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Prevalence of enterotoxigenic Escherichia coli (ETEC) in hospitalized acute diarrhea patients in Denpasar, Bali, Indonesia
Diagnostic Microbiology and Infectious Disease 47 (2003) 399 – 405
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Prevalence of enterotoxigenic Escherichia coli (ETEC) in hospitalized acute diarrhea patients in Denpasar, Bali, Indonesia D. S. Subektia, M. Lesmanaa,b, P. Tjaniadia, N. Machpuda, Sriwatic, Sukarmad, J. C. Daniela, W. K. Alexandera, J. R. Campbella, A. L. Corwina, H. James Beecham IIIa, C. Simanjuntake, B. A. Oyofoa,* a
U.S. Naval Medical Research Unit No. 2, Jakarta, Indonesia b Medical Faculty Trisakti University, Jakarta, Indonesia c Wangaya Hospital, Denpasar, Indonesia d Sanglah Hospital, Denpasar, Indonesia e National Institute of Health Research and Development Ministry of Health, Jakarta, Indonesia
Abstract 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. © 2003 Elsevier Inc. All rights reserved. Keywords: Enterotoxigenic Escherichia coli (ETEC); Acute diarrhea; Hospitalized patients; Denpasar; Indonesia
1. Introduction Enterotoxigenic Escherichia coli (ETEC) is the most frequently isolated enteropathogen, accounting for about 210 million diarrhea episodes and approximately 380,000 deaths annually. The peak of incidence of ETEC diarrhea in a community-based study was reported to occur in the first year of life, with a declining incidence with age, thereafter (WHO, 1999). ETEC is transmitted by food or water contaminated with animal or human feces. Enterotoxigenic Escherichia coli is a leading cause of morbidity and mortality in children up to 5 years of age in developing coun* Corresponding author. Tel.: 011-62-21-4214457/58; fax: 011-6221424-4457. E-mail address: [email protected] (B. A. Oyofo). 0732-8893/03/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0732-8893(03)00120-2
tries and was first recognized as a cause of human diarrheal illness in the 1960s (Black, 1993; Sack et al., 1971). In addition, ETEC is a major bacterial cause of diarrhea among travelers from industrialized to less developed countries (Arduino & DuPont, 1993). Strains of ETEC have two major virulence determinants: the enterotoxins (the heat-labile toxin/LT and heat-stable toxin/ST) and colonization factor antigens (CFAs). To cause diseases, ETEC must adhere to the epithelium of the small intestine by means of CFA. Secretory diarrhea is then produced due to the effects of the enterotoxins (Gaastra & Svennerholm, 1996). Colonization factor antigens on human ETEC strains are usually protein fimbrial antigens, which are capable of agglutinating erythrocytes from different animal species in the presence of D-mannose (Evans et al., 1979). Recognized CFAs associated with human
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ETEC isolates are CFA/I, CFA/II and CFA/IV. Colonization factor antigen I is a single fimbrial antigen (Evans et al., 1975), while CFA/II and CFA/IV have been shown to consist of three distinct fimbrial antigens called Coli Surface (CS) antigens. Colonization factor antigens II is composed of antigens CS1, CS2 and CS3 (Smith et al., 1983). Colonization factor antigens IV is comprised of antigens CS4, CS5 and CS6 (Thomas et al., 1985). The other Coli Surface antigens are CS7, CS8, CS12, CS14, CS11, CS13, CS15, and CS17 (Gaastra & Svennerholm, 1996). In brief, the GM1 immunologic assay has been used to detect enterotoxins of ETEC, ST and LT. This assay is based on the ability of toxin (ST or LT), as present in culture filtrates from ST or LT-producing E. coli, to inhibit specific anti-ST or LT antibody from binding to solid phase bounded ST or LT ganglioside (GM1-bound ST or LT-cholera B subunit). The purified B subunit of cholera toxin (CTB) is then able to bind to GM1 ganglioside, which is the receptor for the CTB absorbed to the ELISA microtiter wells. The presence of the toxin is detected by the addition of a specific antibody, followed by the addition of an enzyme-conjugate antibody. A positive test is indicated by a color reaction. This test has 100% sensitivity and specificity when compared with cell culture assay (Svennerholm & Wiklund, 1983; Svennerholm et al., 1986). This city of Denpasar, Indonesia is a busy commercial center for visitors throughout the year. Due to the diversity of travelers that visit the area, we reasoned that the knowledge of the ETEC and CFAs in the population would provide useful data for the evaluation of ongoing efforts to develop ETEC vaccine. It is suggested that serious efforts should be made to design more effective ETEC vaccines for the prevention of ETEC infection, especially in countries of the world where ETEC is endemic (WHO, 1999). The aim of this study was to determine the prevalence of ETEC and the variation of CFAs among hospitalized diarrheal patients in the tourist and business city of Denpasar, Bali, Indonesia.
