Manoochehr Pourshaban

Quantitative real-time PCR of enteric viruses in influent and effluent samples from wastewater treatment plants in Italy

The prevalence of enteric viruses in wastewater, the efficacy of wastewater treatments in eliminating such viruses, and potential health risks from their release into the environment or by recycling of treated wastewaters, are very important issues in environmental microbiology. In this study we performed a quantitative TaqMan real-time PCR (polymerase chain reaction) analysis of enteric viruses on samples of influents and effluents from 5 wastewater treatment plants in and around Rome. Three epidemiologically important, waterborne enteric viruses were analyzed: adenoviruses, enteroviruses and noroviruses (GI and GII) and compared to classical bacterial indicators of fecal contamination. The concentration of adenoviruses was the highest, in both raw and treated waters. Mean values in influents were ranked as follows: adenovirus > norovirus GI > norovirus GII > enterovirus. In effluents, the ranking was: adenovirus > norovirus GI > enterovirus > norovirus GII. Removal efficiencies ranged from 35% (enterovirus) to 78% (norovirus GI), while removal efficiency for bacterial indicators was up to 99%. Since molecular quantification does not necessarily indicate an actual threat to human health, we proceeded to evaluate the infectivity of enterovirus particles in treated effluents through integrated cell culture and real-time PCR. Infectivity assays detected live virions in treated water, pointing to potential public health risks through the release of these viruses into the environment. A better understanding of viral presence and resistance to sewage purification processes have the potential of contributing to the effective management of risks linked to the recycling of treated wastewater, and its discharge into the environment.

Intestinal Toxemia Botulism in Two Young People, Caused by Clostridium butyricum Type E

Two unconnected cases of type E botulism involving a 19-year-old woman and a 9-year-old child are described. The hospital courses of their illness were similar and included initial acute abdominal pain accompanied by progressive neurological impairment. Both patients were suspected of having appendicitis and underwent laparotomy, during which voluminous Meckels diverticula were resected. Unusual neurotoxigenic Clostridium butyricum strains that produced botulinum-like toxin type E were isolated from the feces of the patients. These isolates were genotypically and phenotypically identical to other neurotoxigenic C. butyricum strains discovered in Italy in 1985-1986. No cytotoxic activity of the strains that might explain the associated gastrointestinal symptoms was demonstrated. The clinical picture of the illness and the persistence of neurotoxigenic clostridia in the feces of these patients suggested a colonization of the large intestine, with in vivo toxin production. The possibility that Meckels diverticulum may predispose to intestinal toxemia botulism may warrant further investigation.

Detection of genogroup IV noroviruses in environmental and clinical samples and partial sequencing through rapid amplification of cDNA ends

Noroviruses (NoVs) give rise to clinically relevant gastroenteritis in all age groups and are widely distributed in both clinical and environmental settings. NoVs are classified into five genogroups (GI to GV), of which GI, GII and GIV infect humans. While data on the epidemiology of human NoVs GI and GII have been steadily increasing, very little information has been published on the spread of GIV in either the health care system or the environment, resulting in a lack of information about its clinical significance and pathogenesis. In order to investigate the distribution of GIV strains in the environment, we analyzed sewage samples collected from five treatment plants, by using newly designed nested RT-PCR assays. A collection of clinical stool samples, originating from pediatric patients with symptoms of acute gastroenteritis, previously analyzed in our laboratory for the presence of NoV GI or GII, was also analyzed for the presence of GIV norovirus. Results of this work attest to the presence of GIV in both clinical and environmental contexts and underline the importance of routinely screening for this genogroup, along with GI and GII, in order to better understand its distribution, prevalence and role during epidemics, which is probably underestimated.

Molecular identification and genetic analysis of Norovirus genogroups I and II in water environments: comparative analysis of different reverse transcription-PCR assays.

