High discordance in blood and genital tract HIV-1 drug resistance in Indian women failing first-line therapy

High discordance in blood and genital tract HIV-1 drug resistance in Indian women failing... Abstract Objectives Examine HIV-1 plasma viral load (PVL) and genital tract (GT) viral load (GVL) and drug resistance in India. Methods At the YRG Centre for AIDS Research and Education, Chennai, we tested: PVL in women on first-line ART for ≥6 months; GVL when PVL >2000 copies/mL; and plasma, genital and proviral reverse transcriptase drug resistance when GVL >2000 copies/mL. Wilcoxon rank-sum and Fisher's exact tests were used to identify failure and resistance associations. Pearson correlations were calculated to evaluate PVL–GVL associations. Inter-compartmental resistance discordance was evaluated using generalized estimating equations. Results Of 200 women, 37% had detectable (>400 copies/mL) PVL and 31% had PVL >1000 copies/mL. Of women with detectable PVL, 74% had PVL >2000 copies/mL, of which 74% had detectable GVL. Higher PVL was associated with higher GVL. Paired plasma and genital sequences were available for 21 women; mean age of 34 years, median ART duration of 33 months, median CD4 count of 217 cells/mm3, median PVL of 5.4 log10 copies/mL and median GVL of 4.6 log10 copies/mL. Drug resistance was detected in 81%–91% of samples and 67%–76% of samples had dual-class resistance. Complete three-compartment concordance was seen in only 10% of women. GT–proviral discordance was significantly larger than plasma–proviral discordance. GT or proviral mutations discordant from plasma led to clinically relevant resistance in 24% and 30%, respectively. Conclusions We identified high resistance and high inter-compartmental resistance discordance in Indian women, which might lead to unrecognized resistance transmission and re-emergence compromising treatment outcomes, particularly relevant to countries like India, where sexual HIV transmission is predominant. Introduction ART is changing the dynamics of the HIV epidemic through suppression of plasma viral load (PVL) and genital tract (GT) viral load (GVL).1–5 Despite viral suppression in plasma, replication can occur in the GT increasing horizontal and vertical transmission risks.6–14 Such compartmentalization and differential evolution between plasma and the GT can be attributed to differences in CD4 or co-receptor expression,15–18 HIV-specific immune pressure,15,19–24 poor genital ART penetration causing selective pressure,25 inflammation from trauma26 or sexually transmitted infections.15,17,20,21,27–31 Resulting tissue-specific lineages can increase the risk of unrecognized drug resistance and tropism,6–12 suggesting compartment-specific HIV replication milieu. With increasing evidence for beneficial early ART to reduce HIV transmission,16,32,33 data on prevalence and correlates of viral replication and of drug resistance mutations (DRMs) in circulating and archived viruses and in the GT among treated individuals are needed, particularly in globally predominant HIV-1 subtypes.34 These measures impact long-term ART management and resistance transmission and can guide development of novel prevention interventions.35–38 Genotyping of circulating plasma virus is the current standard for clinical management before and upon ART failure; however, it may underestimate resistance in settings like treatment interruptions or compromised adherence.39–42 Though clinical significance of DRMs that are archived in cellular reservoirs remains controversial,39–43 proviral genotyping may further inform treatment decisions. Genotyping of GT viruses is uncommon and three-compartment drug resistance concordance and their impacts are unknown. PVL and GVL are higher for the globally predominant HIV-1 subtype C.43–50 In a developing country like India, where subtype C predominates and heterosexual activity is the major mode of transmission, investigating viral shedding and GT resistance upon treatment failure and its discordance from circulating and archived viruses is important, as it carries implications for resistance transmission and clinical outcome. Under the hypothesis of an existing inter-compartmental discordance in viral load and clinically relevant resistance, we examined PVL and GVL, as well as plasma, proviral and genital virus resistance, in South Indian women on first-line ART. Methods Study setting Women were enrolled at the YRG Centre for AIDS Research and Education (YRG-CARE) clinic in Chennai, the largest community-based tertiary HIV care institution in India, with >20 000 registered patients. During the study period (May 2009 to November 2011), 39% of clinic patients were women and 60% were on ART, attending clinic every 3 months. Treatment monitoring was based on WHO immunological and clinical criteria, without virological monitoring. HIV-1 infected women were offered enrolment if they were: (i) ≥18 years old; (ii) on first-line ART (zidovudine/stavudine + lamivudine/emtricitabine + nevirapine/efavirenz, the recommended regimens at the time of the study) for >6 months; (iii) reporting >95% adherence in the past month; (iv) without pelvic surgery in the past 6 months; (v) not pregnant; and (vi) not currently menstruating. Enrolled women had a screening visit during which demographics were collected from interview and chart, including age, WHO stage, adherence in the past month, history of pelvic/cervical surgeries, reproductive history, last menstrual period, CD4 count and ART history. Upon enrolment, PVL was tested and women with PVL >2000 copies/mL were invited for a follow-up visit within 1 month, 1 week prior to their next estimated menstrual period. This PVL threshold was chosen to increase the yield of plasma and genital genotyping in this pilot study. Participants were instructed not to have sexual intercourse, douche or insert any vaginal products for ≤48 h before the second visit. On that visit, additional history was derived, including history of sexually transmitted diseases and use of contraceptives (e.g. oral contraceptive pills, hormonal contraceptive dermal implant, depomedroxyprogesterone acetate injection or intrauterine device); an additional 10 mL of blood was collected, PBMCs were isolated51 and stored at −75 ± 5°C, and genital secretions were collected (as specified below). Ethics The study was approved by Lifespan and YRG-CARE Institutional Review Boards. All participants provided written informed consent. Genital specimen collection and processing During the second visit, pelvic examination was performed to collect endocervical secretions, cervical vaginal lavage (CVL) and vaginal swab samples. Endocervical secretions were collected using four wicks (Tear FloTM), held in place for a 1–3 min wicking period, then removed and cut at the wick neck. Each pair of wicks was placed in a vial containing 500 μL of nucleic acid sequence-based amplification (NASBA) buffer (bioMérieux, Durham, NC, USA) and stored at −75 ± 5°C. CVL samples were collected by bathing the cervix and ectocervix three times with the same 10 mL of PBS, which was then aspirated and placed into a sterile 50 mL conical centrifuge tube. Cervical samples were examined under the microscope for red blood cells and spermatozoa. Genital specimens were aliquotted within 4 h of collection and stored at −75 ± 5°C. Four vaginal swabs were collected prior to CVL collection and wet mount specimens were prepared to diagnose trichomoniasis, candidiasis and bacterial vaginosis. HIV genotyping from plasma, genital Tear FloTM and PBMCs was performed for all women with GVL >2000 copies/mL (to ensure high genotyping yield), who had negative results for co-infection with trichomoniasis, candidiasis and bacterial vaginosis (which may distort results). Laboratory methods All testing was performed at the YRG-CARE laboratory, accredited by the National Accredited Board of Laboratories (ISO 15189:2012). Plasma was separated from blood within 4–6 h of sampling and stored at −75±5°C. CD4 testing was done as part of routine care using a two-colour single-platform flow cytometer, FACS Count (Becton Dickson Immunocytometry Systems, San Jose, USA). PVL was done with the Roche COBAS Amplicor HIV-1 Monitor test, version 1.5 (Roche Diagnostics, Branchburg, USA) as per the manufacturer’s protocol with a 400 copies/mL lower detection limit. The same protocol was applied for GVL with minor modifications: Tear FloTM filter paper wicks were cut and placed in 600 μL of lysis buffer with a quantification standard and incubated at room temperature on a shaker for 45 min at 800 rpm, followed by addition of 600 μL of 100% isopropanol to the supernatant and proceeding as per the manufacturer’s protocol.52 The same 400 copies/mL lower detection limit was used. Reverse transcriptase (RT; amino acid positions 1–230) sequencing from plasma and GT was done as described.53,54 Briefly, RNA was extracted from plasma using the QIAamp viral RNA extraction kit (QIAGEN, Valencia, CA, USA) as per the manufacturer’s protocol. Genital RNA was extracted from Tear FloTM using the Roche COBAS Amplicor, and used for quantification and sequencing. Plasma and genital RNA were then reverse transcribed to cDNA. The RT fragment was amplified from cDNA by nested PCR with RT primers and analysed on 1.5% agarose gel. DNA was extracted from PBMCs using a QIAamp Blood Mini Kit (QIAGEN) as per the manufacturer’s instructions and stored at −75 ± 5°C. Amplification was performed by nested PCR and analysed on 1.5% agarose gel. Amplicons were then column-purified and sequenced with an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems, Foster City, USA) with an ABI sequence analyzing software version 3.7. Sequence analysis Sequences were interpreted for resistance using the Stanford HIV Database tools and clinically significant resistance was defined as intermediate or high levels to ≥1 drug according to the Stanford penalty rules.55 Phylogenetic analyses were performed with Mega version 6.056 by maximum likelihood [1000 bootstraps; HKY model with gamma distribution based on FindModel (http://www.hiv.lanl.gov)]. Sequence quality control and distance estimation were with SQUAT57 and subtyping with REGA.56,58 Genetic distances were estimated to identify the level of evolution between the three compartments and from each compartment to a subtype C consensus. The latter, created from 113 YRG-CARE subtype C sequences from treatment-naive patients available from 2007 to 2011, was used as the ‘reference’. Such analyses are relevant for the understanding of resistance archival and its potential impact on resistance evolution, and of compartmental resistance discordance. Statistical analysis We hypothesized that among women with detectable PVL: (i) some will have suppressed GVL; (ii) some will have clinically relevant DRMs detected in genital samples and/or PBMCs but not in plasma; and (iii) demographic and clinical characteristics will differ according to viral load and DRMs. To evaluate these hypotheses, we compared demographic and clinical measures between several subgroups: (i) women with versus without detectable PVL; (ii) women with detectable PVL ≤2000 versus those with >2000 copies/mL; (iii) women with detectable versus undetectable GVL, among those with PVL >2000 copies/mL; and (iv) women