GENOTYPE AND DRUG RESISTANCE OF CLINICAL AND ENVIRONMENTAL VIBRIO CHOLERAE NON-O1/NON-O139 IN NORTHEASTERN THAILAND.

A total of 124 V cholerae non-O1/non-O139 isolates were collected in Khon Kaen, Thailand from diarrheal patients, asymptomatic carriers and environmental water. The presence of virulence-associated and regulatory genes including ctxA, tcpA, zot, ace, ompU, stn, hlyA and toxR) were examined using multiplex PCR. The genomic diversity of the various V. cholerae isolates were differentiated using the random amplified polymorphic DNA (RAPD) method. Antimicrobial susceptibility was tested using disk diffusion. All of V. cholerae non-O/non-O139 isolates carried hlyA and toxR and none carried ctxA and tcpA. The zot, ace and both genes together were found in 1.6%, 4.7% and 4.7% of 64 clinical V. cholerae non-O1 isolates, respectively, while the environmental ones did not. The stn gene was found in 3.1% (2/64) of the clinical and 3.3% (2/60) of the environmental isolates. The RAPD patterns were differentiated into 45 types (A to 2S). RAPD type A (32.3%) was the most frequently found in both clinical and environmental V cholerae non-O1 strains (34.4% and 30.0%, respectively); indicating that there was a clonal relationship between some clinical and environmental isolates whereas almost all of the environmental isolates belonged to different clones. All strains were sensitive to ciprofloxacin and norfloxacin. The environmental isolates (30%) were more resistant than the clinical ones (21.9%). Resistance to sulfamethoxazole/trimethoprim and tetracycline among the clinical isolates occurred in 9.4% (6/64) in 2007, during which period the prevalence of V cholerae O1 increased. We conclude that V. cholerae non-O1/non-O139 from the aquatic environment are potentially pathogenic and this same aquatic environment may be a source of antimicrobial resistance in V. cholerae.

Application of tetraplex PCR for detection of Vibrio cholerae, V. parahaemolyticus, V. vulnificus and V. mimicus in cockle.

A tetraplex PCR method was developed for simultaneous detection of Vibrio cholerae, V. parahaemolyticus, V. vulnificus and V. mimicus in cockle samples in comparison with conventional culture method. Specific primers targeting ompW of V. cholerae, tl of V. parahaemolyticus, hsp60 of V. vulnificus and sodB of V. mimicus were employed in the same PCR. Detection limit of the tetraplex PCR assay was 104 cfu/ml (400 cfu/PCR reaction) for pure cultures of all four species of Vibrio. In Vibrio spiked cockle samples, the limit of detection after 6 hours enrichment in alkaline peptone water was 1 cfu/10 g of cockle tissue for three Vibrio spp, except for V. mimicus that was 102 cfu/10 g of cockle tissue. When the tetraplex PCR and culture methods were applied to 100 cockle samples, V. parahaemolyticus, V. vulnificus, V. cholerae and V. mimicus were detected in 100, 98, 80 and 9% of the samples by tetraplex PCR and in 76, 42, 0 and 0% by the culture method, respectively. This developed tetraplex PCR method should be suitable for simultaneous and rapid detection of Vibrio species in food samples and for food safety assessment.

Duplex PCR for detection of Salmonella and Shigella spp in cockle samples.

Salmonella and Shigella spp are important causative agents of foodborne diseases. A sensitive, specific and rapid method is essential for detection of these pathogens. In this study, a duplex PCR method was developed for simultaneous detection of Salmonella and Shigella spp in cockle samples and compared with the traditional culture method. Enrichment broths for Salmonella spp recovery were also compared. Sensitivity of the duplex PCR for simultaneous detection of Salmonella and Shigella spp from pure culture was 10(3) CFU/ml (40 CFU/PCR reaction), and that of sterile cockle samples spiked with these two pathogens was 1 CFU/10 g of cockle tissue after 9 hours enrichment [3 hours in buffered peptone water (BPW), followed by 6 hours in Rappaport Vasiliadis (RV) broth or tetrathionate (TT) broth for Salmonella spp and 6 hours enrichment in Shigella broth (SB) for Shigella spp]. There was no significant difference in detection sensitivity between enrichment in RV and TT broths. Salmonella spp detected in cockles in Khon Kaen, Thailand by duplex PCR and culture method was 17% and 13%, respectively but Shigella spp was not detected. The duplex PCR technique developed for simultaneous detection of Salmonella and Shigella spp in cockle samples was highly sensitive, specific and rapid and could serve as a suitable method for food safety assessment.

Molecular analysis and antimicrobial resistance of Vibrio cholerae O1 in northeastern Thailand.

