Tulio Machado Fumian

Molecular Detection and Characterization of Gastroenteritis Viruses Occurring Naturally in the Stream Waters of Manaus, Central Amazônia, Brazil

ABSTRACT To assess the presence of the four main viruses responsible for human acute gastroenteritis in a hydrographic network impacted by a disordered urbanization process, a 1-year study was performed involving water sample collection from streams in the hydrographic basin surrounding the city of Manaus, Amazonas, Brazil. Thirteen surface water sample collection sites, including different areas of human settlement characterized as urban, rural, and primary forest, located in the Tarumã-Açu, São Raimundo, Educandos, and Puraquequara microbasins, were defined with a global positioning system. At least one virus was detected in 59.6% (31/52) of the water samples analyzed, and rotavirus was the most frequent (44.2%), followed by human adenovirus (30.8%), human astrovirus (15.4%), and norovirus (5.8%). The viral contamination observed mainly in the urban streams reflected the presence of a local high-density population and indicated the gastroenteritis burden from pathogenic viruses in the water, principally due to recreational activities such as bathing. The presence of viral genomes in areas where fecal contamination was not demonstrated by bacterial indicators suggests prolonged virus persistence in aquatic environments and emphasizes the enteric virus group as the most reliable for environmental monitoring.

Evaluation of an adsorption–elution method for detection of astrovirus and norovirus in environmental waters

Human astroviruses (HAstV) and noroviruses (NoV) are shed frequently at high concentrations and persist for long periods in the environment, raising a significant health risk of water-related gastroenteritis. The aim of this study was to evaluate an adsorption-elution method with an HA (mixed cellulose esters) negatively charged membrane to determine the best recovery of HAstV and NoV from different environmental waters. As the presence of MgCl(2) affects viral adsorption onto the membrane, three different MgCl(2) concentrations were evaluated. The best recovery of both NoV and HAstV from mineral and river water samples was between 18% and 64%, while recovery from tap water and sea water samples was between 3% and 14%. These results suggest that detection and recovery of each enteric virus with this adsorption-elution method requires a specific MgCl(2) concentration and depends on the source of environmental water tested. The combination of the HA negatively charged membrane to concentrate viruses with quantitative PCR detection allows for the identification of gastroenteritis viruses implicated in acute outbreaks of gastroenteritis.

Viral load and genotypes of noroviruses in symptomatic and asymptomatic children in Southeastern Brazil

BACKGROUND Noroviruses (NoVs) are a major etiological agent of sporadic acute gastroenteritis worldwide. OBJECTIVES To detect, quantify and characterize genogroups and genotypes of NoVs in children with and without gastrointestinal symptoms. STUDY DESIGN NoVs were investigated by RT-PCR in a total of 319 fecal specimens from children up to three years old with (n=229) and without (n=90) acute diarrhea, between February 2003 and June 2004 in the emergency room in Vitória, Southeastern Brazil. NoVs were quantified by real-time PCR and genotyped. RESULTS NoVs were detected in 17% (40/229) and 13% (12/90) of symptomatic and asymptomatic children, respectively. Six NoV-rotavirus A mixed infections were observed. Fifty-one strains were characterized as NoV GII and one as GI. Twenty strains were characterized as GII/4 (9/13), GII/3 (1/13), GII/6 (2/13) and GII/14 (1/13) in symptomatic and GII/3 (6/7) and GII/8 (1/7) in asymptomatic children. The median RNA viral loads were 8.39 and 7.15log(10)copies/g of fecal specimens for symptomatic and asymptomatic children, respectively (p=0.011). NoV load was lower when it was present in a mixed infection with rotavirus A (p=0.0005). CONCLUSIONS This study demonstrates a diversity of NoV strains circulating in this geographic area, and reports GII/8 and GII/14 in the American Continent for the first time. In addition, it confirms GII/4 as the most prevalent genotype in symptomatic children and identified GII/3 in an important frequency, especially in asymptomatic children. Furthermore, preliminary results show that symptomatic patients present a viral load that is significantly greater than asymptomatic children (p=0.011).

