Diagnostic accuracy of tuberculous lymphadenitis fine needle aspiration biopsy confirmed by PCR as gold standard

NASA Astrophysics Data System (ADS)

DSuryadi; Delyuzar; Soekimin

2018-03-01

Indonesia is the second country with the TB (tuberculosis) burden in the world. Improvement in controlling TB and reducing the complications can accelerate early diagnosis and correct treatment. PCR test is a gold standard. However, it is quite expensive for routine diagnosis. Therefore, an accurate and cheaper diagnostic method such as fine needle aspiration biopsy is needed. The study aimsto determine the accuracy of fine needle aspiration biopsy cytology in the diagnosis of tuberculous lymphadenitis. A cross-sectional analytic study was conducted to the samples from patients suspected with tuberculous lymphadenitis. The fine needle aspiration biopsy (FNAB)test was performed and confirmed by PCR test.There is a comparison to the sensitivity, specificity, accuracy, positive predictive value and negative predictive value of both methods. Sensitivity (92.50%), specificity (96.49%), accuracy (94.85%), positive predictive value (94.87%) and negative predictive value (94.83%) were in FNAB test compared to gold standard. We concluded that fine needle aspiration biopsy is a recommendation for a cheaper and accurate diagnostic test for tuberculous lymphadenitis diagnosis.

Diagnostic performance of swab PCR as an alternative to tissue culture methods for diagnosing infections associated with fracture fixation devices.

PubMed

Omar, Mohamed; Suero, Eduardo M; Liodakis, Emmanouil; Reichling, Moritz; Guenther, Daniel; Decker, Sebastian; Stiesch, Meike; Krettek, Christian; Eberhard, Jörg

2016-07-01

Molecular procedures could potentially improve diagnoses of orthopaedic implant-related infections, but are not yet clinically implemented. Analysis of sonication fluid shows the highest sensitivity for diagnosing implant infections in cases of revision surgery with implant removal. However, there remains controversy regarding the best method for obtaining specimens in cases of revision surgery with implant retention. Tissue culture is the most common diagnostic method for pathogen identification in such cases. Here we aimed to assess the diagnostic performance of swab PCR analysis compared to tissue culture from patients undergoing revision surgery of fracture fixation devices. We prospectively investigated 62 consecutive subjects who underwent revision surgery of fracture fixation devices during a two-year period. Tissue samples were collected for cultures, and swabs from the implant surface were obtained for 16S rRNA PCR analysis. Subjects were classified as having an implant-related infection if (1) they presented with a sinus tract or open wound in communication with the implant; or (2) purulence was encountered intraoperatively; or (3) two out of three tissue cultures tested positive for the presence of the same pathogen. Tissue culture and swab PCR results from the subjects were used to calculate the sensitivity, specificity, accuracy, positive predictive value (PPV), negative predictive value (NPV), and area under the ROC curve (AUC) for identifying an orthopaedic implant-related infection. Orthopaedic implant-related infections were detected in 51 subjects. Tissue culture identified infections in 47 cases, and swab PCR in 35 cases. Among the 11 aseptic cases, tissue culture was positive in 2 cases and swab PCR in 4 cases. Tissue culture showed a significantly higher area under the ROC curve for diagnosing infection (AUC=0.89; 95% CI, 0.67-0.96) compared to swab PCR (AUC=0.66; 95% CI, 0.46-0.80) (p=0.033). Compared to swab PCR, tissue culture showed better

Performance of a real-time PCR assay in routine bovine mastitis diagnostics compared with in-depth conventional culture.

PubMed

Hiitiö, Heidi; Riva, Rauna; Autio, Tiina; Pohjanvirta, Tarja; Holopainen, Jani; Pyörälä, Satu; Pelkonen, Sinikka

2015-05-01

Reliable identification of the aetiological agent is crucial in mastitis diagnostics. Real-time PCR is a fast, automated tool for detecting the most common udder pathogens directly from milk. In this study aseptically taken quarter milk samples were analysed with a real-time PCR assay (Thermo Scientific PathoProof Mastitis Complete-12 Kit, Thermo Fisher Scientific Ltd.) and by semi-quantitative, in-depth bacteriological culture (BC). The aim of the study was to evaluate the diagnostic performance of the real-time PCR assay in routine use. A total of 294 quarter milk samples from routine mastitis cases were cultured in the national reference laboratory of Finland and examined with real-time PCR. With BC, 251 out of 294 (85.7%) of the milk samples had at least one colony on the plate and 38 samples were considered contaminated. In the PCR mastitis assay, DNA of target species was amplified in 244 samples out of 294 (83.0%). The most common bacterial species detected in the samples, irrespective of the diagnostic method, was the coagulase negative staphylococci (CNS) group (later referred as Staphylococcus spp.) followed by Staphylococcus aureus. Sensitivity (Se) and specificity (Sp) for the PCR assay to provide a positive Staph. aureus result was 97.0 and 95.8% compared with BC. For Staphylococcus spp., the corresponding figures were 86.7 and 75.4%. Our results imply that PCR performed well as a diagnostic tool to detect Staph. aureus but may be too nonspecific for Staphylococcus spp. in routine use with the current cut-off Ct value (37.0). Using PCR as the only microbiological method for mastitis diagnostics, clinical relevance of the results should be carefully considered before further decisions, for instance antimicrobial treatment, especially when minor pathogens with low amount of DNA have been detected. Introducing the concept of contaminated samples should also be considered.

Use of PCR in resolving diagnostic difficulties potentially caused by genetic variation of hepatitis B virus.

PubMed Central

van Deursen, F J; Hino, K; Wyatt, D; Molyneaux, P; Yates, P; Wallace, L A; Dow, B C; Carman, W F

1998-01-01

AIMS: To assess the relevance of genetic variants of hepatitis B virus (HBV) and to demonstrate the usefulness of the polymerase chain reaction (PCR) in cases of HBV diagnostic difficulty. METHODS: Five serum samples from patients that presented diagnostic difficulty in routine laboratories were sent to a research laboratory for PCR, and if appropriate, S gene sequencing, in vitro expression, and antigenic analysis. RESULTS: The demonstration of HBV in serum by PCR allowed a definitive diagnosis of current infection. One serum sample with poor reactivity in a diagnostic assay had a minor hepatitis B surface antigen (HBsAg) variant and another with very poor reactivity had multiple variants of HBsAg. Transient HBsAg reactivity was observed in a recently vaccinated patient. A hepatitis Be antigen (HBeAg) false positive reaction was noted in a patient from a well defined risk group for HBV. One patient who was strongly HBsAg/HBeAg positive, but anti-hepatitis B core antibody negative, was viraemic. CONCLUSIONS: PCR may become the gold standard for the diagnosis of current HBV infection. HBV variants are responsible for a proportion of diagnostically difficult cases. Modification of commercial assays is necessary to increase the sensitivity of detection of such variants. PMID:9602690

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.

Improved Specificity for Detection of Mycobacterium bovis in Fresh Tissues Using IS6110 Real-time PCR

USDA-ARS?s Scientific Manuscript database

Background: Culture of M. bovis from diagnostic specimens is the gold standard for bovine tuberculosis diagnostics in the US. Detection of M. bovis by PCR in tissue homogenates may provide a simple, rapid method to complement diagnostic culture. A significant impediment to PCR based assays on tissue...

A point-of-care diagnostic system to influenza viruses using chip-based ultra-fast PCR.

PubMed

Kwon, Soon-Hwan; Lee, Sujin; Jang, Jeyoun; Seo, Yujin; Lim, Hee-Young

2018-06-01

In order to diagnose the infectious disease from clinical samples, the various protocols such as culturing microorganism, rapid diagnostic test using chromatographic method, ELISA, conventional PCR are developed. Since a novel strain of avian influenza can be cross-infected human as well as birds and livestock due to genetic reassortment, some strains of influenza such as H7N9 and H5N1 have emerged as a severe virus which can be threaten the health of poultry as well as human. Therefore, we explored the development of simultaneously and rapid diagnostic tool for seasonal influenza (A/H1N1, A/H3N2, B) and highly pathogenic avian influenza (A/H5N1, A/H7N9). We analyzed the unique nucleotide sequences of influenza types including three seasonal influenza, A/H7N9, and A/H5N1, and distinguished each type of influenza and diagnosed through One Step RT-PCR. In the results, Chip-based PCR technique can be diagnosed rapidly and directly from naked eye with EvaGreen the influenza also respiratory specimens within 23 min 15 s, including reverse transcription. The Chip-based PCR is a point-of-care system, and it is expected to reduce diagnosis time and to develop a diagnostic kit. Furthermore the Chip based PCR technique can be used for high risk pathogen in bioterror and/or biological warfare in the field. © 2018 Wiley Periodicals, Inc.

Lyme Borreliosis--the Utility of Improved Real-Time PCR Assay in the Detection of Borrelia burgdorferi Infections.

PubMed

Bil-Lula, Iwona; Matuszek, Patryk; Pfeiffer, Thomas; Woźniak, Mieczysław

2015-01-01

Infections of Borrelia burgdorferi sensu lato reveal clinical manifestations affecting numerous organs and tissues. The standard diagnostic procedure of these infections is quite simple if a positive history of tick exposure or typical erythema migrans appears. Lack of unequivocal clinical symptoms creates the necessity for further evaluation with laboratory tests. This study discusses the utility of a novel, improved, well-optimized, sensitive and highly specific quantitative real-time PCR assay for the diagnostics of infections caused by Borrelia burgdorferi sensu lato. We designed an improved, specific, highly sensitive real-time quantitative polymerase chain reaction (RQ-PCR) assay for the detection and quantification of all Borrelia burgdorferi genotypes. A wide validation effort was undertaken to ensure confidence in the highly sensitive and specific detection of B. burgdorferi. Due to high sensitivity and great specificity, as low as 1.6×10² copies of Borrelia per mL of whole blood could be detected. As much as 12 (3%) negative ELISA IgM results, 14 (2.8%) negative results of Line blot IgM, 11 (3.1%) and 7 (2.7%) of negative ELISA IgG and Line blot IgG results, respectively, were positive in real-time PCR. The data in this study confirms the high positive predictive value of real-time PCR test in the detection of Borrelia infections.

Comparison of microfluidic digital PCR and conventional quantitative PCR for measuring copy number variation.

PubMed

Whale, Alexandra S; Huggett, Jim F; Cowen, Simon; Speirs, Valerie; Shaw, Jacqui; Ellison, Stephen; Foy, Carole A; Scott, Daniel J

2012-06-01

One of the benefits of Digital PCR (dPCR) is the potential for unparalleled precision enabling smaller fold change measurements. An example of an assessment that could benefit from such improved precision is the measurement of tumour-associated copy number variation (CNV) in the cell free DNA (cfDNA) fraction of patient blood plasma. To investigate the potential precision of dPCR and compare it with the established technique of quantitative PCR (qPCR), we used breast cancer cell lines to investigate HER2 gene amplification and modelled a range of different CNVs. We showed that, with equal experimental replication, dPCR could measure a smaller CNV than qPCR. As dPCR precision is directly dependent upon both the number of replicate measurements and the template concentration, we also developed a method to assist the design of dPCR experiments for measuring CNV. Using an existing model (based on Poisson and binomial distributions) to derive an expression for the variance inherent in dPCR, we produced a power calculation to define the experimental size required to reliably detect a given fold change at a given template concentration. This work will facilitate any future translation of dPCR to key diagnostic applications, such as cancer diagnostics and analysis of cfDNA.

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

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

Rapid and sensitive detection of Yersinia pestis using amplification of plague diagnostic bacteriophages monitored by real-time PCR.

PubMed

Sergueev, Kirill V; He, Yunxiu; Borschel, Richard H; Nikolich, Mikeljon P; Filippov, Andrey A

2010-06-28

Yersinia pestis, the agent of plague, has caused many millions of human deaths and still poses a serious threat to global public health. Timely and reliable detection of such a dangerous pathogen is of critical importance. Lysis by specific bacteriophages remains an essential method of Y. pestis detection and plague diagnostics. The objective of this work was to develop an alternative to conventional phage lysis tests--a rapid and highly sensitive method of indirect detection of live Y. pestis cells based on quantitative real-time PCR (qPCR) monitoring of amplification of reporter Y. pestis-specific bacteriophages. Plague diagnostic phages phiA1122 and L-413C were shown to be highly effective diagnostic tools for the detection and identification of Y. pestis by using qPCR with primers specific for phage DNA. The template DNA extraction step that usually precedes qPCR was omitted. phiA1122-specific qPCR enabled the detection of an initial bacterial concentration of 10(3) CFU/ml (equivalent to as few as one Y. pestis cell per 1-microl sample) in four hours. L-413C-mediated detection of Y. pestis was less sensitive (up to 100 bacteria per sample) but more specific, and thus we propose parallel qPCR for the two phages as a rapid and reliable method of Y. pestis identification. Importantly, phiA1122 propagated in simulated clinical blood specimens containing EDTA and its titer rise was detected by both a standard plating test and qPCR. Thus, we developed a novel assay for detection and identification of Y. pestis using amplification of specific phages monitored by qPCR. The method is simple, rapid, highly sensitive, and specific and allows the detection of only live bacteria.

Schistosoma real-time PCR as diagnostic tool for international travellers and migrants.

PubMed

Cnops, Lieselotte; Tannich, Egbert; Polman, Katja; Clerinx, Jan; Van Esbroeck, Marjan

2012-10-01

To evaluate the use of a genus-specific PCR that combines high sensitivity with the detection of different Schistosoma species for diagnosis in international travellers and migrants in comparison to standard microscopy. The genus-specific real-time PCR was developed to target the 28S ribosomal RNA gene of the major human Schistosoma species. It was validated for analytical specificity and reproducibility and demonstrated an analytical sensitivity of 0.2 eggs per gram of faeces. Its diagnostic performance was further evaluated on 152 faecal, 32 urine and 38 serum samples from patients presenting at the outpatient clinic of the Institute of Tropical Medicine in Antwerp (Belgium). We detected Schistosoma DNA in 76 faecal (50.0%) and five urine (15.6%) samples of which, respectively, nine and one were not detected by standard microscopy. Only two of the 38 serum samples of patients with confirmed schistosomiasis were positive with the presently developed PCR. Sequence analysis on positive faecal samples allowed identification of the Schistosoma species complex. The real-time PCR is highly sensitive and may offer added value in diagnosing imported schistosomiasis. The genus-specific PCR can detect all schistosome species that are infectious to humans and performs very well with faeces and urine, but not in serum. © 2012 Blackwell Publishing Ltd.

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.

Preanalytical blood sample workup for cell-free DNA analysis using Droplet Digital PCR for future molecular cancer diagnostics.

PubMed

van Ginkel, Joost H; van den Broek, Daan A; van Kuik, Joyce; Linders, Dorothé; de Weger, Roel; Willems, Stefan M; Huibers, Manon M H

2017-10-01

In current molecular cancer diagnostics, using blood samples of cancer patients for the detection of genetic alterations in plasma (cell-free) circulating tumor DNA (ctDNA) is an emerging practice. Since ctDNA levels in blood are low, highly sensitive Droplet Digital PCR (ddPCR) can be used for detecting rare mutational targets. In order to perform ddPCR on blood samples, a standardized procedure for processing and analyzing blood samples is necessary to facilitate implementation into clinical practice. Therefore, we assessed the technical sample workup procedure for ddPCR on blood plasma samples. Blood samples from healthy individuals, as well as lung cancer patients were analyzed. We compared different methods and protocols for sample collection, storage, centrifugation, isolation, and quantification. Cell-free DNA (cfDNA) concentrations of several wild-type targets and BRAF and EGFR-mutant ctDNA concentrations quantified by ddPCR were primary outcome measurements. Highest cfDNA concentrations were measured in blood collected in serum tubes. No significant differences in cfDNA concentrations were detected between various time points of up to 24 h until centrifugation. Highest cfDNA concentrations were detected after DNA isolation with the Quick cfDNA Serum & Plasma Kit, while plasma isolation using the QIAamp Circulating Nucleic Acid Kit yielded the most consistent results. DdPCR results on cfDNA are highly dependent on multiple factors during preanalytical sample workup, which need to be addressed during the development of this diagnostic tool for cancer diagnostics in the future. © 2017 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Diagnostic accuracy of PCR for detecting ALK gene rearrangement in NSCLC patients: A systematic review and meta-analysis

PubMed Central

Zhang, Xia; Zhou, Jian-Guo; Wu, Hua-Lian; Ma, Hu; Jiang, Zhi-Xia

2017-01-01

Background Anaplastic lymphoma kinase (ALK) gene fusion has been reported in 3∼5% non-small cell lung carcinoma (NSCLC) patients, and polymerase chain reaction (PCR) is commonly used to detecting the gene status, but the diagnostic capacity of it is still controversial. A systematic review and meta-analysis was conducted to clarify the diagnostic accuracy of PCR for detecting ALK gene rearrangement in NSCLC patients. Results 18 articles were enrolled, which included 21 studies, involving 2800 samples from NSCLC patients. The overall pooled parameters were calculated: sensitivity was 92.4% [95% confidence interval (CI): 82.2%–97.0%], specificity was 97.8% [95% CI: 95.1%–99.0%], PLR was 41.51 [95% CI: 18.10–95.22], NLR was 0.08 [95% CI: 0.03–0.19], DOR was 535.72 [95% CI: 128.48–2233.79], AUROC was 0.99 [95% CI: 0.98–1.00]. Materials and Methods Relevant articles were searched from PubMed, EMBASE, Web of Science, Cochrane library, American Society of Clinical Oncology (ASCO), European Society for Medical Oncology (ESMO), China National Knowledge Infrastructure (CNKI), China Wan Fang databases and Chinese biomedical literature database (CBM). Diagnostic capacity of PCR test was assessed by the pooled sensitivity and specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR), diagnostic odds ratio (DOR), area under the summary receiver operating characteristic (AUROC). Conclusions Based on the results from this review, PCR has good diagnostic performance for detecting the ALK gene fusion in NSCLC patients. Moreover, due to the poor methodology quality of the enrolled trials, more well-designed multi-center trials should be performed. PMID:29088875

Harmonization of Bordetella pertussis real-time PCR diagnostics in the United States in 2012.

PubMed

Williams, Margaret M; Taylor, Thomas H; Warshauer, David M; Martin, Monte D; Valley, Ann M; Tondella, M Lucia

2015-01-01

Real-time PCR (rt-PCR) is an important diagnostic tool for the identification of Bordetella pertussis, Bordetella holmesii, and Bordetella parapertussis. Most U.S. public health laboratories (USPHLs) target IS481, present in 218 to 238 copies in the B. pertussis genome and 32 to 65 copies in B. holmesii. The CDC developed a multitarget PCR assay to differentiate B. pertussis, B. holmesii, and B. parapertussis and provided protocols and training to 19 USPHLs. The 2012 performance exercise (PE) assessed the capability of USPHLs to detect these three Bordetella species in clinical samples. Laboratories were recruited by the Wisconsin State Proficiency Testing program through the Association of Public Health Laboratories, in partnership with the CDC. Spring and fall PE panels contained 12 samples each of viable Bordetella and non-Bordetella species in saline. Fifty and 53 USPHLs participated in the spring and fall PEs, respectively, using a variety of nucleic acid extraction methods, PCR platforms, and assays. Ninety-six percent and 94% of laboratories targeted IS481 in spring and fall, respectively, in either singleplex or multiplex assays. In spring and fall, respectively, 72% and 79% of USPHLs differentiated B. pertussis and B. holmesii and 68% and 72% identified B. parapertussis. IS481 cycle threshold (CT) values for B. pertussis samples had coefficients of variation (CV) ranging from 10% to 28%. Of the USPHLs that differentiated B. pertussis and B. holmesii, sensitivity was 96% and specificity was 95% for the combined panels. The 2012 PE demonstrated increased harmonization of rt-PCR Bordetella diagnostic protocols in USPHLs compared to that of the previous survey. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

PCR diagnostics underestimate the prevalence of avian malaria (Plasmodium relictum) in experimentally-infected passerines

USGS Publications Warehouse

Jarvi, Susan I.; Schultz, Jeffrey J.; Atkinson, Carter T.

2002-01-01

Several polymerase chain reaction (PCR)-based methods have recently been developed for diagnosing malarial infections in both birds and reptiles, but a critical evaluation of their sensitivity in experimentally-infected hosts has not been done. This study compares the sensitivity of several PCR-based methods for diagnosing avian malaria (Plasmodium relictum) in captive Hawaiian honeycreepers using microscopy and a recently developed immunoblotting technique. Sequential blood samples were collected over periods of up to 4.4 yr after experimental infection and rechallenge to determine both the duration and detectability of chronic infections. Two new nested PCR approaches for detecting circulating parasites based on P. relictum 18S rRNA genes and the thrombospondin-related anonymous protein (TRAP) gene are described. The blood smear and the PCR tests were less sensitive than serological methods for detecting chronic malarial infections. Individually, none of the diagnostic methods was 100% accurate in detecting subpatent infections, although serological methods were significantly more sensitive (97%) than either nested PCR (61–84%) or microscopy (27%). Circulating parasites in chronically infected birds either disappear completely from circulation or to drop to intensities below detectability by nested PCR. Thus, the use of PCR as a sole means of detection of circulating parasites may significantly underestimate true prevalence.

A diagnostic PCR assay for the detection of an Australian epidemic strain of Pseudomonas aeruginosa

PubMed Central

2010-01-01

Background Chronic lung infection with the bacterium Pseudomonas aeruginosa is one of the hallmarks of cystic fibrosis (CF) and is associated with worsening lung function, increased hospitalisation and reduced life expectancy. A virulent clonal strain of P. aeruginosa (Australian epidemic strain I; AES-I) has been found to be widespread in CF patients in eastern Australia. Methods Suppression subtractive hybridization (SSH) was employed to identify genetic sequences that are present in the AES-I strain but absent from the sequenced reference strain PAO1. We used PCR to evaluate the distribution of several of the AES-I loci amongst a collection of 188 P. aeruginosa isolates which was comprised of 35 AES-I isolates (as determined by PFGE), 78 non-AES-I CF isolates including other epidemic CF strains as well as 69 P. aeruginosa isolates from other clinical and environmental sources. Results We have identified a unique AES-I genetic locus that is present in all 35 AES-I isolates tested and not present in any of the other 153 P. aeruginosa strains examined. We have used this unique AES-I locus to develop a diagnostic PCR and a real-time PCR assay to detect the presence of P. aeruginosa and AES-I in patient sputum samples. Conclusions We have developed diagnostic PCR assays that are 100% sensitive and 100% specific for the P. aeruginosa strain AES-I. We have also shown that Whatman FTA® Elute cards may be used with PCR-based assays to rapidly detect the presence of P. aeruginosa strains in CF sputum. PMID:20637114

Comparison of four PCR methods for efficient detection of Trypanosoma cruzi in routine diagnostics.

PubMed

Seiringer, Peter; Pritsch, Michael; Flores-Chavez, María; Marchisio, Edoardo; Helfrich, Kerstin; Mengele, Carolin; Hohnerlein, Stefan; Bretzel, Gisela; Löscher, Thomas; Hoelscher, Michael; Berens-Riha, Nicole

2017-07-01

Due to increased migration, Chagas disease has become an international health problem. Reliable diagnosis of chronically infected people is crucial for prevention of non-vectorial transmission as well as treatment. This study compared four distinct PCR methods for detection of Trypanosoma cruzi DNA for the use in well-equipped routine diagnostic laboratories. DNA was extracted of T. cruzi-positive and negative patients' blood samples and cultured T. cruzi, T. rangeli as well as Leishmania spp. One conventional and two real-time PCR methods targeting a repetitive Sat-DNA sequence as well as one conventional PCR method targeting the variable region of the kDNA minicircle were compared for sensitivity, intra- and interassay precision, limit of detection, specificity and cross-reactivity. Considering the performance, costs and ease of use, an algorithm for PCR-diagnosis of patients with a positive serology for T. cruzi antibodies was developed. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

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

Isolation of RNA From Peripheral Blood Cells: A Validation Study for Molecular Diagnostics by Microarray and Kinetic RT-PCR Assays - Application in Aerospace Medicine

DTIC Science & Technology

2004-01-01

of RNA From Peripheral Blood Cells: A Validation Study for Molecular Diagnostics by Microarray and Kinetic RT-PCR Assays  Application in...VALIDATION STUDY FOR MOLECULAR DIAGNOSTICS BY MICROARRAY AND KINETIC RT-PCR ASSAYS  APPLICATION IN AEROSPACE MEDICINE INTRODUCTION Extraction of cellular

Diagnostic accuracy of droplet digital PCR for detection of EGFR T790M mutation in circulating tumor DNA

PubMed Central

Tong, Xiang; Wang, Ye; Wang, Chengdi; Jin, Jing; Tian, Panwen; Li, Weimin

2018-01-01

Objectives Although different methods have been established to detect epidermal growth factor receptor (EGFR) T790M mutation in circulating tumor DNA (ctDNA), a wide range of diagnostic accuracy values were reported in previous studies. The aim of this meta-analysis was to provide pooled diagnostic accuracy measures for droplet digital PCR (ddPCR) in the diagnosis of EGFR T790M mutation based on ctDNA. Materials and methods A systematic review and meta-analysis were carried out based on resources from Pubmed, Web of Science, Embase and Cochrane Library up to October 11, 2017. Data were extracted to assess the pooled sensitivity, specificity, positive likelihood ratio, negative likelihood ratio (NLR), diagnostic OR (DOR), and areas under the summary receiver-operating characteristic curve (SROC). Results Eleven of 311 studies identified have met the including criteria. The sensitivity and specificity of ddPCR for the detection of T790M mutation in ctDNA ranged from 0.0% to 100.0% and 63.2% to 100.0%, respectively. For the pooled analysis, ddPCR had a performance of 70.1% (95% CI, 62.7%–76.7%) sensitivity, 86.9 % (95% CI, 80.6%–91.7%) specificity, 3.67 (95% CI, 2.33–5.79) PLR, 0.41 (95% CI, 0.32–0.55) NLR, and 10.83 (95% CI, 5.86–20.03) DOR, with the area under the SROC curve being 0.82. Conclusion The ddPCR harbored a good performance for detection of EGFR T790M mutation in ctDNA. PMID:29844700

Improved assay to detect Plasmodium falciparum using an uninterrupted, semi-nested PCR and quantitative lateral flow analysis

PubMed Central

2013-01-01

Background A rapid, non-invasive, and inexpensive point-of-care (POC) diagnostic for malaria followed by therapeutic intervention would improve the ability to control infection in endemic areas. Methods A semi-nested PCR amplification protocol is described for quantitative detection of Plasmodium falciparum and is compared to a traditional nested PCR. The approach uses primers that target the P. falciparum dihydrofolate reductase gene. Results This study demonstrates that it is possible to perform an uninterrupted, asymmetric, semi-nested PCR assay with reduced assay time to detect P. falciparum without compromising the sensitivity and specificity of the assay using saliva as a testing matrix. Conclusions The development of this PCR allows nucleic acid amplification without the need to transfer amplicon from the first PCR step to a second reaction tube with nested primers, thus reducing both the chance of contamination and the time for analysis to < two hours. Analysis of the PCR amplicon yield was adapted to lateral flow detection using the quantitative up-converting phosphor (UCP) reporter technology. This approach provides a basis for migration of the assay to a POC microfluidic format. In addition the assay was successfully evaluated with oral samples. Oral fluid collection provides a simple non-invasive method to collect clinical samples. PMID:23433252

Rv1458c: a new diagnostic marker for identification of Mycobacterium tuberculosis complex in a novel duplex PCR assay.

PubMed

Shrivastava, Kamal; Garima, Kushal; Narang, Anshika; Bhattacharyya, Kausik; Vishnoi, Ekta; Singh, Roshan Kumar; Chaudhry, Anil; Prasad, Rajendra; Bose, Mridula; Varma-Basil, Mandira

2017-03-01

We explored the efficiency of Rv1458c, the gene encoding a putative ABC drug transporter specific for the Mycobacterium tuberculosis complex (MTBC), as a diagnostic marker. A 190 bp region of Rv1458c and a 300 bp region of hsp65 were targeted in a novel duplex PCR assay and the results were compared with those for PCR restriction analysis(PRA) using the restriction enzymes NruI and BamHI. Species identification of a subset of the isolates (n=50) was confirmed by sequencing. Clinical isolates of M. tuberculosis (n=426) obtained from clinically suspected patients of pulmonary tuberculosis and mycobacterial (n=13) and non-mycobacterial (n=8) reference strains were included in the study. The duplex PCR assay correctly identified 320/426 isolates as MTBC and 106/426 isolates as non-tuberculous mycobacteria(NTM). The test was 100 % specific and sensitive when compared with NruI/BamHI PCR restriction analysis and highlighted the use of Rv1458c as a diagnostic marker for MTBC. The duplex PCR assay could be developed for use as a screening test to identify MTBC in clinical specimens in peripheral laboratories with limited resources.

Improvement of malaria diagnostic system based on acridine orange staining.

PubMed

Kimura, Masatsugu; Teramoto, Isao; Chan, Chim W; Idris, Zulkarnain Md; Kongere, James; Kagaya, Wataru; Kawamoto, Fumihiko; Asada, Ryoko; Isozumi, Rie; Kaneko, Akira

2018-02-07

Rapid diagnosis of malaria using acridine orange (AO) staining and a light microscope with a halogen lamp and interference filter was deployed in some malaria-endemic countries. However, it has not been widely adopted because: (1) the lamp was weak as an excitation light and the set-up did not work well under unstable power supply; and, (2) the staining of samples was frequently inconsistent. The halogen lamp was replaced by a low-cost, blue light-emitting diode (LED) lamp. Using a reformulated AO solution, the staining protocol was revised to make use of a concentration gradient instead of uniform staining. To evaluate this new AO diagnostic system, a pilot field study was conducted in the Lake Victoria basin in Kenya. Without staining failure, malaria infection status of about 100 samples was determined on-site per one microscopist per day, using the improved AO diagnostic system. The improved AO diagnosis had both higher overall sensitivity (46.1 vs 38.9%: p = 0.08) and specificity (99.0 vs 96.3%) than the Giemsa method (N = 1018), using PCR diagnosis as the standard. Consistent AO staining of thin blood films and rapid evaluation of malaria parasitaemia with the revised protocol produced superior results relative to the Giemsa method. This AO diagnostic system can be set up easily at low cost using an ordinary light microscope. It may supplement rapid diagnostic tests currently used in clinical settings in malaria-endemic countries, and may be considered as an inexpensive tool for case surveillance in malaria-eliminating countries.

Development of a pan-Simbu real-time reverse transcriptase PCR for the detection of Simbu serogroup viruses and comparison with SBV diagnostic PCR systems.

PubMed

Fischer, Melina; Schirrmeier, Horst; Wernike, Kerstin; Wegelt, Anne; Beer, Martin; Hoffmann, Bernd

2013-11-05

Schmallenberg virus (SBV), a novel orthobunyavirus of the Simbu serogroup, was first identified in October 2011 in dairy cattle in Germany, where it caused fever, diarrhea and a drop in milk yield. Since then, SBV additionally has been detected in adult sheep and goats. Although symptoms of acute infection were not observed, infection during a vulnerable phase of pregnancy caused congenital malformations and stillbirths. In view of the current situation and the possible emergence of further Simbu serogroup members, a pan-Simbu real-time reverse transcriptase (RT) PCR system for the reliable detection of Simbu serogroup viruses should be developed. In this study a pan-Simbu real-time RT-PCR system was established and compared to several SBV real-time RT-PCR assays. All PCR-systems were tested using a panel of different Simbu serogroup viruses as well as several field samples from diseased cattle, sheep and goats originating from all over Germany. Several pan-Simbu real-time RT-PCR products were sequenced via Sanger sequencing. Furthermore, in silico analyses were performed to investigate suitability for the detection of further orthobunyaviruses. All tested members of the Simbu serogroup (n = 14) as well as most of the field samples were successfully detected by the pan-Simbu real-time RT-PCR system. The comparison of this intercalating dye assay with different TaqMan probe-based assays developed for SBV diagnostics confirmed the functionality of the pan-Simbu assay for screening purposes. However, the SBV-TaqMan-assay SBV-S3 delivered the highest analytical sensitivity of less than ten copies per reaction for duplex systems including an internal control. In addition, for confirmation of SBV-genome detection the highly specific SBV-M1 assay was established. The pan-Simbu real-time RT-PCR system was able to detect all tested members of the Simbu serogroup, most of the SBV field samples as well as three tested Bunyamwera serogroup viruses with a suitable

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

Use of amplicon sequencing to improve sensitivity in PCR-based detection of microbial pathogen in environmental samples.

PubMed

Saingam, Prakit; Li, Bo; Yan, Tao

2018-06-01

DNA-based molecular detection of microbial pathogens in complex environments is still plagued by sensitivity, specificity and robustness issues. We propose to address these issues by viewing them as inadvertent consequences of requiring specific and adequate amplification (SAA) of target DNA molecules by current PCR methods. Using the invA gene of Salmonella as the model system, we investigated if next generation sequencing (NGS) can be used to directly detect target sequences in false-negative PCR reaction (PCR-NGS) in order to remove the SAA requirement from PCR. False-negative PCR and qPCR reactions were first created using serial dilutions of laboratory-prepared Salmonella genomic DNA and then analyzed directly by NGS. Target invA sequences were detected in all false-negative PCR and qPCR reactions, which lowered the method detection limits near the theoretical minimum of single gene copy detection. The capability of the PCR-NGS approach in correcting false negativity was further tested and confirmed under more environmentally relevant conditions using Salmonella-spiked stream water and sediment samples. Finally, the PCR-NGS approach was applied to ten urban stream water samples and detected invA sequences in eight samples that would be otherwise deemed Salmonella negative. Analysis of the non-target sequences in the false-negative reactions helped to identify primer dime-like short sequences as the main cause of the false negativity. Together, the results demonstrated that the PCR-NGS approach can significantly improve method sensitivity, correct false-negative detections, and enable sequence-based analysis for failure diagnostics in complex environmental samples. Copyright © 2018 Elsevier B.V. All rights reserved.

The applicability of real-time PCR in the diagnostic of cutaneous leishmaniasis and parasite quantification for clinical management: Current status and perspectives.

PubMed

Moreira, Otacilio C; Yadon, Zaida E; Cupolillo, Elisa

2017-09-29

Cutaneous leishmaniasis (CL) is spread worldwide and is the most common manifestation of leishmaniasis. Diagnosis is performed by combining clinical and epidemiological features, and through the detection of Leishmania parasites (or DNA) in tissue specimens or trough parasite isolation in culture medium. Diagnosis of CL is challenging, reflecting the pleomorphic clinical manifestations of this disease. Skin lesions vary in severity, clinical appearance, and duration, and in some cases, they can be indistinguishable from lesions related to other diseases. Over the past few decades, PCR-based methods, including real-time PCR assays, have been developed for Leishmania detection, quantification and species identification, improving the molecular diagnosis of CL. This review provides an overview of many real-time PCR methods reported for the diagnostic evaluation of CL and some recommendations for the application of these methods for quantification purposes for clinical management and epidemiological studies. Furthermore, the use of real-time PCR for Leishmania species identification is also presented. The advantages of real-time PCR protocols are numerous, including increased sensitivity and specificity and simpler standardization of diagnostic procedures. However, despite the numerous assays described, there is still no consensus regarding the methods employed. Furthermore, the analytical and clinical validation of CL molecular diagnosis has not followed international guidelines so far. A consensus methodology comprising a DNA extraction protocol with an exogenous quality control and an internal reference to normalize parasite load is still needed. In addition, the analytical and clinical performance of any consensus methodology must be accurately assessed. This review shows that a standardization initiative is essential to guide researchers and clinical laboratories towards the achievement of a robust and reproducible methodology, which will permit further evaluation

Real-time PCR in virology.

PubMed

Mackay, Ian M; Arden, Katherine E; Nitsche, Andreas

2002-03-15

The use of the polymerase chain reaction (PCR) in molecular diagnostics has increased to the point where it is now accepted as the gold standard for detecting nucleic acids from a number of origins and it has become an essential tool in the research laboratory. Real-time PCR has engendered wider acceptance of the PCR due to its improved rapidity, sensitivity, reproducibility and the reduced risk of carry-over contamination. There are currently five main chemistries used for the detection of PCR product during real-time PCR. These are the DNA binding fluorophores, the 5' endonuclease, adjacent linear and hairpin oligoprobes and the self-fluorescing amplicons, which are described in detail. We also discuss factors that have restricted the development of multiplex real-time PCR as well as the role of real-time PCR in quantitating nucleic acids. Both amplification hardware and the fluorogenic detection chemistries have evolved rapidly as the understanding of real-time PCR has developed and this review aims to update the scientist on the current state of the art. We describe the background, advantages and limitations of real-time PCR and we review the literature as it applies to virus detection in the routine and research laboratory in order to focus on one of the many areas in which the application of real-time PCR has provided significant methodological benefits and improved patient outcomes. However, the technology discussed has been applied to other areas of microbiology as well as studies of gene expression and genetic disease.

Diagnostic potential of nested PCR, galactomannan EIA, and beta-D-glucan for invasive aspergillosis in pediatric patients.

PubMed

Badiee, Parisa; Alborzi, Abdolvahab; Karimi, Mahammad; Pourabbas, Bahman; Haddadi, Pedram; Mardaneh, Jalal; Moieni, Mahsa

2012-04-13

Limited specific data and investigations are available for invasive aspergillosis (IA) in pediatric patients. We evaluated the diagnostic potential of three noninvasive tests including the Platelia Aspergillus EIA kit for using galactomannan antigen, (1,3)-β-D-glucan Detection Reagent Kit, and nested-PCR for Aspergillus DNA in sera. We evaluated the diagnostic potential of three noninvasive tests including EIA for galactomannan antigen  (Platelia Aspergillus), nested  PCR assay for Aspergillus DNA and test for (1→3)-β-D-glucan (Glucatell assay Kit). All pediatric patients treated at the hematology/oncology unit who were at increased risk of developing invasive aspergillosis were enrolled. Clinical samples were examined for Aspergillus infections by mycological methods. Serial blood samples were collected twice weekly and evaluated by noninvasive tests. We analyzed 230 consecutive blood samples from 62 pediatric patients. The incidence rate of invasive aspergillosis in the patients was found to be 27.4%, and the etiologic agents were Aspergillus flavus, Aspergillus fumigatus, and Aspergillus spp.  The sensitivity, specificity, positive and negative predictive values, and likelihood ratios for positive and negative results of galactomannan in patients with proven and probable IA were 90%, 92%, 81.8%, 96%, 11.25, and 0.1; for beta-D-glucan they were 50%, 46%, 26%, 70.6%, 0.9, 0.9; and for nested-PCR they were 80%, 96.2%, 88.9%, 92.6%, 21, and 0.2, respectively. The conventional methods are not able to detect IA, due to the lack of valid and proper sampling. Galactomannan and nested-PCR tests in serum, with enough accuracy and reliability, can serve as noninvasive methods for the detection of IA in pediatric patients. However, the beta-D-glucan test cannot serve as an efficient diagnostic tool in those with hematologic disorders. 

Improved safety for molecular diagnosis of classical rabies viruses by use of a TaqMan real-time reverse transcription-PCR "double check" strategy.

PubMed

Hoffmann, B; Freuling, C M; Wakeley, P R; Rasmussen, T B; Leech, S; Fooks, A R; Beer, M; Müller, T

2010-11-01

To improve the diagnosis of classical rabies virus with molecular methods, a validated, ready-to-use, real-time reverse transcription-PCR (RT-PCR) assay was developed. In a first step, primers and 6-carboxyfluorescien-labeled TaqMan probes specific for rabies virus were selected from the consensus sequence of the nucleoprotein gene of 203 different rabies virus sequences derived from GenBank. The selected primer-probe combination was highly specific and sensitive. During validation using a sample set of rabies virus strains from the virus archives of the Friedrich-Loeffler-Institut (FLI; Germany), the Veterinary Laboratories Agency (VLA; United Kingdom), and the DTU National Veterinary Institute (Lindholm, Denmark), covering the global diversity of rabies virus lineages, it was shown that both the newly developed assay and a previously described one had some detection failures. This was overcome by a combined assay that detected all samples as positive. In addition, the introduction of labeled positive controls (LPC) increased the diagnostic safety of the single as well as the combined assay. Based on the newly developed, alternative assay for the detection of rabies virus and the application of LPCs, an improved diagnostic sensitivity and reliability can be ascertained for postmortem and intra vitam real-time RT-PCR analyses in rabies reference laboratories.

Real Time Polymerase Chain Reaction (rt-PCR): A New Patent to Diagnostic Purposes for Paracoccidioidomycosis.

PubMed

Rocha-Silva, Fabiana; Gomes, Luciana I; Gracielle-Melo, Cidiane; Goes, Alfredo M; Caligiorne, Rachel B

2017-01-01

Paracoccidioidomycosis (PCM) is a systemic mycosis caused by dimorphic fungi Paracoccidioides brasiliensis and Paracoccidioides lutzii. It is prevalent in Latin American, mainly in Brazil. Therefore, PCM has fundamental impact on the Brazilian global economy, especially in public health system, since it is affecting economical active population in different country regions. The present study aimed to standardize the Real Time-Polymerase Chain Reaction (rt-PCR) for an efficient and safe PCM diagnosis amplifying the recombinant protein PB27 gene, only expressed by specimens of Paracoccidioides genus. To standardize a methodology of rt-PCR using species-specific primers and probe designed for annealing in this specific region of the fungi´s genome, amplifying the recombinant protein PB27 gene, only expressed by specimens of Paracoccidioides genus. Followed by design in silico, experiments were performed in vitro to determine rt-PCR specificity, efficiency and genome detection limit. The primers and probe sequences were deposited in Brazilian Coordination of Technological Innovation and Transfer (CTIT), under patent reference number BR1020160078830. The present study demonstrated the rt-PCR applicability for support on diagnosis of paracoccidioidomycosis, presenting low cost, which makes it affordable for public health services in developing countries as Brazil. It is noteworthy that it is necessary to validate this methodology using clinical samples before to use as a safe method of diagnosis. A review of all patents related to this topic was performed and it was shown that, to date, there are no records of patent on kits for paracoccidioidomycosis´s diagnostic. Indeed, there is still a lot to go to reach this goal. The reaction developed was standardized and patented, opening perspectives to molecular diagnosis development for paracoccidioidomycosis, since rt-PCR can be applied to a broad spectrum of infectious diseases. It would need to be tested in biological

Diagnostics based on nucleic acid sequence variant profiling: PCR, hybridization, and NGS approaches.

PubMed

Khodakov, Dmitriy; Wang, Chunyan; Zhang, David Yu

2016-10-01

Nucleic acid sequence variations have been implicated in many diseases, and reliable detection and quantitation of DNA/RNA biomarkers can inform effective therapeutic action, enabling precision medicine. Nucleic acid analysis technologies being translated into the clinic can broadly be classified into hybridization, PCR, and sequencing, as well as their combinations. Here we review the molecular mechanisms of popular commercial assays, and their progress in translation into in vitro diagnostics. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Diagnostic accuracy and applicability of a PCR system for the detection of Schistosoma mansoni DNA in human urine samples from an endemic area.

PubMed

Enk, Martin Johannes; Oliveira e Silva, Guilherme; Rodrigues, Nilton Barnabé

2012-01-01

Schistosomiasis caused by Schistosoma mansoni, one of the most neglected human parasitoses in Latin America and Africa, is routinely confirmed by microscopic visualization of eggs in stool. The main limitation of this diagnostic approach is its lack of sensitivity in detecting individual low worm burdens and consequently data on infection rates in low transmission settings are little reliable. According to the scientific literature, PCR assays are characterized by high sensitivity and specificity in detecting parasite DNA in biological samples. A simple and cost effective extraction method for DNA of Schistosoma mansoni from urine samples in combination with a conventional PCR assay was developed and applied in an endemic area. This urine based PCR system was tested for diagnostic accuracy among a population of a small village in an endemic area, comparing it to a reference test composed of three different parasitological techniques. The diagnostic parameters revealed a sensitivity of 100%, a specificity of 91.20%, positive and negative predictive values of 86.25% and 100%, respectively, and a test accuracy of 94.33%. Further statistical analysis showed a k index of 0.8806, indicating an excellent agreement between the reference test and the PCR system. Data obtained from the mouse model indicate the infection can be detected one week after cercariae penetration, opening a new perspective for early detection and patient management during this stage of the disease. The data indicate that this innovative PCR system provides a simple to handle and robust diagnostic tool for the detection of S. mansoni DNA from urine samples and a promising approach to overcome the diagnostic obstacles in low transmission settings. Furthermore the principals of this molecular technique, based on the examination of human urine samples may be useful for the diagnosis of other neglected tropical diseases that can be detected by trans-renal DNA.

Improved serotype-specific dengue virus detection in Trinidad and Tobago using a multiplex, real-time RT-PCR.

PubMed

Waggoner, Jesse J; Sahadeo, Nikita S D; Brown, Arianne; Mohamed-Hadley, Alisha; Hadley, Dexter; Carrington, Leslie; Carrington, Christine V F; Pinsky, Benjamin A

2015-02-01

Dengue virus (DENV) transmission occurs throughout the Caribbean, though laboratory confirmation and epidemiologic surveillance are limited by the availability of serotype-specific molecular diagnostics. In this study, we show that a serotype-specific DENV multiplex, real-time reverse transcriptase-PCR (RT-PCR) detected DENV RNA in significantly more samples (82/182) than a reference hemi-nested RT-PCR (57/182; P=0.01). Copyright © 2015 Elsevier Inc. All rights reserved.

PCR-based verification of positive rapid diagnostic tests for intestinal protozoa infections with variable test band intensity.

PubMed

Becker, Sören L; Müller, Ivan; Mertens, Pascal; Herrmann, Mathias; Zondie, Leyli; Beyleveld, Lindsey; Gerber, Markus; du Randt, Rosa; Pühse, Uwe; Walter, Cheryl; Utzinger, Jürg

2017-10-01

Stool-based rapid diagnostic tests (RDTs) for pathogenic intestinal protozoa (e.g. Cryptosporidium spp. and Giardia intestinalis) allow for prompt diagnosis and treatment in resource-constrained settings. Such RDTs can improve individual patient management and facilitate population-based screening programmes in areas without microbiological laboratories for confirmatory testing. However, RDTs are difficult to interpret in case of 'trace' results with faint test band intensities and little is known about whether such ambiguous results might indicate 'true' infections. In a longitudinal study conducted in poor neighbourhoods of Port Elizabeth, South Africa, a total of 1428 stool samples from two cohorts of schoolchildren were examined on the spot for Cryptosporidium spp. and G. intestinalis using an RDT (Crypto/Giardia DuoStrip; Coris BioConcept). Overall, 121 samples were positive for G. intestinalis and the RDT suggested presence of cryptosporidiosis in 22 samples. After a storage period of 9-10 months in cohort 1 and 2-3 months in cohort 2, samples were subjected to multiplex PCR (BD Max™ Enteric Parasite Panel, Becton Dickinson). Ninety-three percent (112/121) of RDT-positive samples for G. intestinalis were confirmed by PCR, with a correlation between RDT test band intensity and quantitative pathogen load present in the sample. For Cryptosporidium spp., all positive RDTs had faintly visible lines and these were negative on PCR. The performance of the BD Max™ PCR was nearly identical in both cohorts, despite the prolonged storage at disrupted cold chain conditions in cohort 1. The Crypto/Giardia DuoStrip warrants further validation in communities with a high incidence of diarrhoea. Copyright © 2017 Elsevier B.V. All rights reserved.

Simple Repeat-Primed PCR Analysis of the Myotonic Dystrophy Type 1 Gene in a Clinical Diagnostics Environment

PubMed Central

Dryland, Philippa A.; Doherty, Elaine; Love, Jennifer M.; Love, Donald R.

2013-01-01

Myotonic dystrophy type 1 is an autosomal dominant neuromuscular disorder that is caused by the expansion of a CTG trinucleotide repeat in the DMPK gene. The confirmation of a clinical diagnosis of DM-1 usually involves PCR amplification of the CTG repeat-containing region and subsequent sizing of the amplification products in order to deduce the number of CTG repeats. In the case of repeat hyperexpansions, Southern blotting is also used; however, the latter has largely been superseded by triplet repeat-primed PCR (TP-PCR), which does not yield a CTG repeat number but nevertheless provides a means of stratifying patients regarding their disease severity. We report here a combination of forward and reverse TP-PCR primers that allows for the simple and effective scoring of both the size of smaller alleles and the presence or absence of expanded repeat sequences. In addition, the CTG repeat-containing TP-PCR forward primer can target both the DM-1 and Huntington disease genes, thereby streamlining the work flow for confirmation of clinical diagnoses in a diagnostic laboratory. PMID:26317000

Improved Safety for Molecular Diagnosis of Classical Rabies Viruses by Use of a TaqMan Real-Time Reverse Transcription-PCR “Double Check” Strategy▿ †

PubMed Central

Hoffmann, B.; Freuling, C. M.; Wakeley, P. R.; Rasmussen, T. B.; Leech, S.; Fooks, A. R.; Beer, M.; Müller, T.

2010-01-01

To improve the diagnosis of classical rabies virus with molecular methods, a validated, ready-to-use, real-time reverse transcription-PCR (RT-PCR) assay was developed. In a first step, primers and 6-carboxyfluorescien-labeled TaqMan probes specific for rabies virus were selected from the consensus sequence of the nucleoprotein gene of 203 different rabies virus sequences derived from GenBank. The selected primer-probe combination was highly specific and sensitive. During validation using a sample set of rabies virus strains from the virus archives of the Friedrich-Loeffler-Institut (FLI; Germany), the Veterinary Laboratories Agency (VLA; United Kingdom), and the DTU National Veterinary Institute (Lindholm, Denmark), covering the global diversity of rabies virus lineages, it was shown that both the newly developed assay and a previously described one had some detection failures. This was overcome by a combined assay that detected all samples as positive. In addition, the introduction of labeled positive controls (LPC) increased the diagnostic safety of the single as well as the combined assay. Based on the newly developed, alternative assay for the detection of rabies virus and the application of LPCs, an improved diagnostic sensitivity and reliability can be ascertained for postmortem and intra vitam real-time RT-PCR analyses in rabies reference laboratories. PMID:20739489

Molecular diagnostics for human leptospirosis.

PubMed

Waggoner, Jesse J; Pinsky, Benjamin A

2016-10-01

The definitive diagnosis of leptospirosis, which results from infection with spirochetes of the genus Leptospira, currently relies on the use of culture, serological testing (microscopic agglutination testing), and molecular detection. The purpose of this review is to describe new molecular diagnostics for Leptospira and discuss advancements in the use of available methods. Efforts have been focused on improving the clinical sensitivity of Leptospira detection using molecular methods. In this review, we describe a reoptimized pathogenic species-specific real-time PCR (targeting lipL32) that has demonstrated improved sensitivity, findings by two groups that real-time reverse-transcription PCR assays targeting the 16S rrs gene can improve detection, and two new loop-mediated amplification techniques. Quantitation of leptospiremia, detection in different specimen types, and the complementary roles played by molecular detection and microscopic agglutination testing will be discussed. Finally, a protocol for Leptospira strain subtyping using variable number tandem repeat targets and high-resolution melting will be described. Molecular diagnostics have an established role for the diagnosis of leptospirosis and provide an actionable diagnosis in the acute setting. The use of real-time reverse-transcription PCR for testing serum/plasma and cerebrospinal fluid, when available, may improve the detection of Leptospira without decreasing clinical specificity.

Prevalence of tick-borne haemoparasites in small ruminants in Turkey and diagnostic sensitivity of single-PCR and RLB.

PubMed

Bilgic, Huseyin Bilgin; Bakırcı, Serkan; Kose, Onur; Unlu, Ahmet Hakan; Hacılarlıoglu, Selin; Eren, Hasan; Weir, William; Karagenc, Tulin

2017-04-27

Tick-borne haemoparasitic diseases (TBHDs), caused by Theileria, Babesia, Anaplasma and Ehrlichia, are common in regions of the world where the distributions of host, pathogen and vector overlap. Many of these diseases threaten livestock production and some also represent a concern to human public health. The primary aim of this study was to determine the prevalence of the above-mentioned pathogens in a large number of blood samples (n = 1979) collected from sheep (n = 1727) and goats (n = 252) in Turkey. A secondary aim was to assess the diagnostic sensitivity of a number of species-specific polymerase chain reaction (PCR) tests and the reverse line blotting (RLB) assay. DNA samples were screened using species-specific PCR for the presence of Theileria ovis, Theileria sp. MK, T. lestoquardi, T. uilenbergi, T. luwenshuni, Babesia ovis, Anaplasma ovis and A. phagocytophilum while RLB was undertaken to test for the presence of all known Theileria, Babesia, Anaplasma and Ehrlichia species. The diagnostic sensitivity of these two approaches was then compared in terms of their ability to detect single species and mixed infections. Overall, 84 and 74.43% of the small ruminants sampled were identified as hosting one or more pathogen(s) by species-specific PCR and RLB respectively. The presence of Theileria sp. OT1, T. luwenshuni and T. uilenbergi in Turkey was revealed for the first time while the presence of Babesia motasi, B. crassa and T. separata in Turkish small ruminants was confirmed using molecular methods. A high prevalence of mixed infection was evident, with PCR and RLB approaches indicating that 52.24 and 35.42% of animals were co-infected with multiple species, respectively. More than 80% of the mixed infections contained T. ovis and/or A. ovis. The RLB approach was found to be capable of detecting mixed infections with species such as Theileria sp. OT1, Theileria sp. OT3, T. separata, B. crassa and Babesia spp. The results indicated that

Diagnostic evaluation of a multiplexed RT-PCR microsphere array assay for the detection of foot-and-mouth and look-alike disease viruses

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

Hindson, B J; Baker, B R; Bentley Tammero, L F

2007-09-18

A high-throughput multiplexed assay (Multiplex Version 1.0) was developed for the differential laboratory diagnosis of foot-and-mouth disease virus (FMDV) from viruses which cause clinically similar diseases of livestock. This assay simultaneously screens for five RNA and two DNA viruses using multiplexed reverse transcription PCR (mRT-PCR) amplification coupled with a microsphere hybridization array and flow-cytometric detection. Two of the seventeen primer-probe sets included in this multiplex assay were adopted from previously characterized real-time RT-PCR (rRT-PCR) assays for FMDV. The diagnostic accuracy of the mRT-PCR was evaluated using 287 field samples, including 248 (true positive n= 213, true negative n=34) from suspectmore » cases of foot-and-mouth disease collected from 65 countries between 1965 and 2006 and 39 true negative samples collected from healthy animals. The mRT-PCR assay results were compared with two singleplex rRT-PCR assays, using virus isolation with antigen-ELISA as the reference method. The diagnostic sensitivity of the mRT-PCR assay for FMDV was 93.9% [95% C.I. 89.8-96.4%], compared to 98.1% [95% C.I. 95.3-99.3%] for the two singleplex rRTPCR assays used in combination. In addition, the assay could reliably differentiate between FMDV and other vesicular viruses such as swine vesicular disease virus and vesicular exanthema of swine virus. Interestingly, the mRT-PCR detected parapoxvirus (n=2) and bovine viral diarrhea virus (n=2) in clinical samples, demonstrating the screening potential of this mRT-PCR assay to identify viruses in FMDV-negative material not previously recognized using focused single-target rRT-PCR assays.« less

Performance of rapid diagnostic test, blood-film microscopy and PCR for the diagnosis of malaria infection among febrile children from Korogwe District, Tanzania.

PubMed

Mahende, Coline; Ngasala, Billy; Lusingu, John; Yong, Tai-Soon; Lushino, Paminus; Lemnge, Martha; Mmbando, Bruno; Premji, Zul

2016-07-26

Rapid diagnostic tests (RDT) and light microscopy are still recommended for diagnosis to guide the clinical management of malaria despite difficult challenges in rural settings. The performance of these tests may be affected by several factors, including malaria prevalence and intensity of transmission. The study evaluated the diagnostic performance of malaria RDT, light microscopy and polymerase chain reaction (PCR) in detecting malaria infections among febrile children at outpatient clinic in Korogwe District, northeastern Tanzania. The study enrolled children aged 2-59 months with fever and/or history of fever in the previous 48 h attending outpatient clinics. Blood samples were collected for identification of Plasmodium falciparum infection using histidine-rich-protein-2 (HRP-2)-based malaria RDT, light microscopy and conventional PCR. A total of 867 febrile patients were enrolled into the study. Malaria-positive samples were 85/867 (9.8 %, 95 % CI, 7.9-12.0 %) by RDT, 72/867 (8.3 %, 95 % CI, 6.5-10.1 %) by microscopy and 79/677 (11.7 %, 95 % CI, 9.3-14.3 %) by PCR. The performance of malaria RDT compared with microscopy results had sensitivity and positive predictive value (PPV) of 88.9 % (95 % CI, 79.3-95.1 %) and 75.3 % (95 % CI, 64.8-84.0 %), respectively. Confirmation of P. falciparum infection with PCR analysis provided lower sensitivity and PPV of 88.6 % (95 % CI, 79.5-94.7 %) and 84.3 % (95 % CI, 74.7-91.4 %) for RDT compared to microscopy. Diagnosis of malaria infection is still a challenge due to variation in results among diagnostic methods. HRP-2 malaria RDT and microscopy were less sensitive than PCR. Diagnostic tools with high sensitivity are required in areas of low malaria transmission.

Improving the Specificity of Plasmodium falciparum Malaria Diagnosis in High-Transmission Settings with a Two-Step Rapid Diagnostic Test and Microscopy Algorithm.

PubMed

Murungi, Moses; Fulton, Travis; Reyes, Raquel; Matte, Michael; Ntaro, Moses; Mulogo, Edgar; Nyehangane, Dan; Juliano, Jonathan J; Siedner, Mark J; Boum, Yap; Boyce, Ross M

2017-05-01

Poor specificity may negatively impact rapid diagnostic test (RDT)-based diagnostic strategies for malaria. We performed real-time PCR on a subset of subjects who had undergone diagnostic testing with a multiple-antigen (histidine-rich protein 2 and pan -lactate dehydrogenase pLDH [HRP2/pLDH]) RDT and microscopy. We determined the sensitivity and specificity of the RDT in comparison to results of PCR for the detection of Plasmodium falciparum malaria. We developed and evaluated a two-step algorithm utilizing the multiple-antigen RDT to screen patients, followed by confirmatory microscopy for those individuals with HRP2-positive (HRP2 + )/pLDH-negative (pLDH - ) results. In total, dried blood spots (DBS) were collected from 276 individuals. There were 124 (44.9%) individuals with an HRP2 + /pLDH + result, 94 (34.1%) with an HRP2 + /pLDH - result, and 58 (21%) with a negative RDT result. The sensitivity and specificity of the RDT compared to results with real-time PCR were 99.4% (95% confidence interval [CI], 95.9 to 100.0%) and 46.7% (95% CI, 37.7 to 55.9%), respectively. Of the 94 HRP2 + /pLDH - results, only 32 (34.0%) and 35 (37.2%) were positive by microscopy and PCR, respectively. The sensitivity and specificity of the two-step algorithm compared to results with real-time PCR were 95.5% (95% CI, 90.5 to 98.0%) and 91.0% (95% CI, 84.1 to 95.2), respectively. HRP2 antigen bands demonstrated poor specificity for the diagnosis of malaria compared to that of real-time PCR in a high-transmission setting. The most likely explanation for this finding is the persistence of HRP2 antigenemia following treatment of an acute infection. The two-step diagnostic algorithm utilizing microscopy as a confirmatory test for indeterminate HRP2 + /pLDH - results showed significantly improved specificity with little loss of sensitivity in a high-transmission setting. Copyright © 2017 American Society for Microbiology.

Detection of Mycoplasma pneumoniae by real-time PCR.

PubMed

Winchell, Jonas M; Mitchell, Stephanie L

2013-01-01

Mycoplasma pneumoniae is a significant cause of respiratory disease, accounting for approximately 20% of cases of community-acquired pneumonia. Although several diagnostic methods exist to detect M. pneumoniae in respiratory specimens, real-time PCR has emerged as a significant improvement for the rapid diagnosis of this pathogen. The method described herein details the procedure for the detection of M. pneumoniae by real-time PCR (qPCR). The qPCR assay described can be performed with three targets specific for M. pneumoniae (Mp181, Mp3, and Mp7) and one marker for the detection of the RNaseP gene found in human nucleic acid as an internal control reaction. Recent studies have demonstrated the ability of this procedure to reliably identify this agent and facilitate the timely recognition of an outbreak.

