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1.  Hormonal Contraception and the Risk of HIV Acquisition: An Individual Participant Data Meta-analysis 
PLoS Medicine  2015;12(1):e1001778.
In a meta-analysis of individual participant data, Charles Morrison and colleagues explore the association between hormonal contraception use and risk of HIV infection in sub-Saharan Africa.
Observational studies of a putative association between hormonal contraception (HC) and HIV acquisition have produced conflicting results. We conducted an individual participant data (IPD) meta-analysis of studies from sub-Saharan Africa to compare the incidence of HIV infection in women using combined oral contraceptives (COCs) or the injectable progestins depot-medroxyprogesterone acetate (DMPA) or norethisterone enanthate (NET-EN) with women not using HC.
Methods and Findings
Eligible studies measured HC exposure and incident HIV infection prospectively using standardized measures, enrolled women aged 15–49 y, recorded ≥15 incident HIV infections, and measured prespecified covariates. Our primary analysis estimated the adjusted hazard ratio (aHR) using two-stage random effects meta-analysis, controlling for region, marital status, age, number of sex partners, and condom use. We included 18 studies, including 37,124 women (43,613 woman-years) and 1,830 incident HIV infections. Relative to no HC use, the aHR for HIV acquisition was 1.50 (95% CI 1.24–1.83) for DMPA use, 1.24 (95% CI 0.84–1.82) for NET-EN use, and 1.03 (95% CI 0.88–1.20) for COC use. Between-study heterogeneity was mild (I2 < 50%). DMPA use was associated with increased HIV acquisition compared with COC use (aHR 1.43, 95% CI 1.23–1.67) and NET-EN use (aHR 1.32, 95% CI 1.08–1.61). Effect estimates were attenuated for studies at lower risk of methodological bias (compared with no HC use, aHR for DMPA use 1.22, 95% CI 0.99–1.50; for NET-EN use 0.67, 95% CI 0.47–0.96; and for COC use 0.91, 95% CI 0.73–1.41) compared to those at higher risk of bias (pinteraction = 0.003). Neither age nor herpes simplex virus type 2 infection status modified the HC–HIV relationship.
This IPD meta-analysis found no evidence that COC or NET-EN use increases women’s risk of HIV but adds to the evidence that DMPA may increase HIV risk, underscoring the need for additional safe and effective contraceptive options for women at high HIV risk. A randomized controlled trial would provide more definitive evidence about the effects of hormonal contraception, particularly DMPA, on HIV risk.
Editors’ Summary
AIDS has killed about 36 million people since the first recorded case of the disease in 1981. About 35 million people (including 25 million living in sub-Saharan Africa) are currently infected with HIV, the virus that causes AIDS, and every year, another 2.3 million people become newly infected with HIV. At the beginning of the epidemic, more men than women were infected with HIV. Now, about half of all adults infected with HIV are women. In 2013, almost 60% of all new HIV infections among young people aged 15–24 years occurred among women, and it is estimated that, worldwide, 50 young women are newly infected with HIV every hour. Most women become infected with HIV through unprotected intercourse with an infected male partner—biologically, women are twice as likely to become infected through unprotected intercourse as men. A woman’s risk of becoming infected with HIV can be reduced by abstaining from sex, by having one or a few partners, and by always using condoms.
Why Was This Study Done?
Women and societies both benefit from effective contraception. When contraception is available, women can avoid unintended pregnancies, fewer women and babies die during pregnancy and childbirth, and maternal and infant health improves. However, some (but not all) observational studies (investigations that measure associations between the characteristics of participants and their subsequent development of specific diseases) have reported an association between hormonal contraceptive use and an increased risk of HIV acquisition by women. So, does hormonal contraception increase the risk of HIV acquisition among women or not? Here, to investigate this question, the researchers undertake an individual participant data meta-analysis of studies conducted in sub-Saharan Africa (a region where both HIV infection and unintended pregnancies are common) to compare the incidence of HIV infection (the number of new cases in a population during a given time period) among women using and not using hormonal contraception. Meta-analysis is a statistical method that combines the results of several studies; an individual participant data meta-analysis combines the data recorded for each individual involved in the studies rather than the aggregated results from each study.
What Did the Researchers Do and Find?
The researchers included 18 studies that measured hormonal contraceptive use and incident HIV infection among women aged 15–49 years living in sub-Saharan Africa in their meta-analysis. More than 37,000 women took part in these studies, and 1,830 became newly infected with HIV. Half of the women were not using hormonal contraception, a quarter were using depot-medroxyprogesterone acetate (DMPA; an injectable hormonal contraceptive), and the remainder were using combined oral contraceptives (COCs) or norethisterone enanthate (NET-EN, another injectable contraceptive). After adjustment for other factors likely to influence HIV acquisition (for example, condom use), women using DMPA had a 1.5-fold increased risk of HIV acquisition compared to women not using hormonal contraception. There was a slightly increased risk of HIV acquisition among women using NET-EN compared to women not using hormonal contraception, but this increase was not statistically significant (it may have happened by chance alone). There was no increased risk of HIV acquisition associated with COC use. DMPA use was associated with a 1.43-fold and 1.32-fold increased risk of HIV acquisition compared with COC and NET-EN use, respectively. Finally, neither age nor herpes simplex virus 2 infection status modified the effect of hormonal contraceptive use on HIV acquisition.
What Do These Findings Mean?
The findings of this individual patient data meta-analysis provide no evidence that COC or NET-EN use increases a woman’s risk of acquiring HIV, but add to the evidence suggesting that DMPA use increases the risk of HIV acquisition. These findings are likely to be more accurate than those of previous meta-analyses that used aggregated data but are likely to be limited by the quality, design, and representativeness of the studies included in the analysis. These findings nevertheless highlight the need to develop additional safe and effective contraceptive options for women at risk of HIV, particularly those living in sub-Saharan Africa, where although contraceptive use is generally low, DMPA is the most widely used hormonal contraceptive. In addition, these findings highlight the need to initiate randomized controlled trials to provide more definitive evidence of the effects of hormonal contraception, particularly DMPA, on HIV risk.
