Multiregion Hybridization Assay Research Articles

HIV-1 genetic diversity and demographic characteristics in Bulgaria.

HIV-1 strain diversity in Bulgaria is extensive and includes contributions from nearly all major subtypes and the Circulating Recombinant Forms (CRF): 01_AE, 02_AG, and 05_DF. Prior to this study, HIV-1 sequence information from Bulgaria has been based solely on the pro-RT gene, which represent less than 15% of the viral genome. To further characterize HIV-1 in Bulgaria, assess participant risk behaviors, and strengthen knowledge of circulating strains in the region, the study “Genetic Subtypes of HIV-1 in Bulgaria (RV240)” was conducted. This study employed the real time-PCR based Multi-region Hybridization Assay (MHA) B/non-B and HIV-1 sequencing to survey 215 of the approximately 1100 known HIV-1 infected Bulgarian adults (2008–2009) and determine if they were infected with subtype B HIV-1. The results indicated a subtype B prevalence of 40% and demonstrate the application of the MHA B/non-B in an area containing broad HIV-1 strain diversity. Within the assessed risk behaviors, the proportion of subtype B infection was greatest in men who have sex with men and lowest among those with drug use risk factors. During this study, 15 near full-length genomes and 22 envelope sequences were isolated from study participants. Phylogenetic analysis shows the presence of subtypes A1, B, C, F1, and G, CRF01_AE, CRF02_AG, CRF05_DF, and one unique recombinant form (URF). These sequences also show the presence of two strain groups containing participants with similar risk factors. Previous studies in African and Asian cohorts have shown that co-circulation of multiple subtypes can lead to viral recombination within super-infected individuals and the emergence of new URFs. The low prevalence of URFs in the presence of high subtype diversity in this study, may be the result of successful infection prevention and control programs. Continued epidemiological monitoring and support of infection prevention programs will help maintain control of the HIV-1 epidemic in Bulgaria.

Molecular epidemiology of a primarily MSM acute HIV-1 cohort in Bangkok, Thailand and connections within networks of transmission in Asia.

IntroductionThailand plays a substantial role in global HIV‐1 transmission of CRF01_AE. Worldwide, men who have sex with men (MSM) are at elevated risk for HIV‐1 infection. Hence, understanding HIV‐1 diversity in a primarily Thai MSM cohort with acute infection, and its connections to the broader HIV‐1 transmission network in Asia is crucial for research and development of HIV‐1 vaccines, treatment and cure.MethodsSubtypes and diversity of infecting viruses from individuals sampled from 2009 to 2015 within the RV254/SEARCH 010 cohort were assessed by multiregion hybridization assay (MHAbce), multiregion subtype‐specific PCR assay (MSSPbce) and full‐length single‐genome sequencing (SGS). Phylogenetic analysis was performed by maximum likelihood. Pairwise genetic distances of envelope gp160 sequences obtained from the cohort and from Asia (Los Alamos National Laboratory HIV Database) were calculated to identify potential transmission networks.Results MHAbce/MSSPbce results identified 81.6% CRF01_AE infecting strains in RV254. CRF01_AE/B recombinants and subtype B were found at 7.3% and 2.8% respectively. Western subtype B strains outnumbered Thai B′ strains. Phylogenetic analysis revealed one C, one CRF01_AE/CRF02_AG recombinant and one CRF01_AE/B/C recombinant. Asian network analysis identified one hundred and twenty‐three clusters, including five clusters of RV254 participants. None of the RV254 sequences clustered with non‐RV254 sequences. The largest international cluster involved 15 CRF01_AE strains from China and Vietnam. The remaining clusters were mostly intracountry connections, of which 31.7% included Thai nodes and 43.1% included Chinese nodes.ConclusionWhile the majority of strains in Thailand are CRF01_AE and subtype B, emergence of unique recombinant forms (URFs) are found in a moderate fraction of new HIV‐1 infections. Approaches to vaccine design and immunotherapeutics will need to monitor and consider the expanding proportion of recombinants and the increasing genetic diversity in the region. Identified HIV‐1 transmission networks indicate ongoing spread of HIV‐1 among MSM. As HIV‐1 epidemics continue to expand in other Asian countries, transmission network analyses can inform strategies for prevention, intervention, treatment and cure.