Pitfalls in PCR troubleshooting: Expect the unexpected?

PubMed Central

Schrick, Livia; Nitsche, Andreas

2015-01-01

PCR is a well-understood and established laboratory technique often used in molecular diagnostics. Huge experience has been accumulated over the last years regarding the design of PCR assays and their set-up, including in-depth troubleshooting to obtain the optimal PCR assay for each purpose. Here we report a PCR troubleshooting that came up with a surprising result never observed before. With this report we hope to sensitize the reader to this peculiar problem and to save troubleshooting efforts in similar situations, especially in time-critical and ambitious diagnostic settings. PMID:27077041

Measures to Improve Diagnostic Safety in Clinical Practice

PubMed Central

Singh, Hardeep; Graber, Mark L; Hofer, Timothy P

2016-01-01

Timely and accurate diagnosis is foundational to good clinical practice and an essential first step to achieving optimal patient outcomes. However, a recent Institute of Medicine report concluded that most of us will experience at least one diagnostic error in our lifetime. The report argues for efforts to improve the reliability of the diagnostic process through better measurement of diagnostic performance. The diagnostic process is a dynamic team-based activity that involves uncertainty, plays out over time, and requires effective communication and collaboration among multiple clinicians, diagnostic services, and the patient. Thus, it poses special challenges for measurement. In this paper, we discuss how the need to develop measures to improve diagnostic performance could move forward at a time when the scientific foundation needed to inform measurement is still evolving. We highlight challenges and opportunities for developing potential measures of “diagnostic safety” related to clinical diagnostic errors and associated preventable diagnostic harm. In doing so, we propose a starter set of measurement concepts for initial consideration that seem reasonably related to diagnostic safety, and call for these to be studied and further refined. This would enable safe diagnosis to become an organizational priority and facilitate quality improvement. Health care systems should consider measurement and evaluation of diagnostic performance as essential to timely and accurate diagnosis and to the reduction of preventable diagnostic harm. PMID:27768655

Diagnostic PCR assays to unravel food web interactions in cereal crops with focus on biological control of aphids.

PubMed

Staudacher, Karin; Jonsson, Mattias; Traugott, Michael

Successful biological control of agricultural pests is dependent on a thorough understanding of the underlying trophic interactions between predators and prey. Studying trophic interactions can be challenging, particularly when generalist predators that frequently use multiple prey and interact with both pest and alternative prey are considered. In this context, diagnostic PCR proved to be a suitable approach, however at present, prey-specific PCR primers necessary for assessing such interactions across trophic levels are missing. Here we present a new set of 45 primers designed to target a wide range of invertebrate taxa common to temperate cereal crops: cereal aphids, their natural enemies such as carabid beetles, ladybeetles, lacewings, and spiders, and potential alternative prey groups (earthworms, springtails, and dipterans). These primers were combined in three 'ready to use' multiplex PCR assays for quick and cost-effective analyses of large numbers of predator samples. The assays were tested on 560 carabids collected in barley fields in Sweden. Results from this screening suggest that aphids constitute a major food source for carabids in cereal crops (overall DNA detection rate: 51 %), whereas alternative extraguild and intraguild prey appear to be less frequently preyed upon when aphids are present (11 % for springtails and 12 % for earthworms; 1 % for spiders and 4 % for carabids). In summary, the newly developed molecular assays proved reliable and effective in assessing previously cryptic predator-prey trophic interactions, specifically with focus on biological control of aphids. The diagnostic PCR assays will be applicable manifold as the targeted invertebrates are common to many agricultural systems of the temperate region.

Comparative study of diagnostic accuracy of established PCR assays and in-house developed sdaA PCR method for detection of Mycobacterium tuberculosis in symptomatic patients with pulmonary tuberculosis.

PubMed

Nimesh, Manoj; Joon, Deepali; Pathak, Anil Kumar; Saluja, Daman

2013-11-01

Indian contribution to global burden of tuberculosis is about 26%. In the present study we have developed an in-house PCR assay using primers for sdaA gene of Mycobacterium tuberculosis and evaluated against already established primers devR, IS6110, MPB64, rpoB primers for diagnosis of pulmonary tuberculosis. Using universal sample preparation (USP) method, DNA was extracted from sputum specimens of 412 symptomatic patients from Delhi, India. The DNA so extracted was used as template for PCR amplification using primers targeting sdaA, devR, IS6110, MPB64 and rpoB genes. Out of 412, 149 specimens were considered positive based on composite reference standard (CRS) criteria. The in-house designed sdaA PCR showed high specificity (96.5%), the high positive likelihood ratio (28), the high sensitivity (95.9%), and the very low negative likelihood ratio (0.04) in comparison to CRS. Based on our results, the sdaA PCR assay can be considered as one of the most reliable diagnostic tests in comparison to other PCR based detection methods. Copyright © 2013 The British Infection Association. Published by Elsevier Ltd. All rights reserved.

Diagnostic evaluation of a multiplexed RT-PCR microsphere array assay for the detection of foot-and-mouth disease virus and look-alike disease viruses

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

Hindson, B J; Reid, S M; Baker, B R

2007-07-26

A high-throughput multiplexed assay was developed for the differential laboratory diagnosis of foot-and-mouth disease virus (FMDV) from viruses which cause clinically similar diseases of livestock. This assay simultaneously screens for five RNA and two DNA viruses using multiplexed reverse transcription PCR (mRT-PCR) amplification coupled with a microsphere hybridization array and flow-cytometric detection. Two of the seventeen primer-probe sets included in this multiplex assay were adopted from previously characterized real-time RT-PCR (rRT-PCR) assays for FMDV. The diagnostic accuracy of the mRT-PCR was evaluated using 287 field samples, including 248 (true positive n= 213, true negative n=34) from suspect cases of foot-and-mouthmore » disease collected from 65 countries between 1965 and 2006 and 39 true negative samples collected from healthy animals. The mRT-PCR assay results were compared with two singleplex rRT-PCR assays, using virus isolation with antigen-ELISA as the reference method. The diagnostic sensitivity of the mRT-PCR assay for FMDV was 93.9% [95% C.I. 89.8-96.4%], compared to 98.1% [95% C.I. 95.3-99.3%] for the two singleplex rRT-PCR assays used in combination. In addition, the assay could reliably differentiate between FMDV and other vesicular viruses such as swine vesicular disease virus and vesicular exanthema of swine virus. Interestingly, the mRT-PCR detected parapoxvirus (n=2) and bovine viral diarrhea virus (n=2) in clinical samples, demonstrating the screening potential of this mRT-PCR assay to identify viruses in FMDV-negative material not previously recognized using focused single-target rRT-PCR assays.« less

Diagnostic accuracy of a loop-mediated isothermal PCR assay for detection of Orientia tsutsugamushi during acute Scrub Typhus infection.

PubMed

Paris, Daniel H; Blacksell, Stuart D; Nawtaisong, Pruksa; Jenjaroen, Kemajittra; Teeraratkul, Achara; Chierakul, Wirongrong; Wuthiekanun, Vanaporn; Kantipong, Pacharee; Day, Nicholas P J

2011-09-01

There is an urgent need to develop rapid and accurate point-of-care (POC) technologies for acute scrub typhus diagnosis in low-resource, primary health care settings to guide clinical therapy. In this study we present the clinical evaluation of loop-mediated isothermal PCR assay (LAMP) in the context of a prospective fever study, including 161 patients from scrub typhus-endemic Chiang Rai, northern Thailand. A robust reference comparator set comprising following 'scrub typhus infection criteria' (STIC) was used: a) positive cell culture isolate and/or b) an admission IgM titer ≥1∶12,800 using the 'gold standard' indirect immunofluorescence assay (IFA) and/or c) a 4-fold rising IFA IgM titer and/or d) a positive result in at least two out of three PCR assays. Compared to the STIC criteria, all PCR assays (including LAMP) demonstrated high specificity ranging from 96-99%, with sensitivities varying from 40% to 56%, similar to the antibody based rapid test, which had a sensitivity of 47% and a specificity of 95%. The diagnostic accuracy of the LAMP assay was similar to realtime and nested conventional PCR assays, but superior to the antibody-based rapid test in the early disease course. The combination of DNA- and antibody-based detection methods increased sensitivity with minimal reduction of specificity, and expanded the timeframe of adequate diagnostic coverage throughout the acute phase of scrub typhus.

The Diagnostic Utility of Bact/ALERT and Nested PCR in the Diagnosis of Tuberculous Meningitis.

PubMed

Sastry, Apurba Sankar; Bhat K, Sandhya; Kumudavathi

2013-01-01

The early laboratory diagnosis of Tuberculous Meningitis (TBM) is crucial, to start the antitubercular chemotherapy and to prevent its complications. However, the conventional methods are either less sensitive or time consuming. Hence, the diagnostic potentials of BacT/ALERT and Polymerase Chain Reaction (PCR) was evaluated in this study. The study group comprised of 62 cases and 33 controls. The cases were divided according to Ahuja's criteria into the confirmed (two cases), highly probable (19 cases), probable (26 cases) and the possible (15 cases) subgroups. Ziehl Neelsen's (ZN) and Auramine Phenol (AP) staining, Lowenstein Jensen (LJ) medium culture, BacT/ALERT and nested Polymerase Chain Reaction (PCR) which targeted IS6110 were carried out on all the patients. The sensitivity of the LJ culture was 3.22%. BacT/ALERT showed a sensitivity and a specificity of 25.80% and 100% and those of nested PCR were found to be 40.32% and 96.97% respectively. The mean detection time of growth of the LJ culture was 31.28 days, whereas that of BacT/ALERT was 20.68 days. The contamination rate in the LJ culture and BacT/ALERT were 7.2% and 5.8% respectively. Nested PCR was found to be more sensitive, followed by BacT/ALERT as compared to the LJ culture and smear microscopy. As both false negative and false positive results have been reported for nested PCR, so it should not be used alone as a criterion for initiating or terminating the therapy, but it should be supported by clinical, radiological, cytological and other microbiological findings.

The market trend analysis and prospects of cancer molecular diagnostics kits.

PubMed

Seo, Ju Hwan; Lee, Joon Woo; Cho, Daemyeong

2018-01-01

The molecular diagnostics market can be broadly divided into PCR (rt-PCR, d-PCR), NGS(Next Generation Sequencing), Microarray, FISH(Fluorescent in situ-hybridization) and other categories, based on the diagnostic technique. Also, depending on the disease being diagnosed, the market can also be divided into cancer, infectious diseases, HIV/STDs (herpes, syphilis), and women's health issues such as breast cancer, cervical cancer, ovarian cancer, HPV(human papillomavirus), and vaginitis.Chromosome analysis (including Fluorescent In-situ Hybridization) is one type of blood cancer diagnostic method, which involves the direct detection of individual cells with chromosomal translocation, but there have been problems of sensitivity when using this method. PCR targeting individual genes or the RT (reverse transcription)-PCR method offers outstanding sensitivity, but one drawback is the risk of false-positive reaction caused by contamination of samples, etc. Blood cancer molecular diagnostics kits allow us to overcome these shortcomings, and related products have been under development, with a focus on improving detection sensitivity, enabling multiple tests, and reducing the cost and diagnostic time. Blood cancer molecular diagnostics is usually performed based on platforms such as PCR. The global market for blood cancer molecular diagnostics kits is $ 335.9 million as of 2016 and is expected to reach $ 6980 million in 2026 with an average annual growth rate of 32.9%. The market in South Korea is anticipated to grow at an average annual rate of 28.9%, from $ 3.75 million as of 2016 to $ 60.89 million in 2026. The Market for blood cancer molecular diagnostics kits is judged to be higher in growth possibility due to the increase in the number of cancer patients.

Using Rapid Diagnostic Tests as a Source of Viral RNA for Dengue Serotyping by RT-PCR - A Novel Epidemiological Tool

PubMed Central

Vongsouvath, Manivanh; Phommasone, Koukeo; Sengvilaipaseuth, Onanong; Kosoltanapiwat, Nathamon; Chantratita, Narisara; Blacksell, Stuart D.; Lee, Sue J.; de Lamballerie, Xavier; Mayxay, Mayfong; Keomany, Sommay; Newton, Paul N.; Dubot-Pérès, Audrey

2016-01-01

Background Dengue virus infection causes major public health problems in tropical and subtropical areas. In many endemic areas, including the Lao PDR, inadequate access to laboratory facilities is a major obstacle to surveillance and study of dengue epidemiology. Filter paper is widely used for blood collection for subsequent laboratory testing for antibody and nucleic acid detection. For the first time, we demonstrate that dengue viral RNA can be extracted from dengue rapid diagnostic tests (RDT) and then submitted to real-time RT-PCR for serotyping. Methodology/Principal Findings We evaluated the Standard Diagnostics (SD) Bioline Dengue Duo RDT, a commonly used test in dengue endemic areas. First, using the QIAamp RNA kit, dengue RNA was purified from the sample pad of the NS1 RDT loaded with virus isolates of the four serotypes, then quantified by RT-PCR. We observed greater recovery of virus, with a mean of 27 times more RNA recovered from RDT, than from filter paper. Second, we evaluated dengue NS1 RDTs from patients at Mahosot Hospital, Vientiane, (99 patients) and from rural Salavan Provincial Hospital (362 patients). There was good agreement between dengue RT-PCR from NS1 RDT with RT-PCR performed on RNA extracted from patient sera, either using RDT loaded with blood (82.8% and 91.4%, in Vientiane and Salavan, respectively) or serum (91.9% and 93.9%). There was 100% concordance between RDT and serum RT-PCR of infecting dengue serotype. Conclusions/Significance Therefore, the collection of NS1 positive RDTs, which do not require cold storage, may be a novel approach for dengue serotyping by RT-PCR and offers promising prospects for the collection of epidemiological data from previously inaccessible tropical areas to aid surveillance and public health interventions. PMID:27159058

Comparative diagnostics of allergy using quantitative immuno-PCR and ELISA.

PubMed

Simonova, Maria A; Pivovarov, Victor D; Ryazantsev, Dmitry Y; Dolgova, Anna S; Berzhets, Valentina M; Zavriev, Sergei K; Svirshchevskaya, Elena V

2018-05-01

Estimation of specific IgE is essential for the prevention of allergy progression. Quantitative immuno-PCR (qiPCR) can increase the sensitivity of IgE detection. We aimed to develop qiPCR and compare it to the conventional ELISA in identification of IgE to Alt a 1 and Fel d 1 allergens. Single stranded 60-mer DNA conjugated to streptavidin was used to detect antigen-IgE-biotin complex by qiPCR. In semi-logarithmic scale qiPCR data were linear in a full range of serum dilutions resulting in three- to ten-times higher sensitivity of qiPCR in comparison with ELISA in IgE estimation in low titer sera. Higher sensitivity of qiPCR in identification of low titer IgE is a result of a higher linearity of qiPCR data.

[Tropheryma whipplei and Whipple disease: false positive PCR detections of Tropheryma whipplei in diagnostic samples are rare].

PubMed

Le Scanff, J; Gaultier, J B; Durand, D Vital; Durieu, I; Celard, M; Benito, Y; Vandenesch, F; Rousset, H

2008-11-01

PCR can be used to detect T. whipplei (Tw) in samples from variable tissue types and body fluids. We report clinical, evolutive characteristics and final diagnosis in patients with positive Tw PCR assay. Retrospective study of Tw PCR realized since 10years in a microbiology laboratory. Twenty-five Tw PCR assays were positive among 200 realized. Diagnosis was not confirmed in six cases. One patient was missing for follow up. Eighteen patients presented with Whipple's disease. Among these 18 patients, 14 had a classic Whipple's disease, three patients presented an endocarditis and one patient isolated neurological manifestations. Ten patients presented fever, seven a weight loss and 12 joint involvement. Four patients presented cutaneous manifestations, only six had gastrointestinal symptoms. Neurological involvement was reported in five cases, pulmonary symptoms in four cases, cardiac involvement in six cases and ocular signs in two cases. Anemia was reported in four patients and elevated levels of acute-phase reactants in 14 cases. Positive predictive value of Tw PCR for Whipple's disease diagnosis was 75%. Thirteen patients had a good evolution with antibiotics. Three patients presented recurrence and two cases with cardiovascular involvement died. Whipple's disease is rare but often mentioned in internist experience. The diagnosis should be every time confirmed. Tw PCR assay is an important diagnostic tool but is not sufficient to establish the diagnosis and must be interpreted with histopathology and immunohistochemical testing results.

Measures to Improve Diagnostic Safety in Clinical Practice.

PubMed

Singh, Hardeep; Graber, Mark L; Hofer, Timothy P

2016-10-20

Timely and accurate diagnosis is foundational to good clinical practice and an essential first step to achieving optimal patient outcomes. However, a recent Institute of Medicine report concluded that most of us will experience at least one diagnostic error in our lifetime. The report argues for efforts to improve the reliability of the diagnostic process through better measurement of diagnostic performance. The diagnostic process is a dynamic team-based activity that involves uncertainty, plays out over time, and requires effective communication and collaboration among multiple clinicians, diagnostic services, and the patient. Thus, it poses special challenges for measurement. In this paper, we discuss how the need to develop measures to improve diagnostic performance could move forward at a time when the scientific foundation needed to inform measurement is still evolving. We highlight challenges and opportunities for developing potential measures of "diagnostic safety" related to clinical diagnostic errors and associated preventable diagnostic harm. In doing so, we propose a starter set of measurement concepts for initial consideration that seem reasonably related to diagnostic safety and call for these to be studied and further refined. This would enable safe diagnosis to become an organizational priority and facilitate quality improvement. Health-care systems should consider measurement and evaluation of diagnostic performance as essential to timely and accurate diagnosis and to the reduction of preventable diagnostic harm.This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal.

ECM1 and TMPRSS4 Are Diagnostic Markers of Malignant Thyroid Neoplasms and Improve the Accuracy of Fine Needle Aspiration Biopsy

PubMed Central

Kebebew, Electron; Peng, Miao; Reiff, Emily; Duh, Quan-Yang; Clark, Orlo H.; McMillan, Alex

2005-01-01

Objective: The objective of this study was to determine whether genes that regulate cellular invasion and metastasis are differentially expressed and could serve as diagnostic markers of malignant thyroid nodules. Summary and Background Data: Patients whose thyroid nodules have indeterminate or suspicious cytologic features on fine needle aspiration (FNA) biopsy require thyroidectomy because of a 20% to 30% risk of thyroid cancer. Cell invasion and metastasis is a hallmark of malignant phenotype; therefore, genes that regulate these processes might be differentially expressed and could serve as diagnostic markers of malignancy. Methods: Differentially expressed genes (2-fold higher or lower) in malignant versus benign thyroid neoplasms were identified by extracellular matrix and adhesion molecule cDNA array analysis and confirmed by real-time quantitative polymerase chain reaction (PCR). The area under the receiver operating characteristic (AUC) curve was calculated to determine diagnostic accuracy of gene expression level cutoffs established by logistic regression analysis. Results: By cDNA array analysis, ADAMTS8, ECM1, MMP8, PLAU, SELP, and TMPRSS4 were upregulated, and by quantitative PCR, ECM1, SELP, and TMPRSS4 mRNA expression was higher in malignant (n = 57) than in benign (n = 38) thyroid neoplasms (P< 0.002). ECM1 and TMPRSS4 mRNA expression levels were independent predictors of a malignant thyroid neoplasm (P < 0.003). The AUC was 0.956 for ECM1 and 0.926 for TMPRSS4. Combining both markers improved their diagnostic use (AUC 0.985; sensitivity, 91.7%; specificity, 89.8%; positive predictive value, 85.7%; negative predictive value, 82.8%). ECM1 and TMPRSS4 expression analysis improved the diagnostic accuracy of FNA biopsy in 35 of 38 indeterminate or suspicious results. The level of ECM1 mRNA expression was higher in TNM stage I differentiated thyroid cancers than in stage II and III tumors (P ≤ 0.031). Conclusions: ECM1 and TMPRSS4 are excellent

High diagnostic yield by CSF-PCR for entero- and herpes simplex viruses and TBEV serology in adults with acute aseptic meningitis in Stockholm.

PubMed

Franzen-Rohl, Elisabeth; Larsson, Kenny; Skoog, Eva; Tiveljung-Lindell, Annika; Grillner, Lena; Aurelius, Elisabeth; Glimåker, Martin

2008-01-01

Acute aseptic meningitis (AAM) affects 10-20/100,000 inhabitants per years in Sweden. Up to the beginning of the 1980s the diagnoses were made by virus isolation and/or determination of viral antibodies in serum. The development of PCR for detection of viruses in CSF samples has increased the sensitivity and diagnostic efficiency considerably. We investigated the aetiology of AAM and the diagnostic efficiency in an adult population in Stockholm, using a limited first-line combination of microbiological assays. CSF and serum samples, consecutively collected in 419 patients with clinical symptoms of AAM in northern Stockholm during 1999-2004, were included. PCR assays for herpes simplex virus (HSV) DNA and enterovirus (EV) RNA in the CSF as well as ELISA for IgM in serum to tick-borne encephalitis virus (TBEV) were performed routinely. A viral diagnosis was obtained in 255 of the 419 cases (62%) with these routinely performed assays. Clinical findings in combination with additional diagnostic tests resulted in an overall aetiological yield of 72%. EV was the major causative agent (27%) followed by TBEV (21%) and HSV-2 (19%). We conclude that consistent use of CSF-PCR for EV and HSV and TBEV serology established a diagnosis in the majority of AAM patients.

Molecular diagnostics of periodontitis.

PubMed

Korona-Głowniak, Izabela; Siwiec, Radosław; Berger, Marcin; Malm, Anna; Szymańska, Jolanta

2017-01-28

The microorganisms that form dental plaque are the main cause of periodontitis. Their identification and the understanding of the complex relationships and interactions that involve these microorganisms, environmental factors and the host's health status enable improvement in diagnostics and targeted therapy in patients with periodontitis. To this end, molecular diagnostics techniques (both techniques based on the polymerase chain reaction and those involving nucleic acid analysis via hybridization) come increasingly into use. On the basis of a literature review, the following methods are presented: polymerase chain reaction (PCR), real-time polymerase chain reaction (real-time PCR), 16S rRNA-encoding gene sequencing, checkerboard and reverse-capture checkerboard hybridization, microarrays, denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE), as well as terminal restriction fragment length polymorphism (TRFLP) and next generation sequencing (NGS). The advantages and drawbacks of each method in the examination of periopathogens are indicated. The techniques listed above allow fast detection of even small quantities of pathogen present in diagnostic material and prove particularly useful to detect microorganisms that are difficult or impossible to grow in a laboratory.

Diagnostic Value of PCR Analysis of Bacteria and Fungi from Blood in Empiric-Therapy-Resistant Febrile Neutropenia ▿

PubMed Central

Nakamura, Akiko; Sugimoto, Yuka; Ohishi, Kohshi; Sugawara, Yumiko; Fujieda, Atsushi; Monma, Fumihiko; Suzuki, Kei; Masuya, Masahiro; Nakase, Kazunori; Matsushima, Yoshiko; Wada, Hideo; Katayama, Naoyuki; Nobori, Tsutomu

2010-01-01

This study aimed to assess the clinical utility of PCR for the analysis of bacteria and fungi from blood for the management of febrile neutropenic patients with hematologic malignancies. Using a PCR system able to detect a broad range of bacteria and fungi, we conducted a prospective pilot study of periodic analyses of blood from patients following intensive chemotherapy. When fever occurred, it was treated with empirical antibiotic therapy, basically without knowledge of the PCR results. In 23 febrile episodes during the neutropenic period, bacteria were detected by PCR in 11 cases, while the same species were identified by blood culture in 3 cases. In 10 out of 11 PCR-positive cases, fever could be managed by empirical therapy. In the empirical-therapy-resistant case, the identification of Stenotrophomonas maltophilia by PCR led to improvement of fever. No fungi were detected by PCR in febrile cases, while Aspergillus fumigatus was detected in one afebrile patient, several days before a clinical diagnosis was made. In subsequent sporadic PCR analyses in 15 cases of febrile neutropenia, bacteria were detected by both PCR and blood culture in 7 cases and by PCR alone in 6. Fungi were not detected. While fever was improved by empirical therapy in 12 out of the 13 PCR-positive cases, the identification of Pseudomonas aeruginosa by PCR in one therapy-resistant case contributed to the successful treatment of persistent fever. Our results indicate that PCR analysis of bacteria from blood provides essential information for managing empirical-therapy-resistant febrile neutropenia. PMID:20392911

Improved Efficiency and Robustness in qPCR and Multiplex End-Point PCR by Twisted Intercalating Nucleic Acid Modified Primers

PubMed Central

Schneider, Uffe Vest; Mikkelsen, Nikolaj Dam; Lindqvist, Anja; Okkels, Limei Meng; Jøhnk, Nina; Lisby, Gorm

2012-01-01

We introduce quantitative polymerase chain reaction (qPCR) primers and multiplex end-point PCR primers modified by the addition of a single ortho-Twisted Intercalating Nucleic Acid (o-TINA) molecule at the 5′-end. In qPCR, the 5′-o-TINA modified primers allow for a qPCR efficiency of 100% at significantly stressed reaction conditions, increasing the robustness of qPCR assays compared to unmodified primers. In samples spiked with genomic DNA, 5′-o-TINA modified primers improve the robustness by increased sensitivity and specificity compared to unmodified DNA primers. In unspiked samples, replacement of unmodified DNA primers with 5′-o-TINA modified primers permits an increased qPCR stringency. Compared to unmodified DNA primers, this allows for a qPCR efficiency of 100% at lowered primer concentrations and at increased annealing temperatures with unaltered cross-reactivity for primers with single nucleobase mismatches. In a previously published octaplex end-point PCR targeting diarrheagenic Escherichia coli, application of 5′-o-TINA modified primers allows for a further reduction (>45% or approximately one hour) in overall PCR program length, while sustaining the amplification and analytical sensitivity for all targets in crude bacterial lysates. For all crude bacterial lysates, 5′-o-TINA modified primers permit a substantial increase in PCR stringency in terms of lower primer concentrations and higher annealing temperatures for all eight targets. Additionally, crude bacterial lysates spiked with human genomic DNA show lesser formation of non-target amplicons implying increased robustness. Thus, 5′-o-TINA modified primers are advantageous in PCR assays, where one or more primer pairs are required to perform at stressed reaction conditions. PMID:22701644

A Nested-Splicing by Overlap Extension PCR Improves Specificity of this Standard Method.

PubMed

Karkhane, Ali Asghar; Yakhchali, Bagher; Rastgar Jazii, Ferdous; Bambai, Bijan; Aminzadeh, Saeed; Rahimi, Fatemeh

2015-06-01

Splicing by overlap extension (SOE) PCR is used to create mutation in the coding sequence of an enzyme in order to study the role of specific residues in protein's structure and function. We introduced a nested-SOE-PCR (N -SOE-PCR) in order to increase the specificity and generating mutations in a gene by SOE-PCR. Genomic DNA from Bacillus thermocatenulatus was extracted. Nested PCR was used to amplify B. thermocatenulatus lipase gene variants, namely wild type and mutant, using gene specific and mutagenic specific primers, followed by cloning in a suitable vector. Briefly in N-SOE-PCR method, instead of two pairs of primers, three pairs of primers are used to amplify a mutagenic fragment. Moreover, the first and second PCR products are slightly longer than PCR products in a conventional SOE. PCR products obtained from the first round of PCR are used for the second PCR by applying the nested and mutated primers. Following to the purification of the amplified fragments, they will be subject of the further purification and will be used as template to perform the third round of PCR using gene specific primers. In the end, the products will be cloned into a suitable vector for subsequent application. In comparison to the conventional SOE-PCR, the improved method (i.e. N-SOE-PCR) increases the yield and specificity of the products. In addition, the proposed method shows a large reduction in the non-specific products. By applying two more primers in the conventional SOE, the specificity of the method will be improved. This would be in part due to annealing of the primers further inside the amplicon that increases both the efficiency and a better attachment of the primers. Positioning of the primer far from both ends of an amplicon leads to an enhanced binding as well as increased affinity in the third round of amplification in SOE.

Evaluation of an Improved U.S. Food and Drug Administration Method for the Detection of Cyclospora cayetanensis in Produce Using Real-Time PCR.

PubMed

Murphy, Helen R; Lee, Seulgi; da Silva, Alexandre J

2017-07-01

Cyclospora cayetanensis is a protozoan parasite that causes human diarrheal disease associated with the consumption of fresh produce or water contaminated with C. cayetanensis oocysts. In the United States, foodborne outbreaks of cyclosporiasis have been linked to various types of imported fresh produce, including cilantro and raspberries. An improved method was developed for identification of C. cayetanensis in produce at the U.S. Food and Drug Administration. The method relies on a 0.1% Alconox produce wash solution for efficient recovery of oocysts, a commercial kit for DNA template preparation, and an optimized TaqMan real-time PCR assay with an internal amplification control for molecular detection of the parasite. A single laboratory validation study was performed to assess the method's performance and compare the optimized TaqMan real-time PCR assay and a reference nested PCR assay by examining 128 samples. The samples consisted of 25 g of cilantro or 50 g of raspberries seeded with 0, 5, 10, or 200 C. cayetanensis oocysts. Detection rates for cilantro seeded with 5 and 10 oocysts were 50.0 and 87.5%, respectively, with the real-time PCR assay and 43.7 and 94.8%, respectively, with the nested PCR assay. Detection rates for raspberries seeded with 5 and 10 oocysts were 25.0 and 75.0%, respectively, with the real-time PCR assay and 18.8 and 68.8%, respectively, with the nested PCR assay. All unseeded samples were negative, and all samples seeded with 200 oocysts were positive. Detection rates using the two PCR methods were statistically similar, but the real-time PCR assay is less laborious and less prone to amplicon contamination and allows monitoring of amplification and analysis of results, making it more attractive to diagnostic testing laboratories. The improved sample preparation steps and the TaqMan real-time PCR assay provide a robust, streamlined, and rapid analytical procedure for surveillance, outbreak response, and regulatory testing of foods for

Detection of respiratory bacterial pathogens causing atypical pneumonia by multiplex Lightmix® RT-PCR.

PubMed

Wagner, Karoline; Springer, Burkard; Imkamp, Frank; Opota, Onya; Greub, Gilbert; Keller, Peter M

2018-04-01

Pneumonia is a severe infectious disease. In addition to common viruses and bacterial pathogens (e.g. Streptococcus pneumoniae), fastidious respiratory pathogens like Chlamydia pneumoniae, Mycoplasma pneumoniae and Legionella spp. can cause severe atypical pneumonia. They do not respond to penicillin derivatives, which may cause failure of antibiotic empirical therapy. The same applies for infections with B. pertussis and B. parapertussis, the cause of pertussis disease, that may present atypically and need to be treated with macrolides. Moreover, these fastidious bacteria are difficult to identify by culture or serology, and therefore often remain undetected. Thus, rapid and accurate identification of bacterial pathogens causing atypical pneumonia is crucial. We performed a retrospective method evaluation study to evaluate the diagnostic performance of the new, commercially available Lightmix ® multiplex RT-PCR assay that detects these fastidious bacterial pathogens causing atypical pneumonia. In this retrospective study, 368 clinical respiratory specimens, obtained from patients suffering from atypical pneumonia that have been tested negative for the presence of common agents of pneumonia by culture and viral PCR, were investigated. These clinical specimens have been previously characterized by singleplex RT-PCR assays in our diagnostic laboratory and were used to evaluate the diagnostic performance of the respiratory multiplex Lightmix ® RT-PCR. The multiplex RT-PCR displayed a limit of detection between 5 and 10 DNA copies for different in-panel organisms and showed identical performance characteristics with respect to specificity and sensitivity as in-house singleplex RT-PCRs for pathogen detection. The Lightmix ® multiplex RT-PCR assay represents a low-cost, time-saving and accurate diagnostic tool with high throughput potential. The time-to-result using an automated DNA extraction device for respiratory specimens followed by multiplex RT-PCR detection was

Rapid and sensitive multiplex single-tube nested PCR for the identification of five human Plasmodium species.

PubMed

Saito, Takahiro; Kikuchi, Aoi; Kaneko, Akira; Isozumi, Rie; Teramoto, Isao; Kimura, Masatsugu; Hirasawa, Noriyasu; Hiratsuka, Masahiro

2018-06-01

Malaria is caused by five species of Plasmodium in humans. Microscopy is currently used for pathogen detection, requiring considerable training and technical expertise as the parasites are often difficult to differentiate morphologically. Rapid diagnostic tests are as reliable as microscopy and offer faster diagnoses but possess lower detection limits and are incapable of distinguishing among the parasitic species. To improve global health efforts towards malaria control, a rapid, sensitive, species-specific, and economically viable diagnostic method is needed. In this study, we designed a malaria diagnostic method involving a multiplex single-tube nested PCR targeting Plasmodium mitochondrial cytochrome c oxidase III and single-stranded tag hybridization chromatographic printed-array strip. The detection sensitivity was found to be at least 40 times higher than that of agarose gel electrophoresis with ethidium bromide. This system also enables the identification of both single- and mixed-species malaria infections. The assay was validated with 152 Kenyan samples; using nested PCR as the standard, the assay's sensitivity and specificity were 88.7% and 100.0%, respectively. The turnaround time required, from PCR preparation to signal detection, is 90min. Our method should improve the diagnostic speed, treatment efficacy, and control of malaria, in addition to facilitating surveillance within global malaria eradication programs. Copyright © 2018 Elsevier B.V. All rights reserved.

[Mission oriented diagnostic real-time PCR].

PubMed

Tomaso, Herbert; Scholz, Holger C; Al Dahouk, Sascha; Splettstoesser, Wolf D; Neubauer, Heinrich; Pfeffer, Martin; Straube, Eberhard

2007-01-01

In out of area military missions soldiers are potentially exposed to bacteria that are endemic in tropical areas and can be used as biological agents. It can be difficult to culture these bacteria due to sample contamination, low number of bacteria or pretreatment with antibiotics. Commercial biochemical identification systems are not optimized for these agents which can result in misidentification. Immunological assays are often not commercially available or not specific. Real-time PCR assays are very specific and sensitive and can shorten the time required to establish a diagnosis markedly. Therefore, real-time PCRs for the identification of Bacillus anthracis, Brucella spp., Burkholderia mallei und Burkholderia pseudomallei, Francisella tularensis und Yersinia pestis have been developed. PCR results can be false negative due to inadequate clinical samples, low number of bacteria in samples, DNA degradation, inhibitory substances and inappropriate DNA preparation. Hence, it is crucial to cultivate the organisms as a prerequisite for adequate antibiotic therapy and typing of the agent. In a bioterrorist scenario samples have to be treated according to rules applied in forensic medicine and documentation has to be flawless.

[Laboratory diagnostics of urogenital clamidiosis].

PubMed

Churakov, A A; Kulichenko, A N; Kzakova, E S; Serebrianik, N E; Suvorov, A P; Kutyrev, V V; Glybochko, P V

2005-02-01

Laboratory diagnostic tools of urogenital clamidiosis--PCR, ELISA (IgG and IgM) and direct immunofluorescence (DIF)--were comparatively analyzed. The positive PCR result was checked by another PCR test with a different primer; 5 false positive responses were registered (specificity 99.6%). As against PCR, the sensitivity of ELISA made 53%, its specificity -75.5%, the diagnostic value of positive result -58%, the diagnostic value of negative result -71.6% and the diagnostics accuracy -66.7%. The respective DIF parameters were as follows: 36%, 90%, 81.5%, 54.2% and 60.9%. A high rate of detection (above 90%) of the conditionally pathogenic microflora associated with Chlamydia trachomatis (above 110 microbe cells/ml) was pointed out. Hardnerelli and ureaplasms were more often found in female smears, staphylococci and enterococci--in male sperm. It is underlined as important to hold complex examinations for Chlamidia (PCR, ELISA and DIC as an additional test) combined with bacteriological quantification of the conditionally pathogenic microflora and determination of its resistance to antibiotics.

Improving early cycle economic evaluation of diagnostic technologies.

PubMed

Steuten, Lotte M G; Ramsey, Scott D

2014-08-01

The rapidly increasing range and expense of new diagnostics, compels consideration of a different, more proactive approach to health economic evaluation of diagnostic technologies. Early cycle economic evaluation is a decision analytic approach to evaluate technologies in development so as to increase the return on investment as well as patient and societal impact. This paper describes examples of 'early cycle economic evaluations' as applied to diagnostic technologies and highlights challenges in its real-time application. It shows that especially in the field of diagnostics, with rapid technological developments and a changing regulatory climate, early cycle economic evaluation can have a guiding role to improve the efficiency of the diagnostics innovation process. In the next five years the attention will move beyond the methodological and analytic challenges of early cycle economic evaluation towards the challenge of effectively applying it to improve diagnostic research and development and patient value. Future work in this area should therefore be 'strong on principles and soft on metrics', that is, the metrics that resonate most clearly with the various decision makers in this field.

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 and evaluation of a quantitative PCR assay for detection of Hepatozoon sp.

PubMed

Criado-Fornelio, A; Buling, A; Cunha-Filho, N A; Ruas, J L; Farias, N A R; Rey-Valeiron, C; Pingret, J L; Etievant, M; Barba-Carretero, J C

2007-12-25

With the aim to improve current molecular diagnostic techniques of Hepatozoon sp. in carnivore mammals, we developed a quantitative PCR (qPCR) assay with SYBR Green I((R)). The method, consisting of amplification of a 235bp fragment of the 18S rRNA gene, is able to detect at least 0.1fg of parasite DNA. Reproducible quantitative results were obtained over a range of 0.1ng-0.1fg of Hepatozoon sp. DNA. To assess the performance of the qPCR assay, DNA samples from dogs (140) and cats (50) were tested with either standard PCR or qPCR. Positive samples were always confirmed by partial sequencing of the 18S rRNA gene. Quantitative PCR was 15.8% more sensitive than standard PCR to detect H. canis in dogs. In cats, no infections were detected by standard PCR, compared to two positives by qPCR (which were infected by H. canis as shown by sequencing).

PCR Amplification Strategies towards full-length HIV-1 Genome sequencing.

PubMed

Liu, Chao Chun; Ji, Hezhao

2018-06-26

The advent of next generation sequencing has enabled greater resolution of viral diversity and improved feasibility of full viral genome sequencing allowing routine HIV-1 full genome sequencing in both research and diagnostic settings. Regardless of the sequencing platform selected, successful PCR amplification of the HIV-1 genome is essential for sequencing template preparation. As such, full HIV-1 genome amplification is a crucial step in dictating the successful and reliable sequencing downstream. Here we reviewed existing PCR protocols leading to HIV-1 full genome sequencing. In addition to the discussion on basic considerations on relevant PCR design, the advantages as well as the pitfalls of published protocols were reviewed. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Improvement in laboratory diagnosis of wound botulism and tetanus among injecting illicit-drug users by use of real-time PCR assays for neurotoxin gene fragments.

PubMed

Akbulut, D; Grant, K A; McLauchlin, J

2005-09-01

An upsurge in wound infections due to Clostridium botulinum and Clostridium tetani among users of illegal injected drugs (IDUs) occurred in the United Kingdom during 2003 and 2004. A real-time PCR assay was developed to detect a fragment of the neurotoxin gene of C. tetani (TeNT) and was used in conjunction with previously described assays for C. botulinum neurotoxin types A, B, and E (BoNTA, -B, and -E). The assays were sensitive, specific, rapid to perform, and applicable to investigating infections among IDUs using DNA extracted directly from wound tissue, as well as bacteria growing among mixed microflora in enrichment cultures and in pure culture on solid media. A combination of bioassay and PCR test results confirmed the clinical diagnosis in 10 of 25 cases of suspected botulism and two of five suspected cases of tetanus among IDUs. The PCR assays were in almost complete agreement with the conventional bioassays when considering results from different samples collected from the same patient. The replacement of bioassays by real-time PCR for the isolation and identification of both C. botulinum and C. tetani demonstrates a sensitivity and specificity similar to those of conventional approaches. However, the real-time PCR assays substantially improves the diagnostic process in terms of the speed of results and by the replacement of experimental animals. Recommendations are given for an improved strategy for the laboratory investigation of suspected wound botulism and tetanus among IDUs.

Improvement and automation of a real-time PCR assay for vaginal fluids.

PubMed

De Vittori, E; Giampaoli, S; Barni, F; Baldi, M; Berti, A; Ripani, L; Romano Spica, V

2016-05-01

The identification of vaginal fluids is crucial in forensic science. Several molecular protocols based on PCR amplification of mfDNA (microflora DNA) specific for vaginal bacteria are now available. Unfortunately mfDNA extraction and PCR reactions require manual optimization of several steps. The aim of present study was the verification of a partial automatization of vaginal fluids identification through two instruments widely diffused in forensic laboratories: EZ1 Advanced robot and Rotor Gene Q 5Plex HRM. Moreover, taking advantage of 5-plex thermocycler technology, the ForFluid kit performances were improved by expanding the mfDNA characterization panel with a new bacterial target for vaginal fluids and with an internal positive control (IPC) to monitor PCR inhibition. Results underlined the feasibility of a semi-automated extraction of mfDNA using a BioRobot and demonstrated the analytical improvements of the kit. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Multi-site evaluation of the LN34 pan-lyssavirus real-time RT-PCR assay for post-mortem rabies diagnostics

PubMed Central

Dettinger, Lisa; Powell, James W.; Seiders, Melanie; Condori, Rene Edgar Condori; Griesser, Richard; Okogi, Kenneth; Carlos, Maria; Pesko, Kendra; Breckenridge, Mike; Simon, Edson Michael M.; Chu, Maria Yna Joyce V.; Davis, April D.; Brunt, Scott J.; Orciari, Lillian; Yager, Pamela; Carson, William C.; Hartloge, Claire; Saliki, Jeremiah T.; Deldari, Mojgan; Hsieh, Kristina; Wadhwa, Ashutosh; Wilkins, Kimberly; Rabideau, Patricia; Gruhn, Nina; Cadet, Rolain; Isloor, Shrikrishna; Nath, Sujith S.; Joseph, Tomy; Gao, Jinxin; Wallace, Ryan; Reynolds, Mary; Olson, Victoria A.

2018-01-01

Rabies is a fatal zoonotic disease that requires fast, accurate diagnosis to prevent disease in an exposed individual. The current gold standard for post-mortem diagnosis of human and animal rabies is the direct fluorescent antibody (DFA) test. While the DFA test has proven sensitive and reliable, it requires high quality antibody conjugates, a skilled technician, a fluorescence microscope and diagnostic specimen of sufficient quality. The LN34 pan-lyssavirus real-time RT-PCR assay represents a strong candidate for rabies post-mortem diagnostics due to its ability to detect RNA across the diverse Lyssavirus genus, its high sensitivity, its potential for use with deteriorated tissues, and its simple, easy to implement design. Here, we present data from a multi-site evaluation of the LN34 assay in 14 laboratories. A total of 2,978 samples (1,049 DFA positive) from Africa, the Americas, Asia, Europe, and the Middle East were tested. The LN34 assay exhibited low variability in repeatability and reproducibility studies and was capable of detecting viral RNA in fresh, frozen, archived, deteriorated and formalin-fixed brain tissue. The LN34 assay displayed high diagnostic specificity (99.68%) and sensitivity (99.90%) when compared to the DFA test, and no DFA positive samples were negative by the LN34 assay. The LN34 assay produced definitive findings for 80 samples that were inconclusive or untestable by DFA; 29 were positive. Five samples were inconclusive by the LN34 assay, and only one sample was inconclusive by both tests. Furthermore, use of the LN34 assay led to the identification of one false negative and 11 false positive DFA results. Together, these results demonstrate the reliability and robustness of the LN34 assay and support a role for the LN34 assay in improving rabies diagnostics and surveillance. PMID:29768505

Cost-effectiveness of diagnostic strategies using quantitative real-time PCR and bacterial culture to identify contagious mastitis cases in large dairy herds.

PubMed

Murai, Kiyokazu; Lehenbauer, Terry W; Champagne, John D; Glenn, Kathy; Aly, Sharif S

2014-03-01

Diagnostic strategies to detect contagious mastitis caused by Mycoplasma bovis, Staphylococcus aureus, and Streptococcus agalactiae in dairy herds during an outbreak have been minimally studied with regard to cost and diagnostic sensitivity. The objective of this cross-sectional study was to compare the cost-effectiveness of diagnostic strategies for identification of infected cows in two California dairy herds during contagious mastitis outbreaks. M. bovis was investigated in a subset of a herd (n=1210 cows) with an estimated prevalence of 2.8% (95% CI=1.9, 3.7), whereas Staph. aureus and Strep. agalactiae were studied in a second herd (n=351 cows) with an estimated prevalence of 3.4% (95% CI=1.5, 5.3) and 16.8% (95% CI=12.9, 20.7), respectively. Diagnostic strategies involved a combination of testing stages that utilized bacterial culture, quantitative real-time PCR (qPCR), or both. Strategies were applied to individual or pooled samples of 5, 10, 50 or 100 samples. Culture was considered the gold standard for sensitivity estimation of each strategy. The reference strategy was the strategy with the lowest cost per culture-positive cow which for both M. bovis and Strep. agalactiae consisted of 2 stages, culture of samples in pools of 5 followed by culture of individual samples in positive pools with a sensitivity of 73.5% (95% CI: 55.6, 87.1) and 96.6% (95% CI: 27.7, 84.8), respectively. The reference strategy for Staph. aureus consisted of 3 stages, culture of individual samples in pools of 100 (stage 1), culture constituents of those positive from stage 1 in pools of 5 (stage 2), culture constituents of those positive from stage 2 individually (stage 3) which resulted in a sensitivity of 58.3% (95% CI: 88.3, 99.6). The most cost-effective alternative to the reference strategy was whole herd milk culture for all 3 pathogens. QPCR testing was a component of the second most cost-effective alternative for M. bovis and the third most cost-effective alternatives for

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.

Improving qPCR telomere length assays: Controlling for well position effects increases statistical power.

PubMed

Eisenberg, Dan T A; Kuzawa, Christopher W; Hayes, M Geoffrey

2015-01-01

Telomere length (TL) is commonly measured using quantitative PCR (qPCR). Although, easier than the southern blot of terminal restriction fragments (TRF) TL measurement method, one drawback of qPCR is that it introduces greater measurement error and thus reduces the statistical power of analyses. To address a potential source of measurement error, we consider the effect of well position on qPCR TL measurements. qPCR TL data from 3,638 people run on a Bio-Rad iCycler iQ are reanalyzed here. To evaluate measurement validity, correspondence with TRF, age, and between mother and offspring are examined. First, we present evidence for systematic variation in qPCR TL measurements in relation to thermocycler well position. Controlling for these well-position effects consistently improves measurement validity and yields estimated improvements in statistical power equivalent to increasing sample sizes by 16%. We additionally evaluated the linearity of the relationships between telomere and single copy gene control amplicons and between qPCR and TRF measures. We find that, unlike some previous reports, our data exhibit linear relationships. We introduce the standard error in percent, a superior method for quantifying measurement error as compared to the commonly used coefficient of variation. Using this measure, we find that excluding samples with high measurement error does not improve measurement validity in our study. Future studies using block-based thermocyclers should consider well position effects. Since additional information can be gleaned from well position corrections, rerunning analyses of previous results with well position correction could serve as an independent test of the validity of these results. © 2015 Wiley Periodicals, Inc.

Improvement of a PCR test to diagnose infection by Mansonella ozzardi.

PubMed

Vera, Luana Janaína Souza; Basano, Sergio de Almeida; Camargo, Juliana de Souza Almeida Aranha; França, Andonai Krauze de; Ferreira, Ricardo de Godoi Mattos; Casseb, Almeida Andrade; Medeiros, Jansen Fernandes; Fontes, Gilberto; Camargo, Luís Marcelo Aranha

2011-01-01

Mansonelliasis is caused by Mansonella ozzardi. It is widespread in the Amazon region, with a high prevalence. The common exam of thick blood smears stained with Giemsa shows low efficacy levels and has been an obstacle to diagnosing individuals with low blood parasitemia. In order to increase diagnosis efficacy, the PCR technique was improved. PCR demonstrated the best performance, with sensitivity and negative predictive values (NPV) of 100%, followed by blood filtration through membrane filters, which showed a sensitivity of 88.9% and a NPV of 84.6%, when compared to thick blood smears.

Application of COLD-PCR for improved detection of KRAS mutations in clinical samples.

PubMed

Zuo, Zhuang; Chen, Su S; Chandra, Pranil K; Galbincea, John M; Soape, Matthew; Doan, Steven; Barkoh, Bedia A; Koeppen, Hartmut; Medeiros, L Jeffrey; Luthra, Rajyalakshmi

2009-08-01

KRAS mutations have been detected in approximately 30% of all human tumors, and have been shown to predict response to some targeted therapies. The most common KRAS mutation-detection strategy consists of conventional PCR and direct sequencing. This approach has a 10-20% detection sensitivity depending on whether pyrosequencing or Sanger sequencing is used. To improve detection sensitivity, we compared our conventional method with the recently described co-amplification-at-lower denaturation-temperature PCR (COLD-PCR) method, which selectively amplifies minority alleles. In COLD-PCR, the critical denaturation temperature is lowered to 80 degrees C (vs 94 degrees C in conventional PCR). The sensitivity of COLD-PCR was determined by assessing serial dilutions. Fifty clinical samples were used, including 20 fresh bone-marrow aspirate specimens and the formalin-fixed paraffin-embedded (FFPE) tissue of 30 solid tumors. Implementation of COLD-PCR was straightforward and required no additional cost for reagents or instruments. The method was specific and reproducible. COLD-PCR successfully detected mutations in all samples that were positive by conventional PCR, and enhanced the mutant-to-wild-type ratio by >4.74-fold, increasing the mutation detection sensitivity to 1.5%. The enhancement of mutation detection by COLD-PCR inversely correlated with the tumor-cell percentage in a sample. In conclusion, we validated the utility and superior sensitivity of COLD-PCR for detecting KRAS mutations in a variety of hematopoietic and solid tumors using either fresh or fixed, paraffin-embedded tissue.

Analytical validation of a reverse transcriptase droplet digital PCR (RT-ddPCR) for quantitative detection of infectious hematopoietic necrosis virus

USGS Publications Warehouse

Jia, Peng; Purcell, Maureen; Pan, Guang; Wang, Jinjin; Kan, Shifu; Liu, Yin; Zheng, Xiaocong; SHi, Xiujie; He, Junqiang; Yu, Li; Hua, Qunyi; Lu, Tikang; Lan, Wensheng; Winton, James; Jin, Ningyi; Liu, Hong

2017-01-01

Infectious hematopoietic necrosis virus (IHNV) is an important pathogen of salmonid fishes. A validated universal reverse transcriptase quantitative PCR (RT-qPCR) assay that can quantify levels of IHNV in fish tissues has been previously reported. In the present study, we adapted the published set of IHNV primers and probe for use in a reverse-transcriptase droplet digital PCR (RT-ddPCR) assay for quantification of the virus in fish tissue samples. The RT-ddPCR and RT-qPCR assays detected 13 phylogenetically diverse IHNV strains, but neither assay produced detectable amplification when RNA from other fish viruses was used. The RT-ddPCR assay had a limit of detection (LOD) equating to 2.2 plaque forming units (PFU)/μl while the LOD for the RT-qPCR was 0.2 PFU/μl. Good agreement (69.4–100%) between assays was observed when used to detect IHNV RNA in cell culture supernatant and tissues from IHNV infected rainbow trout (Oncorhynchus mykiss) and arctic char (Salvelinus alpinus). Estimates of RNA copy number produced by the two assays were significantly correlated but the RT-qPCR consistently produced higher estimates than the RT-ddPCR. The analytical properties of the N gene RT-ddPCR test indicated that this method may be useful to assess IHNV RNA copy number for research and diagnostic purposes. Future work is needed to establish the within and between laboratory diagnostic performance of the RT-ddPCR assay.

[Improvement of laboratory diagnostics of cholera due to genetically altered (hybrid) variants of cholera Vibrio biovar El Tor].

PubMed

Savel'eva, I V; Khatsukov, K X; Savel'eva, E I; Moskvitina, S I; Kovalev, D A; Savel'ev, V N; Kulichenko, A N; Antonenko, A D; Babenyshev, B V

2015-01-01

Improvement of laboratory diagnostics of cholera taking into the account appearance of hybrid variants of cholera vibrio El Tor biovar in the 1990s. Phenotypic and molecular-genetic properties of typical toxigenic (151 strains) and hybrid (102 strains) variants of El Tor biovar cholera vibrios, isolated in the Caucuses in 1970-1990 and 1993-1998, respectively, were studied. Toxigenicity gene DNA fragments, inherent to El Tor biovars or classic, were detected by using a reagent kit "Genes of Vibrio cholerae variant ctxB-rstR-rstC, REF" developed by us. Reagent kit "Genes of V. cholerae variant ctxB-rstR-rstC, REF" is proposed to be used for laboratory diagnostics of cholera during study of material from humans or environmental objects and for identification of V. cholerae 01 on genome level in PCR-analysis as a necessary addition to the classic scheme of bacteriological analysis. Laboratory diagnostics of cholera due to genetically altered (hybrid) variants of cholera vibrio El Tor biovar is based on a complex study of material from humans and environmental objects by routine bacteriologic and PCR-analysis methods with the aim of detection of gene DNA fragments in the studied material, that determine biovar (classic or El Tor), identification of V. cholerae O1 strains with differentiation of El Tor vibrios into typical and altered, as well as determination of enterotoxin, produced by the specific cholera vibrio strain (by the presence ctxB(El) or ctxB(Cl) gene DNA fragment, coding biosynthesis of CT-2 or CT-1, respectively).

Comparative evaluation of serum, FTA filter-dried blood and oral fluid as sample material for PRRSV diagnostics by RT-qPCR in a small-scale experimental study.

PubMed

Steinrigl, Adolf; Revilla-Fernández, Sandra; Wodak, Eveline; Schmoll, Friedrich; Sattler, Tatjana

2014-01-01

Recently, research into alternative sample materials, such as oral fluid or filter-dried blood has been intensified, in order to facilitate cost-effective and animal-friendly sampling of individuals or groups of pigs for diagnostic purposes. The objective of this study was to compare the sensitivity of porcine reproductive and respiratory syndrome virus (PRRSV)-RNA detection by reverse transcription quantitative real-time PCR (RT-qPCR) in serum, FTA filter-dried blood and oral fluid sampled from individual pigs. Ten PRRSV negative pigs were injected with an EU-type PRRSV live vaccine. Blood and oral fluid samples were taken from each pig before, and 4, 7, 14 and 21 days after vaccination. All samples were then analyzed by PRRSV RT-qPCR. In serum, eight often pigs tested RT-qPCR positive at different time points post infection. Absolute quantification showed low serum PRRSV-RNA loads in most samples. In comparison to serum, sensitivity of PRRSV-RNA detection was strongly reduced in matched FTA filter-dried blood and in oral fluid from the same pigs. These results indicate that with low PRRSV-RNA loads the diagnostic sensitivity of PRRSV-RNA detection by RT-qPCR achieved with serum is currently unmatched by either FTA filter-dried blood or oral fluid.

Diagnostic accuracy of PCR in gambiense sleeping sickness diagnosis, staging and post-treatment follow-up: a 2-year longitudinal study.

PubMed

Deborggraeve, Stijn; Lejon, Veerle; Ekangu, Rosine Ali; Mumba Ngoyi, Dieudonné; Pati Pyana, Patient; Ilunga, Médard; Mulunda, Jean Pierre; Büscher, Philippe

2011-02-22

The polymerase chain reaction (PCR) has been proposed for diagnosis, staging and post-treatment follow-up of sleeping sickness but no large-scale clinical evaluations of its diagnostic accuracy have taken place yet. An 18S ribosomal RNA gene targeting PCR was performed on blood and cerebrospinal fluid (CSF) of 360 T. brucei gambiense sleeping sickness patients and on blood of 129 endemic controls from the Democratic Republic of Congo. Sensitivity and specificity (with 95% confidence intervals) of PCR for diagnosis, disease staging and treatment failure over 2 years follow-up post-treatment were determined. Reference standard tests were trypanosome detection for diagnosis and trypanosome detection and/or increased white blood cell concentration in CSF for staging and detection of treatment failure. PCR on blood showed a sensitivity of 88.4% (84.4-92.5%) and a specificity of 99.2% (97.7-100%) for diagnosis, while for disease staging the sensitivity and specificity of PCR on cerebrospinal fluid were 88.4% (84.8-91.9%) and 82.9% (71.2-94.6%), respectively. During follow-up after treatment, PCR on blood had low sensitivity to detect treatment failure. In cerebrospinal fluid, PCR positivity vanished slowly and was observed until the end of the 2 year follow-up in around 20% of successfully treated patients. For T.b. gambiense sleeping sickness diagnosis and staging, PCR performed better than, or similar to, the current parasite detection techniques but it cannot be used for post-treatment follow-up. Continued PCR positivity in one out of five cured patients points to persistence of living or dead parasites or their DNA after successful treatment and may necessitate the revision of some paradigms about the pathophysiology of sleeping sickness.

Digital PCR on a SlipChip.

PubMed

Shen, Feng; Du, Wenbin; Kreutz, Jason E; Fok, Alice; Ismagilov, Rustem F

2010-10-21

This paper describes a SlipChip to perform digital PCR in a very simple and inexpensive format. The fluidic path for introducing the sample combined with the PCR mixture was formed using elongated wells in the two plates of the SlipChip designed to overlap during sample loading. This fluidic path was broken up by simple slipping of the two plates that removed the overlap among wells and brought each well in contact with a reservoir preloaded with oil to generate 1280 reaction compartments (2.6 nL each) simultaneously. After thermal cycling, end-point fluorescence intensity was used to detect the presence of nucleic acid. Digital PCR on the SlipChip was tested quantitatively by using Staphylococcus aureus genomic DNA. As the concentration of the template DNA in the reaction mixture was diluted, the fraction of positive wells decreased as expected from the statistical analysis. No cross-contamination was observed during the experiments. At the extremes of the dynamic range of digital PCR the standard confidence interval determined using a normal approximation of the binomial distribution is not satisfactory. Therefore, statistical analysis based on the score method was used to establish these confidence intervals. The SlipChip provides a simple strategy to count nucleic acids by using PCR. It may find applications in research applications such as single cell analysis, prenatal diagnostics, and point-of-care diagnostics. SlipChip would become valuable for diagnostics, including applications in resource-limited areas after integration with isothermal nucleic acid amplification technologies and visual readout.

MT-PCR panel detection of canine parvovirus (CPV-2): Vaccine and wild-type CPV-2 can be difficult to differentiate in canine diagnostic fecal samples.

PubMed

Meggiolaro, Maira N; Ly, Anna; Rysnik-Steck, Benjamin; Silva, Carolina; Zhang, Joshua; Higgins, Damien P; Muscatello, Gary; Norris, Jacqueline M; Krockenberger, Mark; Šlapeta, Jan

2017-06-01

Canine parvovirus (CPV-2) remains an important cause of devastating enteritis in young dogs. It can be successfully prevented with live attenuated CPV-2 vaccines when given at the appropriate age and in the absence of maternal antibody interference. Rapid diagnosis of parvoviral enteritis in young dogs is essential to ensuring suitable barrier nursing protocols within veterinary hospitals. The current diagnostic trend is to use multiplexed PCR panels to detect an array of pathogens commonly responsible for diarrhea in dogs. The multiplexed PCR assays do not distinguish wild from vaccine CPV-2. They are highly sensitive and detect even a low level of virus shedding, such as those caused by the CPV-2 vaccine. The aim of this study was to identify the CPV-2 subtypes detected in diagnostic specimens and rule out occult shedding of CPV-2 vaccine strains. For a total of 21 samples that tested positive for CPV-2 in a small animal fecal pathogens diagnostic multiplexed tandem PCR (MT-PCR) panel during 2014-2016 we partially characterized the VP2 gene of CPV-2. Vaccine CPV-2 strain, wild type CPV-2a subtypes and vaccine-like CPV-2b subtypes were detected. High copy number was indicative of wild-type CPV-2a presence, but presence of vaccine-like CPV-2b had a variable copy number in fecal samples. A yardstick approach to a copy number or C t -value to discriminate vaccine strain from a wild type virus of CPV-2 can be, in some cases, potentially misleading. Therefore, discriminating vaccine strain from a wild type subtype of CPV-2 remains ambitious. Copyright © 2017 Elsevier Ltd. All rights reserved.

