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.

Pre-exposure anti-Campylobacter jejuni immunoglobulin a levels associated with reduced risk of Campylobacter diarrhea in adults traveling to Thailand.

Diarrhea history questionnaires were administered to 369 U.S. military volunteers before and after deployment to Thailand. Additionally, blood samples obtained from a subset of 221 volunteers 1-3 weeks previously and 3-4 weeks after their deployment were tested by enzyme-linked immunosorbent assay for immunoglobulin A to Campylobacter jejuni. Stool samples from personnel (including volunteers) contracting diarrhea in Thailand were cultured for enteric pathogens. Overall, 35.2% (130 of 369) of questionnaire respondents reported one or more diarrhea episodes during their trip. Volunteers with pretravel anti-C. jejuni reciprocal titers < or = 450 were 1.6 times as likely to have had diarrhea during their stay in Thailand compared with those with pretravel titers > 450 (39.7% versus 25.3%; P = 0.05). The symptomatic seroconversion, or attributable Campylobacter diarrhea attack rate, for the 1-month exercise was 12.7% (28 of 221). The symptomatic seroconversion rate in nonimmune (titer < or = 450) volunteers was 17.1%, whereas that in immune volunteers was only 4.0% (P = 0.002). Campylobacter jejuni or C. coli were recovered from 32.9% (56 of 170) of stool samples cultured and were the most commonly identified enteropathogens. Campylobacter diarrhea was associated with elevated temperatures, fecal red cells, and fecal white blood cells. The results of this study show that Campylobacter continues to represent a significant health threat to Western travelers to Thailand, but many of these travelers have preexisting Campylobacter immunity that protects them from clinically significant Campylobacter enteritis.

Comparison of direct-plating and enrichment methods for isolation of Vibrio cholerae from diarrhea patients.

A direct-plating method on thiosulfate citrate bile salts sucrose agar (DIR-TCBS) in conjunction with enrichment in alkaline peptone water (APW) incubated for both 6 h and 24 h followed by subculture onto TCBS (APW6h-TCBS and APW24h-TCBS, respectively) was performed on 16,034 rectal swab samples for isolating Vibrio cholerae. A total of 2,932 (18.3%) rectal swab samples were positive for V. cholerae O1 biotype El Tor, with the Ogawa serotype constituting 99.2% of the isolates. There were no significant differences in V. cholerae O1 isolation rates between the three culture systems nor between the combinations of any two systems. However, direct plating plus enrichment demonstrated a significantly higher V. cholerae O1 isolation rate than DIR-TCBS alone (P < 0.02). Conversely, enrichment procedure, alone or in combination with DIR-TCBS, yielded significantly more (P < 0.0001) V. cholerae non-O1 isolates than DIR-TCBS alone. The length of incubation time of the enrichment broth, 6 h, offers no significant advantages over 24 h for the isolation of V. cholerae O1 and non-O1. A 24-h enrichment broth incubation period has the practical advantage of being easy to integrate into a normal laboratory workday, whereas 6-h broth enrichment, although more commonly recommended, requires that arrangements be made for after-hours subculture.

Simple differentiation of Vibrio cholerae O139 from V. cholerae O1 and non-O1, non-O139 by modified CAMP test.

Strong positive CAMP reactions were demonstrated by 121 Vibrio cholerae O139 and 504 El Tor isolates, and weak positive CAMP reactions were shown by 235 non-O1, non O139 isolates when these isolates were tested by a modified CAMP technique. Thirty-five classical biotype V. cholerae O1 isolates included in the tests were all CAMP negative.

Fiber-optic fluorometric sensing of polymerase chain reaction-amplified DNA using an immobilized DNA capture protein.

