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Objective: The aim of this study was to investigate the impact of intersubtype HIV-1 superinfection on viremia, reservoir reseeding, viral evolution and disease progression in HIV controllers (HIC)., Design: A longitudinal analysis of two Brazilian HIC individuals (EEC09 and VC32) previously identified as dually infected with subtypes B and F1 viruses., Methods: Changes in plasma viremia, total HIV-1 DNA levels, CD4+ T-cell counts and HIV-1 quasispecies composition were measured over time. HIV-1 env diversity in peripheral blood mononuclear cell (PBMC) and plasma samples was accessed by single genome amplification and next-generation sequencing approaches, respectively. Viral evolution was evaluated by estimating nucleotide diversity and divergence., Results: Individual EEC09 was probably initially infected with a CCR5-tropic subtype B strain and sequentially superinfected with a CXCR4-tropic subtype B strain and with a subtype F1 variant. Individual VC32 was infected with a subtype B strain and superinfected with a subtype F1 variant. The intersubtype superinfection events lead to a moderate increase in viremia and extensive turnover of viral population in plasma but exhibited divergent impact on the size and composition of cell-associated HIV DNA population. Both individuals maintained virologic control (<2000 copies/ml) and presented no evidence of viral evolution or immunologic progression for at least 2 years after the intersubtype superinfection event., Conclusion: These data revealed that some HIC are able to repeatedly limit replication and evolution of superinfecting viral strains of a different subtype with no signs of disease progression.

Objective: To investigate the origin and to reconstruct the onset date of the HIV-1 subtype C epidemic in Brazil., Design: Three independent datasets of subtype C sequences isolated from HIV-1-positive patients from southern Brazil over a period of 15 years (1991-2006) were analyzed: 82 env V3 sequences (213 nt), 40 env C2-C5 sequences (559 nt), and 72 pol sequences (960 nt)., Methods: Brazilian sequences were compared with other subtype C reference strains from the database using basic local alignment search tool, phylogenetic analyses, and searching of specific amino acid signature patterns. Evolutionary parameters were estimated using a Bayesian coalescent-based method under either strict or relaxed molecular clock models., Results: HIV-1 subtype C sequences from Brazil and Burundi formed a monophyletic cluster at both env and pol regions and shared specific amino acid signatures in the protease region when compared with other viruses of the same subtype from around the world. All Brazilian strains arose as a monophyletic subcluster within the Burundi-Brazilian lineage, whereas isolates from Burundi appeared at the origin of the clade. Evolutionary analyses of both env and pol genomic regions indicate that the age of the most recent common ancestor of the Brazilian subtype C clade dates back to the early 1980s., Conclusion: The subtype C epidemic in the southern Brazilian region was initiated by the introduction of a single founder strain closely related to subtype C strains from Burundi. Our results suggest that this founder event probably took place around the early 1980s, roughly a decade before the previous estimates.

Objective: To reconstruct the onset date of the HIV-1 B and F epidemics in Brazil based on virus diversification over time., Design: We studied HIV-1 env V3 sequences (210 nt) with a known sampling year isolated from HIV-1 positive patients from Brazil between 1989 and 1997: 101 subtype B sequences and 41 subtype F sequences., Methods: HIV-1 V3 env sequences were grouped by year of collection and the relationship between the sampling years of HIV-1 sequences and their genetic distance to the reconstructed common ancestor (intra-population divergence) or to other sequences from the same year (intra-population diversity) was examined by using linear regression analysis., Results: Regression analysis of nucleotide distances, revealed a highly significant positive correlation between sampling years of subtype B and F V3 sequences and their intra-population divergence (P < 0.001) or diversity (P < 0.0001). In both subtype populations, the divergence and diversity increased at a rate of 0.5 and 0.9% per year, respectively. Considering these evolutionary rates, we estimate the onset of the subtype B and F HIV-1 epidemics in Brazil during early 1970s and early 1980s, respectively., Conclusions: The consistent correlation between divergence and diversity of the V3 sequences with their sampling years indicates that the molecular clock is operational in the evolution of the HIV-1 in Brazil's epidemic, and show that subtypes B and F are evolving at a similar rate over time. The dating results suggest a discontinuous introduction of these subtypes in the Brazilian population.

Background: Recent studies have shown substantial increases in the survival of AIDS patients in developed countries and in Brazil as a result of antiretroviral therapy (ART) and prophylaxis for opportunistic infections. This study compares survival rates using the Brazilian Ministry of Health 2004 and Centers for Disease Control and Prevention (CDC) 1993 case definitions in a large HIV/AIDS referral centre in Rio de Janeiro., Methods: Survival after AIDS diagnosis was assessed in a clinic-based cohort of 1415 individuals using the Kaplan-Meier method and Cox proportional hazards models., Results: There were 393 (88%) deaths from AIDS-related causes and 52 (12%) from unrelated or unknown causes. A total of 205 patients (14%) were lost to follow-up and 765 patients (55%) remained alive until the end of the study. Three-quarters of patients (75%) were still alive 22 months [95% confidence interval (CI) 19-26] after the AIDS diagnosis according to the CDC case definition and 31 months (95% CI 26-36) according to the Ministry of Health case definition. Independent predictors of survival included AIDS defined by CD4 cell count and any use of highly active antiretroviral therapy, with either case definition, and initial stage of the case, with the Ministry of Health case definition., Conclusion: Survival observed in this reference centre is comparable or longer than other international studies, although the choice of case definition criterion influenced findings. Adoption of the Ministry of Health case definition may enhance the ability to track the use of and outcomes from ART among AIDS patients.

