Ellis, Giovanina M BS*; Page, Libby C MPH†; Burman, Blaire E MD‡; Buskin, Susan PhD†§; Frenkel, Lisa M MD*‡‖
Transmitted drug-resistant HIV-1 identified in plasma by consensus sequencing has been observed to persist in the plasma for 2-5 years,1-4 and seems to compromise the efficacy of antiretroviral therapy (ART).5,6 HIV-1 drug-resistant mutants selected during ART of chronic infection that persist below the limit of detection of consensus sequencing also seem to compromise subsequent ART.7-10 Low-level concentrations of drug-resistant mutants have been identified at the time of acute seroconversion using assays with a greater sensitivity compared with consensus methods.11 The prevalence and persistence of low-level mutants and their effects on ART have not been thoroughly described; although recently, low-concentration mutants identified by real-time polymerase chain reaction (PCR) were reported to diminish the efficacy of ART.12
Clinical HIV-1 drug resistance is generally assessed by consensus sequencing of the plasma HIV-1 RNA that encodes protease (PR) and reverse transcriptase (RT).13 Currently, the US Department of Health and Human Services recommends testing all people for drug resistance on entry into care.13 Consensus sequencing does not consistently detect genotypes present at concentrations <20%-50% of the viral population.14 In contrast, the oligonucleotide ligation assay (OLA) can detect mutant genotypes present at concentrations as low as 5% among wild-type virus.15,16 HIV-1 replication-competent genotypes are archived in peripheral blood mononuclear cells (PBMCs).17,18 Previously, we found that testing of viral DNA from PBMCs was more sensitive for detecting selected mutations that had regressed to levels below the limit of detection of testing plasma RNA by either consensus sequencing or OLA.19 Others have also observed that PBMCs reveal more mutants selected in the past compared with sequencing of plasma.20,21
Several scenarios could result in low concentrations of drug-resistant viruses in individuals recently diagnosed with HIV-1 infection: First, mixed populations of virus including low concentrations of mutant viruses could be transmitted from individuals failing ART. Second, after transmission of mixed viral populations, a predominately resistant virus population could shift to predominately wild type due to a fitness advantage of the latter.22 Third, after transmission of a pure population of mutant virus, wild-type viruses with greater fitness could evolve. And, fourth, after transmission of mutant virus, superinfection with wild-type virus could overgrow mutant variants with lesser fitness.23 These multiple potential scenarios by which drug-resistant mutants could persist at low levels led us to hypothesize that use of a sensitive assay to assess major drug-resistant genotypes in HIV-1 DNA in peripheral blood cells (PBCs) would increase the detection of HIV-1 drug-resistant mutants in individuals recently diagnosed with HIV-1 compared with consensus sequencing of HIV-1 RNA from sera.
MATERIALS AND METHODS
Study Design and Participant Specimens
Individuals diagnosed as HIV-1 infected after voluntary counseling and testing (VCT) at a Public Health-Seattle and King County (PHSKC) clinic that participated in the “HIV Antiretroviral Drug Resistance Sentinel Surveillance (ARVDRT)” project from 2004 to 2006 were asked to participate in this study, which was approved by the Washington State Committee for Research on Human Subjects. The ARVDRT project utilized the sera remaining after positive HIV-1 enzyme immunoassay and Western blot testing for HIV-1 genotypic resistance testing. Genotyping was performed at either the University of Washington (n = 65) or Stanford University (n = 39), both of which have been certified as proficient through the Genotyping Resistance Proficiency Panel provided by the Virology Quality Assessment (VQA) Program. Exclusion criteria for the ARVDRT project included prior positive HIV-1 serology or use of antiretrovirals. Individuals were consented to this study upon returning to clinic for follow-up, and blood was collected into a 2 mL tube with EDTA. This specimen was assigned the same study identification as the initial VCT specimen. HIV-1 pol was PCR amplified from peripheral blood DNA and tested by OLA.15,16 Demographic, HIV-1 RNA load, and CD4+ lymphocyte count data collected by PHSKC were linked to specimens for the analysis. PHSKC did not have data regarding the timing of infection for the participants in this cohort. Specimens were evaluated by OLA, and results were finalized for this project before comparisons to consensus sequencing. Major drug resistance mutations defined by the International AIDS Society (IAS)-USA and Stanford HIV Drug Resistance Database24 during the course of the study and not evaluated by the OLA were tabulated for consensus sequences.
