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1.  Daily Acyclovir to Decrease Herpes Simplex Virus Type 2 (HSV-2) Transmission from HSV-2/HIV-1 Coinfected Persons: A Randomized Controlled Trial 
The Journal of Infectious Diseases  2013;208(9):1366-1374.
Background. Daily suppressive therapy with valacyclovir reduces risk of sexual transmission of herpes simplex virus type 2 (HSV-2) in HSV-2–serodiscordant heterosexual couples by 48%. Whether suppressive therapy reduces HSV-2 transmission from persons coinfected with HSV-2 and human immunodeficiency virus type 1 (HIV-1) is unknown.
Methods. Within a randomized trial of daily acyclovir 400 mg twice daily in African HIV-1 serodiscordant couples, in which the HIV-1–infected partner was HSV-2 seropositive, we identified partnerships in which HIV-1–susceptible partners were HSV-2 seronegative to estimate the effect of acyclovir on risk of HSV-2 transmission.
Results. We randomly assigned 911 HSV-2/HIV-1–serodiscordant couples to daily receipt of acyclovir or placebo. We observed 68 HSV-2 seroconversions, 40 and 28 in acyclovir and placebo groups, respectively (HSV-2 incidence, 5.1 cases per 100 person-years; hazard ratio [HR], 1.35 [95% confidence interval, .83–2.20]; P = .22). Among HSV-2–susceptible women, vaginal drying practices (adjusted HR, 44.35; P = .004) and unprotected sex (adjusted HR, 9.91; P = .002) were significant risk factors for HSV-2 acquisition; having more children was protective (adjusted HR, 0.47 per additional child; P = .012). Among HSV-2–susceptible men, only age ≤30 years was associated with increased risk of HSV-2 acquisition (P = .016).
Conclusions. Treatment of African HSV-2/HIV-1–infected persons with daily suppressive acyclovir did not decrease risk of HSV-2 transmission to susceptible partners. More-effective prevention strategies to reduce HSV-2 transmission from HIV-1–infected persons are needed.
doi:10.1093/infdis/jit333
PMCID: PMC3789565  PMID: 23901094
HSV-2; HIV-1; acyclovir; transmission; serodiscordant couples; Africa
2.  Dynamics of Viremia in Primary HIV-1 infection in Africans: Insights from Analyses of Host and Viral Correlates 
Virology  2013;449:254-262.
In HIV-1 infection, plasma viral load (VL) has dual implications for pathogenesis and public health. Based on well-known patterns of HIV-1 evolution and immune escape, we hypothesized that VL is an evolving quantitative trait that depends heavily on duration of infection (DOI), demographic features, human leukocyte antigen (HLA) genotypes and viral characteristics. Prospective data from 421 African seroconverters with at least four eligible visits did show relatively steady VL beyond 3 months of untreated infection, but host and viral factors independently associated with cross-sectional and longitudinal VL often varied by analytical approaches and sliding time windows. Specifically, the effects of age, HLA-B*53 and infecting HIV-1 subtypes (A1, C and others) on VL were either sporadic or highly sensitive to time windows. These observations were strengthened by the addition of 111 seroconverters with 2–3 eligible VL results, suggesting that DOI should be a critical parameter in epidemiological and clinical studies.
doi:10.1016/j.virol.2013.11.024
PMCID: PMC3931417  PMID: 24418560
Africa; HIV-1; subtype; HLA; statistical models; viral load
3.  Creating an African HIV Clinical Research and Prevention Trials Network: HIV Prevalence, Incidence and Transmission 
PLoS ONE  2015;10(1):e0116100.
HIV epidemiology informs prevention trial design and program planning. Nine clinical research centers (CRC) in sub-Saharan Africa conducted HIV observational epidemiology studies in populations at risk for HIV infection as part of an HIV prevention and vaccine trial network. Annual HIV incidence ranged from below 2% to above 10% and varied by CRC and risk group, with rates above 5% observed in Zambian men in an HIV-discordant relationship, Ugandan men from Lake Victoria fishing communities, men who have sex with men, and several cohorts of women. HIV incidence tended to fall after the first three months in the study and over calendar time. Among suspected transmission pairs, 28% of HIV infections were not from the reported partner. Volunteers with high incidence were successfully identified and enrolled into large scale cohort studies. Over a quarter of new cases in couples acquired infection from persons other than the suspected transmitting partner.
doi:10.1371/journal.pone.0116100
PMCID: PMC4300215  PMID: 25602351
4.  Transmitted Virus Fitness and Host T Cell Responses Collectively Define Divergent Infection Outcomes in Two HIV-1 Recipients 
PLoS Pathogens  2015;11(1):e1004565.
Control of virus replication in HIV-1 infection is critical to delaying disease progression. While cellular immune responses are a key determinant of control, relatively little is known about the contribution of the infecting virus to this process. To gain insight into this interplay between virus and host in viral control, we conducted a detailed analysis of two heterosexual HIV-1 subtype A transmission pairs in which female recipients sharing three HLA class I alleles exhibited contrasting clinical outcomes: R880F controlled virus replication while R463F experienced high viral loads and rapid disease progression. Near full-length single genome amplification defined the infecting transmitted/founder (T/F) virus proteome and subsequent sequence evolution over the first year of infection for both acutely infected recipients. T/F virus replicative capacities were compared in vitro, while the development of the earliest cellular immune response was defined using autologous virus sequence-based peptides. The R880F T/F virus replicated significantly slower in vitro than that transmitted to R463F. While neutralizing antibody responses were similar in both subjects, during acute infection R880F mounted a broad T cell response, the most dominant components of which targeted epitopes from which escape was limited. In contrast, the primary HIV-specific T cell response in R463F was focused on just two epitopes, one of which rapidly escaped. This comprehensive study highlights both the importance of the contribution of the lower replication capacity of the transmitted/founder virus and an associated induction of a broad primary HIV-specific T cell response, which was not undermined by rapid epitope escape, to long-term viral control in HIV-1 infection. It underscores the importance of the earliest CD8 T cell response targeting regions of the virus proteome that cannot mutate without a high fitness cost, further emphasizing the need for vaccines that elicit a breadth of T cell responses to conserved viral epitopes.