A fiber-optic assay for amplified DNA products has been developed. Modifications of the DNA capture strategy described previously by Kemp et al. [Proc. Natl. Acad. Sci. USA 86, 2423-2427 (1989)] were made that allowed selective binding of DNA labeled during the amplification process to the sensing surface of fused silica fibers. The gene for a chimeric protein composed of the IgG-binding beta 2 subdomain of streptococcal protein G fused with the DNA binding domain of yeast GCN4 was constructed, and this PG/GCN4 protein was overexpressed in Escherichia coli. The purified protein was noncovalently bound to IgG-modified fibers utilizing strong and specific interactions between the protein G beta 2 domain and goat IgG that had been covalently immobilized on the fiber surface. Nanomolar concentrations of amplified DNA labeled with the fluorophore tetramethylrhodamine and the AP-1 consensus nucleotide sequence recognized by GCN4 (5'-ATGACTCAT) were rapidly and selectively bound within the evanescent zone of multimode laser-illuminated fibers. Signal from unincorporated fluorescent PCR primer was negligible. Individual fibers could be used for multiple sequential assays, since the fluorescent double-stranded DNA was rapidly and completely stripped from their surfaces with high salt solutions, leaving the IgG-PG/GCN4 DNA binding complex intact to accept another PCR sample.

Internalization of oligodeoxyribonucleotides by Vibrio parahaemolyticus.

The bacterium Vibrio parahaemolyticus was tested for its ability to internalize unmodified as well as modified DNA oligomers without attempting to permeabilize the cells. These experiments were conducted to establish whether it may be feasible to employ antisense oligomers for control of gene expression in Vibrio species without heat-shocking or electroporating the cells. The bacterium was found to bind radiolabeled synthetic oligodeoxyribonucleotides that were added to culture media. Incorporation of a phosphorothioate oligomer into subcellular regions was determined following cellular fractionation. The phosphorothioate was recovered primarily from the periplasm and peptidoglycan layer of the bacterium; however, a significant fraction was recovered from the bacterial cytosol. The extent of uptake depended on both the concentration of oligomer as well as culture medium selected. A maximum of 2.1 x 10(6) oligomers/cell was achieved when a 12-mer phosphorothioate oligomer (10 microM) was added to bacterial cultures in an artificial seawater (Instant Ocean) medium. Several terminally modified oligomers were found to become associated with bacterial cells, albeit less efficiently than the phosphorothioate. None of the oligomers tested was toxic to the bacteria at 0.1 microM, and the phosphorothioate was only marginally toxic at 10 microM. Stability of the oligomers in extracellular and cell-associated fractions was evaluated by PAGE; even after 8 hr of incubation intact phosphorothioate oligomer could be found in both components.

A comprehensive survey of pediatric diarrhea at a private hospital in Metro Manila.

The epidemiology of diarrhea among Filipino pediatric patients, representing a cross-section of socioeconomic strata, was investigated over a one year period. Rotavirus was detected in 33.9% of the diarrhea stools examined and was the leading cause of diarrhea in the study population. Although proportionately more rotavirus was found during the cold season, most children became infected with rotavirus during the rainy season, when diarrheal disease was at its peak in Metropolitan Manila. Enteric adenovirus types 40 or 41 were associated with only 5.4% of the diarrhea cases. Overall, one or more etiologic agents of diarrhea were detected in 67.2% of the stools examined. Many of these positive stools (21.6%) contained multiple diarrheogenic agents. Bacterial enteric pathogens were isolated from 32.3% of the cases. Nearly 70% of these patients with bacterial gastroenteritis became ill during the rainy season. Etiology specific and general risk factors associated with diarrheal illness in the study population are discussed.

Multiple proteins encoded within the urease gene complex of Proteus mirabilis.

Chromosomal DNA fragments from a uropathogenic isolate of Proteus mirabilis were inserted into the cosmid vector pHC79 to construct a genomic library in Escherichia coli HB101. A urease-positive recombinant cosmid, designated pSKW1, was recovered. Sequential recombinant manipulation of pSKW1 yielded a 10.2-kilobase plasmid, designated pSKW4, which encoded three urease isozymes with electrophoretic mobilities identical to those of the donor P. mirabilis strain. Plasmid pSKW4 gene sequences encode seven proteins designated 68K (apparent molecular weight, of 68,000), 28K, 25K, 22.5K, 18.5K, 7.5K, and 5.2K within the limits of the urease gene complex. Insertion mutations in genes encoding the 68K, 28K, 25K, 22.5K, 7.5K, and 5.2K proteins resulted in complete or partial (22.5K) loss of urease activity. There was no reduction in urease activity when the gene encoding the 18.5K protein was inactivated.