Presence and environmental circulation of enteric viruses in three different wastewater treatment plants.

AIMS: The aim of the work was to evaluate the circulation of the viruses and to determine a correlation between faecal indicators and viruses. METHODS AND RESULTS: Raw wastewater and effluent samples were collected from three wastewater treatment plants, during three sampling periods, and analysed, using cultural and molecular methods, to determine bacteria and virus presence. The results show a removal of bacterial indicators, but a limited reduction of the phages. The viral analysis displays the circulation of cultivable enteroviruses and differences in the seasonal-geographical distribution. Hepatitis A virus was found with only two genotypes: IA-IB. Rotavirus was present in 11.11%, 24.14%, 2.78% of the samples in the 1st, 2nd and 3rd sampling periods; Astrovirus in 33.33%, 6.9%, 25%; Adenovirus in 7.41%, 3.45%, 2.78%; Norovirus in 7.41%, 10.34%, 5.56% respectively. Adenovirus was never identified in plants B and C as Rotavirus in plant C. CONCLUSIONS: The presence of faecal indicators was not predictive of the enteric virus presence, whereas a different circulation of Enteroviruses was found in the wastewater treatment plants. SIGNIFICANCE AND IMPACT OF THE STUDY: The study shows the importance and the usefulness of molecular methods to evaluate the virus circulation and the genetic variability of Enteroviruses.

[AIDS and forensic medicine: II. the particular issues]. / AIDS: attualità intorno al dibattito etico-deontologico e medico-legale. II. Le questioni particolari.

The article deals with some issues of forensic medicine relevance, as well as ethical one, about HIV+/AIDS condition, which still represent open problems in regulations and in the case-law. Particularly, in the light of the specific Italian regulations (namely, the Law 5 June 1990 no. 135), the Authors address the juridical fundaments of the care of HIV+/AIDS patients, reaffirming the obligation for all the healthcare professionals to give care in every case of infection from HIV. Secondly, the issue of the diagnostic tests and the screenings is faced, sustaining the obligatoriness to gain an adequate previous consent by the subject and, however, only for clinical necessity and in his/her best interest. Finally, the delicate matter of the professional secrecy is examined, of the guardianship of confidentiality and of the partner notification is examined. In general, although the doctrine and jurisprudence not justify the transmission of HIV+/AIDS patient's personal data, yet the specific matter is not easily resolvable exclusively in juridical terms, because it is necessary remind also the extra-juridical--that is ethical--nature of deontological norms.

[AIDS and forensic medicine. I. The framework of the debate]. / AIDS: attualità intorno al dibattito etico-deontologico e medico-legale. I. Le coordinate generali.

The ethical debate regarding the AIDS is very rich both at the theoretical and practical level, but--unlike what it is happening for other sectors, e.g. the euthanasia--these two aspects are integrated, shaping a true interdisciplinary job of ethical evaluation, in which clinicians, medical examiners, ethicists, nurses, clinical psychologists, economists and politicians are met. The utilized methodology will be to identify the human act in the light of the patient's good and the common good of the society. These are strong references and broadly shared in general sense, but they become problematic if we want to identify, and we should have to do it, the contents of such "goods". The authors intend to appraise the quality of a Centre for Research and Healthcare for AIDS patients, also considering the "performance measures".

[Enteric viruses and bacteriological parameters in molluscs]. / Parametri virali e batterici in molluschi bivalvi.

Eighty-seven samples of shellfish were collected considering: type of mollusc, origin, growing area, monitoring or for human purpose. The bacteriological parameters were: Fecal Coliforms, Escherichia coli, and Salmonella; whereas the virological parameters included: Hepatitis A and E virus, Rotavirus, Astrovirus and Enterovirus. In total, 63.2% of samples had normal bacteria values, only one sample was Salmonella positive. The percentage of positive samples for Hepatitis A virus was 5.7%, Rotavirus 29.9%, Astrovirus 27.6%, Enterovirus 10.3%. The recovery of hepatitis E virus was always negative, whereas 13 samples (14.9%) were positive for two viruses.

Waterborne gastroenteritis outbreak in Albania.

Three different studies are reported concerning the environmental pollution caused by viruses in Albania. The first study describes an outbreak of gastroenteritis in the capital city, involving 2,722 children attending the Paediatric Unit of Tirana Hospital. The age group with the highest morbidity was 0-5 years, with 89.5%; no fatalities were recorded during the outbreak. Rotavirus was detected in 26/28 faecal samples by RT-PCR, although astrovirus, adenovirus and calicivirus were also present. The second study describes an outbreak of hepatitis A virus involving the city of Lac. Two hundred cases were recorded, with the highest incidence in the age-group 5-9 years. Phylogenetic analysis of the VP1/2A region showed the presence of a unique sequence: genotype IA. Rotavirus was identified in drinking-water samples collected during the outbreak. The third study describes the prevalence of HAV and HEV in 202 sera randomly collected from 12 different cities in Albania. HAV showed a high incidence (66.2%), whereas none was positive for HEV. The genomic analysis of the VP1/2A junction revealed the presence of only one genotype (IA) with few point mutations and just two amino acid substitutions at codons 22 and 34. Additionally, two potential antigenic variants were detected, the first at position 46 of VP3 and the second at position 23 of VP1.

