Simultaneous and sensitive detection of human immunodeficiency virus type 1 (HIV) drug resistant genotypes by multiplex oligonucleotide ligation assay.

Oligonucleotide ligation assay (OLA) is a highly specific and relatively simple method to detect point mutations encoding HIV-1 drug-resistance, which can detect mutants comprising ≥2-5% of the viral population. Nevirapine (NVP), tenofovir (TDF) and lamivudine (3TC) are antiretroviral (ARV) drugs used worldwide for treatment of HIV infection and prevention of mother-to-child-transmission. Adapting the OLA to detect multiple mutations associated with HIV resistance to these ARV simultaneously would provide an efficient tool to monitor drug resistance in resource-limited settings. Known proportions of mutant and wild-type plasmids were used to optimize a multiplex OLA for detection of K103N, Y181C, K65R, and M184V in HIV subtypes B and C, and V106M and G190A in subtype C. Simultaneous detection of two mutations was impaired if probes annealed to overlapping regions of the viral template, but was sensitive to ≥2-5% when testing codons using non-overlapping probes. PCR products from HIV-subtype B- and C-infected individuals were tested by multiplex-OLA and compared to results of single-codon OLA. Multiplex-OLA detected mutations at codon pairs 103/181, 106/190 and 65/184 reliably when compared to singleplex-OLA in clinical specimens. The multiplex-OLA is sensitive and specific and reduces the cost of screening for NVP, TDF and/or 3TC resistance.

Prevalence and impact of minority variant drug resistance mutations in primary HIV-1 infection.

OBJECTIVE: To evaluate minority variant drug resistance mutations detected by the oligonucleotide ligation assay (OLA) but not consensus sequencing among subjects with primary HIV-1 infection. DESIGN/METHODS: Observational, longitudinal cohort study. Consensus sequencing and OLA were performed on the first available specimens from 99 subjects enrolled after 1996. Survival analyses, adjusted for HIV-1 RNA levels at the start of antiretroviral (ARV) therapy, evaluated the time to virologic suppression (HIV-1 RNA<50 copies/mL) among subjects with minority variants conferring intermediate or high-level resistance. RESULTS: Consensus sequencing and OLA detected resistance mutations in 5% and 27% of subjects, respectively, in specimens obtained a median of 30 days after infection. Median time to virologic suppression was 110 (IQR 62-147) days for 63 treated subjects without detectable mutations, 84 (IQR 56-109) days for ten subjects with minority variant mutations treated with ≥3 active ARVs, and 104 (IQR 60-162) days for nine subjects with minority variant mutations treated with <3 active ARVs (p = .9). Compared to subjects without mutations, time to virologic suppression was similar for subjects with minority variant mutations treated with ≥3 active ARVs (aHR 1.2, 95% CI 0.6-2.4, p = .6) and subjects with minority variant mutations treated with <3 active ARVs (aHR 1.0, 95% CI 0.4-2.4, p = .9). Two subjects with drug resistance and two subjects without detectable resistance experienced virologic failure. CONCLUSIONS: Consensus sequencing significantly underestimated the prevalence of drug resistance mutations in ARV-naïve subjects with primary HIV-1 infection. Minority variants were not associated with impaired ARV response, possibly due to the small sample size. It is also possible that, with highly-potent ARVs, minority variant mutations may be relevant only at certain critical codons.

Selection of HIV resistance associated with antiretroviral therapy initiated due to pregnancy and suspended postpartum.

OBJECTIVE: Compare the risk of HIV drug resistance in women stopping suppressive nelfinavir (NFV)-based or Nevirapine (NVP)-based antiretroviral therapy (ART) after pregnancy. METHODS: Specimens collected after stopping ART were tested for drug resistance by an oligonucleotide ligation assay and consensus sequencing. When postpartum drug resistance was detected, specimens obtained at study entry and during ART were evaluated. RESULTS: Sixteen of 38 women with ART-induced suppression of viral replication suspended ART postpartum. Resistance mutations were detected in 75% who stopped NFV-ART and in 50% who stopped NVP-ART. M184V, associated with Lamivudine resistance, was more frequent among those randomized to NFV-ART compared with NVP-ART (6 of 8 versus 1 of 8; P = 0.04), and nonnucleoside reverse transcriptase inhibitor resistance was detected in 4 of 8 stopping NVP-ART. CONCLUSIONS: HIV drug resistance was frequently observed among women who stopped suppressive NVP-ART or NFV-ART postpartum. This suggests that NFV-ART may have suboptimal potency, that staggering discontinuation of NVP-ART may be warranted, and/or ART adherence may be lax in women who choose to stop ART postpartum.

