-E7 PCR detected an additional 7 HPV types and some uncharacterized types. In screening of high-grade squamous intraepithelial lesions (HSILs) and invasive cervical cancer, the sensitivities of HCA-II and LCR-E7 PCR testing the high-risk HPV types were 83% and 81%, respectively, while the specificity of both assays was 93%. The sensitivity of LCR-E7 PCR increased to 87%, which was significantly higher than that in HCA-II, when testing both high-risk and other HPV types. Sixty-eight inconsistent results (17% of total tested) from HCA-II and LCR-E7 PCR were due to (i) low copy number of HPV genome (false-negative for HCA-II, 5.3% and for LCR-E7 PCR, 1.3%), (ii) infection with HPV types undetectable by HCA-II (4.8%), (iii) multiple HPV infections (5%) or (iv) unknown reasons (0.8%). LCR-E7 PCR revealed that infections with HPV-16, -18, -31, -33, -35, -51, -52, -56, -58 or -67 was a high risk for cancer since these types predominated in HSIL and invasive cervical cancer. Samples showing high relative light units (>20) with a high-risk probe in HCA-II also gave positive results in LCR-E7 PCR and were generally associated with abnormal cervical lesions. Thus, we propose that both HCA-II and LCR-E7 PCR are valuable screening tests for premalignant and malignant cervical lesions. Copyright 2001 Wiley-Liss, Inc.

Quant. PCR and bDNA - the search for the truth continues as high viral load are linked to high beta 2 microglobulin levels.

PubMed

1998-01-01

Controversy exists over whether numbers obtained from Quant. PCR & bDNA tests actually represent the HIV virus; however, a researcher is collecting evidence to determine whether both tests have the same prognostic value as the beta 2 microglobulin tests. There is enough evidence to show that beta 2 microglobulin levels correlate directly with high viral loads. The researcher speculates that Quant. PCR or bDNA measures the combined effects of both HIV and HHV-6A infection in destroying cells. To help resolve this and other issues, volunteers with Chronic Fatigue Immune Dysfunction Syndrome (CFIDS) and active HHV-6A infection are being sought. Volunteers will be asked to use Norvir after which its effectiveness against HHV-6A infection will be determined. CFIDS patients will show a viral load for HIV even though they do not have the virus; the research speculates that if the PCR test for HIV is non-detectable in HHV-6A related cases, it will prove that Quant PCR is not measuring HHV-6A titers in persons with AIDS. Other diagnostic tests are available to patients. Five of these tests are briefly summarized and responses to patient questions regarding viral load issues and therapy are included.

Nested-PCR and a new ELISA-based NovaLisa test kit for malaria diagnosis in an endemic area of Thailand.

PubMed

Thongdee, Pimwan; Chaijaroenkul, Wanna; Kuesap, Jiraporn; Na-Bangchang, Kesara

2014-08-01

Microscopy is considered as the gold standard for malaria diagnosis although its wide application is limited by the requirement of highly experienced microscopists. PCR and serological tests provide efficient diagnostic performance and have been applied for malaria diagnosis and research. The aim of this study was to investigate the diagnostic performance of nested PCR and a recently developed an ELISA-based new rapid diagnosis test (RDT), NovaLisa test kit, for diagnosis of malaria infection, using microscopic method as the gold standard. The performance of nested-PCR as a malaria diagnostic tool is excellent with respect to its high accuracy, sensitivity, specificity, and ability to discriminate Plasmodium species. The sensitivity and specificity of nested-PCR compared with the microscopic method for detection of Plasmodium falciparum, Plasmodium vivax, and P. falciparum/P. vivax mixed infection were 71.4 vs 100%, 100 vs 98.7%, and 100 vs 95.0%, respectively. The sensitivity and specificity of the ELISA-based NovaLisa test kit compared with the microscopic method for detection of Plasmodium genus were 89.0 vs 91.6%, respectively. NovaLisa test kit provided comparable diagnostic performance. Its relatively low cost, simplicity, and rapidity enables large scale field application.

Evaluation of PCR for cutaneous leishmaniasis diagnosis and species identification using filter paper samples in Panama, Central America.

PubMed

Miranda, A; Saldaña, A; González, K; Paz, H; Santamaría, G; Samudio, F; Calzada, J E

2012-09-01

Cutaneous leishmaniasis (CL) is a major vectorborne disease in Panama. In this study, the diagnostic performance and usefulness of two DNA extraction procedures from skin scraping samples collected on FTA filter paper for subsequent PCR diagnosis of CL was evaluated. A positive CL laboratory diagnosis was based on a positive parasitological test (Giemsa-stained smears or in vitro culture) and/or positive PCR test performed from skin scrapings collected in TE buffer (PCR-TE). Of 100 patients with skin lesions suggestive of CL, 82 (82%) were confirmed as CL positive. The sensitivity was calculated for each of the PCR approaches from samples collected on filter paper. The highest sensitivity was achieved by PCR-FTA processed by Chelex 100 (PCR-Chelex) (0.94). PCR-FTA extracted using the FTA purification reagent presented a lower sensitivity (0.60). Good concordance between routine PCR-TE and PCR-Chelex was observed (percent agreement=0.88, κ index=0.65). In conclusion, use of FTA filter paper for skin scraping collection combined with PCR is a reliable and convenient method for CL diagnosis in Panama, with comparable performance to the routine PCR method and with improved sensitivity compared with those of conventional parasitological methods. Copyright © 2012 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.

Highly sensitive and quantitative evaluation of the EGFR T790M mutation by nanofluidic digital PCR

PubMed Central

Iwama, Eiji; Takayama, Koichi; Harada, Taishi; Okamoto, Isamu; Ookubo, Fumihiko; Kishimoto, Junji; Baba, Eishi; Oda, Yoshinao; Nakanishi, Yoichi

2015-01-01

The mutation of T790M in EGFR is a major mechanism of resistance to treatment with EGFR-TKIs. Only qualitative detection (presence or absence) of T790M has been described to date, however. Digital PCR (dPCR) analysis has recently been applied to the quantitative detection of target molecules in cancer with high sensitivity. In the present study, 25 tumor samples (13 obtained before and 12 after EGFR-TKI treatment) from 18 NSCLC patients with activating EGFR mutations were evaluated for T790M with dPCR. The ratio of the number of T790M alleles to that of activating mutation alleles (T/A) was determined. dPCR detected T790M in all 25 samples. Although T790M was present in all pre-TKI samples from 13 patients, 10 of these patients had a low T/A ratio and manifested substantial tumor shrinkage during treatment with EGFR-TKIs. In six of seven patients for whom both pre- and post-TKI samples were available, the T/A ratio increased markedly during EGFR-TKI treatment. Highly sensitive dPCR thus detected T790M in all NSCLC patients harboring activating EGFR mutations whether or not they had received EGFR-TKI treatment. Not only highly sensitive but also quantitative detection of T790M is important for evaluation of the contribution of T790M to EGFR-TKI resistance. PMID:26015401

Evaluation of the hyplex® TBC PCR test for detection of Mycobacterium tuberculosis complex in clinical samples

PubMed Central

2010-01-01

Background Tuberculosis (TB) is one of the major public health concerns worldwide. The detection of the pathogen Mycobacterium tuberculosis complex (MTBC) as early as possible has a great impact on the effective control of the spread of the disease. In our study, we evaluated the hyplex® TBC PCR test (BAG Health Care GmbH), a novel assay using a nucleic acid amplification technique (NAAT) with reverse hybridisation and ELISA read out for the rapid detection of M. tuberculosis directly in clinical samples. Results A total of 581 respiratory and non-respiratory specimens from our pneumological hospital and the National TB Institute of Uzbekistan were used for the evaluation of the PCR assay. Of these, 292 were classified as TB samples and 289 as non-TB samples based on the results of the TB cultures as reference method. The PCR results were initially used to optimise the cut-off value of the hyplex® TBC test system by means of a ROC analysis. The overall sensitivity of the assay was determined to be 83.1%. In smear-positive TB samples, the sensitivity of the hyplex® TBC PCR test was estimated to 93.4% versus 45.1% in smear-negative samples. The specificity of the test was 99.25%. Of the two specimens (0.75%) with false-positive PCR results, one yielded a culture positive for non-tuberculous mycobacteria. Based on the assumption of a prevalence of 8% TB positives among the samples in our diagnostic TB laboratory, the positive and negative predictive values were estimated to 90.4% and 98.5%, respectively. Conclusions The hyplex® TBC PCR test is an accurate NAAT assay for a rapid and reliable detection of M. tuberculosis in various respiratory and non-respiratory specimens. Compared to many other conventional NAAT assays, the hyplex® TBC PCR test is in a low price segment which makes it an attractive option for developing and emerging countries with high TB burdens. PMID:20356361

Clinical value of whole-blood interferon-gamma assay in patients with suspected pulmonary tuberculosis and AFB smear- and polymerase chain reaction-negative bronchial aspirates.

PubMed

Lee, Jaehee; Lee, Shin Yup; Yoo, Seung Soo; Cha, Seung Ick; Won, Dong Il; Park, Jae Yong; Lee, Won-Kil; Kim, Chang Ho

2012-07-01

Combining a polymerase chain reaction (PCR) test with bronchoscopy is frequently performed to allow a rapid diagnosis of smear-negative pulmonary tuberculosis (PTB). However, limited data are available concerning clinical judgment in patients with suspected PTB and AFB smear- and PCR-negative bronchial aspirates (BA). The present study evaluated the usefulness of whole-blood QuantiFERON-TB Gold In-Tube (QFT) testing in these patients. Of 166 patients with suspected PTB who had undergone bronchoscopy because of smear-negative sputum or inadequate sputum production, 93 (56%) were diagnosed with culture-positive PTB. Seventy-four patients were either AFB smear- or PCR-positive. In the 75 patients whose BA AFB smear and PCR results were both negative, 19 were finally diagnosed with PTB by culture. The QFT test had a negative predictive value of 91% for PTB. The QFT test may be useful for excluding PTB in patients with suspected PTB whose BA AFB smear and PCR results are both negative. Copyright © 2012 Elsevier Inc. All rights reserved.

Combined evaluation of commonly used techniques, including PCR, for diagnosis of mouse fur mites.

PubMed

Karlsson, Eleanor M; Pearson, Laura M; Kuzma, Kristen M; Burkholder, Tanya H

2014-01-01

Our study evaluated and compared the false-negative rates (FNR) of a wide array of fur-mite diagnostic tests, including 2 postmortem tests (pelt exam and sticky paper) and 3 antemortem tests (adhesive tape, fur pluck, and PCR). Past publications examining fur-mite diagnostic techniques primarily used paired comparisons, evaluating tests by their level of agreement with only one other test. However, different combinations or pairs of diagnostics are used in the different studies, making the results of these comparisons difficult to interpret across all available diagnostics. In the current study, mice from a conventionally maintained colony endemic for Myobia musculi were identified as positive based on at least one positive diagnostic test. From this pool of positive animals, the FNR of all tests were quantified. The PCR assay and the pelt exam performed the best, with 0% and 2% FNR respectively, whereas tape, fur-pluck, and sticky-paper tests showed 24%, 26%, and 36% FNR, respectively. Our study shows that for mice in a colony naturally infested with Myobia musculi, PCR testing can be used for reliable antemortem detection, and pelt exam performed by experienced examiners is reliable for postmortem detection.

Development of a PCR Assay for the Detection of Spironucleus muris

PubMed Central

Jackson, Glenn A; Livingston, Robert S; Riley, Lela K; Livingston, Beth A; Franklin, Craig L

2013-01-01

Spironucleus muris is a protozoan that can colonize the intestinal tract of many rodent species. Although its effects on animal health and research are debated, S. muris is often included on exclusion lists for rodent facilities. Common diagnostic tests for S. muris are insensitive and typically are performed at postmortem examination. We sought to develop a PCR-based diagnostic test with sufficient sensitivity and specificity for use on fecal samples from live rodents. We designed and optimized a PCR assay that targeted the 16S-like rRNA gene of S. muris. The assay was highly specific, given that samples from mice contaminated with S. muris were PCR positive, whereas samples from mice contaminated with other protozoa were negative. The assay also was highly sensitive, detecting as few as 5 template copies per microliter diluent. All mice positive for S. muris on postmortem exams also were positive by fecal PCR. Moreover, S. muris was detected by PCR in mice negative by postmortem examination but from colonies known to be contaminated as well as in rats and hamsters. To assess protozoal loads in mice of differing ages, the PCR assay was adapted to a quantitative format. Fecal loads of S. muris were highest in 4-wk-old mice and declined with age. The PCR assay developed promises to be a highly specific antemortem diagnostic assay with higher sensitivity than that of existing postmortem tests. PMID:23562099

Rapid identification of HPV 16 and 18 by multiplex nested PCR-immunochromatographic test.

PubMed

Kuo, Yung-Bin; Li, Yi-Shuan; Chan, Err-Cheng

2015-02-01

Human papillomavirus (HPV) types 16 and 18 are known to be high-risk viruses that cause cervical cancer. An HPV rapid testing kit that could help physicians to make early and more informed decisions regarding patient care is needed urgently but not yet available. This study aimed to develop a multiplex nested polymerase chain reaction-immunochromatographic test (PCR-ICT) for the rapid identification of HPV 16 and 18. A multiplex nested PCR was constructed to amplify the HPV 16 and 18 genotype-specific L1 gene fragments and followed by ICT which coated with antibodies to identify rapidly the different PCR products. The type-specific gene regions of high-risk HPV 16 and 18 could be amplified successfully by multiplex nested PCR at molecular sizes of approximately 99 and 101bp, respectively. The capture antibodies raised specifically against the moleculars labeled on the PCR products could be detected simultaneously both HPV 16 and 18 in one strip. Under optimal conditions, this PCR-ICT assay had the capability to detect HPV in a sample with as low as 100 copies of HPV viral DNA. The PCR-ICT system has the advantage of direct and simultaneous detection of two high-risk HPV 16 and 18 DNA targets in one sample, which suggested a significant potential of this assay for clinical application. Copyright © 2014. Published by Elsevier B.V.

Estimation of test characteristics of real-time PCR and bacterial culture for diagnosis of subclinical intramammary infections with Streptococcus agalactiae in Danish dairy cattle in 2012 using latent class analysis.

PubMed

Mahmmod, Yasser S; Toft, Nils; Katholm, Jørgen; Grønbæk, Carsten; Klaas, Ilka C

2013-05-01

The misdiagnosis of intramammary infections (IMI) with Streptococcus agalactiae (S. agalactiae) could lead farmers to treat or cull animals unnecessarily. The objective of this field study was to estimate the sensitivity (Se) and specificity (Sp) of real-time PCR at different cut-offs for cycle threshold (Ct) values against bacterial culture (BC) for diagnosis of S. agalactiae IMI using latent class analysis to avoid the assumption of a perfect reference test. A total of 614 dairy cows were randomly selected from 6 herds with bulk tank PCR Ct value ≤ 39 for S. agalactiae and S. aureus. At milk recording, 2456 quarter milk samples were taken aseptically for BC and the routinely taken cow level milk samples were analyzed by PCR. Results showed that 53 cows (8.6%) were positive for S. agalactiae IMI by BC. Sensitivity of PCR at cut-offs; ≤ 39, ≤ 37, ≤ 34, and ≤ 32, was 96.2%, 91.9%, 87.2% and 73.9%, while Se of BC was 25.7%, 29.9%, 59.9% and 72.1%. Specificity of PCR at cut-offs; ≤ 39, ≤ 37, ≤ 34, and ≤ 32, was 96.8%, 96.9%, 96.7%, and 97.22%, while Sp of BC was 99.7%, 99.5%, 99.2%, and 98.9%. The estimated prevalence of S. agalactiae IMI by PCR was higher than the apparent prevalence at the tested cut-offs, indicating under estimation of S. agalactiae IMI in the examined dairy cows. In conclusion, Se of PCR is always higher than Se of BC at all tested cut-offs. The lower cut-off, the more comparable becomes Se of PCR and Se of BC. The changes in Se in both PCR and BC at different Ct-value cut-offs may indicate a change in the definition of the latent infection. The similar Se of both tests at cut-off ≤ 32 may indicate high concentrations of S. agalactiae viable cells, representing a cow truly/heavily infected with S. agalactiae and thus easier to detect with BC. At cut-off ≤ 39 the latent definition of infection may reflect a more general condition of cows being positive for S. agalactiae. Our findings indicate that PCR Ct-value cut-offs should be chosen according to the underlying latent infection definition of interest. Latent class analysis proposes a useful alternative to classic test evaluation of diagnostic tests used for detection of S. agalactiae IMI in milk. Copyright © 2012 Elsevier B.V. All rights reserved.

Malaria surveillance in the Democratic Republic of the Congo: comparison of microscopy, PCR, and rapid diagnostic test.

PubMed

Doctor, Stephanie M; Liu, Yunhao; Whitesell, Amy; Thwai, Kyaw L; Taylor, Steve M; Janko, Mark; Emch, Michael; Kashamuka, Melchior; Muwonga, Jérémie; Tshefu, Antoinette; Meshnick, Steven R

2016-05-01

Malaria surveillance is critical for control efforts, but diagnostic methods frequently disagree. Here, we compare microscopy, PCR, and a rapid diagnostic test in 7137 samples from children in the Democratic Republic of the Congo using latent class analysis. PCR had the highest sensitivity (94.6%) and microscopy had the lowest (76.7%). Copyright © 2016 Elsevier Inc. All rights reserved.

Aspergillus Polymerase Chain Reaction: Systematic Review of Evidence for Clinical Use in Comparison With Antigen Testing

PubMed Central

White, P. Lewis; Wingard, John R.; Bretagne, Stéphane; Löffler, Jürgen; Patterson, Thomas F.; Slavin, Monica A.; Barnes, Rosemary A.; Pappas, Peter G.; Donnelly, J. Peter

2015-01-01

Background. Aspergillus polymerase chain reaction (PCR) was excluded from the European Organisation for the Research and Treatment of Cancer/Mycoses Study Group (EORTC/MSG) definitions of invasive fungal disease because of limited standardization and validation. The definitions are being revised. Methods. A systematic literature review was performed to identify analytical and clinical information available on inclusion of galactomannan enzyme immunoassay (GM-EIA) (2002) and β-d-glucan (2008), providing a minimal threshold when considering PCR. Categorical parameters and statistical performance were compared. Results. When incorporated, GM-EIA and β-d-glucan sensitivities and specificities for diagnosing invasive aspergillosis were 81.6% and 91.6%, and 76.9% and 89.4%, respectively. Aspergillus PCR has similar sensitivity and specificity (76.8%–88.0% and 75.0%–94.5%, respectively) and comparable utility. Methodological recommendations and commercial PCR assays assist standardization. Although all tests have limitations, currently, PCR is the only test with independent quality control. Conclusions. We propose that there is sufficient evidence that is at least equivalent to that used to include GM-EIA and β-d-glucan testing, and that PCR is now mature enough for inclusion in the EORTC/MSG definitions. PMID:26113653

Aspergillus Polymerase Chain Reaction: Systematic Review of Evidence for Clinical Use in Comparison With Antigen Testing.

PubMed

White, P Lewis; Wingard, John R; Bretagne, Stéphane; Löffler, Jürgen; Patterson, Thomas F; Slavin, Monica A; Barnes, Rosemary A; Pappas, Peter G; Donnelly, J Peter

2015-10-15

Aspergillus polymerase chain reaction (PCR) was excluded from the European Organisation for the Research and Treatment of Cancer/Mycoses Study Group (EORTC/MSG) definitions of invasive fungal disease because of limited standardization and validation. The definitions are being revised. A systematic literature review was performed to identify analytical and clinical information available on inclusion of galactomannan enzyme immunoassay (GM-EIA) (2002) and β-d-glucan (2008), providing a minimal threshold when considering PCR. Categorical parameters and statistical performance were compared. When incorporated, GM-EIA and β-d-glucan sensitivities and specificities for diagnosing invasive aspergillosis were 81.6% and 91.6%, and 76.9% and 89.4%, respectively. Aspergillus PCR has similar sensitivity and specificity (76.8%-88.0% and 75.0%-94.5%, respectively) and comparable utility. Methodological recommendations and commercial PCR assays assist standardization. Although all tests have limitations, currently, PCR is the only test with independent quality control. We propose that there is sufficient evidence that is at least equivalent to that used to include GM-EIA and β-d-glucan testing, and that PCR is now mature enough for inclusion in the EORTC/MSG definitions. © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America.

Comparative Performance of Reagents and Platforms for Quantitation of Cytomegalovirus DNA by Digital PCR

PubMed Central

Gu, Z.; Sam, S. S.; Sun, Y.; Tang, L.; Pounds, S.; Caliendo, A. M.

2016-01-01

A potential benefit of digital PCR is a reduction in result variability across assays and platforms. Three sets of PCR reagents were tested on two digital PCR systems (Bio-Rad and RainDance), using three different sets of PCR reagents for quantitation of cytomegalovirus (CMV). Both commercial quantitative viral standards and 16 patient samples (n = 16) were tested. Quantitative accuracy (compared to nominal values) and variability were determined based on viral standard testing results. Quantitative correlation and variability were assessed with pairwise comparisons across all reagent-platform combinations for clinical plasma sample results. The three reagent sets, when used to assay quantitative standards on the Bio-Rad system, all showed a high degree of accuracy, low variability, and close agreement with one another. When used on the RainDance system, one of the three reagent sets appeared to have a much better correlation to nominal values than did the other two. Quantitative results for patient samples showed good correlation in most pairwise comparisons, with some showing poorer correlations when testing samples with low viral loads. Digital PCR is a robust method for measuring CMV viral load. Some degree of result variation may be seen, depending on platform and reagents used; this variation appears to be greater in samples with low viral load values. PMID:27535685

FlindersTechnology Associates (FTA) filter paper-based DNA extraction with polymerase chain reaction (PCR) for detection of Pneumocystis jirovecii from respiratory specimens of immunocompromised patients.

PubMed

Nuchprayoon, Surang; Saksirisampant, Wilai; Jaijakul, Siraya; Nuchprayoon, Issarang

2007-01-01

We evaluated the diagnostic value of Flinders Technology Associates (FTA) filter paper together with polymerase chain reaction (PCR) for detection of Pneumocystis jirovecii (carinii) from induced sputum (IS) and bronchoalveolar lavage fluid (BALF) samples. The study involved 162 patients with clinical diagnosis of pneumocystis pneumonia (PcP) of human immunodeficiency virus/acquired immune deficiency syndrome (HIV/AIDS) patients and other immunocompromised patients. P. jirovecii cysts or trophozoites were detected in IS and BALF by cytological method. The mitochondrial 5S ribosomal ribonucleic acid (rRNA) gene of P. jirovecii was amplified from these samples by using FTA filters together with a one-step PCR method (FTA-PCR). With the FTA-PCR method, the sensitivity and specificity of the test compared to microscopic examination were 67% and 90% for IS, while they were 67% and 91% for BALF, respectively. The sensitivity and specificity of the FTA-PCR test was also comparable to PCR with the conventional deoxyribonucleic acid (DNA) extraction method. We concluded that FTA-PCR is useful to detect P. jirovecii in noninvasive IS.

An optimized method for elution of enteroviral RNA from a cellulose-based substrate.

PubMed

Li, Yan; Yoshida, Hiromu; Wang, Lu; Tao, Zexin; Wang, Haiyan; Lin, Xiaojuan; Xu, Aiqiang

2012-12-01

The Flinders Technology Australia (FTA) Elute Card is a commercial product that facilitates the collection, transport, archiving and processing of nucleic acids from a wide variety of biological samples at room temperature. While the cards have been designed so that sterile/deionized water can elute DNA easily, they are not suitable for some less stable RNAs. This study was undertaken to determine the optimal conditions such as the buffer type, buffer pH and incubation temperature for the elution of enteroviral RNA from FTA Elute Cards prior to quantitative analysis using real-time PCR (qPCR) or consensus degenerate hybrid oligonucleotide primer VP1 RT-semi nested PCR (CODEHOP VP1 RT-snPCR). TE-1 (pH 8.0), rather than sterile water, was the best buffer for high efficiency elution of enteroviral RNA at 95°C. However, as the estimated recovery rate of viral RNA eluted from the cards averaged to be only 6.1%, enterovirus assays using FTA elution should be considered qualitative, especially at low virus titers, and therefore the results of the assay should be interpreted carefully. Copyright © 2012 Elsevier B.V. All rights reserved.

Acid-fast Smear and Histopathology Results Provide Guidance for the Appropriate Use of Broad-Range Polymerase Chain Reaction and Sequencing for Mycobacteria.

PubMed

Miller, Kennon; Harrington, Susan M; Procop, Gary W

2015-08-01

New molecular diagnostic tests are attractive because of the potential they hold for improving diagnostics in microbiology. The value of these tests, which is often assumed, should be investigated to determine the best use of these potentially powerful tools. To investigate the usefulness of broad-range polymerase chain reaction (PCR), followed by sequencing, in mycobacterial infections. We reviewed the test performance of acid-fast bacilli (AFB) PCR and traditional diagnostic methods (histopathology, AFB smear, and culture). We assessed the diagnostic effect and cost of the unrestricted ordering of broad-range PCR for the detection and identification of mycobacteria in clinical specimens. The AFB PCR was less sensitive than culture and histopathology and was less specific than culture, AFB smear, and histopathology. During 18 months, $93 063 was spent on 183 patient specimens for broad-range PCR and DNA sequencing for mycobacteria to confirm one culture-proven Mycobacterium tuberculosis infection that was also known to be positive by AFB smear and histopathology. In this cohort, there was a false-negative AFB PCR for M tuberculosis and a false-positive AFB PCR for Mycobacterium lentiflavum . Testing of AFB smear-negative specimens from patients without an inflammatory response supportive of a mycobacterial infection is costly and has not been proven to improve patient care. Traditional diagnostics (histopathology, AFB smear, and culture) should remain the primary methods for the detection of mycobacteria in clinical specimens.

Comparison between ICT and PCR for diagnosis of Chlamydia trachomatis.

PubMed

Khan, E R; Hossain, M A; Paul, S K; Mahmud, C; Hasan, M M; Rahman, M M; Nahar, K; Kubayashi, N

2012-04-01

Chlamydia trachomatis is an obligate intracellular gram-negative bacterium which is the most prevalent cause of bacterial sexually transmitted infections (STI). The present study was carried to diagnose genital Chlamydia trachomatis infection among women of reproductive age, attending Mymensingh Medical College Hospital, during July 2009 to June 2010 by Immunochromatographic test (ICT) and Polymerase chain reaction (PCR). A total of 70 females were included in this study. Out of 70 cases 56 were symptomatic and 14 asymptomatic. Endocervical swabs were collected from each of the cases and examined by Immunochromatographic test (ICT) for antigen detection and Polymerase chain reaction (PCR) for detection of endogenous plasmid-based nucleic acid. A total 29(41.4%) of the cases were found positive for C. trachomatis either by ICT or PCR. Of the 56 symptomatic cases, 19(33.9%) were found ICT positive and 17(30.4%) were PCR positive. Among 14 asymptomatic females, 2(14.3%) were ICT positive and none were PCR positive. Though PCR is highly sensitive but a total of twelve cases were found ICT positive but PCR negative. It may be due to presence of plasmid deficient strain of C trachomatis which could be amplified by ompA based (Chromosomal gene) multiplex PCR.

Seroprevalence of Toxoplasma gondii in wild kangaroos using an ELISA

PubMed Central

Parameswaran, N.; O'Handley, RM.; Grigg, ME.; Fenwick, SG.; Thompson, RCA.

2009-01-01

Infection with Toxoplasma gondii is a significant problem in Australian marsupials, and can lead to devastating disease and predispose animals to predation. T. gondii infection in kangaroos is also of public health significance due to the kangaroo meat trade. A moderate seroprevalence of T. gondii was observed in a study of western grey kangaroos located in the Perth metropolitan area in Western Australia. Of 219 kangaroos tested, 15.5% (95%CI: 10.7-20.3) were positive for T. gondii antibodies using an ELISA developed to detect T. gondii IgG in macropod marsupials. When compared with the commercially available MAT (modified agglutination test), the ELISA developed was in absolute agreement and yielded a κ coefficient of 1.00. Of 18 kangaroos tested for the presence of T. gondii DNA by PCR, the 9 ELISA positive kangaroos tested PCR positive and the 9 ELISA negative kangaroos tested PCR negative indicating the ELISA protocol was both highly specific and sensitive and correlated 100% with the more labour intensive PCR assay. PMID:19567231

A Real-Time PCR Assay for Bat SARS-Like Coronavirus Detection and Its Application to Italian Greater Horseshoe Bat Faecal Sample Surveys

PubMed Central

Balboni, Andrea; Gallina, Laura; Palladini, Alessandra; Prosperi, Santino; Battilani, Mara

2012-01-01

Bats are source of coronaviruses closely related to the severe acute respiratory syndrome (SARS) virus. Numerous studies have been carried out to identify new bat viruses related to SARS-coronavirus (bat-SARS-like CoVs) using a reverse-transcribed-polymerase chain reaction assay. However, a qualitative PCR could underestimate the prevalence of infection, affecting the epidemiological evaluation of bats in viral ecology. In this work an SYBR Green-real time PCR assay was developed for diagnosing infection with SARS-related coronaviruses from bat guano and was applied as screening tool in a survey carried out on 45 greater horseshoe bats (Rhinolophus ferrumequinum) sampled in Italy in 2009. The assay showed high sensitivity and reproducibility. Its application on bats screening resulted in a prevalence of 42%. This method could be suitable as screening tool in epidemiological surveys about the presence of bat-SARS-like CoVs, consequently to obtain a more realistic scenario of the viral prevalence in the population. PMID:22654650

Microbial examination of anaerobic sludge adaptation to animal slurry.

PubMed

Moset, V; Cerisuelo, A; Ferrer, P; Jimenez, A; Bertolini, E; Cambra-López, M

2014-01-01

The objective of this study was to evaluate changes in the microbial population of anaerobic sludge digesters during the adaptation to pig slurry (PS) using quantitative real-time polymerase chain reaction (qPCR) and qualitative scanning electron microscopy (SEM). Additionally, the relationship between microbial parameters and sludge physicochemical composition and methane yield was examined. Results showed that the addition of PS to an unadapted thermophilic anaerobic digester caused an increase in volatile fatty acids (VFA) concentration, a decrease in removal efficiency and CH4 yield. Additionally, increases in total bacteria and total archaea were observed using qPCR. Scanning electron micrographs provided a general overview of the sludge's cell morphology, morphological diversity and degree of organic matter degradation. A change in microbial morphotypes from homogeneous cell morphologies to a higher morphological diversity, similar to that observed in PS, was observed with the addition of PS by SEM. Therefore, the combination of qPCR and SEM allowed expanding the knowledge about the microbial adaptation to animal slurry in thermophilic anaerobic digesters.

Evaluation of the efficiency of nested q-PCR in the detection of Mycobacterium tuberculosis complex directly from tuberculosis-suspected lesions in post-mortem macroscopic inspections of bovine carcasses slaughtered in the state of Mato Grosso, Brazil.

PubMed

Carvalho, Ricardo César Tavares; Furlanetto, Leone Vinícius; Maruyama, Fernanda Harumy; Araújo, Cristina Pires de; Barros, Sílvia Letícia Bomfim; Ramos, Carlos Alberto do Nascimento; Dutra, Valéria; Araújo, Flábio Ribeiro de; Paschoalin, Vânia Margaret Flosi; Nakazato, Luciano; Figueiredo, Eduardo Eustáquio de Souza

2015-08-01

Bovine tuberculosis (BTB) is a zoonotic disease caused by Mycobacterium bovis, a member of the Mycobacterium tuberculosis complex (MTC). The quick and specific detection of this species is of extreme importance, since BTB may cause economic impacts, in addition to presenting imminent risks to human health. In the present study a nested real-time PCR test (nested q-PCR) was used in post-mortem evaluations to assess cattle carcasses with BTB-suspected lesions. A total of 41,193 cattle slaughtered in slaughterhouses located in the state of Mato Grosso, were examined. Of the examined animals, 198 (0.48%) showed BTB-suspected lesions. M. bovis was isolated in 1.5% (3/198) of the samples. Multiplex-PCR detected MTC in 7% (14/198) of the samples. The nested q-PCR test detected MTC in 28% (56/198) of the BTB-suspected lesions, demonstrating higher efficiency when compared to the multiplex-PCR and conventional microbiology. Nested q-PCR can therefore be used as a complementary test in the national program for control and eradication of bovine tuberculosis. Copyright © 2015 Elsevier Ltd. All rights reserved.

