Although heterosexual contacts represent the main mode of HIV transmission in sub-Saharan Africa, recent reports showed the high vulnerability of HIV infection in men who have sex with men (MSM) in this region.1-6 In Africa, MSM are highly stigmatized and hard to reach because of criminalization of MSM in more than half of the African countries.2,7 Homosexuality is also illegal in Senegal, and male-to-male sex is generally condemned by political and religious authorities and by the general population.7 However, Senegal was the first African country to document6 vulnerability to HIV of MSM as shown through high HIV prevalence (21.5%) in comparison to the general population (1%-2%) (http://www.ancs.sn/Senegal-situation-epidemiologique.htm; in French). Interestingly, a high number of Senegalese MSM (>90%) also reported sexual activity with female partners, which is in accordance with subsequent studies on MSM in Africa.3,6
HIV is characterized by a high genetic variability, and the classification of HIV strains in groups, subtypes, and circulating recombinant forms has helped in tracking the course of the HIV pandemic and to monitor changing patterns.8 Up to now, no data on genetic diversity of HIV in MSM in Senegal and even in Africa are available. In this study, we analyzed the HIV-1 variants that circulate among MSM in Senegal.
MATERIALS AND METHODS
Specimen Collection, pol Region Amplification, and Sequencing
In our previous study, 96 HIV-1-positive samples were identified among 463 MSM. Details on the enrollment and data collection procedures were previously described.6 RNA was extracted from plasma obtained during the epidemiological survey among MSM in 2004 and stored since at −80°C with a QIAmp Viral RNA kit (Qiagen SA, Courtaboeuf, France). After reverse transcription, a nested polymerase chain reaction using IN3/G25rev as outer primers and AV150/PolM4 as inner primers was performed to amplify a fragment of approximately 1860 base pairs (bp) including the entire protease gene and the first 440 amino acids of reverse transcriptase as previously described.9 Polymerase chain reaction products were purified with the Geneclean Turbo Kit (MP Biomedicals, Illkirch, France) and directly sequenced with primers encompassing the pol fragment on an automatic Genetic analyzer (ABI 3130XL; Applied Biosystems, Courtaboeuf, France). Sequences were assembled using SeqMan II 5.08 from the DNAStar package (Lasergene, Konstanz, Germany).
Identification of HIV-1 Subtypes/Circulating Recombinant Forms (CRFs) and Phylogenetic Reconstruction of Transmission Clusters
The newly determined sequences were aligned with known representatives of the different subtypes/CRFs using ClustalX version 1.83. All pure subtypes, subsubtypes, and reference strains for the CRFs available in the Los Alamos database and described in west Africa (CRF01, CRF02, CRF06, CRF09, CRF11, CRF13, CRF18, CRF19, CRF25, CRF36, and CRF37) were included in the analysis. Sites with any gap between the sequences and areas of uncertain alignment were excluded from this analysis. For subtype/CRF designation, phylogenetic trees were constructed using the neighbor-joining method and the reliability of the branching orders was assessed with 100 bootstrap resamplings. To clearly identify the subtype/CRF, this phylogenetic analysis was done for each sequence individually and subtype/CRFs were assigned when bootstrap values were above 80%. The recombination analysis was done with the Simplot version 3.5.1 software (http://sray.med.som.jhmi.edu/SCRoftware) on each sequence. After gapstripping, similarity analyses followed by bootscanning were performed by moving a 400-bp window along the alignment with 20 bp increments and 100 replicates for each phylogeny.
Phylogenetic interrelationships among viral sequences were estimated by the maximum likelihood (ML) heuristic approach using PHYML version 2.4.4 integrated molecular evolutionary genetics analysis software as previously reported.10 The trees were constructed under the Hasegawa-Kishino-Yano (HKY85) model of nucleotide substitution, including gamma-distributed rate heterogeneity among sites. ML bootstrap values were obtained for 1000 replicates. The existence of transmission clusters was ascertained using the statistical robustness of the ML topologies based on high bootstrap values (>98%) with 1000 resamplings and short branch lengths (genetic distances inferior or equal 0.015) of HIV-1 pol gene sequences following the established criteria defined in previous studies.11-13
Genbank Accession Numbers
pol sequences have been submitted to European Molecular Biology Laboratory under the following accession numbers: FM210684 to FM210753.
