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1.  Limited HIV-1 Superinfection in Seroconverters from the CAPRISA 004 Microbicide Trial 
Journal of Clinical Microbiology  2014;52(3):844-848.
HIV-1 superinfection (SI) occurs when an infected individual acquires a distinct new viral strain. The rate of superinfection may be reflective of the underlying HIV risk in a population. The Centre for the AIDS Programme of Research in South Africa (CAPRISA) 004 clinical trial demonstrated that women who used a tenofovir-containing microbicide gel had lower rates of HIV infection than women using a placebo gel. Women who contracted HIV-1 during the trial were screened for the occurrence of superinfection by next-generation sequencing of the viral gag and env genes. There were two cases (one in each trial arm) of subtype C superinfection identified from the 76 women with primary infection screened at two time points (rate of superinfection, 1.5/100 person-years). Both women experienced a >0.5-log increase in viral load during the window when superinfection occurred. The rate of superinfection was significantly lower than the overall primary HIV incidence in the microbicide trial (incidence rate ratio [IRR], 0.20; P = 0.003). The women who seroconverted during the trial reported a significant increase in sexual contact with their stable partner 4 months after seroconversion (P < 0.001), which may have lowered the risk of superinfection in this population. The lower frequency of SI compared to the primary incidence is in contrast to a report from a general heterosexual African population but agrees with a study of high-risk women in Kenya. A better understanding of the rate of HIV superinfection could have important implications for ongoing HIV vaccine research.
doi:10.1128/JCM.03143-13
PMCID: PMC3957790  PMID: 24371237
2.  HIV Type 1 Polymerase Gene Polymorphisms Are Associated With Phenotypic Differences in Replication Capacity and Disease Progression 
Background. Determinants of intersubtype differences in human immunodeficiency virus type 1 (HIV-1) clinical disease progression remain unknown.
Methods. HIV-1 subtype was independently determined for 5 separate genomic regions in 396 HIV-1 seroconverters from Rakai, Uganda, using a multiregion hybridization assay. Replication capacities (RC) in samples from a subset of 145 of these subjects were determined. HIV-1 genomic regions and pol RC were examined for association with disease progression. Amino acid polymorphisms were examined for association with pol RC.
Results. In multivariate analyses, the hazard for progression to the composite end point (defined as a CD4+ T-cell count <250 cells/mm3, antiretroviral therapy initiation, or death) among patients with subtype D pol infection was 2.4 times the hazard for those infected with subtype A pol infection (P = .001). Compared with subtype A pol (the reference group), the hazard for progression to the composite end point for subtype D pol infection with a pol RC >67% (ie, the median pol RC) was significantly greater (HR, 4.6; 95% confidence interval [CI], 1.9–11.0; P = .001), whereas the hazard for progression to the composite end point for subtype D pol infection with a pol RC ≤67% was not significantly different (HR, 2.2; 95% CI, 1.0–4.9; P = .051). Amino acid substitutions at protease positions 62 and 64 and at reverse transcriptase position 272 were associated with significant differences in pol RC.
Conclusions. HIV-1 pol gene intersubtype and RC differences are associated with disease progression and may be influenced by amino acid polymorphisms.
doi:10.1093/infdis/jit425
PMCID: PMC3864385  PMID: 23922373
HIV-1 Subtype; subtype A; subtype D; disease progression; polymerase; replication capacity; amino acid polymorphisms
3.  Liver Stiffness Is Associated With Monocyte Activation in HIV-Infected Ugandans Without Viral Hepatitis 
AIDS Research and Human Retroviruses  2013;29(7):1026-1030.
Abstract
A high prevalence of liver stiffness, as determined by elevated transient elastography liver stiffness measurement, was previously found in a cohort of HIV-infected Ugandans in the absence of chronic viral hepatitis. Given the role of immune activation and microbial translocation in models of liver disease, a shared immune mechanism was hypothesized in the same cohort without other overt causes of liver disease. This study examined whether HIV-related liver stiffness was associated with markers of immune activation or microbial translocation (MT). A retrospective case-control study of subjects with evidence of liver stiffness as defined by a transient elastography stiffness measurement ≥9.3 kPa (cases=133) and normal controls (n=133) from Rakai, Uganda was performed. Cases were matched to controls by age, gender, HIV, hepatitis B virus (HBV), and highly active antiretroviral therapy (HAART) status. Lipopolysaccharide (LPS), endotoxin IgM antibody, soluble CD14 (sCD14), C-reactive protein (CRP), and D-dimer levels were measured. Conditional logistic regression was used to estimate adjusted matched odds ratios (adjMOR) and 95% confidence intervals. Higher sCD14 levels were associated with a 19% increased odds of liver stiffness (adjMOR=1.19, p=0.002). In HIV-infected individuals, higher sCD14 levels were associated with a 54% increased odds of having liver stiffness (adjMOR=1.54, p<0.001); however, the opposite was observed in HIV-negative individuals (adjMOR=0.57, p=0.001). No other biomarker was significantly associated with liver stiffness, and only one subject was found to have detectable LPS. Liver stiffness in HIV-infected Ugandans is associated with increased sCD14 indicative of monocyte activation in the absence of viral hepatitis or microbial translocation, and suggests that HIV may be directly involved in liver disease.
