Wobeser, W L; Krajden, M; Conly, J; Simpson, H; Yim, B; D'costa, M; Fuksa, M; Hian-Cheong, C; Patterson, M; Phillips, A; Bannatyne, R; Haddad, A; Brunton, J L; Krajden, S
The Roche Amplicor Mycobacterium tuberculosis PCR test (RMtb-PCR) was compared with mycobacterial culture, with the BACTEC 460 system and inoculation on Lowenstein-Jensen media. Results were interpreted with an adjusted "gold standard" incorporating clinical diagnosis. A total of 1,480 clinical specimens from 1,155 patients, including tissues and fluids, as well as 141 specimens which demonstrated a positive growth index on the BACTEC 460 system were assessed. The sensitivity, specificity, and positive and negative predictive values of RMtb-PCR compared with the adjusted gold standard for clinical specimens were 79, 99, 93, and 98%, respectively. In smear-positive specimens, the sensitivity of RMtb-PCR was 98% versus 53% for smear-negative specimens. When RMtb-PCR was performed two times per week, PCR results were available an average of 21 days before the culture results. For specimens demonstrating a positive growth index on the BACTEC 460 system, RMtb-PCR had a sensitivity and specificity of 98 and 100%, respectively. This study demonstrates the value of a commercial nucleic acid amplification kit for rapid diagnosis of M. tuberculosis, particularly in smear-positive specimens or BACTEC culture-positive specimens. PMID:8748289
Patel, Vinod B.; Connolly, Cathy; Singh, Ravesh; Lenders, Laura; Matinyenya, Brian; Theron, Grant; Ndung'u, Thumbi
There are no data about the comparative accuracy of commercially available nucleic acid amplification tests (GeneXpert MTB/RIF and Roche Amplicor) for the diagnosis of tuberculous meningitis (TBM). A total of 148 patients with suspected TBM were evaluated, and cultures served as the reference standard. The sensitivities and specificities (95% confidence interval [CI]) for the Amplicor and Xpert MTB/RIF tests were similar: 46 (31–60) versus 50 (33–67) and 99 (93–100) and 94 (84–99), respectively. PMID:25056328
Barlow, K L; Tosswill, J H; Parry, J V; Clewley, J P
Over a 4-year period, the Roche Amplicor kit was used in a United Kingdom reference laboratory for the detection or confirmation of human immunodeficiency virus (HIV) type 1 infection, particularly in infants born to HIV-infected mothers. Of 408 specimens from adults and older children tested, the 122 seronegative specimens were all Amplicor negative. Of the 286 seropositive specimens, 268 were Amplicor positive. On the basis of these results, the Amplicor assay has a specificity of 100% and a sensitivity of 93.7%. In addition, for 247 specimens from infants and young children, serological results may not have been diagnostic because of placental transfer of maternal antibodies. Forty-eight were Amplicor positive, and of the 199 Amplicor-negative specimens, 19 were assumed to be false negative on the basis of clinical data, serological markers (including p24 antigen), and/or results for previous or follow-up specimens. This represents a sensitivity of 75% for the Amplicor test for specimens from patients under 2 years of age. Of these 37 false-negative specimens plus 2 specimens from other laboratories, 31 could be characterized by amplifying extracted material from them by an in-house nested gag PCR spanning the Amplicor target region. The amplicons were sequenced and found to represent subtypes A (35.5%), B (22.6%), C (22.6%), D (16.1%), and G (3.2%). False-negative results by the Amplicor assay may be ascribed to low-target copy number, the physical behavior of one primer (SK462), and sequence variation in the target region of the other primer (SK431). PMID:9350745
Tremblay, Maxime-Antoine; Rodrigue, Marc-André; Deschênes, Louise; Boivin, Guy; Longtin, Jean
We assessed the performance of Abbott RealTime CMV assay (ARC) compared to Roche Cobas Amplicor CMV Monitor Test (RCM) for quantification of CMV in plasma of transplant patients. Commercial panels were used to test linearity, precision and interference and 83 clinical samples were used for the accuracy and precision analyses. All 43 RCM-positive clinical samples tested positive by ARC. The overall concordance between the two tests was good (98%). Based on 17 samples, the inter-assay median coefficient of variation was 13%. A linearity panel ranging from approximately 1 to 7log10copies/mL was used to confirm linearity (R(2)=0.99). CMV viral load measurement was not affected by different concentrations of HSV-1 or EBV DNA. We conclude that The Abbott RealTime CMV assay offers good sensitivity, precision and linearity and is suitable for monitoring CMV viral loads in transplant recipients. Standardization with the WHO CMV standard allows for comparison with other assays. PMID:26341060
Olmedo, E; Costa, J; López-Labrador, F X; Forns, X; Ampurdanés, S; Maluenda, M D; Guilera, M; Sánchez-Tapias, J M; Rodes, J; Jimenez de Anta, M T
A modified competitive RT-PCR (mcRT-PCR) to measure HCV RNA in serum and the Amplicor HCV Monitor assay were compared. For mcRT-PCR, the RNA extracted was retrotranscribed and coamplified in one step with a known amount of a DNA internal control (IC). Digoxigenin-labeled amplified products were hybridized to specific HCV DNA and IC-DNA probes and quantified by colorimetry. HCV RNA concentration was calculated by plotting the ratio of HCV/IC ODs against a calibration curve. Multiple samples were analyzed in the same round and tedious titration of each sample with a competitor was unnecessary. The mcRT-PCR assay was linear from 6 x 10(3) to 6 x 10(7) copies/ml, whereas Amplicor was linear up to 1-2 x 10(6) copies/ml. HCV RNA was measured in samples from 75 carriers. There was agreement between both methods in type 1 infections but not in type 2 or type 3 infections, in which the values measured by Amplicor were, on average, 15 times lower than those measured by the mcRT-PCR. HCV RNA measured by Amplicor was higher in type 1 infections than in type 2 or 3 infections, but no differences were found when viral load was assessed by mcRT-PCR. The binding efficiency of the Amplicor-probe was greater for type 1 than for types 2 or 3, suggesting Amplicor underestimates the viral load in the latter types. In contrast, the mcRT-PCR is not affected by genotype-related variation of HCV. This study suggests that mcRT-PCR assay is reliable for sensitive and accurate measurement of HCV RNA over a broad range of values independently of the HCV genotype.
Gerken, G.; Rothaar, T.; Rumi, M. G.; Soffredini, R.; Trippler, M.; Blunk, M. J.; Butcher, A.; Soviero, S.; Colucci, G.
A clinical evaluation of an automated quantitative PCR assay, the COBAS AMPLICOR HCV MONITOR test, version 2.0 (v2.0), was carried out to assess the performance of this test in comparison with that of the previous, manual version, the AMPLICOR HCV MONITOR test, and with that of nested PCR. Serial dilutions of serum samples infected with genotype 1b, 2a, or 3, as well as synthetic RNA transcripts and serum samples derived from 87 patients with chronic hepatitis C and infected with genotype 1a, 1b, 2a, 2b, 3a, 3b, 4, or 5, were analyzed to determine the ability of the system to efficiently quantify various hepatitis C virus (HCV) genotypes. These experiments showed that the COBAS AMPLICOR HCV MONITOR test, v2.0, has mean intra-assay, interassay, and interoperator coefficients of variation that range from 22 to 34.5% and a 3-logarithm dynamic range, which spans from 103 to 106 copies/ml. Compared to the previous, manual version of the test, the COBAS AMPLICOR HCV MONITOR test, v2.0, showed an improved efficacy for all genotypes, especially genotypes 2, 3, and 4, whose estimated concentrations were on average 1 logarithm higher. When used to monitor patients under treatment, however, both versions showed the same patterns of viremia, indicating that the COBAS AMPLICOR HCV MONITOR test, v2.0, and the AMPLICOR HCV MONITOR test were equally effective at detecting relative viremia changes in serial samples. As expected, the automated test was less sensitive than nested PCR; among specimens from a cohort of patients treated with interferon, nested PCR identified three more viremic specimens, which probably contained very low concentrations of HCV RNA. PMID:10834978
Raboud, J M; Seminari, E; Rae, S L; Harrigan, P R; Hogg, R S; Conway, B; Sherlock, C; Schechter, M T; O'Shaughnessy, M V; Montaner, J S
The costs of four algorithms for monitoring plasma human immunodeficiency virus type 1 RNA were compared. For patients with strong virologic responses, the use of Ultra Direct exclusively was the cheapest strategy. For patients with weak virologic responses, small savings could be obtained by using Amplicor and retesting only samples with values below 500 copies/ml with Ultra Direct.
Raboud, J. M.; Seminari, E.; Rae, S. L.; Harrigan, P. R.; Hogg, R. S.; Conway, B.; Sherlock, C.; Schechter, M. T.; O’Shaughnessy, M. V.; Montaner, J. S. G.
The costs of four algorithms for monitoring plasma human immunodeficiency virus type 1 RNA were compared. For patients with strong virologic responses, the use of Ultra Direct exclusively was the cheapest strategy. For patients with weak virologic responses, small savings could be obtained by using Amplicor and retesting only samples with values below 500 copies/ml with Ultra Direct. PMID:9774597
Stevens, Matthew P.; Rudland, Elice; Garland, Suzanne M.; Tabrizi, Sepehr N.
Roche Molecular Systems recently released two PCR-based assays, AMPLICOR and LINEAR ARRAY (LA), for the detection and genotyping, respectively, of human papillomaviruses (HPVs). The manual specimen processing method recommended for use with both assays, AmpliLute, can be time-consuming and labor-intensive and is open to potential specimen cross-contamination. We evaluated the Roche MagNA Pure LC (MP) as an alternative for specimen processing prior to use with either assay. DNA was extracted from cervical brushings, collected in PreservCyt media, by AmpliLute and MP using DNA-I and Total Nucleic Acid (TNA) kits, from 150 patients with histologically confirmed cervical abnormalities. DNA was amplified and detected by AMPLICOR and the LA HPV test. Concordances of 96.5% (139 of 144) (κ = 0.93) and 95.1% (135 of 142) (κ = 0.90) were generated by AMPLICOR when we compared DNA extracts from AmpliLute to MP DNA-I and TNA, respectively. The HPV genotype profiles were identical in 78.7 and 74.7% of samples between AmpliLute and DNA-I or TNA, respectively. To improve LA concordance, all 150 specimens were extracted by MP DNA-I protocol after the centrifugation of 1-ml PreservCyt samples. This modified approach improved HPV genotype concordance levels between AmpliLute and MP DNA-I to 88.0% (P = 0.043) without affecting AMPLICOR sensitivity. Laboratories that have an automated MP extraction system would find this procedure more feasible and easier to handle than the recommended manual extraction method and could substitute such extractions for AMPLICOR and LA HPV tests once internally validated. PMID:16825360
Tabrizi, Sepehr N; Stevens, Matthew P; Khan, Zaheer A; Chow, Conan; Devitt, Martin A; Garland, Suzanne M
PapType human papillomavirus (HPV) assay was compared to Hybrid Capture 2 (HC2), Amplicor (Amp), and Linear Array (LA) HPV tests in 894 women undergoing management for a high-grade Pap smear abnormality. The sensitivity in detection of underlying high-grade histological diagnosis by PapType was 90.3% and by HC2 was 79.8%, while by Amp and LA it was 92.4% and 91.6%, respectively. The specificities were 52.5%, 55.3%, 49.4%, and 51.7% for PapType, HC2, Amp, and LA, respectively.