Intrapartum tenofovir and emtricitabine reduces low-concentration drug resistance selected by single-dose nevirapine for perinatal HIV prevention.

A single dose of tenofovir/emtricitabine (TDF/FTC) during labor significantly reduces peripartum nevirapine-associated viral drug resistance when measured by consensus HIV sequencing. It is unknown whether this effect extends to HIV subpopulations of <25-50%. We conducted a randomized trial of single-dose TDF/FTC added to peripartum nevirapine to reduce drug resistance associated with nonnucleoside reverse transcriptase inhibitors (NNRTIs). To detect mutations for NNRTIs comprising > or = 2% of the viral population, we used an oligonucleotide ligation assay (OLA) at codons 103, 106, 181, and 190 of HIV reverse transcriptase. To assess development of drug resistance mutations to our study intervention, OLA was also performed at codons 65 and 184. Among the 328 women included in the 2-week analysis, those receiving TDF/FTC were less likely to have NNRTI resistance by OLA (RR = 0.40, 95% CI = 0.21-0.77). A similar trend was observed among the 315 women included in the 6-week analysis (RR = 0.45, 95% CI = 0.31-0.66). Only two (1%) specimens had detectable K65R by OLA. Both were at 6 weeks postpartum; one was detected in the intervention arm and one in the control arm (p = 0.96). M184V was not detected. The ability of single-dose TDF/FTC to protect against peripartum NVP-induced NNRTI resistance extends to minority populations. This efficacy is achieved without significant selection of TDF- or FTC-resistant viruses.

Increased detection of HIV-1 drug resistance at time of diagnosis by testing viral DNA with a sensitive assay.

OBJECTIVE: HIV-1 drug resistance has been detected in 8%-24% of recently infected North Americans when assessed by consensus sequencing of plasma. We hypothesized that rates were likely higher but not detected because drug-resistant mutants are transmitted or regressed to levels below the limit of detection by consensus sequencing of HIV-1 RNA. METHODS: Specimens from antiretroviral-naive individuals recently diagnosed with HIV-1 infection were compared at 15 codons to determine if testing of DNA using a sensitive oligonucleotide ligation assay (OLA) would detect drug resistance mutants not evident by consensus sequencing of serum. RESULTS: HIV-1 drug resistance at 15 major resistance codons was greater by OLA compared with consensus sequencing: 18 of 104 vs. 12 of 104 individuals (P < or = 0.008) and 33 vs. 18 total mutations (P < or = 0.001); increasing the rate of detection at these 15 codons by 83%. Additional mutations were detected by consensus sequencing at L33, M46, D67, V108, and K219 that were not assessed by OLA. CONCLUSIONS: The increased detection of drug-resistant HIV-1 by testing peripheral blood cells with a sensitive assay implies that both low and high levels of drug-resistant mutants are transmitted or persist in antiretroviral-naive individuals, suggesting that the clinical relevance of mutants persisting at both levels should be evaluated.

Transmission cluster of multiclass highly drug-resistant HIV-1 among 9 men who have sex with men in Seattle/King County, WA, 2005-2007.