with versus without GT DRMs. Wilcoxon rank-sum tests were used to compare continuous measures (age, CD4 count, months on ART, PVL and GVL, where applicable) and Fisher's exact test for categorical measures (ART regimen). Pearson correlations were calculated to evaluate the strength of relationship between log10-transformed viral load in the plasma and GT. For comparison of drug resistance concordance, for each compartment comparison (A versus B; e.g. plasma versus GT, GT versus PBMCs and plasma versus PBMCs), patients were classified as ‘concordant’ or ‘discordant’. ‘Concordant’ compartments are those with identical mutation patterns and ‘discordant’ compartments are those with one or more additional DRMs in one compartment that are not detected in the other. Discordance is captured using three categories: additional mutations in compartment A only, additional mutations in compartment B only, or compartments A and B both have additional mutations not found in the other. Patients with detectable GVL were considered ‘shedders’. Viral loads below the detection limit of the assay were provided the lowest rank (≤400 copies/mL, ‘not detected’ as 0; ≤400 copies/mL, ‘detected’ as 1). The presence of any discordance between compartments, measures of genetic distances between genotypes from different compartments, and between each of them and the consensus subtype C sequence, were compared using statistical methods for correlated data. Specifically, we fitted generalized estimating equations with either mutation discordance (yes/no) or genetic distance as the outcome and compartment as the covariate, and assumed an exchangeable correlation structure among the compartments from the same woman. We used models for binomial data for discordance and normally distributed data for genetic distance. Robust standard errors were used to compute 95% CIs and P values. All analyses were performed using R version 3.2.3.59,60 Results Characteristics of enrolled women Two hundred women were enrolled according to the inclusion criteria (Figure S1, available as Supplementary data at JAC Online), with a median age of 33 years, median CD4 count of 422 cells/mm3, median ART duration of 35 months and a most common history of zidovudine/stavudine + lamivudine + nevirapine ART regimens (Table 1). Of the 200 women, 73 (37%) had PVL >400 copies/mL (assay’s lower detection limit) and 62 (31%) had PVL >1000 copies/mL (WHO treatment failure threshold). Compared with women with suppressed PVL, women with detectable PVL presented with lower CD4 counts (median of 246 versus 530 cells/mm3; P < 0.001) and were more likely on zidovudine/stavudine + lamivudine + nevirapine (48% versus 65%; P = 0.02). However, this small study was not designed to compare different regimens and we therefore did not overstate this finding. Of the 73 women, 54 (27% of total cohort) had PVL >2000 copies/mL and were invited for a follow-up second visit for collection of genital secretion samples. Women with PVL >2000 copies/mL had a lower median CD4 count than those with PVL ≤2000 copies/mL (224 versus 383; P = 0.03). Twelve of the 54 women invited for a follow-up visit failed to return. Those 12 were older than those who returned (median age 37 versus 33 years; P = 0.04) but otherwise similar. Table 1. Characteristics of 200 screened South Indian women on first-line ART, overall and stratified by PVL failure status Screened (n = 200) PVL >400 copies/mL (n = 73) PVL ≤400 copies/mL (n = 127) P Age (years) 33 (23–49) 34 (23–49) 33 (24–46) 0.39 PVL (log10 copies/mL) 0 (0–5.88) 4.59 (2.6–5.88) — — CD4 cell count (cells/mm3) 422 (15–1182) 246 (15–832) 530 (27–1182) <0.001 ART duration (months) 35 (6–122) 35 (7–114) 34 (6–122) 0.43 ART regimen  zidovudine/stavudine + lamivudine + efavirenz 51 (26%) 20 (27%) 31 (24%) 0.02  zidovudine/stavudine + lamivudine + nevirapine 118 (59%) 35 (48%) 83 (65%)  tenofovir + lamivudine/emtricitabine + efavirenz 18 (9%) 9 (12%) 9 (7%)  tenofovir + lamivudine + nevirapine 13 (6%) 9 (12%) 4 (3%) Screened (n = 200) PVL >400 copies/mL (n = 73) PVL ≤400 copies/mL (n = 127) P Age (years) 33 (23–49) 34 (23–49) 33 (24–46) 0.39 PVL (log10 copies/mL) 0 (0–5.88) 4.59 (2.6–5.88) — — CD4 cell count (cells/mm3) 422 (15–1182) 246 (15–832) 530 (27–1182) <0.001 ART duration (months) 35 (6–122) 35 (7–114) 34 (6–122) 0.43 ART regimen  zidovudine/stavudine + lamivudine + efavirenz 51 (26%) 20 (27%) 31 (24%) 0.02  zidovudine/stavudine + lamivudine + nevirapine 118 (59%) 35 (48%) 83 (65%)  tenofovir + lamivudine/emtricitabine + efavirenz 18 (9%) 9 (12%) 9 (7%)  tenofovir + lamivudine + nevirapine 13 (6%) 9 (12%) 4 (3%) Continuous measures are presented as median (range) and categorical measures are presented as n (%). Table 1. Characteristics of 200 screened South Indian women on first-line ART, overall and stratified by PVL failure status Screened (n = 200) PVL >400 copies/mL (n = 73) PVL ≤400 copies/mL (n = 127) P Age (years) 33 (23–49) 34 (23–49) 33 (24–46) 0.39 PVL (log10 copies/mL) 0 (0–5.88) 4.59 (2.6–5.88) — — CD4 cell count (cells/mm3) 422 (15–1182) 246 (15–832) 530 (27–1182) <0.001 ART duration (months) 35 (6–122) 35 (7–114) 34 (6–122) 0.43 ART regimen  zidovudine/stavudine + lamivudine + efavirenz 51 (26%) 20 (27%) 31 (24%) 0.02  zidovudine/stavudine + lamivudine + nevirapine 118 (59%) 35 (48%) 83 (65%)  tenofovir + lamivudine/emtricitabine + efavirenz 18 (9%) 9 (12%) 9 (7%)  tenofovir + lamivudine + nevirapine 13 (6%) 9 (12%) 4 (3%) Screened (n = 200) PVL >400 copies/mL (n = 73) PVL ≤400 copies/mL (n = 127) P Age (years) 33 (23–49) 34 (23–49) 33 (24–46) 0.39 PVL (log10 copies/mL) 0 (0–5.88) 4.59 (2.6–5.88) — — CD4 cell count (cells/mm3) 422 (15–1182) 246 (15–832) 530 (27–1182) <0.001 ART duration (months) 35 (6–122) 35 (7–114) 34 (6–122) 0.43 ART regimen  zidovudine/stavudine + lamivudine + efavirenz 51 (26%) 20 (27%) 31 (24%) 0.02  zidovudine/stavudine + lamivudine + nevirapine 118 (59%) 35 (48%) 83 (65%)  tenofovir + lamivudine/emtricitabine + efavirenz 18 (9%) 9 (12%) 9 (7%)  tenofovir + lamivudine + nevirapine 13 (6%) 9 (12%) 4 (3%) Continuous measures are presented as median (range) and categorical measures are presented as n (%). PVL and GVL The 42 women who attended a second visit had pelvic examinations and were negative for genital trichomoniasis, candidiasis and bacterial vaginosis. Demographic, clinical and laboratory characteristics of these 42 women are shown in Table 2; median age of 33 years, median CD4 count of 230 cells/mm3, median PVL of 5.2 log10 copies/mL and median ART duration of 34 months, most commonly zidovudine/stavudine + lamivudine + nevirapine (45%). Of the 42 women, 31 (74%) were shedders, who had significantly higher PVL (P < 0.01) and were less likely to be on a nevirapine-based ART regimen compared with non-shedders (P = 0.02) (Table 2). Table 2. Characteristics of 42 enrolled women with detectable PVL, overall and stratified by detectable and undetectable GVL, and of 21 women with available paired plasma–GT genotypes PVL >2000 copies/mL (N = 42) Women with paired genotypes (n = 21) total shedders (n = 31) non-shedders (n = 11) P Age (years) 33 (23–49) 34 (23–49) 31 (27–38) 0.28 34 (27–49) CD4 cell count (cells/mm3) 230 (51–658) 225 (55–496) 239 (51–658) 0.30 217 (55–485) PVL (log10 copies/mL) 5.17 (3.39–5.88) 5.4 (3.8–5.9) 3.88 (3.4–5.9) 0.01 5.41 (3.8–5.9) GVL (log10 copies/mL) 3.94 (0–5.88) 4.34 (2.6–5.88) — — 4.62 (3.3–5.9) ART duration (months) 33.5 (8–108) 33 (8–76) 35 (18–108) 0.15 33 (8–73) ART regimen  zidovudine/stavudine + lamivudine + efavirenz 11/42 (26%) 11/31 (35%) 0/11 (0%) 6/21 (29%)  zidovudine/stavudine + lamivudine + nevirapine 19/42 (45%) 12/31 (39%) 7/11 (64%) 0.02 10/21 (48%)  tenofovir + lamivudine/emtricitabine + efavirenz 4/42 (10%) 4/31 (13%) 0/11 (0%) 3/21 (14%)  tenofovir + lamivudine + nevirapine 8/42 (19%) 4/31 (13%) 4/11 (36%) 2/21 (10%) PVL >2000 copies/mL (N = 42) Women with paired genotypes (n = 21) total shedders (n = 31) non-shedders (n = 11) P Age (years) 33 (23–49) 34 (23–49) 31 (27–38) 0.28 34 (27–49) CD4 cell count (cells/mm3) 230 (51–658) 225 (55–496) 239 (51–658) 0.30 217 (55–485) PVL (log10 copies/mL) 5.17 (3.39–5.88) 5.4 (3.8–5.9) 3.88 (3.4–5.9) 0.01 5.41 (3.8–5.9) GVL (log10 copies/mL) 3.94 (0–5.88) 4.34 (2.6–5.88) — — 4.62 (3.3–5.9) ART duration (months) 33.5 (8–108) 33 (8–76) 35 (18–108) 0.15 33 (8–73) ART regimen  zidovudine/stavudine + lamivudine + efavirenz 11/42 (26%) 11/31 (35%) 0/11 (0%) 6/21 (29%)  zidovudine/stavudine + lamivudine + nevirapine 19/42 (45%) 12/31 (39%) 7/11 (64%) 0.02 10/21 (48%)  tenofovir + lamivudine/emtricitabine + efavirenz 4/42 (10%) 4/31 (13%) 0/11 (0%) 3/21 (14%)  tenofovir + lamivudine + nevirapine 8/42 (19%) 4/31 (13%) 4/11 (36%) 2/21 (10%) Continuous measures are presented as median (range) and categorical measures are presented as n/N (%). Table 2. Characteristics of 42 enrolled women with detectable PVL, overall and stratified by detectable and undetectable GVL, and of 21 women with available paired plasma–GT genotypes PVL >2000 copies/mL (N = 42) Women with paired genotypes (n = 21) total shedders (n = 31) non-shedders (n = 11) P Age (years) 33 (23–49) 34 (23–49) 31 (27–38) 0.28 34 (27–49) CD4 cell count (cells/mm3) 230 (51–658) 225 (55–496) 239 (51–658) 0.30 217 (55–485) PVL (log10 copies/mL) 5.17 (3.39–5.88) 5.4 (3.8–5.9) 3.88 (3.4–5.9) 0.01 5.41 (3.8–5.9) GVL (log10 copies/mL) 3.94 (0–5.88) 4.34 (2.6–5.88) — — 4.62 (3.3–5.9) ART duration (months) 33.5 (8–108) 33 (8–76) 35 (18–108) 0.15 33 (8–73) ART regimen  zidovudine/stavudine + lamivudine + efavirenz 11/42 (26%) 11/31 (35%) 0/11 (0%) 6/21 (29%)  zidovudine/stavudine + lamivudine + nevirapine 19/42 (45%) 12/31 (39%) 7/11 (64%) 0.02 10/21 (48%)  tenofovir + lamivudine/emtricitabine + efavirenz 4/42 (10%) 4/31 (13%) 0/11 (0%) 3/21 (14%)  tenofovir + lamivudine + nevirapine 8/42 (19%) 4/31 (13%) 4/11 (36%) 2/21 (10%) PVL >2000 copies/mL (N = 42) Women with paired genotypes (n = 21) total shedders (n = 31) non-shedders (n = 11) P Age (years) 33 (23–49) 34 (23–49) 31 (27–38) 0.28 34 (27–49) CD4 cell count (cells/mm3) 230 (51–658) 225 (55–496) 239 (51–658) 0.30 217 (55–485) PVL (log10 copies/mL) 5.17 (3.39–5.88) 5.4 (3.8–5.9) 3.88 (3.4–5.9) 0.01 5.41 (3.8–5.9) GVL (log10 copies/mL) 3.94 (0–5.88) 4.34 (2.6–5.88) — — 4.62 (3.3–5.9) ART duration (months) 33.5 (8–108) 33 (8–76) 35 (18–108) 0.15 33 (8–73) ART regimen  zidovudine/stavudine + lamivudine + efavirenz 11/42 (26%) 11/31 (35%) 0/11 (0%) 6/21 (29%)  zidovudine/stavudine + lamivudine + nevirapine 19/42 (45%) 12/31 (39%) 7/11 (64%) 0.02 10/21 (48%)  tenofovir + lamivudine/emtricitabine + efavirenz 4/42 (10%) 4/31 (13%) 0/11 (0%) 3/21 (14%)  tenofovir + lamivudine + nevirapine 8/42 (19%) 4/31 (13%) 4/11 (36%) 2/21 (10%) Continuous measures are presented as median (range) and categorical measures are presented as n/N (%). The Spearman’s correlation between PVL and GVL for the 42 women with PVL >2000 copies/mL was moderate (r2 = 0.50; P < 0.011), demonstrating that women with higher PVL tend to have higher GVL (Figure 1). A somewhat similar moderate correlation (r2 = 0.44) was also seen in the group of 31 shedders (P = 0.014). Figure 1. View largeDownload slide Relationship between PVL and GVL in 42 South Indian women, and plasma–GT drug resistance concordance in 21/42 women with