Quantitation of HIV-1 RNA Viral Load Using NASBA Methodology and Comparison with other Surrogate Markers for Disease Progression

In this study, HIV-1 viral load quantitation determined by Nucleic Acid Sequence Based Amplification (NASBA) was compared with other surrogate disease progression markers (antigen p24, CD4/CD8 cell counts and b-2 microglobulin) in 540 patients followed up at São Paulo, SP, Brazil. HIV-1 RNA detection was statistically associated with the presence of antigen p24, but the viral RNA was also detected in 68% of the antigen p24 negative samples, confirming that NASBA is much more sensitive than the determination of antigen p24. Regarding other surrogate markers, no statistically significant association with the detection of viral RNA was found. The reproducibility of this viral load assay was assessed by 14 runs of the same sample, using different reagents batches. Viral load values in this sample ranged from 5.83 to 6.27 log (CV = 36 %), less than the range (0.5 log) established to the determination of significant viral load changes.

Evaluation of an enzyme immunoassay for hepatitis C virus antibody detection using a recombinant protein derived from the core region of hepatitis C virus genome

This study was undertaken to evaluate an enzyme immunoassay (EIA) for hepatitis C virus antibody detection (anti-HCV), using just one antigen. Anti-HCV EIA was designed to detect anti-HCV IgG using on the solid-phase a recombinant C22 antigen localized at the N-terminal end of the core region of HCV genome, produced by BioMérieux. The serum samples diluted in phosphate buffer saline were added to wells coated with the C22, and incubated. After washings, the wells were loaded with conjugated anti-IgG, and read in a microtiter plate reader (492 nm). Serum samples of 145 patients were divided in two groups: a control group of 39 patients with non-C hepatitis (10 acute hepatitis A, 10 acute hepatitis B, 9 chronic hepatitis B, and 10 autoimmune hepatitis) and a study group consisting of 106 patients with chronic HCV hepatitis. In the study group all patients had anti-HCV detected by a commercially available EIA (Abbott®), specific for HCV structural and nonstructural polypeptides, alanine aminotransferase elevation or positive serum HCV-RNA detected by nested-PCR. They also had a liver biopsy compatible with chronic hepatitis. The test was positive in 101 of the 106 (95%) sera from patients in the study group and negative in 38 of the 39 (97%) sera from those in the control group, showing an accuracy of 96%. According to these results, our EIA could be used to detect anti-HCV in the serum of patients infected with hepatitis C virus.

Identification of Human T-lymphotropic Virus Type I (HTLV-I) Subtypes Using Restrited Fragment Length Polymorphism in a Cohort of Asymptomatic Carriers and Patients with HTLV-I-associated Myelopathy/tropical Spastic Paraparesis from São Paulo, Brazil

Although human T-lymphotropic virus type I (HTLV-I) exhibits high genetic stability, as compared to other RNA viruses and particularly to human immunodeficiency virus (HIV), genotypic subtypes of this human retrovirus have been characterized in isolates from diverse geographical areas. These are currently believed not to be associated with different pathogenetic outcomes of infection. The present study aimed at characterizing genotypic subtypes of viral isolates from 70 HTLV-I-infected individuals from São Paulo, Brazil, including 42 asymptomatic carriers and 28 patients with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), using restricted fragment length polymorphism (RFLP) analysis of long terminal repeat (LTR) HTLV-I proviral DNA sequences. Peripheral blood mononuclear cell lysates were amplified by nested polymerase chain reaction (PCR) and amplicons submitted to enzymatic digestion using a panel of endonucleases. Among HTLV-I asymptomatic carriers, viral cosmopolitan subtypes A, B, C and E were identified in 73.8%, 7.1%, 7.1% and 12% of tested samples, respectively, whereas among HAM/TSP patients, cosmopolitan A (89.3%), cosmopolitan C (7.1%) and cosmopolitan E (3.6%) subtypes were detected. HTLV-I subtypes were not statistically significant associated with patients' clinical status. We also conclude that RFLP analysis is a suitable tool for descriptive studies on the molecular epidemiology of HTLV-I infections in our environment.

