A possible mechanism of macrolide resistance among multiple resistant Campylobacter jejuni and Campylobacter coli isolated from Thai children during 1991-2000.

A total of 495 Campylobacterjejuni and 122 C. coli isolated from Thai children were screened for macrolide (erythromycin and azithromycin) resistance by disk diffusion assay. Minimum inhibitory concentrations for erythromycin, azithromycin, nalidixic acid, ciprofloxacin, tetracycline, streptomycin, gentamicin and chloramphenicol were further determined for these macrolide-resistant Campylobacter isolates. Presence of known point mutations resulting in reduced susceptibility to macrolides was investigated by PCR and DNA sequencing. Seventeen percent (23/122) of C. coli and 2.4% (12/495) of C. jejuni isolates were resistant to macrolides. By sequencing domain V of the 23S ribosomal DNA from all 35 macrolide-resistant isolates, a known point mutation of 23S rRNA associated with reduced susceptibility to macrolides was detected in all isolates except one. Among the macrolide-resistant isolates, all were multiply resistant to nalidixic acid and ciprofloxacin, of which the latter is the preferred antimicrobial used for diarrheal treatment in Thailand. Furthermore, most macrolide-resistant isolates were also resistant to tetracycline and streptomycin. The spread of macrolide and quinolone resistant Campylobacter should be monitored closely in Thailand and elsewhere as these antimicrobials are preferred drugs for treatment of diarrhea.

Prospective study of the incidence of diarrheal disease and Helicobacter pylori infection among children in an orphanage in Thailand.

To evaluate the hypothesis that gastric infection with Helicobacter pylori increases risk for diarrheal disease in children, we conducted a yearlong prospective study among 160 orphanage children < 5 years of age in Nonthaburi, Thailand. Serum samples collected at six-month intervals were examined by ELISA for antibodies to H. pylori, and children were followed daily for the development of diarrhea. Seven percent of children were seropositive on enrollment, 59% were seronegative, and 34% were indeterminate. Among the seronegative children, seroconversion occurred at a rate of 7% per six months. Forty-six percent of children developed 214 total episodes of diarrhea. By age group, children < 18 months, 18-24 months and > 24 months of age experienced 2.6, 1.1, and 0.2 mean diarrhea episodes per six months. The incidence of diarrhea was not significantly different between children by H. pylori serostatus. We conclude that H. pylori infection was not associated with an increased risk of diarrheal disease.

An observational clinic-based study of diarrheal illness in deployed United States military personnel in Thailand: presentation and outcome of Campylobacter infection.

Campylobacter is a leading cause of traveler's diarrhea in Thailand. Since resistance to quinolones is high among Campylobacter isolates, empiric therapy with quinolones for traveler's diarrhea may be ineffective in this region. We conducted an observational study among 169 U.S. military personnel with acute diarrhea and compared their microbiologic findings to those of 77 asymptomatic personnel deployed to Thailand in May 1998. Of 146 pathogenic bacterial isolates, the most common were nontyphoidal Salmonella (n = 31), enterotoxigenic Escherichia coli (n = 24), and C. jejuni/coli (n = 23). Campylobacter was strongly associated with disease (odds ratio = 5.9; 95% confidence interval = 1.3-37.3), with a more severe clinical presentation, and with a reduced functional ability at presentation (P = 0.02). In vitro resistance to ciprofloxacin was observed in 96% of the Campylobacter isolates. Sub-optimal treatment response to ciprofloxacin was observed in 17% of the cases of Campylobacter infection versus 6% due to other causes. These results highlight the importance of Campylobacter as a cause of severe traveler's diarrhea in Thailand and illustrates the ongoing problem with antibiotic-resistant strains and associated treatment problems.

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.

Bacterial enteric pathogens in children with acute dysentery in Thailand: increasing importance of quinolone-resistant Campylobacter.