Additional Information.
Please access these websites via the online version of this summary at
Information is available from the US National Institute of Allergy and Infectious Diseases on HIV infection and AIDS
NAM/aidsmap provides basic information about HIV/AIDS, and summaries of recent research findings on HIV care and treatment, including personal stories about living with HIV/AIDS and a news report on this meta-analysis
Information is available from Avert, an international AIDS charity, on many aspects of HIV/AIDS, including detailed information on women, HIV, and AIDS, and on HIV and AIDS in South Africa (in English and Spanish); personal stories of women living with HIV are available
The World Health Organization provides information on all aspects of HIV/AIDS (in several languages); information about a 2012 WHO technical consultation about hormonal contraception and HIV
The 2013 UNAIDS World AIDS Day report provides up-to-date information about the AIDS epidemic and efforts to halt it; UNAIDS also provides information about HIV and hormonal contraception
PMCID: PMC4303292  PMID: 25612136
2.  Impact of asymptomatic Herpes simplex virus-2 infection on T cell function in the foreskin of men from Rakai, Uganda 
AIDS (London, England)  2012;26(10):1319-1322.
Herpes simplex type 2 (HSV-2) increases the risk of HIV acquisition in men and overall CD4 T cell density in the foreskin. Using tissues obtained during routine male circumcision, we examined the impact of HSV-2 on the function and phenotype of foreskin T cells in Ugandan men. HSV-2 infection was predominantly associated with a compartmentalized increase in CCR5 expression by foreskin CD4 T cells, which may contribute to HIV susceptibility.
PMCID: PMC4241749  PMID: 22516874
HSV-2; HIV; foreskin; T cells
3.  Effect of Antiretroviral Therapy on HIV Reservoirs in Elite Controllers 
The Journal of Infectious Diseases  2013;208(9):1443-1447.
Elite controllers suppress human immunodeficiency virus (HIV) viremia to below the limit of detection in the absence of antiretroviral therapy (ART). However, precise frequencies of CD4+ T cells carrying replication-competent HIV and/or the dynamics of the infectious viral reservoirs in response to initiation and discontinuation of ART in elite controllers are unknown. We show that the size of the pool of CD4+ T cells harboring infectious HIV diminished significantly after initiation of ART and rebounded to baseline upon cessation of therapy. Our data provide compelling evidence that persistent viral replication occurs in untreated elite controllers even in the absence of detectable plasma viremia.
PMCID: PMC3789563  PMID: 23847057
human immunodeficiency virus; viral reservoirs; antiretroviral therapy; elite controllers
4.  HIV Acquisition Is Associated with Increased Antimicrobial Peptides and Reduced HIV Neutralizing IgA in the Foreskin Prepuce of Uncircumcised Men 
PLoS Pathogens  2014;10(10):e1004416.
The foreskin is the site of most HIV acquisition in uncircumcised heterosexual men. Although HIV-exposed, seronegative (HESN) uncircumcised men demonstrate HIV-neutralizing IgA and increased antimicrobial peptides (AMPs) in the foreskin prepuce, no prospective studies have examined the mucosal immune correlates of HIV acquisition.
To assess the association of foreskin immune parameters with HIV acquisition, antimicrobial peptides and IgA with the capacity to neutralize a primary clade C HIV strain were quantified by blinded investigators, using sub-preputial swabs collected longitudinally during a randomized trial of male circumcision for HIV prevention in Rakai, Uganda.
Participants were 99 men who acquired HIV (cases) and 109 randomly selected controls who remained HIV seronegative. At enrollment, 44.4% of cases vs. 69.7% of controls demonstrated IgA neutralization (adjusted OR = 0.31; 95% CI, 0.16–0.61). IgA neutralization was detected in 38.7% of cases and 70.7% of controls at the last seronegative case visit prior to HIV acquisition and the comparable control visit (adjusted OR 0.21; 95% CI, 0.11–0.39). Levels of the α-defensins and secretory leukocyte protease inhibitor (SLPI) were over ten-fold higher in the foreskin prepuce of cases who acquired HIV, both at enrollment (mean 4.43 vs. 3.03 and 5.98 vs. 4.61 logn pg/mL, P = 0.005 and 0.009, respectively), and at the last seronegative visit (mean 4.81 vs. 3.15 and 6.46 vs. 5.20 logn pg/mL, P = 0.0002 and 0.013).
This prospective, blinded analysis is the first to assess the immune correlates of HIV acquisition in the foreskin. HIV-neutralizing IgA, previously associated with the HESN phenotype, was a biomarker of HIV protection, but other HESN associations correlated with increased HIV acquisition. This emphasizes the importance of prospective epidemiological studies or in vitro tissue studies to define the impact of mucosal parameters on HIV risk.
Author Summary
Randomized trials of male circumcision (MC) for HIV prevention have shown that the foreskin is a major site of HIV acquisition among heterosexual men, but a number of barriers to MC programs mean that many HIV-susceptible men remain uncircumcised. The immune correlates of HIV acquisition in the foreskin are poorly described, and may inform prevention strategies for uncircumcised men. Using swabs collected prospectively during a previous randomized trial of MC for HIV prevention in Uganda, blinded investigators assessed levels of HIV-neutralizing IgA and soluble antimicrobial peptides in the foreskin prepuce of cases who acquired HIV (n = 99) and controls men who did not (n = 109). We show that IgA with the capacity to neutralize HIV was associated with protection against HIV, while levels of α-defensins and secretory leukocyte protease inhibitor (SLPI) were associated with increased acquisition. This is the first study to assess the immune correlates of HIV acquisition in the foreskin. Advantages included its relatively large sample size, rigorous blinding of immune assays, and ability to control for relevant potential confounders.
