Modifications and substitutions of the RNA extraction module in the ViroSeq™ HIV‐1 genotyping system version 2: Effects on sensitivity and complexity of the assay

Abstract

Genotypic testing for HIV-1 resistance to anti-retroviral drugs has become accepted widely as a routine method to guide anti-retroviral therapy. However, implementation into routine high-throughput laboratory diagnosis is difficult due to the complexity of the assay. A commercially available assay is the ViroSeq HIV-1 Genotyping System (Applied Biosystems, Weiterstadt, Germany). We modified and substituted the RNA extraction module to optimize the proportion of samples amplified successfully as follows: 1 ml plasma was concentrated by ultracentrifugation and extracted according to the manufacturer's instructions (Kit), by substituting the lysis buffer (Roche, Roche Diagnostics GmbH, Mannheim, Germany), and by using the QIAamp Viral RNA Kit (Qiagen GmbH, Hilden, Germany) with elution volumes of 60 (Q60) or 50 micro l (Q50). Overall Q50 showed a higher success rate (97%) than the other extraction modules used (range 88-91%). In samples with a viral load range of 1,000-4,999 copies/ml, Q50 was superior (95 vs. 65% to 83%), while in samples with a viral load range of 5,000-9,999 copies/ml or those with 10,000 or more copies/ml, the success rate of the extraction procedures showed no significant differences. In 18 samples, which were negative using the Kit or Roche extraction, Q60 resulted in 7/18 positive results; in addition the Q50 was successful in amplifying 7/10 of the Q60 negative samples. When investigating samples with a measurable viral load of less than 1,000 copies/ml or lower, Q50 had the highest success rate with 80% compared to the other procedures (33-63%). A statistically significant new cut-off could be defined for Q50 at a value of 250 copies/ml. The results showed clearly that the ViroSeq System is suitable for analyzing the HIV-1 genotype over a wide range of viral loads but could be improved significantly when substituting the RNA extraction module with Q50 without using a nested PCR protocol. This is of great importance as it avoids further time- and cost-intensive steps.

Long-Range HIV Genotyping Using Viral RNA and Proviral DNA for Analysis of HIV Drug Resistance and HIV Clustering

“…The extent of HIV clustering was analyzed by using the following subgenomic regions across the HIV-1 genome: (i) amplicon 1, spanning the 3= end of gag and almost the entire pol and corresponding to amplicon 2 in the study of Gall et al (62), nucleotide positions 1486 to 5058; (ii) amplicon 2, spanning vpu, env, nef, and the TATA box in the U3 region of the 3= long terminal repeat (LTR) and corresponding to amplicon 4 in the study of Gall et al (62), nucleotide positions 5967 to 9517; (iii) ViroSeq, a partial pol sequence spanning the region encoding HIV-1 protease and the first 335 amino acids of reverse transcriptase and corresponding to the sequence produced by ViroSeq (39,44,45,78), nucleotide positions 2253 to 3554; and (iv) V1C5, a partial env sequence spanning the region encoding gp120 V1C5 (34,79,80), nucleotide positions 6570 to 7757. In addition, the following combinations of the subgenomic regions included concatenated amplicon 1 plus amplicon 2 and amplicon 1 plus V1C5.…”

“…Both genotyping kits were extensively tested and validated (36)(37)(38)(39)(40)(41)(42)(43)(44)(45). While the ViroSeq HIV-1 kit is still on the market, Siemens discontinued selling and supporting the TruGene HIV-1 kit in 2014.…”

Importance of Viral Sequence Length and Number of Variable and Informative Sites in Analysis of HIV Clustering

“…For example, at the bootstrap threshold of 0.80 (Fig. 1B), 38.9% of pol sequences, 30.7% of env sequences, and 17.2% of gag sequences were found in clusters, while the proportion of viral sequences in clusters in the set of near full-length genome HIV-1C sequences was 58.9% (95% CI 53.8% to 63.7%). The difference in proportions of clustered sequences between the set of near full-length HIV-1C sequences and any of the three structural genes was statistically significant at all targeted bootstrap thresholds (all p-values from < 0.0001, McNemar's test).…”

“…These subgenomic regions included (1) a partial pol sequence spanning the region encoding HIV-1 protease and the first 335 amino acids of reverse transcriptase, which corresponds to the sequence produced by ViroSeq, [38][39][40][41] nt positions 2,253-3,554; (2) partial env sequences spanning the region encoding the gp120 V1C5 region, [42][43][44] nt positions 6,570-7,757; (3) ''product 2'' spanning the 3¢-end of gag and almost the entire pol, 45 nt positions 1,486-5,058; and (4) ''product 4'' spanning vpu, env, nef, and TATA-box in the U3 region of 3¢-LTR, 45 nt positions 5,967-9,517. In addition, combinations of the targeted subregions included gag + pol, gag + env, pol + env, gag + pol + env, and product 2 + product 4.…”

Association of Saquinavir Plasma Concentrations with Side Effects but Not with Antiretroviral Outcome in Patients Infected with Protease Inhibitor-Susceptible Human Immunodeficiency Virus Type 1

“…Baseline genotypic HIV resistance testing using the ViroSeq HIV-1 Genotyping System V2 (Abbott, Wiesbaden, Germany) (22) was negative in all patients with respect to viral mutations conferring resistance against protease inhibitors. Therefore, all patients were eligible for the present analysis, and the exclusion of nonnaïve subjects with respect to therapy as a possible confounder Patients at any CD4 cell count or viral load at baseline of therapy were included.…”