A total of 84 clinical Vibrio cholerae O1 isolates were collected from Khon Kaen (KK), Udon Thani (UT), Loei (LI), and Nong Khai (NK), northeastern Thailand during cholera outbreaks in 2007 and 2008. The majority of V. cholerae O1 strains carried nearly all the virulence-associated genes (ctxA, zot, and ace) except for four isolates and one isolate from UT and NK, respectively, which carried only tcpA, ompU, hlyA and toxR. None of the V. cholerae O1 strains carried sto. Pulsed field gel-electrophoresis (PFGE) profiling of 16 randomly chosen isolates showed the same PFGE pattern, except for one NK isolate, which was sensitive to all seven antibiotics used in the antimicrobial susceptibility tests. The tests revealed that multi-drug resistance to tetracycline and co-trimoxazole were present in KK strains (92%), followed by LI (75%) and UT (52%) strains. All strains were sensitive to norfloxacin but intermediate resistance to ciprofloxacin was found in a single strain from KK and LI. Differences in antimicrobial resistance among V. cholerae strains with the same PFGE pattern reflect differences in the antimicrobial agents used in each area of northeastern Thailand.

Leptospirosis in Takeo Province, Kingdom of Cambodia, 2003.

BACKGROUND: In Cambodia, epidemiology and disease burden of leptospirosis were not addressed as they do not have an existing surveillance system and have limitations on their laboratory diagnosis. OBJECTIVE: Define the existence of leptospirosis and determine the antibodies to serovars of leptospires in Cambodia. MATERIAL AND METHOD: One hundred and twenty-one suspected cases of leptospirosis were enrolled in this cross-sectional study, between September 8 and November 30, 2003 from Takeo Provincial Hospital in Doun Keo District, Cambodia. RESULTS: Common clinical manifestations were fever (96%), headache (92%), and myalgia (87%). Common risk behaviors were throwing garbage on the ground (84%), pulling out sprouts (77%), fertilizing (49%), and plowing (47%). Microscopic agglutination test result confirmed four cases and polymerase chain reaction test result confirmed seven cases. Two cases each showed antibodies to serovars Javanica and Australis. An estimated annual incidence of leptospirosis in Takeo province was 7.65 per 100,000 populations. Further studies to define epidemiology and burden of disease are needed. CONCLUSION: Increasing awareness and knowledge on leptospirosis among people are necessary to decrease the impact of leptospirosis in Cambodia.

Two simple immunoassays using endemic leptospiral antigens for serodiagnosis of human leptospirosis.

Two simple enzyme immunoassays, a conventional microplate and dot-ELISA, were developed to detect specific IgM antibodies using pool sonicated antigen prepared from three of the most reactive serovars of Leptospira associated with disease in Thailand. Both assays were evaluated and compared with the standard microscopic agglutination test (MAT) performed with 343 serum samples. A battery of 16 pathogenic serovars of L. interrogans were used as antigens in the MAT assay. The result of MAT at serum titers > or = 1:400 showed three pathogenic serovars of leptospira, Bratislava (71.88%), Sejroe (63.54%) and Pyrogenes (36.46%), were among the most commonly reacted serovars and they were selected for preparation of pool sonicated antigen for both IgM ELISA tests. The microplate IgM-ELISA, performed with sera at 1:80 dilution using the cutoff OD of 0.60, demonstrated sensitivity, specificity and efficiency of 87.50, 97.57, and 94.75%, respectively. The same values for IgM dot-ELISA performed with sera at 1:160 dilution were 98.96, 93.93, and 95.33%, respectively. Both ELISA methods showed results with statistically significant differences from MAT (p < 0.05). The agreement rate of IgM dot-ELISA compared with microplate IgM-ELISA was 0.85 by Kappa analysis. Both assays offered relatively high negative predictive values (95.26-99.57%), thus making the assays ideally suited for rapid screening. Future applications of the IgM dot-ELISA as a test kit would be suitable for use at the peripheral level as a rapid screening test for human leptospirosis.

An outbreak of leptospirosis, Thailand--the importance of the laboratory.

The reported incidence of leptospirosis increased 30-fold in Thailand between 1995 and 2000. Despite many hypotheses to explain the increase, the true etiology remains unknown. We conducted a review of the national surveillance system for leptospirosis, examining the reporting practices, system attributes, and utilization of laboratory confirmation in two northeastern provinces. Using standard guidelines for evaluation of public health surveillance systems, we assessed the timeliness, completeness, and accuracy of data; the sensitivity and specificity of case ascertainment; and the overall usefulness of the Thai leptospirosis surveillance system. Physicians were interviewed to assess compliance and understanding of the case definition. Capacity for confirmation of leptospirosis by a Thai latex agglutination test was assessed. Completeness for variables critical for linking epidemiologic and laboratory data for leptospirosis was 69%. Twenty-eight percent of 208 provincial surveillance reports were considered timely. Interviewed physicians indicated that the national case definition was difficult to understand and apply, and that laboratory confirmation was infrequently used. Compared to a standardized microscopic agglutination test (MAT) panel, the Thai test was specific, but relatively insensitive. We found that a lack of a standardized case definition for leptospirosis, the infrequent use of confirmatory laboratory testing, and the inability to link clinical, epidemiologic, and laboratory data hindered system utility. This surveillance system for leptospirosis highlights difficulties with surveillance of febrile illnesses in general, and the importance of laboratory confirmation for infections that are difficult to diagnose clinically.