available data. Figure 1. View largeDownload slide Relationship between PVL and GVL in 42 South Indian women, and plasma–GT drug resistance concordance in 21/42 women with available data. Drug resistance in circulating and GT RNA and proviral DNA Twenty-five of 42 women (60%) with PVL >2000 copies/mL had GVL >2000 copies/mL and had RT genotyping attempted in the three compartments. Genotypes were not available for 4/25 women; genital RNA from two women could not be amplified and plasma and genital paired sequences from two other women did not cluster together phylogenetically. Median GVL for the subset with genotype data was significantly higher than for the four without genotypes (4.6 versus 3.7 log10 copies/mL; P < 0.05). Paired plasma and genital sequences were obtained for these 21 women and PBMC genotypes were available for 20 women. All paired sequences were of good quality and clustered well phylogenetically (Figure S2). All but one (subtype A) sample were HIV-1 subtype C. Plasma drug resistance was seen in 91% (19/21), GT drug resistance was seen in 81% (17/21) and PBMC drug resistance was seen in 90% (18/20). Plasma NRTI resistance was seen in 76% (16/21), GT NRTI resistance was seen in 67% (14/21) and PBMC NRTI resistance was seen in 75% (15/20). Plasma NNRTI resistance was seen in 91% (19/21), GT NNRTI resistance was seen in 81% (17/21) and PBMC NNRTI resistance was seen in 90% (18/20). Plasma dual-class resistance was seen in 76% (16/21), GT dual-class resistance was seen in 67% (14/21) and PBMC dual-class resistance was seen in 75% (15/20) (P = not significant). The most common mutations in all three compartments included lamivudine-associated M184V and mutations at NNRTI-associated positions 101, 103 and 190. Thymidine analogue mutations (TAMs) occurred in 12/16 (75%) women on zidovudine/stavudine and K65R occurred in 1/5 (20%) women on tenofovir-based regimens and in one woman on zidovudine + lamivudine + nevirapine. Specific DRMs per patient in the three compartments are demonstrated in Table 3 and in aggregate in Figure 2. Table 3. DRMs in plasma, GT and PBMCs ID ART Plasma GT PBMCs NRTI NNRTI NRTI NNRTI NRTI NNRTI 1 3TC + d4T + NVP L74V, M184V K103N, V108I, Y181C, G190A, H221Y L74V, M184V, T215Y K103N, V108I, Y181C, G190A, H221Y L74LV, M184V, T215SY A98AG, K103N, V108IV, Y181C, G190A, H221HY 2 3TC + ZDV + EFV M184V, T215F K101E, V106M, E138A, G190A M41L, D67DN, M184V, T215F K101E, V106M, E138A, G190A M184V, T215F K101E, V106M, E138A, G190A 3 ZDV + 3TC + NVP V75IMV K103N V75IMV K103N M41L, V75I K103N, F227L 4 ZDV + 3TC + EFV M41L, E44D, D67N, T69D, K70R, V75M, M184V, L210W, T215Y A98G, K101E, G190S M41L, E44D, D67N, T69D, K70R, V75M, M184V, L210W, T215Y A98G, K101E, K103KE, G190S M41LM, E44ED, D67DN, T69ADNT, K70KR, V75IMV, M184V, L210W, T215Y A98G, K101EK, G190S 5 ZDV + 3TC + NVP E44D, D67N, T69D, M184V, T215Y A98AG, K101E, G190S E44D, D67N, T69D, M184V, T215Y A98AG, K101E, G190S, H221Y D67DN, M184MV, T215Y K101EK, K103KN, G190S 6 3TC + d4T + NVP M184V K103N, P225H none M184V K103N, P225HP 7 TDF + 3TC + NVP K65R, M184V K103N, V108I K65R, K70T, M184V K103N, V108I K65R, K70KR, M184V K103N, V108I 8 ZDV + 3TC + NVP K65KR, L74LV, M184V Y181CY, G190A, M230L L74V, M184V G190A M184MV G190AG, F227FL, M230LM 9 d4T + 3TC + NVP K70KR, M184V K101HKNQ, K103KN, Y181CY, G190AG K70R, M184V, K219E K101H, Y181C, G190A M184V, K219EK K101HKNQ, K103KN, Y181CY 10 d4T + 3TC + EFV none 11 ZDV + 3TC + EFV none V106M, F227L none K103N none V106M, F227L 12 FTC + TDF + EFV none none E138EK 13 ZDV + 3TC + NVP M184V, T215Y Y181C M184V, T215Y V106M, Y181C M184MV, T215NSTY K103KN, Y181CY 14 TDF + 3TC + NVP none K101E none K101E none K101E 15 3TC + d4T + EFV M184V V106ILM, V179DV, Y188L M184V V106ILM, Y188L M184V Y188L 16 TDF + 3TC + EFV M184V K103N, Y188L M184V K103N, Y188H M184V K103N, Y188L 17 3TC + d4T + NVP M41L, D67N, V75M, M184V, T215Y A98G, K101E, E138Q, G190A M41L, E44D, D67N, V75I, M184V A98G, K101E, E138Q, G190A M41L, D67DN, V75M, M184V, T215Y A98G, K101E, E138Q, G190A 18 TDF + 3TC + EFV none K101EK, Y188HY none 19 ZDV + 3TC + NVP D67N, K70KR, M184V Y181C D67DN, K70KR, M184MV E138AE D67DN, K70KR, M184MV Y181CY 20 ZDV + 3TC + NVP D67N, M184V A98G, K101E, G190A, P225H none Y181C D67N, K70R, M184V, K219E A98G, K101E, G190A 21 ZDV + 3TC + EFV M41L, D67N, T69D, K70R, M184V, T215Y, K219E Y188L, M230L M41L, D67N, T69D, K70R, M184V, T215Y, K219E Y188L, M230L NA ID ART Plasma GT PBMCs NRTI NNRTI NRTI NNRTI NRTI NNRTI 1 3TC + d4T + NVP L74V, M184V K103N, V108I, Y181C, G190A, H221Y L74V, M184V, T215Y K103N, V108I, Y181C, G190A, H221Y L74LV, M184V, T215SY A98AG, K103N, V108IV, Y181C, G190A, H221HY 2 3TC + ZDV + EFV M184V, T215F K101E, V106M, E138A, G190A M41L, D67DN, M184V, T215F K101E, V106M, E138A, G190A M184V, T215F K101E, V106M, E138A, G190A 3 ZDV + 3TC + NVP V75IMV K103N V75IMV K103N M41L, V75I K103N, F227L 4 ZDV + 3TC + EFV M41L, E44D, D67N, T69D, K70R, V75M, M184V, L210W, T215Y A98G, K101E, G190S M41L, E44D, D67N, T69D, K70R, V75M, M184V, L210W, T215Y A98G, K101E, K103KE, G190S M41LM, E44ED, D67DN, T69ADNT, K70KR, V75IMV, M184V, L210W, T215Y A98G, K101EK, G190S 5 ZDV + 3TC + NVP E44D, D67N, T69D, M184V, T215Y A98AG, K101E, G190S E44D, D67N, T69D, M184V, T215Y A98AG, K101E, G190S, H221Y D67DN, M184MV, T215Y K101EK, K103KN, G190S 6 3TC + d4T + NVP M184V K103N, P225H none M184V K103N, P225HP 7 TDF + 3TC + NVP K65R, M184V K103N, V108I K65R, K70T, M184V K103N, V108I K65R, K70KR, M184V K103N, V108I 8 ZDV + 3TC + NVP K65KR, L74LV, M184V Y181CY, G190A, M230L L74V, M184V G190A M184MV G190AG, F227FL, M230LM 9 d4T + 3TC + NVP K70KR, M184V K101HKNQ, K103KN, Y181CY, G190AG K70R, M184V, K219E K101H, Y181C, G190A M184V, K219EK K101HKNQ, K103KN, Y181CY 10 d4T + 3TC + EFV none 11 ZDV + 3TC + EFV none V106M, F227L none K103N none V106M, F227L 12 FTC + TDF + EFV none none E138EK 13 ZDV + 3TC + NVP M184V, T215Y Y181C M184V, T215Y V106M, Y181C M184MV, T215NSTY K103KN, Y181CY 14 TDF + 3TC + NVP none K101E none K101E none K101E 15 3TC + d4T + EFV M184V V106ILM, V179DV, Y188L M184V V106ILM, Y188L M184V Y188L 16 TDF + 3TC + EFV M184V K103N, Y188L M184V K103N, Y188H M184V K103N, Y188L 17 3TC + d4T + NVP M41L, D67N, V75M, M184V, T215Y A98G, K101E, E138Q, G190A M41L, E44D, D67N, V75I, M184V A98G, K101E, E138Q, G190A M41L, D67DN, V75M, M184V, T215Y A98G, K101E, E138Q, G190A 18 TDF + 3TC + EFV none K101EK, Y188HY none 19 ZDV + 3TC + NVP D67N, K70KR, M184V Y181C D67DN, K70KR, M184MV E138AE D67DN, K70KR, M184MV Y181CY 20 ZDV + 3TC + NVP D67N, M184V A98G, K101E, G190A, P225H none Y181C D67N, K70R, M184V, K219E A98G, K101E, G190A 21 ZDV + 3TC + EFV M41L, D67N, T69D, K70R, M184V, T215Y, K219E Y188L, M230L M41L, D67N, T69D, K70R, M184V, T215Y, K219E Y188L, M230L NA 3TC, lamivudine; d4T, stavudine; EFV, efavirenz; NVP, nevirapine; TDF, tenofovir; ZDV, zidovudine; FTC, emtricitabine; GS, genital secretion; NA, not amplified. Discordant mutations that were detected in GS or in PBMCs and not in plasma are in bold. Discordant mutations that led (alone or in combination) to clinically significant predicted resistance to at least one drug are underlined. Table 3. DRMs in plasma, GT and PBMCs ID ART Plasma GT PBMCs NRTI NNRTI NRTI NNRTI NRTI NNRTI 1 3TC + d4T + NVP L74V, M184V K103N, V108I, Y181C, G190A, H221Y L74V, M184V, T215Y K103N, V108I, Y181C, G190A, H221Y L74LV, M184V, T215SY A98AG, K103N, V108IV, Y181C, G190A, H221HY 2 3TC + ZDV + EFV M184V, T215F K101E, V106M, E138A, G190A M41L, D67DN, M184V, T215F K101E, V106M, E138A, G190A M184V, T215F K101E, V106M, E138A, G190A 3 ZDV + 3TC + NVP V75IMV K103N V75IMV K103N M41L, V75I K103N, F227L 4 ZDV + 3TC + EFV M41L, E44D, D67N, T69D, K70R, V75M, M184V, L210W, T215Y A98G, K101E, G190S M41L, E44D, D67N, T69D, K70R, V75M, M184V, L210W, T215Y A98G, K101E, K103KE, G190S M41LM, E44ED, D67DN, T69ADNT, K70KR, V75IMV, M184V, L210W, T215Y A98G, K101EK, G190S 5 ZDV + 3TC + NVP E44D, D67N, T69D, M184V, T215Y A98AG, K101E, G190S E44D, D67N, T69D, M184V, T215Y A98AG, K101E, G190S, H221Y D67DN, M184MV, T215Y K101EK, K103KN, G190S 6 3TC + d4T + NVP M184V K103N, P225H none M184V K103N, P225HP 7 TDF + 3TC + NVP K65R, M184V K103N, V108I K65R, K70T, M184V K103N, V108I K65R, K70KR, M184V K103N, V108I 8 ZDV + 3TC + NVP K65KR, L74LV, M184V Y181CY, G190A, M230L L74V, M184V G190A M184MV G190AG, F227FL, M230LM 9 d4T + 3TC + NVP K70KR, M184V K101HKNQ, K103KN, Y181CY, G190AG K70R, M184V, K219E K101H, Y181C, G190A M184V, K219EK K101HKNQ, K103KN, Y181CY 10 d4T + 3TC + EFV none 11 ZDV + 3TC + EFV none V106M, F227L none K103N none V106M, F227L 12 FTC + TDF + EFV none none E138EK 13 ZDV + 3TC + NVP M184V, T215Y Y181C M184V, T215Y V106M, Y181C M184MV, T215NSTY K103KN, Y181CY 14 TDF + 3TC + NVP none K101E none K101E none K101E 15 3TC + d4T + EFV M184V V106ILM, V179DV, Y188L M184V V106ILM, Y188L M184V Y188L 16 TDF + 3TC + EFV M184V K103N, Y188L M184V K103N, Y188H M184V K103N, Y188L 17 3TC + d4T + NVP M41L, D67N, V75M, M184V, T215Y A98G, K101E, E138Q, G190A M41L, E44D, D67N, V75I, M184V A98G, K101E, E138Q, G190A M41L, D67DN, V75M, M184V, T215Y A98G, K101E, E138Q, G190A 18 TDF + 3TC + EFV none K101EK, Y188HY none 19 ZDV + 3TC + NVP D67N, K70KR, M184V Y181C D67DN, K70KR, M184MV E138AE D67DN, K70KR, M184MV Y181CY 20 ZDV + 3TC + NVP D67N, M184V A98G, K101E, G190A, P225H none Y181C D67N, K70R, M184V, K219E A98G, K101E, G190A 21 ZDV + 3TC + EFV M41L, D67N, T69D, K70R, M184V, T215Y, K219E Y188L, M230L M41L, D67N, T69D, K70R, M184V, T215Y, K219E Y188L, M230L NA ID ART Plasma GT PBMCs NRTI NNRTI NRTI NNRTI NRTI NNRTI 1 3TC + d4T + NVP L74V, M184V K103N, V108I, Y181C, G190A, H221Y L74V, M184V, T215Y K103N, V108I, Y181C, G190A, H221Y L74LV, M184V, T215SY A98AG, K103N, V108IV, Y181C, G190A, H221HY 2 3TC + ZDV + EFV M184V, T215F K101E, V106M, E138A, G190A M41L, D67DN, M184V, T215F K101E, V106M, E138A, G190A M184V, T215F K101E, V106M, E138A, G190A 3 ZDV + 3TC + NVP V75IMV K103N V75IMV K103N M41L, V75I K103N, F227L 4 ZDV + 3TC + EFV M41L, E44D, D67N, T69D, K70R, V75M, M184V, L210W, T215Y A98G, K101E, G190S M41L, E44D, D67N, T69D, K70R, V75M, M184V, L210W, T215Y A98G, K101E, K103KE, G190S M41LM, E44ED, D67DN, T69ADNT, K70KR, V75IMV, M184V, L210W, T215Y A98G, K101EK, G190S 5 ZDV + 3TC + NVP E44D, D67N, T69D, M184V, T215Y A98AG, K101E, G190S E44D, D67N, T69D, M184V, T215Y A98AG, K101E, G190S, H221Y D67DN, M184MV, T215Y K101EK, K103KN, G190S 6 3TC + d4T + NVP M184V K103N, P225H none M184V K103N, P225HP 7 TDF + 3TC + NVP K65R, M184V K103N, V108I K65R, K70T, M184V K103N, V108I K65R, K70KR, M184V K103N, V108I 8 ZDV + 3TC + NVP K65KR, L74LV, M184V Y181CY, G190A, M230L L74V, M184V G190A M184MV G190AG, F227FL, M230LM 9 d4T + 3TC + NVP K70KR, M184V K101HKNQ, K103KN, Y181CY, G190AG K70R, M184V, K219E K101H, Y181C, G190A M184V, K219EK K101HKNQ, K103KN, Y181CY 10 d4T + 3TC + EFV none 11 ZDV + 3TC + EFV none V106M, F227L none K103N none V106M, F227L 12 FTC + TDF + EFV none none E138EK 13 ZDV + 3TC + NVP M184V, T215Y Y181C M184V, T215Y V106M, Y181C M184MV, T215NSTY K103KN, Y181CY 14 TDF + 3TC + NVP none K101E none K101E none K101E 15 3TC + d4T + EFV M184V V106ILM, V179DV, Y188L M184V V106ILM, Y188L M184V Y188L 16 TDF + 3TC + EFV M184V K103N, Y188L M184V K103N, Y188H M184V K103N, Y188L 17 3TC + d4T + NVP M41L, D67N, V75M, M184V, T215Y A98G, K101E, E138Q, G190A M41L, E44D, D67N, V75I, M184V A98G, K101E, E138Q, G190A