Development of phenotypic HIV-1 drug resistance after exposure to single-dose nevirapine.

OBJECTIVES: Single-dose nevirapine (sdNVP) used to prevent mother-to-child transmission of HIV-1 results in the selection of genotypic drug resistance mutations. To assess the levels of phenotypic resistance conferred by these mutations, we examined the ability of sample-derived HIV-1 reverse transcriptase to function in the presence of nevirapine (NVP). METHODS: Plasma samples from HIV-1 pregnant women before and after exposure to sdNVP were used to extract viral reverse transcriptase for the Cavidi ExaVir Drug susceptibility assay. The fold increases in phenotypic resistance for each sample were compared with the genotypic profiles determined by population-based sequencing. RESULTS: None of the women sampled before sdNVP exposure had phenotypic resistance (median fold increase 0.7). Seven weeks after sdNVP, there was a 16-fold increase in phenotypic resistance among women who had NVP resistance mutations compared with only a 1.9-fold increase among NVP-exposed women with wild-type virus. Phenotypic resistance decayed with time coincident with the fading of genotypic mutations, and by 18 months, all samples were phenotypically susceptible. CONCLUSIONS: Exposure of pregnant women to sdNVP was associated with the transient appearance of viral populations that displayed phenotypic resistance to NVP. Overall, there was good concordance between phenotypic resistance to NVP, as measured with this enzymatic assay, and the presence of NVP genotypic mutations.

Reverse transcriptase viral load correlates with RNA in SIV/SHIV-infected macaques.

Monitoring of viral load in macaques has usually been carried out using in-house PCR-based methods. A novel viral load (VL) kit (ExaVir Load) based on the measurement of lentivirus reverse transcriptase (RT) activity provides a potential alternative to methods that measure plasma viral RNA. RT is a fundamental and conserved activity of all retroviruses and the method should theoretically detect RT from all lentiviruses. To test this we compared VL measured by a commercially available RT kit with an in-house QC RT-PCR in macaques infected with SIV and SHIV. Both RT and RNA levels were measured over time in both sets of macaques. Results indicated that the relationship between both tests was strong for SIV and SHIV (r = 0.95 and r = 0.92, p < 0.0001, respectively). The VL trends also followed each other, indicating that both techniques measured the same process of viral replication. Furthermore, the RT load obtained using standardized control plasma samples supplied by NIBSC gave values close to the designated VL. However, when comparing RT load with QC RT-PCR a consistently three to five time higher level was obtained with the RT assay, highlighting potential differences in assay calibration. Even so, the data suggest that the RT assay is both sensitive and robust for use in the SIV/SHIV macaque model, particularly where molecular-based assays for SIV VL determinations are not easily available. The assay is also a commercially available kit and hence has the potential to reduce the variability seen between laboratories using in-house PCR.

Comparison of two human immunodeficiency virus (HIV) RNA surrogate assays to the standard HIV RNA assay.

Human immunodeficiency virus (HIV) RNA testing is the gold standard for monitoring antiretroviral therapy in HIV-infected patients. However, equipment and reagent costs preclude widespread use of the assay in resource-limited settings. The Perkin-Elmer Ultrasensitive p24 assay and the Cavidi Exavir Load assay both offer potentially simpler, less costly technologies for monitoring viral load. These assays were compared to the Roche Amplicor HIV-1 Monitor Test, v1.5, using panels of clinical samples (subtype B) from HIV-positive subjects and HIV-spiked samples (subtypes A, C, D, CRF_01AE, CRF_02AG, and F). The Ultrasensitive p24 assay detected 100% of the spiked samples with virus loads of >250,000 copies/ml and 61% of the clinical samples with virus loads of 219 to 288,850 copies/ml. Detection rates were improved substantially if an external lysis buffer was added to the procedure. The Cavidi assay detected 54 to 100% of spiked samples with virus loads >10,000 copies/ml and 68% of the clinical samples. These detection rates were also greatly improved with a newly implemented version of this kit. Coefficients of variation demonstrate good reproducibility for each of these kits. The results from the Cavidi v1.0, Cavidi v2.0, and Perkin-Elmer, and the Perkin-Elmer Plus external buffers all correlated well with the results from the Roche Monitor Test (r = 0.83 to 0.96, r = 0.84 to 0.99, r = 0.58 to 0.67, and r = 0.59 to 0.95, respectively). Thus, the use of these two assays for monitoring patients, together with less-frequent confirmation testing, offers a feasible alternative to frequent HIV RNA testing in resource-limited settings.

