Transmission of antiretroviral drug-resistant HIV-1 was first reported in 1993 , and transmission of multi-drug resistant HIV-1 was first reported in 1997 and 1998 [2,3] (reviewed in ). In recent studies from North America, antiretroviral drug resistance has been detected in a significant portion of recently infected antiretroviral drug-naive individuals [5,6]. In 2005, a homosexual man from New York was infected with an HIV-1 strain that was resistant to three antiretroviral drug classes [nucleoside reverse transcriptase inhibitors (nRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), and protease inhibitors (PIs)], and also had CXCR4 tropism, which has been associated with rapid progression of HIV-1 disease .
The EXPLORE Study was a randomized clinical trial of the efficacy of a behavioral intervention to prevent HIV-1 acquisition among men who have sex with men (MSM) . That study provided an opportunity to examine antiretroviral drug resistance soon after infection in a large cohort of seroconverting MSM in the US. We analyzed HIV-1 from the first positive sample from these men for HIV subtype and for evidence of antiretroviral drug resistance and CXCR4 tropism. We also evaluated the association of antiretroviral drug resistance with demographic factors and other factors associated with risk of HIV-1 infection.
The EXPLORE study
HIV-1-negative men who reported engaging in anal sex within the past 12 months (n = 4295) were enrolled between 1999 and 2001 at one of six study sites in the US. Men were randomized to receive a behavioral intervention  versus standard risk reduction counseling. Men were evaluated for HIV-1 infection every 6 months, with a study endpoint of HIV infection. All men were followed for at least 24 months. The longest period of follow-up was 48 months, and the mean follow-up time among all participants was 3.25 years. Trained interviewers collected information on demographics, history of sexually transmitted infections (STIs) and use of post-exposure prophylaxis. Audio-computer-assisted self-interviewing technology was used to collect data on alcohol and drug use, and sexual behaviors. Participants were asked about sexual behaviors in the prior 6 months with partners of each HIV serostatus type (negative, positive, and unknown). After completing the interviews, participants received HIV pre-test counseling and were tested with an enzyme-linked immunosorbent assay (EIA) for the presence of antibodies to HIV. Reactive samples were retested in duplicate. Repeatedly reactive samples were confirmed using a western blot or immunofluorescence assay. Participants with a positive test result at any follow-up visit were referred for medical and social services. Overall, 259 seroconverters were identified in the study.
Study subjects and samples used for analysis
This resistance sub-study included men in the EXPLORE Study with documented HIV-1 seroconversion. Samples used for analysis were collected within 6 months of seroconversion. Samples were available from 220 (85%) of the 259 seroconverters.
HIV-1 genotyping was performed using the ViroSeq HIV-1 Genotyping System, v2.6 (Celera Diagnostics, Alameda, California, USA), using stored plasma or serum samples. GenBank accession numbers of the sequences are: EF136664-EF136858.
Tropism was measured using a replication-defective retroviral vector with a luciferase expression cassette inserted in the env gene . This vector was co-transfected with an env expression vector (containing env DNA from the test samples) into human embryonic kidney cell cultures. After 48 h, recombinant viruses were harvested and were evaluated for their ability to infect CD4-expressing U87 cells that also expressed either the CCR5 or CXCR4 co-receptor. The tropism of the isolates was determined both by the level of luciferase activity (relative light units, RLU) and by the reduction of RLU when the virus was cultured in the presence of high concentrations of CCR5 or CXCR4 antagonists. Isolates were characterized as CCR5-using, CXCR4-using, or dual/mixed (DM, indicating dual and/or mixed-tropic virus).
Other laboratory methods
HIV-1 RNA was measured using the Roche version 1.5 AMPLICOR HIV-1 Monitor Test Kit (Branchburg, New Jersey, USA). EIA and western blot assays were performed to confirm HIV-1 infection in subjects whose samples failed analysis in the genotyping system [Human Immunodeficiency Virus Type I (Viral Lysate and E. coli Recombinant Antigen) Genetics Systems TM rLav EIA, and Human Immunodeficiency Virus Type I Genetics Systems TM HIV-1 Western Blot; Bio-Laboratories, Redmond, Washington, USA].