PMCID: PMC4183701  PMID: 25275513
5.  HIV Transmission among Men Who Have Sex with Men due to Condom Failure 
PLoS ONE  2014;9(9):e107540.
Despite preventive efforts, HIV incidence remains high among men who have sex with men (MSM) in industrialized countries. Condoms are an important element in prevention but, given the high frequency of condom use and their imperfect effectiveness, a substantial number and proportion of HIV transmissions may occur despite condoms. We developed a model to examine this hypothesis.
We used estimates of annual prevalent and incident HIV infections for MSM in Ontario. For HIV-negative men, we applied frequencies of sexual episodes and per-contact HIV transmission risks of receptive and insertive anal sex with and without a condom and oral sex without a condom. We factored in the proportion of HIV-infected partners receiving antiretroviral therapy and its impact in reducing transmissibility. We used Monte-Carlo simulation to determine the plausible range for the proportion of HIV transmissions for each sexual practice.
Among Ontario MSM in 2009, an estimated 92,963 HIV-negative men had 1,184,343 episodes of anal sex with a condom and 117,133 anal sex acts without a condom with an HIV-positive partner. Of the 693 new HIV infections, 51% were through anal sex with a condom, 33% anal sex without a condom and 16% oral sex. For anal sex with a condom, the 95% confidence limits were 17% and 77%.
The proportion of HIV infections related to condom failure appears substantial and higher than previously thought. That 51% of transmissions occur despite condom use may be conservative (i.e. low) since we used a relatively high estimate (87.1%) for condom effectiveness. If condom effectiveness were closer to 70%, a value estimated from a recent CDC study, the number and proportion of HIV transmissions occurring despite condom use would be much higher. Therefore, while condom use should continue to be promoted and enhanced, this alone is unlikely to stem the tide of HIV infection among MSM.
PMCID: PMC4161430  PMID: 25211493
6.  The Semen Microbiome and Its Relationship with Local Immunology and Viral Load in HIV Infection 
PLoS Pathogens  2014;10(7):e1004262.
Semen is a major vector for HIV transmission, but the semen HIV RNA viral load (VL) only correlates moderately with the blood VL. Viral shedding can be enhanced by genital infections and associated inflammation, but it can also occur in the absence of classical pathogens. Thus, we hypothesized that a dysregulated semen microbiome correlates with local HIV shedding. We analyzed semen samples from 49 men who have sex with men (MSM), including 22 HIV-uninfected and 27 HIV-infected men, at baseline and after starting antiretroviral therapy (ART) using 16S rRNA gene-based pyrosequencing and quantitative PCR. We studied the relationship of semen bacteria with HIV infection, semen cytokine levels, and semen VL by linear regression, non-metric multidimensional scaling, and goodness-of-fit test. Streptococcus, Corynebacterium, and Staphylococcus were common semen bacteria, irrespective of HIV status. While Ureaplasma was the more abundant Mollicutes in HIV-uninfected men, Mycoplasma dominated after HIV infection. HIV infection was associated with decreased semen microbiome diversity and richness, which were restored after six months of ART. In HIV-infected men, semen bacterial load correlated with seven pro-inflammatory semen cytokines, including IL-6 (p = 0.024), TNF-α (p = 0.009), and IL-1b (p = 0.002). IL-1b in particular was associated with semen VL (r2 = 0.18, p = 0.02). Semen bacterial load was also directly linked to the semen HIV VL (r2 = 0.15, p = 0.02). HIV infection reshapes the relationship between semen bacteria and pro-inflammatory cytokines, and both are linked to semen VL, which supports a role of the semen microbiome in HIV sexual transmission.
Author Summary
The classical paradigm of HIV infectivity centers on the blood HIV RNA viral load. However, while other fluid compartments such as semen and cerebrospinal fluid can have distinct viral loads from blood, the causes of localized HIV shedding are not fully understood. Since the semen viral load is an independent predictor of HIV transmission risk, it is critical to understand the local factors that trigger increased semen viral shedding in order to develop novel preventative strategies. Here, we evaluated the semen microbiome, bacterial load, and cytokine levels in 22 HIV-uninfected men who have sex with men (MSM) and in 27 HIV-infected MSM before and after initiation of antiretroviral therapy (ART). We found that HIV infection reduces semen microbiome biodiversity, which is restored with ART and immune reconstitution. We also found that semen bacterial load in untreated, HIV-infected men is associated with the levels of seven semen cytokines, relationships not seen in the uninfected controls. In particular, the cytokine IL-1b was uniquely correlated with both semen bacterial and viral load. Our findings support the interaction between semen microbiome and local immunology, and suggest that IL-1b could be a mechanism for semen microbiome to trigger semen viral shedding.
PMCID: PMC4110035  PMID: 25058515
7.  Presence of CD8+ T Cells in the Ectocervical Mucosa Correlates with Genital Viral Shedding in HIV-Infected Women despite a Low Prevalence of HIV RNA–Expressing Cells in the Tissue 
The female genital tract is a portal of entry for sexual HIV transmission and a possible viral reservoir. In this study, the ectocervical CD8+ T cell distribution was explored in situ and was related to expression of CD3 and HLA-DR and presence of HIV RNA. For this purpose, ectocervical tissue samples and genital secretions were collected from HIV-seropositive (HIV+) Kenyan female sex workers (FSWs) (n = 20), HIV-seronegative (HIV−) FSWs (n = 17), and HIV− lower-risk women (n = 21). Cell markers were assessed by in situ staining and by quantitative PCR. HIV RNA expression in tissue was analyzed by in situ hybridization, and viral shedding was assessed by quantitative PCR. The HIV+FSW group had a higher amount of total cells and CD8+, CD3+, and HLA-DR+ cells compared with the HIV−FSW group and HIV− lower-risk women. The majority of CD8+ cells were CD3+ T cells, and the numbers of CD8+ cells correlated significantly with plasma and cervical viral load. HIV RNA expression in situ was found in 4 of the 20 HIV+FSW women but did not correlate with cervical or plasma viral load. Thus, the HIV+ women displayed high numbers of CD8+, CD3+, and HLA-DR+ cells, as well as a limited number of HIV RNA+ cells, in their ectocervical mucosa; hence, this localization cannot be neglected as a potential viral reservoir. The elevated levels of CD8+ T cells may play a role in the immunopathogenesis of HIV in the female genital tract.
