Shiga toxin-producing Escherichia coli in Central Greece: prevalence and virulence genes of O157:H7 and non-O157 in animal feces, vegetables, and humans.

In Greece, Shiga toxin-producing Escherichia coli (STEC) have only been sporadically reported. The objective of this study was to estimate the prevalence of STEC and Escherichia coli O157:H7 in farm animals, vegetables, and humans in Greece. A total number of 1,010 fecal samples were collected from farm animals (sheep, goats, cattle, chickens, pigs), 667 diarrheal samples from humans, and 60 from vegetables, which were cultured in specific media for STEC isolates. Enzyme-linked immunosorbent assay (ELISA) was used to detect toxin-producing colonies, which, subsequently, were subjected to a multiplex polymerase chain reaction (PCR) for stx1, stx2, eae, rfbE O157, and fliC h7 genes. Eighty isolates (7.9 %) from animal samples were found to produce Shiga toxin by ELISA, while by PCR, O157 STEC isolates were detected from 8 (0.8 %) samples and non-O157 STEC isolates from 43 (4.2 %) samples. STEC isolates were recovered mainly from sheep and goats, rarely from cattle, and not from pigs and chickens, suggesting that small ruminants constitute a potential risk for human infections. However, only three human specimens (0.4 %) were positive for the detection of Shiga toxins and all were PCR-negative. Similarly, all 60 vegetable samples were negative for toxin production and for toxin genes, but three samples (two roman rockets and one spinach) were positive by PCR for rfbE O157 and fliC h7 genes. These findings indicate that sheep, goats, cattle, and leafy vegetables can be a reservoir of STEC and Escherichia coli O157:H7 isolates in Greece, which are still rarely detected among humans.

A seroprevalence study of poliovirus antibody against a collection of recombinant and non-recombinant poliovirus vaccine strains in the population of southern Greece.

In this study, the serological status of the southern Greek population in the 1­10-year, 11­20-year, 21­30-year and 31­40-year age groups with regard to Sabin vaccine strains and a collection of 15 recombinant and four non-recombinant poliovirus vaccine strains was determined. For all three poliovirus types, the highest neutralization test (NT) titres were observed in the 1­10- year age group, indicating a good response to vaccination. In general, the serological status of the population of southern Greece with regard to poliovirus is better for types 1 and 2 than for type 3. The presence of the lowest NT titre in the 21­ 30-year age group against poliovirus type 3 suggests the need for a booster dose of monovalent Sabin3 vaccine to ensure personal and herd immunity.

Role of hyperglycemia in isogeneic islet transplantation: an experimental animal study.

OBJECTIVE: Study the role of hyperglycemia-induced beta cell loss on grafted islet destruction. DESIGN: Male inbred rats were made diabetic by streptozotocin administration and used as islet donors and/or isograft recipients to probe directly the role of hyperglycemia as an important determinant of transplanted islet fate, following exclusion of immune-related causes of islet graft destruction like allograft immunity and disease recurrence. RESULTS: Our studies showed that: a) Hyperglycemia destroyed islet but not pituitary isografts and b) Tight control of normoglycemia by sufficient islet mass engraftment prevented graft damage. CONCLUSION: While sustained hyperglycemia caused destruction of transplanted islet isografts, induction of normoglycemia by transplantation of sufficient islet mass to diabetic recipients had a beneficial long term effect on their functional engraftment.

Comparing normal primary endocervical adenoepithelial cells to uninfected and influenza B virus infected human cervical adenocarcinoma HeLa cells.

Human adenocarcinoma HeLa cells surviving infection with low (10(-9) units), medium (10(-6) units), and high (10(-2) units) influenza B titers were compared to their uninfected precursors and to normal endocervical adenoepithelial and metaplastic cells using Papanikolaou-staining method and immunocytochemistry. Normal primary endocervical and infected HeLa cells surviving infection shared similar morphologic, phenotypic, and divisional patterns that differed drastically from those of uninfected HeLa cells. The number of infected hosts surviving 6-7 days of viral exposure did not change during 3-week follow-up period, and their cyclin E levels suggested that they had been arrested to the G1 phase of the cell cycle by viral stress. Our findings suggest that in addition to apoptosis, nononcogenic viral stress activated the expression of endocervical metaplastic-like motifs in surviving hosts. A mechanism of cell response to nononcogenic viral stress was proposed to explain these findings. We conclude that nononcogenic respiratory viruses specifically target and eliminate abnormal cells ectopically overexpressing appropriate receptors and may complement current treatments of cervical cancer.

Laboratory diagnosis of common herpesvirus infections of the central nervous system by a multiplex PCR assay.