HIV-1 subtyping was performed by phylogenetic analysis of the pol region HIV-1 sequences . The genetic relatedness of pol region sequences was determined using PHYLIP. Phylogenetic trees were constructed using the neighbor-joining method. Genetic distances were calculated by the ‘dnadist’ module within PHYLIP using the F84 algorithm.
We used Fisher's exact tests to assess the relationship between antiretroviral drug resistance and demographic and behavioral variables. Statistical significance was assessed using Bonferroni's multiple comparison adjustment. For behavioral variables, we considered baseline measures and measures reflecting the 6 months before documentation of HIV-1 seroconversion. In addition to univariate analyses, we constructed multivariate logistic regression models including demographic variables and behavioral measures during the 6 months prior to HIV-1 seroconversion as covariates. Backward elimination with a stay criteria for covariates set at P = 0.05 was used for model selection. We performed all analyses using SAS®, v9.13 (SAS Institute Inc., Cary, North Carolina, USA).
The EXPLORE study was approved by the institutional review boards at each of the participating institutions, and study participants provided written informed consent. Human experimentation guidelines of the Department of Health and Human Services were followed in the conduct of this research.
Subjects included in the antiretroviral drug resistance sub-study
Samples used for this resistance sub-study were collected within 6 months of HIV-1 seroconversion. Of the 259 men who seroconverted in the EXPLORE study, 220 (85%) had a plasma or serum sample available for genotyping. Genotyping was successful for all but 25 of the available samples. Fourteen of the 25 samples with no genotyping results had low HIV-1 RNA levels (< 400 copies/ml). Assay failure was not likely to be due to subtype diversity, since the ViroSeq system performs well for analysis of diverse HIV-1 strains . EIA and western blot testing confirmed that all of those 14 samples were positive for HIV-1 antibodies. We compared demographic and behavioral characteristics of the 195 seroconverters with genotyping results and the 64 seroconverters for whom results were not available (no sample or genotyping failed, demographic and behavioral variables are described in Tables 1 and 2). We observed no statistically significant differences, after adjusting for multiple comparisons using the Bonferroni correction, for any of the demographic or behavioral variables.
The 195 samples with genotyping results included 135 (69%) collected on the date of documented seroconversion, and 60 (31%) collected at a subsequent study visit. For all 195 subjects, the median number of days between the last study visit with a seronegative test result and sample collection was 189 days [interquartile range (IQR), 56 days]. Among the 60 subjects who had samples collected at a subsequent study visit, the median number of days between documentation of seroconversion and sample collection was 13.5 days (IQR,: 6 days), and the median number of days between the last visit with a seronegative test result and sample collection was 196 days (IQR, 68 days).
The demographic and behavioral characteristics of the 195 seroconverters included in the resistance sub-study are shown in Tables 1 and 2. Over half of the subjects were 31 years of age or older. Sixty-five percent of subjects were white and of non-Hispanic origin. Over 65% of the HIV infections were identified in 2001 or 2002. Nearly 50% of subjects reported light alcohol use, and many reported use of recreational drugs. None reported use of heroin. Eighty-two percent reported use of alcohol or drugs with sex. More than half of the men (56%) reported no primary partner and 79% reported having at least four sexual partners within 6 months of study enrollment. Men most often reported unprotected receptive anal sex with HIV-negative partners or partners with unknown HIV status (46 and 42%, respectively). There was a very low prevalence of self-reported chlamydia (4%), syphilis (1%) or gonorrhea (8%), and also a low prevalence of self-reported use of antiretroviral drug post-exposure prophylaxis, either at enrollment (4%) or in the 6 months prior to seroconversion (3%).
Analysis of antiretroviral drug resistance
Among the 195 subjects with genotyping results, 31 (15.9%) had evidence of resistance to at least one antiretroviral drug (Table 3). In almost all cases, evidence of resistance included detection of at least one mutation recommended for analysis of transmitted drug resistance  (see legend for Table 3). The portion of men with resistance was similar for subjects tested at the time of documented seroconversion (16.7%) versus those tested at a subsequent study visit (15.6%; χ2 = 0.038; P = 0.845). Resistance was observed most frequently for the NNRTIs (delavirdine, nevirapine, efavirenz), stavudine, zidovudine and nelfinavir (Table 3). The highest frequency of resistance to one or more drug of any class was seen in Boston (26.7%), which also had the highest frequency of NRTI resistance (20%) and PI resistance (13.3%). The highest frequency of NNRTI resistance was seen in Seattle (12.5%). However, we did not observe statistically significant differences in resistance levels across the sites.