Subtypes and Risk Behaviors Among Incident HIV Cases in the Bangkok Men Who Have Sex with Men Cohort Study, Thailand, 2006-2014.

HIV-1 incidence and prevalence remain high among men who have sex with men (MSM), and transgender women (TGW), in Thailand. To examine the link between epidemiologic factors and HIV-1 subtype transmission among Thai MSM, we compared covariates of infection with HIV CRF01_AE and other HIV strains among participants in the Bangkok MSM Cohort Study (BMCS). The BMCS was an observational cohort study of Thai MSM and TGW with up to 60 months of follow-up at 4 monthly intervals. Participants underwent HIV/sexually transmitted infections testing and provided behavioral data at each visit. Infecting viral strain was characterized by gene sequencing and/or multiregion hybridization assay. We correlated behavioral/clinical variables with infecting strain using Cox proportional hazards. Among a total of 1372 HIV seronegative enrolled participants with 4,192 person-years of follow-up, we identified 215 seroconverters between April 2006 and December 2014, with 177 infected with CRF01_AE and 38 with non-CRF01_AE subtype. Age 18-21 years (adjusted hazard ratio [AHR] 2.2, 95% confidence interval [CI]: 1.4-3.5), age 22-29 (AHR 1.6, 95% CI: 1.1-2.3), living alone (AHR 1.5, 95% CI: 1.1-2.1), drug use (AHR 2.2, 95% CI: 1.4-3.5), intermittent condom use (AHR 1.7, 95% CI: 1.3-2.3), any receptive anal intercourse (AHR 1.7, 95% CI: 1.2-2.4), group sex (AHR 1.5, 95% CI: 1.1-2.2), anti-herpes simplex virus type 1 (AHR 1.5, 95% CI: 1.1-2.1), and Treponema pallidum antibody positivity (AHR 2.5, 95% CI: 1.4-4.4) were associated with CRF01_AE infection. Age 18-21 years (AHR 5.1, 95% CI: 1.6-16.5), age 22-29 (AHR 3.6, 95% CI: 1.3-10.4), drug use (AHR 3.1, 95% CI: 1.3-7.5), group sex (AHR 2.4, 95% CI: 1.1-5.0), and hepatitis B virus surface antigen (AHR 3.6, 95% CI: 1.3-10.2) were associated with non-CRF01_AE infection. We observed several significant biological and behavioral correlates of infection with CRF01_AE and other HIV strains among Thai MSM. Divergence in correlates by strain may indicate differences in HIV transmission epidemiology between CRF01_AE and other strains. These differences could reflect founder effects, transmission within networks distinguished by specific risk factors, and possibly biological differences between HIV strains.

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.

High frequency of HIV-1 infections with multiple HIV-1 strains in men having sex with men (MSM) in Senegal

Circulating and unique recombinant HIV-1 strains continue to be identified and their number increases over time, suggesting that co-infection with multiple HIV-1 is frequent. In this study we analyzed to what extent dual infections with different HIV-1 variants occur in a population group with high risk behaviour, high HIV-1 prevalence and in an area where multiple HIV-1 subtypes and Circulating Recombinant Forms (CRFs) co-circulate. We studied 69 MSM with our recently developed multi-region hybridization assay (MHA), based on fluorescent probe detection for eight common variants circulating in West and West Central Africa. At least 11 (15.9%) of the 69 patients were simultaneously infected with two different HIV-1 subtypes and/or CRFs. Among the 29 samples identified as subtype C by MHA in gag, 15 (57.7%) reacted with both C1 and C2 probes. Sequence analysis suggests that the majority of the samples reactive with C1 and C2 probes are most likely infected with two different subtype C clades. Single genome amplification and DNA dilutions confirmed dual infection with subtype D and C for MSM1193, triple infection with two different C subtype strains and one CRF02_AG strain in MSM1157 and showed that MSM3017 is at least co-infected with CRF06_cpx and CRF02_AG and another strain that could not be classified. Comparison of all subtype C sequences from the MSM population and from the general population from this and previous studies confirmed the intermixing of HIV-1 variants between low-risk women and high-risk men as shown by the intermixing of subtype C variants from MSM1157 and a female patient (02SN-HALD478). Comparison of dual infection rates between the general population and MSM in Senegal, show also clearly the importance of high HIV prevalence and high risk behavior in dual infections and subsequent intermixing of HIV-1 variants which can lead to emergence and spread of new recombinants (CRFs).

