Prevalence of enterotoxigenic Escherichia coli (ETEC) in hospitalized acute diarrhea patients in Denpasar, Bali, Indonesia.

The relationship between enterotoxigenic Escherichia coli (ETEC) and hospitalized patients with acute diarrhea was examined in a study conducted in two hospitals from June 2000 to May 2001 in Denpasar, Bali, Indonesia. A total of 489 hospitalized patients with acute diarrhea were enrolled, and their rectal swabs were screened for enteric bacterial pathogens. Toxins, colonization factor antigens (CFAs), in vitro antimicrobial susceptibility and seasonal distribution patterns associated with ETEC were ascertained. The diagnosis of ETEC infection and CFAs association were performed with GM-1 ELISA and Dot blot immunoassays. Enterotoxigenic Escherichia coli was isolated from the rectal swabs of 14.9% of the patients. The distribution of toxins among the ETEC strains found was ST in 51 (69.9%), while LT and ST/LT were found in 28.8% and 1.3% respectively. The highest isolation rate for ETEC was found among children between the ages of 1 and 15 years. Colonization factor antigens were identified in 28.8% of the ETEC strains. A high prevalence of CFA was found among the rectal swabs of patients with ST isolates. High frequency of resistance to ampicillin, trimethoprim/sulfamethoxazole, chloramphenicol, tetracycline and cephalothin was displayed among the ETEC strains. All ETEC strains were susceptible to norfloxacin, ciprofloxacin and nalidixic acid. The results of this study document the prevalence of ETEC in hospitalized patients with acute diarrhea in Denpasar, Bali, Indonesia. Data generated in this study depicts the prevalence of ETEC diarrhea and CFA types among diarrhea patients in the tourist city of Denpasar, Bali, Indonesia.

Characterization of Norwalk-like virus associated with gastroenteritis in Indonesia.

Norwalk Virus and Norwalk-like viruses (NLVs) are reportedly responsible for 2.5-4.0% of nonbacterial acute gastroenteritis (NBAG) worldwide. To help clarify the impact of NLVs on NBAG in Indonesia, stool specimens from 102 patients, 74 with NBAG and 28 with BAG, were screened for the presence of NLVs, using a reverse transcription-polymerase chain reaction (RT-PCR) assay. The specimens were subtyped using prototype-specific oligonucleotide probes and were sequenced and compared with published NLV sequences. Of the 102 specimens examined, 31 (30%) were found to be positive for NLVs. Type-specific probe analysis of the RT-PCR products indicated that 31 isolates hybridized to UK1 (Taunton agent) and UK3/4 (Hawaii agent/Snow Mountain agent) prototype strains. The results of this study indicate that prototype strains of NV or NLVs co-circulate in Indonesia and contribute to the overall level of acute gastroenteritis throughout the region.

Incidence of Norwalk-like viruses, rotavirus and adenovirus infection in patients with acute gastroenteritis in Jakarta, Indonesia.

Norwalk-like viruses (NLVs), rotavirus and adenovirus are reportedly responsible from 4 to 42% of non-bacterial acute sporadic gastroenteritis. The incidence of NLVs, adenovirus and rotavirus infections in Indonesia is unclear. A total of 402 symptomatic cases from Indonesian patients with acute gastroenteritis and 102 asymptomatic controls that tested negative for bacteria and parasites were screened for the presence of NLVs, rotavirus and adenovirus using the reverse transcriptase-polymerase chain reaction (RT-PCR), Rotaclone kits and Adenoclone kits. Specific prototype probes were used to ascertain which NLV prototypes were present in the area. NLVs were detected in 45/218 (21%), rotavirus was detected in 170/402 (42%) and adenovirus was detected in 11/273 (4%) samples examined. Genetic analysis of the RT-PCR products using specific prototype probes for NLVs indicated that the prototypes were 42% Taunton agent and 58% Hawaii/Snow Mountain agent. Comparative data on patients showed that the incidence of rotavirus infections was two times greater than the NLVs infections, and that adenovirus infections were the least prevalent. All of the control samples tested were negative for NLVs and adenoviruses, however 8/70 (11%) of the samples were positive for rotaviruses. The high incidence of enteric viral-related infections is a threat among acute diarrheic patients in Jakarta, Indonesia.

Experimental infection of Macaca nemestrina with a Toronto Norwalk-like virus of epidemic viral gastroenteritis.