doi:10.1089/aid.2013.0004
PMCID: PMC3685686  PMID: 23548102
4.  Short Communication: Colony-Forming Hematopoietic Progenitor Cells Are Not Preferentially Infected by HIV Type 1 Subtypes A and D in Vivo 
Abstract
HIV subtype C has previously been shown to infect hematopoietic progenitor cells (HPCs) at a significantly higher rate than subtype B. To better understand the subtype-specific nature of HPC infection, we examined the prevalence of HPC infection in vivo by HIV-1 subtypes A and D. HIV-1 infection of HPC was examined in 40 individuals, 19 infected with subtype A and 21 with subtype D, using a single colony assay format. DNA from 1177 extracted colonies was tested for integrated viral DNA of the p24 gene. Four colonies were found to be stably infected, three of 462 colonies (0.65%) from HIV-1A-infected individuals (1/19 individuals) and one of 715 colonies (0.14%) from HIV-1D-infected individuals (1/22 individuals). These rates of colony infection were comparable to the rates observed in PBMCs from the same subjects. Additionally, no correlation was observed between cell colony density and circulating viral load or proviral load. Our findings suggest that HIV-1 subtypes A and D do not preferentially infect colony-forming HPCs over mature HIV target cells in vivo.
doi:10.1089/aid.2011.0179
PMCID: PMC3423642  PMID: 22149236
5.  Frequency and Implications of HIV Superinfection 
The Lancet infectious diseases  2013;13(7):622-628.
HIV superinfection (HIV-SI) occurs when an HIV infected individual is infected with a new distinct HIV viral strain. HIV-SI has been reported throughout the world, and studies have reported HIV-SI incidence rates of 0% to 7.7% per year. Recent use of next-generation sequencing has improved detection of HIV-SI. Several studies have suggested that HIV-SI may occur at rates comparable to initial HIV infection. HIV-SI has been shown to be transmitted by injecting drug use, heterosexual and homosexual intercourse. Clinicians should encourage safe sexual and injecting drug use practices for HIV-infected patients since HIV-SI has detrimental effects on clinical outcomes, and may pose a concern for large scale antiretroviral treatment plans. The occurrence of HIV-SI has implications for vaccine research since it appears natural HIV infection is not fully protective against a subsequent HIV infection. Additional collaborative HIV-SI research is necessary which might benefit patient care and inform future vaccine design.
doi:10.1016/S1473-3099(13)70066-5
PMCID: PMC3752600  PMID: 23726798
HIV; HIV-1; superinfection; dual infection; double infection; reinfection; re-infection; antiretroviral; next generation sequencing; HIV pathogenesis; HIV clinical care
6.  The Rates of HIV Superinfection and Primary HIV Incidence in a General Population in Rakai, Uganda 
The Journal of Infectious Diseases  2012;206(2):267-274.
Background. Human immunodeficiency virus (HIV) superinfection has been documented in high-risk individuals; however, the rate of superinfection among HIV-infected individuals within a general population remains unknown.
Methods. A novel next-generation ultra-deep sequencing technique was utilized to determine the rate of HIV superinfection in a heterosexual population by examining two regions of the viral genome in longitudinal samples from recent HIV seroconverters (n = 149) in Rakai District, Uganda.
Results. The rate of superinfection was 1.44 per 100 person years (PYs) (95% confidence interval [CI], .4–2.5) and consisted of both inter- and intrasubtype superinfections. This was compared to primary HIV incidence in 20 220 initially HIV-negative individuals in the general population in Rakai (1.15 per 100 PYs; 95% CI, 1.1–1.2; P = .26). Propensity score matching (PS) was used to control for differences in sociodemographic and behavioral characteristics between the HIV-positive individuals at risk for superinfection and the HIV-negative population at baseline and follow-up. After PS matching, the estimated rate of primary incidence was 3.28 per 100 PYs (95% CI, 2.0–5.3; P = .07) controlling for baseline differences and 2.51 per 100 PYs (95% CI, 1.5–4.3; P = .24) controlling for follow-up differences.
Conclusions. This suggests that the rate of HIV superinfection in a general population is substantial, which could have a significant impact on future public health and HIV vaccine strategies.
doi:10.1093/infdis/jis325
PMCID: PMC3415936  PMID: 22675216
7.  Human Papillomavirus Clearance Among Males Is Associated With HIV Acquisition and Increased Dendritic Cell Density in the Foreskin 
The Journal of Infectious Diseases  2013;207(11):1713-1722.
