An acute gastroenteritis outbreak of Vibrio parahaemolyticus O4:K55 in Nursing College, Thailand.

A cluster of acute gastroenteritis among nursing students was noticed on 13th September 2005. Between 13th and 17th September 2005, a retrospective cohort study was then conducted to identify the most likely cause of gastroenteritis at a nursing college in Bangkok, Thailand. Self-administered questionnaires, interviews, environmental investigations, and rectal swabs from all participants were carried out. In the investigation, 98.9% female nursing students were investigated and had completed the questionnaire, 49.4% of the participants were diagnosed to have acute gastroenteritis. The predominant symptoms were watery diarrhoea (90.8%) and abdominal cramps (71.3%). Of 28.9% of rectal swab isolates were identified as Vibrio parahaemolyticus O4:K55 (40.4%), Salmonella spp. (19.2%), Vibrio cholerae non O1/non O139/non O141 (11.5%), Aeromonas trota (3.9%), Vibrio alginolyticus (1.9%) and other co-infections (23.1%). The tdh gene was identified from all V. parahaemolyticus using multiplex PCR. The implicated food risk factor for gastroenteritis was boiled egg (adjusted prevalence rate ratio; PR=1.9, 95% CI, 1.04-3.79). However the bitter melon soup was not significantly associated for gastroenteritis (adjusted PR=1.3, 95% CI, 0.98-1.82). The population attributable fraction analysis indicated that boiled eggs item was an implicated food risk for this outbreak (PAF=45.8%). Vibrio parahaemolyticus O4:K55 was identified as a major causative agent for gastroenteritis in which the contaminated boiled eggs was a vehicle in this outbreak. Cross-contamination control should be emphasized in food operation plans among institutes.

Evaluation and validation of a PulseNet standardized pulsed-field gel electrophoresis protocol for subtyping Vibrio parahaemolyticus: an international multicenter collaborative study.

The pandemic spread of Vibrio parahaemolyticus is an international public health issue. Because of the outbreak potential of the organism, it is critical to establish an internationally recognized molecular subtyping protocol for V. parahaemolyticus that is both rapid and robust as a means to monitor its further spread and to guide control measures in combination with epidemiologic data. Here we describe the results of a multicenter, multicountry validation of a new PulseNet International standardized V. parahaemolyticus pulsed-field gel electrophoresis (PFGE) protocol. The results are from a composite analysis of 36 well-characterized V. parahaemolyticus isolates from six participating laboratories, and the isolates represent predominant serotypes and various genotypes isolated from different geographic regions and time periods. The discriminatory power is very high, as 34 out of 36 sporadic V. parahaemolyticus strains tested fell into 34 distinguishable PFGE groups when the data obtained with two restriction enzymes (SfiI and NotI) were combined. PFGE was further able to cluster members of known pandemic serogroups. The study also identified quality measures which may affect the performance of the protocol. Nonadherence to the recommended procedure may lead to high background in the PFGE gel patterns, partial digestion, and poor fragment resolution. When these quality measures were implemented, the PulseNet V. parahaemolyticus protocol was found to be both robust and reproducible among the collaborating laboratories.

Characteristics of Clostridium difficile strains isolated from asymptomatic individuals and from diarrheal patients.

OBJECTIVES: To characterise genotypes of Clostridium difficile strains isolated from asymptomatic individuals and patients with diarrhea. METHODS: Fecal specimens from 235 asymptomatic infants <12 months, 76 asymptomatic children 1-11 years and 132 adult patients with antibiotic-associated and non-antibiotic-associated diarrhea obtained from Siriraj Hospital, Bangkok from October 1998 to April 1999 were examined for C. difficile by cycloserine-cefoxitin-fructose agar culture. The presence of the C. difficile toxin A gene was determined by specific PCR with the use of primers 5-(CCC AAT AGA AGA TTC AAT ATT AAG CTT)-3 and 5-(GGA AGA AAA GAA CTT CTG GCT CAC TCA GGT)-3. All C. difficile isolates were subsequently genotyped by pulsed-field gel electrophoresis (PFGE). RESULTS: The C. difficile strains were found in 28 (11.9%) asymptomatic infants, 16 (21.1%) asymptomatic children and 33 (25%) adult patients. In total, 14 PFGE types and eight subtypes designated as types A, B, C, D, E, F, G, H, I, J, K, L, M and N, and A1, A2, A3, A4, B1, B2, B3 and E1, respectively, were identified. Only two isolates from infants and 18 isolates from adult patients were toxin A gene positive by PCR. Both isolates of toxigenic C. difficile were from infants in the same ward and were PFGE type B. PFGE type A was the predominant type among all toxigenic isolates (12 of 18 isolates) from adult patients. The other PFGE types of toxigenic C. difficile found in adult patients were: type A1, one isolate; type B, four isolates; and type C, one isolate. Types B2 and D were identified in 38.5% and 46.2%, respectively, of the toxin A gene-negative isolates of C. difficile from infants. CONCLUSIONS: These results revealed the occurrence of three distinct clusters from different wards in Siriraj Hospital. The toxigenic C. difficile of PFGE type A and related subtypes was a predominant infective clone in adult patients, whereas non-toxigenic C. difficile types B2 and D were encountered in asymptomatic infants. This information can be useful in epidemiologic investigations.

A monoclonal antibody-based dot-blot ELISA diagnostic kit for the detection of Vibrio cholerae 01 in stools of diarrheic patients and household contacts.

A "cholera diagnostic kit" was developed for sensitive, specific, rapid, and inexpensive detection of Vibrio cholerae 01. The monoclonal antibody specific to antigen A of Vibrio cholerae 01 was used as an antigen detection reagent and the principle of dot-blot ELISA was adopted. The kits were used in seven Regional Medical Sciences Centres, Ministry of Public Health, located at various regions of Thailand where diarrhea occurs frequently. Diagnostic efficiency of the kits in the detection of Vibrio cholerae 01 from rectal swabs of the diarrheic patients and their household contacts was evaluated in comparison with the conventional culture method. The two methods were found to have excellent degree of agreement (kappa values > 95%). The dot-blot ELISA has several advantages over the culture methods, ie rapid (dot-blot ELISA takes 1-2 hours while the culture method takes at least two days) and inexpensive. It requires no sophisticated equipment. The procedure is not complicated thus it is easy to train personnel. The diagnostic kits are recommended for use in the detection of severe diarrhea caused by V. cholerae 01 not only in hospitals and health centres where adequate treatment of the patients is required as a life-saving measure but also for early recognition of cholera cases and their contacts so that other action, ie prevention and control of outbreaks and surveillance can be promptly implemented.