Human bocavirus in children with acute gastroenteritis in Albania.

Human Bocavirus (HBoV) has been recently identified in association with acute viral gastroenteritis (AGE). The objective of this work was to investigate the prevalence of HBoV in children with AGE in Albania. Stool specimens collected from 142 children were analyzed by amplification of partial NP1 and Vp1/Vp2 genes. HBoV was detected in 13 samples (9.1%), 12 HBoV-1 and one HBoV-2. All HBoV-positive patients were co-infected with rotavirus and/or adenovirus, a finding which might indicate that there is no clear causal association of this agent with diarrhea. Further investigation is needed to assess the pathogenic role of HBoV in childhood diarrhea.

Genetic Diversity of Human Adenovirus in Children with Acute Gastroenteritis, Albania, 2013-2015.

The objectives of the present study were to assess the occurrence of human adenoviruses (HAdVs) in paediatric patients with gastroenteritis in Albania and to characterize HAdV strains. Faecal specimens from children admitted with acute gastroenteritis to the Paediatric Hospital in Tirana were screened for HAdV, using broad-range primers targeting the hexon gene, in combination with species-specific primers targeting the fiber gene. Phylogenetic analysis was then performed to assess the genetic relationships among the different sequences and between the sequences of the samples and those of the prototype strains. Adenovirus DNA was detected in 33/142 samples (23.2%); 14 belonged to species F (13 HAdV-41 and 1 HAdV-40), 13 to species C (1 HAdV-1, 8 HAdV-2, and 4 HAdV-5), 5 to species B (HAdV-3), and 1 to species A (HAdV-12). Rotavirus coinfection was present in 9/33 (27.2%) positive samples. In the remaining 24 positive samples (12 enteric--F species; 12 nonenteric--A, B, or C species), HAdVs were detected as unique viral pathogens, suggesting that HAdV may be an important cause of diarrhoea in children requiring hospitalization. This is the first study investigating the presence of human adenoviruses (species A-G) as etiologic agents of viral gastroenteritis in children in Albania.

First outbreak of norovirus in Albania.

AIMS: Noroviruses (NoVs) represent the most important enteric viruses responsible for acute gastroenteritis world-wide. This study objective is to characterize the first outbreak of NoV that occurred in Ballsh, a small city in Albania. METHODS AND RESULTS: Stool specimens were collected from people attending to the hospital. Samples were also collected from the aqueduct for bacteriological and virological tests. Overall 33 stools and five drinking water samples were collected, respectively, from the hospital in Ballsh and from the municipal aqueduct. No water samples were scored positive whereas ten stool samples (30.3%) were scored GGII NoV positive. All the GGII isolates were identified as GGII·4 genotype, and no GGI was identified. The alignment and protein analysis were performed using, respectively, ClustalV and the mega 4 software. CONCLUSIONS: This is the first report of NoV GGII·4 in Albania causing an outbreak. The genetic analysis showed several point mutations and amino acid substitutions with respect to the international strains. SIGNIFICANCE AND IMPACT OF STUDY: Over the last decades, Albania has suffered from different outbreaks as cholera, poliomyelitis, hepatitis A and now, for the first time, it has been documented an outbreak of NoV.

Are PEI-coated SWCNTs conjugated with hepatitis A virus? A chemical study with SEM, Z-potential, EDXD and RT-PCR.

The conjugation between nanotubes, coated with different doses of polyethylene imine (PEI) and hepatitis A virus (HAV) was investigated by scanning electron microscopy, Z-potential, thermogravimetric and differential thermal analysis, transmission electron microscopy, energy dispersive x-ray diffraction (EDXD) and reverse transcript polymerase chain reaction (RT-PCR). For the first time, to our knowledge, evidence is obtained that conjugation between the nanotubes and the HAV occurs and that it has an (at least a partial) electrostatic character. Since all components of the conjugated systems, nanotubes, coating material and virus are characterized by different peak shapes in the selected q range, it was possible to infer that conjugation occurred. RT-PCR measurements confirmed that the conjugation of the coated nanotubes and HAV occurred and the result was stable. This opens up the prospect of probing the coated nanotubes as intra-cellular carriers in transfection processes of the virus. Further biological applications will concern a possible vaccine especially for non-replicative viruses.