We compared the demographic and behavioral characteristics of the 31 men who had evidence of antiretroviral drug resistance to those of the 164 who did not (Tables 1 and 2). After taking multiple comparisons into account using the Bonferroni correction, we did not find any statistically significant associations between resistance status and the demographic and behavioral risk variables, either in the 6 months prior to study enrollment, or in the 6 months prior to documentation of HIV seroconversion. We also included all demographic and behavioral risk measures as covariates in a multivariate logistic regression model. Backward elimination was used for model selection. Unprotected insertive anal sex with an HIV-positive partner (more frequent among men with resistance) and moderate alcohol use (less frequent among men with resistance) remained in the final model.
Four subjects had evidence of resistance to drugs in two classes, and three subjects had evidence of resistance to drugs in all three classes (Table 3), one of whom had evidence of resistance to 17 of the 18 drugs analyzed. For the three subjects with three-class resistance, seroconversion was documented in one subject in 2000, one in 2001, and one in 2002.
Analysis of HIV-1 tropism
Phenotypic data for HIV-1 co-receptor tropism was obtained for 126 (64.6%) of the 195 men who had genotyping results. Most of the remaining men had insufficient samples for analysis in the tropism assay. Adjusting for multiple comparisons, we observed no statistically significant difference for any of the demographic or behavioral variables shown in Tables 1 and 2 for the 126 seroconverters with tropism results versus the 133 seroconverters without tropism results. All 126 samples with results produced a different level of luciferase expression when CCR5-expressing cells were used in the infection step (range: 1032 to 3 120 708 relative light units, RLU).
Samples from four men were also able to use the CXCR4 co-receptor, demonstrating that they had dual/mixed tropism. In two cases, the level of viral replication was similar on CCR5-bearing and CXCR4-bearing cells (in one man: 803 100 and 812 573 RLU, respectively; in one man: 29 283 and 39 126 RLU, respectively). In the other two cases, viral replication was higher on CCR5-bearing cells than on CXCR4-bearing cells (in one man: 298 606 and 176 RLU, respectively; in one man: 127 062 and 210 RLU, respectively). In the two men with low-level replication on CXCR4-bearing cells, CXCR4 use was confirmed by the reduction of RLU when the virus was cultured in the presence of high concentrations of a CXCR4 antagonist. Each of the four men with CXCR4-using virus was enrolled at a different study site at the time of documentation of seroconversion and each was found to be HIV-1 infected in a different year (Seattle 2000, Chicago 2001, New York 2002, San Francisco 2003). Among those four men, the time between the last seronegative result and documentation of seroconversion ranged from 138 to 187 days (mean = 166 days). Only one of the four men (the study subject enrolled in San Francisco) had evidence of antiretroviral drug resistance. That man had evidence of resistance to all three NNRTIs (delavirdine, efavirenz, nevirapine). All four men had unprotected anal sex with partners of unknown HIV-1 status, and all four reported a history of light alcohol use and recreational drug use. One man reported a history of injection drug use and two reported a history of methamphetamine use.
Phylogenetic analysis of HIV-1 strains
All of the 195 men with genotyping results were confirmed to have subtype B infection. Phylogenetic analysis of sequences from all 195 subjects identified five pairs (two subjects each) and two triplets (three subjects each) who had very similar HIV strains (seven clusters, Fig. 1 and Table 4). In one case, two subjects were infected with a similar HIV-1 strain with two-class resistance (Table 4). In each case, similar HIV-1 strains were detected in individuals who were originally enrolled at the same study site. It was not known whether these individuals were co-enrolled partners, or whether they may have had a common source partner.
