Baseline testing for transmitted drug resistance (TDR) is now routinely performed on presentation to care in the United States and Europe,1,2 where recent estimates suggest TDR prevalence of 11%–21%.3–7 TDR testing is currently not the standard in most resource-limited settings, where relatively low TDR prevalence and limited drug options may lessen potential gains associated with routine testing.8,9 Surveillance of TDR is needed, however, to assess the emergence and spread of drug-resistant strains in order to inform HIV treatment guidelines and decisions by governments regarding initiation of universal TDR testing.10–12
Thailand is experiencing a rapid rise in new HIV infections among men who have sex with men (MSM), with HIV prevalence of 20%–30% in selected populations and new infections occurring at age-disaggregated rates of 5.9–12.2 per 100 person-years.13–16 As access to antiretroviral therapy (ART) for this population increases, there is also potential for increased transmission of drug-resistant viruses.8,17 Although testing for TDR typically occurs on presentation to care in persons who are chronically infected with HIV, testing for TDR during acute HIV infection (AHI) provides increased sensitivity for the detection of drug-resistant strains (primary drug resistance) before the overgrowth of drug-sensitive viral quasi-species.6,18,19
We report the prevalence of TDR in a large cohort of acutely HIV-infected Thais, the majority of whom are MSM. We stratify TDR prevalence according to risk groups and drug classes to inform the need for routine TDR surveillance in Thailand and elsewhere.
The ongoing SEARCH 010/RV 254 study (clinicaltrials.gov NCT00796146) is a prospective cohort study enrolling participants at the Thai Red Cross Anonymous Clinic in Bangkok, the largest voluntary counseling and testing center in Thailand. Clients presenting for HIV testing during AHI are identified in real-time according to a previously published algorithm.20 Briefly, samples are first tested by fourth generation (4thG) enzyme immunoassay (EIA) detecting HIV antigen and HIV IgM. Nonreactive samples undergo pooled nucleic acid testing (NAT) and reactive samples are tested using a less-sensitive second generation (2ndG) EIA. AHI subjects are offered enrollment into the study if they have either a nonreactive 4thG EIA and a positive NAT or a reactive 4thG EIA and a nonreactive 2ndG EIA. AHI subjects are staged according to the published 4thG staging system: stage 1 [NAT+, 4thG EIA−, third generation (3rdG) EIA−], stage 2 (NAT+, 4thG EIA+, 3rdG EIA−) and stage 3 (NAT+, 4thG EIA+, 3rdG EIA+, Western blot, or indeterminate).20 Subjects presenting for HIV screening between April 20, 2009 and September 13, 2013, were eligible for inclusion in this study. The study protocol was reviewed and approved by institutional review boards of Chulalongkorn University, Bangkok, Thailand, and the Walter Reed Army Institute of Research, Bethesda, MD.
HIV genotyping was performed to detect mutations in the reverse transcriptase and protease genes at the time of enrollment into the cohort using the TRUGENE HIV-1 genotyping assay (Siemens Healthcare Diagnostics, Tarrytown, NY) for the first 66 samples and a validated in-house method for the remaining samples.21 Genotype sequences were processed through the Stanford University Drug Resistance Database22,23 and mutations were subsequently categorized according to the World Health Organization surveillance drug resistance mutation (SDRM) list.24 All mutations were classified as conferring resistance to nucleoside/nucleotide analog reverse transcriptase inhibitors (NRTIs), nonnucleoside analog reverse transcriptase inhibitors (NNRTIs), or protease inhibitors (PIs). HIV subtype was determined using the multiregion hybridization assay (MHAbce)25 for the first 63 samples and the HIVSeq26,27 program for the remaining samples.
Prevalence of TDR was calculated by dividing the number of subjects with 1 or more detectable SDRMs by the total number of subjects with available genotyping data. Prevalence of resistance to individual drug classes and among specific risk groups was calculated in a similar fashion. Ninety-five percent confidence intervals (CIs) were calculated using the Clopper–Pearson interval. All analyses were performed using Stata 12.1 (StataCorp LP, College Station, TX).