The general characteristics and factors associated with HIV infection among MSM have been described in detail in the initial study conducted in 2004.6 In our previous study, 96 HIV-1-positive samples were identified among 463 MSM, and our study focused on 70 individuals for whom sufficient plasma was available for subsequent genetic characterization. These samples were derived from MSM from 4 urban sites throughout Senegal: Dakar (n = 51), Mbour (n = 6), Saint-Louis (n = 4) and Thies (n = 9). Sixty-seven (95%) were from Senegalese MSM, mainly unmarried (90%) with a median age of 30 years (range 18-43 years), and 75% of them were less than 30 years. Fifty-five MSM (78.6%) had regular male partners, 63 (90%) reported regular Senegalese partners, but 23 (32.8%), 3 (4.2%), 8 (11.4%), and 2 (2.8%) reported also regular European, American, Lebanese, or Asian male partners, respectively. Interestingly, 39 (55.7%) had also a regular female partner and 82% reported heterosexual contacts.
Subtype/CRF Distribution and Genetic Diversity of HIV-1 Among MSM in Senegal
Phylogenetic and recombination analyses revealed that the overall subtype/CRF distribution among the 70 MSM was as follows: 28 C (40%), 17 CRF02_AG (24.3%), 13 B (18.6%), 6 G (8.6%), 3 CRF09_cpx (4.3%), and 3 (4.3%) unique recombinants (G/CRF02, CRF02/CRF06/CRF09 and CRF02/CRF06/CRF02/CRF06) as shown in Figure 1. There were no significant linkages with any HIV-1 variant pattern and origin of partners; for example, among the predominant variants (C, CRF02, and B), the number of individuals reporting contacts with Europeans and/or Americans was comparable (31%-38%).
The phylogenetic tree showed also several clusters of closely related sequences. Of the 70 pol sequences analyzed, 47 (67.15%) were segregated into 15 clusters, which represent each a transmission cluster based on sequence similarity, high bootstrap values (>98%), and short genetic distances (<0.015). The remaining 23 cases (32.85%) represented nonclustered sequences in the ML pol tree. The transmission clusters were observed among all subtypes or CRFs documented in this MSM population. Six of 15 clusters belonged to subtype C, 4 to CRF02_AG, 3 to subtype B, and 1 for G and CRF09_cpx each. Five clusters with 4-6 individuals were observed, and the remaining had 2-3 individuals per cluster. Interestingly, in 3 of the 15 transmission chains, individuals from 2 different urban sites were present: in cluster 4, 2 individuals were from Dakar and 4 from Mbour (80 km distance); transmission clusters 5 and 12 are represented by 1 individual from Dakar and 1 from Thies (70 km distance) and 1 from Dakar and 1 from Saint-Louis (265 km distance), respectively.
Phylogenetic analysis showed a high genetic diversity of HIV-1 among MSM in Senegal with the predominance of subtype C (40%), followed by CRF02_AG (24.3%) then subtype B (18.6%), and a small proportion of individuals were infected with subtype G, CRF09_cpx, and unique recombinants. Although all these HIV-1 variants have been previously reported to circulate among the general population or female sex workers (FSWs) in Senegal, the proportions are significantly different. Previous studies in the general population and among FSWs showed the predominance of CRF02_AG in Senegal, together with the cocirculation of a high number of different variants, although at low rates each, for example, A3, B, C, D, G, CRF06_cpx, and CRF09_cpx.14,15 The most striking differences were for subtype C with 40% versus <5%, subtype B with 18% versus 3%-6%, and CRF02 with 24% versus 50%-70%, in our MSM population versus the general population and FSWs, respectively.
The phylogenetic tree showed also several clusters of closely related sequences. Recent studies showed that phylogenetic analysis of viral pol gene sequences can be used to reconstruct transmission events in geographically defined populations with acute/primary or chronic HIV-1 infection.11-13 Due to the special procedure, which ensured participants' safety and anonymity, we used this approach and identified 15 transmission clusters that were present among all subtypes or CRFs documented in this MSM population. The high number of transmission chains in this limited sample size among MSM could represent a bias related to convenience sampling, and it is likely that those who were more open about their sexual orientation, more active in networks of MSM and with high-risk behaviors, participated first in the study. Therefore, our findings can also not be extrapolated to the country. The fact that in 3 of the 15 clusters individuals from 2 different sites were present suggests that viruses could spread rapidly and intermix among MSM networks from different cities.
