Prevalence and serodiversity of the pandemic clone among the clinical strains of Vibrio parahaemolyticus isolated in southern Thailand.

We collected diarrhoea specimens in two hospitals in southern Thailand in 1999 to examine whether infection by the Vibrio parahaemolyticus pandemic clone is prevalent. V. parahaemolyticus was isolated from 317 specimens. Seventy-six per cent of the isolated strains had the pandemic clone-specific characteristics (tdh+, trh-, and an unique toxRS sequence detectable by GS-PCR) and an associated characteristic (the ORF8 sequence of f237 phage). These strains belonged to the three pandemic servovars with the O3:K6 strains being dominant and three other serovars (O1:K25, O1:K41 and O4:K12). We also found O1:K25 and O1:K41 strains with the pandemic clone-specific characteristics among the strains isolated from the international travellers who left Thailand and three other Asian countries between 1998 and 1999, verifying pandemic potential of these strains. The results demonstrate prevalence of infection by the pandemic clone in southern Thailand and suggest emergence of various serovariants in this area and their implication in international spread.

Molecular epidemiologic analysis of Vibrio cholerae O1 isolated during the 1997-8 cholera epidemic in southern Thailand.

An unusually high incidence of Vibrio cholerae O1 infection was observed in southern Thailand between late December 1997 and March 1998. Fifty-seven V. cholerae O1 strains were isolated in five provinces during this epidemic and were examined. They were El Tor Ogawa strains exhibiting similar antibiograms. All strains were resistant to tetracycline, which had not been reported in Thailand since 1993. The ribotypes. hybridization patterns with ctx and zot gene probes, arbitrarily primed PCR profiles, and pulsed-field gel electrophoresis profiles of the representative strains were compared with the clinical strains isolated from patients in India and Bangladesh in 1997 and 1998 and from international travellers originating from various Asian countries during the 1992-8 period. All southern Thailand strains and the 1998 international traveller strain of Thai origin showed indistinguishable genetic fingerprinting patterns that were distinct from those of other test strains. The results suggest that a tetracycline-resistant clone newly emerged in late December 1997 caused the large epidemic in southern Thailand and that the variants with a slightly different antibiogram appeared during the course of the spreading epidemic.

An enzyme-linked immunosorbent assay to detect PCR products of the rfbS gene from serogroup D salmonellae: a rapid screening prototype.

We describe a digoxigenin-based enzyme-linked immunosorbent assay (DIG-ELISA) following a PCR to detect the amplified lipopolysaccharide rfbS gene as a means for rapid screening of serogroup D salmonellae in stool specimens. For pure bacterial cultures, the sensitivity of the PCR DIG-ELISA was approximately 10 bacteria. In the presence of stool materials, the salmonellae were first isolated by an immunomagnetic separation technique with an O9-specific monoclonal antibody. MATy-O9, followed by PCR and DIG-ELISA. The corresponding sensitivity was about 10 to 100 bacteria. To evaluate the assay performance clinically, 203 stool samples from patients with diarrhea were subjected to the routine culture techniques and the PCR ELISA method with overnight enrichment. The conventional culture method identified 145 salmonellae (31 serogroup B, 27 serogroup C, 83 serogroup D, and 5 serogroup E isolates) and 58 non-salmonella bacteria. The PCR ELISA method correctly identified all 82 serogroup D salmonellae (A405 by ELISA, 2.54 +/- 0.74) but was negative for the other Salmonella serogroups (A405, 0.26 +/- 0.08; n = 63) and non-Salmonella isolates (A405, 0.16 +/- 0.04; n = 58). In order to obtain a visible result, the assay takes approximately 6 h (PCR, 4 h; ELISA, 2 h), along with brief enrichment cultivation of the samples (from 4 to 16 h). Thus, the PCR DIG-ELISA offers a fast, accurate, semiquantitative means of detecting infectious agents such as salmonellae, and future robotic automation is possible.

Comparison of three stool-processing methods for detection of Salmonella serogroups B, C2, and D by PCR.