[Virus transmission in drinking water]. / La diffusione di virus nelle acque potabili.

Several epidemiological data confirm the presence of enteric viruses in drinking water. The present paper deals with several problems tied to the virological analysis, such as the concentration of the samples, the isolation and the identification of enteric viruses.

A large infantile gastroenteritis outbreak in Albania caused by multiple emerging rotavirus genotypes.

By the end of December 2000, the epidemiological system 'Alert' of the Public Health Institute in Tirane reported an outbreak of acute gastroenteritis. The outbreak involved children in Tirane and in the rural area. In total, 2722 children were seen in Tirane Hospital and 982 (56.4%) were treated for acute gastroenteritis. The age group with the highest morbidity was 0-5 years (89.7%), followed by the 6-9 (6.2%) and 10-15 years age groups (4.1%). The distribution of acute gastroenteritis cases, which occurred along the same water distribution system, suggests a waterborne origin. The nucleic acid amplification confirmed the co-circulation of different genotypes of rotavirus, mainly P[8]G9 and P[8]G3, responsible for the outbreak. Other enteric viruses such as astrovirus serotype 1, adenovirus and Norovirus, genogroups I and II were detected. Co-infections with different rotavirus genotypes and even with different enteric viruses were detected in several samples.

HAV and HEV infection in hospitalised hepatitis patients in Alexandria, Egypt.

A total of 202 serum and stool samples from acute hepatitis patients attending the Fever Hospital of Alexandria, Egypt, have been studied to reveal markers of hepatitis virus infection. Anti-HAV IgM were detected in 21 out of 202 sera (10.4%), whereas 201 sera (99.5%) had anti-HAV IgG. The first age attack was in the class-age 0-9 years with 64.7% of anti-HAV IgM positive sera. Among 202 patients, anti-hepatitis E IgG (sample/over cut off > 1.0) was identified in 90 patients (44.5%). The anti-HEV seropositivity ranged from 17.6% to 60.0% in the different age groups, with the highest level in the class-age 20 29 years. Anti-hepatitis E IgM were identified in 49 patients with the first age attack in the class-age 10-19 years (39.4%). HAV RNA was identified by nested PCR in 7 samples out of 15, whereas HEV RNA was present in 4 out of 75 stool samples. Direct DNA sequence of the latter PCR products confirmed the presence of the HEV genome; comparison of the sequences of the isolates from Egypt with those in data banks revealed the highest homology to the Burma strain. Our data confirm that HAV and HEV are common causes of acute sporadic hepatitis in Alexandria but with different peak age positivity. Occasionally, but not infrequently, dual infections (HAV-HEV and HEV-enteric viruses) were also found. The risk analysis indicates that patients living in rural areas are exposed to a higher risk of hepatitis E infection compared to the urban population, whereas the presence of anti-HEV IgG was significantly associated with consumption of common village water and use of indoor dry pit and oral therapy for schistosomiasis.

Reduction of microorganisms in sewage effluent using hypochlorite and peracetic acid as disinfectants.

A comparative study on peracetic acid and sodium hypochlorite in inactivating bacteria and viruses was carried out. Therefore the disinfection actions of peracetic acid, in comparison with sodium hypochlorite, was evaluated against the usual indicators of faecal contamination, the pathogen Salmonella, Pseudomonas spp., bacteriophages anti-Escherichia coli, F+/phage and the phage of Bactericides fragilis B40-8 and enteroviruses. Under the experimental conditions, no representative results were obtained for enteroviruses and phages because of their low concentration in the sewage effluent. On the other hand, the indicator organisms were reduced substantially by the sodium hypochlorite and peracetic acid concentrations, while more variable results were obtained against Pseudomonas and bacteriophages anti-Escherichia coli.

Serum antibodies to polioviruses in Alexandria, Egypt.

Random sera, in a total of 192, were collected in the Fever Hospital of Alexandria, Egypt, and analysed for the presence of antibodies against polioviruses. The results show good antibody levels, only three sera (1.5%) were negative for poliovirus type 1, 5 (2.6%) for poliovirus type 2 and 10 (5.2%) for poliovirus type 3; one subject was completely negative.