HIV Type 1 Polymerase Gene Polymorphisms Are Associated With Phenotypic Differences in Replication Capacity and Disease Progression

Determinants of intersubtype differences in human immunodeficiency virus type 1 (HIV-1) clinical disease progression remain unknown. 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. In multivariate analyses, the hazard for progression to the composite end point (defined as a CD4(+) T-cell count <250 cells/mm(3), 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. HIV-1 pol gene intersubtype and RC differences are associated with disease progression and may be influenced by amino acid polymorphisms.

Molecular Evolution of the HIV-1 Thai Epidemic between the Time of RV144 Immunogen Selection to the Execution of the Vaccine Efficacy Trial

The RV144 HIV-1 vaccine trial (Thailand, 2003 to 2009), using immunogens genetically matched to the regional epidemic, demonstrated the first evidence of efficacy for an HIV-1 vaccine. Here we studied the molecular evolution of the HIV-1 epidemic from the time of immunogen selection to the execution of the efficacy trial. We studied HIV-1 genetic diversity among 390 volunteers who were deferred from enrollment in RV144 due to preexisting HIV-1 infection using a multiregion hybridization assay, full-genome sequencing, and phylogenetic analyses. The subtype distribution was 91.7% CRF01_AE, 3.5% subtype B, 4.3% B/CRF01_AE recombinants, and 0.5% dual infections. CRF01_AE strains were 31% more diverse than the ones from the 1990s Thai epidemic. Sixty-nine percent of subtype B strains clustered with the cosmopolitan Western B strains. Ninety-three percent of B/CRF01_AE recombinants were unique; recombination breakpoint analysis showed that these strains were highly embedded within the larger network that integrates recombinants from East/Southeast Asia. Compared to Thai sequences from the early 1990s, the distance to the RV144 immunogens increased 52% to 68% for CRF01_AE Env immunogens and 12% to 29% for subtype B immunogens. Forty-three percent to 48% of CRF01_AE sequences differed from the sequence of the vaccine insert in Env variable region 2 positions 169 and 181, which were implicated in vaccine sieve effects in RV144. In conclusion, compared to the molecular picture at the early stages of vaccine development, our results show an overall increase in the genetic complexity of viruses in the Thai epidemic and in the distance to vaccine immunogens, which should be considered at the time of the analysis of the trial results.

A novel multiregion hybridization assay reveals high frequency of dual inter-subtype infections among HIV-positive individuals in Cameroon, West Central Africa

In West and West Central Africa, multiple subtypes, circulating recombinant forms (CRF), and high proportions of unique recombinant forms (URF) are documented. The predominance of recombinants strongly suggests that dual infections occur frequently. In the present study, we adapted the multi-region hybridization assay (MHA), previously developed to identify dual infections in geographic regions where few HIV-1 variants circulate, to identify HIV-1 variants and dual infections.We designed clade-specific probes in three genomic regions (gag p17, vpu, nef) to detect eight different variants that are common in this part of Africa (A, B/D, C, F, G, CRF02_AG, CRF06_cpx, CRF22_01A1). The assay was validated with 163 samples representing the corresponding HIV-1 variants. Depending on the genomic regions, the global sensitivity of the assay ranged from 86% to 94%, and the global specificity was between 85% and 96%. The assay was then applied on 156 antiretroviral treatment-naive patients from Cameroon. The MHA assay identified 79%, 85% and 90% of the strains in nef, gag and vpu regions, respectively. The subtype/CRF distribution and the proportion of inter-region recombinants obtained by the new MHA assay were in accordance with known subtype/CRF distribution in Cameroon. Moreover, the MHA assay identified 35 (22.4%) patients as dually infected, from which 20 were reactive in more than one region and/or with concordant multigenomic recombination pattern.Despite the high genetic diversity, we successfully developed an hybridization assay allowing identification of eight common HIV-1 variants circulating in West and West Central Africa. We documented high rates of dual infection in a low-risk population group, illustrating that the global evolution of HIV diversity is driven by dual infections. This assay could become a useful screening tool for the global surveillance and monitoring of inter-subtype/CRF dual infections in West and West Central Africa.