Author Summary
The length of time taken by HIV-1-infected individuals to develop AIDS varies widely depending on how efficiently virus replication is controlled. Although host cellular immune responses are known to play an important role in viral control, the contributions made by the infecting virus and the host antibody response to this process are less clear. To gain insight into this, we performed a detailed analysis of the interplay between the infecting virus and host immune responses in two HIV-1-infected individuals, one of whom controlled virus replication efficiently while the other did not. We found that the virus infecting the HIV-1 controller replicated much less well in culture than that infecting the progressor. The antibody responses made by both subjects were similar, but early after infection the controller mounted a T cell response targeting many sites in the virus, whilst the progressor's T cell response initially targeted only two sites, one of which rapidly mutated to avoid immune recognition. This study highlights the contribution of the replication capacity of the infecting virus and associated early induction of a broad HIV-specific T cell response, which was less readily undermined by rapid viral escape, to viral control in HIV-1 infection.
doi:10.1371/journal.ppat.1004565
PMCID: PMC4287535  PMID: 25569444
6.  Rwanda 20 years on: investing in life 
Lancet  2014;384(9940):371-375.
Two decades ago, the genocide against the Tutsis in Rwanda led to the deaths of 1 million people, and the displacement of millions more. Injury and trauma were followed by the effects of a devastated health system and economy. In the years that followed, a new course set by a new government set into motion equity-oriented national policies focusing on social cohesion and people-centred development. Premature mortality rates have fallen precipitously in recent years, and life expectancy has doubled since the mid-1990s. Here we reflect on the lessons learned in rebuilding Rwanda’s health sector during the past two decades, as the country now prepares itself to take on new challenges in health-care delivery.
doi:10.1016/S0140-6736(14)60574-2
PMCID: PMC4151975  PMID: 24703831
7.  Effect of Seasonal Variation on Adult Clinical Laboratory Parameters in Rwanda, Zambia, and Uganda: Implications for HIV Biomedical Prevention Trials 
PLoS ONE  2014;9(8):e105089.
Objectives
To investigate the effect of seasonal variation on adult clinical laboratory parameters in Rwanda, Zambia, and Uganda and determine its implications for HIV prevention and other clinical trials.
Methods
Volunteers in a cross-sectional study to establish laboratory reference intervals were asked to return for a seasonal visit after the local season had changed from dry to rainy or vice versa. Volunteers had to be clinically healthy, not pregnant and negative for HIV, Hepatitis B and C, and syphilis infection at both visits. At each visit, blood was taken for measurement of hemoglobin, haematocrit, mean corpuscular volume, red blood cells, platelets, total white blood cells (WBC), neutrophils, lymphocytes, monocytes, eosinophils, basophils, CD4/CD8 T cells, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, direct bilirubin, total bilirubin, total immunoglobulin gamma, total protein, creatinine, total amylase, creatine phosphokinase and lactate dehydrogenase (LDH). Consensus dry season reference intervals were applied to rainy season values (and vice versa) and the proportion of ‘out-of-range’ values determined. Percentage differences between dry and rainy season parameter mean values were estimated.
Results
In this cohort of 903 volunteers, less than 10.0% of consensus parameter (except LDH) values in one season were “out-of-range” in the other. Twenty-two (22) percent of rainy season LDH values fell outside of the consensus dry season interval with the higher values observed in the rainy season. Variability between consensus seasonal means ranged from 0.0% (total WBC, neutrophils, monocytes, basophils, and direct bilirubin) to 40.0% (eosinophils). Within sites, the largest seasonal variations were observed for monocytes (Masaka, 11.5%), LDH (Lusaka, 21.7%), and basophils (Kigali, 22.2%).
Conclusions
Seasonality had minimal impact on adult clinical laboratory parameter values in Rwanda, Zambia, and Uganda. Seasonal variation may not be an important factor in the evaluation of adult clinical laboratory parameters in HIV prevention and other clinical trials in these countries.
doi:10.1371/journal.pone.0105089
PMCID: PMC4132051  PMID: 25118593
8.  Host genetics and viral load in primary HIV-1 infection: clear evidence for gene by sex interactions 
Human Genetics  2014;133(9):1187-1197.