Comparison of real-time SYBR green dengue assay with real-time taqman RT-PCR dengue assay and the conventional nested PCR for diagnosis of primary and secondary dengue infection

PubMed Central

Paudel, Damodar; Jarman, Richard; Limkittikul, Kriengsak; Klungthong, Chonticha; Chamnanchanunt, Supat; Nisalak, Ananda; Gibbons, Robert; Chokejindachai, Watcharee

2011-01-01

Background: Dengue fever and dengue hemorrhagic fever are caused by dengue virus. Dengue infection remains a burning problem of many countries. To diagnose acute dengue in the early phase we improve the low cost, rapid SYBR green real time assay and compared the sensitivity and specificity with real time Taqman® assay and conventional nested PCR assay. Aims: To develop low cost, rapid and reliable real time SYBR green diagnostic dengue assay and compare with Taqman real-time assay and conventional nested PCR (modified Lanciotti). Materials and Methods: Eight cultured virus strains were diluted in tenth dilution down to undetectable level by the PCR to optimize the primer, temperature (annealing, and extension and to detect the limit of detection of the assay. Hundred and ninety three ELISA and PCR proved dengue clinical samples were tested with real time SYBR® Green assay, real time Taqman® assay to compare the sensitivity and specificity. Results: Sensitivity and specificity of real time SYBR® green dengue assay (84% and 66%, respectively) was almost comparable to those (81% and 74%) of Taqman real time PCR dengue assay. Real time SYBR® green RT-PCR was equally sensitive in primary and secondary infection while real time Taqman was less sensitive in the secondary infection. Sensitivity of real time Taqman on DENV3 (87%) was equal to SYBR green real time PCR dengue assay. Conclusion: We developed low cost rapid diagnostic SYBR green dengue assay. Further study is needed to make duplex primer assay for the serotyping of dengue virus. PMID:22363089

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.

Monitoring and improving the sensitivity of dengue nested RT-PCR used in longitudinal surveillance in Thailand.

PubMed

Klungthong, Chonticha; Manasatienkij, Wudtichai; Phonpakobsin, Thipwipha; Chinnawirotpisan, Piyawan; Rodpradit, Prinyada; Hussem, Kittinun; Thaisomboonsuk, Butsaya; Ong-ajchaowlerd, Prapapun; Nisalak, Ananda; Kalayanarooj, Siripen; Buddhari, Darunee; Gibbons, Robert V; Jarman, Richard G; Yoon, In-Kyu; Fernandez, Stefan

2015-02-01

AFRIMS longitudinal dengue surveillance in Thailand depends on the nested RT-PCR and the dengue IgM/IgG ELISA. To examine and improve the sensitivity of the nested RT-PCR using a panel of archived samples collected during dengue surveillance. A retrospective analysis of 16,454 dengue IgM/IgG ELISA positive cases collected between 2000 and 2013 was done to investigate the sensitivity of the nested RT-PCR. From these cases, 318 acute serum specimens or extracted RNA, previously found to be negative by the nested RT-PCR, were tested using TaqMan real-time RT-PCR (TaqMan rRT-PCR). To improve the sensitivity of nested RT-PCR, we designed a new primer based on nucleotide sequences from contemporary strains found to be positive by the TaqMan rRT-PCR. Sensitivity of the new nested PCR was calculated using a panel of 87 samples collected during 2011-2013. The percentage of dengue IgM/IgG ELISA positive cases that were negative by the nested RT-PCR varied from 17% to 42% for all serotypes depending on the year. Using TaqMan rRT-PCR, dengue RNA was detected in 194 (61%) of the 318 acute sera or extracted RNA previously found to be negative by the nested RT-PCR. The newly designed DENV-1 specific primer increased the sensitivity of DENV-1 detection by the nested RT-PCR from 48% to 88%, and of all 4 serotypes from 73% to 87%. These findings demonstrate the impact of genetic diversity and signal erosion on the sensitivity of PCR-based methods. Published by Elsevier B.V.

Development of Nested PCR, Multiplex PCR, and Loop-Mediated Isothermal Amplification Assays for Rapid Detection of Cylindrocladium scoparium on Eucalyptus.

PubMed

Qiao, Tian-Min; Zhang, Jing; Li, Shu-Jiang; Han, Shan; Zhu, Tian-Hui

2016-10-01

Eucalyptus dieback disease, caused by Cylindrocladium scoparium , has occurred in last few years in large Eucalyptus planting areas in China and other countries. Rapid, simple, and reliable diagnostic techniques are desired for the early detection of Eucalyptus dieback of C. scoparium prior to formulation of efficient control plan. For this purpose, three PCR-based methods of nested PCR, multiplex PCR, loop-mediated isothermal amplification (LAMP) were developed for detection of C. scoparium based on factor 1-alpha (tef1) and beta-tubulin gene in this study. All of the three methods showed highly specific to C. scoparium . The sensitivities of the nested PCR and LAMP were much higher than the multiplex PCR. The sensitivity of multiplex PCR was also higher than regular PCR. C. scoparium could be detected within 60 min from infected Eucalyptus plants by LAMP, while at least 2 h was needed by the rest two methods. Using different Eucalyptus tissues as samples for C. scoparium detection, all of the three PCR-based methods showed much better detection results than regular PCR. Base on the results from this study, we concluded that any of the three PCR-based methods could be used as diagnostic technology for the development of efficient strategies of Eucalyptus dieback disease control. Particularly, LAMP was the most practical method in field application because of its one-step and rapid reaction, simple operation, single-tube utilization, and simple visualization of amplification products.

Development of Nested PCR, Multiplex PCR, and Loop-Mediated Isothermal Amplification Assays for Rapid Detection of Cylindrocladium scoparium on Eucalyptus

PubMed Central

Qiao, Tian-Min; Zhang, Jing; Li, Shu-Jiang; Han, Shan; Zhu, Tian-Hui

2016-01-01

Eucalyptus dieback disease, caused by Cylindrocladium scoparium, has occurred in last few years in large Eucalyptus planting areas in China and other countries. Rapid, simple, and reliable diagnostic techniques are desired for the early detection of Eucalyptus dieback of C. scoparium prior to formulation of efficient control plan. For this purpose, three PCR-based methods of nested PCR, multiplex PCR, loop-mediated isothermal amplification (LAMP) were developed for detection of C. scoparium based on factor 1-alpha (tef1) and beta-tubulin gene in this study. All of the three methods showed highly specific to C. scoparium. The sensitivities of the nested PCR and LAMP were much higher than the multiplex PCR. The sensitivity of multiplex PCR was also higher than regular PCR. C. scoparium could be detected within 60 min from infected Eucalyptus plants by LAMP, while at least 2 h was needed by the rest two methods. Using different Eucalyptus tissues as samples for C. scoparium detection, all of the three PCR-based methods showed much better detection results than regular PCR. Base on the results from this study, we concluded that any of the three PCR-based methods could be used as diagnostic technology for the development of efficient strategies of Eucalyptus dieback disease control. Particularly, LAMP was the most practical method in field application because of its one-step and rapid reaction, simple operation, single-tube utilization, and simple visualization of amplification products. PMID:27721691

Combining PCR with Microscopy to Reduce Costs of Laboratory Diagnosis of Buruli Ulcer

PubMed Central

Yeboah-Manu, Dorothy; Asante-Poku, Adwoa; Asan-Ampah, Kobina; Ampadu, Emelia Danso Edwin; Pluschke, Gerd

2011-01-01

The introduction of antibiotic therapy as first-line treatment of Buruli ulcer underlines the importance of laboratory confirmation of clinical diagnosis. Because smear microscopy has very limited sensitivity, the technically demanding and more expensive IS2404 diagnostic polymerase chain reaction (PCR) has become the main method for confirmation. By optimization of the release of mycobacteria from swab specimen and concentration of bacterial suspensions before smearing, we were able to improve the detection rate of acid-fast bacilli by microscopy after Ziehl–Neelsen staining. Compared with IS2404 PCR, which is the gold standard diagnostic method, the sensitivity and specificity of microscopy with 100 concentrated specimens were 58.4% and 95.7%, respectively. We subsequently evaluated a stepwise laboratory confirmation algorithm with detection of AFB as first-line method and IS2404 PCR performed only with those samples that were negative in microscopic analysis. This stepwise approach reduced unit cost by more than 50% to $5.41, and the total costs were reduced from $917 to $433. PMID:22049046

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.

Droplet digital PCR as a novel detection method for quantifying microRNAs in acute myocardial infarction.

PubMed

Robinson, S; Follo, M; Haenel, D; Mauler, M; Stallmann, D; Tewari, M; Duerschmied, D; Peter, K; Bode, C; Ahrens, I; Hortmann, M

2018-04-15

micro-RNAs have shown promise as potential biomarkers for acute myocardial infarction and ischemia-reperfusion injury (I/R). Most recently droplet digital polymerase chain reaction (ddPCR) has been introduced as a more reliable and reproducible method for detecting micro-RNAs. We aimed to demonstrate the improved technical performance and diagnostic potential of ddPCR by measuring micro-RNAs in ST-elevation myocardial infarction (STEMI). A dilution series was performed in duplicate on synthetic Caenorrhabditis elegans-miR-39, comparing quantitative real-time PCR (qRT-PCR) and ddPCR. We used ddPCR and qRT-PCR to quantify the serum levels of miR-21, miR-208a and miR-499 between STEMI patients (n=24) and stable coronary artery disease (CAD) patients (n=20). In STEMI, I/R injury was assessed via measurement of ST-segment resolution. In the dilution series, ddPCR demonstrated superior coefficient of variation (12.1%vs.32.9%) and limit of detection (0.9325 vs.2.425copies/μl). In the patient cohort, ddPCR demonstrated greater differences in miR-21 levels (2190.5 vs. 484.7copies/μl; p=0.0004 for ddPCR and 136.4 vs. 122.8copies/μl; p=0.2273 for qRT-PCR) and in miR-208a (0 vs. 24.1copies/μl, p=0.0013 for ddPCR and 0 vs. 0copies/μl, p=0.0032 for qRT-PCR), with similar differences observed in miR-499 levels (9.4 vs. 81.5copies/μl, p<0.0001 for ddPCR and 0 vs. 19.41copies/μl, p<0.0001 for qRT-PCR). ddPCR also more accurately defined STEMI for all miRNAs (area under the curve (AUC) of 0.8021/0.7740/0.9063 for miR-21/208a/499 with ddPCR vs. AUC of 0.6083/0.6917/0.8417 with qRT-PCR). However, there was no association between miR-21/208a/499 levels and ischemia-reperfusion injury. ddPCR demonstrates superiority in both technical performance and diagnostic potential compared to qRT-PCR. Ultimately, this supports its use as a diagnostic method for quantifying micro-RNAs, particularly in large multi-center trials. Copyright © 2017 Elsevier B.V. All rights reserved.

Strategies to Improve Efficiency and Specificity of Degenerate Primers in PCR.

PubMed

Campos, Maria Jorge; Quesada, Alberto

2017-01-01

PCR with degenerate primers can be used to identify the coding sequence of an unknown protein or to detect a genetic variant within a gene family. These primers, which are complex mixtures of slightly different oligonucleotide sequences, can be optimized to increase the efficiency and/or specificity of PCR in the amplification of a sequence of interest by the introduction of mismatches with the target sequence and balancing their position toward the primers 5'- or 3'-ends. In this work, we explain in detail examples of rational design of primers in two different applications, including the use of specific determinants at the 3'-end, to: (1) improve PCR efficiency with coding sequences for members of a protein family by fully degeneration at a core box of conserved genetic information, with the reduction of degeneration at the 5'-end, and (2) optimize specificity of allelic discrimination of closely related orthologous by 5'-end degenerate primers.

Advances in PCR technology.

PubMed

Lauerman, Lloyd H

2004-12-01

Since the discovery of the polymerase chain reaction (PCR) 20 years ago, an avalanche of scientific publications have reported major developments and changes in specialized equipment, reagents, sample preparation, computer programs and techniques, generated through business, government and university research. The requirement for genetic sequences for primer selection and validation has been greatly facilitated by the development of new sequencing techniques, machines and computer programs. Genetic libraries, such as GenBank, EMBL and DDBJ continue to accumulate a wealth of genetic sequence information for the development and validation of molecular-based diagnostic procedures concerning human and veterinary disease agents. The mechanization of various aspects of the PCR assay, such as robotics, microfluidics and nanotechnology, has made it possible for the rapid advancement of new procedures. Real-time PCR, DNA microarray and DNA chips utilize these newer techniques in conjunction with computer and computer programs. Instruments for hand-held PCR assays are being developed. The PCR and reverse transcription-PCR (RT-PCR) assays have greatly accelerated the speed and accuracy of diagnoses of human and animal disease, especially of the infectious agents that are difficult to isolate or demonstrate. The PCR has made it possible to genetically characterize a microbial isolate inexpensively and rapidly for identification, typing and epidemiological comparison.

Diagnosis of Bubonic Plague by PCR in Madagascar under Field Conditions

PubMed Central

Rahalison, L.; Vololonirina, E.; Ratsitorahina, M.; Chanteau, S.

2000-01-01

The diagnostic value of a PCR assay that amplifies a 501-bp fragment of the Yersinia pestis caf1 gene has been determined in a reference laboratory with 218 bubo aspirates collected from patients with clinically suspected plague managed in a regional hospital in Madagascar. The culture of Y. pestis and the detection of the F1 antigen (Ag) by enzyme-linked immunosorbent assay (ELISA) were used as reference diagnostic methods. The sensitivity of PCR was 89% (57 of 64) for the Y. pestis-positive patients, and 80.7% (63 of 78) for the F1 Ag-positive patients. The specificity of PCR for the culture-, F1 Ag-, and antibody-negative patients (n = 105) was 100%. Because in Madagascar most patients with plague are managed and their clinical samples are collected in remote villages, the usefulness of PCR was evaluated for routine diagnostic use in the operational conditions of the control program. The sensitivity of PCR was 50% (25 of 50) relative to the results of culture and 35.2% (19 of 54) relative to the results of the F1 Ag immunocapture ELISA. The specificity of PCR under these conditions was 96%. In conclusion, the PCR method was found to be very specific but not as sensitive as culture or the F1 Ag detection method. The limitation in sensitivity may have been due to suboptimal field conditions and the small volumes of samples used for DNA extraction. This technique is not recommended as a routine diagnostic test for plague in Madagascar. PMID:10618097

The clinical diagnostic accuracy of rapid detection of healthcare-associated bloodstream infection in intensive care using multipathogen real-time PCR technology

PubMed Central

Dunn, Graham; Chadwick, Paul; Young, Duncan; Bentley, Andrew; Carlson, Gordon; Warhurst, Geoffrey

2011-01-01

Background There is growing interest in the potential utility of real-time PCR in diagnosing bloodstream infection by detecting pathogen DNA in blood samples within a few hours. SeptiFast is a multipathogen probe-based real-time PCR system targeting ribosomal DNA sequences of bacteria and fungi. It detects and identifies the commonest pathogens causing bloodstream infection and has European regulatory approval. The SeptiFast pathogen panel is suited to identifying healthcare-associated bloodstream infection acquired during complex healthcare, and the authors report here the protocol for the first detailed health-technology assessment of multiplex real-time PCR in this setting. Methods/design A Phase III multicentre double-blinded diagnostic study will determine the clinical validity of SeptiFast for the rapid detection of healthcare-associated bloodstream infection, against the current service standard of microbiological culture, in an adequately sized population of critically ill adult patients. Results from SeptiFast and standard microbiological culture procedures in each patient will be compared at study conclusion and the metrics of clinical diagnostic accuracy of SeptiFast determined in this population setting. In addition, this study aims to assess further the preliminary evidence that the detection of pathogen DNA in the bloodstream using SeptiFast may have value in identifying the presence of infection elsewhere in the body. Furthermore, differences in circulating immune-inflammatory markers in patient groups differentiated by the presence/absence of culturable pathogens and pathogen DNA will help elucidate further the patho-physiology of infection developing in the critically ill. Ethics and dissemination Ethical approval has been granted by the North West 6 Research Ethics Committee (09/H1003/109). Based on the results of this first non-commercial study, independent recommendations will be made to The Department of Health (open-access health technology

Diagnostic accuracy of the ROCHE Septifast PCR system for the rapid detection of blood pathogens in neonatal sepsis-A prospective clinical trial.

PubMed

Straub, Julia; Paula, Helga; Mayr, Michaela; Kasper, David; Assadian, Ojan; Berger, Angelika; Rittenschober-Böhm, Judith

2017-01-01

Diagnosis of neonatal sepsis remains a major challenge in neonatology. Most molecular-based methods are not customized for neonatal requirements. The aim of the present study was to assess the diagnostic accuracy of a modified multiplex PCR protocol for the detection of neonatal sepsis using small blood volumes. 212 episodes of suspected neonatal late onset sepsis were analyzed prospectively using the Roche SeptiFast® MGRADE PCR with a modified DNA extraction protocol and software-handling tool. Results were compared to blood culture, laboratory biomarkers and clinical signs of sepsis. Of 212 episodes, 85 (40.1%) were categorized as "not infected". Among these episodes, 1 was false positive by blood culture (1.2%) and 23 were false positive by PCR (27.1%). Of 51 (24.1%) episodes diagnosed as "culture proven sepsis", the same pathogen was detected by blood culture and PCR in 39 episodes (76.5%). In 8 episodes, more pathogens were detected by PCR compared to blood culture, and in 4 episodes the pathogen detected by blood culture was not found by PCR. One of these episodes was caused by Bacillus cereus, a pathogen not included in the PCR panel. In 76/212 (35.8%) episodes, clinical sepsis was diagnosed. Among these, PCR yielded positive results in 39.5% of episodes (30/76 episodes). For culture-positive sepsis, PCR showed a sensitivity of 90.2% (95%CI 86.2-94.2%) and a specificity of 72.9% (95%CI 67.0-79.0%). The Roche SeptiFast® MGRADE PCR using a modified DNA extraction protocol showed acceptable results for rapid detection of neonatal sepsis in addition to conventional blood culture. The benefit of rapid pathogen detection has to be balanced against the considerable risk of contamination, loss of information on antibiotic sensitivity pattern and increased costs.

Detection of Orthopoxvirus DNA by Real-Time PCR and Identification of Variola Virus DNA by Melting Analysis

PubMed Central

Nitsche, Andreas; Ellerbrok, Heinz; Pauli, Georg

2004-01-01

Although variola virus was eradicated by the World Health Organization vaccination program in the 1970s, the diagnosis of smallpox infection has attracted great interest in the context of a possible deliberate release of variola virus in bioterrorist attacks. Obviously, fast and reliable diagnostic tools are required to detect variola virus and to distinguish it from orthopoxviruses that have identical morphological characteristics, including vaccinia virus. The advent of real-time PCR for the clinical diagnosis of viral infections has facilitated the detection of minute amounts of viral nucleic acids in a fast, safe, and precise manner, including the option to quantify and to genotype the target reliably. In this study a complete set of four hybridization probe-based real-time PCR assays for the specific detection of orthopoxvirus DNA is presented. Melting analysis following PCR enables the identification of variola virus by the PCR product's characteristic melting temperature, permitting the discrimination of variola virus from other orthopoxviruses. In addition, an assay for the specific amplification of variola virus DNA is presented. All assays can be performed simultaneously in the same cycler, and results of a PCR run are obtained in less than 1 h. The application of more than one assay for the same organism significantly contributes to the diagnostic reliability, reducing the risk of false-negative results due to unknown sequence variations. In conclusion, the assays presented will improve the speed and reliability of orthopoxvirus diagnostics and variola virus identification. PMID:15004077

Improved Detection of Norovirus and Hepatitis A Virus in Surface Water by Applying Pre-PCR Processing.

PubMed

Borgmästars, Emmy; Jazi, Mehrdad Mousavi; Persson, Sofia; Jansson, Linda; Rådström, Peter; Simonsson, Magnus; Hedman, Johannes; Eriksson, Ronnie

2017-12-01

Quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) detection of waterborne RNA viruses generally requires concentration of large water volumes due to low virus levels. A common approach is to use dead-end ultrafiltration followed by precipitation with polyethylene glycol. However, this procedure often leads to the co-concentration of PCR inhibitors that impairs the limit of detection and causes false-negative results. Here, we applied the concept of pre-PCR processing to optimize RT-qPCR detection of norovirus genogroup I (GI), genogroup II (GII), and hepatitis A virus (HAV) in challenging water matrices. The RT-qPCR assay was improved by screening for an inhibitor-tolerant master mix and modifying the primers with twisted intercalating nucleic acid molecules. Additionally, a modified protocol based on chaotropic lysis buffer and magnetic silica bead nucleic acid extraction was developed for complex water matrices. A validation of the modified extraction protocol on surface and drinking waters was performed. At least a 26-fold improvement was seen in the most complex surface water studied. The modified protocol resulted in average recoveries of 33, 13, 8, and 4% for mengovirus, norovirus GI, GII, and HAV, respectively. The modified protocol also improved the limit of detection for norovirus GI and HAV. RT-qPCR inhibition with C q shifts of 1.6, 2.8, and 3.5 for norovirus GI, GII, and HAV, respectively, obtained for the standard nucleic acid extraction were completely eliminated by the modified protocol. The standard nucleic acid extraction method worked well on drinking water with no RT-qPCR inhibition observed and average recoveries of 80, 124, 89, and 32% for mengovirus, norovirus GI, GII, and HAV, respectively.

Comparison of Established Diagnostic Methodologies and a Novel Bacterial smpB Real-Time PCR Assay for Specific Detection of Haemophilus influenzae Isolates Associated with Respiratory Tract Infections

PubMed Central

Reddington, Kate; Schwenk, Stefan; Tuite, Nina; Platt, Gareth; Davar, Danesh; Coughlan, Helena; Personne, Yoann; Gant, Vanya; Enne, Virve I.; Zumla, Alimuddin

2015-01-01

Haemophilus influenzae is a significant causative agent of respiratory tract infections (RTI) worldwide. The development of a rapid H. influenzae diagnostic assay that would allow for the implementation of infection control measures and also improve antimicrobial stewardship for patients is required. A number of nucleic acid diagnostics approaches that detect H. influenzae in RTIs have been described in the literature; however, there are reported specificity and sensitivity limitations for these assays. In this study, a novel real-time PCR diagnostic assay targeting the smpB gene was designed to detect all serogroups of H. influenzae. The assay was validated using a panel of well-characterized Haemophilus spp. Subsequently, 44 Haemophilus clinical isolates were collected, and 36 isolates were identified as H. influenzae using a gold standard methodology that combined the results of matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) and a fucK diagnostic assay. Using the novel smpB diagnostic assay, 100% concordance was observed with the gold standard, demonstrating a sensitivity of 100% (95% confidence interval [CI], 90.26% to 100.00%) and a specificity of 100% (95% CI, 63.06% to 100.00%) when used on clinical isolates. To demonstrate the clinical utility of the diagnostic assay presented, a panel of lower RTI samples (n = 98) were blindly tested with the gold standard and smpB diagnostic assays. The results generated were concordant for 94/98 samples tested, demonstrating a sensitivity of 90.91% (95% CI, 78.33% to 97.47%) and a specificity of 100% (95% CI, 93.40% to 100.00%) for the novel smpB assay when used directly on respiratory specimens. PMID:26109443

DNA Differential Diagnosis of Taeniasis and Cysticercosis by Multiplex PCR

PubMed Central

Yamasaki, Hiroshi; Allan, James C.; Sato, Marcello Otake; Nakao, Minoru; Sako, Yasuhito; Nakaya, Kazuhiro; Qiu, Dongchuan; Mamuti, Wulamu; Craig, Philip S.; Ito, Akira

2004-01-01

Multiplex PCR was established for differential diagnosis of taeniasis and cysticercosis, including their causative agents. For identification of the parasites, multiplex PCR with cytochrome c oxidase subunit 1 gene yielded evident differential products unique for Taenia saginata and Taenia asiatica and for American/African and Asian genotypes of Taenia solium with molecular sizes of 827, 269, 720, and 984 bp, respectively. In the PCR-based detection of tapeworm carriers using fecal samples, the diagnostic markers were detected from 7 of 14 and 4 of 9 T. solium carriers from Guatemala and Indonesia, respectively. Test sensitivity may have been reduced by the length of time (up to 12 years) that samples were stored and/or small sample volumes (ca. 30 to 50 mg). However, the diagnostic markers were detected by nested PCR in five worm carriers from Guatemalan cases that were found to be negative by multiplex PCR. It was noteworthy that a 720 bp-diagnostic marker was detected from a T. solium carrier who was egg-free, implying that it is possible to detect worm carriers and treat before mature gravid proglottids are discharged. In contrast to T. solium carriers, 827-bp markers were detected by multiplex PCR in all T. saginata carriers. The application of the multiplex PCR would be useful not only for surveillance of taeniasis and cysticercosis control but also for the molecular epidemiological survey of these cestode infections. PMID:14766815

Use of Capillary Blood Samples Leads to Higher Parasitemia Estimates and Higher Diagnostic Sensitivity of Microscopic and Molecular Diagnostics of Malaria than Venous Blood Samples.

PubMed

Mischlinger, Johannes; Pitzinger, Paul; Veletzky, Luzia; Groger, Mirjam; Zoleko-Manego, Rella; Adegnika, Ayola A; Agnandji, Selidji T; Lell, Bertrand; Kremsner, Peter G; Tannich, Egbert; Mombo-Ngoma, Ghyslain; Mordmüller, Benjamin; Ramharter, Michael

2018-05-25

Diagnosis of malaria is usually based on samples of peripheral blood. However, it is unclear whether capillary (CAP) or venous (VEN) blood samples provide better diagnostic performance. Quantitative differences of parasitemia between CAP and VEN blood and diagnostic performance characteristics were investigated. Patients were recruited between September 2015 and February 2016 in Gabon. Light microscopy and qPCR quantified parasitemia of paired CAP and VEN samples, whose preparation followed the exact same methodology. CAP and VEN performance characteristics using microscopy were evaluated against a qPCR gold-standard. Microscopy revealed a median (IQR) parasites/L of 495 (853,243) in CAP and 429 (524,074) in VEN samples manifesting in a +16.6% (p=0.04) higher CAPparasitemia compared with VENparasitemia. Concordantly, qPCR demonstrated that -0.278 (p=0.006) cycles were required for signal detection in CAP samples. CAPsensitivity of microscopy relative to the gold-standard was 81.5% (77.485.6%) versus VENsensitivity of 73.4% (68.878.1%), while CAPspecificity and VENspecificity were 91%. CAPsensitivity and VENsensitivity dropped to 63.3% and 45.9%, respectively for a sub-population of low-level parasitemias while specificities were 92%. CAP sampling leads to higher parasitemias compared to VEN sampling and improves diagnostic sensitivity. These findings may have important implications for routine diagnostics, research and elimination campaigns of malaria.

Overcoming the errors of in-house PCR used in the clinical laboratory for the diagnosis of extrapulmonary tuberculosis.

PubMed

Kunakorn, M; Raksakai, K; Pracharktam, R; Sattaudom, C

1999-03-01

Our experiences from 1993 to 1997 in the development and use of IS6110 base PCR for the diagnosis of extrapulmonary tuberculosis in a routine clinical setting revealed that error-correcting processes can improve existing diagnostic methodology. The reamplification method initially used had a sensitivity of 90.91% and a specificity of 93.75%. The concern was focused on the false positive results of this method caused by product-carryover contamination. This method was changed to single round PCR with carryover prevention by uracil DNA glycosylase (UDG), resulting in a 100% specificity but only 63% sensitivity. Dot blot hybridization was added after the single round PCR, increasing the sensitivity to 87.50%. However, false positivity resulted from the nonspecific dot blot hybridization signal, reducing the specificity to 89.47%. The hybridization of PCR was changed to a Southern blot with a new oligonucleotide probe giving the sensitivity of 85.71% and raising the specificity to 99.52%. We conclude that the PCR protocol for routine clinical use should include UDG for carryover prevention and hybridization with specific probes to optimize diagnostic sensitivity and specificity in extrapulmonary tuberculosis testing.

Validity of a PCR assay in CSF for the diagnosis of neurocysticercosis.

PubMed

Carpio, Arturo; Campoverde, Alfredo; Romo, Matthew L; García, Lorena; Piedra, Luis M; Pacurucu, Mónica; López, Nelson; Aguilar, Jenner; López, Sebastian; Vintimilla, Luis C; Toral, Ana M; Peña-Tapia, Pablo

2017-03-01

To prospectively evaluate the validity of a PCR assay in CSF for the diagnosis of neurocysticercosis (NC). We conducted a multicenter, prospective case-control study, recruiting participants from 5 hospitals in Cuenca, Ecuador, from January 2015 to February 2016. Cases fulfilled validated diagnostic criteria for NC. For each case, a neurosurgical patient who did not fulfill the diagnostic criteria for NC was selected as a control. CT and MRI, as well as a CSF sample, were collected from both cases and controls. The diagnostic criteria to identify cases were used as a reference standard. Overall, 36 case and 36 control participants were enrolled. PCR had a sensitivity of 72.2% (95% confidence interval [CI] 54.8%-85.8%) and a specificity of 100.0% (95% CI 90.3%-100.0%). For parenchymal NC, PCR had a sensitivity of 42.9% (95% CI 17.7%-71.1%), and for extraparenchymal NC, PCR had a sensitivity of 90.9% (95% CI 70.8%-98.9%). This study demonstrated the usefulness of this PCR assay in CSF for the diagnosis of NC. PCR may be particularly helpful for diagnosing extraparenchymal NC when neuroimaging techniques have failed. This study provides Class III evidence that CSF PCR can accurately identify patients with extraparenchymal NC.

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.

Assessment of the Usefulness of Multiplex Real-Time PCR Tests in the Diagnostic and Therapeutic Process of Pneumonia in Hospitalized Children: A Single-Center Experience.

PubMed

Gowin, Ewelina; Bartkowska-Śniatkowska, Alicja; Jończyk-Potoczna, Katarzyna; Wysocka-Leszczyńska, Joanna; Bobkowski, Waldemar; Fichna, Piotr; Sobkowiak, Paulina; Mazur-Melewska, Katarzyna; Bręborowicz, Anna; Wysocki, Jacek; Januszkiewicz-Lewandowska, Danuta

2017-01-01

The aim of the study was assessment of the usefulness of multiplex real-time PCR tests in the diagnostic and therapeutic process in children hospitalized due to pneumonia and burdened with comorbidities. Methods . The study group included 97 children hospitalized due to pneumonia at the Karol Jonscher Teaching Hospital in Poznań, in whom multiplex real-time PCR tests (FTD respiratory pathogens 33; fast-track diagnostics) were used. Results . Positive test results of the test were achieved in 74 patients (76.3%). The average age in the group was 56 months. Viruses were detected in 61 samples (82% of all positive results); bacterial factors were found in 29 samples (39% of all positive results). The presence of comorbidities was established in 90 children (92.78%). On the basis of the obtained results, 5 groups of patients were established: viral etiology of infection, 34 patients; bacterial etiology, 7 patients; mixed etiology, 23 patients; pneumocystis, 9 patients; and no etiology diagnosed, 24 patients. Conclusions . Our analysis demonstrated that the participation of viruses in causing severe lung infections is significant in children with comorbidities. Multiplex real-time PCR tests proved to be more useful in establishing the etiology of pneumonia in hospitalized children than the traditional microbiological examinations.

Better Tests, Better Care: Improved Diagnostics for Infectious Diseases

PubMed Central

Caliendo, Angela M.; Gilbert, David N.; Ginocchio, Christine C.; Hanson, Kimberly E.; May, Larissa; Quinn, Thomas C.; Tenover, Fred C.; Alland, David; Blaschke, Anne J.; Bonomo, Robert A.; Carroll, Karen C.; Ferraro, Mary Jane; Hirschhorn, Lisa R.; Joseph, W. Patrick; Karchmer, Tobi; MacIntyre, Ann T.; Reller, L. Barth; Jackson, Audrey F.

2013-01-01

In this IDSA policy paper, we review the current diagnostic landscape, including unmet needs and emerging technologies, and assess the challenges to the development and clinical integration of improved tests. To fulfill the promise of emerging diagnostics, IDSA presents recommendations that address a host of identified barriers. Achieving these goals will require the engagement and coordination of a number of stakeholders, including Congress, funding and regulatory bodies, public health agencies, the diagnostics industry, healthcare systems, professional societies, and individual clinicians. PMID:24200831

Diagnostic performance of a multiple real-time PCR assay in patients with suspected sepsis hospitalized in an internal medicine ward.

PubMed

Pasqualini, Leonella; Mencacci, Antonella; Leli, Christian; Montagna, Paolo; Cardaccia, Angela; Cenci, Elio; Montecarlo, Ines; Pirro, Matteo; di Filippo, Francesco; Cistaro, Emma; Schillaci, Giuseppe; Bistoni, Francesco; Mannarino, Elmo

2012-04-01

Early identification of causative pathogen in sepsis patients is pivotal to improve clinical outcome. SeptiFast (SF), a commercially available system for molecular diagnosis of sepsis based on PCR, has been mostly used in patients hospitalized in hematology and intensive care units. We evaluated the diagnostic accuracy and clinical usefulness of SF, compared to blood culture (BC), in 391 patients with suspected sepsis, hospitalized in a department of internal medicine. A causative pathogen was identified in 85 patients (22%). Sixty pathogens were detected by SF and 57 by BC. No significant differences were found between the two methods in the rates of pathogen detection (P = 0.74), even after excluding 9 pathogens which were isolated by BC and were not included in the SF master list (P = 0.096). The combination of SF and BC significantly improved the diagnostic yield in comparison to BC alone (P < 0.001). Compared to BC, SF showed a significantly lower contamination rate (0 versus 19 cases; P < 0.001) with a higher specificity for pathogen identification (1.00, 95% confidence interval [CI] of 0.99 to 1.00, versus 0.94, 95% CI of 0.90 to 0.96; P = 0.005) and a higher positive predictive value (1.00, 95% CI of 1.00 to 0.92%, versus 0.75, 95% CI of 0.63 to 0.83; P = 0.005). In the subgroup of patients (n = 191) who had been receiving antibiotic treatment for ≥24 h, SF identified more pathogens (16 versus 6; P = 0.049) compared to BC. These results suggest that, in patients with suspected sepsis, hospitalized in an internal medicine ward, SF could be a highly valuable adjunct to conventional BC, particularly in patients under antibiotic treatment.

Survey of diagnostic and typing capacity for Clostridium difficile infection in Europe, 2011 and 2014.

PubMed

van Dorp, Sofie M; Notermans, Daan W; Alblas, Jeroen; Gastmeier, Petra; Mentula, Silja; Nagy, Elisabeth; Spigaglia, Patrizia; Ivanova, Katiusha; Fitzpatrick, Fidelma; Barbut, Frédéric; Morris, Trefor; Wilcox, Mark H; Kinross, Pete; Suetens, Carl; Kuijper, Ed J

2016-07-21

Suboptimal laboratory diagnostics for Clostridium difficile infection (CDI) impedes its surveillance and control across Europe. We evaluated changes in local laboratory CDI diagnostics and changes in national diagnostic and typing capacity for CDI during the European C. difficile Infection Surveillance Network (ECDIS-Net) project, through cross-sectional surveys in 33 European countries in 2011 and 2014. In 2011, 126 (61%) of a convenience sample of 206 laboratories in 31 countries completed a survey on local diagnostics. In 2014, 84 (67%) of these 126 laboratories in 26 countries completed a follow-up survey. Among laboratories that participated in both surveys, use of CDI diagnostics deemed 'optimal' or 'acceptable' increased from 19% to 46% and from 10% to 15%, respectively (p  < 0.001). The survey of national capacity was completed by national coordinators of 31 and 32 countries in 2011 and 2014, respectively. Capacity for any C. difficile typing method increased from 22/31 countries in 2011 to 26/32 countries in 2014; for PCR ribotyping from 20/31 countries to 23/32 countries, and specifically for capillary PCR ribotyping from 7/31 countries to 16/32 countries. While our study indicates improved diagnostic capability and national capacity for capillary PCR ribotyping across European laboratories between 2011 and 2014, increased use of 'optimal' diagnostics should be promoted. This article is copyright of The Authors, 2016.

Evaluation of Anthelmintic Resistance and Exhaust Air Dust PCR as a Diagnostic Tool in Mice Enzootically Infected with Aspiculuris tetraptera

PubMed Central

Kapoor, Pratibha; Hayes, Yumiko O; Jarrell, Leslie T; Bellinger, Dwight A; Thomas, Rhiannon D; Lawson, Gregory W; Arkema, Jaclyn D; Fletcher, Craig A; Nielsen, Judith N

2017-01-01

diagnostic tool for antemortem detection of A. tetraptera infection, in conjunction with fecal PCR and FCF. PMID:28535863

[Contrast agent improves diagnostic value of dobutamine stress echocardiography].

PubMed

Uehara, H; Yamamoto, T; Hirano, Y; Ozasa, Y; Yamada, S; Ikawa, H; Ishikawa, K

2001-03-01

Suboptimal endocardial definition reduces the diagnostic value of stress echocardiography for coronary artery disease, but intravenous infusion of a left ventricular contrast agent (Albunex) may enhance endocardial border delineation and improve the diagnostic value of dobutamine stress echocardiography. Fifty-six patients, 38 with myocardial infarction, 16 with angina pectoris and two normal subjects, were enrolled in this study. Dobutamine was infused in scalar doses of 5 to 40 micrograms/kg/min. Intravenous infusion of Albunex (0.15 ml/kg) was administered at rest and during peak dobutamine stress during monitoring of the apical four-chamber view. The left ventricle in the apical four-chamber view was divided into six segments and an endocardial delineation score of 0 to 3 (none to excellent visualization) was given to each segment. Endocardial delineation score was increased after Albunex infusion from 2.0 to 2.3 in the basal-septal, 2.0 to 2.4 in the mid-septal, 1.1 to 1.8 in the apical-septal, 0.7 to 1.2 in the apical-lateral, 0.9 to 1.6 in the mid-lateral, and 1.2 to 1.9 in the basal-lateral segments during peak dobutamine administration. Endocardial border resolution in the lateral wall showed greater improvement than in the septal wall after Albunex infusion. Diagnostic values in the left anterior descending artery territory failed to improve with Albunex infusion (sensitivity 82% to 89%, specificity 94% to 89%, and accuracy 86% to 89%), whereas a higher diagnostic accuracy was noted in the left circumflex artery territory with Albunex compared to without Albunex (sensitivity 63% to 81%, specificity 88% to 98%, and accuracy 80% to 93%, p < 0.05). Contrast agent improves the diagnostic accuracy of dobutamine stress echocardiography in the left circumflex artery territory.

A Host-Based RT-PCR Gene Expression Signature to Identify Acute Respiratory Viral Infection

PubMed Central

Zaas, Aimee K.; Burke, Thomas; Chen, Minhua; McClain, Micah; Nicholson, Bradly; Veldman, Timothy; Tsalik, Ephraim L.; Fowler, Vance; Rivers, Emanuel P.; Otero, Ronny; Kingsmore, Stephen F.; Voora, Deepak; Lucas, Joseph; Hero, Alfred O.; Carin, Lawrence; Woods, Christopher W.; Ginsburg, Geoffrey S.

2014-01-01

Improved ways to diagnose acute respiratory viral infections could decrease inappropriate antibacterial use and serve as a vital triage mechanism in the event of a potential viral pandemic. Measurement of the host response to infection is an alternative to pathogen-based diagnostic testing and may improve diagnostic accuracy. We have developed a host-based assay with a reverse transcription polymerase chain reaction (RT-PCR) TaqMan low-density array (TLDA) platform for classifying respiratory viral infection. We developed the assay using two cohorts experimentally infected with influenza A H3N2/Wisconsin or influenza A H1N1/Brisbane, and validated the assay in a sample of adults presenting to the emergency department with fever (n = 102) and in healthy volunteers (n = 41). Peripheral blood RNA samples were obtained from individuals who underwent experimental viral challenge or who presented to the emergency department and had microbiologically proven viral respiratory infection or systemic bacterial infection. The selected gene set on the RT-PCR TLDA assay classified participants with experimentally induced influenza H3N2 and H1N1 infection with 100 and 87% accuracy, respectively. We validated this host gene expression signature in a cohort of 102 individuals arriving at the emergency department. The sensitivity of the RT-PCR test was 89% [95% confidence interval (CI), 72 to 98%], and the specificity was 94% (95% CI, 86 to 99%). These results show that RT-PCR–based detection of a host gene expression signature can classify individuals with respiratory viral infection and sets the stage for prospective evaluation of this diagnostic approach in a clinical setting. PMID:24048524

Point-of-Care Diagnostics for Improving Maternal Health in South Africa

PubMed Central

Mashamba-Thompson, Tivani P.; Sartorius, Benn; Drain, Paul K.

2016-01-01

Improving maternal health is a global priority, particularly in high HIV-endemic, resource-limited settings. Failure to use health care facilities due to poor access is one of the main causes of maternal deaths in South Africa. “Point-of-care” (POC) diagnostics are an innovative healthcare approach to improve healthcare access and health outcomes in remote and resource-limited settings. In this review, POC testing is defined as a diagnostic test that is carried out near patients and leads to rapid clinical decisions. We review the current and emerging POC diagnostics for maternal health, with a specific focus on the World Health Organization (WHO) quality-ASSURED (Affordability, Sensitivity, Specificity, User friendly, Rapid and robust, Equipment free and Delivered) criteria for an ideal point-of-care test in resource-limited settings. The performance of POC diagnostics, barriers and challenges related to implementing POC diagnostics for maternal health in rural and resource-limited settings are reviewed. Innovative strategies for overcoming these barriers are recommended to achieve substantial progress on improving maternal health outcomes in these settings. PMID:27589808

Species-specific diagnostic assays for Bonamia ostreae and B. exitiosa in European flat oyster Ostrea edulis: conventional, real-time and multiplex PCR.

PubMed

Ramilo, Andrea; Navas, J Ignacio; Villalba, Antonio; Abollo, Elvira

2013-05-27

Bonamia ostreae and B. exitiosa have caused mass mortalities of various oyster species around the world and co-occur in some European areas. The World Organisation for Animal Health (OIE) has included infections with both species in the list of notifiable diseases. However, official methods for species-specific diagnosis of either parasite have certain limitations. In this study, new species-specific conventional PCR (cPCR) and real-time PCR techniques were developed to diagnose each parasite species. Moreover, a multiplex PCR method was designed to detect both parasites in a single assay. The analytical sensitivity and specificity of each new method were evaluated. These new procedures were compared with 2 OIE-recommended methods, viz. standard histology and PCR-RFLP. The new procedures showed higher sensitivity than the OIE recommended ones for the diagnosis of both species. The sensitivity of tests with the new primers was higher using oyster gills and gonad tissue, rather than gills alone. The lack of a 'gold standard' prevented accurate estimation of sensitivity and specificity of the new methods. The implementation of statistical tools (maximum likelihood method) for the comparison of the diagnostic tests showed the possibility of false positives with the new procedures, although the absence of a gold standard precluded certainty. Nevertheless, all procedures showed negative results when used for the analysis of oysters from a Bonamia-free area.

Tracking Progress in Improving Diagnosis: A Framework for Defining Undesirable Diagnostic Events.

PubMed

Olson, Andrew P J; Graber, Mark L; Singh, Hardeep

2018-01-29

Diagnostic error is a prevalent, harmful, and costly phenomenon. Multiple national health care and governmental organizations have recently identified the need to improve diagnostic safety as a high priority. A major barrier, however, is the lack of standardized, reliable methods for measuring diagnostic safety. Given the absence of reliable and valid measures for diagnostic errors, we need methods to help establish some type of baseline diagnostic performance across health systems, as well as to enable researchers and health systems to determine the impact of interventions for improving the diagnostic process. Multiple approaches have been suggested but none widely adopted. We propose a new framework for identifying "undesirable diagnostic events" (UDEs) that health systems, professional organizations, and researchers could further define and develop to enable standardized measurement and reporting related to diagnostic safety. We propose an outline for UDEs that identifies both conditions prone to diagnostic error and the contexts of care in which these errors are likely to occur. Refinement and adoption of this framework across health systems can facilitate standardized measurement and reporting of diagnostic safety.

Comparison of Established Diagnostic Methodologies and a Novel Bacterial smpB Real-Time PCR Assay for Specific Detection of Haemophilus influenzae Isolates Associated with Respiratory Tract Infections.

PubMed

Reddington, Kate; Schwenk, Stefan; Tuite, Nina; Platt, Gareth; Davar, Danesh; Coughlan, Helena; Personne, Yoann; Gant, Vanya; Enne, Virve I; Zumla, Alimuddin; Barry, Thomas

2015-09-01

Haemophilus influenzae is a significant causative agent of respiratory tract infections (RTI) worldwide. The development of a rapid H. influenzae diagnostic assay that would allow for the implementation of infection control measures and also improve antimicrobial stewardship for patients is required. A number of nucleic acid diagnostics approaches that detect H. influenzae in RTIs have been described in the literature; however, there are reported specificity and sensitivity limitations for these assays. In this study, a novel real-time PCR diagnostic assay targeting the smpB gene was designed to detect all serogroups of H. influenzae. The assay was validated using a panel of well-characterized Haemophilus spp. Subsequently, 44 Haemophilus clinical isolates were collected, and 36 isolates were identified as H. influenzae using a gold standard methodology that combined the results of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and a fucK diagnostic assay. Using the novel smpB diagnostic assay, 100% concordance was observed with the gold standard, demonstrating a sensitivity of 100% (95% confidence interval [CI], 90.26% to 100.00%) and a specificity of 100% (95% CI, 63.06% to 100.00%) when used on clinical isolates. To demonstrate the clinical utility of the diagnostic assay presented, a panel of lower RTI samples (n = 98) were blindly tested with the gold standard and smpB diagnostic assays. The results generated were concordant for 94/98 samples tested, demonstrating a sensitivity of 90.91% (95% CI, 78.33% to 97.47%) and a specificity of 100% (95% CI, 93.40% to 100.00%) for the novel smpB assay when used directly on respiratory specimens. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Improvement of DGGE analysis by modifications of PCR protocols for analysis of microbial community members with low abundance.

PubMed

Wang, Yong-Feng; Zhang, Fang-Qiu; Gu, Ji-Dong

2014-06-01

Denaturing gradient gel electrophoresis (DGGE) is a powerful technique to reveal the community structures and composition of microorganisms in complex natural environments and samples. However, positive and reproducible polymerase chain reaction (PCR) products, which are difficult to acquire for some specific samples due to low abundance of the target microorganisms, significantly impair the effective applications of DGGE. Thus, nested PCR is often introduced to generate positive PCR products from the complex samples, but one problem is also introduced: The total number of thermocycling in nested PCR is usually unacceptably high, which results in skewed community structures by generation of random or mismatched PCR products on the DGGE gel, and this was demonstrated in this study. Furthermore, nested PCR could not resolve the uneven representative issue with PCR products of complex samples with unequal richness of microbial population. In order to solve the two problems in nested PCR, the general protocol was modified and improved in this study. Firstly, a general PCR procedure was used to amplify the target genes with the PCR primers without any guanine cytosine (GC) clamp, and then, the resultant PCR products were purified and diluted to 0.01 μg ml(-1). Subsequently, the diluted PCR products were utilized as templates to amplify again with the same PCR primers with the GC clamp for 17 cycles, and the products were finally subjected to DGGE analysis. We demonstrated that this is a much more reliable approach to obtain a high quality DGGE profile with high reproducibility. Thus, we recommend the adoption of this improved protocol in analyzing microorganisms of low abundance in complex samples when applying the DGGE fingerprinting technique to avoid biased results.

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

Evaluation of PCR Approaches for Detection of Bartonella bacilliformis in Blood Samples.

PubMed

Gomes, Cláudia; Martinez-Puchol, Sandra; Pons, Maria J; Bazán, Jorge; Tinco, Carmen; del Valle, Juana; Ruiz, Joaquim

2016-03-01

The lack of an effective diagnostic tool for Carrion's disease leads to misdiagnosis, wrong treatments and perpetuation of asymptomatic carriers living in endemic areas. Conventional PCR approaches have been reported as a diagnostic technique. However, the detection limit of these techniques is not clear as well as if its usefulness in low bacteriemia cases. The aim of this study was to evaluate the detection limit of 3 PCR approaches. We determined the detection limit of 3 different PCR approaches: Bartonella-specific 16S rRNA, fla and its genes. We also evaluated the viability of dry blood spots to be used as a sample transport system. Our results show that 16S rRNA PCR is the approach with a lowest detection limit, 5 CFU/μL, and thus, the best diagnostic PCR tool studied. Dry blood spots diminish the sensitivity of the assay. From the tested PCRs, the 16S rRNA PCR-approach is the best to be used in the direct blood detection of acute cases of Carrion's disease. However its use in samples from dry blood spots results in easier management of transport samples in rural areas, a slight decrease in the sensitivity was observed. The usefulness to detect by PCR the presence of low-bacteriemic or asymptomatic carriers is doubtful, showing the need to search for new more sensible techniques.

Validity of a PCR assay in CSF for the diagnosis of neurocysticercosis

PubMed Central

Campoverde, Alfredo; Romo, Matthew L.; García, Lorena; Piedra, Luis M.; Pacurucu, Mónica; López, Nelson; Aguilar, Jenner; López, Sebastian; Vintimilla, Luis C.; Toral, Ana M.; Peña-Tapia, Pablo

2017-01-01

Objective: To prospectively evaluate the validity of a PCR assay in CSF for the diagnosis of neurocysticercosis (NC). Methods: We conducted a multicenter, prospective case-control study, recruiting participants from 5 hospitals in Cuenca, Ecuador, from January 2015 to February 2016. Cases fulfilled validated diagnostic criteria for NC. For each case, a neurosurgical patient who did not fulfill the diagnostic criteria for NC was selected as a control. CT and MRI, as well as a CSF sample, were collected from both cases and controls. The diagnostic criteria to identify cases were used as a reference standard. Results: Overall, 36 case and 36 control participants were enrolled. PCR had a sensitivity of 72.2% (95% confidence interval [CI] 54.8%–85.8%) and a specificity of 100.0% (95% CI 90.3%–100.0%). For parenchymal NC, PCR had a sensitivity of 42.9% (95% CI 17.7%–71.1%), and for extraparenchymal NC, PCR had a sensitivity of 90.9% (95% CI 70.8%–98.9%). Conclusions: This study demonstrated the usefulness of this PCR assay in CSF for the diagnosis of NC. PCR may be particularly helpful for diagnosing extraparenchymal NC when neuroimaging techniques have failed. Classification of evidence: This study provides Class III evidence that CSF PCR can accurately identify patients with extraparenchymal NC. PMID:28105460

Optimal pcr primers for rapid and accurate detection of Aspergillus flavus isolates.

PubMed

Al-Shuhaib, Mohammed Baqur S; Albakri, Ali H; Alwan, Sabah H; Almandil, Noor B; AbdulAzeez, Sayed; Borgio, J Francis

2018-03-01

Aspergillus flavus is among the most devastating opportunistic pathogens of several food crops including rice, due to its high production of carcinogenic aflatoxins. The presence of these organisms in economically important rice strip farming is a serious food safety concern. Several polymerase chain reaction (PCR) primers have been designed to detect this species; however, a comparative assessment of their accuracy has not been conducted. This study aims to identify the optimal diagnostic PCR primers for the identification of A. flavus, among widely available primers. We isolated 122 A. flavus native isolates from randomly collected rice strips (N = 300). We identified 109 isolates to the genus level using universal fungal PCR primer pairs. Nine pairs of primers were examined for their PCR diagnostic specificity on the 109 isolates. FLA PCR was found to be the optimal PCR primer pair for specific identification of the native isolates, over aflP(1), aflM, aflA, aflD, aflP(3), aflP(2), and aflR. The PEP primer pair was found to be the most unsuitable for A. flavus identification. In conclusion, the present study indicates the powerful specificity of the FLA PCR primer over other commonly available diagnostic primers for accurate, rapid, and large-scale identification of A. flavus native isolates. This study provides the first simple, practical comparative guide to PCR-based screening of A. flavus infection in rice strips. Copyright © 2018 Elsevier Ltd. All rights reserved.

Gold Nanorod-based Photo-PCR System for One-Step, Rapid Detection of Bacteria

PubMed Central

Kim, Jinjoo; Kim, Hansol; Park, Ji Ho; Jon, Sangyong

2017-01-01

The polymerase chain reaction (PCR) has been an essential tool for diagnosis of infectious diseases, but conventional PCR still has some limitations with respect to applications to point-of-care (POC) diagnostic systems that require rapid detection and miniaturization. Here we report a light-based PCR method, termed as photo-PCR, which enables rapid detection of bacteria in a single step. In the photo-PCR system, poly(enthylene glycol)-modified gold nanorods (PEG-GNRs), used as a heat generator, are added into the PCR mixture, which is subsequently periodically irradiated with a 808-nm laser to create thermal cycling. Photo-PCR was able to significantly reduce overall thermal cycling time by integrating bacterial cell lysis and DNA amplification into a single step. Furthermore, when combined with KAPA2G fast polymerase and cooling system, the entire process of bacterial genomic DNA extraction and amplification was further shortened, highlighting the potential of photo-PCR for use in a portable, POC diagnostic system. PMID:29071186

Diagnostic decision-making and strategies to improve diagnosis.

PubMed

Thammasitboon, Satid; Cutrer, William B

2013-10-01

A significant portion of diagnostic errors arises through cognitive errors resulting from inadequate knowledge, faulty data gathering, and/or faulty verification. Experts estimate that 75% of diagnostic failures can be attributed to clinician diagnostic thinking failure. The cognitive processes that underlie diagnostic thinking of clinicians are complex and intriguing, and it is imperative that clinicians acquire explicit appreciation and application of different cognitive approaches to make decisions better. A dual-process model that unifies many theories of decision-making has emerged as a promising template for understanding how clinicians think and judge efficiently in a diagnostic reasoning process. The identification and implementation of strategies for decreasing or preventing such diagnostic errors has become a growing area of interest and research. Suggested strategies to decrease diagnostic error incidence include increasing clinician's clinical expertise and avoiding inherent cognitive errors to make decisions better. Implementing Interventions focused solely on avoiding errors may work effectively for patient safety issues such as medication errors. Addressing cognitive errors, however, requires equal effort on expanding the individual clinician's expertise. Providing cognitive support to clinicians for robust diagnostic decision-making serves as the final strategic target for decreasing diagnostic errors. Clinical guidelines and algorithms offer another method for streamlining decision-making and decreasing likelihood of cognitive diagnostic errors. Addressing cognitive processing errors is undeniably the most challenging task in reducing diagnostic errors. While many suggested approaches exist, they are mostly based on theories and sciences in cognitive psychology, decision-making, and education. The proposed interventions are primarily suggestions and very few of them have been tested in the actual practice settings. Collaborative research effort is

Improved detection of Pneumocystis jirovecii in upper and lower respiratory tract specimens from children with suspected pneumocystis pneumonia using real-time PCR: a prospective study

PubMed Central

2011-01-01

Background Pneumocystis pneumonia (PCP) is a major cause of hospitalization and mortality in HIV-infected African children. Microbiologic diagnosis relies predominantly on silver or immunofluorescent staining of a lower respiratory tract (LRT) specimens which are difficult to obtain in children. Diagnosis on upper respiratory tract (URT) specimens using PCR has been reported useful in adults, but data in children are limited. The main objectives of the study was (1) to compare the diagnostic yield of PCR with immunofluorescence (IF) and (2) to investigate the usefulness of upper compared to lower respiratory tract samples for diagnosing PCP in children. Methods Children hospitalised at an academic hospital with suspected PCP were prospectively enrolled. An upper respiratory sample (nasopharyngeal aspirate, NPA) and a lower respiratory sample (induced sputum, IS or bronchoalveolar lavage, BAL) were submitted for real-time PCR and direct IF for the detection of Pneumocystis jirovecii. A control group of children with viral lower respiratory tract infections were investigated with PCR for PCP. Results 202 children (median age 3.3 [inter-quartile range, IQR 2.2 - 4.6] months) were enrolled. The overall detection rate by PCR was higher than by IF [180/349 (52%) vs. 26/349 (7%) respectively; p < 0.0001]. PCR detected more infections compared to IF in lower respiratory tract samples [93/166 (56%) vs. 22/166 (13%); p < 0.0001] and in NPAs [87/183 (48%) vs. 4/183 (2%); p < 0.0001]. Detection rates by PCR on upper (87/183; 48%) compared with lower respiratory tract samples (93/166; 56%) were similar (OR, 0.71; 95% CI, 0.46 - 1.11). Only 2/30 (6.6%) controls were PCR positive. Conclusion Real-time PCR is more sensitive than IF for the detection of P. jirovecii in children with PCP. NPA samples may be used for diagnostic purposes when PCR is utilised. Wider implementation of PCR on NPA samples is warranted for diagnosing PCP in children. PMID:22123076

Nasal PCR assay for the detection of Mycobacterium leprae pra gene to study subclinical infection in a community.

PubMed

Arunagiri, Kamalanathan; Sangeetha, Gopalakrishnan; Sugashini, Padmavathy Krishnan; Balaraman, Sekar; Showkath Ali, M K

2017-03-01

Leprosy is a chronic infectious disease caused by Mycobacterium leprae. Identification of Mycobacterium leprae is difficult in part due to the inability of the leprosy bacillus to grow in vitro. A number of diagnostic methods for leprosy diagnosis have been proposed. Both serological tests and molecular probes have shown certain potential for detection and identification of Mycobacterium leprae in patients. In this study, we have investigated whether Mycobacterium leprae DNA from the nasal secretion of healthy household contacts and the non contacts could be detected through PCR amplification as a method to study the sub clinical infection in a community. A total of 200 samples, 100 each from contacts and non contacts representing all age groups and sex were included in this study. The M. leprae specific primer (proline-rich region) of pra gene was selected and PCR was performed using extracted DNA from the sample. A total of 13 samples were found to be positive for nasal PCR for pra gene among the male and female contacts out of which 7% were males and 6% were females. Even though several diagnostic tools are available to detect the cases of leprosy, they lack the specificity and sensitivity. PCR technology has demonstrated the improved diagnostic accuracy for epidemiological studies and requires minimal time. Although nasal PCR studies have been reported from many countries it is not usually recommended due to the high percentage of negative results in the contact. Copyright © 2017 Elsevier Ltd. All rights reserved.

Novel approach based on one-tube nested PCR and a lateral flow strip for highly sensitive diagnosis of tuberculous meningitis.

PubMed

Sun, Yajuan; Chen, Jiajun; Li, Jia; Xu, Yawei; Jin, Hui; Xu, Na; Yin, Rui; Hu, Guohua

2017-01-01

Rapid and sensitive detection of Mycobacterium tuberculosis (M. Tb) in cerebrospinal fluid is crucial in the diagnosis of tuberculous meningitis (TBM), but conventional diagnostic technologies have limited sensitivity and specificity or are time-consuming. In this work, a novel, highly sensitive molecular diagnostic method, one-tube nested PCR-lateral flow strip test (OTNPCR-LFST), was developed for detecting M. tuberculosis. This one-tube nested PCR maintains the sensitivity of conventional two-step nested PCR and reduces both the chance of cross-contamination and the time required for analysis. The PCR product was detected by a lateral flow strip assay, which provided a basis for migration of the test to a point-of-care (POC) microfluidic format. The developed assay had an improved sensitivity compared with traditional PCR, and the limit of detection was up to 1 fg DNA isolated from M. tuberculosis. The assay was also specific for M. tuberculosis, and no cross-reactions were found in other non-target bacteria. The application of this technique to clinical samples was successfully evaluated, and OTNPCR-LFST showed 89% overall sensitivity and 100% specificity for TBM patients. This one-tube nested PCR-lateral flow strip assay is useful for detecting M. tuberculosis in TBM due to its rapidity, high sensitivity and simple manipulation.