Novel Multiregion Hybridization Assay for the Identification of the Most Prevalent Genetic Forms of the Human Immunodeficiency Virus Type 1 Circulating in Portugal

The most efficient method for HIV-1 genetic characterization involves full-genome sequencing, but the associated costs, technical features, and low throughput preclude it from being routinely used for the analysis of large numbers of viral strains. Multiregion hybridization assays (MHA) represent an alternative for a consistent genetic analysis of large numbers of viral strains. Classically, MHA rely on the amplification by real-time PCR of several regions scattered along the HIV-1 genome, and on their characterization with clade-specific TaqMan probes (also known as hydrolysis probes). In this context, the aim of our study was the development of a technical variant of an MHA (vMHA(B/G/02)) for genotyping the most prevalent genetic forms of HIV-1 circulating in Portugal. Different sets of primers were designed for universal and clade-specific amplifications of several sections of the viral genome: gag, pol(Pr), pol(RT), vpu, env(gp120), and env(gp41). vMHA(B/G/02) was implemented using a real-time PCR-based approach, with detection dependent on the use of SYBR Green I. As an alternative, a technically less demanding strategy based on conventional PCR and agarose gel analysis of the reaction products was also developed. This method performed with overall good sensitivity and specificity (>91%) when a convenience sample of 45 plasma-derived HIV-1 strains was analyzed. Apart from the detection of subtype B, G, CRF02_AG, and CRF14_BG viruses, several unique B/G recombinant were also detected. Curiously, recombinant viruses including CRF02_AG sequences were not detected in the group of samples analyzed.

Socio-demographic and drug use factors associated with HIV-1 recombinants and dual infections in Northern Thai drug users: Associations of risk with genetic complexity

BackgroundDual infection with diverse HIV strains can foster the emergence of recombinants. The resulting increase in viral genetic diversity is a major challenge for vaccine development HIV treatment. In this study we aim to investigate the socio demographic factors associated with an increasing level of genetic diversity among HIV strains in a population of drug-users in Northern Thailand. MethodsFrom 1999 through 2000, 2231 volunteers were enrolled in the Opiate-Users Research in Chiang Mai, Thailand. HIV subtype analysis was conducted among those HIV-1 seropositive (n=347) using a multi-region hybridization assay. Social and demographic variables were assessed using a structured questionnaire. ResultsOverall, 336/347 (96.8%) of the samples could be typed. 81.8% were CRF01_AE, 3.9% were subtype B, 9.2% were recombinants (mostly between CRF01_AE and B) and 5.1% were dual infections. Dual infections were more frequent among those with a lower education level (AOR: 5.2; 95% CI 1.4–20.3), those who have initiated injecting in the last 3 years (AOR: 3.9; 95% CI 1.1–14.6), and those reporting frequent needle sharing in the last 3 months (AOR: 7.0; 95% CI 1.5–34.1). Both recombinant strains and dual infection were more frequent among those reporting frequent needle sharing in the last 3 months (AOR: 5.3; 95% CI 1.6–17.1). ConclusionTo limit the expanding complexity of HIV-1 strains, early intervention should be aimed at reduction in needle sharing, especially among new intravenous drug users.

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.