Current data on pathogen prevalence and drug resistance patterns are important for treatment and vaccine-development strategies. An etiologic study of acute bacterial dysentery was conducted in children up to 12 years of age in 2 major hospitals in and around Bangkok. Stool samples or rectal swabs and clinical data were collected. Standard microbiological methods were used to detect Salmonella, Shigella, Campylobacter, Vibrio, Aeromonas and Plesiomonas. Pathogenic E. coli (ETEC, EIEC, STEC) was identified by digoxigenin-labeled probes. A total of 623 cases were enrolled: median age 11.0 months (range 1 month-12 years). At least one bacterial pathogen was isolated in 55% of cases. Campylobacter was the most common pathogen found (28%), whereas Salmonella, Shigella and ETEC were isolated from 18%, 9% and 6% respectively. EIEC, Vibrio and Plesiomonas were isolated from <1% and no STEC was detected. C. jejuni serotypes 36, 4 and 11 were the most common. The mean age of cases with Campylobacter was significantly lower than with Shigella (17.9 vs 52.8 months, p<0.001). Clinical presentations of Campylobacter and Shigella infections were compared: fever (28% vs 37%), abdominal colic (62% vs 80%, p<0.05), vomiting (38% vs 70%, p<0.001) and bloody stools (52% vs 48%). The Campylobacter isolates (80% C. jejuni, 20% C. coli) were 90% resistant to ciprofloxacin but sensitive to macrolides. All the Shigella isolates (70% S. sonnei) were sensitive to quinolones. Our study illustrates the increasing importance of quinolone-resistant Campylobacter and the decline of Shigella in the etiology of dysentery in Thailand. The clinical presentation of campylobacteriosis is similar to that of shigellosis, except that the patients may be younger and there may be less association with colic and vomiting; having fecal leukocytes will be >10/HPF. The use of macrolide antibiotics rather than quinolones would be reasonable in children <24 months of age; fluoroquinolones will be ineffective in at least half of culture-positive cases.

Emerging fluoroquinolone and macrolide resistance of Campylobacter jejuni and Campylobacter coli isolates and their serotypes in Thai children from 1991 to 2000.

This study investigated fluoroquinolone, macrolide resistances and serotype distributions among Campylobacter jejuni and Campylobacter coli isolated from children in Bangkok and rural settings during 1991-2000. Phenotypic identification, serotyping, and susceptibility testing were performed by standard microbiological procedures. The predominant serotypes of C. jejuni were Lior 36, 2 and 4 and of C. coli were Lior 8, 29 and 55. Resistance to nalidixic acid increased significantly during 1991-2000 and the frequency of isolates resistant to both nalidixic acid and ciprofloxacin in Bangkok was significantly greater than in rural settings. In 1996-2000, a significant trend was observed in C. jejuni isolates resistant to ciprofloxacin from Bangkok but not for macrolide resistance from both settings. In summary, fluoroquinolone resistance among C. jejuni and C. coli isolates became widespread in both Bangkok and rural settings in Thailand in the 1990s while widespread resistance to macrolides was undetected.

Microbiology and diagnosis of infections with Shigella and enteroinvasive Escherichia coli.

The etiology of dysentery in Thailand and the existing methods of diagnosing infections with Shigella and enteroinvasive Escherichia coli (EIEC) are reviewed. The four Shigella species (S. dysenteriae, S. flexneri, S. boydii, and S. sonnei) are classically identified by culture of fecal specimens on selective media and testing of isolates for agglutination in species-specific antisera. DNA probes have been used to identify both lactose-fermenting and non-lactose-fermenting EIEC as well as Shigella isolates that do not agglutinate in antisera. These DNA probes are not necessary for the identification of Shigella if a competent bacteriology laboratory with shigella antisera is available. In Thailand Shigella and EIEC are isolated more often from children greater than 2 years of age than from younger children. The clinical illness associated with EIEC infections is similar to shigellosis. Fewer children with EIEC infections than with shigellosis, however, have occult blood in stool (36% vs. 82%) and more than 10 fecal leukocytes per high-power field (36% vs. 67%). Standard bacteriologic methods and testing of E. coli isolates for hybridization with the shigella/EIEC probe are currently the most sensitive means of diagnosing infections caused by these enteric pathogens. A more rapid method of identifying Shigella and EIEC infections in a situation where a bacteriology laboratory is not available will probably involve immunologic assays.