Validation of the Pockit Dengue Virus Reagent Set for Rapid Detection of Dengue Virus in Human Serum on a Field-Deployable PCR System.

PubMed

Tsai, Jih-Jin; Liu, Li-Teh; Lin, Ping-Chang; Tsai, Ching-Yi; Chou, Pin-Hsing; Tsai, Yun-Long; Chang, Hsiao-Fen Grace; Lee, Pei-Yu Alison

2018-05-01

Dengue virus (DENV) infection, a mosquito-borne disease, is a major public health problem in tropical countries. Point-of-care DENV detection with good sensitivity and specificity enables timely early diagnosis of DENV infection, facilitating effective disease management and control, particularly in regions of low resources. The Pockit dengue virus reagent set (GeneReach Biotech), a reverse transcription insulated isothermal PCR (RT-iiPCR), is available to detect all four serotypes of DENV on the field-deployable Pockit system, which is ready for on-site applications. In this study, analytical and clinical performances of the assay were evaluated. The index assay did not react with 14 non-DENV human viruses, indicating good specificity. Compared to the U.S. CDC DENV-1-4 real-time quantitative RT-PCR (qRT-PCR) assay, testing with serial dilutions of virus-spiked human sera demonstrated that the index assay had detection endpoints that were separately comparable with the 4 serotypes. Excellent reproducibility was observed among repeat tests done by six operators at three sites. In clinical performance, 195 clinical sera collected around Kaohsiung city in 2012 and 21 DENV-4-spiked sera were tested with the RT-iiPCR and qRT-PCR assays in parallel. The 121 (11 DENV-1, 78 DENV-2, 11 DENV-3, and 21 DENV-4) qRT-PCR-positive and 95 qRT-PCR-negative samples were all positive and negative by the RT-iiPCR reagent results, respectively, demonstrating high (100%) interrater agreement (95% confidence interval [CI 95% ], ∼98.81% to 100%; κ = 1). With analytical and clinical performance equivalent to those of the reference qRT-PCR assay, the index PCR assay on the field-deployable system can serve as a highly sensitive and specific on-site tool for DENV detection. Copyright © 2018 American Society for Microbiology.

Comparison of Different Methods to Identify tdh-Positive Pathogenic Vibrio parahaemolyticus Isolates.

PubMed

Zhang, Hongzhi; Chen, Min

2018-01-01

We evaluated the accuracy and ease of operation of three methods to identify tdh-positive Vibrio parahaemolyticus isolates, including the Kanagawa phenomenon test (KP test), a tdh gene PCR test, and a colloidal gold immunochromatographic assay (CGIA). A total of 221 V. parahaemolyticus isolates were collected from patients, freshly harvested seafood, and fresh seawater. Using the KP test, 92% of V. parahaemolyticus isolates from patients were identified tdh-positive, including four weak KP-positive isolates. The PCR test and CGIA also identified 92% of the isolates as tdh-positive. However, PCR and CGIA only confirmed one of the four weak KP-positive isolates. Similar results were obtained using the three methods to identify V. parahaemolyticus isolates from the other sources. Among the three methods, the KP test was the simplest to perform because it lacked any requirement for sample pretreatment, and was low cost, with no equipment requirements. Therefore, the KP test has been applied widely in many first-line quarantine laboratories. However, the sensitivity and accuracy of KP test were lower than those of the other two methods. PCR can identify the tdh rapidly, specifically, and sensitively. However, PCR requires equipment and facilities that are unavailable in first-line quarantine laboratories. The CGIA can compensate for the disadvantages of the other two methods by its higher sensitivity, accuracy, and ease of operation. Therefore, the CGIA has the highest potential to be used to identify tdh-positive V. parahaemolyticus isolates to guarantee food safety.

Development and validation of InnoQuant™, a sensitive human DNA quantitation and degradation assessment method for forensic samples using high copy number mobile elements Alu and SVA.

PubMed

Pineda, Gina M; Montgomery, Anne H; Thompson, Robyn; Indest, Brooke; Carroll, Marion; Sinha, Sudhir K

2014-11-01

There is a constant need in forensic casework laboratories for an improved way to increase the first-pass success rate of forensic samples. The recent advances in mini STR analysis, SNP, and Alu marker systems have now made it possible to analyze highly compromised samples, yet few tools are available that can simultaneously provide an assessment of quantity, inhibition, and degradation in a sample prior to genotyping. Currently there are several different approaches used for fluorescence-based quantification assays which provide a measure of quantity and inhibition. However, a system which can also assess the extent of degradation in a forensic sample will be a useful tool for DNA analysts. Possessing this information prior to genotyping will allow an analyst to more informatively make downstream decisions for the successful typing of a forensic sample without unnecessarily consuming DNA extract. Real-time PCR provides a reliable method for determining the amount and quality of amplifiable DNA in a biological sample. Alu are Short Interspersed Elements (SINE), approximately 300bp insertions which are distributed throughout the human genome in large copy number. The use of an internal primer to amplify a segment of an Alu element allows for human specificity as well as high sensitivity when compared to a single copy target. The advantage of an Alu system is the presence of a large number (>1000) of fixed insertions in every human genome, which minimizes the individual specific variation possible when using a multi-copy target quantification system. This study utilizes two independent retrotransposon genomic targets to obtain quantification of an 80bp "short" DNA fragment and a 207bp "long" DNA fragment in a degraded DNA sample in the multiplex system InnoQuant™. The ratio of the two quantitation values provides a "Degradation Index", or a qualitative measure of a sample's extent of degradation. The Degradation Index was found to be predictive of the observed loss of STR markers and alleles as degradation increases. Use of a synthetic target as an internal positive control (IPC) provides an additional assessment for the presence of PCR inhibitors in the test sample. In conclusion, a DNA based qualitative/quantitative/inhibition assessment system that accurately predicts the status of a biological sample, will be a valuable tool for deciding which DNA test kit to utilize and how much target DNA to use, when processing compromised forensic samples for DNA testing. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Evaluation of the Ortho-Clinical Diagnostics Vitros ECi Anti-HCV test: comparison with three other methods.

PubMed

Watterson, Jeannette M; Stallcup, Paulina; Escamilla, David; Chernay, Patrick; Reyes, Alfred; Trevino, Sylvia C

2007-01-01

After observing a high incidence of low positive hepatitis C virus (HCV) antibody screens by the Ortho-Clinical Vitros ECi test (Orthoclinical Diagnostics, Raritan, NJ), we compared results against those obtained using another chemiluminescent analyzer, as well as two U.S. Food and Drug Administration (FDA)-approved confirmatory methodologies. To ascertain the true anti-HCV status of samples deemed low-positive by the Ortho-Clinical Vitros ECi test, we tested samples using the ADVIA Centaur HCV screen test (Siemens Medical Solutions Diagnostics), the Chiron recombinant immunoblot assay (RIBA) test (Chiron Corp., Emeryville, CA), and the Roche COBAS Amplicor HCV qualitative test (Roche Diagnostics, Indianapolis, IN) in a series of studies. Of 94 specimens positive by Vitros ECi, 19% were observed to be negative by Centaur. A separate study of 91 samples with signal-to-cutoff (s/co) values less than 8.0 showed that all but one was negative for HCV ribonucleic acid (RNA). In comparison with RIBA, 100% (77) samples positive by the Vitros ECi test with s/co values less than 12.0 were negative or indeterminate by RIBA. A final study comparing all four methods side-by-side showed 63% disagreement by Centaur for Vitros ECi low-positive samples, 75% disagreement by RIBA, and 97% disagreement by polymerase chain reaction (PCR). In conclusion, the Ortho-Clinical Vitros ECi Anti-HCV test yields a high rate of false-positive results in the low s/co range in our patient population. (c) 2007 Wiley-Liss, Inc.

Hairy Root Cultures of Gymnema sylvestre R. Br. to Produce Gymnemic Acid.

PubMed

Rajashekar, J; Kumar, Vadlapudi; Veerashree, V; Poornima, D V; Sannabommaji, Torankumar; Gajula, Hari; Giridhara, B

2016-01-01

Gymnema sylvestre R. Br. (Asclepiadaceae) is an endangered species extensively used in the management of diabetes, obesity, and treatment of various diseases. Uncontrolled exploitation to meet the increasing demand and low seed viability hastens the disappearance of the plant from its natural habitat. Hairy root culture provides a suitable alternative for the enhanced production of active principles. The current protocol provides the optimized culture conditions for the establishment of hairy root cultures and elicitation studies and also confirmation of stable integration of A. rhizogenes plasmid T-DNA into host genetic material by PCR and RT-PCR. Furthermore, it also discusses the suitable methods for the extraction procedures, and qualitative and quantitative analysis of gymnemic acid by HPTLC and HPLC.

Enterovirus D68 detection in respiratory specimens: Association with severe disease.

PubMed

Engelmann, Ilka; Fatoux, Marie; Lazrek, Mouna; Alidjinou, Enagnon K; Mirand, Audrey; Henquell, Cécile; Dewilde, Anny; Hober, Didier

2017-07-01

Molecular techniques increased the number of documented respiratory infections. In a substantial number of cases the causative agent remains undetected. Since August 2014, an increase in Enterovirus(EV)-D68 infections was reported. We aimed to investigate epidemiology and clinical relevance of EV-D68. From June to December 2014 and from September to December 2015, 803 and 847 respiratory specimens, respectively, were tested for respiratory viruses with a multiplex RT-PCR. This multiplex RT-PCR does not detect EV-D68. Therefore, 457 (2014) and 343 (2015) specimens with negative results were submitted to an EV-specific-RT-PCR. EV-positive specimens were tested with an EV-D68-specific-RT-PCR and genotyped. Eleven specimens of 2014 tested positive in the EV-specific-RT-PCR and of these seven were positive in the EV-D68-specific-RT-PCR. Typing confirmed these as EV-D68. Median age of EV-D68-positive patients was 3 years (1 month-91 years). Common symptoms included fever (n = 6, 86%), respiratory distress (n = 5, 71%), and cough (n = 4, 57%). All EV-D68-positive patients were admitted to hospital, 4 (57%) were admitted to intensive care units and 6 (86%) received oxygen. One patient suffered from acute flaccid paralysis. Seven specimens of 2015 were positive in the EV-specific-RT-PCR but negative in the EV-D68-specific-RT-PCR. In conclusion, use of an EV-specific-RT-PCR allowed us to detect EV-D68 circulation in autumn 2014 that was not detected by the multiplex RT-PCR and was associated with severe disease. © 2017 Wiley Periodicals, Inc.

Canine parvovirus 2c infection in central Portugal.

PubMed

João Vieira, Maria; Silva, Eliane; Oliveira, João; Luísa Vieira, Ana; Decaro, Nicola; Desario, Costantina; Muller, Alexandra; Carvalheira, Júlio; Buonavoglia, Canio; Thompson, Gertrude

2008-07-01

Canine parvovirus (CPV) has been evolving, generating new genetic and antigenic variants throughout the world. This study was conducted to determine the types of CPV circulating in dogs in Figueira da Foz, Portugal. Thirty fecal samples, collected between 2006 and 2007 from dogs with clinical signs of CPV infection, were tested for CPV by a rapid, in-clinic, enzyme-linked immunosorbent assay (ELISA)/immunomigration test, by conventional real-time polymerase chain reaction (PCR), and by minor-groove binding TaqMan PCR. Of the 29 PCR-positive samples, 15 were identified as CPV-2b and 14 as CPV-2c. No CPV-2a was detected. The sensitivity of the ELISA test was 82.76% compared with the PCR assays. No significant associations were found between CPV type, clinical outcome, breed, vaccination status, or age.

Fundamentals of Counting Statistics in Digital PCR: I Just Measured Two Target Copies-What Does It Mean?

PubMed

Tzonev, Svilen

2018-01-01

Current commercially available digital PCR (dPCR) systems and assays are capable of detecting individual target molecules with considerable reliability. As tests are developed and validated for use on clinical samples, the need to understand and develop robust statistical analysis routines increases. This chapter covers the fundamental processes and limitations of detecting and reporting on single molecule detection. We cover the basics of quantification of targets and sources of imprecision. We describe the basic test concepts: sensitivity, specificity, limit of blank, limit of detection, and limit of quantification in the context of dPCR. We provide basic guidelines how to determine those, how to choose and interpret the operating point, and what factors may influence overall test performance in practice.

Use of Serial Quantitative PCR of the vapA Gene of Rhodococcus equi in Feces for Early Detection of R. equi Pneumonia in Foals.

PubMed

Madrigal, R G; Shaw, S D; Witkowski, L A; Sisson, B E; Blodgett, G P; Chaffin, M K; Cohen, N D

2016-01-01

Current screening tests for Rhodococcus equi pneumonia in foals lack adequate accuracy for clinical use. Real-time, quantitative PCR (qPCR) for virulent R. equi in feces has not been systematically evaluated as a screening test. The objective of this study was to evaluate the accuracy of qPCR for vapA in serially collected fecal samples as a screening test for R. equi pneumonia in foals. One hundred and twenty-five foals born in 2011 at a ranch in Texas. Fecal samples were collected concurrently with thoracic ultrasonography (TUS) screening examinations at ages 3, 5, and 7 weeks. Affected (pneumonic) foals (n = 25) were matched by age and date-of-birth to unaffected (n = 25) and subclinical (ie, having thoracic TUS lesions but no clinical signs of pneumonia) foals (n = 75). DNA was extracted from feces using commercial kits and concentration of virulent R. equi in feces was determined by qPCR. Subsequently affected foals had significantly greater concentrations of vapA in feces than foals that did not develop pneumonia (unaffected and subclinical foals) at 5 and 7 weeks of age. Accuracy of fecal qPCR, however, was poor as a screening test to differentiate foals that would develop clinical signs of pneumonia from those that would remain free of clinical signs (including foals with subclinical pulmonary lesions attributed to R. equi) using receiver operating characteristic (ROC) methods. In the population studied, serial qPCR on feces lacked adequate accuracy as a screening test for clinical R. equi foal pneumonia. Copyright © 2016 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.

Babesia microti real-time polymerase chain reaction testing of Connecticut blood donors: potential implications for screening algorithms.

PubMed

Johnson, Stephanie T; Van Tassell, Eric R; Tonnetti, Laura; Cable, Ritchard G; Berardi, Victor P; Leiby, David A

2013-11-01

Babesia microti, an intraerythrocytic parasite, has been implicated in transfusion transmission. B. microti seroprevalence in Connecticut (CT) blood donors is approximately 1%; however, it is not known what percentage of donors is parasitemic and poses a risk for transmitting infection. Therefore, we determined the prevalence of demonstrable B. microti DNA in donors from a highly endemic area of CT and compared observed rates with concurrent immunofluorescence assay (IFA) testing results. Blood samples from consenting donors in southeastern CT were collected from mid-August through early October 2009 and tested by IFA for immunoglobulin G antibodies and real-time polymerase chain reaction (PCR) for B. microti DNA. IFA specificity was determined using blood donor samples collected in northwestern Vermont (VT), an area nonendemic for Babesia. Of 1002 CT donors, 25 (2.5%) were IFA positive and three (0.3%) were real-time PCR positive. Among the three real-time PCR-positive donors, two were also IFA positive, while one was IFA negative and may represent a window period infection. The two IFA- and real-time PCR-positive donors appeared to subsequently clear infection. The other real-time PCR-positive donor did not provide follow-up samples. Of 1015 VT donors tested by IFA, only one (0.1%) was positive, but may have acquired infection during travel to an endemic area. We prospectively identified several real-time PCR-positive blood donors, including an IFA-negative real-time PCR-positive donor, in an area highly endemic for B. microti. These results suggest the need to include nucleic acid testing in planned mitigation strategies for B. microti. © 2013 American Association of Blood Banks.

Evolution of Specific Antibodies and Proviral DNA in Milk of Small Ruminants Infected by Small Ruminant Lentivirus

PubMed Central

Barquero, Nuria; Gomez-Lucia, Esperanza; Arjona, Alvaro; Toural, Cristina; las Heras, Alfonso; Fernández-Garayzabal, José F.; Domenech, Ana

2013-01-01

The diagnosis of Small Ruminant Lentivirus (SRLV) is based on clinical signs, pathological lesions and laboratory testing. No standard reference test for the diagnosis of maedi visna has been validated up to the present, and it is puzzling that tests which detect antibodies against the virus and tests which detect the proviral genome may render opposite results. The aim of this study was to evaluate the presence in milk throughout a lactation period of specific antibodies by ELISA and of SRLV proviral DNA by a PCR of the highly conserved pol region. A six-month study was conducted with the milk of 28 ewes and 31 goats intensively reared. The percentage of animals with antibodies against SRLV increased throughout the study period. Seroprevalence in sheep was 28% at the beginning of the study and by the end it had increased up to 52.4%. In goats, initial seroprevalence of 5.6% increased to 16%. The percentage of PCR positive ewes was stable throughout the study period. Of the positive sheep, 21.4% were PCR-positive before antibodies could be detected and most of them became PCR-negative shortly after the first detection of antibodies. This might suggest that antibodies have a neutralizing effect. In addition, an equal percentage of sheep were always PCR-negative but either became ELISA-positive or was always ELISA-positive, which might support this hypothesis. On the other hand, the PCR results in goats did not follow any pattern and oscillated between 35.3% and 55.6% depending on the month. Most goats positive by PCR failed to develop antibodies in the 6 months tested. We may conclude that the infection and the antibody response to it follow a different trend in sheep and goats. PMID:24153063

Short communication: Identification of coagulase-negative staphylococcus species from goat milk with the API Staph identification test and with transfer RNA-intergenic spacer PCR combined with capillary electrophoresis.

PubMed

Koop, G; De Visscher, A; Collar, C A; Bacon, D A C; Maga, E A; Murray, J D; Supré, K; De Vliegher, S; Haesebrouck, F; Rowe, J D; Nielen, M; van Werven, T

2012-12-01

Coagulase-negative staphylococci (CNS) are the most commonly isolated bacteria from goat milk, but they have often been identified with phenotypic methods, which may have resulted in misclassification. The aims of this paper were to assess the amount of misclassification of a phenotypic test for identifying CNS species from goat milk compared with transfer RNA intergenic spacer PCR (tDNA-PCR) followed by capillary electrophoresis, and to apply the tDNA-PCR technique on different capillary electrophoresis equipment. Milk samples were collected from 416 does in 5 Californian dairy goat herds on 3 occasions during lactation. In total, 219 CNS isolates were identified at the species level with tDNA-PCR and subjected to the API 20 Staph identification test kit (API Staph; bioMérieux, Durham, NC). If the same species was isolated multiple times from the same udder gland, only the first isolate was used for further analyses, resulting in 115 unique CNS isolates. According to the tDNA-PCR test, the most prevalent CNS species were Staphylococcus epidermidis, Staphylococcus caprae, and Staphylococcus simulans. Typeability with API staph was low (72%). Although the API Staph test was capable of identifying the majority of Staph. epidermidis and Staph. caprae isolates, sensitivity for identification of Staph. simulans was low. The true positive fraction was high for the 3 most prevalent species. It was concluded that the overall performance of API Staph in differentiating CNS species from goat milk was moderate to low, mainly because of the low typeability, and that genotypic methods such as tDNA-PCR are preferred. Copyright © 2012 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Diagnostic accuracy of two multiplex real-time polymerase chain reaction assays for the diagnosis of meningitis in children in a resource-limited setting.

PubMed

Khumalo, Jermaine; Nicol, Mark; Hardie, Diana; Muloiwa, Rudzani; Mteshana, Phindile; Bamford, Colleen

2017-01-01

Accurate etiological diagnosis of meningitis is important, but difficult in resource-limited settings due to prior administration of antibiotics and lack of viral diagnostics. We aimed to develop and validate 2 real-time multiplex PCR (RT-PCR) assays for the detection of common causes of community-acquired bacterial and viral meningitis in South African children. We developed 2 multiplex RT- PCRs for detection of S. pneumoniae, N. meningitidis, H. influenzae, enteroviruses, mumps virus and herpes simplex virus. We tested residual CSF samples from children presenting to a local paediatric hospital over a one-year period, whose CSF showed an abnormal cell count. Results were compared with routine diagnostic tests and the final discharge diagnosis. We calculated accuracy of the bacterial RT-PCR assay compared to CSF culture and using World Health Organisation definitions of laboratory-confirmed bacterial meningitis. From 292 samples, bacterial DNA was detected in 12 (4.1%) and viral nucleic acids in 94 (32%). Compared to CSF culture, the sensitivity and specificity of the bacterial RT-PCR was 100% and 97.2% with complete agreement in organism identification. None of the cases positive by viral RT-PCR had a bacterial cause confirmed on CSF culture. Only 9/90 (10%) of patients diagnosed clinically as bacterial meningitis or partially treated bacterial meningitis tested positive with the bacterial RT-PCR. In this population the use of 2 multiplex RT-PCRs targeting 6 common pathogens gave promising results. If introduced into routine diagnostic testing, these multiplex RT-PCR assays would supplement other diagnostic tests, and have the potential to limit unnecessary antibiotic therapy and hospitalisation.

Diagnostic accuracy of two multiplex real-time polymerase chain reaction assays for the diagnosis of meningitis in children in a resource-limited setting

PubMed Central

Khumalo, Jermaine; Nicol, Mark; Hardie, Diana; Muloiwa, Rudzani; Mteshana, Phindile

2017-01-01

Introduction Accurate etiological diagnosis of meningitis is important, but difficult in resource-limited settings due to prior administration of antibiotics and lack of viral diagnostics. We aimed to develop and validate 2 real-time multiplex PCR (RT-PCR) assays for the detection of common causes of community-acquired bacterial and viral meningitis in South African children. Methods We developed 2 multiplex RT- PCRs for detection of S. pneumoniae, N. meningitidis, H. influenzae, enteroviruses, mumps virus and herpes simplex virus. We tested residual CSF samples from children presenting to a local paediatric hospital over a one-year period, whose CSF showed an abnormal cell count. Results were compared with routine diagnostic tests and the final discharge diagnosis. We calculated accuracy of the bacterial RT-PCR assay compared to CSF culture and using World Health Organisation definitions of laboratory-confirmed bacterial meningitis. Results From 292 samples, bacterial DNA was detected in 12 (4.1%) and viral nucleic acids in 94 (32%). Compared to CSF culture, the sensitivity and specificity of the bacterial RT-PCR was 100% and 97.2% with complete agreement in organism identification. None of the cases positive by viral RT-PCR had a bacterial cause confirmed on CSF culture. Only 9/90 (10%) of patients diagnosed clinically as bacterial meningitis or partially treated bacterial meningitis tested positive with the bacterial RT-PCR. Discussion In this population the use of 2 multiplex RT-PCRs targeting 6 common pathogens gave promising results. If introduced into routine diagnostic testing, these multiplex RT-PCR assays would supplement other diagnostic tests, and have the potential to limit unnecessary antibiotic therapy and hospitalisation. PMID:28346504

Comparison of ELISA, nested PCR and sequencing and a novel qPCR for detection of Giardia isolates from Jordan.

PubMed

Hijjawi, Nawal; Yang, Rongchang; Hatmal, Ma'mon; Yassin, Yasmeen; Mharib, Taghrid; Mukbel, Rami; Mahmoud, Sameer Alhaj; Al-Shudifat, Abdel-Ellah; Ryan, Una

2018-02-01

Little is known about the prevalence of Giardia duodenalis in human patients in Jordan and all previous studies have used direct microscopy, which lacks sensitivity. The present study developed a novel quantitative PCR (qPCR) assay at the β-giardin (bg) locus and evaluated its use as a frontline test for the diagnosis of giardiasis in comparison with a commercially available ELISA using nested PCR and sequencing of the glutamate dehydrogenase (gdh) locus (gdh nPCR) as the gold standard. A total of 96 human faecal samples were collected from 96 patients suffering from diarrhoea from 5 regions of Jordan and were screened using the ELISA and qPCR. The analytical specificity of the bg qPCR assay revealed no cross-reactions with other genera and detected all the Giardia isolates tested. Analytical sensitivity was 1 Giardia cyst per μl of DNA extract. The overall prevalence of Giardia was 64.6%. The clinical sensitivity and specificity of the bg qPCR was 89.9% and 82.9% respectively compared to 76.5 and 68.0% for the ELISA. This study is the first to compare three different methods (ELISA, bg qPCR, nested PCR and sequencing at the gdh locus) to diagnose Jordanian patients suffering from giardiasis and to analyze their demographic data. Copyright © 2018 Elsevier Inc. All rights reserved.

A serotype-specific polymerase chain reaction for identification of Pasteurella multocida serotype 1

USGS Publications Warehouse

Rocke, T.E.; Smith, S.R.; Miyamoto, A.; Shadduck, D.J.

2002-01-01

A serotype-specific polymerase chain reaction (PCR) assay was developed for detection and identification of Pasteurella multocida serotype 1, the causative agent of avian cholera in wild waterfowl. Arbitrarily primed PCR was used to detect DNA fragments that distinguish serotype 1 from the other 15 serotypes of P. multocida (with the exception of serotype 14). Oligonucleotide primers were constructed from these sequences, and a PCR assay was optimized and evaluated. PCR reactions consistently resulted in amplification products with reference strains 1 and 14 and all other serotype 1 strains tested, with cell numbers as low as 2.3 cells/ml. No amplification products were produced with other P. multocida serotypes or any other bacterial species tested. To compare the sensitivity and further test the specificity of this PCR assay with traditional culturing and serotyping techniques, tissue samples from 84 Pekin ducks inoculated with field strains of P. multocida and 54 wild lesser snow geese collected during an avian cholera outbreak were provided by other investigators working on avian cholera. PCR was as sensitive (58/64) as routine isolation (52/64) in detecting and identifying P. multocida serotype 1 from the livers of inoculated Pekins that became sick or died from avian cholera. No product was amplified from tissues of 20 other Pekin ducks that received serotypes other than type 1 (serotype 3, 12 × 3, or 10) or 12 control birds. Of the 54 snow geese necropsied and tested for P. multocida, our PCR detected and identified the bacteria from 44 compared with 45 by direct isolation. The serotype-specific PCR we developed was much faster and less labor intensive than traditional culturing and serotyping procedures and could result in diagnosis of serotype 1 pasteurellosis within 24 hr of specimen submission.

Establishment of a 10-Plex Quantitative Fluorescent-PCR Assay for Rapid Diagnosis of Sex Chromosome Aneuploidies

PubMed Central

Xie, Xingmei; Liang, Qiaoyi

2014-01-01

Sex chromosome aneuploidies occur commonly in the general population, with an incidence of 1 in 400 newborns. However, no tests specifically targeting sex chromosomes have been carried out in prenatal diagnosis or newborn screening, resulting in late recognition of these diseases. In this study, a rapid diagnostic method for sex chromosome aneuploidies was established using Quantitative Fluorescent-PCR (QF-PCR). Ten markers were included in one multiplex QF-PCR assay, including two sex determination genes (AMXY and SRY), five X-linked short tandem repeats (STRs; DXS1053, DXS981, DXS6809, DXS1187, and DXS8377), one X/Y-common STR (X22), and two autosomal STRs (D13S305 and D21S11). Retrospective tests of 70 cases with known cytogenetic results indicated that the 10-plex QF-PCR assay could well determine sex chromosome copy numbers by both allelic peak numbers and a sex chromosome dosage calculation with the autosomal STRs as internal controls. Prospective comparison with cytogenetic karyotyping on 534 cases confirmed that the 10-plex QF-PCR assay could be well employed for sex chromosome aneuploidy diagnosis in at least the Chinese Han population. This is the first QF-PCR test for the diagnosis of sex chromosome aneuploidies in the Chinese population. This test is superior to previous designs by including up to 8 sex-linked markers covering different parts of sex chromosomes as well as employing internal controls for copy number dosage calculation in a single PCR reaction. Due to simple technique and data analysis, as well as easy implementation within routine clinical services, this method is of great clinical application value and could be widely applied. PMID:25207978

Evaluation of Traditional and Contemporary Methods for Detecting Syphacia obvelata and Aspiculuris tetraptera in Laboratory Mice

PubMed Central

Gerwin, Philip M; Arbona, Rodolfo J Ricart; Riedel, Elyn R; Lepherd, Michelle L; Henderson, Ken S; Lipman, Neil S

2017-01-01

There is no consensus regarding the best practice for detecting murine pinworm infections. Initially, we evaluated 7 fecal concentration methods by using feces containing Aspiculuris tetraptera (AT) eggs (n = 20 samples per method). Sodium nitrate flotation, sodium nitrate centrifugation, Sheather sugar centrifugation, and zinc sulfate centrifugation detected eggs in 100% of samples; zinc sulfate flotation and water sedimentation detected eggs in 90%. All had better detection rates than Sheather sugar flotation (50%). To determine optimal detection methods, Swiss Webster mice were exposed to Syphacia obvelata (SO; n = 60) or AT (n = 60). We compared the following methods at days 0, 30, and 90, beginning 21 or 28 d after SO and AT exposure, respectively: fecal concentration (AT only), anal tape test (SO only), direct examination of intestinal contents (cecum and colon), Swiss roll histology (cecum and colon), and PCR analysis (pooled fur swab and feces). Detection rates for SO-exposed mice were: PCR analysis, 45%; Swiss roll histology, 30%; intestinal content exam, 27%; and tape test, 27%. The SO detection rate for PCR analysis was significantly greater than that for the tape test. Detection rates for AT-exposed mice were: intestinal content exam, 53%; PCR analysis, 33%; fecal flotation, 22%; and Swiss roll histology, 17%. The AT detection rate of PCR analysis combined with intestinal content examination was greater than for PCR analysis only and the AT detection rate of intestinal content examination was greater than for Swiss roll histology. Combining PCR analysis with intestinal content examination detected 100% of infected animals. No single test detected all positive animals. We recommend combining PCR analysis with intestinal content examination for optimal pinworm detection. PMID:28905712

Evaluation of a Rapid Fecal PCR Test for Detection of Mycobacterium avium subsp. paratuberculosis in Dairy Cattle▿

PubMed Central

Wells, Scott J.; Collins, Michael T.; Faaberg, Kay S.; Wees, Carrie; Tavornpanich, Saraya; Petrini, Kristine R.; Collins, James E.; Cernicchiaro, Natalia; Whitlock, Robert H.

2006-01-01

A high-throughput TaqMan PCR assay for detection of bovine paratuberculosis was evaluated by using fecal samples from 1,808 dairy cattle in seven naturally infected herds and 347 dairy cattle in seven herds considered free of paratuberculosis. Fecal, blood, and milk samples were submitted to laboratories where the PCR-based assay, three different fecal culture procedures for Mycobacterium avium subsp. paratuberculosis (centrifugation, sedimentation, and the BACTEC filter concentration method), two serologic enzyme-linked immunosorbent assays (ELISAs), and one milk ELISA were performed. Results from testing of dairy cattle in herds free of M. avium subsp. paratuberculosis showed that the PCR assay's specificity was 99.7%. Twenty-three percent of the dairy cows that were fecal culture positive by at least one of the three methods were positive by the PCR assay. By Bayesian non-“gold standard” analysis methods, the TaqMan PCR assay had a higher specificity than the serum ELISAs (99.3%; 95% confidence interval [CI] = 98.6 to 99.7%) and a test sensitivity similar to that of the serum ELISAs (29%; 95% CI = 24 to 35%). By classical methods, the estimated relative sensitivity of the fecal PCR assay was 4% for light and moderate fecal shedders (compared to 12 to 13% for the ELISAs) and 76% for heavy fecal shedders (compared to 67% for the milk ELISA). The PCR assay has higher sensitivity for detection of heavy fecal shedders than the evaluated milk ELISA but lower sensitivity than a serum or milk ELISA for detection of light and moderate fecal shedders. This assay can be used as a quick test for detection of cattle with heavy fecal shedding, those cattle with the highest risk of transmitting infection to susceptible cattle. PMID:16928884

Determining the analytical specificity of PCR-based assays for the diagnosis of IA: What is Aspergillus?