Novel approach based on one-tube nested PCR and a lateral flow strip for highly sensitive diagnosis of tuberculous meningitis

PubMed Central

Sun, Yajuan; Chen, Jiajun; Li, Jia; Xu, Yawei; Jin, Hui; Xu, Na; Yin, Rui

2017-01-01

Rapid and sensitive detection of Mycobacterium tuberculosis (M. Tb) in cerebrospinal fluid is crucial in the diagnosis of tuberculous meningitis (TBM), but conventional diagnostic technologies have limited sensitivity and specificity or are time-consuming. In this work, a novel, highly sensitive molecular diagnostic method, one-tube nested PCR-lateral flow strip test (OTNPCR-LFST), was developed for detecting M. tuberculosis. This one-tube nested PCR maintains the sensitivity of conventional two-step nested PCR and reduces both the chance of cross-contamination and the time required for analysis. The PCR product was detected by a lateral flow strip assay, which provided a basis for migration of the test to a point-of-care (POC) microfluidic format. The developed assay had an improved sensitivity compared with traditional PCR, and the limit of detection was up to 1 fg DNA isolated from M. tuberculosis. The assay was also specific for M. tuberculosis, and no cross-reactions were found in other non-target bacteria. The application of this technique to clinical samples was successfully evaluated, and OTNPCR-LFST showed 89% overall sensitivity and 100% specificity for TBM patients. This one-tube nested PCR-lateral flow strip assay is useful for detecting M. tuberculosis in TBM due to its rapidity, high sensitivity and simple manipulation. PMID:29084241

Investigation of pregnancy-associated malaria by microscopy, rapid diagnostic test and PCR in Bandundu, the Democratic Republic of Congo.

PubMed

Ruh, Emrah; Bateko, Jean Paul; Imir, Turgut; Taylan-Ozkan, Aysegul

2018-01-01

The study was conducted to investigate malaria prevalence among a group of women in the Democratic Republic of Congo (DRC) who received intermittent preventive treatment in pregnancy with sulfadoxine-pyrimethamine (IPTp-SP). A total of 250 women from Bandundu city who received two doses of IPTp-SP were enrolled in the survey. Blood samples were collected at the time of delivery and malaria prevalence was determined using microscopy, rapid diagnostic test (RDT), and nested polymerase chain reaction (PCR). Malaria infection was detected in 81 (32.4%), 93 (37.2%), and 92 (36.8%) samples by microscopy, RDT, and PCR, respectively. Among 92 samples, P. falciparum mono-infection (n=87; 94.5%), P. falciparum+P. vivax (n=2; 2.2%) and P. falciparum+P. malariae (n=1; 1.1%) mixed infections, and P. vivax mono-infection (n=2; 2.2%) were detected. Prevalence of malaria was not affected by age and number of pregnancies (p>0.05). Microscopy and RDT, either alone (κ=0.29; p<0.001) or in combination (κ=0.33; p<0.001) showed a fair agreement with PCR. Our findings indicate that two doses of IPTp-SP did not protect the women against malaria in the DRC, and support the World Health Organization (WHO) guidelines that ensure a minimum of three doses of SP in pregnancy.

Diagnostic accuracy of fungal PCR and β-d-glucan for detection of candidaemia: a preliminary evaluation.

PubMed

McKeating, Cara; White, P Lewis; Posso, Raquel; Palmer, Michael; Johnson, Elizabeth; McMullan, Ronan

2018-05-01

Although treatment for candidaemia is time critical, culture-based tests prolong turnaround times and may promote underdiagnosis. Non-culture-based tests have the potential to overcome these difficulties but are in limited clinical use. The aim of this work was to undertake an initial evaluation of two non-culture-based tests for diagnosis of candidaemia. Patients with candidaemia were identified prospectively over a 4-month period. Sera drawn from case (candidaemic) and control (non-candidaemic) patients on the same day as the positive blood culture were tested with both the Renishaw RenDx Fungiplex test and a commercial β-d-glucan (BDG) assay (Fungitell, Associates of Cape Cod). Sensitivity and specificity were calculated independently and in combination, using paired blood culture as the reference standard. There were 10 eligible case patients and 39 negative controls. PCR sensitivity and specificity were found to be 44.4% (95% CI 18.9% to 73.3%) and 87.2% (72.8% to 94.8%), respectively. BDG sensitivity and specificity were 80% (47.9% to 95.4%) and 89.7% (75.9% to 96.5%), respectively. When combining PCR and BDG, sensitivity was 90% (95% CI 57.4% to 100%) and specificity was 79.5% (64.2% to 89.5%). When two sequential specimens were tested, PCR sensitivity increased to 60% (95% CI 31.2% to 83.3%) and BDG sensitivity to 90% (54.7% to 100%). A combination of tests, or a single test at multiple time points, may be preferable to relying on one test at a single time point. This should be accounted for in design of future diagnostic accuracy studies of tests for invasive candidosis. © 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.

Comparative genome analysis identifies novel nucleic acid diagnostic targets for use in the specific detection of Haemophilus influenzae.

PubMed

Coughlan, Helena; Reddington, Kate; Tuite, Nina; Boo, Teck Wee; Cormican, Martin; Barrett, Louise; Smith, Terry J; Clancy, Eoin; Barry, Thomas

2015-10-01

Haemophilus influenzae is recognised as an important human pathogen associated with invasive infections, including bloodstream infection and meningitis. Currently used molecular-based diagnostic assays lack specificity in correctly detecting and identifying H. influenzae. As such, there is a need to develop novel diagnostic assays for the specific identification of H. influenzae. Whole genome comparative analysis was performed to identify putative diagnostic targets, which are unique in nucleotide sequence to H. influenzae. From this analysis, we identified 2H. influenzae putative diagnostic targets, phoB and pstA, for use in real-time PCR diagnostic assays. Real-time PCR diagnostic assays using these targets were designed and optimised to specifically detect and identify all 55H. influenzae strains tested. These novel rapid assays can be applied to the specific detection and identification of H. influenzae for use in epidemiological studies and could also enable improved monitoring of invasive disease caused by these bacteria. Copyright © 2015 Elsevier Inc. All rights reserved.

A PCR-Based Diagnostic System for Differentiating Two Weevil Species (Coleoptera: Curculionidae) of Economic Importance to the Chilean Citrus Industry.

PubMed

Aguirre, C; Olivares, N; Luppichini, P; Hinrichsen, P

2015-02-01

A PCR-based method was developed to identify Naupactus cervinus (Boheman) and Naupactus xanthographus (Germar), two curculionids affecting the citrus industry in Chile. The quarantine status of these two species depends on the country to which fruits are exported. This identification method was developed because it is not possible to discriminate between these two species at the egg stage. The method is based on the species-specific amplification of sequences of internal transcribed spacers, for which we cloned and sequenced these genome fragments from each species. We designed an identification system based on two duplex-PCR reactions. Each one contains the species-specific primer set and a second generic primer set that amplify a short 18S region common to coleopterans, to avoid false negatives. The marker system is able to differentiate each Naupactus species at any life stage, and with a diagnostic sensitivity to 0.045 ng of genomic DNA. This PCR kit was validated by samples collected from different citrus production areas throughout Chile and showed 100% accuracy in differentiating the two Naupactus species. © The Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Applications of the rep-PCR DNA fingerprinting technique to study microbial diversity, ecology and evolution.

PubMed

Ishii, Satoshi; Sadowsky, Michael J

2009-04-01

A large number of repetitive DNA sequences are found in multiple sites in the genomes of numerous bacteria, archaea and eukarya. While the functions of many of these repetitive sequence elements are unknown, they have proven to be useful as the basis of several powerful tools for use in molecular diagnostics, medical microbiology, epidemiological analyses and environmental microbiology. The repetitive sequence-based PCR or rep-PCR DNA fingerprint technique uses primers targeting several of these repetitive elements and PCR to generate unique DNA profiles or 'fingerprints' of individual microbial strains. Although this technique has been extensively used to examine diversity among variety of prokaryotic microorganisms, rep-PCR DNA fingerprinting can also be applied to microbial ecology and microbial evolution studies since it has the power to distinguish microbes at the strain or isolate level. Recent advancement in rep-PCR methodology has resulted in increased accuracy, reproducibility and throughput. In this minireview, we summarize recent improvements in rep-PCR DNA fingerprinting methodology, and discuss its applications to address fundamentally important questions in microbial ecology and evolution.

Comparison of PCR, culturing and Pap smear microscopy for accurate diagnosis of genital Actinomyces.

PubMed

Kaya, Dilek; Demirezen, Şayeste; Hasçelik, Gülşen; Gülmez Kivanç, Dolunay; Beksaç, Mehmet Sinan

2013-05-01

Members of the genus Actinomyces, Gram-positive, non-spore-forming anaerobic bacteria, are normal inhabitants of the mucosal surfaces of the oral, gastrointestinal and genital tracts. Identification of these bacteria using conventional methods is generally difficult because of their complex transport and growth requirements and their fastidious and slow-growing nature. However, in recent years, the advancement of molecular techniques has provided much improved identification and differentiation of closely related Actinomyces species. The aim of the present study was to evaluate the efficacy of the PCR technique in the diagnosis of genital Actinomyces in comparison with culturing and Papanicolaou (Pap) smear microscopy. Multiple sampling was conducted from 200 women using smear microscopy, culturing and PCR. Cyto-brushes were smeared on glass slides and stained using the routine Pap technique. Culturing was performed from a sterile swab, and Actinomyces were determined using the BBL Crystal ANR ID kit. PCR was performed from a second swab, and the Actinomyces type was determined using type-specific primers designed in our laboratory. Only one vaginal fluid sample (0.5%) revealed Actinomyces-like organisms on Pap smear examination. Actinomyces were detected in nine samples (4.5%) using the BBL Crystal ANR ID kit. Using PCR, eight samples (4%) were found positive for Actinomyces. No specimens that gave positive results by Pap smear microscopy and culturing could be confirmed by PCR. Pap smear microscopy and culturing were both found to have zero sensitivity for Actinomyces. PCR appears to be a sensitive and reliable diagnostic method for the detection of Actinomyces, which are difficult to cultivate from genital samples. PCR can be used for diagnostic confirmation in cases diagnosed by conventional methods, to prevent false-positive results.

Gold nanoparticle-based RT-PCR and real-time quantitative RT-PCR assays for detection of Japanese encephalitis virus

NASA Astrophysics Data System (ADS)

Huang, Su-Hua; Yang, Tsuey-Ching; Tsai, Ming-Hong; Tsai, I.-Shou; Lu, Huang-Chih; Chuang, Pei-Hsin; Wan, Lei; Lin, Ying-Ju; Lai, Chih-Ho; Lin, Cheng-Wen

2008-10-01

Virus isolation and antibody detection are routinely used for diagnosis of Japanese encephalitis virus (JEV) infection, but the low level of transient viremia in some JE patients makes JEV isolation from clinical and surveillance samples very difficult. We describe the use of gold nanoparticle-based RT-PCR and real-time quantitative RT-PCR assays for detection of JEV from its RNA genome. We tested the effect of gold nanoparticles on four different PCR systems, including conventional PCR, reverse-transcription PCR (RT-PCR), and SYBR green real-time PCR and RT-PCR assays for diagnosis in the acute phase of JEV infection. Gold nanoparticles increased the amplification yield of the PCR product and shortened the PCR time compared to the conventional reaction. In addition, nanogold-based real-time RT-PCR showed a linear relationship between Ct and template amount using ten-fold dilutions of JEV. The nanogold-based RT-PCR and real-time quantitative RT-PCR assays were able to detect low levels (1-10 000 copies) of the JEV RNA genomes extracted from culture medium or whole blood, providing early diagnostic tools for the detection of low-level viremia in the acute-phase infection. The assays described here were simple, sensitive, and rapid approaches for detection and quantitation of JEV in tissue cultured samples as well as clinical samples.

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.

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

Comparison of a conventional and nested PCR for diagnostic confirmation and genotyping of Orientia tsutsugamushi.

PubMed

Janardhanan, Jeshina; Prakash, John Antony Jude; Abraham, Ooriapadickal C; Varghese, George M

2014-05-01

A nested polymerase chain reaction (PCR) targeting the 56-kDa antigen gene is currently the most commonly used molecular technique for confirmation of scrub typhus and genotyping of Orientia tsutsugamushi. In this study, we have compared the commonly used nested PCR (N-PCR) with a single-step conventional PCR (C-PCR) for amplification and genotyping. Eschar samples collected from 24 patients with scrub typhus confirmed by IgM enzyme-linked immunosorbent assay were used for DNA extraction following which amplifications were carried out using nested and C-PCR methods. The amplicons were sequenced and compared to other sequences in the database using BLAST. Conventional PCR showed a high positivity rate of 95.8% compared to the 75% observed using N-PCR. On sequence analysis, the N-PCR amplified region showed more variation among strains than the C-PCR amplified region. The C-PCR, which is more economical, provided faster and better results compared to N-PCR. Copyright © 2014 Elsevier Inc. 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

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.

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

Detection of Mycobacterium tuberculosis in extrapulmonary biopsy samples using PCR targeting IS6110, rpoB, and nested-rpoB PCR Cloning

PubMed Central

Meghdadi, Hossein; Khosravi, Azar D.; Ghadiri, Ata A.; Sina, Amir H.; Alami, Ameneh

2015-01-01

Present study was aimed to examine the diagnostic utility of polymerase chain reaction (PCR) and nested PCR techniques for the detection of Mycobacterium tuberculosis (MTB) DNA in samples from patients with extra pulmonary tuberculosis (EPTB). In total 80 formalin-fixed, paraffin-embedded (FFPE) samples comprising 70 samples with definite diagnosis of EPTB and 10 samples from known non- EPTB on the basis of histopathology examination, were included in the study. PCR amplification targeting IS6110, rpoB gene and nested PCR targeting the rpoB gene were performed on the extracted DNAs from 80 FFPE samples. The strong positive samples were directly sequenced. For negative samples and those with weak band in nested-rpoB PCR, TA cloning was performed by cloning the products into the plasmid vector with subsequent sequencing. The 95% confidence intervals (CI) for the estimates of sensitivity and specificity were calculated for each method. Fourteen (20%), 34 (48.6%), and 60 (85.7%) of the 70 positive samples confirmed by histopathology, were positive by rpoB-PCR, IS6110-PCR, and nested-rpoB PCR, respectively. By performing TA cloning on samples that yielded weak (n = 8) or negative results (n = 10) in the PCR methods, we were able to improve their quality for later sequencing. All samples with weak band and 7 out of 10 negative samples, showed strong positive results after cloning. So nested-rpoB PCR cloning revealed positivity in 67 out of 70 confirmed samples (95.7%). The sensitivity of these combination methods was calculated as 95.7% in comparison with histopathology examination. The CI for sensitivity of the PCR methods were calculated as 11.39–31.27% for rpoB-PCR, 36.44–60.83% for IS6110- PCR, 75.29–92.93% for nested-rpoB PCR, and 87.98–99.11% for nested-rpoB PCR cloning. The 10 true EPTB negative samples by histopathology, were negative by all tested methods including cloning and were used to calculate the specificity of the applied methods. The CI for 100

Detection of Mycobacterium tuberculosis in extrapulmonary biopsy samples using PCR targeting IS6110, rpoB, and nested-rpoB PCR Cloning.

PubMed

Meghdadi, Hossein; Khosravi, Azar D; Ghadiri, Ata A; Sina, Amir H; Alami, Ameneh

2015-01-01

Present study was aimed to examine the diagnostic utility of polymerase chain reaction (PCR) and nested PCR techniques for the detection of Mycobacterium tuberculosis (MTB) DNA in samples from patients with extra pulmonary tuberculosis (EPTB). In total 80 formalin-fixed, paraffin-embedded (FFPE) samples comprising 70 samples with definite diagnosis of EPTB and 10 samples from known non- EPTB on the basis of histopathology examination, were included in the study. PCR amplification targeting IS6110, rpoB gene and nested PCR targeting the rpoB gene were performed on the extracted DNAs from 80 FFPE samples. The strong positive samples were directly sequenced. For negative samples and those with weak band in nested-rpoB PCR, TA cloning was performed by cloning the products into the plasmid vector with subsequent sequencing. The 95% confidence intervals (CI) for the estimates of sensitivity and specificity were calculated for each method. Fourteen (20%), 34 (48.6%), and 60 (85.7%) of the 70 positive samples confirmed by histopathology, were positive by rpoB-PCR, IS6110-PCR, and nested-rpoB PCR, respectively. By performing TA cloning on samples that yielded weak (n = 8) or negative results (n = 10) in the PCR methods, we were able to improve their quality for later sequencing. All samples with weak band and 7 out of 10 negative samples, showed strong positive results after cloning. So nested-rpoB PCR cloning revealed positivity in 67 out of 70 confirmed samples (95.7%). The sensitivity of these combination methods was calculated as 95.7% in comparison with histopathology examination. The CI for sensitivity of the PCR methods were calculated as 11.39-31.27% for rpoB-PCR, 36.44-60.83% for IS6110- PCR, 75.29-92.93% for nested-rpoB PCR, and 87.98-99.11% for nested-rpoB PCR cloning. The 10 true EPTB negative samples by histopathology, were negative by all tested methods including cloning and were used to calculate the specificity of the applied methods. The CI for 100

PCR on yeast colonies: an improved method for glyco-engineered Saccharomyces cerevisiae

PubMed Central

2013-01-01

Background Saccharomyces cerevisiae is extensively used in bio-industries. However, its genetic engineering to introduce new metabolism pathways can cause unexpected phenotypic alterations. For example, humanisation of the glycosylation pathways is a high priority pharmaceutical industry goal for production of therapeutic glycoproteins in yeast. Genomic modifications can lead to several described physiological changes: biomass yields decrease, temperature sensitivity or cell wall structure modifications. We have observed that deletion of several N-mannosyltransferases in Saccharomyces cerevisiae, results in strains that can no longer be analyzed by classical PCR on yeast colonies. Findings In order to validate our glyco-engineered Saccharomyces cerevisiae strains, we developed a new protocol to carry out PCR directly on genetically modified yeast colonies. A liquid culture phase, combined with the use of a Hot Start DNA polymerase, allows a 3-fold improvement of PCR efficiency. The results obtained are repeatable and independent of the targeted sequence; as such the protocol is well adapted for intensive screening applications. Conclusions The developed protocol enables by-passing of many of the difficulties associated with PCR caused by phenotypic modifications brought about by humanisation of the glycosylation in yeast and allows rapid validation of glyco-engineered Saccharomyces cerevisiae cells. It has the potential to be extended to other yeast strains presenting cell wall structure modifications. PMID:23688076

Improved diagnostics of chronic inflammatory prostatitis.

PubMed

Kulchavenya, E; Azizoff, A; Brizhatyuk, E; Khomyakov, V; Kholtobin, D; Breusoff, A; Naber, K G

2012-12-01

Prostatitis is a prevalent condition that encompasses a large array of clinical symptoms with significant impacts on men's life. The diagnosis and treatment of this disorder presents numerous challenges for urologists, most notably, a lack of specific and effective diagnostic methods. To improve the diagnostics the comparison of classic 4-glass test Meares and Stamey, 2-glass tests and 3-glass test was conducted in 177 men suspicious for chronic prostatitis. Four-glass test is uncomfortable both for patients and doctors, and leads to contamination of urine with prostatic secretion. Two-glass test is insufficiently effective too. Three-glass test (three urine specimens obtained from one continuous micturition stream) gives more adequate results and may be used for screening. Three-glass test as screening test with the option of an additional EPS investigation in those patients the final diagnosis of chronic prostatitis has to be confirmed is more convenient for patients and doctors than the standard M&S 4-glass test and "false-positive" (contaminated with EPS) midstream urine results are avoided thus improving discrimination of urethritis, cystitis and prostatitis. Therefore, we recommend the KE 3-glass test as a new standard for screening patients with signs and symptoms of chronic inflammatory prostatitis.

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

Blood culture-PCR to optimise typhoid fever diagnosis after controlled human infection identifies frequent asymptomatic cases and evidence of primary bacteraemia.

PubMed

Darton, Thomas C; Zhou, Liqing; Blohmke, Christoph J; Jones, Claire; Waddington, Claire S; Baker, Stephen; Pollard, Andrew J

2017-04-01

Improved diagnostics for typhoid are needed; a typhoid controlled human infection model may accelerate their development and translation. Here, we evaluated a blood culture-PCR assay for detecting infection after controlled human infection with S. Typhi and compared test performance with optimally performed blood cultures. Culture-PCR amplification of blood samples was performed alongside daily blood culture in 41 participants undergoing typhoid challenge. Study endpoints for typhoid diagnosis (TD) were fever and/or bacteraemia. Overall, 24/41 (59%) participants reached TD, of whom 21/24 (86%) had ≥1 positive blood culture (53/674, 7.9% of all cultures) or 18/24 (75%) had ≥1 positive culture-PCR assay result (57/684, 8.3%). A further five non-bacteraemic participants produced culture-PCR amplicons indicating infection; overall sensitivity/specificity of the assay compared to the study endpoints were 70%/65%. We found no significant difference between blood culture and culture-PCR methods in ability to identify cases (12 mismatching pairs, p = 0.77, binomial test). Clinical and stool culture metadata demonstrated that additional culture-PCR amplification positive individuals likely represented true cases missed by blood culture, suggesting the overall attack rate may be 30/41 (73%) rather than 24/41 (59%). Several participants had positive culture-PCR results soon after ingesting challenge providing new evidence for occurrence of an early primary bacteraemia. Overall the culture-PCR assay performed well, identifying extra typhoid cases compared with routine blood culture alone. Despite limitations to widespread field-use, the benefits of increased diagnostic yield, reduced blood volume and faster turn-around-time, suggest that this assay could enhance laboratory typhoid diagnostics in research applications and high-incidence settings. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Rapid and effective processing of blood specimens for diagnostic PCR using filter paper and Chelex-100.

PubMed Central

Polski, J M; Kimzey, S; Percival, R W; Grosso, L E

1998-01-01

AIM: To provide a more efficient method for isolating DNA from peripheral blood for use in diagnostic DNA mutation analysis. METHODS: The use of blood impregnated filter paper and Chelex-100 in DNA isolation was evaluated and compared with standard DNA isolation techniques. RESULTS: In polymerase chain reaction (PCR) based assays of five point mutations, identical results were obtained with DNA isolated routinely from peripheral blood and isolated using the filter paper and Chelex-100 method. CONCLUSION: In the clinical setting, this method provides a useful alternative to conventional DNA isolation. It is easily implemented and inexpensive, and provides sufficient, stable DNA for multiple assays. The potential for specimen contamination is reduced because most of the steps are performed in a single microcentrifuge tube. In addition, this method provides for easy storage and transport of samples from the point of acquisition. PMID:9893748

Rapid and effective processing of blood specimens for diagnostic PCR using filter paper and Chelex-100.

PubMed

Polski, J M; Kimzey, S; Percival, R W; Grosso, L E

1998-08-01

To provide a more efficient method for isolating DNA from peripheral blood for use in diagnostic DNA mutation analysis. The use of blood impregnated filter paper and Chelex-100 in DNA isolation was evaluated and compared with standard DNA isolation techniques. In polymerase chain reaction (PCR) based assays of five point mutations, identical results were obtained with DNA isolated routinely from peripheral blood and isolated using the filter paper and Chelex-100 method. In the clinical setting, this method provides a useful alternative to conventional DNA isolation. It is easily implemented and inexpensive, and provides sufficient, stable DNA for multiple assays. The potential for specimen contamination is reduced because most of the steps are performed in a single microcentrifuge tube. In addition, this method provides for easy storage and transport of samples from the point of acquisition.

Real-time PCR detection chemistry.

PubMed

Navarro, E; Serrano-Heras, G; Castaño, M J; Solera, J

2015-01-15

Real-time PCR is the method of choice in many laboratories for diagnostic and food applications. This technology merges the polymerase chain reaction chemistry with the use of fluorescent reporter molecules in order to monitor the production of amplification products during each cycle of the PCR reaction. Thus, the combination of excellent sensitivity and specificity, reproducible data, low contamination risk and reduced hand-on time, which make it a post-PCR analysis unnecessary, has made real-time PCR technology an appealing alternative to conventional PCR. The present paper attempts to provide a rigorous overview of fluorescent-based methods for nucleic acid analysis in real-time PCR described in the literature so far. Herein, different real-time PCR chemistries have been classified into two main groups; the first group comprises double-stranded DNA intercalating molecules, such as SYBR Green I and EvaGreen, whereas the second includes fluorophore-labeled oligonucleotides. The latter, in turn, has been divided into three subgroups according to the type of fluorescent molecules used in the PCR reaction: (i) primer-probes (Scorpions, Amplifluor, LUX, Cyclicons, Angler); (ii) probes; hydrolysis (TaqMan, MGB-TaqMan, Snake assay) and hybridization (Hybprobe or FRET, Molecular Beacons, HyBeacon, MGB-Pleiades, MGB-Eclipse, ResonSense, Yin-Yang or displacing); and (iii) analogues of nucleic acids (PNA, LNA, ZNA, non-natural bases: Plexor primer, Tiny-Molecular Beacon). In addition, structures, mechanisms of action, advantages and applications of such real-time PCR probes and analogues are depicted in this review. Copyright © 2014 Elsevier B.V. All rights reserved.

Epidemiology of meningitis with a negative CSF Gram stain: under-utilization of available diagnostic tests.

PubMed

Nesher, L; Hadi, C M; Salazar, L; Wootton, S H; Garey, K W; Lasco, T; Luce, A M; Hasbun, R

2016-01-01

Meningitis with a negative cerebrospinal fluid Gram stain (CSF-GS) poses a diagnostic challenge as more than 50% of patients remain without an aetiology. The introduction of polymerase chain reaction (PCR) and arboviral serologies have increased diagnostic capabilities, yet large scale epidemiological studies evaluating their use in clinical practice are lacking. We conducted a prospective observational study in New Orleans between November 1999 and September 2008 (early era) when PCR was not widely available, and in Houston between November 2008 and June 2013 (modern era), when PCR was commonly used. Patients presenting with meningitis and negative CSF-GS were followed for 4 weeks. All investigations, PCR used, and results were recorded as they became available. In 323 patients enrolled, PCR provided the highest diagnostic yield (24·2%) but was ordered for 128 (39·6%) patients; followed by serology for arboviruses (15%) that was ordered for 100 (31%) of all patients. The yield of blood cultures was (10·3%) and that of CSF cultures was 4%; the yield for all other tests was <10%. Overall, 65% of the patients remained without a diagnosis at 4 weeks: 72·1% in early era vs. 53·4% (P < 0·01) in modern era; this change was attributed to diagnosing more viral pathogens, 8·3% and 26·3% (P < 0·01), respectively. The introduction of PCR and arboviral serologies has improved the yield of diagnosing patients with meningitis and a negative CSF-GS, but both tests are being under-utilized.

Introduction of a dermatophyte polymerase chain reaction assay to the diagnostic mycology service in Scotland.

PubMed

Alexander, C L; Shankland, G S; Carman, W; Williams, C

2011-05-01

Dermatophytes are the major cause of superficial mycoses in samples submitted to Clinical Mycology, Glasgow. The most prevalent species is Trichophyton rubrum as identified classically by microscopy and culture. Recent advances in polymerase chain reaction (PCR) technology were examined for the feasibility of introducing a T. rubrum real-time PCR assay into a routine diagnostic service. To improve the diagnostic mycology service by the introduction of a real-time PCR test for T. rubrum. The DNA from 4972 nail and skin samples was obtained using the Qiagen QIAsymphony automated extractor. This DNA was subjected to real-time PCR using T. rubrum-specific primers and a probe. During phase 1 of the study, 862 samples were analysed; 446 of 470 specimens that grew T. rubrum were detected by PCR. Out of 4110 samples analysed during phase 2, 753 T. rubrum infections were diagnosed and reported within 72 h. A total of 3357 samples were negative for a fungal infection by PCR and microscopy; these were also reported within 72 h. A vast reduction in the turnaround times can be achieved using this technique as opposed to classical methods. Samples which are PCR negative but microscopy positive are still subjected to culture. Screening samples for their suitability for PCR prior to processing eliminates the application of PCR for T. rubrum on inappropriate samples such those from the scalp or pityriasis versicolor. © 2011 The Authors. BJD © 2011 British Association of Dermatologists.

Direct PCR Improves the Recovery of DNA from Various Substrates.

PubMed

Templeton, Jennifer E L; Taylor, Duncan; Handt, Oliva; Skuza, Pawel; Linacre, Adrian

2015-11-01

This study reports on the comparison of a standard extraction process with the direct PCR approach of processing low-level DNA swabs typical in forensic investigations. Varying concentrations of control DNA were deposited onto three commonly encountered substrates, brass, plastic, and glass, left to dry, and swabbed using premoistened DNA-free nylon FLOQswabs(™) . Swabs (n = 90) were either processed using the DNA IQ(™) kit or, for direct PCR, swab fibers (~2 mm(2) ) were added directly to the PCR with no prior extraction. A significant increase in the height of the alleles (p < 0.005) was observed when using the direct PCR approach over the extraction methodology when controlling for surface type and mass of DNA deposited. The findings indicate the potential use of direct PCR for increasing the PCR product obtained from low-template DNA samples in addition to minimizing contamination and saving resources. © 2015 American Academy of Forensic Sciences.

Optimization and Evaluation of a PCR Assay for Detecting Toxoplasmic Encephalitis in Patients with AIDS

PubMed Central

Joseph, Priya; Calderón, Maritza M.; Gilman, Robert H.; Quispe, Monica L.; Cok, Jaime; Ticona, Eduardo; Chavez, Victor; Jimenez, Juan A.; Chang, Maria C.; Lopez, Martín J.; Evans, Carlton A.

2002-01-01

Toxoplasma gondii is a common life-threatening opportunistic infection. We used experimental murine T. gondii infection to optimize the PCR for diagnostic use, define its sensitivity, and characterize the time course and tissue distribution of experimental toxoplasmosis. PCR conditions were adjusted until the assay reliably detected quantities of DNA derived from less than a single parasite. Forty-two mice were inoculated intraperitoneally with T. gondii tachyzoites and sacrificed from 6 to 72 h later. Examination of tissues with PCR and histology revealed progression of infection from blood to lung, heart, liver, and brain, with PCR consistently detecting parasites earlier than microscopy and with no false-positive results. We then evaluated the diagnostic value of this PCR assay in human patients. We studied cerebrospinal fluid and serum samples from 12 patients with AIDS and confirmed toxoplasmic encephalitis (defined as positive mouse inoculation and/or all of the Centers for Disease Control clinical diagnostic criteria), 12 human immunodeficiency virus-infected patients with suspected cerebral toxoplasmosis who had neither CDC diagnostic criteria nor positive mouse inoculation, 26 human immunodeficiency virus-infected patients with other opportunistic infections and no signs of cerebral toxoplasmosis, and 18 immunocompetent patients with neurocysticercosis. Eleven of the 12 patients with confirmed toxoplasmosis had positive PCR results in either blood or cerebrospinal fluid samples (6 of 9 blood samples and 8 of 12 cerebrospinal fluid samples). All samples from control patients were negative. This study demonstrates the high sensitivity, specificity, and clinical utility of PCR in the diagnosis of toxoplasmic encephalitis in a resource-poor setting. PMID:12454142

Multiplex real-time PCR using temperature sensitive primer-supplying hydrogel particles and its application for malaria species identification

PubMed Central

Byoun, Mun Sub; Yoo, Changhoon; Sim, Sang Jun; Lim, Chae Seung; Kim, Sung Woo

2018-01-01

Real-time PCR, also called quantitative PCR (qPCR), has been powerful analytical tool for detection of nucleic acids since it developed. Not only for biological research but also for diagnostic needs, qPCR technique requires capacity to detect multiple genes in recent years. Solid phase PCR (SP-PCR) where one or two directional primers are immobilized on solid substrates could analyze multiplex genetic targets. However, conventional SP-PCR was subjected to restriction of application for lack of PCR efficiency and quantitative resolution. Here we introduce an advanced qPCR with primer-incorporated network (PIN). One directional primers are immobilized in the porous hydrogel particle by covalent bond and the other direction of primers are temporarily immobilized at so-called 'Supplimers'. Supplimers released the primers to aqueous phase in the hydrogel at the thermal cycling of PCR. It induced the high PCR efficiency over 92% with high reliability. It reduced the formation of primer dimers and improved the selectivity of qPCR thanks to the strategy of 'right primers supplied to right place only'. By conducting a six-plex qPCR of 30 minutes, we analyzed DNA samples originated from malaria patients and successfully identified malaria species in a single reaction. PMID:29293604

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.

Cholera Rapid Test with Enrichment Step Has Diagnostic Performance Equivalent to Culture

PubMed Central

Ontweka, Lameck N.; Deng, Lul O.; Rauzier, Jean; Debes, Amanda K.; Tadesse, Fisseha; Parker, Lucy A.; Wamala, Joseph F.; Bior, Bior K.; Lasuba, Michael; But, Abiem Bona; Grandesso, Francesco; Jamet, Christine; Cohuet, Sandra; Ciglenecki, Iza; Serafini, Micaela; Sack, David A.; Quilici, Marie-Laure; Azman, Andrew S.; Luquero, Francisco J.

2016-01-01

Cholera rapid diagnostic tests (RDT) could play a central role in outbreak detection and surveillance in low-resource settings, but their modest performance has hindered their broad adoption. The addition of an enrichment step may improve test specificity. We describe the results of a prospective diagnostic evaluation of the Crystal VC RDT (Span Diagnostics, India) with enrichment step and of culture, each compared to polymerase chain reaction (PCR), during a cholera outbreak in South Sudan. RDTs were performed on alkaline peptone water inoculated with stool and incubated for 4–6 hours at ambient temperature. Cholera culture was performed from wet filter paper inoculated with stool. Molecular detection of Vibrio cholerae O1 by PCR was done from dry Whatman 903 filter papers inoculated with stool, and from wet filter paper supernatant. In August and September 2015, 101 consecutive suspected cholera cases were enrolled, of which 36 were confirmed by PCR. The enriched RDT had 86.1% (95% CI: 70.5–95.3) sensitivity and 100% (95% CI: 94.4–100) specificity compared to PCR as the reference standard. The sensitivity of culture versus PCR was 83.3% (95% CI: 67.2–93.6) for culture performed on site and 72.2% (95% CI: 54.8–85.8) at the international reference laboratory, where samples were tested after an average delay of two months after sample collection, and specificity was 98.5% (95% CI: 91.7–100) and 100% (95% CI: 94.5–100), respectively. The RDT with enrichment showed performance comparable to that of culture and could be a sustainable alternative to culture confirmation where laboratory capacity is limited. PMID:27992488

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.

Clinical Implications of Quantitative JAK2 V617F Analysis using Droplet Digital PCR in Myeloproliferative Neoplasms

PubMed Central

Lee, Eunyoung; Lee, Kyoung Joo; Park, Hyein; Chung, Jin Young; Lee, Mi-Na; Chang, Myung Hee; Yoo, Jongha; Lee, Hyewon

2018-01-01

Background JAK2 V617F is the most common mutation in myeloproliferative neoplasms (MPNs) and is a major diagnostic criterion. Mutation quantification is useful for classifying patients with MPN into subgroups and for prognostic prediction. Droplet digital PCR (ddPCR) can provide accurate and reproducible quantitative analysis of DNA. This study was designed to verify the correlation of ddPCR with pyrosequencing results in the diagnosis of MPN and to investigate clinical implications of the mutational burden. Methods Peripheral blood or bone marrow samples were obtained from 56 patients newly diagnosed with MPN or previously diagnosed with MPN but not yet indicated for JAK2 inhibitor treatment between 2012 and 2016. The JAK2 V617F mutation was detected by pyrosequencing as a diagnostic work-up. The same samples were used for ddPCR to determine the correlation between assays and establish a detection sensitivity cut-off. Clinical and hematologic aspects were reviewed. Results Forty-two (75%) and 46 (82.1%) patients were positive for JAK2 V617F by pyrosequencing and ddPCR, respectively. The mean mutated allele frequency at diagnosis was 37.5±30.1% and was 40.7±31.2% with ddPCR, representing a strong correlation (r=0.9712, P<0.001). Follow-up samples were available for 12 patients, including eight that were JAK2 V617F-positive. Of these, mutational burden reduction after treatment was observed in six patients (75%), consistent with trends of hematologic improvement. Conclusions Quantitative analysis of the JAK2 V617F mutation using ddPCR was highly correlated with pyrosequencing data and may reflect the clinical response to treatment. PMID:29214759

Sampling and RNA quality for successful diagnostics using quantitative PCR

USDA-ARS?s Scientific Manuscript database

Diagnostic analyses of RNA targets are widely used in honey bee pathology. These diagnostics can be compromised by the actions of endogenous RNA-degrading enzymes activated upon bee death. RNA degradation can be minimized by storage at ultra-cold temperatures or by immersion in high-salt buffers. H...

Comparison of synovial fluid culture and 16S rRNA PCR in dogs with suspected septic arthritis.

PubMed

Scharf, V F; Lewis, D D; Wellehan, J F; Wamsley, H L; Richardson, R

2015-06-01

To prospectively compare the sensitivity and specificity of 16S rRNA PCR with culture for identifying the causative organism in synovial fluid obtained from dogs with suspected septic arthritis. Synovial fluid cytology, PCR analysis and aerobic, anaerobic and Mycoplasma culture of samples from the affected joints of 18 dogs presenting with suspected septic arthritis were performed. Synovial fluid samples from the corresponding contralateral joints of 7 dogs were also analysed as negative controls. There was no significant difference between the sensitivity of bacterial detection via culture (63.2%) versus PCR (73.7%) of synovial fluid (P=0.728) or between culture and combined PCR and culture (89.5%) of synovial fluid (P=0.124). The specificity of PCR (42.9%) was significantly lower than culture specificity (100%) (P=0.07). Although 16S PCR may hold potential as an ancillary diagnostic test for identifying the causative organism in dogs with septic arthritis, our study failed to demonstrate improved accuracy compared with traditional synovial fluid culture. © 2015 Australian Veterinary Association.

Quantitative Real-Time PCR using the Thermo Scientific Solaris qPCR Assay

PubMed Central

Ogrean, Christy; Jackson, Ben; Covino, James

2010-01-01

The Solaris qPCR Gene Expression Assay is a novel type of primer/probe set, designed to simplify the qPCR process while maintaining the sensitivity and accuracy of the assay. These primer/probe sets are pre-designed to >98% of the human and mouse genomes and feature significant improvements from previously available technologies. These improvements were made possible by virtue of a novel design algorithm, developed by Thermo Scientific bioinformatics experts. Several convenient features have been incorporated into the Solaris qPCR Assay to streamline the process of performing quantitative real-time PCR. First, the protocol is similar to commonly employed alternatives, so the methods used during qPCR are likely to be familiar. Second, the master mix is blue, which makes setting the qPCR reactions easier to track. Third, the thermal cycling conditions are the same for all assays (genes), making it possible to run many samples at a time and reducing the potential for error. Finally, the probe and primer sequence information are provided, simplifying the publication process. Here, we demonstrate how to obtain the appropriate Solaris reagents using the GENEius product search feature found on the ordering web site (www.thermo.com/solaris) and how to use the Solaris reagents for performing qPCR using the standard curve method. PMID:20567213

Comparative diagnostic evaluation of OMP31 gene based TaqMan® real-time PCR assay with visual LAMP assay and indirect ELISA for caprine brucellosis.

PubMed

Saini, Suman; Gupta, V K; Gururaj, K; Singh, D D; Pawaiya, R V S; Gangwar, N K; Mishra, A K; Dwivedi, Deepak; Andani, Dimple; Kumar, Ashok; Goswami, T K

2017-08-01

Brucellosis is one of the leading causes of abortion in domestic animals that imposes costs on both economy and society. The disease is highly zoonotic and poses risk to animal handlers due to its zoonotic nature. It causes stillbirth, loss of kids and abortion in last term of pregnancy. Reproductive damage includes infertility in does and orchitis and epididymitis in breeding bucks, which result in high financial losses to farmers and the agriculture industry as a whole. It requires highly sensitive and specific assays to diagnose the disease at field level. In the current study, a visual loop-mediated isothermal amplification (LAMP) assay and the TaqMan® real-time PCR were developed with high sensitivity and specificity. For the TaqMan® probe, real-time PCR primers were developed using Omp31 gene as target and primers were designed using discontiguous conserved sequences of Omp31 gene. The Omp31 probes were designed by attaching 6-FAM reporter dye at the 5' end and BHQ-1 quencher at the 3' end. Published primers were used for visual LAMP assay targeting the Omp25 gene. Sensitivity of the standardized visual LAMP assay and TaqMan® real-time PCR assay was determined by serial dilution of positive Brucella melitensis DNA (10 2 to 10 -4  ng) obtained from standard culture. The TaqMan® probe real-time assay can detect as low as 100 fg of B. melitensis DNA, whereas culture from vaginal swab washings has a limit of detection (LOD) of only 1 cfu/ml. Similarly, the visual LAMP assay can detect as low as 10 fg of B. melitensis DNA as compared to an LOD of 30 cfu/ml from culture of vaginal swab washings. Both assays were compared with serological tests (serum tube agglutination test (STAT) and indirect enzyme-linked immunosorbent assay (iELISA)) for diagnostic sensitivity and specificity. Diagnostic sensitivities and specificities for TaqMan® real-time PCR vs. LAMP assays were 98 and 100% vs. 100 and 97.8%, respectively. Results of visual LAMP assay indicated that

High-resolution melting PCR assay, applicable for diagnostics and screening studies, allowing detection and differentiation of several Babesia spp. infecting humans and animals.

PubMed

Rozej-Bielicka, Wioletta; Masny, Aleksander; Golab, Elzbieta

2017-10-01

The goal of the study was to design a single tube PCR test for detection and differentiation of Babesia species in DNA samples obtained from diverse biological materials. A multiplex, single tube PCR test was designed for amplification of approximately 400 bp region of the Babesia 18S rRNA gene. Universal primers were designed to match DNA of multiple Babesia spp. and to have low levels of similarity to DNA sequences of other intracellular protozoa and Babesia hosts. The PCR products amplified from Babesia DNA isolated from human, dog, rodent, deer, and tick samples were subjected to high-resolution melting analysis for Babesia species identification. The designed test allowed detection and differentiation of four Babesia species, three zoonotic (B. microti, B. divergens, B. venatorum) and one that is generally not considered zoonotic-Babesia canis. Both detection and identification of all four species were possible based on the HRM curves of the PCR products in samples obtained from the following: humans, dogs, rodents, and ticks. No cross-reactivity with DNA of Babesia hosts or Plasmodium falciparum and Toxoplasma gondii was observed. The lack of cross-reactivity with P. falciparum DNA might allow using the assay in endemic malaria areas. The designed assay is the first PCR-based test for detection and differentiation of several Babesia spp. of medical and veterinary importance, in a single tube reaction. The results of the study show that the designed assay for Babesia detection and identification could be a practical and inexpensive tool for diagnostics and screening studies of diverse biological materials.

Missed diagnostic opportunities within South Africa's early infant diagnosis program, 2010-2015.

PubMed

Haeri Mazanderani, Ahmad; Moyo, Faith; Sherman, Gayle G

2017-01-01

Samples submitted for HIV PCR testing that fail to yield a positive or negative result represent missed diagnostic opportunities. We describe HIV PCR test rejections and indeterminate results, and the associated delay in diagnosis, within South Africa's early infant diagnosis (EID) program from 2010 to 2015. HIV PCR test data from January 2010 to December 2015 were extracted from the National Health Laboratory Service Corporate Data Warehouse, a central data repository of all registered test-sets within the public health sector in South Africa, by laboratory number, result, date, facility, and testing laboratory. Samples that failed to yield either a positive or negative result were categorized according to the rejection code on the laboratory information system, and descriptive analysis performed using Microsoft Excel. Delay in diagnosis was calculated for patients who had a missed diagnostic opportunity registered between January 2013 and December 2015 by means of a patient linking-algorithm employing demographic details. Between 2010 and 2015, 2 178 582 samples were registered for HIV PCR testing of which 6.2% (n = 134 339) failed to yield either a positive or negative result, decreasing proportionally from 7.0% (n = 20 556) in 2010 to 4.4% (n = 21 388) in 2015 (p<0.001). Amongst 76 972 coded missed diagnostic opportunities, 49 585 (64.4%) were a result of pre-analytical error and 27 387 (35.6%) analytical error. Amongst 49 694 patients searched for follow-up results, 16 895 (34.0%) had at least one subsequent HIV PCR test registered after a median of 29 days (IQR: 13-57), of which 8.4% tested positive compared with 3.6% of all samples submitted for the same period. Routine laboratory data provides the opportunity for near real-time surveillance and quality improvement within the EID program. Delay in diagnosis and wastage of resources associated with missed diagnostic opportunities must be addressed and infants actively followed-up as South Africa works towards

Polymerase chain reaction: A molecular diagnostic tool in periodontology

PubMed Central

Maheaswari, Rajendran; Kshirsagar, Jaishree Tukaram; Lavanya, Nallasivam

2016-01-01

This review discusses the principles of polymerase chain reaction (PCR) and its application as a diagnostic tool in periodontology. The relevant MEDLINE and PubMed indexed journals were searched manually and electronically by typing PCR, applications of PCR, PCR in periodontics, polymorphism studies in periodontitis, and molecular techniques in periodontology. The searches were limited to articles in English language and the articles describing PCR process and its relation to periodontology were collected and used to prepare a concise review. PCR has now become a standard diagnostic and research tool in periodontology. Various studies reveal that its sensitivity and specificity allow it as a rapid, efficient method of detecting, identifying, and quantifying organism. Different immune and inflammatory markers can be identified at the mRNA expression level, and also the determination of genetic polymorphisms, thus providing the deeper insight into the mechanisms underlying the periodontal disease. PMID:27143822

Polymerase chain reaction: A molecular diagnostic tool in periodontology.

PubMed

Maheaswari, Rajendran; Kshirsagar, Jaishree Tukaram; Lavanya, Nallasivam

2016-01-01

This review discusses the principles of polymerase chain reaction (PCR) and its application as a diagnostic tool in periodontology. The relevant MEDLINE and PubMed indexed journals were searched manually and electronically by typing PCR, applications of PCR, PCR in periodontics, polymorphism studies in periodontitis, and molecular techniques in periodontology. The searches were limited to articles in English language and the articles describing PCR process and its relation to periodontology were collected and used to prepare a concise review. PCR has now become a standard diagnostic and research tool in periodontology. Various studies reveal that its sensitivity and specificity allow it as a rapid, efficient method of detecting, identifying, and quantifying organism. Different immune and inflammatory markers can be identified at the mRNA expression level, and also the determination of genetic polymorphisms, thus providing the deeper insight into the mechanisms underlying the periodontal disease.

Novel Multiplex Real-Time PCR Diagnostic Assay for Identification and Differentiation of Mycobacterium tuberculosis, Mycobacterium canettii, and Mycobacterium tuberculosis Complex Strains▿†

PubMed Central

Reddington, Kate; O'Grady, Justin; Dorai-Raj, Siobhan; Maher, Majella; van Soolingen, Dick; Barry, Thomas

2011-01-01

Tuberculosis (TB) in humans is caused by members of the Mycobacterium tuberculosis complex (MTC). Rapid detection of the MTC is necessary for the timely initiation of antibiotic treatment, while differentiation between members of the complex may be important to guide the appropriate antibiotic treatment and provide epidemiological information. In this study, a multiplex real-time PCR diagnostics assay using novel molecular targets was designed to identify the MTC while simultaneously differentiating between M. tuberculosis and M. canettii. The lepA gene was targeted for the detection of members of the MTC, the wbbl1 gene was used for the differentiation of M. tuberculosis and M. canettii from the remainder of the complex, and a unique region of the M. canettii genome, a possible novel region of difference (RD), was targeted for the specific identification of M. canettii. The multiplex real-time PCR assay was tested using 125 bacterial strains (64 MTC isolates, 44 nontuberculosis mycobacteria [NTM], and 17 other bacteria). The assay was determined to be 100% specific for the mycobacteria tested. Limits of detection of 2.2, 2.17, and 0.73 cell equivalents were determined for M. tuberculosis/M. canettii, the MTC, and M. canettii, respectively, using probit regression analysis. Further validation of this diagnostics assay, using clinical samples, should demonstrate its potential for the rapid, accurate, and sensitive diagnosis of TB caused by M. tuberculosis, M. canettii, and the other members of the MTC. PMID:21123525

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.

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.

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.

Ureaplasma parvum prosthetic joint infection detected by PCR.

PubMed

Farrell, John J; Larson, Joshua A; Akeson, Jeffrey W; Lowery, Kristin S; Rounds, Megan A; Sampath, Rangarajan; Bonomo, Robert A; Patel, Robin

2014-06-01

We describe the first reported case of Ureaplasma parvum prosthetic joint infection (PJI) detected by PCR. Ureaplasma species do not possess a cell wall and are usually associated with colonization and infection of mucosal surfaces (not prosthetic material). U. parvum is a relatively new species name for certain serovars of Ureaplasma urealyticum, and PCR is useful for species determination. Our patient presented with late infection of his right total knee arthroplasty. Intraoperative fluid and tissue cultures and pre- and postoperative synovial fluid cultures were all negative. To discern the pathogen, we employed PCR coupled with electrospray ionization mass spectrometry (PCR/ESI-MS). Our patient's failure to respond to empirical antimicrobial treatment and our previous experience with PCR/ESI-MS in culture-negative cases of infection prompted us to use this approach over other diagnostic modalities. PCR/ESI-MS detected U. parvum in all samples. U. parvum-specific PCR testing was performed on all synovial fluid samples to confirm the U. parvum detection. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Application of droplet digital PCR for quantitative detection of Spiroplasma citri in comparison with real time PCR

USDA-ARS?s Scientific Manuscript database

Droplet digital Polymerase chain reaction (ddPCR) is a unique approach to measure the absolute copy number of nucleic acid targets without the need of external standards. It is a promising DNA quantification technology for medical diagnostics but there are only a few reports of its use for plant pat...

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.

Utility of dengue NS1 antigen rapid diagnostic test for use in difficult to reach areas and its comparison with dengue NS1 ELISA and qRT-PCR.

PubMed

Shukla, Mohan K; Singh, Neeru; Sharma, Ravendra K; Barde, Pradip V

2017-07-01

The objective of this study was to demonstrate the utility of dengue virus (DENV) non structural protein 1 (NS1) based rapid diagnostic test (RDT) for use in tribal and difficult to reach areas for early dengue (DEN) diagnosis in acute phase patients and evaluate its sensitivity and specificity against DENV NS1 enzyme linked immune sorbent assay (ELISA) and real time reverse transcriptase polymerase chain reaction (qRT-PCR). The DENV NS1 RDT was used for preliminary diagnosis during outbreaks in difficult to reach rural and tribal areas. The diagnosis was confirmed by DENV NS1 ELISA in the laboratory. The samples were also tested and serotyped by qRT-PCR. The results were evaluated using statistical tests. The DENV NS1 RDT showed 99.2% sensitivity and 96.0% specificity when analyzed using DENV NS1 ELISA as standard. The specificity and sensitivity of the RDT when compared with qRT-PCR was 93.6% and 91.1%, respectively. The serotype specific evaluation showed more than 90% sensitivity and specificity for DENV-1, 2, and 3. The RDT proved a good diagnostic tool in difficult to reach rural and tribal areas. Further evaluation studies with different commercially available RDTs in different field conditions are essential, that will help clinicians and patients for treatment and programme managers for timely intervention. © 2017 Wiley Periodicals, Inc.

Minimally Invasive Molecular Staging (MIMS) RT-PCR Breast Cancer Study

DTIC Science & Technology

2007-03-31

per se. 15. SUBJECT TERMS Breast cancer, real-time PCR, molecular diagnostics , micrometastases 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF 18...a superior molecular marker for detection of non- small cell lung cancer in peripheral blood. Journal of Molecular Diagnostics . 5:23 7-42, 2003. With...Journal of Molecular Diagnostics . 5:237-42, 2003 10. Mikhitarian K, Allen A, Reott S, Hoover L, Allen A, Cole DJ, Gillanders WE, and Mitas M. Enhanced

Detection and Differentiation of Lyme Spirochetes and Other Tick-Borne Pathogens from Blood Using Real-Time PCR with Molecular Beacons.

PubMed

Schlachter, Samantha; Chan, Kamfai; Marras, Salvatore A E; Parveen, Nikhat

2017-01-01

Real-time PCR assays have recently been implemented in diagnostics for many bacterial pathogens, allowing rapid and accurate detection, which ultimately results in improved clinical intervention. Here, we describe a sensitive method of detection for three common tick-borne pathogens Borrelia burgdorferi, Anaplasma phagocytophilum, and Babesia microti since coinfections with these pathogens have started occurring with increasing frequency over the last several years in both North America and Europe. A shared geographic region, the same tick vectors, and similar transmission cycle all favor simultaneous transmission of these three tick-borne pathogens. Furthermore, early symptoms of the diseases are often similar and somewhat nonspecific leading to poor clinical identification. The multiplex real-time PCR assay we describe here utilizes gene-specific primers, molecular beacon probes tagged with different fluorophores, and optimized PCR conditions to detect even small amounts of specific pathogen DNA without interference. Application of this detection method will offer better diagnostics for acute and persistent infection compared to the two-tier serological tests that are currently approved in North America and Europe, which do not necessarily detect active infection.

Can rapid integrated polymerase chain reaction-based diagnostics for gastrointestinal pathogens improve routine hospital infection control practice? A diagnostic study.

PubMed

Pankhurst, Louise; Macfarlane-Smith, Louissa; Buchanan, James; Anson, Luke; Davies, Kerrie; O'Connor, Lily; Ashwin, Helen; Pike, Graham; Dingle, Kate E; Peto, Timothy Ea; Wordsworth, Sarah; Walker, A Sarah; Wilcox, Mark H; Crook, Derrick W

2014-08-01

Every year approximately 5000-9000 patients are admitted to a hospital with diarrhoea, which in up to 90% of cases has a non-infectious cause. As a result, single rooms are 'blocked' by patients with non-infectious diarrhoea, while patients with infectious diarrhoea are still in open bays because of a lack of free side rooms. A rapid test for differentiating infectious from non-infectious diarrhoea could be very beneficial for patients. To evaluate MassCode multiplex polymerase chain reaction (PCR) for the simultaneous diagnosis of multiple enteropathogens directly from stool, in terms of sensitivity/specificity to detect four common important enteropathogens: Clostridium difficile, Campylobacter spp., Salmonella spp. and norovirus. A retrospective study of fixed numbers of samples positive for C. difficile (n = 200), Campylobacter spp. (n = 200), Salmonella spp. (n = 100) and norovirus (n = 200) plus samples negative for all these pathogens (n = 300). Samples were sourced from NHS microbiology laboratories in Oxford and Leeds where initial diagnostic testing was performed according to Public Health England methodology. Researchers carrying out MassCode assays were blind to this information. A questionnaire survey, examining current practice for infection control teams and microbiology laboratories managing infectious diarrhoea, was also carried out. MassCode assays were carried out at Oxford University Hospitals NHS Trust. Further multiplex assays, carried out using Luminex, were run on the same set of samples at Leeds Teaching Hospitals NHS Trust. The questionnaire was completed by various NHS trusts. Sensitivity and specificity to detect C. difficile, Campylobacter spp., Salmonella spp., and norovirus. Nucleic acids were extracted from 948 clinical samples using an optimised protocol (200 Campylobacter spp., 199 C. difficile, 60 S. enterica, 199 norovirus and 295 negative samples; some samples contained more than one pathogen). Using the Mass

Longitudinal monitoring of bottlenose dolphins leukocyte cytokine mRNA responsiveness by qPCR

USDA-ARS?s Scientific Manuscript database

Both veterinarians caring for bottlenose dolphins (Tursiops truncatus) in managed populations and researchers monitoring wild populations use blood-based diagnostics to monitor bottlenose dolphin health. Quantitative PCR (qPCR) can be used to assess cytokine expression patterns of peripheral blood m...

Longitudinal monitoring of bottlenose dolphin leukocyte cytokine mRNA responsiveness by qPCR

USDA-ARS?s Scientific Manuscript database

Both veterinarians caring for bottlenose dolphins (Tursiops truncatus) in managed populations and researchers monitoring wild populations use blood-based diagnostics to monitor bottlenose dolphin health. Quantitative PCR (qPCR) can be used to assess cytokine expression patterns of peripheral blood m...

Pathological findings and diagnostic implications of a rhesus macaque (Macacca mulatta) model of aerosol exposure to Burkholderia mallei (glanders).

PubMed

Yingst, Samuel L; Facemire, Paul; Chuvala, Lara; Norwood, David; Wolcott, Mark; Huzella, Louis

2015-06-01

Burkholderia mallei is a Gram-negative bacillus that causes a pneumonic disease known as glanders in equids and humans, and a lymphatic infection known as farcy, primarily in equids. With the potential to infect humans by the respiratory route, aerosol exposure can result in severe, occasionally fatal, pneumonia. Today, glanders infections in humans are rare, likely due to less frequent contact with infected equids than in the past. Acutely ill humans often have non-specific clinical signs and in order to diagnose cases, especially in scenarios of multiple cases in an unexpected setting, rapid diagnostics for B. mallei may be critical. The pathogenesis of acute glanders in the rhesus macaque (Macaca mulatta) was studied as an initial effort to improve diagnostic methods. In the study described here, the diagnostic techniques of PCR, culture and histopathology were compared. The results indicated that PCR may provide rapid, non-invasive diagnosis of glanders in some cases. As expected, PCR results were positive in lung tissue in 11/12 acutely infected rhesus macaques, but more importantly in terms of diagnostic algorithm development, PCR results were frequently positive in non-invasive samples such as broncho-alveolar lavage or nasal swabs (7/12) and occasionally in blood (3/12). However, conventional bacterial culture failed to recover bacteria in many of these samples. The study showed that the clinical presentation of aerosol-exposed rhesus macaques is similar to descriptions of human glanders and that PCR has potential for rapid diagnosis of outbreaks, if not individual cases.

Pathological findings and diagnostic implications of a rhesus macaque (Macacca mulatta) model of aerosol exposure to Burkholderia mallei (glanders)

PubMed Central

Yingst, Samuel L.; Chuvala, Lara; Norwood, David; Wolcott, Mark; Huzella, Louis

2015-01-01

Burkholderia mallei is a Gram-negative bacillus that causes a pneumonic disease known as glanders in equids and humans, and a lymphatic infection known as farcy, primarily in equids. With the potential to infect humans by the respiratory route, aerosol exposure can result in severe, occasionally fatal, pneumonia. Today, glanders infections in humans are rare, likely due to less frequent contact with infected equids than in the past. Acutely ill humans often have non-specific clinical signs and in order to diagnose cases, especially in scenarios of multiple cases in an unexpected setting, rapid diagnostics for B. mallei may be critical. The pathogenesis of acute glanders in the rhesus macaque (Macaca mulatta) was studied as an initial effort to improve diagnostic methods. In the study described here, the diagnostic techniques of PCR, culture and histopathology were compared. The results indicated that PCR may provide rapid, non-invasive diagnosis of glanders in some cases. As expected, PCR results were positive in lung tissue in 11/12 acutely infected rhesus macaques, but more importantly in terms of diagnostic algorithm development, PCR results were frequently positive in non-invasive samples such as broncho-alveolar lavage or nasal swabs (7/12) and occasionally in blood (3/12). However, conventional bacterial culture failed to recover bacteria in many of these samples. The study showed that the clinical presentation of aerosol-exposed rhesus macaques is similar to descriptions of human glanders and that PCR has potential for rapid diagnosis of outbreaks, if not individual cases. PMID:25850696

Diagnostic devices for isothermal nucleic acid amplification.

PubMed

Chang, Chia-Chen; Chen, Chien-Cheng; Wei, Shih-Chung; Lu, Hui-Hsin; Liang, Yang-Hung; Lin, Chii-Wann

2012-01-01

Since the development of the polymerase chain reaction (PCR) technique, genomic information has been retrievable from lesser amounts of DNA than previously possible. PCR-based amplifications require high-precision instruments to perform temperature cycling reactions; further, they are cumbersome for routine clinical use. However, the use of isothermal approaches can eliminate many complications associated with thermocycling. The application of diagnostic devices for isothermal DNA amplification has recently been studied extensively. In this paper, we describe the basic concepts of several isothermal amplification approaches and review recent progress in diagnostic device development.