Three different stool sample-processing methods (centrifugation, immunomagnetic separation, and selective enrichment cultivation) for the identification of Salmonella serogroups by PCR were studied. The corresponding sensitivities in an ethidium bromide stained-agarose gel were 10(5), 10(3), and 10 bacteria, respectively. The PCR assay with overnight enrichment performed as well as, or even better than, the conventional culture technique. Of 485 clinical stool samples, PCR correctly identified all 230 culture-positive samples as well as mixed Salmonella infections in four cases.

Selective amplification of abequose and paratose synthase genes (rfb) by polymerase chain reaction for identification of Salmonella major serogroups (A, B, C2, and D).

Many parts of the Salmonella rfb gene clusters which are responsible for biosynthesis of the oligosaccharide-repeating units of the O-antigenic lipopolysaccharide have recently been cloned and sequenced. On the basis of this knowledge, three sets of nucleotide primers were selected to target defined regions of the abequose and paratose synthase genes: rfbJ of Salmonella serogroup B, rfbJ of Salmonella serogroup C2, and rfbS of Salmonella serogroup D (also present in serogroup A). For good differentiation among these major serogroups, the primers were designed not only to give precise specificity in priming but also to give DNA products with different sizes in polymerase chain reactions (product sizes, approximately 720 bp for both serogroups A and D, approximately 820 bp for serogroup C2, and approximately 882 bp for serogroup B). In a polymerase chain reaction assay utilizing these rfb-specific primers, all of the 40 salmonellae belonging to serogroups B, C2, and D plus A were accurately identified among a total of 123 clinical isolates tested (including 55 salmonellae from 36 different serotypes and 68 strains from 10 other members of the family Enterobacteriaceae). No false-positive reactions were detected. The selected rfb gene sequences were proved for the first time to be useful DNA-based markers for identification of and differentiation among Salmonella serogroups A, B, C2, and D.

Studies on shiga-like toxin produced by enterohemorrhagic Escherichia coli: purification and characterization of the toxin and development of methods for identifying the toxin.

A simple purification method using DEAE cellulose column chromatography and immunoaffinity column chromatography was developed for purifying Shiga-like toxin produced by Escherichia coli O157:H7. About 0.75 mg of purified toxin was obtained from 5 liters of culture (62% recovery). The purified toxin was demonstrated to be immunologically, biologically and structurally indistinguishable from Shiga toxin. A sandwich enzyme-linked immunosorbent assay (ELISA) was developed for detection of Shiga-like toxin. In the ELISA assay, Shigella dysenteriae type 1, Escherichia coli O157:H7 and some strains of Escherichia coli isolated from traveller's diarrhea were positive. Shiga toxin-resistant Vero cells were isolated by treatment of the cells with nitrosoguanidine. Immunofluorescence studies showed that the mutant Vero cells had lost toxin binding capacity. Samples of S. dysenteriae type 1 and E. coli O157:H7 showed cytotoxicity to the parent cells, but not to the mutant cells. Samples of other organisms showed either no cytotoxicity or cytotoxicity to both cell lines. The results suggested that (1) the presence of a receptor for Shiga-like toxin on Vero cells is essential for expression of cytotoxicity of the toxin, (2) the mutant Vero cells could be used to identify Shiga-like toxin producing organisms.

Noise exposure and permanent hearing loss of textile workers in Thailand.

Hearing loss induced by noise exposure in a large scale textile mill (number of workers = 1,611) in Thailand was investigated on the basis of interviews, noise measurements, and audiometric tests. The frequency of subjective symptoms relating to noise exposure was higher in the weavers than among other mill workers and office workers. The average noise levels in the weaving sections and other sections were 101.3 +/- 2.7 dBA and 89.8 +/- 5.3 dBA, respectively. The results of the audiometric tests revealed the significantly higher noise-induced hearing loss among workers in the weaving section compared to other mill workers and office workers (P less than 0.01). Among weavers, hearing levels decreased with the longer years of work. Concerning personal noise protective devices, 38.6% of the weavers never used them. It was concluded that hearing loss status in the workers of the mill was serious. Improvements by means of integrated work organization activities were recommended.