The EXPLORE cohort provided an opportunity to evaluate HIV drug resistance, HIV-1 co-receptor tropism, and HIV-1 subtype in recent infections of MSM in major US cities. Evidence of antiretroviral drug resistance was found in 15.9% of the men studied. This most likely reflects the transmission of antiretroviral drug-resistant strains, since the men were tested using samples collected at or near the time of documented seroconversion. It is important to note that the EXPLORE study population was recruited using behavioral eligibility criteria that specifically identified men who were at high risk of HIV infection. Thus the seroconverters in this study do not necessarily represent all HIV-infected MSM. Other studies of recently infected individuals found resistance rates of 22.7% (1999–2000 in North America, n = 88) , 27.4% (2000–2001 in San Francisco, n = 91) , and 8.3% (1997–2001 in 10 US cities, n = 1082) . In the latter study, the rate of resistance among 482 MSM was similar to what we observed in the EXPLORE cohort (11.6 versus 15.9% in this study) . The resistance rates detected in recently infected individuals may be higher than those detected in individuals with chronic HIV infection. In the SPREAD study in Europe, drug resistance mutations were detected more frequently among drug-naive individuals with recent rather than chronic HIV infection . A similar trend was observed, both among MSM and among all study subjects, in a large surveillance study from the US .
In the EXPLORE cohort, 6.7% of the 195 men had resistance to an NNRTI, 8.7% had resistance to an NRTI, and 5.6% had resistance to a PI. Multi-class resistance was detected in seven (3.6%) of the men, including four (2.1%) with two-class resistance and three (1.5%) with resistance to all three antiretroviral drug classes. In the studies cited above, multi-class resistance (two- or three-class resistance) was found at rates of 1.3% , 10.2% , and 13.2% . One study reported three-class resistance in one (1.2%) of 91 subjects identified between 2000 and 2001 .
Few studies have examined associations between antiretroviral drug resistance and risk factors for HIV-1 infection. In a study from the US, antiretroviral drug resistance was more frequently detected among MSM (11.6%) than either women (6.1%) or heterosexual men (4.7%) . In Italy, a higher rate of resistance was also seen among MSM (21.6%), than among heterosexuals, intravenous drug users and others, however the difference was not statistically significant . In Canada, a similar rate of antiretroviral drug resistance was seen among individuals recently infected by intravenous drug use or sexual transmission . A preliminary report of 214 recently infected MSM in California found an association between antiretroviral drug resistance and methamphetamine use [odds ratio (OR), 4.29: P = 0.01), but not with use of other substances or with income level . In that study, methamphetamine use was reported in 19.5% of men with resistance versus 9.5% of men without resistance. Methamphetamine use was more frequent in the EXPLORE study (32% of men) than in the cohort described above (approximately 10% of men) , but there was no association between antiretroviral drug resistance and methamphetamine use among the men in EXPLORE (Table 2).
In the US, a higher rate of resistance has been noted among whites, compared to African Americans and Hispanics . We did not find an association between race/ethnicity and resistance in the EXPLORE cohort, which may reflect the smaller sample size of our study (195 seroconverters) compared to the sample size of the previous study (n > 1000). We also did not find any association of antiretroviral drug resistance with age, city, number or HIV status of sexual partners, sexual practices, self-reported STIs, or use of antiretroviral drug post-exposure prophylaxis. All of the men analyzed had subtype B HIV infection, indicating a low incidence of non-subtype B among MSM in major US cities who were recently infected with HIV-1.
HIV-1 tropism data were available for 126 men, providing the first large-scale descriptive analysis of HIV-1 tropism in recent HIV-1 seroconverters. We identified four men, each enrolled at a different study site, who were infected with dual or mixed tropic HIV-1 strains. None of those men had multi-class antiretroviral drug resistance, but one had resistance to all three NNRTIs. Two of the four men reported methamphetamine use. Only one of the men reported using intravenous drugs. Therefore, the CXCR4-using strains were likely to have been sexually transmitted in the other three men. Men in the EXPLORE study were not followed after documentation of seroconversion, so we do not have data on disease progression in the four men with CXCR4-using strains.
We identified seven groups of two or three men who were infected with genetically similar HIV-1 strains. One pair of men was infected with a nearly identical strain that had two-class resistance. The 16 men in these groups represented 8.2% of those tested. In each case, the group of two or three men may have become infected from contact with a single individual, or may have passed the infection from one to another. This finding was not surprising, since 31% of EXPLORE subjects at one study site reported having sex with another study participant .