An in-house real-time polymerase chain reaction: standardisation and comparison with the Cobas Amplicor HBV monitor and Cobas AmpliPrep/Cobas TaqMan HBV tests for the quantification of hepatitis B virus DNA.
Santos, Ana Paula de Torres; Levi, José Eduardo; Lemos, Marcilio Figueiredo; Calux, Samira Julien; Oba, Isabel Takano; Moreira, Regina Célia
This study aimed to standardise an in-house real-time polymerase chain reaction (rtPCR) to allow quantification of hepatitis B virus (HBV) DNA in serum or plasma samples, and to compare this method with two commercial assays, the Cobas Amplicor HBV monitor and the Cobas AmpliPrep/Cobas TaqMan HBV test. Samples from 397 patients from the state of São Paulo were analysed by all three methods. Fifty-two samples were from patients who were human immunodeficiency virus and hepatitis C virus positive, but HBV negative. Genotypes were characterised, and the viral load was measure in each sample. The in-house rtPCR showed an excellent success rate compared with commercial tests; inter-assay and intra-assay coefficients correlated with commercial tests (r = 0.96 and r = 0.913, p < 0.001) and the in-house test showed no genotype-dependent differences in detection and quantification rates. The in-house assay tested in this study could be used for screening and quantifying HBV DNA in order to monitor patients during therapy.
An in-house real-time polymerase chain reaction: standardisation and comparison with the Cobas Amplicor HBV monitor and Cobas AmpliPrep/Cobas TaqMan HBV tests for the quantification of hepatitis B virus DNA
Santos, Ana Paula de Torres; Levi, José Eduardo; Lemos, Marcilio Figueiredo; Calux, Samira Julien; Oba, Isabel Takano; Moreira, Regina Célia
This study aimed to standardise an in-house real-time polymerase chain reaction (rtPCR) to allow quantification of hepatitis B virus (HBV) DNA in serum or plasma samples, and to compare this method with two commercial assays, the Cobas Amplicor HBV monitor and the Cobas AmpliPrep/Cobas TaqMan HBV test. Samples from 397 patients from the state of São Paulo were analysed by all three methods. Fifty-two samples were from patients who were human immunodeficiency virus and hepatitis C virus positive, but HBV negative. Genotypes were characterised, and the viral load was measure in each sample. The in-house rtPCR showed an excellent success rate compared with commercial tests; inter-assay and intra-assay coefficients correlated with commercial tests (r = 0.96 and r = 0.913, p < 0.001) and the in-house test showed no genotype-dependent differences in detection and quantification rates. The in-house assay tested in this study could be used for screening and quantifying HBV DNA in order to monitor patients during therapy. PMID:26872342
Multicenter comparison of Roche COBAS AMPLICOR MONITOR version 1.5, Organon Teknika NucliSens QT with Extractor, and Bayer Quantiplex version 3.0 for quantification of human immunodeficiency virus type 1 RNA in plasma.
Murphy, D G; Côté, L; Fauvel, M; René, P; Vincelette, J
The performance and characteristics of Roche COBAS AMPLICOR HIV-1 MONITOR version 1.5 (CA MONITOR 1.5) UltraSensitive (usCA MONITOR 1. 5) and Standard (stCA MONITOR 1.5) procedures, Organon Teknika NucliSens HIV-1 RNA QT with Extractor (NucliSens), and Bayer Quantiplex HIV RNA version 3.0 (bDNA 3.0) were compared in a multicenter trial. Samples used in this study included 460 plasma specimens from human immunodeficiency virus (HIV) type 1 (HIV-1)-infected persons, 100 plasma specimens from HIV antibody (anti-HIV)-negative persons, and culture supernatants of HIV-1 subtype A to E isolates diluted in anti-HIV-negative plasma. Overall, bDNA 3.0 showed the least variation in RNA measures upon repeat testing. For the Roche assays, usCA MONITOR 1.5 displayed less variation in RNA measures than stCA MONITOR 1.5. NucliSens, at an input volume of 2 ml, showed the best sensitivity. Deming regression analysis indicated that the results of all three assays were significantly correlated (P < 0.0001). However, the mean difference in values between CA MONITOR 1.5 and bDNA 3.0 (0.274 log(10) RNA copies/ml; 95% confidence interval, 0.192 to 0.356) was significantly different from 0, indicating that CA MONITOR 1.5 values were regularly higher than bDNA 3.0 values. Upon testing of 100 anti-HIV-negative plasma specimens, usCA MONITOR 1.5 and NucliSens displayed 100% specificity, while bDNA 3.0 showed 98% specificity. NucliSens quantified 2 of 10 non-subtype B viral isolates at 1 log(10) lower than both CA MONITOR 1.5 and bDNA 3.0. For NucliSens, testing of specimens with greater than 1,000 RNA copies/ml at input volumes of 0.1, 0.2, and 2.0 ml did not affect the quality of results. Additional factors differing between assays included specimen throughput and volume requirements, limit of detection, ease of execution, instrument work space, and costs of disposal. These characteristics, along with assay performance, should be considered when one is selecting a viral load assay.
Piersimoni, C; Callegaro, A; Nista, D; Bornigia, S; De Conti, F; Santini, G; De Sio, G
Two commercial assays detecting the presence of Mycobacterium tuberculosis complex in clinical specimens by rRNA target amplification (Gen-Probe Amplified M. tuberculosis Direct Test [AMTD]) and PCR (Amplicor) were evaluated. The tests were applied to 327 digested, decontaminated respiratory specimens collected from 236 patients. Results were compared with those of acid-fast staining and culture. The combination of culture and clinical diagnosis was considered the "gold standard." A total of 60 specimens were collected from 27 patients with a diagnosis of pulmonary tuberculosis. Thirteen of these specimens were from patients receiving standard antituberculosis therapy and therefore were not included in the comparison. Of the remaining 47 specimens, 33 were smear positive, 40 were culture positive, 45 were AMTD positive, and 39 were Amplicor positive. After resolution of discrepant results, the overall sensitivities, specificities, and positive and negative predictive values were 77, 100, 100, and 95 for staining; 87, 100, 100, and 97.4 for culture; 95.9, 98.9, 94, and 99.2 for AMTD; and 85.4, 99.6, 97.9, and 97.1 for Amplicor, respectively. Agreement between AMTD and Amplicor assay results was 96.8%. It is concluded that although both nucleic acid amplification methods are rapid and specific for the detection of M. tuberculosis complex in respiratory specimens, AMTD appeared to be more sensitive than Amplicor. PMID:8968906
Elbeik, Tarek; Loftus, Richard A; Beringer, Scott
This article will compare the VERSANT HIV-1 RNA 3.0 (bDNA 3.0) assay with other HIV-1 viral load assays, particularly the AMPLICOR HIV-1 MONITOR (Amplicor 1.5), the industry standard. The discussion will cover the history of viral load assay development and challenges to the field. It will finish with a description of the evolving markets for viral load assays in the developing world and the impact of variations in the different assays on their ability to reach those markets and patient populations.
Portilho, Moyra Machado; Baptista, Marcia Leite; da Silva, Messias; de Sousa, Paulo Sérgio Fonseca; Lewis-Ximenez, Lia Laura; Lampe, Elisabeth; Villar, Livia Melo
The aim of the present study was to evaluate the performance of three in-house PCR techniques for HBV DNA detection and compare it with commercial quantitative methods to evaluate the usefulness of in-house methods for HBV diagnosis. Three panels of HBsAg reactive sera samples were evaluated: (i) 50 samples were examined using three methods for in-house qualitative PCR and the Cobas Amplicor HBV Monitor Assay; (ii) 87 samples were assayed using in-house semi-nested PCR and the Cobas TaqMan HBV test; (iii) 11 serial samples obtained from 2 HBV-infected individuals were assayed using the Cobas Amplicor HBV test and semi-nested PCR. In panel I, HBV DNA was detected in 44 samples using the Cobas Amplicor HBV test, 42 samples using semi-nested PCR (90% concordance with Cobas Amplicor), 22 samples using PCR for the core gene (63.6% concordance) and 29 samples using single-round PCR for the pre-S/S gene (75% concordance). In panel II, HBV DNA was quantified in 78 of the 87 HBsAg reactive samples using Cobas TaqMan but 52 samples using semi-nested PCR (67.8% concordance). HBV DNA was detected in serial samples until the 17th and 26th week after first donation using in-house semi-nested PCR and the Cobas Amplicor HBV test, respectively. In-house semi-nested PCR presented adequate concordance with commercial methods as an alternative method for HBV molecular diagnosis in low-resource settings.
Jalal, Hamid; Stephen, Hannah; Curran, Martin D; Burton, Janet; Bradley, Michelle; Carne, Christopher
A multitarget real-time PCR (MRT-PCR) for detection of Chlamydia trachomatis DNA was developed and validated. There were three targets for amplification in a single reaction: the cryptic plasmid (CP), the major outer membrane protein (MOMP) gene, and an internal control. The assay had the following characteristics: (i) detection and confirmation of the presence of C. trachomatis DNA in a single reaction, (ii) detection of all genovars of C. trachomatis without any cross-reactivity with pathogenic bacteria or commensal organisms of the oropharynx and genital tract, (iii) a 95% probability of detection with three copies of MOMP and one copy of CP per reaction mixture, (iv) identification of the inhibition of amplification, (v) a quantitative dynamic range of 25 to 250,000 genome copies per reaction mixture, (vi) high intra- and interassay reproducibilities, and (vii) correct identification of all samples in the validation panel. There were 146 COBAS Amplicor PCR (Amplicor PCR)-positive samples and 122 Amplicor PCR-negative samples in the panel. MRT-PCR detected CP DNA alone in 6 (4%) Amplicor PCR-positive samples and both CP and MOMP DNAs in 140 (96%) of 146 Amplicor PCR-positive samples. The quantity of MOMP DNA in 95 (68%) of 140 samples was within the dynamic range of the assay. The median C. trachomatis load in these samples was 321 genome copies per reaction mixture (range, 26 to 40,137 genome copies per reaction mixture). Due to the inclusion of two different C. trachomatis-specific targets, the assay confirmed 259 (97%) of 268 results in a single reaction. This assay could be used in the qualitative format for the routine detection of C. trachomatis and in the quantitative format for study of the pathogenesis of C. trachomatis-associated diseases. The assay demonstrated the potential to eliminate the need for confirmatory testing in almost all samples, thus reducing the turnaround time and the workload.
Kim, Jeong Hyun; Kim, Young Jae; Ki, Chang-Seok; Kim, Ji-Youn; Lee, Nam Yong
Nucleic acid-based amplification tests allow the rapid detection of Mycobacterium tuberculosis. Recently, a real-time PCR assay for M. tuberculosis complex, the Cobas TaqMan MTB test (Roche Diagnostics, Basel, Switzerland), was introduced. We performed a prospective study to evaluate the diagnostic performance of the Cobas TaqMan MTB test system. A total of 406 specimens collected from 247 patients were simultaneously tested by conventional culture, Cobas Amplicor MTB PCR, and TaqMan MTB PCR. The cross-reactivity with other Mycobacterium species and the detection limit were also evaluated. Among 406 specimens, a total of 24 specimens (5.9%) were culture positive: 14 specimens were positive by both TaqMan and Amplicor MTB PCRs, while 5 specimens were positive by only TaqMan PCR. The remaining five specimens were negative by both PCR methods. Seven specimens with negative culture results were positive by TaqMan PCR, but five of these were negative by Amplicor MTB PCR. The sensitivity, specificity, and positive (PPV) and negative (NPV) predictive values were 79.1%, 98.2%, 73.1%, and 98.7% for TaqMan and 58.3%, 99.5%, 87.5%, and 97.4% for the Amplicor MTB PCR test, respectively. There was no cross-reactivity with M. tuberculosis and nontuberculous mycobacterial species. The detection limit for the Cobas TaqMan MTB PCR test was 4.0 copies/μl. The Cobas TaqMan MTB PCR test showed higher sensitivity for detection of the M. tuberculosis complex without disturbing the specificity and NPV than the Amplicor MTB PCR test.
