Hybrid & El Tor variant biotypes of Vibrio cholerae O1 in Thailand.

BACKGROUND & OBJECTIVES: El Tor Vibrio cholerae O1 carrying ctxB C trait, so-called El Tor variant that causes more severe symptoms than the prototype El Tor strain, first detected in Bangladesh was later shown to have emerged in India in 1992. Subsequently, similar V. cholerae strains were isolated in other countries in Asia and Africa. Thus, it was of interest to investigate the characteristics of V. cholerae O1 strains isolated chronologically (from 1986 to 2009) in Thailand. METHODS: A total of 330 V. cholerae O1 Thailand strains from hospitalized patients with cholera isolated during 1986 to 2009 were subjected to conventional biotyping i.e., susceptibility to polymyxin B, chicken erythrocyte agglutination (CCA) and Voges-Proskauer (VP) test. The presence of ctxA, ctxB, zot, ace, toxR, tcpA C , tcpA E, hlyA C and hlyA E were examined by PCR. Mismatch amplification mutation assay (MAMA) - and conventional- PCRs were used for differentiating ctxB and rstR alleles. RESULTS: All 330 strains carried the El Tor virulence gene signature. Among these, 266 strains were typical El Tor (resistant to 50 units of polymyxin B and positive for CCA and VP test) while 64 had mixed classical and El Tor phenotypes (hybrid biotype). Combined MAMA-PCR and the conventional biotyping methods revealed that 36 strains of 1986-1992 were either typical El Tor, hybrid, El Tor variant or unclassified biotype. The hybrid strains were present during 1986-2004. El Tor variant strains were found in 1992, the same year when the typical El Tor strains disappeared. All 294 strains of 1993-2009 carried ctxBC ; 237 were El Tor variant and 57 were hybrid. INTERPRETATION & CONCLUSIONS: In Thailand, hybrid V. cholerae O1 (mixed biotypes), was found since 1986. Circulating strains, however, are predominantly El Tor variant (El Tor biotype with ctxB C).

Indicators for sexual HIV transmission risk among people in Thailand attending HIV care: the importance of positive prevention.

BACKGROUND: Almost half of all new HIV infections in Thailand occur among low-risk partners of people infected with HIV, so it is important to include people infected with HIV in prevention efforts. METHODS: Risk for HIV transmission was assessed among people with HIV attending routine care at the National Infectious Disease Institute in Thailand. Sexual risk behaviour, sexually transmitted infection (STI-syphilis, gonorrhoea, chlamydia, trichomoniasis and genital ulcers) prevalence and HIV disclosure status were assessed. Patients were provided with STI care, risk-reduction and HIV disclosure counselling. RESULTS: Baseline data were assessed among 894 consecutive people with HIV (395 men and 499 women) from July 2005 to September 2006. Unprotected last sex with a partner of unknown or negative HIV status (unsafe sex) was common (33.2%) and more likely with casual, commercial or male-to-male sex partners than with steady heterosexual partners (p = 0.03). People receiving antiretroviral treatment were less likely to report unsafe sex (p<0.001). Overall, 10.7% of men and 7.2% of women had a STI (p = 0.08). More women than men had disclosed HIV status to their steady partners (82.5% vs 65.9%; p = 0.05). CONCLUSION: Indicators for HIV transmission risk were common among people attending HIV care in Bangkok. Efforts need to be strengthened to reduce unsafe casual and commercial sex and to increase HIV disclosure from men to their partners. A strategy for STI screening and treatment for people with HIV in Thailand should be developed.

Impact of drug-resistant tuberculosis on the survival of HIV-infected patients.