Genotypic Testing of Sera
RNA extracted from the sera sent to the University of Washington was reverse transcribed, PCR amplified, and sequenced using dideoxynucleotide chain terminators, with consensus sequence of HIV-1 pol determined as previously described.19 The specimens shipped to Stanford were processed in a similar fashion25; both laboratories utilized the in-house genotyping method certified by the VQA Program. The close similarity between the 2 laboratories' methodologies and quality assurance results allowed genotyping results to be considered equivalent. Mutations and HIV-1 group M subtype were identified using the Stanford HIV Drug Resistance Database.24 The nucleic acid sequence, resistance mutations, and subtype data were reported to the PHSKC and the Centers for Disease Control and Prevention by study code. Consensus sequences spanned from PR codon 2 through RT codon 225.
OLA Testing of PBCs
DNA was extracted from 0.6 to 1.2 mL of whole blood using the Puregene Cell and Tissue Kit (Gentra Systems, Minneapolis, MN). DNA concentrations were measured by optical density (OD) at 260 nm and stored at −20oC until PCR amplified.
PCR was carried out as previously described,19 except the first round primers were NEF10 (5′-GARAGACAGGCTAATTTTTTAGGGA-3′) and NER10 (5′-AAYTTCTGTATATCATTGACAGTCCA-3′). Second round primers were NEF11 (5′-CAAATCACTCTTTGGCArCGACC-3′) and NER11 (5′-CAYTTGTCAGGATGGAGTTCATA-3′). The amplicon, a 1009-base pair DNA fragment encoding codon 2 in PR to codon 238 in RT, was visualized in a 1.2% agarose gel with ethidium bromide staining.
The OLA was performed on all amplicons derived from whole blood DNA extraction using validated probes for major drug resistance mutations selected by protease inhibitors (PIs) at codons D30N (AAT), I50V (GTT), V82A/S/T (GCC, AGC, ACC), I84V (GTA), N88D (GAT), and L90M (ATG); nucleoside reverse transcriptase inhibitors (NRTIs) at codons M41L (TTG, CTG), K65R (AGA, AGG), K70R (AGA), L74V (GTA), M184V (GTG), and T215F/Y (TTC, TAC); and nonnucleoside reverse transcriptase inhibitors (NNRTIs) at codons K103N (AAC), Y181C (TGT), and G190A (GCA) as described.15,16,19,26 Briefly, the amplicon was added to a ligation reaction containing a probe specific for the wild type and the mutant codons labeled at the 5′ end and a probe for the region downstream of the codon of interest that was biotinylated at the 3′ end. After the ligation reaction, the products were bound to a streptavidin-coated microtiter plate, and an enzyme-linked immunosorbent assay was performed using alkaline phosphatase and horseradish peroxidase-labeled antibodies to develop color for the mutant and wild-type codons, respectively. All participant specimens and assay controls were analyzed in duplicate. Control plasmids are described in the OLA manual.26
Amplicon derived from 1 μg of DNA from each specimen was tested by OLA in batches. To confirm detection of mutant, OLA of the specimen was repeated if the mutant reaction was less than 10% of HIV-1 mutant control OD but greater than the nonreactive cutoff. OLA was completed for all codons before comparison to the consensus sequencing results from each participant's serum.