Diagnostic Devices for Isothermal Nucleic Acid Amplification

PubMed Central

Chang, Chia-Chen; Chen, Chien-Cheng; Wei, Shih-Chung; Lu, Hui-Hsin; Liang, Yang-Hung; Lin, Chii-Wann

2012-01-01

Since the development of the polymerase chain reaction (PCR) technique, genomic information has been retrievable from lesser amounts of DNA than previously possible. PCR-based amplifications require high-precision instruments to perform temperature cycling reactions; further, they are cumbersome for routine clinical use. However, the use of isothermal approaches can eliminate many complications associated with thermocycling. The application of diagnostic devices for isothermal DNA amplification has recently been studied extensively. In this paper, we describe the basic concepts of several isothermal amplification approaches and review recent progress in diagnostic device development. PMID:22969402

Cost-Effectiveness Analysis of Diagnostic Options for Pneumocystis Pneumonia (PCP)

PubMed Central

Harris, Julie R.; Marston, Barbara J.; Sangrujee, Nalinee; DuPlessis, Desiree; Park, Benjamin

2011-01-01

Background Diagnosis of Pneumocystis jirovecii pneumonia (PCP) is challenging, particularly in developing countries. Highly sensitive diagnostic methods are costly, while less expensive methods often lack sensitivity or specificity. Cost-effectiveness comparisons of the various diagnostic options have not been presented. Methods and Findings We compared cost-effectiveness, as measured by cost per life-years gained and proportion of patients successfully diagnosed and treated, of 33 PCP diagnostic options, involving combinations of specimen collection methods [oral washes, induced and expectorated sputum, and bronchoalveolar lavage (BAL)] and laboratory diagnostic procedures [various staining procedures or polymerase chain reactions (PCR)], or clinical diagnosis with chest x-ray alone. Our analyses were conducted from the perspective of the government payer among ambulatory, HIV-infected patients with symptoms of pneumonia presenting to HIV clinics and hospitals in South Africa. Costing data were obtained from the National Institutes of Communicable Diseases in South Africa. At 50% disease prevalence, diagnostic procedures involving expectorated sputum with any PCR method, or induced sputum with nested or real-time PCR, were all highly cost-effective, successfully treating 77–90% of patients at $26–51 per life-year gained. Procedures using BAL specimens were significantly more expensive without added benefit, successfully treating 68–90% of patients at costs of $189–232 per life-year gained. A relatively cost-effective diagnostic procedure that did not require PCR was Toluidine Blue O staining of induced sputum ($25 per life-year gained, successfully treating 68% of patients). Diagnosis using chest x-rays alone resulted in successful treatment of 77% of patients, though cost-effectiveness was reduced ($109 per life-year gained) compared with several molecular diagnostic options. Conclusions For diagnosis of PCP, use of PCR technologies, when combined with

History, epidemiology and diagnostics of dengue in the American and Brazilian contexts: a review.

PubMed

Salles, Tiago Souza; da Encarnação Sá-Guimarães, Thayane; de Alvarenga, Evelyn Seam Lima; Guimarães-Ribeiro, Victor; de Meneses, Marcelo Damião Ferreira; de Castro-Salles, Patricia Faria; Dos Santos, Carlucio Rocha; do Amaral Melo, Ana Claudia; Soares, Marcia Regina; Ferreira, Davis Fernandes; Moreira, Monica Ferreira

2018-04-24

Dengue virus (DENV), an arbovirus transmitted by mosquitoes, has become a major threat to American human life, reaching approximately 23 million cases from 1980 to 2017. Brazil is among the countries most affected by this terrible viral disease, with 13.6 million cases. DENV has four different serotypes, DENV1-4, which show a broad clinical spectrum. Dengue creates a staggering epidemiological and economic burden for endemic countries. Without a specific therapy and with a commercial vaccine that presents some problems relative to its full effectiveness, initiatives to improve vector control strategies, early disease diagnostics and the development of vaccines and antiviral drugs are priorities. In this study, we present the probable origins of dengue in America and the trajectories of its spread. Overall, dengue diagnostics are costly, making the monitoring of dengue epidemiology more difficult and affecting physicians' therapeutic decisions regarding dengue patients, especially in developing countries. This review also highlights some recent and important findings regarding dengue in Brazil and the Americas. We also summarize the existing DENV polymerase chain reaction (PCR) diagnostic tests to provide an improved reference since these tests are useful and accurate at discriminating DENV from other flaviviruses that co-circulate in the Americas. Additionally, these DENV PCR assays ensure virus serotyping, enabling epidemiologic monitoring.

An Efficient Multistrategy DNA Decontamination Procedure of PCR Reagents for Hypersensitive PCR Applications

PubMed Central

Pruvost, Mélanie; Bennett, E. Andrew; Grange, Thierry; Geigl, Eva-Maria

2010-01-01

Background PCR amplification of minute quantities of degraded DNA for ancient DNA research, forensic analyses, wildlife studies and ultrasensitive diagnostics is often hampered by contamination problems. The extent of these problems is inversely related to DNA concentration and target fragment size and concern (i) sample contamination, (ii) laboratory surface contamination, (iii) carry-over contamination, and (iv) contamination of reagents. Methodology/Principal Findings Here we performed a quantitative evaluation of current decontamination methods for these last three sources of contamination, and developed a new procedure to eliminate contaminating DNA contained in PCR reagents. We observed that most current decontamination methods are either not efficient enough to degrade short contaminating DNA molecules, rendered inefficient by the reagents themselves, or interfere with the PCR when used at doses high enough to eliminate these molecules. We also show that efficient reagent decontamination can be achieved by using a combination of treatments adapted to different reagent categories. Our procedure involves γ- and UV-irradiation and treatment with a mutant recombinant heat-labile double-strand specific DNase from the Antarctic shrimp Pandalus borealis. Optimal performance of these treatments is achieved in narrow experimental conditions that have been precisely analyzed and defined herein. Conclusions/Significance There is not a single decontamination method valid for all possible contamination sources occurring in PCR reagents and in the molecular biology laboratory and most common decontamination methods are not efficient enough to decontaminate short DNA fragments of low concentration. We developed a versatile multistrategy decontamination procedure for PCR reagents. We demonstrate that this procedure allows efficient reagent decontamination while preserving the efficiency of PCR amplification of minute quantities of DNA. PMID:20927390

Panel of 23S rRNA Gene-Based Real-Time PCR Assays for Improved Universal and Group-Specific Detection of Phytoplasmas▿ †

PubMed Central

Hodgetts, Jennifer; Boonham, Neil; Mumford, Rick; Dickinson, Matthew

2009-01-01

Primers and probes based on the 23S rRNA gene have been utilized to design a range of real-time PCR assays for routine phytoplasma diagnostics. These assays have been authenticated as phytoplasma specific and shown to be at least as sensitive as nested PCR. A universal assay to detect all phytoplasmas has been developed, along with a multiplex assay to discriminate 16SrI group phytoplasmas from members of all of the other 16Sr groups. Assays for the 16SrII, 16SrIV, and 16SrXII groups have also been developed to confirm that the 23S rRNA gene can be used to design group-specific assays. PMID:19270148

Applied Genomics: Data Mining Reveals Species-Specific Malaria Diagnostic Targets More Sensitive than 18S rRNA▿†‡

PubMed Central

Demas, Allison; Oberstaller, Jenna; DeBarry, Jeremy; Lucchi, Naomi W.; Srinivasamoorthy, Ganesh; Sumari, Deborah; Kabanywanyi, Abdunoor M.; Villegas, Leopoldo; Escalante, Ananias A.; Kachur, S. Patrick; Barnwell, John W.; Peterson, David S.; Udhayakumar, Venkatachalam; Kissinger, Jessica C.

2011-01-01

Accurate and rapid diagnosis of malaria infections is crucial for implementing species-appropriate treatment and saving lives. Molecular diagnostic tools are the most accurate and sensitive method of detecting Plasmodium, differentiating between Plasmodium species, and detecting subclinical infections. Despite available whole-genome sequence data for Plasmodium falciparum and P. vivax, the majority of PCR-based methods still rely on the 18S rRNA gene targets. Historically, this gene has served as the best target for diagnostic assays. However, it is limited in its ability to detect mixed infections in multiplex assay platforms without the use of nested PCR. New diagnostic targets are needed. Ideal targets will be species specific, highly sensitive, and amenable to both single-step and multiplex PCRs. We have mined the genomes of P. falciparum and P. vivax to identify species-specific, repetitive sequences that serve as new PCR targets for the detection of malaria. We show that these targets (Pvr47 and Pfr364) exist in 14 to 41 copies and are more sensitive than 18S rRNA when utilized in a single-step PCR. Parasites are routinely detected at levels of 1 to 10 parasites/μl. The reaction can be multiplexed to detect both species in a single reaction. We have examined 7 P. falciparum strains and 91 P. falciparum clinical isolates from Tanzania and 10 P. vivax strains and 96 P. vivax clinical isolates from Venezuela, and we have verified a sensitivity and specificity of ∼100% for both targets compared with a nested 18S rRNA approach. We show that bioinformatics approaches can be successfully applied to identify novel diagnostic targets and improve molecular methods for pathogen detection. These novel targets provide a powerful alternative molecular diagnostic method for the detection of P. falciparum and P. vivax in conventional or multiplex PCR platforms. PMID:21525225

Comparison of a real-time PCR method with a culture method for the detection of Salmonella enterica serotype enteritidis in naturally contaminated environmental samples from integrated poultry houses.

PubMed

Lungu, Bwalya; Waltman, W Douglas; Berghaus, Roy D; Hofacre, Charles L

2012-04-01

Conventional culture methods have traditionally been considered the "gold standard" for the isolation and identification of foodborne bacterial pathogens. However, culture methods are labor-intensive and time-consuming. A Salmonella enterica serotype Enteritidis-specific real-time PCR assay that recently received interim approval by the National Poultry Improvement Plan for the detection of Salmonella Enteritidis was evaluated against a culture method that had also received interim National Poultry Improvement Plan approval for the analysis of environmental samples from integrated poultry houses. The method was validated with 422 field samples collected by either the boot sock or drag swab method. The samples were cultured by selective enrichment in tetrathionate broth followed by transfer onto a modified semisolid Rappaport-Vassiliadis medium and then plating onto brilliant green with novobiocin and xylose lysine brilliant Tergitol 4 plates. One-milliliter aliquots of the selective enrichment broths from each sample were collected for DNA extraction by the commercial PrepSEQ nucleic acid extraction assay and analysis by the Salmonella Enteritidis-specific real-time PCR assay. The real-time PCR assay detected no significant differences between the boot sock and drag swab samples. In contrast, the culture method detected a significantly higher number of positive samples from boot socks. The diagnostic sensitivity of the real-time PCR assay for the field samples was significantly higher than that of the culture method. The kappa value obtained was 0.46, indicating moderate agreement between the real-time PCR assay and the culture method. In addition, the real-time PCR method had a turnaround time of 2 days compared with 4 to 8 days for the culture method. The higher sensitivity as well as the reduction in time and labor makes this real-time PCR assay an excellent alternative to conventional culture methods for diagnostic purposes, surveillance, and research studies

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).

Rapid screening of toxigenic vibrio cholerae O1 strains from south Iran by PCR-ELISA.

PubMed

Mousavi, Seyed Latif; Nazarian, Shahram; Amani, Jafar; Rahgerdi, Ahmad Karimi

2008-01-01

The ability to sensitively detect Vibrio cholera with PCR-ELISA method represents a considerable advancement over alternative more time-consuming methods for detection of this pathogen. The aim of this research is to evaluate the suitability of a PCR-enzyme-linked immunosorbent assay for sensitive and rapid detection of V. cholera O1. The 398-bp sequence of a gene that codes for the cholera toxin B subunit was amplified by PCR. The digoxigenin-labeled amplified products were coated on microplates and detected by ELISA. The PCR product was also hybridized with biotin labelled probe and detected by ELISA using streptavidin. The specificity of the PCR was determined using 10 bacterial strains and 50 samples from south Iran. The detection limit was 0.5 pg of the genomic DNA and five bacterial cells. Adaptation of PCR into PCR-ELISA assay format facilitates specific and sensitive detection and diagnosis of human cholera disease. We conclude that this PCR-ELISA is a diagnostic method that specifically detects toxin genes in V. cholera O1 strains. It is more rapid and less cumbersome than other diagnostic methods for detection of toxicity in these strains.

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.

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.

Advances in molecular-based diagnostics in meeting crop biosecurity and phytosanitary issues.

PubMed

Schaad, Norman W; Frederick, Reid D; Shaw, Joe; Schneider, William L; Hickson, Robert; Petrillo, Michael D; Luster, Douglas G

2003-01-01

Awareness of crop biosecurity and phytosanitation has been heightened since 9/11 and the unresolved anthrax releases in October 2001. Crops are highly vulnerable to accidental or deliberate introductions of crop pathogens from outside U.S. borders. Strategic thinking about protection against deliberate or accidental release of a plant pathogen is an urgent priority. Rapid detection will be the key to success. This review summarizes recent progress in the development of rapid real-time PCR protocols and evaluates their effectiveness in a proposed nationwide network of diagnostic laboratories that will facilitate rapid diagnostics and improved communication.

Sample-ready multiplex qPCR assay for detection of malaria.

PubMed

Kamau, Edwin; Alemayehu, Saba; Feghali, Karla C; Juma, Dennis W; Blackstone, George M; Marion, William R; Obare, Peter; Ogutu, Bernhards; Ockenhouse, Christian F

2014-04-25

Microscopy and antigen detecting rapid diagnostic tests are the diagnostic tests of choice in management of clinical malaria. However, due to their limitations, the need to utilize more sensitive methods such as real-time PCR (qPCR) is evident as more studies are now utilizing molecular methods in detection of malaria. Some of the challenges that continue to limit the widespread utilization of qPCR include lack of assay standardization, assay variability, risk of contamination, and the need for cold-chain. Lyophilization of molecular assays can overcome some of these limitations and potentially enable widespread qPCR utilization. A recently published multiplex malaria qPCR assay was lyophilized by freezing drying into Sample-Ready™ format (MMSR). MMSR assay contained all the required reagents for qPCR including primers and probes, requiring only the addition of water and sample to perform qPCR. The performance of the MMSR assay was compared to the non-freeze dried, "wet" assay. Stability studies were done by maintaining the MMSR assays at four different ambient temperatures of 4°C, room temperature (RT), 37°C and 42°C over a period of 42 days, tested at seven-day intervals. Plasmodium falciparum and Plasmodium vivax DNAs were used for analysis of the MMSR assay either as single or mixed parasites, at two different concentrations. The CT values and the standard deviations (SD) were used in the analysis of the assay performance. The limit of detection for the MMSR assay was 0.244 parasites/μL for Plasmodium spp. (PLU) and P. falciparum (FAL) assay targets compared to "wet" assay which was 0.39 and 3.13 parasites/μL for PLU and FAL assay targets, respectively. The MMSR assay performed with high efficiencies similar to those of the "wet" assay and was stable at 37°C for 42 days, with estimated shelf-life of 5 months. When used to analyse field clinical samples, MMSR assay performed with 100% sensitivity and specificity compared to the "wet" assay. The MMSR

New, Improved Version of the mCOP-PCR Screening System for Detection of Spinal Muscular Atrophy Gene (SMN1) Deletion.

PubMed

Shinohara, Masakazu; Ar Rochmah, Mawaddah; Nakanishi, Kenta; Harahap, Nur Imma Fatimah; Niba, Emma Tabe Eko; Saito, Toshio; Saito, Kayoko; Takeuchi, Atsuko; Bouike, Yoshihiro; Nishio, Hisahide

2017-09-07

Spinal muscular atrophy (SMA) is a frequent autosomal recessive disorder, characterized by lower motor neuron loss in the spinal cord. More than 95% of SMA patients show homozygous survival motor neuron 1 (SMN1) deletion. We previously developed a screening system for SMN1 deletion based on a modified competitive oligonucleotide priming-PCR (mCOP-PCR) technique. However, non-specific amplification products were observed with mCOP-PCR, which might lead to erroneous interpretation of the screening results. To establish an improved version of the mCOP-PCR screening system without non-specific amplification. DNA samples were assayed using a new version of the mCOP-PCR screening system. DNA samples had already been genotyped by PCR-restriction fragment length polymorphism (PCR-RFLP), showing the presence or absence of SMN1 exon 7. The new mCOP-PCR method contained a targeted pre-amplification step of the region, including an SMN1-specific nucleotide, prior to the mCOP-PCR step. mCOP-PCR products were electrophoresed on agarose gels. No non-specific amplification products were detected in electrophoresis gels with the new mCOP-PCR screening system. An additional targeted pre-amplification step eliminated non-specific amplification from mCOP-PCR screening.

Advances in digital polymerase chain reaction (dPCR) and its emerging biomedical applications.

PubMed

Cao, Lei; Cui, Xingye; Hu, Jie; Li, Zedong; Choi, Jane Ru; Yang, Qingzhen; Lin, Min; Ying Hui, Li; Xu, Feng

2017-04-15

Since the invention of polymerase chain reaction (PCR) in 1985, PCR has played a significant role in molecular diagnostics for genetic diseases, pathogens, oncogenes and forensic identification. In the past three decades, PCR has evolved from end-point PCR, through real-time PCR, to its current version, which is the absolute quantitive digital PCR (dPCR). In this review, we first discuss the principles of all key steps of dPCR, i.e., sample dispersion, amplification, and quantification, covering commercialized apparatuses and other devices still under lab development. We highlight the advantages and disadvantages of different technologies based on these steps, and discuss the emerging biomedical applications of dPCR. Finally, we provide a glimpse of the existing challenges and future perspectives for dPCR. Copyright © 2016 Elsevier B.V. All rights reserved.

Determining Fungi rRNA Copy Number by PCR

EPA Science Inventory

The goal of this project is to improve the quantification of indoor fungal pollutants via the specific application of quantitative PCR (qPCR). Improvement will be made in the controls used in current qPCR applications. This work focuses on the use of two separate controls within ...

SMA Diagnosis: Detection of SMN1 Deletion with Real-Time mCOP-PCR System Using Fresh Blood DNA.

PubMed

Niba, Emma Tabe Eko; Ar Rochmah, Mawaddah; Harahap, Nur Imma Fatimah; Awano, Hiroyuki; Morioka, Ichiro; Iijima, Kazumoto; Saito, Toshio; Saito, Kayoko; Takeuchi, Atsuko; Lai, Poh San; Bouike, Yoshihiro; Nishio, Hisahide; Shinohara, Masakazu

2017-12-18

Spinal muscular atrophy (SMA) is one of the most common autosomal recessive disorders. The symptoms are caused by defects of lower motor neurons in the spinal cord. More than 95% of SMA patients are homozygous for survival motor neuron 1 (SMN1) deletion. We previously developed a screening system for SMN1 deletion based on a modified competitive oligonucleotide priming-PCR (mCOP-PCR) technique using dried blood spot (DBS) on filter paper. This system is convenient for mass screening in the large population and/or first-tier diagnostic method of the patients in the remote areas. However, this system was still time-consuming and effort-taking, because it required pre-amplification procedure to avoid non-specific amplification and gel-electrophoresis to detect the presence or absence of SMN1 deletion. When the fresh blood samples are used instead of DBS, or when the gel-electrophoresis is replaced by real-time PCR, we may have a simpler and more rapid diagnostic method for SMA. To establish a simpler and more rapid diagnostic method of SMN1 deletion using fresh blood DNA. DNA samples extracted from fresh blood and stored at 4 ℃ for 1 month. The samples were assayed using a real-time mCOP-PCR system without pre-amplification procedures. DNA samples had already been genotyped by PCR-restriction fragment length polymorphism (PCR-RFLP), showing the presence or absence of SMN1 exon 7. The DNA samples were directly subjected to the mCOP-PCR step. The amplification of mCOP-PCR was monitored in a real-time PCR apparatus. The genotyping results of the real-time mCOP-PCR system using fresh blood DNA were completely matched with those of PCR-RFLP. In this real-time mCOP-PCR system using fresh blood-DNA, it took only four hours from extraction of DNA to detection of the presence or absence of SMN1 deletion, while it took more than 12 hours in PCR-RFLP. Our real-time mCOP-PCR system using fresh blood DNA was rapid and accurate, suggesting it may be useful for the first

3D printing and milling a real-time PCR device for infectious disease diagnostics.

PubMed

Mulberry, Geoffrey; White, Kevin A; Vaidya, Manjusha; Sugaya, Kiminobu; Kim, Brian N

2017-01-01

Diagnosing infectious diseases using quantitative polymerase chain reaction (qPCR) offers a conclusive result in determining the infection, the strain or type of pathogen, and the level of infection. However, due to the high-cost instrumentation involved and the complexity in maintenance, it is rarely used in the field to make a quick turnaround diagnosis. In order to provide a higher level of accessibility than current qPCR devices, a set of 3D manufacturing methods is explored as a possible option to fabricate a low-cost and portable qPCR device. The key advantage of this approach is the ability to upload the digital format of the design files on the internet for wide distribution so that people at any location can simply download and feed into their 3D printers for quick manufacturing. The material and design are carefully selected to minimize the number of custom parts that depend on advanced manufacturing processes which lower accessibility. The presented 3D manufactured qPCR device is tested with 20-μL samples that contain various concentrations of lentivirus, the same type as HIV. A reverse-transcription step is a part of the device's operation, which takes place prior to the qPCR step to reverse transcribe the target RNA from the lentivirus into complementary DNA (cDNA). This is immediately followed by qPCR which quantifies the target sequence molecules in the sample during the PCR amplification process. The entire process of thermal control and time-coordinated fluorescence reading is automated by closed-loop feedback and a microcontroller. The resulting device is portable and battery-operated, with a size of 12 × 7 × 6 cm3 and mass of only 214 g. By uploading and sharing the design files online, the presented low-cost qPCR device may provide easier access to a robust diagnosis protocol for various infectious diseases, such as HIV and malaria.

3D printing and milling a real-time PCR device for infectious disease diagnostics

PubMed Central

Mulberry, Geoffrey; White, Kevin A.; Vaidya, Manjusha; Sugaya, Kiminobu

2017-01-01

Diagnosing infectious diseases using quantitative polymerase chain reaction (qPCR) offers a conclusive result in determining the infection, the strain or type of pathogen, and the level of infection. However, due to the high-cost instrumentation involved and the complexity in maintenance, it is rarely used in the field to make a quick turnaround diagnosis. In order to provide a higher level of accessibility than current qPCR devices, a set of 3D manufacturing methods is explored as a possible option to fabricate a low-cost and portable qPCR device. The key advantage of this approach is the ability to upload the digital format of the design files on the internet for wide distribution so that people at any location can simply download and feed into their 3D printers for quick manufacturing. The material and design are carefully selected to minimize the number of custom parts that depend on advanced manufacturing processes which lower accessibility. The presented 3D manufactured qPCR device is tested with 20-μL samples that contain various concentrations of lentivirus, the same type as HIV. A reverse-transcription step is a part of the device’s operation, which takes place prior to the qPCR step to reverse transcribe the target RNA from the lentivirus into complementary DNA (cDNA). This is immediately followed by qPCR which quantifies the target sequence molecules in the sample during the PCR amplification process. The entire process of thermal control and time-coordinated fluorescence reading is automated by closed-loop feedback and a microcontroller. The resulting device is portable and battery-operated, with a size of 12 × 7 × 6 cm3 and mass of only 214 g. By uploading and sharing the design files online, the presented low-cost qPCR device may provide easier access to a robust diagnosis protocol for various infectious diseases, such as HIV and malaria. PMID:28586401

The use of PCR/Electrospray Ionization-Time-of-Flight-Mass Spectrometry (PCR/ESI-TOF-MS) to detect bacterial and fungal colonization in healthy military service members.

PubMed

Vetor, Ryan; Murray, Clinton K; Mende, Katrin; Melton-Kreft, Rachel; Akers, Kevin S; Wenke, Joseph; Spirk, Tracy; Guymon, Charles; Zera, Wendy; Beckius, Miriam L; Schnaubelt, Elizabeth R; Ehrlich, Garth; Vento, Todd J

2016-07-22

The role of microbial colonization in disease is complex. Novel molecular tools to detect colonization offer theoretical improvements over traditional methods. We evaluated PCR/Electrospray Ionization-Time-of-Flight-Mass Spectrometry (PCR/ESI-TOF-MS) as a screening tool to study colonization of healthy military service members. We assessed 101 healthy Soldiers using PCR/ESI-TOF-MS on nares, oropharynx, and groin specimens for the presence of gram-positive and gram-negative bacteria (GNB), fungi, and antibiotic resistance genes. A second set of swabs was processed by traditional culture, followed by identification using the BD Phoenix automated system; comparison between PCR/ESI-TOF-MS and culture was carried out only for GNB. Using PCR/ESI-TOF-MS, at least one colonizing organism was found on each individual: mean (SD) number of organisms per subject of 11.8(2.8). The mean number of organisms in the nares, groin and oropharynx was 3.8(1.3), 3.8(1.4) and 4.2(2), respectively. The most commonly detected organisms were aerobic gram-positive bacteria: primarily coagulase-negative Staphylococcus (101 subjects: 341 organisms), Streptococcus pneumoniae (54 subjects: 57 organisms), Staphylococcus aureus (58 subjects: 80 organisms) and Nocardia asteroides (45 subjects: 50 organisms). The mecA gene was found in 96 subjects. The most commonly found GNB was Haemophilus influenzae (20 subjects: 21 organisms) and the most common anaerobe was Propionibacterium acnes (59 subjects). Saccharomyces species (30 subjects) were the most common fungi detected. Only one GNB (nares E. coli) was identified in the same subject by both diagnostic systems. PCR/ESI-TOF-MS detected common colonizing organisms and identified more typically-virulent bacteria in asymptomatic, healthy adults. PCR/ESI-TOF-MS appears to be a useful method for detecting bacterial and fungal organisms, but further clinical correlation and validation studies are needed.

Comparison of droplet digital PCR and quantitative real-time PCR for examining population dynamics of bacteria in soil.

PubMed

Kim, Tae Gwan; Jeong, So-Yeon; Cho, Kyung-Suk

2014-07-01

The newly developed droplet digital PCR (DD-PCR) has shown promise as a DNA quantification technology in medical diagnostic fields. This study evaluated the applicability of DD-PCR as a quantitative tool for soil DNA using quantitative real-time PCR (qRT-PCR) as a reference technology. Cupriavidus sp. MBT14 and Sphingopyxis sp. MD2 were used, and a primer/TaqMan probe set was designed for each (CupMBT and SphMD2, respectively). Standard curve analyses on tenfold dilution series showed that both qRT-PCR and DD-PCR exhibited excellent linearity (R (2) = 1.00) and PCR efficiency (≥92 %) across their detectable ranges. However, DD-PCR showed a tenfold greater sensitivity than qRT-PCR. MBT14 and MD2 were added to non-sterile soil at 0 ~ 5 × 10(8) and 0 ~ 5 × 10(7) cells per gram of soil, respectively (n = 5). This bacterial load test indicated that DD-PCR was more sensitive and discriminating than qRT-PCR. For instance, DD-PCR showed a gradual DNA increase from 14 to 141,160 MBT14 rDNA copies μL DNA extract(-1) as the bacterial load increased, while qRT-PCR could quantify the DNA (6,432 copies μL DNA(-1)) at ≥5 × 10(5) MBT14 per gram of soil. When temporal DNA changes were monitored for 3 weeks in the amended soils, the two technologies exhibited nearly identical changes over time. Linearity tests (y = a · x) revealed excellent quantitative agreement between the two technologies (a = 0.98, R (2) = 0.97 in the CupMBT set and a = 0.90, R (2) = 0.94 in the SphMD2 set). These results suggest that DD-PCR is a promising tool to examine temporal dynamics of microorganisms in complex environments.

Clinical evaluation of a Mucorales-specific real-time PCR assay in tissue and serum samples.

PubMed

Springer, Jan; Lackner, Michaela; Ensinger, Christian; Risslegger, Brigitte; Morton, Charles Oliver; Nachbaur, David; Lass-Flörl, Cornelia; Einsele, Hermann; Heinz, Werner J; Loeffler, Juergen

2016-12-01

Molecular diagnostic assays can accelerate the diagnosis of fungal infections and subsequently improve patient outcomes. In particular, the detection of infections due to Mucorales is still challenging for laboratories and physicians. The aim of this study was to evaluate a probe-based Mucorales-specific real-time PCR assay (Muc18S) using tissue and serum samples from patients suffering from invasive mucormycosis (IMM). This assay can detect a broad range of clinically relevant Mucorales species and can be used to complement existing diagnostic tests or to screen high-risk patients. An advantage of the Muc18S assay is that it exclusively detects Mucorales species allowing the diagnosis of Mucorales DNA without sequencing within a few hours. In paraffin-embedded tissue samples this PCR-based method allowed rapid identification of Mucorales in comparison with standard methods and showed 91 % sensitivity in the IMM tissue samples. We also evaluated serum samples, an easily accessible material, from patients at risk from IMM. Mucorales DNA was detected in all patients with probable/proven IMM (100 %) and in 29 % of the possible cases. Detection of IMM in serum could enable an earlier diagnosis (up to 21 days) than current methods including tissue samples, which were gained mainly post-mortem. A screening strategy for high-risk patients, which would enable targeted treatment to improve patient outcomes, is therefore possible.

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

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 digital PCR for absolute RNA quantification.

PubMed

Sanders, Rebecca; Mason, Deborah J; Foy, Carole A; Huggett, Jim F

2013-01-01

Gene expression measurements detailing mRNA quantities are widely employed in molecular biology and are increasingly important in diagnostic fields. Reverse transcription (RT), necessary for generating complementary DNA, can be both inefficient and imprecise, but remains a quintessential RNA analysis tool using qPCR. This study developed a Transcriptomic Calibration Material and assessed the RT reaction using digital (d)PCR for RNA measurement. While many studies characterise dPCR capabilities for DNA quantification, less work has been performed investigating similar parameters using RT-dPCR for RNA analysis. RT-dPCR measurement using three, one-step RT-qPCR kits was evaluated using single and multiplex formats when measuring endogenous and synthetic RNAs. The best performing kit was compared to UV quantification and sensitivity and technical reproducibility investigated. Our results demonstrate assay and kit dependent RT-dPCR measurements differed significantly compared to UV quantification. Different values were reported by different kits for each target, despite evaluation of identical samples using the same instrument. RT-dPCR did not display the strong inter-assay agreement previously described when analysing DNA. This study demonstrates that, as with DNA measurement, RT-dPCR is capable of accurate quantification of low copy RNA targets, but the results are both kit and target dependent supporting the need for calibration controls.

Development and application of a universal Hemoplasma screening assay based on the SYBR green PCR principle.

PubMed

Willi, Barbara; Meli, Marina L; Lüthy, Ruedi; Honegger, Hanspeter; Wengi, Nicole; Hoelzle, Ludwig E; Reusch, Claudia E; Lutz, Hans; Hofmann-Lehmann, Regina

2009-12-01

Hemotropic mycoplasmas (hemoplasmas) are the causative agents of infectious anemia in several mammalian species. Their zoonotic potential has recently been substantiated by the identification of a feline hemoplasma isolate in an immunocompromised human patient. Although species-specific diagnostic molecular methods have been developed, their application as screening tools is limited due to the species diversity of hemoplasmas. The goals of this study were to develop a universal hemoplasma screening assay with broad specificity based on the SYBR green PCR principle, to compare the assay with hemoplasma-specific TaqMan PCR, and to analyze potential tick vectors and human blood samples to address the zoonotic potential. The newly developed PCR assay based on the 16S rRNA gene amplified feline, canine, bovine, porcine, camelid, and murine hemoplasmas, as well as Mycoplasma penetrans and Mycoplasma pneumoniae. The lower detection limit for feline and canine hemoplasmas was 1 to 10 copies/PCR. The assay exhibited 98.2% diagnostic sensitivity and 92.1% diagnostic specificity for feline hemoplasmas. All 1,950 Ixodes ticks were PCR negative, suggesting that Ixodes ticks are not relevant vectors for the above-mentioned hemoplasma species in Switzerland. None of the 414 blood samples derived from anemic or immunocompromised human patients revealed a clear positive result. The SYBR green PCR assay described here is a suitable tool to screen for known and so-far-undiscovered hemoplasma species. Positive results should be confirmed by specific TaqMan PCR or sequencing.

Diagnostic accuracy of quantitative real-time PCR assay versus clinical and Gram stain identification of bacterial vaginosis.

PubMed

Menard, J-P; Mazouni, C; Fenollar, F; Raoult, D; Boubli, L; Bretelle, F

2010-12-01

The purpose of this investigation was to determine the diagnostic accuracy of quantitative real-time polymerase chain reaction (PCR) assay in diagnosing bacterial vaginosis versus the standard methods, the Amsel criteria and the Nugent score. The Amsel criteria, the Nugent score, and results from the molecular tool were obtained independently from vaginal samples of 163 pregnant women who reported abnormal vaginal symptoms before 20 weeks gestation. To determine the performance of the molecular tool, we calculated the kappa value, sensitivity, specificity, and positive and negative predictive values. Either or both of the Amsel criteria (≥3 criteria) and the Nugent score (score ≥7) indicated that 25 women (15%) had bacterial vaginosis, and the remaining 138 women did not. DNA levels of Gardnerella vaginalis or Atopobium vaginae exceeded 10(9) copies/mL or 10(8) copies/mL, respectively, in 34 (21%) of the 163 samples. Complete agreement between both reference methods and high concentrations of G. vaginalis and A. vaginae was found in 94.5% of women (154/163 samples, kappa value = 0.81, 95% confidence interval 0.70-0.81). The nine samples with discordant results were categorized as intermediate flora by the Nugent score. The molecular tool predicted bacterial vaginosis with a sensitivity of 100%, a specificity of 93%, a positive predictive value of 73%, and a negative predictive value of 100%. The quantitative real-time PCR assay shows excellent agreement with the results of both reference methods for the diagnosis of bacterial vaginosis.

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.

Improved diagnostic testing and malaria treatment practices in Zambia.

PubMed

Hamer, Davidson H; Ndhlovu, Micky; Zurovac, Dejan; Fox, Matthew; Yeboah-Antwi, Kojo; Chanda, Pascalina; Sipilinyambe, Naawa; Simon, Jonathon L; Snow, Robert W

2007-05-23

Improving the accuracy of malaria diagnosis with rapid antigen-detection diagnostic tests (RDTs) has been proposed as an approach for reducing overtreatment of malaria in the current era of widespread implementation of artemisinin-based combination therapy in sub-Saharan Africa. To assess the association between use of microscopy and RDT and the prescription of antimalarials. Cross-sectional, cluster sample survey, carried out between March and May 2006, of all outpatients treated during 1 working day at government and mission health facilities in 4 sentinel districts in Zambia. Proportions of patients undergoing malaria diagnostic procedures and receiving antimalarial treatment. Seventeen percent of the 104 health facilities surveyed had functional microscopy, 63% had RDTs available, and 73% had 1 or more diagnostics available. Of patients with fever (suspected malaria), 27.8% (95% confidence interval [CI], 13.1%-42.5%) treated in health facilities with malaria diagnostics were tested and 44.6% had positive test results. Of patients with negative blood smear results, 58.4% (95% CI, 36.7%-80.2%) were prescribed an antimalaria drug, as were 35.5% (95% CI, 16.0%-55.0%) of those with a negative RDT result. Of patients with fever who did not have diagnostic tests done, 65.9% were also prescribed antimalarials. In facilities with artemether-lumefantrine in stock, this antimalarial was prescribed to a large proportion of febrile patients with a positive diagnostic test result (blood smear, 75.0% [95% CI, 51.7%-98.3%]; RDT, 70.4% [95% CI, 39.3%-100.0%]), but also to some of those with a negative diagnostic test result (blood smear, 30.4% [95% CI, 8.0%-52. 9%]; RDT, 26.7% [95% CI, 5.7%-47.7%]). Despite efforts to expand the provision of malaria diagnostics in Zambia, they continue to be underused and patients with negative test results frequently receive antimalarials. Provision of new tools to reduce inappropriate use of new expensive antimalarial treatments must be

Calibration-free assays on standard real-time PCR devices

PubMed Central

Debski, Pawel R.; Gewartowski, Kamil; Bajer, Seweryn; Garstecki, Piotr

2017-01-01

Quantitative Polymerase Chain Reaction (qPCR) is one of central techniques in molecular biology and important tool in medical diagnostics. While being a golden standard qPCR techniques depend on reference measurements and are susceptible to large errors caused by even small changes of reaction efficiency or conditions that are typically not marked by decreased precision. Digital PCR (dPCR) technologies should alleviate the need for calibration by providing absolute quantitation using binary (yes/no) signals from partitions provided that the basic assumption of amplification a single target molecule into a positive signal is met. Still, the access to digital techniques is limited because they require new instruments. We show an analog-digital method that can be executed on standard (real-time) qPCR devices. It benefits from real-time readout, providing calibration-free assessment. The method combines advantages of qPCR and dPCR and bypasses their drawbacks. The protocols provide for small simplified partitioning that can be fitted within standard well plate format. We demonstrate that with the use of synergistic assay design standard qPCR devices are capable of absolute quantitation when normal qPCR protocols fail to provide accurate estimates. We list practical recipes how to design assays for required parameters, and how to analyze signals to estimate concentration. PMID:28327545

Calibration-free assays on standard real-time PCR devices

NASA Astrophysics Data System (ADS)

Debski, Pawel R.; Gewartowski, Kamil; Bajer, Seweryn; Garstecki, Piotr

2017-03-01

Quantitative Polymerase Chain Reaction (qPCR) is one of central techniques in molecular biology and important tool in medical diagnostics. While being a golden standard qPCR techniques depend on reference measurements and are susceptible to large errors caused by even small changes of reaction efficiency or conditions that are typically not marked by decreased precision. Digital PCR (dPCR) technologies should alleviate the need for calibration by providing absolute quantitation using binary (yes/no) signals from partitions provided that the basic assumption of amplification a single target molecule into a positive signal is met. Still, the access to digital techniques is limited because they require new instruments. We show an analog-digital method that can be executed on standard (real-time) qPCR devices. It benefits from real-time readout, providing calibration-free assessment. The method combines advantages of qPCR and dPCR and bypasses their drawbacks. The protocols provide for small simplified partitioning that can be fitted within standard well plate format. We demonstrate that with the use of synergistic assay design standard qPCR devices are capable of absolute quantitation when normal qPCR protocols fail to provide accurate estimates. We list practical recipes how to design assays for required parameters, and how to analyze signals to estimate concentration.

Treatment of PCR products with exonuclease I and heat-labile alkaline phosphatase improves the visibility of combined bisulfite restriction analysis

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

Watanabe, Kousuke; Emoto, Noriko; Sunohara, Mitsuhiro

2010-08-27

Research highlights: {yields} Incubating PCR products at a high temperature causes smears in gel electrophoresis. {yields} Smears interfere with the interpretation of methylation analysis using COBRA. {yields} Treatment with exonuclease I and heat-labile alkaline phosphatase eliminates smears. {yields} The elimination of smears improves the visibility of COBRA. -- Abstract: DNA methylation plays a vital role in the regulation of gene expression. Abnormal promoter hypermethylation is an important mechanism of inactivating tumor suppressor genes in human cancers. Combined bisulfite restriction analysis (COBRA) is a widely used method for identifying the DNA methylation of specific CpG sites. Here, we report that exonucleasemore » I and heat-labile alkaline phosphatase can be used for PCR purification for COBRA, improving the visibility of gel electrophoresis after restriction digestion. This improvement is observed when restriction digestion is performed at a high temperature, such as 60 {sup o}C or 65 {sup o}C, with BstUI and TaqI, respectively. This simple method can be applied instead of DNA purification using spin columns or phenol/chloroform extraction. It can also be applied to other situations when PCR products are digested by thermophile-derived restriction enzymes, such as PCR restriction fragment length polymorphism (RFLP) analysis.« less

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

Diagnostic performance of rapid diagnostic tests for the diagnosis of malaria at public health facilities in north-west Ethiopia.

PubMed

Getnet, Gebeyaw; Getie, Sisay; Srivastava, Mitaly; Birhan, Wubet; Fola, Abebe A; Noedl, Harald

2015-11-01

To assess the performance of RDTs against nested polymerase chain reaction (nPCR) for the diagnosis of malaria in public health facilities in north-western Ethiopia. Cross-sectional study at public health facilities in North Gondar, Ethiopia, of 359 febrile patients with signs and symptoms consistent with malaria. Finger prick blood samples were collected for testing in a P. falciparum/pan-malaria RDTs and for molecular analysis. Sensitivity, specificity and predictive values were determined for the RDTs using nPCR as reference diagnostic method. Kappa value was determined to demonstrate the consistency of the results between the diagnostic tools. By RDTs, 22.28% (80/359) of patients tested positive for malaria, and by nPCR, 27.02% (97/359) did. In nPCR, 1.67% (6/359) and 0.28% (1/359) samples were positive for P. ovale and P. malariae, which had almost all tested negative in the RDTs. The sensitivity, specificity, positive and negative predictive values of RDTs for the diagnosis of malaria were 62.9%, 92.7%, 76.3% and 87.1%, respectively, with 0.589 measurement agreement between RDTs and nPCR. The sensitivity and specificity of RDTs for P. falciparum identification only were 70.8% and 95.2%, and 65.2% and 93.1% for P. vivax. Although RDTs are commonly used at health posts in resource-limited environments, their sensitivity and specificity for the detection and species identification of Plasmodium parasites were poor compared to nPCR, suggesting caution in interpreting RDTs results. Particularly, in the light of expanded efforts to eliminate malaria in the country, more sensitive diagnostic procedures will be needed. © 2015 John Wiley & Sons Ltd.

Cytomegalovirus infection in immunocompetent wheezy infants: the diagnostic value of CMV PCR in bronchoalveolar lavage fluid.

PubMed

Cinel, G; Pekcan, S; Ozçelik, U; Alp, A; Yalçın, E; Doğru Ersöz, D; Kiper, N

2014-08-01

Cytomegalovirus (CMV) pneumonitis in immunocompetent hosts is uncommon but is being recognized more frequently, particularly when presenting as severe viral pneumonia. The objective of this study was to examine lower respiratory tract CMV infection in immunocompetent wheezy infants, based on polymerase chain reaction (PCR) in bronchoalveolar lavage (BAL) fluid, to compare CMV PCR results in BAL and in blood samples and to evaluate the benefits of antiviral ganciclovir therapy in these patients. Retrospective review of the records of patients referred to our tertiary care hospital between January 2000 and July 2010 who had unexplained persistent wheezing and underwent fibreoptic flexible bronchoscopy (FFB). Fibreoptic flexible bronchoscopy was applied to 102 infants with persistent wheezing and diffuse interstitial infiltration on radiological investigations; so CMV PCR in BAL fluid was performed. CMV PCR in BAL fluid was positive in 51 patients. Retrospectively, we had access to the files of 25 of these patients. The mean CMV PCR in BAL fluid was 334 840 copies/mL. Only eight patients had CMV PCR positivity in their blood samples (mean: 2026·3 copies/mL). There was not a relationship between BAL and blood CMV PCR values based on Spearman's correlation analysis (r = -0·008). Fourteen patients had severe respiratory symptoms and received ganciclovir therapy. Twelve of them fully recovered. Bronchoalveolar lavage fluid CMV PCR was superior to blood CMV PCR in diagnosing lower respiratory tract infections caused by CMV in immunocompetent infants. Ganciclovir therapy may be effective in selected immunocompetent wheezy infants with CMV PCR positivity in BAL fluid. © 2014 John Wiley & Sons Ltd.

Latent class analysis of diagnostic tests for adenovirus, Bordetella pertussis and influenza virus infections in German adults with longer lasting coughs.

PubMed

Sobotzki, C; Riffelmann, M; Kennerknecht, N; Hülsse, C; Littmann, M; White, A; Von Kries, R; Wirsing VON König, C H

2016-03-01

Laboratory tests in adult outpatients with longer lasting coughs to identify a potential causal pathogen are rarely performed, and there is no gold standard for these diagnostic tests. While the diagnostic validity of serological tests for pertussis is well established their potential contribution for diagnosing adenovirus and influenza virus A and B infections is unclear. A sentinel study into the population-based incidence of longer lasting coughs in adults was done in Rostock (former East Germany) and Krefeld (former West Germany). A total of 971 outpatients who consulted general practitioners or internists were included. Inclusion criteria were coughing for ⩾1 week and no chronic respiratory diseases. We evaluated the performance of polymerase chain reaction (PCR) as well as IgG and IgA serology, applying a latent class model for diagnosing infections with adenovirus, B. pertussis, and influenza virus A and B. The adult outpatients first sought medical attention when they had been coughing for a median of 3 weeks. In this situation, direct detection of infectious agents by PCR had a low sensitivity. Modelling showed that additional serological tests equally improved sensitivity and specificity for diagnosis for adenovirus, B. pertussis and influenza virus A and B infections. The combination of serology and PCR may improve the overall performance of diagnostic tests for B. pertussis and also for adenovirus, and influenza virus A and B infections.

Virtual PCR

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

Gardner, S N; Clague, D S; Vandersall, J A

2006-02-23

The polymerase chain reaction (PCR) stands among the keystone technologies for analysis of biological sequence data. PCR is used to amplify DNA, to generate many copies from as little as a single template. This is essential, for example, in processing forensic DNA samples, pathogen detection in clinical or biothreat surveillance applications, and medical genotyping for diagnosis and treatment of disease. It is used in virtually every laboratory doing molecular, cellular, genetic, ecologic, forensic, or medical research. Despite its ubiquity, we lack the precise predictive capability that would enable detailed optimization of PCR reaction dynamics. In this LDRD, we proposed tomore » develop Virtual PCR (VPCR) software, a computational method to model the kinetic, thermodynamic, and biological processes of PCR reactions. Given a successful completion, these tools will allow us to predict both the sequences and concentrations of all species that are amplified during PCR. The ability to answer the following questions will allow us both to optimize the PCR process and interpret the PCR results: What products are amplified when sequence mixtures are present, containing multiple, closely related targets and multiplexed primers, which may hybridize with sequence mismatches? What are the effects of time, temperature, and DNA concentrations on the concentrations of products? A better understanding of these issues will improve the design and interpretation of PCR reactions. The status of the VPCR project after 1.5 years of funding is consistent with the goals of the overall project which was scoped for 3 years of funding. At half way through the projected timeline of the project we have an early beta version of the VPCR code. We have begun investigating means to improve the robustness of the code, performed preliminary experiments to test the code and begun drafting manuscripts for publication. Although an experimental protocol for testing the code was developed, the

Human papillomavirus detection and typing using a nested-PCR-RFLP assay.

PubMed

Coser, Janaina; Boeira, Thaís da Rocha; Fonseca, André Salvador Kazantzi; Ikuta, Nilo; Lunge, Vagner Ricardo

2011-01-01

It is clinically important to detect and type human papillomavirus (HPV) in a sensitive and specific manner. Development of a nested-polymerase chain reaction-restriction fragment length polymorphism (nested-PCR-RFLP) assay to detect and type HPV based on the analysis of L1 gene. Analysis of published DNA sequence of mucosal HPV types to select sequences of new primers. Design of an original nested-PCR assay using the new primers pair selected and classical MY09/11 primers. HPV detection and typing in cervical samples using the nested-PCR-RFLP assay. The nested-PCR-RFLP assay detected and typed HPV in cervical samples. Of the total of 128 clinical samples submitted to simple PCR and nested-PCR for detection of HPV, 37 (28.9%) were positive for the virus by both methods and 25 samples were positive only by nested-PCR (67.5% increase in detection rate compared with single PCR). All HPV positive samples were effectively typed by RFLP assay. The method of nested-PCR proved to be an effective diagnostic tool for HPV detection and typing.

PCR in laboratory diagnosis of human Borrelia burgdorferi infections.

PubMed

Schmidt, B L

1997-01-01

The laboratory diagnosis of Lyme borreliosis, the most prevalent vector-borne disease in the United States and endemic in parts of Europe and Asia, is currently based on serology with known limitations. Direct demonstration of Borrelia burgdorferi by culture may require weeks, while enzyme-linked immunosorbent assays for antigen detection often lack sensitivity. The development of the PCR has offered a new dimension in the diagnosis. Capable of amplifying minute amounts of DNA into billions of copies in just a few hours, PCR facilitates the sensitive and specific detection of DNA or RNA of pathogenic organisms. This review is restricted to applications of PCR methods in the diagnosis of human B. burgdorferi infections. In the first section, methodological aspects, e.g., sample preparation, target selection, primers and PCR methods, and detection and control of inhibition and contamination, are highlighted. In the second part, emphasis is placed on diagnostic aspects, where PCR results in patients with dermatological, neurological, joint, and ocular manifestations of the disease are discussed. Here, special attention is given to monitoring treatment efficacy by PCR tests. Last, specific guidelines on how to interpret PCR results, together with the advantages and limitations of these new techniques, are presented.

Competitive PCR-High Resolution Melting Analysis (C-PCR-HRMA) for large genomic rearrangements (LGRs) detection: A new approach to assess quantitative status of BRCA1 gene in a reference laboratory.

PubMed

Minucci, Angelo; De Paolis, Elisa; Concolino, Paola; De Bonis, Maria; Rizza, Roberta; Canu, Giulia; Scaglione, Giovanni Luca; Mignone, Flavio; Scambia, Giovanni; Zuppi, Cecilia; Capoluongo, Ettore

2017-07-01

Evaluation of copy number variation (CNV) in BRCA1/2 genes, due to large genomic rearrangements (LGRs), is a mandatory analysis in hereditary breast and ovarian cancers families, if no pathogenic variants are found by sequencing. LGRs cannot be detected by conventional methods and several alternative methods have been developed. Since these approaches are expensive and time consuming, identification of alternative screening methods for LGRs detection is needed in order to reduce and optimize the diagnostic procedure. The aim of this study was to investigate a Competitive PCR-High Resolution Melting Analysis (C-PCR-HRMA) as molecular tool to detect recurrent BRCA1 LGRs. C-PCR-HRMA was performed on exons 3, 14, 18, 19, 20 and 21 of the BRCA1 gene; exons 4, 6 and 7 of the ALB gene were used as reference fragments. This study showed that it is possible to identify recurrent BRCA1 LGRs, by melting peak height ratio between target (BRCA1) and reference (ALB) fragments. Furthermore, we underline that a peculiar amplicon-melting profile is associated to a specific BRCA1 LGR. All C-PCR-HRMA results were confirmed by Multiplex ligation-dependent probe amplification. C-PCR-HRMA has proved to be an innovative, efficient and fast method for BRCA1 LGRs detection. Given the sensitivity, specificity and ease of use, c-PCR-HRMA can be considered an attractive and powerful alternative to other methods for BRCA1 CNVs screening, improving molecular strategies for BRCA testing in the context of Massive Parallel Sequencing. Copyright © 2017 Elsevier B.V. All rights reserved.

Digital PCR Improves Mutation Analysis in Pancreas Fine Needle Aspiration Biopsy Specimens

PubMed Central

Court, Colin M.; Kim, Stephen; Braxton, David R.; Hou, Shuang; Muthusamy, V. Raman; Watson, Rabindra R.; Sedarat, Alireza; Tseng, Hsian-Rong; Tomlinson, James S.

2017-01-01

Applications of precision oncology strategies rely on accurate tumor genotyping from clinically available specimens. Fine needle aspirations (FNA) are frequently obtained in cancer management and often represent the only source of tumor tissues for patients with metastatic or locally advanced diseases. However, FNAs obtained from pancreas ductal adenocarcinoma (PDAC) are often limited in cellularity and/or tumor cell purity, precluding accurate tumor genotyping in many cases. Digital PCR (dPCR) is a technology with exceptional sensitivity and low DNA template requirement, characteristics that are necessary for analyzing PDAC FNA samples. In the current study, we sought to evaluate dPCR as a mutation analysis tool for pancreas FNA specimens. To this end, we analyzed alterations in the KRAS gene in pancreas FNAs using dPCR. The sensitivity of dPCR mutation analysis was first determined using serial dilution cell spiking studies. Single-cell laser-microdissection (LMD) was then utilized to identify the minimal number of tumor cells needed for mutation detection. Lastly, dPCR mutation analysis was performed on 44 pancreas FNAs (34 formalin-fixed paraffin-embedded (FFPE) and 10 fresh (non-fixed)), including samples highly limited in cellularity (100 cells) and tumor cell purity (1%). We found dPCR to detect mutations with allele frequencies as low as 0.17%. Additionally, a single tumor cell could be detected within an abundance of normal cells. Using clinical FNA samples, dPCR mutation analysis was successful in all preoperative FNA biopsies tested, and its accuracy was confirmed via comparison with resected tumor specimens. Moreover, dPCR revealed additional KRAS mutations representing minor subclones within a tumor that were not detected by the current clinical gold standard method of Sanger sequencing. In conclusion, dPCR performs sensitive and accurate mutation analysis in pancreas FNAs, detecting not only the dominant mutation subtype, but also the additional rare

Digital PCR Improves Mutation Analysis in Pancreas Fine Needle Aspiration Biopsy Specimens.

PubMed

Sho, Shonan; Court, Colin M; Kim, Stephen; Braxton, David R; Hou, Shuang; Muthusamy, V Raman; Watson, Rabindra R; Sedarat, Alireza; Tseng, Hsian-Rong; Tomlinson, James S

2017-01-01

Applications of precision oncology strategies rely on accurate tumor genotyping from clinically available specimens. Fine needle aspirations (FNA) are frequently obtained in cancer management and often represent the only source of tumor tissues for patients with metastatic or locally advanced diseases. However, FNAs obtained from pancreas ductal adenocarcinoma (PDAC) are often limited in cellularity and/or tumor cell purity, precluding accurate tumor genotyping in many cases. Digital PCR (dPCR) is a technology with exceptional sensitivity and low DNA template requirement, characteristics that are necessary for analyzing PDAC FNA samples. In the current study, we sought to evaluate dPCR as a mutation analysis tool for pancreas FNA specimens. To this end, we analyzed alterations in the KRAS gene in pancreas FNAs using dPCR. The sensitivity of dPCR mutation analysis was first determined using serial dilution cell spiking studies. Single-cell laser-microdissection (LMD) was then utilized to identify the minimal number of tumor cells needed for mutation detection. Lastly, dPCR mutation analysis was performed on 44 pancreas FNAs (34 formalin-fixed paraffin-embedded (FFPE) and 10 fresh (non-fixed)), including samples highly limited in cellularity (100 cells) and tumor cell purity (1%). We found dPCR to detect mutations with allele frequencies as low as 0.17%. Additionally, a single tumor cell could be detected within an abundance of normal cells. Using clinical FNA samples, dPCR mutation analysis was successful in all preoperative FNA biopsies tested, and its accuracy was confirmed via comparison with resected tumor specimens. Moreover, dPCR revealed additional KRAS mutations representing minor subclones within a tumor that were not detected by the current clinical gold standard method of Sanger sequencing. In conclusion, dPCR performs sensitive and accurate mutation analysis in pancreas FNAs, detecting not only the dominant mutation subtype, but also the additional rare

Incidence of pulmonary aspergillosis and correlation of conventional diagnostic methods with nested PCR and real-time PCR assay using BAL fluid in intensive care unit patients.

PubMed

Zarrinfar, Hossein; Makimura, Koichi; Satoh, Kazuo; Khodadadi, Hossein; Mirhendi, Hossein

2013-05-01

Although the incidence of invasive aspergillosis in the intensive care unit (ICU) is scarce, it has emerged as major problems in critically ill patients. In this study, the incidence of pulmonary aspergillosis (PA) in ICU patients has evaluated and direct microscopy and culture has compared with nested polymerase chain reaction (PCR) and real-time PCR for detection of Aspergillus fumigatus and A. flavus in bronchoalveolar lavage (BAL) samples of the patients. Thirty BAL samples obtained from ICU patients during a 16-month period were subjected to direct examinations on 20% potassium hydroxide (KOH) and culture on two culture media. Nested PCR targeting internal transcribed spacer ribosomal DNA and TaqMan real-time PCR assay targeting β-tubulin gene were used for the detection of A. fumigatus and A. flavus. Of 30 patients, 60% were men and 40% were women. The diagnosis of invasive PA was probable in 1 (3%), possible in 11 (37%), and not IPA in 18 (60%). Nine samples were positive in nested PCR including seven samples by A. flavus and two by A. fumigatus specific primers. The lowest amount of DNA that TaqMan real-time PCR could detect was ≥40 copy numbers. Only one of the samples had a positive result of A. flavus real-time PCR with Ct value of 37.5. Although a significant number of specimens were positive in nested PCR, results of this study showed that establishment of a correlation between the conventional methods with nested PCR and real-time PCR needs more data confirmed by a prospective study with a larger sample group. © 2013 Wiley Periodicals, Inc.

Molecular diagnosis of bacterial vaginosis: Does adjustment for total bacterial load or human cellular content improve diagnostic performance?

PubMed

Plummer, E L; Garland, S M; Bradshaw, C S; Law, M G; Vodstrcil, L A; Hocking, J S; Fairley, C K; Tabrizi, S N

2017-02-01

We investigated the utility of quantitative PCR assays for diagnosis of bacterial vaginosis and found that while the best model utilized bacterial copy number adjusted for total bacterial load (sensitivity=98%, specificity=93%, AUC=0.95[95%CI=0.93,0.97]), adjusting for total bacterial or human cell load did not consistently increase the diagnostic performance of the assays. Copyright © 2017 Elsevier B.V. All rights reserved.

Sample-ready multiplex qPCR assay for detection of malaria

PubMed Central

2014-01-01

Background Microscopy and antigen detecting rapid diagnostic tests are the diagnostic tests of choice in management of clinical malaria. However, due to their limitations, the need to utilize more sensitive methods such as real-time PCR (qPCR) is evident as more studies are now utilizing molecular methods in detection of malaria. Some of the challenges that continue to limit the widespread utilization of qPCR include lack of assay standardization, assay variability, risk of contamination, and the need for cold-chain. Lyophilization of molecular assays can overcome some of these limitations and potentially enable widespread qPCR utilization. Methods A recently published multiplex malaria qPCR assay was lyophilized by freezing drying into Sample-Ready™ format (MMSR). MMSR assay contained all the required reagents for qPCR including primers and probes, requiring only the addition of water and sample to perform qPCR. The performance of the MMSR assay was compared to the non-freeze dried, “wet” assay. Stability studies were done by maintaining the MMSR assays at four different ambient temperatures of 4°C, room temperature (RT), 37°C and 42°C over a period of 42 days, tested at seven-day intervals. Plasmodium falciparum and Plasmodium vivax DNAs were used for analysis of the MMSR assay either as single or mixed parasites, at two different concentrations. The CT values and the standard deviations (SD) were used in the analysis of the assay performance. Results The limit of detection for the MMSR assay was 0.244 parasites/μL for Plasmodium spp. (PLU) and P. falciparum (FAL) assay targets compared to “wet” assay which was 0.39 and 3.13 parasites/μL for PLU and FAL assay targets, respectively. The MMSR assay performed with high efficiencies similar to those of the “wet” assay and was stable at 37°C for 42 days, with estimated shelf-life of 5 months. When used to analyse field clinical samples, MMSR assay performed with 100% sensitivity and specificity

A new diagnostic real-time PCR method for huanglongbing detection in citrus root tissue

USDA-ARS?s Scientific Manuscript database

Citrus fibrous root tissue was evaluated as an alternative source material for Huanglongbing (HLB) diagnosis by real-time PCR using primer-probe set TXCChlb, developed in the present study based on 16S rDNA of “Candidatus Liberibacter asiaticus” (CLas). Real-time PCR data obtained with DNA samples p...

Improving the existing diagnostic strategy by accounting for characteristics of the women in the diagnostic work up for postmenopausal bleeding.

