Enteric viruses' dissemination in a private reserve of natural heritage.

This study aimed to assess anthropogenic impact of surrounding population in the Private Reserve of Natural Heritage at Pantanal, the world's largest freshwater wetland ecosystem located in the centre of South America. Viral aetiological agents of acute gastroenteritis as rotavirus A (RVA), noroviruses, human adenoviruses, klassevirus and of hepatitis, as hepatitis A virus, were investigated in different aquatic matrices. Annual collection campaigns were carried out from 2009 to 2012, alternating dry and rainy seasons. Viral particles present in the samples were concentrated by the adsorption-elution method, with negatively charged membranes, and detected by qualitative and quantitative PCR. From a total of 43 samples at least one virus was detected in 65% (28) of them. Viruses were detected in all matrices with concentrations ranging from 2 × 102 to 8·3 × 104 genome copies per litre. A significant higher RVA frequency was observed in the dry season. Our data revealing dissemination of human enteric viruses in water matrices both inside and outside the reserve could be useful to trace faecal contamination in the environment and to minimize the risk of infection by exposure of susceptible individuals. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is part of a collaborative project designed to investigate the environmental and health conditions of the Private Reserve of Natural Heritage at Pantanal, the largest seasonally flooded wetland in the world. The project aimed to promote health and quality of human and wildlife extending technical-scientific knowledge about pathogens present in the region. By assessing the occurrence of human enteric viruses in different water matrices we demonstrated the anthropogenic impact of surrounding population and pointed out the potential risk of infection by exposure of susceptible individuals.

Injectable ß-TCP/MCPM cement associated with mesoporous silica for bone regeneration: characterization and toxicity evaluation.

Calcium phosphate cement has been widely investigated as a bone graft substitute due to its excellent self-setting ability, biocompatibility, osteoconductivity and moldability. In addition, mesoporous materials have been studied as potential materials for application in medical devices due to their large surface area, which is capable of loading numerous biological molecules, besides being bioactive. In this study, bone ß-TCP-MCPM-based injectable cement with mesoporous silica particles was synthesized and characterized in terms of its mechanical properties, microstructure, porosity, injectability, in vitro bioactivity and degradability; together with toxicity effects in CHO-K1 cell culture. The results showed that the ß-TCP-MCPM cement is bioactive after soaking in simulated body fluid solution, and mesoporous silica particles provided better physicochemical properties compared with silica-free cement. Toxicity assays showed low CHO-K1 cell viability after treatment with more concentrated extracts (200 mg ml-1). However, this behavior did not compromise the reproductive capacity and did not promote significant DNA damage in those cells. In conclusion, the ß-TCP-MCPM cement associated with mesoporous silica might be considered as a potential bone substitute for the repair and regeneration of bone defects.

Bacterial cellulose-hydroxyapatite nanocomposites for bone regeneration.

The aim of this study was to develop and to evaluate the biological properties of bacterial cellulose-hydroxyapatite (BC-HA) nanocomposite membranes for bone regeneration. Nanocomposites were prepared from bacterial cellulose membranes sequentially incubated in solutions of CaCl(2) followed by Na(2)HPO(4). BC-HA membranes were evaluated in noncritical bone defects in rat tibiae at 1, 4, and 16 weeks. Thermogravimetric analyses showed that the amount of the mineral phase was 40%-50% of the total weight. Spectroscopy, electronic microscopy/energy dispersive X-ray analyses, and X-ray diffraction showed formation of HA crystals on BC nanofibres. Low crystallinity HA crystals presented Ca/P a molar ratio of 1.5 (calcium-deficient HA), similar to physiological bone. Fourier transformed infrared spectroscopy analysis showed bands assigned to phosphate and carbonate ions. In vivo tests showed no inflammatory reaction after 1 week. After 4 weeks, defects were observed to be completely filled in by new bone tissue. The BC-HA membranes were effective for bone regeneration.

Dynamical transition of protein-hydration water.

Thin layers of water on biomolecular and other nanostructured surfaces can be supercooled to temperatures not accessible with bulk water. Chen et al. [Proc. Natl. Acad. Sci. U.S.A. 103, 9012 (2006)]10.1073/pnas.0602474103 suggested that anomalies near 220 K observed by quasielastic neutron scattering can be explained by a hidden critical point of bulk water. Based on more sensitive measurements of water on perdeuterated phycocyanin, using the new neutron backscattering spectrometer SPHERES, and an improved data analysis, we present results that show no sign of such a fragile-to-strong transition. The inflection of the elastic intensity at 220 K has a dynamic origin that is compatible with a calorimetric glass transition at 170 K. The temperature dependence of the relaxation times is highly sensitive to data evaluation; it can be brought into perfect agreement with the results of other techniques, without any anomaly.

