Comparative study of two extraction methods for enteric virus recovery from sewage sludge by molecular methods.
ABSTRACT 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.
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ABSTRACT: 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. 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). Reverse transcription (RT)-PCR was performed to detect the presence of HAV genome in serum samples collected from anti-HAV negative, susceptible subjects. 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). 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.Journal of Clinical Virology 05/2004; 29(4):254-9. · 3.29 Impact Factor
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ABSTRACT: A reverse transcription-PCR (RT-PCR) was established to amplify a 379-bp cDNA fragment (nucleotides 747 to 1126, coding for amino acids 241 to 367) of the VP6 gene of group A rotaviruses associated with subgroup (SG) specificity. Thirty-eight human rotavirus strains characterized with SG-specific monoclonal antibodies were subjected to VP6-specific RT-PCR, and PCR amplicons were used for sequencing. Nucleic acid sequencing and phylogenetic analysis of the VP6 amplicons revealed two clusters, or genogroups. Two genetic lineages were distinguished within genogroup I, consisting of strains serologically characterized as SG I, and three genetic lineages were distinguished within genogroup II, composed of strains serologically characterized as SG II, SG I + II, and SG non-I, non-II. Subgrouping of rotaviruses by means of serological methods may result in strains not being assigned the correct SG or in a failure of strains to subgroup. Molecular characterization of the SG-defining region of VP6 provided evidence for independent segregation of the rotavirus genes encoding VP4, VP6, and VP7.Journal of Virology 08/2002; 76(13):6596-601. · 5.08 Impact Factor