Jussara Silveira

HIV-1 subtype C dissemination in southern Brazil

Objectives:To describe the molecular and epidemiological profile of HIV-1 in patients followed at the University Hospital of Rio Grande, Brazil. Design and methods:A cross-sectional study was conducted from September to December 2002. Plasma viral RNA of 85 patients was extracted and protease and reverse transcriptase genes were polymerase chain reaction-amplified and sequenced. Sequences were subtyped and examined to antiretroviral resistance mutations. Laboratory data and past history of antiretroviral treatment were also collected. Results:Most viruses were either subtype B (42%) or subtype C (45%). No risk behaviour, sexual orientation or laboratory parameter was associated with any specific subtype, but subtype C tended to be more frequently found in women (P = 0.06). The prevalence of subtype C has increased over the HIV/AIDS epidemic, accounting for almost 60% of cases diagnosed in 2002. Intra-subtype genetic distances were smaller in subtype C than in subtype B, suggesting a more recent introduction of the former in the epidemic. Of patients under treatment, 60% had at least one antiretroviral drug resistance mutation, but no mutation was specifically associated with any HIV-1 subtype. Only one resistance mutation each was found in drug-naive patients with subtypes B and C. Conclusion:Despite the fact that subtype C appeared in southern Brazil more recently than subtype B, it is now the predominant strain in Rio Grande. The epidemic spread of subtype C could be taking place in Brazil, and possibly in south America, a phenomenon similar to that seen in other countries where this subtype is now totally dominant.

Differential Drug Resistance Acquisition in HIV-1 of Subtypes B and C

Background Subtype C is the most prevalent HIV-1 subtype in the world, mainly in countries with the highest HIV prevalence. However, few studies have evaluated the impact of antiretroviral therapy on this subtype. In southern Brazil, the first developing country to offer free and universal treatment, subtypes B and C co-circulate with equal prevalence, allowing for an extensive evaluation of this issue. Methods and Findings Viral RNA of 160 HIV-1+ patients was extracted, and the protease and reverse transcriptase genes were sequenced, subtyped and analyzed for ARV mutations. Sequences were grouped by subtype, and matched to type (PI, NRTI and NNRTI) and time of ARV exposure. Statistical analyses were performed to compare differences in the frequency of ARV-associated mutations. There were no significant differences in time of treatment between subtypes B and C groups, although they showed distinct proportions of resistant strains at different intervals for two of three ARV classes. For PI, 26% of subtype B strains were resistant, compared to only 8% in subtype C (p = 0.0288, Fishers exact test). For NRTI, 54% of subtype B strains were resistant versus 23% of subtype C (p = 0.0012). Differences were significant from 4 years of exposure, and remained so until the last time point analyzed. The differences observed between both subtypes were independent of time under rebound viremia in cases of virologic failure and of the number of HAART regimens used by treated patients. Conclusions Our results pointed out to a lower rate of accumulation of mutations conferring resistance to ARV in subtype C than in subtype B. These findings are of crucial importance for current initiatives of ARV therapy roll-out in developing countries, where subtype is C prevalent.

Conservation Patterns of HIV-1 RT Connection and RNase H Domains: Identification of New Mutations in NRTI-Treated Patients

Background Although extensive HIV drug resistance information is available for the first 400 amino acids of its reverse transcriptase, the impact of antiretroviral treatment in C-terminal domains of Pol (thumb, connection and RNase H) is poorly understood. Methods and Findings We wanted to characterize conserved regions in RT C-terminal domains among HIV-1 group M subtypes and CRF. Additionally, we wished to identify NRTI-related mutations in HIV-1 RT C-terminal domains. We sequenced 118 RNase H domains from clinical viral isolates in Brazil, and analyzed 510 thumb and connection domain and 450 RNase H domain sequences collected from public HIV sequence databases, together with their treatment status and histories. Drug-naïve and NRTI-treated datasets were compared for intra- and inter-group conservation, and differences were determined using Fishers exact tests. One third of RT C-terminal residues were found to be conserved among group M variants. Three mutations were found exclusively in NRTI-treated isolates. Nine mutations in the connection and 6 mutations in the RNase H were associated with NRTI treatment in subtype B. Some of them lay in or close to amino acid residues which contact nucleic acid or near the RNase H active site. Several of the residues pointed out herein have been recently associated to NRTI exposure or increase drug resistance to NRTI. Conclusions This is the first comprehensive genotypic analysis of a large sequence dataset that describes NRTI-related mutations in HIV-1 RT C-terminal domains in vivo. The findings into the conservation of RT C-terminal domains may pave the way to more rational drug design initiatives targeting those regions.

Epidemiologic and evolutionary trends of HIV-1 CRF31_BC-related strains in southern Brazil.