Background. The association between human papillomavirus (HPV) infection and the risk of human immunodeficiency virus (HIV) seroconversion is unclear, and the genital cellular immunology has not been evaluated.
Methods. A case-control analysis nested within a male circumcision trial was conducted. Cases consisted of 44 male HIV seroconverters, and controls were 787 males who were persistently negative for HIV. The Roche HPV Linear Array Genotype Test detected high-risk HPV (HR-HPV) and low-risk HPV (LR-HPV) genotypes. Generalized estimating equations logistic regression was used to estimate adjusted odds ratios (aORs) of HIV seroconversion. In addition, densities of CD1a+ dendritic cells, CD4+ T cells, and CD8+ T cells were measured using immunohistochemistry analysis in foreskins of 79 males randomly selected from participants in the circumcision trial.
Results. HR-HPV or LR-HPV acquisition was not significantly associated with HIV seroconversion, after adjustment for sexual behaviors. However, HR-HPV and LR-HPV clearance was significantly associated with HIV seroconversion (aOR, 3.25 [95% confidence interval {CI}, 1.11–9.55] and 3.18 [95% CI, 1.14–8.90], respectively). The odds of HIV seroconversion increased with increasing number of HPV genotypes cleared (P < .001, by the test for trend). The median CD1a+ dendritic cell density in the foreskin epidermis was significantly higher among males who cleared HPV (72.0 cells/mm2 [interquartile range {IQR}, 29.4–138.3 cells/mm2]), compared with males who were persistently negative for HPV (32.1 cells/mm2 [IQR, 3.1–96.2 cells/mm2]; P = .047), and increased progressively with the number of HPV genotypes cleared (P = .05).
Conclusions. HPV clearance was associated with subsequent HIV seroconversion and also with increased epidermal dendritic cell density, which potentially mediates HIV seroconversion.
doi:10.1093/infdis/jit035
PMCID: PMC3636782  PMID: 23345339
human papillomavirus (HPV); male circumcision; HIV; AIDS; Uganda; penile cancer; cervical cancer; sexually transmitted infections
8.  Comparison of a High-Resolution Melting Assay to Next-Generation Sequencing for Analysis of HIV Diversity 
Journal of Clinical Microbiology  2012;50(9):3054-3059.
Next-generation sequencing (NGS) has recently been used for analysis of HIV diversity, but this method is labor-intensive, costly, and requires complex protocols for data analysis. We compared diversity measures obtained using NGS data to those obtained using a diversity assay based on high-resolution melting (HRM) of DNA duplexes. The HRM diversity assay provides a single numeric score that reflects the level of diversity in the region analyzed. HIV gag and env from individuals in Rakai, Uganda, were analyzed in a previous study using NGS (n = 220 samples from 110 individuals). Three sequence-based diversity measures were calculated from the NGS sequence data (percent diversity, percent complexity, and Shannon entropy). The amplicon pools used for NGS were analyzed with the HRM diversity assay. HRM scores were significantly associated with sequence-based measures of HIV diversity for both gag and env (P < 0.001 for all measures). The level of diversity measured by the HRM diversity assay and NGS increased over time in both regions analyzed (P < 0.001 for all measures except for percent complexity in gag), and similar amounts of diversification were observed with both methods (P < 0.001 for all measures except for percent complexity in gag). Diversity measures obtained using the HRM diversity assay were significantly associated with those from NGS, and similar increases in diversity over time were detected by both methods. The HRM diversity assay is faster and less expensive than NGS, facilitating rapid analysis of large studies of HIV diversity and evolution.
doi:10.1128/JCM.01460-12
PMCID: PMC3421787  PMID: 22785188
9.  Reply to Wertheim et al 
The Journal of Infectious Diseases  2013;207(7):1182-1183.
doi:10.1093/infdis/jit022
PMCID: PMC3611769  PMID: 23315325
10.  The Role of Viral Introductions in Sustaining Community-Based HIV Epidemics in Rural Uganda: Evidence from Spatial Clustering, Phylogenetics, and Egocentric Transmission Models 
PLoS Medicine  2014;11(3):e1001610.
Using different approaches to investigate HIV transmission patterns, Justin Lessler and colleagues find that extra-community HIV introductions are frequent and likely play a role in sustaining the epidemic in the Rakai community.
Please see later in the article for the Editors' Summary
Background
It is often assumed that local sexual networks play a dominant role in HIV spread in sub-Saharan Africa. The aim of this study was to determine the extent to which continued HIV transmission in rural communities—home to two-thirds of the African population—is driven by intra-community sexual networks versus viral introductions from outside of communities.