PubMed

Opmeer, B C; van Doorn, H C; Heintz, A P M; Burger, C W; Bossuyt, P M M; Mol, B W J

2007-01-01

The aim of this study was to evaluate whether the efficiency of the current diagnostic work up following postmenopausal bleeding could be improved by diagnostic strategies that take into account characteristics of the women in addition to the currently recommended transvaginal measurement of endometrial thickness to determine for subsequent histological assessment. Multicenter, prospective cohort study. A university hospital and seven teaching hospitals in the Netherlands. Consecutive women not using hormone replacement therapy, presenting with postmenopausal bleeding. Five hundred and forty women underwent transvaginal sonography, and in case of endometrial thickness (double layer) above 4 mm, subsequent endometrial sampling was performed. Presence of carcinoma was ruled out by the absence of abnormalities in histological specimen or by an uneventful follow up of at least 6 months. Probability of endometrial carcinoma was estimated by multivariable logistic regression models. For each diagnostic strategy, we calculated diagnostic accuracy (area under receiver operating characteristic curve [AUC]), negative predictive value (NPV) and the number of diagnostic procedures. A strategy with transvaginal sonography alone with a fixed threshold incorrectly classified 0.7% of the women as nonmalignant (NPV: 99.3% [98.5-100%]), with 97% sensitivity and 56% specificity. A strategy integrating characteristics of the women with transvaginal sonography could result in less false reassurances (NPV: 99.6% [99.2-100%]), with only marginal decrease in diagnostic procedures, or a minor increase in false reassurances (NPV: 99.0% [98.3-100%]), with a substantial reduction (15-20%) in the procedures. AUCs associated with these strategies could improve from 0.76 (0.73-0.79) for transvaginal sonography alone to 0.90 (0.87-0.93) for the integrated strategy. Taking into account the characteristics of the women could increase the efficiency of the diagnostic work up for postmenopausal

Portable low-power thermal cycler with dual thin-film Pt heaters for a polymeric PCR chip.

PubMed

Jeong, Sangdo; Lim, Juhun; Kim, Mi-Young; Yeom, JiHye; Cho, Hyunmin; Lee, Hyunjung; Shin, Yong-Beom; Lee, Jong-Hyun

2018-01-29

Polymerase chain reaction (PCR) has been widely used for major definite diagnostic tool, but very limited its place used only indoor such as hospital or diagnosis lab. For the rapid on-site detection of pathogen in an outdoor environment, a low-power cordless polymerase chain reaction (PCR) thermal cycler is crucial module. At this point of view, we proposed a low-power PCR thermal cycler that could be operated in an outdoor anywhere. The disposable PCR chip was made of a polymeric (PI/PET) film to reduce the thermal mass. A dual arrangement of the Pt heaters, which were positioned on the top and bottom of the PCR chip, improved the temperature uniformity. The temperature sensor, which was made of the same material as the heater, utilized the temperature dependence of the Pt resistor to ensure simple fabrication of the temperature sensor. Cooling the PCR chip using dual blower fans enabled thermal cycling to operate with a lower power than that of a Peltier element with a high power consumption. The PCR components were electrically connected to a control module that could be operated with a Li-ion battery (12 V), and the PCR conditions (temperature, time, cycle, etc.) were inputted on a touch screen. For 30 PCR cycles, the accumulated power consumption of heating and cooling was 7.3 Wh, which is easily available from a compact battery. Escherichia coli genomic DNA (510 bp) was amplified using the proposed PCR thermal cycler and the disposable PCR chip. A similar DNA amplification capability was confirmed using the proposed portable and low-power thermal cycler compared with a conventional thermal cycler.

DNA Extraction from Protozoan Oocysts/Cysts in Feces for Diagnostic PCR

PubMed Central

2014-01-01

PCR detection of intestinal protozoa is often restrained by a poor DNA recovery or by inhibitors present in feces. The need for an extraction protocol that can overcome these obstacles is therefore clear. QIAamp® DNA Stool Mini Kit (Qiagen) was evaluated for its ability to recover DNA from oocysts/cysts directly from feces. Twenty-five Giardia-positive, 15 Cryptosporidium-positive, 15 Entamoeba histolytica-positive, and 45 protozoa-free samples were processed as control by microscopy and immunoassay tests. DNA extracts were amplified using 3 sets of published primers. Following the manufacturer's protocol, the kit showed sensitivity and specificity of 100% towards Giardia and Entamoeba. However, for Cryptosporidium, the sensitivity and specificity were 60% (9/15) and 100%, respectively. A series of optimization experiments involving various steps of the kit's protocol were conducted using Cryptosporidium-positive samples. The best DNA recoveries were gained by raising the lysis temperature to the boiling point for 10 min and the incubation time of the InhibitEX tablet to 5 min. Also, using a pre-cooled ethanol for nucleic acid precipitation and small elution volume (50-100 µl) were valuable. The sensitivity of the amended protocol to Cryptosporidium was raised to 100%. Cryptosporidium DNA was successfully amplified by either the first or the second primer set. When applied on parasite-free feces spiked with variable oocysts/cysts counts, ≈ 2 oocysts/cysts were theoretically enough for detection by PCR. To conclude, the Qiagen kit with the amended protocol was proved to be suitable for protozoan DNA extraction directly from feces and support PCR diagnosis. PMID:25031466

DNA extraction from protozoan oocysts/cysts in feces for diagnostic PCR.

PubMed

Hawash, Yousry

2014-06-01

PCR detection of intestinal protozoa is often restrained by a poor DNA recovery or by inhibitors present in feces. The need for an extraction protocol that can overcome these obstacles is therefore clear. QIAamp® DNA Stool Mini Kit (Qiagen) was evaluated for its ability to recover DNA from oocysts/cysts directly from feces. Twenty-five Giardia-positive, 15 Cryptosporidium-positive, 15 Entamoeba histolytica-positive, and 45 protozoa-free samples were processed as control by microscopy and immunoassay tests. DNA extracts were amplified using 3 sets of published primers. Following the manufacturer's protocol, the kit showed sensitivity and specificity of 100% towards Giardia and Entamoeba. However, for Cryptosporidium, the sensitivity and specificity were 60% (9/15) and 100%, respectively. A series of optimization experiments involving various steps of the kit's protocol were conducted using Cryptosporidium-positive samples. The best DNA recoveries were gained by raising the lysis temperature to the boiling point for 10 min and the incubation time of the InhibitEX tablet to 5 min. Also, using a pre-cooled ethanol for nucleic acid precipitation and small elution volume (50-100 µl) were valuable. The sensitivity of the amended protocol to Cryptosporidium was raised to 100%. Cryptosporidium DNA was successfully amplified by either the first or the second primer set. When applied on parasite-free feces spiked with variable oocysts/cysts counts, ≈ 2 oocysts/cysts were theoretically enough for detection by PCR. To conclude, the Qiagen kit with the amended protocol was proved to be suitable for protozoan DNA extraction directly from feces and support PCR diagnosis.

Sensitive, microliter PCR with consensus degenerate primers for Epstein Barr virus amplification

PubMed Central

Oh, Kyudam; Pak, Nikita; Saunders, D. Curtis; Conrardy, Christina; Landers, James P.; Tong, Suxiang; Forest, Craig R.

2016-01-01

Sensitive identification of the etiology of viral diseases is key to implementing appropriate prevention and treatment. The gold standard for virus identification is the polymerase chain reaction (PCR), a technique that allows for highly specific and sensitive detection of pathogens by exponentially amplifying a specific region of DNA from as little as a single copy through thermocycling a biochemical cocktail. Today, molecular biology laboratories use commercial instruments that operate in 0.5–2 h/analysis using reaction volumes of 5–50 μL contained within polymer tubes or chambers. Towards reducing this volume and maintaining performance, we present a semi-quantitative, systematic experimental study of how PCR yield is affected by tube/chamber substrate, surface-area-to-volume ratio (SA:V), and passivation methods. We perform PCR experiments using traditional PCR tubes as well as using disposable polymer microchips with 1 μL reaction volumes thermocycled using water baths. We report the first oil encapsulation microfluidic PCR method without fluid flow and its application to the first microfluidic amplification of Epstein Barr virus using consensus degenerate primers, a powerful and broad PCR method to screen for both known and novel members of a viral family. The limit of detection is measured as 140 starting copies of DNA from a starting concentration of 3×105 copies/mL, regarded as an accepted sensitivity threshold for diagnostic purposes, and reaction specificity was improved as compared to conventional methods. Also notable, these experiments were conducted with conventional reagent concentrations, rather than commonly spiked enzyme and/or template mixtures. This experimental study of the effects of substrate, SA:V, and passivation, together with sensitive and specific microfluidic PCR with consensus degenerate primers represent advances towards lower cost and higher throughput pathogen screening. PMID:23080522

Direct Multiplex PCR (dmPCR) for the Identification of Six Phlebotomine Sand Fly Species (Diptera: Psychodidae), Including Major Leishmania Vectors of the Mediterranean.

PubMed

Giantsis, Ioannis A; Chaskopoulou, Alexandra; Claude Bon, Marie

2017-02-01

Sand flies (Diptera: Psychodidae, subfamily Phlebotominae) are hematophagous insects that are known to transmit several anthroponotic and zoonotic diseases. Reliable identification of sand flies at species level is crucial for their surveillance, the detection and spread of their pathogens, and the implementation of targeted pest control strategies. Here, we designed a novel, time-saving, cost-effective and easy-to-apply molecular methodology, which avoids sequencing, for the identification of the following six Eastern Mediterranean sand fly species: Phebotomus perfiliewi Parrot, Phebotomus simici Theodor, Phebotomus tobbi Adler and Theodor, Phebotomus papatasi Scopoli, Sergentomyia dentata Sinton, and Sergentomyia minuta Theodor. This methodology, which is a multiplex PCR assay using one common and six diagnostic primers, is based on species-specific single-nucleotide polymorphisms of the nuclear 18S rRNA gene. Amplification products were easily and reliably separated in agarose gel yielding one single clear band of diagnostic size for each species. Further, we verified its successful application on tissue samples that were immersed directly to the PCR mix, skipping DNA extraction. The direct multiplex PCR can be completed in < 3 h, including all operating procedures, and costing no more than a simple PCR. The applicability of this methodology in the detection of hybrids is an additional considerable benefit. © The Authors 2016. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Targeted RNA-Sequencing with Competitive Multiplex-PCR Amplicon Libraries

PubMed Central

Blomquist, Thomas M.; Crawford, Erin L.; Lovett, Jennie L.; Yeo, Jiyoun; Stanoszek, Lauren M.; Levin, Albert; Li, Jia; Lu, Mei; Shi, Leming; Muldrew, Kenneth; Willey, James C.

2013-01-01

Whole transcriptome RNA-sequencing is a powerful tool, but is costly and yields complex data sets that limit its utility in molecular diagnostic testing. A targeted quantitative RNA-sequencing method that is reproducible and reduces the number of sequencing reads required to measure transcripts over the full range of expression would be better suited to diagnostic testing. Toward this goal, we developed a competitive multiplex PCR-based amplicon sequencing library preparation method that a) targets only the sequences of interest and b) controls for inter-target variation in PCR amplification during library preparation by measuring each transcript native template relative to a known number of synthetic competitive template internal standard copies. To determine the utility of this method, we intentionally selected PCR conditions that would cause transcript amplification products (amplicons) to converge toward equimolar concentrations (normalization) during library preparation. We then tested whether this approach would enable accurate and reproducible quantification of each transcript across multiple library preparations, and at the same time reduce (through normalization) total sequencing reads required for quantification of transcript targets across a large range of expression. We demonstrate excellent reproducibility (R2 = 0.997) with 97% accuracy to detect 2-fold change using External RNA Controls Consortium (ERCC) reference materials; high inter-day, inter-site and inter-library concordance (R2 = 0.97–0.99) using FDA Sequencing Quality Control (SEQC) reference materials; and cross-platform concordance with both TaqMan qPCR (R2 = 0.96) and whole transcriptome RNA-sequencing following “traditional” library preparation using Illumina NGS kits (R2 = 0.94). Using this method, sequencing reads required to accurately quantify more than 100 targeted transcripts expressed over a 107-fold range was reduced more than 10,000-fold, from 2.3×109 to 1

Evaluation of TaqMan qPCR System Integrating Two Identically Labelled Hydrolysis Probes in Single Assay

PubMed Central

Nagy, Alexander; Vitásková, Eliška; Černíková, Lenka; Křivda, Vlastimil; Jiřincová, Helena; Sedlák, Kamil; Horníčková, Jitka; Havlíčková, Martina

2017-01-01

Ongoing evolution of viral pathogens is a significant issue in diagnostic virology employing TaqMan qPCR/RT-qPCR. Specific concerns are related to false negativity due to probe binding failure. One option for compensating for such deficiency is to integrate a second identically labelled probe in the assay. However, how this alteration influences the reaction parameters has not been comprehensively demonstrated. In the present study, we evaluate a TaqMan protocol using two identically labelled hydrolysis probes (simple, LNA (locked-nucleic-acid)) and MGB (minor-groove-binder) modified probes and combinations thereof in a single assay. Our results based on a synthetic amplicon suggest that the second probe does not compromise the TaqMan qPCR/RT-qPCR parameters, which repeatedly and reproducibly remained comparable to those of the corresponding single-probe assays, irrespective of the relative probe orientation, whether opposite or tandem, and probe modifications or combinations thereof. On the other hand, the second probe additively contributed to the overall fluorescence signal. The utility of the dual-probe approach was demonstrated on practical examples by using field specimens. We hope that the present study might serve as a theoretical basis for the development or improvement of TaqMan qPCR/RT-qPCR assays for the detection of highly variable nucleic acid templates. PMID:28120891

The Utility of Malaria Rapid Diagnostic Tests as a Tool in Enhanced Surveillance for Malaria Elimination in Vanuatu

PubMed Central

Guintran, Jean-Olivier; Iata, Harry; Anderson, Karen; Nausien, Johnny; Gresty, Karryn J; Waters, Norman C.; Vestergaard, Lasse S.; Taleo, George; Cheng, Qin

2016-01-01

Background As part of efforts to eliminate malaria, Vanuatu has piloted the implementation of enhanced malaria surveillance and response strategies since 2011. This involves passive case detection (PCD) in health facilities, proactive case detection (Pro-ACD) and reactive case detection (Re-ACD) in communities using malaria rapid diagnostic tests (RDTs). While RDTs improve case management, their utility for detection of malaria infections in ACDs in this setting is unclear. Methods The utility of malaria RDTs as diagnostic tools was evaluated in PCD, in five rounds of Pro-ACDs and five rounds of Re-ACDs conducted in Tafea and Torba Provinces between 2011 and 2014. The number of malaria infections detected by RDTs was compared to that detected by PCR from collected used-RDTs. Results PCD in Tafea Province (2013) showed a RDT-positive rate of 0.21% (2/939) and a PCR-positive rate of 0.44% (2/453), indicating less than 1% of suspected malaria cases in Tafea Province were due to malaria. In Pro-ACDs conducted in Tafea and Torba Provinces, RDT-positive rates in 2013 and 2014 were 0.14% (3/2145) and 0% (0/2823), respectively, while the corresponding PCR-positive rates were 0.72% (9/1242) and 0.79% (9/1141). PCR identified villages in both provinces appearing to be transmission foci with a small number of low-density infections, mainly P. falciparum infections. In five rounds of Re-ACD, RDTs did not identify any additional infections while PCR detected only one among 173 subjects screened. Conclusions PCD and Pro-ACDs demonstrate that both Tafea and Torba Provinces in Vanuatu has achieved very low malaria prevalence. In these low-transmission areas, conducting Pro-ACD and Re-ACDs using RDTs appears not cost-effective and may have limited impact on interrupting malaria transmission due to the small number of infections identified by RDTs and considerable operational resources invested. More sensitive, field deployable and affordable diagnostic tools will improve malaria

PCR diagnostics and monitoring of adenoviral infections in hematopoietic stem cell transplantation recipients

PubMed Central

Ussowicz, Marek; Rybka, Blanka; Wendycz-Domalewska, Danuta; Ryczan, Renata; Gorczyńska, Ewa; Kałwak, Krzysztof; Woźniak, Mieczysław

2010-01-01

After stem cell transplantation, human patients are prone to life-threatening opportunistic infections with a plethora of microorganisms. We report a retrospective study on 116 patients (98 children, 18 adults) who were transplanted in a pediatric bone marrow transplantation unit. Blood, urine and stool samples were collected and monitored for adenovirus (AdV) DNA using polymerase chain reaction (PCR) and real-time PCR (RT-PCR) on a regular basis. AdV DNA was detected in 52 (44.8%) patients, with mortality reaching 19% in this subgroup. Variables associated with adenovirus infection were transplantations from matched unrelated donors and older age of the recipient. An increased seasonal occurrence of adenoviral infections was observed in autumn and winter. Analysis of immune reconstitution showed a higher incidence of AdV infections during periods of low T-lymphocyte count. This study also showed a strong interaction between co-infections of AdV and BK polyomavirus in patients undergoing hematopoietic stem cell transplantations. PMID:20848295

Evaluation of digital real-time PCR assay as a molecular diagnostic tool for single-cell analysis.

PubMed

Chang, Chia-Hao; Mau-Hsu, Daxen; Chen, Ke-Cheng; Wei, Cheng-Wey; Chiu, Chiung-Ying; Young, Tai-Horng

2018-02-21

In a single-cell study, isolating and identifying single cells are essential, but these processes often require a large investment of time or money. The aim of this study was to isolate and analyse single cells using a novel platform, the PanelChip™ Analysis System, which includes 2500 microwells chip and a digital real-time polymerase chain reaction (dqPCR) assay, in comparison with a standard PCR (qPCR) assay. Through the serial dilution of a known concentration standard, namely pUC19, the accuracy and sensitivity levels of two methodologies were compared. The two systems were tested on the basis of expression levels of the genetic markers vimentin, E-cadherin, N-cadherin and GAPDH in A549 lung carcinoma cells at two known concentrations. Furthermore, the influence of a known PCR inhibitor commonly found in blood samples, heparin, was evaluated in both methodologies. Finally, mathematical models were proposed and separation method of single cells was verified; moreover, gene expression levels during epithelial-mesenchymal transition in single cells under TGFβ1 treatment were measured. The drawn conclusion is that dqPCR performed using PanelChip™ is superior to the standard qPCR in terms of sensitivity, precision, and heparin tolerance. The dqPCR assay is a potential tool for clinical diagnosis and single-cell applications.

Molecular detection of Plasmodium knowlesi in a Dutch traveler by real-time PCR.

PubMed

Link, Lonneke; Bart, Aldert; Verhaar, Nienke; van Gool, Tom; Pronk, Marjolijn; Scharnhorst, Volkher

2012-07-01

Plasmodium knowlesi infection with low parasitemia presents a diagnostic challenge, as rapid diagnostic tests are often negative and identification to the species level by microscopy is difficult. P. knowlesi malaria in a traveler is described, and real-time PCR is demonstrated to support fast and reliable diagnosis and identification to the species level.

Design and verification of a highly reliable Linear-After-The-Exponential PCR (LATE-PCR) assay for the detection of African swine fever virus.

PubMed

Ronish, B; Hakhverdyan, M; Ståhl, K; Gallardo, C; Fernandez-Pinero, J; Belák, S; Leblanc, N; Wangh, L

2011-03-01

African swine fever virus (ASFV) is a highly pathogenic DNA virus that is the causative agent of African swine fever (ASF), an infectious disease of domestic and wild pigs of all breeds and ages, causing a range of syndromes. Acute disease is characterized by high fever, haemorrhages in the reticuloendothelial system, and a high mortality rate. A powerful novel diagnostic assay based on the Linear-After-The-Exponential-PCR (LATE-PCR) principle was developed to detect ASFV. LATE-PCR is an advanced form of asymmetric PCR which results in direct amplification of large amount of single-stranded DNA. Fluorescent readings are acquired using endpoint analysis after PCR amplification. Amplification of the correct product is verified by melting curve analysis. The assay was designed to amplify the VP72 gene of ASFV genome. Nineteen ASFV DNA cell culture virus strains and three tissue samples (spleen, tonsil, and liver) from infected experimental pigs were tested. Virus was detected in all of the cell culture and tissue samples. None of five ASFV-related viruses tested produced a positive signal, demonstrating the high specificity of the assay. The sensitivity of the LATE-PCR assay was determined in two separate real-time monoplex reactions using samples of synthetic ASFV and synthetic control-DNA targets that were diluted serially from 10⁹ to 1 initial copies per reaction. The detection limit was 1 and 10 copies/reaction, respectively. The sensitivity of the assay was also tested in a duplex end-point reactions comprised of a constant level of 150 copies of synthetic control-DNA and a clinical sample of spleen tissue diluted serially from 10⁻¹ to 10⁻⁵. The detection limit was 10⁻⁵ dilution which corresponds to approximately 1 copy/reaction. Since the assay is designed to be used in either laboratory settings or in a portable PCR machine (Bio-Seeq Portable Veterinary Diagnostics Laboratory; Smiths Detection, Watford UK), the LATE-PCR provides a robust and novel

Multiplex Amplification Refractory Mutation System PCR (ARMS-PCR) provides sequencing independent typing of canine parvovirus.

PubMed

Chander, Vishal; Chakravarti, Soumendu; Gupta, Vikas; Nandi, Sukdeb; Singh, Mithilesh; Badasara, Surendra Kumar; Sharma, Chhavi; Mittal, Mitesh; Dandapat, S; Gupta, V K

2016-12-01

Canine parvovirus-2 antigenic variants (CPV-2a, CPV-2b and CPV-2c) ubiquitously distributed worldwide in canine population causes severe fatal gastroenteritis. Antigenic typing of CPV-2 remains a prime focus of research groups worldwide in understanding the disease epidemiology and virus evolution. The present study was thus envisioned to provide a simple sequencing independent, rapid, robust, specific, user-friendly technique for detecting and typing of presently circulating CPV-2 antigenic variants. ARMS-PCR strategy was employed using specific primers for CPV-2a, CPV-2b and CPV-2c to differentiate these antigenic types. ARMS-PCR was initially optimized with reference positive controls in two steps; where first reaction was used to differentiate CPV-2a from CPV-2b/CPV-2c. The second reaction was carried out with CPV-2c specific primers to confirm the presence of CPV-2c. Initial validation of the ARMS-PCR was carried out with 24 sequenced samples and the results were matched with the sequencing results. ARMS-PCR technique was further used to screen and type 90 suspected clinical samples. Randomly selected 15 suspected clinical samples that were typed with this technique were sequenced. The results of ARMS-PCR and the sequencing matched exactly with each other. The developed technique has a potential to become a sequencing independent method for simultaneous detection and typing of CPV-2 antigenic variants in veterinary disease diagnostic laboratories globally. Copyright © 2016 Elsevier B.V. All rights reserved.

Stabilization of biothreat diagnostic samples through vitrification matrices.

PubMed

Minogue, Timothy Devin; Kalina, Warren Vincent; Coyne, Susan Rajnik

2014-06-01

Diagnostics for biothreat agents require sample shipment to reference labs for diagnosis of disease; however high/fluctuating temperatures during sample transport negatively affect sample quality and results. Vitrification additives preserve sample integrity for molecular-based assay diagnostics in the absence of refrigeration by imparting whole molecule stability to a plethora of environmental insults. Therefore, we have evaluated commercially available vitrification matrices' (Biomatrica's CloneStable® and RNAStable®) ability to stabilize samples of Yersinia pestis and Venezuelan Equine Encephalitis Virus. When heated to 95°C in RNAStable®, Y. pestis had a 13-fold improvement in detection via real-time PCR compared to heated samples in buffer. VEEV, in RNAStable® at 55°C, had a ~10-fold improved detection versus heated samples in buffer. CloneStable® also preserved Y. pestis antigens for 7days after exposure to cycling temperatures. Overall, RNAStable® and CloneStable® respectively offered superior stabilization to nucleic acids and proteins in response to temperature fluctuations. Copyright © 2014. Published by Elsevier B.V.

Solar thermal polymerase chain reaction for smartphone-assisted molecular diagnostics.

PubMed

Jiang, Li; Mancuso, Matthew; Lu, Zhengda; Akar, Gunkut; Cesarman, Ethel; Erickson, David

2014-02-20

Nucleic acid-based diagnostic techniques such as polymerase chain reaction (PCR) are used extensively in medical diagnostics due to their high sensitivity, specificity and quantification capability. In settings with limited infrastructure and unreliable electricity, however, access to such devices is often limited due to the highly specialized and energy-intensive nature of the thermal cycling process required for nucleic acid amplification. Here we integrate solar heating with microfluidics to eliminate thermal cycling power requirements as well as create a simple device infrastructure for PCR. Tests are completed in less than 30 min, and power consumption is reduced to 80 mW, enabling a standard 5.5 Wh iPhone battery to provide 70 h of power to this system. Additionally, we demonstrate a complete sample-to-answer diagnostic strategy by analyzing human skin biopsies infected with Kaposi's Sarcoma herpesvirus (KSHV/HHV-8) through the combination of solar thermal PCR, HotSHOT DNA extraction and smartphone-based fluorescence detection. We believe that exploiting the ubiquity of solar thermal energy as demonstrated here could facilitate broad availability of nucleic acid-based diagnostics in resource-limited areas.

Solar thermal polymerase chain reaction for smartphone-assisted molecular diagnostics

PubMed Central

Jiang, Li; Mancuso, Matthew; Lu, Zhengda; Akar, Gunkut; Cesarman, Ethel; Erickson, David

2014-01-01

Nucleic acid-based diagnostic techniques such as polymerase chain reaction (PCR) are used extensively in medical diagnostics due to their high sensitivity, specificity and quantification capability. In settings with limited infrastructure and unreliable electricity, however, access to such devices is often limited due to the highly specialized and energy-intensive nature of the thermal cycling process required for nucleic acid amplification. Here we integrate solar heating with microfluidics to eliminate thermal cycling power requirements as well as create a simple device infrastructure for PCR. Tests are completed in less than 30 min, and power consumption is reduced to 80 mW, enabling a standard 5.5 Wh iPhone battery to provide 70 h of power to this system. Additionally, we demonstrate a complete sample-to-answer diagnostic strategy by analyzing human skin biopsies infected with Kaposi's Sarcoma herpesvirus (KSHV/HHV-8) through the combination of solar thermal PCR, HotSHOT DNA extraction and smartphone-based fluorescence detection. We believe that exploiting the ubiquity of solar thermal energy as demonstrated here could facilitate broad availability of nucleic acid-based diagnostics in resource-limited areas. PMID:24553130

Solar thermal polymerase chain reaction for smartphone-assisted molecular diagnostics

NASA Astrophysics Data System (ADS)

Jiang, Li; Mancuso, Matthew; Lu, Zhengda; Akar, Gunkut; Cesarman, Ethel; Erickson, David

2014-02-01

Nucleic acid-based diagnostic techniques such as polymerase chain reaction (PCR) are used extensively in medical diagnostics due to their high sensitivity, specificity and quantification capability. In settings with limited infrastructure and unreliable electricity, however, access to such devices is often limited due to the highly specialized and energy-intensive nature of the thermal cycling process required for nucleic acid amplification. Here we integrate solar heating with microfluidics to eliminate thermal cycling power requirements as well as create a simple device infrastructure for PCR. Tests are completed in less than 30 min, and power consumption is reduced to 80 mW, enabling a standard 5.5 Wh iPhone battery to provide 70 h of power to this system. Additionally, we demonstrate a complete sample-to-answer diagnostic strategy by analyzing human skin biopsies infected with Kaposi's Sarcoma herpesvirus (KSHV/HHV-8) through the combination of solar thermal PCR, HotSHOT DNA extraction and smartphone-based fluorescence detection. We believe that exploiting the ubiquity of solar thermal energy as demonstrated here could facilitate broad availability of nucleic acid-based diagnostics in resource-limited areas.

Towards Modernizing the Characterization of Shock and Detonation Physics Performance via Novel Diagnostics and Tests

NASA Astrophysics Data System (ADS)

Salyer, Terry

2017-06-01

For the bulk of detonation performance experiments, a fairly basic set of diagnostic techniques has evolved as the standard for acquiring the necessary measurements. Gold standard techniques such as pin switches and streak cameras still produce the high-quality data required, yet much room remains for improvement with regard to ease of use, cost of fielding, breadth of data, and diagnostic versatility. Over the past several years, an alternate set of diagnostics has been under development to replace many of these traditional techniques. Pulse Correlation Reflectometry (PCR) is a capable substitute for pin switches with the advantage of obtaining orders of magnitude more data at a small fraction of the cost and fielding time. Spectrally Encoded Imaging (SEI) can replace most applications of streak camera with the advantage of imaging surfaces through a single optical fiber that are otherwise optically inaccessible. Such diagnostics advance the measurement state of the art, but even further improvements may come through revamping the standardized tests themselves such as the copper cylinder expansion test. At the core of this modernization, the aforementioned diagnostics play a significant role in revamping and improving the standard test suite for the present era. This research was performed under the auspices of the United States Department of Energy.

ddpcr: an R package and web application for analysis of droplet digital PCR data.

PubMed

Attali, Dean; Bidshahri, Roza; Haynes, Charles; Bryan, Jennifer

2016-01-01

Droplet digital polymerase chain reaction (ddPCR) is a novel platform for exact quantification of DNA which holds great promise in clinical diagnostics. It is increasingly popular due to its digital nature, which provides more accurate quantification and higher sensitivity than traditional real-time PCR. However, clinical adoption has been slowed in part by the lack of software tools available for analyzing ddPCR data. Here, we present ddpcr - a new R package for ddPCR visualization and analysis. In addition, ddpcr includes a web application (powered by the Shiny R package) that allows users to analyze ddPCR data using an interactive graphical interface.

A Unique Capsule Locus in the Newly Designated Actinobacillus pleuropneumoniae Serovar 16 and Development of a Diagnostic PCR Assay.

PubMed

Bossé, Janine T; Li, Yanwen; Sárközi, Rita; Gottschalk, Marcelo; Angen, Øystein; Nedbalcova, Katerina; Rycroft, Andrew N; Fodor, László; Langford, Paul R

2017-03-01

Actinobacillus pleuropneumoniae causes pleuropneumonia, an economically significant lung disease of pigs. Recently, isolates of A. pleuropneumoniae that were serologically distinct from the previously characterized 15 serovars were described, and a proposal was put forward that they comprised a new serovar, serovar 16. Here we used whole-genome sequencing of the proposed serovar 16 reference strain A-85/14 to confirm the presence of a unique capsular polysaccharide biosynthetic locus. For molecular diagnostics, primers were designed from the capsule locus of strain A-85/14, and a PCR was formulated that differentiated serovar 16 isolates from all 15 known serovars and other common respiratory pathogenic/commensal bacteria of pigs. Analysis of the capsule locus of strain A-85/14 combined with the previous serological data show the existence of a sixteenth serovar-designated serovar 16-of A. pleuropneumoniae . Copyright © 2017 Bossé et al.

Detection of malaria infection in blood transfusion: a comparative study among real-time PCR, rapid diagnostic test and microscopy: sensitivity of Malaria detection methods in blood transfusion.

PubMed

Hassanpour, Gholamreza; Mohebali, Mehdi; Raeisi, Ahmad; Abolghasemi, Hassan; Zeraati, Hojjat; Alipour, Mohsen; Azizi, Ebrahim; Keshavarz, Hossein

2011-06-01

The transmission of malaria by blood transfusion was one of the first transfusion-transmitted infections recorded in the world. Transfusion-transmitted malaria may lead to serious problems because infection with Plasmodium falciparum may cause rapidly fatal death. This study aimed to compare real-time polymerase chain reaction (real-time PCR) with rapid diagnostic test (RDT) and light microscopy for the detection of Plasmodium spp. in blood transfusion, both in endemic and non-endemic areas of malaria disease in Iran. Two sets of 50 blood samples were randomly collected. One set was taken from blood samples donated in blood bank of Bandar Abbas, a city located in a malarious-endemic area, and the other set from Tehran, a non-endemic one. Light microscopic examination on both thin and thick smears, RDTs, and real-time PCR were performed on the blood samples and the results were compared. Thin and thick light microscopic examinations of all samples as well as RDT results were negative for Plasmodium spp. Two blood samples from endemic area were positive only with real-time PCR. It seems that real-time PCR as a highly sensitive method can be helpful for the confirmation of malaria infection in different units of blood transfusion organization especially in malaria-endemic areas where the majority of donors may be potentially infected with malaria parasites.

Development and validation of a real-time PCR assay for the detection of anguillid herpesvirus 1.

PubMed

van Beurden, S J; Voorbergen-Laarman, M A; Roozenburg, I; van Tellingen, J; Haenen, O L M; Engelsma, M Y

2016-01-01

Anguillid herpesvirus 1 (AngHV1) causes a haemorrhagic disease with increased mortality in wild and farmed European eel, Anguilla anguilla (L.) and Japanese eel Anguilla japonica, Temminck & Schlegel). Detection of AngHV1 is currently based on virus isolation in cell culture, antibody-based typing assays or conventional PCR. We developed, optimized and concisely validated a diagnostic TaqMan probe based real-time PCR assay for the detection of AngHV1. The primers and probe target AngHV1 open reading frame 57, encoding the capsid protease and scaffold protein. Compared to conventional PCR, the developed real-time PCR is faster, less labour-intensive and has a reduced risk of cross-contamination. The real-time PCR assay was shown to be analytically sensitive and specific and has a high repeatability, efficiency and r(2) -value. The diagnostic performance of the assay was determined by testing 10% w/v organ suspensions and virus cultures from wild and farmed European eels from the Netherlands by conventional and real-time PCR. The developed real-time PCR assay is a useful tool for the rapid and sensitive detection of AngHV1 in 10% w/v organ suspensions from wild and farmed European eels. © 2015 John Wiley & Sons Ltd.

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.

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.

A novel PCR-based system for the detection of four species of human malaria parasites and Plasmodium knowlesi

PubMed Central

Komaki-Yasuda, Kanako; Vincent, Jeanne Perpétue; Nakatsu, Masami; Kato, Yasuyuki; Ohmagari, Norio

2018-01-01

A microscopy-based diagnosis is the gold standard for the detection and identification of malaria parasites in a patient’s blood. However, the detection of cases involving a low number of parasites and the differentiation of species sometimes requires a skilled microscopist. Although PCR-based diagnostic methods are already known to be very powerful tools, the time required to apply such methods is still much longer in comparison to traditional microscopic observation. Thus, improvements to PCR systems are sought to facilitate the more rapid and accurate detection of human malaria parasites Plasmodium falciparum, P. vivax, P. ovale, and P. malariae, as well as P. knowlesi, which is a simian malaria parasite that is currently widely distributed in Southeast Asia. A nested PCR that targets the small subunit ribosomal RNA genes of malaria parasites was performed using a “fast PCR enzyme”. In the first PCR, universal primers for all parasite species were used. In the second PCR, inner-specific primers, which targeted sequences from P. falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi, were used. The PCR reaction time was reduced with the use of the “fast PCR enzyme”, with only 65 minutes required to perform the first and second PCRs. The specific primers only reacted with the sequences of their targeted parasite species and never cross-reacted with sequences from other species under the defined PCR conditions. The diagnoses of 36 clinical samples that were obtained using this new PCR system were highly consistent with the microscopic diagnoses. PMID:29370297

A novel PCR-based system for the detection of four species of human malaria parasites and Plasmodium knowlesi.

PubMed

Komaki-Yasuda, Kanako; Vincent, Jeanne Perpétue; Nakatsu, Masami; Kato, Yasuyuki; Ohmagari, Norio; Kano, Shigeyuki

2018-01-01

A microscopy-based diagnosis is the gold standard for the detection and identification of malaria parasites in a patient's blood. However, the detection of cases involving a low number of parasites and the differentiation of species sometimes requires a skilled microscopist. Although PCR-based diagnostic methods are already known to be very powerful tools, the time required to apply such methods is still much longer in comparison to traditional microscopic observation. Thus, improvements to PCR systems are sought to facilitate the more rapid and accurate detection of human malaria parasites Plasmodium falciparum, P. vivax, P. ovale, and P. malariae, as well as P. knowlesi, which is a simian malaria parasite that is currently widely distributed in Southeast Asia. A nested PCR that targets the small subunit ribosomal RNA genes of malaria parasites was performed using a "fast PCR enzyme". In the first PCR, universal primers for all parasite species were used. In the second PCR, inner-specific primers, which targeted sequences from P. falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi, were used. The PCR reaction time was reduced with the use of the "fast PCR enzyme", with only 65 minutes required to perform the first and second PCRs. The specific primers only reacted with the sequences of their targeted parasite species and never cross-reacted with sequences from other species under the defined PCR conditions. The diagnoses of 36 clinical samples that were obtained using this new PCR system were highly consistent with the microscopic diagnoses.

Steps to achieve quantitative measurements of microRNA using two step droplet digital PCR.

PubMed

Stein, Erica V; Duewer, David L; Farkas, Natalia; Romsos, Erica L; Wang, Lili; Cole, Kenneth D

2017-01-01

Droplet digital PCR (ddPCR) is being advocated as a reference method to measure rare genomic targets. It has consistently been proven to be more sensitive and direct at discerning copy numbers of DNA than other quantitative methods. However, one of the largest obstacles to measuring microRNA (miRNA) using ddPCR is that reverse transcription efficiency depends upon the target, meaning small RNA nucleotide composition directly effects primer specificity in a manner that prevents traditional quantitation optimization strategies. Additionally, the use of reagents that are optimized for miRNA measurements using quantitative real-time PCR (qRT-PCR) appear to either cause false positive or false negative detection of certain targets when used with traditional ddPCR quantification methods. False readings are often related to using inadequate enzymes, primers and probes. Given that two-step miRNA quantification using ddPCR relies solely on reverse transcription and uses proprietary reagents previously optimized only for qRT-PCR, these barriers are substantial. Therefore, here we outline essential controls, optimization techniques, and an efficacy model to improve the quality of ddPCR miRNA measurements. We have applied two-step principles used for miRNA qRT-PCR measurements and leveraged the use of synthetic miRNA targets to evaluate ddPCR following cDNA synthesis with four different commercial kits. We have identified inefficiencies and limitations as well as proposed ways to circumvent identified obstacles. Lastly, we show that we can apply these criteria to a model system to confidently quantify miRNA copy number. Our measurement technique is a novel way to quantify specific miRNA copy number in a single sample, without using standard curves for individual experiments. Our methodology can be used for validation and control measurements, as well as a diagnostic technique that allows scientists, technicians, clinicians, and regulators to base miRNA measures on a single

Steps to achieve quantitative measurements of microRNA using two step droplet digital PCR

PubMed Central

Duewer, David L.; Farkas, Natalia; Romsos, Erica L.; Wang, Lili; Cole, Kenneth D.

2017-01-01

Droplet digital PCR (ddPCR) is being advocated as a reference method to measure rare genomic targets. It has consistently been proven to be more sensitive and direct at discerning copy numbers of DNA than other quantitative methods. However, one of the largest obstacles to measuring microRNA (miRNA) using ddPCR is that reverse transcription efficiency depends upon the target, meaning small RNA nucleotide composition directly effects primer specificity in a manner that prevents traditional quantitation optimization strategies. Additionally, the use of reagents that are optimized for miRNA measurements using quantitative real-time PCR (qRT-PCR) appear to either cause false positive or false negative detection of certain targets when used with traditional ddPCR quantification methods. False readings are often related to using inadequate enzymes, primers and probes. Given that two-step miRNA quantification using ddPCR relies solely on reverse transcription and uses proprietary reagents previously optimized only for qRT-PCR, these barriers are substantial. Therefore, here we outline essential controls, optimization techniques, and an efficacy model to improve the quality of ddPCR miRNA measurements. We have applied two-step principles used for miRNA qRT-PCR measurements and leveraged the use of synthetic miRNA targets to evaluate ddPCR following cDNA synthesis with four different commercial kits. We have identified inefficiencies and limitations as well as proposed ways to circumvent identified obstacles. Lastly, we show that we can apply these criteria to a model system to confidently quantify miRNA copy number. Our measurement technique is a novel way to quantify specific miRNA copy number in a single sample, without using standard curves for individual experiments. Our methodology can be used for validation and control measurements, as well as a diagnostic technique that allows scientists, technicians, clinicians, and regulators to base miRNA measures on a single

A Pentaplex PCR Assay for the Detection and Differentiation of Shigella Species

PubMed Central

Ojha, Suvash Chandra; Yean Yean, Chan; Ismail, Asma; Banga Singh, Kirnpal-Kaur

2013-01-01

The magnitude of shigellosis in developing countries is largely unknown because an affordable detection method is not available. Current laboratory diagnosis of Shigella spp. is laborious and time consuming and has low sensitivity. Hence, in the present study, a molecular-based diagnostic assay which amplifies simultaneously four specific genes to identify invC for Shigella genus, rfc for S. flexneri, wbgZ for S. sonnei, and rfpB for S. dysenteriae, as well as one internal control (ompA) gene, was developed in a single reaction to detect and differentiate Shigella spp. Validation with 120 Shigella strains and 37 non-Shigella strains yielded 100% specificity. The sensitivity of the PCR was 100 pg of genomic DNA, 5.4 × 104 CFU/ml, or approximately 120 CFU per reaction mixture of bacteria. The sensitivity of the pentaplex PCR assay was further improved following preincubation of the stool samples in Gram-negative broth. A preliminary study with 30 diarrhoeal specimens resulted in no cross-reaction with other non-Shigella strains tested. We conclude that the developed pentaplex PCR assay is robust and can provide information about the four target genes that are essential for the identification of the Shigella genus and the three Shigella species responsible for the majority of shigellosis cases. PMID:23509722

Diagnostic efficacy of microscopy, rapid diagnostic test and polymerase chain reaction for malaria using bayesian latent class analysis.

PubMed

Saha, Sreemanti; Narang, Rahul; Deshmukh, Pradeep; Pote, Kiran; Anvikar, Anup; Narang, Pratibha

2017-01-01

The diagnostic techniques for malaria are undergoing a change depending on the availability of newer diagnostics and annual parasite index of infection in a particular area. At the country level, guidelines are available for selection of diagnostic tests; however, at the local level, this decision is made based on malaria situation in the area. The tests are evaluated against the gold standard, and if that standard has limitations, it becomes difficult to compare other available tests. Bayesian latent class analysis computes its internal standard rather than using the conventional gold standard and helps comparison of various tests including the conventional gold standard. In a cross-sectional study conducted in a tertiary care hospital setting, we have evaluated smear microscopy, rapid diagnostic test (RDT), and polymerase chain reaction (PCR) for diagnosis of malaria using Bayesian latent class analysis. We found the magnitude of malaria to be 17.7% (95% confidence interval: 12.5%-23.9%) among the study subjects. In the present study, the sensitivity of microscopy was 63%, but it had very high specificity (99.4%). Sensitivity and specificity of RDT and PCR were high with RDT having a marginally higher sensitivity (94% vs. 90%) and specificity (99% vs. 95%). On comparison of likelihood ratios (LRs), RDT had the highest LR for positive test result (175) and the lowest LR for negative test result (0.058) among the three tests. In settings like ours conventional smear microscopy may be replaced with RDT and as we move toward elimination and facilities become available PCR may be roped into detect cases with lower parasitaemia.

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.

Single-Cell Quantitative PCR: Advances and Potential in Cancer Diagnostics.

PubMed

Ok, Chi Young; Singh, Rajesh R; Salim, Alaa A

2016-01-01

Tissues are heterogeneous in their components. If cells of interest are a minor population of collected tissue, it would be difficult to obtain genetic or genomic information of the interested cell population with conventional genomic DNA extraction from the collected tissue. Single-cell DNA analysis is important in the analysis of genetics of cell clonality, genetic anticipation, and single-cell DNA polymorphisms. Single-cell PCR using Single Cell Ampligrid/GeXP platform is described in this chapter.

A Pan-Lyssavirus Taqman Real-Time RT-PCR Assay for the Detection of Highly Variable Rabies virus and Other Lyssaviruses

PubMed Central

Wadhwa, Ashutosh; Wilkins, Kimberly; Gao, Jinxin; Condori Condori, Rene Edgar; Gigante, Crystal M.; Zhao, Hui; Ma, Xiaoyue; Ellison, James A.; Greenberg, Lauren; Velasco-Villa, Andres; Orciari, Lillian

2017-01-01

Rabies, resulting from infection by Rabies virus (RABV) and related lyssaviruses, is one of the most deadly zoonotic diseases and is responsible for up to 70,000 estimated human deaths worldwide each year. Rapid and accurate laboratory diagnosis of rabies is essential for timely administration of post-exposure prophylaxis in humans and control of the disease in animals. Currently, only the direct fluorescent antibody (DFA) test is recommended for routine rabies diagnosis. Reverse-transcription polymerase chain reaction (RT-PCR) based diagnostic methods have been widely adapted for the diagnosis of other viral pathogens, but there is currently no widely accepted rapid real-time RT-PCR assay for the detection of all lyssaviruses. In this study, we demonstrate the validation of a newly developed multiplex real-time RT-PCR assay named LN34, which uses a combination of degenerate primers and probes along with probe modifications to achieve superior coverage of the Lyssavirus genus while maintaining sensitivity and specificity. The primers and probes of the LN34 assay target the highly conserved non-coding leader region and part of the nucleoprotein (N) coding sequence of the Lyssavirus genome to maintain assay robustness. The probes were further modified by locked nucleotides to increase their melting temperature to meet the requirements for an optimal real-time RT-PCR assay. The LN34 assay was able to detect all RABV variants and other lyssaviruses in a validation panel that included representative RABV isolates from most regions of the world as well as representatives of 13 additional Lyssavirus species. The LN34 assay was successfully used for both ante-mortem and post-mortem diagnosis of over 200 clinical samples as well as field derived surveillance samples. This assay represents a major improvement over previously published rabies specific RT-PCR and real-time RT-PCR assays because of its ability to universally detect RABV and other lyssaviruses, its high

A Pan-Lyssavirus Taqman Real-Time RT-PCR Assay for the Detection of Highly Variable Rabies virus and Other Lyssaviruses.

PubMed

Wadhwa, Ashutosh; Wilkins, Kimberly; Gao, Jinxin; Condori Condori, Rene Edgar; Gigante, Crystal M; Zhao, Hui; Ma, Xiaoyue; Ellison, James A; Greenberg, Lauren; Velasco-Villa, Andres; Orciari, Lillian; Li, Yu

2017-01-01

Rabies, resulting from infection by Rabies virus (RABV) and related lyssaviruses, is one of the most deadly zoonotic diseases and is responsible for up to 70,000 estimated human deaths worldwide each year. Rapid and accurate laboratory diagnosis of rabies is essential for timely administration of post-exposure prophylaxis in humans and control of the disease in animals. Currently, only the direct fluorescent antibody (DFA) test is recommended for routine rabies diagnosis. Reverse-transcription polymerase chain reaction (RT-PCR) based diagnostic methods have been widely adapted for the diagnosis of other viral pathogens, but there is currently no widely accepted rapid real-time RT-PCR assay for the detection of all lyssaviruses. In this study, we demonstrate the validation of a newly developed multiplex real-time RT-PCR assay named LN34, which uses a combination of degenerate primers and probes along with probe modifications to achieve superior coverage of the Lyssavirus genus while maintaining sensitivity and specificity. The primers and probes of the LN34 assay target the highly conserved non-coding leader region and part of the nucleoprotein (N) coding sequence of the Lyssavirus genome to maintain assay robustness. The probes were further modified by locked nucleotides to increase their melting temperature to meet the requirements for an optimal real-time RT-PCR assay. The LN34 assay was able to detect all RABV variants and other lyssaviruses in a validation panel that included representative RABV isolates from most regions of the world as well as representatives of 13 additional Lyssavirus species. The LN34 assay was successfully used for both ante-mortem and post-mortem diagnosis of over 200 clinical samples as well as field derived surveillance samples. This assay represents a major improvement over previously published rabies specific RT-PCR and real-time RT-PCR assays because of its ability to universally detect RABV and other lyssaviruses, its high

Digital PCR methods improve detection sensitivity and measurement precision of low abundance mtDNA deletions.

PubMed

Belmonte, Frances R; Martin, James L; Frescura, Kristin; Damas, Joana; Pereira, Filipe; Tarnopolsky, Mark A; Kaufman, Brett A

2016-04-28

Mitochondrial DNA (mtDNA) mutations are a common cause of primary mitochondrial disorders, and have also been implicated in a broad collection of conditions, including aging, neurodegeneration, and cancer. Prevalent among these pathogenic variants are mtDNA deletions, which show a strong bias for the loss of sequence in the major arc between, but not including, the heavy and light strand origins of replication. Because individual mtDNA deletions can accumulate focally, occur with multiple mixed breakpoints, and in the presence of normal mtDNA sequences, methods that detect broad-spectrum mutations with enhanced sensitivity and limited costs have both research and clinical applications. In this study, we evaluated semi-quantitative and digital PCR-based methods of mtDNA deletion detection using double-stranded reference templates or biological samples. Our aim was to describe key experimental assay parameters that will enable the analysis of low levels or small differences in mtDNA deletion load during disease progression, with limited false-positive detection. We determined that the digital PCR method significantly improved mtDNA deletion detection sensitivity through absolute quantitation, improved precision and reduced assay standard error.

Digital PCR methods improve detection sensitivity and measurement precision of low abundance mtDNA deletions

PubMed Central

Belmonte, Frances R.; Martin, James L.; Frescura, Kristin; Damas, Joana; Pereira, Filipe; Tarnopolsky, Mark A.; Kaufman, Brett A.

2016-01-01

Mitochondrial DNA (mtDNA) mutations are a common cause of primary mitochondrial disorders, and have also been implicated in a broad collection of conditions, including aging, neurodegeneration, and cancer. Prevalent among these pathogenic variants are mtDNA deletions, which show a strong bias for the loss of sequence in the major arc between, but not including, the heavy and light strand origins of replication. Because individual mtDNA deletions can accumulate focally, occur with multiple mixed breakpoints, and in the presence of normal mtDNA sequences, methods that detect broad-spectrum mutations with enhanced sensitivity and limited costs have both research and clinical applications. In this study, we evaluated semi-quantitative and digital PCR-based methods of mtDNA deletion detection using double-stranded reference templates or biological samples. Our aim was to describe key experimental assay parameters that will enable the analysis of low levels or small differences in mtDNA deletion load during disease progression, with limited false-positive detection. We determined that the digital PCR method significantly improved mtDNA deletion detection sensitivity through absolute quantitation, improved precision and reduced assay standard error. PMID:27122135

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.

Improving the molecular diagnosis of Chlamydia psittaci and Chlamydia abortus infection with a species-specific duplex real-time PCR.

PubMed

Opota, Onya; Jaton, Katia; Branley, James; Vanrompay, Daisy; Erard, Veronique; Borel, Nicole; Longbottom, David; Greub, Gilbert

2015-10-01

Chlamydia psittaci and Chlamydia abortus are closely related intracellular bacteria exhibiting different tissue tropism that may cause severe but distinct infection in humans. C. psittaci causes psittacosis, a respiratory zoonotic infection transmitted by birds. C. abortus is an abortigenic agent in small ruminants, which can also colonize the human placenta and lead to foetal death and miscarriage. Infections caused by C. psittaci and C. abortus are underestimated mainly due to diagnosis difficulties resulting from their strict intracellular growth. We developed a duplex real-time PCR to detect and distinguish these two bacteria in clinical samples. The first PCR (PCR1) targeted a sequence of the 16S-23S rRNA operon allowing the detection of both C. psittaci and C. abortus. The second PCR (PCR2) targeted the coding DNA sequence CPSIT_0607 unique to C. psittaci. The two PCRs showed 100 % detection for ≥ 10 DNA copies per reaction (1000 copies ml(- 1)). Using a set of 120 samples, including bacterial reference strains, clinical specimens and infected cell culture material, we monitored 100 % sensitivity and 100 % specificity for the detection of C. psittaci and C. abortus for PCR1. When PCR1 was positive, PCR2 could discriminate C. psittaci from C. abortus with a positive predictive value of 100 % and a negative predictive value of 88 %. In conclusion, this new duplex PCR represents a low-cost and time-saving method with high-throughput potential, expected to improve the routine diagnosis of psittacosis and pregnancy complication in large-scale screening programs and also during outbreaks.

Broad-Range Detection of Microorganisms Directly from Bronchoalveolar Lavage Specimens by PCR/Electrospray Ionization-Mass Spectrometry

PubMed Central

Ullberg, Måns; Lüthje, Petra; Mölling, Paula; Strålin, Kristoffer

2017-01-01

The clinical demand on rapid microbiological diagnostic is constantly increasing. PCR coupled to electrospray ionization-mass spectrometry, PCR/ESI-MS, offers detection and identification of over 750 bacteria and Candida species directly from clinical specimens within 6 hours. In this study, we investigated the clinical performance of the IRIDICA BAC LRT Assay for detection of bacterial pathogens in 121 bronchoalveolar lavage (BAL) samples that were received consecutively at our bacterial laboratory for BAL culture. Commensal or pathogenic microorganisms were detected in 118/121 (98%) BAL samples by PCR/ESI-MS, while in 104/121 (86%) samples by routine culture (P<0.01). Detection of potentially pathogenic microorganisms by PCR/ESI-MS was evaluated in comparison with conventional culture-based or molecular methods. The agreement between positive findings was overall good. Most Staphylococcus aureus-positive PCR/ESI-MS results were confirmed by culture or species-specific PCR (27/33, 82%). The identity of Streptococcus pneumoniae could however be confirmed for only 6/17 (35%) PCR/ESI-MS-positive samples. Non-cultivable and fastidious pathogens, which were not covered by standard culture procedures were readily detected by PCR/ESI-MS, including Legionella pneumophila, Bordetella pertussis, Norcadia species and Mycoplasma pneumoniae. In conclusion, PCR/ESI-MS detected a broad range of potential pathogens with equal or superior sensitivity compared to conventional methods within few hours directly from BAL samples. This novel method might thus provide a relevant tool for diagnostics in critically ill patients. PMID:28085931

Improved DNA hybridization parameters by Twisted Intercalating Nucleic Acid (TINA).

PubMed

Schneider, Uffe Vest

2012-01-01

This thesis establishes oligonucleotide design rules and applications of a novel group of DNA stabilizing molecules collectively called Twisted Intercalating Nucleic Acid - TINA. Three peer-reviewed publications form the basis for the thesis. One publication describes an improved and rapid method for determination of DNA melting points and two publications describe the effects of positioning TINA molecules in parallel triplex helix and antiparallel duplex helix forming DNA structures. The third publication establishes that TINA molecules containing oligonucleotides improve an antiparallel duplex hybridization based capture assay's analytical sensitivity compared to conventionel DNA oligonucleotides. Clinical microbiology is traditionally based on pathogenic microorganisms' culture and serological tests. The introduction of DNA target amplification methods like PCR has improved the analytical sensitivity and total turn around time involved in clinical diagnostics of infections. Due to the relatively weak hybridization between the two strands of double stranded DNA, a number of nucleic acid stabilizing molecules have been developed to improve the sensitivity of DNA based diagnostics through superior binding properties. A short introduction is given to Watson-Crick and Hoogsteen based DNA binding and the derived DNA structures. A number of other nucleic acid stabilizing molecules are described. The stabilizing effect of TINA molecules on different DNA structures is discussed and considered in relation to other nucleic acid stabilizing molecules and in relation to future use of TINA containing oligonucleotides in clinical diagnostics and therapy. In conclusion, design of TINA modified oligonucleotides for antiparallel duplex helixes and parallel triplex helixes follows simple purpose dependent rules. TINA molecules are well suited for improving multiplex PCR assays and can be used as part of novel technologies. Future research should test whether combinations of TINA

Detection of Rare Drug Resistance Mutations by Digital PCR in a Human Influenza A Virus Model System and Clinical Samples

PubMed Central

Bushell, Claire A.; Grant, Paul R.; Cowen, Simon; Gutierrez-Aguirre, Ion; O'Sullivan, Denise M.; Žel, Jana; Milavec, Mojca; Foy, Carole A.; Nastouli, Eleni; Garson, Jeremy A.; Huggett, Jim F.

2015-01-01

Digital PCR (dPCR) is being increasingly used for the quantification of sequence variations, including single nucleotide polymorphisms (SNPs), due to its high accuracy and precision in comparison with techniques such as quantitative PCR (qPCR) and melt curve analysis. To develop and evaluate dPCR for SNP detection using DNA, RNA, and clinical samples, an influenza virus model of resistance to oseltamivir (Tamiflu) was used. First, this study was able to recognize and reduce off-target amplification in dPCR quantification, thereby enabling technical sensitivities down to 0.1% SNP abundance at a range of template concentrations, a 50-fold improvement on the qPCR assay used routinely in the clinic. Second, a method was developed for determining the false-positive rate (background) signal. Finally, comparison of dPCR with qPCR results on clinical samples demonstrated the potential impact dPCR could have on clinical research and patient management by earlier (trace) detection of rare drug-resistant sequence variants. Ultimately this could reduce the quantity of ineffective drugs taken and facilitate early switching to alternative medication when available. In the short term such methods could advance our understanding of microbial dynamics and therapeutic responses in a range of infectious diseases such as HIV, viral hepatitis, and tuberculosis. Furthermore, the findings presented here are directly relevant to other diagnostic areas, such as the detection of rare SNPs in malignancy, monitoring of graft rejection, and fetal screening. PMID:26659206

Detection of Rare Drug Resistance Mutations by Digital PCR in a Human Influenza A Virus Model System and Clinical Samples.

PubMed

Whale, Alexandra S; Bushell, Claire A; Grant, Paul R; Cowen, Simon; Gutierrez-Aguirre, Ion; O'Sullivan, Denise M; Žel, Jana; Milavec, Mojca; Foy, Carole A; Nastouli, Eleni; Garson, Jeremy A; Huggett, Jim F

2016-02-01

Digital PCR (dPCR) is being increasingly used for the quantification of sequence variations, including single nucleotide polymorphisms (SNPs), due to its high accuracy and precision in comparison with techniques such as quantitative PCR (qPCR) and melt curve analysis. To develop and evaluate dPCR for SNP detection using DNA, RNA, and clinical samples, an influenza virus model of resistance to oseltamivir (Tamiflu) was used. First, this study was able to recognize and reduce off-target amplification in dPCR quantification, thereby enabling technical sensitivities down to 0.1% SNP abundance at a range of template concentrations, a 50-fold improvement on the qPCR assay used routinely in the clinic. Second, a method was developed for determining the false-positive rate (background) signal. Finally, comparison of dPCR with qPCR results on clinical samples demonstrated the potential impact dPCR could have on clinical research and patient management by earlier (trace) detection of rare drug-resistant sequence variants. Ultimately this could reduce the quantity of ineffective drugs taken and facilitate early switching to alternative medication when available. In the short term such methods could advance our understanding of microbial dynamics and therapeutic responses in a range of infectious diseases such as HIV, viral hepatitis, and tuberculosis. Furthermore, the findings presented here are directly relevant to other diagnostic areas, such as the detection of rare SNPs in malignancy, monitoring of graft rejection, and fetal screening. Copyright © 2016 Whale et al.

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.