2. Materials and methods 2.1. Study population Two hospitals (Wangaya and Sanglah) in Denpasar, Bali, Indonesia, were selected for the study that was conducted from June 2000 through May 2001. Denpasar is the biggest city in Bali Island. Bali is the most popular island for travelers among the 17,000 islands in Indonesia, and it is located between 8 to 9 degrees South of the equator at longitude 115 degree East. The two hospitals are close to each other about 1 mile distance. Both are also located in the center of the city. They were selected based on the fact that they are the two largest hospitals in Denpasar, both in terms of hospital beds and the number of diarrhea cases seen per
year. The other factor in their selection was their willingness to participate in the study. 2.2. Subjects and specimen collection During the period of June 2000 through May 2001, a total of 489 rectal swabs were collected from acute ill diarrhea patients hospitalized at Sanglah and Wangaya hospitals in Denpasar, Bali, Indonesia. All patients presenting with diarrhea and admitted to the hospitals were included in this study, regardless of sex and age. The participating hospital staff was instructed to obtain rectal swab samples from each diarrhea patient on the day of admission before antibiotics were given. Personal data and symptoms of the illness including the type of diarrhea were obtained. Specimens were transported to the U.S. Naval Medical Research Unit No.2 (NAMRU-2) laboratory in Cary-Blair transport medium and processed immediately. Specimens obtained in the evening or at night were kept in a refrigerator at 2-8°C and transported in a cool box the following day. 2.3. Laboratory processing Rectal swabs collected were stored at 2-8°C in CaryBlair media and processed immediately after arrival at the Microbiology Laboratory located at NAMRU-2. Upon arrival at the laboratory, rectal swabs were routinely screened for enteric pathogens including Vibrio spp., Salmonella enterica, Shigella spp., and Campylobacter jejuni by standard techniques (Tison, 1999; Bopp et al., 1999; Goosens & Butzle, 1992; Lesmana et al., 1997). For the detection of ETEC, the rectal swabs were plated onto MacConkey agar and incubated at 37°C for 18-24 h. Five lactose-fermenting individual colonies morphologically resembling E. coli were tested immediately after isolation for the presence of toxins and CFAs as described below. 2.4. Detection of toxin types and CFAs Rectal swabs obtained from patients were cultured on MacConkey agar plates for selection of E. coli isolates. After overnight culture, five E. coli lactose-fermenting colonies from each patient were picked and stored on nutrient agar and analyzed for heat-labile (LT) and heat-stable (ST) enterotoxin by ganglioside GM1-enzyme-linked immunosorbent assay (Svennerholm et al., 1986). For these purposes E. coli colonies were inoculated into separate wells of a GM1 coated microtiter plate containing Luria–Bertani broth with lincomycin and glucose and incubated overnight at 37°C with agitation (150 rpm). After incubation, the broth culture was transferred to fresh GM1 coated plates and tested for ST by an inhibition ELISA procedure with the ST-cholera toxin B subunit (CTB) conjugate (ST-CTB) as described earlier (Svennerholm et al., 1986). Luria–Bertani broth from the original plate used for culture was analyzed for GM1 bound LT by using a specific-mouse monoclonal
D.S. Subekti et al / Diagnostic Microbiology and Infectious Disease 47 (2003) 399 – 405 Table 1 Enteropathogens found among 489 hospitalized diarrhea patients in Denpasar, Bali, Indonesia Enteropathogens
No. positives (%)
ETEC* ETEC* ⫹ Salmonella enterica ETEC* ⫹ S. flexneri Vibrio cholerae Vibrio parahaemolyticus Salmonella enterica Shigella flexneri Shigella sonnei Campylobacter jejuni Total identified Enteropathogens
71 (14.5) 1 (0.2) 1 (0.2) 1 (0.2) 2 (0.4) 12 (2.5) 12 (2.5) 4 (0.8) 1 (0.2) 105 (21.5)
* ETEC: Enterotoxigenic Escherichia coli.