OBJECTIVE: To determine the effect of drug-resistant tuberculosis (TB) on the survival of human immunodeficiency virus (HIV) infected patients in an area with a high prevalence of TB. DESIGN: Retrospective cohort study. RESULTS: Of 225 HIV-TB patients with a mean age of 35.8 years, 72.4% were male. The median CD4 cell count at TB diagnosis was 44 cells/mm3. Sixty per cent presented with extra-pulmonary TB (EPTB). Sixty-three (28%) patients were infected with Mycobacterium tuberculosis resistant to at least one drug; respectively 16.4%, 9.3%, 5.3% and 12.9% were resistant to isoniazid (INH), rifampicin (RMP), ethambutol and streptomycin, and 14 (6.2%) had multidrug-resistant TB (MDR-TB). During a median follow-up of 11.5 months, 4% died. From Kaplan-Meier analysis, INH resistance, RMP resistance and MDR-TB were associated with shorter survival (log-rank test, P < 0.005). Cox's proportional hazard model showed that MDR-TB (hazard ratio [HR] 11.7; 95% CI 2.1-64.9), not receiving antiretroviral therapy (ART) (HR 7.9; 95% CI 1.5-43.1) and EPTB (HR 5.1; 95% CI 1.9-25.9) were significant risk factors for death. CONCLUSION: MDR-TB and EPTB substantially reduce survival among patients co-infected with HIV and TB. Early detection and optimal treatment of MDR-TB are crucial. ART significantly prolongs survival and should be initiated in HIV-TB co-infected patients.

Genetic variations in Aeromonas hydrophila isolates from clinical and environmental sources in Thailand.

Aeromonas hydrophila, a widely distributed human pathogen causing a variety of diseases, can be isolated from clinical and environmental sources. Analysis in Thailand of 110 isolates of Aeromonas hydrophila by randomly amplified polymorphic DNA-PCR (RAPD-PCR) revealed one specific RAPD pattern group (G) that was associated only with strains from environmental sources. Cytotoxic activity, adhesion to epithelial cells and exoenzyme secretions of A. hydrophila were also investigated. A comparison of isolates with pattern group G with a set of isolates derived from human blood showed low induction of cytotoxicity from those with RAPD pattern group G suggesting low virulence of these strains.

Clinical disease associated with HIV-1 subtype B' and E infection among 2104 patients in Thailand.

BACKGROUND: Two HIV-1 envelope subtypes have accounted for virtually all infections in Thailand: subtype B' (Thai B), found mainly in injection drug users (IDU), and subtype E, found in over 90% of sexually infected persons and an increasing proportion of IDU in recent years. It remains unclear whether there are differences in pathogenesis associated with these HIV-1 subtypes. METHODS: From November 1993 to June 1996, demographic, risk, clinical, and laboratory data were collected by enhanced surveillance from HIV-infected inpatients (> or =14 years) at an infectious disease hospital near Bangkok. HIV-1 subtype was determined by V3-loop peptide enzyme immunoassay (EIA). Because of confounding, multivariate analyses were stratified by risk category and controlled for sex and age. RESULTS: The infecting HIV-1 subtype was determined for 2104 (94.9%) of 2217 HIV-infected patients with complete data: 284 (13.5%) were subtype B', 1820 (86.5%) were E. Specimens from 113 (5.1%) patients were non-reactive or dually reactive on peptide EIA and were excluded. Among IDU, 199 (67.2%) had subtype B', and 97 (32.7%) had E. IDU accounted for 70.1% (199/284) of patients with subtype B' and 5.3% (97/1820) of those with E. Patients infected with HIV-1 subtypes B' or E had similar spectrums of opportunistic infections (OI), levels of immunosuppression, and in-hospital mortality rates. Of patients who did not inject drugs, more patients infected with subtype E had extrapulmonary cryptococcosis than those with subtype B' (adjusted odds ratio, 2.6; 95% confidence interval, 1.28-5.37). CONCLUSION: HIV-1 subtypes B' and E seem to be associated with similar degrees of immunosuppression and, with one exception, with similar OI patterns. These data do not suggest an association between HIV-1 subtype and differences in pathogenicity.

Molecular characterization of Vibrio cholerae O139 isolates from Asia.