Statistical correlation between enterovirus genome copy numbers and infectious viral particles in wastewater samples.

AIMS: Classic virological tests are time consuming and labour-intensive; real-time RT-PCR has proven to be a fast method to detect and quantify enterovirus genomes in clinical and environmental samples. This method is unable to discriminate between infective and noninfective enterovirus particles; few clinical studies have compared real-time RT-PCR and viral culture. We wondered if the enterovirus genome quantification could be correlated to the infectivity. METHODS AND RESULTS: We used the statistical approach to verify our hypotheses to correlate data, obtained by the standard method (most probable number of cytopathic units-MPNCU) and molecular test (real-time RT-PCR), on wastewater treatment plant samples. Chi-squared test was used, considering several cut-off values ('50'-'100'-'200' genome copy numbers), to determine statistical significance in comparison of the two methods. Chi-square value was not significant when cut-off of 50 (P = 0.103) and 100 (P = 0.178) was assumed but was significant with cut-off of 200 (P = 0.044). CONCLUSION: This limit, 200 genome copy, could be used as cut-off value to indicate enterovirus survival in environmental monitoring. SIGNIFICANT AND IMPACT OF THE STUDY: To introduce a fast procedure that is able to compensate for disadvantages of cell culture method for viral environmental analyses.

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.

Prevalence and genotyping of human isolates of Giardia duodenalis from Albania.

Microscopical and PCR-based techniques were performed in order to investigate the prevalence of infection and the genotypes of Giardia duodenalis from 125 stool samples collected from children living in the urban and the rural areas of Tirana (Albania) and hospitalized with acute gastroenteritis. 7 out of 125 samples resulted positive for Giardia at the microscopic examination (5.6%). In 50 selected samples including the 7 samples positive for Giardia by microscopy, 3 and 15 additional positive samples were detected by immunofluorescence and PCR, respectively. Seasonality appeared as an important parameter to be evaluated in order to better understand the prevalence of infection. Sequence analysis revealed both human Assemblage A and B. This result represents the first data on G. duodenalis genotypes in Albania.

Use of armored RNA as a standard to construct a calibration curve for real-time RT-PCR.

Armored Enterovirus RNA was used to standardize a real-time reverse transcription (RT)-PCR for environmental testing. Armored technology is a system to produce a robust and stable RNA standard, trapped into phage proteins, to be used as internal control. The Armored Enterovirus RNA protected sequence includes 263 bp of highly conserved sequences in 5' UTR region. During these tests, Armored RNA has been used to produce a calibration curve, comparing three different fluorogenic chemistry: TaqMan system, Syber Green I and Lux-primers. The effective evaluation of three amplifying commercial reagent kits, in use to carry out real-time RT-PCR, and several extraction procedures of protected viral RNA have been carried out. The highest Armored RNA recovery was obtained by heat treatment while chemical extraction may decrease the quantity of RNA. The best sensitivity and specificity was obtained using the Syber Green I technique since it is a reproducible test, easy to use and the cheapest one. TaqMan and Lux-primer assays provide good RT-PCR efficiency in relationship to the several extraction methods used, since labelled probe or primer request in these chemistry strategies, increases the cost of testing.

[Hygienic aspects of the Tiber river: presence of pathogenic protozoa and correlation with microbiological and physico-chemical parameters]. / Aspetti igienico-sanitari delle acque del flume Tevere: presenza di protozoi patogeni e rapporti di correlazione con parametri microbiologici e chimico-fisici.

An investigation on the hygienic quality of the Tiber river was conducted with the aim both to enumerate Giardia cysts and Cryptosporidium oocysts in the water and to determine possible correlations between them and bacterial indicators, pathogens and physico-chemical parameters. A low hygienic water quality was evidenced, with high counts of microorganisms. Furthermore, variable concentrations of Giardia and Cryptosporidium were observed. No correlation was found between the protozoa concentrations and that of the other microorganisms, whilst a significant correlation with redox potential and Giardia cysts was calculated.

