Detection of enteric viruses in recreational waters of an urban lagoon in the city of Rio de Janeiro, Brazil

The aim of this study was to determine the occurrences of the group A rotavirus (RVA), norovirus (NoV) and human adenovirus (HAdV) in the surface waters of an urban lagoon (Rodrigo de Freitas Lagoon) in the city of Rio de Janeiro, Brazil. During one year of surveillance, water samples were obtained from the lagoon and other interconnected ecosystems (river and beach). The samples were concentrated using an adsorption-elution method with a negatively charged membrane and tested by qualitative and quantitative polymerase chain reaction assays. RVA was the most prevalent virus detected (24.3%) with a viral load ranging from 3.0 x 10¹-5.6 x 10(4) genome copies/L, followed by NoV (18.8%) and HAdV (16.7%). Considering water samples suitable for bathing, according to Escherichia coli criterion (< 2,000 most probable number/100 mL), viruses were detected in 50% (57/114) of them. Physicochemical parameters were also measured and showed possible correlations between turbidity and RVA presence and between pH and NoV presence. These data demonstrate the importance of considering viral parameters to ensure water quality and the utilisation of these parameters as additional tools for the characterisation of environmental contamination.

The efficiency of concentration methods used to detect enteric viruses in anaerobically digested sludge

The presence of enteric viruses in biosolids can be underestimated due to the inefficient methods (mainly molecular methods) used to recover the viruses from these matrices. Therefore, the goal of this study was to evaluate the different methods used to recover adenoviruses (AdV), rotavirus species A (RVA), norovirus genogroup II (NoV GII) and the hepatitis A virus (HAV) from biosolid samples at a large urban wastewater treatment plant in Brazil after they had been treated by mesophilic anaerobic digestion. Quantitative polymerase chain reaction (PCR) was used for spiking experiments to compare the detection limits of feasible methods, such as beef extract elution and ultracentrifugation. Tests were performed to detect the inhibition levels and the bacteriophage PP7 was used as an internal control. The results showed that the inhibitors affected the efficiency of the PCR reaction and that beef extract elution is a suitable method for detecting enteric viruses, mainly AdV from biosolid samples. All of the viral groups were detected in the biosolid samples: AdV (90%), RVA, NoV GII (45%) and HAV (18%), indicating the viruses' resistance to the anaerobic treatment process. This is the first study in Brazil to detect the presence of RVA, AdV, NoV GII and HAV in anaerobically digested sludge, highlighting the importance of adequate waste management.

Acute gastroenteritis and enteric viruses in hospitalised children in southern Brazil: aetiology, seasonality and clinical outcomes

Viral acute gastroenteritis (AG) is a significant cause of hospitalisation in children younger than five years. Group A rotavirus (RVA) is responsible for 30% of these cases. Following the introduction of RVA immunisation in Brazil in 2006, a decreased circulation of this virus has been observed. However, AG remains an important cause of hospitalisation of paediatric patients and only limited data are available regarding the role of other enteric viruses in these cases. We conducted a prospective study of paediatric patients hospitalised for AG. Stool samples were collected to investigate human adenovirus (HAdV), RVA, norovirus (NoV) and astrovirus (AstV). NoV typing was performed by nucleotide sequencing and phylogenetic analysis. From the 225 samples tested, 60 (26%) were positive for at least one viral agent. HAdV, NoV, RVA and AstV were detected in 16%, 8%, 6% and 0% of the samples, respectively. Mixed infections were found in nine patients: HAdV/RVA (5), HAdV/NoV (3) and HAdV/NoV/RVA (1). The frequency of fever and lymphocytosis was significantly higher in virus-infected patients. Phylogenetic analysis of NoV indicated that all of these viruses belonged to genotype GII.4. The significant frequency of these pathogens in patients with AG highlights the need to routinely implement laboratory investigations.

Comparative study of two extraction methods for enteric virus recovery from sewage sludge by molecular methods

The aim of this study was to compare two nucleic acid extraction methods for the recovery of enteric viruses from activated sludge. Test samples were inoculated with human adenovirus (AdV), hepatitis A virus (HAV), poliovirus (PV) and rotavirus (RV) and were then processed by an adsorption-elution-precipitation method. Two extraction methods were used: an organic solvent-based method and a silica method. The organic-based method was able to recoup 20% of the AdV, 90% of the RV and 100% of both the PV and HAV from seeded samples. The silica method was able to recoup 1.8% of the AdV and 90% of the RV. These results indicate that the organic-based method is more suitable for detecting viruses in sewage sludge.