A test strip IgM dot-ELISA assay using leptospiral antigen of endemic strains for serodiagnosis of acute leptospirosis.

A test strip IgM dot-ELISA assay for the detection of leptospire-specific IgM antibodies in human sera was developed. Antigen dotted on a nitrocellulose paper strip was the pool sonicated antigen prepared from three predominant reactive Leptospira serovars currently in endemic area, i.e., Bratislava, Sejroe and Pyrogenes. The ability of the test to diagnose acute leptospiral infection was assessed by testing 343 single serum samples from 96 laboratory-confirmed leptospirosis case patients with positive result in the standard microscopic agglutination test (MAT), 55 serum samples from patients with various diseases other than leptospirosis, and 192 serum samples from healthy individuals. Using the results of the MAT as a gold standard, the sensitivity and specificity of the test strip IgM dot-ELISA assay were 98.96 and 93.93 per cent, respectively. The assay offered relatively high negative predictive values (99.57%) thus making the assay ideally suited for rapid screening. The stability of the test strip was assessed with a panel of five positive and five negative control sera after storage at 4 degrees C and -20 degrees C at different times. The results showed a good performance of the test strip at both storage temperatures for up to one year. In conclusion, the test strip IgM dot-ELISA assay was sufficiently sensitive for use as a screening test for serodiagnosis of acute leptospirosis. The assay was simple, inexpensive, and easy to perform for both a single test format and a large number of specimens. However, further studies are still needed to improve the stability of the test strip and assay reagents at ambient temperature, and to make the assay more rapidly and more user friendly.

Traveler's Diarrhea in Foreign Travelers in Southeast Asia: A Cross-Sectional Survey Study in Bangkok, Thailand.

The effect of origin and destination country on traveler's diarrhea incidence rates in Southeast Asia is poorly understood, and research generally only addresses diarrhea in travelers from the developed world. This study evaluated the attack rate and effects of traveler's diarrhea by origin and destination and analyzed key risk factors. A self-administered questionnaire was provided to foreign travelers departing Southeast Asia from Suvarnabhumi Airport, Bangkok, Thailand. It evaluated traveler demographics, relevant knowledge and practices, experiences of diarrhea, and the details and consequences of each diarrheal episode. A total of 7,963 questionnaires were completed between April 2010 and July 2011. Respondents were 56% male (mean age 35) with a mean and median duration of stay of 28 days and 10 days, respectively. Most respondents were from Europe (36.8%) or East Asia (33.4%). The attack rate of traveler's diarrhea was 16.1%, with an incidence rate of 32.05 per 100 person months. Travelers' origin and destination countries significantly related to diarrhea risk. Oceanians had the highest risk (32.9%) and East Asians the lowest (2.6%). Vietnam and Indonesia were the highest risk destinations (19.3%). Other significant factors were youth, trip duration, number of countries visited, and frequently drinking beverages with ice.

Incidence and spectrum of health problems among travelers to Laos.

BACKGROUND: The number of travelers visiting Laos has more than doubled in the last 5 years. Little is known about their pre-travel preparations and the incidence of health problems during their trips. METHODS: At three border posts between Laos and Thailand, travelers were invited to complete a study questionnaire. They were asked about their demographic profile, travel characteristics, pre-travel health preparations, and health problems during their stay in Laos. RESULTS: From September 2011 to April 2012, 1,205 questionnaires from Thais and 1,191 from foreigners were collected. Approximately 60% of the travelers were male; the overall median age among the Thais was 43 years, and among the foreigners was 32 years. Most foreign visitors were from Europe (66.8%), followed by other Asian countries (19.0%) and North America (7.1%). Almost half of the foreigners (47.8%) traveled as individual backpackers, whereas the majority of Thais traveled as package tourists. Foreigners were more likely to trek, cycle, and swim during their trips. There were also significant differences in the average length of stay between foreigners (16.1 days) and Thais (3.8 days, p < 0.001). Health problems were reported in 24.9% of foreigners, the most common being diarrhea, cough, animal exposure, and fever. Only 6.1% of Thais reported health problems, the most common being cough and diarrhea. The relative risk adjusted for duration of stay was 1.63 (95% confidence interval: 1.01-2.64). Occurrence of health problems was associated with younger age group, non-Asian nationality, length of stay, and participation in adventurous activities. CONCLUSIONS: Health problems are rather frequent among non-Thai travelers in contrast to Thais during their trips to Lao People's Democratic Republic. There were significant differences in terms of risk profile and prevalence of health problems between these two groups.