Takahashi, Masahiko; Saito, Hidetsugu; Higashimoto, Makiko; Atsukawa, Kazuhiro; Ishii, Hiromasa
A highly sensitive second-generation hepatitis C virus (HCV) core antigen assay has recently been developed. We compared viral disappearance and first-phase kinetics between commercially available core antigen (Ag) assays, Lumipulse Ortho HCV Ag (Lumipulse-Ag), and a quantitative HCV RNA PCR assay, Cobas Amplicor HCV Monitor test, version 2 (Amplicor M), to estimate the predictive benefit of a sustained viral response (SVR) and non-SVR in 44 genotype 1b patients treated with interferon (IFN) and ribavirin. HCV core Ag negativity could predict SVR on day 1 (sensitivity = 100%, specificity = 85.0%, accuracy = 86.4%), whereas RNA negativity could predict SVR on day 7 (sensitivity = 100%, specificity = 87.2%, accuracy = 88.6%). None of the patients who had detectable serum core Ag or RNA on day 14 achieved SVR (specificity = 100%). The predictive accuracy on day 14 was higher by RNA negativity (93.2%) than that by core Ag negativity (75.0%). The combined predictive criterion of both viral load decline during the first 24 h and basal viral load was also predictive for SVR; the sensitivities of Lumipulse-Ag and Amplicor-M were 45.5 and 47.6%, respectively, and the specificity was 100%. Amplicor-M had better predictive accuracy than Lumipulse-Ag in 2-week disappearance tests because it had better sensitivity. On the other hand, estimates of kinetic parameters were similar regardless of the detection method. Although the correlations between Lumipulse-Ag and Amplicor-M were good both before and 24 h after IFN administration, HCV core Ag seemed to be relatively lower 24 h after IFN administration than before administration. Lumipulse-Ag seems to be useful for detecting the HCV concentration during IFN therapy; however, we still need to understand the characteristics of the assay.
Bestetti, Arabella; Pierotti, Chiara; Terreni, Mariarosa; Zappa, Alessandra; Vago, Luca; Lazzarin, Adriano; Cinque, Paola
The diagnostic reliabilities of three cytomegalovirus (CMV) nucleic acid amplification assays of cerebrospinal fluid (CSF) were compared by using CSF samples from human immunodeficiency virus-infected patients with a postmortem histopathological diagnosis of CMV encephalitis (n = 15) or other central nervous system conditions (n = 16). By using a nested PCR assay, the quantitative COBAS AMPLICOR CMV MONITOR PCR, and the NucliSens CMV pp67 nucleic acid sequence-based amplification assay, sensitivities were 93.3, 86.6, and 93.3%, respectively, and specificities were 93.7, 93.7, and 87.5%, respectively. The COBAS AMPLICOR assay revealed significantly higher CMV DNA levels in patients with diffuse ventriculoencephalitis than in patients with focal periventricular lesions. PMID:11230445
Tortoli, Enrico; Urbano, Pasquale; Marcelli, Fiorella; Simonetti, Tullia M; Cirillo, Daniela M
Cobas Amplicor MTB and later Cobas TaqMan MTB were used to test a very large series of consecutive specimens received for tuberculosis diagnosis. Performance parameters were estimated and compared overall and for separate specimen categories. Both systems showed excellent specificity, and that of TaqMan was the higher. The sensitivities were similar but satisfactory only with respiratory specimens and smear-positive samples.
Gamboa, F; Manterola, J M; Lonca, J; Matas, L; Viñado, B; Giménez, M; Cardona, P J; Padilla, E; Ausina, V
A sodium dodecyl (lauryl) sulfate method was evaluated for the preparation of blood specimens and bone marrow aspirates for use in two amplification procedures (Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test [AMTDT] and Roche Amplicor M. avium/M. intracellulare [MAI] Test) for the detection and identification of Mycobacterium tuberculosis and M. avium and M. intracellulare, respectively. The AMTDT is based on amplification of rRNA, whereas the Amplicor MAI Test amplifies a specific DNA region of the 16S rRNA gene. The results of amplification techniques were compared with those of standard culture and culture in BACTEC 13A and BACTEC 12B liquid media. A total of 121 blood specimens and 15 bone marrow aspirates were collected from 136 AIDS patients. Mycobacterial growth was recovered for 103 specimens; 35 yielded M. tuberculosis, 62 yielded M. avium, 5 yielded M. genavense, and 1 yielded M. kansasii. The values of sensitivity and specificity in pretreated specimens for detection of M. tuberculosis by the AMTDT were 94.3 and 100%, respectively, and those for detection of M. avium by the Amplicor MAI Test were 91.9 and 100%, respectively. The simple lysis method described in the present work allows the recovery of mycobacteria from blood specimens and bone marrow aspirates and may be used in combination with the AMTDT and the Amplicor MAI Test to detect and identify different members of the genus Mycobacterium. This method might also be applicable for the identification of mycobacteria from blood culture fluids with acridinium-ester-labeled DNA probes. PMID:9230395
Cornall, A M; Poljak, M; Garland, S M; Phillips, S; Machalek, D A; Tan, J H; Quinn, M A; Tabrizi, S N
The purpose of this study was to evaluate the performance of the EUROIMMUN EUROArray HPV genotyping assay against the Roche Cobas 4800, Roche HPV Amplicor, Roche Linear Array and Qiagen Hybrid Capture 2 assays in the detection of high-risk HPV (HR-HPV) from liquid based cervical cytology samples collected from women undergoing follow-up for abnormal cervical cytology results. Cervical specimens from 404 women undergoing management of high-grade cytological abnormality were evaluated by EUROarray HPV for detection of HR-HPV genotypes and prediction of histologically-confirmed cervical intraepithelial neoplasia grade 2 or higher (≥CIN2). The results were compared to Hybrid Capture 2, Cobas 4800 HPV, Amplicor and Linear Array HPV. Positivity for 14 HR-HPV types was 80.0 % for EUROarray (95 % CI; 75.7-83.8 %). Agreement (κ, 95 % CI) between the EUROarray and other HPV tests for detection of HR-HPV was good to very good [Hybrid Capture κ = 0.62 (0.54-0.71); Cobas κ = 0.81 (0.74-0.88); Amplicor κ = 0.68 (0.60-0.77); Linear Array κ = 0.77 (0.70-0.85)]. For detection of HR-HPV, agreement with EUROarray was 87.90 % (Hybrid Capture), 93.58 % (Cobas), 92.84 % (Amplicor) and 92.59 % (Linear Array). Detection of HR-HPV was not significantly different between EUROarray and any other test (p < 0.001). EUROarray was concordant with other assays evaluated for detection of high-risk HPV and showed sensitivity and specificity for detection of ≥ CIN2 of 86 % and 71 %, respectively. PMID:27048314
Tabrizi, Sepehr N; Unemo, Magnus; Limnios, Athena E; Hogan, Tiffany R; Hjelmevoll, Stig-Ove; Garland, Susanne M; Tapsall, John
Molecular detection of Neisseria gonorrhoeae in extragenital samples may result in false-positive results due to cross-reaction with commensal Neisseria species or Neisseria meningitidis. This study examined 450 characterized clinical culture isolates, comprising 216 N. gonorrhoeae isolates and 234 isolates of nongonococcal Neisseria species (n = 218) and 16 isolates of other closely related bacteria, with six commercial nucleic acid amplification tests (NAATs). The six NAATs tested were Gen-Probe APTIMA COMBO 2 and APTIMA GC, Roche COBAS Amplicor CT/NG and COBAS 4800 CT/NG tests, BD ProbeTec GC Qx amplified DNA assay, and Abbott RealTime CT/NG test. All assays except COBAS Amplicor CT/NG test where four (1.9%) isolates were not detected showed a positive result with all N. gonorrhoeae isolates (n = 216). Among the 234 nongonococcal isolates examined, initial results from all assays displayed some false-positive results due to cross-reactions. Specifically, the COBAS Amplicor and ProbeTec tests showed the highest number of false-positive results, detecting 33 (14.1%) and 26 (11%) nongonococcal Neisseria isolates, respectively. On the first testing, APTIMA COMBO 2, APTIMA GC, Abbott RealTime, and Roche COBAS 4800 showed lower level of cross-reactions with five (2.1%), four (1.7%), two (1%), and two (1%) of the isolates showing low-level positivity, respectively. Upon retesting of these nine nongonococcal isolates using freshly cultured colonies, none were positive by the APTIMA COMBO 2, Abbott RealTime, or COBAS 4800 test. In conclusion, the COBAS Amplicor and ProbeTec tests displayed high number of false-positive results, while the remaining NAATs showed only sporadic low-level false-positive results. Supplementary testing for confirmation of N. gonorrhoeae NAATs remains recommended with all samples tested, in particular those from extragenital sites.
Becquart, Pierre; Foulongne, Vincent; Willumsen, Juana; Rouzioux, Christine; Segondy, Michel; Van de Perre, Philippe
HIV-1 RNA in breast milk is a strong predictor of HIV-1 transmission through breastfeeding. In the present report, breast milk samples from HIV-1 uninfected donors were spiked with dilution of quantified culture supernatant from HIV-1(NDK) infected PBMC. Two RNA extraction techniques based on silica extraction, Nuclisens (BioMerieux) and Triazol (Qiagen), two techniques based on guanidine thiocynanate/chloroforme extraction, TRIzol (Life Technologie) and Amplicor HIV-1 Monitor (Roche Diagnostic Systems), and one technique based on electrostatic adsorption on iron oxide micro beads (Promega) were compared. HIV-1 RNA was quantitated by real time PCR (LTR gene) and Amplicor HIV-1 Monitor. Combining magnetic micro beads extraction and real time PCR quantitation allowed to correctly quantify breast milk HIV-1 RNA, with a difference between the expected and measured HIV-1 RNA levels always lower than 0.3 log copies/ml. The same combination was confirmed on 25 breast milk samples from HIV-1 infected women collected in Kwazulu-Natal, South Africa, by comparing measurements with those obtained by the Amplicor HIV-1 Monitor (r(2)=0.88). Nucleic acid extraction by magnetic micro beads followed by real time PCR is a reliable, sensitive, rapid and simple procedure to quantify HIV-1 RNA in breast milk and allows for PCR inhibitors found frequently in these samples.