Prevalence of hepatitis C Virus infection among hemophiliacs in Central Brazil

In order to investigate the hepatitis C virus (HCV) infection prevalence and risk factors in hemophiliacs in Central Brazil, 90 patients were interviewed and serum samples tested for HCV RNA and anti-HCV antibodies. An overall prevalence of 63.3% (CI 95%: 53.0-72.7) was found. Multivariate analysis of risk factors showed that number of blood transfusions was significantly associated with this infection. Most hemophiliacs received locally produced cryoprecipitate. All infected patients were transfused before the screening of blood units for anti-HCV. However, hemophiliacs who received exclusively screened cryoprecipitate were HCV negative. It confirms the expected decline in transfusion-acquired hepatitis C.

Hepatitis C and hepatitis B virus infection in different hemodialysis units in Belo Horizonte, Minas Gerais, Brazil

The prevalence, virological and epidemilogical aspects of the hepatitis C virus (HCV) and the hepatitis B virus (HBV) infections vary among hemodialysis patients in different countries. Aiming at analyzing these aspects of HCV and HBV infections in hemodialysis patients in Belo Horizonte, MG, Brazil, we studied three hemodialysis units including 434 patients. Serology was used to detect anti-HCV and HBsAg. Reverse trancriptase nested polymerase chain reaction (RT-nested-PCR) of the 5'-noncoding region was used to detect circulating HCV RNA and restriction fragment length polymorphism analysis for genotyping. Seroprevalence varied from 26.5% to 11.1% for hepatitis C and from 5.9% to 0% for hepatitis B. Risk factors observed for HBV and/or HCV infections were the number of patients per dialysis unit, duration of treatment, number of clinics attended, number of blood units transfused, and lower level scholarity. Alanine aminotransferase levels were altered with a higher frequency in HBV or HCV seropositive patients. Half of ten patients, negative for anti-HCV, had detectable viremia by RT-nested-PCR, indicating that this technique should be used to confirm infections in this group of patients. The HCV genotype 1 was the most frequently observed, followed by the genotype 2, but no correlation was detected between genotype and clinical or epidemiological data.

Prevalence and genotypes of GB Virus C/Hepatitis G virus among blood donors in Central Brazil

A survey was conducted in a blood donor population of Central Brazil aiming to investigate the prevalence of GB virus C (GBV-C)/hepatitis G virus (HGV) infection and also to analyze the virus genotypes distribution. A total of 241 voluntary blood donors were interviewed at the State Blood Bank in Goiânia, State of Goiás, Brazil. Blood samples were collected and serum samples tested for GBV-C/HGV RNA by polymerase chain reaction. Genotypes were determined by restriction fragment length polymorphism (RFLP) analysis. Seventeen samples were GBV-C/HGV RNA-positive, resulting in a prevalence of 7.1% (95% CI: 4.2-11.1). A significant trend of GBV-C/HGV RNA positivity in relation to age was observed, with the highest prevalence in donors between 29-39 years old. Ten infected individuals were characterized by reporting parenteral (30%), sexual (18%), both (6%) and intrafamiliar (6%) transmission. However, 7 (40%) GBV-C/HGV RNA-positive donors did not mention any potential transmission route. RFLP analysis revealed the presence of genotypes 1 and 2 of GBV-C/HGV; more precisely, 10 (58.9%) samples were found belonging to the 2b subtype, 4 (23.5%) to the 2a subtype, and 3 (17.6%) to genotype 1. The present data indicate an intermediate endemicity of GBV-C/HGV infection among this blood donor population, and a predominant circulation of genotype 2 (subtype 2b) in Central Brazil.

