Subtypes of human immunodeficiency virus type-1 (HIV) vary in geographic distribution. Subtype B is predominant in North and South America, Western Europe, and Australia, whereas subtypes A and C are predominant in Africa.1,2 In the regions with 2 or more circulating subtypes, individuals with different subtypes are at risk for recombinant HIV infection (RHI).2 Implications of RHI include treatment response to combined antiretroviral therapy (CART), HIV vaccine development, and patient education.3,4
In 1988 in Thailand, HIV subtype B transmission primarily occurred among injecting drug users (IDUs), whereas HIV subtype E or CRF01_AE transmission occurred primarily among commercial sex workers (CSW).5 The AE subtype subsequently became the predominant HIV subtype in the Thai population, although a small proportion of newly infected HIV cases continued to be HIV subtype B concurrent with detection of RHI among the heterosexual and IDU populations.5–9 To date, the reports of RHI from Thailand have been descriptive in nature and did not include assessment of risks for and sequelae from RHI.5–10 This study was conducted to determine the risk factors associated with RHI and to assess the impact of RHI on HIV outcomes.
Between January and December 2005, patients >15 years of age with HIV infection and in established HIV care at the Thammasat University Hospital were eligible for study participation. The hospital has an estimated 3850 HIV-related annual outpatient visits. All patients who gave consent were enrolled and prospectively followed for 18 months. This study was approved by the Thammasat University Hospital Institutional Review Board.
At the initial clinic visit, data on patient characteristics were collected. HIV infection was confirmed by 3 positive enzyme immunoassays or 2 positive enzyme immunoassays and a confirmatory Western blot. Baseline CD4 cell count, HIV RNA, and HIV genotype were obtained. As part of a prior study to assess antiretroviral drug resistance, all enrolled patients' sera were analyzed for pol gene sequences through bootscan.11 In addition, heteroduplex mobility assays and differential PCR were performed to detect the recombinants.12 A positive RHI screening test was evidence of subtype discordance or >1 subtype in different parts of the HIV genome by these assays. Confirmatory testing for RHI included analysis of the entire gag/pol/env genes of HIV-1 proviruses from DNA extracted from primary peripheral blood mononuclear cells.5,6,13 The control experiments of these assays showed 100% accuracy and reproducibility. At the second clinic visit 2 weeks later, CART, opportunistic infection prophylaxis, and routine HIV care were initiated according to established HIV guidelines.14,15 CD4 cell counts and HIV RNA were obtained at the 6-, 12-, and 18-month follow-up visits.
All statistical analyses were performed using SPSS version 15.0 (SPSS, Chicago, IL). Categorical variables were compared using Pearson χ2 or Fisher exact test, as appropriate. Continuous variables were compared using Mann-Whitney U test. Variables that were present at a significance level of P < 0.20 were entered into logistic regression models. Potential confounding variables, including age, duration of HIV infection, baseline CD4 count, and HIV RNA, were adjusted for measurement of outcome, if appropriate. Adjusted odd ratios (aORs) and 95% confidence intervals (CIs) were calculated in the multivariate logistic regression analyses.
In all, 303 patients were prospectively enrolled during calendar year 2005. The majority of participants (55%) were women, 83% identified a high-risk sex partner, 7% had coinfection with hepatitis B virus, and 1% had coinfection with hepatitis C virus (Table 1). Ten participants (3%) screened positive for RHI, 4 (1.3%) of whom had confirmed RHI. The distribution of HIV genotypes among patients without RHI was A/E (297 [98%]) and B (2 [0.7%]), whereas RHI genotypes included A/AE (1 [0.3%]), B/AE (1 [0.3%]), A/AE/B/D (1 [0.3%]), and B/D/G/AE (1 [0.3%]). RHI patients were more likely than non-RHI patients to report one or more non-Thai sex partner (P < 0.001), exchange of sex for money (P < 0.001), and a higher number of sex partners within the prior 6 months (5 vs. 1; P < 0.001). By multivariate regression analysis, the independent risk factors for RHI were exchange of sex for money (aOR, 49.03; 95% CI: 2.55–941.50; P = 0.01) and more than one sex partner within the prior 6 months (aOR, 41.52; 95% CI: 2.17–796.02; P = 0.01).
