Pre-screening HIV-1 Reverse Transcriptase Resistance Mutations in Subtype B Patients Using a Novel Multiplex Primer Extension Assay
ABSTRACT Antiretroviral therapy is standard treatment for HIV-infected patients. Such therapy has decreased mortality and morbidity, but treatment success is often jeopardized by the emergence of viral drug resistance. Moreover, in recent years there has been a reported rise in the incidence of transmitted drug resistance, highlighting the importance of pre-treatment resistance screening. In this report, we describe the development and utility of a sensitive multiplex approach for detecting mutations conferring drug resistance to HIV-1 reverse transcriptase inhibitors. This protocol, termed HIV-SNaPshot, utilizes a multiplex primer extension assay with capillary electrophoresis reporting altered nucleotides at nine important drug resistance mutation positions. Mutations were successfully detected to levels of 5% in viral quasispecies populations. Furthermore, although developed and optimised for HIV-1 subtype B, drug resistance mutations could also be detected in most non-B subtypes. Comparison of the HIV-SNaPshot with the commercial Viroseq genotyping system in 10 patients gave similar results, but importantly, additional resistance mutations were identified in several patients by the HIV-SNaPshot assay. Thus, the HIV-SNaPshot is a method capable to support standard genotyping for the determination of minority HIV-1 resistance mutations, with equivalent and perhaps greater sensitivity than Viroseq.
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ABSTRACT: Human immunodeficiency virus type 1 (HIV-1) drug resistance is an important threat to the overall success of antiretroviral therapy (ART). Because of the limited sensitivity of commercial assays, transmitted drug resistance (TDR) may be underestimated; thus, the effect that TDR has on treatment outcome needs to be investigated. The objective of this study was to investigate the prevalence of TDR in HIV-infected patients and to evaluate the significance of TDR with respect to treatment outcome by analyzing plasma viral RNA and peripheral blood mononuclear cell proviral DNA for the presence of drug resistance mutations. In a prospective study, we investigated the level of TDR in 61 patients by comparing the results of a sensitive multiplex-primer-extension approach (termed HIV-SNaPshot) that is capable of screening for 9 common nucleoside reverse-transcriptase inhibitor and nonnucleotide reverse-transcriptase inhibitor mutations with those of a commercial genotyping kit, ViroSeq (Abbott). Twenty-two patients were found to carry mutations. More patients with TDR were identified by the HIV-SNaPshot assay than by ViroSeq analysis (33% vs 13%; [P=.015). There was no significant difference in the time from initiation of ART to virological suppression between susceptible patients and those carrying low- or high-level resistance mutations (mean +/- standard deviation, 128 +/- 59.1 vs 164.9 +/- 120.4; P=.147). Furthermore, analyses of CD4 cell counts showed no significant difference between these 2 groups 1 year after the initiation of ART (mean, 184 vs 219 cells/microL; P=.267). We found the prevalence of TDR in recently infected ART-naive patients to be higher than that estimated by ViroSeq genotyping alone. Follow-up of patients after treatment initiation showed a trend toward there being more clinical complications for patients carrying TDR, although a significant effect on treatment outcome could not be demonstrated. Therefore, the clinical relevance of low-abundance resistant quasispecies in early infection is still in question.Clinical Infectious Diseases 02/2010; 50(4):566-73. DOI:10.1086/650001 · 9.42 Impact Factor
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ABSTRACT: Clinical chemistry also known as chemical pathology is a discipline that is generally concerned with analysis of biological fluids for diagnosis of human diseases. Capillary electrophoresis (CE) is electrophoretic separation run in narrow-bore capillary format, which has become a highly efficient and versatile separation technique available for analysis of a wide variety of biologically active molecules. Implementation of CE in clinical laboratories will enhance the capability of the current clinical diagnostic system and improve the efficiency and quality of routine clinical testing. Owing to the nature of clinical diagnostics, the adoption of CE in clinical laboratories has been slow. However, CE has begun to replace some older and antiquated conventional techniques with unique applications in some pioneering clinical laboratories. This review covers a brief introduction to the CE technique (i.e. principle of separation, modes of operation, instrumentation, and practical considerations), and a summary of its applications in clinical chemistry (i.e. proteins, nucleic acids, drugs, and organic and inorganic ions), as well as the future prospects of this field.Encyclopedia of Analytical Chemistry, 12/2012; , ISBN: 0471976709
Martin R Jakobsen