M41L, D67DN, V75M, M184V, T215Y A98G, K101E, E138Q, G190A 18 TDF + 3TC + EFV none K101EK, Y188HY none 19 ZDV + 3TC + NVP D67N, K70KR, M184V Y181C D67DN, K70KR, M184MV E138AE D67DN, K70KR, M184MV Y181CY 20 ZDV + 3TC + NVP D67N, M184V A98G, K101E, G190A, P225H none Y181C D67N, K70R, M184V, K219E A98G, K101E, G190A 21 ZDV + 3TC + EFV M41L, D67N, T69D, K70R, M184V, T215Y, K219E Y188L, M230L M41L, D67N, T69D, K70R, M184V, T215Y, K219E Y188L, M230L NA 3TC, lamivudine; d4T, stavudine; EFV, efavirenz; NVP, nevirapine; TDF, tenofovir; ZDV, zidovudine; FTC, emtricitabine; GS, genital secretion; NA, not amplified. Discordant mutations that were detected in GS or in PBMCs and not in plasma are in bold. Discordant mutations that led (alone or in combination) to clinically significant predicted resistance to at least one drug are underlined. Figure 2. View largeDownload slide Prevalence of DRMs in plasma, GT and PBMCs. Figure 2. View largeDownload slide Prevalence of DRMs in plasma, GT and PBMCs. Drug resistance discordance Complete DRM concordance in all three compartments occurred in only 2/20 (10%) women for which sequences from all compartments were available, one of whom had no DRMs; 5/21 (24%) between plasma and GT, 8/20 (40%) between plasma and PBMCs and 3/20 (15%) between GT and PBMCs (Table 3). Odds of any discordance between GT and PBMCs was significantly larger than any discordance between plasma and PBMCs (OR = 5.35; 95% CI = 1.09–26.18; P = 0.04). The odds of discordance between plasma and GT was also larger than between plasma and PBMCs, but not significantly so (OR = 2.56; 95% CI = 0.46–14.10; P = 0.28). Of the 16 women with plasma–GT discordance, 12 (75%) had, overall, 14 additional DRMs in GT not detected in plasma (bold in Table 3). Of these, eight discordant mutations (underlined in Table 3) led to clinically relevant higher predicted resistance in GT versus plasma to at least one drug in 5/21 (24%) women. Of the 12 women with plasma–PBMC discordance, 10 (83%) had, overall, 15 DRMs in PBMCs not detected in plasma (bold in Table 3). Of these, 12 discordant mutations (underlined in Table 3) led to clinically relevant higher predicted resistance in PBMCs than plasma to at least one drug in 6/20 (30%) women. There was no statistically significant difference in genital shedding between the plasma–GT concordant and discordant groups, which may be due to small sample sizes (Figure 1). However, women with concordant mutations between the two compartments (black circles in Figure 1) did appear in the higher ends of both PVL and GVL. Table S1 demonstrates characteristics of women according to sequence concordance among compartments. While not statistically significant, higher plasma–PBMC discordance was seen in women with longer time on ART, and among all compartments with zidovudine/stavudine + lamivudine + nevirapine regimens. Genetic distances Genetic distances between pairs of sequences from the three compartments were similar overall (mean 1.78% plasma–GT, 2.01% plasma–PBMCs and 2.27% PBMC–GT). On average, compared with plasma–GT, plasma–PBMC distances were 0.22 units higher (95% CI = −0.38–0.81; P = 0.471) and PBMC–GT distances were 0.48 units higher (95% CI = −0.18–1.14; P = 0.155). Models comparing genetic distances between each compartment and the subtype C reference sequence showed that on average, compared with PBMCs (mean 8.27%), genetic distances of GT sequences were 8.98% (0.77 units higher; 95% CI = 0.16–1.37; P = 0.013) and genetic distances of plasma sequences were 9.01% (0.80 units higher; 95% CI = 0.30–1.29; P = 0.002). Discussion We investigated the compartmentalization of HIV-1 in plasma, GT and PBMCs and determined virological failure, GT viral shedding and inter-compartmental drug resistance concordance in HIV-1 subtype C-infected South Indian women on first-line ART. We found high resistance levels and, supporting our hypotheses, we demonstrated a fair, not perfect, plasma–genital viral load concordance, and high drug resistance discordance among the three compartments, with 71% (15/21) of women having DRMs in genital and/or PBMCs that were not detected in plasma, which might impact clinical care. Depending on the threshold, virological failure was detected in 31%–37% of women and, in line with previous reports, was associated with lower CD4 counts61 and higher use of nevirapine-based regimens. Consistent with the literature,27,48,62,63 74% of women with PVL >2000 copies/mL were genital shedders with a moderate but significant PVL–GVL concordance. This finding is supportive of PVL serving as a surrogate marker for genital shedding.15,64 Though we did not quantitate GVL in women with undetectable PVL, which might have underestimated genital shedding, this finding supports previous studies.13,65,66 Suppression of GT viral replication is essential to prevent evolution and transmission of resistance, and would have an epidemiological impact in places like India, where sexual contact is the main transmission cause. To prevent compartmentalization of HIV replication in the GT, 14,31,67,68 all ART components should reach adequate concentrations in that compartment. Though further such studies are needed,65 some suggest that failure to suppress PVL is the main determinant of GT shedding.69–72 As our study participants were more immunocompromised and had higher GVL compared with other studies,50 the risk of vertical and horizontal resistance transmission is presumably greater.73 High, fairly similar, levels of resistance (any in 81%–91%; dual-class in 67%–76%) were detected in all compartments, but with high discordance: only 10% of women had full DRM pattern concordance among the three compartments, with slightly higher rates between plasma and GT (24%) and between plasma and PBMCs (40%). These results are consistent with previous findings, implying the potential inadequacy of plasma genotyping as a representative for other anatomical sites.65 Moreover, these discordant mutations led to clinically relevant GT resistance that was not detected in plasma in 24% of women, and in PBMCs in 30%. These differences may suggest distinct viral evolution, possibly attributed to variable drug penetration/concentration, not measured here,14,74–76 but could also be the consequence of sampling bias between compartments, associated for example with different viral loads.77 Additionally, ART reduces viral load in plasma and the GT but does not diminish proviral DNA,78 which, even in the absence of detectable plasma RNA, can promote both perinatal and heterosexual resistance transmission.66,79 Observed DRM patterns were, overall, as expected,80 with lamivudine-associated M184V most frequently observed in all three compartments. Reported good GT penetration of lamivudine64,75 may explain our observed decreased frequency of M184V in that compartment compared with plasma (62% versus 71%). Regarding zidovudine/stavudine-associated TAMs, previous reports suggest that zidovudine can penetrate the GT in equal or higher concentrations than the blood plasma, whereas stavudine concentrations are much lower in the GT.75 In our participants, GT TAM prevalence was slightly higher (48%) compared with plasma (43%) despite zidovudine use in 5/9 women with TAMs in genital secretions. Drug concentration measurements in these compartments is necessary to substantiate the occurrence of DRMs as a result of decreased drug penetration in the GT. Regarding NNRTIs, the predominance of efavirenz/nevirapine-associated G190A was observed, confirming its fitness advantage on drug pressure, despite its relatively low-level resistance.81–83 However, G190A confers intermediate-level resistance to etravirine in synergism with Y181C, a concerning combination found in plasma (14%), PBMCs (5%) and genital secretions (10%), that could interfere with its use in third-line regimens.84 Similar concerns were observed in 10% (plasma), 15% (PBMCs) and 14% (genital secretions) of women with E138A/K/Q, conferring resistance to rilpivirine.85 Genotyping of proviral DNA can shed light on archived viruses and resistance to previous drugs which might impact resistance evolution.85–88 Indeed, genetic distances between PBMCs and the consensus sequence were lower than other compartments suggesting earlier sequence archival. Higher genetic distances between compartments shows that observed discordances were caused by differential evolution as a consequence of compartment-specific genetic differentiation among anatomically separate viral populations. DRMs in PBMCs that are not detected in plasma have been previously observed and may be transmitted or re-emerge upon ART to which they provide selective advantage.35,73–75 Increased HIV transmission risk is seen with higher genital HIV-1 RNA and our data suggest that resistance mutation accumulation in women with undetected virological failure could lead to increased risk of transmission of resistant HIV-1 variants.89 The main limitations of this study are its small sample size and cross-sectional design, restricting our ability to strongly address hypotheses. In addition, plasma and GT drug concentrations were not measured, which might explain resistance discordances; GVLs for women with suppressed PVLs and pre-treatment resistance were unavailable; drug resistance testing was population-based; Gram stain was not done to diagnose bacterial vaginosis, only wet mount was used to diagnose sexually transmitted infections, molecular-based testing was not done for trichomoniasis and herpes, and syphilis testing was also not done. Lastly, though tenofovir-based first-line regimens are currently preferred, zidovudine/stavudine-based regimens, more commonly reported here, are still in use in India as alternative regimens, making results relevant. In conclusion, GT viral shedding and archival, high-level resistance and discordance across compartments with genital/proviral DRMs not detected in plasma of South Indian women on first-line ART may result in their hidden transmission and/or re-emergence, which is likely to have a negative impact on treatment outcome. Whether such high resistance levels and discordances would be minimized by routine viral load monitoring, whether they should lead to incorporation of plasma/proviral genotyping or whether they suggest the need to monitor GVL and genital resistance to reduce the risk of ART failure and resistance acquisition and transmission, remains to be determined. Acknowledgements Part of this work was presented as a poster at the Twentieth International AIDS Conference, Melbourne, Australia, 2014 (Poster no. MOPDA0106). We are most grateful to the clinical and laboratory staff at YRG-CARE, VHS, Chennai, India, for their facilitation of the study. Funding Funding was provided by: the Indian Council for Medical Research (ICMR), New Delhi, India; the Providence/Boston Center for AIDS Research (CFAR) (P30AI042853); RO1AI108441; and the Brown Tufts AIDS International Training and Research Program (AITRP; D43TW000237). Transparency declarations None to declare. Supplementary data Figures S1 and S2 and Table S1 are available as Supplementary data at JAC Online. References 1 Cu-Uvin S , Caliendo AM , Reinert S et al. Effect of highly active antiretroviral therapy on cervicovaginal HIV-1 RNA . AIDS 2000 ; 14 : 415 – 21 . Google Scholar CrossRef Search ADS PubMed 2 Graham SM , Holte SE , Peshu NM et al. Initiation of antiretroviral therapy leads to a rapid decline in cervical and vaginal HIV-1 shedding . AIDS 2007 ; 21 : 501 – 7 . Google Scholar CrossRef Search ADS PubMed 3 Chun TW , Engel D , Mizell SB. Induction of HIV-1 replication in latently infected CD4+ T cells using a combination of cytokines . J Exp Med 1998 ; 188 : 83 – 91 . 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Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Antimicrobial Chemotherapy Oxford University Press