Kinetics of hepatitis A virus replication in vivo and in vitro using negative-strand quantitative PCR.

The replication of hepatitis A virus (HAV) is via a complementary negative-strand RNA. Each negative strand may serve as a template for the synthesis of many positive strands. The aim of this study was to detect the intermediate replicative (negative strand) of HAV in order to monitor its replication in vitro and in vivo. Real-time polymerase chain reaction (PCR) was standardized to detect the intermediate replicative of HAV in cell culture and liver from non-human primates infected experimentally. HAV primers from the 5' non-translated region and VP3 were used in the cDNA synthesis of negative-strand RNA. The negative strand was detected in the infected cell lines and liver by highly strand-specific rTth recombinant Thermus thermophilus DNA polymerase reverse transcription followed by quantitative PCR. The results indicate that the negative-strand HAV RNA can be detected in vivo and in vitro. This model is an approach for assessing the dynamic patterns of replication and should represent a valuable tool for the monitoring of HAV replications in cell cultures and for the evaluation of experimental infections in animal models.

Fixation of autogenous bone grafts with ethyl-cyanoacrylate glue or titanium screws in the calvaria of rabbits.

This study compared the fixation of autogenous onlay bone grafts with cyanoacrylate glue (Super Bonder) and with titanium screws. Twenty rabbits underwent bilateral parietal ostectomies. Bone segments were fixed anteriorly to the resulting bone defect. In group I, the grafts were fixed with 4 mm long, 1.5 mm diameter screws; in group II, adhesive was used. The animals were killed after 5, 15, 30, 60 and 120 days. Histomorphometric analysis was used to quantify the maintenance of the graft area. Discrete areas of inflammatory reaction were seen in both groups after 5 days and for group II after 15 days. After 30 days, new bone formation was seen at the interface of the grafts. After 120 days, the graft was incorporated into the host bed in group I and partially incorporated in group II. There was a significant statistical difference regarding the mean graft areas between 15 and 120 days (p<0.001) and between fixation methods (p<0.002). Fixation with adhesive promoted a significantly greater area of bone graft than screw fixation, independent of time period. The adhesive was biocompatible, presented similar stability to the screw and maintained the bone area, although there was a delay in graft incorporation.

Changing epidemiology of hepatitis A in Brazil: reassessing immunization policy.

Recent studies have shown that the prevalence of antibody to hepatitis A virus (HAV) is decreasing in several Latin American countries. Brazil is a very large and heterogeneous country, showing striking regional differences. With regard to sanitary facilities, 81.7% of the districts in the south-eastern region have sewage systems, compared with only 5.8% in the northern region. Results of sero-epidemiological studies and reported hepatitis A outbreaks indicate a change in the epidemiological pattern of hepatitis A in the country. Individuals, especially those under the age of 10, are mostly unprotected from HAV infection, regardless of their socioeconomic status. During 2000-2005, approximately 14 000-21 000 cases of hepatitis A were reported annually in Brazil, a rate of 7.5-11 cases per 100 000 population. Nationwide, hepatitis A mortality rates declined progressively from 1980 to 2002. As fatal cases constitute a small, but predictable, portion of all acute hepatitis A cases, which are in turn part of the total number of HAV infections, these data suggest that there has been a decline in HAV circulation in all Brazilian regions over the last two decades. Taken together these facts point out that the epidemiological pattern of hepatitis A is changing in Brazil. Besides improvements in sanitary conditions in the poorest Brazilian regions, the introduction of hepatitis A vaccination of young children could be a strategy for controlling HAV infection in the country.

Isoleucine and valine supplementation of a low-protein corn-wheat-soybean meal-based diet for piglets: growth performance and nitrogen balance.