Gueye, Sokhna Bousso; Diop-Ndiaye, Halimatou; Diallo, Mamadou Malick; Ly, Omar; Sow-Ndoye, Aissatou; Diagne-Gueye, Ndèye Diabou; Kébé-Fall, Khady; Diop, Fatou; Gaye-Diallo, Aïssatou; Belec, Laurent; Mboup, Souleymane; Touré-Kane, Coumba
In the context of early infant diagnosis (EID) decentralization in sub-Saharan Africa, dried blood spot (DBS) is now widely used for HIV proviral DNA detection in resource-limited settings. A new version of CAP/CTM (version 2) has been introduced, recently by Roche Diagnosis as a new real-time PCR assay to replace previous technologies on qualitative detection of HIV-1 DNA using whole blood and DBS samples. The objective of this study was to evaluate CAP/CTM version 2 compared to CAP/CTM version 1 and Amplicor on DBS. A total of 261 DBS were collected from children aged 4 weeks to 17 months born from HIV-seropositive mothers and tested by the three techniques. CAP/CTM version 2 showed 100% of agreement with Amplicor including 74 positive results and 187 negative results. CAP/CTM version 2 versus CAP/CTM version 1 as well as CAP/CTM version 1 versus Amplicor showed two discordant results giving a sensitivity of 98.6%, specificity of 99.5%, positive predictive value of 98.6% and negative predictive value of 99.5%. The concordance was 99.12% (95% of confidence interval) giving a Kappa coefficient of 0.97 (p<0.001). These findings confirmed the expected good performance of CAP/CTM version 2 for HIV-1 EID.
Impact of HIV-1 genetic diversity on plasma HIV-1 RNA Quantification: usefulness of the Agence Nationale de Recherches sur le SIDA second-generation long terminal repeat-based real-time reverse transcriptase polymerase chain reaction test.
Rouet, François; Chaix, Marie-Laure; Nerrienet, Eric; Ngo-Giang-Huong, Nicole; Plantier, Jean-Christophe; Burgard, Marianne; Peeters, Martine; Damond, Florence; Ekouevi, Didier Koumavi; Msellati, Philippe; Ferradini, Laurent; Rukobo, Sandra; Maréchal, Valérie; Schvachsa, Nilda; Wakrim, Lahcen; Rafalimanana, Christian; Rakotoambinina, Benjamin; Viard, Jean-Paul; Seigneurin, Jean-Marie; Rouzioux, Christine
The high genetic diversity of HIV-1 has a major impact on the quantification of plasma HIV-1 RNA, representing an increasingly difficult challenge. A total of 898 plasma specimens positive for HIV-1 RNA by commercial assays (Amplicor v1.5; Roche Diagnostic Systems, Alameda, CA or Versant v3.0; Bayer Diagnostics, Emeryville, CA) were tested using the Agence Nationale de Recherches sur le SIDA second-generation (G2) real-time reverse transcriptase polymerase chain reaction (RT-PCR) test: 518 samples containing HIV-1 of known subtype, including 88 from 2 subtype panels and 430 harboring B (n = 266) and non-B (n = 164) group M HIV-1 subtypes from patients followed up in 2002 through 2005 at Necker Hospital (Paris, France), and 380 samples from 10 different countries (Argentina, Cambodia, Cameroon, Central African Republic, France, Ivory Coast, Madagascar, Morocco, Thailand, and Zimbabwe). HIV-1 RNA values obtained by G2 real-time PCR were highly correlated with those obtained by the Amplicor v1.5 for B and non-B subtypes (R = 0.892 and 0.892, respectively) and for samples from diverse countries (R = 0.867 and 0.893 for real-time PCR vs. Amplicor v1.5 and real-time PCR vs. Versant v3.0, respectively). Approximately 30% of specimens harboring non-B subtypes were underquantified by at least -0.51 log10 in Amplicor v1.5 versus 5% underquantified in G2 real-time PCR. Discrepant results were also obtained with subtype B samples (14% underquantified by Amplicor v1.5 vs. 7% by G2 real-time PCR). Similar percentages were observed when comparing results obtained with the G2 real-time PCR assay with those obtained using the Versant assay. Addressing HIV-1 diversity, continual monitoring of HIV-1 RNA assays, together with molecular epidemiology studies, is required to improve the accuracy of all HIV RNA assays.
Ikegame, Satoshi; Sakoda, Yoritake; Fujino, Nao; Taguchi, Kazuhito; Kawasaki, Masayuki; Kajiki, Akira
A retrospective observational study was performed to determine the sensitivity and limitation of PCR test for the detection of Mycobacterium tuberculosis and M. avium complex. We obtained clinical specimens collected from the respiratory tract, cultured M. tuberculosis or M. avium complex, and performed PCR analysis. A total of 299 samples (M. tuberculosis, 177; M. avium, 35; M. intracellulare, 87) were analyzed by COBAS TaqMan PCR from April 2007 to March 2011. The PCR positivity rates were 50–55%, 70–100%, 88–98%, and 100% in smear-negative, smear 1+, 2+, and 3+ groups, respectively. The PCR positivity of tuberculosis in smear 1+ was 80.6%, which was statistically significantly (P < 0.001) lower than that of smear 2+ (97.3%). From January 2005 to March 2007, we collected an additional 138 samples (M. tuberculosis, 74; M. avium, 21; M. intracellulare, 43), which were analyzed by COBAS Amplicor PCR. The PCR positivity rates obtained using COBAS TaqMan PCR and COBAS Amplicor PCR were not significantly different. The sensitivity of PCR test for mycobacteria is not sufficient in case of smear 1+. Careful consideration must be given to the interpretation of negative PCR test results in smear 1+, because smear-positive tuberculosis is the criterion for isolation. PMID:23029612
Diaz-Mitoma, Francisco; Leger, Chantal; Miller, Helen; Giulivi, Antonio; Frost, Rita; Shaw, Laura; Huebsch, Lothar
A total of 676 specimens from 63 recipients of bone marrow allografts were tested for cytomegalovirus (CMV) by the following assays: CMV pp67 NucliSens (NS), AMPLICOR CMV MONITOR (RA), and the Digene CMV DNA test (DG). In a consensus analysis, the sensitivities and specificities were 60 and 99% (NS), 96 and 98% (RA), and 90 and 76% (DG), respectively; for detection of symptomatic CMV infection, they were 60 and 97% (NS), 65 and 97% (RA), and 95 and 77% (DG), respectively. In multivariate analysis, the major risk factor for symptomatic CMV infection was an increase in the viral load in the DG assay; in univariate analyses, maximum viral loads in both DG and RA assays and a rising viral load in the RA assay were also significant. The earliest detection of CMV replication was provided by the RA assay (mean, 39 days posttransplantation), followed by the DG assay (mean, 48 days) and the NS assay (mean, 58 days). PMID:14605153
Cox, J. Thomas; Johnson, Kristin; Quint, Wim; Ridder, Ruediger; Sideri, Mario; Wright, Thomas C.; Behrens, Catherine M.
A post hoc analysis of the ATHENA study was performed to determine whether true HPV‐negative cervical lesions occur and whether they have clinical relevance. The ATHENA database was searched for all CIN2 or worse (CIN2+) cases with cobas HPV‐negative results and comparison was made with Linear Array (LA) and Amplicor to detect true false‐negative HPV results. Immunostaining with p16 was performed on these cases to identify false‐positive histology results. H&E slides were re‐reviewed by the study pathologists with knowledge of patient age, HPV test results and p16 immunostaining. Those with positive p16 immunostaining and/or a positive histopathology review underwent whole tissue section HPV PCR by the SPF10/LiPA/RHA system. Among 46,887 eligible women, 497 cases of CIN2+ were detected, 55 of which tested negative by the cobas® HPV Test (32 CIN2, 23 CIN3/ACIS). By LA and/or Amplicor, 32 CIN2+ (20 CIN2, 12 CIN3/ACIS) were HPV positive and categorized as false‐negatives by cobas HPV; nine of 12 false‐negative CIN3/ACIS cases were p16+. There were 23 cases (12 CIN2, 11 CIN3/ACIS) negative by all HPV tests; seven of 11 CIN3/ACIS cases were p16+. H&E slides were available for six cases for re‐review and all were confirmed as CIN3/ACIS. Tissue PCR was performed on the six confirmed CIN3/ACIS cases (and one without confirmation): four were positive for HPV types not considered oncogenic, two were positive for oncogenic genotypes and one was indeterminate. In summary, subanalysis of a large cervical cancer screening study did not identify any true CIN3/ACIS not attributable to HPV. PMID:26851121
Unemo, M; Rossouw, A; James, V; Jenkins, C
In 2006, a new variant of Chlamydia trachomatis (nvCT) was reported in Sweden. The nvCT contains a deletion that includes the targets for the C. trachomatis genetic diagnostic single-target systems from Roche Diagnostics and Abbott Laboratories. Roche and Abbott have now developed certified dual-target assays that can detect the nvCT. This study examined the nucleic acid amplification tests (NAATs) currently used (in 2009) for C. trachomatis detection in laboratories from 17 European countries and five countries/regions outside Europe that are participating in the United Kingdom (UK) National External Quality Assessment Service (NEQAS). It further examined changes in these laboratories' testing strategy during the period from 2006 to 2009, and their performance regarding nvCT detection. A UK NEQAS blinded nvCT specimen was distributed to all 283 participating laboratories, which were asked to analyse the specimen according to their routine C. trachomatis diagnostic protocols for endocervical swabs. BD ProbeTec was the most commonly used NAAT, followed by Cobas Amplicor, Cobas TaqMan, and Aptima. From 2006 to 2009, the use of Cobas Amplicor, which does not detect the nvCT, decreased, but it was still used by 22% (n=57) of responding participants in 59% of the countries, 54 of these 57 used it as first assay. Virtually all of the other participants detected the nvCT correctly. Laboratories using commercial or in house NAATs that do not detect the nvCT are encouraged to carefully monitor their C. trachomatis incidence, participate in effective internal and external quality assurance and controls schemes, and to consider changing their testing system. PMID:19442398
Husic-Selimovic, Azra; Sofic, Amela; Huskic, Jasminko; Bulja, Deniz
Introduction: Renin-angiotenzin system (RAS) is frequently activated in patients with chronic liver disease. Angiotenzin - II (AT-II), produced by angiotenzin converting enzyme (ACE), has many physiological effects, including an important role in liver fibrogenesis. Combined antiviral therapy with PEG-IFN and ribavirin besides its antiviral effect also leads to a reduction in liver parenchyma fibrosis. Aim of the study: Determining the value of ACE in serum of patients with chronic hepatitis C before and after combined antiviral therapy, as well as the value of ACE activities in sera of the control group. Materials and methods: We studied 50 patients treated at Gastroenterohepatology Department, in the time-period of four years. Value of ACE in serum was determined by Olympus AU 400 device, with application of kit “Infinity TN ACE Liquid Stable Reagent”. HCV RNA levels in sera were measured by real time PCR. HCV RNA test was performed with modular analysis of AMPLICOR and COBAS AMPLICOR HCV MONITOR test v2.0, which has proved infection and was used for quantification of the viruses and monitoring of the patients’ response to therapy. Liver histology was evaluated in accordance with the level of necroinflammation activity and stage of fibrosis. Results: Serum activities of ACE in chronic hepatitis C patients is statistically higher than the values in the control group (p=0.02). Antiviral therapy in chronic hepatitis C patients statistically decreases serum activities of ACE (p= 0.02) and indirectly affects fibrogenesis of the liver parenchyma. Correlation between ACE and ALT activity after the therapy was proved (0.3934). Conclusion: Our findings suggest that the activity of ACE in serum is a good indirect parameter of the liver damage, and could be used as an indirect prognostic factor of the level of liver parenchyma damage. Serum activity of ACE can be used as a parameter for non-invasive assessment of intensity of liver damage. PMID:27147779
Petry, Karl Ulrich; Cox, J Thomas; Johnson, Kristin; Quint, Wim; Ridder, Ruediger; Sideri, Mario; Wright, Thomas C; Behrens, Catherine M
A post hoc analysis of the ATHENA study was performed to determine whether true HPV-negative cervical lesions occur and whether they have clinical relevance. The ATHENA database was searched for all CIN2 or worse (CIN2+) cases with cobas HPV-negative results and comparison was made with Linear Array (LA) and Amplicor to detect true false-negative HPV results. Immunostaining with p16 was performed on these cases to identify false-positive histology results. H&E slides were re-reviewed by the study pathologists with knowledge of patient age, HPV test results and p16 immunostaining. Those with positive p16 immunostaining and/or a positive histopathology review underwent whole tissue section HPV PCR by the SPF10/LiPA/RHA system. Among 46,887 eligible women, 497 cases of CIN2+ were detected, 55 of which tested negative by the cobas(®) HPV Test (32 CIN2, 23 CIN3/ACIS). By LA and/or Amplicor, 32 CIN2+ (20 CIN2, 12 CIN3/ACIS) were HPV positive and categorized as false-negatives by cobas HPV; nine of 12 false-negative CIN3/ACIS cases were p16+. There were 23 cases (12 CIN2, 11 CIN3/ACIS) negative by all HPV tests; seven of 11 CIN3/ACIS cases were p16+. H&E slides were available for six cases for re-review and all were confirmed as CIN3/ACIS. Tissue PCR was performed on the six confirmed CIN3/ACIS cases (and one without confirmation): four were positive for HPV types not considered oncogenic, two were positive for oncogenic genotypes and one was indeterminate. In summary, subanalysis of a large cervical cancer screening study did not identify any true CIN3/ACIS not attributable to HPV. PMID:26851121
Seu, Lillian; Mwape, Innocent; Guffey, M Bradford
The ability to evaluate individual HIV-1 virions from the quasispecies of vertically infected infants was evaluated in a field setting at the Centre for Infectious Disease Research in Zambia. Infant heel-prick blood specimens were spotted onto dried blood spot (DBS) filter paper cards at government health clinics. Nucleic acid was extracted and used as a template for HIV-1 proviral DNA detection by a commercial Amplicor HIV-1 PCR test (Roche, version 1.5). On samples that tested positive by commercial diagnostic assay, amplification of DNA was performed using an in-house assay of the 5' and 3' region of the HIV-1 genome. Additionally, fragments covering 1200 nucleotides within pol (full length protease and partial reverse transcriptase) and 1400 nucleotides within env (variable 1-variable 5 region) were further analyzed by single genome amplification (SGA). In summary, we have demonstrated an in-house assay for amplifying the 5' and 3' proviral HIV-1 DNA as well as pol and env proviral DNA fragments from DBS cards collected and analyzed entirely in Zambia. In conclusion, this study shows the feasibility of utilizing DBS cards to amplify the whole proviral HIV-1 genome as well as perform SGA on key HIV-1 genes.