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Abstract

Abstract Objectives Examine HIV-1 plasma viral load (PVL) and genital tract (GT) viral load (GVL) and drug resistance in India. Methods At the YRG Centre for AIDS Research and Education, Chennai, we tested: PVL in women on first-line ART for ≥6 months; GVL when PVL >2000 copies/mL; and plasma, genital and proviral reverse transcriptase drug resistance when GVL >2000 copies/mL. Wilcoxon rank-sum and Fisher's exact tests were used to identify failure and resistance associations. Pearson correlations were calculated to evaluate PVL–GVL associations. Inter-compartmental resistance discordance was evaluated using generalized estimating equations. Results Of 200 women, 37% had detectable (>400 copies/mL) PVL and 31% had PVL >1000 copies/mL. Of women with detectable PVL, 74% had PVL >2000 copies/mL, of which 74% had detectable GVL. Higher PVL was associated with higher GVL. Paired plasma and genital sequences were available for 21 women; mean age of 34 years, median ART duration of 33 months, median CD4 count of 217 cells/mm3, median PVL of 5.4 log10 copies/mL and median GVL of 4.6 log10 copies/mL. Drug resistance was detected in 81%–91% of samples and 67%–76% of samples had dual-class resistance. Complete three-compartment concordance was seen in only 10% of women. GT–proviral discordance was significantly larger than plasma–proviral discordance. GT or proviral mutations discordant from plasma led to clinically relevant resistance in 24% and 30%, respectively. Conclusions We identified high resistance and high inter-compartmental resistance discordance in Indian women, which might lead to unrecognized resistance transmission and re-emergence compromising treatment outcomes, particularly relevant to countries like India, where sexual HIV transmission is predominant. Introduction ART is changing the dynamics of the HIV epidemic through suppression of plasma viral load (PVL) and genital tract (GT) viral load (GVL).1–5 Despite viral suppression in plasma, replication can occur in the GT increasing horizontal and vertical transmission risks.6–14 Such compartmentalization and differential evolution between plasma and the GT can be attributed to differences in CD4 or co-receptor expression,15–18 HIV-specific immune pressure,15,19–24 poor genital ART penetration causing selective pressure,25 inflammation from trauma26 or sexually transmitted infections.15,17,20,21,27–31 Resulting tissue-specific lineages can increase the risk of unrecognized drug resistance and tropism,6–12 suggesting compartment-specific HIV replication milieu. With increasing evidence for beneficial early ART to reduce HIV transmission,16,32,33 data on prevalence and correlates of viral replication and of drug resistance mutations (DRMs) in circulating and archived viruses and in the GT among treated individuals are needed, particularly in globally predominant HIV-1 subtypes.34 These measures impact long-term ART management and resistance transmission and can guide development of novel prevention interventions.35–38 Genotyping of circulating plasma virus is the current standard for clinical management before and upon ART failure; however, it may underestimate resistance in settings like treatment interruptions or compromised adherence.39–42 Though clinical significance of DRMs that are archived in cellular reservoirs remains controversial,39–43 proviral genotyping may further inform treatment decisions. Genotyping of GT viruses is uncommon and three-compartment drug resistance concordance and their impacts are unknown. PVL and GVL are higher for the globally predominant HIV-1 subtype C.43–50 In a developing country like India, where subtype C predominates and heterosexual activity is the major mode of transmission, investigating viral shedding and GT resistance upon treatment failure and its discordance from circulating and archived viruses is important, as it carries implications for resistance transmission and clinical outcome. Under the hypothesis of an existing inter-compartmental discordance in viral load and clinically relevant resistance, we examined PVL and GVL, as well as plasma, proviral and genital virus resistance, in South Indian women on first-line ART. Methods Study setting Women were enrolled at the YRG Centre for AIDS Research and Education (YRG-CARE) clinic in Chennai, the largest community-based tertiary HIV care institution in India, with >20 000 registered patients. During the study period (May 2009 to November 2011), 39% of clinic patients were women and 60% were on ART, attending clinic every 3 months. Treatment monitoring was based on WHO immunological and clinical criteria, without virological monitoring. HIV-1 infected women were offered enrolment if they were: (i) ≥18 years old; (ii) on first-line ART (zidovudine/stavudine + lamivudine/emtricitabine + nevirapine/efavirenz, the recommended regimens at the time of the study) for >6 months; (iii) reporting >95% adherence in the past month; (iv) without pelvic surgery in the past 6 months; (v) not pregnant; and (vi) not currently menstruating. Enrolled women had a screening visit during which demographics were collected from interview and chart, including age, WHO stage, adherence in the past month, history of pelvic/cervical surgeries, reproductive history, last menstrual period, CD4 count and ART history. Upon enrolment, PVL was tested and women with PVL >2000 copies/mL were invited for a follow-up visit within 1 month, 1 week prior to their next estimated menstrual period. This PVL threshold was chosen to increase the yield of plasma and genital genotyping in this pilot study. Participants were instructed not to have sexual intercourse, douche or insert any vaginal products for ≤48 h before the second visit. On that visit, additional history was derived, including history of sexually transmitted diseases and use of contraceptives (e.g. oral contraceptive pills, hormonal contraceptive dermal implant, depomedroxyprogesterone acetate injection or intrauterine device); an additional 10 mL of blood was collected, PBMCs were isolated51 and stored at −75 ± 5°C, and genital secretions were collected (as specified below). Ethics The study was approved by Lifespan and YRG-CARE Institutional Review Boards. All participants provided written informed consent. Genital specimen collection and processing During the second visit, pelvic examination was performed to collect endocervical secretions, cervical vaginal lavage (CVL) and vaginal swab samples. Endocervical secretions were collected using four wicks (Tear FloTM), held in place for a 1–3 min wicking period, then removed and cut at the wick neck. Each pair of wicks was placed in a vial containing 500 μL of nucleic acid sequence-based amplification (NASBA) buffer (bioMérieux, Durham, NC, USA) and stored at −75 ± 5°C. CVL samples were collected by bathing the cervix and ectocervix three times with the same 10 mL of PBS, which was then aspirated and placed into a sterile 50 mL conical centrifuge tube. Cervical samples were examined under the microscope for red blood cells and spermatozoa. Genital specimens were aliquotted within 4 h of collection and stored at −75 ± 5°C. Four vaginal swabs were collected prior to CVL collection and wet mount specimens were prepared to diagnose trichomoniasis, candidiasis and bacterial vaginosis. HIV genotyping from plasma, genital Tear FloTM and PBMCs was performed for all women with GVL >2000 copies/mL (to ensure high genotyping yield), who had negative results for co-infection with trichomoniasis, candidiasis and bacterial vaginosis (which may distort results). Laboratory methods All testing was performed at the YRG-CARE laboratory, accredited by the National Accredited Board of Laboratories (ISO 15189:2012). Plasma was separated from blood within 4–6 h of sampling and stored at −75±5°C. CD4 testing was done as part of routine care using a two-colour single-platform flow cytometer, FACS Count (Becton Dickson Immunocytometry Systems, San Jose, USA). PVL was done with the Roche COBAS Amplicor HIV-1 Monitor test, version 1.5 (Roche Diagnostics, Branchburg, USA) as per the manufacturer’s protocol with a 400 copies/mL lower detection limit. The same protocol was applied for GVL with minor modifications: Tear FloTM filter paper wicks were cut and placed in 600 μL of lysis buffer with a quantification standard and incubated at room temperature on a shaker for 45 min at 800 rpm, followed by addition of 600 μL of 100% isopropanol to the supernatant and proceeding as per the manufacturer’s protocol.52 The same 400 copies/mL lower detection limit was used. Reverse transcriptase (RT; amino acid positions 1–230) sequencing from plasma and GT was done as described.53,54 Briefly, RNA was extracted from plasma using the QIAamp viral RNA extraction kit (QIAGEN, Valencia, CA, USA) as per the manufacturer’s protocol. Genital RNA was extracted from Tear FloTM using the Roche COBAS Amplicor, and used for quantification and sequencing. Plasma and genital RNA were then reverse transcribed to cDNA. The RT fragment was amplified from cDNA by nested PCR with RT primers and analysed on 1.5% agarose gel. DNA was extracted from PBMCs using a QIAamp Blood Mini Kit (QIAGEN) as per the manufacturer’s instructions and stored at −75 ± 5°C. Amplification was performed by nested PCR and analysed on 1.5% agarose gel. Amplicons were then column-purified and sequenced with an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems, Foster City, USA) with an ABI sequence analyzing software version 3.7. Sequence analysis Sequences were interpreted for resistance using the Stanford HIV Database tools and clinically significant resistance was defined as intermediate or high levels to ≥1 drug according to the Stanford penalty rules.55 Phylogenetic analyses were performed with Mega version 6.056 by maximum likelihood [1000 bootstraps; HKY model with gamma distribution based on FindModel (http://www.hiv.lanl.gov)]. Sequence quality control and distance estimation were with SQUAT57 and subtyping with REGA.56,58 Genetic distances were estimated to identify the level of evolution between the three compartments and from each compartment to a subtype C consensus. The latter, created from 113 YRG-CARE subtype C sequences from treatment-naive patients available from 2007 to 2011, was used as the ‘reference’. Such analyses are relevant for the understanding of resistance archival and its potential impact on resistance evolution, and of compartmental resistance discordance. Statistical analysis We hypothesized that among women with detectable PVL: (i) some will have suppressed GVL; (ii) some will have clinically relevant DRMs detected in genital samples and/or PBMCs but not in