Methods and Findings
We analyzed the spatial dynamics of HIV transmission in rural Rakai District, Uganda, using data from a cohort of 14,594 individuals within 46 communities. We applied spatial clustering statistics, viral phylogenetics, and probabilistic transmission models to quantify the relative contribution of viral introductions into communities versus community- and household-based transmission to HIV incidence. Individuals living in households with HIV-incident (n = 189) or HIV-prevalent (n = 1,597) persons were 3.2 (95% CI: 2.7–3.7) times more likely to be HIV infected themselves compared to the population in general, but spatial clustering outside of households was relatively weak and was confined to distances <500 m. Phylogenetic analyses of gag and env genes suggest that chains of transmission frequently cross community boundaries. A total of 95 phylogenetic clusters were identified, of which 44% (42/95) were two individuals sharing a household. Among the remaining clusters, 72% (38/53) crossed community boundaries. Using the locations of self-reported sexual partners, we estimate that 39% (95% CI: 34%–42%) of new viral transmissions occur within stable household partnerships, and that among those infected by extra-household sexual partners, 62% (95% CI: 55%–70%) are infected by sexual partners from outside their community. These results rely on the representativeness of the sample and the quality of self-reported partnership data and may not reflect HIV transmission patterns outside of Rakai.
Conclusions
Our findings suggest that HIV introductions into communities are common and account for a significant proportion of new HIV infections acquired outside of households in rural Uganda, though the extent to which this is true elsewhere in Africa remains unknown. Our results also suggest that HIV prevention efforts should be implemented at spatial scales broader than the community and should target key populations likely responsible for introductions into communities.
Please see later in the article for the Editors' Summary
Editors' Summary
Background
About 35 million people (25 million of whom live in sub-Saharan Africa) are currently infected with HIV, the virus that causes AIDS, and about 2.3 million people become newly infected every year. HIV destroys immune system cells, leaving infected individuals susceptible to other infections. HIV infection can be controlled by taking antiretroviral drugs (antiretroviral therapy, or ART) daily throughout life. Although originally available only to people living in wealthy countries, recent political efforts mean that 9.7 million people in low- and middle-income countries now have access to ART. However, ART does not cure HIV infection, so prevention of viral transmission remains extremely important. Because HIV is usually transmitted through unprotected sex with an infected partner, individuals can reduce their risk of infection by abstaining from sex, by having one or a few partners, and by using condoms. Male circumcision also reduces HIV transmission. In addition to reducing illness and death among HIV-positive people, ART also reduces HIV transmission.
Why Was This Study Done?
Effective HIV control requires an understanding of how HIV spreads through sexual networks. These networks include sexual partnerships between individuals in households, between community members in different households, and between individuals from different communities. Local sexual networks (household and intra-community sexual partnerships) are sometimes assumed to be the dominant driving force in HIV spread in sub-Saharan Africa, but are viral introductions from sexual partnerships with individuals outside the community also important? This question needs answering because the effectiveness of interventions such as ART as prevention partly depends on how many new infections in an intervention area are attributable to infection from partners residing in that area and how many are attributable to infection from partners living elsewhere. Here, the researchers use three analytical methods—spatial clustering statistics, viral phylogenetics, and egocentric transmission modeling—to ask whether HIV transmission in rural Uganda is driven predominantly by intra-community sexual networks. Spatial clustering analysis uses the geographical coordinates of households to measure the tendency of HIV-infected people to cluster spatially at scales consistent with community transmission. Viral phylogenetic analysis examines the genetic relatedness of viruses; if transmission is through local networks, viruses in newly infected individuals should more closely resemble viruses in other community members than those in people outside the community. Egocentric transmission modelling uses information on the locations of recent sexual partners to estimate the proportions of new transmissions from household, intra-community, and extra-community partners.
What Did the Researchers Do and Find?
The researchers applied their three analytical methods to data collected from 14,594 individuals living in 46 communities (governmental administrative units) in Rakai District, Uganda. Spatial clustering analysis indicated that individuals who lived in households with individuals with incident HIV (newly diagnosed) or prevalent HIV (previously diagnosed) were 3.2 times more likely than the general population to be HIV-positive themselves. Spatial clustering outside households was relatively weak, however, and was confined to distances of less than half a kilometer. Viral phylogenetic analysis indicated that 44% of phylogenetic clusters (viruses with related genetic sequences found in more than one individual) were within households, but that 40% of clusters crossed community borders. Finally, analysis of the locations of self-reported sexual partners indicated that 39% of new viral transmissions occurred within stable household partnerships, but that among people newly infected by extra-household partners, nearly two-thirds were infected by partners from outside their community.
What Do These Findings Mean?
The results of all three analyses suggest that HIV introductions into communities are frequent and are likely to play an important role in sustaining HIV transmission in the Rakai District. Specifically, within this rural HIV-endemic region (a region where HIV infection is always present), viral introductions combined with intra-household transmission account for the majority of new infections, although community-based sexual networks also play a critical role in HIV transmission. These findings may not be generalizable to the broader Ugandan population or to other regions of Africa, and their accuracy is likely to be limited by the use of self-reported sexual partner data. Nevertheless, these findings indicate that the dynamics of HIV transmission in rural Uganda (and probably elsewhere) are complex. Consequently, to halt the spread of HIV, prevention efforts will need to be implemented at spatial scales broader than individual communities, and key populations that are likely to introduce HIV into communities will need to be targeted.