The effects of Ile and Val supplementation of a low-CP, corn-wheat-soybean meal-based piglet diet on growth performance, incidence of diarrhea, and N balance were studied using 60 Landrace x Duroc male piglets in a 4-wk experiment. The 60 individually caged piglets were divided into 5 dietary treatments, each consisting of 12 piglets. Diet 1 was a positive control diet (20% CP); diet 2 was a low-CP negative control diet (17% CP); diets 3, 4, and 5 were low-CP diets to which Ile, Val, or the combination of Ile and Val were added, respectively. All diets were supplemented with Lys, Met, Thr, and Trp to provide the required concentrations of these AA according to the 1998 NRC. Average daily gain and ADFI were similar among pigs fed the positive control, Val-added, and the Val plus Ile-added diets. On wk-2 and wk-4, fecal score was greater (softer feces) in piglets fed the 20% CP level compared with the remaining treatments (P < 0.01). Nitrogen intake was decreased (P < 0.0001) in pigs fed diets containing low levels of CP compared with pigs fed the 20% CP diet. Fecal N excretion (g/d) was decreased (P < 0.05) in piglets fed low-CP diets at wk 1 and wk 4 of feeding, and in urine at wk 4 of feeding. Crude protein levels or AA supplementation had no effect on N retention efficiencies. These results indicate that the supplementation of Val alone, or in combination with Ile, to a low-CP piglet diet with adequate levels of Lys, Met, Thr, and Trp is necessary to achieve maximum performance in pigs consuming corn-wheat-soybean meal-based diets.

Changes in hepatitis A virus seroepidemiology in HIV-infected Brazilian patients.

Shifting of hepatitis A virus (HAV) epidemiology from a high towards an intermediate endemicity pattern and use of antiretroviral therapy increased the risk of HIV/HAV coinfection in developing countries. The aim of this study was to investigate the presence of HAV markers in a cohort of HIV-infected patients from 1988 to 2004. The presence of serum anti-HAV antibodies and HAV-RNA by real-time polymerase chain reaction was investigated in 581 patients. Total anti-HAV antibodies was found in 464/581 (79.8%) patients, however, a changing epidemiologic pattern of hepatitis A among HIV-infected patients from 1988 to 2004 was observed. Among patients susceptible to HAV (n = 117), 5 (4.2%) were coinfected with HAV, all of them had IgM anti-HAV antibodies and were serum HAV-RNA-positive. The high prevalence of anti-HAV antibodies in HIV-infected patients suggests that screening tests for anti-HAV antibodies should be performed before implementation of hepatitis A vaccination, especially in those patients from endemic countries.

Dynamics of well-folded and natively disordered proteins in solution: a time-of-flight neutron scattering study.

Casein proteins belong to the class of natively disordered proteins. The existence of disordered biologically active proteins questions the assumption that a well-folded structure is required for function. A hypothesis generally put forward is that the unstructured nature of these proteins results from the functional need of a higher flexibility. This interplay between structure and dynamics was investigated in a series of time-of-flight neutron scattering experiments, performed on casein proteins, as well as on three well-folded proteins with distinct secondary structures, namely, myoglobin (alpha), lysozyme (alpha/beta) and concanavalin A (beta). To illustrate the subtraction of the solvent contribution from the scattering spectra, we used the dynamic susceptibility spectra emphasizing the high frequency part of the spectrum, where the solvent dominates. The quality of the procedure is checked by comparing the corrected spectra to those of the dry and hydrated protein with negligible solvent contamination. Results of spectra analysis reveal differences in motional amplitudes of well-folded proteins, where beta-sheet structures appear to be more rigid than a cluster of alpha-helices. The disordered caseins display the largest conformational displacements. Moreover their global diffusion rates deviate from the expected dependence, suggesting further large-scale conformational motions.

Molecular detection of hepatitis A virus in urban sewage in Rio de Janeiro, Brazil.

AIMS: A one-year survey was conducted to examine hepatitis A virus (HAV) prevalence, distribution of genotypes and their relationship to bacterial indicators in raw and treated sewage samples. METHODS AND RESULTS: Fifty sewage samples (raw = 25 and treated = 25) were collected twice monthly from one sewage treatment plant in Rio de Janeiro. Virus concentration was performed by adsorption to an electronegative membrane followed by ultrafiltration. Viral RNA was detected by nested reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time PCR and positive products were directly sequenced. Total and faecal coliform concentrations were also determined. By nested RT-PCR, HAV RNA was detected in 16/50 (32%) and eight (16%) of them were found in treated sewage samples. By real-time PCR, HAV RNA was detected in 46/50 (92%) samples and 24 were from treated sewage. Phylogenetic analyses classified nine isolates (56%) as subgenotype IA and seven (44%) as IB. CONCLUSIONS: Real-time PCR was more sensitive than nested RT-PCR; the presence of subgenotypes IA and IB was described and bacterial indicators cannot be used to predict HAV presence in sewage. SIGNIFICANCE AND IMPACT OF THE STUDY: These results demonstrated that HAV still remains in the environment after sewage treatment and could play an important role in maintaining the endemicity of HAV infection.

