Prevalence and incidence of anal high-grade squamous intraepithelial lesions in a cohort of cisgender men and transgender women who have sex with men diagnosed and treated during acute HIV acquisition in Bangkok, Thailand.

INTRODUCTION: Men who have sex with men (MSM), especially those living with HIV, are at an increased risk of anal cancer. The prevalence and incidence of its precursor, anal high-grade squamous intraepithelial lesions (HSILs), among MSM who started antiretroviral therapy during acute HIV acquisition are yet to be explored. METHODS: Participants in an acute HIV acquisition cohort in Bangkok, Thailand, who agreed to take part in this study, were enrolled. All participants were diagnosed and started antiretroviral therapy during acute HIV acquisition. Human papillomavirus (HPV) genotyping and high-resolution anoscopy, followed by anal biopsy as indicated, were done at baseline and 6-monthly visits. RESULTS: A total of 89 MSM and four transgender women were included in the analyses. Median age at enrolment was 26 years. Baseline prevalence of histologic anal HSIL was 11.8%. With a total of 147.0 person-years of follow-up, the incidence of initial histologic anal HSIL was 19.7 per 100 person-years. Factors associated with incident anal HSIL were anal HPV 16 (adjusted hazards ratio [aHR] 4.33, 95% CI 1.03-18.18), anal HPV 18/45 (aHR 6.82, 95% CI 1.57-29.51), other anal high-risk HPV (aHR 4.23, 95% CI 1.27-14.14), syphilis infection (aHR 4.67, 95% CI 1.10-19.90) and CD4 count <350 cells/mm3 (aHR 3.09, 95% CI 1.28-7.48). CONCLUSIONS: With antiretroviral therapy initiation during acute HIV acquisition, we found the prevalence of anal HSIL among cisgender men and transgender women who have sex with men to be similar to those without HIV. Subsequent anal HSIL incidence, although lower than that of those with chronic HIV acquisition, was still higher than that of those without HIV. Screening for and management of anal HSIL should be a crucial part of long-term HIV care for all MSM.

RV144 vaccine imprinting constrained HIV-1 evolution following breakthrough infection.

The scale of the HIV-1 epidemic underscores the need for a vaccine. The multitude of circulating HIV-1 strains together with HIV-1's high evolvability hints that HIV-1 could adapt to a future vaccine. Here, we wanted to investigate the effect of vaccination on the evolution of the virus post-breakthrough infection. We analyzed 2,635 HIV-1 env sequences sampled up to a year post-diagnosis from 110 vaccine and placebo participants who became infected in the RV144 vaccine efficacy trial. We showed that the Env signature sites that were previously identified to distinguish vaccine and placebo participants were maintained over time. In addition, fewer sites were under diversifying selection in the vaccine group than in the placebo group. These results indicate that HIV-1 would possibly adapt to a vaccine upon its roll-out.

Factors influencing estimates of HIV-1 infection timing using BEAST.

While large datasets of HIV-1 sequences are increasingly being generated, many studies rely on a single gene or fragment of the genome and few comparative studies across genes have been done. We performed genome-based and gene-specific Bayesian phylogenetic analyses to investigate how certain factors impact estimates of the infection dates in an acute HIV-1 infection cohort, RV217. In this cohort, HIV-1 diagnosis corresponded to the first RNA positive test and occurred a median of four days after the last negative test, allowing us to compare timing estimates using BEAST to a narrow window of infection. We analyzed HIV-1 sequences sampled one week, one month and six months after HIV-1 diagnosis in 39 individuals. We found that shared diversity and temporal signal was limited in acute infection, and insufficient to allow timing inferences in the shortest HIV-1 genes, thus dated phylogenies were primarily analyzed for env, gag, pol and near full-length genomes. There was no one best-fitting model across participants and genes, though relaxed molecular clocks (73% of best-fitting models) and the Bayesian skyline (49%) tended to be favored. For infections with single founders, the infection date was estimated to be around one week pre-diagnosis for env (IQR: 3-9 days) and gag (IQR: 5-9 days), whilst the genome placed it at a median of 10 days (IQR: 4-19). Multiply-founded infections proved problematic to date. Our ability to compare timing inferences to precise estimates of HIV-1 infection (within a week) highlights that molecular dating methods can be applied to within-host datasets from early infection. Nonetheless, our results also suggest caution when using uniform clock and population models or short genes with limited information content.

Molecular dating and viral load growth rates suggested that the eclipse phase lasted about a week in HIV-1 infected adults in East Africa and Thailand.