Research in the past two decades has generated unequivocal evidence that host genetic variations substantially account for the heterogeneous outcomes following human immunodeficiency virus type 1 (HIV-1) infection. In particular, genes encoding human leukocyte antigens (HLA) have various alleles, haplotypes, or specific motifs that can dictate the set-point (a relatively steady state) of plasma viral load (VL), although rapid viral evolution driven by innate and acquired immune responses can obscure the long-term relationships between HLA genotypes and HIV-1-related outcomes. In our analyses of VL data from 521 recent HIV-1 seroconverters enrolled from eastern and southern Africa, HLA-A*03:01 was strongly and persistently associated with low VL in women (frequency = 11.3 %, P < 0.0001) but not in men (frequency = 7.7 %, P = 0.66). This novel sex by HLA interaction (P = 0.003, q = 0.090) did not extend to other frequent HLA class I alleles (n = 34), although HLA-C*18:01 also showed a weak association with low VL in women only (frequency = 9.3 %, P = 0.042, q > 0.50). In a reduced multivariable model, age, sex, geography (clinical sites), previously identified HLA factors (HLA-B*18, B*45, B*53, and B*57), and the interaction term for female sex and HLA-A*03:01 collectively explained 17.0 % of the overall variance in geometric mean VL over a 3-year follow-up period (P < 0.0001). Multiple sensitivity analyses of longitudinal and cross-sectional VL data yielded consistent results. These findings can serve as a proof of principle that the gap of “missing heritability” in quantitative genetics can be partially bridged by a systematic evaluation of sex-specific associations.
doi:10.1007/s00439-014-1465-x
PMCID: PMC4127002  PMID: 24969460
9.  Syphilis Treatment Response Among HIV-Discordant Couples in Zambia and Rwanda 
In 2 large human immunodeficiency virus (HIV)–discordant couple cohorts in Africa, time to treatment response after penicillin therapy for a positive rapid plasma reagin test result was similar irrespective of HIV status. Despite effective therapy, serofast state and syphilis reinfection were common.
Background. Syphilis continues to be a common sexually transmitted infection, despite the availability of inexpensive and effective treatment. Infection in human immunodeficiency virus (HIV)–discordant couples is important because syphilis increases the risk of HIV acquisition. Current US treatment guidelines recommend 1 dose of benzathine penicillin for early syphilis, irrespective of HIV status, but data from coinfected patients are limited.
Methods. Retrospective analysis of 1321 individuals in 2 African HIV-discordant couple cohorts was performed. Cox proportional hazards analysis and multivariable modeling were used to assess predictors of serologic response to treatment at 180 days and 400 days. Modeling was performed for all episodes of positive rapid plasma reagin (RPR) test results and on a subset with higher RPR titers (≥1:4).
Results. A total of 1810 episodes of syphilis among 1321 individuals were treated with penicillin between 2002 and 2008. Although a positive RPR was more common in the HIV-infected partners, HIV infection did not impact the likelihood of serologic response to therapy (odds ratio [OR], 1.001; P = .995). By 400 days, 67% had responded to therapy, 27% were serofast, and 6.5% had documented reinfection. Prevalent infections were more likely to remain serofast than incident infections (33% vs 20% at 400 days).
Conclusions. In 2 HIV-serodiscordant couple cohorts in Africa, incident syphilis had a very good likelihood of response to penicillin therapy, irrespective of HIV infection. This supports current Centers for Disease Control and Prevention treatment guidelines. A high proportion of prevalent RPR-positive infections remain serofast despite treatment.
doi:10.1093/cid/cit146
PMCID: PMC3658364  PMID: 23487377
HIV/AIDS; syphilis; discordant couples; Zambia; Rwanda
10.  Plasma Viral Loads During Early HIV-1 Infection Are Similar in Subtype C– and Non-Subtype C–Infected African Seroconverters 
The Journal of Infectious Diseases  2013;207(7):1166-1170.
Recent data suggest that infection with human immunodeficiency virus type 1 (HIV-1) subtype C results in prolonged high-level viremia (>5 log10 copies/mL) during early infection. We examined the relationship between HIV-1 subtype and plasma viremia among 153 African seroconverters. Mean setpoint viral loads were similar for C and non-C subtypes: 4.36 vs 4.42 log10 copies/mL (P = .61). The proportion of subtype C–infected participants with viral loads >5 log10 copies/mL was not greater than the proportion for those with non-C infection. Our data do not support the hypothesis that higher early viral load accounts for the rapid spread of HIV-1 subtype C in southern Africa.
doi:10.1093/infdis/jit015
PMCID: PMC3583276  PMID: 23315322
HIV-1; group M subtype; plasma viral load; early infection; Africa
11.  Molecular identification, cloning and characterization of transmitted/founder HIV-1 subtype A, D and A/D infectious molecular clones 
Virology  2012;436(1):33-48.
We report the molecular identification, cloning and initial biological characterization of 12 full-length HIV-1 subtype A, D and A/D recombinant transmitted/founder (T/F) genomes. T/F genomes contained intact canonical open reading frames and all T/F viruses were replication competent in primary human T-cells, although subtype D virus replication was more efficient (p<0.05). All 12 viruses utilized CCR5 but not CXCR4 as a co-receptor for entry and exhibited a neutralization profile typical of tier 2 primary virus strains, with significant differences observed between subtype A and D viruses with respect to sensitivity to monoclonal antibodies VRC01, PG9 and PG16 and polyclonal subtype C anti-HIV IgG (p<0.05 for each). The present report doubles the number of T/F HIV-1 clones available for pathogenesis and vaccine research and extends their representation to include subtypes A, B, C and D.
doi:10.1016/j.virol.2012.10.009
PMCID: PMC3545109  PMID: 23123038
HIV-1; transmitted/founder virus; single genome sequencing; HIV-1 transmission; HIV-1 subtype A; HIV-1 subtype D; neutralizing antibodies
12.  A gp41-Based Heteroduplex Mobility Assay Provides Rapid and Accurate Assessment of Intrasubtype Epidemiological Linkage in HIV Type 1 Heterosexual Transmission Pairs 
AIDS Research and Human Retroviruses  2012;28(12):1745-1755.