Molecular characterization and antibiotic susceptibility of Vibrio cholerae non-O1.

A collection of 64 clinical and environmental Vibrio cholerae non-O1 strains isolated in Asia and Peru were characterized by molecular methods and antibiotic susceptibility testing. All strains were resistant to at least 1 and 80% were resistant to two or more antibiotics. Several strains showed multiple antibiotic resistance (> or = three antibiotics). Plasmids most often of low molecular weight were found in 21/64 (33%) strains. The presence of plasmids did not correlate with antibiotic resistance or influence ribotype patterns. In colony hybridization studies 63/64 (98%) V. cholerae non-O1 strains were cholera toxin negative, whereas only strains recovered from patients were heat-stable enterotoxin positive. Forty-seven Bgl I ribotypes were observed. No correlation was shown between ribotype and toxin gene status. Ribotype similarity was compared by cluster analysis and two main groups of 13 and 34 ribotypes was found. Ribotyping is apparently a useful epidemiological tool in investigations of V. cholerae non-O1 infections.

Detection of Shigella and enteroinvasive Escherichia coli by PCR in the stools of patients with dysentery in Thailand.

The rate of detection of Shigella and enteroinvasive Escherichia coli (EIEC) using a PCR technique was compared with the rate detected by standard microbiological methods (bacteriology plus hybridization of E. coli colonies with a 17 kb EIEC probe) among patients with dysentery before and after antibiotic therapy. The PCR amplified DNA sequences encoding IpaH, a multiple copy sequence located on the chromosome and the invasion plasmid. Shigella or EIEC were detected using the IpaH PCR system among 72 (61%) of 119 patients with dysentery on the first day they were seen at hospital, compared to 50 (42%) using standard microbiological methods (p = 0.006). After three days of antibiotic therapy, IpaH sequences were detected in stools from 38 percent of patients, compared to 10 percent using standard microbiology (p < 0.001). After seven days of therapy, the rates were 26 percent vs. 8 percent respectively (p < 0.001). The IpaH PCR system appeared to be specific for Shigella or EIEC based on low rates of positive reactions among non-diarrhoea controls, and a strong correlation between persistently positive reactions and antibiotic resistance of bacterial isolates. IpaH sequences were detected in 10 (8%) of 119 drinking water samples from homes of patients with disease; none of these specimens were positive for Shigella or EIEC by standard microbiology. In conclusion, PCR amplification of IpaH sequences and detection of target DNA with a non-radioactive probe increased the rates of identification of Shigella and EIEC by 45% in initial clinical specimens and by nearly 300% in specimens obtained from patients receiving antibiotic therapy.

Isolation of group 2 aerotolerant Campylobacter species from Thai children with diarrhea.

Campylobacter species were isolated from 93 (15%) of 631 Thai children with diarrhea using the membrane filter technique on nonselective blood agar incubated at 37 degrees C. Campylobacter jejuni was isolated from 62 (10%), Campylobacter coli from 14 (2%), and atypical campylobacters from 17 (3%). The 17 atypical strains were first characterized biochemically and by dot blot DNA hybridization. Catalase-negative strains also were characterized by DNA hybridization and ribotype pattern. One strain was a catalase-negative "Campylobacter upsaliensis" and another was a nitrate-negative Campylobacter jejuni doylei. Fifteen isolates were aerotolerant strains most closely resembling Campylobacter cryaerophila or "C. upsaliensis" by dot hybridization. These aerotolerant strains, designated group 2 ("Campylobacter butzleri"), had ribotypes distinct from C. cryaerophila and have previously been shown to be related by DNA hybridization at the species level to the group 2 aerotolerant Campylobacter type strain (D2686). Group 2 aerotolerant Campylobacter were the atypical Campylobacter species most frequently isolated from Thai children with diarrhea.

Influence of strain characteristics and immunity on the epidemiology of Campylobacter infections in Thailand.