Detection and quantification of classic and emerging viruses by skimmed-milk flocculation and PCR in river water from two geographical areas

Molecular techniques and virus concentration methods have shown that previously unknown viruses are shed by humans and animals, and may be transmitted by sewage-contaminated water. In the present study, 10-L river-water samples from urban areas in Barcelona, Spain and Rio Janeiro, Brazil, have been analyzed to evaluate the viral dissemination of human viruses, validating also a low-cost concentration method for virus quantification in fresh water. Three viral groups were analyzed: (i) recently reported viruses, klassevirus (KV), asfarvirus-like virus (ASFLV), and the polyomaviruses Merkel cell (MCPyV), KI (KIPyV) and WU (WUPyV); (ii) the gastroenteritis agents noroviruses (NoV) and rotaviruses (RV); and (iii) the human fecal viral indicators in water, human adenoviruses (HAdV) and JC polyomaviruses (JCPyV). Virus detection was based on nested and quantitative PCR assays. For KV and ASFLV, nested PCR assays were developed for the present study. The method applied for virus concentration in fresh water samples is a one-step procedure based on a skimmed-milk flocculation procedure described previously for seawater. Using spiked river water samples, inter- and intra-laboratory assays showed a viral recovery rate of about 50% (20-95%) for HAdV, JCPyV, NoV and RV with a coefficient of variation ≤ 50%. HAdV and JCPyV were detected in 100% (12/12) of the river samples from Barcelona and Rio de Janeiro. Moreover, NoV GGII was detected in 83% (5/6) and MCPyV in 50% (3/6) of the samples from Barcelona, whereas none of the other viruses tested were detected. NoV GGII was detected in 33% (2/6), KV in 33% (2/6), ASFLV in 17% (1/6) and MCPyV in 50% (3/6) of the samples from Rio de Janeiro, whereas KIPyV and WUPyV were not detected. RV were only analyzed in Rio de Janeiro and resulted positive in 67% (4/6) of the samples. The procedure applied here to river water represents a useful, straightforward and cost-effective method that could be applied in routine water quality testing. The results of the assays expand our understanding of the global distribution of the viral pathogens studied here and their persistence in the environment.

A rapid procedure for detecting noroviruses from cheese and fresh lettuce

Noroviruses (NoVs) are recognized as the most common agents of outbreaks of food-borne viral gastroenteritis and the efficiency of different methods for detection of NoVs from food matrices have been tested in several laboratories worldwide. The aim of this study was to develop a rapid and sensitive method for recovery of NoVs by using a filtration concentration method followed by PCR amplification for detection of NoVs from cheese and fresh lettuce. Experimentally, a fecal suspension containing different number of NoVs copies was spiked in the food surface and extracted by a direct elution using a Stomacher apparatus. An Ozone-Safe solvent Vertrel XF treatment was included for cheese samples for removing particulate matter. The watery phase was collected and the viral concentration was performed by the adsorption-elution method using negatively charged membranes with inorganic solvents in a Stericup and afterwards ultrafiltered using a Centriprep Concentrator 50 to obtain a final volume of 2ml. RNA isolation was carried out with the QIAamp Viral RNA Mini Kit available commercially and reverse transcription was carried out with a Pd(N6) random primer. Real time quantitative PCR (TaqMan) and qualitative PCR were used for molecular detection of NoVs. The recovery rate of NoVs ranged from 5.2 to 72.3% in lettuce and from 6 to 56.3% in cheese. The results indicate that this method is suitable for detection of NoVs contamination in food and will help establish the cause and source of NoVs outbreaks of food-borne illness.

One year monitoring of norovirus in a sewage treatment plant in Rio de Janeiro, Brazil.

Norovirus (NoV) is one of the most important aetiological agents of acute gastroenteritis both in developed and developing countries. NoV is shed in high concentrations by infected persons and contaminates recreational and drinking water through sewage discharge into the environment. The aim of this study was to determine the prevalence, genotypes and removal ratio of NoV by PCR, seminested-PCR and quantitative PCR (qPCR) assays in a sewage treatment plant in Rio de Janeiro city, Brazil, during one year of surveillance. NoV was detected in 7 (15%), 14 (29%) and 28 (58%) samples using PCR, seminested-PCR and qPCR, respectively. The mean removal ratio for the activated sludge process was 0.6 log10 for NoV genogroup I (GI) and 0.32 log10 for NoV genogroup II (GII). The peak NoV concentration was detected in the coldest months, with 53,300 genomic copies/litre. Nucleotide sequencing and phylogenetic analysis revealed that five strains clustered with GI strains and six with GII strains. This study demonstrates that NoV spreads into the environment despite the sewage treatment process and remains a source of waterborne outbreaks of acute gastroenteritis.