antibody to the toxin (Svennerholm & Wiklund, 1983). After the addition of rabbit anti-mouse immunoglubolin conjugated to horse-radish peroxidase (Jackson, West Grove, PA), the enzyme substrates and hydrogen peroxide together with ortho-phenylenediamine were added and the optical density was measured at 450 nm. Reference strains were used in this assay as ST, LT positive and negative controls (ST 64111 for ST positive control, 286 C2 for LT positive control, VM 756888 for ST/LT positive control and E 34420 C for negative control). The colonies tested for toxin production were also plated onto colonization factor antigen (CFA) agar plates with and without bile salt and the plates were incubated overnight at 37°C. Enterotoxin positive E. coli colonies from CFA agar plates were tested for the expression of CFA/1, CS1, CS2, CS3, CS4 and CS6; and such colonies from CFA agar plus bile plates were tested for expression of CS5, CS7, CS17, CS8, CS12 and CS14 by monoclonal antibody-based dot blot assays as previously described (Viboud et al., 1993; Lopez-Vidal & Svennerholm, 1990). The antibiotic susceptibility of ETEC isolates was performed by the disk diffusion against antibiotic consisted of ampicillin, trimethoprim/sulfamethoxazole, chloramphenicol, tetracycline, cephalothin, ceftriaxone, nalidixic acid, norfloxacin and ciprofloxacin (BBL Microbiology Systems, Cockeysville, MD). Susceptibility to ceftriaxone was confirmed by the MIC test. The test was done as recommended
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by the National Committee for Clinical Laboratory Standards (NCCLS, 1997; NCCLS, 1999). Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 259213 were used as control strains.
3. Results Although ETEC was the organism of primary interest, other bacterial pathogens were identified as illustrated in Table 1. The following pathogens were found in decreasing order: Shigella spp. (3.3%), Salmonella enterica (2.5%), Vibrio spp. (0.6%) and Campylobacter spp. (0.2%). Mixed infections were found in the rectal swab of two patients with isolated cases of Salmonella enterica and Shigella spp. (Table 1). Of all the 489 rectal swabs processed, 73 (14.9%) were positive for ETEC. The prevalence of ETEC in the age Group 0-1 year was 7.8%, while the prevalence of ETEC in the age Group 1-5 years, 5-15 years and ⬎15 years was 20.4%, 20.8% and 15.6%, respectively. A total of 51(69%) ST and 21(28.8%) LT producers associated ETEC cases were detected in the rectal swabs of 489 patients (Table 2). Only 1(1.4%) ST/LT producer was detected in the rectal swabs of a patient in the age group of 1-5 years old. Out of 73 ETEC isolated, 8 (10.9%) expressed CFA/I, 3 (4.1%) expressed CS1⫹CS3, 1 (1.4%) expressed CS5⫹CS6, 6 (8.2%) expressed CS6, 1 (1.4%) expressed CS17, 1 (1.4%) expressed CS14 and 1 (1.4%) expressed CS5. No CFAs were expressed by 52 (71.2%) of the ETEC (Table 3). Analysis of the sex distribution among the patients with ETEC infection, showed that of the 228 female patients, 43 (18.9%) were positive for ETEC, while of 261 male patients, 30 (11.5%) were positive for ETEC (Table 4). Patients with ETEC demonstrated a wide range of clinical profiles. The most common symptoms were watery stools (98.1%), vomiting (62.1%), fever (55%) and abdominal cramp (80%). The frequency of resistance to conventional antimicrobial agents such as ampicillin, trimethoprim-sulfamethoxazole, chloramphenicol and tetracycline was high throughout this study. Multiple antibiotic resistances to three or
Table 2 Isolation and age distribution of enterotoxigenic Escherichia coli (ETEC) in hospitalized diarrhea patients in Denpasar, Bali, Indonesia ETEC Positive Isolation 0-1 years (n ⫽ 167) ST LT ST/LT All
6 7 0 13 (7.8%)
ST: heat-stable toxin, LT: heat-labile toxin.
1-5 years (n ⫽ 147) 25 4 1 30 (20.4%)
5-15 years (n ⫽ 53) 7 4 0 11 (20.8%)
⬎15 years (n ⫽ 122)
All age
13 6 0
51 21 1
19 (15.6%)
73 (14.9%)
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Table 3 Occurrence and association of Toxins and Colonization Factor Antigens (CFAs) in enterotoxigenic Escherichia coli (ETEC) isolates
Table 5 The percentage of antimicrobial resistance patterns of enterotoxigenic Escherichia coli (ETEC) isolated from Denpasar, Bali, Indonesia
Toxin
No. of isolates
CFA types
No. of isolates (%)
ETEC
ST
51
CFA/I CS1 ⫹ CS3 CS5 ⫹ CS6 CS6 CS17 CS14 None CFA/I CS5 None None
6 (11.8) 3 (5.9) 1 (2.0) 6 (11.8) 1 (2.0) 1 (2.0) 33 (64.7) 2 (9.5) 1 (4.8) 18 (85.7) 1 (100)
LT
21
ST and LT
1
LT (n⫽21) ST (n⫽46)
% of Isolates resistant toa AM
SXT
C
Te
CF
NA
NOR
CIP
CRO
67
48
62
95
86
0
0
0
0
83
70
61
85
87
0
0
0
0
a
Abbreviations: ETEC: enterotoxigenic Escherichia coli; LT: the heatlabile toxin; ST: heat-stable toxin; AM: Ampicillin; SXT: Trimethoprim/ sulfamethoxazole; C: Chloramphenicol; Te: Tetracycline; CF: Cephalothin; NA: Nalidixic Acid; NOR: Norfloxacin; CIP: Ciprofloxacin; CRO: Ceftriaxone.