Drug response and genetic properties of Vibrio cholerae associated with endemic cholera in north-eastern Thailand, 2003-2011.

Cholera, caused by Vibrio cholerae, results in significant morbidity and mortality worldwide, including Thailand. Representative V. cholerae strains associated with endemic cholera (n = 32), including strains (n = 3) from surface water sources, in Khon Kaen, Thailand (2003-2011), were subjected to microbiological, molecular and phylogenetic analyses. According to phenotypic and related genetic data, all tested V. cholerae strains belonged to serogroup O1, biotype El Tor (ET), Inaba (IN) or Ogawa (OG). All of the strains were sensitive to gentamicin and ciprofloxacin, while multidrug-resistant (MDR) strains showing resistance to erythromycin, tetracycline, trimethoprim/sulfamethoxazole and ampicillin were predominant in 2007. V. cholerae strains isolated before and after 2007 were non-MDR. All except six diarrhoeal strains possessed ctxA and ctxB genes and were toxigenic altered ET, confirmed by MAMA-PCR and DNA sequencing. Year-wise data revealed that V. cholerae INET strains isolated between 2003 and 2004, plus one strain isolated in 2007, lacked the RS1 sequence (rstC) and toxin-linked cryptic plasmid (TLC)-specific genetic marker, but possessed CTX(CL) prophage genes ctxB(CL) and rstR(CL). A sharp genetic transition was noted, namely the majority of V. cholerae strains in 2007 and all in 2010 and 2011 were not repressor genotype rstR(CL) but instead were rstR(ET), and all ctx(+) strains possessed RS1 and TLC-specific genetic markers. DNA sequencing data revealed that strains isolated since 2007 had a mutation in the tcpA gene at amino acid position 64 (N→S). Four clonal types, mostly of environmental origin, including subtypes, reflected genetic diversity, while distinct signatures were observed for clonally related, altered ET from Thailand, Vietnam and Bangladesh, confirmed by distinct subclustering patterns observed in the PFGE (NotI)-based dendrogram, suggesting that endemic cholera is caused by V. cholerae indigenous to Khon Kaen.

Risk of potentially rabid animal exposure among foreign travelers in Southeast Asia.

BACKGROUND: Each year millions of travelers visit Southeast Asia where rabies is still prevalent. This study aimed to assess the risk of rabies exposure, i.e., by being bitten or licked by an animal, among travelers in Southeast Asia. The secondary objective was to assess their attitudes and practices related to rabies. METHODOLOGY/PRINCIPAL FINDINGS: Foreign travelers departing to the destination outside Southeast Asia were invited to fill out the study questionnaire in the departure hall of Bangkok International Airport. They were asked about their demographic profile, travel characteristics, pre-travel health preparations, their possible exposure and their practices related to rabies during this trip. From June 2010 to February 2011, 7,681 completed questionnaires were collected. Sixty-two percent of the travelers were male, and the median age was 32 years. 34.0% of the participants were from Western/Central Europe, while 32.1% were from East Asia. Up to 59.3% had sought health information before this trip. Travel clinics were the source of information for 23.6% of travelers. Overall, only 11.6% of the participants had completed their rabies pre-exposure prophylaxis, and 15.3% had received only 1-2 shots, while 73.1% had not been vaccinated at all. In this study, the risk of being bitten was 1.11 per 100 travelers per month and the risk of being licked was 3.12 per 100 travelers per month. Among those who were bitten, only 37.1% went to the hospital to get post exposure treatment. Travelers with East Asian nationalities and longer duration of stay were significantly related to higher risk of animal exposure. Reason for travel was not related to the risk of animal exposure. CONCLUSIONS: Travelers were at risk of being exposed to potentially rabid animals while traveling in Southeast Asia. Many were inadequately informed and unprepared for this life-threatening risk. Rabies prevention advice should be included in every pre-travel visit.

Hantaan virus antibody prevalence in rodent populations of several provinces of northeastern Thailand.

We conducted a serological survey of 632 rodents from the northeast region of Thailand in order to assess the presence of Hantaan-like viruses that may be a risk to the human population. Rodents were collected from rice fields, houses and domestic gardens in five northeastern provinces and tested for IgG reacting sera to Hantaan antigen using enzyme-linked immunoassays. The overall prevalence of Hantavirus infection in rodents was 2.1% (13/632). Species that tested positive included Bandicota indica (4.3% positive within species), Rattus exulans (2.1%), R. losea (1.6%) and R. rattus (0.9%). Species such as R. exulans and R. losea are candidate hosts of unidentified Hantaan-like viruses in Thailand.