HIV-1 DNA Quantification
If a sufficient quantity of DNA remained after PCR for OLA, HIV-1 DNA concentrations were assessed in each subject's specimen by real-time PCR of the gag region. Real-time PCR reactions contained iQ supermix, subtype B (FAM-AAAGAGACCATCAATGAGGAAGCTGCAGAA-TAM) or subtype C (FAM-ACCATCAATGAGGAGGCTGCAGAATGGGA-TAM) probe, forward and reverse primers (HXB2gagFmod 5′-GACATCAAGCAGCCATGCAAATGTT-3′ and SK431 5′-TGCTATGTCACTTCCCCTTGGTTCTCT-3′), and 1-4 μg of extracted DNA. DNA specimens were assayed in duplicate under the following conditions: 95oC for 3 minutes, 95oC for 15 seconds, and 60oC for 1 minute for 45 cycles. Standard curves, specific for the HIV-1 subtype being tested, were run in duplicate with every assay.
McNemar's χ2 P values were calculated to determine the significance of the associations, with a P value of ≤0.05 considered statistically significant.
Participants' Demographics, CD4 Cell, and HIV-1 RNA and DNA Concentrations
Recently 113 HIV-1 diagnosed antiretroviral-naive participants were enrolled into this study. Consensus sequence was not obtained for 9 individuals due to insufficient volume of serum or no amplification of virus in PCR for sequencing. Analysis was limited to 104 participants with both OLA and consensus sequence data. Participants were predominately male (94%), 20-40 years old (65%), and most were caucasian (69%) (Table 1). The median interval between the VCT and OLA specimen was 10 days (range 0-324).
HIV-1 RNA loads were available for 62 of 104 (60%) with an average of 4.94 log10 copies per milliliter (median: 4.51 log10 copies/mL, range: 2.75-5.73 log10 copies/mL). Average CD4+ lymphocyte count, calculated from only 41 participants (39%), was 508 cells per microliter (median: 507 cells/μL, range: 6-1236 cells/μL). The median HIV-1 DNA load, assessed in 96 of 104 participants, was 194 copies per million PBCs.
Ninety-eight participants (94%) had subtype B virus. The non-subtype B specimens consisted of subtype C (n = 3), CRF01_AE (n = 1), CRF02_AG (n = 1), and CRF01_AE/A for PR/RT (n = 1). No major drug resistance mutations were detected in the non-subtype B specimens by either assay.
Detection of Mutants by Consensus Sequencing
Resistance by consensus sequencing of HIV-1 RNA from sera was detected in 17 of 104 participants (16.3%) when interpreted by the IAS-USA guidelines27 for major drug resistance. However, when restricted to Stanford's major drug resistance guidelines,24 only 15 of 104 participants (14.4%) had major resistance detected. This discrepancy was due to tallying codons at PR L33 and D67, V108, L210, and K219 by the former but not the latter criteria. The final resistance tally by Stanford's surveillance guideline24 yields resistance mutations in 16 of 104 participants due to the inclusion of RT T215D.
Detection of Mutants by OLA
Analyses first compared detection of resistance at the codons assessed by OLA: PR D30N, I50V, V82A/S/T, I84V, D88N, and L90M and RT M41L, K65R, K70R, L74V, K103N, Y181C, M184V, G190A, and T215F/Y (Fig. 1). OLA of HIV-1 DNA detected major drug resistance mutations in 18 of 104 participants (17.3%), including 11, 8, and 14 drug resistance mutations to PI, NRTI, and NNRTI, respectively, for a total of 33 mutations. Consensus sequencing of HIV-1 RNA from sera detected major drug resistance mutations at these codons in 12 of 104 individuals (11.5%), including 4, 3, and 11 mutations to PI, NRTI, NNRTI, respectively, for a total of 18 mutations (Fig. 1). The proportion of individuals with drug-resistant mutants by OLA was greater than by sequencing, 18 of 104 vs. 12 of 104, P ≤ 0.008, suggesting that individuals had resistance at these codons at concentrations below the limit of detection by consensus sequencing. The proportion with a major mutation to each class of drug was also greater by OLA compared with sequencing 29 of 312 vs. 16 of 312, P ≤ 0.001. OLA identified all major drug resistance mutations identified by sequencing at the 15 codons assessed by both assays (GenBank accession numbers EF195268-332 with 39 in progress).