ST: heat-stable toxin, LT: heat-labile toxin.
more antimicrobial agents were observed (Table 5). Of interest, is that the only female patient who expressed ST/LT toxin, displayed resistance to ampicillin, trimethoprim-sulfamethoxazole, chloramphenicol, tetracycline and cephalothin. None of the ETEC isolates were found to be resistant to norfloxacin, ciprofloxacin or nalidixic acid (Table 5). All enterotoxigenic Escherichia coli isolates were susceptible to ceftriaxone by MIC test. The MIC was ⬍ 0.5 g/ml. The MIC50 was 0.2 g/ml with a range of 0.125-0.25 g/ml. The MIC90 was 0.4 g/ml with a range of 0.125-0.5 g/ml. MIC result showed that ETEC isolates are susceptible to ceftriaxone. A review of seasonal patterns revealed that ETEC diarrhea occurred all year-round in patients. The isolation rate of ETEC increased during the start of the rainy season in October (Fig. 1). The distribution of ETEC shows that the isolation rate of ETEC increased during the months of October through March (Fig. 1). All the patients who participating on this study came from urban area, no travelers among them and no underlying diseases accompanied the diarrhea.
4. Discussion This study was conducted in the city of Denpasar, Bali, Indonesia, which is frequently visited by travelers. The aim of the study was to determine the prevalence of ETEC and Table 4 Distribution of enterotoxigenic Escherichia coli (ETEC) among sex group in hospitalized diarrhea patients in Denpasar, Bali, Indonesia Sex group
Female Male
No. of Positive Patients
No. of ETEC Positive Patients ST
LT
ST/LT
Total (ETEC)
228 261
29 (12.7%) 22 (8.4%)
13 (5.7%) 8 (3.1%)
1 (0.4%) 0
43 (18.9%) 30 (11.5%)
ST: heat-stable toxin, LT: heat-labile toxin.
CFA types expressed by hospitalized patients with acute diarrhea. The proportion of ETEC isolated from the rectal swabs obtained from hospitalized patients was 14.9%. A previous study in hospitalized children less than 5 years of age in Jakarta showed that the prevalence of ETEC was 8.2% (Subekti et al., 1993). Other studies in Ujung Pandang (South Sulawesi), Kebumen (Central Java) and Jakarta, Indonesia, showed that the prevalence of ETEC were 8%, 19% and 19%, respectively (Maidin, 1983; Orndorff et al., 1996; Oyofo et al., 2001). A study in Bangladesh showed that the prevalence of ETEC was 14% (Qadri et al., 2000). Heat stable (ST) toxin was predominant in 51 (69.9%) of ETEC isolates, while 21 (28.7%) produced heat labile toxin (LT). LT and ST toxins were found in 1(1.4%) ETEC isolates. This distribution concurs with a previous study in Jakarta that showed 72% of the isolates elaborating ST, while LT alone was produced by 23% of the isolates (Oyofo et al., 2001). An examination of the age distribution among patients with ETEC showed isolation from patients of different age groups (from less than 1 month to over 60 years of age). The highest proportion was among patients 1-5 years and 5-15 years of age. This finding was in agreement with the results of a study that was conducted among patients with diarrhea in a hospital located in Dacca. The study showed that diarrhea due to ETEC is a disease of all ages, and that the organism was isolated more frequently from patients older than 1 year (Sack et al., 1977). World Health Organization (1999) also reported ETEC to be a childhood disease, due to its substantially higher incidence in early childhood than in older age groups. Surveillance of hospitalized cases of ETEC diarrhea showed that almost half of such cases occur in person aged ⬎10 years, due to the large denominator population at risk in these older groups (WHO, 1999). Sutoto et al. (1982) reported that 1.8% of all diarrheal cases needed hospitalization and that ETEC was the major cause (15.9%) of bacterial diarrhea in Jakarta, with the highest prevalence (50%) in children ⬍ 2 years old. Although, our study focused only in patients who were hospitalized for diarrheal illness, our results were in agreement with the above community-based study results obtained by Sutoto et
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403
Fig. 1.
