Comparing Peripheral Blood Mononuclear Cell DNA and Circulating Plasma viral RNA pol Genotypes of Subtype C HIV-1

Abstract

Introduction Drug resistance mutations (DRM) in viral RNA are important in defining to provide effective antiretroviral therapy (ART) in HIV-1 infected patients. Detection of DRM in peripheral blood mononuclear cell (PBMC) DNA is another source of information, although the clinical significance of DRMs in proviral DNA is less clear. Materials and Methods From 25 patients receiving ART at a center in Zimbabwe, 32 blood samples were collected. Dideoxy-sequencing of gag-pol identified subtype and resistance mutations from plasma viral RNA and proviral DNA. Drug resistance was estimated using the calibrated population resistance tool on www.hivdb.stanford.edu database. Numerical resistance scores were calculated for all antiretroviral drugs and for the subjects’ reported regimen. Phylogenetic analysis as maximum likelihood was performed to determine the evolutionary distance between sequences. Results Of the 25 patients, 4 patients (2 of which had given 2 blood samples) were not known to be on ART (NA) and had exclusively wild-type virus, 17 had received Protease inhibitors (PI), 18, non-nucleoside reverse transcriptase inhibitors (NNRTI) and 19, two or more nucleoside reverse transcriptase inhibitors (NRTI). Of the 17 with history of PI, 10 had PI mutations, 5 had minor differences between mutations in RNA and DNA. Eighteen samples had NNRTI mutations, six of which demonstrated some discordance between DNA and RNA mutations. Although NRTI resistance mutations were frequently different between analyses, mutations resulted in very similar estimated phenotypes as measured by resistance scores. The numerical resistance scores from RNA and DNA for PIs differed between 2/10, for NNRTIs between 8/18, and for NRTIs between 17/32 pairs. When calculated resistance scores were collapsed, 3 pairs showed discordance between RNA and DNA for at least one PI, 6 were discordant for at least one NNRTI and 11 for at least one NRTI. Regarding phylogenetic evolutionary analysis, all RNA and DNA sequence pairs clustered closely in a maximum likelihood tree. Conclusion PBMC DNA could be useful for testing drug resistance in conjunction with plasma RNA where the results of each yielded complementary information about drug resistance. Identification of DRM, archived in proviral DNA, could be used to provide for sustainable public health surveillance among subtype C infected patients.

Use of Proviral DNA to Investigate Virus Resistance Mutations in HIV-infected Zimbabweans

“…The most frequently occurring mutation was M184V, which was observed in five out of the six sequences (Table 4 ). This is consistent with various studies done in Zimbabwe [17, 19, 20]. M184V causes high level resistance to 3TC [15, 19], a drug that is recommended in all 1 st and 2 nd line ART regimens in Zimbabwe [5].…”

“…Although sequencing of plasma viral RNA (vRNA) remains the gold standard in genotypic resistance testing [19], the method has been shown to miss the presence of drug resistance if there has been poor suppression of the virus due to interruption in adherence. Moreover, some studies have shown analysis of the alternative proviral DNA to give relatively similar results to plasma vRNA [19, 20] which warrants the investigative use of proviral DNA in drug resistance testing. Hence the main objective of this study was to determine HIVDR mutations using proviral DNA from blood specimens of HIV infected ART-naïve and ART-experienced patients presenting to an HIV treatment clinic in Harare, Zimbabwe.…”

Proviral DNA as a Target for HIV-1 Resistance Analysis

“…In the samples from the TE patients, the majority of DRMs appeared in viral RNA (99%) whereas those from the TN patients displayed a significantly higher proportion of DRMs in proviral DNA (33%). These data are in line with other published studies investigating information on resistance in viral RNA and proviral DNA of identical blood samples [17,18,19,20,21,22]. …”

“…Although this analysis revealed an overall higher frequency of DRMs in viral RNA than in proviral DNA, 75% of the DRMs were detected in both materials. Similar results were published in a study by Banks et al [17], comparing the pol genotypes of the circulating viral RNA and proviral DNA in the peripheral blood mononuclear cells in 32 blood samples of 25 subtype C-infected patients receiving ART. They also reported similar mutation patterns in viral RNA and proviral DNA, with unique mutations in >50% of cases.…”

HIV drug resistance testing among patients failing second line antiretroviral therapy. Comparison of in-house and commercial sequencing

“…The two samples which failed to amplify had viral loads of 1247 and 901 copies/ml, around the lower recommended levels of plasma virus for genotyping with either ViroSeq [16] or TruGene [17]. Drug resistance mutations in patients with low-level viremia (50 – 1000 copies/ml) may be obtained from proviral DNA [18–20] or by concentrating virus from large volumes of plasma [21], although successful amplification is reduced among samples with lower virus copy numbers [22]. However, it may be important to investigate viral genotypes in such patients, as resistance at low-level viremia is associated with an increased risk of future virologic failure (i.e.…”