HIV-1 epidemiology and circulating subtypes in the countryside of South Brazil

INTRODUCTION: Human immunodeficiency virus type 1 (HIV-1) has spread worldwide, with several subtypes and circulating recombinant forms. Brazil has an incidence of 20.5 HIV-1/acquired immunodeficiency syndrome (AIDS) patients per 100,000 inhabitants; however, the Southernmost State of Rio Grande do Sul (RS) has more than twice the number of HIV-1-infected people (41.3/100,000 inhabitants) and a different pattern of subtype frequencies, as previously reported in studies conducted in the capital (Porto Alegre) and its metropolitan region. This study examined HIV-1/AIDS epidemiological and molecular aspects in the countryside of Rio Grande do Sul. METHODS: Socio-demographic, clinical and risk behavioral characteristics were obtained from HIV-1-positive adult patients using a structured questionnaire. HIV-1 subtypes were determined by nested-polymerase chain reaction (PCR) and sequencing of the pol and env genes. RESULTS: The study sample included 149 (55% women) patients with a mean age of 41.8 ± 11.9 years. Most (73.8%) patients had a low education level and reported heterosexual practices as the most (91.9%) probable transmission route. HIV-1 subtypes were detected in 26 patients: 18 (69.2%) infected with subtype C, six (23.1%) infected with subtype B and two (7.7%) infected with BC recombinant forms. CONCLUSIONS: These data highlight the increasing number of HIV-1 subtype C infections in the countryside of South Brazil. .

Distribution of human immunodeficiency virus type 1 subtypes in the state of Amazonas, Brazil, and subtype C identification

Few studies have reported the molecular epidemiological characterization of HIV-1 in the Northern region of Brazil. The present study reports the molecular and epidemiological characterization of 31 HIV-1 isolates from blood donors from the State of Amazonas who donated blood between April 2006 and March 2007. Serum/plasma samples from all donors were screened for HIV antibodies by ELISA and the results confirmed by Western blot analysis. Genomic DNA was extracted from the buffy coat using the Super Quik-Gene-DNA Isolation kit. Nested PCR was performed on the env, gag, and pol regions of HIV-1 using the Gene Amp PCR System 9700. Sequencing reactions were performed using the inner PCR primers and the DYEnamic™ ET Dye Terminator Kit, and phylogenetic analysis was performed using the gag, pol, and env gene sequences. We collected samples from 31 blood donors who tested positive for HIV-1 in confirmatory experiments. The male:female ratio of blood donors was 3.4:1, and the mean age was 32.4 years (range: 19 to 61 years). Phylogenetic analysis showed that subtype B is the most prevalent among Northern Brazilian HIV-1-seropositive blood donors. One HIV-1 subtype C and one circulating recombinant form (CRF_BF) of HIV-1 were identified in the State of Amazonas. This is the first study showing the occurrence of a possible "homogenous" subtype C in this region of Brazil. This finding could contribute to a better characterization of the HIV-1 strains that circulate in the country.

Fixations of the HIV-1 env gene refute neutralism: New evidence for pan-selective evolution

We examined 103 nucleotide sequences of the HIV-1 env gene, sampled from 35 countries and tested: I) the random (neutral) distribution of the number of nucleotide changes; II) the proportion of bases at molecular equilibrium; III) the neutral expected homogeneity of the distribution of new fxated bases; IV) the hypothesis of the neighbor infuence on the mutation rates in a site. The expected random number of fxations per site was estimated by Bose-Einstein statistics, and the expected frequencies of bases by matrices of mutation-fxation rates. The homogeneity of new fxations was analyzed using χ2 and trinomial tests for homogeneity. Fixations of the central base in trinucleotides were used to test the neighbor infuence on base substitutions. Neither the number of fxations nor the frequencies of bases ftted the expected neutral distribution. There was a highly signifcant heterogeneity in the distribution of new fxations, and several sites showed more transversions than transitions, showing that each nucleotide site has its own pattern of change. These three independent results make the neutral theory, the nearly neutral and the neighbor infuence hypotheses untenable and indicate that evolution of env is rather highly selective.

