Human immunodeficiency virus type 1 (HIV-1) genotyping in Rio de Janeiro, Brazil: assessing subtype and drug-resistance associated mutations in HIV-1 infected individuals failing highly active antiretroviral therapy

In order to assess the human immunodeficiency virus type 1 (HIV-1) drug resistance mutation profiles and evaluate the distribution of the genetic subtypes in the state of Rio de Janeiro, Brazil, blood samples from 547 HIV-1 infected patients failing antiretroviral (ARV) therapy, were collected during the years 2002 and 2003 to perform the viral resistance genotyping at the Renageno Laboratory from Rio de Janeiro (Oswaldo Cruz Foundation). Viral resistance genotyping was performed using ViroSeqTM Genotyping System (Celera Diagnostic-Abbott, US). The HIV-1 subtyping based on polymerase (pol) gene sequences (protease and reverse transcriptase-RT regions) was as follows: subtype B (91.2%), subtype F (4.9%), and B/F viral recombinant forms (3.3%). The subtype C was identified in two patients (0.4%) and the recombinant CRF_02/AG virus was found infecting one patient (0.2%). The HIV-1 genotyping profile associated to the reverse transcriptase inhibitors has shown a high frequency of the M184V mutation followed by the timidine-associated mutations. The K103N mutation was the most prevalent to the non-nucleoside RT inhibitor and the resistance associated to protease inhibitor showed the minor mutations L63P, L10F/R, and A71V as the more prevalent. A large proportion of subtype B was observed in HIV-1 treated patients from Rio de Janeiro. In addition, we have identified the circulation of drug-resistant HIV-1 subtype C and are presenting the first report of the occurrence of an African recombinant CRF_02/AG virus in Rio de Janeiro, Brazil. A clear association between HIV-1 subtypes and protease resistance mutations was observed in this study. The maintenance of resistance genotyping programs for HIV-1 failing patients is important to the management of ARV therapies and to attempt and monitor the HIV-1 subtype prevalence in Brazil.

The human immunodeficiency virus preventive vaccine research at the French National Agency for acquired immunodeficiency syndrome research

The human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS) epidemic is of unprecedented gravity and is spreading rapidly, notably in the most disadvantaged regions of the world. The search for a preventive vaccine is thus an absolute priority. For over 10 years the French National Agency for AIDS research (ANRS) has been committed to an original program combining basic science and clinical research. The HIV preventive vaccine research program run by the ANRS covers upstream research for the definition of immunogens, animal models, and clinical research to evaluate candidate vaccines. Most researchers in 2004 believe that it should be possible to obtain partial vaccine protection through the induction of a strong and multiepitopic cellular response. Since 1992, the ANRS has set up 15 phases I and II clinical trials in order to evaluate the safety and the capacity of the candidate vaccines for inducing cellular immune responses. The tested candidate vaccines were increasingly complex recombinant canarypox viruses (Alvac) containing sequences coding for certain viral proteins, utilized alone or combined with other immunogens (whole or truncated envelope proteins). ANRS has also been developing an original strategy based on the utilization of lipopeptides. These comprise synthetic fragments of viral proteins associated with lipids that facilitate the induction of a cellular immune response. These approaches promptly allowed the assessment of a prime-boost strategy combining a viral vector and lipopeptides.

Human immunodeficiency virus type 1 neutralization by plasma from B or F genotype infected individuals

Anti-human immunodeficiency virus type 1 (HIV-1) "binding antibodies" (antibodies capable of binding to synthetic peptides or proteins) occur throughout HIV-1 infection, are high-titered and highly cross-reactive, as confirmed in this study by analyzing plasma from B and F genotype HIV-1 infected individuals. Plasma from individuals infected with clade F HIV-1 displayed the most frequent cross-reactivity, in high titers, while Bbr plasma showed much higher specificity. Similarly, neutralization of a reference HIV-1 isolate (HIV-1 MN) was more frequently observed by plasma from F than B genotype infected individuals. No significant difference was seen in neutralization susceptibility of primary B, Bbr or F clade HIV-1 by plasma from individuals infected with the classical B (GPGR) or F HIV-1, but Bbr (GWGR) plasma were less likely to neutralize the F genotype primary HIV-1 isolates. The data indicate that both B and F genotype derived vaccines would be equally effective against B and F HIV-1 infection, with a slightly more probable effectiveness for F than B genotype. Although the Bbr variant appears to induce a much more specific humoral immune response, the susceptibility in neutralizing the Brazilian HIV-1 B genotype Bbr variant is similar to that observed with the classical B genotype HIV-1.