Plasma virion reverse transcriptase activity and heat dissociation-boosted p24 assay for HIV load in Burkina Faso, West Africa.

BACKGROUND: In resource-limited settings, the requirement for inexpensive, easy-to-perform viral load monitoring has increased with greater antiretroviral drug availability. OBJECTIVES: To evaluate feasibility, in Burkina Faso, of a simple assay for plasma HIV reverse transcriptase (RT) activity quantification compared to heat dissociation-boosted (HDB) p24 antigen and RNA-based quantifications in plasma samples from HIV-infected patients. METHODS: : Plasma viraemia was quantified by RT activity, HDB-p24 and RNA copies in 84 samples from 70 HIV-1 group M-infected patients (82% non-B subtype, 93% treatment naive), including serial samples from nine patients. RESULTS: RT activity detected 86% of plasma samples containing measurable RNA copies; corresponding to 0, 93 and 100% of samples with 1.7-4.0 log(10), 4.1-4.8 log(10) and 4.9-6.7 log(10) RNA copies/ml, respectively. HDB-p24 detected 77% of plasma samples containing measurable RNA copies; corresponding to 27, 80 and 86% of samples with 1.7-4.0 log(10), 4.1-4.8 log(10) and 4.9-6.7 log(10) RNA copies/ml, respectively. Measurement error based on one-way analysis of variance between RT activity and HDB-p24 values with RNA copies showed good agreement with RT activity (ME, <10%), however poorer agreement was obtained with HDB-p24 values (ME, >10%). Patient follow up showed a similar pattern of viraemia with RNA and RT activity assays. CONCLUSION: Field trials in Burkina Faso support the practical use of plasma RT activity assay as an affordable alternative for HIV viral load determination in regions where RNA detection remains difficult to perform. HDB-p24 use requires further evaluation before being considered as an alternative method in African HIV-infected patient follow up.

Evaluation of a low cost reverse transcriptase assay for plasma HIV-1 viral load monitoring.

We evaluated a low cost manual reverse transcriptase assay (ExaVir Load V.1 and V.2; Cavidi Tech AB) against commercially available HIV RNA assays that quantify viral load to assess its suitability for use in resource-constrained settings. Frozen plasma samples previously tested for RNA by RT-PCR (Roche Diagnostics) and bDNA (Bayer Diagnostics) were retested for RT activity. Text sequence obtained from HIV genotype analysis was submitted to the Stanford HIV Resistance Database V.3.9 and were examined for resistant virus. Detectable RT was present in 98% of samples (V.1; n=127) and in 95% of samples (V.2; n=69) with RNA >10,000 and >1,000 copies/ml respectively. Positive association was found between the log10 RNA copies/ml and log10 RT copies/ml equivalents variables using Pearson's correlation (V.1: r=0.89, n=189; V.2: r=0.89, n=85). The RT activity over time closely followed the trend for RNA levels in samples from 10 HIV seropositive patients with progressive disease. A strong association between RT and RNA was also found with paired samples from 19 patients taken at initiation or change of antiretroviral therapy and again within 2 months. Current (n=40) or no (n=119) exposure to efavirenz therapy had no effect on RT assay performance despite efavirenz binding tightly to the RT enzyme. Samples that demonstrated resistance to the non-nucleoside RT inhibitors (n=112) had a decrease in RT of 0.20 log10 indicating a possible decrease in RT fitness. The RT assay showed good association with current molecular assays, and V.2 is sufficiently sensitive for monitoring HIV viral load in resource-constrained settings.

Phenotypic susceptibility to nonnucleoside inhibitors of virion-associated reverse transcriptase from different HIV types and groups.