HIV-1 subtypes and differences in heterosexual HIV transmission among HIV-discordant couples in Rakai, Uganda

To determine whether heterosexual transmission of HIV differs according to HIV-1 subtype. A retrospective observational cohort. HIV-1 subtype effects on heterosexual HIV-1 transmission were determined among 268 HIV-discordant couples retrospectively identified from a population cohort in Rakai, Uganda. HIV-1 subtype (gag and gp41 sequencing and multiregion hybridization assay) and viral loads (reverse transcriptase PCR) were determined. Adjusted incidence rate ratios (adj IRR) of HIV transmission by subtype were estimated by multivariable Poisson regression adjusting for characteristics of index HIV-positive and HIV-negative partners. Adjusting for index HIV-positive partners' age, viral load, stage of disease, genital ulcer disease, and HIV-negative partners' genital ulcer disease and nonuse of condoms, subtype A viruses were associated with a higher rate of transmission than subtype D [adj.IRR 1.98, 95% confidence interval (CI) 1.17-3.34], but no differences in transmission were observed between recombinant viruses and subtype D (aIRR 1.53, P = 0.25). Index-positive partners' age less than 30 years (adj.IRR 3.44, 95% CI 1.75-6.78) and viral load (adj.IRR 2.37, 95% CI 1.75-3.21), and index-negative partners' genital ulcer disease (adj.IRR 1.71, 95% CI 1.08-2.70) and nonuse of condoms (adj.IRR 1.94, 95% CI 1.15-3.28) were significant determinants of HIV transmission. In Rakai, Uganda, subtype A viruses have a significantly higher rate of heterosexual transmission than subtype D viruses. Differential subtype transmission efficiency may be important for HIV vaccine evaluation and could contribute to subtype-specific HIV epidemics in sub-Saharan Africa.

Short Communication: HIV Type 1 Genetic Diversity among Tea Plantation Workers in Kericho, Kenya

In preparation for HIV-1 vaccine trials in Kenya, 2801 study volunteers, from a tea plantation in Kericho, were recruited as part of a prospective vaccine cohort development study. Cryopreserved plasma was available from 401 HIV-positive volunteers, and was the source of viral RNA for genotyping by the multiregion hybridization assay (MHA). Logistic regression was performed to determine association of risk factors and HIV-1 recombinant and dual infections. At baseline, HIV-1 subtype A was the dominant circulating pure subtype (56%), followed by subtype D (10%) and C (5%). Recombinant HIV-1 strains accounted for almost one-third of all infections (29%), with 7% infected with a dual strain of the HIV-1 variants described. A higher number of HIV-1 recombinant and dual infections was observed among volunteers who were 18-24 and 25-29 years of age, affiliated with the Luo tribe, had been married two or more times, reported not being circumcised, and had STI symptoms in the past 6 months. Adjusted odds ratios (AOR) significantly associated with HIV-1 recombinant and dual infection were age difference from current spouse (5-9 years; AOR = 2.5, 95% CI = 1.2-5.3 and > or = 10 years; AOR = 3.1, 95% CI = 1.5-6.4) and reported STI symptoms in the past 6 months (AOR = 4.8, 95% CI = 2.0-11.6), respectively. In conclusion, our results suggest that there is considerable heterogeneity with respect to HIV-1 subtype diversity in this population that should be considered in the planning for future vaccine trials in the region.

Implementation of a Multiregion Hybridization Assay to Characterize HIV-1 Strains Detected among Injecting Drug Users in Manipur, India

We have implemented the latest technology of a multiregion hybridization assay (MHAbce, version 2) for the molecular characterization of HIV-1 among injecting drug users (IDUs) of Manipur, India. This study provides a more detailed analysis on the basis of probes designed from eight different genomic regions of HIV-1, to achieve a clear picture of HIV-1 genomic diversity in Manipur. Out of 30 samples, 15 were found to be of subtype C, 1 of subtype B, 5 with dual-probe reactivity, 8 with multigenomic recombination pattern and 1 sample showed both dual-probe reactivity and multigenomic variations. In contrast, the heteroduplex mobility assay (HMA) with respect to gag and env genes revealed 21 samples to be of subtype C (gag C/env C), 3 samples of subtype B (gag B/env B) and 6 samples of B/C recombinants (gag C/env B). MHAbce illustrates the occurrence of inter- and intragenomic variants and dual infection in an IDU population from India. It also indicates the possibility of the presence of new circulating recombinant forms of HIV-1 strains, which might have been difficult to trace by HMA alone.