plasma; and (iii) demographic and clinical characteristics will differ according to viral load and DRMs. To evaluate these hypotheses, we compared demographic and clinical measures between several subgroups: (i) women with versus without detectable PVL; (ii) women with detectable PVL ≤2000 versus those with >2000 copies/mL; (iii) women with detectable versus undetectable GVL, among those with PVL >2000 copies/mL; and (iv) women with versus without GT DRMs. Wilcoxon rank-sum tests were used to compare continuous measures (age, CD4 count, months on ART, PVL and GVL, where applicable) and Fisher's exact test for categorical measures (ART regimen). Pearson correlations were calculated to evaluate the strength of relationship between log10-transformed viral load in the plasma and GT. For comparison of drug resistance concordance, for each compartment comparison (A versus B; e.g. plasma versus GT, GT versus PBMCs and plasma versus PBMCs), patients were classified as ‘concordant’ or ‘discordant’. ‘Concordant’ compartments are those with identical mutation patterns and ‘discordant’ compartments are those with one or more additional DRMs in one compartment that are not detected in the other. Discordance is captured using three categories: additional mutations in compartment A only, additional mutations in compartment B only, or compartments A and B both have additional mutations not found in the other. Patients with detectable GVL were considered ‘shedders’. Viral loads below the detection limit of the assay were provided the lowest rank (≤400 copies/mL, ‘not detected’ as 0; ≤400 copies/mL, ‘detected’ as 1). The presence of any discordance between compartments, measures of genetic distances between genotypes from different compartments, and between each of them and the consensus subtype C sequence, were compared using statistical methods for correlated data. Specifically, we fitted generalized estimating equations with either mutation discordance (yes/no) or genetic distance as the outcome and compartment as the covariate, and assumed an exchangeable correlation structure among the compartments from the same woman. We used models for binomial data for discordance and normally distributed data for genetic distance. Robust standard errors were used to compute 95% CIs and P values. All analyses were performed using R version 3.2.3.59,60 Results Characteristics of enrolled women Two hundred women were enrolled according to the inclusion criteria (Figure S1, available as Supplementary data at JAC Online), with a median age of 33 years, median CD4 count of 422 cells/mm3, median ART duration of 35 months and a most common history of zidovudine/stavudine + lamivudine + nevirapine ART regimens (Table 1). Of the 200 women, 73 (37%) had PVL >400 copies/mL (assay’s lower detection limit) and 62 (31%) had PVL >1000 copies/mL (WHO treatment failure threshold). Compared with women with suppressed PVL, women with detectable PVL presented with lower CD4 counts (median of 246 versus 530 cells/mm3; P < 0.001) and were more likely on zidovudine/stavudine + lamivudine + nevirapine (48% versus 65%; P = 0.02). However, this small study was not designed to compare different regimens and we therefore did not overstate this finding. Of the 73 women, 54 (27% of total cohort) had PVL >2000 copies/mL and were invited for a follow-up second visit for collection of genital secretion samples. Women with PVL >2000 copies/mL had a lower median CD4 count than those with PVL ≤2000 copies/mL (224 versus 383; P = 0.03). Twelve of the 54 women invited for a follow-up visit failed to return. Those 12 were older than those who returned (median age 37 versus 33 years; P = 0.04) but otherwise similar. Table 1. Characteristics of 200 screened South Indian women on first-line ART, overall and stratified by PVL failure status Screened (n = 200) PVL >400 copies/mL (n = 73) PVL ≤400 copies/mL (n = 127) P Age (years) 33 (23–49) 34 (23–49) 33 (24–46) 0.39 PVL (log10 copies/mL) 0 (0–5.88) 4.59 (2.6–5.88) — — CD4 cell count (cells/mm3) 422 (15–1182) 246 (15–832) 530 (27–1182) <0.001 ART duration (months) 35 (6–122) 35 (7–114) 34 (6–122) 0.43 ART regimen  zidovudine/stavudine + lamivudine + efavirenz 51 (26%) 20 (27%) 31 (24%) 0.02  zidovudine/stavudine + lamivudine + nevirapine 118 (59%) 35 (48%) 83 (65%)  tenofovir + lamivudine/emtricitabine + efavirenz 18 (9%) 9 (12%) 9 (7%)  tenofovir + lamivudine + nevirapine 13 (6%) 9 (12%) 4 (3%) Screened (n = 200) PVL >400 copies/mL (n = 73) PVL ≤400 copies/mL (n = 127) P Age (years) 33 (23–49) 34 (23–49) 33 (24–46) 0.39 PVL (log10 copies/mL) 0 (0–5.88) 4.59 (2.6–5.88) — — CD4 cell count (cells/mm3) 422 (15–1182) 246 (15–832) 530 (27–1182) <0.001 ART duration (months) 35 (6–122) 35 (7–114) 34 (6–122) 0.43 ART regimen  zidovudine/stavudine + lamivudine + efavirenz 51 (26%) 20 (27%) 31 (24%) 0.02  zidovudine/stavudine + lamivudine + nevirapine 118 (59%) 35 (48%) 83 (65%)  tenofovir + lamivudine/emtricitabine + efavirenz 18 (9%) 9 (12%) 9 (7%)  tenofovir + lamivudine + nevirapine 13 (6%) 9 (12%) 4 (3%) Continuous measures are presented as median (range) and categorical measures are presented as n (%). Table 1. Characteristics of 200 screened South Indian women on first-line ART, overall and stratified by PVL failure status Screened (n = 200) PVL >400 copies/mL (n = 73) PVL ≤400 copies/mL (n = 127) P Age (years) 33 (23–49) 34 (23–49) 33 (24–46) 0.39 PVL (log10 copies/mL) 0 (0–5.88) 4.59 (2.6–5.88) — — CD4 cell count (cells/mm3) 422 (15–1182) 246 (15–832) 530 (27–1182) <0.001 ART duration (months) 35 (6–122) 35 (7–114) 34 (6–122) 0.43 ART regimen  zidovudine/stavudine + lamivudine + efavirenz 51 (26%) 20 (27%) 31 (24%) 0.02  zidovudine/stavudine + lamivudine + nevirapine 118 (59%) 35 (48%) 83 (65%)  tenofovir + lamivudine/emtricitabine + efavirenz 18 (9%) 9 (12%) 9 (7%)  tenofovir + lamivudine + nevirapine 13 (6%) 9 (12%) 4 (3%) Screened (n = 200) PVL >400 copies/mL (n = 73) PVL ≤400 copies/mL (n = 127) P Age (years) 33 (23–49) 34 (23–49) 33 (24–46) 0.39 PVL (log10 copies/mL) 0 (0–5.88) 4.59 (2.6–5.88) — — CD4 cell count (cells/mm3) 422 (15–1182) 246 (15–832) 530 (27–1182) <0.001 ART duration (months) 35 (6–122) 35 (7–114) 34 (6–122) 0.43 ART regimen  zidovudine/stavudine + lamivudine + efavirenz 51 (26%) 20 (27%) 31 (24%) 0.02  zidovudine/stavudine + lamivudine + nevirapine 118 (59%) 35 (48%) 83 (65%)  tenofovir + lamivudine/emtricitabine + efavirenz 18 (9%) 9 (12%) 9 (7%)  tenofovir + lamivudine + nevirapine 13 (6%) 9 (12%) 4 (3%) Continuous measures are presented as median (range) and categorical measures are presented as n (%). PVL and GVL The 42 women who attended a second visit had pelvic examinations and were negative for genital trichomoniasis, candidiasis and bacterial vaginosis. Demographic, clinical and laboratory characteristics of these 42 women are shown in Table 2; median age of 33 years, median CD4 count of 230 cells/mm3, median PVL of 5.2 log10 copies/mL and median ART duration of 34 months, most commonly zidovudine/stavudine + lamivudine + nevirapine (45%). Of the 42 women, 31 (74%) were shedders, who had significantly higher PVL (P < 0.01) and were less likely to be on a nevirapine-based ART regimen compared with non-shedders (P = 0.02) (Table 2). Table 2. Characteristics of 42 enrolled women with detectable PVL, overall and stratified by detectable and undetectable GVL, and of 21 women with available paired plasma–GT genotypes PVL >2000 copies/mL (N = 42) Women with paired genotypes (n = 21) total shedders (n = 31) non-shedders (n = 11) P Age (years) 33 (23–49) 34 (23–49) 31 (27–38) 0.28 34 (27–49) CD4 cell count (cells/mm3) 230 (51–658) 225 (55–496) 239 (51–658) 0.30 217 (55–485) PVL (log10 copies/mL) 5.17 (3.39–5.88) 5.4 (3.8–5.9) 3.88 (3.4–5.9) 0.01 5.41 (3.8–5.9) GVL (log10 copies/mL) 3.94 (0–5.88) 4.34 (2.6–5.88) — — 4.62 (3.3–5.9) ART duration (months) 33.5 (8–108) 33 (8–76) 35 (18–108) 0.15 33 (8–73) ART regimen  zidovudine/stavudine + lamivudine + efavirenz 11/42 (26%) 11/31 (35%) 0/11 (0%) 6/21 (29%)  zidovudine/stavudine + lamivudine + nevirapine 19/42 (45%) 12/31 (39%) 7/11 (64%) 0.02 10/21 (48%)  tenofovir + lamivudine/emtricitabine + efavirenz 4/42 (10%) 4/31 (13%) 0/11 (0%) 3/21 (14%)  tenofovir + lamivudine + nevirapine 8/42 (19%) 4/31 (13%) 4/11 (36%) 2/21 (10%) PVL >2000 copies/mL (N = 42) Women with paired genotypes (n = 21) total shedders (n = 31) non-shedders (n = 11) P Age (years) 33 (23–49) 34 (23–49) 31 (27–38) 0.28 34 (27–49) CD4 cell count (cells/mm3) 230 (51–658) 225 (55–496) 239 (51–658) 0.30 217 (55–485) PVL (log10 copies/mL) 5.17 (3.39–5.88) 5.4 (3.8–5.9) 3.88 (3.4–5.9) 0.01 5.41 (3.8–5.9) GVL (log10 copies/mL) 3.94 (0–5.88) 4.34 (2.6–5.88) — — 4.62 (3.3–5.9) ART duration (months) 33.5 (8–108) 33 (8–76) 35 (18–108) 0.15 33 (8–73) ART regimen  zidovudine/stavudine + lamivudine + efavirenz 11/42 (26%) 11/31 (35%) 0/11 (0%) 6/21 (29%)  zidovudine/stavudine + lamivudine + nevirapine 19/42 (45%) 12/31 (39%) 7/11 (64%) 0.02 10/21 (48%)  tenofovir + lamivudine/emtricitabine + efavirenz 4/42 (10%) 4/31 (13%) 0/11 (0%) 3/21 (14%)  tenofovir + lamivudine + nevirapine 8/42 (19%) 4/31 (13%) 4/11 (36%) 2/21 (10%) Continuous measures are presented as median (range) and categorical measures are presented as n/N (%). Table 2. Characteristics of 42 enrolled women with detectable PVL, overall and stratified by detectable and undetectable GVL, and of 21 women with available paired plasma–GT genotypes PVL >2000 copies/mL (N = 42) Women with paired genotypes (n = 21) total shedders (n = 31) non-shedders (n = 11) P Age (years) 33 (23–49) 34 (23–49) 31 (27–38) 0.28 34 (27–49) CD4 cell count (cells/mm3) 230 (51–658) 225 (55–496) 239 (51–658) 0.30 217 (55–485) PVL (log10 copies/mL) 5.17 (3.39–5.88) 5.4 (3.8–5.9) 3.88 (3.4–5.9) 0.01 5.41 (3.8–5.9) GVL (log10 copies/mL) 3.94 (0–5.88) 4.34 (2.6–5.88) — — 4.62 (3.3–5.9) ART duration (months) 33.5 (8–108) 33 (8–76) 35 (18–108) 0.15 33 (8–73) ART regimen  zidovudine/stavudine + lamivudine + efavirenz 11/42 (26%) 11/31 (35%) 0/11 (0%) 6/21 (29%)  zidovudine/stavudine + lamivudine + nevirapine 19/42 (45%) 12/31 (39%) 7/11 (64%) 0.02 10/21 (48%)  tenofovir + lamivudine/emtricitabine + efavirenz 4/42 (10%) 4/31 (13%) 0/11 (0%) 3/21 (14%)  tenofovir + lamivudine + nevirapine 8/42 (19%) 4/31 (13%) 4/11 (36%) 2/21 (10%) PVL >2000 copies/mL (N = 42) Women with paired genotypes (n = 21) total shedders (n = 31) non-shedders (n = 11) P Age (years) 33 (23–49) 34 (23–49) 31 (27–38) 0.28 34 (27–49) CD4 cell count (cells/mm3) 230 (51–658) 225 (55–496) 239 (51–658) 0.30 217 (55–485) PVL (log10 copies/mL) 5.17 (3.39–5.88) 5.4 (3.8–5.9) 3.88 (3.4–5.9) 0.01 5.41 (3.8–5.9) GVL (log10 copies/mL) 3.94 (0–5.88) 4.34 (2.6–5.88) — — 4.62 (3.3–5.9) ART duration (months) 33.5 (8–108) 33 (8–76) 35 (18–108) 0.15 33 (8–73) ART regimen  zidovudine/stavudine + lamivudine + efavirenz 11/42 (26%) 11/31 (35%) 0/11 (0%) 6/21 (29%)  zidovudine/stavudine + lamivudine + nevirapine 19/42 (45%) 12/31 (39%) 7/11 (64%) 0.02 10/21 (48%)  tenofovir + lamivudine/emtricitabine + efavirenz 4/42 (10%) 4/31 (13%) 0/11 (0%) 3/21 (14%)  tenofovir + lamivudine + nevirapine 8/42 (19%) 4/31 (13%) 4/11 (36%) 2/21 (10%) Continuous measures are presented as median (range) and categorical measures are presented as n/N (%). The Spearman’s correlation between