In 1992, a serologically novel clone of Vibrio cholerae, designated O139, caused large epidemics of diarrhea in India and Bangladesh. To determine the extent of the spread of V. cholerae O139 worldwide, 484 V. cholerae non-O1 strains isolated from different patients with diarrhea in Thailand, Indonesia, the Philippines, and Peru in 1993 were tested for agglutination in O139 antisera. One hundred fifty-one of these 484 isolates were examined for genes encoding cholera toxin, zonula occlulans toxin, the repetitive sequence 1, and the toxin coregulated pilin A (the V. cholerae virulence gene complex). Thirty-three percent (122 of 364) of V. cholerae non-O1 strains isolated from different patients with diarrhea in Thailand agglutinated in O139 antisera. Ninety-eight percent (120 of 122) of V. cholerae O139 contained the V. cholerae virulence gene complex. None of the 104 V. cholerae non-O1 strains isolated from patients with diarrhea in Indonesia or the 14 strains from patients with diarrhea in the Philippines were serotype O139. Four different ribotypes were found in V. cholerae O139 isolated in Asia. Twenty-three (47%) of 49 Thai O139 strains examined were of different ribotypes than isolates from India and Bangladesh; V. cholerae strains that were not O1 or O139 that were isolated from flies and water in Thailand 11 years previously in 1981 contained the same V. cholerae virulence gene complex found in V. cholerae O1 and O139. This suggests that other unidentified virulence determinants are involved in V. cholerae O139 pathogenesis.

A longitudinal study of the prevalence of bacterial enteric pathogens among adults with diarrhea in Bangkok, Thailand.

Six hundred sixty adults with diarrhea treated at Bamrasnaradura hospital, Bangkok, Thailand were investigated to determine the prevalence, seasonality, and severity of diarrhea associated with bacterial enteric pathogens in 1980 and 1981. Shigella were isolated from 27% and Vibrio parahaemolyticus from 19% of the patients studied. Enterotoxigenic Escherichia coli (5%), Salmonella (3%), non-01 Vibrio cholerae (3%), Campylobacter jejuni (1%), and Group F vibrio (less than 1%) were isolated from a smaller proportion of the patients. Shigella infections were most common in July-September, during the period of maximum rainfall in Thailand, while V. parahaemolyticus was isolated most frequently in September and October at the end of the rainy season. 0-1 Vibrio cholerae was isolated from 25% of 104 patients studied in the hot, dry spring of 1980, but was not isolated throughout 1981. Patients with cholera passed more watery stools, while those with Salmonella and Shigella most frequently had headaches, and those with Shigella more often had blood in their stools than those with other infections, or in whom no bacterial enteric pathogens were identified. Annual, seasonal, and, from a comparison with other reported surveys, geographical differences exist in the prevalence of bacterial enteric pathogens in adults with diarrhea in tropical developing countries.

Causes of chronic diarrhea in patients with AIDS in Thailand: a prospective clinical and microbiological study.

A prospective study was designed to investigate the causes of chronic diarrhea in AIDS patients in Thailand. Forty-five patients from Bamrasnaradura Infectious Diseases Hospital were enrolled. Extensive investigations included multiple stool examinations for ova and parasites, using the stool formalin-ether concentration method, stool culture, stool acid-fast bacilli (AFB) stain, stool modified AFB stain, esophagogastroduoscopy with duodenal aspirate and biopsy, and colonoscopy with biopsy. Biopsied specimens were examined with H&E, Giemsa, Gram, Periodic acid Schiff, and AFB stains. Definitive causes were found in 29 patients (64.4%). Of these 29, 7 patients were found to habor more than 1 pathogen (15.5%). The most commonly found enteric pathogen was Cryptosporidium parvum (20.0%). Less frequently found pathogens were Mycobacterium tuberculosis (17.8%), Salmonella spp. (15.5%), Cytomegalovirus (11.1%), Mycobacterium avium intracellulare (6.6%), Strongyloides stercoralis (4.4%), Giardia lamblia (4.4%), Cryptococcus neoformans (2.2%), Histoplasma capsulatum (2.2%), Campylobacter jejun (2.2%), and Cyclospora cayetanensis (2.2%). Salmonella spp., Mycobacterium tuberculosis, and Mycobacterium avium intracellulare infections were shown to be more common in Thailand than in African countries.

Comparison of the efficacy of ceftibuten and norfloxacin in the treatment of acute gastrointestinal infection in children.

