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Background: Great efforts have been made to increase accessibility of HIV antiretroviral therapy (ART) in low and middle-income countries. The threat of wide-scale emergence of drug resistance could severely hamper ART scale-up efforts. Population-based surveillance of transmitted HIV drug resistance ensures the use of appropriate first-line regimens to maximize efficacy of ART programs where drug options are limited. However, traditional HIV genotyping is extremely expensive, providing a cost barrier to wide-scale and frequent HIV drug resistance surveillance., Methods/results: We have developed a low-cost laboratory-scale next-generation sequencing-based genotyping method to monitor drug resistance. We designed primers specifically to amplify protease and reverse transcriptase from Brazilian HIV subtypes and developed a multiplexing scheme using multiplex identifier tags to minimize cost while providing more robust data than traditional genotyping techniques. Using this approach, we characterized drug resistance from plasma in 81 HIV infected individuals collected in São Paulo, Brazil. We describe the complexities of analyzing next-generation sequencing data and present a simplified open-source workflow to analyze drug resistance data. From this data, we identified drug resistance mutations in 20% of treatment naïve individuals in our cohort, which is similar to frequencies identified using traditional genotyping in Brazilian patient samples., Conclusion: The developed ultra-wide sequencing approach described here allows multiplexing of at least 48 patient samples per sequencing run, 4 times more than the current genotyping method. This method is also 4-fold more sensitive (5% minimal detection frequency vs. 20%) at a cost 3-5× less than the traditional Sanger-based genotyping method. Lastly, by using a benchtop next-generation sequencer (Roche/454 GS Junior), this approach can be more easily implemented in low-resource settings. This data provides proof-of-concept that next-generation HIV drug resistance genotyping is a feasible and low-cost alternative to current genotyping methods and may be particularly beneficial for in-country surveillance of transmitted drug resistance.

Introduction: Primary HIV infection is usually caused by R5 viruses, and there is an association between the emergence of CCXR4-utilizing strains and faster disease progression. We characterized HIV-1 from a cohort of recently infected individuals in Brazil, predicted the virus's co-receptor use based on the env genotype and attempted to correlate virus profiles with disease progression., Methods: A total of 72 recently infected HIV patients were recruited based on the Serologic Testing Algorithm for Recent HIV Seroconversion and were followed every three to four months for up to 78 weeks. The HIV-1 V3 region was characterized by sequencing nine to twelve weeks after enrollment. Disease progression was characterized by CD4+ T-cell count decline to levels consistently below 350 cells/µL., Results: Twelve out of 72 individuals (17%) were predicted to harbor CXCR4-utilizing strains; a baseline CD4<350 was more frequent among these individuals (p = 0.03). Fifty-seven individuals that were predicted to have CCR5-utilizing viruses and 10 individuals having CXCR4-utilizing strains presented with baseline CD4>350; after 78 weeks, 33 individuals with CCR5 strains and one individual with CXCR4 strains had CD4>350 (p = 0.001). There was no association between CD4 decline and demographic characteristics or HIV-1 subtype., Conclusions: Our findings confirm the presence of strains with higher in vitro pathogenicity during early HIV infection, suggesting that even among recently infected individuals, rapid progression may be a consequence of the early emergence of CXCR4-utilizing strains. Characterizing the HIV-1 V3 region by sequencing may be useful in predicting disease progression and guiding treatment initiation decisions.

Background: Genetic variability is a major feature of human immunodeficiency virus type 1 (HIV-1) and is considered the key factor frustrating efforts to halt the HIV epidemic. A proper understanding of HIV-1 genomic diversity is a fundamental prerequisite for proper epidemiology, genetic diagnosis, and successful drugs and vaccines design. Here, we report on the partial and near full-length genomic (NFLG) variability of HIV-1 isolates from a well-characterized cohort of recently infected patients in São Paul, Brazil., Methodology: HIV-1 proviral DNA was extracted from the peripheral blood mononuclear cells of 113 participants. The NFLG and partial fragments were determined by overlapping nested PCR and direct sequencing. The data were phylogenetically analyzed., Results: Of the 113 samples (90.3% male; median age 31 years; 79.6% homosexual men) studied, 77 (68.1%) NFLGs and 32 (29.3%) partial fragments were successfully subtyped. Of the successfully subtyped sequences, 88 (80.7%) were subtype B sequences, 12 (11%) BF1 recombinants, 3 (2.8%) subtype C sequences, 2 (1.8%) BC recombinants and subclade F1 each, 1 (0.9%) CRF02 AG, and 1 (0.9%) CRF31 BC. Primary drug resistance mutations were observed in 14/101 (13.9%) of samples, with 5.9% being resistant to protease inhibitors and nucleoside reverse transcriptase inhibitors (NRTI) and 4.9% resistant to non-NRTIs. Predictions of viral tropism were determined for 86 individuals. X4 or X4 dual or mixed-tropic viruses (X4/DM) were seen in 26 (30.2%) of subjects. The proportion of X4 viruses in homosexuals was detected in 19/69 (27.5%)., Conclusions: Our results confirm the existence of various HIV-1 subtypes circulating in São Paulo, and indicate that subtype B account for the majority of infections. Antiretroviral (ARV) drug resistance is relatively common among recently infected patients. The proportion of X4 viruses in homosexuals was significantly higher than the proportion seen in other study populations.