al. (1982). We found that the prevalence of ETEC was 13.7% in children ⬍5 years old, and 20.8% in children between 5-15 years old. In a hospital-based study conducted by Khusmith et al. (1980), a similar result was reported. The high number of ETEC among children may reflect the fact that children usually receive more medical attention than adults. However, in our study, the prevalence of ETEC in adults was 15.6%, higher than that in children ⬍15 years old (14.7%), although this difference was not significant. Since ETEC was endemic in Indonesia and comprised the most prevalent cause of bacterial diarrhea, this strain posed an important role in the etiology of traveler’s diarrhea. Since 71.2% of the ETEC isolated had undetectable CFA/toxins by the existing monoclonal antibodies, it is suggested here that, continued efforts must be made to screen for CFAs for inclusion in future vaccine testing. The loss of plasmid during storage and processing may have attributed to the high number of undetectable CFAs/toxins. However, plasmid loss has been reported among ETEC isolates (Murray et al., 1983). The prevalence of CFAs has been observed to vary from one region to another (Nirdnoy et al., 1997). Previous reports showed that CFAs were identified in 59% of the ETEC isolates examined (Serichantalergs et al., 1997). In a study in Indonesia (Nirdnoy et al., 1997) showed variance in the expression of CFAs by ETEC among children less then 5 years old. Result of this study showed that 47% of the ETEC expressed CFAs. In another study conducted in Jakarta by Oyofo et al. (2001) among adults and children, result showed that CFA was expressed
in 18.2% of the ETEC isolates. In Argentinean children, 48% of the ETEC had no detectable CFAs (Binsztein et al., 1991). In a study conducted in Israel, 34.5% of the ETEC isolates had CFA, while 65.5% had no detectable CFAs (Wolk et al., 1997). In our study, ETEC disease peaked in the beginning of rainy seasons in November and decreased during the dry seasons. Contrary to our finding, a study conducted in Bangkok, Thailand, reported that ETEC was detected only in a certain period of the year (Maidin et al., 1982). The antimicrobial susceptibility test for ETEC isolates showed a high degree of resistance to ampicillin, trimethoprim-sulfamethoxazole, chloramphenicol and tetracycline (Table 5). The resistance of ETEC to trimethoprimsulfamethoxazole and tetracycline were 48% and 95% in ETEC that produced LT; 70% and 85% in ETEC that produced ST, ETEC isolates in this study had greater resistance to trimethoprim-sulfamethoxazole and tetracycline when compared to the ETEC strains that was isolated in a study that was conducted in Mexico (Jiang et al., 2000). This study showed that 31% of the ETEC strains were resistance to trimethoprim-sulfamethoxazole and 81% resistance to tetracycline (Jiang et al., 2000). The high frequency of resistance of ETEC strains to trimethoprim-sulfamethoxazole, ampicillin and tetracycline has been documented in a study among patients with traveler’s diarrhea in tropical and subtropical areas (Villa et al., 2000). The study found that ETEC strains were resistant to nalidixic acid and ciprofloxacin among patients who traveled to India.
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The current study supports the fact that ETEC is a major cause of disease in children less than 15 years of age in developing countries. The relatively high prevalence of ETEC, types of CFAs and the antimicrobial resistance patterns in this region, may be useful for future investigations in reducing morbidity attributable to ETEC infection in Southeast Asia.
Acknowledgments We thank Dr. A. M. Svennerholm for supplying the monoclonal antibodies used for the GM1 ELISA. The contributions of the Microbiology staff (NAMRU-2), the staff of Wangaya hospital and the staff of Sanglah hospital in Denpasar, Bali, Indonesia, are all appreciated. Disclaimer: The opinions or assertions expressed herein are the private views of the authors and are not to be construed as representing those of the U.S. Navy, the Department of Defense, or the Indonesian Ministry of Health. This work was supported by the Global Emerging Infections Surveillance (D0016) program of the US Department of Defense.
