Lamivudine resistance of hepatitis B virus masked by coemergence of mutations in probe region of the COBAS AMPLICOR assay.
Ontology highlight
ABSTRACT:
SUBMITTER: Lindh M
PROVIDER: S-EPMC1489515 | biostudies-literature | 2006 Jul
REPOSITORIES: biostudies-literature
Publications
Lamivudine resistance of hepatitis B virus masked by coemergence of mutations in probe region of the COBAS AMPLICOR assay.
Lindh Magnus M Hannoun Charles C Malmström Sebastian S Lindberg Johan J Norkrans Gunnar G
Journal of clinical microbiology 20060701 7
The COBAS AMPLICOR hepatitis B virus assay targets a conserved region of the genome and is widely used to monitor treatment of hepatitis B in order to identify emerging resistance. However, the assay failed to recognize increasing viremia levels when YMDD mutations were paralleled by mutations in the segment targeted by the COBAS AMPLICOR probe. ...[more]
Similar Datasets
Project description:With the availability of more potent nucleotide/nucleoside analogues, the early detection of drug-resistant mutants of hepatitis B virus (HBV) is important for the strategic treatment of chronic hepatitis B. We studied 336 serum samples from 80 patients chronically infected with HBV who were receiving lamivudine treatment for the presence of lamivudine resistance mutations at codons 80, 173, 180, and 204 of the HBV polymerase. The sequencing data were compared with the results generated with the INNO-LiPA HBV DR (drug resistance) v2 strip, a line probe assay (LiPA) covering wild-type and mutant motifs, for resistance mutations to lamivudine and adefovir dipivoxil. This method provided at least the same information as sequencing for 99.1% of all codons analyzed. On the basis of the LiPA results, 20 of 80 patients developed a lamivudine resistance mutation after 1 year. In all 20 patients, the mutation occurred in the YMDD motif at reverse transcriptase position 204 (rt204; M204V/I) either with or without the compensatory mutation at position rt180 (L180M). A compensatory mutation at position rt80 (L80V/I) was detected in half of these patients. After 36 months, a compensatory mutation was seen at position rt173 (V173L) in 3/15 patients. Time-to-event survival analysis indicated a 2.8 times greater chance for LiPA to detect a given mutation than sequencing at any moment in time (hazard ratio, 2.8, 95% confidence interval, 1.79, 4.41; P < 0.0001). These results demonstrate that a highly sensitive and specific assay such as the INNO-LiPA HBV DR v2 can precociously detect and monitor the emergence of primary and compensatory lamivudine resistance mutations in patients chronically infected with HBV and is more sensitive than sequencing.
| S-EPMC2168556 | biostudies-literature
Project description:Screening assays for Neisseria gonorrhoeae exhibit low positive predictive values, particularly in low-prevalence populations. A new real-time PCR assay that detects and identifies individual Neisseria spp. using melt curve analysis was compared to two previously published supplementary assays. NsppID, a 16S rRNA real-time PCR/melt curve assay developed to distinguish N. gonorrhoeae from other Neisseria spp., was compared to real-time PCR assays targeting genes reportedly specific for N. gonorrhoeae, the cppB gene and the porA pseudogene. A total of 408 clinical specimens (324 female endocervical swabs and 84 male urine or urogenital swab specimens) were screened using the COBAS Amplicor assay for Chlamydia trachomatis and N. gonorrhoeae (CT/NG) (Roche Diagnostics, Indianapolis, IN) followed by confirmatory testing via real-time PCR. The NsppID assay detected Neisseria spp. in 150/181 COBAS-positive specimens (82%), including six dual infections, and identified N. gonorrhoeae in 102 (56%) specimens. Sixty-nine of 181 (38%) specimens were positive for N. gonorrhoeae by porA pseudogene, and 115/181 (64%) were positive for cppB. However, cppB was also positive in 15% of COBAS-negative specimens, more than either NsppID (4%) or porA pseudogene (2%) assays. The porA pseudogene assay had the highest specificity for both genders but the lowest sensitivity, especially in female specimens. NsppID had a slightly lower specificity but greater sensitivity and overall accuracy. The least desirable confirmatory assay was cppB, due to poor specificity. The NsppID assay is an accurate confirmatory assay for N. gonorrhoeae detection. In addition, the NsppID assay can identify the non-N. gonorrhoeae species responsible for the majority of false-positive results from the COBAS Amplicor CT/NG assay.
| S-EPMC1865884 | biostudies-literature
Project description:Screening guidelines for men who have sex with men (MSM) recommend testing of extragenital sites (pharyngeal and rectal) for gonorrhoea and chlamydia. Testing of specimens from these sites is not validated by most commercial nucleic amplification tests, such as the COBAS Amplicor assay. To investigate the utility of the COBAS Amplicor assay for detection of Chlamydia trachomatis in extragenital specimens, this study developed and evaluated confirmatory tests using the omp1 gene as an alternative target for amplification by PCR. Of anal and throat swabs collected from men in male-only saunas, 52 swabs that tested C. trachomatis positive by COBAS Amplicor and 30 swabs that tested as negative were included for confirmatory omp1 PCR testing. A total of 49 (94%) COBAS Amplicor-positive samples were confirmed by the omp1 PCR. A substantial proportion of specimens were confirmed by using a nested omp1 PCR (27%). Not confirmed by any omp1 PCR were three anal swabs (6%). It is most probable that these samples contained lower bacterial levels that were near or below the detection level of the omp1 PCR assays. The findings of this study support the confident reporting of C. trachomatis detected by COBAS Amplicor in extragenital specimens and support the utility of this assay as a screening test for MSM.