Additional Information
Please access these websites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001610.
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
Information is available from Avert, an international AIDS charity, on many aspects of HIV/AIDS, including information on HIV and AIDS in Uganda and on HIV prevention strategies (in English and Spanish)
The UNAIDS Report on the Global AIDS Epidemic 2013 provides up-to-date information about the AIDS epidemic and efforts to halt it
The Center for AIDS Prevention Studies (University of California, San Francisco) has a fact sheet about sexual networks and HIV prevention
Wikipedia provides information on spatial clustering analysis (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
A PLOS Computational Biology Topic Page (a review article that is a published copy of record of a dynamic version of the article as found in Wikipedia) about viral phylodynamics is available
Personal stories about living with HIV/AIDS are available through Avert, NAM/aidsmap, and Healthtalkonline
doi:10.1371/journal.pmed.1001610
PMCID: PMC3942316  PMID: 24595023
12.  Previously Transmitted HIV-1 Strains Are Preferentially Selected During Subsequent Sexual Transmissions 
The Journal of Infectious Diseases  2012;206(9):1433-1442.
Background. A genetic bottleneck is known to exist for human immunodeficiency virus (HIV) at the point of sexual transmission. However, the nature of this bottleneck and its effect on viral diversity over time is unclear.
Methods. Interhost and intrahost HIV diversity was analyzed in a stable population in Rakai, Uganda, from 1994 to 2002. HIV-1 envelope sequences from both individuals in initially HIV-discordant relationships in which transmission occurred later were examined using Sanger sequencing of bulk polymerase chain reaction (PCR) products (for 22 couples), clonal analysis (for 3), and next-generation deep sequencing (for 9).
Results. Intrahost viral diversity was significantly higher than changes in interhost diversity (P < .01). The majority of HIV-1–discordant couples examined via bulk PCR (16 of 22 couples), clonal analysis (3 of 3), and next-generation deep sequencing (6 of 9) demonstrated that the viral populations present in the newly infected recipient were more closely related to the donor partner's HIV-1 variants found earlier during infection as compared to those circulating near the estimated time of transmission (P = .03).
Conclusions. These findings suggest that sexual transmission constrains viral diversity at the population level, partially because of the preferential transmission of ancestral as opposed to contemporary strains circulating in the transmitting partner. Future successful vaccine strategies may need to target these transmitted ancestral strains.
doi:10.1093/infdis/jis503
PMCID: PMC3466994  PMID: 22997233
13.  T-cell enumeration from dried blood spots by quantifying rearranged T-cell receptor-β genes 
Journal of immunological methods  2010;354(1-2):40-44.
Significant hurdles remain to large-scale implementation of medical interventions in the developing world due to the lack of a modern diagnostic infrastructure. This is especially pertinent to the international roll-out of antiretroviral drugs to treat HIV, which ideally includes a CD4 t-cell to determine eligibility. We designed a novel technique to estimate mature t-cell numbers by calculating the amount of rearranged t-cell receptor β genes from dried blood spots of HIV-infected individuals in the United States and Uganda. It was observed that the rearranged t-cell receptor β count correlated well with total lymphocyte counts from both study populations (Baltimore R=0.602, Uganda R=0.497; p<0.001) and the ability for this measurement to determine antiretroviral initiation was similar to total lymphocyte counts, which can be used to determine eligibility in HIV+ children. This technique as well as other dried blood spot based technologies could increase the diagnostic and monitoring capabilities in resource-limited settings.
doi:10.1016/j.jim.2010.01.008
PMCID: PMC2844472  PMID: 20109463
14.  HIV Type 1 Genetic Variation in Foreskin and Blood from Subjects in Rakai, Uganda 
Abstract
The foreskin contains a subset of dendritic cells, macrophages, and CD4+ and CD8+ T cells that may be targets for initial HIV infection in female-to-male sexual transmission of HIV-1. We present analyses comparing HIV-1 sequences isolated from foreskin DNA and serum RNA in 12 heterosexual men enrolled in an adult male circumcision trial performed in Rakai, Uganda. Phylogenetic analysis demonstrated three topologies: (1) little divergence between foreskin and serum, (2) multiple genetic bottlenecks occurring in both foreskin and serum, and (3) complete separation of foreskin and serum populations. The latter tree topology provided evidence that foreskin may serve as a reservoir for distinct HIV-1 strains. Distance and recombination analysis also demonstrated that viral genotypes in the foreskin might segregate independently from the circulating pool of viruses.
