Detection of hepatitis A virus in sewage sludge by antigen capture polymerase chain reaction.

Antigen capture polymerase chain reaction (PCR) was tested as a sensitive and rapid method for detecting hepatitis A virus (HAV) in raw sewage sludge. The antigen capture PCR was performed both with and without solid-phase virus-catching monoclonal antibodies. Similar results proved that both methods were equally sensitive. Sewage sludge samples from different regions in Germany were examined for evidence of HAV contamination by antigen capture PCR. This method of detection was compared with that used in a previous study of these sewage sludge samples, in which the HAV was detected through indirect immunofluorescence after cell culture inoculation. The results obtained by antigen capture PCR matched those obtained in the earlier cell culture investigations, when HAV was detected in raw as well as digested sewage sludge samples. The advantage of the PCR method, however, lies in the fact that it needs only two days while the cell culture propagation of HAV takes about 8 to 10 weeks.     

Nested real-time PCR for hepatitis A detection

Aims:  The hepatitis A virus (HAV) is one of the most important human foodborne pathogens causing a number of worldwide outbreaks each year. The detection of HAV in food samples remains a complex issue, because commonly used detection tools, such as conventional or even real-time PCR assays, are often unable to detect HAV with sufficient sensitivity. The aims of this study were to develop highly sensitive and specific nested real-time PCR (NRT-PCR)-based method for HAV detection in food and to compare it with currently available methods.
Methods and Results:  By combining conventional PCR, nested PCR and real-time PCR techniques, we have developed a specific NRT-PCR assay for the detection of HAV. The procedure involves two consecutive PCRs, the first of which is performed as a conventional RT-PCR using primers specific for HAV 5' noncoding region. The second reaction involves a real-time PCR using a nested primer pair specific for the first PCR product and a TaqMan probe.
Conclusions:  We have developed a novel NRT-PCR method capable of detecting as little as 0·2 PFU of HAV, which is significantly more sensitive than any other PCR technique tested in our system.
Significance and Impact of the Study:  NRT-PCR provides a potentially useful method for detecting HAV at extremely low levels, as frequently found in food samples, and can be potentially adopted as a regulatory method to ensure food safety.     

Comparison of immunological and molecular hybridization detection methods for the detection of hepatitis A virus in sewage

Immune electron microscopy (IEM), radioimmunoassay (RIA) and molecular hybridization with a digoxigenin-labelled cDNA probe were compared for the detection of wild-type human hepatitis A virus (HAV) in raw and treated sewage. In the same experiments, classic tests for culturable enteroviruses were carried out. With the hybridization probes, HAV was detected in three of the 13 amuent samples (23%) and in eight out of 13 effluent samples (61%). For four of the emuent samples, positivity revealed by IEM was confirmed by the cDNA probe. In contrast, two of the samples shown as positive by IEM were negative with the probes. Detection of HAV by RIA was negative in all cases. Demonstration of HAV was higher in emuent than in affluent. No particular relationship was established between demonstration of HAV, on the one hand, and the various concentrations of enteroviruses observed in the same samples on the other. Overall, if all the results, irrespective of the type of water (affluent or effluent), are taken together, 50% of the sewage samples tested were found to contain HAV by one or another method of detection.     

Detection and characterization of hepatitis A virus and Norovirus in estuarine water samples using ultrafiltration – RT-PCR integrated methods

Aims:  Waterborne outbreaks of hepatitis A and Norovirus disease have been reported and associated with contaminated water supply in various countries. However, in Mexico, there are no studies that report HAV and NV presence in water. This study reports the application of ultrafiltration and RT-nested PCR methods to concentrate and identify these viruses.
Methods and Results:  Forty estuarine water samples were collected from the Huizache Caimanero Lagunary Complex. Samples were concentrated by ultrafiltration system (UFS) and RT-nested PCR was performed for HAV and NV identification. These viruses were found in 80% and 70% of the samples collected respectively and both were present in 57·5%. The DNA sequences analysis showed that 21 estuarine water samples were associated with HAV and 13 with NV. Faecal coliforms were isolated in 48·57% of the samples, while Escherichia coli were found in 34·28%.
Conclusions:  DNA sequencing showed that the genotype IB for HAV and GII for NV were predominant in México. No significant relationships were detected between indicators and viruses ( P  < 0·05).
Significance and Impact of the Study:  This study shows that the UFS is adequate for viral concentration. This is the first study analysing the genetic sequence of HAV and NV isolated from Mexican estuarine water.     