ABSTRACT Noroviruses have received increased attention in recent years because their role as etiologic agents in acute gastroenteritis outbreaks is now clearly established. Our inability to grow them in cell culture and the lack of an animal model hinder the characterization of these viruses. More recently, molecular approaches have been used to study the genetic relationships that exist among them. In the present study, environmental samples from seawater, estuarine water, and effluents of sewage treatment plants were analyzed in order to evaluate the role of environmental surface contamination as a possible vehicle for transmission of norovirus genogroups I and II. Novel broad-range reverse transcription-PCR/nested assays targeting the region coding for the RNA-dependent RNA polymerase were developed, amplifying fragments of 516 bp and 687 bp in the nested reactions for genogroups II and I, respectively. The assays were evaluated and compared against widely used published assays. The newly designed assays provide long regions for high-confidence BLAST searches in public databases and therefore are useful diagnostic tools for molecular diagnosis and typing of human noroviruses in clinical and environmental samples, as well as for the study of molecular epidemiology and the evolution of these viruses.

Molecular Detection of Hepatitis E Virus in Sewage Samples

ABSTRACT Human hepatitis E virus (HEV) is considered an emerging pathogen in industrialized countries. In Italy, the true burden of HEV infection is unknown. Molecular HEV screening of raw sewage samples from 11 wastewater treatment plants yielded 19 positives (16%; 18 genotype I, 1 genotype III) evenly distributed throughout Italy. Evidence that HEV could be establishing itself in our region is accumulating and may justify more active surveillance to monitor its spread.

Detection and molecular characterization of noroviruses from five sewage treatment plants in central Italy.

Noroviruses (NoVs) are the most frequent etiological agents of non-bacterial gastroenteritis. These viruses are transmitted through the fecal-oral route, leading to high viral loads in sewages. The objective of this paper was to study the environmental occurrence of the most prevalent NoV strains in different wastewater treatment plants. In addition, molecular characterization of the isolated strains was performed. Two different PCR-based methods were carried out and a novel strategy was used to verify the level of RT-PCR inhibition. From May to September 2007, a total of 97 inflow and outflow samples were collected from five wastewater treatment plants in central Italy. We detected NoV by nested PCR in 96.9% of influent samples: 89.1% contained both genogroups; 4.7% contained only GI and 3.1% only GII. In effluents, we detected NoV in 78.8% of samples: 30.3% contained both genogroups, and 48.5% contained only GI. The major genotypes detected by sequencing analyses were GI/2, GI/5, GII/b, GII/4 and GII/6. This work confirms the wide circulation of NoVs in Italy with a predominance of GI strains, and the widespread distribution of NoV variants in both raw and treated wastewater.

Molecular detection and genetic diversity of norovirus genogroup IV: a yearlong monitoring of sewage throughout Italy

Noroviruses cause acute viral gastroenteritis worldwide. They are classified in five genogroups, of which GI, GII, and GIV infect humans. Little information is available on the prevalence and clinical effects of GIV noroviruses. We conducted a large-scale molecular-epidemiological investigation, a yearlong monitoring of 11 wastewater treatment plants throughout Italy, with the aim of studying the circulation of GIV NoV, as well as its genetic diversity. Eight percent of samples tested positive, and sequence analysis showed a considerable degree of genetic variability. These results imply the need for further studies to elucidate the role of this virus as a gastroenteritis-causing pathogen.

Genetic typing of human and food isolates of Listeria monocytogenes from episodes of listeriosis.

Ten clinical and food Listeria monocytogenes strains isolated during the epidemiological investigations of episodes of listeriosis (one outbreak and two sporadic cases) that occurred in northern Italy during 1993–1995 have been examined by DNA macrorestriction pattern analysis obtained by PFGE and RAPD typing, in order to confirm the food vehicle of infections. The same DNA profiles within the isolates from the three episodes were obtained by both techniques. The ApaI and SmaI PFGE profiles and RAPD patterns with primer OPM-01 confirmed the close relationship between strains from two distinct episodes. However, RAPD analysis with primer UBC-127 distinguished between these L. monocytogenes isolates.