Is human immunodeficiency virus/acquired immunodeficiency syndrome decreasing among Brazilian injection drug users? Recent findings and how to interpret them

We briefly review findings from Brazilian settings where the human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS) epidemic among injection drug users (IDUs) seems to be decreasing, highlighting recent findings from Rio de Janeiro and discussing methodological alternatives. Former analyses using serologic testing algorithm for recent HIV seroconversion have shown that HIV incidence has been low in IDUs recruited by two different surveys carried out in Rio, where low injection frequencies and infection rates have been found among new injectors. The proportion of AIDS cases among IDUs in Rio has been fairly modest, compared to São Paulo and especially to the southernmost states. Notwithstanding, the interpretation of findings from serial surveys constitutes a challenge, magnified in the assessment of HIV spread among IDUs due to the dynamic nature of the drug scenes and limitations of sampling strategies targeting hard-to-reach populations. Assessment of epidemic trends may profit from the triangulation of data, but cannot avert biases associated with sampling errors. Efforts should be made to triangulate data from different sources, besides exploring specific studies from different perspectives. In an attempt to further assess the observed trends, we carried out original analyses using data from Brazilian AIDS databank.

Antiretroviral resistance mutations in human immunodeficiency virus type 1 infected patients enrolled in genotype testing at the Central Public Health Laboratory, São Paulo, Brazil: preliminary results

Antiretroviral resistance mutations (ARM) are one of the major obstacles for pharmacological human immunodeficiency virus (HIV) suppression. Plasma HIV-1 RNA from 306 patients on antiretroviral therapy with virological failure was analyzed, most of them (60%) exposed to three or more regimens, and 28% of them have started therapy before 1997. The most common regimens in use at the time of genotype testing were AZT/3TC/nelfinavir, 3TC/D4T/nelfinavir and AZT/3TC/efavirenz. The majority of ARM occurred at protease (PR) gene at residue L90 (41%) and V82 (25%); at reverse transcriptase (RT) gene, mutations at residue M184 (V/I) were observed in 64%. One or more thymidine analogue mutations were detected in 73%. The number of ARM at PR gene increased from a mean of four mutations per patient who showed virological failure at the first ARV regimens to six mutations per patient exposed to six or more regimens; similar trend in RT was also observed. No differences in ARM at principal codon to the three drug classes for HIV-1 clades B or F were observed, but some polymorphisms in secondary codons showed significant differences. Strategies to improve the cost effectiveness of drug therapy and to optimize the sequencing and the rescue therapy are the major health priorities.

Dried blood spots as a practical and inexpensive source for human immunodeficiency virus and hepatitis C virus surveillance

Passive surveillance of infectious diseases with a high percentage of asymptomatic cases or long incubation periods, such as acquired immunodeficiency syndrome (AIDS), does not reflect the current transmission dynamics. Thus, a multi-strategic surveillance, such as the human immunodeficiency virus (HIV) sentinel surveillance proposed by the World Health Organization (WHO), is necessary. The Brazilian HIV sentinel surveillance was started in May 1992 with this purpose. The objectives of this study were to evaluate the feasibility and costs of HIV and hepatitis C virus (HCV) surveillance using dried blood spots (DBS) collected for neonatal screening of metabolic diseases in the state of Minas Gerais, Brazil. This was accomplished through the comparison of HIV and HCV seroprevalence with previous Brazilian studies. From December 2001 to June 2002, 24,905 newborns were tested for HIV and 4211 for HCV. HIV seroprevalence was 0.25% and the 95% confidence interval (CI) was 0.18, 0.31%; and HCV seroprevalence was 0.71% and the 95% CI was 0.46, 0.97%. These numbers are similar to previous Brazilian studies. Cost in this study was approximately US$ 3.10 per sample, which was roughly one third of the cost of the same exam at the Brazilian HIV sentinel surveillance. We conclude that it is possible and more cost-effective to use DBS for infectious diseases surveillance, albeit it is still necessary to compare these results with the usual sentinel methodology in a concomitant trial.

Molecular characterization of human T-cell lymphotropic virus coinfecting human immunodeficiency virus 1 infected patients in the Amazon region of Brazil

The present work evaluated the epidemiology of human immunodeficiency virus 1/human T-cell lymphotropic virus (HIV-1/HTLV) coinfection in patients living in Belém (state of Pará) and Macapá (state of Amapá), two cities located in the Amazon region of Brazil. A total of 169 blood samples were collected. The sera were tested by enzyme-linked immunosorbent assay to determine the presence of antibodies anti-HTLV-1/2. Confirmation of infection and discrimination of HTLV types and subtypes was performed using a nested polymerase chain reaction targeting the pX and 5' LTR regions, followed by restriction fragment length polymorphism and sequencing analysis. The presence of anti-HTLV1/2 was detected in six patients from Belém. The amplification of the pX region followed by RFLP analysis, demonstrated the presence of HTLV-1 and HTLV-2 infections among two and four patients, respectively. Sequencing HTLV-1 5' LTR indicated that the virus is a member of the Cosmopolitan Group, Transcontinental subgroup. HTLV-2 strains isolated revealed a molecular profile of subtype HTLV-2c. These results are a reflex of the epidemiological features of HIV-1/HTLV-1/2 coinfection in the North region of Brazil, which is distinct from other Brazilian regions, as reported by previous studies.