PubMed

Morton, C Oliver; White, P Lewis; Barnes, Rosemary A; Klingspor, Lena; Cuenca-Estrella, Manuel; Lagrou, Katrien; Bretagne, Stéphane; Melchers, Willem; Mengoli, Carlo; Caliendo, Angela M; Cogliati, Massimo; Debets-Ossenkopp, Yvette; Gorton, Rebecca; Hagen, Ferry; Halliday, Catriona; Hamal, Petr; Harvey-Wood, Kathleen; Jaton, Katia; Johnson, Gemma; Kidd, Sarah; Lengerova, Martina; Lass-Florl, Cornelia; Linton, Chris; Millon, Laurence; Morrissey, C Orla; Paholcsek, Melinda; Talento, Alida Fe; Ruhnke, Markus; Willinger, Birgit; Donnelly, J Peter; Loeffler, Juergen

2017-06-01

A wide array of PCR tests has been developed to aid the diagnosis of invasive aspergillosis (IA), providing technical diversity but limiting standardisation and acceptance. Methodological recommendations for testing blood samples using PCR exist, based on achieving optimal assay sensitivity to help exclude IA. Conversely, when testing more invasive samples (BAL, biopsy, CSF) emphasis is placed on confirming disease, so analytical specificity is paramount. This multicenter study examined the analytical specificity of PCR methods for detecting IA by blind testing a panel of DNA extracted from a various fungal species to explore the range of Aspergillus species that could be detected, but also potential cross reactivity with other fungal species. Positivity rates were calculated and regression analysis was performed to determine any associations between technical specifications and performance. The accuracy of Aspergillus genus specific assays was 71.8%, significantly greater (P < .0001) than assays specific for individual Aspergillus species (47.2%). For genus specific assays the most often missed species were A. lentulus (25.0%), A. versicolor (24.1%), A. terreus (16.1%), A. flavus (15.2%), A. niger (13.4%), and A. fumigatus (6.2%). There was a significant positive association between accuracy and using an Aspergillus genus PCR assay targeting the rRNA genes (P = .0011). Conversely, there was a significant association between rRNA PCR targets and false positivity (P = .0032). To conclude current Aspergillus PCR assays are better suited for detecting A. fumigatus, with inferior detection of most other Aspergillus species. The use of an Aspergillus genus specific PCR assay targeting the rRNA genes is preferential. © The Author 2016. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Role of PCR method using IS6110 primer in detecting Mycobacterium tuberculosis among the clinically diagnosed childhood tuberculosis patients at an urban hospital in Dhaka, Bangladesh.

PubMed

Kabir, Senjuti; Uddin, Mohammad Khaja Mafij; Chisti, Mohammod Jobayer; Fannana, Tilka; Haque, Mohammad Enamul; Uddin, Muhammad Reaj; Banu, Sayera; Ahmed, Tahmeed

2018-03-01

Better methods are needed for the accurate detection of child tuberculosis (TB). This study compared different laboratory tests and evaluated IS6110 PCR for the detection of Mycobacterium tuberculosis (MTB) among clinically diagnosed child TB patients. A total of 102 paediatric patients (<15 years old) with clinically diagnosed TB were enrolled in this study. The patients were admitted to the icddr,b hospital in Dhaka between 2003 and 2005. Sputum/gastric lavage samples were collected for smear microscopy, culture (solid/Lowenstein-Jensen medium and liquid/MGIT), and IS6110 PCR testing. The sensitivity, specificity, and positive and negative predictive values (PPV, NPV) of smear microscopy and PCR were compared to the two culture methods. Three patients were positive on smear microscopy (2.9%). MTB was detected by conventional culture in 15.7% (16/102), liquid culture in 14% (14/100), and IS6110 PCR in 61.8% (63/102). PCR detected an additional 45 patients who were undetected with the three other tests. Compared to conventional and liquid culture, respectively, smear microscopy showed sensitivity of 18.8% and 21.4%, specificity of 100% individually, PPV of 100% individually, and NPV of 86.9% and 88.7%, whereas PCR had sensitivity of 87.5% and 92.9%, specificity of 43% individually, PPV of 22.2% and 21%, and NPV of 94.9% and 97.4%. PCR can be useful compared to smear microscopy and culture methods and is applicable as a rapid screening test for child TB. A larger scale study is required to determine its diagnostic efficacy in improving the detection of child TB in the presence and absence of severe malnutrition. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

Sensitivity and specificity of the AdenoPlus point-of-care system in detecting adenovirus in conjunctivitis patients at an ophthalmic emergency department: a diagnostic accuracy study.

PubMed

Kam, K Y Ronald; Ong, Hon Shing; Bunce, Catey; Ogunbowale, Lola; Verma, Seema

2015-09-01

To estimate the diagnostic accuracy (sensitivity and specificity) of the AdenoPlus point-of-care adenoviral test compared to PCR in an ophthalmic accident and emergency service. These findings were compared with those of a previous study. This was a prospective diagnostic accuracy study on 121 patients presenting to an emergency eye unit with a clinical picture of acute adenoviral conjunctivitis. AdenoPlus testing was carried out on one eye of each patient and a PCR analysis was also performed on a swab taken from the same eye. AdenoPlus and PCR results were interpreted by masked personnel. Sensitivity and specificity for the AdenoPlus test were calculated using PCR results as the reference standard. 121 patients were enrolled and 109 met the inclusion criteria. 43 patients (39.4%) tested positive for adenovirus by PCR analysis. The sensitivity of the AdenoPlus swab in detecting adenovirus was 39.5% (17/43, 95% CI 26% to 54%) and specificity was 95.5% (63/66, 95% CI 87% to 98%) compared to PCR. The AdenoPlus test has a high specificity for diagnosing adenoviral conjunctivitis, but in this clinical setting, we could not reproduce the high sensitivity that has been previously published. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Comparative application of IS711-based polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) for canine brucellosis diagnosis.

PubMed

Batinga, Maria Cryskely Agra; de Lima, Julia Teresa Ribeiro; Gregori, Fabio; Diniz, Jaqueline Assumpção; Muner, Kerstin; Oliveira, Trícia M F S; Ferreira, Helena Lage; Soares, Rodrigo Martins; Keid, Lara Borges

2018-06-01

Canine brucellosis is caused by Brucella canis, a gram negative and facultative intracellular bacterium that is commonly associated with reproductive failures in dogs. The accurate diagnosis of the infection relies on the use of serological tests associated with blood culturing to guarantee sensitivity. The polymerase chain reaction (PCR) can replace the culturing procedure for the direct diagnosis of the infection because of its speed, high specificity and sensitivity values; however, it depends on some laboratory infrastructure to be conducted. The loop-mediated isothermal amplification (LAMP) may be an alternative method for DNA amplification in a shorter period, using simpler equipment, and with a lower cost. This study evaluated the potential of molecular tools based on PCR and LAMP using primers targeting the insertion sequence IS711 for Brucella detection in three groups of dogs (infected, non-infected and suspected of brucellosis), which were determined according to the results of blood culturing and clinical examination. The performance of the three diagnostic tests was also determined using McNemar test and Kappa coefficient. The proportion of positive samples detected by blood culturing, PCR and LAMP was respectively 31.57% (18/57), 33.34% (19/57), and 14.03% (8/57). The agreement between blood culturing and PCR was almost perfect, while the agreement of PCR and blood culturing compared to LAMP was fair. The diagnostic sensitivity of PCR and LAMP was respectively 100% (18/18) and 44.44% (8/18), while the diagnostic specificity of both tests was 100% (21/21). LAMP performance was not satisfactory for canine brucellosis diagnosis because of the low diagnostic sensitivity of the test. The IS711 based PCR, otherwise, showed high values of sensitivity and specificity, which makes it a good alternative for use for the rapid diagnosis of canine brucellosis. Copyright © 2018 Elsevier Ltd. All rights reserved.

Evaluation of a new amplified enzyme immunoassay (EIA) for the detection of Chlamydia trachomatis in male urine, female endocervical swab, and patient obtained vaginal swab specimens

PubMed Central

Tanaka, M.; Nakayama, H.; Sagiyama, K.; Haraoka, M.; Yoshida, H.; Hagiwara, T.; Akazawa, K.; Naito, S.

2000-01-01

Aims—To compare the performance of a new generation dual amplified enzyme immunoassay (EIA) with a molecular method for the diagnosis of Chlamydia trachomatis, using a range of urogenital samples, and to assess the reliability of testing self collected vaginal specimens compared with clinician collected vaginal specimens. Methods—Two population groups were tested. For the first population group, first void urine samples were collected from 193 male patients with urethritis, and endocervical swabs were collected from 187 high risk commercial sex workers. All urine and endocervical specimens were tested by a conventional assay (IDEIA chlamydia), a new generation amplified immunoassay (IDEIA PCE chlamydia), and the Amplicor polymerase chain reaction (PCR). Discrepant results obtained among the three sample types were confirmed using a nested PCR test with a different plasmid target region. For the second population group, four swab specimens, including one patient obtained vaginal swab, two clinician obtained endocervical swabs, and one clinician obtained vaginal swab, were collected from 91 high risk sex workers. Self collected and clinician collected vaginal swabs were tested by IDEIA PCE chlamydia. Clinician obtained endocervical swabs were assayed by IDEIA PCE chlamydia and Amplicor PCR. Results—The performance of the IDEIA PCE chlamydia test was comparable to that of the Amplicor PCR test when male urine and female endocervical swab specimens were analysed. The relative sensitivities of IDEIA, IDEIA PCE, and Amplicor PCR on male first void urine specimens were 79.3%, 91.4%, and 100%, respectively. The relative sensitivities of the three tests on female endocervical specimens were 85.0%, 95.0%, and 100%, respectively. The positivity rates for patient collected vaginal specimens and clinician collected vaginal specimens by IDEIA PCE were 25.2% and 23.1%, respectively, whereas those for clinician collected endocervical swabs by PCR and IDEIA PCE were both 27.5%. Conclusions—IDEIA PCE chlamydia is a lower cost but sensitive alternative test to PCR for testing male urine samples and female endocervical swabs. In addition, self collected or clinician collected vaginal specimens tested by IDEIA PCE chlamydia are a reliable alternative to analysing endocervical specimens. Key Words: Chlamydia trachomatis • enzyme immunoassay • clinical specimens PMID:10889816

Amplification of ST50 gene using dry-reagent-based polymerase chain reaction for the detection of Salmonella typhi.

PubMed

Aziah, Ismail; Ravichandran, Manickam; Ismail, Asma

2007-12-01

Conventional polymerase chain reaction (PCR) testing requires many pipetting steps and has to be transported and stored in cold chain. To overcome these limitations, we designed a ready-to-use PCR test for Salmonella typhi using PCR reagents, primers against the ST50 gene of S. typhi, a built-in internal amplification control (IAC), and gel loading dye mixed and freeze-dried in a single tube. The 2-step dry-reagent-based assay was used to amplify a 1238-bp target gene and an 810-bp IAC gene from 73 BACTEC blood culture broths (33 true positives for S. typhi and 40 true negatives for non-S. typhi). The sensitivity, specificity, positive predictive value, and negative predictive value of the PCR assay were 87.9%, 100%, 100%, and 90.9%, respectively. We suggest that this rapid 2-step PCR test could be used for the rapid diagnosis of typhoid fever.

Health Care Resource Utilization and Costs in Patients with Chronic Myeloid Leukemia with Better Adherence to Tyrosine Kinase Inhibitors and Increased Molecular Monitoring Frequency.

PubMed

Latremouille-Viau, Dominick; Guerin, Annie; Gagnon-Sanschagrin, Patrick; Dea, Katherine; Cohen, Benjamin G; Joseph, George J

2017-02-01

Frequent molecular monitoring (qPCR tests), as recommended by evidence-based monitoring guidelines, is associated with higher adherence to tyrosine kinase inhibitors (TKIs) in the management of chronic myeloid leukemia (CML); both factors have been associated with better clinical and economic outcomes. To (a) estimate the effect of more frequent qPCR tests on health care resource utilization (HRU) and associated costs, including direct (effect of qPCR test frequency on HRU) and indirect (through TKI adherence) effects, and (b) develop an economic model applicable to multiple clinical practice scenarios. Adult patients newly diagnosed with CML who started TKI firstline therapy were identified from U.S. administrative claims data (2010-2015). TKI adherence (medication possession ratio [MPR]), number of inpatient days, emergency room (ER) visits, outpatient service days, and mean costs per HRU event were measured during the first year of CML treatment. Direct and indirect effects of qPCR test frequency were estimated using multivariate regression models. Subsequently, an economic model was developed to assess the overall effect of varying qPCR test frequency on HRU and associated costs during the first year of CML treatment under different clinical practice scenarios; the scenario reported is the increase from 1 to 2 qPCR tests. Of the 1,431 patients included, 36% had no qPCR tests, the average qPCR test frequency was 1.6, and the average MPR was 0.86 during the first year of CML treatment. The direct effect of increasing qPCR test frequency by 1 was associated with 13.0% fewer inpatient days (adjusted incidence rate ratio [adjusted IRR] = 0.87; P = 0.010); 8.3% fewer ER visits (adjusted IRR = 0.92; P = 0.043); and 3.0% more outpatient service days (adjusted IRR = 1.03; P = 0.002). Each increase of 1 test was associated with an increase in TKI adherence by 2.2 percentage points (adjusted MPR difference = 0.022; P < 0.001). When considering the indirect effect of qPCR test frequency through TKI adherence, an increase of 1 qPCR test combined with an increase in TKI adherence by 2.2 percentage points was associated with a greater reduction of inpatient days from 13.0% to 15.2%, ER visits from 8.3% to 8.6%, and a smaller increase of outpatient service days from 3.0% to 2.6%. Based on the economic model, an increase from 1 to 2 qPCR tests, considering the increase in TKI adherence, was associated with a reduction of 0.87 (95% CI = -1.49, -0.18) inpatient days and 0.06 (95% CI = -0.12, 0.05) ER visits, an increase of 0.98 (95% CI = 0.25, 1.60) outpatient service days and a cost savings of $2,918 (95% CI = -5,213, -349) per patient per year. Closer alignment with the monitoring guidelines' recommended qPCR test frequency and better adherence to TKIs were associated with lower HRU and medical service costs. Managed care initiatives to increase qPCR test frequency and TKI adherence might benefit from an enhanced reduction because of the interaction between both factors. This study was funded by Novartis Pharmaceuticals, which was involved in all stages of the study and in the decision to submit the report for publication. Latremouille-Viau, Guerin, Gagnon-Sanschagrin, and Dea are employees of Analysis Group, which received consulting fees from Novartis Pharmaceuticals for work on this study. Joseph is an employee of Novartis Pharmaceuticals and owns stock in Amgen and Pfizer. Cohen was an employee of Novartis Pharmaceuticals at the time of this study. Portions of this study were presented online (beginning May 20, 2016) as part of the American Society of Clinical Oncology (ASCO) Annual Meeting in Chicago, Illinois, on June 3-7, 2016, and as a poster at the American Society of Hematology (ASH) Annual Meeting in San Diego, California, on December 3-6, 2016. Study concept and design were contributed by Latremouille-Viau and Guerin, along with the other authors. Gagnon-Sanschagrin and Dea took the lead in data collection, assisted by the other authors, and data interpretation was performed by Cohen and Joseph, along with the other authors. The manuscript was written by Latremouille-Viau, along with the other authors, and revised by Joseph, along with the other authors.

Rapid detection of fungal keratitis with DNA-stabilizing FTA filter paper.

PubMed

Menassa, Nardine; Bosshard, Philipp P; Kaufmann, Claude; Grimm, Christian; Auffarth, Gerd U; Thiel, Michael A

2010-04-01

Purpose. Polymerase chain reaction (PCR) is increasingly important for the rapid detection of fungal keratitis. However, techniques of specimen collection and DNA extraction before PCR may interfere with test sensitivity. The purpose of this study was to investigate the use of DNA-stabilizing FTA filter paper (Indicating FTA filter paper; Whatman International, Ltd., Maidstone, UK) for specimen collection without DNA extraction in a single-step, nonnested PCR for fungal keratitis. Methods. Specimens were collected from ocular surfaces with FTA filter discs, which automatically lyse collected cells and stabilize nucleic acids. Filter discs were directly used in single-step PCR reactions to detect fungal DNA. Test sensitivity was evaluated with serial dilutions of Candida albicans, Fusarium oxysporum, and Aspergillus fumigatus cultures. Test specificity was analyzed by comparing 196 and 155 healthy individuals from Switzerland and Egypt, respectively, with 15 patients with a diagnosis of microbial keratitis. Results. PCR with filter discs detected 3 C. albicans, 25 F. oxysporum, and 125 A. fumigatus organisms. In healthy volunteers, fungal PCR was positive in 1.0% and 8.4% of eyes from Switzerland and Egypt, respectively. Fungal PCR remained negative in 10 cases of culture-proven bacterial keratitis, became positive in 4 cases of fungal keratitis, but missed 1 case of culture-proven A. fumigatus keratitis. Conclusions. FTA filter paper for specimen collection together with direct PCR is a promising method of detecting fungal keratitis. The analytical sensitivity is high without the need for a semi-nested or nested second PCR, the clinical specificity is 91.7% to 99.0%, and the method is rapid and inexpensive.

Promising Nucleic Acid Lateral Flow Assay Plus PCR for Shiga Toxin-Producing Escherichia coli.

PubMed

Terao, Yoshitaka; Takeshita, Kana; Nishiyama, Yasutaka; Morishita, Naoki; Matsumoto, Takashi; Morimatsu, Fumiki

2015-08-01

Shiga toxin (Stx)-producing Escherichia coli (STEC) is a frequent cause of foodborne infections, and methods for rapid and reliable detection of STEC are needed. A nucleic acid lateral flow assay (NALFA) plus PCR was evaluated for detecting STEC after enrichment. When cell suspensions of 45 STEC strains, 14 non-STEC strains, and 13 non-E. coli strains were tested with the NALFA plus PCR, all of the STEC strains yielded positive results, and all of the non-STEC and non-E. coli strains yielded negative results. The lower detection limit for the STEC strains ranged from 0.1 to 1 pg of genomic DNA (about 20 to 200 CFU) per test, and the NALFA plus PCR was able to detect Stx1- and Stx2-producing E. coli strains with similar sensitivities. The ability of the NALFA plus PCR to detect STEC in enrichment cultures of radish sprouts, tomato, raw ground beef, and beef liver inoculated with 10-fold serially diluted STEC cultures was comparable to that of a real-time PCR assay (at a level of 100 to 100,000 CFU/ml in enrichment culture). The bacterial inoculation test in raw ground beef revealed that the lower detection limit of the NALFA plus PCR was also comparable to that obtained with a real-time PCR assay that followed the U.S. Department of Agriculture guidelines. Although further evaluation is required, these results suggest that the NALFA plus PCR is a specific and sensitive method for detecting STEC in a food manufacturing plant.

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. Part III. Virus Detection by RT-qPCR

EPA Science Inventory

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. The viral ribonucleic acid (RNA) from water sample concentrates is extracted and tested for enterovirus and norovirus RNA using reverse transcription-quantitative PCR (RT-qPCR). V...

Use of FTA gene guard filter paper for the storage and transportation of tumor cells for molecular testing.

PubMed

Dobbs, Larry J; Madigan, Merle N; Carter, Alexis B; Earls, Lori

2002-01-01

Efficient methods of storing tumor specimens for molecular testing are needed in the modern surgical pathology laboratory. The FTA Gene Guard system is a novel method for the collection and room temperature storage of blood samples for DNA testing. The method uses index card-sized filter papers that provide an ideal medium on which to store tumor specimens for DNA testing. To determine whether FTA filter paper can be used in the surgical pathology laboratory to store tumor cells for DNA testing. Cell suspensions were prepared from 60 surgical specimens, and DNA was extracted either immediately or after storage on FTA paper. The DNA extracted by each method was tested by polymerase chain reaction (PCR) for the beta-globin and interferon gamma genes, and the results were compared. Fifteen lymph node specimens stored on FTA paper were then tested for immunoglobulin heavy chain (IgH) gene rearrangement by PCR, and these results were compared with those obtained for immediately extracted DNA. University medical center. The DNA extracted from cells stored on FTA paper performed as well in the PCR as the freshly extracted DNA in nearly all cases (>95%). The results of tests for IgH gene rearrangements showed 100% concordance between the 2 methods of DNA extraction.Conclusion.-Cells from surgical specimens can be stored on FTA paper for extended lengths of time, and DNA can be extracted from these cells for PCR-based testing. FTA filter paper is a reliable medium for the storage and/or transport of tumor cells for PCR-based DNA analysis.

Enhanced resistance to blister blight in transgenic tea (Camellia sinensis [L.] O. Kuntze) by overexpression of class I chitinase gene from potato (Solanum tuberosum).

PubMed

Singh, H Ranjit; Deka, Manab; Das, Sudripta

2015-07-01

Tea is the second most consumed beverage in the world. A crop loss of up to 43 % has been reported due to blister blight disease of tea caused by a fungus, Exobasidium vexans. Thus, it directly affects the tea industry qualitatively and quantitatively. Solanum tuberosum class I chitinase gene (AF153195) is a plant pathogenesis-related gene. It was introduced into tea genome via Agrobacterium-mediated transformation with hygromycin phosphotransferase (hpt) gene conferring hygromycin resistance as plant selectable marker. A total of 41 hygromycin resistant plantlets were obtained, and PCR analysis established 12 plantlets confirming about the stable integration of transgene in the plant genome. Real-time PCR detected transgene expression in four transgenic plantlets (T28, C57, C9, and T31). Resistance to biotrophic fungal pathogen, E. vexans, was tested by detached leaf infection assay of greenhouse acclimated plantlets. An inhibitory activity against the fungal pathogen was evident from the detached leaves from the transformants compared with the control. Fungal lesion formed on control plantlet whereas the transgenic plantlets showed resistance to inoculated fungal pathogen by the formation of hypersensitivity reaction area. This result suggests that constitutive expression of the potato class I chitinase gene can be exploited to improve resistance to fungal pathogen, E. vexans, in economical perennial plantation crop like tea.

Development of an improved PCR-ICT hybrid assay for direct detection of Legionellae and Legionella pneumophila from cooling tower water specimens.

PubMed

Horng, Yu-Tze; Soo, Po-Chi; Shen, Bin-Jon; Hung, Yu-Li; Lo, Kai-Yin; Su, Hsun-Pi; Wei, Jun-Rong; Hsieh, Shang-Chen; Hsueh, Po-Ren; Lai, Hsin-Chih

2006-06-01

A novelly improved polymerase chian reaction and immunochromatography test (PCR-ICT) hybrid assay comprising traditional multiplex-nested PCR and ICT, (a lateral-flow device) was developed for direct detection of Legionella bacteria from environmental cooling tower samples. The partial 16S rDNA (specific for Legionella spp.) and dnaJ (specific for Legionella pneumophila) genes from Legionella chromosome were first specifically amplified by multiplex-nested PCR, respectively, followed by detection using ICT strip. Reading of results was based on presence or absence of the two test lines on the strips. Presence of test line 1 indicated existence of Legionella spp. specific 16S rDNA and identified Legionella spp. Presence of test line 2 further indicated existence of dnaJ and thus specifically identified L. pneumophila. In contrast, for non-Legionellae bacteria no test line formation was observed. Results of direct detection of Legionella bacteria and L. pneumophila from water tower specimens by this assay showed 100% sensitivity, and 96.6% and 100% specificity, respectively compared with traditional culture, biochemical and serological identification methods. The PCR-ICT hybrid assay does not require sophisticated equipment and was proved to be practically useful in rapid and direct Legionellae detection from environmental water samples.

Validation of a new algorithm for a quick and easy RT-PCR-based ALK test in a large series of lung adenocarcinomas: Comparison with FISH, immunohistochemistry and next generation sequencing assays.

PubMed

Marchetti, Antonio; Pace, Maria Vittoria; Di Lorito, Alessia; Canarecci, Sara; Felicioni, Lara; D'Antuono, Tommaso; Liberatore, Marcella; Filice, Giampaolo; Guetti, Luigi; Mucilli, Felice; Buttitta, Fiamma

2016-09-01

Anaplastic Lymphoma Kinase (ALK) gene rearrangements have been described in 3-5% of lung adenocarcinomas (ADC) and their identification is essential to select patients for treatment with ALK tyrosine kinase inhibitors. For several years, fluorescent in situ hybridization (FISH) has been considered as the only validated diagnostic assay. Currently, alternative methods are commercially available as diagnostic tests. A series of 217 ADC comprising 196 consecutive resected tumors and 21 ALK FISH-positive cases from an independent series of 702 ADC were investigated. All specimens were screened by IHC (ALK-D5F3-CDx-Ventana), FISH (Vysis ALK Break-Apart-Abbott) and RT-PCR (ALK RGQ RT-PCR-Qiagen). Results were compared and discordant cases subjected to Next Generation Sequencing. Thirty-nine of 217 samples were positive by the ALK RGQ RT-PCR assay, using a threshold cycle (Ct) cut-off ≤35.9, as recommended. Of these positive samples, 14 were negative by IHC and 12 by FISH. ALK RGQ RT-PCR/FISH discordant cases were analyzed by the NGS assay with results concordant with FISH data. In order to obtain the maximum level of agreement between FISH and ALK RGQ RT-PCR data, we introduced a new scoring algorithm based on the ΔCt value. A ΔCt cut-off level ≤3.5 was used in a pilot series. Then the algorithm was tested on a completely independent validation series. By using the new scoring algorithm and FISH as reference standard, the sensitivity and the specificity of the ALK RGQ RT-PCR(ΔCt) assay were 100% and 100%, respectively. Our results suggest that the ALK RGQ RT-PCR test could be useful in clinical practice as a complementary assay in multi-test diagnostic algorithms or even, if our data will be confirmed in independent studies, as a standalone or screening test for the selection of patients to be treated with ALK inhibitors. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

A Cytolethal Distending Toxin Gene-Based Multiplex PCR Assay for Campylobacter jejuni, C. fetus, C. coli, C. upsaliensis, C. hyointestinalis, and C. lari.

PubMed

Kamei, Kazumasa; Kawabata, Hiroki; Asakura, Masahiro; Samosornsuk, Worada; Hinenoya, Atsushi; Nakagawa, Shinsaku; Yamasaki, Shinji

2016-05-20

In this study, we devised a multiplex PCR assay based on the gene of cytolethal distending toxin (cdt) B subunit to simultaneously detect and discriminate Campylobacter jejuni, C. fetus, C. coli, C. upsaliensis, C. hyointestinalis, and C. lari. Species-specific PCR products were successfully obtained from all 38 C. jejuni, 12 C. fetus, 39 C. coli, 22 C. upsaliensis, 24 C. hyointestinalis, and 7 C. lari strains tested. On the other hand, no specific PCR products were obtained from other campylobacters and bacterial species tested (41 strains in total). The proposed multiplex PCR assay is a valuable tool for detection and descrimination of 6 major Campylobacter species, that are associated with gastrointestinal diseases in humans.

Evaluation of sensitivity of TaqMan RT-PCR for rubella virus detection in clinical specimens.

PubMed

Okamoto, Kiyoko; Mori, Yoshio; Komagome, Rika; Nagano, Hideki; Miyoshi, Masahiro; Okano, Motohiko; Aoki, Yoko; Ogura, Atsushi; Hotta, Chiemi; Ogawa, Tomoko; Saikusa, Miwako; Kodama, Hiroe; Yasui, Yoshihiro; Minagawa, Hiroko; Kurata, Takako; Kanbayashi, Daiki; Kase, Tetsuo; Murata, Sachiko; Shirabe, Komei; Hamasaki, Mitsuhiro; Kato, Takashi; Otsuki, Noriyuki; Sakata, Masafumi; Komase, Katsuhiro; Takeda, Makoto

2016-07-01

An easy and reliable assay for detection of the rubella virus is required to strengthen rubella surveillance. Although a TaqMan RT-PCR assay for detection of the rubella virus has been established in Japan, its utility for diagnostic purposes has not been tested. To allow introduction of the TaqMan RT-PCR into the rubella surveillance system in Japan, the sensitivity of the assay was determined using representative strains for all genotypes and clinical specimens. The detection limits of the method for individual genotypes were examined using viral RNA extracted from 13 representative strains. The assay was also tested at 10 prefectural laboratories in Japan, designated as local reference laboratories for measles and rubella, to allow nationwide application of the assay. The detection limits and amplification efficiencies of the assay were similar among all the representative strains of the 13 genotypes. The TaqMan RT-PCR could detect approximately 90% of throat swab and urine samples taken up to 5days of illness. These samples were determined positive by a highly sensitive nested RT-PCR. The TaqMan RT-PCR could detect at least 10 pfu of rubella virus. Although the sensitivity was somewhat lower than that of the conventional nested RT-PCR, the TaqMan RT-PCR could be more practical to routine tests for rubella laboratory diagnosis and detection in view of the rapid response and reducing risks of contamination. Copyright © 2016 Elsevier B.V. All rights reserved.

Use of Multiplex Real-Time PCR To Diagnose Scrub Typhus.

PubMed

Tantibhedhyangkul, Wiwit; Wongsawat, Ekkarat; Silpasakorn, Saowaluk; Waywa, Duangdao; Saenyasiri, Nuttawut; Suesuay, Jintapa; Thipmontree, Wilawan; Suputtamongkol, Yupin

2017-05-01

Scrub typhus, caused by Orientia tsutsugamushi , is a common cause of acute undifferentiated febrile illness in the Asia-Pacific region. However, its nonspecific clinical manifestation often prevents early diagnosis. We propose the use of PCR and serologic tests as diagnostic tools. Here, we developed a multiplex real-time PCR assay using hydrolysis (TaqMan) probes targeting O. tsutsugamushi 47-kDa, groEL , and human interferon beta (IFN-β gene) genes to improve early diagnosis of scrub typhus. The amplification efficiency was higher than 94%, and the lower detection limit was 10 copies per reaction. We used a human gene as an internal DNA quality and quantity control. To determine the sensitivity of this PCR assay, we selected patients with confirmed scrub typhus who exhibited a clear 4-fold increase in the level of IgG and/or IgM. The PCR assay result was positive in 45 of 52 patients, indicating a sensitivity of 86.5% (95% confidence interval [CI]: 74.2 to 94.4). The PCR assessment was negative for all 136 non-scrub typhus patients, indicating a specificity of 100% (95% CI: 97.3 to 100). In addition, this test helped diagnose patients with inconclusive immunofluorescence assay (IFA) results and using single blood samples. In conclusion, the real-time PCR assay proposed here is sensitive and specific in diagnosing scrub typhus. Combining PCR and serologic tests will improve the diagnosis of scrub typhus among patients presenting with acute febrile illness. Copyright © 2017 American Society for Microbiology.