Abstract
A critical step in HIV-1 transmission studies is the rapid and accurate identification of epidemiologically linked transmission pairs. To date, this has been accomplished by comparison of polymerase chain reaction (PCR)-amplified nucleotide sequences from potential transmission pairs, which can be cost-prohibitive for use in resource-limited settings. Here we describe a rapid, cost-effective approach to determine transmission linkage based on the heteroduplex mobility assay (HMA), and validate this approach by comparison to nucleotide sequencing. A total of 102 HIV-1-infected Zambian and Rwandan couples, with known linkage, were analyzed by gp41-HMA. A 400-base pair fragment within the envelope gp41 region of the HIV proviral genome was PCR amplified and HMA was applied to both partners' amplicons separately (autologous) and as a mixture (heterologous). If the diversity between gp41 sequences was low (<5%), a homoduplex was observed upon gel electrophoresis and the transmission was characterized as having occurred between partners (linked). If a new heteroduplex formed, within the heterologous migration, the transmission was determined to be unlinked. Initial blind validation of gp-41 HMA demonstrated 90% concordance between HMA and sequencing with 100% concordance in the case of linked transmissions. Following validation, 25 newly infected partners in Kigali and 12 in Lusaka were evaluated prospectively using both HMA and nucleotide sequences. Concordant results were obtained in all but one case (97.3%). The gp41-HMA technique is a reliable and feasible tool to detect linked transmissions in the field. All identified unlinked results should be confirmed by sequence analyses.
doi:10.1089/aid.2012.0023
PMCID: PMC3505061  PMID: 22587371
13.  HLA-B*57 versus HLA-B*81 in HIV-1 Infection: Slow and Steady Wins the Race? 
Journal of Virology  2013;87(7):4043-4051.
Two human leukocyte antigen (HLA) variants, HLA-B*57 and -B*81, are consistently known as favorable host factors in human immunodeficiency virus type 1 (HIV-1)-infected Africans and African-Americans. In our analyses of prospective data from 538 recent HIV-1 seroconverters and cross-sectional data from 292 subjects with unknown duration of infection, HLA-B*57 (mostly B*57:03) and -B*81 (exclusively B*81:01) had mostly discordant associations with virologic and immunologic manifestations before antiretroviral therapy. Specifically, relatively low viral load (VL) in HLA-B*57-positive subjects (P ≤ 0.03 in various models) did not translate to early advantage in CD4+ T-cell (CD4) counts (P ≥ 0.37). In contrast, individuals with HLA-B*81 showed little deviation from the normal set point VL (P > 0.18) while maintaining high CD4 count during early and chronic infection (P = 0.01). These observations suggest that discordance between VL and CD4 count can occur in the presence of certain HLA alleles and that effective control of HIV-1 viremia is not always a prerequisite for favorable prognosis (delayed immunodeficiency). Of note, steady CD4 count associated with HLA-B*81 in HIV-1-infected Africans may depend on the country of origin, as observations differed slightly between subgroups enrolled in southern Africa (Zambia) and eastern Africa (Kenya, Rwanda, and Uganda).
doi:10.1128/JVI.03302-12
PMCID: PMC3624227  PMID: 23365442
14.  Low Primary and Secondary HIV Drug-Resistance after 12 Months of Antiretroviral Therapy in Human Immune-Deficiency Virus Type 1 (HIV-1)-Infected Individuals from Kigali, Rwanda 
PLoS ONE  2013;8(8):e64345.
Treatment outcomes of HIV patients receiving antiretroviral therapy (ART) in Rwanda are scarcely documented. HIV viral load (VL) and HIV drug-resistance (HIVDR) outcomes at month 12 were determined in a prospective cohort study of antiretroviral–naïve HIV patients initiating first-line therapy in Kigali. Treatment response was monitored clinically and by regular CD4 counts and targeted HIV viral load (VL) to confirm drug failure. VL measurements and HIVDR genotyping were performed retrospectively on baseline and month 12 samples. One hundred and fifty-eight participants who completed their month 12 follow-up visit had VL data available at month 12. Most of them (88%) were virologically suppressed (VL≤1000 copies/mL) but 18 had virological failure (11%), which is in the range of WHO-suggested targets for HIVDR prevention. If only CD4 criteria had been used to classify treatment response, 26% of the participants would have been misclassified as treatment failure. Pre-therapy HIVDR was documented in 4 of 109 participants (3.6%) with an HIVDR genotyping results at baseline. Eight of 12 participants (66.7%) with virological failure and HIVDR genotyping results at month 12 were found to harbor mutation(s), mostly NNRTI resistance mutations, whereas 4 patients had no HIVDR mutations. Almost half (44%) of the participants initiated ART at CD4 count ≤200cell/µl and severe CD4 depletion at baseline (<50 cells/µl) was associated with virological treatment failure (p = 0.008).
Although the findings may not be generalizable to all HIV patients in Rwanda, our data suggest that first-line ART regimen changes are currently not warranted. However, the accumulation of acquired HIVDR mutations in some participants underscores the need to reinforce HIVDR prevention strategies, such as increasing the availability and appropriate use of VL testing to monitor ART response, ensuring high quality adherence counseling, and promoting earlier identification of HIV patients and enrollment into HIV care and treatment programs.
doi:10.1371/journal.pone.0064345
PMCID: PMC3741294  PMID: 23950859
15.  Fertility goal-based counseling increases contraceptive implant and IUD use in HIV discordant couples in Rwanda and Zambia 
Contraception  2012;88(1):74-82.