PMCID: PMC4098133  PMID: 24639358
8.  Epigenetic analysis of HIV-1 proviral genomes from infected individuals: Predominance of unmethylated CpG’s 
Virology  2013;449:181-189.
Efforts to cure HIV-1 infections aim at eliminating proviral DNA. Integrated DNA from various viruses often becomes methylated de novo and transcriptionally inactivated. We therefore investigated CpG methylation profiles of 55 of 94 CpG’s (58.5%) in HIV-1 proviral genomes including ten CpG’s in each LTR and additional CpG’s in portions of gag, env, nef, rev, and tat genes. We analyzed 33 DNA samples from PBMC’s of 23 subjects representing a broad spectrum of HIV-1 disease. In 22 of 23 HIV-1-infected individuals, there were only unmethylated CpG’s regardless of infection status. In one long term nonprogressor, however, methylation of proviral DNA varied between 0 and 75% over an 11-year period although the CD4+ counts remained stable. Hence levels of proviral DNA methylation can fluctuate. The preponderance of unmethylated CpG’s suggests that proviral methylation is not a major factor in regulating HIV-1 proviral activity in PBMC’s. Unmethylated CpG’s may play a role in HIV-1 immunopathogenesis.
PMCID: PMC4060985  PMID: 24418551
Epigenetics of HIV-1 proviral DNA; Integrated HIV-1 DNA in PBMC’s from infected individuals; Wide spectrum of infection outcome; Bisulfite sequencing; Methylation analysis of integrated HIV-1 genomes; Predominance of unmethylated CpG’s in PBMC’s; Escape from proviral DNA methylation; Fluctuation of CpG methylation in one LTNP individual
9.  Canadian consensus statement on HIV and its transmission in the context of criminal law 
Many individuals involved in the legal process regarding prosecution of HIV-positive individuals who do not disclose their HIV status lack a thorough understanding of the risk for HIV transmission in different circumstances; this lack of knowledge may have a significant effect on the application of the criminal law to individuals with HIV. This consensus statement, intended as a reference for individuals involved in the criminal justice system and endorsed by >70 leading HIV experts in Canada, summarizes the risks for transmission of HIV under various circumstances, on a continuum ranging from low possibility to zero possibility of transmission.
A poor appreciation of the science related to HIV contributes to an overly broad use of the criminal law against individuals living with HIV in cases of HIV nondisclosure.
To promote an evidence-informed application of the law in Canada, a team of six Canadian medical experts on HIV and transmission led the development of a consensus statement on HIV sexual transmission, HIV transmission associated with biting and spitting, and the natural history of HIV infection. The statement is based on a literature review of the most recent and relevant scientific evidence (current as of December 2013) regarding HIV and its transmission. It has been endorsed by >70 additional Canadian HIV experts and the Association of Medical Microbiology and Infectious Disease Canada.
Scientific and medical evidence clearly indicate that HIV is difficult to transmit during sex. For the purpose of informing the justice system, the per-act possibility of HIV transmission through sex, biting or spitting is described along a continuum from low possibility, to negligible possibility, to no possibility of transmission. This possibility takes into account the impact of factors such as the type of sexual acts, condom use, antiretroviral therapy and viral load. Dramatic advances in HIV therapy have transformed HIV infection into a chronic manageable condition.
HIV physicians and scientists have a professional and ethical responsibility to assist those in the criminal justice system to understand and interpret the science regarding HIV. This is critical to prevent miscarriage of justice and to remove unnecessary barriers to evidence-based HIV prevention strategies.
PMCID: PMC4173974  PMID: 25285108
Chronic manageable condition; Consensus statement; Criminal law; HIV risks of transmission
10.  IL-10-Producing B Cells Are Induced Early in HIV-1 Infection and Suppress HIV-1-Specific T Cell Responses 
PLoS ONE  2014;9(2):e89236.
A rare subset of IL-10-producing B cells, named regulatory B cells (Bregs), suppresses adaptive immune responses and inflammation in mice. In this study, we examined the role of IL-10-producing B cells in HIV-1 infection. Compared to uninfected controls, IL-10-producing B cell frequencies were elevated in both blood and sigmoid colon during the early and chronic phase of untreated HIV-1 infection. Ex vivo IL-10-producing B cell frequency in early HIV-1 infection directly correlated with viral load. IL-10-producing B cells from HIV-1 infected individuals were enriched in CD19+TIM-1+ B cells and were enriched for specificity to trimeric HIV-1 envelope protein. Anti-retroviral therapy was associated with reduced IL-10-producing B cell frequencies. Treatment of B cells from healthy donors with microbial metabolites and Toll-like receptor (TLR) agonists could induce an IL-10 producing phenotype, suggesting that the elevated bacterial translocation characteristic of HIV-1 infection may promote IL-10-producing B cell development. Similar to regulatory B cells found in mice, IL-10-producing B cells from HIV-1-infected individuals suppressed HIV-1-specific T cell responses in vitro, and this suppression is IL-10-dependent. Also, ex vivo IL-10-producing B cell frequency inversely correlated with contemporaneous ex vivo HIV-1-specific T cell responses. Our findings show that IL-10-producing B cells are induced early in HIV-1 infection, can be HIV-1 specific, and are able to inhibit effective anti-HIV-1 T cell responses. HIV-1 may dysregulate B cells toward Bregs as an immune evasion strategy.