Ramia, S.; Ramlawi, F.; Kanaan, M.; Klayme, S.; Naman, R.
During a 2-year period, blood samples from 2505 Lebanese blood donors were chosen at random, at various periods of time at one blood donation centre (Hotel Dieu de France, Beirut, Lebanon) and were screened for markers of HBV infection (HBsAg, anti-HBc and anti-HBs). The study showed HBsAg positivity of 0.6% and an overall exposure rate to HBV of 10.0%. Out of the 2505 blood donors screened, 56 (22%) were found to be 'anti-HBc alone' positive which is almost four times the HBsAg positivity. The 56 'anti-HBc alone' samples were retested by another ELISA kit commercially available and 54 samples were 'anti-HBc alone' positive by both assays. The 54 samples had no serological markers as evidence of infection with human immunodeficiency virus (HIV) or hepatitis C virus (HCV). Only seven (13%) out of the 54 samples were HBV DNA positive by PCR and all were HBV genotype D. All seven HBV DNA-positive samples had HBV DNA levels below 400 copies/ml. Although any circulating HBV DNA among our 'anti-HBc alone' blood donors was below the detection limit of our Amplicor Monitor assay, some of these samples had circulating virus. A national study, where a larger number of blood donors from different blood donation centres across the country will perhaps determine whether screening for anti-HBc in addition to HBsAg detection is needed in Lebanese blood donors. PMID:16050516
Sension, M G; Farthing, C; Shaffer, A G; Graham, E; Siemon-Hryczyk, P; Pilson, R S
This is an open-label, single-arm, phase 3b study (part of phase 3 development) to evaluate the efficacy and safety of Fortovase-soft gelatin formulation (saquinavir-SGC), combined with zidovudine (ZDV) and lamivudine (3TC), human immune deficiency virus type 1 in (HIV-1)-positive, antiretroviral-naive individuals. Forty-two HIV-1-positive adults with plasma HIV RNA >10,000 copies per milliliter (Roche Amplicor HIV Monitor assay) and CD4 cell count >100 cells/mm(3) were treated with SQV-SGC, 1200 mg three times per day; ZDV, 300 mg; and 3TC, 150 mg each twice per day for 48 weeks. High proportions were drug users (26%), demonstrated psychiatric disorders (alcohol abuse [14%]/depression [14%]), or were inadequately housed (5%). At 48 weeks, 50% of patients achieved viral suppression <400 copies per milliliter with 43% <20 copies per milliliter using an intent-to-treat analysis (missing values counted as virological failures). Corresponding proportions for patients remaining on therapy at 48 weeks were 91% <400 copies per milliliter and 78% <20 copies per milliliter. Most adverse events were mild. Saquinavir-SGC combined with ZDV and 3TC, achieved potent and durable HIV RNA suppression and was well tolerated over 48 weeks in an antiretroviral-naive population including high proportions of individuals considered difficult to treat, such as drug users, people with psychiatric problems and homeless individuals. PMID:11313025
Seu, Lillian; Mwape, Innocent; Guffey, M Bradford
The ability to evaluate individual HIV-1 virions from the quasispecies of vertically infected infants was evaluated in a field setting at the Centre for Infectious Disease Research in Zambia. Infant heel-prick blood specimens were spotted onto dried blood spot (DBS) filter paper cards at government health clinics. Nucleic acid was extracted and used as a template for HIV-1 proviral DNA detection by a commercial Amplicor HIV-1 PCR test (Roche, version 1.5). On samples that tested positive by commercial diagnostic assay, amplification of DNA was performed using an in-house assay of the 5' and 3' region of the HIV-1 genome. Additionally, fragments covering 1200 nucleotides within pol (full length protease and partial reverse transcriptase) and 1400 nucleotides within env (variable 1-variable 5 region) were further analyzed by single genome amplification (SGA). In summary, we have demonstrated an in-house assay for amplifying the 5' and 3' proviral HIV-1 DNA as well as pol and env proviral DNA fragments from DBS cards collected and analyzed entirely in Zambia. In conclusion, this study shows the feasibility of utilizing DBS cards to amplify the whole proviral HIV-1 genome as well as perform SGA on key HIV-1 genes. PMID:24667303
Seu, Lillian; Mwape, Innocent; Guffey, M. Bradford
The ability to evaluate individual HIV-1 virions from the quasispecies of vertically infected infants was evaluated in a field setting at the Centre for Infectious Disease Research in Zambia. Infant heel-prick blood specimens were spotted onto dried blood spot (DBS) filter paper cards at government health clinics. Nucleic acid was extracted and used as a template for HIV-1 proviral DNA detection by a commercial Amplicor HIV-1 PCR test (Roche, version 1.5). On samples that tested positive by commercial diagnostic assay, amplification of DNA was performed using an in-house assay of the 5′ and 3′ region of the HIV-1 genome. Additionally, fragments covering 1200 nucleotides within pol (full length protease and partial reverse transcriptase) and 1400 nucleotides within env (variable 1-variable 5 region) were further analyzed by single genome amplification (SGA). In summary, we have demonstrated an in-house assay for amplifying the 5′ and 3′ proviral HIV-1 DNA as well as pol and env proviral DNA fragments from DBS cards collected and analyzed entirely in Zambia. In conclusion, this study shows the feasibility of utilizing DBS cards to amplify the whole proviral HIV-1 genome as well as perform SGA on key HIV-1 genes. PMID:24667303
Hussein, Magdi M; Mooij, Jaap M; Hegazy, Mohamed S; Bamaga, Mohammed S
Hepatitis C virus (HCV) infection is most often diagnosed by detection of antibodies against the virus (HCV Ab). However, it has been reported that some HCV Ab negative patients test positive for HCV-RNA. Over a study period of 30 months, all patients on hemodialysis at the Al Hada Armed Forces Hospital in Taif, Saudi Arabia were tested monthly for HCV Ab and twice per year for HCV-RNA. HCV Ab was tested by a third generation microparticle enzyme immunoassay (MEIA), and HCV-RNA by a qualitative hepatitis-RNA assay, second version (COBAS Amplicor PCR), which was recently introduced in the Molecular Pathology Laboratory of our hospital. Of the 180 patients studied, 34 (18.9%) had positive HCV Ab, and of the 146 HCV Ab negative patients, five patients tested positive for HCV-RNA (3.42%). Our study further finds that, when applying HCV Ab testing only, some patients with HCV viremia may be undetected. For better HCV infection control, routine HCV-RNA testing of dialysis patients should be considered, particularly in areas where the infection is common and in units applying isolation policies.
Farma, E; Boeri, E; Bettini, P; Repetto, C M; McDermott, J; Lillo, F B; Varnier, O E
The diagnostic utility of two PCR systems and three PCR detection methods for hepatitis C virus (HCV) RNA was evaluated in serum samples. A nested PCR was considered the reference assay and was compared with two single-step PCR methods: the first is based on the detection of PCR products by liquid hybridization with a 32P-end-labeled probe, and the second is the Roche Amplicor colorimetric assay using microwell plate hybridization with a specific nucleic acid probe. Using the Pelicheck HCV RNA Eurohep genotype 1 proficiency panel, our laboratory achieved medium-high levels of performance with all three methods. The highest sensitivity was, however, observed with the isotopic single-step PCR (ss-PCR) method. The analytical sensitivity of ss-PCR with isotopic detection and ss-PCR with colorimetric detection was identical to that of nested PCR, with a 100% result concordance. Comparison of ss-PCR with enzyme-linked immunosorbent and RIBA assays in the analysis of clinical samples showed a high concordance. ss-PCR methods appear more suitable for diagnostic application. Nevertheless, HCV RNA PCR cannot be considered a screening assay; it should be requested in the presence of reactive serology or specific clinical symptomatology with altered liver parameters, and it is a potential tool for the follow-up of patients with HCV infection.