A prospective randomized study was conducted at an infectious disease hospital in Thailand. Ceftibuten was compared with norfloxacin, both given orally for five days for treatment of acute gastroenteritis in children. One hundred and seventy cases were included in the study. Eighty-eight cases were treated with ceftibuten and eighty-two cases with norfloxacin. The baseline characteristics of the patients in both treatment groups were similar. The results showed that mean durations of diarrhea in the ceftibuten and norfloxacin groups were 2.48 days and 2.29 days, respectively, but there was no statistically significant difference between the two groups (p > 0.05). There were Salmonella spp and Shigella spp isolated in both treatment groups and all were susceptible to both antibiotics. The mean durations of Salmonella diarrhea in the ceftibuten and norfloxacin groups were 2.7 and 2.2 days, respectively, while those of Shigella diarrhea were 2.3 days and 2.0 days, respectively. There were no statistically significant differences in either comparison (p > 0.05). Neither complications nor clinical relapses were observed after both antibiotics' treatment.

Comparison of the efficacy of tetracycline and norfloxacin in the treatment of acute severe watery diarrhea.

Antibiotic treatment appears to shorten the duration of diarrhea and eradicate Vibrio cholerae. The objective of this study was to compare the efficacy of tetracycline with norfloxacin therapy in patients (adults and children) with acute severe watery diarrhea caused by VC 01 and VC 0139. Patients (adults and children) with acute severe watery diarrhea admitted to Bamrasnaradura Infectious Disease Hospital, Thailand were randomized to receive either tetracycline (500 mg qid in adults and 12.5 mg/kg qid in children) or norfloxacin (400 mg bid in adults and 7.5 mg/kg bid in children) for 3 days each. The duration of diarrhea and the fecal shedding were comparable between two groups. Thirteen cases were treated with tetracycline and twelve cases with norfloxacin. The results showed the mean duration of diarrhea in tetracycline-treated and norfloxacin-treated groups were 1.31 and 1.25 days, respectively. The mean fecal shedding in tetracycline-treated and norfloxacin-treated group were 1.38 and 1.33 days, respectively. However, there were no statistically significant differences between two groups of both comparisons (p > 0.05). All isolates (VC 01 and VC 0139) in this study were susceptible to both antibiotics. Tetracycline therapy is as good as norfloxacin therapy for quick recovery and time for bacterial eradication in patients with acute severe watery diarrhea caused by Vibrio cholerae. Children aged less than 8 years should not use tetracycline therapy because of its toxic effects.

Cryptosporidiosis in HIV infected patients in Thailand.

Diarrhea, mostly chronic diarrhea and weight loss are common in patients with AIDS. Cryptosporidium had been identified as responsible for chronic, debilitating secretory diarrhea in HIV infected patients. We performed a retrospective study of the prevalence, clinical features and laboratory findings of cryptosporidiosis in HIV infected patients (adults and children), in the period of 6 years from January 1988 to December 1993 at Bamrasnaradura Hospital in Nonthaburi, Thailand. In this study, Cryptosporidium was found in 22 (8.8%) by detection in stool specimens of 250 HIV infected patients with diarrhea and was found throughout the year. The prevalence rates of cryptosporidiosis in this study among children and adults were 19% and 7.9%, respectively. The common features were chronic diarrhea (84.6%), mostly watery diarrhea and weight loss/malnutrition (100%). A few fecal leukocytes were found in 15.4%.

Bacterial causes of AIDS-associated diarrhea in Thailand.

The incidence of bacterial diarrhea in AIDS patients has increased steadily and has led to enormous medical and public health problems. In this study, the clinical data together with 350 rectal swab samples each from AIDS patients with diarrhea (APD) and non-AIDS patients with diarrhea (NAPD), were collected and examined for bacterial enteropathogens at the Bamrasnaradura Infectious Diseases Hospital (BIDH), Nonthaburi, Thailand from May to December 1996. Patients were matched by age and sex. The majority of these patients were male (79%, 554/700), aged between 15 and 34 years (70.9%). The study found that the isolation rates of bacterial enteropathogens causing diarrhea in APD (18%, 62/350) were considerably lower than those in NAPD (43%, 152/350) (p<0.05). The infection rate with Salmonella group B (19.7%, 12/61) in APD was found to be significantly higher than that in NAPD (14.3%, 2/14) (p<0.05). Vibrio parahaemolyticus (53.3%, 81/152), Plesiomonas shigelloides (27%, 41/152), Aeromonas spp (19.1%, 29/152) and V. cholerae O1 (15.1%, 23/152), were more frequently detected in NAPD than in APD (p<0.05). Only nine Escherichia coli strains were isolated from APD, of which six were enteroinvasive E. coli, two enterotoxigenic E. coli and one enterohemorrhagic E. coli (non O157) possessing both vtl and vt2. No V. cholerae strains were detected in APD. The least effective antibiotics were ampicillin, tetracycline and cotrimoxazole. Antibiotic resistant patterns of the isolated organisms were similar from both groups. The results from this study might be useful in Thailand in the diagnosis and management of clinical cases of bacterial diarrhea, especially APD.