Background: HIV-1-infected individuals who spontaneously control viral replication represent an example of successful containment of the AIDS virus. Understanding the anti-viral immune responses in these individuals may help in vaccine design. However, immune responses against HIV-1 are normally analyzed using HIV-1 consensus B 15-mers that overlap by 11 amino acids. Unfortunately, this method may underestimate the real breadth of the cellular immune responses against the autologous sequence of the infecting virus., Methodology and Principal Findings: Here we compared cellular immune responses against nef and vif-encoded consensus B 15-mer peptides to responses against HLA class I-predicted minimal optimal epitopes from consensus B and autologous sequences in six patients who have controlled HIV-1 replication. Interestingly, our analysis revealed that three of our patients had broader cellular immune responses against HLA class I-predicted minimal optimal epitopes from either autologous viruses or from the HIV-1 consensus B sequence, when compared to responses against the 15-mer HIV-1 type B consensus peptides., Conclusion and Significance: This suggests that the cellular immune responses against HIV-1 in controller patients may be broader than we had previously anticipated.

Background: Human herpesvirus 8 (HHV-8) is the etiological agent for Kaposi Sarcoma, which occurs especially in HIV-infected subjects. HHV-8 infection and its clinical correlates have not been well characterized in recently HIV-1-infected subjects, especially men who have sex with men (MSM)., Methodology/ Principal Findings: We assessed the HHV-8 seroprevalence, clinical correlates, and incidence after one year of follow-up in a cohort of 228 recently HIV-1-infected individuals, of whom 83.6% were MSM, using indirect immunofluorescence assay. The prevalence of HHV-8 infection at the time of cohort enrollment was 25.9% (59/228). In the univariate model, there were significant associations with male gender, black ethnicity, MSM practice, and previous hepatitis B virus and syphilis infections. In the multivariate model we could still demonstrate association with MSM, hepatitis B, and black ethnicity. No differences in mean CD4+ cell counts or HIV viral load according to HHV-8 status were found. In terms of incidence, there were 23/127 (18.1%) seroconversions in the cohort after 1 year., Conclusions: HHV-8 is highly prevalent among recently HIV-1-infected subjects. Correlations with other sexually transmitted infections suggest common transmission routes.

Introduction: HIV-1 is often acquired in the presence of pre-existing co-infections, such as Herpes Simplex Virus 2 (HSV-2). We examined the impact of HSV-2 status at the time of HIV-1 acquisition for its impact on subsequent clinical course, and total CD4+ T cell phenotypes., Methods: We assessed the relationship of HSV-1/HSV-2 co-infection status on CD4+ T cell counts and HIV-1 RNA levels over time prior in a cohort of 186 treatment naïve adults identified during early HIV-1 infection. We assessed the activation and differentiation state of total CD4+ T cells at study entry by HSV-2 status., Results: Of 186 recently HIV-1 infected persons, 101 (54%) were sero-positive for HSV-2. There was no difference in initial CD8+ T cell count, or differences between the groups for age, gender, or race based on HSV-2 status. Persons with HIV-1/HSV-2 co-infection sustained higher CD4+ T cell counts over time (+69 cells/ul greater (SD = 33.7, p = 0.04) than those with HIV-1 infection alone (Figure 1), after adjustment for HIV-1 RNA levels (-57 cells per 1 log(10) higher HIV-1 RNA, p<0.0001). We did not observe a relationship between HSV-2 infection status with plasma HIV-1 RNA levels over time. HSV-2 acquisition after HIV-1 acquisition had no impact on CD4+ count or viral load. We did not detect differences in CD4+ T cell activation or differentiation state by HSV-2+ status., Discussion: We observed no effect of HSV-2 status on viral load. However, we did observe that treatment naïve, recently HIV-1 infected adults co-infected with HSV-2+ at the time of HIV-1 acquisition had higher CD4+ T cell counts over time. If verified in other cohorts, this result poses a striking paradox, and its public health implications are not immediately clear.