Evidence of Hepatitis A Virus Person-to-Person Transmission in Household Outbreaks

The person-to-person transmission of the hepatitis A virus primarily occurs in enclosed spaces, particularly in the presence of inadequate hygiene conditions and a high proportion of susceptible individuals. Thus, intimate family contact stands out as a risk factor for HAV infection dissemination. The present study aimed to evaluate the occurrence of household HAV transmission. Blood samples were collected from patients with hepatitis A (index cases) and their family members (contacts) that were referred to an ambulatory care clinic specializing in viral hepatitis. A total of 97 samples were collected from 30 families with a confirmed hepatitis A case (index case). Serological and molecular techniques for the diagnosis of hepatitis A were conducted on all samples. HAV infection (anti-HAV IgM + and/or HAV RNA +) was detected in 34.3% (23/67) of the contacts; 34.3% (23/67) of the contacts were immune to HAV, and 31.4% (21/67) were susceptible. In the household contacts, HAV immunity was significantly associated with older age; susceptibility to infection and HAV infection were associated with younger age. Household outbreaks were detected in 16/30 families studied. Co-circulation of subgenotypes IA and IB was found in the household outbreaks, and person-to-person transmission was evidenced in six of the household outbreaks, with 100% homology between the index case and contact strains. The results demonstrated the relevance of HAV household transmission, reaffirming the need for hepatitis A vaccine administration in susceptible contacts and effective infection control procedures to prevent the extension of household outbreaks.     

Detecting the presence of infectious hepatitis A virus in molluscs positive to RT-nested-PCR

AIMS: The objective of this study was to determine the presence of infectious hepatitis A virus (HAV) in molluscs naturally contaminated with viral HAV-RNA. METHODS AND RESULTS: One hundred and forty-two mollusc samples were analysed for the presence of viral HAV-RNA using RT-nested-PCR; positive samples were then analysed with an integrated method, cell-culture RT-PCR, to detect infectious virus. Viral HAV-RNA was detected in 34.5% of the samples while 12.7% of the total samples were positive for the presence of infectious virus. CONCLUSIONS: The results demonstrate the validity of the screening method (RT-nested-PCR) and the necessity of applying a method that is capable of detecting the presence of infectious HAV. SIGNIFICANCE AND IMPACT OF THE STUDY: The study demonstrates that in any case, to determine the safety for human consumption, the results of RT-nested-PCR must be confirmed with an integrated cell-culture PCR method.     

The composition and persistence of faecal coliforms and enterococcal populations in sewage treatment plants

AIMS: The changes in structure and composition of faecal coliforms and enterococcal populations in sewage from different treatment plants, and the elimination of vancomycin- and erythromycin-resistant enterococci (VRE and ERE, respectively) in these treatment plants was analysed to determine any selective reduction. METHODS AND RESULTS: Faecal coliforms, enterococci, VRE, ERE and spores of sulphite-reducing bacteria were enumerated using standard methods. Samples were enriched where necessary in order to isolate antibiotic resistant strains. The structure and composition of these bacterial populations were determined by biochemical fingerprinting and clustering analysis. High diversity and similarity indexes were detected among all the bacterial populations in raw and treated sewage, independently of their origin and the treatment processes employed. Antibiotic resistant strains were detected in all sewage tested and no selective reduction was observed. CONCLUSIONS: The faecal coliforms and enterococci populations did not differ in the sewage samples studied. The vancomycin and erythromycin resistances of the enterococcal populations were similar in the sewage samples. Resistance to both antibiotics persisted after the treatment process independently of raw sewage flow, faecal origin or size of the human population contributing to sewage. However, sewage of mixed origin (human and animal) presented a lower similarity index for the two bacterial populations compared with that of the other human sewage analysed. SIGNIFICANCE AND IMPACT OF THE STUDY: Although a significant reduction in bacterial populations was observed, the persistence of VRE and ERE strains in the same proportions in sewage suggests that there is no selective elimination of bacterial populations during the treatment processes. The ability of antibiotic resistance strains to survive sewage treatment systems should be considered in certain water reuse programmes.     