HIV‐1 Subtype as a Determinant of Disease Progression

Although the average time from infection with HIV type 1 (HIV-1) to the development of AIDS is 1 decade, the rate of disease progression varies considerably among infected persons. Numerous factors, including plasma HIV-1 RNA level, CD4 cell count, degree of immune activation, age, socioeconomic status, and host genetics, contribute to determining the rate of progression in the individual patient [1]. Intrinsic viral properties, such as coreceptor use and replication capacity, may also influence progression rates. Data have been accumulating to suggest that viral subtype is another contributing factor [2– 4]. The report by Kiwanuka et al. [5] in this issue of the journal provides compelling evidence that infection with HIV-1 subtype A (HIV-1A) progresses more slowly than infection with HIV-1 subtype D (HIV-1D) or with recombinant or multiple HIV-1 subtypes. As summarized in the report by Kiwanuka and colleagues, HIV is classified into types (HIV-1 or HIV type 2), groups (HIV-1 M, N, and O), and subtypes (or clades) on the basis of genetic relatedness. At present, 9 subtypes are recognized (A–D, F–H, J, and K), along with several circulating recombinant forms, which contain sequences from 1 subtype. Because the different HIV-1 subtypes are not uniformly dispersed, comparisons of virulence and transmissibility are hampered by potential confounders, such as ethnic, socioeconomic, and other epidemiological factors. The cocirculation of HIV-1A, HIV-1D, and several intersubtype recombinants in the Rakai district of Uganda provides an opportunity to compare rates of disease progression associated with these different subtypes within a similar population. In the current report, Kiwanuka et al. [5] compared rates of progression among HIV-1 seroconverters who were followed as part of the Rakai Health Sciences Program. Subjects identified during 1997–2002 were followed through 2004, when antiretroviral therapy became available in Rakai district. The HIV-1 subtype was determined by a multiregion hybridization assay in which gag, pol, vpu, env, and gp41 amplicons from polymerase chain reaction (PCR) of plasma HIV-1 RNA were tested in a second-round PCR with subtype-specific Taqman probes. This approach allowed the authors to categorize samples as comprising a single subtype, mixtures of 2 subtypes, or intersubtype recombinants. The primary end point was the time to achievement of a CD4 cell count of 250 cells/mm3 or death due to AIDS. Of the 350 seroconverters enrolled, nearly 60% were infected with HIV-1D, 15% with HIV-1A, 21% with recombinant subtypes, and 4% with multiple subtypes. Progression to a CD4 cell count of 250 cells/mm3 was significantly less common among subjects infected with HIV-1A (20%), compared with subjects infected with HIV-1D (40%), recombinant forms (40%), or multiple subtypes (53%) (P .03). Death from AIDS was also less common among subjects infected with HIV-1A. These differences were reflected in the longer time to AIDS onset for HIV-1A–infected subjects (8.05 years), compared with those infected with HIV-1D (6.49 years), recombinant forms (5.57 years), or multiple subtypes (5.80 years). In multivariable models that controlled for virus load, age, and sex, subjects infected with multiple subtypes, HIV-1D, or intersubtype recombinants were 4.40, 2.13, and 2.16 times, respectively, more likely to progress to AIDS, compared with those infected with HIV1A. Similarly, subjects infected with non-A subtypes had an approximately 6 – 8-fold greater risk of death from AIDS. Previous studies involving cohorts of persons with seroprevalent infection also found that the risk of disease progression is greater for persons with HIV-1D infection than for those with HIV-1A infection [3, 4]. Although seroprevalent cohorts can provide important information about late events, such as death after AIDS, they potentially suffer from survival bias (i.e., subjects with rapid progression tend to be Received 26 November 2007; accepted 26 November 2007; electronically published 11 February 2008. Potential conflicts of interest: none reported. Financial support: National Institutes of Health (grants R37 AI-055357 and K24 RR016482). Reprints or correspondence: Dr. Daniel R. Kuritzkes, Sect. of Retroviral Therapeutics, Brigham and Women’s Hospital, 65 Landsdowne St., Rm. 449, Cambridge, MA 02139 (dkuritzkes@partners.org). The Journal of Infectious Diseases 2008; 197:638 –9 © 2008 by the Infectious Diseases Society of America. All rights reserved. 0022-1899/2008/19705-0002$15.00 DOI: 10.1086/527417 E D I T O R I A L C O M M E N T A R Y