Human immunodeficiency virus type 1 Brazilian subtype B variant showed an increasing avidity of the anti-V3 antibodies over time compared to the subtype B US/European strain in São Paulo, Brazil

The Brazilian variant of human immunodeficiency virus type 1 (HIV-1) subtype B, (serotype B"-GWGR), has a tryptophan replacing the proline in position 328 the HIV-1 envelope. A longer median time period from infection to acquired immunodeficiency syndrome (AIDS) for serotype B (B"-GWGR) infected subjects compared to the B-GPGR US/European strain was reported. In a cohort study, in São Paulo city, 10 B"-GWGR patients had a statistically significant increased avidity of the anti-V3 antibodies, from 79% ± 33% to 85% ± 75%, versus from 48% ± 59% to 32% ± 17% for the 10 B-GPGR subjects (p = 0.02). The T CD4+ cells showed a mean increase of + 0.45 cells/month for the B-GPGR subjects and for B"-GWGR the slope was + 1.24 cells/month (p = 0.06), for 62 and 55 months of follow up, respectively. RNA plasma viral load decreased from 3.98 ± 1.75 to 2.16 ± 1.54 log10 in the B"-GWGR group while B-GPGR patients showed one log10 reduction in viral load from 4.09 ± 0.38 to 3.17 ± 1.47 log10 over time (p = 0.23), with a decreasing slope of 0.0042 ± log10,/month and 0.0080 ± log10/month, for B-GPGR and B"-GWGR patients, respectively (p = 0.53). Neither group presented any AIDS defining events during the study, according to Center for Diseases Control criteria. Although the sample size is small, these results may indicate that differences in the pathogenicity of the 2 HIV-1 B serotypes which co-circulate in Brazil may be correlated to the avidity of anti-V3 antibodies.

GB virus C/hepatitis G virus infection in dialysis patients and kidney transplant recipients in central Brazil

In order to investigate the prevalence of GB virus C (GBV-C)/hepatitis G virus (HGV) infection in dialysis patients and kidney transplant recipients in Central Brazil and also to analyze the virus genotypes distribution, a total of 123 patients including 98 on hemodialysis, 13 on continuous ambulatory peritoneal dialysis treatment, and 12 who received kidney transplantation were interviewed in one unit of dialysis treatment in Goiânia city. Blood samples were collected and serum samples tested for GBV-C/HGV RNA by polymerase chain reaction. Genotypes were determined by restriction fragment length polymorphism (RFLP) analysis. Eighteen samples were GBV-C/HGV RNA-positive, resulting in an overall prevalence of 14.6% (95% CI: 9.2-21.7). A high positivity for GBV-C/HGV RNA was observed in patients who had received kidney transplant (16.7%), followed by those on hemodialysis (15.3%), and peritoneal dialysis (7.7%). RFLP analysis revealed the presence of genotypes 1, 2, and 3 of GBV-C/HGV; more precisely, 9 (50%) samples were found belonging to the 2b subtype, 4 (22%) to the 2a subtype, 3 (17%) to genotype 1, and 2 (11%) to genotype 3. The present data indicate an intermediate prevalence of GBV-C/HGV infection among dialysis patients and kidney transplant recipients in Central Brazil. Genotype 2 (subtype 2b) seems to be the most prevalent GBV-C/HGV genotype in our region.

Hepatitis C virus genotypes in blood donors from the Federal District, Central Brazil

The objective of this study was to characterize hepatitis C virus (HCV) genotypes in blood donors from the Federal District, Central Brazil, and to compare HCV screening by serological assays and reverse transcriptase polymerase chain reaction (RT-PCR). Plasma samples from 57 individuals with reactive or indeterminate results in serological anti-HCV screening assays (ELISA or EIA) were tested for HCV RNA by RT-PCR. The results from a confirmatory LIA serological assay were also evaluated. The 5' non-coding region of the HCV genome was amplified from 41 PCR positive samples (71.9%), which were further characterized by nucleotide sequencing analysis. Of these, 60.9% were of HCV genotype 1 and 39.1% of genotype 3.

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.

Prevalence and risk factors of hepatitis C virus infection in hemodialysis patients from one center in Recife, Brazil

A hemodialysis population from a dialysis unit in the city of Recife, Northeastern Brazil, was screened to assess the prevalence of hepatitis C virus (HCV) infection and to investigate the associated risk factors. Hemodialysis patients (n = 250) were interviewed and serum samples tested for anti-HCV antibodies by enzyme-linked immunosorbent assay (ELISA). All samples were also tested for HCV RNA by reverse transcriptase nested polymerase chain reaction (RT-nested-PCR). Out of 250 patients, 21 (8.4%) were found to be seropositive by ELISA, and 19 (7.6%) patients were HCV RNA positive. HCV viraemia was present in 90.5% of the anti-HCV positive patients. The predominant genotype was HCV 1a (8/19), followed by 3a (7/19), and 1b (4/19). None of the anti-HCV negative patients were shown to be viraemic by the PCR. Univariate analysis of risk factors showed that time spent on hemodialysis, the number of blood transfusions and a blood transfusion before November 1993 were associated with HCV positivity. However, multivariate analysis revealed that blood transfusions before November 1993 were significantly associated with HCV infection in this population. Low prevalence levels were encountered in this center, however prospective studies are necessary to confirm these findings.