PMCID: PMC3931714  PMID: 24586620
11.  Antiretroviral therapy is not associated with reduced herpes simplex virus shedding in HIV coinfected adults: an observational cohort study 
BMJ Open  2014;4(1):e004210.
Herpes simplex virus types 1 and 2 (HSV-1/2) may have adverse consequences on HIV type 1 infection. We quantified the frequency of HSV reactivations in highly active antiretroviral therapy (HAART)-treated adults with HIV, and compared it with that in HAART-naïve patients.
2 academic hospital sites in Toronto, Canada.
Asymptomatic HAART-naive (n=44) or treated (with HIV RNA <50 copies/mL, n=41) adults with HSV-1 and/or 2, HIV coinfection.
Outcome measures
HSV-1 and HSV-2 shedding as measured by PCR on oral, genital and anal swabs self-collected daily for 28 days.
Of the 85 participants, 88%, 67% and 53% were coinfected with HSV-1, HSV-2 and both HSV types, respectively. Median (IQR) CD4 count was 516 (382, 655) cells/mm3. HSV (type 1 and/or 2) shedding occurred on a median (IQR) of 7.1% (0, 17.9%) of days in HAART users and 3.6% (0, 10.7%) of days in non-HAART users. No significant relationship was observed between HAART and HSV-1/2 shedding in univariable (OR=1.55, 95% CI 0.83 to 2.87) or multivariable negative binomial models adjusted for sex, baseline CD4 count, recent immigrant status and time since HIV diagnosis (adjusted OR, aOR=1.05, 95% CI 0.43 to 2.58). Similar null results were observed for HSV-2 shedding in HSV-2 seropositive participants (aOR=1.16, 95% CI 0.40 to 3.36) and HSV-1 shedding in HSV-1 seropositive participants (aOR=0.70, 95% CI 0.14 to 3.47).
HSV reactivations persist despite suppressive HAART among adults coinfected with HSV and HIV. Clinical trials of suppressive anti-HSV therapy are warranted in this population.
PMCID: PMC3902380  PMID: 24464523
Virology; Sexual Medicine
12.  Optimizing Viable Leukocyte Sampling from the Female Genital Tract for Clinical Trials: An International Multi-Site Study 
PLoS ONE  2014;9(1):e85675.
Functional analysis of mononuclear leukocytes in the female genital mucosa is essential for understanding the immunologic effects of HIV vaccines and microbicides at the site of HIV exposure. However, the best female genital tract sampling technique is unclear.
Methods and Findings
We enrolled women from four sites in Africa and the US to compare three genital leukocyte sampling methods: cervicovaginal lavages (CVL), endocervical cytobrushes, and ectocervical biopsies. Absolute yields of mononuclear leukocyte subpopulations were determined by flow cytometric bead-based cell counting. Of the non-invasive sampling types, two combined sequential cytobrushes yielded significantly more viable mononuclear leukocytes than a CVL (p<0.0001). In a subsequent comparison, two cytobrushes yielded as many leukocytes (∼10,000) as one biopsy, with macrophages/monocytes being more prominent in cytobrushes and T lymphocytes in biopsies. Sample yields were consistent between sites. In a subgroup analysis, we observed significant reproducibility between replicate same-day biopsies (r = 0.89, p = 0.0123). Visible red blood cells in cytobrushes increased leukocyte yields more than three-fold (p = 0.0078), but did not change their subpopulation profile, indicating that these leukocytes were still largely derived from the mucosa and not peripheral blood. We also confirmed that many CD4+ T cells in the female genital tract express the α4β7 integrin, an HIV envelope-binding mucosal homing receptor.
CVL sampling recovered the lowest number of viable mononuclear leukocytes. Two cervical cytobrushes yielded comparable total numbers of viable leukocytes to one biopsy, but cytobrushes and biopsies were biased toward macrophages and T lymphocytes, respectively. Our study also established the feasibility of obtaining consistent flow cytometric analyses of isolated genital cells from four study sites in the US and Africa. These data represent an important step towards implementing mucosal cell sampling in international clinical trials of HIV prevention.
PMCID: PMC3893217  PMID: 24454917
13.  Correction: Enumeration of Sex Workers in the Central Business District of Nairobi, Kenya 
PLoS ONE  2013;8(11):10.1371/annotation/29718092-c94c-4c98-b5a8-253a6252493a.
PMCID: PMC3847187
14.  The epidemiology of sexually transmitted co-infections in HIV-positive and HIV-negative African-Caribbean women in Toronto 
BMC Infectious Diseases  2013;13:550.
HIV disproportionately affects African-Caribbean women in Canada but the frequency and distribution of sexually transmitted infections in this community have not been previously studied.
We recruited women based on HIV status through a Toronto community health centre. Participants completed a socio-behavioural questionnaire using Audio Computer Assisted Self-Interview (ACASI) and provided blood for syphilis, HIV, hepatitis B and C, herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), and human cytomegalovirus (CMV) serology, urine for chlamydia and gonorrhea molecular testing and vaginal secretions for bacterial vaginosis (BV) and human papillomavirus (HPV). Differences in prevalence were assessed for statistical significance using chi-square.
We recruited 126 HIV-positive and 291 HIV-negative women, with a median age of 40 and 31 years, respectively (p < 0.001). Active HBV infection and lifetime exposure to HBV infection were more common in HIV-positive women (4.8% vs. 0.34%, p = 0.004; and 47.6% vs. 21.2%, p < 0.0001), as was a self-reported history of HBV vaccination (66.1% vs. 44.0%, p = 0.0001). Classical STIs were rare in both groups; BV prevalence was low and did not vary by HIV status. HSV-2 infection was markedly more frequent in HIV-positive (86.3%) than HIV-negative (46.6%) women (p < 0.0001). Vaginal HPV infection was also more common in HIV-positive than in HIV-negative women (50.8% vs. 22.6%, p < 0.0001) as was infection with high-risk oncogenic HPV types (48.4% vs. 17.3%, p < 0.0001).