Most HIV-1 infected individuals do not know their infection dates. Precise infection timing is crucial information for studies that document transmission networks or drug levels at infection. To improve infection timing, we used the prospective RV217 cohort where the window when plasma viremia becomes detectable is narrow: the last negative visit occurred a median of four days before the first detectable HIV-1 viremia with an RNA test, referred below as diagnosis. We sequenced 1,280 HIV-1 genomes from 39 participants at a median of 4, 32 and 170 days post-diagnosis. HIV-1 infections were dated by using sequence-based methods and a viral load regression method. Bayesian coalescent and viral load regression estimated that infections occurred a median of 6 days prior to diagnosis (IQR: 9-3 and 11-4 days prior, respectively). Poisson-Fitter, which analyzes the distribution of hamming distances among sequences, estimated a median of 7 days prior to diagnosis (IQR: 15-4 days) based on sequences sampled 4 days post-diagnosis, but it did not yield plausible results using sequences sampled at 32 days. Fourteen participants reported a high-risk exposure event at a median of 8 days prior to diagnosis (IQR: 12 to 6 days prior). These different methods concurred that HIV-1 infection occurred about a week before detectable viremia, corresponding to 20 days (IQR: 34-15 days) before peak viral load. Together, our methods comparison helps define a framework for future dating studies in early HIV-1 infection.

Sieve analysis of breakthrough HIV-1 sequences in HVTN 505 identifies vaccine pressure targeting the CD4 binding site of Env-gp120.

Although the HVTN 505 DNA/recombinant adenovirus type 5 vector HIV-1 vaccine trial showed no overall efficacy, analysis of breakthrough HIV-1 sequences in participants can help determine whether vaccine-induced immune responses impacted viruses that caused infection. We analyzed 480 HIV-1 genomes sampled from 27 vaccine and 20 placebo recipients and found that intra-host HIV-1 diversity was significantly lower in vaccine recipients (P ≤ 0.04, Q-values ≤ 0.09) in Gag, Pol, Vif and envelope glycoprotein gp120 (Env-gp120). Furthermore, Env-gp120 sequences from vaccine recipients were significantly more distant from the subtype B vaccine insert than sequences from placebo recipients (P = 0.01, Q-value = 0.12). These vaccine effects were associated with signatures mapping to CD4 binding site and CD4-induced monoclonal antibody footprints. These results suggest either (i) no vaccine efficacy to block acquisition of any viral genotype but vaccine-accelerated Env evolution post-acquisition; or (ii) vaccine efficacy against HIV-1s with Env sequences closest to the vaccine insert combined with increased acquisition due to other factors, potentially including the vaccine vector.

Correction: Rare HIV-1 transmitted/founder lineages identified by deep viral sequencing contribute to rapid shifts in dominant quasispecies during acute and early infection.

[This corrects the article DOI: 10.1371/journal.ppat.1006510.].

Rare HIV-1 transmitted/founder lineages identified by deep viral sequencing contribute to rapid shifts in dominant quasispecies during acute and early infection.

In order to inform the rational design of HIV-1 preventive and cure interventions it is critical to understand the events occurring during acute HIV-1 infection (AHI). Using viral deep sequencing on six participants from the early capture acute infection RV217 cohort, we have studied HIV-1 evolution in plasma collected twice weekly during the first weeks following the advent of viremia. The analysis of infections established by multiple transmitted/founder (T/F) viruses revealed novel viral profiles that included: a) the low-level persistence of minor T/F variants, b) the rapid replacement of the major T/F by a minor T/F, and c) an initial expansion of the minor T/F followed by a quick collapse of the same minor T/F to low frequency. In most participants, cytotoxic T-lymphocyte (CTL) escape was first detected at the end of peak viremia downslope, proceeded at higher rates than previously measured in HIV-1 infection, and usually occurred through the exploration of multiple mutational pathways within an epitope. The rapid emergence of CTL escape variants suggests a strong and early CTL response. Minor T/F viral strains can contribute to rapid and varied profiles of HIV-1 quasispecies evolution during AHI. Overall, our results demonstrate that early, deep, and frequent sampling is needed to investigate viral/host interaction during AHI, which could help identify prerequisites for prevention and cure of HIV-1 infection.

Significant contribution of subtype G to HIV-1 genetic complexity in Nigeria identified by a newly developed subtyping assay specific for subtype G and CRF02_AG.

While abundant sequence information is available from human immunodeficiency virus type 1 (HIV-1) subtypes A, B, C and CRF01_AE for HIV-1 vaccine design, sequences from West Africa are less represented. We sought to augment our understanding of HIV-1 variants circulating in 6 Nigerian cities as a step to subsequent HIV-1 vaccine development.The G/CRF02_AG multi-region hybridization assay (MHA) was developed to differentiate subtype G, CRF02_AG and their recombinants from other subtypes based on 7 HIV-1 segments. Plasma from 224 HIV-1 infected volunteers enrolled in a cohort examining HIV-1 prevalence, risk factor, and subtype from Makurdi (30), Abuja (18), Enugu (11), Kaduna (12), Tafa (95), and Ojo/Lagos (58) was analyzed using MHA. HIV-1 genomes from 42 samples were sequenced to validate the MHA and fully explore the recombinant structure of G and CRF02_AG variants.The sensitivity and specificity of MHA varied between 73-100% and 90-100%, respectively. The subtype distribution as identified by MHA among 224 samples revealed 38% CRF02_AG, 28% G, and 26% G/CRF02_AG recombinants while 8% remained nontypeable strains. In envelope (env) gp120, 38.84% of the samples reacted to a G probe while 31.25% reacted to a CRF02 (subtype A) probe. Full genome characterization of 42 sequences revealed the complexity of Nigerian HIV-1 variants.CRF02_AG, subtype G, and their recombinants were the major circulating HIV-1 variants in 6 Nigerian cities. High proportions of samples reacted to a G probe in env gp120 confirms that subtype G infections are abundant and should be considered in strategies for global HIV-1 vaccine development.