Difficulties in the diagnosis of HTLV-2 infection in HIV/AIDS patients from Brazil: comparative performances of serologic and molecular assays, and detection of HTLV-2b subtype

The current diagnosis of human T-lymphotropic virus type-2 (HTLV-2) infection is based on the search of specific antibodies; nevertheless, several studies conducted in Brazil pointed deficiencies of the commercially available kits in detecting HTLV-2, mostly in HIV/AIDS patients. This study searched for the presence of HTLV-1 and -2 in 758 HIV/AIDS patients from Londrina, Paraná, Brazil. Serum samples were screened for HTLV-1/2 antibodies using two EIA kits (Vironostika and Murex), and confirmed by WB (HTLV Blot 2.4, Genelabs). The results obtained by EIA disclosed 49 (6.5 percent) reactive sera: 43 positive by both EIA kits, and six with discordant results. WB confirmed HTLV-1 infection in seven samples (0.9 percent) and HTLV-2 in 21 sera (2.8 percent). Negative and indeterminate results were detected in four (0.5 percent) and 16 (2.1 percent) sera, respectively. Blood from 47 out of 49 HTLV seroreactive patients were collected and analyzed for the presence of env, LTR and tax genomic segments of HTLVs by PCR. PCR confirmed six cases of HTLV-1 and 37 cases of HTLV-2 infection (14 out of 16 that were found to be WB indeterminate). Restriction analysis of the env PCR products of HTLV-2 disclosed 36 isolates of HTLV-2a/c subtype, and one of HTLV-2b subtype. These results emphasize the need of improving serologic tests for detecting truly HTLV-2 infected patients from Brazil, and confirm the presence of HTLV-2b subtype in the South of this country.

O diagnóstico de infecção por HTLV-2 se baseia na pesquisa de anticorpos específicos, entretanto, vários estudos conduzidos no Brasil têm apontado falhas nos kits sorológicos disponíveis no mercado em detectar HTLV-2, principalmente nos pacientes com HIV/aids. Este trabalho avaliou a presença de infecção por HTLV-1 e -2 em 758 pacientes HIV/aids de Londrina, Paraná, Brasil. Amostras de soro foram analisadas quanto à presença de anticorpos anti-HTLV-1/2 por dois kits de EIA (Vironostika e Murex) e confirmados por WB (HTLV Blot 2.4, Genelabs). Os resultados obtidos pelos testes sorológicos mostraram 49 (6,5 por cento) soros reagentes: 43 positivos para ambos os kits e seis com resultados discordantes. O WB confirmou infecção por HTLV-1 em sete soros (0,9 por cento) e HTLV-2 em 21 soros (2,8 por cento). Resultados negativos e indeterminados foram detectados, respectivamente, em quatro (0,5 por cento) e 16 (2,1 por cento) soros. Amostras de sangue de 47 dos 49 pacientes com sorologia reagente foram avaliadas quanto à presença de segmentos do genoma dos HTLVs (env, LTR e tax), usando a técnica de PCR. As PCRs confirmaram seis casos de infecção por HTLV-1 e 37 casos por HTLV-2 (14 dos 16 cuja sorologia resultou WB indeterminada). A subtipagem de HTLV-2 por análise de restrição enzimática de produtos da PCR env mostrou 36 isolados de subtipo HTLV-2a/c e um HTLV-2b. Esses resultados reforçam a necessidade de melhorar o diagnóstico de infecção por HTLV-2 no Brasil e confirmam a presença do subtipo HTLV-2b na região sul do país.