Classical STIs were infrequent in this clinic-based population of African-Caribbean women in Toronto. However, HSV-2 prevalence was higher than that reported in previous studies in the general Canadian population and was strongly associated with HIV infection, as was infection with hepatitis B and HPV.
PMCID: PMC3835625  PMID: 24238493
Sexually transmitted infections; HIV; Epidemiology; African-Caribbean women; Toronto
15.  Patterns of syphilis testing in a large cohort of HIV patients in Ontario, Canada, 2000–2009 
BMC Infectious Diseases  2013;13:246.
Since 2000, reported syphilis cases increased ten-fold in Canada, particularly among men who have sex with men (MSM) co-infected with HIV. We characterized temporal patterns of of syphilis testing in a large cohort of HIV patients in Ontario, Canada.
We analyzed data from a multi-site cohort of people in HIV care from 2000 to 2009. Data were obtained from medical charts, interviews and record linkage with the syphilis test database at the Public Health Ontario Laboratories. We estimated the proportion that had syphilis testing at least once per year and the period and annual prevalence of reactive tests.
Among 4232 participants, the annual proportion tested rose from 2.7% (95%CI 1.9, 3.5) in 2000 to 54.6% (95%CI 52.9, 56.3) in 2009. Testing was most common for participants who were men who have sex with men (MSM), aged <30, recently diagnosed with HIV, were antiretroviral treatment naive, had routine HIV lab testing at least twice in that year, or tested for syphilis in the preceding year. The proportion with at least one reactive test in 2000–09 was 21.0% (95%CI 19.4, 22.7) for MSM, 5.3% (95%CI 3.3, 7.4) for non-MSM males, and 2.6% (95%CI 1.2, 4.0) for women. Among MSM, the annual prevalence of reactive syphilis tests with high RPR titre (≥1:16) peaked at 3.8% in 2009.
The burden of syphilis co-infection rose considerably among HIV-positive MSM, such that by 2009, at least 1 in 5 men had laboratory evidence of current or past infection. Interventions may be needed to boost syphilis testing to achieve goals set by guidelines even in settings with universal health care.
PMCID: PMC3668135  PMID: 23710699
Syphilis; HIV co-infections; Screening and diagnosis; Epidemiology
16.  Measurement of mucosal biomarkers in a phase 1 trial of intravaginal 3% SPL 7013 gel (VivaGel®) to assess expanded safety 
The aim of this study was to examine the effect of the 3% SPL 7013 gel (VivaGel®) on mucosal immune markers hypothesized to be associated with HIV-1 acquisition.
Phase 1, placebo-controlled, randomized, double-blind clinical trial was performed in 54 young women in the U.S. and Kenya. Participants used carbopol gel with and without (placebo) SPL 7013 twice daily over 14 days. Cervical specimens were collected for cytokines, chemokines, T-cells, and dendritic cells at day (D) 0, 7, 14, and 21. A negative binomial regression model was used to assess differences between study arms.
Several mucosal immune parameters were increased in the VivaGel® arm compared to placebo. For cytokines: D 7, IL-6 (p=0.05); D 14, IFN-γ (p=0.03), IL-2 (p=0.04), IL-5 (p=0.003) and IL-10 (p=0.001) were increased. On D 7, CD8+/CD69+ T-cells tended to be increased (p<0.08); limiting analysis to visits without blood or bacterial vaginosis, these findings were stronger: at D7, CD8+/CD69+ T-cells were increased in the VivaGel® arm (p<0.005), as were CD4+/CD69+ cells (p=0.001) and CD4+/CCR5+ T-cells (p=0.01). The changes described for D7 and 14 were no longer seen at D21.
Markers associated with inflammation and epithelial damage were reversibly elevated in the VivaGel® arm compared to the placebo arm after 7–14 days of twice daily product use.
PMCID: PMC3261360  PMID: 22067666
Vaginal microbicide; mucosal immune markers
17.  Enumeration of Sex Workers in the Central Business District of Nairobi, Kenya 
PLoS ONE  2013;8(1):e54354.
Accurate program planning for populations most at risk for HIV/STI acquisition requires knowledge of the size and location where these populations can best be reached. To obtain this information for sex workers operating at 137 hotspots in the central business district (CBD) in Nairobi, Kenya, we utilized a combined mapping and capture-recapture enumeration exercise. The majority of identified hotspots in this study were bars. Based on this exercise, we estimate that 6,904 male and female sex workers (95% confidence intervals, 6690 and 7118) were working nightly in the Nairobi CBD in April 2009. Wide ranges of captures per spot were obtained, suggesting that relatively few hot spots (18%) contain a relatively high proportion of the area's sex workers (65%). We provide geographic data including relatively short distances from hotspots to our dedicated sex worker outreach program in the CBD (mean<1 km), and clustering of hotspots within a relatively small area. Given the size covered and areas where sex work is likely taking place in Nairobi, the estimate is several times lower than what would be obtained if the entire metropolitan area was enumerated. These results have important practical and policy implications for enhancing HIV/STI prevention efforts.
PMCID: PMC3556081  PMID: 23372713
18.  HIV-1 Clade D Is Associated with Increased Rates of CD4 Decline in a Kenyan Cohort 
PLoS ONE  2012;7(11):e49797.
HIV-1 is grouped phylogenetically into clades, which may impact rates of HIV-1 disease progression. Clade D infection in particular has been shown to be more pathogenic. Here we confirm in a Nairobi-based prospective female sex worker cohort (1985–2004) that Clade D (n = 54) is associated with a more rapid CD4 decline than clade A1 (n = 150, 20.6% vs 13.4% decline per year, 1.53-fold increase, p = 0.015). This was independent of “protective” HLA and country of origin (p = 0.053), which in turn were also independent predictors of the rate of CD4 decline (p = 0.026 and 0.005, respectively). These data confirm that clade D is more pathogenic than clade A1. The precise reason for this difference is currently unclear, and requires further study. This is first study to demonstrate difference in HIV-1 disease progression between clades while controlling for protective HLA alleles.