Detecting Malaria Hotspots: A Comparison of Rapid Diagnostic Test, Microscopy, and Polymerase Chain Reaction

PubMed Central

Mogeni, Polycarp; Williams, Thomas N; Omedo, Irene; Kimani, Domtila; Ngoi, Joyce M; Mwacharo, Jedida; Morter, Richard; Nyundo, Christopher; Wambua, Juliana; Nyangweso, George; Kapulu, Melissa; Fegan, Gregory; Bejon, Philip

2017-01-01

Abstract Background Malaria control strategies need to respond to geographical hotspots of transmission. Detection of hotspots depends on the sensitivity of the diagnostic tool used. Methods We conducted cross-sectional surveys in 3 sites within Kilifi County, Kenya, that had variable transmission intensities. Rapid diagnostic test (RDT), microscopy, and polymerase chain reaction (PCR) were used to detect asymptomatic parasitemia, and hotspots were detected using the spatial scan statistic. Results Eight thousand five hundred eighty-one study participants were surveyed in 3 sites. There were statistically significant malaria hotspots by RDT, microscopy, and PCR for all sites except by microscopy in 1 low transmission site. Pooled data analysis of hotspots by PCR overlapped with hotspots by microscopy at a moderate setting but not at 2 lower transmission settings. However, variations in degree of overlap were noted when data were analyzed by year. Hotspots by RDT were predictive of PCR/microscopy at the moderate setting, but not at the 2 low transmission settings. We observed long-term stability of hotspots by PCR and microscopy but not RDT. Conclusion Malaria control programs may consider PCR testing to guide asymptomatic malaria hotspot detection once the prevalence of infection falls. PMID:28973672

Effects of topical anaesthetics and fluorescein on the real‐time PCR used for the diagnosis of Herpesviruses and Acanthamoeba keratitis

PubMed Central

Goldschmidt, P; Rostane, H; Saint‐Jean, C; Batellier, L; Alouch, C; Zito, E; Bourcier, T; Laroche, L; Chaumeil, C

2006-01-01

Background The early microbiological diagnosis of corneal infections may prevent the condition from worsening. Aim To study the potential interferences of oxybuprocain and fluorescein solutions used by ophthalmologists on the performances of the real‐time polymerase chain reaction (PCR) carried out as routine test for diagnosis of keratitis. Methods Quantified suspensions of Herpes simplex virus (HSV1), Varicella zoster virus (VZV), Cytomegalovirus (CMV) and Acanthamoeba with and without oxybuprocain or fluorescein added before DNA extraction were tested by real‐time PCR. Results The capacities of the real‐time PCR to detect HSV, VZV, CMV and Acanthamoeba were reduced by oxybuprocain and fluorescein. Both products diluted to 1/16 reduced the PCR detection capacities for more than 2 logs (DNA copies/sample). Conclusions The simultaneous introduction of fluorescein or topical anaesthetics into the tubes containing the specimens to be tested by PCR may lead to false negative results. Because corneal specimens for microbiological diagnosis of keratitis are obtained after topical administration of anaesthetics and corneal staining with fluorescein, ophthalmologists should be aware to rinse the eye surface intensively with appropriate eye solutions to minimise the risks of misdiagnosis. PMID:16899529

Effects of topical anaesthetics and fluorescein on the real-time PCR used for the diagnosis of Herpesviruses and Acanthamoeba keratitis.

PubMed

Goldschmidt, P; Rostane, H; Saint-Jean, C; Batellier, L; Alouch, C; Zito, E; Bourcier, T; Laroche, L; Chaumeil, C

2006-11-01

The early microbiological diagnosis of corneal infections may prevent the condition from worsening. To study the potential interferences of oxybuprocain and fluorescein solutions used by ophthalmologists on the performances of the real-time polymerase chain reaction (PCR) carried out as routine test for diagnosis of keratitis. Quantified suspensions of Herpes simplex virus (HSV1), Varicella zoster virus (VZV), Cytomegalovirus (CMV) and Acanthamoeba with and without oxybuprocain or fluorescein added before DNA extraction were tested by real-time PCR. The capacities of the real-time PCR to detect HSV, VZV, CMV and Acanthamoeba were reduced by oxybuprocain and fluorescein. Both products diluted to 1/16 reduced the PCR detection capacities for more than 2 logs (DNA copies/sample). The simultaneous introduction of fluorescein or topical anaesthetics into the tubes containing the specimens to be tested by PCR may lead to false negative results. Because corneal specimens for microbiological diagnosis of keratitis are obtained after topical administration of anaesthetics and corneal staining with fluorescein, ophthalmologists should be aware to rinse the eye surface intensively with appropriate eye solutions to minimise the risks of misdiagnosis.

Real-time PCR for rapidly detecting aniline-degrading bacteria in activated sludge.

PubMed

Kayashima, Takakazu; Suzuki, Hisako; Maeda, Toshinari; Ogawa, Hiroaki I

2013-05-01

We developed a detection method that uses quantitative real-time PCR (qPCR) and the TaqMan system to easily and rapidly assess the population of aniline-degrading bacteria in activated sludge prior to conducting a biodegradability test on a chemical compound. A primer and probe set for qPCR was designed by a multiple alignment of conserved amino acid sequences encoding the large (α) subunit of aniline dioxygenase. PCR amplification tests showed that the designed primer and probe set targeted aniline-degrading strains such as Acidovorax sp., Gordonia sp., Rhodococcus sp., and Pseudomonas putida, thereby suggesting that the developed method can detect a wide variety of aniline-degrading bacteria. There was a strong correlation between the relative copy number of the α-aniline dioxygenase gene in activated sludge obtained with the developed qPCR method and the number of aniline-degrading bacteria measured by the Most Probable Number method, which is the conventional method, and a good correlation with the lag time of the BOD curve for aniline degradation produced by the biodegradability test in activated sludge samples collected from eight different wastewater treatment plants in Japan. The developed method will be valuable for the rapid and accurate evaluation of the activity of inocula prior to conducting a ready biodegradability test. Copyright © 2013 Elsevier Ltd. All rights reserved.

Usefulness of the (GTG)4-PCR for typing of monophasic Salmonella enterica isolates with antigenic shame l,4,[5],12:i:-.

PubMed

Wołkowicz, Tomasz; Januszkiewicz, Aleksandra; Chróst, Anna; Wolaniuk, Natalia; Kubiak, Anna B; Majchrzak, Marta; Szych, Jolanta; Parniewski, Paweł

2015-01-01

Monophasic Salmonella enterica strains presenting the antigenic shame 1,4,[5],12:i:- are becoming more prevalent. Accurate identification of such strains is hard with routine using biochemical and serological tests. Such strains can be identified with molecular tests. In this study we have tested the usefulness of(GTG)4-PCR for the diagnostic of such monophasic strains. This usefulness of this method was previously confirmed for genoserotyping of S. Enterica, Typhimurium, Infantis, Virchow, Hadar, Newport and Anatum. 76 strains with antigenic shame l,4,[5],12:i:-, isolated in Poland in years 2007-12 were tested. Additionally (GTG)4-PCR patterns were obtained for reference strains of serotypes S. Lagos, S. Agama, S. Farsta, S. Tsevie, S. Glocester and S. Tumodi. (GTG)4-PCR was performed with DreamTaq DNA polymerase. Obtained patterns were analysed with BioNumerics software. No pattern specific for monophasic pattern was identified. Additionally it was also impossible to differentiate patterns obtained for S. Typhimurium, S. Farsta, S. Tsevie and S. Glocester. Only reference strains of serotypes S. Tumodi, Farsta and Agama has the distinguishable patterns of (GTG)4-PCR. Analysed (GTG)4-PCR method do not show the ability to distinguish S. enterica serotypes from group 04, H:i, including monophasic strains with the antigenic shame 1,4,[5],12:i:-.

Pregnancy rates of beef cattle are not affected by Campylobacter fetus subsp. venerealis real-time PCR-positive breeding sires in New Zealand.

PubMed

Sanhueza, J M; Heuer, C; Jackson, R; Hughes, P; Anderson, P; Kelly, K; Walker, G

2014-09-01

Campylobacter fetus subspecies venerealis (C. fetus venerealis) is the causal agent of bovine genital campylobacteriosis, a venereal disease that is asymptomatic in bulls but responsible for reproductive wastage in female cattle. In New Zealand, a commercial real-time PCR assay was introduced in 2007 to identify the DNA of this pathogen in preputial scrapings; however, concerns were raised about the specificity of the test following anecdotal reports of a high number of test-positive bulls with no apparent relationship to reproductive performance. The objective of this study, therefore, was to examine the association between real-time PCR assay results from beef breeding bulls and pregnancy rates in beef herds using these bulls. Veterinarians from four veterinary practices selected beef cattle herds with relatively high and low pregnancy rates between December 2008 and February 2009. Preputial scrapings were collected from bulls used for mating in those herds. Samples were tested using the real-time PCR assay under consideration. Bivariable and multivariable analyses were used to assess the relationship between pregnancy rates in each mob (15-month-old heifers, 27-month-old heifers and mixed-age cows) and the percentage of real-time PCR-positive bulls in each mob. Sixty-four (28.8%) of 222 bulls tested positive, 130 (58.6%) tested negative, and 28 (12.6%) returned an inconclusive result to the real-time PCR assay. The percentage of bulls testing real-time PCR-positive in these mobs was not associated with pregnancy rates (p=0.757) after controlling for mob, average body condition score of cows, cow to bull ratio, length of the mating period, and farm. Real-time PCR assay results were not associated with pregnancy rates, suggesting that the specificity of the real-time PCR assay was too low to be used to reliably detect C. fetus venerealis. This study adds to a growing body of evidence indicating that C. fetus venerealis strains are either absent from, or present at clinically insignificant levels of endemicity among, beef breeding herds in New Zealand. The real-time PCR assay that was assessed in this study should not be used for the detection of C. fetus venerealis in bulls or for investigations of low conception rates in cattle in New Zealand. During the course of this survey, sequencing analysis of an apparent C. fetus venerealis isolate from the intestines of a Friesian bull turned out to be Campylobacter hyointestinalis. As a consequence, this real-time PCR assay for C. fetus venerealis is no longer being offered by diagnostic laboratories in New Zealand.

Sporulation properties and antimicrobial susceptibility in endemic and rare Clostridium difficile PCR ribotypes.

PubMed

Zidaric, Valerija; Rupnik, Maja

2016-06-01

Increased sporulation and antibiotic resistance have been proposed to be associated with certain Clostridium difficile epidemic strains such as PCR ribotype 027. In this study we examined these properties in another widespread PCR ribotype, 014/020, in comparison to prevalent PCR ribotype 002 and a group of rarely represented PCR ribotypes. Highest sporulation was observed in 014/020 strains at 24 h, while after 72 h PCR ribotype 002 and rare PCR ribotypes formed higher total number of spores. PCR ribotype 014/020 strains exhibited slightly higher resistance to tested antimicrobials, followed by group of rare PCR ribotypes and less common PCR ribotype 002. Neither sporulation properties nor antibiotic resistance clearly differed in endemic and rare strains. Copyright © 2016 Elsevier Ltd. All rights reserved.

Analytical Performance of Four Polymerase Chain Reaction (PCR) and Real Time PCR (qPCR) Assays for the Detection of Six Leishmania Species DNA in Colombia

PubMed Central

León, Cielo M.; Muñoz, Marina; Hernández, Carolina; Ayala, Martha S.; Flórez, Carolina; Teherán, Aníbal; Cubides, Juan R.; Ramírez, Juan D.

2017-01-01

Leishmaniasis comprises a spectrum of parasitic diseases caused by protozoans of the genus Leishmania. Molecular tools have been widely employed for the detection of Leishmania due to its high sensitivity and specificity. However, the analytical performance of molecular platforms as PCR and real time PCR (qPCR) including a wide variety of molecular markers has never been evaluated. Herein, the aim was to evaluate the analytical performance of 4 PCR-based assays (designed on four different targets) and applied on conventional and real-time PCR platforms. We evaluated the analytical performance of conventional PCR and real time PCR, determining exclusivity and inclusivity, Anticipated Reportable Range (ARR), limit of detection (LoD) and accuracy using primers directed to kDNA, HSP70, 18S and ITS-1 targets. We observed that the kDNA was the most sensitive but does not meet the criterion of exclusivity. The HSP70 presented a higher LoD in conventional PCR and qPCR in comparison with the other markers (1 × 101 and 1 × 10-1 equivalent parasites/mL respectively) and had a higher coefficient of variation in qPCR. No statistically significant differences were found between the days of the test with the four molecular markers. The present study revealed that the 18S marker presented the best performance in terms of analytical sensitivity and specificity for the qPCR in the species tested (species circulating in Colombia). Therefore, we recommend to explore the analytical and diagnostic performance in future studies using a broader number of species across America. PMID:29046670

Clinical Assessment of a Nocardia PCR-Based Assay for Diagnosis of Nocardiosis.

PubMed

Rouzaud, Claire; Rodriguez-Nava, Véronica; Catherinot, Emilie; Méchaï, Frédéric; Bergeron, Emmanuelle; Farfour, Eric; Scemla, Anne; Poirée, Sylvain; Delavaud, Christophe; Mathieu, Daniel; Durupt, Stéphane; Larosa, Fabrice; Lengelé, Jean-Philippe; Christophe, Jean-Louis; Suarez, Felipe; Lortholary, Olivier; Lebeaux, David

2018-06-01

The diagnosis of nocardiosis, a severe opportunistic infection, is challenging. We assessed the specificity and sensitivity of a 16S rRNA Nocardia PCR-based assay performed on clinical samples. In this multicenter study (January 2014 to April 2015), patients who were admitted to three hospitals and had an underlying condition favoring nocardiosis, clinical and radiological signs consistent with nocardiosis, and a Nocardia PCR assay result for a clinical sample were included. Patients were classified as negative control (NC) (negative Nocardia culture results and proven alternative diagnosis or improvement at 6 months without anti- Nocardia treatment), positive control (PC) (positive Nocardia culture results), or probable nocardiosis (positive Nocardia PCR results, negative Nocardia culture results, and no alternative diagnosis). Sixty-eight patients were included; 47 were classified as NC, 8 as PC, and 13 as probable nocardiosis. PCR results were negative for 35/47 NC patients (74%). For the 12 NC patients with positive PCR results, the PCR assay had been performed with respiratory samples. These NC patients had chronic bronchopulmonary disease more frequently than did the NC patients with negative PCR results (8/12 patients [67%] versus 11/35 patients [31%]; P = 0.044). PCR results were positive for 7/8 PC patients (88%). There were 13 cases of probable nocardiosis, diagnosed solely using the PCR results; 9 of those patients (69%) had lung involvement (consolidation or nodule). Nocardia PCR testing had a specificity of 74% and a sensitivity of 88% for the diagnosis of nocardiosis. Nocardia PCR testing may be helpful for the diagnosis of nocardiosis in immunocompromised patients but interpretation of PCR results from respiratory samples is difficult, because the PCR assay may also detect colonization. Copyright © 2018 American Society for Microbiology.

Analytical Performance of Four Polymerase Chain Reaction (PCR) and Real Time PCR (qPCR) Assays for the Detection of Six Leishmania Species DNA in Colombia.

PubMed

León, Cielo M; Muñoz, Marina; Hernández, Carolina; Ayala, Martha S; Flórez, Carolina; Teherán, Aníbal; Cubides, Juan R; Ramírez, Juan D

2017-01-01

Leishmaniasis comprises a spectrum of parasitic diseases caused by protozoans of the genus Leishmania . Molecular tools have been widely employed for the detection of Leishmania due to its high sensitivity and specificity. However, the analytical performance of molecular platforms as PCR and real time PCR (qPCR) including a wide variety of molecular markers has never been evaluated. Herein, the aim was to evaluate the analytical performance of 4 PCR-based assays (designed on four different targets) and applied on conventional and real-time PCR platforms. We evaluated the analytical performance of conventional PCR and real time PCR, determining exclusivity and inclusivity, Anticipated Reportable Range (ARR), limit of detection (LoD) and accuracy using primers directed to kDNA, HSP70, 18S and ITS-1 targets. We observed that the kDNA was the most sensitive but does not meet the criterion of exclusivity. The HSP70 presented a higher LoD in conventional PCR and qPCR in comparison with the other markers (1 × 10 1 and 1 × 10 -1 equivalent parasites/mL respectively) and had a higher coefficient of variation in qPCR. No statistically significant differences were found between the days of the test with the four molecular markers. The present study revealed that the 18S marker presented the best performance in terms of analytical sensitivity and specificity for the qPCR in the species tested (species circulating in Colombia). Therefore, we recommend to explore the analytical and diagnostic performance in future studies using a broader number of species across America.

Real-time PCR assay for the diagnosis of pleural tuberculosis

PubMed Central

Cárdenas Bernal, Ana María; Giraldo-Cadavid, Luis Fernando; Prieto Diago, Enrique; Santander, Sandra Paola

2017-01-01

Abstract Introduction: The diagnosis of pleural tuberculosis requires an invasive and time-consuming reference method. Polymerase chain reaction (PCR) is rapid, but validation in pleural tuberculosis is still weak. Objective: To establish the operating characteristics of real-time polymerase chain reaction (RT-PCR) hybridization probes for the diagnosis of pleural tuberculosis. Methods: The validity of the RT-PCR hybridization probes was evaluated compared to a composite reference method by a cross-sectional study at the Hospital Universitario de la Samaritana. 40 adults with lymphocytic pleural effusion were included. Pleural tuberculosis was confirmed (in 9 patients) if the patient had at least one of three tests using the positive reference method: Ziehl-Neelsen or Mycobacterium tuberculosis culture in fluid or pleural tissue, or pleural biopsy with granulomas. Pleural tuberculosis was ruled out (in 31 patients) if all three tests were negative. The operating characteristics of the RT-PCR, using the Mid-P Exact Test, were determined using the OpenEpi 2.3 Software (2009). Results: The RT-PCR hybridization probes showed a sensitivity of 66.7% (95% CI: 33.2%-90.7%) and a specificity of 93.5% (95% CI: 80.3%-98.9%). The PPV was 75.0% (95% CI: 38.8%-95.6%) and a NPV of 90.6% (95% CI: 76.6%-97.6%). Two false positives were found for the test, one with pleural mesothelioma and the other with chronic pleuritis with mesothelial hyperplasia. Conclusions: The RT-PCR hybridization probes had good specificity and acceptable sensitivity, but a negative value cannot rule out pleural tuberculosis. PMID:29021638

Impact of a Rapid Herpes Simplex Virus PCR Assay on Duration of Acyclovir Therapy

PubMed Central

Van, Tam T.; Mongkolrattanothai, Kanokporn; Arevalo, Melissa; Lustestica, Maryann

2017-01-01

ABSTRACT Herpes simplex virus (HSV) infections of the central nervous system (CNS) are associated with significant morbidity and mortality rates in children. This study assessed the impact of a direct HSV (dHSV) PCR assay on the time to result reporting and the duration of acyclovir therapy for children with signs and symptoms of meningitis and encephalitis. A total of 363 patients with HSV PCR results from cerebrospinal fluid (CSF) samples were included in this retrospective analysis, divided into preimplementation and postimplementation groups. For the preimplementation group, CSF testing was performed using a laboratory-developed real-time PCR assay; for the postimplementation group, CSF samples were tested using a direct sample-to-answer assay. All CSF samples were negative for HSV. Over 60% of patients from both groups were prescribed acyclovir. The average HSV PCR test turnaround time for the postimplementation group was reduced by 14.5 h (23.6 h versus 9.1 h; P < 0.001). Furthermore, 79 patients (43.6%) in the postimplementation group had dHSV PCR results reported <4 h after specimen collection. The mean time from specimen collection to acyclovir discontinuation was 17.1 h shorter in the postimplementation group (31.1 h versus 14 h; P < 0.001). The median duration of acyclovir therapy was also significantly reduced in the postimplementation group (29.2 h versus 14.3 h; P = 0.01). Our investigation suggests that implementation of rapid HSV PCR testing can decrease turnaround times and the duration of unnecessary acyclovir therapy. PMID:28275080

Impact of a Rapid Herpes Simplex Virus PCR Assay on Duration of Acyclovir Therapy.

PubMed

Van, Tam T; Mongkolrattanothai, Kanokporn; Arevalo, Melissa; Lustestica, Maryann; Dien Bard, Jennifer

2017-05-01

Herpes simplex virus (HSV) infections of the central nervous system (CNS) are associated with significant morbidity and mortality rates in children. This study assessed the impact of a direct HSV (dHSV) PCR assay on the time to result reporting and the duration of acyclovir therapy for children with signs and symptoms of meningitis and encephalitis. A total of 363 patients with HSV PCR results from cerebrospinal fluid (CSF) samples were included in this retrospective analysis, divided into preimplementation and postimplementation groups. For the preimplementation group, CSF testing was performed using a laboratory-developed real-time PCR assay; for the postimplementation group, CSF samples were tested using a direct sample-to-answer assay. All CSF samples were negative for HSV. Over 60% of patients from both groups were prescribed acyclovir. The average HSV PCR test turnaround time for the postimplementation group was reduced by 14.5 h (23.6 h versus 9.1 h; P < 0.001). Furthermore, 79 patients (43.6%) in the postimplementation group had dHSV PCR results reported <4 h after specimen collection. The mean time from specimen collection to acyclovir discontinuation was 17.1 h shorter in the postimplementation group (31.1 h versus 14 h; P < 0.001). The median duration of acyclovir therapy was also significantly reduced in the postimplementation group (29.2 h versus 14.3 h; P = 0.01). Our investigation suggests that implementation of rapid HSV PCR testing can decrease turnaround times and the duration of unnecessary acyclovir therapy. Copyright © 2017 American Society for Microbiology.

Comparison of traditional microbiological culture and 16S polymerase chain reaction analyses for identification of preoperative airway colonization for patients undergoing lung resection.

PubMed

Howitt, Samuel H; Blackshaw, Diana; Fontaine, Eustace; Hassan, Ibrahim; Malagon, Ignacio

2018-04-28

Preoperative airway colonization is associated with increased risk of postoperative respiratory complications following lung resection. This study compares the rates of preoperative lower respiratory tract colonization identified by traditional culture and novel 16S polymerase chain reaction (PCR) tests. Preoperative sputum and bronchoalveolar lavage (BAL) samples for 49 lung resection patients underwent culture and 16S PCR analyses. Rates of positive test results were determined and relationships between test results and suspected postoperative respiratory tract infection and hospital length of stay (LOS) were investigated. Preoperative BAL cultures were positive for 29 (59.2%) patients (population estimate 95%CI 45.2%-71.8%). 16S PCR tests were positive for 28 (57.1%) patients (population estimate 95%CI 43.3%-70.0%). 17 (34.7%) patients suffered suspected postoperative respiratory tract infection (population estimate 95%CI 22.9%-48.7%). Positive 16S PCR results tended to be associated with longer LOS (median 7.5 days vs 4.0 days for negative, p = 0.08) and increased risk of suspected postoperative respiratory tract infection (46.4% for positive vs 19.0% for negative, p = 0.07). Rates of colonization identified by culture and 16S PCR analyses of BAL samples were similar. Future research should attempt to clarify associations between airway colonization identified by 16S PCR and outcomes. 16S PCR may be useful when stratifying risk of postoperative respiratory complications. Copyright © 2018 Elsevier Inc. All rights reserved.

Evaluation and Comparison of Multiple Test Methods, Including Real-time PCR, for Legionella Detection in Clinical Specimens

PubMed Central

Peci, Adriana; Winter, Anne-Luise; Gubbay, Jonathan B.

2016-01-01

Legionella is a Gram-negative bacterium that can cause Pontiac fever, a mild upper respiratory infection and Legionnaire’s disease, a more severe illness. We aimed to compare the performance of urine antigen, culture, and polymerase chain reaction (PCR) test methods and to determine if sputum is an acceptable alternative to the use of more invasive bronchoalveolar lavage (BAL). Data for this study included specimens tested for Legionella at Public Health Ontario Laboratories from 1st January, 2010 to 30th April, 2014, as part of routine clinical testing. We found sensitivity of urinary antigen test (UAT) compared to culture to be 87%, specificity 94.7%, positive predictive value (PPV) 63.8%, and negative predictive value (NPV) 98.5%. Sensitivity of UAT compared to PCR was 74.7%, specificity 98.3%, PPV 77.7%, and NPV 98.1%. Out of 146 patients who had a Legionella-positive result by PCR, only 66 (45.2%) also had a positive result by culture. Sensitivity for culture was the same using either sputum or BAL (13.6%); sensitivity for PCR was 10.3% for sputum and 12.8% for BAL. Both sputum and BAL yield similar results regardless testing methods (Fisher Exact p-values = 1.0, for each test). In summary, all test methods have inherent weaknesses in identifying Legionella; therefore, more than one testing method should be used. Obtaining a single specimen type from patients with pneumonia limits the ability to diagnose Legionella, particularly when urine is the specimen type submitted. Given ease of collection and similar sensitivity to BAL, clinicians are encouraged to submit sputum in addition to urine when BAL submission is not practical from patients being tested for Legionella. PMID:27630979

Concordance of polymerase chain reaction with human immunodeficiency virus antibody detection.

PubMed

Horsburgh, C R; Ou, C Y; Jason, J; Holmberg, S D; Lifson, A R; Moore, J L; Ward, J W; Seage, G R; Mayer, K H; Evatt, B L

1990-08-01

To evaluate the correlation of detection of human immunodeficiency virus (HIV) by polymerase chain reaction (PCR) with detection of HIV antibody, 271 simultaneous serum and peripheral blood mononuclear cell samples were examined from 242 persons whose activities placed them at increased risk for HIV infection: 142 from homosexual men, 86 from hemophilic men, and 43 from heterosexual partners of HIV-infected persons. PCR was performed using the gag region primer pair SK38/39 and the env region primer pairs SK68/69 and CO71/72. Amplified HIV DNA was detected using specific oligomer probes. Of 63 HIV antibody-positive samples, 58 (92%) had HIV DNA by PCR. Of 208 HIV antibody-negative samples, 7 (3.4%) had HIV DNA by PCR. On follow-up, 4 of the latter persons were seropositive when next tested; 2 were well and antibody- and PCR-negative; 1 had died of a stroke before retesting. Thus, PCR detects HIV in most antibody-positive persons; detection is increased by use of multiple primer pairs. PCR-positive antibody-negative specimens may indicate HIV infection in which antibody has not yet developed or may be false-positive PCR results. When PCR is discordant with HIV antibody, testing of additional specimens and clinical follow-up are necessary to assess HIV infection status.

Could Digital PCR Be an Alternative as a Non-Invasive Prenatal Test for Trisomy 21: A Proof of Concept Study.

PubMed

El Khattabi, Laïla Allach; Rouillac-Le Sciellour, Christelle; Le Tessier, Dominique; Luscan, Armelle; Coustier, Audrey; Porcher, Raphael; Bhouri, Rakia; Nectoux, Juliette; Sérazin, Valérie; Quibel, Thibaut; Mandelbrot, Laurent; Tsatsaris, Vassilis; Vialard, François; Dupont, Jean-Michel

2016-01-01

NIPT for fetal aneuploidy by digital PCR has been hampered by the large number of PCR reactions needed to meet statistical requirements, preventing clinical application. Here, we designed an octoplex droplet digital PCR (ddPCR) assay which allows increasing the number of available targets and thus overcomes statistical obstacles. After technical optimization of the multiplex PCR on mixtures of trisomic and euploid DNA, we performed a validation study on samples of plasma DNA from 213 pregnant women. Molecular counting of circulating cell-free DNA was performed using a mix of hydrolysis probes targeting chromosome 21 and a reference chromosome. The results of our validation experiments showed that ddPCR detected trisomy 21 even when the sample's trisomic DNA content is as low as 5%. In a validation study of plasma samples from 213 pregnant women, ddPCR discriminated clearly between the trisomy 21 and the euploidy groups. Our results demonstrate that digital PCR can meet the requirements for non-invasive prenatal testing of trisomy 21. This approach is technically simple, relatively cheap, easy to implement in a diagnostic setting and compatible with ethical concerns regarding access to nucleotide sequence information. These advantages make it a potential technique of choice for population-wide screening for trisomy 21 in pregnant women.

Standardization of a TaqMan-based real-time PCR for the detection of Mycobacterium tuberculosis-complex in human sputum.

PubMed

Barletta, Francesca; Vandelannoote, Koen; Collantes, Jimena; Evans, Carlton A; Arévalo, Jorge; Rigouts, Leen

2014-10-01

Real-time polymerase chain reaction (qPCR) was optimized for detecting Mycobacterium tuberculosis in sputum. Sputum was collected from patients (N = 112) with suspected pulmonary tuberculosis, tested by smear microscopy, decontaminated, and split into equal aliquots that were cultured in Löwenstein-Jensen medium and tested by qPCR for the small mobile genetic element IS6110. The human ERV3 sequence was used as an internal control. 3 of 112 (3%) qPCR failed. For the remaining 109 samples, qPCR diagnosed tuberculosis in 79 of 84 patients with culture-proven tuberculosis, and sensitivity was greater than microscopy (94% versus 76%, respectively, P < 0.05). The qPCR sensitivity was similar (P = 0.9) for smear-positive (94%, 60 of 64) and smear-negative (95%, 19 of 20) samples. The qPCR was negative for 24 of 25 of the sputa with negative microscopy and culture (diagnostic specificity 96%). The qPCR had 99.5% sensitivity and specificity for 211 quality control samples including 84 non-tuberculosis mycobacteria. The qPCR cost ∼5US$ per sample and provided same-day results compared with 2-6 weeks for culture. © The American Society of Tropical Medicine and Hygiene.

Diagnostic performance of influenza viruses and RSV rapid antigen detection tests in children in tertiary care.

PubMed

Moesker, F M; van Kampen, J J A; Aron, G; Schutten, M; van de Vijver, D A M C; Koopmans, M P G; Osterhaus, A D M E; Fraaij, P L A

2016-06-01

Rapid antigen detection tests (RADTs) are increasingly used to detect influenza viruses and respiratory syncytial virus (RSV). However, their sensitivity and specificity are a matter of debate, challenging their clinical usefulness. Comparing diagnostic performances of BinaxNow Influenza AB(®) (BNI) and BinaxNow RSV(®) (BNR), to those of real-time reverse transcriptase PCR (RT-PCR), virus isolation and direct immunofluorescence (D-IF) in paediatric patients. Between November 2005 and September 2013, 521 nasal washings from symptomatic children (age <5 years) attending our tertiary care centre were tested, with a combination of the respective assays using RT-PCR as gold standard. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of BNI were 69% (confidence interval [CI] [51-83]), 96% [94-97], 55% [39-70] and 98% [96-99] respectively. Of eleven false-negative samples, RT-PCR Ct-values were higher than all RT-PCR positive test results (27 vs 22, p=0.012). Of twenty false-positive samples, none were culture positive and two tested positive in D-IF. Sensitivity, specificity, PPV and NPV for BNR were 79% [73-85], 98% [96-99], 97% [93-99] and 88% [84-91]. Of the 42 false-negative samples the median Ct-value was higher than that of all RT-PCR positive samples (31 vs 23, p<0.0001). Five false-positive samples were detected. Three of these tested positive for RSV in virus isolation and D-IF. RADTs have a high specificity with BNR being superior to BNI. However, their relative low sensitivity limits their usefulness for clinical decision making in a tertiary care paediatric hospital. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Evaluation of culture- and PCR-based detection methods for Escherichia coli O157:H7 in inoculated ground beeft.

PubMed

Arthur, Terrance M; Bosilevac, Joseph M; Nou, Xiangwu; Koohmaraie, Mohammad

2005-08-01

Currently, several beef processors employ test-and-hold systems for increased quality control of ground beef. In such programs, each lot of product must be tested and found negative for Escherichia coli O157:H7 prior to release of the product into commerce. Optimization of three testing attributes (detection time, specificity, and sensitivity) is critical to the success of such strategies. Because ground beef is a highly perishable product, the testing methodology used must be as rapid as possible. The test also must have a low false-positive result rate so product is not needlessly discarded. False-negative results cannot be tolerated because they would allow contaminated product to be released and potentially cause disease. In this study, two culture-based and three PCR-based methods for detecting E. coli O157:H7 in ground beef were compared for their abilities to meet the above criteria. Ground beef samples were individually spiked with five genetically distinct strains of E. coli O157: H7 at concentrations of 17 and 1.7 CFU/65 g and then subjected to the various testing methodologies. There was no difference (P > 0.05) in the abilities of the PCR-based methods to detect E. coli O157:H7 inoculated in ground beef at 1.7 CFU/65 g. The culture-based systems detected more positive samples than did the PCR-based systems, but the detection times (21 to 48 h) were at least 9 h longer than those for the PCR-based methods (7.5 to 12 h). Ground beef samples were also spiked with potentially cross-reactive strains. The PCR-based systems that employed an immunomagnetic separation step prior to detection produced fewer false-positive results.

An exploratory study of the impact of hypermedia-based approach and science-in-fiction approach for instruction on the polymerase chain reaction

NASA Astrophysics Data System (ADS)

Britton, Lynda A.