Nontyphoidal Salmonella diarrhea in Thai children: a study at Bamrasnaradura Hospital, Nonthaburi, Thailand.

From July 1, 1994 to June 30, 1996, 394 children with Nontyphoidal Salmonella diarrhea were studied at Bamrasnaradura Infectious Disease Hospital in Thailand. The ages ranged from 1 month to 12 years (mean, 1.4 years). Eighty-seven per cent of patients were in the first 2 years of life. Diarrhea, mostly acute with watery stool and fever were the most common presenting symptoms. The duration of diarrhea ranged from 1 to 20 days (mean, 5.2 days). Salmonella group B was the most common serogroup (56.1%). Most isolates were multiresistant strains, however, they were all sensitive to norfloxacin. Four (1%) patients were HIV-infected. Pneumonia found in 2 patients (0.5%) and septicemia in 1 patient (0.3%). None of the patients died. We conclude that nontyphoidal Salmonella diarrhea in children is still endemic in Thailand, especially among infants and high multidrug resistance occurs.

Oral rehydration therapy for invasive diarrhea.

Current guidelines for the management of diarrheal diseases focus on the use of oral rehydration therapy (ORT). Clinically evident dehydration of a moderate degree is not uncommon with invasive diarrhea, and when it occurs, the response to ORT is satisfactory. Studies from hospitals and the community each document the effectiveness of ORT for rehydration of patients with invasive diarrhea. This has been confirmed in a clinical trial of oral rehydration solution (ORS) for the treatment of diarrheal diseases in children less than 5 years of age. Children with moderate dehydration benefited most from ORS, especially those who had culture-proven invasive diarrhea. Significant early weight gain was observed for this group of patients alone after rehydration with ORS. In developing countries, there is no reason to withhold ORT at the first signs of watery or dysenteric diarrhea regardless of the cause and independent of the decision to treat the patient with antimicrobial agents.

Detection of Shigellae and enteroinvasive Escherichia coli by amplification of the invasion plasmid antigen H DNA sequence in patients with dysentery.

Detection of Shigella organisms and enteroinvasive Escherichia coli (EIEC) by polymerase chain reaction (PCR) was evaluated in 20 patients with dysentery before and in 17 of the 20 after treatment with ciprofloxacin. DNA sequences coding for IpaH antigen, a multiple copy sequence found on the chromosome, and the invasion plasmid locus (ial) was detected after DNA amplification in 13 stools from patients from whom shigellae or EIEC were isolated but not in 21 nondysenteric stools containing other enteric bacteria. Although shigellae or EIEC were not isolated from any patient with dysentery after ciprofloxacin treatment, IpaH and ial sequences were found after PCR amplification in 7 patients after treatment with ciprofloxacin. IpaH sequences alone were detected in 4 patients; DNA augmentation of IpaH in stools in a specific way to identify Shigella or EIEC infection in persons from whom cultures cannot be obtained promptly after the onset of diarrhea or who have received antibiotics.

Recovery of Cryptococcus neoformans from the nasopharynx of AIDS patients.

Nasopharyngeal swabbings, obtained from AIDS patients, were plated onto Niger seed agar containing antibiotics Cryptococcus neoformans was isolated from 35 out of 84 patients (41.7%) diagnosed as primary cryptococcal cases before antifungal administration, and 8 out of 86 (9.3%) cryptococcosis patients on antifungal therapy. The fungus could not be isolated from any of 447 samples from 194 AIDS patients not diagnosed with cryptococcosis. These findings are novel in that the presence of C. neoformans in AIDS patients at this site has never been looked at previously.

Serotypes of Cryptococcus neoformans isolated from patients prior to and during the AIDS era in Thailand.

