Evaluation of an affordable HIV-1 virological failure assay and antiretroviral drug resistance genotyping protocol.

HIV-1 RNA viral load is the preferred tool to monitor virological failure during antiretroviral therapy (ART) exposure. Timely detection of virological failure can reduce the prevalence and complexity of HIV-1 drug resistance. This field evaluation further characterizes a two-step approach to identify virological failure, as a measure of ART adherence, and detect HIVDR mutations in the reverse transcriptase (RT) gene of HIV-1. Two hundred and forty-eight (248) samples were tested; 225 from South African HIV-1 participants enrolled in the PharmAccess African Studies to Evaluate Resistance (PASER) cohort, forty of which had paired dried blood spot (DBS) samples and 23 HIV-1 negative samples. A newly developed virological failure assay (ARTA-VFA) was used on all samples, and those with a viral load >5000 RNA copies/ml were genotyped with a shortened RT protocol to detect HIVDR (ARTA-HIVDR(ultralight)). The ARTA-VFA showed good precision and linearity as compared to a commercial reference assay (NucliSENS EasyQ v1.2, Roche) with an R(2) of 0.99. Accuracy studies illustrated standard deviations of <1 log RNA copies/ml for plasma and DBS ARTA-VFA results compared to the reference method. The ARTA-VFA's intended use was to deliver qualitative results either < or >5000 RNA copies/ml. No significant differences in the proportion of results < or > either the 5000 RNA copies/ml or 1000 RNA copies/ml cut-off were noted for plasma indicating either cut-off to be useful. Significant differences were noted in these proportions when DBS were used (P=0.0002), where a 5000 RNA copies/ml cut-off was deemed more appropriate. The sensitivity and specificity of the ARTA-VFA with plasma were 95% and 93% and 91% and 95% for DBS using a 5000 RNA copies/ml cut-off. The ARTA HIVDR(ultralight) assay was reliable for plasma and DBS samples with a viral load >5000 RNA copies/ml, with amplification and sequencing success rates of 91% and 92% respectively for plasma, and 95% and 80% respectively for DBS. HIVDR profiles for plasma and DBS were 100% concordant with the reference assay. This study evaluated a previously described combination of two assays potentially useful in assessing HIV-1 virological failure and resistance, showing good concordance with reference assays. These assays are simple to perform and are affordable, viable options to detect virological failures in certain resource limited settings. The assays' compatibility with DBS sampling extends the access of HIV-1 virological monitoring to more remote settings.

Cross-resistance profile determination of two second-generation HIV-1 integrase inhibitors using a panel of recombinant viruses derived from raltegravir-treated clinical isolates.

The integrase inhibitor raltegravir (RAL) is currently used for the treatment of both treatment-naïve and treatment-experienced HIV-1-infected patients. Elvitegravir (EVG) is in late phases of clinical development. Since significant cross-resistance between RAL and EVG is observed, there is a need for second-generation integrase inhibitors (INIs) with a higher genetic barrier and limited cross-resistance to RAL/EVG. A panel of HIV-1 integrase recombinants, derived from plasma samples from raltegravir-treated patients (baseline and follow-up samples), were used to study the cross-resistance profile of two second-generation integrase inhibitors, MK-2048 and compound G. Samples with Q148H/R mutations had elevated fold change values with all compounds tested. Although samples with the Y143R/C mutation had reduced susceptibility to RAL, they remained susceptible to MK-2048 and compound G. Samples with the N155H mutation had no reduced susceptibility to compound G. In conclusion, our results allowed ranking of the INIs on the basis of the antiviral activities using recombinant virus stocks from RAL-treated patient viruses. The order according to decreasing susceptibility is compound G, MK-2048, and EVG.

Nomenclature for antiviral-resistant human hepatitis B virus mutations in the polymerase region.

There is currently no universally accepted numbering convention for the antiviral drug-related resistance mutations in the reverse transcriptase (rt) domain of the human hepatitis B virus (HBV) polymerase. The published inconsistencies have resulted from different HBV genotypes. A standardized numbering system for HBV polymerase is proposed. The new system is based on functional observations of HBV surface gene proteins (preS1, preS2, and HBsAg) and on the current convention used for human immunodeficiency virus type 1 (HIV-1) polymerase proteins (protease, rt, and integrase), in which the amino acid numbering restarts at the first codon position of each domain. The HBV polymerase protein can be divided into 4 domains (terminal protein, spacer, rt, ribonuclease H) and each of these can be numbered separately. In this proposal, the HBV rt domain starts with the highly conserved EDWGPCDEHG motif, contains 344 amino acids, and the lamivudine-related resistance mutations are found at amino acid rtL180M (previously amino acid 528, 526, 515, or 525) and rtM204V/I (previously 552, 550, 539, or 549). The new consensus rt domain numbering system is genotype independent and allows investigators to number any previously and newly discovered antiviral-related amino acid change in a standardized manner.