To determine how strain differences and immunity affect the clinical expression of Campylobacter infections, we conducted a study of acute diarrheal disease in Thailand in which specimens from children with Campylobacter infections were cultured weekly for up to 12 weeks to determine the serotype-specific length of time of convalescent-phase excretion and rate of reinfection. Levels of immunoglobulin G to cell-surface antigens of C. jejuni were determined in another population of healthy children who were closely related by age and location to the children in the diarrheal disease study. Campylobacter species were initially isolated from 18% of 586 children under 5 years old with diarrhea; most isolates in Thailand belonged to serotypes commonly found in developed countries. C. coli was significantly less often associated with symptomatic infections and with bloody diarrhea than C. jejuni (P less than 0.001 and P = 0.045, respectively). The peak age of isolation and the peak level of immunoglobulin G to Campylobacter species occurred before 2 years of age. The mean duration of convalescent-phase excretion was 14 +/- 2 (standard error of the mean) days for children less than 1 year old and 8 +/- 2 days for children 1 to 5 years old (P = 0.02, t test). Infection with another Campylobacter serotype was found in 34% of 105 children during the 12-week follow-up period. The rate of reinfection in these children was 15% (range, 8 to 22%) each week. Hyperendemic exposure to Campylobacter species in Thailand confers immunity to infection that is associated with an early peak in specific serum antibodies and an age-related decrease in the case-to-infection ratio and duration of convalescent-phase excretion but does not prevent asymptomatic infections.

Trends in antibiotic resistance among diarrheal pathogens isolated in Thailand over 15 years.

Antibiotic resistance trends were examined for Shigella species, nontyphoidal Salmonella species, enterotoxigenic Escherichia coli (ETEC), and Campylobacter species isolates from indigenous persons and travelers in Thailand for up to 15 years. Resistance to trimethoprim-sulfamethoxazole was found in >90% of Shigella and 40% of ETEC and nontyphoidal Salmonella isolates. Resistance to nalidixic acid was found in 97%-100% of Shigella dysenteriae 1 strains isolated between 1992 and 1995. Ciprofloxacin resistance was detected in 1% of ETEC isolates in 1994 and 1995 and in one of 349 nontyphoidal Salmonella isolates in 1995. Ciprofloxacin resistance among Campylobacter species increased from zero before 1991 to 84% in 1995 (P < .0001). Azithromycin resistance was found in 7%-15% of Campylobacter isolates in 1994 and 1995, as well as 15% of ETEC and 3% of Salmonella isolates in 1995. Enteric pathogens in Thailand have developed resistance to virtually all antibiotics routinely used in the treatment of diarrhea, as well as the newer fluoroquinolone and macrolide classes of drugs.

Phenotypic and molecular characterization of Vibrio cholerae O1 isolated in Samutsakorn, Thailand before, during and after the emergence of V. cholerae O139.

Seventy clinical strains of Vibrio cholerae O1 isolated from 1982-96 in Samutsakorn, a port city 30 km southwest of Bangkok where cholera occurs at low levels with regular seasonality, were characterized to investigate if there were any differences among the O1 strains isolated before, during and after the 0139 epidemic. Pulsed-field gel electrophoresis (PFGE) typing, ribotyping and southern blot hybridization with a cholera toxin probe (CT genotyping) demonstrated several genotypes among O1 strains isolated before the emergence of V. cholerae 0139. However, O1 strains isolated during and after the advent of 0139 showed identical ribotypes which were distinctly different from the types identified in strains isolated before the emergence of 0139. Ribotypes identified in strains during and after the advent of 0139 were also demonstrated by O1 strains isolated immediately before the emergence of 0139. Considering the seasonality of cholera in Samutsakorn, the identical ribotype and CT genotype and the closely related PFGE types shown by all O1 strains isolated during and after the appearance of 0139 is remarkable and suggest that the V. cholerae O1 strain may reemerge from an environmental source. A subgroup of V. cholerae O1 strains isolated before the emergence of the 0139 epidemic had a ribotype identical to a type demonstrated by 0139 strains isolated in Thailand. Our results support similar findings in Bangladesh and India that a distinct O1 strain appeared during the 0139 epidemic. However, compared with the apparent identical strain which replaced 0139 in Bangladesh and India, the emerged O1 strain in Samutsakorn showed a different ribotype and CT genotype.