Norwalk virus (NV) and Norwalk-like viruses (NLVs) are common etiologic agents of viral gastroenteritis. Viral gastroenteritis is a common disease that is highly transmissible, spreading rapidly through families, institutions, and communities. Because methods for in vitro cultivation of Norwalk etiologic agents are not available, information regarding this syndrome has come largely from studies in human volunteers. Sequential passaging of an NLV through an immunoincompetent newborn pigtail macaque (Macaca nemestrina) may allow for the adaptation of a human NLV to a primate host, thus providing an animal model for investigating this disease. A fecal filtrate of human origin containing NLV, Toronto virus P2-A, was obtained from a patient during an epidemic of viral gastroenteritis. The filtrate was administered via nasogastric tube to three newborn pigtailed macaques. Clinical illness, which was characterized by diarrhea, dehydration, and vomiting, occurred in three monkeys. Reverse transcription-polymerase chain reaction (RT-PCR) and oligonucleotide probe analysis of RNA extracted from the stool samples following infection revealed viral RNA in all inoculated monkeys. Infection was also transmitted experimentally by feeding two additional newborn macaques a fecal filtrate prepared from the three previously infected animals. Detection of viral RNA in the stools of animals that received the fecal filtrate indicates that viral replication occurred in association with clinical illness. The susceptibility of Macaca nemestrina to infection with a Norwalk-like agent will facilitate the study of the mechanisms of the pathogenesis of NLV. This system may also have the potential to serve as a vaccine test model for human epidemic viral gastroenteritis.

Toxins and colonization factor antigens of enterotoxigenic Escherichia coli among residents of Jakarta, Indonesia.

Infection caused by enterotoxigenic Escherichia coli (ETEC) poses a serious health problem among children and adults in developing countries. Colonization of the small intestinal mucosa by ETEC strains is mediated by antigenically specific fimbriae, also known as colonization factor antigens (CFA). The significance of this study arises from reports that active and passive immunization with ETEC strains harboring CFAs has previously been shown to induce protective immunity against diarrhea in animal models. The aim of this study was to determine toxin-associated CFAs of ETEC isolated from a diarrheal disease case-control study in Jakarta, Indonesia. Thirteen hundred and twenty-three diarrheic and control patients with lactose-fermenting colonies were screened by ganglioside GM1-enzyme-linked immunosorbent assay (GM1-ELISA) for heat-labile (LT) and heat-stable (ST) toxins. Two hundred and forty-six (19%) ETEC isolates identified by GM1-ELISA for the LT/ST toxins were screened for CFAs by Dot blot assay using monoclonal antibodies against CFA/I, II, and IV and against the putative colonization antigens (PCF) PCFO159, PCFO166, CS7, and CS17. Of the 246 ETEC isolates, 177 (72%) elaborated ST, 56 (23%) produced LT, while 13 (5%) elicited both the ST and LT toxins. CFA testing of the 246 ETEC isolates showed that 21 (8%) expressed CFA/I, 3 (1%) exhibited CFA/II, 14 (6%) elaborated CFA/IV, while 7 (3%) expressed PCFO159 and PCFO159 plus CS5. No CFAs or PCFs could be associated with 201 (82%) of the ETEC strains. This report documents the types of CFAs associated with ETEC strains in Jakarta, Indonesia. These data may help current research efforts on the development of CFA-based vaccines for humans against ETEC and provide additional information for future ETEC vaccine trials in Southeast Asia.

Shigella spp. surveillance in Indonesia: the emergence or reemergence of S. dysenteriae.

From June 1998 through November 1999, Shigella spp. were isolated in 5% of samples from 3,848 children and adults with severe diarrheal illness in hospitals throughout Indonesia. S. dysenteriae has reemerged in Bali, Kalimantan, and Batam and was detected in Jakarta after a hiatus of 15 years.

Vibrio parahaemolyticus associated with cholera-like diarrhea among patients in North Jakarta, Indonesia.