Six specimens with no resistance by consensus sequencing at codons assessed by OLA had resistance to 1 (n = 3), 2 (n = 1), or 3 classes (n = 2) of ARV detected as mixtures of wild-type and resistant virus by OLA. Of these 6, consensus sequencing detected 2 mutations that could confer major resistance at sites not assessed by OLA. ID #5 had a V108I mutation (impacting NNRTI susceptibility; whereas OLA detected only mixtures of mutants and wild-type virus at sites impacting susceptibility to PI and NRTI). ID #1 had T215D by consensus sequencing, which is not assessed by OLA, but OLA detected a mix of wild type and mutant for T215Y. Among 9 participants with resistance to one drug class detected by sequencing, OLA identified resistance to a second drug class in 1 of the 9. Two specimens with resistance to 2 drug classes by sequencing were also detected by OLA, plus an additional mutation to a third class was detected by OLA in 1 of these 2.
The overall rate of indeterminate OLA calls for this study was 5.5%. Genetic variation in non-B HIV-1 subtypes accounted for 27% of the indeterminate calls, with the majority of indeterminate calls due to polymorphisms near the ligation site as evident by the participant's consensus sequence. No mutations associated with resistance were detected by consensus sequencing at codons with indeterminate OLA calls.
Detection of Mutants by Consensus Sequencing or OLA But Not by Both Methods
Discrepant results between consensus sequencing and OLA were primarily due to detection of low-level mutants by OLA that were not detected in consensus sequences. At the codons that could be directly compared, there were no mutations detected by sequencing that were not detected by OLA. Although the most common resistance mutations identified by both methods were RT K103N and Y181C, the OLA detected Y181C in 3 participants of IDs #6, #9, and #11 that have wild-type codons in corresponding consensus sequences. OLA alone detected resistance in RT at M184V in 2 participants, #9 and #11, and in PR at I50V in 4 participants: #2, #5, #9, and #11 (Fig. 1).
Overall, consensus sequencing detected a total of 20 mutations in 10 participants at sites not assessed by OLA when resistance was interpreted with both IAS-USA and Stanford resistance guidelines (Fig. 2). Restricted to IAS-USA interpretation guidelines, consensus sequencing detected a total of 16 resistance mutations in 9 participants at codons not submitted to OLA. By Stanford's resistance interpretation, sequencing detected 8 major mutations in 6 participants at sites not tested by OLA. However, using Stanford's surveillance guideline, sequencing detected 19 mutations in 8 participants at codons not assessed by OLA. Codons PR L33 and M46 and RT D67, T69, V108, L210, and K219 are classified as major drug resistance codons by IAS-USA27; however, these codons were not assessed by OLA in this study. Two participants had resistance at RT D67 and K219 by consensus sequencing but not at any other codons by either method. Four participants had resistance at M46 by sequencing, and 2 of those patients had no resistance at other sites by either method.
The OLA used in this study was not designed to detect intermediates between wild-type and mutant codons (eg, codon RT T215C/D/E/I/V). Intermediate mutations at RT T215 were identified by sequencing in 3 of the 104 participants, and OLA of these specimens yielded indeterminate (neither wild type nor mutant) results (n = 1), wild type (n = 1), and a mix of wild type and 215Y (n = 1).
Consensus sequence genotypes interpreted by IAS-USA guidelines combined with OLA genotypes yield major resistance in 22 of the 104 participants (21.2%).
Analysis of HIV-1 DNA by OLA increased the detection of drug resistance mutations at 15 codons known to confer major resistance to antiretrovirals by 83% (33 vs. 18 mutations) compared with consensus sequencing of HIV-1 RNA. These data suggest that testing of PBCs, known to include archived viruses,7,18,19,29 and using a more sensitive assay, such as OLA, increase the rate at which drug-resistant HIV-1 viruses are detected in newly diagnosed individuals. This more sensitive testing strategy presumably detected drug-resistant mutants that were either transmitted at a low level or outcompeted by wild-type variants and persisted at levels below the limit of detection of consensus sequencing of virus in serum.