Comparative study of ceftriaxone and trimethoprim-sulfamethoxazole for the treatment of chancroid in Thailand.

A single dose of ceftriaxone (250 mg) administered intramuscularly was compared with trimethoprim-sulfamethoxazole (TMP-SMZ; 160/800 mg) administered orally twice daily for seven days or with a single dose of TMP-SMZ (640/3,200 mg) administered orally for the treatment of chancroid in men in Thailand. Haemophilus ducreyi was isolated from 79 (48%) of 164 men with a clinical diagnosis of chancroid. For men with ulcers that were culture positive for H. ducreyi, rates of cure were 100% in 25 men treated with ceftriaxone, 87% in 23 men given TMP-SMZ for seven days, and 55% in 31 men given TMP-SMZ in a single dose. For men with ulcers that were culture negative for H. ducreyi, rates of cure were 100% in 29 men treated with ceftriaxone, 66% in 32 men given TMP-SMZ for seven days, and 63% in 24 men given TMP-SMZ in a single dose. The MIC50 of the three antibiotics for 94 isolates of H. ducreyi were as follows: 0.004 micrograms/ml for ceftriaxone, 16 micrograms/ml for trimethoprim, and greater than 512 micrograms/ml for sulfamethoxazole. Our study indicates that ceftriaxone in a single dose of 250 mg is effective, but that TMP-SMZ, even when given in a standard seven-day regimen, is not effective treatment for chancroid in Thailand.

Antimicrobial susceptibility and characterization of outer membrane proteins of Haemophilus ducreyi isolated in Thailand.

One hundred strains of Haemophilus ducreyi isolated in Thailand from patients with chancroid were tested by the agar dilution method against 10 antimicrobial agents and typed by outer membrane protein pattern by using sodium dodecyl sulfate gel electrophoresis. All strains produced beta-lactamase and were resistant to tetracycline, kanamycin, and sulfonamides. Most had a decreased susceptibility to trimethoprim (MIC for 50% of the strains [MIC50], 0.5 micrograms/ml) and chloramphenicol (MIC50, 8 micrograms/ml). Strains were susceptible to ciprofloxacin (MIC90, 0.001 micrograms/ml), ceftriaxone (MIC50, 0.0015 micrograms/ml), erythromycin (MIC50, 0.015 micrograms/ml), rosoxacin (MIC50, 0.03 micrograms/ml), and spectinomycin (MIC50, 8 micrograms/ml). The degree of antimicrobial resistance found in Thailand is higher than that reported for H. ducreyi isolated in other regions. Five different outer membrane protein patterns were found by analyzing proteins in the range of 29 to 61 kilodaltons, but 98% of the Thai strains fell into three patterns which did not differ greatly. Outer membrane protein patterns of Thai strains were also seen in strains from other geographic areas. A new outer membrane protein type was found among nine strains isolated in Singapore.

Vibrio cholerae O139 in Thailand in 1994.

Vibrio cholerae O139 first appeared in India and Bangladesh in 1992. Surveillance for O139 was started at three hospitals in Thailand in 1993. By 1994 all three hospitals surveyed in Thailand had experienced an increase in Vibrio cholerae O139 infections.

Erythromycin-resistant Campylobacter infections in Thailand.

Erythromycin therapy was compared with no treatment in a prospective trial of acute diarrheal disease among 100 infants in an orphanage in Bangkok. Within 24 h of the onset of diarrhea, 50 children received erythromycin ethylsuccinate (40 mg/kg per day) in four divided doses for 5 days. Campylobacter jejuni isolated from 31, Campylobacter coli isolated from 21, and Shigella spp. isolated from 21 of 100 children were the most commonly recognized pathogens; use of a sensitive, nonselective method substantially increased Campylobacter isolation. Treatment with erythromycin had no effect on the duration of diarrhea caused by Campylobacter spp., Shigella spp., or other agents; 37% of the treatment group and 35% of the control group had diarrhea for 1 week. Of 23 Campylobacter strains isolated from the treatment group before treatment, 15 (65%) were resistant (MIC, greater than or equal to 8 micrograms/ml) to erythromycin. Among orphanage-acquired strains, 53% of 43 C. jejuni strains and 91% of 23 C. coli strains were resistant to erythromycin compared with 11% of 114 C. jejuni strains and 46% of 35 C. coli strains that were community acquired. Erythromycin resistance is common among Campylobacter strains in Bangkok, especially in an institutional setting, which may account for the lack of efficacy of erythromycin for treatment of acute diarrheal illnesses.

