Comparison of oligonucleotide ligation assay and consensus sequencing for detection of drug-resistant mutants of human immunodeficiency virus type 1 in peripheral blood mononuclear cells and plasma.

Department of Pediatrics, University of Washington, Seattle, USA.
Journal of Clinical Microbiology (Impact Factor: 4.07). 09/2004; 42(8):3670-4. DOI:10.1128/JCM.42.8.3670-3674.2004
Source: PubMed

ABSTRACT Drug-resistant mutants of human immunodeficiency virus type 1 (HIV-1) recede below the limit of detection of most assays applied to plasma when selective pressure is altered due to changes in antiretroviral treatment (ART). Viral variants with different mutations are selected by the new ART when replication is not suppressed or wild-type variants with greater replication fitness outgrow mutants following the cessation of ART. Mutants selected by past ART appear to persist in reservoirs even when not detected in the plasma, and when conferring cross-resistance they can compromise the efficacy of novel ART. Oligonucleotide ligation assay (OLA) of virus in plasma and peripheral blood mononuclear cells (PBMC) was compared to consensus sequence dideoxynucleotide chain terminator sequencing for detection of 91 drug resistance mutations that had receded below the limit of detection by sequencing of plasma. OLA of plasma virus detected 27.5% (95% confidence interval [CI], 19 to 39%) of mutant genotypes; consensus sequencing of the PBMC amplicon from the same specimen detected 23.1% (95% CI, 14 to 34%); and OLA of PBMC detected 53.8% (95% CI, 44 to 64%). These data suggest that concentrations of drug-resistant mutants were greater in PBMC than in plasma after changes in ART and indicate that the OLA was more sensitive than consensus sequencing in detecting low levels of select drug-resistant mutants.

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    ABSTRACT: The simple method for amplifying RNA targets (SMART) was used to detect K103N, the most common HIV-1 reverse transcriptase drug-resistance mutation. Novel amplifiable SMART probes served as reporter molecules for RNA sequences that are captured and separated on a microfluidic platform under zero-flow conditions. Assays were performed both off chip and in a microchip reservoir using a modified version of real-time nucleic acid sequence-based amplification, without the noncyclic phase, and 65°C preheat. A total of 6000 copies/mL of the synthetic sequences were detected within 180 minutes of amplification. Although the sensitivity of research platforms is higher, SMART has the potential to offer comparable sensitivity and speed to commercially available viral load and HIV detection kits. Furthermore, SMART uses an inexpensive, practical, and more accurate isothermal exponential amplification technique. The use of molecular beacons resulted in relatively fast real-time detection (<180 minutes); however, they were also shown to hinder the amplification process when compared with end point detection. Finally, SMART probes were used for modeling of K103N concentrations within an unknown sample. Only 1% of the SMART probes was detected within the wild-type population (6 × 10(8) copies/mL). These results establish the groundwork for point-of-care drug resistance and viral load monitoring in clinical samples, which can revolutionize HIV patient care globally.
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