There were 102 of 303 participants (34%) who did not meet the clinical criteria for CART initiation (<200 cells/μL) after the initial clinic visit. In this group, 3 persons with RHI and 76 of 99 persons (76.8%) without RHI were observed without CART exposure for 18 months; 23 of 99 persons (23.2%) without RHI were lost to follow-up. There were no significant differences in the median time since HIV diagnosis (1 vs. 2 years; P = 0.25), baseline CD4 cell count (median, 573 vs. 417 cells/μL; P = 0.21), or HIV RNA (median, 17,000 vs. 4000 copies/mL; P = 0.08) between the RHI and non-RHI participants. However, the 3 participants with RHI had greater interval reduction in CD4 count than the 76 non-RHI patients at 6 months (−77 vs. −8 cells/μL; P = 0.02), 12 months (−125 vs. +10 cells/μL; P = 0.03), and 18 months (−160 vs. −24 cells/μL; P = 0.06). There was no significant difference in the interval change of HIV RNA between RHI and non-RHI patients at 6 months (−300 vs. −3000 copies/mL; P = 0.14), 12 months (+6000 vs. −8000 copies/mL; P = 0.50), and 18 months (+7000 vs. −8000 copies/mL; P = 0.92). Anecdotally, the sole patient with RHI who was treated with CART had a typical treatment response.
RHIs have an estimated 10% global prevalence,16 whereas the reported prevalence of RHI in Thailand ranged from 1.7% to 13.2% depending on the geographic regions and study populations.5,7–10 We report a low-RHI prevalence of 1.3% in a primarily heterosexual population from central Thailand, an estimate comparable with another Thai cohort.9 In northern Thailand, the reported RHI prevalence was 9.2% to 13.2% among drug users (DUs),8,10 compared to a lower prevalence of 1.7% among general populations from other regions.9 The higher RHI prevalence (especially AE/B) among DUs could be explained by the predominance of subtype B infection among DUs and increased risk for subtype AE acquisition through heterosexual sex and sharing needles.8,10 The RHI with subtypes A, D, and G detected in this study may have been associated with sex encounters among persons/travelers from other geographic regions such as Europe, Australia, and/or the Americas.
An additional finding in this study was the association of RHI with exchange of sex for money and more than 1 sex partner within the prior 6 months. Differential behavioral risks for RHI by geographic region indicate unmet needs in behavioral health and education focused on safer sex practices (Table 2). In African countries, RHI has been most frequently reported in individuals who were CSW or lived in urban settings.17–19 The genetic complexity of HIV was reported in urban areas with high-HIV prevalence, suggesting transmission dynamics attributed to an increased number of social interactions and sex partners.18,19 In Europe, RHI has been prevalent among immigrants from countries where subtype non-B is predominant, multiple subtypes of HIV cocirculate, and barriers to HIV care exist.4,20 In the United States, RHI has been reported among young military personnel after contact with CSW when deployed overseas.3
Although the clinical course of RHI disease progression has not been fully characterized, it is hypothesized that recombination may allow for the simultaneous introduction of a large number of genetic changes in viral quasispecies which can alter antiretroviral drug susceptibility and disease progression.2 A few prior studies have reported that in comparison to persons infected with HIV subtype B, those infected with subtypes non-B have similar immunologic and virological responses to first-line CART for up to 18 months.21,22 A study from Africa demonstrated no difference in disease progression or CD4 decline among individuals with a recombinant subtype, CRF02_AG, infection, and those with non-RHI.23 In our study, the 3 patients with RHI and CD4 counts >200 cells/μL who were not treated with CART had a significantly greater decline in CD4 count during the period of 18 months compared to 76 patients without RHI. Interpretations of this finding are limited given the small sample size of the RHI group, difference in genetics of HIV coreceptor type which was not evaluated in this study, and potential ongoing differential high-risk behaviors among persons in the 2 groups. Nonetheless, more frequent monitoring of CD4 count in patients with RHI who are not treated with CART may be prudent.
Our study had notable limitations. First, the study participants were from a single HIV clinic, and the findings may not be generalizable to other Thai and non-Thai populations. Second, the sensitivity and specificity of the diagnostic assays we used to assess RHI have not been fully validated. To minimize misclassification bias for RHI, screening and confirmatory laboratory testing included env sequencing, heteroduplex mobility assays, and differential PCR. Finally, the small sample size limits any in-depth interpretations of the identified risks and outcomes.
In conclusion, our findings suggest that RHI was associated with high-risk sex behaviors. Selective implementation of further HIV genotype tests based on these characteristics may be clinically relevant and eventually feasible in resource-limited settings. The relevance of RHI to HIV diagnostic technology, clinical monitoring in routine care, and vaccine research requires further evaluation.
GENBANK ACCESSION NUMBERS
GenBank accession numbers of recombinant HIV nucleotide sequences reported in this article are JF922855-JF922857 for patient 1 (TU-065); JF939054-JF939057 for patient 2 (TU-174); JF939058-JF939060 for patient 3 (TU-230); and JF939061-JF939064 for patient 4 (TU-240).
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