| S-EPMC321736 | biostudies-literature
Project description:A duplex quantitative real-time PCR (qPCR) assay was designed to detect both the polymerase gene (pol) and the glycoprotein gene (gB) of cytomegalovirus (CMV). The detection limit of the qPCR was determined to be 1 to 3 copies/reaction and the linear measure interval was 10(3) to 10(8) copies/ml. The qPCR system was compared to the COBAS Amplicor CMV Monitor test (COBAS) by an analysis of 138 plasma samples. Both systems detected CMV in 71 cases and had negative results for 33 samples. In addition, 34 samples were positive by qPCR and negative by the COBAS assay, but in no case was the COBAS result positive and the qPCR result negative. Thus, qPCR detected 48% more positive cases than the COBAS method. For samples with > or = 10(5) copies/ml by qPCR, a saturation effect was seen in the COBAS assay and quantification required dilution. Copy numbers for pol and gB by qPCR generally agreed. However, the reproducibility of qPCR assays and the need for an international standard are discussed. Discrepant copy numbers for pol and gB by qPCR were found for samples from two patients, and sequence analysis revealed that the corresponding CMV strains were mismatched at four nucleotide positions compared with the gB fragment primer sequences. In conclusion, a duplex qPCR assay in a real-time format facilitates quantitative measurements and minimizes the risk of false-negative results.
| S-EPMC404600 | biostudies-literature
Project description:Hepatitis C virus (HCV) RNA viral load (VL) monitoring is a well-established diagnostic tool for the management of chronic hepatitis C patients. HCV RNA VL results are used to make treatment decisions with the goal of therapy to achieve an undetectable VL result. Therefore, a sensitive assay with high specificity in detecting and accurately quantifying HCV RNA across genotypes is critical. Additionally, a lower sample volume requirement is desirable for the laboratory and the patient. This study evaluated the performance characteristics of a second-generation real-time PCR assay, the Cobas AmpliPrep/Cobas TaqMan HCV quantitative test, version 2.0 (CAP/CTM HCV test, v2.0), designed with a novel dual-probe approach and an optimized automated extraction and amplification procedure. The new assay demonstrated a limit of detection and lower limit of quantification of 15 IU/ml across all HCV genotypes and was linear from 15 to 100,000,000 IU/ml with high accuracy (<0.2-log(10) difference) and precision (standard deviation of 0.04 to 0.22 log(10)). A specificity of 100% was demonstrated with 600 HCV-seronegative specimens without cross-reactivity or interference. Correlation to the Cobas AmpliPrep/Cobas TaqMan HCV test (version 1) was good (n = 412 genotype 1 to 6 samples, R(2) = 0.88; R(2) = 0.94 without 105 genotype 4 samples). Paired plasma and serum samples showed similar performance (n = 25, R(2) = 0.99). The sample input volume was reduced from 1 to 0.65 ml in the second version. The CAP/CTM HCV test, v2.0, demonstrated excellent performance and sensitivity across all HCV genotypes with a smaller sample volume. The new HCV RNA VL assay has performance characteristics that make it suitable for use with currently available direct-acting antiviral agents.
| S-EPMC3553902 | biostudies-literature
Project description:Treatment of chronic hepatitis B virus (HBV) infection with lamivudine-monotherapy rapidly selects mutant variants in a high proportion of individuals. Monitoring lamivudine resistance by consensus sequencing is costly and insensitive for detection of minority variants. An oligonucleotide ligation assay (OLA) for HBV lamivudine-resistance was developed and compared to consensus sequencing. Both assays detected drug resistance mutations in 35/64 (54.7%) specimens evaluated, and OLA detected minority mutants in an additional six (9.4%). OLA may offer a sensitive and inexpensive alternative to consensus sequencing for detection of HBV drug resistance in resource-limited settings.
| S-EPMC5074383 | biostudies-literature
Project description:Lamivudine was the first approved inhibitor of hepatitis B virus (HBV) reverse transcriptase (RT). Lamivudine resistance develops in 53% to 76% of patients after 3 years of treatment. We extensively characterized the dynamics of HBV quasispecies variant populations in four HBV-infected patients who developed lamivudine resistance. Virological breakthrough was preceded by 2 to 4 months by the emergence of quasispecies variants bearing amino acid substitutions at RT position 204, i.e., within the YMDD catalytic motif (rtM204V/I). Three patients had a gradual switch from a YMDD variant population at baseline to a 100% lamivudine-resistant variant population, whereas the remaining patient had a fluctuating pattern of resistance variant dynamics. Careful analysis of amino acid substitutions located outside domain C of HBV RT, including those known to partially restore replication capacities in vitro, showed that the in vivo replication of HBV variants is driven by multiple forces, including intrinsic replicative advantages conferred by mutations accumulating outside domain C and the changing environment in which these variants replicate. Our findings also suggest that individual treatment optimization will require sensitive methods capable of detecting the emergence of viral resistance before the relevant variants acquire optimal replicative capacities.