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D.S. Subekti et al / Diagnostic Microbiology and Infectious Disease 47 (2003) 399 – 405 Sutoto, Mochtar M. A., Karyadi, & Wasisto, B. (1982). Morbidity and mortality study on diarrhoeal diseases in north Jakarta-an urban area. Southeast Asian J Trop Med Public Health 13, 405– 411. Svennerholm, A.-M., & Wiklund, G. (1983). Rapid GM1- enzyme-linked immunosorbent assay with visual reading for identification of Escherichia coli heat- labile enterotoxin. J Clin Microbiol 17, 596 – 600. Svennerholm, A.-M., Wikstrom, M., Lindblad, M., & Holmgren, J. (1986). Monoclonal antibodies against E. coli heat-stable toxin (Sta) and their use in a diagnostic ST ganglioside GM1-enzyme-linked immunosorbent assay. J Clin Microbiol 24, 585–590. Thomas, L. V., McConnell, M. M., Rowe, B., & Field, A. M. (1985). The possession of three novel coli surface antigens by enterotoxigenic Escherichia coli strains positive for the putative colonization factor PCF 8775. J Gen Microbiol 131, 2319 –2326. Tison, D. L. (1999). Vibrio. In: P. R. Murray, E. J. Baron, M. A. Pfaller,
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F. C. Tenover, & R. H. Yolken (Eds), Manual of Clinical Microbiology, 7th ed. Washington DC: Am Soc Microbiol, p. 497–506. Viboud, G. I., Binsztein, N., & Svennerholm, A.-M. (1993). Characterization of monoclonal antibodies against putative colonization factors of enterotoxigenic Escherichia coli and their use in an epidemiological study. J Clin Microbiol 31, 558 –564. Villa, J., Vargas, M., Ruiz, J., Corachan, M., de Anta, M. T. J., & Gascon, J. (2000). Quinolone resistance in enterotoxigenic Escherichia coli causing diarrhea in travelers to India in comparison with other geographical areas. Antimicrob Agents Chemo 44, 1731–1733. Wolk, M., Ohad, E., Shafran, R., Schmid, I., & Jarjoui, E. (1997). Enterotoxigenic Escherichia coli (ETEC) in Hospitalized Arab infants from Judea area –west bank, Israel. Public Health 11, 11–17. World Health Organization. (1999). New frontiers in the development of vaccines against enterotoxigenic (ETEC) and enterohaemorrhagic (EHEC) E. coli infections. Weekly Epidemiology Rec 13, 98 –100.
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Prevalence of enterotoxigenic Escherichia coli (ETEC) in hospitalized acute diarrhea patients in Denpasar, Bali, Indonesia D. S. Subektia, M. Lesmanaa,b, P. Tjaniadia, N. Machpuda, Sriwatic, Sukarmad, J. C. Daniela, W. K. Alexandera, J. R. Campbella, A. L. Corwina, H. James Beecham IIIa, C. Simanjuntake, B. A. Oyofoa,* a
U.S. Naval Medical Research Unit No. 2, Jakarta, Indonesia b Medical Faculty Trisakti University, Jakarta, Indonesia c Wangaya Hospital, Denpasar, Indonesia d Sanglah Hospital, Denpasar, Indonesia e National Institute of Health Research and Development Ministry of Health, Jakarta, Indonesia
Abstract 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. © 2003 Elsevier Inc. All rights reserved. Keywords: Enterotoxigenic Escherichia coli (ETEC); Acute diarrhea; Hospitalized patients; Denpasar; Indonesia
1. Introduction Enterotoxigenic Escherichia coli (ETEC) is the most frequently isolated enteropathogen, accounting for about 210 million diarrhea episodes and approximately 380,000 deaths annually. The peak of incidence of ETEC diarrhea in a community-based study was reported to occur in the first year of life, with a declining incidence with age, thereafter (WHO, 1999). ETEC is transmitted by food or water contaminated with animal or human feces. Enterotoxigenic Escherichia coli is a leading cause of morbidity and mortality in children up to 5 years of age in developing coun* Corresponding author. Tel.: 011-62-21-4214457/58; fax: 011-6221424-4457. E-mail address: [email protected] (B. A. Oyofo). 0732-8893/03/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0732-8893(03)00120-2
tries and was first recognized as a cause of human diarrheal illness in the 1960s (Black, 1993; Sack et al., 1971). In addition, ETEC is a major bacterial cause of diarrhea among travelers from industrialized to less developed countries (Arduino & DuPont, 1993). Strains of ETEC have two major virulence determinants: the enterotoxins (the heat-labile toxin/LT and heat-stable toxin/ST) and colonization factor antigens (CFAs). To cause diseases, ETEC must adhere to the epithelium of the small intestine by means of CFA. Secretory diarrhea is then produced due to the effects of the enterotoxins (Gaastra & Svennerholm, 1996). Colonization factor antigens on human ETEC strains are usually protein fimbrial antigens, which are capable of agglutinating erythrocytes from different animal species in the presence of D-mannose (Evans et al., 1979). Recognized CFAs associated with human