A diarrhea study was conducted in North Jakarta, Indonesia from December 1996 through December 1997. Vibrio parahaemolyticus was isolated from 333 (6.1%) of 5442 rectal swab samples collected from patients with cholera-like diarrhea. Vibrio cholerae O1 was isolated from 545 (10.0%) and V. cholerae non-O1 from 183 samples (3.4%), respectively. Patients positive for V. parahaemolyticus were mostly adults between 20 and 40 years of age, with males constituting 62%. A majority (65%) of these patients demonstrated watery diarrhea with a frequency of fewer than 10 episodes per 24 hour. A large number of the patients had abdominal pain (83%) and vomiting (76%) and were non-febrile (90%). The highest isolation rate (9.6%) of V. parahaemolyticus was found during the dry season (June, July) and the lowest (4.5%) in the rainy season (December, January, February). All of the V. parahaemolyticus isolates were hemolytic on human blood agar (positive Kanagawa) but none was urease positive. Disk diffusion antibiotic susceptibility tests performed on the isolates demonstrated resistance to ampicillin (98%), cephalothin (24%), kanamycin (15%), colistin (97%), neomycin (2%) and ceftriaxone (0.3%). All isolates (100%) were sensitive to chloramphenicol, tetracycline, trimethoprim-sulfamethoxazole, gentamicin, and ciprofloxacin.

In vitro antibiotic susceptibility of Neisseria gonorrhoeae in Jakarta, Indonesia.

Antibiotic susceptibilities were determined for 122 Neisseria gonorrheae isolates obtained from 400 sex workers in Jakarta, Indonesia, and susceptibilities to ciprofloxacin, cefuroxime, cefoxitin, cefotaxime, ceftriaxone, chloramphenicol, and spectinomycin were found. All isolates were resistant to tetracycline. A number of the isolates demonstrated decreased susceptibilities to erythromycin (MIC >/= 1.0 microg/ml), thiamphenicol (MIC >/= 1.0 microg/ml), kanamycin (MIC >/= 16.0 microg/ml), penicillin (MIC >/= 2.0 microg/ml), gentamicin (MIC >/= 16.0 microg/ml), and norfloxacin (MIC = 0.5 microg/ml). These data showed that certain antibiotics previously used in the treatment of gonorrhea are no longer effective.

Norwalk-like virus and bacterial pathogens associated with cases of gastroenteritis onboard a US Navy ship.

Acute gastroenteritis is a potential cause of substantial morbidity in U.S. military personnel during deployment. This study investigated the microbial causes of diarrhea in U.S. troops on exercises in Southeast Asia aboard the U.S.S. Germantown from March through May 1996. A total of 49 (7%) patients with diarrhea reported to sick call during a 3-month deployment involving 721 personnel. Diarrheal samples from 49 patients were subjected to bacterial and parasitologic examination, but sufficient samples from only 47 of 49 were available for analysis of the presence of Norwalk-like virus (NLV). Of the 49 diarrhea cases, 10 (20.4%) appeared to be due to bacterial etiology alone, 10 (20.4%) due to bacteria and the prototype Taunton agent (TNA), 11 (22.4%) due to TNA only, and 4 (8.0%) due to parasites. Norwalk-like virus RNA was present in 21 (45%) of 47 stool samples from the diarrhea cases, 10 with bacterial etiologies and 11 without bacterial or parasitic etiologies. No pathogen was detected in 14 (29%) of the cases. Four of the controls showed the presence of parasitic organisms. Of the 11 cases in which enterotoxigenic Escherichia coli was isolated, 8 were positive for colonization factor antigen (CFA/IV), and 3 were CFA-negative. The bacterial pathogens tested were all susceptible to gentamicin, and furadantin, but were resistant to ceftriaxone and norfloxacin, including 75% of the Campylobacter spp. These data support the view that the major cause of diarrhea for troops deployed in this geographic area is most likely NLVs.

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.

Persistent, recurring diarrhea in a colony of orangutans (Pongo pygmaeus) caused by multiple strains of Campylobacter spp.

A colony of 10 orangutans (Pongo pygmaeus) experienced persistent, recurring diarrhea caused by multiple infections with Campylobacter jejuni and C. coli. Infections appeared to have occurred through several mechanisms, including fecal-oral transmission between orangutans, and possibly transmission by houseflies contaminated with the organisms from nearby chicken feces. Among the 14 fecal and environmental C. jejuni isolates, 4 different antibiotic susceptibility profiles were detected; there were also 4 different profiles among the 8 isolates of C. coli. In 5 orangutans, there were back-to-back infections by different strains of C. jejuni, suggesting that a single C. jejuni infection may not confer protective immunity against heterologous strains circulating in the same vicinity. Transmission was effectively interrupted by environmental modifications and a 7-day course of oral erythromycin.

Modified CAMP test for biogrouping Vibrio cholerae O1 strains and distinguishing them from strains of V. cholerae non-O1.