PMCID: PMC3504142  PMID: 23185441
19.  Feasibility and Safety of Cervical Biopsy Sampling for Mucosal Immune Studies in Female Sex Workers from Nairobi, Kenya 
PLoS ONE  2012;7(10):e47570.
There is an urgent need to improve our understanding of the mucosal immuno-pathogenesis of HIV acquisition in the female genital tract, particularly in high-risk women such as female sex workers (FSWs). Cervical biopsy samples offer technical advantages over cytobrush sampling, but there are concerns that this might increase HIV acquisition, particularly if healing is slow and/or women do not abstain from sex during healing.
Methodology/Principal Findings
Cervical biopsy samples and cervico-vaginal swabs for co-infection diagnostics, prostate specific antigen (PSA) and immune studies were collected from 59 women, including HIV seropositive and HIV-exposed seronegative (HESN) FSWs as well as lower risk women from Nairobi, Kenya. A clinical-demographic questionnaire was administered and women were instructed to avoid sexual intercourse, douching and the insertion of tampons for 14 days. All participants underwent a repeat exam to assess healing within the 14 days, and had HIV diagnostics at six months. Cervical sampling was well tolerated, and 82% of participants had healed macroscopically by 5 days. Both self-report and PSA screening suggested high levels of compliance with pre- and post-procedure abstinence. Delayed healing was associated with vulvovaginal candidiasis (VVC) and HESN status. At six-month follow up all low-risk and HESN participants remained HIV seronegative.
Cervical biopsy sampling is a safe and well-tolerated method to obtain cervical biopsies in this context, particularly if participants with VVC are excluded. As healing could be delayed up to 11 days, it is important to support (both financially and with rigorous counseling) a period of post-procedure abstinence to minimize HIV risk.
PMCID: PMC3471881  PMID: 23077640
20.  Blunted IL17/IL22 and Pro-Inflammatory Cytokine Responses in the Genital Tract and Blood of HIV-Exposed, Seronegative Female Sex Workers in Kenya 
PLoS ONE  2012;7(8):e43670.
Identifying the immune correlates of reduced susceptibility to HIV remains a key goal for the HIV vaccine field, and individuals who are HIV-exposed, seronegative (HESN) may offer important clues. Reduced systemic immune activation has been described in HESN individuals. Conversely, pro-inflammatory T cell subsets, particularly CD4+ T cells producing the cytokine IL17 (Th17 cells), may represent a highly susceptible target for HIV infection after sexual exposure. Therefore, we characterized the cellular pro-inflammatory and IL17/IL22 cytokine immune milieu in the genital mucosa and blood of HESN female sex workers (FSWs).
Methods and Results
Blinded lab personnel characterized basal and mitogen-induced gene and cytokine immune responses in the cervix and blood of HESN FSWs (n = 116) and non-FSW controls (n = 17) using qPCR and ELISA. IL17 and IL22 production was significantly reduced in both the cervix and blood of HESNs, both in resting cells and after mitogen stimulation. In addition, HESN participants demonstrated blunted production of both pro-inflammatory cytokines and β-chemokines.
Discussion and Conclusions
We conclude that HIV exposure without infection was associated with blunted IL17/IL22 and pro-inflammatory responses, both systemically and at the site of mucosal HIV exposure. It will be important for further studies to examine the causal nature of the association and to define the cell subsets responsible for these differences.
PMCID: PMC3425491  PMID: 22928014
21.  No Difference in Keratin Thickness between Inner and Outer Foreskins from Elective Male Circumcisions in Rakai, Uganda 
PLoS ONE  2012;7(7):e41271.
It has been hypothesized that increased HIV acquisition in uncircumcised men may relate to a more thinly keratinized inner foreskin. However, published data are contradictory and potentially confounded by medical indications for circumcision. We tested the hypothesis that the inner foreskin was more thinly keratinized than the outer foreskin using tissues from 19 healthy, HIV-uninfected men undergoing routine prophylactic circumcision in Rakai, Uganda. Sections from 3 foreskin anatomic sites (inner, outer, and frenar band) were snap-frozen separately. Two independent laboratories each separately stained, imaged, and measured keratin thicknesses in a blinded fashion. There was no significant difference in keratin thickness between the inner (mean = 14.67±7.48 µm) and outer (mean = 13.30±8.49 µm) foreskin, or between the inner foreskin and the frenar band (mean = 16.91±12.42 µm). While the frenar band showed the greatest intra-individual heterogeneity in keratin thickness, there was substantial inter-individual variation seen in all regions. Measurements made by the two laboratories showed high correlation (r = 0.741, 95% CI, 0.533–0.864). We conclude that, despite inter- and intra-individual variability, keratin thickness was similar in the inner and outer foreskin of healthy Ugandan men, and that reduced keratin thickness is not likely to make the inner foreskin more susceptible to HIV acquisition.
PMCID: PMC3399815  PMID: 22815984
22.  Tim-3 Negatively Regulates Cytotoxicity in Exhausted CD8+ T Cells in HIV Infection 
PLoS ONE  2012;7(7):e40146.