In summary, 31 (15.9%) of 195 MSM with recent HIV-1 infection had drug resistance, including seven (3.6%) with multi-class resistance, and three (1.5%) with resistance to all three drug classes. We found no association of antiretroviral drug resistance with demographic factors, clinical factors, or specific risk behaviors. Four (3.2%) of 126 men tested had CXCR4-using HIV-1 strains, including one man with NNRTI resistance. All 195 men had HIV-1 subtype B. The frequent detection of antiretroviral drug resistant HIV-1 strains in recently infected MSM emphasizes the importance of antiretroviral drug resistance testing in similar patient populations prior to initiation of antiretroviral drug therapy. Identification of CXCR4-using strains in some men emphasizes the need for further studies defining the prevalence of mixed or dual tropic HIV-stains in individuals with recent HIV-1 infection, and for studies investigating the relationship between acquisition of CXCR4-using strains and HIV-1 disease progression.
The authors thank the EXPLORE study team and study subjects. The team also acknowledges Kayla Stratton (SCHARP) and Rufeng Xu-Friedman (Frontier Science Foundation) for assistance with data management. The authors thank Neil Parkin (Monogram Biosciences) for critical review of the manuscript.
Role of the funding source: The sponsor (HPTN) supported the original EXPLORE Study (HPTN 015) and provided support for the individuals responsible for design and conduct of the study; collection, management, analysis and interpretation of the data; preparation, review, and approval of the manuscript. The sponsor also provided support for HIV genotyping and subtyping, which was performed at the HPTN Central Laboratory under the direction of S.H.E.
Authors’ contributions: This manuscript has more than 10 authors. Each author contributed to the manuscript, helped to write and review the manuscript, and approved the final version. Individual roles of the authors are described as follows: S.H.E. designed and coordinated the study, drafted the original manuscript, handled all revisions and was responsible for HIV genotyping studies. M.H. was the lead data analyst for the EXPLORE study. S.H. performed HIV genotyping and analyzed genotyping results. D.D. was the lead statistician for the EXPLORE study and was responsible for the statistical analysis for this study. Y.H. assisted with the statistical analysis. W.H. performed the HIV tropism assays and reviewed all tropism results. S.H. performed the phylogenetic analysis of HIV subtypes and linked infections. J.B.J. was the HPTN Network Laboratory Representative for the EXPLORE study and helped draft the original study plan. T.C., M.C. and B.K. were EXPLORE study Protocol Co-Chairs and B.K. assisted with the study design and data presentation.
Sponsorship: This work was supported by the HIV Network for Prevention Trials and sponsored by the U.S. National Institute of Allergy and Infectious Diseases (NIAID) and the National Institute on Alcohol Abuse and Alcoholism, of the National Institutes of Health (NIH), U.S. Department of Health and Human Services (DHHS), through contract N01 AI35176 with Abt Associates Inc; contract N01 AI45200 with the Fred Hutchinson Cancer Research Center; and subcontracts with the Denver Public Health, the Fenway Community Health Center, the Howard Brown Health Center, the New York Blood Center, the Public Health Foundation Inc., and the Univ. of Washington. In addition, this work was supported by the HIV Prevention Trials Network (HPTN) and sponsored by the NIAID, the National Institute of Child Health and Human Development, the National Institute on Drug Abuse, the National Institute of Mental Health, and the Office of AIDS Research, of the NIH, DHHS, through a cooperative agreement with Family Health International (cooperative agreement 5 U01 AI46749) with a subsequent subcontract to Abt Associates Inc. with subcontracts to the Howard Brown Health Center and Denver Public Health; cooperative agreement U01 AI48040 to the Fenway Community Health Center, cooperative agreement U01 AI48016 to Columbia Univ. (including a sub-agreement with the New York Blood Center); cooperative agreement U01 AI47981 to the Univ. of Washington; and cooperative agreement U01 AI47995 to the Univ. of California, San Francisco; as well as HPTN contracts U01-AI-46745 and U01-AI-068613 with Johns Hopkins University.
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