Serologic evidence of human metapneumovirus circulation in Uruguay

First identified in 2001, the human metapneumovirus (hMPV), is a respiratory tract pathogen that affects young children, elderly, and immunocompromised patients. The present work represents the first serologic study carried out in Uruguay. It was performed with the purpose of obtaining serological evidence of hMPV circulation in Uruguay and to contribute to the few serologic reports described until now. Sixty nine serum samples collected between 1998 and 2001 by vein puncture from patients without respiratory symptoms or underlying pathology aged 6 days to 60 years were examined using an indirect immunofluorescence assay (IFA). The global seropositivity rate of the samples was 80% (55/69). Rates of 60% (15/25) and 91% (40/44) were observed for the pediatric and adult cohorts, respectively. Results obtained from a longitudinal analysis of 6 children aged 6 days to 18 months are discussed. These results are a clear evidence of hMPV circulation in Uruguay, at least since 1998, and reinforce the previous data on worldwide circulation of this virus.

A genotyping study of human immunodeficiency virus type-1 drug resistance in a small Brazilian municipality

In Brazil, surveillance studies on antiretroviral drug resistance among drug-naïve and treatment-experienced patients have focused primarily on patients living in large urban centers. As the epidemic spreads towards small municipalities and the innermost parts of the country, it will be essential to monitor the prevalence of antiretroviral drug resistance in these areas. We report the first survey on the prevalence of antiretroviral drug resistance in a small Brazilian municipality. Between July 1999 and March 2005, 72 adult human immunodeficiency virus type-1(HIV-1)-infected patients received care at the Municipal HIV/AIDS Program of the small, southeastern municipality of Miracema, state of Rio de Janeiro. A genotyping study of antiretroviral drug resistance was performed in 54 patients. Among 27 samples from treatment-experienced patients, 9 (33.3%) harbored strains with reduced drug susceptibility. Among these, 6 had reduced susceptibility to reverse transcriptase (RT) inhibitors and 3 to both RT and protease inhibitors. No primary antiretroviral drug resistance was recorded among 27 drug-naïve subjects. The relatively low prevalence of resistance mutations in the Miracema cohort argues against the concern that resource-poor settings should not implement widespread accessibility to standard of care antiretroviral combinations due to the possibility of sub-optimal adherence leading to the emergence and spread of drug-resistant strains.

Frequency of viruses associated with acute respiratory infections in children younger than five years of age at a locality of Mexico City

A locality in the district of Tlalpan, Mexico City, was selected in order to identify the viral agents in children younger than 5 years of age with acute respiratory infection (ARI). A total of 300 children were randomly selected and were included in this study for a period of 13 months. During this period nasopharyngeal exudates were collected for the isolation of viral agents. Monoclonal fluorescent antibodies were used for viral identification after cell culture. Viral infection was detected in 65% of the specimens. The respiratory syncytial virus (RSV) was the most common virus agent detected. Children required an average of two consultations during the study period. Two high incidence peaks were observed, one during the summer and the other during winter; the most frequent viruses during these seasons were influenza A and RSV, respectively. The largest number of viruses was isolated in the group of children between 1 and 2 years of age and in the group between 4 and 5 years of age. This study demonstrated the presence of ARI and of different viruses in a period of 13 months, as well as the most frequent viruses in children younger than 5 years of age from a community of Mexico City.

Genetic characterization of St. Louis encephalitis virus isolated from human in São Paulo, Brazil

The molecular characterization of SPH253157, a new strain of St. Louis encephalitis virus (SLEV), isolated in 2004 from the first case of human infection recognized in the state of São Paulo, Brazil, is reported. The patient, presenting a febrile illness without neurological involvement, was hospitalized as a probable case of dengue fever. Genomic RNA was isolated from the supernatant of C6/36 cells infected with acute phase-serum specimen of the patient and the envelope gene was amplified by reverse-transcription-polymerase chain reaction. The complete nucleotide sequence of the envelope gene of this isolate was directly sequenced from the amplified products and compared with other Brazilian and American SLEV strains. Phylogenetic analyses were carried out under maximum likelihood criterion with outgroups both included and excluded. Outgroups comprised four flavivirus of the Japanese encephalitis group. Phylogeny also included Bayesian analysis. The results indicated that the new SLEV isolate belongs to lineage III, being closely related to an Argentinean strain recovered from Culex sp. in 1979. It is concluded that there are at least 3 lineages of SLEV in Brazil.