PVL and GVL for the 42 women with PVL >2000 copies/mL was moderate (r2 = 0.50; P < 0.011), demonstrating that women with higher PVL tend to have higher GVL (Figure 1). A somewhat similar moderate correlation (r2 = 0.44) was also seen in the group of 31 shedders (P = 0.014). Figure 1. View largeDownload slide Relationship between PVL and GVL in 42 South Indian women, and plasma–GT drug resistance concordance in 21/42 women with available data. Figure 1. View largeDownload slide Relationship between PVL and GVL in 42 South Indian women, and plasma–GT drug resistance concordance in 21/42 women with available data. Drug resistance in circulating and GT RNA and proviral DNA Twenty-five of 42 women (60%) with PVL >2000 copies/mL had GVL >2000 copies/mL and had RT genotyping attempted in the three compartments. Genotypes were not available for 4/25 women; genital RNA from two women could not be amplified and plasma and genital paired sequences from two other women did not cluster together phylogenetically. Median GVL for the subset with genotype data was significantly higher than for the four without genotypes (4.6 versus 3.7 log10 copies/mL; P < 0.05). Paired plasma and genital sequences were obtained for these 21 women and PBMC genotypes were available for 20 women. All paired sequences were of good quality and clustered well phylogenetically (Figure S2). All but one (subtype A) sample were HIV-1 subtype C. Plasma drug resistance was seen in 91% (19/21), GT drug resistance was seen in 81% (17/21) and PBMC drug resistance was seen in 90% (18/20). Plasma NRTI resistance was seen in 76% (16/21), GT NRTI resistance was seen in 67% (14/21) and PBMC NRTI resistance was seen in 75% (15/20). Plasma NNRTI resistance was seen in 91% (19/21), GT NNRTI resistance was seen in 81% (17/21) and PBMC NNRTI resistance was seen in 90% (18/20). Plasma dual-class resistance was seen in 76% (16/21), GT dual-class resistance was seen in 67% (14/21) and PBMC dual-class resistance was seen in 75% (15/20) (P = not significant). The most common mutations in all three compartments included lamivudine-associated M184V and mutations at NNRTI-associated positions 101, 103 and 190. Thymidine analogue mutations (TAMs) occurred in 12/16 (75%) women on zidovudine/stavudine and K65R occurred in 1/5 (20%) women on tenofovir-based regimens and in one woman on zidovudine + lamivudine + nevirapine. Specific DRMs per patient in the three compartments are demonstrated in Table 3 and in aggregate in Figure 2. Table 3. DRMs in plasma, GT and PBMCs ID ART Plasma GT PBMCs NRTI NNRTI NRTI NNRTI NRTI NNRTI 1 3TC + d4T + NVP L74V, M184V K103N, V108I, Y181C, G190A, H221Y L74V, M184V, T215Y K103N, V108I, Y181C, G190A, H221Y L74LV, M184V, T215SY A98AG, K103N, V108IV, Y181C, G190A, H221HY 2 3TC + ZDV + EFV M184V, T215F K101E, V106M, E138A, G190A M41L, D67DN, M184V, T215F K101E, V106M, E138A, G190A M184V, T215F K101E, V106M, E138A, G190A 3 ZDV + 3TC + NVP V75IMV K103N V75IMV K103N M41L, V75I K103N, F227L 4 ZDV + 3TC + EFV M41L, E44D, D67N, T69D, K70R, V75M, M184V, L210W, T215Y A98G, K101E, G190S M41L, E44D, D67N, T69D, K70R, V75M, M184V, L210W, T215Y A98G, K101E, K103KE, G190S M41LM, E44ED, D67DN, T69ADNT, K70KR, V75IMV, M184V, L210W, T215Y A98G, K101EK, G190S 5 ZDV + 3TC + NVP E44D, D67N, T69D, M184V, T215Y A98AG, K101E, G190S E44D, D67N, T69D, M184V, T215Y A98AG, K101E, G190S, H221Y D67DN, M184MV, T215Y K101EK, K103KN, G190S 6 3TC + d4T + NVP M184V K103N, P225H none M184V K103N, P225HP 7 TDF + 3TC + NVP K65R, M184V K103N, V108I K65R, K70T, M184V K103N, V108I K65R, K70KR, M184V K103N, V108I 8 ZDV + 3TC + NVP K65KR, L74LV, M184V Y181CY, G190A, M230L L74V, M184V G190A M184MV G190AG, F227FL, M230LM 9 d4T + 3TC + NVP K70KR, M184V K101HKNQ, K103KN, Y181CY, G190AG K70R, M184V, K219E K101H, Y181C, G190A M184V, K219EK K101HKNQ, K103KN, Y181CY 10 d4T + 3TC + EFV none 11 ZDV + 3TC + EFV none V106M, F227L none K103N none V106M, F227L 12 FTC + TDF + EFV none none E138EK 13 ZDV + 3TC + NVP M184V, T215Y Y181C M184V, T215Y V106M, Y181C M184MV, T215NSTY K103KN, Y181CY 14 TDF + 3TC + NVP none K101E none K101E none K101E 15 3TC + d4T + EFV M184V V106ILM, V179DV, Y188L M184V V106ILM, Y188L M184V Y188L 16 TDF + 3TC + EFV M184V K103N, Y188L M184V K103N, Y188H M184V K103N, Y188L 17 3TC + d4T + NVP M41L, D67N, V75M, M184V, T215Y A98G, K101E, E138Q, G190A M41L, E44D, D67N, V75I, M184V A98G, K101E, E138Q, G190A M41L, D67DN, V75M, M184V, T215Y A98G, K101E, E138Q, G190A 18 TDF + 3TC + EFV none K101EK, Y188HY none 19 ZDV + 3TC + NVP D67N, K70KR, M184V Y181C D67DN, K70KR, M184MV E138AE D67DN, K70KR, M184MV Y181CY 20 ZDV + 3TC + NVP D67N, M184V A98G, K101E, G190A, P225H none Y181C D67N, K70R, M184V, K219E A98G, K101E, G190A 21 ZDV + 3TC + EFV M41L, D67N, T69D, K70R, M184V, T215Y, K219E Y188L, M230L M41L, D67N, T69D, K70R, M184V, T215Y, K219E Y188L, M230L NA ID ART Plasma GT PBMCs NRTI NNRTI NRTI NNRTI NRTI NNRTI 1 3TC + d4T + NVP L74V, M184V K103N, V108I, Y181C, G190A, H221Y L74V, M184V, T215Y K103N, V108I, Y181C, G190A, H221Y L74LV, M184V, T215SY A98AG, K103N, V108IV, Y181C, G190A, H221HY 2 3TC + ZDV + EFV M184V, T215F K101E, V106M, E138A, G190A M41L, D67DN, M184V, T215F K101E, V106M, E138A, G190A M184V, T215F K101E, V106M, E138A, G190A 3 ZDV + 3TC + NVP V75IMV K103N V75IMV K103N M41L, V75I K103N, F227L 4 ZDV + 3TC + EFV M41L, E44D, D67N, T69D, K70R, V75M, M184V, L210W, T215Y A98G, K101E, G190S M41L, E44D, D67N, T69D, K70R, V75M, M184V, L210W, T215Y A98G, K101E, K103KE, G190S M41LM, E44ED, D67DN, T69ADNT, K70KR, V75IMV, M184V, L210W, T215Y A98G, K101EK, G190S 5 ZDV + 3TC + NVP E44D, D67N, T69D, M184V, T215Y A98AG, K101E, G190S E44D, D67N, T69D, M184V, T215Y A98AG, K101E, G190S, H221Y D67DN, M184MV, T215Y K101EK, K103KN, G190S 6 3TC + d4T + NVP M184V K103N, P225H none M184V K103N, P225HP 7 TDF + 3TC + NVP K65R, M184V K103N, V108I K65R, K70T, M184V K103N, V108I K65R, K70KR, M184V K103N, V108I 8 ZDV + 3TC + NVP K65KR, L74LV, M184V Y181CY, G190A, M230L L74V, M184V G190A M184MV G190AG, F227FL, M230LM 9 d4T + 3TC + NVP K70KR, M184V K101HKNQ, K103KN, Y181CY, G190AG K70R, M184V, K219E K101H, Y181C, G190A M184V, K219EK K101HKNQ, K103KN, Y181CY 10 d4T + 3TC + EFV none 11 ZDV + 3TC + EFV none V106M, F227L none K103N none V106M, F227L 12 FTC + TDF + EFV none none E138EK 13 ZDV + 3TC + NVP M184V, T215Y Y181C M184V, T215Y V106M, Y181C M184MV, T215NSTY K103KN, Y181CY 14 TDF + 3TC + NVP none K101E none K101E none K101E 15 3TC + d4T + EFV M184V V106ILM, V179DV, Y188L M184V V106ILM, Y188L M184V Y188L 16 TDF + 3TC + EFV M184V K103N, Y188L M184V K103N, Y188H M184V K103N, Y188L 17 3TC + d4T + NVP M41L, D67N, V75M, M184V, T215Y A98G, K101E, E138Q, G190A M41L, E44D, D67N, V75I, M184V A98G, K101E, E138Q, G190A M41L, D67DN, V75M, M184V, T215Y A98G, K101E, E138Q, G190A 18 TDF + 3TC + EFV none K101EK, Y188HY none 19 ZDV + 3TC + NVP D67N, K70KR, M184V Y181C D67DN, K70KR, M184MV E138AE D67DN, K70KR, M184MV Y181CY 20 ZDV + 3TC + NVP D67N, M184V A98G, K101E, G190A, P225H none Y181C D67N, K70R, M184V, K219E A98G, K101E, G190A 21 ZDV + 3TC + EFV M41L, D67N, T69D, K70R, M184V, T215Y, K219E Y188L, M230L M41L, D67N, T69D, K70R, M184V, T215Y, K219E Y188L, M230L NA 3TC, lamivudine; d4T, stavudine; EFV, efavirenz; NVP, nevirapine; TDF, tenofovir; ZDV, zidovudine; FTC, emtricitabine; GS, genital secretion; NA, not amplified. Discordant mutations that were detected in GS or in PBMCs and not in plasma are in bold. Discordant mutations that led (alone or in combination) to clinically significant predicted resistance to at least one drug are underlined. Table 3. DRMs in plasma, GT and PBMCs ID ART Plasma GT PBMCs NRTI NNRTI NRTI NNRTI NRTI NNRTI 1 3TC + d4T + NVP L74V, M184V K103N, V108I, Y181C, G190A, H221Y L74V, M184V, T215Y K103N, V108I, Y181C, G190A, H221Y L74LV, M184V, T215SY A98AG, K103N, V108IV, Y181C, G190A, H221HY 2 3TC + ZDV + EFV M184V, T215F K101E, V106M, E138A, G190A M41L, D67DN, M184V, T215F K101E, V106M, E138A, G190A M184V, T215F K101E, V106M, E138A, G190A 3 ZDV + 3TC + NVP V75IMV K103N V75IMV K103N M41L, V75I K103N, F227L 4 ZDV + 3TC + EFV M41L, E44D, D67N, T69D, K70R, V75M, M184V, L210W, T215Y A98G, K101E, G190S M41L, E44D, D67N, T69D, K70R, V75M, M184V, L210W, T215Y A98G, K101E, K103KE, G190S M41LM, E44ED, D67DN, T69ADNT, K70KR, V75IMV, M184V, L210W, T215Y A98G, K101EK, G190S 5 ZDV + 3TC + NVP E44D, D67N, T69D, M184V, T215Y A98AG, K101E, G190S E44D, D67N, T69D, M184V, T215Y A98AG, K101E, G190S, H221Y D67DN, M184MV, T215Y K101EK, K103KN, G190S 6 3TC + d4T + NVP M184V K103N, P225H none M184V K103N, P225HP 7 TDF + 3TC + NVP K65R, M184V K103N, V108I K65R, K70T, M184V K103N, V108I K65R, K70KR, M184V K103N, V108I 8 ZDV + 3TC + NVP K65KR, L74LV, M184V Y181CY, G190A, M230L L74V, M184V G190A M184MV G190AG, F227FL, M230LM 9 d4T + 3TC + NVP K70KR, M184V K101HKNQ, K103KN, Y181CY, G190AG K70R, M184V, K219E K101H, Y181C, G190A M184V, K219EK K101HKNQ, K103KN, Y181CY 10 d4T + 3TC + EFV none 11 ZDV + 3TC + EFV none V106M, F227L none K103N none V106M, F227L 12 FTC + TDF + EFV none none E138EK 13 ZDV + 3TC + NVP M184V, T215Y Y181C M184V, T215Y V106M, Y181C M184MV, T215NSTY K103KN, Y181CY 14 TDF + 3TC + NVP none K101E none K101E none K101E 15 3TC + d4T + EFV M184V V106ILM, V179DV, Y188L M184V V106ILM, Y188L M184V Y188L 16 TDF + 3TC + EFV M184V K103N, Y188L M184V K103N, Y188H M184V K103N, Y188L 17 3TC + d4T + NVP M41L, D67N, V75M, M184V, T215Y A98G, K101E, E138Q, G190A M41L, E44D, D67N, V75I, M184V A98G, K101E, E138Q, G190A M41L, D67DN, V75M, M184V, T215Y A98G, K101E, E138Q, G190A 18 TDF + 3TC + EFV none K101EK, Y188HY none 19 ZDV + 3TC + NVP D67N, K70KR, M184V Y181C D67DN, K70KR, M184MV E138AE D67DN, K70KR, M184MV Y181CY 20 ZDV + 3TC + NVP D67N, M184V A98G, K101E, G190A, P225H none Y181C D67N, K70R, M184V, K219E A98G, K101E, G190A 21 ZDV + 3TC + EFV M41L, D67N, T69D, K70R, M184V, T215Y, K219E Y188L, M230L M41L, D67N, T69D, K70R, M184V, T215Y, K219E Y188L, M230L NA ID ART Plasma GT PBMCs NRTI NNRTI NRTI NNRTI NRTI NNRTI 1 3TC + d4T + NVP L74V, M184V K103N, V108I, Y181C, G190A, H221Y L74V, M184V, T215Y K103N, V108I, Y181C, G190A, H221Y L74LV, M184V, T215SY A98AG, K103N, V108IV, Y181C, G190A, H221HY 2 3TC + ZDV + EFV M184V, T215F K101E, V106M, E138A, G190A M41L, D67DN, M184V, T215F K101E, V106M, E138A, G190A M184V, T215F K101E, V106M, E138A, G190A 3 ZDV + 3TC + NVP V75IMV K103N V75IMV K103N M41L, V75I K103N, F227L 4 ZDV + 3TC + EFV M41L, E44D, D67N, T69D, K70R, V75M, M184V, L210W, T215Y A98G, K101E, G190S M41L, E44D, D67N, T69D, K70R, V75M, M184V, L210W, T215Y A98G, K101E, K103KE, G190S M41LM, E44ED, D67DN, T69ADNT, K70KR, V75IMV, M184V, L210W, T215Y A98G, K101EK, G190S 5 ZDV + 3TC + NVP E44D, D67N, T69D, M184V, T215Y A98AG, K101E, G190S E44D, D67N, T69D, M184V, T215Y A98AG, K101E, G190S, H221Y D67DN, M184MV, T215Y K101EK, K103KN, G190S 6 3TC + d4T + NVP M184V K103N, P225H none M184V K103N, P225HP 7 TDF + 3TC + NVP K65R, M184V K103N, V108I K65R, K70T, M184V K103N, V108I K65R, K70KR, M184V K103N, V108I 8 ZDV + 3TC + NVP K65KR, L74LV, M184V Y181CY, G190A, M230L L74V, M184V G190A M184MV G190AG, F227FL, M230LM 9 d4T + 3TC + NVP K70KR, M184V K101HKNQ, K103KN, Y181CY, G190AG K70R, M184V, K219E K101H, Y181C, G190A M184V, K219EK K101HKNQ, K103KN, Y181CY 10 d4T + 3TC + EFV none 11 ZDV + 3TC + EFV none V106M, F227L none K103N none V106M, F227L 12 FTC + TDF + EFV none none E138EK 13 ZDV + 