Polymeric LabChip Real-Time PCR as a Point-of-Care-Potential Diagnostic Tool for Rapid Detection of Influenza A/H1N1 Virus in Human Clinical Specimens

PubMed Central

Song, Hyun-Ok; Kim, Je-Hyoung; Ryu, Ho-Sun; Lee, Dong-Hoon; Kim, Sun-Jin; Kim, Deog-Joong; Suh, In Bum; Choi, Du Young; In, Kwang-Ho; Kim, Sung-Woo; Park, Hyun

2012-01-01

It is clinically important to be able to detect influenza A/H1N1 virus using a fast, portable, and accurate system that has high specificity and sensitivity. To achieve this goal, it is necessary to develop a highly specific primer set that recognizes only influenza A viral genes and a rapid real-time PCR system that can detect even a single copy of the viral gene. In this study, we developed and validated a novel fluidic chip-type real-time PCR (LabChip real-time PCR) system that is sensitive and specific for the detection of influenza A/H1N1, including the pandemic influenza strain A/H1N1 of 2009. This LabChip real-time PCR system has several remarkable features: (1) It allows rapid quantitative analysis, requiring only 15 min to perform 30 cycles of real-time PCR. (2) It is portable, with a weight of only 5.5 kg. (3) The reaction cost is low, since it uses disposable plastic chips. (4) Its high efficiency is equivalent to that of commercially available tube-type real-time PCR systems. The developed disposable LabChip is an economic, heat-transferable, light-transparent, and easy-to-fabricate polymeric chip compared to conventional silicon- or glass-based labchip. In addition, our LabChip has large surface-to-volume ratios in micro channels that are required for overcoming time consumed for temperature control during real-time PCR. The efficiency of the LabChip real-time PCR system was confirmed using novel primer sets specifically targeted to the hemagglutinin (HA) gene of influenza A/H1N1 and clinical specimens. Eighty-five human clinical swab samples were tested using the LabChip real-time PCR. The results demonstrated 100% sensitivity and specificity, showing 72 positive and 13 negative cases. These results were identical to those from a tube-type real-time PCR system. This indicates that the novel LabChip real-time PCR may be an ultra-fast, quantitative, point-of-care-potential diagnostic tool for influenza A/H1N1 with a high sensitivity and specificity

Molecular diagnosis of Plasmodium ovale by photo-induced electron transfer fluorogenic primers: PET-PCR

PubMed Central

Akerele, David; Ljolje, Dragan; Talundzic, Eldin; Udhayakumar, Venkatachalam

2017-01-01

Accurate diagnosis of malaria infections continues to be challenging and elusive, especially in the detection of submicroscopic infections. Developing new malaria diagnostic tools that are sensitive enough to detect low-level infections, user friendly, cost effective and capable of performing large scale diagnosis, remains critical. We have designed novel self-quenching photo-induced electron transfer (PET) fluorogenic primers for the detection of P. ovale by real-time PCR. In our study, a total of 173 clinical samples, consisting of different malaria species, were utilized to test this novel PET-PCR primer. The sensitivity and specificity were calculated using nested-PCR as the reference test. The novel primer set demonstrated a sensitivity of 97.5% and a specificity of 99.2% (95% CI 85.2–99.8% and 95.2–99.9% respectively). Furthermore, the limit of detection for P. ovale was found to be 1 parasite/μl. The PET-PCR assay is a new molecular diagnostic tool with comparable performance to other commonly used PCR methods. It is relatively easy to perform, and amiable to large scale malaria surveillance studies and malaria control and elimination programs. Further field validation of this novel primer will be helpful to ascertain the utility for large scale malaria screening programs. PMID:28640824

Molecular diagnosis of Plasmodium ovale by photo-induced electron transfer fluorogenic primers: PET-PCR.

PubMed

Akerele, David; Ljolje, Dragan; Talundzic, Eldin; Udhayakumar, Venkatachalam; Lucchi, Naomi W

2017-01-01

Accurate diagnosis of malaria infections continues to be challenging and elusive, especially in the detection of submicroscopic infections. Developing new malaria diagnostic tools that are sensitive enough to detect low-level infections, user friendly, cost effective and capable of performing large scale diagnosis, remains critical. We have designed novel self-quenching photo-induced electron transfer (PET) fluorogenic primers for the detection of P. ovale by real-time PCR. In our study, a total of 173 clinical samples, consisting of different malaria species, were utilized to test this novel PET-PCR primer. The sensitivity and specificity were calculated using nested-PCR as the reference test. The novel primer set demonstrated a sensitivity of 97.5% and a specificity of 99.2% (95% CI 85.2-99.8% and 95.2-99.9% respectively). Furthermore, the limit of detection for P. ovale was found to be 1 parasite/μl. The PET-PCR assay is a new molecular diagnostic tool with comparable performance to other commonly used PCR methods. It is relatively easy to perform, and amiable to large scale malaria surveillance studies and malaria control and elimination programs. Further field validation of this novel primer will be helpful to ascertain the utility for large scale malaria screening programs.

PCR thermocycler

DOEpatents

Benett, William J.; Richards, James B.

2003-01-01

A sleeve-type silicon polymerase chain reaction (PCR) chamber or thermocycler having improved thermal performance. The silicon sleeve reaction chamber is improved in thermal performance by etched features therein that reduce thermal mass and increase the surface area of the sleeve for cooling. This improved thermal performance of the thermocycler enables an increase in speed and efficiency of the reaction chamber. The improvement is accomplished by providing grooves in the faces of the sleeve and a series of grooves on the interior surfaces that connect with grooves on the faces of the sleeve. The grooves can be anisotropically etched in the silicon sleeve simultaneously with formation of the chamber.

PCR thermocycler

DOEpatents

Benett, William J.; Richards, James B.

2005-05-17

A sleeve-type silicon polymerase chain reaction (PCR) chamber or thermocycler having improved thermal performance. The silicon sleeve reaction chamber is improved in thermal performance by etched features therein that reduce thermal mass and increase the surface area of the sleeve for cooling. This improved thermal performance of the thermocycler enables an increase in speed and efficiency of the reaction chamber. The improvement is accomplished by providing grooves in the faces of the sleeve and a series of grooves on the interior surfaces that connect with grooves on the faces of the sleeve. The grooves can be anisotropically etched in the silicon sleeve simultaneously with formation of the chamber.

DNA decontamination methods for internal quality management in clinical PCR laboratories.

PubMed

Wu, Yingping; Wu, Jianyong; Zhang, Zhihui; Cheng, Chen

2018-03-01

The polymerase chain reaction (PCR) technique, one of the most commonly applied methods in diagnostic and molecular biology, has a frustrating downside: the occurrence of false-positive signals due to contamination. In previous research, various DNA decontamination methods have been developed to overcome this limitation. Unfortunately, the use of random or poorly focused sampling methods for monitoring air and/or object surfaces leads to the incomplete elimination during decontamination procedures. We herein attempted to develop a novel DNA decontamination method (environmental surveillance, including surface and air sampling) and quality management program for clinical molecular diagnostic laboratories (or clinical PCR laboratories). Here, we performed a step-by-step evaluation of current DNA decontamination methods and developed an effective procedure for assessing the presence of decontaminating DNA via PCR analysis. Performing targeted environmental surveillance by sampling, which reached optimal performance over 2 weeks, and the decontamination process had been verified as reliable. Additionally, the process was validated to not affect PCR amplification efficiency based on a comparative study. In this study, effective guidelines for DNA decontamination were developed. The method employed ensured that surface DNA contamination could be effectively identified and eliminated. Furthermore, our study highlighted the importance of overall quality assurance and good clinical laboratory practices for preventing contamination, which are key factors for compliance with regulatory or accreditation requirements. Taken together, we provided the evidence that the presented scheme ranged from troubleshooting to the elimination of surface contamination, could serve as critical foundation for developing regular environmental surveillance guidelines for PCR laboratories. © 2017 Wiley Periodicals, Inc.

Comparison of three human papillomavirus DNA detection methods: Next generation sequencing, multiplex-PCR and nested-PCR followed by Sanger based sequencing.

PubMed

da Fonseca, Allex Jardim; Galvão, Renata Silva; Miranda, Angelica Espinosa; Ferreira, Luiz Carlos de Lima; Chen, Zigui

2016-05-01

To compare the diagnostic performance for HPV infection using three laboratorial techniques. Ninty-five cervicovaginal samples were randomly selected; each was tested for HPV DNA and genotypes using 3 methods in parallel: Multiplex-PCR, the Nested PCR followed by Sanger sequencing, and the Next_Gen Sequencing (NGS) with two assays (NGS-A1, NGS-A2). The study was approved by the Brazilian National IRB (CONEP protocol 16,800). The prevalence of HPV by the NGS assays was higher than that using the Multiplex-PCR (64.2% vs. 45.2%, respectively; P = 0.001) and the Nested-PCR (64.2% vs. 49.5%, respectively; P = 0.003). NGS also showed better performance in detecting high-risk HPV (HR-HPV) and HPV16. There was a weak interobservers agreement between the results of Multiplex-PCR and Nested-PCR in relation to NGS for the diagnosis of HPV infection, and a moderate correlation for HR-HPV detection. Both NGS assays showed a strong correlation for detection of HPVs (k = 0.86), HR-HPVs (k = 0.91), HPV16 (k = 0.92) and HPV18 (k = 0.91). NGS is more sensitive than the traditional Sanger sequencing and the Multiplex PCR to genotype HPVs, with promising ability to detect multiple infections, and may have the potential to establish an alternative method for the diagnosis and genotyping of HPV. © 2015 Wiley Periodicals, Inc.

Comparison of rapid diagnostic test Plasmotec Malaria-3, microscopy, and quantitative real-time PCR for diagnoses of Plasmodium falciparum and Plasmodium vivax infections in Mimika Regency, Papua, Indonesia.

PubMed

Fransisca, Liony; Kusnanto, Josef Hari; Satoto, Tri Baskoro T; Sebayang, Boni; Supriyanto; Andriyan, Eko; Bangs, Michael J

2015-03-05

The World Health Organization recommends malaria be diagnosed by standard microscopy or rapid diagnostic test (RDT) before treatment. RDTs have been used with greater frequency in the absence of matching blood slide confirmation in the majority of RDT reported cases in Mimika Regency, Papua Province, Indonesia. Given the importance of RDT in current health system as point-of-care tool, careful validation of RDT product performance for providing accurate malaria diagnosis is critical. Plasmotec Malaria-3 (XW-P07) performance was evaluated by comparing it with paired blood film microscopy and quantitative real-time PCR (qPCR). Consecutive whole blood samples were derived from one clinic in Mimika as part of routine passive malaria case detection. RDT results were read by two trained technicians and interpreted by consensus. Expert microscopic examination of blood slides was cross-checked by observer-blinded second reader and a third examiner if discordant between examinations. qPCR was used as the 'gold standard', followed by microscopy for the outcome/disease variable. Comparison analysis included sensitivity (Sn), specificity (Sp), positive and negative predictive values (PPV & NPV), and other diagnostic screening performance measures for detecting Plasmodium falciparum and Plasmodium vivax infections. Overall malaria positive samples from qPCR was 42.2% (175/415 samples); and from matching blood slides 40.5% (168/415) of which those infections with relatively low parasite densities ≤100/μl blood was 5.7% of P. falciparum and 16.5% of P. vivax samples examined. Overall RDT performance when compared with microscopy for detecting P. falciparum was Sn:92%, Sp:96.6%, PPV:88%, NPV:97.8%, Kappa:0.87; and for P. vivax Sn:72.9%, Sp:99.1%, PPV:95.4%, NPV:93.4%, Kappa:0.79. Overall RDT performance when compared with qPCR for detecting P. falciparum was Sn:92%, Sp:96.6%, PPV:88%, NPV:97.8%, Kappa:0.87; and for P. vivax Sn:66%, Sp:99.1%, PPV:95.4%, NPV:90.9%, Kappa:0

Competitive allele-specific TaqMan PCR (Cast-PCR) is a sensitive, specific and fast method for BRAF V600 mutation detection in Melanoma patients

PubMed Central

Barbano, Raffaela; Pasculli, Barbara; Coco, Michelina; Fontana, Andrea; Copetti, Massimiliano; Rendina, Michelina; Valori, Vanna Maria; Graziano, Paolo; Maiello, Evaristo; Fazio, Vito Michele; Parrella, Paola

2015-01-01

BRAF codon 600 mutation testing of melanoma patients is mandatory for the choice of the most appropriate therapy in the clinical setting. Competitive allele specific TaqMan PCR (Cast-PCR) technology allows not only the selective amplification of minor alleles, but it also blocks the amplification of non-mutant allele. We genotyped codon 600 of the BRAF gene in 54 patients’ samples by Cast-PCR and bidirectional direct sequence analysis. All the mutations detected by sequencing were also identified by Cast-PCR. In addition, Cast-PCR assay detected four samples carrying mutations and was able to clearly identify two mutations of uncertain interpretation by Sanger sequencing. The limit of detection of Cast-PCR was evaluated by constructing dilution curves of BRAFV600E and BRAFV600K mutated clinical samples mixed with a not-mutated specimens. Both mutations could be detected until a 1:100 mutated/not mutated ratio. Cloning and sequencing of the clones was used to confirm mutations on representative discrepant cases. Cast PCR performances were not affected by intratumour heterogeneity, and less affected by melanin content. Our results indicate that Cast-PCR is a reliable diagnostic tool for the identification of melanoma patients as eligible to be treated with TKIs and might be implemented in the clinical setting as elective screening method. PMID:26690267

Adenovirus 2, Bordetella bronchiseptica, and Parainfluenza Molecular Diagnostic Assay Results in Puppies After vaccination with Modified Live Vaccines.

PubMed

Ruch-Gallie, R; Moroff, S; Lappin, M R

2016-01-01

Canine adenovirus 2, parainfluenza, and Bordetella bronchiseptica cause respiratory disease in dogs, and each has a modified live intranasal vaccine available. Molecular diagnostic assays to amplify specific nucleic acids are available for each of these agents. If positive molecular diagnostic assay results are common after vaccination, the positive predictive value of the diagnostic assays for disease would be decreased. To determine the impact of administration of commercially available modified live topical adenovirus 2, B. bronchiseptica, and parainfluenza vaccine has on the results of a commercially available PCR panel. Eight puppies from a research breeding facility negative for these pathogens. Blinded prospective pilot study. Puppies were vaccinated with a single dose of modified live topical adenovirus 2, B. bronchiseptica, and parainfluenza and parenteral dose of adenovirus 2, canine distemper virus, and parvovirus. Nasal and pharyngeal swabs were collected on multiple days and submitted for PCR assay. Nucleic acids of all 3 organisms contained in the topical vaccine were detected from both samples multiple times through 28 days after vaccination with higher numbers of positive samples detected between days 3 and 10 after vaccination. Vaccine status should be considered when interpreting respiratory agent PCR results if modified live vaccines have been used. Development of quantitative PCR and wild-type sequencing are necessary to improve positive predictive value of these assays by distinguishing vaccinate from natural infection. Copyright © 2015 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.

Diagnostic Utility of the Social Skills Improvement System Performance Screening Guide

ERIC Educational Resources Information Center

Krach, S. Kathleen; McCreery, Michael P.; Wang, Ye; Mohammadiamin, Houra; Cirks, Christen K.

2017-01-01

Researchers investigated the diagnostic utility of the Social Skills Improvement System: Performance Screening Guide (SSIS-PSG). Correlational, regression, receiver operating characteristic (ROC), and conditional probability analyses were run to compare ratings on the SSIS-PSG subscales of Prosocial Behavior, Reading Skills, and Math Skills, to…

Multicategory reclassification statistics for assessing improvements in diagnostic accuracy

PubMed Central

Li, Jialiang; Jiang, Binyan; Fine, Jason P.

2013-01-01

In this paper, we extend the definitions of the net reclassification improvement (NRI) and the integrated discrimination improvement (IDI) in the context of multicategory classification. Both measures were proposed in Pencina and others (2008. Evaluating the added predictive ability of a new marker: from area under the receiver operating characteristic (ROC) curve to reclassification and beyond. Statistics in Medicine 27, 157–172) as numeric characterizations of accuracy improvement for binary diagnostic tests and were shown to have certain advantage over analyses based on ROC curves or other regression approaches. Estimation and inference procedures for the multiclass NRI and IDI are provided in this paper along with necessary asymptotic distributional results. Simulations are conducted to study the finite-sample properties of the proposed estimators. Two medical examples are considered to illustrate our methodology. PMID:23197381

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.

Molecular diagnostic methods for invasive fungal disease: the horizon draws nearer?

PubMed

Halliday, C L; Kidd, S E; Sorrell, T C; Chen, S C-A

2015-04-01

Rapid, accurate diagnostic laboratory tests are needed to improve clinical outcomes of invasive fungal disease (IFD). Traditional direct microscopy, culture and histological techniques constitute the 'gold standard' against which newer tests are judged. Molecular diagnostic methods, whether broad-range or fungal-specific, have great potential to enhance sensitivity and speed of IFD diagnosis, but have varying specificities. The use of PCR-based assays, DNA sequencing, and other molecular methods including those incorporating proteomic approaches such as matrix-assisted laser desorption ionisation-time of flight mass spectroscopy (MALDI-TOF MS) have shown promising results. These are used mainly to complement conventional methods since they require standardisation before widespread implementation can be recommended. None are incorporated into diagnostic criteria for defining IFD. Commercial assays may assist standardisation. This review provides an update of molecular-based diagnostic approaches applicable to biological specimens and fungal cultures in microbiology laboratories. We focus on the most common pathogens, Candida and Aspergillus, and the mucormycetes. The position of molecular-based approaches in the detection of azole and echinocandin antifungal resistance is also discussed.

Novel potential diagnostic test for Mycobacterium tuberculosis complex using combined immunomagnetic separation (IMS) and PCR-CTPP.

PubMed

Intorasoot, S; Tharinjaroen, C S; Phunpae, P; Butr-Indr, B; Anukool, U; Intachai, K; Orrapin, S; Apiratmateekul, N; Arunothong, S; Suthachai, V; Saengsawang, K; Khamnoi, P; Pata, S; Kasinrerk, W; Tragoolpua, K

2016-08-01

To exploit immunomagnetic separation combined with PCR with confronting two-pair primers (IMS-PCR-CTPP) as a rapid method for detection of Mycobacterium tuberculosis complex (MTC) and identification of Mycobacterium bovis from sputum specimens. Monoclonal antibody (mAb) against the mycobacterial antigen, 85B (Ag85B), was coupled with magnetic particles for specific immunomagnetic separation (IMS) of Mycobacterium spp. Immunofluorescence assay indicated the capability of mAb to bind to Ag85B in both the recombinant and the native form. The IMS combined with PCR-CTPP targeting the mycobacterial lep B gene was further implemented using 133 sputum samples with acid-fast bacilli grading from negative to 3+. The results showed the sensitivity and specificity of IMS-PCR-CTPP vs gold standard culture method were 89·9 and 88·6% respectively. The IMS-PCR-CTPP method shortens the time for tuberculosis (TB) diagnosis from months to a day. This method is also suitable for investigation of MTC and epidemiological study of Myco. bovis in sputum specimens. This study is the first report emphasizing the combination of IMS and PCR-CTPP for the detection of MTC and simultaneous identification of Myco. bovis from sputum. It could be used for TB diagnosis in resource-limited countries with high TB burden. © 2016 The Society for Applied Microbiology.

False negative PCR despite high levels of JC virus DNA in spinal fluid: Implications for diagnostic testing

PubMed Central

Landry, Marie L.; Eid, Tore; Bannykh, Serguei; Major, Eugene

2009-01-01

Genome amplification methods such as polymerase chain reaction (PCR) have revolutionized our ability to detect viruses in spinal fluids of patients with neurologic diseases. It is not as well appreciated among clinicians that PCR protocols, quality assurance, and technical expertise vary significantly among laboratories. In a multi-laboratory blinded study of herpes simplex virus PCR, the most widely used and best validated CSF PCR assay, low-level positives were often missed and false positives were not uncommon [Schloss L, van Loon AM, Cinque P, Cleator G, Echevarria JM, Falk KI, et al. An international external quality assessment of nucleic acid amplification of herpes simplex virus. J Clin Virol 2003;28(2):175–85]. In addition, genome variability and mutations, which are increasingly recognized for a number of different viruses, can lead to falsely low or negative results. Both clinicians and laboratories must recognize the limitations of PCR, since misleading results may have serious consequences. We present here a case of a rapidly progressive, fatal neurologic illness in a young mother, whose CSF JCV DNA PCR at a reference laboratory was falsely negative. Ultimately, brain biopsy established the diagnosis of progressive multifocal leukoencephalopathy (PML). Repeat PCR testing of the same CSF targeting a different region of the genome yielded a high positive result. PMID:18701345

Improvement of diagnostic agreement among pathologists in resolving an "atypical glands suspicious for cancer" diagnosis in prostate biopsies using a novel "Disease-Focused Diagnostic Review" quality improvement process.

PubMed

Shah, Rajal B; Leandro, Gioacchino; Romerocaces, Gloria; Bentley, James; Yoon, Jiyoon; Mendrinos, Savvas; Tadros, Yousef; Tian, Wei; Lash, Richard

2016-10-01

One of the major goals of an anatomic pathology laboratory quality program is to minimize unwarranted diagnostic variability and equivocal reporting. This study evaluated the utility of Miraca Life Sciences' "Disease-Focused Diagnostic Review" (DFDR) quality program in improving interobserver diagnostic reproducibility associated with classification of "atypical glands suspicious for adenocarcinoma" (ATYP) in prostate biopsies. Seventy-one selected prostate biopsies with a focus of ATYP were reviewed by 8 pathologists. Participants were blinded to the original diagnosis and were first asked to classify the ATYP as benign, atypical, or limited adenocarcinoma. DFDR comprised a "theoretical consensus" (in which pathologists first reached consensus on the morphological features they considered relevant for the diagnosis of limited prostatic adenocarcinoma), a didactic review including relevant literature, and "practical consensus" (pathologists performed joint microscopic sessions, reconciling each other's observations and positions evaluating a separate unique slide set). Participants were finally asked to reclassify the original 71 ATYP cases based on knowledge gleaned from DFDR. Pre- and post-DFDR interobserver reproducibility of overall diagnostic agreement was assessed. Interobserver reproducibility measured by Fleiss κ values of pre- and post-DFDR was 0.36 and 0.59, respectively (P=.006). Post-DFDR, there were significant improvement for "100% concordance" (P=.011) and reduction for "no consensus" (P=.0004) categories. Despite a lower pre-DFDR reproducibility for non-uropathology fellowship-trained (n=3, κ=0.38) versus uropathology fellowship-trained (n=5, κ=0.43) pathologists, both groups achieved similarly high post-DFDR κ levels (κ=0.58 and 0.56, respectively). DFDR represents an effective tool to formally achieve diagnostic consensus and reduce variability associated with critical diagnoses in an anatomic pathology practice. Copyright © 2016 Elsevier

Benefits and challenges of molecular diagnostics for childhood tuberculosis.

PubMed

Gutierrez, Cristina

2016-12-01

pathophysiological-response differences relative to those observed in adults. Several recent meta-analyses showed low sensitivity estimates of PCR-based TB assays for paucibacillary forms of TB (extrapulmonary TB and smear-negative pulmonary disease), which represent the vast majority of childhood TB cases. Despite the lack of evidence regarding use of the rapid molecular assays to identify TB and detect DR in children, and due to the clinical nature of childhood TB, TB-expert groups recommend including rapid methods for TB identification and DR detection in diagnostic algorithms for children suspected of both smear-positive and -negative pulmonary or extrapulmonary TB, both with or without human immunodeficiency virus (HIV)-coinfection, when combined with standard methods (including clinical, microbiological, and radiological assessment) for diagnosing active TB and conventional DR. Since 2011, the WHO has specifically recommended use of the Xpert MTB/RIF test as an initial diagnostic tool for children with suspected HIV-associated TB or multidrug-resistant TB based on successful treatment data related to adults. Implementation of the rapid molecular assays for rapid detection of TB and DR should occur in laboratories with proven capability to run molecular tests and where quality control systems are implemented. Molecular approaches should be more largely tested in children, given their status as the group in whom the diagnostic dilemma is most pronounced. These tests should also be included in specific childhood TB diagnostic algorithms adapted to the local/national context in combination with other strategies for improving diagnostics, including more effective specimen collection. Copyright © 2016.

Detection of Respiratory Viruses in Sputum from Adults by Use of Automated Multiplex PCR

PubMed Central

Walsh, Edward E.; Formica, Maria A.; Falsey, Ann R.

2014-01-01

Respiratory tract infections (RTI) frequently cause hospital admissions among adults. Diagnostic viral reverse transcriptase PCR (RT-PCR) of nose and throat swabs (NTS) is useful for patient care by informing antiviral use and appropriate isolation. However, automated RT-PCR systems are not amenable to utilizing sputum due to its viscosity. We evaluated a simple method of processing sputum samples in a fully automated respiratory viral panel RT-PCR assay (FilmArray). Archived sputum and NTS samples collected in 2008-2012 from hospitalized adults with RTI were evaluated. A subset of sputum samples positive for 10 common viruses by a uniplex RT-PCR was selected. A sterile cotton-tip swab was dunked in sputum, swirled in 700 μL of sterile water (dunk and swirl method) and tested by the FilmArray assay. Quantitative RT-PCR was performed on “dunked” sputum and NTS samples for influenza A (Flu A), respiratory syncytial virus (RSV), coronavirus OC43 (OC43), and human metapneumovirus (HMPV). Viruses were identified in 31% of 965 illnesses using a uniplex RT-PCR. The sputum sample was the only sample positive for 105 subjects, including 35% (22/64) of influenza cases and significantly increased the diagnostic yield of NTS alone (302/965 [31%] versus 197/965 [20%]; P = 0.0001). Of 108 sputum samples evaluated by the FilmArray assay using the dunk and swirl method, 99 (92%) were positive. Quantitative RT-PCR revealed higher mean viral loads in dunked sputum samples compared to NTS samples for Flu A, RSV, and HMPV (P = 0.0001, P = 0.006, and P = 0.011, respectively). The dunk and swirl method is a simple and practical method for reliably processing sputum samples in a fully automated PCR system. The higher viral loads in sputa may increase detection over NTS testing alone. PMID:25056335

A Research Agenda for Helminth Diseases of Humans: Diagnostics for Control and Elimination Programmes

PubMed Central

McCarthy, James S.; Lustigman, Sara; Yang, Guo-Jing; Barakat, Rashida M.; García, Héctor H.; Sripa, Banchob; Willingham, Arve Lee; Prichard, Roger K.; Basáñez, María-Gloria

2012-01-01

Diagnostic tools appropriate for undertaking interventions to control helminth infections are key to their success. Many diagnostic tests for helminth infection have unsatisfactory performance characteristics and are not well suited for use in the parasite control programmes that are being increasingly implemented. Although the application of modern laboratory research techniques to improve diagnostics for helminth infection has resulted in some technical advances, uptake has not been uniform. Frequently, pilot or proof of concept studies of promising diagnostic technologies have not been followed by much needed product development, and in many settings diagnosis continues to rely on insensitive and unsatisfactory parasitological or serodiagnostic techniques. In contrast, PCR-based xenomonitoring of arthropod vectors, and use of parasite recombinant proteins as reagents for serodiagnostic tests, have resulted in critical advances in the control of specific helminth parasites. The Disease Reference Group on Helminths Infections (DRG4), established in 2009 by the Special Programme for Research and Training in Tropical Diseases (TDR) was given the mandate to review helminthiases research and identify research priorities and gaps. In this review, the diagnostic technologies relevant to control of helminth infections, either available or in development, are reviewed. Critical gaps are identified and opportunities to improve needed technologies are discussed. PMID:22545166

New immuno-PCR assay for detection of low concentrations of shiga toxin 2 and its variants.

PubMed

Zhang, Wenlan; Bielaszewska, Martina; Pulz, Matthias; Becker, Karsten; Friedrich, Alexander W; Karch, Helge; Kuczius, Thorsten

2008-04-01

Shiga toxin (Stx)-producing Escherichia coli (STEC) strains secrete toxins that are major virulence factors and diagnostic targets, but some STEC strains secrete Stx in amounts that cannot be detected using conventional cell cytotoxicity or immunological assays. Therefore, there is an urgent need for more-sensitive Stx detection methods. We describe the development of an assay that can detect low concentrations of Stx2 and its variants. An immuno-PCR Stx2 assay was developed based on an enzyme immunoassay (EIA) combining antibody capture and DNA amplification to increase the signal. The immuno-PCR assay detected 10 pg/ml of purified Stx2, compared to 1 ng/ml Stx2 detected by commercial EIA. Consequently, immuno-PCR detected Stx2 and its variants in STEC strains that produce the toxins at levels that are nondetectable by using the EIA, as well as the Stx2 in EIA-negative enriched stool cultures from patients. Our data demonstrate that the immuno-PCR developed here is a highly sensitive and specific method for the detection of trace amounts of Stx2 and Stx2 variants. It is therefore suitable for use by clinical microbiological laboratories to improve the toxin detection in clinical samples.

New Immuno-PCR Assay for Detection of Low Concentrations of Shiga Toxin 2 and Its Variants▿

PubMed Central

Zhang, Wenlan; Bielaszewska, Martina; Pulz, Matthias; Becker, Karsten; Friedrich, Alexander W.; Karch, Helge; Kuczius, Thorsten

2008-01-01

Shiga toxin (Stx)-producing Escherichia coli (STEC) strains secrete toxins that are major virulence factors and diagnostic targets, but some STEC strains secrete Stx in amounts that cannot be detected using conventional cell cytotoxicity or immunological assays. Therefore, there is an urgent need for more-sensitive Stx detection methods. We describe the development of an assay that can detect low concentrations of Stx2 and its variants. An immuno-PCR Stx2 assay was developed based on an enzyme immunoassay (EIA) combining antibody capture and DNA amplification to increase the signal. The immuno-PCR assay detected 10 pg/ml of purified Stx2, compared to 1 ng/ml Stx2 detected by commercial EIA. Consequently, immuno-PCR detected Stx2 and its variants in STEC strains that produce the toxins at levels that are nondetectable by using the EIA, as well as the Stx2 in EIA-negative enriched stool cultures from patients. Our data demonstrate that the immuno-PCR developed here is a highly sensitive and specific method for the detection of trace amounts of Stx2 and Stx2 variants. It is therefore suitable for use by clinical microbiological laboratories to improve the toxin detection in clinical samples. PMID:18272709

Molecular Diagnosis of Malaria by Photo-Induced Electron Transfer Fluorogenic Primers: PET-PCR

PubMed Central

Lucchi, Naomi W.; Narayanan, Jothikumar; Karell, Mara A.; Xayavong, Maniphet; Kariuki, Simon; DaSilva, Alexandre J.; Hill, Vincent; Udhayakumar, Venkatachalam

2013-01-01

There is a critical need for developing new malaria diagnostic tools that are sensitive, cost effective and capable of performing large scale diagnosis. The real-time PCR methods are particularly robust for large scale screening and they can be used in malaria control and elimination programs. We have designed novel self-quenching photo-induced electron transfer (PET) fluorogenic primers for the detection of P. falciparum and the Plasmodium genus by real-time PCR. A total of 119 samples consisting of different malaria species and mixed infections were used to test the utility of the novel PET-PCR primers in the diagnosis of clinical samples. The sensitivity and specificity were calculated using a nested PCR as the gold standard and the novel primer sets demonstrated 100% sensitivity and specificity. The limits of detection for P. falciparum was shown to be 3.2 parasites/µl using both Plasmodium genus and P. falciparum-specific primers and 5.8 parasites/µl for P. ovale, 3.5 parasites/µl for P. malariae and 5 parasites/µl for P. vivax using the genus specific primer set. Moreover, the reaction can be duplexed to detect both Plasmodium spp. and P. falciparum in a single reaction. The PET-PCR assay does not require internal probes or intercalating dyes which makes it convenient to use and less expensive than other real-time PCR diagnostic formats. Further validation of this technique in the field will help to assess its utility for large scale screening in malaria control and elimination programs. PMID:23437209

Evaluation of urine for Leishmania infantum DNA detection by real-time quantitative PCR.

PubMed

Pessoa-E-Silva, Rômulo; Mendonça Trajano-Silva, Lays Adrianne; Lopes da Silva, Maria Almerice; da Cunha Gonçalves-de-Albuquerque, Suênia; de Goes, Tayná Correia; Silva de Morais, Rayana Carla; Lopes de Melo, Fábio; de Paiva-Cavalcanti, Milena

2016-12-01

The availability of some sorts of biological samples which require noninvasive collection methods has led to an even greater interest in applying molecular biology on visceral leishmaniasis (VL) diagnosis, since these samples increase the safety and comfort of both patients and health professionals. In this context, this work aimed to evaluate the suitability of the urine as a specimen for Leishmania infantum kinetoplast DNA detection by real-time quantitative PCR (qPCR). Subsequent to the reproducibility analysis, the detection limit of the qPCR assay was set at 5fg (~0.025 parasites) per μL of urine. From the comparative analysis performed with a set of diagnostic criteria (serological and molecular reference tests), concordance value of 96.08% was obtained (VL-suspected and HIV/AIDS patients, n=51) (P>0.05). Kappa coefficient (95% CI) indicated a good agreement between the test and the set of diagnostic criteria (k=0.778±0.151). The detection of Leishmania DNA in urine by qPCR was possible in untreated individuals, and in those with or without suggestive renal impairment. Fast depletion of the parasite's DNA in urine after treatment (from one dose of meglumine antimoniate) was suggested by negative qPCR results, thus indicating it as a potential alternative specimen to follow up the efficacy of therapeutic approaches. Even when evaluated in a clinically heterogeneous set of patients, the urine showed good prospect as sample for VL diagnosis by qPCR, also indicating a good negative predictive value for untreated suspected patients. Copyright © 2016 Elsevier B.V. All rights reserved.

Molecular diagnosis of malaria by photo-induced electron transfer fluorogenic primers: PET-PCR.

PubMed

Lucchi, Naomi W; Narayanan, Jothikumar; Karell, Mara A; Xayavong, Maniphet; Kariuki, Simon; DaSilva, Alexandre J; Hill, Vincent; Udhayakumar, Venkatachalam

2013-01-01

There is a critical need for developing new malaria diagnostic tools that are sensitive, cost effective and capable of performing large scale diagnosis. The real-time PCR methods are particularly robust for large scale screening and they can be used in malaria control and elimination programs. We have designed novel self-quenching photo-induced electron transfer (PET) fluorogenic primers for the detection of P. falciparum and the Plasmodium genus by real-time PCR. A total of 119 samples consisting of different malaria species and mixed infections were used to test the utility of the novel PET-PCR primers in the diagnosis of clinical samples. The sensitivity and specificity were calculated using a nested PCR as the gold standard and the novel primer sets demonstrated 100% sensitivity and specificity. The limits of detection for P. falciparum was shown to be 3.2 parasites/µl using both Plasmodium genus and P. falciparum-specific primers and 5.8 parasites/µl for P. ovale, 3.5 parasites/µl for P. malariae and 5 parasites/µl for P. vivax using the genus specific primer set. Moreover, the reaction can be duplexed to detect both Plasmodium spp. and P. falciparum in a single reaction. The PET-PCR assay does not require internal probes or intercalating dyes which makes it convenient to use and less expensive than other real-time PCR diagnostic formats. Further validation of this technique in the field will help to assess its utility for large scale screening in malaria control and elimination programs.

Improving Inspection and Maintenance Performance and On-board Diagnostics Monitor Readiness Memo

EPA Pesticide Factsheets

This EPA memorandum transmits an updated list of vehicles that exhibit issues related to OBD (on board diagnostics) monitor readiness and makes suggestions for how Inspection/Maintenance (I/M) programs can improve operational performance by addressing

A vibroacoustic diagnostic system as an element improving road transport safety.

PubMed

Komorska, Iwona

2013-01-01

Mechanical defects of a vehicle driving system can be dangerous on the road. Diagnostic systems, which monitor operations of electric and electronic elements and devices of vehicles, are continuously developed and improved, while defects of mechanical systems are still not managed properly. This article proposes supplementing existing on-board diagnostics with a system of diagnosing selected defects to minimize their impact. It presents a method of diagnosing mechanical defects of the engine, gearbox and other elements of the driving system on the basis of a model of the vibration signal obtained adaptively. This method is suitable for engine valves, engine head gasket, main gearbox, joints, etc.

Pulmonary toxoplasmosis in immunocompromised patients with interstitial pneumonia: a single-centre prospective study assessing PCR-based diagnosis.

PubMed

Desoubeaux, Guillaume; Cabanne, Églantine; Franck-Martel, Claire; Gombert, Martin; Gyan, Emmanuel; Lissandre, Séverine; Renaud, Marc; Monjanel, Hélène; Dartigeas, Caroline; Bailly, Éric; Van Langendonck, Nathalie; Chandenier, Jacques

2016-08-01

Pulmonary toxoplasmosis has become a very rare parasitic infection since the advent of highly active antiretroviral therapies. It is generally diagnosed by the direct microscopic observation of Toxoplasma gondii tachyzoites in bronchoalveolar lavage fluid (BALF). The aim of this study was to assess possible improvements in diagnostic performance associated with the use of real-time PCR. This prospective study was carried out on BALFs obtained from immunocompromised patients over a 2-year period. We systematically compared the results of conventional staining with those of molecular detection. Two cases of pulmonary toxoplasmosis were diagnosed for a total of 336 samples. PCR did not detect any additional cases and was more time-consuming than conventional staining. Conventional staining is a reliable technique and is probably the most appropriate method for experienced microbiology laboratories, whereas T. gondii-specific PCR may be useful for laboratories with less experience in parasitology. 2015_030, May 27th 2015. 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/

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

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

Detection of bacterial pathogens in Mongolia meningitis surveillance with a new real-time PCR assay to detect Haemophilus influenzae.

PubMed

Wang, Xin; Mair, Raydel; Hatcher, Cynthia; Theodore, M Jordan; Edmond, Karen; Wu, Henry M; Harcourt, Brian H; Carvalho, Maria da Gloria S; Pimenta, Fabiana; Nymadawa, Pagbajab; Altantsetseg, Dorjpurev; Kirsch, Mariah; Satola, Sarah W; Cohn, Amanda; Messonnier, Nancy E; Mayer, Leonard W

2011-04-01

Since the implementation of Haemophilus influenzae (Hi) serotype b vaccine, other serotypes and non-typeable strains have taken on greater importance as a cause of Hi diseases. A rapid and accurate method is needed to detect all Hi regardless of the encapsulation status. We developed 2 real-time PCR (rt-PCR) assays to detect specific regions of the protein D gene (hpd). Both hpd assays are very specific and sensitive for detection of Hi. Of the 63 non-Hi isolates representing 21 bacterial species, none was detected by the hpd #1 assay, and only one of 2 H. aphrophilus isolates was detected by the hpd #3 assay. The hpd #1 and #3 assays detected 97% (229/237) and 99% (234/237) of Hi isolates, respectively, and were superior for detection of both typeable and non-typeable Hi isolates, as compared to previously developed rt-PCR targeting ompP2 or bexA. The diagnostic sensitivity and specificity of these rt-PCR assays were assessed on cerebrospinal fluid specimens collected as part of meningitis surveillance in Ulaanbaatar, Mongolia. The etiology (Neisseria meningitidis, Hi, and Streptococcus pneumoniae) of 111 suspected meningitis cases was determined by conventional methods (culture and latex agglutination), previously developed rt-PCR assays, and the new hpd assays. The rt-PCR assays were more sensitive for detection of meningitis pathogens than other classical methods and improved detection from 50% (56/111) to 75% (83/111). The hpd #3 assay identified a non-b Hi that was missed by the bexA assay and other methods. A sensitive rt-PCR assay to detect both typeable and non-typeable Hi is a useful tool for improving Hi disease surveillance especially after Hib vaccine introduction. Published by Elsevier GmbH.

A Rapid and Low-Cost PCR Thermal Cycler for Low Resource Settings.

PubMed

Wong, Grace; Wong, Isaac; Chan, Kamfai; Hsieh, Yicheng; Wong, Season

2015-01-01

Many modern molecular diagnostic assays targeting nucleic acids are typically confined to developed countries or to the national reference laboratories of developing-world countries. The ability to make technologies for the rapid diagnosis of infectious diseases broadly available in a portable, low-cost format would mark a revolutionary step forward in global health. Many molecular assays are also developed based on polymerase chain reactions (PCR), which require thermal cyclers that are relatively heavy (>20 pounds) and need continuous electrical power. The temperature ramping speed of most economical thermal cyclers are relatively slow (2 to 3 °C/s) so a polymerase chain reaction can take 1 to 2 hours. Most of all, these thermal cyclers are still too expensive ($2k to $4k) for low-resource setting uses. In this article, we demonstrate the development of a low-cost and rapid water bath based thermal cycler that does not require active temperature control or continuous power supply during PCR. This unit costs $130 to build using commercial off-the-shelf items. The use of two or three vacuum-insulated stainless-steel Thermos food jars containing heated water (for denaturation and annealing/extension steps) and a layer of oil on top of the water allow for significantly stabilized temperatures for PCR to take place. Using an Arduino-based microcontroller, we automate the "archaic" method of hand-transferring PCR tubes between water baths. We demonstrate that this innovative unit can deliver high speed PCR (17 s per PCR cycle) with the potential to go beyond the 1,522 bp long amplicons tested in this study and can amplify from templates down to at least 20 copies per reaction. The unit also accepts regular PCR tubes and glass capillary tubes. The PCR efficiency of our thermal cycler is not different from other commercial thermal cyclers. When combined with a rapid nucleic acid detection approach, the thermos thermal cycler (TTC) can enable on-site molecular

A Rapid and Low-Cost PCR Thermal Cycler for Low Resource Settings

PubMed Central

Wong, Grace; Wong, Isaac; Chan, Kamfai; Hsieh, Yicheng; Wong, Season

2015-01-01

Background Many modern molecular diagnostic assays targeting nucleic acids are typically confined to developed countries or to the national reference laboratories of developing-world countries. The ability to make technologies for the rapid diagnosis of infectious diseases broadly available in a portable, low-cost format would mark a revolutionary step forward in global health. Many molecular assays are also developed based on polymerase chain reactions (PCR), which require thermal cyclers that are relatively heavy (>20 pounds) and need continuous electrical power. The temperature ramping speed of most economical thermal cyclers are relatively slow (2 to 3°C/s) so a polymerase chain reaction can take 1 to 2 hours. Most of all, these thermal cyclers are still too expensive ($2k to $4k) for low-resource setting uses. Methodology/Principal Findings In this article, we demonstrate the development of a low-cost and rapid water bath based thermal cycler that does not require active temperature control or continuous power supply during PCR. This unit costs $130 to build using commercial off-the-shelf items. The use of two or three vacuum-insulated stainless-steel Thermos food jars containing heated water (for denaturation and annealing/extension steps) and a layer of oil on top of the water allow for significantly stabilized temperatures for PCR to take place. Using an Arduino-based microcontroller, we automate the “archaic” method of hand-transferring PCR tubes between water baths. Conclusions/Significance We demonstrate that this innovative unit can deliver high speed PCR (17 s per PCR cycle) with the potential to go beyond the 1,522 bp long amplicons tested in this study and can amplify from templates down to at least 20 copies per reaction. The unit also accepts regular PCR tubes and glass capillary tubes. The PCR efficiency of our thermal cycler is not different from other commercial thermal cyclers. When combined with a rapid nucleic acid detection approach

Detection of adenoviruses in shellfish by means of conventional-PCR, nested-PCR, and integrated cell culture PCR (ICC/PCR).

PubMed

Rigotto, C; Sincero, T C M; Simões, C M O; Barardi, C R M

2005-01-01

We tested three PCR based methodologies to detect adenoviruses associated with cultivated oysters. Conventional-PCR, nested-PCR, and integrated cell culture-PCR (ICC/PCR) were first optimized using oysters seeded with know amounts of Adenovirus serotype 5 (Ad5). The maximum sensitivity for Ad5 detection was determined for each method, and then used to detect natural adenovirus contamination in oysters from three aquiculture farms in Florianopolis, Santa Catarina State, Brazil, over a period of 6 months. The results showed that the nested-PCR was more sensitive (limit of detection: 1.2 PFU/g of tissue) than conventional-PCR and ICC-PCR (limit of detection for both: 1.2 x 10(2)PFU/g of tissue) for detection of Ad5 in oyster extracts. Nested-PCR was able to detect 90% of Ad5 contamination in harvested oyster samples, while conventional-PCR was unable to detect Ad5 in any of the samples. The present work suggests that detection of human adenoviruses can be used as a tool to monitor the presence of human viruses in marine environments where shellfish grow, and that nested-PCR is the method of choice.

Developing high throughput quantitative PCR assays for diagnosing Ikeda and other Theileria orientalis types common to New Zealand in bovine blood samples.

PubMed

Pulford, D J; Gias, E; Bueno, I M; McFadden, Amj

2016-01-01

To develop rapid, quantitative PCR (qPCR) assays using high resolution melt (HRM) analysis and type-specific TaqMan assays for identifying the prevalent types of Theileria orientalis found in New Zealand cattle; and to evaluate their analytical and diagnostic characteristics compared with other assays for T. orientalis. Nucleotide sequences aligned with T. orientalis Buffeli, Chitose and Ikeda types, obtained from DNA extracted from blood samples from infected cattle, were used to design HRM and type-specific probe-based qPCR assays. The three type-specific assays were also incorporated into a single-tube multiplex qPCR assay. These assays were validated using DNA extracted from blood samples from cattle in herds with or without clinical signs of T. orientalis infection, other veterinary laboratory samples, as well as plasmids containing T. orientalis type-specific sequences. Diagnostic specificity (DSp) and sensitivity (DSe) estimates for the qPCR assays were compared to blood smear piroplasm results, and other PCR assays for T. orientalis. Copy number estimates of Ikeda DNA in blood were determined from cattle exhibiting anaemia using the Ikeda-specific qPCR assay. The T. orientalis type-specific and the HRM qPCR assays displayed 100% analytical specificity. The Ikeda-specific qPCR assay exhibited linearity (R(2) = 0.997) with an efficiency of 94.3%. Intra-assay CV were ≤0.08 and inter-assay CV were ≤0.095. For blood samples from cows with signs of infection with T. orientalis, the DSp and DSe of the multiplex probe qPCR assay were 93 and 96%, respectively compared with blood smears, and 97 and 100%, respectively compared with conventional PCR assays. For the Ikeda-specific qPCR assay, the number of positive samples (n=66) was slightly higher than a conventional PCR assay (n=64). The concentration of Ikeda genomes in blood samples from 41 dairy cows with signs of infection with T. orientalis ranged between 5.6 × 10(4) and 3.3 × 10(6) genomes per

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.

Molecular method for the detection of Andes hantavirus infection: validation for clinical diagnostics

PubMed Central

Vial, Cecilia; Martinez-Valdebenito, Constanza; Rios, Susana; Martinez, Jessica; Vial, Pablo; Ferres, Marcela; Rivera, Juan Carlos; Perez, Ruth; Valdivieso, Francisca

2016-01-01

Hantavirus Cardiopulmonary Syndrome is a severe disease caused by exposure to New World hantaviruses. Early diagnosis is difficult due to the lack of specific initial symptoms. Anti-hantavirus antibodies are usually negative until late in the febrile prodrome or the beginning of cardiopulmonary phase while Andes hantavirus (ANDV) RNA genome can be detected before symptoms onset. We analyzed the effectiveness of RTqPCR as a diagnostic tool detecting ANDV-Sout genome in peripheral blood cells from 78 confirmed hantavirus patients and 166 negative controls. Our results indicate that RTqPCR had a low detection limit (~10 copies), with a specificity of 100% and a sensitivity of 94.9%. This suggests the potential for establishing RT-qPCR as the assay of choice for early diagnosis, promoting early effective care of patients and improve other important aspects of ANDV infection management, such as compliance of biosafety recommendations for health personnel in order to avoid nosocomial transmission. PMID:26508102

Molecular diagnostic toolkit for Rhizophagus irregularis isolate DAOM-197198 using quantitative PCR assay targeting the mitochondrial genome.

PubMed

Badri, Amine; Stefani, Franck O P; Lachance, Geneviève; Roy-Arcand, Line; Beaudet, Denis; Vialle, Agathe; Hijri, Mohamed

2016-10-01

Rhizophagus irregularis (previously named Glomus irregulare) is one of the most widespread and common arbuscular mycorrhizal fungal (AMF) species. It has been recovered worldwide in agricultural and natural soils, and the isolate DAOM-197198 has been utilized as a commercial inoculant for two decades. Despite the ecological and economical importance of this taxon, specific markers for quantification of propagules by quantitative real-time PCR (qPCR) are extremely limited and none have been rigorously validated for quality control of manufactured products such as biofertilizers. From the sequencing of 14 complete AMF mitochondrial (mt) genomes, a qPCR assay using a hydrolysis probe designed in the single copy cox3-rnl intergenic region was tested and validated to specifically and accurately quantify the spores of R. irregularis isolate DAOM-197198. Specificity tests were performed using standard PCR and qPCR, and results clearly showed that the primers specifically amplified the isolate DAOM-197198, yielding a PCR product of 106 bp. According to the qPCR analyses on spores produced in vitro, the average copy number of mt genomes per spore was 3172 ± 304 SE (n = 6). Quantification assays were successfully undertaken on known and unknown samples in liquid suspensions and commercial dry formulations to show the accuracy, precision, robustness, and reproducibility of the qPCR assay. This study provides a powerful molecular toolkit specifically designed to quantify spores of the model AMF isolate DAOM-197198. The approach of molecular toolkit used in our study could be applied to other AMF taxa and will be useful to research institutions and governmental and industrial laboratories running routine quality control of AMF-based products.

Assessment of Clinically Suspected Tubercular Lymphadenopathy by Real-Time PCR Compared to Non-Molecular Methods on Lymph Node Aspirates.

PubMed

Gupta, Vivek; Bhake, Arvind

2018-01-01

The diagnosis of tubercular lymphadenitis (TBLN) is challenging. This study assesses the role of diagnostic intervention with real-time PCR in clinically suspected tubercular lymphadenopathy in relation to cytology and microbiological methods. The cross-sectional study involved 214 patients, and PCR, cytology, and Ziehl-Neelsen (ZN) staining was performed on aspirates. The findings were compared with culture on Lowenstein-Jensen medium. The overall concordance of cytology and PCR, both individually and combined, was calculated. χ2 and Phi values were assessed between cytology, PCR, and culture. A cytological diagnosis of tuberculosis (TB), reactive lymphoid hyperplasia, and suppurative lymphadenitis was made in 71, 112, and 6 patients, respectively. PCR and culture were positive in 40% of the cases. Among the TBLN patients, PCR showed higher positivity in necrosis and culture showed higher positivity in necrotizing granuloma. Positive ZN staining was observed in 29.6% of the TBLN cases, with an overall positivity of 11%. PCR could additionally detect 82 cases missed by ZN staining. The overall concordance rate for either diagnostic modality, i.e., PCR or cytology, was highest (75%), and for PCR alone was 74%. Phi values were observed to be 0.47 between PCR and culture. Real-time PCR for Mycobacterium tuberculosis complex on aspirates offers a definitive and comparable diagnosis of TBLN. Including this approach as the primary investigation in the work-up of TBLN could reduce the burden of TB. © 2017 S. Karger AG, Basel.

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.

The diagnostic accuracy of three rapid diagnostic tests for typhoid fever at Chittagong Medical College Hospital, Chittagong, Bangladesh.

PubMed

Maude, Rapeephan R; de Jong, Hanna K; Wijedoru, Lalith; Fukushima, Masako; Ghose, Aniruddha; Samad, Rasheda; Hossain, Mohammed Amir; Karim, Mohammed Rezaul; Faiz, Mohammed Abul; Parry, Christopher M

2015-10-01

To determine the diagnostic accuracy of three rapid diagnostic tests (RDTs) for typhoid fever in febrile hospitalised patients in Bangladesh. Febrile adults and children admitted to Chittagong Medical College Hospital, Bangladesh, were investigated with Bact/Alert(®) blood cultures and real-time PCR to detect Salmonella enterica Typhi and Paratyphi A and assays for Rickettsia, leptospirosis and dengue fever. Acute serum samples were examined with the LifeAssay (LA) Test-it™ Typhoid IgM lateral flow assay detecting IgM antibodies against S. Typhi O antigen, CTKBiotech Onsite Typhoid IgG/IgM Combo Rapid-test cassette lateral flow assay detecting IgG and IgM antibodies against S. Typhi O and H antigens and SD Bioline line assay for IgG and IgM antibodies against S. Typhi proteins. In 300 malaria smear-negative febrile patients [median (IQR) age of 13.5 (5-31) years], 34 (11.3%) had confirmed typhoid fever: 19 positive by blood culture for S. Typhi (three blood PCR positive) and 15 blood culture negative but PCR positive for S. Typhi in blood. The respective sensitivity and specificity of the three RDTs in patients using a composite reference standard of blood culture and/or PCR-confirmed typhoid fever were 59% and 61% for LifeAssay, 59% and 74% for the CTK IgM and/or IgG, and 24% and 96% for the SD Bioline RDT IgM and/or IgG. The LifeAssay RDT had a sensitivity of 63% and a specificity of 91% when modified with a positive cut-off of ≥2+ and analysed using a Bayesian latent class model. These typhoid RDTs demonstrated moderate diagnostic accuracies, and better tests are needed. © 2015 The Authors. Tropical Medicine & International Health Published by John Wiley & Sons Ltd.

Field-Deployable Reverse Transcription-Insulated Isothermal PCR (RT-iiPCR) Assay for Rapid and Sensitive Detection of Foot-and-Mouth Disease Virus.

PubMed

Ambagala, A; Fisher, M; Goolia, M; Nfon, C; Furukawa-Stoffer, T; Ortega Polo, R; Lung, O

2017-10-01

Foot-and-mouth disease (FMD) is a highly contagious viral disease of cloven-hoofed animals, which can decimate the livestock industry and economy of countries previously free of this disease. Rapid detection of foot-and-mouth disease virus (FMDV) is critical to containing an FMD outbreak. Availability of a rapid, highly sensitive and specific, yet simple and field-deployable assay would support local decision-making during an FMDV outbreak. Here we report validation of a novel reverse transcription-insulated isothermal PCR (RT-iiPCR) assay that can be performed on a commercially available, compact and portable POCKIT ™ analyser that automatically analyses data and displays '+' or '-' results. The FMDV RT-iiPCR assay targets the 3D region of the FMDV genome and was capable of detecting 9 copies of in vitro-transcribed RNA standard with 95% confidence. It accurately identified 63 FMDV strains belonging to all seven serotypes and showed no cross-reactivity with viruses causing similar clinical diseases in cloven-hoofed animals. The assay was able to identify FMDV RNA in multiple sample types including oral, nasal and lesion swabs, epithelial tissue suspensions, vesicular and oral fluid samples, even before the appearance of clinical signs. Clinical sensitivity of the assay was comparable or slightly higher than the laboratory-based real-time RT-PCR assay in use. The assay was able to detect FMDV RNA in vesicular fluid samples without nucleic acid extraction. For RNA extraction from more complex sample types, a commercially available taco ™ mini transportable magnetic bead-based, automated extraction system was used. This assay provides a potentially useful field-deployable diagnostic tool for rapid detection of FMDV in an outbreak in FMD-free countries or for routine diagnostics in endemic countries with less structured laboratory systems. © 2016 Her Majesty the Queen in Right of Canada.

Development of 2, 7-Diamino-1, 8-Naphthyridine (DANP) Anchored Hairpin Primers for RT-PCR Detection of Chikungunya Virus Infection.

PubMed

Chen, Huixin; Parimelalagan, Mariya; Takei, Fumie; Hapuarachchi, Hapuarachchige Chanditha; Koay, Evelyn Siew-Chuan; Ng, Lee Ching; Ho, Phui San; Nakatani, Kazuhiko; Chu, Justin Jang Hann

2016-08-01

A molecular diagnostic platform with DANP-anchored hairpin primer was developed and evaluated for the rapid and cost-effective detection of Chikungunya virus (CHIKV) with high sensitivity and specificity. The molecule 2, 7-diamino-1, 8-naphthyridine (DANP) binds to a cytosine-bulge and emits fluorescence at 450 nm when it is excited by 400 nm light. Thus, by measuring the decline in fluorescence emitted from DANP-primer complexes after PCR reaction, we could monitor the PCR progress. By adapting this property of DANP, we have previously developed the first generation DANP-coupled hairpin RT-PCR assay. In the current study, we improved the assay performance by conjugating the DANP molecule covalently onto the hairpin primer to fix the DANP/primer ratio at 1:1; and adjusting the excitation emission wavelength to 365/430 nm to minimize the background signal and a 'turn-on' system is achieved. After optimizing the PCR cycle number to 30, we not only shortened the total assay turnaround time to 60 minutes, but also further reduced the background fluorescence. The detection limit of our assay was 0.001 PFU per reaction. The DANP-anchored hairpin primer, targeting nsP2 gene of CHIKV genome, is highly specific to CHIKV, having no cross-reactivity to a panel of other RNA viruses tested. In conclusion, we report here a molecular diagnostic assay that is sensitive, specific, rapid and cost effective for CHIKV detection and can be performed where no real time PCR instrumentation is required. Our results from patient samples indicated 93.62% sensitivity and 100% specificity of this method, ensuring that it can be a useful tool for rapid detection of CHIKV for outbreaks in many parts of the world.

Development of 2, 7-Diamino-1, 8-Naphthyridine (DANP) Anchored Hairpin Primers for RT-PCR Detection of Chikungunya Virus Infection

PubMed Central

Chen, Huixin; Parimelalagan, Mariya; Takei, Fumie; Hapuarachchi, Hapuarachchige Chanditha; Koay, Evelyn Siew-Chuan; Ng, Lee Ching; Ho, Phui San; Nakatani, Kazuhiko; Chu, Justin Jang Hann

2016-01-01

A molecular diagnostic platform with DANP-anchored hairpin primer was developed and evaluated for the rapid and cost-effective detection of Chikungunya virus (CHIKV) with high sensitivity and specificity. The molecule 2, 7-diamino-1, 8-naphthyridine (DANP) binds to a cytosine-bulge and emits fluorescence at 450 nm when it is excited by 400 nm light. Thus, by measuring the decline in fluorescence emitted from DANP—primer complexes after PCR reaction, we could monitor the PCR progress. By adapting this property of DANP, we have previously developed the first generation DANP-coupled hairpin RT-PCR assay. In the current study, we improved the assay performance by conjugating the DANP molecule covalently onto the hairpin primer to fix the DANP/primer ratio at 1:1; and adjusting the excitation emission wavelength to 365/430 nm to minimize the background signal and a ‘turn-on’ system is achieved. After optimizing the PCR cycle number to 30, we not only shortened the total assay turnaround time to 60 minutes, but also further reduced the background fluorescence. The detection limit of our assay was 0.001 PFU per reaction. The DANP-anchored hairpin primer, targeting nsP2 gene of CHIKV genome, is highly specific to CHIKV, having no cross-reactivity to a panel of other RNA viruses tested. In conclusion, we report here a molecular diagnostic assay that is sensitive, specific, rapid and cost effective for CHIKV detection and can be performed where no real time PCR instrumentation is required. Our results from patient samples indicated 93.62% sensitivity and 100% specificity of this method, ensuring that it can be a useful tool for rapid detection of CHIKV for outbreaks in many parts of the world. PMID:27571201

Specific primers for rapid detection of Microsporum audouinii by PCR in clinical samples.

PubMed

Roque, H D; Vieira, R; Rato, S; Luz-Martins, M

2006-12-01

This report describes application of PCR fingerprinting to identify common species of dermatophytes using the microsatellite primers M13, (GACA)4, and (GTG)5. The initial PCR analysis rendered a specific DNA fragment for Microsporum audouinii, which was cloned and sequenced. Based on the sequencing data of this fragment, forward (MA_1F) and reverse (MA_1R) primers were designed and verified by PCR to establish their reliability in the diagnosis of M. audouinii. These primers produced a singular PCR band of 431 bp specific only to strains and isolates of M. audouinii, based on a global test of 182 strains/isolates belonging to 11 species of dermatophytes. These findings indicate these primers are reliable for diagnostic purposes, and we recommend their use in laboratory analysis.

A traditionally administered short course failed to improve medical students' diagnostic performance. A quantitative evaluation of diagnostic thinking.

PubMed

Noguchi, Yoshinori; Matsui, Kunihiko; Imura, Hiroshi; Kiyota, Masatomo; Fukui, Tsuguya

2004-05-01

Quite often medical students or novice residents have difficulty in ruling out diseases even though they are quite unlikely and, due to this difficulty, such students and novice residents unnecessarily repeat laboratory or imaging tests. To explore whether or not a carefully designed short training course teaching Bayesian probabilistic thinking improves the diagnostic ability of medical students. Ninety students at 2 medical schools were presented with clinical scenarios of coronary artery disease corresponding to high, low, and intermediate pretest probabilities. The students' estimates of test characteristics of exercise stress test, and pretest and posttest probability for each scenario were evaluated before and after the short course. The pretest probability estimates by the students, as well as their proficiency in applying Bayes's theorem, were improved in the high pretest probability scenario after the short course. However, estimates of pretest probability in the low pretest probability scenario, and their proficiency in applying Bayes's theorem in the intermediate and low pretest probability scenarios, showed essentially no improvement. A carefully designed, but traditionally administered, short course could not improve the students' abilities in estimating pretest probability in a low pretest probability setting, and subsequently students remained incompetent in ruling out disease. We need to develop educational methods that cultivate a well-balanced clinical sense to enable students to choose a suitable diagnostic strategy as needed in a clinical setting without being one-sided to the "rule-in conscious paradigm."