Higher HIV-1 Incidence and Genetic Complexity Along Main Roads in Rakai District, Uganda

To determine the association between the incidence of HIV-1 infection and the genetic complexity of HIV-1 strains in 2 geographic strata within Rakai District, Uganda. Study volunteers with recent HIV-1 infections during the period 1997 through 2003 were recruited from 10 communities that were geographically stratified as a main road trading center (n = 5) or a secondary road trading village (n = 5). Cryopreserved plasma was available from 384 volunteers and was the source of viral RNA for genotyping by the multiregion hybridization assay. Hazard ratios (HRs) for a single HIV subtype, a recombinant form, or dual infection for gender and geographic strata were obtained using Cox proportional hazards analysis. The HIV-1 incidence rate during the period 1999 through 2002 was 1.3 per 100 person-years (PYs) in the trading centers and 1.1 per 100 PYs in the trading villages. The HR for infection with an HIV-1 recombinant strain in trading centers relative to trading villages was 2.3 (95% confidence interval [CI]: 1.0 to 6.7). Among those who changed residence between village strata, the HR for a recombinant HIV-1 infection was 8.1 (95% CI: 0.4 to 47.7). HIV-1 incidence and genetic complexity are associated with geographic strata and population mobility in Rakai District and are important variables to be considered in planning and recruitment for vaccine trials.

Distinguishing molecular forms of HIV-1 in Asia with a high-throughput, fluorescent genotyping assay, MHAbce v.2

High-resolution HIV-1 genotyping of large sample sets is crucial to define the evolving and dynamic epidemics in Asia. Here we present MHAbce v.2, a multi-region hybridization assay that individually discriminates subtypes B, C, CRF01_AE, and virtually all of their described recombinants, based on real-time PCR using subtype-specific TaqMan probes in 8 regions throughout the viral genome. In a validation panel ( n = 70), the assay performed with a sensitivity of 95.7% and specificity of 99.8%. The assay was field-tested on samples from a retrospective MTCT cohort ( n = 180; Lampang Province, Northern Thailand; 1996–1998). 177/180 of the samples were typeable, and 94.4% were typed as CRF01_AE. The remaining strains represented even proportions of subtype B and B/CRF01_AE recombinants and were confirmed by sequencing, revealing early links between the heterosexual and IDU HIV-1 epidemics in Thailand. MHAbce v.2, with an area of application including China, India, Southeast Asia, and the Pacific Rim, can be used to develop a comprehensive and detailed picture of this important component of the HIV/AIDS pandemic.