| S-EPMC1346832 | biostudies-literature
Project description:The high genetic variability of hepatitis B virus (HBV) can impair DNA quantification. Here, we investigate a major underquantification of HBV by the cobas TaqMan HBV assay (CTM; Roche). In France, between 2005 and 2017, HBV DNA was detected in 3,102 blood donations by use of the CTM (95% limit of detection [LOD95], 4.8?IU/ml). HBV strains were sequenced in the S region (LOD95, ?30?IU/ml). Concordant (n?=?120) and discordant (n?=?45) samples were identified according to the agreement between the plasma viral load (pVL) determined by the CTM and sequencing; all samples were also quantified using the RealTime HBV assay (RTH; Abbott). The viral signature, cloning, and mutagenesis were used to characterize the polymorphism responsible for CTM misquantification. A CTM-RTH discordance (>1 log IU/ml) was found in 14/45 samples that had low pVLs and were successfully genotyped (pVLlow genoS+). PreC/C clones of concordant (C1, C2) and discordant (D1, D2) strains were used to challenge the CTM. Strains D1 and D2 were highly underquantified (42- and 368-fold). In clones, mutating the region corresponding to the CTM reverse primer from a discordant sequence to a concordant sequence restored the levels of quantification to 24% (D1?C1) and 59% (D2?C1) of theoretical levels, while mutating the sequence of a concordant strain to that of a discordant strain led to 78-fold (C1?D1) and 146-fold (C1?D2) decreases in quantification. Moreover, mutating positions 1961 and 1962 was enough to induce a 5-fold underquantification. We conclude that the CTM underestimates pVLs for HBV strains with mutations in the reverse primer target. Specifically, the polymorphism at nucleotides 1961 and 1962 is naturally present in 4.79 and 4.22% of genotype A and D strains, which are highly frequent in Europe, leading to a 5-fold decrease in quantification. Quantification using the new-generation Roche C4800 assay is not affected by this polymorphism.
| S-EPMC7448625 | biostudies-literature
Project description:We have recently developed a PCR assay for detection of Mycobacterium spp. at the genus level based on the Cobas Amplicor platform. The sensitivities for smear-positive and smear-negative specimens were found to be 100% and 47.9%, respectively. The specificity was 97.7%, the positive predictive value 84.6%, and the negative predictive value 93.1%. In a follow-up study, we have systematically evaluated the Mycobacterium genus assay in parallel with the Cobas Amplicor Mycobacterium tuberculosis assay on 2,169 clinical specimens, including respiratory and nonrespiratory specimens. Based on the genus assay, nontuberculous mycobacteria were readily detected and identified to the species level by PCR-mediated sequencing. In addition, our data point to a limited specificity of the Cobas Amplicor M. tuberculosis assay.
| S-EPMC3020847 | biostudies-literature
Project description:BACKGROUND: Lamivudine is an oral nucleoside analogue widely used for the treatment of chronic hepatitis B. The main limitation of lamivudine use is the selection of resistant mutations that increases with time of utilization. Hepatitis B virus (HBV) isolates have been classified into eight genotypes (A to H) with distinct geographical distributions. HBV genotypes may also influence pathogenic properties and therapeutic features. Here, we analyzed the HBV genotype distribution and the nature and frequency of lamivudine resistant mutations among 36 patients submitted to lamivudine treatment for 12 to 84 months. RESULTS: Half of the patients were homosexual men. Only 4/36 (11%) patients were HBV DNA negative. As expected for a Brazilian group, genotypes A (24/32 positive individuals, 75%), D (3/32, 9.3%) and F (1/32, 3%) were present. One sample was from genotype C, which is a genotype rarely found in Brazil. Three samples were from genotype G, which had not been previously detected in Brazil. Lamivudine resistance mutations were identified in 20/32 (62%) HBV DNA positive samples. Mean HBV loads of patients with and without lamivudine resistance mutations were not very different (2.7 x 107 and 6.9 x 107 copies/mL, respectively). Fifteen patients showed the L180M/M204V lamivudine resistant double mutation. The triple mutant rt173V/180M/204V, which acts as a vaccine escape mutant, was found in two individuals. The three isolates of genotype G were entirely sequenced. All three showed the double mutation L180M/M204V and displayed a large genetic divergence when compared with other full-length genotype G isolates. CONCLUSION: A high (55%) proportion of patients submitted to long term lamivudine therapy displayed resistant mutations, with elevated viral load. The potential of transmission of such HBV mutants should be monitored. The identification of genotypes C and G, rarely detected in South America, seems to indicate a genotype distribution different to that observed in non treated patients. Disparities in routes of transmission (genotype G seems to be linked to homosexual behavior) and in pathogenic properties (genotype C is very aggressive) among HBV genotypes may explain the presence of rare genotypes in the present work.
| S-EPMC2245951 | biostudies-literature