Background
HIV discordant heterosexual couples are faced with the dual challenge of preventing sexual HIV transmission and unplanned pregnancies with the attendant risk of perinatal HIV transmission. Our aim was to examine uptake of two long-acting reversible contraceptive (LARC) methods – intrauterine devices (IUDs) and hormonal implants – among HIV discordant couples in Rwanda and Zambia.
Study Design
Women were interviewed alone or with their partner during routine cohort study follow-up visits to ascertain fertility goals; those not pregnant, not infertile, not already using LARC, and wishing to limit or delay fertility for ≥3 years were counseled on LARC methods and offered an IUD and implant on-site.
Results
Among 409 fertile Rwandan women interviewed (126 alone, 283 with partners), 365 (89%) were counseled about LARC methods and 130 (36%) adopted a method (100 implant, 30 IUD). Of 787 fertile Zambian women interviewed (457 alone, 330 with partners), 528 (67%) received LARC counseling, of whom 177 (34%) adopted a method (139 implant, 38 IUD). In both countries, a woman’s younger age was predictive of LARC uptake. LARC users reported fewer episodes of unprotected sex than couples using only condoms.
Conclusions
Integrated fertility-goal based family planning counseling and access to LARC methods with reinforcement of dual-method use prompted uptake of IUDs and implants and reduced unprotected sex among HIV-discordant couples in two African capital cities.
doi:10.1016/j.contraception.2012.10.004
PMCID: PMC3625675  PMID: 23153896
contraception; family planning; HIV; intrauterine devices; implant
16.  INFLUENCE NETWORK AGENT EFFECTIVENESS IN PROMOTING COUPLES’ HIV COUNSELING AND TESTING IN KIGALI, RWANDA 
AIDS (London, England)  2012;26(2):217-227.
Objective
To identify predictors of promotion of couples’ voluntary counseling and testing (CVCT) in Kigali, Rwanda
Design
Analysis of CVCT promotional agent (influential network leaders, INLs; influential network agents, INAs), and couple/invitation-level predictors of CVCT uptake.
Methods
Number of invitations and couples tested were evaluated by INL, INA, and couple/contextual factors. Multivariable logistic regression accounting for two-level clustering analyzed factors predictive of couples’ testing.
Results
26 INLs recruited and mentored 118 INAs who delivered 24,991 invitations. 4,513 couples sought CVCT services after invitation. INAs distributed an average of 212 invitations resulting in an average of 38 couples tested/agent. Characteristics predictive of CVCT in multivariate analyses included the invitee and INA being socially acquainted (aOR=1.4;95%CI:1.2–1.6); invitations delivered after public endorsement (aOR=1.3;95%CI:1.1–1.5); and presence of a mobile testing unit (aOR=1.4;95%CI:1.0–2.0). In stratified analyses, predictors significant among cohabiting couples included invitation delivery to the couple (aOR=1.2;95%CI:1.0–1.4) in the home (aOR=1.3;95%CI:1.1–1.4), while among non-cohabiting couples predictors included invitations given by unemployed INAs (aOR=1.7;95%CI:1.1–2.7). Cohabiting couples with older men were more likely to test, while younger age was associated with testing among men in non-cohabiting unions.
Conclusions
Invitations distributed by influential people were successful in prompting couples to seek joint HIV testing, particularly if the invitation was given in the home to someone known to the INA, and accompanied by a public endorsement of CVCT. Mobile units also increased the number of couples tested. Country-specific strategies to promote CVCT programs are needed to reduce HIV transmission among those at highest risk for HIV in sub-Saharan Africa.
doi:10.1097/QAD.0b013e32834dc593
PMCID: PMC3679893  PMID: 22008653
Community Workers; Couples’ Voluntary Counseling and Testing; HIV; Rwanda
17.  Viral Escape from Neutralizing Antibodies in Early Subtype A HIV-1 Infection Drives an Increase in Autologous Neutralization Breadth 
PLoS Pathogens  2013;9(2):e1003173.
Antibodies that neutralize (nAbs) genetically diverse HIV-1 strains have been recovered from a subset of HIV-1 infected subjects during chronic infection. Exact mechanisms that expand the otherwise narrow neutralization capacity observed during early infection are, however, currently undefined. Here we characterized the earliest nAb responses in a subtype A HIV-1 infected Rwandan seroconverter who later developed moderate cross-clade nAb breadth, using (i) envelope (Env) glycoproteins from the transmitted/founder virus and twenty longitudinal nAb escape variants, (ii) longitudinal autologous plasma, and (iii) autologous monoclonal antibodies (mAbs). Initially, nAbs targeted a single region of gp120, which flanked the V3 domain and involved the alpha2 helix. A single amino acid change at one of three positions in this region conferred early escape. One immunoglobulin heavy chain and two light chains recovered from autologous B cells comprised two mAbs, 19.3H-L1 and 19.3H-L3, which neutralized the founder Env along with one or three of the early escape variants carrying these mutations, respectively. Neither mAb neutralized later nAb escape or heterologous Envs. Crystal structures of the antigen-binding fragments (Fabs) revealed flat epitope contact surfaces, where minimal light chain mutation in 19.3H-L3 allowed for additional antigenic interactions. Resistance to mAb neutralization arose in later Envs through alteration of two glycans spatially adjacent to the initial escape signatures. The cross-neutralizing nAbs that ultimately developed failed to target any of the defined V3-proximal changes generated during the first year of infection in this subject. Our data demonstrate that this subject's first recognized nAb epitope elicited strain-specific mAbs, which incrementally acquired autologous breadth, and directed later B cell responses to target distinct portions of Env. This immune re-focusing could have triggered the evolution of cross-clade antibodies and suggests that exposure to a specific sequence of immune escape variants might promote broad humoral responses during HIV-1 infection.