Background:To evaluate the impact of HIV-1 CRF31_BC in the southern Brazilian HIV epidemic. Methods:Blood plasma from 284 patients was collected from July 2002 to January 2003 at 2 reference HIV/AIDS centers in southern Brazil. Viral protease and reverse transcriptase (RT) genomic regions were amplified by RT polymerase chain reaction, sequenced, and subtyped. Evolutionary analyses were performed to estimate the CRF31_BC most recent common ancestor and its population growth rate with BEAST version 1.3. Results:CRF31_BC was responsible for 7.4% of infections. The average time of HIV diagnosis and the proportion of patients on antiretroviral treatment were shorter for CRF31_BC and subtype C than for subtype B. CRF31_BC was found as early as in 1990 in the Brazilian epidemic. Evolutionary analysis of CRF31_BC revealed that it appeared immediately after the introduction of subtype C in Brazil and has been growing at a similar rate as subtype C. Conclusions:CRF31_BC plays an important role in the HIV epidemic of southern Brazil, and its prevalence has increased throughout the years. This circulating recombinant form corresponds to approximately 25% of total HIV isolates in this region in 2004. Understanding the cause of this spread is important for public health strategies in Brazil and in Latin America.

Molecular epidemiology of HIV-1 in Rio Grande, RS, Brazil

We conducted a molecular epidemiological study to investigate HIV-1 strains in Rio Grande, southern Brazil, searching for an association with transmission mode and risk behavior. Patients (185) identified at an AIDS treatment reference Hospital, from 1994 to 1997, were included; from which 107 blood samples were obtained. Nested PCR was realized once for each sample; for amplified samples (69) HIV subtypes were classified using the heteroduplex mobility assay. Subtypes identified were B (75%), C (22%) and F (3%). All infections with C were diagnosed after 1994. Comparing patients with B and C, no differences were detected regarding demographic, clinical and laboratory characteristics; survival analysis did not reveal differences in HIV to AIDS evolution. A higher proportion of injecting drug users, IDU (not significant, p < .07) was found among those with C. This suggests that C may have been introduced in this area through IDU, and is being spread, probably by their sexual partners, to persons with other risk practices.

Heterosexual transmission of human immunodeficiency virus type 1 subtype C in southern Brazil.

BACKGROUND Human immunodeficiency virus type 1 (HIV-1) subtype B predominates in Brazil, but in the southern region subtype C is the most frequent, followed by subtypes B, F1 and recombinant forms. In southern Brazil, these subtypes co-circulate in subjects with homogeneous demographic and clinical features, enabling a better understanding of the role of HIV-1 subtypes on the characteristics of infection. OBJECTIVES To evaluate the prevalence of different HIV-1 subtypes in subjects with recent diagnosis for HIV infection in the extreme south of Brazil, and to study their association with demographic, behavioral, clinical and laboratorial characteristics. STUDY DESIGN We have determined the genetic sequence of viral protease and reverse transcriptase (polymerase, connection and RNase H domains) isolated from studied subjects. Viral subtype was inferred by comparison with reference HIV sequences, and recombination was determined with Simplot analysis. The association of HIV-1 subtypes with studied characteristics was evaluated by chi-square, Fishers exact, Students t and Kruskal-Wallis tests. RESULTS Two hundred and forty-five HIV isolates were molecularly characterized, and the association with variables was studied for 233 (95.1%) patients. Of those, 46.8% followed AIDS defining criteria. HIV-1C was responsible for 56.3% of infections, and was associated with heterosexual transmission (p=0.001) and with higher CD4(+) T-cell counts (p=0.02). CONCLUSIONS The molecular epidemiology of HIV-1 in the southernmost Brazil is currently steady with predominance of HIV-1C. This is the first study showing a robust association of the infection by this subtype and heterosexual transmission in the state of Rio Grande do Sul, Brazil.

Connection subdomain mutations in HIV-1 subtype-C treatment-experienced patients enhance NRTI and NNRTI drug resistance.

Mutations in the connection subdomain (CN) and RNase H domain (RH) of HIV-1 reverse transcriptase (RT) from subtype B-infected patients enhance nucleoside and nonnucleoside RT inhibitor (NRTI and NNRTI) resistance by affecting the balance between polymerization and RNase H activity. To determine whether CN mutations in subtype C influence drug sensitivity, single genome sequencing was performed on Brazilian subtype C-infected patients failing RTI therapy. CN mutations identified were similar to subtype B, including A376S, A400T, Q334D, G335D, N348I, and A371V, and increased AZT resistance in the presence of thymidine analog mutations. CN mutations also enhanced NNRTI resistance in the presence of classical NNRTI mutations: etravirine resistance was enhanced 6- to 11-fold in the presence of L100I/K103N/Y181C. These results indicate that selection of CN mutations in treatment-experienced patients also occurs in subtype-C-infected patients and are likely to provide valuable information in predicting clinical RTI resistance.