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ETEC isolates are CFA/I, CFA/II and CFA/IV. Colonization factor antigen I is a single fimbrial antigen (Evans et al., 1975), while CFA/II and CFA/IV have been shown to consist of three distinct fimbrial antigens called Coli Surface (CS) antigens. Colonization factor antigens II is composed of antigens CS1, CS2 and CS3 (Smith et al., 1983). Colonization factor antigens IV is comprised of antigens CS4, CS5 and CS6 (Thomas et al., 1985). The other Coli Surface antigens are CS7, CS8, CS12, CS14, CS11, CS13, CS15, and CS17 (Gaastra & Svennerholm, 1996). In brief, the GM1 immunologic assay has been used to detect enterotoxins of ETEC, ST and LT. This assay is based on the ability of toxin (ST or LT), as present in culture filtrates from ST or LT-producing E. coli, to inhibit specific anti-ST or LT antibody from binding to solid phase bounded ST or LT ganglioside (GM1-bound ST or LT-cholera B subunit). The purified B subunit of cholera toxin (CTB) is then able to bind to GM1 ganglioside, which is the receptor for the CTB absorbed to the ELISA microtiter wells. The presence of the toxin is detected by the addition of a specific antibody, followed by the addition of an enzyme-conjugate antibody. A positive test is indicated by a color reaction. This test has 100% sensitivity and specificity when compared with cell culture assay (Svennerholm & Wiklund, 1983; Svennerholm et al., 1986). This city of Denpasar, Indonesia is a busy commercial center for visitors throughout the year. Due to the diversity of travelers that visit the area, we reasoned that the knowledge of the ETEC and CFAs in the population would provide useful data for the evaluation of ongoing efforts to develop ETEC vaccine. It is suggested that serious efforts should be made to design more effective ETEC vaccines for the prevention of ETEC infection, especially in countries of the world where ETEC is endemic (WHO, 1999). The aim of this study was to determine the prevalence of ETEC and the variation of CFAs among hospitalized diarrheal patients in the tourist and business city of Denpasar, Bali, Indonesia.
2. Materials and methods 2.1. Study population Two hospitals (Wangaya and Sanglah) in Denpasar, Bali, Indonesia, were selected for the study that was conducted from June 2000 through May 2001. Denpasar is the biggest city in Bali Island. Bali is the most popular island for travelers among the 17,000 islands in Indonesia, and it is located between 8 to 9 degrees South of the equator at longitude 115 degree East. The two hospitals are close to each other about 1 mile distance. Both are also located in the center of the city. They were selected based on the fact that they are the two largest hospitals in Denpasar, both in terms of hospital beds and the number of diarrhea cases seen per
year. The other factor in their selection was their willingness to participate in the study. 2.2. Subjects and specimen collection During the period of June 2000 through May 2001, a total of 489 rectal swabs were collected from acute ill diarrhea patients hospitalized at Sanglah and Wangaya hospitals in Denpasar, Bali, Indonesia. All patients presenting with diarrhea and admitted to the hospitals were included in this study, regardless of sex and age. The participating hospital staff was instructed to obtain rectal swab samples from each diarrhea patient on the day of admission before antibiotics were given. Personal data and symptoms of the illness including the type of diarrhea were obtained. Specimens were transported to the U.S. Naval Medical Research Unit No.2 (NAMRU-2) laboratory in Cary-Blair transport medium and processed immediately. Specimens obtained in the evening or at night were kept in a refrigerator at 2-8°C and transported in a cool box the following day. 2.3. Laboratory processing Rectal swabs collected were stored at 2-8°C in CaryBlair media and processed immediately after arrival at the Microbiology Laboratory located at NAMRU-2. Upon arrival at the laboratory, rectal swabs were routinely screened for enteric pathogens including Vibrio spp., Salmonella enterica, Shigella spp., and Campylobacter jejuni by standard techniques (Tison, 1999; Bopp et al., 1999; Goosens & Butzle, 1992; Lesmana et al., 1997). For the detection of ETEC, the rectal swabs were plated onto MacConkey agar and incubated at 37°C for 18-24 h. Five lactose-fermenting individual colonies morphologically resembling E. coli were tested immediately after isolation for the presence of toxins and CFAs as described below. 2.4. Detection of toxin types and CFAs Rectal swabs obtained from patients were cultured on MacConkey agar plates for selection of E. coli isolates. After overnight culture, five E. coli lactose-fermenting colonies from each patient were picked and stored on nutrient agar and analyzed for heat-labile (LT) and heat-stable (ST) enterotoxin by ganglioside GM1-enzyme-linked immunosorbent assay (Svennerholm et al., 1986). For these purposes E. coli colonies were inoculated into separate wells of a GM1 coated microtiter plate containing Luria–Bertani broth with lincomycin and glucose and incubated overnight at 37°C with agitation (150 rpm). After incubation, the broth culture was transferred to fresh GM1 coated plates and tested for ST by an inhibition ELISA procedure with the ST-cholera toxin B subunit (CTB) conjugate (ST-CTB) as described earlier (Svennerholm et al., 1986). Luria–Bertani broth from the original plate used for culture was analyzed for GM1 bound LT by using a specific-mouse monoclonal
D.S. Subekti et al / Diagnostic Microbiology and Infectious Disease 47 (2003) 399 – 405 Table 1 Enteropathogens found among 489 hospitalized diarrhea patients in Denpasar, Bali, Indonesia Enteropathogens
No. positives (%)
ETEC* ETEC* ⫹ Salmonella enterica ETEC* ⫹ S. flexneri Vibrio cholerae Vibrio parahaemolyticus Salmonella enterica Shigella flexneri Shigella sonnei Campylobacter jejuni Total identified Enteropathogens
71 (14.5) 1 (0.2) 1 (0.2) 1 (0.2) 2 (0.4) 12 (2.5) 12 (2.5) 4 (0.8) 1 (0.2) 105 (21.5)
* ETEC: Enterotoxigenic Escherichia coli.