Cytotoxic CD8+ T cells (CTLs) contain virus infections through the release of granules containing both perforin and granzymes. T cell ‘exhaustion’ is a hallmark of chronic persistent viral infections including HIV. The inhibitory regulatory molecule, T cell Immunoglobulin and Mucin domain containing 3 (Tim-3) is induced on HIV-specific T cells in chronic progressive infection. These Tim-3 expressing T cells are dysfunctional in terms of their capacities to proliferate or to produce cytokines. In this study, we evaluated the effect of Tim-3 expression on the cytotoxic capabilities of CD8+ T cells in the context of HIV infection. We investigated the cytotoxic capacity of Tim-3 expressing T cells by examining 1) the ability of Tim-3+ CD8+ T cells to make perforin and 2) the direct ability of Tim-3+ CD8+ T cells to kill autologous HIV infected CD4+ target cells. Surprisingly, Tim-3+ CD8+ T cells maintain higher levels of perforin, which was mainly in a granule-associated (stored) conformation, as well as express high levels of T-bet. However, these cells were also defective in their ability to degranulate. Blocking the Tim-3 signalling pathway enhanced the cytotoxic capabilities of HIV specific CD8+ T cells from chronic progressors by increasing; a) their degranulation capacity, b) their ability to release perforin, c) their ability to target activated granzyme B to HIV antigen expressing CD4+ T cells and d) their ability to suppress HIV infection of CD4+ T cells. In this latter effect, blocking the Tim-3 pathway enhances the cytotoxcity of CD8+ T cells from chronic progressors to the level very close to that of T cells from viral controllers. Thus, the Tim-3 receptor, in addition to acting as a terminator for cytokine producing and proliferative functions of CTLs, can also down-regulate the CD8+ T cell cytotoxic function through inhibition of degranulation and perforin and granzyme secretion.
PMCID: PMC3390352  PMID: 22792231
23.  Does antiretroviral therapy initiation increase sexual risk taking in Kenyan female sex workers? A retrospective case–control study 
BMJ Open  2012;2(2):e000565.
Although antiretroviral therapy (ART) prolongs life and reduces infectiousness, in some contexts, it has been associated with increased sexual risk taking.
Retrospective case–control study.
Nairobi-based dedicated female sex worker (FSW) clinic.
HIV-infected FSWs before and after ART initiation (n=62); HIV-infected and -uninfected control FSWs not starting ART during the same follow-up period (n=40).
Initiation of ART.
Primary outcome measures
Self-reported condom use, client numbers and sexually transmitted infection incidence over the study period (before and after ART initiation in cases).
Sexual risk-taking behaviour with casual clients did not increase after ART initiation; condom use increased and sexually transmitted infection incidence decreased in both cases and controls, likely due to successful cohort-wide HIV prevention efforts.
ART provision was not associated with increases in unsafe sex in this FSW population.
Article summary
Article focus
Impact of starting ART on sexual risk-taking behaviour in FSWs, which could have an important impact on HIV transmission to clients.
Key messages
ART initiation was not associated with increased risk taking or sexually transmitted infection incidence.
Both of these declined over time, most likely as a result of risk reduction counselling.
Strengths and limitations of this study
Strengths include a relevant population, longitudinal follow-up and inclusion of biological measures of risk taking (sexually transmitted infection incidence).
Limitations include relatively small study groups and limited sampling time points.
PMCID: PMC3323809  PMID: 22466157
24.  Increased Levels of Immune Activation in the Genital Tract of Healthy Young Women from sub-Saharan Africa 
AIDS (London, England)  2010;24(13):2069-2074.
To determine if healthy, young women in sub-Saharan Africa have a more activated immune milieu in the genital tract (i.e. activated CD4+ T-cells) than a similar population in the US.
Cross-sectional study nested in a phase 1 microbicide trial.
Cervical cytobrushes were collected from 18–24 year old women in San Francisco, CA (n=18) and Kisumu, Kenya (n=36) at enrollment into a phase 1 microbicide trial. All participants tested negative for HIV, HSV-2, gonorrhea, chlamydia and trichomonas, and had abstained from sex for at least seven days prior to enrollment. Cryopreserved T-cell populations were assayed by flow cytometry in a central laboratory. SLPI levels were assayed in cervicovaginal lavage samples. The Wilcoxon rank-sum test was used to compare immune parameters between sites.
The total number of endocervical CD4+ T-cells was slightly higher in San Francisco, but participants from Kisumu had a substantially higher number and proportion of CD4+ T-cells expressing the early activation marker CD69, with and without the HIV-coreceptor CCR5, and a greater proportion of activated CD8+ T-cells. Median [interquartile] genital levels of SLPI were lower in participants from Kisumu compared to those from San Francisco (190 pg/mL [96, 519] vs. 474 pg/mL [206, 817]; p<0.03).
Activated mucosal T-cells were increased in the genital tract of young, STI/HIV-free Kenyan women, independent of common genital co-infections, and SLPI levels were reduced. The cause of these mucosal immune differences is not known, but could partly explain the high HIV incidence in young women from sub-Saharan Africa.
PMCID: PMC2914808  PMID: 20588163
HIV transmission; mucosal immunology; female genital tract; sub-Saharan Africa; CD4+ T-cells; SLPI
25.  Impact of Collection Method on Assessment of Semen HIV RNA Viral Load 
PLoS ONE  2011;6(8):e23654.
The blood HIV RNA viral load is the best-defined predictor of HIV transmission, in part due to ease of measurement and the correlation of blood and genital tract (semen or cervico-vaginal) viral load, although recent studies found semen HIV RNA concentration to be a stronger predictor of HIV transmission. There is currently no standardized method for semen collection when measuring HIV RNA concentration. Therefore, we compared two collection techniques in order to study of the impact of antiretroviral therapy on the semen viral load.
Methodology/Principal Findings
Semen was collected by masturbation from HIV-infected, therapy-naïve men who have sex with men (MSM) either undiluted (Visit 1) or directly into transport medium (Visit 2). Seminal plasma was then isolated, and the HIV RNA concentration obtained with each collection technique was measured and corrected for dilution if necessary. Collection of semen directly into transport medium resulted in a median HIV RNA viral load that was 0.4 log10 higher than undiluted samples.
The method of semen collection is an important consideration when quantifying the HIV RNA viral load in this compartment.
PMCID: PMC3158791  PMID: 21886808

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