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.

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.

Fate of bacterial indicators, viruses and protozoan parasites in a wastewater multi-component treatment system.

The extent of reduction in selected microrganisms was tested at a multi-component wastewater treatment plant that treats sewage for a potential re-use in agriculture. The aim of the investigation was to evaluate possible reciprocal correlation among the different microrganisms and to compare the removal of two encysted pathogenic protozoa with that of microbial indicators, Clostridium perfringens spores, enteroviruses and bacteriophages. Samples collected included the raw wastewater, the chlorinated effluent and the effluent after an ultraviolet light treatment. All of the raw sewage samples were positive for Cryptosporidium oocysts and Giardia cysts, as well as for the other microorganisms tested but the bacteriophage B40-8. The data obtained confirm the removal efficiency of the entire process for indicator bacteria but also show the low and variable removal efficiency for the other microbial parameters, such as Giardia and Cryptosporidium, enteroviruses and Clostridium perfringens spores. Reciprocal correlation between Cryptosporidium and Giardia (oo)cysts and the other microbial groups was not demonstrated. The results confirm the resistance of Clostridium perfringens spores, enteroviruses and protozoa to chlorination and demonstrate the relative persistence of these organisms in the effluents even during the ultraviolet light treatment. The yields also emphasise the influence of the analytical method for the determination of protozoan parasites.

Determination of enteroviruses, hepatitis A virus, bacteriophages and Escherichia coli in Adriatic Sea mussels.

The aim of the present study was to evaluate the incidence of enteric viruses in mussels and to verify the possibility of using phages as indirect indicators of mussel viral contamination. Mussels (36 samples) collected from three different areas of the Adriatic Sea were analysed to determine the following parameters: Escherichia coli, somatic coliphage (T6 phage), F-Plus (MS2 phage), B40-8 (phage of Bacteroides fragilis), enteroviruses and hepatitis A virus. Most of the results of the bacteriological analysis (most probable number (MPN) ml-1) were in accordance with the bacteriological limits established by European law, with the exception of seven samples. The bacteriophage analyses were always negative for F-Plus and B40-8, with the exception of a few samples, whereas the somatic coliphages were generally between 0 and 20 MPN g-1, with the exception of two samples (110 MPN g-1). The virological analysis showed five samples positive for the presence of enteroviruses and 13 for the presence of hepatitis A virus (in three samples both viruses were present). Most of these samples presented acceptable bacteriological parameters and the bacteriophages were absent or their value was generally very low. The results show that the detection of E. coli and phages does not seem to be a good indicator of viral contamination.

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.

Genomic characterization of human and environmental polioviruses isolated in Albania.

Between April and December 1996, a serious outbreak of poliomyelitis occurred in Albania; almost 140 subjects were involved, and the episode presented an unusually high mortality rate (12%). During the outbreak, water samples from the Lana River in Tirana, Albania, and stool samples from two cases of paralytic poliomyelitis were collected and analyzed for the presence of polioviruses. Six polioviruses were isolated from the environmental and human samples, according to standard methods. All the samples were characterized by partial genomic sequencing of 330 bases across the 5' untranslated region (5'-UTR) (nucleotide positions 200 to 530) and of 300 bases across the VP1 region (nucleotide positions 2474 to 2774). Comparison of these sequences with those present in data banks permitted the identification of environmental isolates Lana A and Lana B as, respectively, a Sabin-like type 2 poliovirus and an intertypic recombinant poliovirus (Sabin-like type 2/wild type 1), both bearing a G instead of an A at nucleotide position 481. The two other environmental polioviruses were similar to the isolates from the paralytic cases. They were characterized by a peculiar 5'-UTR and by a VP1 region showing 98% homology with the Albanian epidemic type 1 isolates reported by other authors. This study confirms the environmental circulation in Albania of recombinant poliovirus strains, likely sustained by a massive vaccination effort and by the presence in the environment of a type 1 poliovirus, as isolated from the Lana River in Tirana about 2 months before the first case of symptomatic acute flaccid paralysis was reported in this town.

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.