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ABSTRACT |
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The genetic subtypes of HIV-1 in most of South America resemble those in North America: subtype B is the predominant subtype.5 In the countries of the Southern Cone of South America, however, BF recombinants of a wide range of subtype structures circulate.5,10 While it is known that they have been present in the epidemic since 1986,11 the relative proportions among recently infected persons is unknown. Knowing whether the proportion of recombinants in the epidemic is increasing over time would be extremely valuable because recombination has an impact on the prevalence and types of drug resistance.
In the baseline seroprevalence study we conducted in the capital city of Montevideo, Uruguay in 1999,12 male transvestite CSWs were reported to have a rather high HIV prevalence (21.5%; 95% confidence interval [CI] = 16.0–27.8%); a prior IDU history and older age (
26 years) were found to be associated with HIV. Despite their documented elevated HIV prevalences, no cohort-based, HIV incidence studies have been conducted in this high-risk core group. The objectives of this epidemiologic study were to determine observed HIV incidence rates and to evaluate potential risk factors associated with HIV seroconversion among initially seronegative male transvestite CSWs enrolled in the original study.
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MATERIALS AND METHODS |
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18 years old and HIV negative at baseline were deemed to be eligible and were invited to participate in this cohort study and were followed-up until May 2001. Serologic test results were linked to the questionnaire only by a unique numeric code to preserve confidentiality and anonymity of study participants. The study protocol was reviewed and approved by the local ethical and scientific review boards, as well as by the U.S. Naval Medical Research Center’s institutional review board, in compliance with all federal regulations governing the protection of human subjects. We defined a transvestite as a subject whose biologic sex was male and who self-identified as a cross-dresser or as a male-female transgendered person, irrespective of his prior history of hormonal use or gender reassignment surgery. This definition is consistent with that applied in other studies of transgendered individuals.13 Volunteers were recruited in streets, bathhouses, nightclubs, and brothels by trained health care personnel from the Uruguayan Ministry of Health. The HIV-negative participants who accepted to participate in this prospective study were briefed on its purpose, provided written informed consent and a blood sample (7–10 mL), and were interviewed using a standardized questionnaire format that contained information regarding sociodemographic characteristics (such as age and occupation), sexual practices (such as numbers of sexual contacts per week, workplace, time as a CSW, frequency of condom use with clients and steady partners, sexual contact with foreigners and others), and current use of illegal drugs. All study subjects were provided with HIV and STI prevention information, which included counseling, and printed and oral information on infectious agents, as well as with condoms and lubricant gel as requested. Additional details of subject enrollment procedures have been published elsewhere.12 Study participants were recalled after one year of the initial (baseline) study visit and followed-up approximately every six months until study closure or until lost to follow-up.
Laboratory sampling. Serum samples were subjected to screening for antibodies to HIV by laboratory-based enzyme-linked immunosorbent assay (ELISA) testing (HIV 1/2, MEIA-IMx; Abbott Laboratories, Chicago, IL) of rapid test-positive samples. Samples found to be repeatedly reactive on ELISA screening were subsequently subjected to immunoblot confirmation (New LAV Blot 1; Sanofi-Pasteur, Marnes-La-Coquette, France). Peripheral blood mono-nuclear cells (PBMCs) were isolated from whole heparinized blood samples by the Ficoll gradient method (lymphocyte separation medium; ICN/Cappel, Aurora, OH), and DNA was extracted from approximately 1 x 106 infected PBMCs by the QIAmp DNA extraction technique (Qiagen, Valencia, CA).
Genetic characterization of HIV-1 strains. The DNA samples were genotyped using the envelope heteroduplex mobility assay (env HMA) as previously described.14,15 Briefly, after two rounds of a polymerase chain reaction (PCR) with primers ED14 and ED3 (first round) and ED31 and ED33 primers (second round), HMA electrophoresis was performed with second-round PCR products using nine reference standards in the formation of heteroduplexes.
Partial pol PCR amplification from PBMC DNA was performed on 37 randomly selected samples from the sample set using a nested strategy. The first-round amplification was done in a volume of 50 µL with primers Pro5F (5'-AGAAATTGCAGGGCCCCTAGGAA-3') and RT3474R (5'-GAATCTCTCTGTTTTCTGCCAG-3') and Amplitaq Gold Taq polymerase (Applied Biosystems, Foster City, CA). The second-round amplification was completed using 1 µL of the first-round product and primers Pro3F (5'-AGAICAG-AGCCAACAGCCCCACCA-3') and ProRT (5'-TTCCCC-ACTAACTTCTGTATGTCATTGACA-3').