Author Summary
Since cases were first recognized in the United States in 1981, human immunodeficiency virus (HIV-1) has infected over one million Americans. Globally, this scale reaches into the tens of millions, but no effective vaccine exists. Of those infected, approximately 20–30% of patients will develop broadly neutralizing antibodies. The reasons for maturation of these potentially protective responses are presently unknown, but being able to elicit such antibodies via vaccination could curb the pandemic. Here, we defined the earliest neutralizing antibody targets and the consequent routes of viral escape in one subtype A HIV-1 infected subject who developed modest breadth. We also determined the genetic and structural characteristics of early neutralizing monoclonal antibodies circulating in this subject and found that subtle light chain alteration enhanced target contact and neutralization. Overall, our data support the idea that exposure to a specific sequence of viral variants, which have escaped from immune pressure, could program long-term potential for antibody breadth.
doi:10.1371/journal.ppat.1003173
PMCID: PMC3585129  PMID: 23468623
18.  Molecular and Phylogeographic Analysis of Human Immuno-deficiency Virus Type 1 Strains Infecting Treatment-naive Patients from Kigali, Rwanda 
PLoS ONE  2012;7(8):e42557.
This study aimed at describing the genetic subtype distribution of HIV-1 strains circulating in Kigali and their epidemiological link with the HIV-1 strains from the five countries surrounding Rwanda. One hundred and thirty eight pol (RT and PR) sequences from 116 chronically- and 22 recently-infected antiretroviral therapy (ART)-naïve patients from Kigali were generated and subjected to HIV drug resistance (HIV-DR), phylogenetic and recombinant analyses in connection with 366 reference pol sequences from Rwanda, Burundi, Kenya, Democratic Republic of Congo, Tanzania and Uganda (Los Alamos database). Among the Rwandan samples, subtype A1 predominated (71.7%), followed by A1/C recombinants (18.1%), subtype C (5.8%), subtype D (2.9%), one A1/D recombinant (0.7%) and one unknown subtype (0.7%). Thirteen unique and three multiple A1/C recombinant forms were identified. No evidence for direct transmission events was found within the Rwandan strains. Molecular characteristics of HIV-1 were similar between chronically and recently-infected individuals and were not significantly associated with demographic or social factors. Our report suggests that the HIV-1 epidemic in Kigali is characterized by the emergence of A1/C recombinants and is not phylogenetically connected with the HIV-1 epidemic in the five neighboring countries. The relatively low level of transmitted HIV-DR mutations (2.9%) reported here indicates the good performance of the ART programme in Rwanda. However, the importance of promoting couples' counseling, testing and disclosure during HIV prevention strategies is highlighted.
doi:10.1371/journal.pone.0042557
PMCID: PMC3419187  PMID: 22905148
19.  Failure of A Novel, Rapid Antigen and Antibody Combination Test to Detect Antigen-Positive HIV Infection in African Adults with Early HIV Infection 
PLoS ONE  2012;7(6):e37154.
Background
Acute HIV infection (prior to antibody seroconversion) represents a high-risk window for HIV transmission. Development of a test to detect acute infection at the point-of-care is urgent.
Methods
Volunteers enrolled in a prospective study of HIV incidence in four African cities, Kigali in Rwanda and Ndola, Kitwe and Lusaka in Zambia, were tested regularly for HIV by rapid antibody test and p24 antigen ELISA. Five subgroups of samples were also tested by the Determine Ag/Ab Combo test 1) Antigen positive, antibody negative (acute infection); 2) Antigen positive, antibody positive; 3) Antigen negative, antibody positive; 4) Antigen negative, antibody negative; and 5) Antigen false positive, antibody negative (HIV uninfected). A sixth group included serial dilutions from a p24 antigen-positive control sample. Combo test results were reported as antigen positive, antibody positive, or both.
Results
Of 34 group 1 samples with VL between 5x105 and >1.5x107 copies/mL (median 3.5x106), 1 (2.9%) was detected by the Combo antigen component, 7 (20.6%) others were positive by the Combo antibody component. No group 2 samples were antigen positive by the Combo test (0/18). Sensitivity of the Combo antigen test was therefore 1.9% (1/52, 95% CI 0.0, 9.9). One false positive Combo antibody result (1/30, 3.3%) was observed in group 4. No false-positive Combo antigen results were observed. The Combo antigen test was positive in group 6 at concentrations of 80 pg/mL, faintly positive at 40 and 20 pg/mL, and negative thereafter. The p24 ELISA antigen test remained positive at 5 pg/mL.