Genetic polymorphisms in estrogen receptors and sexual dimorphism in fat redistribution in HIV-infected patients on HAART.

Objective:To investigate genetic single nucleotide polymorphisms (SNPs) in estrogen receptor-&agr; (ER&agr;) (ESR1, rs2234693, rs1801132, rs7757956 and rs2813544) and ER&bgr; (ESR2, rs3020450, rs7154455 and rs4986938) genes and relate them to the adverse effects lipodystrophy, dyslipidemia and metabolic syndrome as well as to differences in their prevalence between sexes in HIV-infected individuals on HAART. Design:Cross-sectional study. Methods:Blood samples and anthropometric measurements were collected from 614 patients at reference services in the cities of Porto Alegre, Pelotas and Rio Grande in Brazil. The SNPs were genotyped by real-time PCR. Results:The lipodystrophy subtype frequencies in patients of different sexes showed statistically significant differences; the atrophic pattern was more prevalent in men, and the hypertrophic pattern was more prevalent in women. Furthermore, metabolic syndrome prevalence was higher in women than in men. The ESR1 rs2813544 G-allele was associated with higher measurements of several anthropometric variables in women: BMI, total subcutaneous fat and subcutaneous fat of limbs. Additionally, patients who were AA homozygous for ESR2 rs3020450 presented an increased risk for developing lipoatrophy (prevalence ratio 1.37, 95% confidence interval 1.09–1.73, P = 0.007). Conclusion:Significant differences in lipodystrophy and metabolic syndrome prevalence were detected between sexes. Moreover, the ESR1 gene (rs2813544) presented significant sex-specific associations with anthropometric variables, and the ESR2 gene (rs3020450) was associated with an increased risk of developing lipoatrophy. Our results suggest that these genes are in part responsible for the sexual dimorphism in fat tissue redistribution and patterns of lipodystrophy.

Virologic and immunologic effectiveness at 48 weeks of darunavir-ritonavir-based regimens in treatment-experienced persons living with HIV-1 infection in clinical practice: a multicenter Brazilian cohort.

Introduction: Published data addressing the effectiveness of darunavir–ritonavir (DRV/r)-based therapy for multiexperienced patients in developing countries are scarce. This study evaluated the 48-week virologic and immunologic effectiveness of salvage therapy based on DRV/r for the treatment of multidrug-experienced HIV-1-infected adults in Brazil. Materials and Methods: A multicenter retrospective cohort study was carried out with multidrug-experienced adults who were on a failing antiretroviral therapy and started a DRV/r-based salvage therapy between 2008 and 2010. The primary effectiveness end point was the proportion of patients with virologic success (plasma HIV-1 RNA <50 copies/mL at week 48). Results: At 48 weeks, 73% of the patients had HIV-RNA <50 copies/mL and a mean increase of 108 CD4 cells/mm3. Higher baseline viral load, lower baseline CD4 count, younger age, and 3 or more DRV/r-associated resistance mutations were significantly predictive of virologic failure. Concomitant use of raltegravir was strongly associated with virologic success. Conclusion: The use of DRV/r-based regimens for salvage therapy is an effective strategy in the clinical care setting of a developing country.

Genetic Markers Associated to Dyslipidemia in HIV-Infected Individuals on HAART

This study evaluated the impact of 9 single nucleotide polymorphisms (SNPs) in 6 candidate genes (APOB, APOA5, APOE, APOC3, SCAP, and LDLR) over dyslipidemia in HIV-infected patients on stable antiretroviral therapy (ART) with undetectable viral loads. Blood samples were collected from 614 patients at reference services in the cities of Porto Alegre, Pelotas, and Rio Grande in Brazil. The SNPs were genotyped by conventional polymerase chain reaction (PCR) and real-time PCR. The prevalence of dyslipidemia was particularly high among the protease inhibitors-treated patients (79%). APOE (rs429358 and rs7412) genotypes and APOA5 −1131T>C (rs662799) were associated with plasma triglycerides (TG) and low-density-lipoprotein cholesterol levels (LDL-C). The APOA5 −1131T>C (rs662799) and SCAP 2386A>G (rs12487736) polymorphisms were significantly associated with high-density-lipoprotein cholesterol levels. The mean values of the total cholesterol and LDL-C levels were associated with both the APOB SP Ins/Del (rs17240441) and APOB XbaI (rs693) polymorphisms. In conclusion, our data support the importance of genetic factors in the determination of lipid levels in HIV-infected individuals. Due to the relatively high number of carriers of these risk variants, studies to verify treatment implications of genotyping before HAART initiation may be advisable to guide the selection of an appropriate antiretroviral therapy regimen.