During the study period, 83,475 samples were prospectively screened and 149 subjects were diagnosed with AHI. Of these, 123 enrolled in the study and 120 genotypes were obtained at the time of inclusion. Samples from 3 participants failed to amplify during genotypic testing. The mean time from AHI diagnosis to HIV genotyping was 2.3 days (SD, 1.02). Participants in this study had a median age of 28 years (Table 1). They were mostly male (93%) and MSM (90%). Their median income was 40,000 Thai Bhat (1231 USD) per month, which is over 4 times the minimum wage in Thailand, and 62% had a college-level or higher education. The mean duration since reported last HIV exposure was 18 days. Median CD4 was 376 cells per cubic millimeter and median HIV RNA 5.6 log10 copies per milliliter. Most subjects (82%) were infected by the CRF01_AE clade.
Baseline genotypes from 11 subjects included mutations from the WHO SDRM list, yielding an overall TDR prevalence estimate of 9.2% (95% CI: 4.7 to 15.8). Prevalence of SDRMs conferring resistance to specific drug classes included NRTI 5.0% (1.9–10.7), NNRTI 3.4% (0.9–8.4), and PI 3.4% (0.9–8.6). The most common SDRMs were M41L, K103N, and Y181C, with each mutation found in 2 participants. TDR prevalence among MSM was 9.3% (4.5–16.4) and among heterosexual females 11.1% (0.3–48.2). None of the 3 heterosexual males enrolled in the study demonstrated any TDR.
Characteristics of the 11 subjects with SDRMs are summarized in Table 2. One study participant had TDR to 2 drug classes and another study participant had TDR to 3 drug classes. Of note, subjects 1 and 2 were linked HIV transmission cases. They were sexual partners whose plasma viruses were matched by full-length sequencing and were found to be CRF01_AE clade. Both had the K103N mutation in their plasma samples. When genotyping was done on the seminal plasma of subject 1 (the anal insertive partner) and the anal wash of subject 2 (the anal receptive partner), K103N was detected in both specimens.
In this prospective cohort of HIV IgG nonreactive AHI subjects in Thailand, we observed a TDR prevalence of 9.2% using WHO criteria for surveillance. The majority of subjects were MSM, among whom TDR prevalence was 9.3%. Among HIV drug classes, resistance to NRTIs was most common.
The prevalence of TDR in this study is higher than in previous reports from Thai subjects, in which an estimated TDR prevalence of 5% or less was observed, although CIs between our estimate and prior estimates do overlap.28–30 Estimation of TDR prevalence is complicated by the challenge of capturing subjects early after infection, before overgrowth of more fit, drug-sensitive viral quasi-species renders genotype testing less sensitive for the detection of drug resistance.6,18,19 Therefore, most studies of TDR enroll participants who are newly diagnosed with HIV but have usually entered the chronic stage of infection. Our study has the advantage of genotypic resistance testing performed, on average, 2.3 days after AHI diagnosis in a large cohort of such subjects. The prevalence of TDR among the largely MSM Thai population in this cohort meets the WHO criterion for moderate transmission of HIV drug resistance and actions to be taken should include (1) evaluation of early warning indicators at clinics, such as on-time pill pick-up, retention in care, pharmacy stock-outs, dispensing practices, and viral suppression rates, (2) review of HIV transmission prevention programs for individuals who are aware of their infection, and (3) assessment of the coverage of HIV screening programs.31
MSM in Thailand are disproportionately newly infected with HIV.15,32 The presence of TDR in the genital compartment of our cases of linked MSM transmission further highlights the ongoing risk of TDR spread through unprotected anal intercourse.