Conclusions
Although the antibody component of the Combo test detected antibodies to HIV earlier than the comparison antibody tests used, less than 2% of the cases of antigen-positive HIV infection were detected by the Combo antigen component. The development of a rapid point-of-care test to diagnose acute HIV infection remains an urgent goal.
doi:10.1371/journal.pone.0037154
PMCID: PMC3371037  PMID: 22715363
20.  Performance of the Focus HerpeSelect-2 EIA for the detection of herpes simplex virus type 2 antibodies in seven African countries 
Sexually transmitted infections  2011;87(3):238-241.
Objective
To compare the performance of the Focus HerpeSelect-2 enzyme immunoassay (EIA) to the gold standard HSV-2 Western blot, among HIV-1 uninfected men and women in East and Southern Africa.
Methods
3399 HIV-1 uninfected women and men from 7 countries in East and Southern Africa were tested for HSV-2 antibody using Focus HerpeSelect-2 EIA. The performance of the HerpesSelect-2 EIA was compared with the gold standard HSV-2 specific Western blot.
Results
Two-thirds (2294/3399) of participants were male and two-thirds (2242/3399) were from East Africa. By Western blot testing, HSV-2 prevalence was 68%, 59% in men and 85% in women. At the manufacturer’s recommended cut-off value of greater than 1.1, the HerpeSelect-2 EIA had a sensitivity of 98.3% and specificity 80.3%. Receiver operating characteristic (ROC) plot analysis indicated that the optimum cut-off was 2.1 or greater with sensitivity 93.9% and specificity 90.5%. Diagnostic accuracy was modestly higher for Southern Africa (AUC=0.979, 95% CI: 0.970-0.988) compared with East Africa (AUC=0.954, 95% CI: 0.942-0.965; p<0.001 for Southern vs. East Africa).
Conclusions
The Focus HerpeSelect-2 EIA has acceptable diagnostic accuracy for determination of HSV-2 serostatus in African HIV-1 uninfected adults. An assay cut-off value of 2.1 or greater results in approximately 90% sensitivity and specificity, against a gold standard HSV-2 Western blot. Diagnostic accuracy differed slightly by geographical region.
doi:10.1136/sti.2010.047415
PMCID: PMC3066402  PMID: 21307152
HSV-2; Focus HerpeSelect-2 EIA; Western blot; HIV-1; Africa
21.  Differences in HIV Natural History among African and Non-African Seroconverters in Europe and Seroconverters in Sub-Saharan Africa 
PLoS ONE  2012;7(3):e32369.
Introduction
It is unknown whether HIV treatment guidelines, based on resource-rich country cohorts, are applicable to African populations.
Methods
We estimated CD4 cell loss in ART-naïve, AIDS-free individuals using mixed models allowing for random intercept and slope, and time from seroconversion to clinical AIDS, death and antiretroviral therapy (ART) initiation by survival methods. Using CASCADE data from 20 European and 3 sub-Saharan African (SSA) cohorts of heterosexually-infected individuals, aged ≥15 years, infected ≥2000, we compared estimates between non-African Europeans, Africans in Europe, and Africans in SSA.
Results
Of 1,959 (913 non-Africans, 302 Europeans - African origin, 744 SSA), two-thirds were female; median age at seroconversion was 31 years. Individuals in SSA progressed faster to clinical AIDS but not to death or non-TB AIDS. They also initiated ART later than Europeans and at lower CD4 cell counts. In adjusted models, Africans (especially from Europe) had lower CD4 counts at seroconversion and slower CD4 decline than non-African Europeans. Median (95% CI) CD4 count at seroconversion for a 15–29 year old woman was 607 (588–627) (non-African European), 469 (442–497) (European - African origin) and 570 (551–589) (SSA) cells/µL with respective CD4 decline during the first 4 years of 259 (228–289), 155 (110–200), and 199 (174–224) cells/µL (p<0.01).
Discussion
Despite differences in CD4 cell count evolution, death and non-TB AIDS rates were similar across study groups. It is therefore prudent to apply current ART guidelines from resource-rich countries to African populations.
doi:10.1371/journal.pone.0032369
PMCID: PMC3295758  PMID: 22412867
22.  Human Leukocyte Antigen Variants B*44 and B*57 Are Consistently Favorable during Two Distinct Phases of Primary HIV-1 Infection in Sub-Saharan Africans with Several Viral Subtypes▿† 
Journal of Virology  2011;85(17):8894-8902.
As part of an ongoing study of early human immunodeficiency virus type 1 (HIV-1) infection in sub-Saharan African countries, we have identified 134 seroconverters (SCs) with distinct acute-phase (peak) and early chronic-phase (set-point) viremias. SCs with class I human leukocyte antigen (HLA) variants B*44 and B*57 had much lower peak viral loads (VLs) than SCs without these variants (adjusted linear regression beta values of −1.08 ± 0.26 log10 [mean ± standard error] and −0.83 ± 0.27 log10, respectively; P < 0.005 for both), after accounting for several nongenetic factors, including gender, age at estimated date of infection, duration of infection, and country of origin. These findings were confirmed by alternative models in which major viral subtypes (A1, C, and others) in the same SCs replaced country of origin as a covariate (P ≤ 0.03). Both B*44 and B*57 were also highly favorable (P ≤ 0.03) in analyses of set-point VLs. Moreover, B*44 was associated with relatively high CD4+ T-cell counts during early chronic infection (P = 0.02). Thus, at least two common HLA-B variants showed strong influences on acute-phase as well as early chronic-phase VL, regardless of the infecting viral subtype. If confirmed, the identification of B*44 as another favorable marker in primary HIV-1 infection should help dissect mechanisms of early immune protection against HIV-1 infection.