Hepatitis A virus in environmental water samples from the Amazon Basin.

Hepatitis A virus (HAV) is a significant waterborne human pathogen. Of the global supply of potable water, Brazil retains 13%, of which 75% resides in the Amazon Basin. Although hepatitis A morbidity has declined progressively in Brazil as a whole, it remains high in the Amazon region. We used nested and real-time reverse-transcription polymerase chain reaction (RT-PCR) to detect and quantify the viral load in water samples from the Amazon Basin. Most samples tested positive (92%), with viral loads varying from 60 to 5500 copies /L, depending on sanitary conditions and the degree of flooding. Nested RT-PCR of the VP1-2A region detected HAV RNA in 23% of the samples. In low viral load samples, HAV was detected only with real-time RT-PCR, suggesting that this technique is useful for monitoring HAV contamination. The presence of HAV in water samples constitutes a serious public health problem.

Co-circulation of genotypes IA and IB of hepatitis A virus in Northeast Brazil.

The Northeast region is the location of most cases of acute hepatitis A virus (HAV) in Brazil. In the present study, the genotypes of HAV strains from Pernambuco State, one of most populous states in the Northeast region, were characterized. Blood samples positive for anti-HAV IgM from 145 individuals (mean age = 29.1 years), collected during 2002 and 2003, were submitted to nested RT-PCR for amplification of the 5'non-translated region (5'NTR) and VP1/2A regions of the HAV genome. The VP1/2A and 5'NTR regions were amplified in 39 and 21% of the samples, respectively. Nucleotide sequencing was carried out in 46% of VP1/2A and in 53% of 5'NTR isolates. The identity in nucleotide sequence of the VP1/2A region ranged from 93.6 to 100.0%. Phylogenetic analysis of the VP1/2A sequences showed that 65% belong to sub-genotype IA and 35% to sub-genotype IB. Co-circulation of both sub-genotypes was observed in the two years studied. Distinct clusters of highly related sequences were observed in both sub-genotypes, suggesting endemic circulation of HAV strains in this area. In the 5'NTR isolates, 92.7-99.2% identity was observed and two isolates presented one deletion at position 413. Phylogenetic analysis showed that genotype IA strains cluster in the tree in the same way as genotype IB strains, but one IIIA isolate from Spain clusters with genotype IB strains. These results do not allow us to state that 5'NTR could be used to genotype HAV sequences. This is the first report of co-circulation of sub-genotypes IA and IB in this region, providing additional information about the molecular epidemiology of HAV strains in Brazil.

Co-circulation of genotypes IA and IB of hepatitis A virus in Northeast Brazil

The Northeast region is the location of most cases of acute hepatitis A virus (HAV) in Brazil. In the present study, the genotypes of HAV strains from Pernambuco State, one of most populous states in the Northeast region, were characterized. Blood samples positive for anti-HAV IgM from 145 individuals (mean age = 29.1 years), collected during 2002 and 2003, were submitted to nested RT-PCR for amplification of the 5'non-translated region (5'NTR) and VP1/2A regions of the HAV genome. The VP1/2A and 5'NTR regions were amplified in 39 and 21 percent of the samples, respectively. Nucleotide sequencing was carried out in 46 percent of VP1/2A and in 53 percent of 5'NTR isolates. The identity in nucleotide sequence of the VP1/2A region ranged from 93.6 to 100.0 percent. Phylogenetic analysis of the VP1/2A sequences showed that 65 percent belong to sub-genotype IA and 35 percent to sub-genotype IB. Co-circulation of both sub-genotypes was observed in the two years studied. Distinct clusters of highly related sequences were observed in both sub-genotypes, suggesting endemic circulation of HAV strains in this area. In the 5'NTR isolates, 92.7-99.2 percent identity was observed and two isolates presented one deletion at position 413. Phylogenetic analysis showed that genotype IA strains cluster in the tree in the same way as genotype IB strains, but one IIIA isolate from Spain clusters with genotype IB strains. These results do not allow us to state that 5'NTR could be used to genotype HAV sequences. This is the first report of co-circulation of sub-genotypes IA and IB in this region, providing additional information about the molecular epidemiology of HAV strains in Brazil.