Viral and bacterial contamination in recreational waters: a case study in the Lisbon bay area

Aims: To assess the presence of viral pathogens in bathing water samples and to evaluate the interdependency of bacterial indicator counts and viral detection. Methods and Results: Bathing water samples of 16 beaches collected along a Portuguese Coastal area were screened for the hepatitis A virus (HAV) and norovirus genogroup I (NVGI) using RT‐PCR technique. Bacteriological water quality was also assessed, according to European regulations. HAV and NVGI were detected in 95% and 27% of the water samples, respectively, whereas bacteriological quality was good in all but one sample, according to current water quality regulations. Conclusions: All water samples would be considered of excellent quality according to the most recent European regulations. No relationship between viral detection and regulatory‐based bacterial indicators was found. Significance and Impact of the Study: The current results reinforce the importance of increased surveillance for pathogenic viruses in bathing waters.     

A real-time polymerase chain reaction assay for quantitative detection of the human-specific enterococci surface protein marker in sewage and environmental waters

A real-time polymerase chain reaction (PCR) assay using SYBR Green I dye was developed to quantify the Enterococcus faecium enterococci surface protein (esp) marker in sewage (n = 16) and environmental waters (n = 16). The concentration of culturable enterococci in raw sewage samples ranged between 1.3 x 10(5) and 5.6 x 10(5) colony-forming units (cfu) per 100 ml. The real-time PCR detected 9.8 x 10(3)-3.8 x 10(4) gene copies of the esp marker per 100 ml of sewage. However, the concentration of culturable enterococci and the esp marker in secondary effluent was two orders of magnitude lower than raw sewage. Surface water samples were collected from a non-sewered catchment after storm events and the real-time PCR was applied to quantify the esp marker. Of the 16 samples tested, 6 (38%) were PCR-positive and the concentration of the esp marker ranged between 1.1 x 10(2) and 5.3 x 10(2) gene copies per 100 ml of water samples. The newly developed real-time PCR method was successfully used to quantify the esp marker in samples collected from sewage and environmental waters. The presence of the esp marker in water samples immediately after storm events not only indicated human faecal pollution but also provided evidence of the degree of human faecal pollution. To our knowledge, this is the first study that reports the use of a real-time PCR assay to quantify the esp marker in sewage and surface waters. Such study would provide valuable information for managers for the improved management of water quality.     

Genogroup distribution of F-specific coliphages in wastewater and river water in the Kofu basin in Japan

Aims: To determine the genogroup distribution of F‐specific coliphages in aquatic environments using the plaque isolation procedure combined with genogroup‐specific real‐time PCR. Methods and Results: Thirty water samples were collected from a wastewater treatment plant and a river in the Kofu basin in Japan on fine weather days. F‐specific coliphages were detected in all tested samples, 187 (82%) of 227 phage plaques isolated were classified into one of the 4 F‐specific RNA (F‐RNA) coliphage genogroups and 24 (11%) plaques were F‐specific DNA coliphages. Human genogroups II and III F‐RNA coliphages were more abundant in raw sewage than animal genogroups I and IV, excluding one sample that was suspected to be heavily contaminated with sporadic heavy animal faeces. The secondary‐treated sewage samples were highly contaminated with genogroup I F‐RNA coliphages, probably because of different behaviours among the coliphage genogroups during wastewater treatment. The river water samples were expected to be mainly contaminated with human faeces, independent of rainfall effects. Conclusions: A wide range of F‐specific coliphage genogroups were successfully identified in wastewater and river water samples. Significance and Impact of the Study: Our results clearly show the usefulness of the genogroup‐specific real‐time PCR for determining the genogroups of F‐specific coliphages present in aquatic environments.