1998-12-01

Exploration of meaningful learning of the polymerase chain reaction (PCR) followed instruction by a researcher-developed hypermedia computer program that incorporated human constructivist principles and a "science-in-fiction" chapter of a novel that described PCR. Human constructivism is the Ausubel-Novak-Gowin (1997) meaningful learning theory that supports science learning through graphic representations and multiple examples. Science-in-fiction is a new genre of fiction introduced by the prominent scientist, Carl Djerassi, to engender an appreciation for science, and its ethical dilemmas. Chapter 19 of Djerassi's 1994 novel, The Bourbaki Gambit, was placed into hypermedia format to standardize the presentation. As part of a clinical microbiology course in the medical technology curriculum at a major medical center in the Deep South, 10 undergraduates participated in this study. Each first read The Bourbaki Gambit, and then half of the participants experienced the human constructivist approach first (the PCR group) while the others first encountered the science-in-fiction approach (the Chapter 19 group). For the rest, the order of presentation was reversed, so that all experienced both programs. Students' explanations while using the computer were videotaped. Students were tested and interviewed before experiencing either program, after their first instructional session, and again after the second instructional session. These students were also assessed on their knowledge of the nature of science by taking the Nature of Science Questionnaire, before and after instruction (Roach, 1993) and interviewed as a cross-check on its reliability. Students' preferred learning approaches were determined using Schmeck's Inventory of Learning Processes (Schmeck, Ribich, & Ramanaiah, 1977). Data were collected and analyzed both qualitatively and quantitatively using appropriate verbal analysis techniques (Chi, 1997). All but three students reached a structural level of PCR biological literacy. A mean of 79% of the concepts identified as necessary was attained by participants after experiencing both approaches. The Chapter 19 science-in-fiction group scored slightly better than those who experienced the PCR program first, indicating that the chapter served as an advance organizer when used first, but inhibited mastery when used second. Significant conceptual change about the nature of science was not detected, even though most students demonstrated deep and/or elaborative learning styles.

Application of denaturing gradient gel electrophoresis (DGGE) to the analysis of microbial communities of subgingival plaque.

PubMed

Fujimoto, C; Maeda, H; Kokeguchi, S; Takashiba, S; Nishimura, F; Arai, H; Fukui, K; Murayama, Y

2003-08-01

Denaturing gradient gel electrophoresis (DGGE) was applied to the microbiologic examination of subgingival plaque. The PCR primers were designed from conserved nucleotide sequences on 16S ribosomal RNA gene (16SrDNA) with GC rich clamp at the 5'-end. Polymerase chain reaction (PCR) was performed using the primers and genomic DNAs of typical periodontal bacteria. The generated 16SrDNA fragments were separated by denaturing gel. Although the sizes of the amplified DNA fragments were almost the same among the species, 16SrDNAs of the periodontal bacteria were distinguished according to their specific sequences. The microflora of clinical plaque samples were profiled by the PCR-DGGE method, and the dominant 16SrDNA bands were cloned and sequenced. Simultaneously, Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and Prevotella intermedia were detected by an ordinary PCR method. In the deep periodontal pockets, the bacterial community structures were complicated and P. gingivalis was the most dominant species, whereas the DGGE profiles were simple and Streptococcus or Neisseria species were dominant in the shallow pockets. The species-specific PCR method revealed the presence of A. actinomycetemcomitans, P. gingivalis and P. intermedia in the clinical samples. However, corresponding bands were not always observed in the DGGE profiles, indicating a lower sensitivity of the DGGE method. Although the DGGE method may have a lower sensitivity than the ordinary PCR methods, it could visualize the bacterial qualitative compositions and reveal the major species of the plaque. The DGGE analysis and following sequencing may have the potential to be a promising bacterial examination procedure in periodontal diseases.

Interlaboratory Comparison of Quantitative PCR Test Results for Dehalococcoides

EPA Science Inventory

Quantitative PCR (qPCR) techniques have been widely used to measure Dehalococcoides (Dhc) DNA in the groundwater at field sites for several years. Interpretation of these data may be complicated when different laboratories using alternate methods conduct the analysis. An...

Validation of a commercial kit aimed to the detection of pathogenic anisakid nematodes in fish products.

PubMed

Cavallero, Serena; Bruno, Alessandro; Arletti, Enrico; Caffara, Monica; Fioravanti, Maria Letizia; Costa, Antonella; Cammilleri, Gaetano; Graci, Stefania; Ferrantelli, Vincenzo; D'Amelio, Stefano

2017-09-18

Anisakids are parasitic nematodes responsible for a zoonosis that occurs following the ingestion of fish and fish products infected with larvae belonging to the genera Anisakis and Pseudoterranova. Rarely Contracaecum is found in association with gastric/intestinal illness, while Hysterothylacium is commonly considered not pathogenic. Although Real Time PCR assays have been recently used with the aim to detect and quantify these parasites in food products, methods applied did not undergo through extensive validation process, a feature highly desirable or mandatory in the case of testing laboratories accredited for the ISO EN 17025:2005. Here, a comprehensive study has been performed to validate a commercial kit based on multiplex real time PCR for the qualitative detection of Anisakis and Pseudoterranova. Inclusivity/exclusivity trials were carried out on DNA from species of the genera Anisakis, Pseudoterranova, Contracaecum, Hysterothylacium and Ascaris, on fish intentionally contaminated with Anisakis spp. and Pseudoterranova spp. and on marine organisms as fish, crustacean and squid to test the commercial kit on a large sample. The assay gave positive amplification for several Anisakis and Pseudoterranova species, while providing no signal for the members of the remaining genera. Each sample was correctly assigned either to Anisakis or Pseudoterranova, thus indicating that no cross-reaction occurred. The LOD was determined using two independent standard curves. Robustness was assayed by using two different thermocyclers in three distinct laboratories with different operators. The establishment of a validation dossier will permit the use of the commercial kit for the detection of Anisakis and Pseudoterranova DNA in fish and fish products intended for human consumption by public or private laboratories, following the requirements regarding the quality assurance processes described in the ISO EN 17025:2005. Copyright © 2017 Elsevier B.V. All rights reserved.

Prevalence and risk factors of Echinococcus granulosus infection in dogs in Moroto and Bukedea districts in Uganda.

PubMed

Oba, Peter; Ejobi, Francis; Omadang, Leonard; Chamai, Martin; Okwi, Andrew Livex; Othieno, Emmanuel; Inangolet, Francis Olaki; Ocaido, Michael

2016-02-01

A cross sectional study was conducted in Moroto and Bukedea districts of Uganda from May to September 2013 to determine the prevalence and risk factors of Echinococcus granulosus infection in dogs. Fresh dog faecal samples were collected, preserved in 70 % ethanol, and later screened for presence of taeniid eggs using zinc chloride floatation method. Positive samples were confirmed by a copro-PCR (polymerase chain reaction) for E. granulosus using NADH dehydrogenase sub-unit 1 gene (NADH1) as a target molecular marker. Structured questionnaires and focus group discussions were used to collect quantitative and qualitative data for risk factor identification. Study sub-counties were selected by simple random sampling. Overall apparent prevalence of taeniid infection in dogs of 14.9 % (39/261, confidence interval 10.6-19.2) in both districts was recorded using the faecal floatation test. The sensitivity of the faecal floatation test was found to be 78 % (25/32), while the specificity was 93 % (215/229). Copro-PCR results revealed a true prevalence of 14.4 % (9.91-19.0, 95 % CI) in dogs in Moroto district and 7.4 % (2.14-12.60, 95 % CI) in Bukedea district. The overall true prevalence of cystic echinococcosis (CE) was 12.2 % (8.70-15.76, 95 % CI) in both districts. The major risk factors identified using logistic regression were uncontrolled access of dogs to animal slaughter facilities, higher cattle herd sizes and lack of knowledge about the disease. It was recommended that restricting dog access to infected tissues and public health education about epidemiology of CE should be done.

Quantitative Detection and Genotyping of Helicobacter pylori from Stool using Droplet Digital PCR Reveals Variation in Bacterial Loads that Correlates with cagA Virulence Gene Carriage.

PubMed

Talarico, Sarah; Safaeian, Mahboobeh; Gonzalez, Paula; Hildesheim, Allan; Herrero, Rolando; Porras, Carolina; Cortes, Bernal; Larson, Ann; Fang, Ferric C; Salama, Nina R

2016-08-01

Epidemiologic studies of the carcinogenic stomach bacterium Helicobacter pylori have been limited by the lack of noninvasive detection and genotyping methods. We developed a new stool-based method for detection, quantification, and partial genotyping of H. pylori using droplet digital PCR (ddPCR), which allows for increased sensitivity and absolute quantification by PCR partitioning. Stool-based ddPCR assays for H. pylori 16S gene detection and cagA virulence gene typing were tested using a collection of 50 matched stool and serum samples from Costa Rican volunteers and 29 H. pylori stool antigen-tested stool samples collected at a US hospital. The stool-based H. pylori 16S ddPCR assay had a sensitivity of 84% and 100% and a specificity of 100% and 71% compared to serology and stool antigen tests, respectively. The stool-based cagA genotyping assay detected cagA in 22 (88%) of 25 stools from CagA antibody-positive individuals and four (16%) of 25 stools from CagA antibody-negative individuals from Costa Rica. All 26 of these samples had a Western-type cagA allele. Presence of serum CagA antibodies was correlated with a significantly higher load of H. pylori in the stool. The stool-based ddPCR assays are a sensitive, noninvasive method for detection, quantification, and partial genotyping of H. pylori. The quantitative nature of ddPCR-based H. pylori detection revealed significant variation in bacterial load among individuals that correlates with presence of the cagA virulence gene. These stool-based ddPCR assays will facilitate future population-based epidemiologic studies of this important human pathogen. © 2015 John Wiley & Sons Ltd.

Detection of respiratory viruses and bacteria in children using a twenty-two target reverse-transcription real-time PCR (RT-qPCR) panel.

PubMed

Ellis, Chelsey; Misir, Amita; Hui, Charles; Jabbour, Mona; Barrowman, Nicholas; Langill, Jonathan; Bowes, Jennifer; Slinger, Robert

2016-05-01

Rapid detection of the wide range of viruses and bacteria that cause respiratory infection in children is important for patient care and antibiotic stewardship. We therefore designed and evaluated a ready-to-use 22 target respiratory infection reverse-transcription real-time polymerase chain reaction (RT-qPCR) panel to determine if this would improve detection of these agents at our pediatric hospital. RT-qPCR assays for twenty-two target organisms were dried-down in individual wells of 96 well plates and saved at room temperature. Targets included 18 respiratory viruses and 4 bacteria. After automated nucleic acid extraction of nasopharyngeal aspirate (NPA) samples, rapid qPCR was performed. RT-qPCR results were compared with those obtained by the testing methods used at our hospital laboratories. One hundred fifty-nine pediatric NPA samples were tested with the RT-qPCR panel. One or more respiratory pathogens were detected in 132/159 (83%) samples. This was significantly higher than the detection rate of standard methods (94/159, 59%) (P<0.001). This difference was mainly due to improved RT-qPCR detection of rhinoviruses, parainfluenza viruses, bocavirus, and coronaviruses. The panel internal control assay performance remained stable at room temperature storage over a two-month testing period. The RT-qPCR panel was able to identify pathogens in a high proportion of respiratory samples. The panel detected more positive specimens than the methods in use at our hospital. The pre-made panel format was easy to use and rapid, with results available in approximately 90 minutes. We now plan to determine if use of this panel improves patient care and antibiotic stewardship.

Virus detection by PCR following vaccination of naive calves with intranasal or injectable multivalent modified-live viral vaccines.

PubMed

Walz, Paul H; Newcomer, Benjamin W; Riddell, Kay P; Scruggs, Daniel W; Cortese, Victor S

2017-09-01

We evaluated duration of PCR-positive results following administration of modified-live viral (MLV) vaccines to beef calves. Twenty beef calves were randomly assigned to either group 1 and vaccinated intranasally with a MLV vaccine containing bovine alphaherpesvirus 1 (BoHV-1), bovine respiratory syncytial virus (BRSV), and bovine parainfluenza virus 3 (BPIV-3), or to group 2 and vaccinated subcutaneously with a MLV vaccine containing bovine viral diarrhea virus 1 and 2 (BVDV-1, -2), BoHV-1, BRSV, and BPIV-3. Deep nasopharyngeal swabs (NPS) and transtracheal washes (TTW) were collected from all calves, and whole blood was collected from group 2 calves and tested by PCR. In group 1, the proportions of calves that tested PCR-positive to BVDV, BoHV-1, BRSV, and BPIV-3 on any sample at any time were 0%, 100%, 100%, and 10%, respectively. In group 1 calves, 100% of calves became PCR-positive for BoHV-1 by day 3 post-vaccination and 100% of calves became PCR-positive for BRSV by day 7 post-vaccination. In group 2, the proportions of calves that tested positive to BVDV, BoHV-1, BRSV, and BPIV-3 on any sample at any time were 50%, 40%, 10%, and 0%, respectively. All threshold cycle (Ct) values were >30 in group 2 calves, irrespective of virus; however, Ct values <25 were observed in group 1 calves from PCR-positive results for BoHV-1 and BRSV. All calves were PCR-negative for all viruses after day 28. Following intranasal MLV viral vaccination, PCR results and Ct values for BRSV and BoHV-1 suggest that attempts to differentiate vaccine virus from natural infection is unreliable.

Detection of fungal DNA in human body fluids and tissues during a multistate outbreak of fungal meningitis and other infections.

PubMed

Gade, Lalitha; Scheel, Christina M; Pham, Cau D; Lindsley, Mark D; Iqbal, Naureen; Cleveland, Angela Ahlquist; Whitney, Anne M; Lockhart, Shawn R; Brandt, Mary E; Litvintseva, Anastasia P

2013-05-01

Exserohilum rostratum was the major cause of an outbreak of fungal infections linked to injections of contaminated methylprednisolone acetate. Because almost 14,000 persons were exposed to product that was possibly contaminated with multiple fungal pathogens, there was unprecedented need for a rapid throughput diagnostic test that could detect both E. rostratum and other unusual agents of fungal infection. Here we report development of a novel PCR test that allowed for rapid and specific detection of fungal DNA in cerebrospinal fluid (CSF), other body fluids and tissues of infected individuals. The test relied on direct purification of free-circulating fungal DNA from fluids and subsequent PCR amplification and sequencing. Using this method, we detected Exserohilum rostratum DNA in 123 samples from 114 case-patients (28% of 413 case-patients for whom 627 samples were available), and Cladosporium DNA in one sample from one case-patient. PCR with novel Exserohilum-specific ITS-2 region primers detected 25 case-patients with samples that were negative using broad-range ITS primers. Compared to fungal culture, this molecular test was more sensitive: of 139 case-patients with an identical specimen tested by culture and PCR, E. rostratum was recovered in culture from 19 (14%), but detected by PCR in 41 (29%), showing a diagnostic sensitivity of 29% for PCR compared to 14% for culture in this patient group. The ability to rapidly confirm the etiologic role of E. rostratum in these infections provided an important contribution in the public health response to this outbreak.

Restriction-Site-Specific PCR as a Rapid Test To Detect Enterohemorrhagic Escherichia coli O157:H7 Strains in Environmental Samples

PubMed Central

Kimura, Richard; Mandrell, Robert E.; Galland, John C.; Hyatt, Doreene; Riley, Lee W.

2000-01-01

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an important food-borne pathogen in industrialized countries. We developed a rapid and simple test for detecting E. coli O157:H7 using a method based on restriction site polymorphisms. Restriction-site-specific PCR (RSS-PCR) involves the amplification of DNA fragments using primers based on specific restriction enzyme recognition sequences, without the use of endonucleases, to generate a set of amplicons that yield “fingerprint” patterns when resolved electrophoretically on an agarose gel. The method was evaluated in a blinded study of E. coli isolates obtained from environmental samples collected at beef cattle feedyards. The 54 isolates were all initially identified by a commonly used polyclonal antibody test as belonging to O157:H7 serotype. They were retested by anti-O157 and anti-H7 monoclonal antibody enzyme-linked immunosorbent assay (ELISA). The RSS-PCR method identified all 28 isolates that were shown to be E. coli O157:H7 by the monoclonal antibody ELISA as belonging to the O157:H7 serotype. Of the remaining 26 ELISA-confirmed non-O157:H7 strains, the method classified 25 strains as non-O157:H7. The specificity of the RSS-PCR results correlated better with the monoclonal antibody ELISA than with the polyclonal antibody latex agglutination tests. The RSS-PCR method may be a useful test to distinguish E. coli O157:H7 from a large number of E. coli isolates from environmental samples. PMID:10831431

Evaluation of Polymerase Chain Reaction for Detecting Coliform Bacteria in Drinking Water Sources.

PubMed

Isfahani, Bahram Nasr; Fazeli, Hossein; Babaie, Zeinab; Poursina, Farkhondeh; Moghim, Sharareh; Rouzbahani, Meisam

2017-01-01

Coliform bacteria are used as indicator organisms for detecting fecal pollution in water. Traditional methods including microbial culture tests in lactose-containing media and enzyme-based tests for the detection of β-galactosidase; however, these methods are time-consuming and less specific. The aim of this study was to evaluate polymerase chain reaction (PCR) for detecting coliform. Totally, 100 of water samples from Isfahan drinking water source were collected. Coliform bacteria and Escherichia coli were detected in drinking water using LacZ and LamB genes in PCR method performed in comparison with biochemical tests for all samples. Using phenotyping, 80 coliform isolates were found. The results of the biochemical tests illustrated 78.7% coliform bacteria and 21.2% E. coli . PCR results for LacZ and LamB genes were 67.5% and 17.5%, respectively. The PCR method was shown to be an effective, sensitive, and rapid method for detecting coliform and E. coli in drinking water from the Isfahan drinking water sources.

Comparison of real-time PCR with disk diffusion, agar screen and E-test methods for detection of methicillin-resistant Staphylococcus aureus.

PubMed

Shariati, Laleh; Validi, Majid; Tabatabaiefar, Mohammad Amin; Karimi, Ali; Nafisi, Mohammad Reza

2010-12-01

Methicillin-resistant Staphylococcus aureus (MRSA) is a nosocomial pathogen. Our main objective was to compare oxacillin disk test, oxacillin E-test, and oxacillin agar screen for detection of methicillin resistance in S. aureus, using real-time PCR for mecA as the "gold standard" comparison assay. 196 S. aureus isolates were identified out of 284 Staphylococcus isolates. These isolates were screened for MRSA with several methods: disk diffusion, agar screen (6.0 μg/ml), oxacillin E-test, and real-time PCR for detection of mecA gene. Of the 196 S. aureus isolates tested, 96 isolates (49%) were mecA-positive and 100 isolates (51%) mecA-negative. All methods tested had a statistically significant agreement with real-time PCR. E-test was 100% sensitive and specific for mecA presence. The sensitivity and specificity of oxacillin agar screen method were 98 and 99%, respectively and sensitivity and specificity of oxacillin disk diffusion method were 95 and 93%, respectively. In the present study, oxacillin E-test is proposed as the best phenotypic method. For economic reasons, the oxacillin agar screen method (6.0 μg/ml), which is suitable for the detection of MRSA, is recommended due to its accuracy and low cost.

Assessment of the influence of test characteristics on the clinical and cost impacts of methicillin-resistant Staphylococcus aureus screening programs in US hospitals.

PubMed

Olchanski, Natalia; Mathews, Charles; Fusfeld, Lauren; Jarvis, William

2011-03-01

To compare the impacts of different methicillin-resistant Staphylococcus aureus (MRSA) screening test options (eg, polymerase chain reaction [PCR], rapid culture) and program characteristics on the clinical outcomes and budget of a typical US hospital. We developed an Excel-based decision-analytic model, using published literature to calculate and compare hospital costs and MRSA infection rates for PCR- or culture-based MRSA screening and then used multivariate sensitivity analysis to evaluate key variables. Same-day PCR testing for a representative 370-bed teaching hospital in the United States was assessed in different populations (high-risk patients, intensive care unit [ICU] patients, or all patients) and compared with other test options. Different screening program populations (all patients, high-risk patients, ICU patients, or patients with previous MRSA colonization or infection only) represented a potential savings of $12,158-$76,624 per month over no program ($188,618). Analysis of multiple test options in high-risk population screening indicated that same-day PCR testing of high-risk patients resulted in fewer infections over 1,720 patient-days (2.9, compared with 3.5 for culture on selective media and 3.8 for culture on nonselective media) and the lowest total cost ($112,012). The costs of other testing approaches ranged from $113,742 to $123,065. Sensitivity analysis revealed that variations in transmission rate, conversion to infection, prevalence increases, and hospital size are important to determine program impact. Among test characteristics, turnaround time is highly influential. All screening options showed reductions in infection rates and cost impact improvement over no screening program. Among the options, same-day PCR testing for high-risk patients slightly edges out the others in terms of fewest infections and greatest potential cost savings.

Evaluation of Genotypic and Phenotypic Protease Virulence Tests for Dichelobacter nodosus Infection in Sheep

PubMed Central

McPherson, Andrew S.; Dhungyel, Om P.

2017-01-01

ABSTRACT Dichelobacter nodosus is a fastidious, strictly anaerobic bacterium, an obligate parasite of the ruminant hoof, and the essential causative agent of virulent ovine footrot. The clinical disease results from a complex interplay between the pathogen, the environment, and the host. Sheep flocks diagnosed with virulent but not benign footrot in Australia may be quarantined and required to undergo a compulsory eradication program, with costs met by the farmer. Virulence of D. nodosus at least partially depends on the elaboration of a protease encoded by aprV2 and manifests as elastase activity. Laboratory virulence tests are used to assist diagnosis because clinical differentiation of virulent and benign footrot can be challenging during the early stages of disease or when the disease is not fully expressed due to unfavorable pasture conditions. Using samples collected from foot lesions from 960 sheep from 40 flocks in four different geographic regions, we evaluated the analytical characteristics of qPCR tests for the protease gene alleles aprV2 and aprB2, and compared these with results from phenotypic protease (elastase and gelatin gel) tests. There was a low level of agreement between clinical diagnosis and quantitative PCR (qPCR) test outcomes at both the flock and sample levels and poor agreement between qPCR test outcomes and the results of phenotypic virulence tests. The diagnostic specificity of the qPCR test was low at both the flock and individual swab levels (31.3% and 18.8%, respectively). By contrast, agreement between the elastase test and clinical diagnosis was high at both the flock level (diagnostic sensitivity [DSe], 100%; diagnostic specificity [DSp], 78.6%) and the isolate level (DSe, 69.5%; DSp, 80.5%). PMID:28202796

Evaluation of Genotypic and Phenotypic Protease Virulence Tests for Dichelobacter nodosus Infection in Sheep.

PubMed

McPherson, Andrew S; Dhungyel, Om P; Whittington, Richard J

2017-05-01

Dichelobacter nodosus is a fastidious, strictly anaerobic bacterium, an obligate parasite of the ruminant hoof, and the essential causative agent of virulent ovine footrot. The clinical disease results from a complex interplay between the pathogen, the environment, and the host. Sheep flocks diagnosed with virulent but not benign footrot in Australia may be quarantined and required to undergo a compulsory eradication program, with costs met by the farmer. Virulence of D. nodosus at least partially depends on the elaboration of a protease encoded by aprV2 and manifests as elastase activity. Laboratory virulence tests are used to assist diagnosis because clinical differentiation of virulent and benign footrot can be challenging during the early stages of disease or when the disease is not fully expressed due to unfavorable pasture conditions. Using samples collected from foot lesions from 960 sheep from 40 flocks in four different geographic regions, we evaluated the analytical characteristics of qPCR tests for the protease gene alleles aprV2 and aprB2 , and compared these with results from phenotypic protease (elastase and gelatin gel) tests. There was a low level of agreement between clinical diagnosis and quantitative PCR (qPCR) test outcomes at both the flock and sample levels and poor agreement between qPCR test outcomes and the results of phenotypic virulence tests. The diagnostic specificity of the qPCR test was low at both the flock and individual swab levels (31.3% and 18.8%, respectively). By contrast, agreement between the elastase test and clinical diagnosis was high at both the flock level (diagnostic sensitivity [DSe], 100%; diagnostic specificity [DSp], 78.6%) and the isolate level (DSe, 69.5%; DSp, 80.5%). Copyright © 2017 McPherson et al.

Development of a highly sensitive real-time nested RT-PCR assay in a single closed tube for detection of enterovirus 71 in hand, foot, and mouth disease.

PubMed

Niu, Peihua; Qi, Shunxiang; Yu, Benzhang; Zhang, Chen; Wang, Ji; Li, Qi; Ma, Xuejun

2016-11-01

Enterovirus 71 (EV71) is one of the major causative agents of outbreaks of hand, foot, and mouth disease (HFMD). A commercial TaqMan probe-based real-time PCR assay has been widely used for the differential detection of EV71 despite its relatively high cost and failure to detect samples with a low viral load (Ct value > 35). In this study, a highly sensitive real-time nested RT-PCR (RTN RT-PCR) assay in a single closed tube for detection of EV71 in HFMD was developed. The sensitivity and specificity of this assay were evaluated using a reference EV71 stock and a panel of controls consisting of coxsackievirus A16 (CVA16) and common respiratory viruses, respectively. The clinical performance of this assay was evaluated and compared with those of a commercial TaqMan probe-based real-time PCR (qRT-PCR) assay and a traditional two-step nested RT-PCR assay. The limit of detection for the RTN RT-PCR assay was 0.01 TCID50/ml, with a Ct value of 38.3, which was the same as that of the traditional two-step nested RT-PCR assay and approximately tenfold lower than that of the qRT-PCR assay. When testing the reference strain EV71, this assay showed favorable detection reproducibility and no obvious cross-reactivity. The testing results of 100 clinical throat swabs from HFMD-suspected patients revealed that 41 samples were positive for EV71 by both RTN RT-PCR and traditional two-step nested RT-PCR assays, whereas only 29 were EV71 positive by qRT-PCR assay.

Evidence of a louse-borne outbreak involving typhus in Douai, 1710-1712 during the war of Spanish succession.

PubMed

Nguyen-Hieu, Tung; Aboudharam, Gérard; Signoli, Michel; Rigeade, Catherine; Drancourt, Michel; Raoult, Didier

2010-10-27

The new field of paleomicrobiology allows past outbreaks to be identified by testing dental pulp of human remains with PCR. We identified a mass grave in Douai, France dating from the early XVIII(th) century. This city was besieged during the European war of Spanish succession. We tested dental pulp from 1192 teeth (including 40 from Douai) by quantitative PCR (qPCR) for R. prowazekii and B. quintana. We also used ultra-sensitive suicide PCR to detect R. prowazekii and genotyped positive samples. In the Douai remains, we identified one case of B. quintana infection (by qPCR) and R. prowazekii (by suicide PCR) in 6/21 individuals (29%). The R. prowazekii was genotype B, a genotype previously found in a Spanish isolate obtained in the first part of the XX(th) century. Louse-borne outbreaks were raging during the XVIII(th) century; our results support the hypothesis that typhus was imported into Europe by Spanish soldiers from America.

Evidence of a Louse-Borne Outbreak Involving Typhus in Douai, 1710-1712 during the War of Spanish Succession

PubMed Central

Nguyen-Hieu, Tung; Aboudharam, Gérard; Signoli, Michel; Rigeade, Catherine; Drancourt, Michel; Raoult, Didier

2010-01-01

Background The new field of paleomicrobiology allows past outbreaks to be identified by testing dental pulp of human remains with PCR. Methods We identified a mass grave in Douai, France dating from the early XVIIIth century. This city was besieged during the European war of Spanish succession. We tested dental pulp from 1192 teeth (including 40 from Douai) by quantitative PCR (qPCR) for R. prowazekii and B. quintana. We also used ultra-sensitive suicide PCR to detect R. prowazekii and genotyped positive samples. Results and Discussion In the Douai remains, we identified one case of B. quintana infection (by qPCR) and R. prowazekii (by suicide PCR) in 6/21 individuals (29%). The R. prowazekii was genotype B, a genotype previously found in a Spanish isolate obtained in the first part of the XXth century. Conclusion Louse-borne outbreaks were raging during the XVIIIth century; our results support the hypothesis that typhus was imported into Europe by Spanish soldiers from America. PMID:21060879

Discrimination of gastrointestinal nematode eggs from crude fecal egg preparations by inhibitor-resistant conventional and real-time PCR.

PubMed

Demeler, Janina; Ramünke, Sabrina; Wolken, Sonja; Ianiello, Davide; Rinaldi, Laura; Gahutu, Jean Bosco; Cringoli, Giuseppe; von Samson-Himmelstjerna, Georg; Krücken, Jürgen

2013-01-01

Diagnosis of gastrointestinal nematodes relies predominantly on coproscopic methods such as flotation, Kato-Katz, McMaster or FLOTAC. Although FLOTAC allows accurate quantification, many nematode eggs can only be differentiated to genus or family level. Several molecular diagnostic tools discriminating closely related species suffer from high costs for DNA isolation from feces and limited sensitivity since most kits use only small amounts of feces (<1 g). A direct PCR from crude egg preparations was designed for full compatibility with FLOTAC to accurately quantify eggs per gram feces (epg) and determine species composition. Eggs were recovered from the flotation solution and concentrated by sieving. Lysis was achieved by repeated boiling and freezing cycles - only Trichuris eggs required additional mechanic disruption. Egg lysates were directly used as template for PCR with Phusion DNA polymerase which is particularly resistant to PCR inhibitors. Qualitative results were obtained with feces of goats, cattle, horses, swine, cats, dogs and mice. The finally established protocol was also compatible with quantitative real-time PCR in the presence of EvaGreen and no PCR inhibition was detectable when extracts were diluted at least fourfold. Sensitivity was comparable to DNA isolation protocols and spiked samples with five epg were reliably detected. For Toxocara cati a detection limit below one epg was demonstrated. It was possible to distinguish T. cati and Toxocara canis using high resolution melt (HRM) analysis, a rapid tool for species identification. In human samples, restriction fragment length polymorphism (RFLP) and HRM analysis were used to discriminate Necator americanus and Ancylostoma duodenale. The method is able to significantly improve molecular diagnosis of gastrointestinal nematodes by increasing speed and sensitivity while decreasing overall costs. For identification of species or resistance alleles, analysis of PCR products with many different post PCR methods can be used such as RFLP, reverse-line-blot, Sanger sequencing and HRM.

Discrimination of Gastrointestinal Nematode Eggs from Crude Fecal Egg Preparations by Inhibitor-Resistant Conventional and Real-Time PCR

PubMed Central

Demeler, Janina; Ramünke, Sabrina; Wolken, Sonja; Ianiello, Davide; Rinaldi, Laura; Gahutu, Jean Bosco; Cringoli, Giuseppe; von Samson-Himmelstjerna, Georg; Krücken, Jürgen

2013-01-01

Diagnosis of gastrointestinal nematodes relies predominantly on coproscopic methods such as flotation, Kato-Katz, McMaster or FLOTAC. Although FLOTAC allows accurate quantification, many nematode eggs can only be differentiated to genus or family level. Several molecular diagnostic tools discriminating closely related species suffer from high costs for DNA isolation from feces and limited sensitivity since most kits use only small amounts of feces (<1 g). A direct PCR from crude egg preparations was designed for full compatibility with FLOTAC to accurately quantify eggs per gram feces (epg) and determine species composition. Eggs were recovered from the flotation solution and concentrated by sieving. Lysis was achieved by repeated boiling and freezing cycles – only Trichuris eggs required additional mechanic disruption. Egg lysates were directly used as template for PCR with Phusion DNA polymerase which is particularly resistant to PCR inhibitors. Qualitative results were obtained with feces of goats, cattle, horses, swine, cats, dogs and mice. The finally established protocol was also compatible with quantitative real-time PCR in the presence of EvaGreen and no PCR inhibition was detectable when extracts were diluted at least fourfold. Sensitivity was comparable to DNA isolation protocols and spiked samples with five epg were reliably detected. For Toxocara cati a detection limit below one epg was demonstrated. It was possible to distinguish T. cati and Toxocara canis using high resolution melt (HRM) analysis, a rapid tool for species identification. In human samples, restriction fragment length polymorphism (RFLP) and HRM analysis were used to discriminate Necator americanus and Ancylostoma duodenale. The method is able to significantly improve molecular diagnosis of gastrointestinal nematodes by increasing speed and sensitivity while decreasing overall costs. For identification of species or resistance alleles, analysis of PCR products with many different post PCR methods can be used such as RFLP, reverse-line-blot, Sanger sequencing and HRM. PMID:23620739

Detection of Mycoplasma hyopneumoniae by polymerase chain reaction in swine presenting respiratory problems

PubMed Central

Yamaguti, M.; Muller, E.E.; Piffer, A.I.; Kich, J.D.; Klein, C.S.; Kuchiishi, S.S.