doi:10.1089/aid.2011.0176
PMCID: PMC3380386  PMID: 21902587
15.  Identification of New CRF51_01B in Singapore Using Full Genome Analysis of Three HIV Type 1 Isolates 
Abstract
A recent HIV-1 molecular epidemiology survey in Singapore identified a novel CRF01_AE/B recombinant form, which accounted for 13 (11.9%) of 109 patient samples. Peripheral blood mononuclear cell DNA from three of these 13 patients was used to generate near full-length sequences to characterize the novel CRF01_AE/B recombinant form. The three isolates had a recombinant structure composed of CRF01_AE and subtype B, and shared identical breakpoints. As the three patients were not epidemiologically linked, this recombinant form has been designated CRF51_01B. Identification of the novel recombinant forms indicates ongoing active HIV-1 transmission in Singapore.
doi:10.1089/aid.2011.0177
PMCID: PMC3332370  PMID: 21902588
16.  Frequency of Long-Term Nonprogressors in HIV-1 Seroconverters From Rakai Uganda 
Objective
Studies on long-term nonprogressors (LTNP) have been conducted in the USA and Europe. This study examined the frequency of LTNPs and HIV controllers among 637 HIV-1 seroconverters in rural Uganda.
Design and Methods
LTNPs were defined as being infected for more than 7 years with a CD4+ T-cell count above 600 cells per microliter, and HIV controllers as having undetectable viral loads on 3 separate occasions without antiretroviral treatment. HIV-1 viral load and subtype distribution between LTNP and non-LTNP populations were determined.
Results
Of the HIV seroconverters, 9.1% (58/637) were LTNPs and 1.4% (9/637) were HIV controllers. LTNPs had a significantly lower viral load at set point than non-LTNP participants (P < 0.001). The Kaplan–Meier joint probability of surviving to 7 years with a CD4 count >600 was 19.2%. Individuals who survived 7 years had a significantly higher frequency of HIV-1 subtype A (P < 0.05), but seroconverters infected with HIV-1A did not have a significantly higher probability of becoming an LTNP.
Conclusions
The frequency of LTNPs appears to be relatively high in Uganda and it may be important to take this into account when designing studies of viral pathogenesis and performing HIV vaccine trials in sub-Saharan Africa.
doi:10.1097/QAI.0b013e3181bc08f5
PMCID: PMC2837462  PMID: 19726998
Africa; HIV; HIV disease progression; long-term nonprogressors
17.  Efficiency of CCR5 Coreceptor Utilization by the HIV Quasispecies Increases over Time, But Is Not Associated with Disease Progression 
Abstract
CCR5 is the primary coreceptor for HIV entry. Early after infection, the HIV viral population diversifies rapidly into a quasispecies. It is not known whether the initial efficiency of the viral quasispecies to utilize CCR5 is associated with HIV disease progression or if it changes in an infected individual over time. The CCR5 and CXCR4 utilization efficiencies (R5-UE and X4-UE) of the HIV quasispecies were examined using a pseudovirus, single-round infection assay for samples obtained from known seroconverters from Rakai district, Uganda (n=88). Initial and longitudinal R5-UE values were examined to assess the association of R5-UE with HIV disease progression using multivariate Cox proportional hazard models. Longitudinal samples were analyzed for 35 seroconverters who had samples available from multiple time points. There was no association between initial or longitudinal changes in R5-UE and the hazard of HIV disease progression (p=0.225 and p=0.942, respectively). In addition, R5-UE increased significantly over time after HIV seroconversion (p<0.001), regardless of HIV subtype or the emergence of CXCR4-tropic virus. These data demonstrate that the R5-UE of the viral quasispecies early in HIV infection is not associated with disease progression, and that R5-UE levels increase in HIV-infected individuals over time.
doi:10.1089/aid.2011.0006
PMCID: PMC3292754  PMID: 21663455
18.  Male Circumcision and Herpes Simplex Virus Type 2 Infection in Female Partners: A Randomized Trial in Rakai, Uganda 
The Journal of Infectious Diseases  2011;205(3):486-490.
Male circumcision reduces acquisition of herpes simplex virus type 2 (HSV-2) in men. We assessed whether male circumcision reduces HSV-2 infection among female partners. HSV-2–negative, human immunodeficiency virus–negative female partners of 368 males who were and 372 males who were not randomized to receive male circumcision were enrolled. The incidence of HSV-2 infection among females over a period of 2 years was 6.09 cases per 100 person-years in the intervention arm and 6.32 cases per 100 person-years in the control arm (incidence rate ratio [IRR], 0.96 [95% confidence interval {CI}, .62–1.49]; P = .87). Among female partners of HSV-2–positive males, the incidence of HSV-2 infection was 9.55 cases per 100 person-years in the intervention arm and 11.17 cases per 100 person-years in the control arm (IRR, 0.85 [95% CI, .44–1.67]; P = .62). Contrary to findings in males, male circumcision did not affect HSV-2 acquisition among female partners.
doi:10.1093/infdis/jir767
PMCID: PMC3256952  PMID: 22147796
19.  Analysis of Genetic Linkage of HIV From Couples Enrolled in the HIV Prevention Trials Network 052 Trial 
The Journal of Infectious Diseases  2011;204(12):1918-1926.