Hepatitis C genotype distribution and homology among geographically disparate injecting drug users in Afghanistan.

Hepatitis C virus (HCV) prevalence is high among injecting drug users in Afghanistan, but transmission dynamics are poorly understood. Samples from HCV-infected injecting drug users were sequenced to determine circulating genotypes and potential transmission linkages. Serum samples were obtained from injecting drug user participants in Hirat, Jalalabad, and Mazar-i-Sharif between 2006 and 2008 with reactive anti-HCV rapid tests. Specimens with detected HCV viremia were amplified and underwent sequence analysis. Of 113 samples evaluated, 25 samples (35.2%) were only typeable in NS5B, nine samples (12.7%) were only typeable in CE1, and 37 samples (52.1%) were genotyped in both regions. Of those with typeable HCV, all were Afghan males with a mean age of 31.1 (standard deviation [SD] ± 8.0) years and mean duration of injecting of 3.9 (SD ± 4.3) years. Most reported residence outside Afghanistan in the last decade (90.1%) and prior incarceration (76.8%). HCV genotypes detected were: 1a, (35.2%, n = 25), 3a (62.0%, n = 44), and 1b (2.8%, n = 2). Cluster formation was detected in NS5B and CE1 and were generally from within the same city. All participants within clusters reported being a refugee in Iran compared to 93.5% of those outside clusters. Only 22.2% (4/11) of those within clusters had been refugees in Pakistan and these four individuals had also been refugees in Iran. Predominance of genotype 3a and the association between HCV viremia and having been a refugee in Iran potentially reflects migration between Afghanistan and Iran among IDUs from Mazar-i-Sharif and Hirat and carry implications for harm reduction programs for this migratory population.

Use of dried blood spots for HIV-1 genotyping in Southeast Asia: Thailand experience.

The multi-region hybridization assay (MHAbce) for genotyping HIV-1 subtypes B, C and circulating recombinant form (CRF01_AE) was evaluated on paired plasma and dried blood spots (DBS) collected from 68 HIV-1 infected individuals in Thailand. CRF01_AE was the predominant subtype identified using plasma samples (51/62) and DBS (24/27). There was no discordance in subtype designations between plasma and DBS.

Two unique recombinant forms identified in incident HIV type 1 infections in Thai blood donors.

HIV-1 genetic diversity of recently seroconverting (<12 months) Thai repeated blood donors attending the National Blood Centre, Thai Red Cross Society (NBC, TRCS) from September 2007 until March 2008 was assessed. Ten HIV-1 recent seroconvertors (10/239,134 donations) were identified during the study period. The estimated median time to seroconversion was 67.3 days (range: 45.5-102.0 days), and viral load ranged from 307 to 341,805 copies HIV-1 RNA/ml. MHAbce, a real-time-based PCR genotyping assay, identified six CRF01_AE, two CRF01_AE/B recombinants, one subtype B, and one CRF01_AE/B dual infection. Nine samples were further characterized by full genome sequencing, identifying CRF01_AE (N=6), unique CRF01_AE/B recombinants (N=2), and subtype B (N=1). One recombinant contained 13 breakpoints located in gag, pol, vif, vpr, env, and nef while the other recombinant contained 10 breakpoints located in pol, vif, env, and nef. This study found two unique CRF01B recombinants circulating in 10 recent HIV-1-positive subjects from a blood donor population in Thailand.

HIV type 1 molecular epidemiology among high-risk clients attending the Thai Red Cross Anonymous Clinic in Bangkok, Thailand.

Several studies have reported an increasing number of non-CRF01_AE infections in high-risk groups in Thailand suggesting a more complex HIV-1 epidemic. This study assessed the complexity of the HIV epidemic among high-risk clients tested for HIV-1 at the Thai Red Cross Anonymous Clinic (TRCAC) between July 1, 2006 and February 28, 2007. HIV-1 genotypes were determined from plasma of infected subjects (n = 401) by the multiregion hybridization assay (MHAbce, v.2). Univariate and multivariate logistic regression analyses were used to determine risk factors associated with HIV prevalence and non-CRF01_AE infection. The estimated overall HIV prevalence was 14.1%: 25.3% among men who have sex with men (MSM), 18.4% among heterosexual women, and 9.6% among heterosexual men. Among the risk factors found to be associated with HIV prevalence were age (25-29 years), risk behavior (MSM), marital status (not single), education (less than high school), and inconsistent condom use. Overall, non-CRF01_AE strains accounted for 18.9% of the infections: 25.3% among MSM and 14.8% and 20.4% among heterosexual women and men, respectively. Our results indicate a concentrated and genetically complex HIV epidemic among Thai MSM. These findings advocate for targeted intervention and prevention measures among high-risk populations in Thailand.