Evidence of hepatitis E virus replication on cell cultures.

Several human and animal cell lines have been used to grow hepatitis E virus. The strain SAR-55 was adapted only on PLF/PLC/5 cell line without any visible cytopathic effect. The growth of the SAR-55 was monitored by examining the positive and the negative strands of HEV-RNA. Stool samples, obtained from hospitalised acute hepatitis patients at the Fever Hospital of Alexandria (Egypt), were used to confirm the susceptibility of PLF/PLC/5 cells. After more than one-week's cultivation, three stool samples out of 17 IgM anti-HEV positive and 1 from 52 IgG anti-HEV positive patients showed a specific RT-PCR amplification product. The nucleotide sequences of the methyltransferase region of the genome in the isolates revealed the maximum homology with Burma strain with several point mutations.

Concomitant poliovirus infection during an outbreak of hepatitis A.

AIM OF THE STUDY: The present study was designed to evaluate the possible co-infection, with other enteric viruses, during an outbreak of hepatitis A (HA). MATERIAL AND METHODS: Forty-two stool samples and sera were collected during an outbreak of hepatitis A. Sera were analysed by the Abbott test for IgG-IgM anti-HAV antibodies. Stool samples were used to identify the presence of enteric viruses. HAV genome was identified by a RT-PCR test, other enteric viruses were identified, after cell passage and seroneutralization test on BGM cells, by RT-PCR and RFLP assay. RESULTS: The samples were obtained from 27 employees of an industrial plant, nine household contacts and six non-employee controls. The attack rate was 12.5%, whereas the overall prevalence was 63%. In the employee group, 12 out of 27 stool samples were positive for the presence of HAV by reverse transcriptase polymerase chair reaction (RT-PCR). All the other samples (30) were negative. Five samples from employees, three from household contacts and one from non-employees were also found positive for enteroviruses. These viruses were classified by seroneutralization as poliovirus and RFLP assay as Sabin poliovirus type 1. Four samples were positive both for HAV and poliovirus. CONCLUSIONS: This study confirms co-infection with different enteric viruses may occur and also emphasizes the wide circulation of HAV and the existence of silent infection with poliovirus.

Microbiological quality of coastal sea water of Alexandria, Egypt.

The aim of the present study was to evaluate the quality of the seawater in Alexandria, Egypt. Samples were collected in 6 different points: Kayet Bay, El Shatby, Camp Cesar, Sporting, Beir Massoud and El Max. In total, 24 samples were analyzed. For each point the analysis included estimation of the following parameters: Esherichia coli, total coliform and fecal streptococci, Yersinia, Shigella, Salmonella, bacteriophages and enteric viruses. Just one sample (El Max) was positive for the presence of Salmonella, neither Shigella or Yersinia were isolated from any of the analyzed points. E. coli was identified in 10 samples while the ratio between total coliform and fecal streptococci showed variable results with the exception of El Max that resulted constantly high. Three samples were positive for the presence of enteric viruses: El Shatby, Beir Massoud and Sporting. The analysis of phages showed a variable pollution values.

PIP: The bacteriological virological parameters were evaluated on seawater samples taken at different points on the coast of Alexandria, Egypt. Samples were collected at 6 different points: Kayet Bay, El Shatby, Camp Cesar, Sporting, Beir Massoud, and El Max. A total of 24 samples were analyzed by estimation of the following parameters: Escherichia coli, total coliform and fecal streptococci, Yersinia, Shigella, Salmonella, bacteriophages, and enteric viruses. The virological analysis included the isolation and identification of cytopathogenic enteroviruses and three phages: somatic coliphage, F-specific, and B 40-8. The bacteriophage analysis was performed by the plaque assay method using the double-layer method, whereas the membrane filtration method was used to estimate bacterial populations in the samples. During the summer period no E. coli could be isolated from any point during the study, whereas in autumn E. coli were identified in all the points except for Sporting. E. coli was identified in 65% of the qualitative analyses. The limit was exceeded in 12 samples out of 24; for fecal streptococci, in 15 samples out of 24. The ratio over 4.4 relating to fecal coli and fecal streptococci indicated human fecal pollution. The El Max sample was positive for the presence of Salmonella; neither Shigella nor Yersinia were isolated from any of the analyzed points. In the El Max sample (autumn period), total coliform and fecal streptococci exceeded the European Community (EC) limit, whereas the ratio was 10, confirming human fecal pollution. E. coli was identified in 10 samples, while the ratio between total coliform and fecal streptococci showed variable results with the exception of El Max, which was constantly high. Three samples were positive for the presence of enteric viruses: El Shatby, Beir Massoud, and Sporting. The enteric viruses were confirmed by a secondary passage on cell culture. The analysis of phages showed variable pollution values.