A non-enteric adenovirus A12 gastroenteritis outbreak in Rio de Janeiro, Brazil

A gastroenteritis outbreak that occurred in 2013 in a low-income community in Rio de Janeiro was investigated for the presence of enteric viruses, including species A rotavirus (RVA), norovirus (NoV), astrovirus (HAstV), bocavirus (HBoV), aichivirus (AiV), and adenovirus (HAdV). Five of nine stool samples (83%) from patients were positive for HAdV, and no other enteric viruses were detected. Polymerase chain reaction products were sequenced and subjected to phylogenetic analysis, which revealed four strains and one strain of non-enteric HAdV-A12 and HAdV-F41, respectively. The HAdV-A12 nucleotide sequences shared 100% nucleotide similarity. Viral load was assessed using a TaqMan real-time PCR assay. Stool samples that were positive for HAdV-A12 had high viral loads (mean 1.9 X 107 DNA copies/g stool). All four patients with HAdV-A12 were < 25 months of age and had symptoms of fever and diarrhoea. Evaluation of enteric virus outbreaks allows the characterisation of novel or unique diarrhoea-associated viruses in regions where RVA vaccination is routinely performed.

The epidemiology of acute viral gastroenteritis in hospitalized children in Cordoba city, Argentina: an insight of disease burden

Information concerning the disease burden of viral gastroenteritis has important implications for the use and monitoring the impact of public health policies. The present study, carried out in Córdoba city, Argentina, documents the epidemiology of severe viral diarrhea as well as the burden of viral gastrointestinal disease in the hospital children admission. A total of 133 stools were collected from hospitalized children (Town Childhood Hospital) suffering from acute diarrhea and studied for the presence of Group A rotavirus, astrovirus and adenovirus 40/41 by enzyme-immuno assay, between November 1997 and October 1998. Enteric viruses accounted for 42.1% of the total diarrheal cases analyzed. Group A rotaviruses, astroviruses, adenoviruses 40/41 and mixed infections were found in 35.3, 4.5, 1.5, and 0.8% studied specimens respectively. We estimated that 1 in 27 children in the 0-35 month-old cohort/range would be annually hospitalized for a viral gastroenteritis illness. The major impact on viral diarrhea lies on rotaviral infection, accouting for 84.0% of the viral diarrheal cases analyzed and for approximately one third of severe diarrheas requiring hospital admission in Córdoba City, Argentina.

Echovirus 4 associated to hand, foot and mouth disease

Hand, foot and mouth disease (HFMD) is a contagious enteroviral infection occurring primarily in children and characterized by vesicular palmoplantar eruptions and erosive stomatitis. Echovirus 4 (EV-4) has been commonly associated with aseptic meningitis. The association of HFMD with EV-4 has not been reported previously. Two samples of a 14-month child who presented mild fever, sores in the mouth, rash with blisters on the palm of hands and soles of feet were sent to Enteric Viruses Laboratory of Adolfo Lutz Institute. Clinical samples were inoculated in three different cell lines, and those which presented cytopathic effect (CPE), were submitted to Indirect Immunofluorescence Assay (IFA) and "one step" RT-PCR. Agarose gel electrophoresis from RT-PCR product, showed a product with 437 bp, which is characteristic of Enterovirus group. Echovirus 4 was identified by IFA. Although HFMD is a viral infection associated mainly with Enterovirus 71 (HEV-71) and Coxsackievirus A16 (CV-A16), our results demonstrate a diversity of serotype related to HFMD and stress the importance of epidemiological surveillance to this disease and its complications.

Rotavirus and adenovirus in Rondônia

Acute gastroenteritis is one of the most common diseases in humans worldwide. Viral gastroenteritis is a global problem in infants and young children. In this study the incidence of diarrhea was assessed in 877 hospitalized children under five years old, over a period of 24 months and distributed in 470 cases of diarrhea and 407 age-matched group with other pathologies, as control group. Two antigen detection techniques based on enzyme immunoassay (EIA) and latex particles were used for detection of rotavirus and adenovirus. Rotavirus A was a major cause of gastroenteritis with 23.6% of cases, being 90% of these cases in young children. Adenovirus infections was detected by EIA with frequency of 6.4%. Rotavirus and adenovirus were detected in 10.1 and 1.7% of stools from control group, respectively. Interestingly, the frequency of the youngest children in the control group excreting Rotavirus A was comparable to that detected in stools from diarrheic children. We cannot rule out the existence of other enteric viruses because the etiology of 171 cases of diarrhea was not determined and active search for astrovirus and calicivirus was not done. This is the first study that shows the presence of enteric viruses in the infantile population from Western Brazilian Amazonia and it was important to help physicians in the treatment of viral gastroenteritis.

Adenovirus, calicivirus and astrovirus detection in fecal samples of hospitalized children with acute gastroenteritis from Campo Grande, MS, Brazil

We analyzed fecal samples from hospitalized children up to three years of age with acute gastroenteritis at Campo Grande, Mato Grosso do Sul, Brazil, from May 2000-January 2004. Astrovirus and calicivirus were detected by Reverse Transcription-Polymerase Chain Reaction and adenovirus was detected using the Rotavirus and Adenovirus combined immunoenzyme assay. Astrovirus, adenovirus and calicivirus were detected at rates of 3.1%, 3.6% and 7.6%, respectively. These results re-emphasize the need for the establishment of regional vigilance systems to evaluate the impact of enteric viruses on viral gastroenteritis.