BACKGROUND: From 2005 through 2007, Seattle health care providers identified cases of primary multiclass drug-resistant (MDR) HIV-1 with common patterns of resistance to antiretrovirals (ARVs). Through surveillance activities and genetic analysis, the local Health Department and the University of Washington identified phylogenetically linked cases among ARV treatment-naive and -experienced individuals. METHODS: HIV-1 pol nucleotide consensus sequences submitted to the University of Washington Clinical Virology Laboratory were assessed for phylogenetically related MDR HIV. Demographic and clinical data collected included HIV diagnosis date, ARV history, and laboratory results. RESULTS: Seven ARV-naive men had phylogenetically linked MDR strains with resistance to most ARVs; these were linked to 2 ARV-experienced men. All 9 men reported methamphetamine use and multiple anonymous male partners. Primary transmissions were diagnosed for more than a 2-year period, 2005-2007. Three, including the 2 ARV-experienced men, were prescribed ARVs. CONCLUSIONS: This cluster of 9 men with phylogenetically related highly drug-resistant MDR HIV strains and common risk factors but without reported direct epidemiologic links may have important implications to public health. This cluster demonstrates the importance of primary resistance testing and of collaboration between the public and private medical community in identifying MDR outbreaks. Public health interventions and surveillance are needed to reduce transmission of MDR HIV-1.

Comparison of oligonucleotide ligation assay and consensus sequencing for detection of drug-resistant mutants of human immunodeficiency virus type 1 in peripheral blood mononuclear cells and plasma.

Drug-resistant mutants of human immunodeficiency virus type 1 (HIV-1) recede below the limit of detection of most assays applied to plasma when selective pressure is altered due to changes in antiretroviral treatment (ART). Viral variants with different mutations are selected by the new ART when replication is not suppressed or wild-type variants with greater replication fitness outgrow mutants following the cessation of ART. Mutants selected by past ART appear to persist in reservoirs even when not detected in the plasma, and when conferring cross-resistance they can compromise the efficacy of novel ART. Oligonucleotide ligation assay (OLA) of virus in plasma and peripheral blood mononuclear cells (PBMC) was compared to consensus sequence dideoxynucleotide chain terminator sequencing for detection of 91 drug resistance mutations that had receded below the limit of detection by sequencing of plasma. OLA of plasma virus detected 27.5% (95% confidence interval [CI], 19 to 39%) of mutant genotypes; consensus sequencing of the PBMC amplicon from the same specimen detected 23.1% (95% CI, 14 to 34%); and OLA of PBMC detected 53.8% (95% CI, 44 to 64%). These data suggest that concentrations of drug-resistant mutants were greater in PBMC than in plasma after changes in ART and indicate that the OLA was more sensitive than consensus sequencing in detecting low levels of select drug-resistant mutants.

Multiple viral genetic analyses detect low-level human immunodeficiency virus type 1 replication during effective highly active antiretroviral therapy.

To evaluate human immunodeficiency virus type 1 (HIV-1) replication and selection of drug-resistant viruses during seemingly effective highly active antiretroviral therapy (HAART), multiple HIV-1 env and pol sequences were analyzed and viral DNA levels were quantified from nucleoside analog-experienced children prior to and during a median of 5.1 (range, 1.8 to 6.4) years of HAART. Viral replication was detected at different rates, with apparently increasing sensitivity: 1 of 10 by phylogenetic analysis; 2 of 10 by viral evolution with increasing genetic distances from the most recent common ancestor (MRCA) of infection; 3 of 10 by selection of drug-resistant mutants; and 6 of 10 by maintenance of genetic distances from the MRCA. When four- or five-drug antiretroviral regimens were given to these children, persistent plasma viral rebound did not occur despite the accumulation of highly drug-resistant genotypes. Among the four children without genetic evidence of viral replication, a statistically significant decrease in the genetic distance to the MRCA was detected in three, indicating the persistence of a greater number of early compared to recent viruses, and their HIV-1 DNA decreased by > or =0.9 log(10), resulting in lower absolute DNA levels (P = 0.007). This study demonstrates the variable rates of viral replication when HAART has suppressed plasma HIV-1 RNA for years to a median of <50 copies/ml and that combinations of four or five antiretroviral drugs suppress viral replication even after short-term virologic failure of three-drug HAART and despite ongoing accumulation of drug-resistant mutants. Furthermore, the decrease of cellular HIV-1 DNA to low absolute levels in those without genetic evidence of viral replication suggests that monitoring viral DNA during HAART may gauge low-level replication.