Background. The HIV Prevention Trials Network (HPTN) 052 trial demonstrated that early initiation of antiretroviral therapy (ART) reduces human immunodeficiency virus (HIV) transmission from HIV-infected adults (index participants) to their HIV-uninfected sexual partners. We analyzed HIV from 38 index-partner pairs and 80 unrelated index participants (controls) to assess the linkage of seroconversion events.
Methods. Linkage was assessed using phylogenetic analysis of HIV pol sequences and Bayesian analysis of genetic distances between pol sequences from index-partner pairs and controls. Selected samples were also analyzed using next-generation sequencing (env region).
Results. In 29 of the 38 (76.3%) cases analyzed, the index was the likely source of the partner’s HIV infection (linked). In 7 cases (18.4%), the partner was most likely infected from a source other than the index participant (unlinked). In 2 cases (5.3%), linkage status could not be definitively established.
Conclusions. Nearly one-fifth of the seroconversion events in HPTN 052 were unlinked. The association of early ART and reduced HIV transmission was stronger when the analysis included only linked events. This underscores the importance of assessing the genetic linkage of HIV seroconversion events in HIV prevention studies involving serodiscordant couples.
doi:10.1093/infdis/jir651
PMCID: PMC3209811  PMID: 21990420
20.  Molecular Epidemiology of HIV Type 1 in Singapore and Identification of Novel CRF01_AE/B Recombinant Forms 
AIDS Research and Human Retroviruses  2011;27(10):1135-1137.
Abstract
To investigate HIV-1 molecular epidemiology in Singapore, we sequenced portions of three regions of the HIV-1 genome (protease HXB2: 2163 to 2620, gp120 HXB2: 6904 to 7628, and gp41 HXB2: 7817 to 8264) from 212 plasma samples collected between February 2008 and August 2009. From these samples, 109 (51.4%) generated interpretable data in all regions. Sixty-one (56.0%) were identified as CRF01_AE, 26 (23.9%) as subtype B and 14 (12.8%) as possible novel recombinant forms. The main novel recombinant pattern, detected in 13 sequences, had subtype B in protease and gp41 and CRF01_AE in gp120. There was intermixing of subtypes within transmission risk groups. However, 85% of subjects infected with the novel recombinant forms self-identified as men who have sex with men or bisexuals compared with only 41% of individuals infected with CRF01_AE and 62% infected with subtype B (p = 0.001).
doi:10.1089/aid.2010.0364
PMCID: PMC3186691  PMID: 21235306
21.  Performance of Euroline Western Blot Assay in the Detection of Herpes Simplex Virus type 2 Antibody in Uganda, China, and the United States 
Herpes simplex virus type 2 (HSV-2) infection is one of the most commonly sexually transmitted infections worldwide. While glycoprotein G-2 ELISA based assays are commonly used for the serologic detection of HSV-2 infections, they have low specificity in developing countries. Euroline Western blot (WB) is a commercially available assay that is easy to perform; however, little is known about its performance characteristics. This study evaluated Euroline WB for the detection of HSV-2 antibodies compared to University of Washington Western blot in three geographically different regions, Baltimore, Maryland, Rakai, Uganda, and Kunming, China. Among the 135 American men attending an STD clinic in Baltimore, Maryland, 72% (n=97) were HSV-2 positive by Euroline WB. The Euroline WB had a sensitivity of 97.8% and a specificity of 81.8%. Among the 273 commercial sex workers in Kunming, 62.3% were HSV-2 positive by Euroline WB. The Euroline WB had a sensitivity of 96.9% and a specificity of 89.1%. Among the 437 Ugandans in Rakai, 67.3% were HSV-2 positive by Euroline WB. The Euroline WB had a sensitivity of 98.7% and a specificity of 65.4%. The Euroline WB has a consistently high sensitivity, but specificity varied significantly among the different locations.
doi:10.1258/ijsa.2009.009327
PMCID: PMC3262047  PMID: 21680672
22.  Effects of HIV-1 and Herpes Simplex Virus Type 2 Infection on Lymphocyte and Dendritic Cell Density in Adult Foreskins from Rakai, Uganda 
The Journal of Infectious Diseases  2011;203(5):602-609.
Background. Male circumcision reduces human immunodeficiency virus (HIV) and herpes simplex virus type 2 (HSV-2) acquisition, and HSV-2 infection is associated with an increased risk of HIV acquisition. To assess the cellular basis for these associations, we estimated immunologic cellular densities in foreskin tissue.