doi:10.1128/JVI.00439-11
PMCID: PMC3165830  PMID: 21715491
23.  Transmitted HIV Type 1 Drug Resistance Among Individuals with Recent HIV Infection in East and Southern Africa 
Abstract
To characterize WHO-defined transmitted HIV drug resistance mutation (TDRM) data from recently HIV-infected African volunteers, we sequenced HIV (pol) and evaluated for TDRM the earliest available specimens from ARV-naive volunteers diagnosed within 1 year of their estimated date of infection at eight research centers in sub-Saharan Africa. TDRMs were detected in 19/408 (5%) volunteers. The prevalence of TDRMs varied by research center, from 5/26 (19%) in Entebbe, 6/78 (8%) in Kigali, 2/49 (4%) in Kilifi, to 3/106 (3%) in Lusaka. One of five volunteers from Cape Town (20%) had TDRMs. Despite small numbers, our data suggest an increase in DRMs by year of infection in Zambia (p = 0.004). The prevalence observed in Entebbe was high across the entire study. ARV history data from 12 (63%) HIV-infected sexual partners were available; 3 reported ARV use prior to transmission. Among four partners with sequence data available, transmission linkage was confirmed and two had the same TDRMs as the newly infected volunteer (both K103N). As ARV therapy continues to increase in availability throughout Africa, monitoring incident virus strains for the presence of TDRMs should be a priority. Early HIV infection cohorts provide an excellent and important platform to monitor the development of TDRMs to inform treatment guidelines, drug choices, and strategies for secondary prevention of TDRM transmission.
doi:10.1089/aid.2010.0030
PMCID: PMC3045073  PMID: 21091377
24.  Knowledge and Perceptions of Couples' Voluntary Counseling and Testing in Urban Rwanda and Zambia: A Cross-Sectional Household Survey 
PLoS ONE  2011;6(5):e19573.
Background
Most incident HIV infections in sub-Saharan Africa occur between cohabiting, discordant, heterosexual couples. Though couples' voluntary HIV counseling and testing (CVCT) is an effective, well-studied intervention in Africa, <1% of couples have been jointly tested.
Methods
We conducted cross-sectional household surveys in Kigali, Rwanda (n = 600) and Lusaka, Zambia (n = 603) to ascertain knowledge, perceptions, and barriers to use of CVCT.
Results
Compared to Lusaka, Kigali respondents were significantly more aware of HIV testing sites (79% vs. 56%); had greater knowledge of HIV serodiscordance between couples (83% vs. 43%); believed CVCT is good (96% vs. 72%); and were willing to test jointly (91% vs. 47%). Stigma, fear of partner reaction, and distance/cost/logistics were CVCT barriers.
Conclusions
Though most respondents had positive attitudes toward CVCT, the majority were unaware that serodiscordance between cohabiting couples is possible. Future messages should target gaps in knowledge about serodiscordance, provide logistical information about CVCT services, and aim to reduce stigma and fear.
doi:10.1371/journal.pone.0019573
PMCID: PMC3090401  PMID: 21573068
25.  Indeterminate and discrepant rapid HIV test results in couples' HIV testing and counselling centres in Africa 
Background
Many HIV voluntary testing and counselling centres in Africa use rapid antibody tests, in parallel or in sequence, to establish same-day HIV status. The interpretation of indeterminate or discrepant results between different rapid tests on one sample poses a challenge. We investigated the use of an algorithm using three serial rapid HIV tests in cohabiting couples to resolve unclear serostatuses.
Methods
Heterosexual couples visited the Rwanda Zambia HIV Research Group testing centres in Kigali, Rwanda, and Lusaka, Zambia, to assess HIV infection status. Individuals with unclear HIV rapid antibody test results (indeterminate) or discrepant results were asked to return for repeat testing to resolve HIV status. If either partner of a couple tested positive or indeterminate with the screening test, both partners were tested with a confirmatory test. Individuals with indeterminate or discrepant results were further tested with a tie-breaker and monthly retesting. HIV-RNA viral load was determined when HIV status was not resolved by follow-up rapid testing. Individuals were classified based on two of three initial tests as "Positive", "Negative" or "Other". Follow-up testing and/or HIV-RNA viral load testing determined them as "Infected", "Uninfected" or "Unresolved".
Results
Of 45,820 individuals tested as couples, 2.3% (4.1% of couples) had at least one discrepant or indeterminate rapid result. A total of 65% of those individuals had follow-up testing and of those individuals initially classified as "Negative" by three initial rapid tests, less than 1% were resolved as "Infected". In contrast, of those individuals with at least one discrepant or indeterminate result who were initially classified as "Positive", only 46% were resolved as "Infected", while the remainder was resolved as "Uninfected" (46%) or "Unresolved" (8%). A positive HIV serostatus of one of the partners was a strong predictor of infection in the other partner as 48% of individuals who resolved as "Infected" had an HIV-infected spouse.
Conclusions
In more than 45,000 individuals counselled and tested as couples, only 5% of individuals with indeterminate or discrepant rapid HIV test results were HIV infected. This represented only 0.1% of all individuals tested. Thus, algorithms using screening, confirmatory and tie-breaker rapid tests are reliable with two of three tests negative, but not when two of three tests are positive. False positive antibody tests may persist. HIV-positive partner serostatus should prompt repeat testing.
doi:10.1186/1758-2652-14-18
PMCID: PMC3086828  PMID: 21477317

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