Ekiel, Alicja; Pietrzak, Bronisława; Aptekorz, Małgorzata; Mazanowska, Natalia; Kamiński, Paweł; Martirosian, Gayane
Bacterial infections, especially endogenous, are the frequent complications among hemodialyzed and renal transplant patients. In this study we assumed the prevalence of urogenital mycoplasmas and HPV among hemodialysed women. We examined 32 hemodialysed women aged 20–48 (mean 35.6 ± 8.23) and 100 healthy controls of the same ages. Two swabs were collected for detection of mycoplasmas and HPV. Culture of Ureaplasma spp. and M. hominis was performed using Mycoplasma IST2 (bioMérieux, France), Identificaton of U. parvum and U. urealyticum was performed by Kong. Primers described by Jensen were used for M. genitalium. For detection of high-risk HPV types Amplicor HPV (Roche Molecular System, CA) was used. Prevalence of urogenital mycoplasmas in the hemodialysed women (53.1%) was significantly higher (P = 0.0059), compared with controls (25%). In both groups, U. parvum was the most frequently isolated. Cooccurrence of urogenital mycoplasmas was shown in 75% of the HPV-positive hemodialysed women and in 30.4% of HPV-positive controls (P = 0.0461). Cooccurrence of urogenital mycoplasmas with HPV was significantly higher in hemodialysed women. The need to take into account these microorganisms in routine diagnostic, especially for hemodialysed patients, was demonstrated. Further studies to demonstrate the role of this cooccurrence in etiopathogenesis of infection in hemodialysed patients are required. PMID:24363622
Reischl, U; Straube, E; Unemo, M
The May 2009 round of INSTAND's ring trial "Chlamydia trachomatis detection PCR/NAT" included a sample with high amount of the Swedish new variant of C. trachomatis (nvCT). A spectrum of at least 12 different commercial diagnostic nucleic acid amplification tests (NAATs) and many different in house NAATs were applied by the 128 participating laboratories which reported 152 results. Approximately 80% of the results correctly reported the presence of C. trachomatis in the nvCT specimen. The nvCT sample was mainly missed, as expected, by participants using the Roche COBAS Amplicor CT/NG (15.5% of reported results) but also by several participants using in house NAATs. The trend towards using nvCT-detecting NAATs is obvious and in addition to the new dual-target NAATs from Roche and Abbott, and BD ProbeTec ET, also a number of new CE mark-certified commercial tests from smaller diagnostic companies as well as many different in house NAATs were used. Laboratories using commercial or in house NAATs that do not detect the nvCT are encouraged to carefully monitor their C. trachomatis incidence, participate in appropriate external quality assurance and controls schemes, and consider altering their testing system. The reliable detection of low amounts of the wildtype C. trachomatis strain in other samples of the ring trial set indicates a good diagnostic performance of all applied commercial NAATs while also detecting the nvCT strain. PMID:19679035
Sachdev, Divya; Patel, Achchhe Lal; Sonkar, Subash Chandra; Kumari, Indu; Saluja, Daman
Neisseria gonorrhoeae is an important sexually transmitted diseases (STD) causing pathogen worldwide. Due to absence of an affordable diagnostic assay, routine screening of gonococcal infection becomes impossible in developing countries where infection rates are maximum. Treatment is given on the basis of symptoms alone which leads to spread of infection. Thus, development of a rapid, sensitive, specific, and PCR based visual diagnostic assay suitable for developing countries, required for better disease management, is aimed at in present study. Endocervical swabs were collected from patients visiting gynecology department of various hospitals in Delhi. In-house PCR based assay was developed and modified to visual assay using molecular beacon for end-point detection. It was evaluated against Roche AMPLICOR NG kit and rmp gene. Specificity of beacon was confirmed by competition experiments. Diagnostic test was 98.21% specific and 99.59% sensitive whereas negative and positive predicted value were 99.40% and 98.78%, respectively. We also observed that twice the concentration (2X) of premix was stable at 4°C for 4 months and dry swab samples gave concordant results with that of wet swabs. These features make the test best suitable for routine diagnosis of genital infections in developing countries.
Sum, Simon Siu-Man; Wong, Danny Ka-Ho; Yuen, Man-Fung; Yuan, He-Jun; Yu, Jian; Lai, Ching-Lung; Ho, David; Zhang, Linqi
Levels of hepatitis B virus (HBV) DNA in the blood serve as an important marker in monitoring the disease progression and treatment efficacy of chronic HBV infection. Several commercial assays are available for accurate measurement of HBV genomic DNA, but many of them are hampered by relatively low sensitivity and limited dynamic range. The aim of this study was to develop a sensitive and accurate assay for measuring HBV genomic DNA using real-time PCR with a molecular beacon (HBV beacon assay). The performance of this assay was validated by testing serial dilutions of the two EUROHEP HBV DNA standards (ad and ay subtypes) of known concentrations. The assay showed low intra-assay (<7%) and interassay (<5%) variations for both subtypes. Its dynamic range was found to be 10(1) to 10(7) copies per reaction (1.0 x 10(2) to 1.0 x 10(9) copies ml(-1)). The assay was further evaluated clinically using serum samples from 175 individuals with chronic hepatitis B. The HBV DNA level measured by this assay showed good correlation with that measured by the commercially available COBAS AMPLICOR HBV Monitor test (r = 0.901; P < 0.001). The higher sensitivity and broader dynamic range of this assay compared to the existing commercial assays will provide an ideal tool for monitoring disease progression and treatment efficacy in HBV-infected patients, in particular for those with low levels of HBV viremia.
Huang, Shihai; Erickson, Brian; Mak, Wai Bing; Salituro, John; Abravaya, Klara
Abbott RealTime HIV-1 Qualitative is an in vitro real-time PCR assay for detecting HIV-1 nucleic acids in human plasma and dried blood spots (DBS). The assay was designed to be used in diagnosis of HIV-1 infections in pediatric and adult patients, with an emphasis on the applicability in resource-limited settings. Use of DBS facilitates specimen collection from remote areas and transportation to testing laboratories. Small sample input requirement facilitates testing of specimens with limited collection volume. The Abbott RealTime HIV-1 Qualitative assay is capable of detecting HIV-1 group M subtypes A-H, group O and group N samples. HIV-1 virus concentrations detected with 95% probability were 80 copies/mL of plasma using the plasma protocol, and 2469 copies/mL of whole blood using the DBS protocol. The assay detected HIV-1 infection in 13 seroconversion panels an average 10.5 days earlier than an HIV-1 antibody test and 4.9 days earlier than a p24 antigen test. For specimens collected from 6 weeks to 18 months old infants born to HIV-1 positive mothers, assay results using both the DBS and plasma protocols agreed well with the Roche Amplicor HIV-1 DNA Test version 1.5 (95.5% agreement for DBS and 97.8% agreement for plasma).
Fiscus, Susan A; McMillion, Takesha; Nelson, Julie A E; Miller, William C
The qualitative Roche HIV-1 DNA Amplicor assay has been used for the past 20 years to diagnose HIV infection in infants and young children but is being phased out; hence, alternative assays must be found. The Gen-Probe Aptima qualitative HIV-1 RNA assay is currently the only FDA-cleared HIV-1 nucleic acid assay approved for diagnosis, but data on the use of this assay with infant plasma are limited. We assessed Aptima's performance using control material for reproducibility and limit of detection and 394 plasma samples (0.2 to 0.5 ml) from HIV-exposed infected and uninfected infants and children for analytical sensitivity and specificity. Assays to assess within-run repeatability and between-run reproducibility indicated that the controls with 10,000 (5 of 5), 200 (5 of 5), 100 (16 of 16), 50 (12 of 12), and 25 (20 of 20) HIV-1 RNA copies/ml (cp/ml) were always positive, and negatives were always negative (20 of 20). The limit of detection was 14 cp/ml, as determined by probit analysis. The analytic sensitivity of the assay was 99.5% (189/190 samples; 95% confidence interval [CI], 97.1 to 99.9%) and specificity was 99.5% (199/200 samples; 95% CI, 97.2 to 99.9%). These results suggest that the assay is suitable for early infant diagnosis of HIV-1.
Higashimoto, Makiko; Takahashi, Masahiko; Jokyu, Ritsuko; Saito, Hidetsugu
A highly sensitive second generation HCV core antigen assay has recently been developed. We compared viral disappearance and kinetics data between commercially available core antigen assays, Lumipulse Ortho HCV Ag, and a quantitative HCV RNA PCR assay, Cobas Amplicor HCV Monitor Test, Version 2 to estimate the predictive benefit of sustained viral response (SVR) and non-SVR in 59 patients treated with interferon and ribavirin combination therapy. We found a good correlation between HCV core Ag and HCV RNA level regardless of genotype. Although the sensitivity of the core antigen assay was lower than PCR, the dynamic range was broader than that of the PCR assay, so that we did not need to dilute the samples in 59 patients. We detected serial decline of core Ag levels in 24 hrs, 7 days and 14 days after interferon combination therapy. The decline of core antigen levels was significant in SVR patients compared to non-SVR as well as in genotype 2a, 2b patients compared to 1b. Core antigen-negative on day 1 could predict all 10 SVR patients (PPV = 100%), whereas RNA-negative could predict 22 SVR out of 25 on day 14 (PPV = 88.0%). None of the patients who had detectable serum core antigen on day 14 became SVR(NPV = 100%), although NPV was 91.2% on RNA negativity. An easy, simple, low cost new HCV core antigen detecting system seems to be useful for assessing and monitoring IFN treatment for HCV.