Methods. Immunostained CD1a+ dendritic cell and CD4+ and CD8+ T cell densities were quantified in foreskin samples obtained from medical circumcision in Rakai, Uganda (35 HIV-infected, HSV-2-infected men; 5 HIV-infected, HSV-2-uninfected men; 22 HIV-uninfected, HSV-2-infected men; and 29 HIV-uninfected, HSV-2-uninfected men.
Results. CD1A+ dendritic cell densities did not vary by HIV or HSV-2 status. Compared with densities in HIV-uninfected, HSV-2-uninfected men (mean, 26.8 cells/mm2), CD4+ T cell densities were similar in the HIV-infected, HSV-2-infected group (mean, 28.7 cells/mm2), were significantly decreased in the HIV-infected, HSV-2-uninfected group (mean, 11.2; P < .05), and were increased in the HIV-uninfected, HSV-2-infected group (mean, 68.7; P < .05). Dermal CD8+ T cell densities were higher in the HIV and HSV-2-coinfected group (mean, 102.9) than in the HIV-uninfected, HSV-2-uninfected group (mean, 10.0; P < .001), the HIV-infected, HSV-2-uninfected group (mean, 27.3; P < .001), and the HIV-uninfected, HSV-2-infected group (mean, 25.3; P < .005).
Discussion. The increased CD4+ cellular density in the HIV-uninfected, HSV-2-infected men may help to explain why HSV-2–infected men are at increased risk of HIV acquisition. The absence of this increase in men coinfected with both HIV and HSV-2 is likely in part the result of the progressive loss of CD4+ cells in HIV infection. Conversely, HIV and HSV-2 coinfection appears to synergistically increase CD8+ T cell densities.
doi:10.1093/infdis/jiq091
PMCID: PMC3071278  PMID: 21220779
24.  Identification of HIV Superinfection in Seroconcordant Couples in Rakai, Uganda, by Use of Next-Generation Deep Sequencing ▿ †  
Journal of Clinical Microbiology  2011;49(8):2859-2867.
HIV superinfection, which occurs when a previously infected individual acquires a new distinct HIV strain, has been described in a number of populations. Previous methods to detect superinfection have involved a combination of labor-intensive assays with various rates of success. We designed and tested a next-generation sequencing (NGS) protocol to identify HIV superinfection by targeting two regions of the HIV viral genome, p24 and gp41. The method was validated by mixing control samples infected with HIV subtype A or D at different ratios to determine the inter- and intrasubtype sensitivity by NGS. This amplicon-based NGS protocol was able to consistently identify distinct intersubtype strains at ratios of 1% and intrasubtype variants at ratios of 5%. By using stored samples from the Rakai Community Cohort Study (RCCS) in Uganda, 11 individuals who were HIV seroconcordant but virally unlinked from their spouses were then tested by this method to detect superinfection between 2002 and 2005. Two female cases of HIV intersubtype superinfection (18.2%) were identified. These results are consistent with other African studies and support the hypothesis that HIV superinfection occurs at a relatively high rate. Our results indicate that NGS can be used for detection of HIV superinfection within large cohorts, which could assist in determining the incidence and the epidemiologic, virologic, and immunological correlates of this phenomenon.
doi:10.1128/JCM.00804-11
PMCID: PMC3147722  PMID: 21697329
25.  Changes in the Distribution of HIV Type 1 Subtypes D and A in Rakai District, Uganda Between 1994 and 2002 
AIDS Research and Human Retroviruses  2010;26(10):1087-1091.
Abstract
HIV-1 subtype D (HIV-1D) progresses to disease faster and has lower transmissibility than subtype A (HIV-1A). We examined whether these differences could lead to a population level change in the distribution of these subtypes over time. HIV-1 viral RNA was extracted from stored serum samples from HIV-positive subjects participating in a population-based cohort study in Rakai, Uganda in 1994 and 2002. Portions of the viral proteins gag and gp41 were sequenced and subtyped. HIV-1 subtype assignments were generated for 773 subjects in 1994 and 812 subjects in 2002. The change in subtype distribution of the population as a whole as well as quartile age groups were examined for significant changes using a linear model. There was a significant decrease in the proportion of subjects infected with HIV-1D from 70.2% to 62.4% and a significant increase in subjects infected with HIV-1A from 16.7% to 23.3% over the 8-year period (p = 0.005). The most marked changes in proportion of HIV-1D and A were seen in the younger individuals (<25 and 25–30 years; p < 0.05). The percentages of subjects infected with HIV-1C and recombinant subtypes did not change significantly. Over this 8-year period, the overall viral population in this region evolved toward the less virulent HIV-1A strain, most likely as a consequence of the faster disease progression and lower transmissibility of HIV-1D.
doi:10.1089/aid.2010.0054
PMCID: PMC2965693  PMID: 20925575

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