Molecular Epidemiology of HIV Type 1 in Preparation for a Phase III Prime-Boost Vaccine Trial in Thailand and a New Approach to HIV Type 1 Genotyping

To characterize HIV-1 genotypes in candidate populations for a prime-boost phase III vaccine trial in Thailand, specimens from prevalent and incident HIV-1 infections from a family planning clinic population in Rayong Province and a community cohort in Chon Buri Province, collected from 1998 to 2001, were genotyped. A new multiregion hybridization assay, MHAbce, capable of distinguishing HIV-1 CRF01_AE, subtype B, and subtype C and their recombinants, was developed and applied to prevalent infections. Most incident and selected prevalent infections were studied by complete genome sequencing. By MHAbce, 168 of 194 prevalent infections were genotyped. Of these, 90.5% were CRF01_AE, 2.4% were subtype B, and 7.2% showed discordant or dual probe reactivity, indicative of recombination or dual infection, respectively. Among 23 incident infections, 20 were sequenced. Eighteen CRF01_AE, one subtype B, and one CRF01/B recombinant strains were seen. Two CRF01/B and one CRF01/C recombinant were identified among selected prevalent infections. These results indicate that incident and prevalent HIV-1 infections in Rayong and Chon Buri during 1998-2001 were 90% CRF01_AE, 3% subtype B, and 7% either recombinant or dual. This study frames the genetic diversity of HIV-1 in these cohorts in their preparatory phase for the ongoing ALVACHIV (vCP1521) prime, AIDSVAX B/E boost, phase III vaccine trial and will provide a benchmark for interpretation and analysis.

Frequency of HIV Type 1 Dual Infection and HIV Diversity: Analysis of Low- and High-Risk Populations in Mbeya Region, Tanzania

HIV-1 diversity, frequency of recombinants, and dual infection were determined in two populations with different HIV risk behavior. A high-risk cohort of 600 female bar workers and a normal-risk population of 1,108 antenatal clinic attendees and blood donors were recruited. Behavioral data were assessed and blood for HIV- 1 diagnosis and genotyping was sampled. HIV-1 subtypes were defined through the multiregion hybridization assay (MHA(acd)). HIV-1 prevalence differed significantly among the two populations. The prevalence was 67.8% in the population of bar workers and 17% in the normal-risk population (antenatal care attendees and blood donors). Within the normal-risk population the HIV-1 prevalence was lowest in the group of volunteer blood donors. The frequency of HIV-1 infection in women was 1.7 times higher than in men. The overall subtype distribution was A (8.5%), C (40.8%), D (3.8%), AC (25.4%), AD (5.4%), CD (8.8%), and ACD (7.3%). In the high-risk population there was a higher percentage of HIV-1 recombinant strains (54% vs. 40%, p < 0.05) and a higher frequency of dual infections (19% vs. 9%, p < 0.02) compared to the normal-risk population. High-risk populations may play an important role in the evolution of HIV, as they can provide an opportunity for the virus to coinfect, recombine, and adapt to the host-specific genetic background.

HIV-1 diversity and prevalence differ between urban and rural areas in the Mbeya region of Tanzania

To characterize HIV-1 strains in a potential vaccine trial cohort (CODE) in the Mbeya region of southwest Tanzania. Study volunteers (n = 3096) were recruited from urban areas in Mbeya Town, using two different recruitment strategies, and in a nearby rural village. Cryopreserved plasma from 507 HIV-1 prevalent cases was the source of viral RNA for HIV-1 genotyping by the Multi-region Hybridization Assay, the MHA(acd), and selected strains were confirmed by complete genome sequencing. The overall HIV-1 prevalence was 16.6% [95% confidence interval (CI), 15.3-17.9] within the cohort. HIV-1 prevalence was higher among women, and in urban areas. Recruitment through advertisement targeted a high-risk urban male population for HIV-1 infection [adjusted odds ratio (adj. OR), 1.68; 95% CI, 1.13-2.51] when compared with men recruited door-to-door. The complexity of the HIV-1 epidemic was also higher in urban areas evidenced by the high-risk of HIV-1 infection with a recombinant strain (adj. OR, 2.69; 95% CI, 1.08-6.69) and HIV-1 dual infection (adj. OR, 5.16; 95% CI, 1.07-24.9), mainly driven by urban men recruited through advertisement. Overall the urban epidemic was more genetically complex, with higher prevalence and more recombinants and dual infections. Vaccine trials in Mbeya region can assess a complex HIV-1 population dynamic and determine vaccine efficacy in relationship to the genetic diversity of HIV-1 strains that challenge vaccines.