Genetic variability of hepatitis A virus isolates in Rio de Janeiro: implications for the vaccination of school children

The epidemiology of hepatitis A virus (HAV) infection is shifting from high to intermediate endemicity in Brazil, resulting in increased numbers of susceptible individuals and a greater potential for the emergence of outbreaks. Universal vaccination against HAV has been recommended for children, but updated sero-epidemiological data are necessary to analyze the level of natural immunity and to identify candidates for preventive measures. In addition, more molecular studies are necessary to characterize the genotypes involved in HAV infections and outbreaks. Sera from 299 school children (5-15 years old) and 25 school staff members, collected during an outbreak of HAV at a rural public school in June 2000, were tested for IgM and total anti-HAV antibodies (ELISA). Viral RNA was amplified by RT-PCR from anti-HAV IgM-positive sera and from 19 fecal samples. Direct nucleotide sequencing of the VP1/2A region was carried out on 18 PCR-positive samples. Acute HAV infection was detected by anti-HAV IgM in 93/299 children and in 3/25 adult staff members. The prevalence of total anti-HAV antibodies in IgM-negative children under 5 years of age was only 10.5 percent. HAV-RNA was detected in 46 percent IgM-positive serum samples and in 16 percent stool samples. Sequence analysis showed that half the isolates belonged to subgenotype IA and the other half to IB. On the basis of these data, mass vaccination against HAV is recommended without prevaccination screening, especially for children before they enter school, since nearly 90 percent of the children under 5 years were susceptible. Molecular characterization indicated the endemic circulation of specific HAV strains belonging to subgenotypes IA and IB.

Genetic variability of hepatitis A virus isolates in Rio de Janeiro: implications for the vaccination of school children.

The epidemiology of hepatitis A virus (HAV) infection is shifting from high to intermediate endemicity in Brazil, resulting in increased numbers of susceptible individuals and a greater potential for the emergence of outbreaks. Universal vaccination against HAV has been recommended for children, but updated sero-epidemiological data are necessary to analyze the level of natural immunity and to identify candidates for preventive measures. In addition, more molecular studies are necessary to characterize the genotypes involved in HAV infections and outbreaks. Sera from 299 school children (5-15 years old) and 25 school staff members, collected during an outbreak of HAV at a rural public school in June 2000, were tested for IgM and total anti-HAV antibodies (ELISA). Viral RNA was amplified by RT-PCR from anti-HAV IgM-positive sera and from 19 fecal samples. Direct nucleotide sequencing of the VP1/2A region was carried out on 18 PCR-positive samples. Acute HAV infection was detected by anti-HAV IgM in 93/299 children and in 3/25 adult staff members. The prevalence of total anti-HAV antibodies in IgM-negative children under 5 years of age was only 10.5%. HAV-RNA was detected in 46% IgM-positive serum samples and in 16% stool samples. Sequence analysis showed that half the isolates belonged to subgenotype IA and the other half to IB. On the basis of these data, mass vaccination against HAV is recommended without prevaccination screening, especially for children before they enter school, since nearly 90% of the children under 5 years were susceptible. Molecular characterization indicated the endemic circulation of specific HAV strains belonging to subgenotypes IA and IB.

In situ enzyme immunoassay for titration of a Brazilian hepatitis A virus strain (HAF-203).

Hepatitis A virus (HAV) replicates relatively slowly in cell culture without a cytopathic effect, a fact that limits the use of tissue culture assays. The radioimmunofocus assay is the standard method for HAV titration, although it is labor intensive and requires the use of radioisotopes. A simple, rapid and objective infectivity assay based on an in situ enzyme immunoassay (EIA) is described here for a Brazilian cell culture-adapted HAV strain (HAF-203). The assay uses a peroxidase-labeled polyclonal antibody to fixed monolayers as an indicator of infection. EIA may be completed within 7 days using serial 5-fold dilutions of the virus, yielding a titer of 5.024 log 50% tissue culture infective dose (TCID50)/ml for HAF-203. This technique had a detection limit of 1.1 log TCID50/ml and the specificity was demonstrated by detecting no reaction on the columns of uninfected wells. The reproducibility (with intra- and inter-assay coefficients of variation ranging from 1.9 to 3.8% and from 3.5 to 9.9%, respectively) and quantitation of the assay were demonstrated by close agreement in virus infectivity titers among different assays of the same amount of virus and between assays of different amounts of virus. Furthermore, this assay does not require the use of radiolabeled antibodies. We describe here an efficient EIA that is highly reproducible and that could be used to monitor HAV growth in cell culture and to determine the quantity of HAV antigen needed for diagnostic assays. This is the first report of the infectious titer of the Brazilian cell culture-adapted HAV strain (HAF-203).