This nested strategy amplified 1.1 kb of the HIV genome. The amplified product was sequenced with Big Dye terminators using an ABI 3100 automated sequencer (Applied Bio-systems). The region sequenced included all of the protease region and the carboxy terminus of the reverse transcriptase (RT) protein.
Nearly full genomes were also amplified directly from the PBMC DNA of each subject using a nested PCR strategy that amplified the region from gag to the end of the R region of the 3'-long terminal repeat.16,17 Sequences were aligned in a multiple alignment with standard subtype references and phylogenetic analyses were conducted using Neighbor-joining with Kimura’s two-parameter model of distance calculation and a bootstrap computed with parsimony.18,19 Analysis for potential recombination was done using Simplot version 3.4.20,21
Statistical analysis. Participants were defined as HIV sero-converters if they were tested seronegative at baseline (beginning of cross-sectional study) and then were found to be HIV seropositive at some point during the 24-month follow-up period. Participants were defined as non-HIV seroconverters if they were tested seronegative throughout the whole study period. The date of seroconversion was estimated using the time of last negative test during the baseline enrollment period in 1999 and the first positive test result in the cohort study. Person-years of observation were calculated by recording the total number of surveillance days for each participant. For individuals lost to follow-up, person-time denominator data was censored at the time of the last visit. Continuous variables were compared using the Student’s t-test or the Mann-Whitney U test. Kaplan-Meier survival analysis was applied to show and compare time to HIV seroconversion. The magnitude of associations of potential risk factors were expressed as hazard ratios. Cox proportional hazards regression analysis was performed to evaluate risk factors associated with HIV seroconversion. Ninety-five percent confidence intervals (CIs) were calculated for the incidence-density rates based on person-time denominator data by using Fisher’s exact formula. All hazard ratios were adjusted for age group and workplace in a multiple Cox regression analysis; all P values were two-sided and a P < 0.15 was considered to be marginally significant. All statistical analyses were carried out using EGRET version 2.0 (Cytel Software Corporation, Cambridge, MA) and SPSS version 10.1 (SPSS Corporation, Chicago, IL).
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RESULTS |
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Incidence-density rate analysis. The 60 male transvestite CSWs enrolled contributed a total of 99.5 person-years of observation. The mean ± SD duration of follow-up was 20 ± 2.4 months with a range from 14 to 24 months. Six (10%) participants seroconverted to HIV infection during the study follow-up period. The overall incidence-density rate was estimated at 6.03 (95% CI = 2.21–13.12) per 100-person years. The median time to HIV seroconversion was 23 months and all seroconversions occurred after the 18-month follow-up timeframe. Five (83%) of the six seroconverters reported being street-based with an observed incidence-density rate of 12.2 (95% CI = 2.4–17.2) per 100-person years.
Risk factor analysis.
All six seroconverters reported receiving money-for-sex. Two (33%) of them never used condoms with their steady partner, three (50%) reported using intra-nasal cocaine, and none gave a prior IDU history. The mean ± SD age in years of seroconverters was found to be slightly higher (but not statistically significant) than that for non-seroconverters (32.0 ± 7.6 versus 29.4 ± 8.7; P = 0.29). Four marginally-significant risk factors were found to be associated with HIV seroconversion by multiple Cox regression analysis after controlling for age and workplace (Table 1
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Genetic characterization.
The env HMA was completed on five (83.3%) of the six seroconverters’ strains; three were found to be subtype B and two were subtype F. Partial pol sequencing of these five strains, followed by phylogenetic analysis, confirmed that three were subtype B and two were BF recombinants (Figure 1). Both env HMA subtype F strains were found to be BF recombinants, as has been observed consistently in this region of the world. Full-genome analysis of all of the strains identified as subtype B in partial pol analysis confirmed that all were non-recombinant throughout the genome. One of the BF recombinant strains was sequenced in full and found to be a mosaic of subtypes B and F in an arrangement not yet documented elsewhere (Figure 2).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Available research seems to indicate that there is an elevated risk of HIV infection among male transvestite CSWs due to the myriad of high-risk sexual activities they engage in, as well the aforementioned inconsistent use of protective measures.7–9,22 We found suggestive evidence that inconsistent condom use during client sex (AHR = 6.7), during oral sex (AHR = 5.6), and at last sexual encounter (AHR = 7.8) increased the risk of HIV infection in this high-risk core group. The implementation of socially accepted HIV and STI counseling efforts, which includes condom use promotion, has been proven to effectively diminish high-risk, unprotected sexual behavior among MSM in the United States,23 as well as among female CSWs in Thailand.24 Similar interventional efforts appear to be needed in Uruguay at this time.