Understanding the magnitude of TDR in the MSM population is critical for Thailand and other countries with similar epidemics. In Asia, data suggest that TDR may be more common among MSM than among persons with other risk factors for HIV.33 Thailand has the longest experience with widely available ART in Asia and is therefore an important sentinel setting for the region. Duration of ART availability has been an important predictor of TDR emergence in other resource-limited situations.9 Among MSM in China, recent estimates of TDR prevalence remain around 5%.34,35 The moderate TDR prevalence detected among Thai MSM in this study may signal similar findings to emerge among MSM in other countries.
In the developed world, baseline genotype testing is routine and PI-based therapy is recommended as first-line treatment for patients who require ART initiation before genotype results are known, such as those presenting during AHI.2,36 This recommendation reflects the preponderance of NNRTI resistance mutations among TDR in the developed world.3–7 Implementation of such an approach in resource-limited settings would be challenging, due to the relative expense of PI-based regimens. Our data also suggest that NNRTI resistance may not be the major driver of TDR among Thai MSM (prevalence 3.4%). Empiric selection of PIs as first-line therapy may not yet be a cost-effective alternative in these environments. It is unclear at what threshold it might become cost-effective for resource-limited settings to undertake universal screening of newly diagnosed subjects for drug resistance, but TDR rates above 1%–5% have been proposed.37 The rate in our Thai cohort of predominantly MSM subjects exceeds this threshold.
This study was performed among subjects enrolled in a single clinic in Bangkok, which may limit the generalizability of our findings. However, the Thai Red Cross Anonymous Clinic is the largest voluntary counseling and testing center in Thailand and diagnoses approximately 18% of the country's new HIV infections in MSM.38 Because subjects were tested for resistance during AHI, our findings reflect an increased sensitivity for the detection of drug-resistant strains as compared with studies conducted during chronic infection. This increased sensitivity may contribute to the higher rate of TDR observed relative to prior studies among chronically infected Thais. The study population was predominantly comprised of MSM, so the findings of this study may not be representative of the epidemic among persons with other HIV risk factors. Importantly, a single heterosexual female presented with TDR. With only 9 females in the cohort, this corresponds to a TDR rate of 11.1%, which is higher than the 9.2% observed in the overall cohort. It is not possible to draw strong conclusions about TDR among females due to the small sample size in our study, but it is possible that the high rate of TDR is not a phenomenon that is limited to Thai MSM.
In summary, we observed a TDR prevalence of 9.2% among a predominantly MSM cohort of Thai AHI subjects. As CD4 cell thresholds for ART initiation increase and ART utilization is expanded, this figure is likely to increase and TDR prevalence may soon approach rates seen in developed countries. With moderate overall TDR prevalence and similar rates of resistance to all 3 drug classes evaluated in our study, our data suggest that baseline drug resistance testing may be warranted, particularly among newly diagnosed MSM. Furthermore, baseline genotype testing for all Thai subjects diagnosed during AHI will help inform both international and national treatment and management guidelines. Future research should focus on investigating the feasibility and cost-effectiveness of universal drug resistance screening among persons living with HIV in Thailand and other countries with similar epidemics around the world.
The RV 254/SEARCH 010 Study Group includes from SEARCH/TRCARC/HIV-NAT: Nipat Teeratakulpisarn, Eugene Kroon, Thep Chalermchai, Duanghathai Sutthichom, Somprartthana Rattanamanee, Peeriya Mangu, Putthachard Sangtawan, Somporn Tipsuk, Siriporn Sangthong, Sasiwimol Ubolyam, Apicha Mahanontharit, Tippawan Pankam, Supanit Pattanachaiwit; from AFRIMS: Robert J. O′ Connell, Siriwat Akapirat, Vatcharain Assawadarachai, Bessara Nuntapinit, Kultida Poltavee, Nantana Tantibul, Susan Mason, Panadda Sawangsinth; from the US Military HIV Research Program: Sodsai Tovanabutra, Eric Sanders-Buell; from Monogram: Laura Napolitano, Yolanda Lie and the R&D and PDO groups.
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