Isolation of Shiga toxin-resistant Vero cells and their use for easy identification of the toxin.

Shiga toxin-resistant Vero cells were isolated by treatment of the cells with nitrosoguanidine. These mutant cells were not affected by Shiga toxin at more than 1 microgram/ml, although the parent Vero cells were sensitive to 25 pg of the toxin per ml. Immunofluorescence studies showed that all the mutant cells had lost toxin-binding capacity. The cytotoxic activities of various bacterial cultures against the parent and mutant cells were compared. All samples from 10 strains of Shigella dysenteriae type 1 and all three strains of Escherichia coli O157:H7 tested showed cytotoxicity to the parent cells but not to the mutant cells. Samples from other organisms, such as Shigella flexneri, Shigella sonnei, Clostridium difficile, Aeromonas hydrophila, Aeromonas sobria, and other E. coli strains, either had no effect or were cytotoxic on both the parent and mutant cells. Thus, these mutant cells could be used to identify Shiga-like toxin and distinguish it from other cytotoxins. The results also suggest the presence of a receptor for Shiga-like toxin on Vero cells that is essential for expression of the cytotoxicity of Shiga toxin but is not essential for growth of Vero cells.

Enzyme-linked immunosorbent assay to detect Shiga toxin of Shigella dysenteriae and related toxins.

A sandwich enzyme-linked immunosorbent assay (ELISA) was developed for detection of Shiga toxin. Four species of Shigella, Escherichia coli, and Vibro parahaemolyticus were tested for production of Shiga or Shiga-like toxin by ELISA and Vero cell bioassay. In the ELISA, most strains of S. dysenteriae and some strains of E. coli isolated from traveler's diarrhea were positive. These ELISA-positive strains were positive by Vero cell bioassay without exception. Some E. coli strains and most V. parahaemolyticus strains were toxic to Vero cells, although they were negative in the ELISA. Much of the cytotoxic activity was not neutralized by anti-Shiga toxin antiserum. The newly developed sandwich ELISA is specific and can be a substitute for the cumbersome Vero cell bioassay.

Enzyme-linked immunosorbent assays for detection of thermostable direct hemolysin of Vibrio parahaemolyticus.

Several systems for enzyme-linked immunosorbent assay (ELISA) of thermostable direct hemolysin (TDH) of Vibrio parahaemolyticus were tested, and single-antibody sandwich ELISA systems gave satisfactory results. ELISA was able to detect as little as several nanograms of purified TDH per milliliter. The method of De Jong (J. Clin. Microbiol. 17:928-930, 1983) and the glutaraldehyde method were successful for preparing conjugates of alkaline phosphatase and anti-TDH antibody. TDH in fluids in intestinal loops of experimental animals challenged with living V. parahaemolyticus was accurately detectable by ELISA.

Glucose-6-phosphate dehydrogenase deficiency in South Vietnamese.

7 out of 534 South Vietnamese males showed erythrocyte glucose-6-phosphate dehydrogenase (G-6-PD) deficiency, giving a 1.31% incidence of G-6-PD deficiency. Partially purified erythrocyte enzyme was studied in 6 of the 7 G-6-PD deficient males. Three variants were found: G-6-PD Mahidol (3), Canton (2), and Long Xuyen (1).

Aerobic and anaerobic cervical flora of healthy Thai women.

The bacteriologic study of the cervical flora in 50 healthy women revealed that 70% harbored mixed aerobic and anaerobic organisms. Aerobic organisms alone were recovered in 15 women (30%). Polymicrobial organisms were found in all but five women (in these only one aerobe was isolated per patient). The majority of patients had 1-2 anaerobes, with more than two aerobes. The common aerobes were alpha-hemolytic streptococci, Staphylococcus epidermidis and lactobacilli. The most common anaerobes were Peptococcus asaccharolyticus isolated in 21 women (42%), P prevotii in 13 (26%) and Bacteroides in 10 (20%). These organisms are frequently found in pelvic infection, suggesting the pathogenic potential of the normal flora of the cervix.