Marihuana consumption appears to have increased in recent years in Uruguay; general population sampling shows an increase from 4.2% in 1994 to 5.3% in 2001 (Viñoles J, unpublished data). It is possible that the five-fold increase in the incidence of HIV infection seen among marihuana users in this study may be related to an increase in associated high-risk behaviors, which may increase susceptibility to HIV as has been seen to occur in the past few years among injecting drug users in eastern Europe and the former Soviet Union.22,25
Recent increases in the incidence of HIV infections and STIs, such as hepatitis B, among populations of MSM have been noted in the United States in the past few years.26,27 It is very likely that a similar phenomenon may be taking place in Uruguay and other regions of South America where gay and male transvestite/transgendered communities exist and may contribute to the high HIV incidence noted in this population.
The HIV subtype analysis of five of the six seroincident cases showed that both subtype B and BF recombinants were circulating in this population. The BF recombinants have been previously documented in this region of the Southern Cone of South America;5,10,11 thus, their presence in Uruguay is not unexpected. At least one circulating recombinant form (CRF) has been identified in samples from both Argentina and Uruguay: CRF12_BF.28 However, the incident BF recombinant fully sequenced in this analysis is not the CRF12_BF previously described by our group, nor is it similar to previously identified BF recombinants.10,11,15
The relatively high HIV infection rate detected in this study among male transvestite CSWs (6.03 per 100 person-years) raises serious concerns regarding the progression of this epidemic in Uruguay and clearly demonstrates the need for conduction of future, larger prospective studies to better define sex- and drug-related transmission of HIV among male transvestite CSWs in this region. An increased emphasis in the area of HIV/STI prevention and education geared specifically to male transvestite CSWs and other transgendered populations is needed now.13 These high-risk core groups represent socially marginalized communities that are often ignored by public health authorities in the provision of prevention education and services for the control of HIV and other STIs.
Received August 24, 2004. Accepted for publication October 26, 2004.
Acknowledgments: We thank Gloria Chauca, Merly Sovero, and Maria Ester Gamero for their invaluable assistance with sample preparation instructions and handling. We also thank the administrative and field support personnel at the Uruguayan Ministry of Health for their invaluable time and advice.
Financial support: This work was supported by the U.S. Military HIV Research Program, Walter Reed Army Institute of Research (Rockville, MD) and by the U.S. Naval Medical Research Command (Silver Spring, MD) Work Unit No. (WUN) 62787A 873 H B0002.
Disclaimer: The opinions and assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the U.S. Department of the Navy or Army, the Department of Defense, or the Uruguayan Ministry of Health.
Author’s addresses: Jose Viñoles and Margarita Serra, HIV/AIDS National Control Program, Ministry of Health, Av. 18 de Julio 1892, 4to piso 11200, Montevideo, Uruguay. Jose C. Russi and Dora Ruchansky, Department of Laboratories of Public Health, Ministry of Health, Av. 8 de Octubre 2720 1er piso 11600, Montevideo, Uruguay, Telephone: 598-2-487-2516, Fax: 598-2-480-7014. Sergio Sosa-Estani and Mercedes Weissenbacher, Microbiology Department, School of Medicine, University of Buenos Aires, Paraguay 2155, Piso 11 (1121), Buenos Aires, Argentina, Telephone: 54-11-5950-9633 or 54-11-4508-3689, Fax: 54-11-4508-3705. Silvia M. Montano, Gladys Carrion, and James G. Olson, U.S. Naval Medical Research Center Detachment-Lima, Unit 3800, American Embassy-Lima, APO AA 34031-3800, Telephone: 51-1-561-2733, Fax: 51-1-561-3042. Lindsay M. Eyzaguirre, Jean K. Carr, Christian T. Bautista, and Jose L. Sanchez, U.S. Military HIV Research Program, Walter Reed Army Institute of Research and Henry M. Jackson Foundation, Rockville, Maryland. 1 Taft Court, Suite 250, Rockville, MD 20850, Telephone: 301-251-5077, Fax: 301-294-1898, E-mail: jsanchez{at}hivresearch.org.
Reprint requests: Jose L. Sanchez, U.S. Military HIV Research Program, Walter Reed Army Institute of Research, 1 Taft Court, Suite 250, Rockville, MD 20850.
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