Human immunodeficiency virus type- 1 subtypes of infected patients in Espírito Santo, Brazil

Genetic variability of human immunodeficiency virus type - 1(HIV-1) is a potential threat for both diagnosis and treatment of HIV/AIDS, as well as the development of effective vaccines. Up to now, HIV subtypes circulating among HIV-positive patients in the state of Espírito Santo were not known. In the present study, blood samples from 100 therapy-naïve HIV-1 infected patients were collected and the HIV subtype was determined through the Heteroduplex Mobility Assay (HMA). Ninety-seven out of 100 studied samples were subtyped by HMA, 73 samples (75.2 percent) were from subtype B, 9 (9.3 percent) from subtype F, 3 (3.1 percent) from subtype C, 6 (6.2 percent) Benv/Fgag, and another 6 (6.2 percent) Fenv/Bgag, what suggests that recombinant viruses were present in the studied samples. Twenty-eight percent of the subtype B samples were represented by the Brazilian B" subtype, which were identified by RFLP with Fok I. Data presented here demonstrate that the epidemiological characteristics of the HIV epidemic in the state of Espírito Santo are similar to those from the other Southeastern states and helped to better understand the genetic polymorphism of HIV in Brazil.

HIV-1 subtyping using gag/env heteroduplex mobility assay and peptide enzyme-linked immunosorbent assay.

Two HIV-1 subtypes have accounted for virtually all infections in Thailand: subtype B', found mainly in injection drug users (IDUs), and CRF01_AE (initially subtype E), found in over 90% of sexually infected persons and increasingly in IDUs in recent years. During 1997-1998, 227 blood samples were collected from HIV-1 infected individuals consisting of 92 mothers, 35 children and 100 IDUs. The blood samples were subtyped by heteroduplex mobility assay (HMA) and peptide enzyme-linked immunosorbent assay (PEIA). Using gag and env HMA, CRF01_AE and subtype B' accounted for 96-97% and 3-4% of both the mothers and the children, respectively. In the IDU group, 10% of the plasma samples could only be performed by gag HMA and gave the result as CRF01_AE. CRF01_AE and subtype B' using PEIA accounted for 67% and 33% of the IDUs. There was 100% concordance of the results between gag HMA and env HMA. Ninety-five percentages of concordant results were observed between HMA and PEIA. Of the 6/134 (5%) subjects with discordant results, nucleotide sequencing, used as a gold standard, confirmed the HMA result. In this study, HIV-1 was successfully genotyped by HMA and PEIA. However, a comparison of the subtyping results between HMA and PEIA revealed that HMA was slightly more accurate than PEIA.

Early detection of HIV-1 in infants by PCR.

Infants of HIV-infected mothers are at great risk of becoming infected with HIV during childbirth. Many infants acquire HIV during labor and delivery. Others can acquire HIV through the mixing of fetal and maternal blood as the placenta separates. The duration of membrane rupture, acute chorioamnionitis and invasive delivery techniques that increase the baby's contact with the mother's blood have been associated with higher risks of MTCT during labor and delivery. HIV is present in breast milk and risk of its transmission during breastfeeding depends on several factors, including: infant age, pattern of breastfeeding, breastfeeding duration, breast health, maternal viral load and maternal immune status. Infants born to HIV infected mothers carry their mother's antibodies in their blood into the second year of life, even if the infants themselves are not infected. For this reason, standard HIV antibody tests cannot reliably confirm HIV infection in infants until after the maternal antibodies have disappeared. Tests that can diagnose pediatric HIV infection accurately during the first year of life include HIV-PCR, CD4/CD8 counts, P24 antigen tests, and viral cultures. PCR offers an effective tool to reliably diagnose HIV in a pediatric age group. Nineteen infants born by normal delivery to HIV-1 seropositive mothers were studied by PCR for the HIV1 env gene. Thirteen babies (68.5%) were negative whereas 6 babies were found to be positive (31.5%). Although PCR is one of the most useful tests for this clinical situation, it is not definitive. Therefore, PCR should be interpreted with caution and repeated at regular defined intervals, preferably lasting until the HIV antibody status of the infant is resolved.