A sensitive multiplex PCR assay for single-tube amplification that detects simultaneous herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), varicella-zoster virus (VZV), human cytomegalovirus (CMV), and Epstein-Barr virus (EBV) is reported with particular emphasis on how the method was optimized and carried out and its sensitivity was compared to previously described assays. The assay has been used on a limited number of clinical samples and must be thoroughly evaluated in the clinical context. A total of 86 cerebrospinal fluid (CSF) specimens from patients which had the clinical symptoms of encephalitis, meningitis or meningoencephalitis were included in this study. The sensitivity of the multiplex PCR was determined to be 0.01 and 0.03 50% tissue culture infective doses/the reciprocal of the highest dilution positive by PCR for HSV-1 and HSV-2 respectively, whereas for VZV, CMV and EBV, 14, 18, and 160 ag of genomic DNA were detected corresponding to 48, 66, and 840 genome copies respectively. Overall, 9 (10.3%) of the CSF samples tested were positive in the multiplex PCR. HSV-1 was detected in three patients (3.5%) with encephalitis, VZV was detected in four patients (4.6%) with meningitis, HSV-2 was detected in one neonate (1.16%), and CMV was also detected in one neonate (1.16%). None of the samples tested was positive for the EBV genome. None of the nine positive CSF samples presented herpesvirus coinfection in the central nervous system. Failure of DNA extraction or failure to remove any inhibitors of DNA amplification from CSF samples was avoided by the inclusion in the present multiplex PCR assay of alpha-tubulin primers. The present multiplex PCR assay detects simultaneously five different herpesviruses and sample suitability for PCR in a single amplification round of 40 cycles with an excellent sensitivity and can, therefore, provide an early, rapid, reliable noninvasive diagnostic tool allowing the application of antiviral therapy on the basis of a specific viral diagnosis. The results of this preliminary study should prompt a more exhaustive analysis of the clinical value of the present multiplex PCR assay.

Molecular and phylogenetic analysis of haemagglutinin and neuraminidase sequences from recent human influenza type A (H3N2) viral isolates in Southern Greece.

Eighteen haemagglutinin (HA1) gene segments and eleven neuraminidase (NA) genes of human influenza type A (H3N2) viruses isolated from non-vaccinated individuals presenting severe influenza-like illness at peak influenza activity in Southern Greece during the surveillance period 1996-1999, were subjected to sequence and phylogenetic analyses following propagation in embryonated hen's eggs. The HA1 gene segment of the clinical isolates differed from the recent reference influenza type A (H3N2) vaccine strains in an Ile at residue 186, a Val at residue 194 and a Val at residue 226 for one, two and thirteen isolates of the 1996-1997 and 1996-1999 periods, respectively. The analogous differences in the NA gene were confined in an Asp to Asn substitution at residue 198 in one A/Wuhan/359/95 (H3N2)-like isolate of the 1996-1997 period, primarily. In addition, phylogenetic analysis revealed that an isolate of the 1997-1998 period was a recombinant with its HA1 gene segment being closely related to that of A/Wuhan/359/95-like viruses and its NA to viruses of the A/Sydney/5/97 (H3N2) lineage. These findings confirmed the profound genetic instability of influenza type A (H3N2) viruses and underscored the importance for periodic molecular surveys of HA and NA in the effective prevention and management of viral outbreaks. Most importantly, however, they contributed the first complete epidemiological material for influenza in Southern Greece, the archival nature of which constitutes valuable reference for future surveys.

Development of a quadriplex polymerase chain reaction for human cytomegalovirus detection.

The development of a quadriplex PCR method with amplification of HCMV in a single-step procedure using primers taken from four different regions of the viral genome is described. Different concentrations of dNTPs and MgCl2 were assayed in order to optimize the constitution of the buffer for the multiplex PCR. The specificity of the PCR was tested with 100 ng, 10 ng, and 1 ng of genomic MRC-5 cell DNA infected with CMV in the presence of 10 microg of uninfected MRC-5 cell DNA. The sensitivity of the PCR was evaluated by the amplification of various amounts (100 ng, 10 ng, 1 ng, and 0.1 ng) of genomic MRC-5 cell DNA infected with CMV. The specificity and sensitivity assays were performed for each pair of primers and for the combined four primer pairs in the multiplex PCR. CMV was consistently detected from 10 ng of genomic MRC-5 cell DNA with each primer pair. When all four sets of primers were combined in a single reaction tube, the sensitivity of the assay was equivalent to 10 ng of genomic MRC-5 cell DNA, whereas amplification from 1 ng genomic MRC-5 cell DNA produced only a subset of the amplimers. By amplifying four target-sequences of HCMV simultaneously with minimum incubation time at each temperature, a quadriplex, highly sensitive PCR assay was performed. The use of four primer sets designed in different genomic regions of HCMV allowed the detection of variants and achieved maximal sensitivity and specificity which are essential for a diagnostic utilization.