2008-01-01

Since Mycoplasma hyopneumoniae isolation in appropriate media is a difficult task and impractical for daily routine diagnostics, Nested-PCR (N-PCR) techniques are currently used to improve the direct diagnostic sensitivity of Swine Enzootic Pneumonia. In a first experiment, this paper describes a N-PCR technique optimization based on three variables: different sampling sites, sample transport media, and DNA extraction methods, using eight pigs. Based on the optimization results, a second experiment was conducted for testing validity using 40 animals. In conclusion, the obtained results of the N-PCR optimization and validation allow us to recommend this test as a routine monitoring diagnostic method for Mycoplasma hyopneumoniae infection in swine herds. PMID:24031248

77 FR 27738 - Proposed Collection; Comment Request

Federal Register 2010, 2011, 2012, 2013, 2014

2012-05-11

... qualitative testing mortgage servicing related model forms and disclosures. DATES: Written comments must be...: Qualitative Testing of Mortgage Servicing Related Model Forms and Disclosures. OMB Number: 3170-0018. Abstract: The Bureau seeks to allow for qualitative testing of mortgage servicing related model forms and...

Accuracy of polimerase chain reaction for the diagnosis of pleural tuberculosis.

PubMed

Trajman, Anete; da Silva Santos Kleiz de Oliveira, Elen Fabricia; Bastos, Mayara Lisboa; Belo Neto, Epaminondas; Silva, Edgar Manoel; da Silva Lourenço, Maria Cristina; Kritski, Afrânio; Oliveira, Martha Maria

2014-06-01

Polymerase chain reaction (PCR)-based techniques to detect Mycobacterium tuberculosis DNA in respiratory specimens have been increasingly used to diagnose pulmonary tuberculosis. Their use in non-respiratory specimens to diagnose extrapulmonary tuberculosis is, however, controversial. In this study, we estimated the accuracy of three in-country commercialized PCR-based diagnostic techniques in pleural fluid samples for the diagnosis of pleural tuberculosis. Patients underwent thoracenthesis for diagnosis purposes; pleural fluid aliquots were frozen and subsequently submitted to two real time PCR tests (COBAS(®)TAQMAN(®)MTB and Xpert(®)MTB/Rif) and one conventional PCR test (Detect-TB(®)). Two different reference standards were considered: probable tuberculosis (based on clinical grounds) and confirmed tuberculosis (bacteriologically or histologically). Ninety-three patients were included, of whom 65 with pleural tuberculosis, 35 of them confirmed. Sensitivities were 29% for COBAS(®)TAQMAN(®)MTB, 3% for Xpert(®)MTB/Rif and 3% for Detect-TB(®); specificities were 86%, 100% and 97% respectively, considering confirmed tuberculosis. Considering all cases, sensitivities were 16%, 3% and 2%, and specificities, 86%, 100%, and 97%. Compared to the 95% sensitivity of adenosine deaminase, the most sensitive test for pleural tuberculosis, the sensitivities of the three PCR-based tests were very low. We conclude that at present, there is no major place for such tests in routine clinical use. Copyright © 2014 Elsevier Ltd. All rights reserved.

Sensitivity and specificity of real-time PCR and bacteriological culture for francisellosis in farm-raised Nile tilapia (Oreochromis niloticus L.).

PubMed

Assis, G B N; de Oliveira, T F; Gardner, I A; Figueiredo, H C P; Leal, C A G

2017-06-01

Despite the worldwide occurrence of Francisella noatunensis subsp. orientalis (Fno) infection in farmed tilapia, sensitivity and specificity estimates of commonly used diagnostic tests have not been reported. This study aimed to estimate the sensitivity and specificity of bacteriological culture and qPCR to detect Fno infection. We tested 559 fish, sampled from four farms with different epidemiological scenarios: (i) healthy fish in a hatchery free of Fno; (ii) targeted sampling of diseased fish with suggestive external clinical signs of francisellosis during an outbreak; (iii) convenience sampling of diseased and clinically healthy fish during an outbreak; and (iv) sampling of healthy fish in a cage farm without a history of outbreaks, but with francisellosis reported in other farms in the same reservoir. The qPCR had higher median sensitivity (range, 48.8-99.5%) than culture (range, 1.6-74.4%). Culture had a substantially lower median sensitivity (1.6%) than qPCR (48.8%) to detect Fno in carrier tilapia (farm 4). Median specificity estimates for both tests were >99.2%. The qPCR is the superior test for use in surveillance and monitoring programmes for francisellosis in farmed Nile tilapia, but both tests have high sensitivity and specificity which make them fit for use in the diagnosis of Fno outbreaks. © 2016 John Wiley & Sons Ltd.

Polymerase chain reaction technology as analytical tool in agricultural biotechnology.

PubMed

Lipp, Markus; Shillito, Raymond; Giroux, Randal; Spiegelhalter, Frank; Charlton, Stacy; Pinero, David; Song, Ping

2005-01-01

The agricultural biotechnology industry applies polymerase chain reaction (PCR) technology at numerous points in product development. Commodity and food companies as well as third-party diagnostic testing companies also rely on PCR technology for a number of purposes. The primary use of the technology is to verify the presence or absence of genetically modified (GM) material in a product or to quantify the amount of GM material present in a product. This article describes the fundamental elements of PCR analysis and its application to the testing of grains. The document highlights the many areas to which attention must be paid in order to produce reliable test results. These include sample preparation, method validation, choice of appropriate reference materials, and biological and instrumental sources of error. The article also discusses issues related to the analysis of different matrixes and the effect they may have on the accuracy of the PCR analytical results.

Multiplex PCR for Rapid Detection of Genes Encoding Class A Carbapenemases

PubMed Central

Hong, Sang Sook; Kim, Kyeongmi; Huh, Ji Young; Jung, Bochan; Kang, Myung Seo

2012-01-01

In recent years, there have been increasing reports of KPC-producing Klebsiella pneumoniae in Korea. The modified Hodge test can be used as a phenotypic screening test for class A carbapenamase (CAC)-producing clinical isolates; however, it does not distinguish between carbapenemase types. The confirmation of type of CAC is important to ensure optimal therapy and to prevent transmission. This study applied a novel multiplex PCR assay to detect and differentiate CAC genes in a single reaction. Four primer pairs were designed to amplify fragments encoding 4 CAC families (SME, IMI/NMC-A, KPC, and GES). The multiplex PCR detected all genes tested for 4 CAC families that could be differentiated by fragment size according to gene type. This multiplex PCR offers a simple and useful approach for detecting and distinguishing CAC genes in carbapenem-resistant strains that are metallo-β-lactamase nonproducers. PMID:22950072

Multiplex PCR for rapid detection of genes encoding class A carbapenemases.

PubMed

Hong, Sang Sook; Kim, Kyeongmi; Huh, Ji Young; Jung, Bochan; Kang, Myung Seo; Hong, Seong Geun

2012-09-01

In recent years, there have been increasing reports of KPC-producing Klebsiella pneumoniae in Korea. The modified Hodge test can be used as a phenotypic screening test for class A carbapenamase (CAC)-producing clinical isolates; however, it does not distinguish between carbapenemase types. The confirmation of type of CAC is important to ensure optimal therapy and to prevent transmission. This study applied a novel multiplex PCR assay to detect and differentiate CAC genes in a single reaction. Four primer pairs were designed to amplify fragments encoding 4 CAC families (SME, IMI/NMC-A, KPC, and GES). The multiplex PCR detected all genes tested for 4 CAC families that could be differentiated by fragment size according to gene type. This multiplex PCR offers a simple and useful approach for detecting and distinguishing CAC genes in carbapenem-resistant strains that are metallo-β-lactamase nonproducers.

Merkel cell polyomavirus detection in Merkel cell cancer tumors in Northern Germany using PCR and protein expression.

PubMed

Leitz, Miriam; Stieler, Kristin; Grundhoff, Adam; Moll, Ingrid; Brandner, Johanna M; Fischer, Nicole

2014-10-01

Merkel cell carcinoma is a highly malignant skin cancer which predominantly occurs in elderly and immunocompromised persons. The identification of the Merkel cell polyomavirus (MCPyV) has inaugurated a new understanding of Merkel cell carcinoma pathogenesis. The frequent detection of the virus in Merkel cell carcinoma tissue (70-90%), its monoclonal integration in the tumor cells and the expression of viral oncogenes highly suggest that MCPyV is causally linked to the pathogenesis of the majority of Merkel cell cancer (MCC) cases. Using qualitative and quantitative PCR together with immunohistochemical staining this study aimed at characterizing the presence of MCPyV sequences and viral early gene expression in a cohort of MCC cases (n = 32) selected in Northern Germany. 40-57% of the cases were identified as MCPyV positive with 40.6% of the cases positive by immunohistochemical staining and 51.6-57.6% positive by PCR. Interestingly, in the majority (64%) of LT-Antigen positive tumors only 25-50% of tumor cells express LT-Antigen. These data are in accord with published studies describing heterogeneity in MCPyV viral loads and suggest that detection of MCPyV in Merkel cell carcinoma by PCR should be undertaken using multiple primer pairs. © 2013 Wiley Periodicals, Inc.

Novel PCR Assays Complement Laser Biosensor-Based Method and Facilitate Listeria Species Detection from Food.

PubMed

Kim, Kwang-Pyo; Singh, Atul K; Bai, Xingjian; Leprun, Lena; Bhunia, Arun K

2015-09-08

The goal of this study was to develop the Listeria species-specific PCR assays based on a house-keeping gene (lmo1634) encoding alcohol acetaldehyde dehydrogenase (Aad), previously designated as Listeria adhesion protein (LAP), and compare results with a label-free light scattering sensor, BARDOT (bacterial rapid detection using optical scattering technology). PCR primer sets targeting the lap genes from the species of Listeria sensu stricto were designed and tested with 47 Listeria and 8 non-Listeria strains. The resulting PCR primer sets detected either all species of Listeria sensu stricto or individual L. innocua, L. ivanovii and L. seeligeri, L. welshimeri, and L. marthii without producing any amplified products from other bacteria tested. The PCR assays with Listeria sensu stricto-specific primers also successfully detected all species of Listeria sensu stricto and/or Listeria innocua from mixed culture-inoculated food samples, and each bacterium in food was verified by using the light scattering sensor that generated unique scatter signature for each species of Listeria tested. The PCR assays based on the house-keeping gene aad (lap) can be used for detection of either all species of Listeria sensu stricto or certain individual Listeria species in a mixture from food with a detection limit of about 10⁴ CFU/mL.

Novel PCR Assays Complement Laser Biosensor-Based Method and Facilitate Listeria Species Detection from Food

PubMed Central

Kim, Kwang-Pyo; Singh, Atul K.; Bai, Xingjian; Leprun, Lena; Bhunia, Arun K.

2015-01-01

The goal of this study was to develop the Listeria species-specific PCR assays based on a house-keeping gene (lmo1634) encoding alcohol acetaldehyde dehydrogenase (Aad), previously designated as Listeria adhesion protein (LAP), and compare results with a label-free light scattering sensor, BARDOT (bacterial rapid detection using optical scattering technology). PCR primer sets targeting the lap genes from the species of Listeria sensu stricto were designed and tested with 47 Listeria and 8 non-Listeria strains. The resulting PCR primer sets detected either all species of Listeria sensu stricto or individual L. innocua, L. ivanovii and L. seeligeri, L. welshimeri, and L. marthii without producing any amplified products from other bacteria tested. The PCR assays with Listeria sensu stricto-specific primers also successfully detected all species of Listeria sensu stricto and/or Listeria innocua from mixed culture-inoculated food samples, and each bacterium in food was verified by using the light scattering sensor that generated unique scatter signature for each species of Listeria tested. The PCR assays based on the house-keeping gene aad (lap) can be used for detection of either all species of Listeria sensu stricto or certain individual Listeria species in a mixture from food with a detection limit of about 104 CFU/mL. PMID:26371000

Validation of a Real Time PCR for Classical Swine Fever Diagnosis

PubMed Central

Dias, Natanael Lamas; Fonseca Júnior, Antônio Augusto; Oliveira, Anapolino Macedo; Sales, Érica Bravo; Alves, Bruna Rios Coelho; Dorella, Fernanda Alves

2014-01-01

The viral disease classical swine fever (CSF), caused by a Pestivirus, is one of the major causes of economic losses for pig farming. The aim of this work was to validate a RT-qPCR using Taqman for detection of CSF in swine tissues. The parameters for the validation followed the specifications of the Manual of Diagnostic Tests and Vaccines for Terrestrial Animals of the World Organization for Animal Health (OIE) and the guide ABNT NBR ISO/IEC 17025:2005. The analysis of the 5′NTR region of CSF virus was performed in 145 samples from 29 infected pigs and in 240 samples from 80 pigs originated in the Brazilian CSF-free zone. The tissues tested were spleen, kidney, blood, tonsils, and lymph nodes. Sequencing of the positive samples for 5′NTR region was performed to evaluate the specificity of the RT-qPCR. Tests performed for the RT-qPCR validation demonstrated that the PCR assay was efficient in detecting RNA from CSF virus in all materials from different tissues of infected animals. Furthermore, RNA from CSF virus was not detected in samples of swine originated from the Brazilian CSF-free zone. Hence, it is concluded that RT-qPCR can be used as a complementary diagnostic for CSF. PMID:24818039

Validation of a real time PCR for classical Swine Fever diagnosis.

PubMed

Dias, Natanael Lamas; Fonseca Júnior, Antônio Augusto; Oliveira, Anapolino Macedo; Sales, Erica Bravo; Alves, Bruna Rios Coelho; Dorella, Fernanda Alves; Camargos, Marcelo Fernandes

2014-01-01

The viral disease classical swine fever (CSF), caused by a Pestivirus, is one of the major causes of economic losses for pig farming. The aim of this work was to validate a RT-qPCR using Taqman for detection of CSF in swine tissues. The parameters for the validation followed the specifications of the Manual of Diagnostic Tests and Vaccines for Terrestrial Animals of the World Organization for Animal Health (OIE) and the guide ABNT NBR ISO/IEC 17025:2005. The analysis of the 5'NTR region of CSF virus was performed in 145 samples from 29 infected pigs and in 240 samples from 80 pigs originated in the Brazilian CSF-free zone. The tissues tested were spleen, kidney, blood, tonsils, and lymph nodes. Sequencing of the positive samples for 5'NTR region was performed to evaluate the specificity of the RT-qPCR. Tests performed for the RT-qPCR validation demonstrated that the PCR assay was efficient in detecting RNA from CSF virus in all materials from different tissues of infected animals. Furthermore, RNA from CSF virus was not detected in samples of swine originated from the Brazilian CSF-free zone. Hence, it is concluded that RT-qPCR can be used as a complementary diagnostic for CSF.

Standardisation of polymerase chain reaction for the detection of Salmonella typhi in typhoid fever.

PubMed Central

Chaudhry, R; Laxmi, B V; Nisar, N; Ray, K; Kumar, D

1997-01-01

To improve the diagnosis of Salmonella typhi infection, a polymerase chain reaction (PCR) assay was developed for the amplification of the dH flagellin gene of S typhi. Primers were designed from dH flagellin gene sequence which will give an amplification product of 486 base pairs. In tests to study the specificity of the assay, no amplification was seen in non-salmonella strains or salmonella strains with flagellar gene other than "d". Sensitivity tests determined that 28 pg of S typhi target DNA or 3 x 10(2) target bacteria could be detected by the PCR assay. Subsequently, the PCR technique was used for detection of S typhi in blood or clot cultures from 84 patients clinically suspected of having typhoid fever, and from 20 healthy control subjects. Twenty five of 84 samples from clinically suspected cases were positive by PCR; four of which were culture negative. No amplification was seen in samples from patients who were culture positive for organisms other than S typhi or from controls. The time taken for each sample for PCR analysis was less than 48 hours compared with three to five days for blood or clot culture. PCR appeared to be a promising diagnostic test for typhoid fever. Images PMID:9215131

Oncogenic Viral Prevalence in Invasive Vulvar Cancer Specimens from HIV Positive and Negative Women in Botswana

PubMed Central

Tesfalul, Martha; Simbiri, Kenneth; Wheat, Chikoti M.; Motsepe, Didintle; Goldbach, Hayley; Armstrong, Kathleen; Hudson, Kathryn; Kayembe, Mukendi K.; Robertson, Erle; Kovarik, Carrie

2014-01-01

Objective The primary aim of this study is to describe the prevalence of select oncogenic viruses within vulvar squamous cell carcinoma (VSCC) and their association with Human Immunodeficiency Virus (HIV) status in women in Botswana, where the national HIV prevalence is the third highest in the world. Methods/materials A cross-sectional study of biopsy-confirmed VSCC specimens and corresponding clinical data was conducted in Gaborone, Botswana. Polymerase Chain Reaction (PCR) and Immunohistochemistry (IHC) viral testing were done for Epstein-Barr Virus (EBV), Human Papilloma Virus (HPV) strains, and Kaposi's Sarcoma Herpesvirus (KSHV), and PCR viral testing alone was done for John Cunningham Virus (JCV). Results HPV prevalence by PCR was 100% (39/39 35/35) among tested samples. HPV16 was the most prevalent HPV strain (82.9% by PCR, 94.7% by either PCR or IHC). KSHV prevalence by PCR had a significant association with HIV status (p = 0.013), but not by IHC (p = 0.650). Conclusions The high burden of HPV, specifically HPV16, in VSCC in Botswana suggests a distinct HPV profile that differs from other studied populations, which provides increased motivation for HPV vaccination efforts. Oncogenic viruses KSHV and EBV were also more prevalent in our study population though their potential role in VSCC pathology is unclear. PMID:24651632

Comparison of pulsed-field gel electrophoresis and enterobacterial repetitive intergenic consensus PCR and biochemical tests to characterize Lactococcus garvieae.

PubMed

Ture, M; Altinok, I; Capkin, E

2015-01-01

Biochemical test, pulsed-field gel electrophoresis (PFGE) and enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) were used to compare 42 strains of Lactococcus garvieae isolated from different regions of Turkey, Italy, France and Spain. Twenty biotypes of L. garvieae were formed based on 54 biochemical tests. ERIC-PCR of genomic DNA from different L. garvieae strains resulted in amplification of multiple fragments of DNA in sizes ranging between 200 and 5000 bp with various band intensities. After cutting DNA with ApaI restriction enzyme and running on the PFGE, 11–22 resolvable bands ranging from 2 to 194 kb were observed. Turkish isolates were grouped into two clusters, and only A58 (Italy) strain was connected with Turkish isolates. Similarities between Turkish, Spanish, Italian and French isolates were <50% except 216-6 Rize strain. In Turkey, first lactococcosis occurred in Mugla, and then, it has been spread all over the country. Based on ERIC-PCR, Spanish and Italian strains of L. garvieae were related to Mugla strains. Therefore, after comparing PFGE profiles, ERIC-PCR profiles and phenotypic characteristics of 42 strains of L. garvieae, there were no relationships found between these three typing methods. PFGE method was more discriminative than the other methods. © 2014 John Wiley & Sons Ltd.

A Simple, High-Throughput Assay for Fragile X Expanded Alleles Using Triple Repeat Primed PCR and Capillary Electrophoresis

PubMed Central

Lyon, Elaine; Laver, Thomas; Yu, Ping; Jama, Mohamed; Young, Keith; Zoccoli, Michael; Marlowe, Natalia

2010-01-01

Population screening has been proposed for Fragile X syndrome to identify premutation carrier females and affected newborns. We developed a PCR-based assay capable of quickly detecting the presence or absence of an expanded FMR1 allele with high sensitivity and specificity. This assay combines a triplet repeat primed PCR with high-throughput automated capillary electrophoresis. We evaluated assay performance using archived samples sent for Fragile X diagnostic testing representing a range of Fragile X CGG-repeat expansions. Two hundred five previously genotyped samples were tested with the new assay. Data were analyzed for the presence of a trinucleotide “ladder” extending beyond 55 repeats, which was set as a cut-off to identify expanded FMR1 alleles. We identified expanded FMR1 alleles in 132 samples (59 premutation, 71 full mutation, 2 mosaics) and normal FMR1 alleles in 73 samples. We found 100% concordance with previous results from PCR and Southern blot analyses. In addition, we show feasibility of using this assay with DNA extracted from dried-blood spots. Using a single PCR combined with high-throughput fragment analysis on the automated capillary electrophoresis instrument, we developed a rapid and reproducible PCR-based laboratory assay that meets many of the requirements for a first-tier test for population screening. PMID:20431035

Development of real-time RT-PCR for the detection of low concentrations of Rift Valley fever virus.

PubMed

Maquart, Marianne; Temmam, Sarah; Héraud, Jean-Michel; Leparc-Goffart, Isabelle; Cêtre-Sossah, Catherine; Dellagi, Koussay; Cardinale, Eric; Pascalis, Hervé

2014-01-01

In recent years, Madagascar and the Comoros archipelago have been affected by epidemics of Rift Valley fever (RVF), however detection of Rift Valley fever virus (RVFV) in zebu, sheep and goats during the post epidemic periods was frequently unsuccessful. Thus, a highly sensitive real-time RT-PCR assay was developed for the detection of RVFV at low viral loads. A new RVF SYBR Green RT-PCR targeting the M segment was tested on serum from different RVF seronegative ruminant species collected from May 2010 to August 2011 in Madagascar and the Comoros archipelago and compared with a RVF specific quantitative real time RT-PCR technique, which is considered as the reference technique. The specificity was tested on a wide range of arboviruses or other viruses giving RVF similar clinical signs. A total of 38 out of 2756 serum samples tested positive with the new RT-PCR, whereas the reference technique only detected 5 out of the 2756. The described RT-PCR is an efficient diagnostic tool for the investigation of enzootic circulation of the RVF virus. It allows the detection of low viral RNA loads adapted for the investigations of reservoirs or specific epidemiological situations such as inter-epizootic periods. Copyright © 2013 Elsevier B.V. All rights reserved.

Detecting Malaria Hotspots: A Comparison of Rapid Diagnostic Test, Microscopy, and Polymerase Chain Reaction.

PubMed

Mogeni, Polycarp; Williams, Thomas N; Omedo, Irene; Kimani, Domtila; Ngoi, Joyce M; Mwacharo, Jedida; Morter, Richard; Nyundo, Christopher; Wambua, Juliana; Nyangweso, George; Kapulu, Melissa; Fegan, Gregory; Bejon, Philip

2017-11-27

Malaria control strategies need to respond to geographical hotspots of transmission. Detection of hotspots depends on the sensitivity of the diagnostic tool used. We conducted cross-sectional surveys in 3 sites within Kilifi County, Kenya, that had variable transmission intensities. Rapid diagnostic test (RDT), microscopy, and polymerase chain reaction (PCR) were used to detect asymptomatic parasitemia, and hotspots were detected using the spatial scan statistic. Eight thousand five hundred eighty-one study participants were surveyed in 3 sites. There were statistically significant malaria hotspots by RDT, microscopy, and PCR for all sites except by microscopy in 1 low transmission site. Pooled data analysis of hotspots by PCR overlapped with hotspots by microscopy at a moderate setting but not at 2 lower transmission settings. However, variations in degree of overlap were noted when data were analyzed by year. Hotspots by RDT were predictive of PCR/microscopy at the moderate setting, but not at the 2 low transmission settings. We observed long-term stability of hotspots by PCR and microscopy but not RDT. Malaria control programs may consider PCR testing to guide asymptomatic malaria hotspot detection once the prevalence of infection falls. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America.

A nested polymerase chain reaction for the detection of genomic DNA of Myxobolus cerebralis in rainbow trout Oncorhynchus mykiss.

PubMed

Andree, K B; MacConnell, E; Hedrick, R P

1998-10-08

A nested polymerase chain reaction (PCR) test was developed to amplify a segment of the 18S rRNA gene from Myxobolus cerebralis, the agent causing whirling disease in salmonid fish. The PCR amplifies a 415 bp amplicon that was identified by dideoxynucleotide terminated sequencing to be identical to the known 18S rDNA sequence of M. cerebralis. There was no amplification of genomic DNA from 4 other myxosporean parasites of salmonid fish from the genus Myxobolus including M. arcticus, M. insidiosus, M. neurobius, and M. squamalis. The efficacy of the PCR test to detect early infections was demonstrated by amplification of the 415 bp fragment from experimentally exposed rainbow trout Oncorhynchus mykiss at 2 h and at 1, 2, and 3 wk postexposure to actinosporean stages (triactinomyxons) of M. cerebralis. In contrast, standard microscopic examinations of stained tissue sections of the same fish used for PCR were less reliable in detecting the presence of the parasite. Additional examinations of fish 5 mo postexposure, after sporogenesis had occurred, found the PCR to be a more reliable indicator of infection than pepsin-trypsin digest (PTD) method, particularly when trout were experimentally exposed to low levels of the infectious stages of the parasite. The PCR was able to amplify to detectable levels the equivalent of a single sporoplasm of M. cerebralis as found in a tissue sample. This test improves the detection of M. cerebralis because it can detect the presence of the parasite: (1) in both hosts, (2) in all known stages of its life cycle, and (3) at lower thresholds than currently used diagnostic methods. Lastly, the PCR test is less susceptible to morphological misidentifications of the spores that can occur with current microscopic procedures.

Development and validation of a Pneumocystis jirovecii real-time polymerase chain reaction assay for diagnosis of Pneumocystis pneumonia

PubMed Central

Church, Deirdre L; Ambasta, Anshula; Wilmer, Amanda; Williscroft, Holly; Ritchie, Gordon; Pillai, Dylan R; Champagne, Sylvie; Gregson, Daniel G

2015-01-01

BACKGROUND: Pneumocystis jirovecii (PJ), a pathogenic fungus, causes severe interstitial Pneumocystis pneumonia (PCP) among immunocompromised patients. A laboratory-developed real-time polyermase chain reaction (PCR) assay was validated for PJ detection to improve diagnosis of PCP. METHODS: Forty stored bronchoalveolar lavage (BAL) samples (20 known PJ positive [PJ+] and 20 known PJ negative [PJ−]) were initially tested using the molecular assay. Ninety-two sequentially collected BAL samples were then analyzed using an immunofluorescence assay (IFA) and secondarily tested using the PJ real-time PCR assay. Discrepant results were resolved by retesting BAL samples using another real-time PCR assay with a different target. PJ real-time PCR assay performance was compared with the existing gold standard (ie, IFA) and a modified gold standard, in which a true positive was defined as a sample that tested positive in two of three methods in a patient suspected to have PCP. RESULTS: Ninety of 132 (68%) BAL fluid samples were collected from immunocompromised patients. Thirteen of 92 (14%) BALs collected were PJ+ when tested using IFA. A total of 40 BAL samples were PJ+ in the present study including: all IFA positive samples (n=13); all referred PJ+ BAL samples (n=20); and seven additional BAL samples that were IFA negative, but positive using the modified gold standard. Compared with IFA, the PJ real-time PCR had sensitivity, specificity, and positive and negative predictive values of 100%, 91%, 65% and 100%, respectively. Compared with the modified gold standard, PJ real-time PCR had a sensitivity, specificity, and positive and negative predictive values of 100%. CONCLUSION: PJ real-time PCR improved detection of PJ in immunocompromised patients. PMID:26600815

TaqMan 5′-Nuclease Human Immunodeficiency Virus Type 1 PCR Assay with Phage-Packaged Competitive Internal Control for High-Throughput Blood Donor Screening

PubMed Central

Drosten, C.; Seifried, E.; Roth, W. K.

2001-01-01

Screening of blood donors for human immunodeficiency virus type 1 (HIV-1) infection by PCR permits the earlier diagnosis of HIV-1 infection compared with that by serologic assays. We have established a high-throughput reverse transcription (RT)-PCR assay based on 5′-nuclease PCR. By in-tube detection of HIV-1 RNA with a fluorogenic probe, the 5′-nuclease PCR technology (TaqMan PCR) eliminates the risk of carryover contamination, a major problem in PCR testing. We outline the development and evaluation of the PCR assay from a technical point of view. A one-step RT-PCR that targets the gag genes of all known HIV-1 group M isolates was developed. An internal control RNA detectable with a heterologous 5′-nuclease probe was derived from the viral target cDNA and was packaged into MS2 coliphages (Armored RNA). Because the RNA was protected against digestion with RNase, it could be spiked into patient plasma to control the complete sample preparation and amplification process. The assay detected 831 HIV-1 type B genome equivalents per ml of native plasma (95% confidence interval [CI], 759 to 936 HIV-1 B genome equivalents per ml) with a ≥95% probability of a positive result, as determined by probit regression analysis. A detection limit of 1,195 genome equivalents per ml of (individual) donor plasma (95% CI, 1,014 to 1,470 genome equivalents per ml of plasma pooled from individuals) was achieved when 96 samples were pooled and enriched by centrifugation. Up to 4,000 plasma samples per PCR run were tested in a 3-month trial period. Although data from the present pilot feasibility study will have to be complemented by a large clinical validation study, the assay is a promising approach to the high-throughput screening of blood donors and is the first noncommercial test for high-throughput screening for HIV-1. PMID:11724836

A New Single-Step PCR Assay for the Detection of the Zoonotic Malaria Parasite Plasmodium knowlesi

PubMed Central

Lucchi, Naomi W.; Poorak, Mitra; Oberstaller, Jenna; DeBarry, Jeremy; Srinivasamoorthy, Ganesh; Goldman, Ira; Xayavong, Maniphet; da Silva, Alexandre J.; Peterson, David S.; Barnwell, John W.; Kissinger, Jessica; Udhayakumar, Venkatachalam

2012-01-01

Background Recent studies in Southeast Asia have demonstrated substantial zoonotic transmission of Plasmodium knowlesi to humans. Microscopically, P. knowlesi exhibits several stage-dependent morphological similarities to P. malariae and P. falciparum. These similarities often lead to misdiagnosis of P. knowlesi as either P. malariae or P. falciparum and PCR-based molecular diagnostic tests are required to accurately detect P. knowlesi in humans. The most commonly used PCR test has been found to give false positive results, especially with a proportion of P. vivax isolates. To address the need for more sensitive and specific diagnostic tests for the accurate diagnosis of P. knowlesi, we report development of a new single-step PCR assay that uses novel genomic targets to accurately detect this infection. Methodology and Significant Findings We have developed a bioinformatics approach to search the available malaria parasite genome database for the identification of suitable DNA sequences relevant for molecular diagnostic tests. Using this approach, we have identified multi-copy DNA sequences distributed in the P. knowlesi genome. We designed and tested several novel primers specific to new target sequences in a single-tube, non-nested PCR assay and identified one set of primers that accurately detects P. knowlesi. We show that this primer set has 100% specificity for the detection of P. knowlesi using three different strains (Nuri, H, and Hackeri), and one human case of malaria caused by P. knowlesi. This test did not show cross reactivity with any of the four human malaria parasite species including 11 different strains of P. vivax as well as 5 additional species of simian malaria parasites. Conclusions The new PCR assay based on novel P. knowlesi genomic sequence targets was able to accurately detect P. knowlesi. Additional laboratory and field-based testing of this assay will be necessary to further validate its utility for clinical diagnosis of P. knowlesi. PMID:22363751

Field evaluation of Abbott Real Time HIV-1 Qualitative test for early infant diagnosis using dried blood spots samples in comparison to Roche COBAS Ampliprep/COBAS TaqMan HIV-1 Qual Test in Kenya

PubMed Central

Chang, Joy; Omuomo, Kenneth; Anyango, Emily; Kingwara, Leonard; Basiye, Frank; Morwabe, Alex; Shanmugam, Vedapuri; Nguyen, Shon; Sabatier, Jennifer; Zeh, Clement; Ellenberger, Dennis

2016-01-01

Timely diagnosis and treatment of infants infected with HIV are critical for reducing infant mortality. High-throughput automated diagnostic tests like Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 Qual Test (Roche CAPCTM Qual) and the Abbott Real Time HIV-1 Qualitative (Abbott Qualitative) can be used to rapidly expand early infant diagnosis testing services. In this study, the performance characteristics of the Abbott Qualitative were evaluated using two hundred dried blood spots (DBS) samples (100 HIV-1 positive and 100 HIV-1 negative) collected from infants attending the antenatal facilities in Kisumu, Kenya. The Abbott Qualitative results were compared to the diagnostic testing completed using the Roche CAPCTM Qual in Kenya. The sensitivity and specificity of the Abbott Qualitative were 99.0% (95% CI: 95.0–100.0) and 100.0% (95% CI: 96.0–100.0), respectively, and the overall reproducibility was 98.0% (95% CI: 86.0–100.0). The limits of detection for the Abbott Qualitative and Roche CAPCTM Qual were 56.5 and 6.9 copies/mL at 95% CIs (p = 0.005), respectively. The study findings demonstrate that the Abbott Qualitative test is a practical option for timely diagnosis of HIV in infants. PMID:24726703

Field evaluation of Abbott Real Time HIV-1 Qualitative test for early infant diagnosis using dried blood spots samples in comparison to Roche COBAS Ampliprep/COBAS TaqMan HIV-1 Qual test in Kenya.

