| 1. |
- Lindstrom, A., et al.
(författare)
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Pyrosequencing for detection of lamivudine-resistant hepatitis B virus
- 2004
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Ingår i: Journal of Clinical Microbiology. - : American Society for Microbiology. - 0095-1137 .- 1098-660X. ; 42:10, s. 4788-4795
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Tidskriftsartikel (refereegranskat)abstract
- Chronic hepatitis B virus (HBV) infection can cause severe liver disease, including cirrhosis and hepatocellular carcinoma. Lamivudine is a relatively recent alternative to alpha interferon for the treatment of HBV infection, but unfortunately, resistance to lamivudine commonly develops during monotherapy. Lamivudine-resistant HBV mutants display specific mutations in the YMDD (tyrosine, methionine, aspartate, aspartate) motif of the viral polymerase (reverse transcriptase [rt]), which is the catalytic site of the enzyme, i.e., methionine 204 to isoleucine (rtM204I) or valine (rtM204V). The latter mutation is often accompanied by a compensatory leucine-to-methionine change at codon 180 (rtL180M). In the present study, a novel sequencing method, pyrosequencing, was applied to the detection of lamivudine resistance mutations and was compared with direct Sanger sequencing. The new pyrosequencing method had advantages in terms of throughput. Experiments with mixtures of wild-type and resistant viruses indicated that pyrosequencing can detect minor sequence variants in heterogeneous virus populations. The new pyrosequencing method was evaluated with a small number of patient samples, and the results showed that the method could be a useful tool for the detection of lamivudine resistance in the clinical setting.
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| 2. |
- Nygren, M., et al.
(författare)
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Polymorphism in the pertussis toxin promoter region affecting the DNA-based diagnosis of Bordetella infection
- 2000
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Ingår i: Journal of Clinical Microbiology. - 0095-1137 .- 1098-660X. ; 38:1, s. 55-60
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Tidskriftsartikel (refereegranskat)abstract
- The pertussis toxin (PT) promoter region is a frequently used target for DNA-based diagnosis of pertussis and perapertussis infections. The reported polymorphism in this region has also allowed discrimination of species in mixtures with several Bordetella species by their specific PCR amplicon restriction patterns. In the present study, we investigated the degree of polymorphism in order to confirm the reliability of the assay, Five different sequence types of the amplified 239- or 249-bp region were found among the 33 Bordetella pertussis, B. parapertussis, and B. bronchiseptica American Type Culture Collection reference strains and patient isolates analyzed. According to the sequences that were obtained and according to the PT promoter sequences already available in the databases, restriction enzyme analysis with TaqI, Bg/I, and HaeII, which gave four different patterns, can be performed to reliably identify B. pertussis, B. papapeptussis, and B. bronchiseptica.
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| 3. |
- O'Meara, D., et al.
(författare)
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Monitoring resistance to human immunodeficiency virus type 1 protease inhibitors by pyrosequencing
- 2001
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Ingår i: Journal of Clinical Microbiology. - 0095-1137 .- 1098-660X. ; 39:2, s. 464-473
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Tidskriftsartikel (refereegranskat)abstract
- The emergence of drug-resistant viral variants is the inevitable consequence of incomplete suppression of human immunodeficiency virus type 1 (HIV-1) replication during treatment with antiretroviral drugs. Sequencing to determine the resistance profiles of these variants has become increasingly important in the clinical management of HIV-1 patients, both in the initial design of a therapeutic plan and in selecting a salvage regimen. Here we have developed a pyrosequencing assay for the rapid characterization of resistance to HIV-1 protease inhibitors (PIs). Twelve pyrosequencing primers were designed and were evaluated on the MN strain and on viral DNA from peripheral blood mononuclear cells from eight untreated HN-l infected individuals. The method had a limit of detection of 20 to 25% for minor sequence variants. Pattern recognition (i.e., comparing actual sequence data with expected wild-type and mutant sequence patterns) simplified the identification of minor sequence variants. This real-time pyrosequencing method was applied in a longitudinal study monitoring the development of PI resistance in plasma samples obtained from four patients over a 2 1/2-year period. Pyrosequencing identified eight primary PI resistance mutations as well as several secondary mutations. This sequencing approach allows parallel analysis of 96 reactions in 1 h, facilitating the monitoring of drug resistance in eight patients simultaneously and, in combination with viral load analysis, should be a useful tool in the future to monitor HIV-1 during therapy.
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| 4. |
- Weibull, Emilie, et al.
(författare)
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Bacterial Nanoscale Cultures for Phenotypic Multiplexed Antibiotic Susceptibility Testing
- 2014
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Ingår i: Journal of Clinical Microbiology. - 0095-1137 .- 1098-660X. ; 52:9, s. 3310-3317
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Tidskriftsartikel (refereegranskat)abstract
- An optimal antimicrobial drug regimen is the key to successful clinical outcomes of bacterial infections. To direct the choice of antibiotic, access to fast and precise antibiotic susceptibility profiling of the infecting bacteria is critical. We have developed a high-throughput nanowell antibiotic susceptibility testing (AST) device for direct, multiplexed analysis. By processing in real time the optical recordings of nanoscale cultures of reference and clinical uropathogenic Escherichia coli strains with a mathematical algorithm, the time point when growth shifts from lag phase to early logarithmic phase (T-lag) was identified for each of the several hundreds of cultures tested. Based on T-lag, the MIC could be defined within 4 h. Heatmap presentation of data from this high-throughput analysis allowed multiple resistance patterns to be differentiated at a glance. With a possibility to enhance multiplexing capacity, this device serves as a high-throughput diagnostic tool that rapidly aids clinicians in prescribing the optimal antibiotic therapy.
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