OBJECTIVES: To evaluate a phenotype assay based on plasma reverse transcriptase (RT) to assess HIV susceptibility to nonnucleoside RT inhibitors (NNRTIs). To compare RT-based phenotype with recombinant virus assay (RVA) phenotype- and genotype-based analysis. To assess group O and HIV-2 susceptibility to NNRTIs in correlation with genotype polymorphisms. METHODS: RT activity was quantified and its susceptibility to efavirenz, nevirapine, and delavirdine measured as drug concentration resulting in 50% inhibition. RT phenotype was compared with genotype analysis. Eighteen plasma samples from 14 group M- and culture supernatants from 4 group M-, 9 group O-, and 7 HIV-2-infected patients were investigated. RT-based and RVA-based phenotypes were compared for identical plasma from 9 group M-infected patients. RESULTS: RT-based and RVA-based phenotypes were in complete agreement. RT-based phenotype- and genotype-predicted susceptibility were concordant for all but 1 group M samples. One plasma showed susceptibility to 3 NNRTIs by phenotypes, despite the presence of 101E and 106I/V residues. The HIV-2 RTs were totally resistant to the NNRTIs tested. Among HIV-1 group O, 6 were totally resistant to NNRTIs independently of the presence of the 181C mutation and 3 were susceptible to some NNRTIs. CONCLUSION: Plasma RT-based phenotype could be useful as a simple alternative for monitoring resistance to NNRTIs. This assay is suitable for highly divergent strains. It would be particularly useful for large epidemiologic survey of the natural HIV polymorphism and the potential impact in emergence of drug resistance, particularly to nevirapine, widely used to prevent mother-to-child transmission.

HIV-1 viral load determination based on reverse transcriptase activity recovered from human plasma.

We describe a procedure (ExaVir Load) to carry out human immunodeficiency virus-1 (HIV-1) viral load testing using reverse transcriptase (RT) recovered from HIV-1 virions in plasma. Samples from individuals infected with HIV-1 were treated with a sulphydryl-reactive agent to inactivate endogenous polymerases. Virions were then immobilised on a gel and washed in individual mini columns to remove RT-inhibiting antibodies, antiviral drugs, and other RT inhibitors. Immobilised virions were lysed finally, and the viral RT eluted. The amount of RT recovered was quantified by a sensitive RT activity assay using either colorimetry or fluorimetry to detect DNA produced by RT. The "RT load" values of 390 samples from 302 HIV-1 patients living in Sweden were compared to results obtained with an HIV-1 RNA viral load assay. The correlation between the two tests was r = 0.90, P < 0.0001. Four of 202 samples from healthy blood donors gave low positive values in the RT test. All samples in a panel with 10 HIV-1 subtypes were positive by the RT load. The RT load test provides a technically less demanding and cost-effective alternative to methods based on nucleic acid amplification. Being insensitive to genetic drift occurring in HIV, the assay should be of particular use in resource-limited settings, where different subtypes and recombinant HIV strains occur.

A new quantitative HIV load assay based on plasma virion reverse transcriptase activity for the different types, groups and subtypes.

BACKGROUND: Plasma viral load monitoring is an integral part of the standard of care for HIV-infected patients in industrialized countries. In developing countries, viral load assay is either unaffordable or hindered by on-site maintenance and/or technical problems. OBJECTIVES: To evaluate a new and simple quantitative assay for plasma HIV reverse transcriptase (RT) activity; and to compare RT activity-based and RNA-based quantification in plasma samples from patients infected by different subtypes of HIV-1 group-M, HIV-1 group-O and HIV-2. METHODS: The RT-based viral load assay involves separation of the virion-protected RT and quantification of its activity with an enzyme immunoassay. Plasma viraemia was quantified both by RT activity and by RNA copies in 322 samples from 236 HIV-1 group M-infected patients, including serial samples from 54 patients. Samples from 49 patients infected by HIV-1 group O or HIV-2 were also tested. RESULTS: RT activity and RNA copies were detected in 70% of plasma samples; respectively 25% and 1% of samples contained detectable RNA copies or RT activity alone. Measured RT activity corresponded to 48%, 96% and 100% of samples with 1.7-4.0 log(10), 4.1-4.8 log(10) and 4.9-6.7 log(10) RNA copies/ml, respectively. The values of the two assays correlated independently of the HIV subtype (P < 0.0001) and group/type (P < 0.03). Patient follow-up showed a similar pattern of viraemia with the two assays. CONCLUSION: Plasma RT activity assay is a simple, cheap and reliable alternative for HIV viral load determination. As such, it could be particularly valuable for diagnosis and treatment monitoring in developing countries.