Detection of rotavirus A in sewage samples using multiplex qPCR and an evaluation of the ultracentrifugation and adsorption-elution methods for virus concentration

Group A rotaviruses (RV-A) are the most common agents of viral gastroenteritis in children worldwide. The goal of this study was to compare two different methods to concentrate RV-A from sewage samples and to improve the detection and quantification of RV-A using a multiplex quantitative PCR assay with an internal control. Both RV-A and the internal control virus, bacteriophage PP7, were seeded into wastewater and then concentrated using either an ultrafiltration-based adsorption-elution protocol or an ultracentrifugation-based protocol. Real time multiplex quantitative PCR was used to quantify the purified RV-A and PP7, and the results of the multiplex assay were compared with the results of the monoplex assays. The ultracentrifugation-based method had a mean recovery rate of 47% (range: 34-60%), while the ultrafiltration-based adsorption-elution method had a mean recovery rate of 3.5% (range: 1.5-5.5%). These results demonstrate that ultracentrifugation is a more appropriate method for recovering RV-A from wastewater. This method together with the multiplex qPCR assay may be suitable for routine laboratory use.

One year environmental surveillance of rotavirus specie A (RVA) genotypes in circulation after the introduction of the Rotarix® vaccine in Rio de Janeiro, Brazil

Rotavirus specie A (RVA) infection is the leading cause of severe acute diarrhea among young children worldwide. To reduce this major RVA health impact, the Rotarix® vaccine (GlaxoSmithKline, Rixensart, Belgium) was introduced in the Brazilian Expanded Immunization Program in March 2006 and became available to the entire birth cohort. The aim of this study was to evaluate the spread of RVA in the environment after the introduction of Rotarix® in Brazil. For this purpose, a Wastewater Treatment Plant (WTP) in Rio de Janeiro was monitored for one year to detect, characterize and discriminate RVA genotypes and identify possible circulation of vaccine strains. Using TaqMan® quantitative PCR (qPCR), RVA was detected in 100% (mean viral loads from 2.40×10(5) to 1.16×10(7) genome copies (GC)/L) of sewage influent samples and 71% (mean viral loads from 1.35×10(3) to 1.64×10(5)GC/L) of sewage effluent samples. The most prevalent RVA genotypes were P[4], P[6] and G2, based on VP4 and VP7 classification. Direct nucleotide sequencing (NSP4 fragment) and restriction enzyme digestion (NSP3) analysis did not detect RVA vaccine-like strains from the sewage samples. These data on RVA detection, quantification and molecular characterization highlight the importance of environmental monitoring as a tool to study RVA epidemiology in the surrounding human population and may be useful on ongoing vaccine monitoring programs, since sewage may be a good screening option for a rapid and economical overview of the circulating genotypes.

Molecular detection, quantification and characterization of human polyomavirus JC from waste water in Rio De Janeiro, Brazil

Polyomavirus JC (JCPyV) is largely excreted by the human population through the urinary route and has been recognized as a potential viral marker for human waste contamination. This study aims to investigate the dissemination of JCPyV in waste water from a sewage treatment plant (STP) located in Rio de Janeiro, Brazil, and to describe the prevalence of JCPyV subtypes currently present in this population. Raw and treated sewage samples were collected bimonthly during one year, and examined for the presence of JCPyV using nested polymerase chain reaction (nPCR) and quantitative real time PCR (qPCR). JCPyV was detected by nPCR in 96% and 43% of raw and treated sewage samples, respectively. The concentration of JCPyV present in the samples ranged from 1.2x10(3) to 3.2x10(5) and 2.6x10(2) to 6.2x10(3) genome copies per 2 ml of concentrated raw and treated sewage sample, respectively. The strains were characterized and the obtained nucleotide sequences indicated that the detected JCPyV strains clustered with subtypes of East African, West African and European origin. To our knowledge, this is the first study describing the incidence and diversity of JCPyV strains in raw and treated sewage in Brazil.

Assessment of burden of virus agents in an urban sewage treatment plant in Rio de Janeiro, Brazil

Sewage discharge is considered to be the main source of virus contamination in aquatic environments. There is no correlation between the presence of viruses and the presence of fecal coliforms in water; therefore virological markers are needed when monitoring contamination. This study investigates DNA and RNA virus concentrations in wastewater and evaluates a potential virus marker of human contamination. Influent and effluent samples were collected twice a month throughout a 1-year period. Viruses were detected using quantitative polymerase chain reaction protocols; nucleotide sequencing was carried out for virus genotyping. Human adenovirus (HAdV) and polyomavirus JC (JCPyV) were the most prevalent viruses found in influent samples (100%) with a virus load that ranged from 10(6) to 10(5) genome copies per liter (gc l(-1)). Norovirus genogroup II (NoV GII) and human astrovirus (HAstV) were less prevalent, and ranged from 10(4) to 10(3)gc l(-1). Quantitative data on virus profiles in wastewaters stress the high level of rotavirus species A environmental dissemination and address the potential of HAdV as a useful virological marker of virus contamination in aquatic environments. This study corroborates other studies performed in developed countries on DNA viruses as good markers of human fecal contamination.