Identification of human T-cell lymphotropic virus infection in a semi-isolated Afro-Brazilian quilombo located in the Marajó Island (Pará, Brazil)

Antibodies to human T-cell lymphotropic virus-1 and 2 (HTLV-1 and 2) were tested in 259 inhabitants (98 males and 161 females) of four villages of the Marajó Island (Pará, Brazil) using enzyme immunoassays (ELISA and Western blot). Types and subtypes of HTLV were determined by nested polymerase chain reaction (PCR) targeting the pX, env and 5´LTR regions. HTLV-1 infection was detected in Santana do Arari (2.06%) and Ponta de Pedras (1%). HTLV-2 was detected only in Santana do Arari (1.06%). Sequencing of the 5´LTR region of HTLV-1 and the phylogenetic analysis identified the virus as a member of the Cosmopolitan Group, subgroup Transcontinental. Santana do Arari is an Afro-Brazilian community and the current results represent the first report of HTLV-1 infection in a mocambo located in the Brazilian Amazon region.

Lack of tax diversity for tropical spastic paraparesis/human T-cell lymphotropic virus type 1 (HTLV-I) associated myelopathy development in HTLV-I-infected subjects in São Paulo, Brazil

The product of human T-cell lymphotropic virus type 1 (HTLV-1) tax gene has a transactivating effect of the viral and cellular gene expression. Genetic variations in this gene have been correlated with differences in clinical outcomes. Based upon its diversity, two closely related substrains, namely tax A and tax B, have been described. The tax A substrain has been found at a higher frequency among human T-cell leukemia virus type 1 (TSP/HAM) patients than among healthy HTLV-I-infected asymptomatic subjects in Japan. In this study, we determined the distribution of tax substrains in HTLV-I-infected subjects in the city of São Paulo, Brazil. Using the ACCII restriction enzyme site, we detected only tax A substrain from 48 TSP/HAM patients and 28 healthy HTLV-I carriers. The sequenced tax genes from nine TSP/HAM patients and five asymptomatic HTLV-I carriers showed a similar pattern of mutation, which characterizes tax A. Our results indicate that HTLV-I tax subtypes have no significant influences on TSP/HAM disease progression. Furthermore, monophyletic introduction of HTLV-I to Brazil probably occurred during the African slave trade many years ago.

Patterns of hepatitis B virus infection in Brazilian human immunodeficiency virus infected patients: high prevalence of occult infection and low frequency of lamivudine resistant mutations

Hepatitis B virus (HBV) molecular profiles were determined for 44 patients who were infected with human immunodeficiency virus (HIV) type 1 and had antibodies to the hepatitis B core antigen (anti-HBc), with and without other HBV serological markers. In this population, 70% of the patients were under lamivudine treatment as a component of antiretroviral therapy. HBV DNA was detected in 14 (32%) patients. Eight out of 12 (67%) HBsAg positive samples, 3/10 (30%) anti-HBc only samples, and 3/22 (14%) anti-HBs positive samples were HBV DNA positive. HBV DNA loads, measured by real time polymerase chain reaction, were much higher in the HBsAg positive patients (mean, 2.5 × 10(9) copies/ml) than in the negative ones (HBV occult infection; mean, 2.7 × 10(5) copies/ml). Nine out of the 14 HBV DNA positive patients were under lamivudine treatment. Lamivudine resistant mutations in the polymerase gene were detected in only three patients, all of them belonging to the subgroup of five HBsAg positive, HBV DNA positive patients. A low mean HBV load (2.7 × 10(5) copies/ml) and an absence of lamivudine resistant mutations were observed among the cases of HBV occult infection.

Primary resistance of human immunodeficiency virus type 1 in a reference center in Recife, Pernambuco, Brazil

To assess the prevalence of primary resistance of human immunodeficiency virus type 1 (HIV-1) to antiretrovirals, 84 patients chronically infected with HIV without prior antiretroviral treatment from Northeast Brazil were studied. Genotyping was performed using the ViroSeqTM Genotyping System. Thimidine analog mutations occurred in 3 (3.6%) patients. Accessory mutations related to NRTI occurred in 6 (7.1%) and related to PI in 67 (79.8%). Subtypes B (72.6%), F (22.6%), B/F 3 (3.6%), and C (1.2%) were detected. A low prevalence of major mutations related to NRTI in patients chronically infected by HIV was observed.