In situ enzyme immunoassay for titration of a Brazilian hepatitis A virus strain (HAF-203)

Hepatitis A virus (HAV) replicates relatively slowly in cell culture without a cytopathic effect, a fact that limits the use of tissue culture assays. The radioimmunofocus assay is the standard method for HAV titration, although it is labor intensive and requires the use of radioisotopes. A simple, rapid and objective infectivity assay based on an in situ enzyme immunoassay (EIA) is described here for a Brazilian cell culture-adapted HAV strain (HAF-203). The assay uses a peroxidase-labeled polyclonal antibody to fixed monolayers as an indicator of infection. EIA may be completed within 7 days using serial 5-fold dilutions of the virus, yielding a titer of 5.024 log 50 percent tissue culture infective dose (TCID50)/ml for HAF-203. This technique had a detection limit of 1.1 log TCID50/ml and the specificity was demonstrated by detecting no reaction on the columns of uninfected wells. The reproducibility (with intra- and inter-assay coefficients of variation ranging from 1.9 to 3.8 percent and from 3.5 to 9.9 percent, respectively) and quantitation of the assay were demonstrated by close agreement in virus infectivity titers among different assays of the same amount of virus and between assays of different amounts of virus. Furthermore, this assay does not require the use of radiolabeled antibodies. We describe here an efficient EIA that is highly reproducible and that could be used to monitor HAV growth in cell culture and to determine the quantity of HAV antigen needed for diagnostic assays. This is the first report of the infectious titer of the Brazilian cell culture-adapted HAV strain (HAF-203).

Detection of hepatitis A virus RNA in serum during the window period of infection.

BACKGROUND: Hepatitis A virus (HAV) infection is the leading cause of clinically apparent viral hepatitis in many parts of the world, including developed and developing countries. Only limited information is available regarding the seronegative viremic window that follows HAV infection, and no systematic search has been reported for HAV RNA positive, IgM anti-HAV negative serum samples during hepatitis A outbreaks. OBJECTIVES: To determine the proportion of HAV infected individuals among (i) children who were tested negative for anti-HAV antibodies during hepatitis A outbreaks which occurred in a public school (n = 157) and a child care center (n = 38); (ii) subjects (n = 46) initially classified as acute non-A-C hepatitis patients after clinical examination and serological tests (sporadic cases). STUDY DESIGN: Reverse transcription (RT)-PCR was performed to detect the presence of HAV genome in serum samples collected from anti-HAV negative, susceptible subjects. RESULTS: HAV RNA was detected in 19/157 (12%) and 5/38 (13%) anti-HAV negative children from the public school and child care center, respectively. Twelve (26%) out of the 46 acute hepatitis patients (sporadic cases) were also HAV RNA positive. From nine of these 12 patients, a second blood sample was obtained 18-34 days after the first one: all nine had seroconverted to IgM anti-HAV, and their serum transaminases had reached elevated levels (mean ALT, 418; mean AST, 241). CONCLUSIONS: Detection of HAV RNA before IgM anti-HAV seroconversion may be used as an early diagnosis method during hepatitis A outbreaks. HAV RNA testing should also help to elucidate acute hepatitis cases of unknown etiology.

Hepatitis A virus infection in hepatitis C Brazilian patients.

OBJECTIVE: HAV infection in patients with pre-existing chronic liver disease has been associated with increased rate of fulminant hepatitis and mortality. The aim of this study was to investigate the presence of serological and molecular HAV markers in a population of HCV infected patients. PATIENTS AND METHODS: The presence of total and IgM anti-HAV antibodies was investigated in 197 patients (mean age 44.8+/-12.5 years) referred to the Brazilian Reference Center for Viral Hepatitis and who tested positive for anti-HCV antibodies and HCV RNA. HAV RNA was investigated by reverse transcription-nested PCR in these patients.Results. One hundred seventy patients (86%) had total, but not IgM anti-HAV antibodies, being therefore, immune to hepatitis A, while 27 (14%) were not. A high proportion (6/27, 22%) of the susceptible patients presented markers of recent HAV infection: One patient was IgM anti-HAV positive, three were HAV RNA positive, and two presented both markers. By nucleotide sequencing, it was demonstrated that the HAV isolates infecting these patients belonged to subgenotypes 1A and 1B. CONCLUSIONS: Superinfection with HAV was a common event in the group of HCV infected patients under study. Implementation of hepatitis A vaccination should be considered for this population.