Blending DNA binding dyes to improve detection in real-time PCR.

PubMed

Jansson, Linda; Koliana, Marianne; Sidstedt, Maja; Hedman, Johannes

2017-03-01

The success of real-time PCR (qPCR) analysis is partly limited by the presence of inhibitory compounds in the nucleic acid samples. For example, humic acid (HA) from soil and aqueous sediment interferes with amplification and also quenches the fluorescence of double-stranded (ds) DNA binding dyes, thus hindering amplicon detection. We aimed to counteract the HA fluorescence quenching effect by blending complementary dsDNA binding dyes, thereby elevating the dye saturation levels and increasing the fluorescence signals. A blend of the four dyes EvaGreen, ResoLight, SYBR Green and SYTO9 gave significantly higher fluorescence intensities in the presence and absence of HA, compared with the dyes applied separately and two-dye blends. We propose blending of dyes as a generally applicable means for elevating qPCR fluorescence signals and thus enabling detection in the presence of quenching substances.

Nested PCR Assay for Eight Pathogens: A Rapid Tool for Diagnosis of Bacterial Meningitis.

PubMed

Bhagchandani, Sharda P; Kubade, Sushant; Nikhare, Priyanka P; Manke, Sonali; Chandak, Nitin H; Kabra, Dinesh; Baheti, Neeraj N; Agrawal, Vijay S; Sarda, Pankaj; Mahajan, Parikshit; Ganjre, Ashish; Purohit, Hemant J; Singh, Lokendra; Taori, Girdhar M; Daginawala, Hatim F; Kashyap, Rajpal S

2016-02-01

Bacterial meningitis is a dreadful infectious disease with a high mortality and morbidity if remained undiagnosed. Traditional diagnostic methods for bacterial meningitis pose a challenge in accurate identification of pathogen, making prognosis difficult. The present study is therefore aimed to design and evaluate a specific and sensitive nested 16S rDNA genus-based polymerase chain reaction (PCR) assay using clinical cerebrospinal fluid (CSF) for rapid diagnosis of eight pathogens causing the disease. The present work was dedicated to development of an in-house genus specific 16S rDNA nested PCR covering pathogens of eight genera responsible for causing bacterial meningitis using newly designed as well as literature based primers for respective genus. A total 150 suspected meningitis CSF obtained from the patients admitted to Central India Institute of Medical Sciences (CIIMS), India during the period from August 2011 to May 2014, were used to evaluate clinical sensitivity and clinical specificity of optimized PCR assays. The analytical sensitivity and specificity of our newly designed genus-specific 16S rDNA PCR were found to be ≥92%. With such a high sensitivity and specificity, our in-house nested PCR was able to give 100% sensitivity in clinically confirmed positive cases and 100% specificity in clinically confirmed negative cases indicating its applicability in clinical diagnosis. Our in-house nested PCR system therefore can diagnose the accurate pathogen causing bacterial meningitis and therefore be useful in selecting a specific treatment line to minimize morbidity. Results are obtained within 24 h and high sensitivity makes this nested PCR assay a rapid and accurate diagnostic tool compared to traditional culture-based methods.

Use of a nested PCR-enzyme immunoassay with an internal control to detect Chlamydophila psittaci in turkeys

PubMed Central

Van Loock, Marnix; Verminnen, Kristel; Messmer, Trudy O; Volckaert, Guido; Goddeeris, Bruno M; Vanrompay, Daisy

2005-01-01

Background Laboratory diagnosis of Chlamydophila psittaci, an important turkey respiratory pathogen, is difficult. To facilitate the diagnosis, a nested PCR-enzyme immunoassay (PCR-EIA) was developed to detect the Cp. psittaci outer membrane protein A (ompA) gene in pharyngeal swabs. Methods The fluorescein-biotin labelled PCR products were immobilized on streptavidin-coated microtiter plates and detected with anti-fluorescein peroxidase conjugate and a colorimetric substrate. An internal inhibition control was included to rule out the presence of inhibitors of DNA amplification. The diagnostic value of the ompA nested PCR-EIA in comparison to cell culture and a 16S-rRNA based nested PCR was assessed in pharyngeal turkey swabs from 10 different farms experiencing respiratory disease. Results The sensitivity of the nested PCR-EIA was established at 0.1 infection forming units (IFU). Specificity was 100%. The ompA nested PCR-EIA was more sensitive than the 16S-rRNA based nested PCR and isolation, revealing 105 out of 200 (52.5%) positives against 13 and 74 for the latter two tests, respectively. Twenty-nine (23.8%) out of 122 ompA PCR-EIA negatives showed the presence of inhibitors of DNA amplification, although 27 of them became positive after diluting (1/10) the specimens in PCR buffer or after phenol-chloroform extraction and subsequent ethanol precipitation. Conclusion The present study stresses the need for an internal control to confirm PCR true-negatives and demonstrates the high prevalence of chlamydiosis in Belgian turkeys and its potential zoonotic risk. The ompA nested PCR-EIA described here is a rapid, highly sensitive and specific diagnostic assay and will help to facilitate the diagnosis of Cp. psittaci infections in both poultry and man. PMID:16185353

Field evaluation of the photo-induced electron transfer fluorogenic primers (PET) real-time PCR for the detection of Plasmodium falciparum in Tanzania

PubMed Central

2014-01-01

Background Accurate diagnosis of malaria infections remains challenging, especially in the identification of submicroscopic infections. New molecular diagnostic tools that are inexpensive, sensitive enough to detect low-level infections and suitable in laboratory settings of resource-limited countries are required for malaria control and elimination programmes. Here the diagnostic potential of a recently developed photo-induced electron transfer fluorogenic primer (PET) real-time polymerase chain reaction (PCR) called PET-PCR was investigated. This study aimed to (i) evaluate the use of this assay as a method for the detection of both Plasmodium falciparum and other Plasmodium species infections in a developing country’s diagnostic laboratory; and, (ii) determine the assay’s sensitivity and specificity compared to a nested 18S rRNA PCR. Methods Samples used in this study were obtained from a previous study conducted in the region of Iringa, Tanzania. A total of 303 samples from eight health facilities in Tanzania were utilized for this evaluation. All samples were screened using the multiplex PET-PCR assay designed to detect Plasmodium genus and P. falciparum initially in laboratory in Tanzania and then repeated at a reference laboratory at the CDC in the USA. Microscopy data was available for all the 303 samples. A subset of the samples were tested in a blinded fashion to find the sensitivity and specificity of the PET-PCR compared to the nested 18S rRNA PCR. Results Compared to microscopy, the PET-PCR assay was 59% more sensitive in detecting P. falciparum infections. The observed sensitivity and specificity were 100% (95% confidence interval (CI0.95) = 94-100%) and (CI0.95 = 96-100%), respectively, for the PET-PCR assay when compared to nested 18S rRNA PCR. When compared to 18S rRNA PCR, microscopy had a low sensitivity of 40% (CI0.95 = 23-61%) and specificity of 100% (CI0.95 = 96-100%). The PET-PCR results performed in the field laboratory

Field evaluation of the photo-induced electron transfer fluorogenic primers (PET) real-time PCR for the detection of Plasmodium falciparum in Tanzania.

PubMed

Talundzic, Eldin; Maganga, Mussa; Masanja, Irene M; Peterson, David S; Udhayakumar, Venkatachalam; Lucchi, Naomi W

2014-01-27

Accurate diagnosis of malaria infections remains challenging, especially in the identification of submicroscopic infections. New molecular diagnostic tools that are inexpensive, sensitive enough to detect low-level infections and suitable in laboratory settings of resource-limited countries are required for malaria control and elimination programmes. Here the diagnostic potential of a recently developed photo-induced electron transfer fluorogenic primer (PET) real-time polymerase chain reaction (PCR) called PET-PCR was investigated. This study aimed to (i) evaluate the use of this assay as a method for the detection of both Plasmodium falciparum and other Plasmodium species infections in a developing country's diagnostic laboratory; and, (ii) determine the assay's sensitivity and specificity compared to a nested 18S rRNA PCR. Samples used in this study were obtained from a previous study conducted in the region of Iringa, Tanzania. A total of 303 samples from eight health facilities in Tanzania were utilized for this evaluation. All samples were screened using the multiplex PET-PCR assay designed to detect Plasmodium genus and P. falciparum initially in laboratory in Tanzania and then repeated at a reference laboratory at the CDC in the USA. Microscopy data was available for all the 303 samples. A subset of the samples were tested in a blinded fashion to find the sensitivity and specificity of the PET-PCR compared to the nested 18S rRNA PCR. Compared to microscopy, the PET-PCR assay was 59% more sensitive in detecting P. falciparum infections. The observed sensitivity and specificity were 100% (95% confidence interval (CI0.95) = 94-100%) and (CI0.95 = 96-100%), respectively, for the PET-PCR assay when compared to nested 18S rRNA PCR. When compared to 18S rRNA PCR, microscopy had a low sensitivity of 40% (CI0.95 = 23-61%) and specificity of 100% (CI0.95 = 96-100%). The PET-PCR results performed in the field laboratory in Tanzania were in 100

Improved Diagnostic Validity of the ADOS Revised Algorithms: A Replication Study in an Independent Sample

ERIC Educational Resources Information Center

Oosterling, Iris; Roos, Sascha; de Bildt, Annelies; Rommelse, Nanda; de Jonge, Maretha; Visser, Janne; Lappenschaar, Martijn; Swinkels, Sophie; van der Gaag, Rutger Jan; Buitelaar, Jan

2010-01-01

Recently, Gotham et al. ("2007") proposed revised algorithms for the Autism Diagnostic Observation Schedule (ADOS) with improved diagnostic validity. The aim of the current study was to replicate predictive validity, factor structure, and correlations with age and verbal and nonverbal IQ of the ADOS revised algorithms for Modules 1 and 2…

Improved diagnostic differentiation of renal cystic lesions with phase-contrast computed tomography (PCCT)

NASA Astrophysics Data System (ADS)

Noel, Peter B.; Willner, Marian; Fingerle, Alexander; Herzen, Julia; Münzel, Daniela; Hahn, Dieter; Rummeny, Ernst J.; Pfeiffer, Franz

2012-03-01

The diagnostic quality of phase-contrast computed tomography (PCCT) is one the unexplored areas in medical imaging; at the same time, it seems to offer the opportunity as a fast and highly sensitive diagnostic tool. Conventional computed tomography (CT) has had an enormous impact on medicine, while it is limited in soft-tissue contrast. One example that portrays this challenge is the differentiation between benign and malignant renal cysts. In this work we report on a feasibility study to determine the usefulness of PCCT in differentiation of renal cysts. A renal phantom was imaged with a grating-based PCCT system consisting of a standard rotating anode x-ray tube (40 kV, 70 mA) and a Pilatus II photoncounting detector (pixel size: 172 μm). The phantom is composed of a renal equivalent soft-tissue and cystic lesions grouped in non-enhancing cyst and hemorrhage series and an iodine enhancing series. The acquired projection images (absorption and phase-contrast) are reconstructed with a standard filtered backprojection algorithm. For evaluation both reconstructions are compared in respect to contrast-to-noise ratio (CNR), signal-to-noise ratio (SNR), and subjective image quality. We found that with PCCT a significantly improved differentiation between hemorrhage renal cysts from contrast enhancing malignant cysts is possible. If comparing PCCT and CT with respect to CNR and SNR, PCCT shows significant improvements. In conclusion, PCCT has the potential to improve the diagnostics and characterization of renal cysts without using any contrast agents. These results in combination with a non-synchrotron setup indicate a future paradigm shift in diagnostic computed tomography.

Predicting Gene Structures from Multiple RT-PCR Tests

NASA Astrophysics Data System (ADS)

Kováč, Jakub; Vinař, Tomáš; Brejová, Broňa

It has been demonstrated that the use of additional information such as ESTs and protein homology can significantly improve accuracy of gene prediction. However, many sources of external information are still being omitted from consideration. Here, we investigate the use of product lengths from RT-PCR experiments in gene finding. We present hardness results and practical algorithms for several variants of the problem and apply our methods to a real RT-PCR data set in the Drosophila genome. We conclude that the use of RT-PCR data can improve the sensitivity of gene prediction and locate novel splicing variants.

Quantitative PCR for detection of Shigella improves ascertainment of Shigella burden in children with moderate-to-severe diarrhea in low-income countries.

PubMed

Lindsay, Brianna; Ochieng, John B; Ikumapayi, Usman N; Toure, Aliou; Ahmed, Dilruba; Li, Shan; Panchalingam, Sandra; Levine, Myron M; Kotloff, Karen; Rasko, David A; Morris, Carolyn R; Juma, Jane; Fields, Barry S; Dione, Michel; Malle, Dramane; Becker, Stephen M; Houpt, Eric R; Nataro, James P; Sommerfelt, Halvor; Pop, Mihai; Oundo, Joe; Antonio, Martin; Hossain, Anowar; Tamboura, Boubou; Stine, O Colin

2013-06-01

Estimates of the prevalence of Shigella spp. are limited by the suboptimal sensitivity of current diagnostic and surveillance methods. We used a quantitative PCR (qPCR) assay to detect Shigella in the stool samples of 3,533 children aged <59 months from the Gambia, Mali, Kenya, and Bangladesh, with or without moderate-to-severe diarrhea (MSD). We compared the results from conventional culture to those from qPCR for the Shigella ipaH gene. Using MSD as the reference standard, we determined the optimal cutpoint to be 2.9 × 10(4) ipaH copies per 100 ng of stool DNA for set 1 (n = 877). One hundred fifty-eight (18%) specimens yielded >2.9 × 10(4) ipaH copies. Ninety (10%) specimens were positive by traditional culture for Shigella. Individuals with ≥ 2.9 × 10(4) ipaH copies have 5.6-times-higher odds of having diarrhea than those with <2.9 × 10(4) ipaH copies (95% confidence interval, 3.7 to 8.5; P < 0.0001). Nearly identical results were found using an independent set of samples. qPCR detected 155 additional MSD cases with high copy numbers of ipaH, a 90% increase from the 172 cases detected by culture in both samples. Among a subset (n = 2,874) comprising MSD cases and their age-, gender-, and location-matched controls, the fraction of MSD cases that were attributable to Shigella infection increased from 9.6% (n = 129) for culture to 17.6% (n = 262) for qPCR when employing our cutpoint. We suggest that qPCR with a cutpoint of approximately 1.4 × 10(4) ipaH copies be the new reference standard for the detection and diagnosis of shigellosis in children in low-income countries. The acceptance of this new standard would substantially increase the fraction of MSD cases that are attributable to Shigella.

Touch-down reverse transcriptase-PCR detection of IgV(H) rearrangement and Sybr-Green-based real-time RT-PCR quantitation of minimal residual disease in patients with chronic lymphocytic leukemia.

PubMed

Peková, Sona; Marková, Jana; Pajer, Petr; Dvorák, Michal; Cetkovský, Petr; Schwarz, Jirí

2005-01-01

Patients with chronic lymphocytic leukemia (CLL) can relapse even after aggressive therapy and autografts. It is commonly assumed that to prevent relapse the level of minimal residual disease (MRD) should be as low as possible. To evaluate MRD, highly sensitive quantitative assays are needed. The aim of the study was to develop a robust and sensitive method for detection of the clonal immunoglobulin heavy-chain variable (IgV(H)) rearrangement in CLL and to introduce a highly sensitive and specific methodology for MRD monitoring in patients with CLL who undergo intensive treatment. As a prerequisite for MRD detection, touch-down reverse transcriptase (RT)-PCR using degenerate primers were used for the diagnostic identification of (H) gene rearrangement(s). For quantitative MRD detection in 18 patients, we employed a real-time RT-PCR assay (RQ-PCR) making use of patient-specific primers and the cost-saving Sybr-Green reporter dye (SG). For precise calibration of RQ-PCR, patient-specific IgV(H) sequences were cloned. Touch-down RT-PCR with degenerate primers allowed the successful detection of IgV(H) clonal rearrangement(s) in 252 of 257 (98.1%) diagnostic samples. Biallelic rearrangements were found in 27 of 252 (10.7%) cases. Degenerate primers used for the identification of clonal expansion at diagnosis were not sensitive enough for MRD detection. In contrast, our RQ-PCR assay using patient-specific primers and SG reached the sensitivity of 10(-)(6). We demonstrated MRD in each patient tested, including four of four patients in complete remission following autologous hematopoietic stem cell transplantation (HSCT) and three of three following allogeneic 'mini'-HSCT. Increments in MRD might herald relapse; aggressive chemotherapy could induce molecular remission. Our touch-down RT-PCR has higher efficiency to detect clonal IgV(H) rearrangements including the biallelic ones. MRD quantitation of IgV(H) expression using SG-based RQ-PCR represents a highly specific

Sample pooling for real-time PCR detection and virulence determination of the footrot pathogen Dichelobacter nodosus.

PubMed

Frosth, Sara; König, Ulrika; Nyman, Ann-Kristin; Aspán, Anna

2017-09-01

Dichelobacter nodosus is the principal cause of ovine footrot and strain virulence is an important factor in disease severity. Therefore, detection and virulence determination of D. nodosus is important for proper diagnosis of the disease. Today this is possible by real-time PCR analysis. Analysis of large numbers of samples is costly and laborious; therefore, pooling of individual samples is common in surveillance programs. However, pooling can reduce the sensitivity of the method. The aim of this study was to develop a pooling method for real-time PCR analysis that would allow sensitive detection and simultaneous virulence determination of D. nodosus. A total of 225 sheep from 17 flocks were sampled using ESwabs within the Swedish Footrot Control Program in 2014. Samples were first analysed individually and then in pools of five by real-time PCR assays targeting the 16S rRNA and aprV2/B2 genes of D. nodosus. Each pool consisted of four negative and one positive D. nodosus samples with varying amounts of the bacterium. In the individual analysis, 61 (27.1%) samples were positive in the 16S rRNA and the aprV2/B2 PCR assays and 164 (72.9%) samples were negative. All samples positive in the aprV2/B2 PCR-assay were of aprB2 variant. The pooled analysis showed that all 41 pools were also positive for D. nodosus 16S rRNA and the aprB2 variant. The diagnostic sensitivity for pooled and individual samples was therefore similar. Our method includes concentration of the bacteria before DNA-extraction. This may account for the maintenance of diagnostic sensitivity. Diagnostic sensitivity in the real-time PCR assays of the pooled samples were comparable to the sensitivity obtained for individually analysed samples. Even sub-clinical infections were able to be detected in the pooled PCR samples which is important for control of the disease. This method may therefore be implemented in footrot control programs where it can replace analysis of individual samples.

Molecular diagnostics of the honey bee parasites Lotmaria passim and Crithidia spp. (Trypanosomatidae) using multiplex PCR

USDA-ARS?s Scientific Manuscript database

Lotmaria passim Schwarz is a recently described trypanosome parasite of honey bees in continental United States, Europe, and Japan. We developed a multiplex PCR technique using a PCR primer specific for L. passim to distinguish this species from C. mellificae. We report the presence of L. passim in ...

Rapid group-, serotype-, and vaccine strain-specific identification of poliovirus isolates by real-time reverse transcription-PCR using degenerate primers and probes containing deoxyinosine residues.

PubMed

Kilpatrick, David R; Yang, Chen-Fu; Ching, Karen; Vincent, Annelet; Iber, Jane; Campagnoli, Ray; Mandelbaum, Mark; De, Lina; Yang, Su-Ju; Nix, Allan; Kew, Olen M

2009-06-01

We have adapted our previously described poliovirus diagnostic reverse transcription-PCR (RT-PCR) assays to a real-time RT-PCR (rRT-PCR) format. Our highly specific assays and rRT-PCR reagents are designed for use in the WHO Global Polio Laboratory Network for rapid and large-scale identification of poliovirus field isolates.

Semiquantitative Multiplexed Tandem PCR for Detection and Differentiation of Four Theileria orientalis Genotypes in Cattle

PubMed Central

Perera, Piyumali K.; Gasser, Robin B.; Firestone, Simon M.; Smith, Lee; Roeber, Florian

2014-01-01

Oriental theileriosis is an emerging, tick-borne disease of bovines in the Asia-Pacific region and is caused by one or more genotypes of the Theileria orientalis complex. This study aimed to establish and validate a multiplexed tandem PCR (MT-PCR) assay using three distinct markers (major piroplasm surface protein, 23-kDa piroplasm membrane protein, and the first internal transcribed spacer of nuclear DNA), for the simultaneous detection and semiquantification of four genotypes (Buffeli, Chitose, Ikeda, and type 5) of the T. orientalis complex. Analytical specificity, analytical sensitivity, and repeatability of the established MT-PCR assay were assessed in a series of experiments. Subsequently, the assay was evaluated using 200 genomic DNA samples collected from cattle from farms on which oriental theileriosis outbreaks had occurred, and 110 samples from a region where no outbreaks had been reported. The results showed the MT-PCR assay specifically and reproducibly detected the expected genotypes (i.e., genotypes Buffeli, Chitose, Ikeda, and type 5) of the T. orientalis complex, reliably differentiated them, and was able to detect as little as 1 fg of genomic DNA from each genotype. The diagnostic specificity and sensitivity of the MT-PCR were estimated at 94.0% and 98.8%, respectively. The MT-PCR assay established here is a practical and effective diagnostic tool for the four main genotypes of T. orientalis complex in Australia and should assist studies of the epidemiology and pathophysiology of oriental theileriosis in the Asia-Pacific region. PMID:25339402

Cytological diagnostic of lymphadenitis tuberculosis by eosinophilic material

NASA Astrophysics Data System (ADS)

Delyuzar; Amir, Z.; Kusumawati, L.

2018-03-01

AFB sputum and chest X-ray are used to identify patients with pulmonary TB. For extrapulmonary TB, fine needle aspiration cytology is needed, even though occasionally found not atypical feature in the form of eosinophilic material with dark brown particles, suspected as TB. This research was to show that eosinophilic material with dark brown particles is accurate as new criteria for the cytological diagnosis of TB. By performing fine needle aspiration biopsy stained with Giemsa, if an eosinophilic material with dark brown particles was encountered, we continued with Ziehl-Neelsen AFB stain and confirmed with PCR. To assess accuracy, we used a diagnostic test to evaluate sensitivity and specificity of eosinophilic material with dark brown particles by using AFB and PCR as the gold standard. The sensitivity and specificity of cytological diagnosis in tuberculosis of eosinophilic material with dark brown particles were 93.65% and 70.99%, respectively if confirmed with AFB. On the other hand, if confirmed with PCR using Mycobacterium tuberculosis DNA, the sensitivity and specificity were 98.95% and 96.79%, respectively. In conclusion, eosinophilic masses with dark brown particles is accurate as new criteria of TB diagnostic cytology with high sensitivity and specificity confirmed with AFB and PCR test.

A novel quantitative reverse-transcription PCR (qRT-PCR) for the enumeration of total bacteria, using meat micro-flora as a model.

PubMed

Dolan, Anthony; Burgess, Catherine M; Barry, Thomas B; Fanning, Seamus; Duffy, Geraldine

2009-04-01

A sensitive quantitative reverse-transcription PCR (qRT-PCR) method was developed for enumeration of total bacteria. Using two sets of primers separately to target the ribonuclease-P (RNase P) RNA transcripts of gram positive and gram negative bacteria. Standard curves were generated using SYBR Green I kits for the LightCycler 2.0 instrument (Roche Diagnostics) to allow quantification of mixed microflora in liquid media. RNA standards were used and extracted from known cell equivalents and subsequently converted to cDNA for the construction of standard curves. The number of mixed bacteria in culture was determined by qRT-PCR, and the results correlated (r(2)=0.88, rsd=0.466) with the total viable count over the range from approx. Log(10) 3 to approx. Log(10) 7 CFU ml(-1). The rapid nature of this assay (8 h) and its potential as an alternative method to the standard plate count method to predict total viable counts and shelf life are discussed.

Quantitative phase imaging to improve the diagnostic accuracy of urine cytology.

PubMed

Pham, Hoa V; Pantanowitz, Liron; Liu, Yang

2016-09-01

A definitive diagnosis of urothelial carcinoma in urine cytology is often challenging and subjective. Many urine cytology samples receive an indeterminate diagnosis. Ancillary techniques such as fluorescence in situ hybridization (FISH) have been used to improve the diagnostic sensitivity, but FISH is not approved as a routine screening test, and the complex fluorescent staining protocol also limits its widespread clinical use. Quantitative phase imaging (QPI) is an emerging technology allowing accurate measurements of the single-cell dry mass. This study was undertaken to explore the ability of QPI to improve the diagnostic accuracy of urine cytology for malignancy. QPI was performed on unstained, ThinPrep-prepared urine cytology slides from 28 patients with 4 categories of cytological diagnoses (negative, atypical, suspicious, and positive for malignancy). The nuclear/cell dry mass, the entropy, and the nucleus-to-cell mass ratio were calculated for several hundred cells for each patient, and they were then correlated with the follow-up diagnoses. The nuclear mass and nuclear mass entropy of urothelial cells showed significant differences between negative and positive groups. These data showed a progressive increase from patients with negative diagnosis, to patients with atypical/suspicious and positive cytologic diagnosis. Most importantly, among the patients in the atypical and suspicious diagnosis, the nuclear mass and its entropy were significantly higher for those patients with a follow-up diagnosis of malignancy than those patients without a subsequent follow-up diagnosis of malignancy. QPI shows potential for improving the diagnostic accuracy of urine cytology, especially for indeterminate cases, and should be further evaluated as an ancillary test for urine cytology. Cancer Cytopathol 2016;124:641-50. © 2016 American Cancer Society. © 2016 American Cancer Society.

Development and evaluation of tailored specific real-time RT-PCR assays for detection of foot-and-mouth disease virus serotypes circulating in East Africa.

PubMed

Bachanek-Bankowska, Katarzyna; Mero, Herieth R; Wadsworth, Jemma; Mioulet, Valerie; Sallu, Raphael; Belsham, Graham J; Kasanga, Christopher J; Knowles, Nick J; King, Donald P

2016-11-01

Rapid, reliable and accurate diagnostic methods provide essential support to programmes that monitor and control foot-and-mouth disease (FMD). While pan-specific molecular tests for FMD virus (FMDV) detection are well established and widely used in endemic and FMD-free countries, current serotyping methods mainly rely either on antigen detection ELISAs or nucleotide sequencing approaches. This report describes the development of a panel of serotype-specific real-time RT-PCR assays (rRT-PCR) tailored to detect FMDV lineages currently circulating in East Africa. These assays target sequences within the VP1-coding region that share high intra-lineage identity, but do not cross-react with FMD viruses from other serotypes that circulate in the region. These serotype-specific assays operate with the same thermal profile as the pan-diagnostic tests making it possible to run them in parallel to produce C T values comparable to the pan-diagnostic test detecting the 3D-coding region. These assays were evaluated alongside the established pan-specific molecular test using field samples and virus isolates collected from Tanzania, Kenya and Ethiopia that had been previously characterised by nucleotide sequencing. Samples (n=71) representing serotype A (topotype AFRICA, lineage G-I), serotype O (topotypes EA-2 and EA-4), serotype SAT 1 (topotype I (NWZ)) and serotype SAT2 (topotype IV) were correctly identified with these rRT-PCR assays. Furthermore, FMDV RNA from samples that did not contain infectious virus could still be serotyped using these assays. These serotype-specific real-time RT-PCR assays can detect and characterise FMDVs currently circulating in East Africa and hence improve disease control in this region. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Clinical utility of an optimised multiplex real-time PCR assay for the identification of pathogens causing sepsis in Vietnamese patients.

PubMed

Tat Trung, Ngo; Van Tong, Hoang; Lien, Tran Thi; Van Son, Trinh; Thanh Huyen, Tran Thi; Quyen, Dao Thanh; Hoan, Phan Quoc; Meyer, Christian G; Song, Le Huu

2018-02-01

For the identification of bacterial pathogens, blood culture is still the gold standard diagnostic method. However, several disadvantages apply to blood cultures, such as time and rather large volumes of blood sample required. We have previously established an optimised multiplex real-time PCR method in order to diagnose bloodstream infections. In the present study, we evaluated the diagnostic performance of this optimised multiplex RT-PCR in blood samples collected from 110 septicaemia patients enrolled at the 108 Military Central Hospital, Hanoi, Vietnam. Positive results were obtained by blood culture, the Light Cylcler-based SeptiFast ® assay and our multiplex RT-PCR in 35 (32%), 31 (28%), and 31 (28%) samples, respectively. Combined use of the three methods confirmed 50 (45.5%) positive cases of bloodstream infection, a rate significantly higher compared to the exclusive use of one of the three methods (P=0.052, 0.012 and 0.012, respectively). The sensitivity, specificity and area under the curve (AUC) of our assay were higher compared to that of the SeptiFast ® assay (77.4%, 86.1% and 0.8 vs. 67.7%, 82.3% and 0.73, respectively). Combined use of blood culture and multiplex RT-PCR assay showed a superior diagnostic performance, as the sensitivity, specificity, and AUC reached 83.3%, 100%, and 0.95, respectively. The concordance between blood culture and the multiplex RT-PCR assay was highest for Klebsiella pneumonia (100%), followed by Streptococcus spp. (77.8%), Escherichia coli (66.7%), Staphylococcus spp. (50%) and Salmonella spp. (50%). In addition, the use of the newly established multiplex RT-PCR assay increased the spectrum of identifiable agents (Acintobacter baumannii, 1/32; Proteus mirabilis, 1/32). The combination of culture and the multiplex RT-PCR assay provided an excellent diagnostic accomplishment and significantly supported the identification of causative pathogens in clinical samples obtained from septic patients. Copyright © 2017 The

Aspergillus Galactomannan Enzyme Immunoassay and Quantitative PCR for Diagnosis of Invasive Aspergillosis with Bronchoalveolar Lavage Fluid

PubMed Central

Musher, Benjamin; Fredricks, David; Leisenring, Wendy; Balajee, S. Arunmozhi; Smith, Caitlin; Marr, Kieren A.

2004-01-01

Invasive pulmonary aspergillosis (IPA) is frequent and often fatal in hematopoietic stem cell transplant patients. Diagnosis requires microbiological or histopathologic demonstration of the organism in tissues; however, cultivation of Aspergillus species from respiratory secretions has low diagnostic sensitivity. Assays to detect Aspergillus antigen or DNA in bronchoalveolar lavage (BAL) fluid could facilitate earlier diagnosis, thereby guiding optimal therapy and obviating the need for additional costly and potentially morbid diagnostic evaluation. We evaluated the performance of a galactomannan enzyme immunoassay (GM EIA; Bio-Rad) by using a range of index cutoffs to define positivity and a quantitative PCR (qPCR) assay for the detection of Aspergillus species from BAL samples of patients with proven and probable IPA (case patients; n = 49) and without IPA (control patients; n = 50). The sensitivity of the GM EIA was 61% with an index cutoff of 1.0 and 76% with an index cutoff of 0.5; the corresponding specificities were 98 and 94%, respectively. The sensitivity and specificity of qPCR assay were 67 and 100%, respectively. The sensitivity with 22 culture-negative BAL specimens from patients with IPA was 41% for GM EIA with an index cutoff of 1.0, 59% for GM EIA with an index cutoff of 0.5, and 36% for qPCR assay. GM EIA indices and DNA quantities corresponded to BAL fungal burdens, with culture-positive samples having larger amounts of antigen and DNA compared to culture-negative samples. GM EIA and qPCR assay add to the sensitivity of BAL for diagnosing IPA in high-risk patients, with excellent specificity. Adjunctive use of these tests may reduce dependence on invasive diagnostic procedures. PMID:15583275

Improving statistical inference on pathogen densities estimated by quantitative molecular methods: malaria gametocytaemia as a case study.

PubMed

Walker, Martin; Basáñez, María-Gloria; Ouédraogo, André Lin; Hermsen, Cornelus; Bousema, Teun; Churcher, Thomas S

2015-01-16

Quantitative molecular methods (QMMs) such as quantitative real-time polymerase chain reaction (q-PCR), reverse-transcriptase PCR (qRT-PCR) and quantitative nucleic acid sequence-based amplification (QT-NASBA) are increasingly used to estimate pathogen density in a variety of clinical and epidemiological contexts. These methods are often classified as semi-quantitative, yet estimates of reliability or sensitivity are seldom reported. Here, a statistical framework is developed for assessing the reliability (uncertainty) of pathogen densities estimated using QMMs and the associated diagnostic sensitivity. The method is illustrated with quantification of Plasmodium falciparum gametocytaemia by QT-NASBA. The reliability of pathogen (e.g. gametocyte) densities, and the accompanying diagnostic sensitivity, estimated by two contrasting statistical calibration techniques, are compared; a traditional method and a mixed model Bayesian approach. The latter accounts for statistical dependence of QMM assays run under identical laboratory protocols and permits structural modelling of experimental measurements, allowing precision to vary with pathogen density. Traditional calibration cannot account for inter-assay variability arising from imperfect QMMs and generates estimates of pathogen density that have poor reliability, are variable among assays and inaccurately reflect diagnostic sensitivity. The Bayesian mixed model approach assimilates information from replica QMM assays, improving reliability and inter-assay homogeneity, providing an accurate appraisal of quantitative and diagnostic performance. Bayesian mixed model statistical calibration supersedes traditional techniques in the context of QMM-derived estimates of pathogen density, offering the potential to improve substantially the depth and quality of clinical and epidemiological inference for a wide variety of pathogens.

Comparison of PCR methods for the detection of genetic variants of carp edema virus.

PubMed

Adamek, Mikolaj; Matras, Marek; Jung-Schroers, Verena; Teitge, Felix; Heling, Max; Bergmann, Sven M; Reichert, Michal; Way, Keith; Stone, David M; Steinhagen, Dieter

2017-09-20

The infection of common carp and its ornamental variety, koi, with the carp edema virus (CEV) is often associated with the occurrence of a clinical disease called 'koi sleepy disease'. The disease may lead to high mortality in both koi and common carp populations. To prevent further spread of the infection and the disease, a reliable detection method for this virus is required. However, the high genetic variability of the CEV p4a gene used for PCR-based diagnostics could be a serious obstacle for successful and reliable detection of virus infection in field samples. By analysing 39 field samples from different geographical origins obtained from koi and farmed carp and from all 3 genogroups of CEV, using several recently available PCR protocols, we investigated which of the protocols would allow the detection of CEV from all known genogroups present in samples from Central European carp or koi populations. The comparison of 5 different PCR protocols showed that the PCR assays (both end-point and quantitative) developed in the Centre for Environment, Fisheries and Aquaculture Science exhibited the highest analytical inclusivity and diagnostic sensitivity. Currently, this makes them the most suitable protocols for detecting viruses from all known CEV genogroups.

Malaria diagnosis by PCR revealed differential distribution of mono and mixed species infections by Plasmodium falciparum and P. vivax in India

PubMed Central

Dash, Manoswini; Kar, Sonalika; Rani, Swati; Rawal, Charu; Singh, Rajkumar; Anvikar, Anupkumar R.; Pande, Veena

2018-01-01

Malaria is a vector-borne infectious disease, caused by five different species of the genus Plasmodium, and is endemic to many tropical and sub-tropical countries of the globe. At present, malaria diagnosis at the primary health care level in India is conducted by either microscopy or rapid diagnostic test (RDT). In recent years, molecular diagnosis (by PCR assay), has emerged as the most sensitive method for malaria diagnosis. India is highly endemic to malaria and shoulders the burden of two major malaria parasites, Plasmodium falciparum and P. vivax. Previous studies using PCR diagnostic assay had unraveled several interesting facts on distribution of malaria parasites in India. However, these studies had several limitations from small sample size to limited geographical areas of sampling. In order to mitigate these limitations, we have collected finger-prick blood samples from 2,333 malaria symptomatic individuals in nine states from 11 geographic locations, covering almost the entire malaria endemic regions of India and performed all the three diagnostic tests (microscopy, RDT and PCR assay) and also have conducted comparative assessment on the performance of the three diagnostic tests. Since PCR assay turned out to be highly sensitive (827 malaria positive cases) among the three types of tests, we have utilized data from PCR diagnostic assay for analyses and inferences. The results indicate varied distributional prevalence of P. vivax and P. falciparum according to locations in India, and also the mixed species infection due to these two species. The proportion of P. falciparum to P. vivax was found to be 49:51, and percentage of mixed species infections due to these two parasites was found to be 13% of total infections. Considering India is set for malaria elimination by 2030, the present malaria epidemiological information is of high importance. PMID:29565981

Malaria diagnosis by PCR revealed differential distribution of mono and mixed species infections by Plasmodium falciparum and P. vivax in India.

PubMed

Siwal, Nisha; Singh, Upasana Shyamsunder; Dash, Manoswini; Kar, Sonalika; Rani, Swati; Rawal, Charu; Singh, Rajkumar; Anvikar, Anupkumar R; Pande, Veena; Das, Aparup

2018-01-01

Malaria is a vector-borne infectious disease, caused by five different species of the genus Plasmodium, and is endemic to many tropical and sub-tropical countries of the globe. At present, malaria diagnosis at the primary health care level in India is conducted by either microscopy or rapid diagnostic test (RDT). In recent years, molecular diagnosis (by PCR assay), has emerged as the most sensitive method for malaria diagnosis. India is highly endemic to malaria and shoulders the burden of two major malaria parasites, Plasmodium falciparum and P. vivax. Previous studies using PCR diagnostic assay had unraveled several interesting facts on distribution of malaria parasites in India. However, these studies had several limitations from small sample size to limited geographical areas of sampling. In order to mitigate these limitations, we have collected finger-prick blood samples from 2,333 malaria symptomatic individuals in nine states from 11 geographic locations, covering almost the entire malaria endemic regions of India and performed all the three diagnostic tests (microscopy, RDT and PCR assay) and also have conducted comparative assessment on the performance of the three diagnostic tests. Since PCR assay turned out to be highly sensitive (827 malaria positive cases) among the three types of tests, we have utilized data from PCR diagnostic assay for analyses and inferences. The results indicate varied distributional prevalence of P. vivax and P. falciparum according to locations in India, and also the mixed species infection due to these two species. The proportion of P. falciparum to P. vivax was found to be 49:51, and percentage of mixed species infections due to these two parasites was found to be 13% of total infections. Considering India is set for malaria elimination by 2030, the present malaria epidemiological information is of high importance.

[REAL TIME POLYMERASE CHAIN REACTION IN TULAREMIA LABORATORY DIAGNOSTICS].

PubMed

Kormilitsyna, M I; Mescheryakova, I S; Mikhailova, T V; Dobrovolsky, A A

2015-01-01

Enhancement of tularemia laboratory diagnostics by F. tularensis DNA determination in blood sera of patients using real time polymerase chain reaction (RT-PCR). 39 blood sera of patients obtained during transmissive epidemic outbreak of tularemia in Khanty-Mansiysk in 2013 were studied in agglutination reaction, passive hemagglutination, RT-PCR. Specific primers and fluorescent probes were used: ISFTu2F/R+ISFTu2P, Tu14GF/R+tul4-PR2. Advantages of using RT-PCR for early diagnostics of tularemia, when specific antibodies are not detected using traditional immunologic methods, were established. Use of a combination of primers and ISFTu2F/R+ISFTu2P probe allowed to detect F. tularensis DNA in 100% of sera, whereas Tul4G F/R+tul4-PR2 combination--92% of sera. The data were obtained when DNA was isolated from sera using "Proba Rapid" express method. Clinical-epidemiologic diagnosis oftularemia was confirmed by both immune-serologic and RT-PCR methods when sera were studied 3-4 weeks after the onset of the disease. RT-PCR with ISFTu2F/R primers and fluorescent probe ISFTu2P, having high sensitivity and specificity, allows to determine F. tularensis DNA in blood sera of patients at both the early stage and 3-4 weeks after the onset of the disease.

Concordance of IHC, FISH and RT-PCR for EML4-ALK rearrangements.

PubMed

Teixidó, Cristina; Karachaliou, Niki; Peg, Vicente; Gimenez-Capitan, Ana; Rosell, Rafael

2014-04-01

The echinoderm microtubule-associated protein-like 4 anaplastic lymphoma kinase (EML4-ALK) has emerged as the second most important driver oncogene in lung cancer and the first targetable fusion oncokinase to be identified in 4-6% of lung adenocarcinomas. Crizotinib, along with a diagnostic test-the Vysis ALK Break Apart fluorescence in situ hybridization (FISH) Probe Kit-is approved for the treatment of ALK positive advanced non-small cell lung cancer (NSCLC). However, the success of a targeted drug is critically dependent on a sensitive and specific screening assay to detect the molecular drug target. In our experience, reverse transcription polymerase chain reaction (RT-PCR)-based detection of EML4-ALK is a more sensitive and reliable approach compared to FISH and immunohistochemistry (IHC). Although ALK FISH is clinically validated, the assay can be technically challenging and other diagnostic modalities, including IHC and RT-PCR should be further explored.

Diagnostic needle arthroscopy and the economics of improved diagnostic accuracy: a cost analysis.

PubMed

Voigt, Jeffrey D; Mosier, Michael; Huber, Bryan

2014-10-01

Hundreds of thousands of surgical arthroscopy procedures are performed annually in the United States (US) based on MRI findings. There are situations where these MRI findings are equivocal or indeterminate and because of this clinicians commonly perform the arthroscopy in order not to miss pathology. Recently, a less invasive needle arthroscopy system has been introduced that is commonly performed in the physician office setting and that may help improve the accuracy of diagnostic findings. This in turn may prevent unnecessary follow-on arthroscopy procedures from being performed. The purpose of this analysis is to determine whether the in-office diagnostic needle arthroscopy system can provide cost savings by reducing unnecessary follow on arthroscopy procedures. Data obtained from a recent trial and from a systematic review were used in comparing the accuracy of MRI and VisionScope needle arthroscopy (VSI) with standard arthroscopy (gold standard). The resultant false positive and false negative findings were then used to evaluate the costs of follow-on procedures. These differences were then modeled for the US patient population diagnosed and treated for meniscal knee pathology (most common disorder) to determine if a technology such as VSI could save the US healthcare system money. Data on surgical arthroscopy procedures in the US for meniscal knee pathology were used (calendar year [CY] 2010). The costs of performing diagnostic and surgical arthroscopy procedures (using CY 2013 Medicare reimbursement amounts), costs associated with false negative findings, and the costs for treating associated complications arising from diagnostic and therapeutic arthroscopy procedures were assessed. In patients presenting with medial meniscal pathology (International Classification of Diseases, 9th edition, Clinical Modification [ICD9CM] diagnosis 836.0), VSI in place of MRI (standard of care) resulted in a net cost savings to the US system of US$115-US$177 million (CY 2013

Identification of Erwinia species isolated from apples and pears by differential PCR.

PubMed

Gehring, I; Geider, K

2012-04-01

Many pathogenic and epiphytic bacteria isolated from apples and pears belong to the genus Erwinia; these include the species E. amylovora, E. pyrifoliae, E. billingiae, E. persicina, E. rhapontici and E. tasmaniensis. Identification and classification of freshly isolated bacterial species often requires tedious taxonomic procedures. To facilitate routine identification of Erwinia species, we have developed a PCR method based on species-specific oligonucleotides (SSOs) from the sequences of the housekeeping genes recA and gpd. Using species-specific primers that we report here, differentiation was done with conventional PCR (cPCR) and quantitative PCR (qPCR) applying two consecutive primer annealing temperatures. The specificity of the primers depends on terminal Single Nucleotide Polymorphisms (SNPs) that are characteristic for the target species. These PCR assays enabled us to distinguish eight Erwinia species, as well as to identify new Erwinia isolates from plant surfaces. When performed with mixed bacterial cultures, they only detected a single target species. This method is a novel approach to classify strains within the genus Erwinia by PCR and it can be used to confirm other diagnostic data, especially when specific PCR detection methods are not already available. The method may be applied to classify species within other bacterial genera. Copyright © 2012 Elsevier B.V. All rights reserved.

One time quantitative PCR detection of Pseudomonas aeruginosa to discriminate intermittent from chronic infection in cystic fibrosis.

PubMed

Boutin, Sébastien; Weitnauer, Michael; Hassel, Selina; Graeber, Simon Y; Stahl, Mirjam; Dittrich, A Susanne; Mall, Marcus A; Dalpke, Alexander H

2018-05-01

Chronic airway infection with Pseudomonas aeruginosa is a major risk factor of progression of lung disease in patients with cystic fibrosis (CF). Chronic P. aeruginosa infection evolves from intermittent infection that is amenable to antibiotic eradication, whereas chronically adapted P. aeruginosa becomes resistant to antibiotic therapy. Discrimination of intermittent versus chronic infection is therefore of high therapeutic relevance, yet the available diagnostic methods are only partly satisfactory. The aim of the present study was, therefore, to evaluate the usage of quantitative PCR (qPCR) to measure pathogen abundance and to discriminate between intermittent and chronic Pseudomonas infection in patients with CF. Using an established qPCR protocol, we analyzed the abundance of P. aeruginosa in 141 throats swabs and 238 sputa from CF patients with intermittent or chronic infection with P. aeruginosa, as determined by standard culture based diagnostics. We observed a large increase of abundance of P. aeruginosa in throat swabs and sputum samples from patients with chronic compared to intermittent infections with P. aeruginosa. The data show that abundance of P. aeruginosa as measured by qPCR is a valuable tool to discriminate intermittent from chronic infection. Of note, P. aeruginosa burden seems more sensitive than mucoidity phenotype to discriminate chronic from intermittent strains. Furthermore we observed that molecular detection in throat swabs was linked to a viable culture in the sputum when sputum was available. This result is of special interest in young patients with cystic fibrosis that often cannot expectorate sputum. We also observed that qPCR in comparison to culture detected the infection earlier. The results suggest that qPCR detection and quantification of P. aeruginosa is a precious tool to be added to the diagnostic toolbox in cystic fibrosis. Copyright © 2018 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

A Review of Conventional PCR Assays for the Detection of Selected Phytopathogens of Wheat.

PubMed

Kuzdraliński, Adam; Kot, Anna; Szczerba, Hubert; Nowak, Michał; Muszyńska, Marta

2017-01-01

Infection of phyllosphere (stems, leaves, husks, and grains) by pathogenic fungi reduces the wheat yield and grain quality. Detection of the main wheat pathogenic fungi provides information about species composition and allows effective and targeted plant treatment. Since conventional procedures for the detection of these organisms are unreliable and time consuming, diagnostic DNA-based methods are required. Nucleic acid amplification technologies are independent of the morphological and biochemical characteristics of fungi. Microorganisms do not need to be cultured. Therefore, a number of PCR-based methodologies have been developed for the identification of key pathogenic fungi, such as Fusarium spp., Puccinia spp., Zymoseptoria tritici, Parastagonospora nodorum, Blumeria graminis f. sp. tritici, and Pyrenophora tritici-repentis. This article reviews frequently used DNA regions for fungus identification and discusses already known PCR assays for detection of the aforementioned wheat pathogens. We demonstrate that PCR-based wheat pathogen identification assays require further research. In particular, the number of diagnostic tests for Fusarium graminearum, Puccinia spp., and P. tritici-repentis are insufficient. © 2017 S. Karger AG, Basel.

Specific Primers for Rapid Detection of Microsporum audouinii by PCR in Clinical Samples▿

PubMed Central

Roque, H. D.; Vieira, R.; Rato, S.; Luz-Martins, M.

2006-01-01

This report describes application of PCR fingerprinting to identify common species of dermatophytes using the microsatellite primers M13, (GACA)4, and (GTG)5. The initial PCR analysis rendered a specific DNA fragment for Microsporum audouinii, which was cloned and sequenced. Based on the sequencing data of this fragment, forward (MA_1F) and reverse (MA_1R) primers were designed and verified by PCR to establish their reliability in the diagnosis of M. audouinii. These primers produced a singular PCR band of 431 bp specific only to strains and isolates of M. audouinii, based on a global test of 182 strains/isolates belonging to 11 species of dermatophytes. These findings indicate these primers are reliable for diagnostic purposes, and we recommend their use in laboratory analysis. PMID:17005755

Current status and future perspectives on molecular and serological methods in diagnostic mycology.

PubMed

Lau, Anna; Chen, Sharon; Sleiman, Sue; Sorrell, Tania

2009-11-01

Invasive fungal infections are an important cause of infectious morbidity. Nonculture-based methods are increasingly used for rapid, accurate diagnosis to improve patient outcomes. New and existing DNA amplification platforms have high sensitivity and specificity for direct detection and identification of fungi in clinical specimens. Since laboratories are increasingly reliant on DNA sequencing for fungal identification, measures to improve sequence interpretation should support validation of reference isolates and quality control in public gene repositories. Novel technologies (e.g., isothermal and PNA FISH methods), platforms enabling high-throughput analyses (e.g., DNA microarrays and Luminex xMAP) and/or commercial PCR assays warrant further evaluation for routine diagnostic use. Notwithstanding the advantages of molecular tests, serological assays remain clinically useful for patient management. The serum Aspergillus galactomannan test has been incorporated into diagnostic algorithms of invasive aspergillosis. Both the galactomannan and the serum beta-D-glucan test have value for diagnosing infection and monitoring therapeutic response.

A PCR-based tool for the cultivation-independent monitoring of Pandora neoaphidis.

PubMed

Fournier, A; Enkerli, J; Keller, S; Widmer, F

2008-09-01

Pandora neoaphidis is one of the most important fungal pathogens of aphids and has a great potential for use in biocontrol. Little is known on how this fungus persists in an area and in particular on its overwintering strategies. It is hypothesized that natural areas play an important role for survival and that soil may serve as a source of inoculum for new aphid populations in spring. To test these hypotheses, a cultivation-independent PCR-based diagnostic tool was developed, that allows the detection of P. neoaphidis in the environment. Two P. neoaphidis specific PCR primer pairs were designed, targeting sequences in the ribosomal RNA gene cluster. Specificity of both primer pairs was demonstrated with P. neoaphidis and non-target close entomophthoralean relatives. Moreover, single amplicons of expected sizes were obtained with both primer pairs from various environmental sample types, including aphid cadavers, plant material, and soil. The PCR-based diagnostic tool was applied to investigate the persistence of P. neoaphidis in soil samples obtained in 2004/2005 from a nettle field harboring infected aphids in fall 2004. P. neoaphidis was detected in every sample collected in November 2004 and March 2005, suggesting an overwintering stage of P. neoaphidis in top soil layers. The developed cultivation-independent PCR-based tool will be valuable for further investigation of the ecology of P. neoaphidis and for the development and future implementation of management strategies against aphids involving conservation biocontrol.

EQUAL-quant: an international external quality assessment scheme for real-time PCR.

PubMed

Ramsden, Simon C; Daly, Sarah; Geilenkeuser, Wolf-Jochen; Duncan, Graeme; Hermitte, Fabienne; Marubini, Ettore; Neumaier, Michael; Orlando, Claudio; Palicka, Vladimir; Paradiso, Angelo; Pazzagli, Mario; Pizzamiglio, Sara; Verderio, Paolo

2006-08-01

Quantitative gene expression analysis by real-time PCR is important in several diagnostic areas, such as the detection of minimum residual disease in leukemia and the prognostic assessment of cancer patients. To address quality assurance in this technically challenging area, the European Union (EU) has funded the EQUAL project to develop methodologic external quality assessment (EQA) relevant to diagnostic and research laboratories among the EU member states. We report here the results of the EQUAL-quant program, which assesses standards in the use of TaqMan probes, one of the most widely used assays in the implementation of real-time PCR. The EQUAL-quant reagent set was developed to assess the technical execution of a standard TaqMan assay, including RNA extraction, reverse transcription, and real-time PCR quantification of target DNA copy number. The multidisciplinary EQA scheme included 137 participating laboratories from 29 countries. We demonstrated significant differences in performance among laboratories, with 20% of laboratories reporting at least one result lacking in precision and/or accuracy according to the statistical procedures described. No differences in performance were observed for the >10 different testing platforms used by the study participants. This EQA scheme demonstrated both the requirement and demand for external assessment of technical standards in real-time PCR. The reagent design and the statistical tools developed within this project will provide a benchmark for defining acceptable working standards in this emerging technology.

Combining functional and structural tests improves the diagnostic accuracy of relevance vector machine classifiers

PubMed Central

Racette, Lyne; Chiou, Christine Y.; Hao, Jiucang; Bowd, Christopher; Goldbaum, Michael H.; Zangwill, Linda M.; Lee, Te-Won; Weinreb, Robert N.; Sample, Pamela A.

2009-01-01

Purpose To investigate whether combining optic disc topography and short-wavelength automated perimetry (SWAP) data improves the diagnostic accuracy of relevance vector machine (RVM) classifiers for detecting glaucomatous eyes compared to using each test alone. Methods One eye of 144 glaucoma patients and 68 healthy controls from the Diagnostic Innovations in Glaucoma Study were included. RVM were trained and tested with cross-validation on optimized (backward elimination) SWAP features (thresholds plus age; pattern deviation (PD); total deviation (TD)) and on Heidelberg Retina Tomograph II (HRT) optic disc topography features, independently and in combination. RVM performance was also compared to two HRT linear discriminant functions (LDF) and to SWAP mean deviation (MD) and pattern standard deviation (PSD). Classifier performance was measured by the area under the receiver operating characteristic curves (AUROCs) generated for each feature set and by the sensitivities at set specificities of 75%, 90% and 96%. Results RVM trained on combined HRT and SWAP thresholds plus age had significantly higher AUROC (0.93) than RVM trained on HRT (0.88) and SWAP (0.76) alone. AUROCs for the SWAP global indices (MD: 0.68; PSD: 0.72) offered no advantage over SWAP thresholds plus age, while the LDF AUROCs were significantly lower than RVM trained on the combined SWAP and HRT feature set and on HRT alone feature set. Conclusions Training RVM on combined optimized HRT and SWAP data improved diagnostic accuracy compared to training on SWAP and HRT parameters alone. Future research may identify other combinations of tests and classifiers that can also improve diagnostic accuracy. PMID:19528827

PCR analysis is superior to histology for diagnosis of Whipple's disease mimicking seronegative rheumatic diseases.

PubMed

Lehmann, P; Ehrenstein, B; Hartung, W; Dragonas, C; Reischl, U; Fleck, M

2017-03-01

The diagnosis of Whipple's disease (WD) is commonly confirmed by histology demonstrating Periodic Acid Schiff (PAS)-positive macrophages in the duodenal mucosa. Analysis of intestinal tissue or other specimens using polymerase chain reaction (PCR) is a more sensitive method. However, the relevance of positive PCR findings is still controversial. Therefore, we evaluated the relevance of histology and PCR findings to establishing the diagnosis of WD in a series of WD patients initially presenting with suspected rheumatic diseases. Between 2006 and 2014, 20 patients with seronegative rheumatic diseases tested positive for Tropheryma whipplei (Tw) by PCR and/or histology and were enrolled in a retrospective analysis of the diagnostic value of both procedures. Seven of the 20 cases (35%) were diagnosed with 'classic' WD as indicated by PAS-positive macrophages. In the remaining 13 patients, the presence of Tw was detected by intestinal (n = 10) or synovial PCR analysis (n = 3). Two of the 20 patients (10%) with evidence of Tw did not respond to antibiotic therapy. They were not considered to suffer from WD. Therefore, relying only on histological findings of intestinal biopsies would have missed 11 (61%) of the 18 patients with WD in our cohort. In comparison, PCR of intestinal biopsies detected Tw-DNA in 14 (93%) of the 15 WD patients evaluated. Patients with a positive histology did not differ from PCR-positive patients with regard to sex, age, or duration of disease, but more often presented with gastrointestinal symptoms. A substantial number of WD patients present without typical intestinal histology findings. Additional PCR analysis of intestinal tissue or synovial fluid increased the sensitivity of the diagnostic evaluation and should be considered particularly in patients presenting with atypical seronegative rheumatic diseases and a high-risk profile for WD.

Evaluation of PCR electrospray-ionization mass spectrometry for rapid molecular diagnosis of bovine mastitis.

PubMed

Perreten, Vincent; Endimiani, Andrea; Thomann, Andreas; Wipf, Juliette R K; Rossano, Alexandra; Bodmer, Michèle; Raemy, Andreas; Sannes-Lowery, Kristin A; Ecker, David J; Sampath, Rangarajan; Bonomo, Robert A; Washington, Cicely

2013-06-01

Bovine mastitis, an inflammatory disease of the mammary gland, is one of the most costly diseases affecting the dairy industry. The treatment and prevention of this disease is linked heavily to the use of antibiotics in agriculture and early detection of the primary pathogen is essential to control the disease. Milk samples (n=67) from cows suffering from mastitis were analyzed for the presence of pathogens using PCR electrospray-ionization mass spectrometry (PCR/ESI-MS) and were compared with standard culture diagnostic methods. Concurrent identification of the primary mastitis pathogens was obtained for 64% of the tested milk samples, whereas divergent results were obtained for 27% of the samples. The PCR/ESI-MS failed to identify some of the primary pathogens in 18% of the samples, but identified other pathogens as well as microorganisms in samples that were negative by culture. The PCR/ESI-MS identified bacteria to the species level as well as yeasts and molds in samples that contained a mixed bacterial culture (9%). The sensitivity of the PCR/ESI-MS for the most common pathogens ranged from 57.1 to 100% and the specificity ranged from 69.8 to 100% using culture as gold standard. The PCR/ESI-MS also revealed the presence of the methicillin-resistant gene mecA in 16.2% of the milk samples, which correlated with the simultaneous detection of staphylococci including Staphylococcus aureus. We demonstrated that PCR/ESI-MS, a more rapid diagnostic platform compared with bacterial culture, has the significant potential to serve as an important screening method in the diagnosis of bovine clinical mastitis and has the capacity to be used in infection control programs for both subclinical and clinical disease. Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Contribution of PCR Denaturing Gradient Gel Electrophoresis Combined with Mixed Chromatogram Software Separation for Complex Urinary Sample Analysis.

PubMed

Kotásková, Iva; Mališová, Barbora; Obručová, Hana; Holá, Veronika; Peroutková, Tereza; Růžička, Filip; Freiberger, Tomáš

2017-01-01

Complex samples are a challenge for sequencing-based broad-range diagnostics. We analysed 19 urinary catheter, ureteral Double-J catheter, and urine samples using 3 methodological approaches. Out of the total 84 operational taxonomic units, 37, 61, and 88% were identified by culture, PCR-DGGE-SS (PCR denaturing gradient gel electrophoresis followed by Sanger sequencing), and PCR-DGGE-RM (PCR- DGGE combined with software chromatogram separation by RipSeq Mixed tool), respectively. The latter approach was shown to be an efficient tool to complement culture in complex sample assessment. © 2017 S. Karger AG, Basel.

PCR performance of a thermostable heterodimeric archaeal DNA polymerase

PubMed Central

Killelea, Tom; Ralec, Céline; Bossé, Audrey; Henneke, Ghislaine

2014-01-01

DNA polymerases are versatile tools used in numerous important molecular biological core technologies like the ubiquitous polymerase chain reaction (PCR), cDNA cloning, genome sequencing, and nucleic acid based diagnostics. Taking into account the multiple DNA amplification techniques in use, different DNA polymerases must be optimized for each type of application. One of the current tendencies is to reengineer or to discover new DNA polymerases with increased performance and broadened substrate spectra. At present, there is a great demand for such enzymes in applications, e.g., forensics or paleogenomics. Current major limitations hinge on the inability of conventional PCR enzymes, such as Taq, to amplify degraded or low amounts of template DNA. Besides, a wide range of PCR inhibitors can also impede reactions of nucleic acid amplification. Here we looked at the PCR performances of the proof-reading D-type DNA polymerase from P. abyssi, Pab-polD. Fragments, 3 kilobases in length, were specifically PCR-amplified in its optimized reaction buffer. Pab-polD showed not only a greater resistance to high denaturation temperatures than Taq during cycling, but also a superior tolerance to the presence of potential inhibitors. Proficient proof-reading Pab-polD enzyme could also extend a primer containing up to two mismatches at the 3' primer termini. Overall, we found valuable biochemical properties in Pab-polD compared to the conventional Taq, which makes the enzyme ideally suited for cutting-edge PCR-applications. PMID:24847315

Microfluidic Purification and Concentration of Malignant Pleural Effusions for Improved Molecular and Cytomorphological Diagnostics