Purification and primary structure of metallothioneins induced by cadmium in the protists Tetrahymena pigmentosa and Tetrahymena pyriformis.

Tetrahymena pyriformis and Tetrahymena pigmentosa grown in the presence of a non-toxic dose of cadmium, accumulate the metal in the cytosol. Purification by gel-permeation, ion-exchange and reverse-phase high-performance liquid chromatography showed that the metal is bound principally to newly formed proteins with ultraviolet spectra and cysteine contents similar to those of Cd(2+)-metallothioneins from multicellular organisms. The isolated proteins revealed that the two species of ciliates each express two Cd(2+)-isothioneins. The primary structures determined by both Edman degradation and mass spectrometry revealed that the equivalent proteins from T. pyriformis and T. pigmentosa have identical sequences and that the two isoforms in each species differ only by the presence or absence of a lysine residue at the N-terminus. The development of automated mass spectrometric sequence analysis algorithms combined with an accurate determination of the molecular mass allowed the rapid confirmation of the sequences. The Tetrahymena metallothionein sequences are unusually long (105 and 104 amino acids) and show a unique internal homology which suggests that the proteins arose by gene duplication. The chains contain 31 cysteine residues, 15 of which are arranged in motifs characteristic of the mammalian metallothioneins; the remaining residues show several unique repeating motifs, which could have interesting consequences for the tertiary structure of the metal-binding sites. Amino acid sequences of Tetrahymena metallothioneins have some similarity with other eukaryotic metallothioneins. A comparison on the basis of optimised FASTA scores, shows a closer relationship with horse metallothionein-1B.

Influence of polyions on the early steps of enterovirus infection.

The influence of electric charged molecules on the early phases of enterovirus infection was studied in order to select antiviral compounds able to prevent viral attachment. The effect of different polyelectrolytes on the multiplication of coxsackie virus B3, echovirus 6 and hepatitis A virus was investigated in susceptible cells by adding the drug before, during or after the viral adsorption period. Among polyanions, the polysaccharides heparin and dextran sulfate inhibited viral infectivity, dextran sulfate being the most effective mainly towards hepatitis A virus infection. DEAE-dextran and protamine sulfate, generally recognized as enhancers of infectivity of naked and enveloped viruses, exhibited an inhibitory effect towards the three picornaviruses tested. Only in the case of hepatitis A did DEAE-dextran slightly improve viral antigen synthesis. The inhibitory effect shown by compounds belonging to positive and negative polyions suggests that the electric charge is not sufficient by itself to explain the antiviral activity of these drugs.

Depuration of Mytilus galloprovincialis experimentally contaminated with hepatitis A virus.

Mussels (Mytilus galloprovincialis) were contaminated with known amounts of laboratory strains of hepatitis A virus and Poliovirus 1 and the effectiveness of a self-cleansing mechanism was studied using a pilot depuration system. Both viruses were rapidly bioaccumulated by mussels and the maximal concentration of about 10(4) TCID50/ml was reached within 1.5 hours. Depuration was carried out up to 24 h; infectivity titer decreased to 10(2) TCID50/ml and 10(3.2) TCID50/ml within 6 h in hepatitis A virus and Poliovirus 1 contaminated mussels, respectively, but only a very slight further decrease was obtained after 24 h. E. coli was used as a control; within 24 h the concentration decreased from 40 to 2 bacteria/ml of mussel (MPN). The elimination of bacteria is not a reliable parameter to control the effectiveness of viral depuration.

Identification of enteroviruses isolated from sea-water: indirect immunofluorescence (IIF).

In this research the Enteroviruses presence in sea water was studied. In previous studies presumptive Enteroviruses were revealed in 43% and in 77% of sea water samples analyzed. It was necessary to identify viral particles isolated from marine water because the detection of this kind of virus was performed only on the basis of cytopathic effect appearance on cell cultures. The aim of this research was to verify the suitability of Indirect Immunofluorescence for identification of presumptive Enteroviruses isolated from marine waters. 13 field strains from RC 37 cells (Cercopithecus Kidney cell line) were tested. Pools of Horse immune sera against COXS, POLIO and ECHO, and anti-horse antibodies conjugated with fluorescein were used. The results revealed the presence of Coxsackie virus and Echo virus. There were many problems related with ECHO identification for the presence of cross-reactions with COXS. The IIF method cannot be performed routinely, because it did not show high level of specificity and sensibility, and it is expensive. This methodology can be used only in particular cases and in laboratories with competent virologists and related facilities. In the future, the only suitable methodology for identification of Enteroviruses isolated from field samples is based on the use of RNA and DNA probes.