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.

Early infection and asymptomatic spread of hepatitis A virus in a public child care center in Rio de Janeiro, Brazil: should attending children under two years of age be vaccinated?

A cross-sectional study was conducted in order to identify hepatitis A virus (HAV) serological markers in 418 individuals (mean age, 16.4 years; range, 1 month-80 years) at a public child care center in Rio de Janeiro, Brazil, as well as to analyze risk factors and determine circulating genotypes. Serum samples were tested using an enzyme immunoassay. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect and characterize HAV RNA, and sequencing was performed. Anti-HAV antibodies and IgM anti-HAV antibodies were detected, respectively, in 89.5% (374/418) and 10.5% (44/418) of the individuals tested. Acute HAV infection in children was independently correlated with crawling (p < 0.05). In 56.8% (25/44) of the IgM anti-HAV-positive individuals and in 33.3% (5/15) of the IgM anti-HAV-negative individuals presenting clinical symptoms, HAV RNA was detected. Phylogenetic analysis revealed co-circulation of subgenotypes IA and IB in 93.3% (28/30) of the amplified samples. In present study, we verify that 79% (30/38) of children IgM anti-HAV-positive were asymptomatic. In child care centers, this asymptomatic spread is a more serious problem, promoting the infection of young children, who rarely show signs of infection. Therefore, vaccinating children below the age of two might prevent the asymptomatic spread of hepatitis A.

Prevalence of hepatitis A virus infection in Goiânia, Goiás, Brazil, by molecular and serological procedures, 1995-2002

In this study, a total of 865 serum samples were collected between 1995 and 2002 from individuals living in Goiânia, Central Brazil, and clinically suspected of hepatitis. After exclusion of 162 samples which were positive for hepatitis B virus or hepatitis C virus, 703 samples were tested for anti-hepatitis A virus (anti-HAV) IgM antibodies by enzyme immunoassay. In addition, 588 of these samples and 22 fecal samples were analyzed by reverse transcription-nested PCR for HAV RNA detection, with positivity indices of 13.1% (77/588) and 54.5% (12/22), respectively. A similar index of viral RNA detection in anti-HAV-IgM positive or negative samples was observed in serum samples. HAV infection is a public health problem worldwide and this study underscores the extent of HAV circulation in our region.

Low prevalence of hepatitis C virus infection in Amerindians from Western Venezuela

Previous studies have not found hepatitis C virus (HCV) infection in Amerindians from Western Venezuela. A survey of 254 Bari and Yukpa natives aged 10-60 years (mean ± SD age = 35 ± 5.4 years) from four communities, two Bari and two Yukpa, in this area were studied to assess the prevalence of antibodies to HCV (anti-HCV) and HCV RNA among these indigenous populations. Serum samples were examined initially for anti-HCV by a four generation enzyme-linked immunosorbent assay (ELISA). Reactive samples were then tested using a third generation recombinant immunoblot assay (RIBA-3). Viral RNA was investigated in all immunoblot-reactive samples by a nested polymerase chain reaction (PCR) method. Six (2.3%) of 254 natives were positive by ELISA, one (2.2%) of these reactive samples were positive by RIBA, and four (1.5%) were indeterminate. Only two (0.8%) were positive by PCR, corresponding to 1 (2.1%) of 47 inhabitants of a Yukpa community and to 1 (2.2%) of 45 subjects of a Bari community. Iatrogenic is thought to play a role in acquisition of the infection. The findings indicate a HCV focus of low endemicity and are compatible with a low degree of exposures of the natives to the virus. Studies are necessary to assess the risk factors for infection in these Amerindians.