PubMed

Chang, Joy; Omuomo, Kenneth; Anyango, Emily; Kingwara, Leonard; Basiye, Frank; Morwabe, Alex; Shanmugam, Vedapuri; Nguyen, Shon; Sabatier, Jennifer; Zeh, Clement; Ellenberger, Dennis

2014-08-01

Timely diagnosis and treatment of infants infected with HIV are critical for reducing infant mortality. High-throughput automated diagnostic tests like Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 Qual Test (Roche CAPCTM Qual) and the Abbott Real Time HIV-1 Qualitative (Abbott Qualitative) can be used to rapidly expand early infant diagnosis testing services. In this study, the performance characteristics of the Abbott Qualitative were evaluated using two hundred dried blood spots (DBS) samples (100 HIV-1 positive and 100 HIV-1 negative) collected from infants attending the antenatal facilities in Kisumu, Kenya. The Abbott Qualitative results were compared to the diagnostic testing completed using the Roche CAPCTM Qual in Kenya. The sensitivity and specificity of the Abbott Qualitative were 99.0% (95% CI: 95.0-100.0) and 100.0% (95% CI: 96.0-100.0), respectively, and the overall reproducibility was 98.0% (95% CI: 86.0-100.0). The limits of detection for the Abbott Qualitative and Roche CAPCTM Qual were 56.5 and 6.9copies/mL at 95% CIs (p=0.005), respectively. The study findings demonstrate that the Abbott Qualitative test is a practical option for timely diagnosis of HIV in infants. Published by Elsevier B.V.

HIV RNA testing in the context of nonoccupational postexposure prophylaxis.

PubMed

Roland, Michelle E; Elbeik, Tarek A; Kahn, James O; Bamberger, Joshua D; Coates, Thomas J; Krone, Melissa R; Katz, Mitchell H; Busch, Michael P; Martin, Jeffrey N

2004-08-01

The specificity and positive predictive value of human immunodeficiency virus (HIV) RNA assays have not been evaluated in the setting of postexposure prophylaxis (PEP). Plasma from subjects enrolled in a nonoccupational PEP study was tested with 2 branched-chain DNA (bDNA) assays, 2 polymerase chain reaction (PCR) assays, and a transcription-mediated amplification (TMA) assay. Assay specificity and positive predictive value were determined for subjects who remained negative for HIV antibody for >or=3 months. In 329 subjects examined, the lowest specificities (90.1%-93.7%) were seen for bDNA testing performed in real time. The highest specificities were seen with batched bDNA version 3.0 (99.1%), standard PCR (99.4%), ultrasensitive PCR (100%), and TMA (99.6%) testing. Only the 2 assays with the highest specificities had positive predictive values >40%. For the bDNA assays, increasing the cutoff point at which a test is called positive (e.g., from 50 copies/mL to 500 copies/mL for version 3.0) increased both specificity and positive predictive values to 100%. The positive predictive value of HIV RNA assays in individuals presenting for PEP is unacceptably low for bDNA-based testing and possibly acceptable for PCR- and TMA-based testing. Routine use of HIV RNA assays in such individuals is not recommended.

Droplet digital PCR-based EGFR mutation detection with an internal quality control index to determine the quality of DNA.

PubMed

Kim, Sung-Su; Choi, Hyun-Jeung; Kim, Jin Ju; Kim, M Sun; Lee, In-Seon; Byun, Bohyun; Jia, Lina; Oh, Myung Ryurl; Moon, Youngho; Park, Sarah; Choi, Joon-Seok; Chae, Seoung Wan; Nam, Byung-Ho; Kim, Jin-Soo; Kim, Jihun; Min, Byung Soh; Lee, Jae Seok; Won, Jae-Kyung; Cho, Soo Youn; Choi, Yoon-La; Shin, Young Kee

2018-01-11

In clinical translational research and molecular in vitro diagnostics, a major challenge in the detection of genetic mutations is overcoming artefactual results caused by the low-quality of formalin-fixed paraffin-embedded tissue (FFPET)-derived DNA (FFPET-DNA). Here, we propose the use of an 'internal quality control (iQC) index' as a criterion for judging the minimum quality of DNA for PCR-based analyses. In a pre-clinical study comparing the results from droplet digital PCR-based EGFR mutation test (ddEGFR test) and qPCR-based EGFR mutation test (cobas EGFR test), iQC index ≥ 0.5 (iQC copies ≥ 500, using 3.3 ng of FFPET-DNA [1,000 genome equivalents]) was established, indicating that more than half of the input DNA was amplifiable. Using this criterion, we conducted a retrospective comparative clinical study of the ddEGFR and cobas EGFR tests for the detection of EGFR mutations in non-small cell lung cancer (NSCLC) FFPET-DNA samples. Compared with the cobas EGFR test, the ddEGFR test exhibited superior analytical performance and equivalent or higher clinical performance. Furthermore, iQC index is a reliable indicator of the quality of FFPET-DNA and could be used to prevent incorrect diagnoses arising from low-quality samples.

Identification of Histoplasma capsulatum from culture extracts by real-time PCR.

PubMed

Martagon-Villamil, Jose; Shrestha, Nabin; Sholtis, Mary; Isada, Carlos M; Hall, Gerri S; Bryne, Terry; Lodge, Barbara A; Reller, L Barth; Procop, Gary W

2003-03-01

We designed and tested a real-time LightCycler PCR assay for Histoplasma capsulatum that correctly identified the 34 H. capsulatum isolates in a battery of 107 fungal isolates tested and also detected H. capsulatum in clinical specimens from three patients that were culture positive for this organism.

A PCR Based Microbial Monitoring Alternative Method of Detection and Identification of Microbes Aboard ISS

NASA Technical Reports Server (NTRS)

Khodadad, Christina; Oubre, Cherie; Castro, Victoria; Flint, Stephanie; Ott, Mark; Roman, Monserrate; Wheeler, Ray; Melendez, Orlando

2017-01-01

Previous research has shown that microorganisms and potential human pathogens have been detected on the International Space Station (ISS) with additional introduction of new microflora occurring with every exchange of crew or addition of equipment and supplies. These microbes are readily transferred between crew and subsystems (i.e. ECLSS, environmental control and life support systems). As this can be detrimental to astronaut health and optimal performance of ISS systems, monitoring of systems such as ECLSS to include identification of microbial contaminants could prevent adverse effects on human health and life support systems. Current monitoring on ISS is laborious and utilizes culture based methods followed by sample return to Earth for complete analysis. Future, long-distance spaceflight missions will require real-time monitoring capabilities that enable efficient and rapid assessments of the microbial environment allowing for expedited decisions and more targeted response to cope with anomalies. Polymerase chain reaction (PCR), a molecular microbial monitoring method was chosen and numerous PCR instruments investigated for their potential to perform in microgravity conditions. Using ISS as a test bed for PCR verification in microgravity will enable NASA to assess whether molecular based microbiological sensors may be components of reliable, closed-loop life support and habitation systems in spacecraft, enhancing infrastructure capabilities through increased efficiency, reliability, and time savings by enabling sample analysis on orbit. NASA selected the Water Monitoring Suite as one of the rapid spaceflight hardware demonstration activities utilizing a streamlined process to minimize the time required to fly experimental flight hardware. The RAZOR EX (BioFire Defense, Salt Lake City, UT) system was part of the water monitoring suite and is a commercial off-the-shelf (COTS) real-time PCR instrument designed for field work. The RAZOR EX was originally designed for Department of Defense (DoD) under a small business innovative research (SBIR) grant and is ruggedized, compact and provides a rapid, sample to answer in less than an hour. PCR assays using a fluorescent probe were optimized and spiked with known concentrations of DNA (Pseudomonas aeruginosa) ranging from 0.002 to 20 ng. PCR reagents were lyophilized and configured in customized pouches and tested for flight readiness. Three types of water were used to rehydrate the reagents and demonstrate the fidelity of the PCR reaction in microgravity. Molecular grade deionized water served as a control while filtered and unfiltered ISS potable water served to test for chemical or biological inhibitors. All three types were compared to parallel ground test results. Nine tests were run on ISS (3 of each water type) and the critical threshold cycle (Ct) was compared to parallel ground tests completed at Kennedy Space Center, FL and Johnson Space Center, TX. All concentrations of Pseudomonas aeruginosa DNA were detected. A comparison of the Ct produced in real time PCR indicated similarity between flight and ground samples. There appeared to be no significant difference between flight or ground PCR reactions or between any of the three water types. This testing demonstrated the ability to perform molecular testing during spaceflight operations with similar sensitivity. It will allow for future ground development of molecular protocols and minimize the need for spaceflight testing. Future testing will include development of additional targets including environmental and health related organisms.

A serotype-specific polymerase chain reaction for identification of Pasteurella multocida serotype 1

USGS Publications Warehouse

Rocke, Tonie E.; Smith, Susan R.; Miyamoto, Amy; Shadduck, Daniel J.

2002-01-01

A serotype-specific polymerase chain reaction (PCR) assay was developed for detection and identification of Pasteurella multocida serotype 1, the causative agent of avian cholera in wild waterfowl. Arbitrarily primed PCR was used to detect DNA fragments that distinguish serotype 1 from the other 15 serotypes of P. multocida (with the exception of serotype 14). Oligonucleotide primers were constructed from these sequences, and a PCR assay was optimized and evaluated. PCR reactions consistently resulted in amplification products with reference strains 1 and 14 and all other serotype 1 strains tested, with cell numbers as low as 2.3 cells/ml. No amplification products were produced with other P. multocida serotypes or any other bacterial species tested. To compare the sensitivity and further test the specificity of this PCR assay with traditional culturing and serotyping techniques, tissue samples from 84 Pekin ducks inoculated with field strains of P. multocida and 54 wild lesser snow geese collected during an avian cholera outbreak were provided by other investigators working on avian cholera. PCR was as sensitive (58/64) as routine isolation (52/64) in detecting and identifying P. multocida serotype 1 from the livers of inoculated Pekins that became sick or died from avian cholera. No product was amplified from tissues of 20 other Pekin ducks that received serotypes other than type 1 (serotype 3, 12 × 3, or 10) or 12 control birds. Of the 54 snow geese necropsied and tested for P. multocida, our PCR detected and identified the bacteria from 44 compared with 45 by direct isolation. The serotype-specific PCR we developed was much faster and less labor intensive than traditional culturing and serotyping procedures and could result in diagnosis of serotype 1 pasteurellosis within 24 hr of specimen submission.

Testing of candidate non-lethal sampling methods for detection of Renibacterium salmoninarum in juvenile Chinook salmon Oncorhynchus tshawytscha

USGS Publications Warehouse

Elliott, Diane G.; McKibben, Constance L.; Conway, Carla M.; Purcell, Maureen K.; Chase, Dorothy M.; Applegate, Lynn M.

2015-01-01

Non-lethal pathogen testing can be a useful tool for fish disease research and management. Our research objectives were to determine if (1) fin clips, gill snips, surface mucus scrapings, blood draws, or kidney biopsies could be obtained non-lethally from 3 to 15 g Chinook salmon Oncorhynchus tshawytscha, (2) non-lethal samples could accurately discriminate between fish exposed to the bacterial kidney disease agent Renibacterium salmoninarum and non-exposed fish, and (3) non-lethal samples could serve as proxies for lethal kidney samples to assess infection intensity. Blood draws and kidney biopsies caused ≥5% post-sampling mortality (Objective 1) and may be appropriate only for larger fish, but the other sample types were non-lethal. Sampling was performed over 21 wk following R. salmoninarum immersion challenge of fish from 2 stocks (Objectives 2 and 3), and nested PCR (nPCR) and real-time quantitative PCR (qPCR) results from candidate non-lethal samples were compared with kidney tissue analysis by nPCR, qPCR, bacteriological culture, enzyme-linked immunosorbent assay (ELISA), fluorescent antibody test (FAT) and histopathology/immunohistochemistry. R. salmoninarum was detected by PCR in >50% of fin, gill, and mucus samples from challenged fish. Mucus qPCR was the only non-lethal assay exhibiting both diagnostic sensitivity and specificity estimates >90% for distinguishing between R. salmoninarum-exposed and non-exposed fish and was the best candidate for use as an alternative to lethal kidney sample testing. Mucus qPCR R. salmoninarum quantity estimates reflected changes in kidney bacterial load estimates, as evidenced by significant positive correlations with kidney R. salmoninaruminfection intensity scores at all sample times and in both fish stocks, and were not significantly impacted by environmentalR. salmoninarum concentrations.

What assay is optimal for the diagnosis of measles virus infection? An evaluation of the performance of a measles virus real-time reverse transcriptase PCR using the Cepheid SmartCycler(®) and antigen detection by immunofluorescence.

PubMed

Chua, Kyra Y L; Thapa, Kiran; Yapa, Chaturangi M; Somerville, Lucy K; Chen, Sharon C-A; Dwyer, Dominic E; Sheppeard, Vicky; Kok, Jen

2015-09-01

Despite the World Health Organization (WHO)-reported elimination of measles in Australia, importation of cases especially in travellers from Asia continues in Sydney, Australia's largest city. Laboratory confirmation supports clinico-epidemiological evidence of measles virus infection, and is needed to establish elimination. To evaluate the performance of a random access real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay using the moderate complexity SmartCycler(®) platform, and measles antigen detection by immunofluorescence (IFA), for the detection of measles virus in patient samples. One hundred samples comprising nose and throat swabs, nasopharyngeal aspirates and urine, collected from patients with suspected measles were tested in parallel using IFA and nucleic acid testing using the SmartCycler(®) and LightCycler(®) RT-PCR platforms. The LightCycler(®) RT-PCR was used as the reference assay against which the SmartCycler(®) RT-PCR and IFA were compared. Using the LightCycler(®) RT-PCR, measles virus was detected in 35 clinical samples. There was 100% concordance between the results of the SmartCycler(®) and the LightCycler(®)-based RT-PCR. Measles genotypes detected included B3, D8, and D9. Testing urine in addition to NTS did not improve diagnostic yield. In contrast, the sensitivity and specificity of IFA compared to the reference LightCycler(®) RT-PCR was 34.3% and 96.7%, respectively. The performance of the SmartCycler(®) is comparable to the LightCycler(®) for the detection of measles virus. However, IFA had poor sensitivity and should not be used to confirm measles virus infection where nucleic acid testing is available. Copyright © 2015 Elsevier B.V. All rights reserved.

Accurate clinical genetic testing for autoinflammatory diseases using the next-generation sequencing platform MiSeq.

PubMed

Nakayama, Manabu; Oda, Hirotsugu; Nakagawa, Kenji; Yasumi, Takahiro; Kawai, Tomoki; Izawa, Kazushi; Nishikomori, Ryuta; Heike, Toshio; Ohara, Osamu

2017-03-01

Autoinflammatory diseases occupy one of a group of primary immunodeficiency diseases that are generally thought to be caused by mutation of genes responsible for innate immunity, rather than by acquired immunity. Mutations related to autoinflammatory diseases occur in 12 genes. For example, low-level somatic mosaic NLRP3 mutations underlie chronic infantile neurologic, cutaneous, articular syndrome (CINCA), also known as neonatal-onset multisystem inflammatory disease (NOMID). In current clinical practice, clinical genetic testing plays an important role in providing patients with quick, definite diagnoses. To increase the availability of such testing, low-cost high-throughput gene-analysis systems are required, ones that not only have the sensitivity to detect even low-level somatic mosaic mutations, but also can operate simply in a clinical setting. To this end, we developed a simple method that employs two-step tailed PCR and an NGS system, MiSeq platform, to detect mutations in all coding exons of the 12 genes responsible for autoinflammatory diseases. Using this amplicon sequencing system, we amplified a total of 234 amplicons derived from the 12 genes with multiplex PCR. This was done simultaneously and in one test tube. Each sample was distinguished by an index sequence of second PCR primers following PCR amplification. With our procedure and tips for reducing PCR amplification bias, we were able to analyze 12 genes from 25 clinical samples in one MiSeq run. Moreover, with the certified primers designed by our short program-which detects and avoids common SNPs in gene-specific PCR primers-we used this system for routine genetic testing. Our optimized procedure uses a simple protocol, which can easily be followed by virtually any office medical staff. Because of the small PCR amplification bias, we can analyze simultaneously several clinical DNA samples with low cost and can obtain sufficient read numbers to detect a low level of somatic mosaic mutations.

One-step cross-genogroup multiplex RT-qPCR with an internal control system for the detection of infectious pancreatic necrosis virus (IPNV).

PubMed

Hoferer, Marc; Braun, Anne; Skrypski, Julia; Bock, Sabine; Thalheim, Sabine; Sting, Reinhard

2017-09-01

Infectious pancreatic necrosis virus (IPNV) causes great losses in fish hatcheries world-wide. The detection of IPNV can be challenging in certain circumstances, particularly due to low viral load and the genetic variability of this RNA virus. For the first time, this project created a quantitative triplex real-time reverse transcription PCR (RT-qPCR), including an endogenous control system, for specific, sensitive and rapid detection of IPNV in routine diagnostics. Multiple sequence alignment of 46 nucleotide sequences of the segment A genome obtained from the NCBI database allowed the design of two RT-qPCR systems covering the IPNV genogroup 1 and genogroups 2-5, respectively. The completed triplex RT-qPCR including a salmonid-specific endogenous control showed high specificity and an analytical sensitivity of 20-40 oligonucleotide copies. Testing of dilution series of virus-loaded cell culture suspensions proved equality of the triplex RT-qPCR with virus detection in cell culture and a higher sensitivity than conventional RT-PCR in field samples. In comparative studies of a total of 77 field samples tested, 51 showed identical positive and 19 identical negative results in cell culture and the triplex RT-qPCR. However, seven other samples yielded positive results in the triplex RT-qPCR, but negative results in cell culture. Copyright © 2017 Elsevier B.V. All rights reserved.

Could Digital PCR Be an Alternative as a Non-Invasive Prenatal Test for Trisomy 21: A Proof of Concept Study

PubMed Central

El Khattabi, Laïla Allach; Rouillac-Le Sciellour, Christelle; Le Tessier, Dominique; Luscan, Armelle; Coustier, Audrey; Porcher, Raphael; Bhouri, Rakia; Nectoux, Juliette; Sérazin, Valérie; Quibel, Thibaut; Mandelbrot, Laurent; Tsatsaris, Vassilis

2016-01-01

Objective NIPT for fetal aneuploidy by digital PCR has been hampered by the large number of PCR reactions needed to meet statistical requirements, preventing clinical application. Here, we designed an octoplex droplet digital PCR (ddPCR) assay which allows increasing the number of available targets and thus overcomes statistical obstacles. Method After technical optimization of the multiplex PCR on mixtures of trisomic and euploid DNA, we performed a validation study on samples of plasma DNA from 213 pregnant women. Molecular counting of circulating cell-free DNA was performed using a mix of hydrolysis probes targeting chromosome 21 and a reference chromosome. Results The results of our validation experiments showed that ddPCR detected trisomy 21 even when the sample’s trisomic DNA content is as low as 5%. In a validation study of plasma samples from 213 pregnant women, ddPCR discriminated clearly between the trisomy 21 and the euploidy groups. Conclusion Our results demonstrate that digital PCR can meet the requirements for non-invasive prenatal testing of trisomy 21. This approach is technically simple, relatively cheap, easy to implement in a diagnostic setting and compatible with ethical concerns regarding access to nucleotide sequence information. These advantages make it a potential technique of choice for population-wide screening for trisomy 21 in pregnant women. PMID:27167625

Development, application, and results of routine monitoring of Marek's disease virus in broiler house dust using real-time quantitative PCR.

PubMed

Walkden-Brown, Stephen W; Islam, A F Aminul; Groves, Peter J; Rubite, Ambrosio; Sharpe, Sue M; Burgess, Susan K

2013-06-01

Results are presented from four studies between 2002 and 2011 into the feasibility of routinely monitoring Marek's disease virus serotype 1 (MDV-1) in broiler house dust using real-time quantitative PCR (qPCR) measurement. Study 1 on two farms showed that detection of MDV-1 occurred earlier on average in dust samples tested using qPCR than standard PCR and in spleen samples from five birds per shed assayed for MDV-1 by qPCR or standard PCR. DNA quality following extraction from dust had no effect on detection of MDV-1. Study 2 demonstrated that herpesvirus of turkeys (HVT) and MDV serotype 2 (MDV-2) in addition to MDV-1 could be readily amplified from commercial farm dust samples, often in mixtures. MDV-2 was detected in 11 of 20 samples despite the absence of vaccination with this serotype. Study 3 investigated the reproducibility and sensitivity of the qPCR test and the presence of inhibitors in the samples. Samples extracted and amplified in triplicate showed a high level of reproducibility except at very low levels of virus near the limit of detection. Mixing of samples prior to extraction provided results consistent with the proportions in the mixture. Tests for inhibition showed that if the template contained DNA in the range 0.5-20 ng/microl no inhibition of the reaction was detectable. The sensitivity of the tests in terms of viral copy number (VCN) per milligram of dust was calculated to be in the range 24-600 VCN/mg for MDV-1, 48-1200 VCN/mg for MDV-2, and 182-4560 VCN/mg for HVT. In study 4 the results of 1976 commercial tests carried out for one company were analyzed. Overall 23.1% of samples were positive for MDV-1, 26.1% in unvaccinated and 16.4% in vaccinated chickens. There was marked regional and temporal variation in the proportion of positive samples and the MDV-1 load. The tests were useful in formulating Marek's disease vaccination strategies. The number of samples submitted has increased recently, as has the incidence of positive samples. These studies provide strong evidence that detection and quantitation of MDV-1, HVT, and MDV-2 in poultry house dust using qPCR is robust, sensitive, reproducible, and meaningful, both biologically and commercially. Tactical vaccination based on monitoring of MDV-1 rather than routine vaccination may reduce selection pressure for increased virulence in MDV-1.

Culture-Independent Identification of Mycobacterium avium Subspecies paratuberculosis in Ovine Tissues: Comparison with Bacterial Culture and Histopathological Lesions

PubMed Central

Acharya, Kamal R.; Dhand, Navneet K.; Whittington, Richard J.; Plain, Karren M.

2017-01-01

Johne’s disease is a chronic debilitating enteropathy of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP). Current abattoir surveillance programs detect disease via examination of gross lesions and confirmation by histopathological and/or tissue culture, which is time-consuming and has relatively low sensitivity. This study aimed to investigate whether a high-throughput quantitative PCR (qPCR) test is a viable alternative for tissue testing. Intestine and mesenteric lymph nodes were sourced from sheep experimentally infected with MAP and the DNA extracted using a protocol developed for tissues, comprised enzymatic digestion of the tissue homogenate, chemical and mechanical lysis, and magnetic bead-based DNA purification. The extracted DNA was tested by adapting a previously validated qPCR for fecal samples, and the results were compared with culture and histopathology results of the corresponding tissues. The MAP tissue qPCR confirmed infection in the majority of sheep with gross lesions on postmortem (37/38). Likewise, almost all tissue culture (61/64) or histopathology (52/58) positives were detected with good to moderate agreement (Cohen’s kappa statistic) and no significant difference to the reference tests (McNemar’s Chi-square test). Higher MAP DNA quantities corresponded to animals with more severe histopathology (odds ratio: 1.82; 95% confidence interval: 1.60, 2.07). Culture-independent strain typing on tissue DNA was successfully performed. This MAP tissue qPCR method had a sensitivity equivalent to the reference tests and is thus a viable replacement for gross- and histopathological examination of tissue samples in abattoirs. In addition, the test could be validated for testing tissue samples intended for human consumption. PMID:29312970

Culture-Independent Identification of Mycobacterium avium Subspecies paratuberculosis in Ovine Tissues: Comparison with Bacterial Culture and Histopathological Lesions.

PubMed

Acharya, Kamal R; Dhand, Navneet K; Whittington, Richard J; Plain, Karren M

2017-01-01

Johne's disease is a chronic debilitating enteropathy of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP). Current abattoir surveillance programs detect disease via examination of gross lesions and confirmation by histopathological and/or tissue culture, which is time-consuming and has relatively low sensitivity. This study aimed to investigate whether a high-throughput quantitative PCR (qPCR) test is a viable alternative for tissue testing. Intestine and mesenteric lymph nodes were sourced from sheep experimentally infected with MAP and the DNA extracted using a protocol developed for tissues, comprised enzymatic digestion of the tissue homogenate, chemical and mechanical lysis, and magnetic bead-based DNA purification. The extracted DNA was tested by adapting a previously validated qPCR for fecal samples, and the results were compared with culture and histopathology results of the corresponding tissues. The MAP tissue qPCR confirmed infection in the majority of sheep with gross lesions on postmortem (37/38). Likewise, almost all tissue culture (61/64) or histopathology (52/58) positives were detected with good to moderate agreement (Cohen's kappa statistic) and no significant difference to the reference tests (McNemar's Chi-square test). Higher MAP DNA quantities corresponded to animals with more severe histopathology (odds ratio: 1.82; 95% confidence interval: 1.60, 2.07). Culture-independent strain typing on tissue DNA was successfully performed. This MAP tissue qPCR method had a sensitivity equivalent to the reference tests and is thus a viable replacement for gross- and histopathological examination of tissue samples in abattoirs. In addition, the test could be validated for testing tissue samples intended for human consumption.

[Trypanosoma cruzi in triatomines from Nuevo Leon, Mexico].

PubMed

Molina-Garza, Zinnia Judith; Rosales-Encina, José Luis; Galaviz-Silva, Lucio; Molina-Garza, Daniel

2007-01-01

To determine the prevalence of Trypanosoma cruzi in triatomines from Nuevo León using the standardization of an improved enzyme-linked immunosorbent assay test. From July to September 2005, 52 triatomines were captured in General Terán, a municipality located in Nuevo León. They were analyzed using optical microscopy (OM) and a polymerase chain reaction (PCR), as standards of reference, to develop a technique for detecting the parasite using enzyme-linked immunosorbent assay (ELISA). Using OM and PCR, 31 triatomines were found to be positive and 21 negative. Using ELISA, 27 samples were identified as positive and 25 negative (specificity 100%, sensitivity 87%, negative predictive value 84%, and positive predictive value 100%). The prevalence of infected triatomines was 59.61% with OM and PCR, and 51.92% with ELISA. Our data confirm that the ELISA assay in triatomines is a fast, reliable and useful tool. Since it was possible to simultaneously analyze a large number of samples with high sensibility and specificity values, the ELISA test proves to be useful for new epidemiologic studies having a high number of vectors. It is also less expensive than PCR. It is therefore recommended for epidemiological and preventive surveillance programs as a first screening test before conducting a confirmatory test using PCR.

A prospective study of a quantitative PCR ELISA assay for the diagnosis of CMV pneumonia in lung and heart-transplant recipients.

PubMed

Barber, L; Egan, J J; Lomax, J; Haider, Y; Yonan, N; Woodcock, A A; Turner, A J; Fox, A J

2000-08-01

Qualitative polymerase chain reaction (PCR) for the identification of cytomegalovirus (CMV) infection has a low predictive value for the identification of CMV pneumonia. This study prospectively evaluated the application of a quantitative PCR Enzyme-Linked Immuno-Sorbent Assay (ELISA) assay in 9 lung- and 18 heart-transplant recipients who did not receive ganciclovir prophylaxis. DNA was collected from peripheral blood polymorphonuclear leucocytes (PMNL) posttransplantation. Oligonucleotide primers for the glycoprotein B gene (149 bp) were used in a PCR ELISA assay using an internal standard for quantitation. CMV disease was defined as histological evidence of end organ damage. The median level CMV genome equivalents in patients with CMV disease was 2665/2 x 10(5) PMNL (range 1,200 to 61,606) compared to 100 x 10(5) PMNL (range 20 to 855) with infection but no CMV disease (p = 0.036). All patients with CMV disease had genome equivalents levels of >1200/2 x 10(5) PMNL. A cut-off level of 1,200 PMNL had a positive predictive value for CMV disease of 100% and a negative predictive value of 100%. The first detection of levels of CMV genome equivalents above a level of 1200/2 x 10(5) PMNL was at a median of 58 days (range 47 to 147) posttransplant. Quantitative PCR assays for the diagnosis of CMV infection may predict patients at risk of CMV disease and thereby direct preemptive treatment to high-risk patients.

Revealing the microbiota of marketed edible insects through PCR-DGGE, metagenomic sequencing and real-time PCR.

PubMed

Osimani, Andrea; Milanović, Vesna; Garofalo, Cristiana; Cardinali, Federica; Roncolini, Andrea; Sabbatini, Riccardo; De Filippis, Francesca; Ercolini, Danilo; Gabucci, Claudia; Petruzzelli, Annalisa; Tonucci, Franco; Clementi, Francesca; Aquilanti, Lucia

2018-07-02

The present study aimed to identify the microbiota present in six species of processed edible insects produced in Thailand and marketed worldwide via the internet, namely, giant water bugs (Belostoma lutarium), black ants (Polyrhachis), winged termites (alates, Termitoidae), rhino beetles (Hyboschema contractum), mole crickets (Gryllotalpidae), and silkworm pupae (Bombyx mori). For each species, two samples of boiled, dried and salted insects were purchased. The microbial DNA was extracted from the insect samples and subjected to polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), high-throughput sequencing and qualitative real-time PCR assays. The microbiota of the analyzed samples were widely characterized by the presence of spore-forming bacteria mainly represented by the genera Bacillus and Clostridium. Moreover, the genera Anaerobacillus, Paenibacillus, Geobacillus, Pseudomonas, Stenotrophomonas, Massilia, Delftia, Lactobacillus, Staphylococcus, Streptococcus, Vagococcus, and Vibrio were also detected. Real-time PCR allowed for ascertainment of the absence of Coxiella burnetii, Shiga toxin-producing E. coli (STEC), and Pseudomonas aeruginosa in all samples. The results of this study confirm the importance of combining different molecular techniques to characterize the biodiversity of complex ecosystems such as edible insects. The presence of potential human pathogens suggests the need for a careful application of good manufacturing practices during insect processing. This study provides further data that will be useful in risk analyses of edible insects as a novel food source. Copyright © 2018 Elsevier B.V. All rights reserved.

Antigenic typing of canine parvovirus using differential PCR.

PubMed

Kaur, Gurpreet; Chandra, Mudit; Dwivedi, P N; Sharma, N S

2014-12-01

Canine parvovirus (CPV) is an enteric pathogen causing hemorrhagic enteritis in pups of 3-6 months of age and is mainly transmitted via feco-oral route. In the present study, a total of 85 animals rectal swabs suspected of CPV were tested using a PCR, nested PCR and a newly designed differential PCR. Using PCR 7 (8.23 %) animals were positive whereas 39 (45.88 %) were positive by using nested PCR and 40 (47.05 %) were positive for either one or more than one antigenic types of CPV using differential PCR. Using differential PCR it was found that CPV-2a and CPV-2b were the most prevailing antigenic types. Also it was found that dogs that were vaccinated too yielded positive CPV indicating a possible presence of additional CPV antigenic types. Thus, the primers used in differential PCR can be used in a single PCR reaction to detect various antigenic types of CPV.

Diagnostic value of nested-PCR for identification of Malassezia species in dandruff

NASA Astrophysics Data System (ADS)

Jusuf, N. K.; Nasution, T. A.; Ullyana, S.