3TC + NVP M184V, T215Y Y181C M184V, T215Y V106M, Y181C M184MV, T215NSTY K103KN, Y181CY 14 TDF + 3TC + NVP none K101E none K101E none K101E 15 3TC + d4T + EFV M184V V106ILM, V179DV, Y188L M184V V106ILM, Y188L M184V Y188L 16 TDF + 3TC + EFV M184V K103N, Y188L M184V K103N, Y188H M184V K103N, Y188L 17 3TC + d4T + NVP M41L, D67N, V75M, M184V, T215Y A98G, K101E, E138Q, G190A M41L, E44D, D67N, V75I, M184V A98G, K101E, E138Q, G190A M41L, D67DN, V75M, M184V, T215Y A98G, K101E, E138Q, G190A 18 TDF + 3TC + EFV none K101EK, Y188HY none 19 ZDV + 3TC + NVP D67N, K70KR, M184V Y181C D67DN, K70KR, M184MV E138AE D67DN, K70KR, M184MV Y181CY 20 ZDV + 3TC + NVP D67N, M184V A98G, K101E, G190A, P225H none Y181C D67N, K70R, M184V, K219E A98G, K101E, G190A 21 ZDV + 3TC + EFV M41L, D67N, T69D, K70R, M184V, T215Y, K219E Y188L, M230L M41L, D67N, T69D, K70R, M184V, T215Y, K219E Y188L, M230L NA 3TC, lamivudine; d4T, stavudine; EFV, efavirenz; NVP, nevirapine; TDF, tenofovir; ZDV, zidovudine; FTC, emtricitabine; GS, genital secretion; NA, not amplified. Discordant mutations that were detected in GS or in PBMCs and not in plasma are in bold. Discordant mutations that led (alone or in combination) to clinically significant predicted resistance to at least one drug are underlined. Figure 2. View largeDownload slide Prevalence of DRMs in plasma, GT and PBMCs. Figure 2. View largeDownload slide Prevalence of DRMs in plasma, GT and PBMCs. Drug resistance discordance Complete DRM concordance in all three compartments occurred in only 2/20 (10%) women for which sequences from all compartments were available, one of whom had no DRMs; 5/21 (24%) between plasma and GT, 8/20 (40%) between plasma and PBMCs and 3/20 (15%) between GT and PBMCs (Table 3). Odds of any discordance between GT and PBMCs was significantly larger than any discordance between plasma and PBMCs (OR = 5.35; 95% CI = 1.09–26.18; P = 0.04). The odds of discordance between plasma and GT was also larger than between plasma and PBMCs, but not significantly so (OR = 2.56; 95% CI = 0.46–14.10; P = 0.28). Of the 16 women with plasma–GT discordance, 12 (75%) had, overall, 14 additional DRMs in GT not detected in plasma (bold in Table 3). Of these, eight discordant mutations (underlined in Table 3) led to clinically relevant higher predicted resistance in GT versus plasma to at least one drug in 5/21 (24%) women. Of the 12 women with plasma–PBMC discordance, 10 (83%) had, overall, 15 DRMs in PBMCs not detected in plasma (bold in Table 3). Of these, 12 discordant mutations (underlined in Table 3) led to clinically relevant higher predicted resistance in PBMCs than plasma to at least one drug in 6/20 (30%) women. There was no statistically significant difference in genital shedding between the plasma–GT concordant and discordant groups, which may be due to small sample sizes (Figure 1). However, women with concordant mutations between the two compartments (black circles in Figure 1) did appear in the higher ends of both PVL and GVL. Table S1 demonstrates characteristics of women according to sequence concordance among compartments. While not statistically significant, higher plasma–PBMC discordance was seen in women with longer time on ART, and among all compartments with zidovudine/stavudine + lamivudine + nevirapine regimens. Genetic distances Genetic distances between pairs of sequences from the three compartments were similar overall (mean 1.78% plasma–GT, 2.01% plasma–PBMCs and 2.27% PBMC–GT). On average, compared with plasma–GT, plasma–PBMC distances were 0.22 units higher (95% CI = −0.38–0.81; P = 0.471) and PBMC–GT distances were 0.48 units higher (95% CI = −0.18–1.14; P = 0.155). Models comparing genetic distances between each compartment and the subtype C reference sequence showed that on average, compared with PBMCs (mean 8.27%), genetic distances of GT sequences were 8.98% (0.77 units higher; 95% CI = 0.16–1.37; P = 0.013) and genetic distances of plasma sequences were 9.01% (0.80 units higher; 95% CI = 0.30–1.29; P = 0.002). Discussion We investigated the compartmentalization of HIV-1 in plasma, GT and PBMCs and determined virological failure, GT viral shedding and inter-compartmental drug resistance concordance in HIV-1 subtype C-infected South Indian women on first-line ART. We found high resistance levels and, supporting our hypotheses, we demonstrated a fair, not perfect, plasma–genital viral load concordance, and high drug resistance discordance among the three compartments, with 71% (15/21) of women having DRMs in genital and/or PBMCs that were not detected in plasma, which might impact clinical care. Depending on the threshold, virological failure was detected in 31%–37% of women and, in line with previous reports, was associated with lower CD4 counts61 and higher use of nevirapine-based regimens. Consistent with the literature,27,48,62,63 74% of women with PVL >2000 copies/mL were genital shedders with a moderate but significant PVL–GVL concordance. This finding is supportive of PVL serving as a surrogate marker for genital shedding.15,64 Though we did not quantitate GVL in women with undetectable PVL, which might have underestimated genital shedding, this finding supports previous studies.13,65,66 Suppression of GT viral replication is essential to prevent evolution and transmission of resistance, and would have an epidemiological impact in places like India, where sexual contact is the main transmission cause. To prevent compartmentalization of HIV replication in the GT, 14,31,67,68 all ART components should reach adequate concentrations in that compartment. Though further such studies are needed,65 some suggest that failure to suppress PVL is the main determinant of GT shedding.69–72 As our study participants were more immunocompromised and had higher GVL compared with other studies,50 the risk of vertical and horizontal resistance transmission is presumably greater.73 High, fairly similar, levels of resistance (any in 81%–91%; dual-class in 67%–76%) were detected in all compartments, but with high discordance: only 10% of women had full DRM pattern concordance among the three compartments, with slightly higher rates between plasma and GT (24%) and between plasma and PBMCs (40%). These results are consistent with previous findings, implying the potential inadequacy of plasma genotyping as a representative for other anatomical sites.65 Moreover, these discordant mutations led to clinically relevant GT resistance that was not detected in plasma in 24% of women, and in PBMCs in 30%. These differences may suggest distinct viral evolution, possibly attributed to variable drug penetration/concentration, not measured here,14,74–76 but could also be the consequence of sampling bias between compartments, associated for example with different viral loads.77 Additionally, ART reduces viral load in plasma and the GT but does not diminish proviral DNA,78 which, even in the absence of detectable plasma RNA, can promote both perinatal and heterosexual resistance transmission.66,79 Observed DRM patterns were, overall, as expected,80 with lamivudine-associated M184V most frequently observed in all three compartments. Reported good GT penetration of lamivudine64,75 may explain our observed decreased frequency of M184V in that compartment compared with plasma (62% versus 71%). Regarding zidovudine/stavudine-associated TAMs, previous reports suggest that zidovudine can penetrate the GT in equal or higher concentrations than the blood plasma, whereas stavudine concentrations are much lower in the GT.75 In our participants, GT TAM prevalence was slightly higher (48%) compared with plasma (43%) despite zidovudine use in 5/9 women with TAMs in genital secretions. Drug concentration measurements in these compartments is necessary to substantiate the occurrence of DRMs as a result of decreased drug penetration in the GT. Regarding NNRTIs, the predominance of efavirenz/nevirapine-associated G190A was observed, confirming its fitness advantage on drug pressure, despite its relatively low-level resistance.81–83 However, G190A confers intermediate-level resistance to etravirine in synergism with Y181C, a concerning combination found in plasma (14%), PBMCs (5%) and genital secretions (10%), that could interfere with its use in third-line regimens.84 Similar concerns were observed in 10% (plasma), 15% (PBMCs) and 14% (genital secretions) of women with E138A/K/Q, conferring resistance to rilpivirine.85 Genotyping of proviral DNA can shed light on archived viruses and resistance to previous drugs which might impact resistance evolution.85–88 Indeed, genetic distances between PBMCs and the consensus sequence were lower than other compartments suggesting earlier sequence archival. Higher genetic distances between compartments shows that observed discordances were caused by differential evolution as a consequence of compartment-specific genetic differentiation among anatomically separate viral populations. DRMs in PBMCs that are not detected in plasma have been previously observed and may be transmitted or re-emerge upon ART to which they provide selective advantage.35,73–75 Increased HIV transmission risk is seen with higher genital HIV-1 RNA and our data suggest that resistance mutation accumulation in women with undetected virological failure could lead to increased risk of transmission of resistant HIV-1 variants.89 The main limitations of this study are its small sample size and cross-sectional design, restricting our ability to strongly address hypotheses. In addition, plasma and GT drug concentrations were not measured, which might explain resistance discordances; GVLs for women with suppressed PVLs and pre-treatment resistance were unavailable; drug resistance testing was population-based; Gram stain was not done to diagnose bacterial vaginosis, only wet mount was used to diagnose sexually transmitted infections, molecular-based testing was not done for trichomoniasis and herpes, and syphilis testing was also not done. Lastly, though tenofovir-based first-line regimens are currently preferred, zidovudine/stavudine-based regimens, more commonly reported here, are still in use in India as alternative regimens, making results relevant. In conclusion, GT viral shedding and archival, high-level resistance and discordance across compartments with genital/proviral DRMs not detected in plasma of South Indian women on first-line ART may result in their hidden transmission and/or re-emergence, which is likely to have a negative impact on treatment outcome. Whether such high resistance levels and discordances would be minimized by routine viral load monitoring, whether they should lead to incorporation of plasma/proviral genotyping or whether they suggest the need to monitor GVL and genital resistance to reduce the risk of ART failure and resistance acquisition and transmission, remains to be determined. Acknowledgements Part of this work was presented as a poster at the Twentieth International AIDS Conference, Melbourne, Australia, 2014 (Poster no. MOPDA0106). We are most grateful to the clinical and laboratory staff at YRG-CARE, VHS, Chennai, India, for their facilitation of the study. 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Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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