Evaluation of HA negatively charged membranes in the recovery of human adenoviruses and hepatitis A virus in different water matrices

Human adenoviruses (HAdV) and hepatitis A virus (HAV) are shed in the faeces and consequently may be present in environmental waters, resulting in an increase in pathogen concentration that can affect water quality and human health. The aim of this study was to evaluate an adsorption-elution method which utilizes negatively charged membrane HA to determine the efficient recovery of HAdV and HAV from different water matrices and to combine this procedure with a qualitative molecular method (nested RT-PCR and nested PCR). The best efficiency recovery was achieved in distilled water and treated wastewater effluent (100%) for both viruses and in recreational lagoon water for HAV (100%). The efficiency recovery was 10% for HAdV and HAV in seawater and 10% for HAdV in lagoon water. The viral detection limit by nested PCR for HAV in water samples ranged between 20-0.2 FFU/mL and 250 and 25 TCID50/mL for HAdV. In conclusion, these results suggest that the HA negatively charged membranes vary their efficiency for recovery of viral concentration depending upon the types of both enteric viruses and water matrices.

Serological analysis reveals circulation of influenza C viruses, Brazil

The circulation of influenza C viruses in Rio de Janeiro, Brazil, was studied when significant levels of antibodies were detected (56.7%) with hemagglutination inhibition test, used as a standard methodology for influenza virus studies.

Longitudinal study of acute respiratory diseases in Rio de Janeiro: occurrence of respiratory viruses during four consecutive years

The occurrence of different viruses in nasopharyngeal secretions from children less than 5 years old with acute respiratory infections (ARI) was investigated over a period of 4 years (1982-1985) in Rio de Janeiro. Of the viruses known to be associated with ARI, all but influenza C and parainfluenza types 1, 2 and 4 were found. Viruses were found more frequently in children attending emergency or pediatric wards than in outpatients. This was clearly related to the high incidence of respiratory syncytial virus (RSV) in the more severe cases of ARI. RSV positive specimens appeared mainly during the fall, over four consecutive years, showing a clear seasonal ocurrence of this virus. Emergency wards provide the best source of data for RSV surveillance, showing sharp increase in the number of positive cases coinciding with increased incidence of ARI cases. Adenovirus were the second most frequent viruses isolated and among these serotypes 1,2 and 7 were predominant. Influenza virus and parainfluenza virus type 3 were next in frequency. Influenza A virus were isolated with equal frequency in outpatient departments, emergency and pediatric wards. Influenza B was more frequent among outpatients. Parainfluenza type 3 caused outbreaks in the shanty town population annually during the late winter or spring and were isolated mainly from outpatients. Herpesvirus, enterovi-rus and rhinovirus were found less frequently. Other viruses than RSV and parainfluenza type 3 did not show a clear seasonal incidence.

Circulation of Eastern equine encephalitis, Western equine encephalitis, Ilhéus, Maguari and Tacaiuma viruses in equines of the Brazilian Pantanal, South America

Neutralizing antibodies to EEE (6.7%), WEE (1.2%), ILH (26.6%), MAG (28.2%) and TCM (15.7%) viruses were found in sera of 432 equines of the Brazilian Pantanal, area where undiagnosed horse deaths are frequently observed. A 4-fold rise in CF titer to EEE virus was detected in acute and convalescent sera of an encephalitis horse sacrified in 1992. Antibodies to EEE, ILH, MAG and TCM viruses were detected in horses less than 2 years old indicating recent circulation of these viruses in the Pantanal. The evidence of recent equine encephalitis associated with rising CF titer to EEE warrants a more intensive study with attempts to isolate virus from horses with clinical manifestations of encephalitis.

Lysotypes and plasmidial profile of Salmonella Serovar Typhimurium isolated from children with enteric processes in the cities of Rio de Janeiro, RJ, and Salvador, BA - Brazil

The lysotypes, plasmidial profiles, and profiles of resistance to antimicrobial agents were determined in 111 Salmonella Typhimurium strains isolated from feces and blood of children treated in Rio de Janeiro and in Salvador. Six distinct lysotypes (19, 41, 97, 105, 120 and 193) were recognized, with a predominance of lysotype 193 (59.7%) in Rio de Janeiro and of phage type 105 (38.4) in Salvador. Approximately 86.7% of the lysotype 193 strains presented multiple resistance to more than six antimicrobial agents, whereas 93% of lysotype 105 strains were fully susceptible. More than 90% of the strains presented plasmids distributed into 36 different profiles in Rio de Janeiro and into 10 profiles in Salvador. A 40 MDa plasmid was the most frequent (47%) in the strains from Rio de Janeiro, whereas a 61 MDa plasmid predominated (14.5%) in Salvador. Combined analysis of plasmid profile and classification into lysotypes (especially those belonging to types 105 and 103, proved to be more discriminatory than the other methods applied).