2018-03-01

Dandruff or pityriasis simplex is a condition of abnormal occurrence of formation of yellowish white scales from the scalp. Many factors play a role in the pathogenesis of dandruff, i.e.colonization of Malassezia species. Examination of Malassezia species previously done by culture as the gold standard. However, there are various difficulties in doing the culture. Identification method with anested-polymerase chain reaction (nested-PCR) is expected to provide quickly and easily detected. This study aimedto determine the diagnostic value of nested-PCR in the identification of Malassezia species in dandruff. From 21 subjects, scales from the scalp were taken and sent to the laboratory for nested-PCR identification. Statistical analysis of diagnostic test carried out to determine sensitivity, specificity, positive predictive value, and negative predictive value. The results showed nested-PCR detected 10 sample (47.6%) positive for Malassezia species consist of M. sympodialis (23.8%); M. slooffiae (9.5%); M. furfur (4.8%); M. globosa and M. furfur (4.8%); and M. restricta and M. sympodialis (4.8%). Detection of Malassezia species by nested-PCR has 100% in sensitivity whereas the specificity was 55%. Nested-PCR test has high sensitivity. Therefore nested-PCR may be considered for a faster and simpler alternative examination in identification for Malassezia species in dandruff.

Urinary PCR as an increasingly useful tool for an accurate diagnosis of leptospirosis in livestock.

PubMed

Hamond, C; Martins, G; Loureiro, A P; Pestana, C; Lawson-Ferreira, R; Medeiros, M A; Lilenbaum, W

2014-03-01

The aim of the present study was to consider the wide usage of urinary PCR as an increasingly useful tool for an accurate diagnosis of leptospirosis in livestock. A total of 512 adult animals (300 cattle, 138 horses, 59 goats and 15 pigs), from herds/flocks with reproductive problems in Rio de Janeiro, Brazil was studied by serology and urinary PCR. From the 512 serum samples tested, 223 (43.5 %) were seroreactive (cattle: 45.6 %, horses: 41.3 %, goats: 34%and pigs: 60 %). PCR detected leptospiral DNA in 32.4 % (cattle: 21.6 %, horses: 36.2 %, goats: 77.4 % and pigs: 33.3 %. To our knowledge there is no another study including such a large number of samples (512) from different species, providing a comprehensive analysis of the usage of PCR for detecting leptospiral carriers in livestock. Serological and molecular results were discrepant, regardless the titre, what was an expected outcome. Nevertheless, it is impossible to establish agreement between these tests, since the two methodologies are conducted on different samples (MAT - serum; PCR - urine). Additionally, the MAT is an indirect method and PCR is a direct one. In conclusion, we have demonstrated that urinary PCR should be considered and encouraged as an increasingly useful tool for an accurate diagnosis of leptospirosis in livestock.

Development of a multiplex PCR assay for detection and discrimination of Theileria annulata and Theileria sergenti in cattle.

PubMed

Junlong, Liu; Li, Youquan; Liu, Aihong; Guan, Guiquan; Xie, Junren; Yin, Hong; Luo, Jianxun

2015-07-01

Aim to construct a simple and efficient diagnostic assay for Theileria annulata and Theileria sergenti, a multiplex polymerase chain reaction (PCR) method was developed in this study. Following the alignment of the related sequences, two primer sets were designed specific targeting on T. annulata cytochrome b (COB) gene and T. sergenti internal transcribed spacer (ITS) sequences. It was found that the designed primers could react in one PCR system and generating amplifications of 818 and 393 base pair for T. sergenti and T. annulata, respectively. The standard genomic DNA of both species Theileria was serial tenfold diluted for testing the sensitivity, while specificity test confirmed both primer sets have no cross-reaction with other Theileria and Babesia species. In addition, 378 field samples were used for evaluation of the utility of the multiplex PCR assay for detection of the pathogens infection. The detection results were compared with the other two published PCR methods which targeting on T. annulata COB gene and T. sergenti major piroplasm surface protein (MPSP) gene, respectively. The developed multiplex PCR assay has similar efficient detection with COB and MPSP PCR, which indicates this multiplex PCR may be a valuable assay for the epidemiological studies for T. annulata and T. sergenti.

Qualitative Examination of Children's Naming Skills through Test Adaptations.

ERIC Educational Resources Information Center

Fried-Oken, Melanie

1987-01-01

The Double Administration Naming Technique assists clinicians in obtaining qualitative information about a client's visual confrontation naming skills through administration of a standard naming test; readministration of the same test; identification of single and double errors; cuing for double naming errors; and qualitative analysis of naming…

[Contribution of nested PCR in the diagnosis of imported malaria in southern Algeria].

PubMed

Bouiba, L; Gassen, B; Gasmi, M; Hammadi, D; Harrat, Z

2016-12-01

The nested PCR was used to estimate its inputs in malaria diagnosis and in the performance of the microscope operators involved in the surveillance of malaria in remote areas of South Algeria. For the period 2010 to 2015, 112 patients (93 febrile and 19 asymptomatic) coming from sub-Saharan Africa were tested for malaria in the hospital of Tamanrasset. One part of the blood taken from fingertip was used for blood smears and the second part was absorbed in filter paper for molecular diagnosis. Overall, the infection was detected by nested PCR in 63 samples versus 53 by direct examination. In addition, 11 mixed infections and 6 positive asymptomatic cases not detected by microscopy were diagnosed by PCR. Moreover, two negative samples in nested PCR were tested positive by direct examination. The molecular tool is more sensitive than the direct examination in detecting infra-microscopic parasitaemia and mixed infections...

Virology: The Next Generation from Digital PCR to Single Virion Genomics

DOE Office of Scientific and Technical Information (OSTI.GOV)

White, Richard A.; Brazelton De Cardenas, Jessica N.; Hayden, Randall T.

In the past 25 years, virology has had major technology breakthroughs stemming first from the introduction of nucleic acid amplification testing, but more recently from the use of next-generation sequencing, digital PCR, and the possibility of single virion genomics. These technologies have and will improve diagnosis and disease state monitoring in clinical settings, aid in environmental monitoring, and reveal the vast genetic potential of viruses. Using the principle of limiting dilution, digital PCR amplifies single molecules of DNA in highly partitioned endpoint reactions and reads each of those reactions as either positive or negative based on the presence or absencemore » of target fluorophore. In this review, digital PCR will be highlighted along with current studies, advantages/disadvantages, and future perspectives with regard to digital PCR, viral load testing, and the possibility of single virion genomics.« less

Real-time PCR assay in differentiating Entamoeba histolytica, Entamoeba dispar, and Entamoeba moshkovskii infections in Orang Asli settlements in Malaysia.

PubMed

Lau, Yee Ling; Anthony, Claudia; Fakhrurrazi, Siti Aminah; Ibrahim, Jamaiah; Ithoi, Init; Mahmud, Rohela

2013-08-28

Amebiasis caused by Entamoeba histolytica is the third leading cause of death worldwide. This pathogenic amoeba is morphologically indistinguishable from E. dispar and E. moshkovskii, the non-pathogenic species. Polymerase chain reaction is the current method of choice approved by World Health Organization. Real-time PCR is another attractive molecular method for diagnosis of infectious diseases as post-PCR analyses are eliminated and turnaround times are shorter. The present work aimed to compare the results of Entamoeba species identification using the real-time assay against the established nested PCR method. In this study, a total of 334 human faecal samples were collected from different Orang Asli settlements. Faecal samples were processed by direct wet smear and formalin ethyl acetate concentration methods followed by iodine staining and was microscopically examined for Entamoeba species and other intestinal parasites. Microscopically positive samples were then subject to nested PCR and real-time PCR. The overall prevalence of Entamoeba infection was 19.5% (65/334). SK Posh Piah recorded highest Entamoeba prevalence (63.3%) while Kampung Kemensah had the lowest prevalence (3.7%) of Entamoeba. Microscopically positive samples were then tested by real-time PCR and nested PCR for the presence of Entamoeba histolytica, Entamoeba dispar, and Entamoeba moshkovskii infection. Real-time PCR showed higher Entamoeba detection (86.2%) compared to nested PCR (80%), although the McNemar test value showed no significant difference between the two methods (p = 0.221). This study is the first in Malaysia to report the use of real-time PCR in identifying and differentiating the three Entamoeba infections. It is also proven to be more effective compared to the conventional nested PCR molecular method.

Real-time PCR assay in differentiating Entamoeba histolytica, Entamoeba dispar, and Entamoeba moshkovskii infections in Orang Asli settlements in Malaysia

PubMed Central

2013-01-01

Background Amebiasis caused by Entamoeba histolytica is the third leading cause of death worldwide. This pathogenic amoeba is morphologically indistinguishable from E. dispar and E. moshkovskii, the non-pathogenic species. Polymerase chain reaction is the current method of choice approved by World Health Organization. Real-time PCR is another attractive molecular method for diagnosis of infectious diseases as post-PCR analyses are eliminated and turnaround times are shorter. The present work aimed to compare the results of Entamoeba species identification using the real-time assay against the established nested PCR method. Methods In this study, a total of 334 human faecal samples were collected from different Orang Asli settlements. Faecal samples were processed by direct wet smear and formalin ethyl acetate concentration methods followed by iodine staining and was microscopically examined for Entamoeba species and other intestinal parasites. Microscopically positive samples were then subject to nested PCR and real-time PCR. Results The overall prevalence of Entamoeba infection was 19.5% (65/334). SK Posh Piah recorded highest Entamoeba prevalence (63.3%) while Kampung Kemensah had the lowest prevalence (3.7%) of Entamoeba. Microscopically positive samples were then tested by real-time PCR and nested PCR for the presence of Entamoeba histolytica, Entamoeba dispar, and Entamoeba moshkovskii infection. Real-time PCR showed higher Entamoeba detection (86.2%) compared to nested PCR (80%), although the McNemar test value showed no significant difference between the two methods (p = 0.221). Conclusions This study is the first in Malaysia to report the use of real-time PCR in identifying and differentiating the three Entamoeba infections. It is also proven to be more effective compared to the conventional nested PCR molecular method. PMID:23985047

Application of real-time PCR and melting curve analysis in rapid Diego blood group genotyping.

PubMed

Novaretti, M C Z; Ruiz, A S; Dorlhiac-Llacer, P E; Chamone, D A F

2010-01-01

The paucity of appropriate reagents for serologic typing of the Diego blood group antigens has prompted the development of a real-time PCR and melting curve analysis for Diego blood group genotyping. In this study, we phenotyped 4326 donor blood samples for Di(a) using semiautomated equipment. All 157 Di(a+) samples were then genotyped by PCR using sequence-specific primers (PCR-SSP) for DI*02 because of anti-Di(b) scarcity. Of the 4326 samples, we simultaneously tested 160 samples for Di(a) and Di(b) serology, and DI*01 and DI*02 by PCR-SSP and by real-time PCR. We used the same primers for Diego genotyping by real-time PCR and PCR-SSP. Melting curve profiles obtained using the dissociation software of the real-time PCR apparatus enabled the discrimination of Diego alleles. Of the total samples tested, 4169 blood donors, 96.4 percent (95% confidence interval [CI], 95.8-96.9%), were homozygous for DI*02 and 157, 3.6 percent (95% CI, 3.1%-4.2%), were heterozygous DI*01/02. No blood donor was found to be homozygous for DI*01 in this study. The calculated DI*01 and DI*02 allele frequencies were 0.0181 (95% CI, 0.0173-0.0189) and 0.9819 (95% CI, 0.9791-0.9847), respectively, showing a good fit for the Hardy-Weinberg equilibrium. There was full concordance among Diego phenotype results by PCR-SSP and real-time PCR. DI*01 and DI*02 allele determination with SYBR Green I and thermal cycler technology are useful methods for Diego determination. The real-time PCR with SYBR Green I melting temperature protocol can be used as a rapid screening tool for DI*01 and DI*02 blood group genotyping.

Highly Sensitive Assay for Detection of Enterovirus in Clinical Specimens by Reverse Transcription-PCR with an Armored RNA Internal Control

PubMed Central

Beld, Marcel; Minnaar, René; Weel, Jan; Sol, Cees; Damen, Marjolein; van der Avoort, Harry; Wertheim-van Dillen, Pauline; Breda, Alex van; Boom, René

2004-01-01

The objective of the present study was the development of a diagnostic reverse transcription (RT)-PCR for the specific detection of enterovirus (EV) RNA in clinical specimens controlled by an internal control (IC) RNA. The IC RNA contains the same primer binding sites as EV RNA but has a different probe region. The IC RNA was packaged into an MS2 phage core particle (armored) and was added to the clinical sample to allow monitoring of both extraction efficiency and RT-PCR efficiency. Serial dilutions of the IC RNA were made, and the detection limit of the RT-PCR was tested in a background of EV RNA-negative cerebrospinal fluid. The sensitivity and specificity of the RT-PCR assay were tested by using all 64 known EV serotypes, several non-EV serotypes, and two Quality Control for Molecular Diagnostics (QCMD) Program EV proficiency panels from 2001 and 2002. In total, 322 clinical specimens were tested by RT-PCR, and to establish the clinical utility of the RT-PCR, a comparison of the results of viral culture and RT-PCR was done with 87 clinical specimens. The lower limit of sensitivity was reached at about 150 copies of IC RNA/ml. All 64 EV serotypes were positive, while all non-EV serotypes were negative. All culture-positive samples of the 2001 QCMD proficiency panel (according to the 50% tissue culture infective doses per milliliter) were positive by RT-PCR. Invalid results, i.e., negativity for both EV RNA and IC RNA, due to inhibition of RT-PCR were observed for 33.3% of the members of the 2002 QCMD proficiency panel and 3.1% of the clinical specimens. Inhibition of RT-PCR could be relieved by the addition of 400 ng of bovine α-casein per μl to both the RT reaction mixture and the PCR mixture. With this optimized protocol, the results for all samples of the 2002 QCMD proficiency panel and all clinical specimens except one fecal sample (0.3%) were valid. Evaluation of the clinical samples demonstrated that EV infection could be detected in 12 of 87 samples (13.8%) by RT-PCR, while viral culture was negative. Our data show that the RT-PCR with armored IC RNA offers a very reliable and rapid diagnostic tool for the detection of EV in clinical specimens and that the addition of bovine α-casein relieved inhibition of the RT-PCR for 99.7% of clinical specimens. PMID:15243060

[Contamination with genetically modified maize MON863 of processed foods on the market].

PubMed

Ohgiya, Yoko; Sakai, Masaaki; Miyashita, Taeko; Yano, Koichi

2009-06-01

Genetically modified maize MON863 (MON863), which has passed a safety examination in Japan, is commercially cultivated in the United States as a food and a resource for fuel. Maize is an anemophilous flower, which easily hybridizes. However, an official method for quantifying the content of MON863 has not been provided yet in Japan. We here examined MON863 contamination in maize-processed foods that had no labeling indicating of the use of genetically modified maize.From March 2006 to July 2008, we purchased 20 frozen maize products, 8 maize powder products, 7 canned maize products and 4 other maize processed foods. Three primer pairs named MON 863 primer, MON863-1, and M3/M4 for MON863-specific integrated cassette were used for qualitative polymerase chain reaction (PCR). A primer pair "SSIIb-3" for starch synthase gene was used to confirm the quality of extracted DNA. The starch synthase gene was detected in all samples. In qualitative tests, the MON863-specific fragments were detected in 7 (18%) maize powder products out of the 39 processed foods with all the three primer pairs.We concluded that various maize processed foods on the market were contaminated with MON863. It is important to accumulate further information on MON863 contamination in maize-processed foods that have no label indication of the use of genetically modified maize.

Development of an In-House Multiplex Nested RT-PCR Method for Detecting Acute HIV-1 Infection in High Risk Populations.

PubMed

Liu, Zhiying; Li, Wei; Xu, Meng; Sheng, Bo; Yang, Zixuan; Jiao, Yanmei; Zhang, Tong; Mou, Danlei; Chen, Dexi; Wu, Hao

2015-01-01

The detection of acute HIV infection (AHI) among high risk populations can help reduce secondary transmission of HIV. The nucleic acid testing (NAT) can shorten the test window period by up to 7-12 days. In this study, we describe an in-house NAT based on the multiplex nested RT-PCR method to detect the HIV RNA. We also evaluated it in a high risk cohort in Beijing. Four primer pairs were designed and evaluated for the detection of different HIV-1 subtypes in group M. Multiplex RT-PCR and nested PCR were performed. The sensitivity, specialty, primers compatibility among HIV subtypes were evaluated simultaneously. In an MSM cohort in Beijing during a 3-year period, a total of 11,808 blood samples that were negative by ELISA or indeterminate by Western blot were analyzed by this multiplex nested RT-PCR with pooling strategy. The multiplex nested RT-PCR was successfully applied for the detection of at least six HIV-1 subtypes. The sensitivity was 40 copies/ml and the specificity was 100%. A total of 29 people were tested HIV-1 positive with acute infection in a MSM cohort of Beijing during a 3 years period. This multiplex nested RT-PCR provides a useful tool for the rapid detection of acute HIV-1 infection. When used in combination with the 3(rd) generation ELISA, it can improve the detection rate of HIV infection, especially in the source limited regions.

The agony of choice in dermatophyte diagnostics-performance of different molecular tests and culture in the detection of Trichophyton rubrum and Trichophyton interdigitale.

PubMed

Kupsch, C; Ohst, T; Pankewitz, F; Nenoff, P; Uhrlaß, S; Winter, I; Gräser, Y

2016-08-01

Dermatophytosis caused by dermatophytes of the genera Trichophyton and Microsporum belong to the most frequent mycoses worldwide. Molecular detection methods proved to be highly sensitive and enable rapid and accurate detection of dermatophyte species from clinical specimens. For the first time, we compare the performance of different molecular methods with each other and with conventional diagnostics in the detection of dermatophytoses caused by Trichophyton rubrum and Trichophyton interdigitale in clinical specimens (nail, skin and hair). The compared molecular methods comprise two already published PCR-ELISAs, a published quantitative RT-PCR as well as a newly developed PCR-ELISA targeting the internal transcribed spacer region. We investigated the sensitivity of the assays by analysing 375 clinical samples. In 148 specimens (39.5%) a positive result was gained in at least one of the four molecular tests or by culture, but the number of detected agents differed significantly between some of the assays. The most sensitive assay, a PCR-ELISA targeting a microsatellite region, detected 81 T. rubrum infections followed by an internal transcribed spacer PCR-ELISA (60), quantitative RT-PCR (52) and a topoisomerase II PCR-ELISA (51), whereas cultivation resulted in T. rubrum identification in 37 samples. The pros and cons of all four tests in routine diagnostics are discussed. Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

A multiplex PCR method for the identification of commercially important salmon and trout species (Oncorhynchus and Salmo) in North America.

PubMed

Rasmussen Hellberg, Rosalee S; Morrissey, Michael T; Hanner, Robert H

2010-09-01

The purpose of this study was to develop a species-specific multiplex polymerase chain reaction (PCR) method that allows for the detection of salmon species substitution on the commercial market. Species-specific primers and TaqMan® probes were developed based on a comprehensive collection of mitochondrial 5' cytochrome c oxidase subunit I (COI) deoxyribonucleic acid (DNA) "barcode" sequences. Primers and probes were combined into multiplex assays and tested for specificity against 112 reference samples representing 25 species. Sensitivity and linearity tests were conducted using 10-fold serial dilutions of target DNA (single-species samples) and DNA admixtures containing the target species at levels of 10%, 1.0%, and 0.1% mixed with a secondary species. The specificity tests showed positive signals for the target DNA in both real-time and conventional PCR systems. Nonspecific amplification in both systems was minimal; however, false positives were detected at low levels (1.2% to 8.3%) in conventional PCR. Detection levels were similar for admixtures and single-species samples based on a 30 PCR cycle cut-off, with limits of 0.25 to 2.5 ng (1% to 10%) in conventional PCR and 0.05 to 5.0 ng (0.1% to 10%) in real-time PCR. A small-scale test with food samples showed promising results, with species identification possible even in heavily processed food items. Overall, this study presents a rapid, specific, and sensitive method for salmon species identification that can be applied to mixed-species and heavily processed samples in either conventional or real-time PCR formats. This study provides a newly developed method for salmon and trout species identification that will assist both industry and regulatory agencies in the detection and prevention of species substitution. This multiplex PCR method allows for rapid, high-throughput species identification even in heavily processed and mixed-species samples. An inter-laboratory study is currently being carried out to assess the ability of this method to identify species in a variety of commercial salmon and trout products.

Diagnosis of Helicobacter pylori: What should be the gold standard?

PubMed Central

Patel, Saurabh Kumar; Pratap, Chandra Bhan; Jain, Ashok Kumar; Gulati, Anil Kumar; Nath, Gopal

2014-01-01

Since the discovery of Helicobacter pylori (H. pylori) in 1983, numerous detection methods for the presence of the bacterium have been developed. Each one of them has been associated with advantages and disadvantages. Noninvasive tests such as serology, 13C urea breath test (UBT) and stool antigen tests are usually preferred by the clinicians. Serology has its own limitation especially in endemic areas while 13C UBT is technically very demanding. The stool antigen detection method, although specific, is usually associated with poor sensitivity. The 13C UBT is believed to be specific, but with present revelation of the fact that stomach is colonized by many other urease producing bacteria makes it questionable. Histology, culture, rapid urease test and polymerase chain reaction (PCR) are the tests which are carried out on antral biopsies collected by invasive means. Histology has been proposed to be very sensitive and specific but the question is how by simply looking the morphology of the bacteria in the microscope, one can claim that the curved bacterium is exclusively H. pylori. Rapid urease test (RUT), the doctor’s test, is also challenged because the presence of other urease producing bacteria in the stomach cannot be denied. Moreover, RUT has been reported with poor sensitivity specially, when density of the bacterium is low. Isolation of H. pylori is essential to investigate its growth requirements, antibiotic susceptibility testing, studying virulence factor to develop vaccine and many more explorations. It has also got several disadvantages i.e., special condition for transporting, media, incubation and few days waiting for the colonies to appear, apart from the speed essentially needed to process the specimens. Till date, majority of the microbiological laboratories in the world are not equipped and trained to isolate such fastidious bacterium. The option left is PCR methods to detect H. pylori’s DNA in gastric mucosa, gastric juice, saliva, dental plaques and environmental specimens. There are speculations for false positivity due to detection of non-pylori Helicobacters due to genetic sharing; and false negativity due to low bacterial counts and presence of PCR inhibitors. However, specimen collection, transportation and processing do not require speed and special conditions. PCR based diagnosis may be considered as gold standard by designing primers extremely specific to H. pylori and targeting at least more than one conserved genes. Similarly specificity of PCR may be improved by use of internal Primers. Further, nested PCR will take care of false negatives by countering the effect of PCR inhibitors and low bacterial counts. Therefore, nested PCR based methods if performed properly, may be proposed as gold standard test. PMID:25278682

African Swine Fever Diagnosis Adapted to Tropical Conditions by the Use of Dried-blood Filter Papers.

PubMed

Randriamparany, T; Kouakou, K V; Michaud, V; Fernández-Pinero, J; Gallardo, C; Le Potier, M-F; Rabenarivahiny, R; Couacy-Hymann, E; Raherimandimby, M; Albina, E

2016-08-01

The performance of Whatman 3-MM filter papers for the collection, drying, shipment and long-term storage of blood at ambient temperature, and for the detection of African swine fever virus and antibodies was assessed. Conventional and real-time PCR, viral isolation and antibody detection by ELISA were performed on paired samples (blood/tissue versus dried-blood 3-MM filter papers) collected from experimentally infected pigs and from farm pigs in Madagascar and Côte d'Ivoire. 3-MM filter papers were used directly in the conventional and real-time PCR without previous extraction of nucleic acids. Tests that performed better with 3-MM filter papers were in descending order: virus isolation, real-time UPL PCR and conventional PCR. The analytical sensitivity of real-time UPL PCR on filter papers was similar to conventional testing (virus isolation or conventional PCR) on organs or blood. In addition, blood-dried filter papers were tested in ELISA for antibody detection and the observed sensitivity was very close to conventional detection on serum samples and gave comparable results. Filter papers were stored up to 9 months at 20-25°C and for 2 months at 37°C without significant loss of sensitivity for virus genome detection. All tests on 3-MM filter papers had 100% specificity compared to the gold standards. Whatman 3-MM filter papers have the advantage of being cheap and of preserving virus viability for future virus isolation and characterization. In this study, Whatman 3-MM filter papers proved to be a suitable support for the collection, storage and use of blood in remote areas of tropical countries without the need for a cold chain and thus provide new possibilities for antibody testing and virus isolation. © 2014 Blackwell Verlag GmbH.

Detection of Mycobacterium bovis in formalin-fixed, paraffin-embedded tissues of cattle and elk by PCR amplification of an IS6110 sequence specific for Mycobacterium tuberculosis complex organisms.

PubMed

Miller, J; Jenny, A; Rhyan, J; Saari, D; Suarez, D

1997-07-01

A presumptive diagnosis of tuberculosis can be made if a tissue has characteristic histopathologic changes and acid-fast organisms. However, definitive diagnosis requires culture and species identification of the causative mycobacterium, a process that takes several weeks to complete. The purpose of work reported here was to determine if formalin-fixed, paraffin-embedded tissues could be tested by polymerase chain reaction (PCR) to provide a more rapid diagnosis of tuberculosis. Nondecalcified tissues from cases of tuberculosis in cattle and elk (Cervus elaphus) were examined. The primers used for PCR amplified a 123-bp fragment of IS6110, an insertion sequence that is specific for organisms in the Mycobacterium tuberculosis complex (M. tuberculosis, M. bovis, M. microti, M. africanum). The PCR test detected this sequence in tissues from 92 of 99 (93%) tuberculosis cases, including 3 of 4 elk. In 80 tissues, the positive results were obtained using material prepared by immersion of paraffin sections in water containing a detergent, followed by alternating boil/freeze cycles. The remaining positive results were obtained with DNA isolated from the crude tissue extracts by proteinase K digestion and phenol/chloroform purification. Accuracy of the IS6110 PCR test was demonstrated by negative test results on 31 tissues that had either nonmycobacterial granulomas or granulomatous lesions caused by other mycobacteria (M. paratuberculosis or M. avium). The findings of this study show that a PCR test usually can provide a rapid diagnosis of tuberculosis when it is applied to paraffin sections that have characteristic lesions and acid-fast organisms.

Diagnostic Yield of Laboratory Methods and Value of Viral Genotyping during an Outbreak of Mumps in a Partially Vaccinated Population in British Columbia, Canada.

PubMed

Nunn, Alexandra; Masud, Shazia; Krajden, Mel; Naus, Monika; Jassem, Agatha N

2018-05-01

Mumps remains endemic in North America despite routine use of the measles, mumps, and rubella (MMR) vaccine. In 2016, an outbreak of mumps in British Columbia, Canada, provided an opportunity to determine the diagnostic utility of laboratory testing methods. Specimens from patients with clinical mumps were tested for infection using a commercial enzyme-linked immunosorbent assay (ELISA) for antibody detection and an in-house reverse transcriptase PCR (RT-PCR) targeting viral fusion and small hydrophobic (SH) genes. Viral genotyping was performed by SH gene sequencing. Laboratory data was linked with epidemiologic case data. Of the 139 confirmed cases, 94 (68%) had reported or documented history of MMR vaccination. Specimens were typically collected 1 day (for buccal and IgM tests) or 2 days (for urine tests) after symptom onset. Most confirmed cases (69%) were confirmed by buccal swab RT-PCR. Among cases tested by multiple methods, the percent positivity for buccal swab RT-PCR was 90% (96/107) compared to 43% (30/69) for both IgM ELISA and urine RT-PCR. Mumps IgM detection was higher in confirmed cases with no history of vaccination than in those with history (64% versus 34%, P = 0.02). The outbreak strain was identified as genotype G related to MuVi/Sheffield.GBR/1.05 but with conserved variations in five nucleotides within the SH gene that allowed linkage of geographically distinct cases. In conclusion, RT-PCR of buccal specimens had the highest diagnostic yield during a mumps outbreak in a partially vaccinated population. To optimize mumps diagnostic potential, clinicians should collect specimens depending on when the patient presents for care and their immunization history. © Crown copyright 2018.

Validation of Geno-Sen's Scrub Typhus Real Time Polymerase Chain Reaction Kit by its Comparison with a Serological ELISA Test

PubMed Central

Anitharaj, Velmurugan; Stephen, Selvaraj; Pradeep, Jothimani; Pooja, Pratheesh; Preethi, Sridharan

2017-01-01

Background: In the recent past, scrub typhus (ST) has been reported from different parts of India, based on Weil-Felix/enzyme-linked immunosorbent assay (ELISA)/indirect immunofluorescence assay (IFA). Molecular tests are applied only by a few researchers. Aims: Evaluation of a new commercial real time polymerase chain reaction (PCR) kit for molecular diagnosis of ST by comparing it with the commonly used IgM ELISA is our aim. Settings and Design: ST has been reported all over India including Puducherry and surrounding Tamil Nadu and identified as endemic for ST. This study was designed to correlate antibody detection by IgM ELISA and Orientia tsutsugamushi DNA in real time PCR. Materials and Methods: ST IgM ELISA (InBios Inc., USA) was carried out for 170 consecutive patients who presented with the symptoms of acute ST during 11 months (November, 2015– September, 2016). All 77 of these patients with IgM ELISA positivity and 49 of 93 IgM ELISA negative patients were subjected to real time PCR (Geno-Sen's ST real time PCR, Himachal Pradesh, India). Statistical Analysis: Statistical analysis for clinical and laboratory results was performed using IBM SPSS Statistics 17 for Windows (SPSS Inc., Chicago, USA). Chi-square test with Yates correction (Fisher's test) was employed for a small number of samples. Results and Conclusion: Among 77 suspected cases of acute ST with IgM ELISA positivity and 49 IgM negative patients, 42 and 7 were positive, respectively, for O. tsutsugamushi 56-kDa type-specific gene in real time PCR kit. Until ST IFA, the gold standard diagnostic test, is properly validated in India, diagnosis of acute ST will depend on both ELISA and quantitative PCR. PMID:28878522

Detection of Japanese Encephalitis Virus RNA in Human Throat Samples in Laos - A Pilot study.

PubMed

Bharucha, Tehmina; Sengvilaipaseuth, Onanong; Seephonelee, Malee; Vongsouvath, Malavanh; Vongsouvath, Manivanh; Rattanavong, Sayaphet; Piorkowski, Géraldine; Lecuit, Marc; Gorman, Christopher; Pommier, Jean-David; Newton, Paul N; de Lamballerie, Xavier; Dubot-Pérès, Audrey

2018-05-22

Japanese encephalitis virus (JEV) is the most commonly identified cause of acute encephalitis syndrome (AES) in Asia. The WHO recommended test is anti-JEV IgM-antibody-capture-enzyme-linked-immunosorbent-assay (JEV MAC-ELISA). However, data suggest this has low positive predictive value, with false positives related to other Flavivirus infections and vaccination. JEV RT-PCR in cerebrospinal fluid (CSF) and/or serum is highly specific, but is rarely positive; 0-25% of patients that fulfil the WHO definition of JE (clinical Acute Encephalitis Syndrome (AES) and JEV MAC-ELISA positive). Testing other body fluids by JEV RT-qPCR may improve the diagnosis. As a pilot study thirty patients admitted to Mahosot Hospital 2014-2017, recruited to the South-East-Asia-Encephalitis study, were tested by JEV MAC-ELISA and two JEV real-time RT-PCR (RT-qPCR) assays (NS2A and NS3). Eleven (36.7%) were JEV MAC-ELISA positive. Available CSF and serum samples of these patients were JEV RT-qPCR negative but 2 (7%) had JEV RNA detected in their throat swabs. JEV RNA was confirmed by re-testing, and sequencing of RT-qPCR products. As the first apparent report of JEV RNA detection in human throat samples, the provides new perspectives on human JEV infection, potentially informing improving JEV detection. We suggest that testing patients' throat swabs for JEV RNA is performed, in combination with molecular and serological CSF and serum investigations, on a larger scale to investigate the epidemiology of the presence of JEV in human throats. Throat swabs are an easy and non-invasive tool that could be rolled out to a wider population to improve knowledge of JEV molecular epidemiology.