Drug-Associated Resistance Mutations in Plasma and Peripheral Blood Mononuclear Cells of Human Immunodeficiency Virus Type 1-Infected Patients for Whom Highly Active Antiretroviral Therapy Is Failing

Abstract

In 32 patients for whom highly active antiretroviral therapy was failing, a good agreement between drug resistance-associated mutations in plasma and peripheral blood mononuclear cells (PBMCs) was found (k ‫؍‬ 0.85). The mutations with the lowest agreement were 20R, 63P, and 84V in the protease gene and 184V in the reverse transcriptase gene. In eight patients, primary drug resistance mutations were detected only in PBMCs.The presence of resistant viral strains is routinely verified in plasma samples only, since circulating virus variants are considered representative of the viral population escaping the drug pressure. Nevertheless, archival human immunodeficiency virus (HIV) DNA present in PBMCs might represent the reservoir of additional drug-resistant viral variants (8).This study was designed to assess the level of agreement between the drug resistance-associated mutations in plasma and peripheral blood mononuclear cells (PBMCs) in 32 patients for whom highly active antiretroviral therapy (HAART) was failing.A commercial kit was used to identify mutations in the pol gene of HIV type 1 (HIV-1) (ViroSeq HIV-1 V2 genotyping system; PE Biosystems, Foster City, Calif.). DNA was extracted from 3 ϫ 10 6 PBMCs with a High Pure PCR template preparation kit (Roche Diagnostics GmbH, Mannheim, Germany).The Cohen k test was used to determine the correlation between the presence of HIV-1 drug resistance in plasma and PBMCs. Cohen k agreement is defined as poor if k is Յ0.20, fair if k is Ն0.21 and Յ0.60, substantial if k is Ն0.61 and Յ0.80, and good if k is Ͼ0.80 (5). The association between determinant factors and discordance in plasma and PBMC genotypic analysis was assessed by crude and adjusted odds ratios (OR) and their 95% confidence intervals (CI) through univariate and multivariate models.Patients received a mean of 5.5 antiretroviral drugs (range, 3 to 8) during a mean of 57 months of treatment (range, 9 to 126). Twenty-eight (87%) and 16 (50%) of 32 subjects were exposed to protease inhibitors (PI) and nonnucleoside reverse transcriptase inhibitors (NNRTI), respectively.In 492 (21.9%) of 2,240 codons analyzed, there was evidence of drug resistance. In plasma and PBMC genotypic analysis, means of 7.4 (Ϯ4.7 [standard deviation]) and 7.9 (Ϯ5.5) drug resistance mutations were detected, respectively. When total numbers of mutations were calculated for each sample, they were higher in plasma than in PBMCs in 9 of 32 samples (28%), higher in PBMCs than plasma in 14 of 32 samples (43%), and the same in plasma and PBMCs in 9 of 32 samples (28%). A significant correlation between the time of the last ART and the number of protease (PR)-related drug resistance mutations detected in plasma (r ϭ 0.42; P ϭ 0.024) and in PBMCs (r ϭ 0.52; P ϭ 0.006) was found. No significant correlation between mutations in the reverse transcriptase (RT) gene in PBMCs and plasma and the time of drug exposure was detected.The agreement values (mean Cohen k) between drug resistance mutations in plasma and PBMCs are shown in Table 1. The mean of t...

Both Human Immunodeficiency Virus Cellular DNA Sequencing and Plasma RNA Sequencing Are Useful for Detection of Drug Resistance Mutations in Blood Samples from Antiretroviral-Drug-Naive Patients

“…A reasonable hypothesis is that DRMs persist at detectable levels longer in PBMC DNA than in plasma RNA due to the different rates of turnover of the virus in the two compartments (20). Indeed, discrepancies between drug resistance mutations in virus populations harbored in plasma RNA and PBMC DNA have been reported for subjects failing therapy as well as following cessation of treatment (24,27). Furthermore, drug resistance mutations were virtually identical in plasma RNA and PBMC DNA in the only study published so far on drugnaive subjects (5).…”

Feasibility of Detecting Human Immunodeficiency Virus Type 1 Drug Resistance in DNA Extracted from Whole Blood or Dried Blood Spots

“…Some studies reported more mutations in the plasma RNA (6,10,21,24), and others reported more mutations in the cellular DNA (14,34). Most of these discordances may be attributed to differences in the selection of the samples used for the evaluation.…”

“…Results of published reports in this regard are very inconsistent (6,10,14,21,24,34). The present study aims to investigate both the possibility of DNA sequencing from whole blood cells or from DBS and the usefulness of DNA sequencing for determination of drug resistance in patients from a resource-poor setting with a high level of HIV-1 subtype diversity.…”

A Novel Assay Allows Genotyping of the Latent Reservoir for Human Immunodeficiency Virus Type 1 in the Resting CD4+T Cells of Viremic Patients

“…Previous studies that have attempted to characterize the latent reservoir in viremic patients have not excluded the unintegrated viral DNA that predominates in the setting of high levels of viral replication (18,34,56). HIV-1 can enter resting CD4 ϩ T cells and undergo reverse transcription, but it cannot enter the nucleus to undergo integration into the host genome (6,53,63,71).…”