The prevalence of major drug resistance mutations at 15 directly comparable codons in this antiretroviral-naive population was 11.5% by consensus genotyping methods and 17.3% by OLA. This is consistent with resistance rates currently being reported by standard genotyping across North America and western Europe.30-33 The prevalence of major drug resistance from combined consensus sequencing and OLA data was 21.2%, which is closer to recently reported rates in ARV-naive patients in San Diego and New York City.34,35
OLA of HIV-1 DNA increased both the overall detection of mutations and the number of drug classes to which participants' viruses were resistant. Based on previous observations that low-level mutants can be associated with virologic failure,8,12 the choice of initial treatment, based on consensus sequencing, would be anticipated to result in increased rate of treatment failure compared with OLA.
Codons M46 in PR and D67, T69, T215 intermediates and K219 in RT were not evaluated by OLA in this project. However, the detection of mutations at these sites in this and other studies28,36 suggests that the OLA would be more valuable if reagents were included to evaluate these codons when screening ARV-naive individuals for drug resistance.
The sensitivity of the OLA may have been limited in this study by the use of whole blood as a source of HIV-1 DNA. One microgram of DNA extracted from whole blood had a median of 29 copies of HIV-1 DNA. Use of PBMC is preferred due to the relatively higher concentrations of HIV-1. In addition, replicate PCR to sample at least 200 copies of HIV-1 DNA would improve detection of viral variants at low concentrations.
Multiple methods detect minor populations of drug-resistant mutants: real-time allele-specific PCR, pyrosequencing, line probe assay, single-genome sequencing, LigAmp, and parallel allele-specific sequencing.11,37-42 The advantages of OLA include relatively inexpensive reagents and equipment, availability of validated reagents for multiple codons associated with HIV-1 drug resistance (http://www.aidsreagent.org, catalog numbers 7643-5), and ease of interpretation.
One disadvantage of the OLA compared with consensus sequencing is that new oligonucleotide probes must be developed as new drug resistance mutations are identified. OLA probes have been designed only against mutations conferring major drug resistance in PR and RT and need to be expanded to include more recently described variants and transmitted mutations. To address this need, new probes for L24I, M46I/L, I50L, I54V, and G73S in PR and D67N, L210W, T215C/E/I, and K219Q/E in RT are currently being evaluated. The high specificity of the ligation reaction for the 4 bases flanking the ligation site also necessitates optimization of the OLA probes for different HIV-1 subtypes, as completed for NNRTI codons.26,43 The codons tested by OLA in this study detect the most common variants of primary resistance mutations in subtype B that have also been defined as surveillance mutations by Shafer et al.35
The thresholds below which drug-resistant mutant populations are clinically inconsequential have not been defined. Such cutoffs will likely be affected by multiple factors that determine the longevity of mutants in the viral reservoir, including whether a specific mutation was transmitted or selected. If selected, both the duration of selection and viral load during selection44 as well as the interval between selection of the mutation and initiation of the ART45 seem to be relevant to the virologic outcome of subsequent ART. Detection of minority populations of drug-resistant mutants among individuals recently diagnosed with HIV-1 remains an important focus of research aimed at optimizing the virologic success of ART. Importantly, our data suggest that testing of HIV-1 DNA in PBCs and use of sensitive assays may improve detection of transmitted drug-resistant virus.
The authors would like to thank the individuals who participated in this study. We would also like to gratefully acknowledge the contributions of Drs. Sharon Hopkins, Matthew Golden, and Robert Wood; Sandra Dross, Gregory Pepper, the staff at the PHSKC Clinic and Laboratory, and Dr. Robert Shafer and his staff at Stanford University.
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