In conclusion, despite the massive stigma, it is important to note that MSM are not completely isolated from the general population because more than 80% recognized having sex with women, which might modify HIV-1 subtype/CRF distribution in this low-risk population. Although the factors associated with the rapid spread of subtype C and its predominance in the global epidemic are not entirely known, the high prevalence among MSM could also lead to an increase over time of subtype C in the general population. HIV_1 subtype/CRF distribution can change over time as shown in FSWs in Senegal; actually, subsubtype A3 predominates in new infections.16 Regular sentinel surveys to monitor HIV prevalences and distribution of HIV-1 variants among different population groups are recommended in order to follow-up whether subtype C will increase in other population groups. Taking into account the high prevalence of HIV in MSM, their unsafe sexual relationship, and their contact with women, an enhanced nationwide HIV/AIDS education campaign is also urgently needed for these groups. These interventions may reduce stigma, increase diagnosis and access to treatment, and eventually hold back the epidemic.
1. Adebajo S, Meyers T, Allman D, et al. HIV and sexually transmitted infections among men who have sex with men (MSM) in Nigeria. Presented at: XVII International AIDS Conference; August 3-8, 2008; Mexico. Abstract MOPE0411.
2. Baral S, Sifakis F, Cleghorn F, et al. Elevated risk for HIV infection among men who have sex with men in low- and middle-income countries 2000-2006: a systematic review [review]. PLoS Med
3. Cáceres CF, Konda K, Segura ER, et al. Epidemiology of male same-sex behaviour and associated sexual health indicators in low- and middle-income countries: 2003-2007 estimates [review]. Sex Transm Infect
. 2008;84(Suppl 1):i49-i56.
4. Sanders EJ, Graham SM, Okuku HS, et al. HIV-1 infection in high risk men who have sex with men in Mombasa, Kenya. AIDS
5. Umar E, Trapence G, Chibwezo W, et al. A cross-sectional evaluation of the HIV prevalence and HIV-related risk factors of men who have sex with men (MSM) in Malawi. Presented at: XVII International AIDS Conference; August 3-8, 2008; Mexico. Abstract MOPE0412.
6. Wade AS, Kane CT, Diallo PA, et al. HIV infection and sexually transmitted infections among men who have sex with men in Senegal. AIDS
7. Niang CI, Tapsoba P, Weiss E, et al. "It's raining stones'': stigma, violence and HIV vulnerability among men who have sex with men in Dakar, Senegal. Cult Health Sex
8. McCutchan FE. Global epidemiology of HIV. J Med Virol
. 2006;78(Suppl 1):S7-S12.
9. Vergne L, Kane CT, Laurent C, et al. Low rate of genotypic HIV-1 drug-resistant strains in the Senegalese government initiative of access to antiretroviral therapy. AIDS
. 2003;17(Suppl 3):S31-S8.
10. Guindon S, Gascuel O. A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol
11. Hue S, Pillay D, Clewley JP, et al. Genetic analysis reveals the complex structure of HIV-1 transmission within defined risk groups. Proc Natl Acad Sci U S A
12. Brenner BG, Roger M, Routy JP, et al. Quebec Primary HIV Infection Study Group. High rates of forward transmission events after acute/early HIV-1 infection. J Infect Dis
13. Bao L, Vidal N, Fang H, et al. Molecular tracing of sexual HIV type 1 transmission in the southwest border of China. AIDS Res Hum Retroviruses
14. Toure-Kane C, Montavon C, Faye MA, et al. Identification of all HIV type 1 group M subtypes in Senegal, a country with low and stable seroprevalence. AIDS Res Hum Retroviruses
15. Meloni ST, Sankalé JL, Hamel DJ, et al. Molecular epidemiology of human immunodeficiency virus type 1 sub-subtype A3 in Senegal from 1988 to 2001. J Virol
16. Hamel DJ, Sankalé JL, Eisen G, et al. Twenty years of prospective molecular epidemiology in Senegal: changes in HIV diversity. AIDS Res Hum Retroviruses