Jordan, Jeanne A.; Lowery, Donna; Trucco, Massimo
A double-labeled fluorescent probe was designed and evaluated for detecting Trichomonas vaginalis DNA in a 5′ nuclease (TaqMan) assay. The T. vaginalis-specific probe contains a 5′-fluorescein (5′-FAM) and a 3′-rhodamine (TAMRA) derivative. Female genital secretions were collected on Amplicor (Roche Molecular, Indianapolis, Ind.) swabs and by a transport system used for Chlamydia trachomatis and/or Neisseria gonorrhoeae DNA detection by PCR. Five hundred fifty-two female genital specimens, of which 248 (45%) were vaginal specimens and 304 (55%) were introital, were tested for both T. vaginalis DNA and viable microorganisms using the 5′ nuclease assay and broth culture, respectively. Of these, 304 of 552 (55%) were also evaluated by direct microscopic examination for the characteristic motile organism. After resolving discrepancies, the comparisons produced an analytical sensitivity and specificity for the TaqMan-based PCR assay of 97.8 and 97.4%, respectively. As a result, ΔRQ values (differences in fluorescence due to probe hybridization and resulting 5′-FAM cleavage from the specific PCR product) of ≥2.0 and ≤1.5 were established for T. vaginalis-positive and -negative cutoffs, respectively. ΔRQ values between 1.5 and 2.0 were considered indeterminate. Overall findings revealed a high level of agreement between PCR and culture for detecting T. vaginalis. Potential benefits of the 5′ nuclease assay include a greater sensitivity compared to direct microscopic examination and the ease of testing large numbers of clinical specimens in a significantly shorter turnaround time compared to culture. PMID:11682492
Ostergaard, L; Møller, J K
A procedure for use of the Amplicor Chlamydia PCR with the Syva MicroTrak enzyme immunoassay (EIA) medium was developed, and the performance of the Syva MicroTrak EIA was evaluated by use of PCR and the Syva MicroTrak direct immunofluorescence assay (DFA) as confirmatory methods. PCR detected Chlamydia organisms at a 10-fold greater dilution than did DFA. Of 366 specimens, 119 specimens were positive by both PCR and DFA, 6 specimens were positive only by PCR, and 241 specimens were negative by both PCR and DFA. Subsequently, DFA and the developed PCR procedure were used prospectively for confirmation of EIA results in a defined negative gray zone between the cutoff value and 30% of the cutoff value (70% below the cutoff value). All specimens with results above the EIA cutoff value were also subjected to confirmation with DFA and PCR. EIA was performed on 7,748 endocervical swab specimens, of which 494 (6.4%) were subjected to confirmation, and on 968 male urethral swab specimens, of which 185 (19.1%) were subjected to confirmation. A "gold standard" was based on the findings by DFA and PCR, and divergent results were resolved by a major outer membrane protein-based PCR. Forty-five of 160 female specimens (28.1%) and 11 of 93 male specimens (11.8%) within the defined negative gray zone were found to be positive. Of 334 female specimens having absorbance unit (AU) values above the EIA cutoff value, 258 could be confirmed, thereby giving a positive predictive value of 77% (258/334). Accordingly, the positive predictive value with male specimens was 95% (87/92). The prevalence of Chlamydia trachomatis-positive specimens was 3.9% (303/7,748) in females and 10.1% (98/968) in males.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8567894
Cassol, S; Gill, M J; Pilon, R; Cormier, M; Voigt, R F; Willoughby, B; Forbes, J
To assess dried plasma spots (DPSs) as a source of material for virus quantification, human immunodeficiency virus type 1 (HIV-1) RNA levels were quantified in matched DPS and liquid plasma samples from 73 infected patients, including 5 neonates and 4 adult patients with acute HIV-1 infection. Quantifications were performed by commercially available assays (NASBA [nucleic acid sequence-based amplification] or Amplicor, or both). There was a strong correlation between HIV-1 RNA levels in plasma and DPSs. More importantly, there was no decline in HIV-1 RNA levels in DPSs stored for as long as 2 weeks at 20 degrees C. Similarly, storage of DPSs for 3 days at 37 degrees C resulted in no decrease in viral RNA levels. For patients with primary infection, the DPS method allowed for the measurement of RNA levels in plasma during the initial spike in the level of viremia and in the subsequent period of suppressed viral replication. DPS quantification was equally informative in the neonatal setting, with all five newborns showing HIV-1 RNA loads of greater than 4.991 log10 copies/ml. We conclude that the viral RNA levels in DPSs are equivalent to those measured in fresh-frozen plasma. The ease and economy of DPS sampling, the minute volumes required, and the unexpected stability of dried RNA suggest that the use of DPSs will be particularly valuable for small-volume neonatal samples and large, population-based studies in which cold storage and transportation present special problems, as is often the case in developing countries. The ability to measure viral changes during primary infection suggests that the method will be useful for assessing vaccine efficacy in large field trials. PMID:9350736
Yohannan, Jithin; He, Bing; Wang, Jiangxia; Greene, Gregory; Schein, Yvette; Mkocha, Harran; Munoz, Beatriz; Quinn, Thomas C.; Gaydos, Charlotte; West, Sheila K.
Purpose. We detected spatial clustering of households with Chlamydia trachomatis infection (CI) and active trachoma (AT) in villages undergoing mass treatment with azithromycin (MDA) over time. Methods. We obtained global positioning system (GPS) coordinates for all households in four villages in Kongwa District, Tanzania. Every 6 months for a period of 42 months, our team examined all children under 10 for AT, and tested for CI with ocular swabbing and Amplicor. Villages underwent four rounds of annual MDA. We classified households as having ≥1 child with CI (or AT) or having 0 children with CI (or AT). We calculated the difference in the K function between households with and without CI or AT to detect clustering at each time point. Results. Between 918 and 991 households were included over the 42 months of this analysis. At baseline, 306 households (32.59%) had ≥1 child with CI, which declined to 73 households (7.50%) at 42 months. We observed borderline clustering of households with CI at 12 months after one round of MDA and statistically significant clustering with growing cluster sizes between 18 and 24 months after two rounds of MDA. Clusters diminished in size at 30 months after 3 rounds of MDA. Active trachoma did not cluster at any time point. Conclusions. This study demonstrates that CI clusters after multiple rounds of MDA. Clusters of infection may increase in size if the annual antibiotic pressure is removed. The absence of growth after the three rounds suggests the start of control of transmission. PMID:24906862
Kuritzkes, Daniel R.; Grant, Robert M.; Feorino, Paul; Griswold, Marshal; Hoover, Marie; Young, Russell; Day, Stephen; Lloyd, Jr., Robert M.; Reid, Caroline; Morgan, Gillian F.; Winslow, Dean L.
The TRUGENE HIV-1 Genotyping Kit and OpenGene DNA Sequencing System are designed to sequence the protease (PR)- and reverse transcriptase (RT)-coding regions of human immunodeficiency virus type 1 (HIV-1) pol. Studies were undertaken to determine the accuracy of this assay system in detecting resistance-associated mutations and to determine the effects of RNA extraction methods, anticoagulants, specimen handling, and potentially interfering substances. Samples were plasma obtained from HIV-infected subjects or seronegative plasma to which viruses derived from wild-type and mutant infectious molecular clones (IMC) of HIV-1 were added. Extraction methods tested included standard and UltraSensitive AMPLICOR HIV-1 MONITOR, QIAGEN viral RNA extraction mini kit, and QIAGEN Ultra HIV extraction kit, and NASBA manual HIV-1 quantitative NucliSens. Sequence data from test sites were compared to a “gold standard” reference sequence to determine the percent agreement. Comparisons between test and reference sequences at the nucleotide level showed 97.5 to 100% agreement. Similar results were obtained regardless of extraction method, regardless of use of EDTA or acid citrate dextrose as anticoagulant, and despite the presence of triglycerides, bilirubin, hemoglobin, antiretroviral drugs, HIV-2, hepatitis C virus (HCV), HBV, cytomegalovirus, human T-cell leukemia virus type 1 (HTLV-1), or HTLV-2. Samples with HIV-1 RNA titers of ≥1,000 copies/ml gave consistent results. The TRUGENE HIV-1 Genotyping Kit and OpenGene DNA Sequencing System consistently generate highly accurate sequence data when tested with IMC-derived HIV and patient samples. PMID:12682150
O'Connell, Emer; Brennan, Wendy; Cormican, Martin; Glacken, Marita; O'Donovan, Diarmuid; Vellinga, Akke; Cahill, Niall; Lysaght, Fionnguala; O'Donnell, Joan
Background There are no prevalence data on Chlamydia trachomatis relating to female students attending higher education available for the Republic of Ireland. This information is required to guide on the necessity for Chlamydia screening programmes in higher education settings. This research aimed to determine the prevalence of and predictive risk factors for Chlamydia trachomatis genital infection among female higher education students in Ireland. Methods All females presenting during one-day periods at Student Health Units in three higher education institutions in two cities in the Republic of Ireland were invited to participate. Participants completed a questionnaire on lifestyle and socio-demographic factors and provided a urine sample. Samples were tested for C. trachomatis DNA by a PCR based technique (Cobas Amplicor, Roche). To examine possible associations between a positive test and demographic and lifestyle risk factors, a univariate analysis was performed. All associations with a p value < 0.05 were included in a multivariate logistic regression analysis. Results Of the 460 sexually active participants 22 tested positive (prevalence 4.8%; 95% CI 3.0 to 7.1%). Variables associated with significantly increased risk were current suggestive symptoms, two or more one-night stands and three or more lifetime sexual partners. The students displayed high-risk sexual behaviour. Conclusion The prevalence of C. trachomatis infection and the lack of awareness of the significance of suggestive symptoms among sexually experienced female students demonstrate the need for a programme to test asymptomatic or non-presenting higher education students. The risk factors identified by multivariate analysis may be useful in identifying those who are most likely to benefit from screening. Alcohol abuse, condom use, sexual behaviour (at home and abroad) and, knowledge of sexually transmitted infections (STIs) (including asymptomatic nature or relevant symptoms) were identified as
Ikezaki, Hiroaki; Furusyo, Norihiro; Ihara, Takeshi; Hayashi, Takeo; Ogawa, Eiichi; Toyoda, Kazuhiro; Taniai, Hiroaki; Kainuma, Mosaburo; Murata, Masayuki; Hayashi, Jun
This study was done to evaluate the utility of the Abbott RealTime PCR assay (ART) for the monitoring of chronic hepatitis C patients. The serum samples of 183 patients infected with hepatitis C virus (HCV) genotype 1b who had completed a 48-week period of pegylated interferon (PEG-IFN) alpha-2b plus ribavirin treatment were prospectively analyzed. Serum HCV RNA levels were measured both by ART and by the Roche COBAS Amplicor Monitor test, version2.0 (CAM) at baseline and at weeks 4, 12, 24, 36, and 48 of treatment, and at 24 weeks after the end of treatment (EOT). A significant positive correlation of pretreatment HCV RNA levels was found between ART and CAM (r = 0.595, P < 0.0001). Of the 183 patients, 66 (36.0%) achieved a sustained virological response (SVR). The logarithmic decline of the HCV RNA level from the pretreatment level determined by ART in SVR patients was significantly higher than that in non-SVR patients at all time points tested. The logarithmic decline determined by CAM in SVR patients was significantly higher than that in non-SVR patients only at week 4, but there was no significant difference at other weeks. Of 124 patients who were HCV RNA-negative at EOT by ART, 58 (46.8%) had a relapse of viremia at 24 weeks after EOT, whereas 77 of 143 patients (53.8%) who were HCV RNA-negative at EOT by CAM had a relapse. The relapse rate was lower when determined by ART than by CAM, but not significantly so. ART is more useful than CAM for evaluating the virological response to antiviral treatment for chronic hepatitis C. PMID:21528383
Grennan, J. Troy; Loutfy, Mona R.; Su, DeSheng; Harrigan, P. Richard; Cooper, Curtis; Klein, Marina; Machouf, Nima; Montaner, Julio S. G.; Rourke, Sean; Tsoukas, Christos; Hogg, Bob
(See the editorial commentary by Taiwo and Bosch, on pages 1189–91.) Background. The importance of human immunodeficiency virus (HIV) blip magnitude on virologic rebound has been raised in clinical guidelines relating to viral load assays. Methods. Antiretroviral-naive individuals initiating combination antiretroviral therapy (cART) after 1 January 2000 and achieving virologic suppression were studied. Negative binomial models were used to identify blip correlates. Recurrent event models were used to determine the association between blips and rebound by incorporating multiple periods of virologic suppression per individual. Results. 3550 participants (82% male; median age, 40 years) were included. In a multivariable negative binomial regression model, the Amplicor assay was associated with a lower blip rate than branched DNA (rate ratio, 0.69; P < .01), controlling for age, sex, region, baseline HIV-1 RNA and CD4 count, AIDS-defining illnesses, year of cART initiation, cART type, and HIV-1 RNA testing frequency. In a multivariable recurrent event model controlling for age, sex, intravenous drug use, cART start year, cART type, assay type, and HIV-1 RNA testing frequency, blips of 500–999 copies/mL were associated with virologic rebound (hazard ratio, 2.70; P = .002), whereas blips of 50–499 were not. Conclusions. HIV-1 RNA assay was an important determinant of blip rates and should be considered in clinical guidelines. Blips ≥500 copies/mL were associated with increased rebound risk. PMID:22438396
Because cultures of mycobacteria and viruses take a great number of days, the results are delayed in these clinical laboratory tests. For this reason, rapid diagnostic tests without these cultures are required. Among the laboratory tests of mycobacteria, acid-fast stain as a rapid diagnostic test is the first to give significant information to laboratory staffs and physicians. There are mainly two procedures, directly smearing clinical specimens and smearing concentrated specimens treated with NALC-NaOH on slide glass. The former is performed for specimens from emergency patients. When a patient is suspected of tuberculosis, a DNA-amplification test such as COBAS AMPLICOR TEST (PCR test) is performed for directly detecting Mycobacterium tuberculosis in clinical specimens, regardless of whether the specimens show positive or negative by acid-fast stain. Sixty-one of 1,023 specimens tested in Showa University Fujigaoka Hospital in February 2000-May 2002 were found positive by both PCR and culture tests. Fourteen were found negative by PCR test and positive by culture test. Five were found positive by PCR test and negative by culture test. Alternatively, MTB-64 (Capilia TB) is a kit for rapidly identifying M. tuberculosis using colonies. The time necessary for completion of the test was approximately 15 minutes per sample. In our study, the identification test showed weak-positive for 2 strains of M. marinum (ATCC927 and one clinical isolate), compared with positive for 28 clinical isolates and strain H37Ra of M. tuberculosis. Recently, rapid diagnostic kits for detecting various pathogenic viruses have been developed. The kits for detecting influenza virus are in especially great demand with the spread of newly-developed antiviral agents. There was an epidemic of the type A virus in the winters 2001-2002. 101 (32%) of 316 clinical specimens tested in the hospital were found positive with InfluA. B-Quick [SEIKEN] (Denka) or Directigen Flu A (Becton Dickinson). These
Madico, Guillermo; Quinn, Thomas C.; Boman, Jens; Gaydos, Charlotte A.