One hundred and eighty-seven strains of Cryptococcus neoformans isolated from patients in Thailand were characterized by biochemical varieties relating to serogroups. Canavanine-glycine-bromothymol blue (CGB) agar was used for differentiating the varieties of C. neoformans. Slide agglutination tests were performed with Crypto Check (Iatron, Inc., Tokyo) to determine their serotypes. Fifty-five percent (10 out of 18) of the pre-AIDS isolates were serotype B, 28% were serotype A, 5% were serotype D, and an unexpected 11% (2 out of 18) were serotype C. These are the first to be recorded in Asia. In contrast, among the 169 clinical isolates obtained between January 1993 and March 1995 (AIDS epidemic), serotype A was outstandingly predominant--93% (157 out of 169), serotype B was relatively low (3.6%) and both serotypes D and AD were 1.8%. The pattern of serotypes of the 59 isolates from known HIV-positive patients was closely similar to the total isolates during the AIDS epidemic. In determining the varieties of C. neoformans by CGB, only 1 of the 187 isolates gave a false reaction. On the basis of our findings, we believe that in the pre-AIDS era either C. neoformans var. gattii serotype B or serotype C were the common causative agents of cryptococcosis in Thailand. The advent of AIDS changed the pattern of serotypes with serotype A becoming predominant as has been reported world wide.

Molecular typing of Penicillium marneffei isolates from Thailand by NotI macrorestriction and pulsed-field gel electrophoresis.

Penicillium marneffei is recognized as one of the most frequently detected opportunistic pathogens of AIDS patients in northern Thailand. We undertook a genomic epidemiology study of 64 P. marneffei isolates collected from immunosuppressed patients by pulsed-field gel electrophoresis (PFGE) with restriction enzyme NotI. Among the 69 isolates fingerprinted by PFGE, 17 were compared by HaeIII restriction endonuclease typing. The PFGE method demonstrated a higher degree of discriminatory power than restriction endonuclease typing with HaeII. Moreover, an impressive diversity of P. marneffei isolates was observed, as there were 54 distinct macrorestriction profiles among the 69 isolates of P. marneffei. These profiles were grouped into two large clusters by computer-assisted similarity analysis: macrorestriction pattern I (MPI) and MPII, with nine subprofiles (MPIa to MPIf and MPIIa to MPIIc). We observed no significant correlation between the macrorestriction patterns of the P. marneffei isolates and geographical region or specimen source. It is interesting that all isolates obtained before 1995 were MPI, and we found an increase in the incidence of infections with MPII isolates after 1995. We conclude that PFGE is a highly discriminatory typing method and is well suited for computer-assisted analysis. Together, PFGE and NotI macrorestriction allow reliable identification and epidemiological characterization of isolates as well as generate a manageable database that is convenient for expansion with information on additional P. marneffei isolates.

Urease production correlates with possession of the trh gene in Vibrio parahaemolyticus strains isolated in Thailand.

A total of 489 Vibrio parahaemolyticus isolates from patients in Thailand with diarrhea was examined for the presence of thermostable direct hemolysin (TDH) and TDH-related hemolysin genes (tdh and trh, respectively), their serovars, TDH production, and urease activity. Of the strains, 81% were positive only for the tdh gene, 6% for both trh and tdh genes, and 2% for the trh gene only. Thirty-seven (8%) of the 489 isolates were positive for urease production. Of special interest, all urease-positive strains possessed the trh gene, and conversely, urease-negative strains lacked the gene, indicating that urease production by V. parahaemolyticus strains strongly correlates with the possession of the trh gene. Thus, the urease-positive phenotype of V. parahaemolyticus can be considered an indication of virulent (trh-possessing) V. parahaemolyticus strains in clinical diagnosis.

Colonization factors associated with enterotoxigenic Escherichia coli isolated in Thailand.

Eighty-six percent (72 of 84) of heat-labile and heat-stable, none of 141 heat-labile, and 24% (27 of 111) heat-stable enterotoxigenic Escherichia coli isolates from Thailand aggregated in less than 1 M (NH4)2SO4, hemagglutinated human group A and bovine erythrocytes in 1% D-mannose, and possessed either colonization factor I or colonization factor II. No other colonization factors were identified by these two methods.