The role of Haemophilus ducreyi in penile ulcers in Bangkok, Thailand.

In October 1982, penile ulcers were identified in 248 (19%) of 1282 men at Bangrak Hospital, Bangkok, Thailand. The etiology of the ulcers was determined for 120 (48%) of these patients. Haemophilus ducreyi was isolated from 45 (38%), herpes simplex virus from 14 (12%), and Neisseria gonorrhoeae from two (2%) of 120 patients. Syphilis was detected in one patient. Two patients had both H. ducreyi and herpes simplex virus isolated from the same ulcer. Thirty-five per cent of patients had a single ulcer, 56% had two to four ulcers, and 9% had more than four. Ulcers were located on the prepuce or coronal sulcus (69%), frenulum (18%), shaft (9%), and glans (4%). Inguinal adenopathy was present in only 11% of patients. H. ducreyi infection was diagnosed by gram stain in 28 (62%) of 45 patients from whom H. ducreyi was isolated and in only one of 75 patients from whom H. ducreyi was not isolated. Of the 45 isolates of H. ducreyi, 42 (93%) were isolated with use of media containing horse blood and 34 (76%) with use of media containing rabbit blood.

Polymicrobial aetiology of travellers' diarrhoea.

Of 35 US Peace Corps volunteers in Thailand, 20 (57%) had a total of 30 episodes of diarrhoea during their first 6 weeks in the country. Enteric pathogens were associated with 90% of the episodes. A single pathogen was identified in 17 (57%) episodes, 2-4 pathogens were identified in 10 (33%) episodes, and there were 15 symptomless infections. Enterotoxigenic Escherichia coli (ETEC) was identified in 37% of these episodes, and various salmonella serotypes were isolated in 33%. Infections with 9 other enteric pathogens were also identified: Campylobacter jejuni (17%), Plesiomonas shigelloides (13%), Aeromonas hydrophila (10%), Blastocystis hominis (7%), Norwalk virus (7%), Vibrio parahaemolyticus (3%), non-O1 Vibrio cholerae (3%), Vibrio fluvialis (3%), and rotavirus (3%). In total, 56 enteric infections were documented in 35 volunteers.

Enteropathogenicity of Aeromonas hydrophila and Plesiomonas shigelloides: prevalence among individuals with and without diarrhea in Thailand.

To evaluate the enteropathogenicity of Aeromonas hydrophila and Plesiomonas shigelloides, the rate of isolation of these organisms was compared among individuals with and without diarrhea in Thailand. In two groups of American travelers, A. hydrophila, but not P. shigelloides, was associated with episodes of travelers diarrhea more often than when individuals did not have diarrhea (P less than 0.025). Among three populations of Thais, A. hydrophila and P. shigelloides were isolated with similar frequencies from individuals with and without diarrhea. The biochemical characteristics, production of cytotoxin, and ability to distend suckling mouse intestine were similar among A. hydrophila isolates from individuals with and without diarrhea. However, cytotoxic A. hydrophila strains distended rabbit and suckling mouse intestine and produced destructive lesions in intestinal mucosa of both species of animal. P. shigelloides strains produced neither cytotoxin nor distended intestine. Oral administration of whole cultures (10(9)) of cytotoxic A. hydrophila or P. shigelloides failed to cause diarrhea in rhesus monkeys. Volunteer studies or intestinal biopsies of patients with diarrhea may be required to establish whether A. hydrophila is a gastrointestinal pathogen in humans.