Three touchdown enzyme time release (TETR)-PCR assays were used to amplify different DNA sequences in the variable regions of the 16S and 16S-23S spacer rRNA genes specific for Chlamydia trachomatis, Chlamydia pneumoniae, and Chlamydia psittaci as improved tests for sensitive diagnosis and rapid species differentiation. The TETR-PCR protocol used 60 cycles of amplification, which provided improved analytical sensitivity (0.004 to 0.063 inclusion-forming unit of Chlamydia species per PCR). The sensitivity of TETR-PCR with primer set CTR 70-CTR 71 was 96.7%, and the specificity was 99.6%, compared to those of the AMPLICOR PCR for the detection of C. trachomatis in vaginal swab samples. TETR-PCR for C. pneumoniae with primer set CPN 90-CPN 91 was 90% sensitive and 93.3% specific compared with a nested PCR with primer set CP1/2-CPC/D for clinical respiratory samples. TETR-PCR for C. psittaci with primer set CPS 100-CPS 101 showed substantial agreement with cell culturing (κ, 0.78) for animal tissue samples. Primer sets were then combined into a single multiplex TETR-PCR test. The respective 315-, 195-, and 111-bp DNA target products were precisely amplified when DNA from each of the respective Chlamydia species or combinations of them was used. Multiplex chlamydia TETR-PCR correctly identified one strain of each of the 15 serovars of C. trachomatis, 22 isolates of C. pneumoniae, and 20 isolates of C. psittaci. The primer sets were specific for each species. No target products were amplified when DNA from C. pecorum or a variety of other microorganisms was tested for specificity. TETR-PCR with primers selected for specific sequences in the 16S and 16S-23S spacer rRNA genes is a valuable test that could be used either with individual primers or in a multiplex assay for the identification and differentiation of Chlamydia species from culture isolates or for the detection of chlamydiae in clinical samples. PMID:10699002
Background Viral hepatitis is a serious public health problem affecting billions of people globally. Limited information is available on this issue in Morocco. This cross-sectional study was undertaken with the aim of determining the seroprevalence and risk factors of hepatitis B virus (HBV) and hepatitis C virus (HCV) among the general population and among blood donors. Methods Blood samples from volunteers, have been screened with ELISA tests for detecting the hepatitis-B surface antigen (HBsAg) and anti-HCV. Within the seroreactive patients for HCV in the general population, RT-PCR was performed by the Cobas Ampliprep/Cobas Amplicor. Results HCV and HBV-seropositivity was documented in 1.58% and 1.81% out of 41269 and 23578 participants respectively from the general population. Two patients were found to be co-infected. HCV-RNA was detected by PCR in 70.9% of the 195 anti-HCV positive subjects. The anti-HCV prevalence was not different among males and females (P = 0.3). It increased with age; the highest prevalence was observed among subjects with >50 years old (3.12%). Various risk factors for acquiring HCV infection were identified; age, dental treatment, use of glass syringes and surgical history. In addition to these factors, gender and sexual risk behaviors were found to be associated with higher prevalence of hepatitis B. The HBV positivity was significantly higher among males than females participants in all age groups (P < 0.01). The peak was noticed among males aged 30–49 years (2.4%). None of the 152 persons younger than 20 years had HBsAg or anti-HCV. The prevalence of anti-HCV and HBsAg among 169605 blood donors was 0.62% and 0.96% respectively. Conclusions Our study provided much important information concerning hepatitis B and C prevalence and risk factors; it confirmed the intermediate endemicity for HCV infection and pointed to a decreasing trend of HBV incidence, which might reclassify Morocco in low HBV endemicity area. This could be
Pant, Bidya Prasad; Bhatta, Ramesh C.; Chaudhary, J. S. P.; Awasthi, Suresh; Mishra, Sailesh; Sharma, Shekhar; Cuddapah, Puja A.; Gwyn, Sarah E.; Stoller, Nicole E.; Martin, Diana L.; Keenan, Jeremy D.; Lietman, Thomas M.; Gaynor, Bruce D.
Background The WHO seeks to control trachoma as a public health problem in endemic areas. Achham District in western Nepal was found to have TF (trachoma follicular) above 20% in a 2006 government survey, triggering 3 annual mass drug administrations finishing in 2010. Here we assess the level of control that has been achieved using surveillance for clinical disease, ocular chlamydia trachomatis infection, and serology for antibodies against chlamydia trachomatis protein antigens. Methods We conducted a cross-sectional survey of children aged 1–9 years in communities in Achham District in early 2014 including clinical examination validated with photographs, conjunctival samples for Chlamydia trachomatis (Amplicor PCR), and serological testing for antibodies against chlamydia trachomatis protein antigens pgp3 and CT694 using the Luminex platform. Findings In 24 randomly selected communities, the prevalence of trachoma (TF and/or TI) in 1–9 year olds was 3/1124 (0.3%, 95% CI 0.1 to 0.8%), and the prevalence of ocular chlamydia trachomatis infection was 0/1124 (0%, 95% CI 0 to 0.3%). In 18 communities selected because they had the highest prevalence of trachoma in a previous survey, the prevalence of TF and/or TI was 7/716 (1.0%, 95% CI 0.4 to 2.0%) and the prevalence of ocular chlamydia trachomatis infection was 0/716 (0%, 95% CI 0 to 0.5%). In 3 communities selected for serological testing, the prevalence of trachoma was 0/68 (0%, 95% CI 0 to 5.3%), the prevalence of ocular chlamydia trachomatis infection was 0/68 (0%, 95% CI 0 to 0.5%), the prevalence of antibodies against chlamydia trachomatis protein antigen pgp3 was 1/68 (1.5%, 95% CI 0.04% to 7.9%), and the prevalence of antibodies against chlamydia trachomatis protein antigen CT694 was 0/68 (0%, 95% CI 0 to 5.3%). Conclusion/Significance This previously highly endemic district in Nepal has little evidence of recent clinical disease, chlamydia trachomatis infection, or serological evidence of trachoma
Ramjee, Gita; van der Straten, Ariane; Chipato, Tsungai; de Bruyn, Guy; Blanchard, Kelly; Shiboski, Stephen; Cheng, Helen; Montgomery, Elizabeth; Padian, Nancy
Background We evaluated the effectiveness of the Ortho All-Flex Diaphragm, lubricant gel (Replens®) and condoms compared to condoms alone on the incidence of chlamydial and gonococcal infections in an open-label randomized controlled trial among women at risk of HIV/STI infections. Methods We randomized 5045 sexually-active women at three sites in Southern Africa. Participants who tested positive for curable STIs were treated prior to enrollment as per local guidelines. Women were followed quarterly and tested for Chlamydia trachomatis (CT) or Neisseria gonorrhoeae (GC) infection by nucleic-acid amplification testing (Roche Amplicor®) using first-catch urine specimens. STIs detected at follow-up visits were treated. We compared the incidence of first infection after randomization between study arms in both intent-to-treat (ITT) and per-protocol populations. Findings Baseline demographic, behavioral and clinical characteristics were balanced across study arms. Nearly 80% of participants were under 35 years of age. Median follow-up time was 21 months and the retention rate was over 93%. There were 471 first chlamydia infections, 247 in the intervention arm and 224 in the control arm with an overall incidence of 6.2/100 woman-years (wy) (relative hazard (RH) 1.11, 95% Confidence Interval (CI): 0.93–1.33; p = 0.25) and 192 first gonococcal infections, 95 in the intervention arm and 97 in the control arm with an overall incidence of 2.4/100wy (RH 0.98, 95%CI: 0.74–1.30; p = 0.90). Per protocol results indicated that when diaphragm adherence was defined as “always use” since the last visit, there was a significant reduction in the incidence of GC infection among women randomized to the intervention arm (RH 0.61, 95%CI: 0.41–0.91, P = 0.02). Interpretation There was no difference by study arm in the rate of acquisition of CT or GC. However, our per-protocol results suggest that consistent use of the diaphragm may reduce acquisition of GC. Trial
Weber, B; Melchior, W; Gehrke, R; Doerr, H W; Berger, A; Rabenau, H
Isolated reactivity to hepatitis B virus (HBV) core antigen (anti-HBc) is observed relatively frequently in immunocompromised individuals, intravenous drug abusers (IVDA), and in the presence of HCV infection. The reason for the lack of HBsAg is not clear. The aim of the present study was to investigate which factors (genetic variability of S gene, low-level HBsAg, and immune complexes may be responsible for the failure of HBsAg detection with commercial HBsAg screening assays. Dilution series of two recombinant HBsAg escape mutants and dilutions of serum samples from chronic HBV carriers with multiple insertions in the a determinant and different HBsAg subtypes were tested with a highly sensitive assay that detects wild-type HBsAg (Elecsys HBsAg, Roche Diagnostics, Penzberg, Germany) and two assays that detect HBV wild-type and escape mutants (Murex HBsAg Version 3, Murex and Enzygnost HBsAg 5.0, Dade Behring, Marburg, Germany). Elecsys HBsAg showed in comparison to Murex HBsAg Version 3 and Enzygnost HBsAg 5.0 a reduced sensitivity for escape mutant detection. On the other hand, the best performance for HBsAg subtype detection was obtained with Elecsys HBsAg. In the second part of the study, a selected panel of isolated anti-HBc reactive (n = 104) serum samples (AxSYM Core) was submitted to testing by Elecsys HBsAg, Murex HBsAg Version 3, Enzygnost HBsAg 5.0, and HBsAg detection after immune complex dissociation (ICD) and anti-HBs determination with two different assays (AxSYM Ausab and Elecsys Anti-HBs). To assess the specificity of anti-HBc test results, all the samples were tested by a second anti-HBc assay (Elecsys Anti-HBc). Quantitative HBV DNA detection was undertaken with a commercially available HBV PCR assay (Amplicor HBV Monitor). HCV infection was present in 65.4% of anti-HBc only reactive individuals. Five AxSYM Core positive samples were negative by Elecsys Anti-HBc. Overall, 15 (14.4%) AxSYM Ausab negative samples gave positive results with Elecsys