A modified CAMP test was used to identify 973 Vibrio cholerae isolates by phenotype. Eltor and non-O1 strains were CAMP positive; classical strains were CAMP negative. Sausage-shaped zones of hemolysis of eltor strains were easily distinguished from narrower bands of non-O1 isolates. For O1 isolates, there was 100% agreement between the CAMP test and inhibition by polymyxin B.

Enterotoxigenic Escherichia coli and other causes of infectious pediatric diarrheas in Jakarta, Indonesia.

A hospital stool survey of Indonesian children less than 5 years of age determined the prevalence of diarrhea caused by enterotoxigenic Escherichia coli (ETEC) and other bacterial enteropathogens, compared to non-diarrheic control patients. ETEC were the second most frequent cause of diarrhea, isolated from 16 of 194 (8.2%) of patient's stools compared to 2 of 97 (2.1%) of control stools. The highest prevalence was in infants 12 to 23 months of age (17.9%).

Use of vaginal tampons in sewer surveys for non-O1. Vibrio cholerae.

Vaginal tampons were shown to be a practical alternative to conventional Moore swabs for isolating Vibrio cholerae from sewage. Associated laboratory investigations demonstrated improved isolation of V. cholerae by using 12- or 18-h enrichments in alkaline peptone water, in comparison with 6-h enrichments, when cultures were incubated at ambient temperatures.

The use of a coagglutination test to presumptively identify Salmonella typhi in bone marrow-oxgall medium cultures from typhoid fever patients.

A study was conducted to test a coagglutination procedure for detection of Salmonella typhi in bone marrow cultures from suspected typhoid patients admitted to Friendship Hospital, Jakarta, Indonesia. The results of the coagglutination tests were compared to the results from standard cultural isolation and identification. Bone marrow aspirates (356) were cultured in oxgall medium and aliquots subcultured daily for 7 days while simultaneously testing for the presence of Salmonella group D and Vi antigens using coagglutination (COAG). S. typhi was isolated from 220 (62%) of the cultures and the D- and Vi-COAG tests were positive for those same cultures. The COAG test was also negative for 6 cultures containing S. paratyphi A. The COAG results were available within 10 minutes after 18 to 24 hours incubation of the primary cultures whereas the isolation and confirmed identification took 2 to 3 days longer. The COAG test is valuable as an aid to rapidly identify S. typhi in bone marrow-oxgall cultures.

Rapid diagnosis of typhoid fever through identification of Salmonella typhi within 18 hours of specimen acquisition by culture of the mononuclear cell-platelet fraction of blood.

Detection of Salmonella typhi in blood by culture of the mononuclear cell-platelet layer was compared with other methods currently used for the diagnosis of typhoid fever. Colonies of S. typhi were present in all mononuclear cell-platelet layer-positive cultures within 18 h of plating and were identified within an additional 10 min by a coagglutination technique. In contrast, identification of all positive cultures by conventional blood culture required 3 days.

A penicillinase test incorporated in the rapid carbohydrate fermentation method for rapid detection of beta-lactamase producing Neisseria gonorrhoeae.

A modification of the acidometric (phenol red) test for penicillinase producing N. gonorrhoeae was incorporated into the rapid fermentation method for rapid screening and identification of PPNG strains. Two hundred and twenty-four non-penicillinase-producing N. gonorrhoeae, 55 penicillinase-producing N. gonorrhoeae, 87 N. meningitidis and 89 N. lactamica were included in this study. Results of the modified test were comparable with the iodometric and penicillin disk diffusion susceptibility and were obtainable within 1 to 5 minutes.

Isolation of Salmonella typhi from standard whole-blood culture vs blood-clot cultures.

The use of 10% oxgall and bile broth medium, both supplemented with freshly prepared 100 u/ml streptokinase, for isolating Salmonella typhi by clot culture technique was evaluated and compared against whole blood culture systems (3 ml blood in 9 ml media and 8 ml blood in 72 ml media). These gave a 1:4 and 1:10 blood to medium ratio, respectively. Clot cultures in 10% oxgall (CLOX) gave a 57% positive isolation rate for S. typhi. A similar result was obtained from clot cultures in bile broth medium (CLBB). A total of 184 samples identified as positive for S. typhi were tested. There was no significant difference between the use of 10% oxgall or bile broth medium when used for clot culture. The whole blood culture systems still showed a significantly better rate of isolation than the clot culture methods.