Objective: To study the prevalence of HIV drug resistance mutations and subtype distribution in a Brazilian drug-naive population. Asymptomatic, drug-naive HIV-1-infected individuals were targeted in 13 voluntary counseling and testing centers spread around the country., Methods: Plasma viral RNA was extracted from 535 HIV-1-positive subjects. Protease (PR) and reverse transcriptase (RT) genomic regions were sequenced for subtype determination and analysis of drug resistance mutations., Results: Eight samples (2.24 %) showed primary mutations related to protease inhibitor (PI) resistance, eight (2.36%) to nucleoside reverse transcriptase inhibitors (NRTI) and seven (2.06%) to non-nucleoside reverse transcriptase inhibitors (NNRTI). Accessory mutations were found in the PR gene at the following positions: L63P/V/T/A/I [153/345 (44.3%)], M36I/L [149/345 (43.2%)], L10I/F/V [82/345 (23.8%)], V77I [60/345 (17.4%)], A71V/T [11/345 (3.2%)], K20M/R [10/345 (2.9%)], and V82I [4/345 (1.2%)]. Mutations known to be associated with reduced sensitivity to NRTI or NNRTI (V118I, E44D, K219R, T69A, and V75L) were found in a low prevalence (0.6-2.4%). A high proportion of the isolates from subtype C was found in the southern states. Subtype F-related viruses were the main non-B variant in the rest of the country., Conclusions: Brazil has a low prevalence of drug-resistant strains circulating among recently diagnosed individuals. However, there was an increase in these rates compared with similar studies performed with samples collected in Brazil from 1996 to 1998. Continued surveys are required to detect trends in these rates, but routine genotypic testing in the drug-naive population prior to antiretroviral initiation is not required in Brazil.

Objective: To characterize the subtype C strains of HIV type 1 that circulate in Brazil, especially those originated from the southern part of the country., Design and Methods: One hundred and twelve HIV-1-positive subjects had their plasma viral RNA extracted. Protease (PR) and reverse transcriptase (RT) genomic regions were polymerase chain reaction-amplified and sequenced for subtype determination. Subtype C strains were selected and compared to other strains of this subtype from the database, and specific amino acid signature patterns were searched., Results: Brazilian subtype C viruses form a very strong monophyletic group when compared to subtype C viruses from other countries and presented specific signature amino acids. Recombinants between subtype C and B viruses have been documented in areas of co-circulation. The incidence of primary PR and RT inhibitor resistance mutations in drug-naïve subjects was observed. An increasing number of secondary resistance mutations was also seen, some of which are characteristic of subtype C-related sequences., Conclusions: Introduction of subtype C of HIV-1 in Brazil was likely a single event of one or a mixture of similarly related strains. Recombination between subtype C and B viruses is an ongoing process in the country. Primary and secondary drug resistance mutations were observed, although some of the secondary mutations could be associated with subtype C molecular signatures. Subtype-specific polymorphisms of PR and RT sequences found in this subtype C Brazilian variant might influence this emergence and have an impact on HIV treatment and on vaccine development in the country.

HIV-1-positive individuals were recruited from January 1993 to December 1996 from several cohorts receiving follow-up in the city of Rio de Janeiro, Brazil, to evaluate HIV-1 genetic variability and the potential association with modes of transmission. HIV-1 subtyping was carried out using the heteroduplex mobility assay (HMA), and those samples corresponding to the typical Brazilian subtype B variant were further identified based on the Fok I restriction fragment length polymorphism (RFLP). DNA sequencing was performed to evaluate one case of subtype D infection. From the 131 HIV-1-positive individuals analyzed, 106 (80.9%) could be identified as infected by subtype B and 20 (15.3%) by subtype F. One of the samples (0.8%) was classified as subtype D. DNA samples from 4 patients (3.0%) did not yield polymerase chain reaction (PCR)-amplified products to be typed. Based on the Fok I RFLP, 39 of the 106 subtype B samples (37%) were identified as corresponding to the typical Brazilian subtype B variant containing the GWGR motif at the tip of the V3 loop. No statistically significant association could be detected between HIV-I subtypes and modes of transmission, exposure categories, or gender. This is the first reported case of HIV-1 subtype D infection in Brazil.

Objective: To study the susceptibility of primary HIV-1 isolates towards autologous and heterologous neutralizing antibodies (NAb)., Design: Blood was collected and primary HIV-1 isolated from individuals residing in Rio de Janeiro, Brazil, in all phases of disease., Methods: Primary HIV-1 isolates were incubated with autologous or heterologous plasma and neutralization of infection of freshly pre-stimulated normal human peripheral blood mononuclear cells was assayed in parallel to median infectious dose determinations in the absence of antibodies. Levels of HIV-1 p24 antigen were used for evaluation of viral neutralization., Results: Autologous neutralization (75%) was observed for 13 (52%) out of 25 of the primary HIV-1 isolates, and 15 (71%) out of 21 isolates were susceptible to 75% heterologous neutralization by at least one-half of the heterologous plasma tested. Primary HIV-1 isolates susceptible to autologous NAb showed a higher susceptibility towards neutralization by heterologous NAb than isolates that could not be neutralized by the autologous plasma (P = 0.049). The susceptibility of the primary HIV-1 isolates towards neutralization by heterologous NAb was significantly higher for isolates derived from men (P = 0.001), and for isolates obtained from individuals infected through homo-/bisexual risk behaviour in comparison with those infected through heterosexual HIV-1 transmission (P = 0.03)., Conclusions: Susceptibility of primary HIV-1 isolates to autologous and heterologous neutralization was significantly correlated, indicating that escape mutants may become resistant not only to autologous but also to heterologous NAb.