antibody to the toxin (Svennerholm & Wiklund, 1983). After the addition of rabbit anti-mouse immunoglubolin conjugated to horse-radish peroxidase (Jackson, West Grove, PA), the enzyme substrates and hydrogen peroxide together with ortho-phenylenediamine were added and the optical density was measured at 450 nm. Reference strains were used in this assay as ST, LT positive and negative controls (ST 64111 for ST positive control, 286 C2 for LT positive control, VM 756888 for ST/LT positive control and E 34420 C for negative control). The colonies tested for toxin production were also plated onto colonization factor antigen (CFA) agar plates with and without bile salt and the plates were incubated overnight at 37°C. Enterotoxin positive E. coli colonies from CFA agar plates were tested for the expression of CFA/1, CS1, CS2, CS3, CS4 and CS6; and such colonies from CFA agar plus bile plates were tested for expression of CS5, CS7, CS17, CS8, CS12 and CS14 by monoclonal antibody-based dot blot assays as previously described (Viboud et al., 1993; Lopez-Vidal & Svennerholm, 1990). The antibiotic susceptibility of ETEC isolates was performed by the disk diffusion against antibiotic consisted of ampicillin, trimethoprim/sulfamethoxazole, chloramphenicol, tetracycline, cephalothin, ceftriaxone, nalidixic acid, norfloxacin and ciprofloxacin (BBL Microbiology Systems, Cockeysville, MD). Susceptibility to ceftriaxone was confirmed by the MIC test. The test was done as recommended
401
by the National Committee for Clinical Laboratory Standards (NCCLS, 1997; NCCLS, 1999). Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 259213 were used as control strains.
3. Results Although ETEC was the organism of primary interest, other bacterial pathogens were identified as illustrated in Table 1. The following pathogens were found in decreasing order: Shigella spp. (3.3%), Salmonella enterica (2.5%), Vibrio spp. (0.6%) and Campylobacter spp. (0.2%). Mixed infections were found in the rectal swab of two patients with isolated cases of Salmonella enterica and Shigella spp. (Table 1). Of all the 489 rectal swabs processed, 73 (14.9%) were positive for ETEC. The prevalence of ETEC in the age Group 0-1 year was 7.8%, while the prevalence of ETEC in the age Group 1-5 years, 5-15 years and ⬎15 years was 20.4%, 20.8% and 15.6%, respectively. A total of 51(69%) ST and 21(28.8%) LT producers associated ETEC cases were detected in the rectal swabs of 489 patients (Table 2). Only 1(1.4%) ST/LT producer was detected in the rectal swabs of a patient in the age group of 1-5 years old. Out of 73 ETEC isolated, 8 (10.9%) expressed CFA/I, 3 (4.1%) expressed CS1⫹CS3, 1 (1.4%) expressed CS5⫹CS6, 6 (8.2%) expressed CS6, 1 (1.4%) expressed CS17, 1 (1.4%) expressed CS14 and 1 (1.4%) expressed CS5. No CFAs were expressed by 52 (71.2%) of the ETEC (Table 3). Analysis of the sex distribution among the patients with ETEC infection, showed that of the 228 female patients, 43 (18.9%) were positive for ETEC, while of 261 male patients, 30 (11.5%) were positive for ETEC (Table 4). Patients with ETEC demonstrated a wide range of clinical profiles. The most common symptoms were watery stools (98.1%), vomiting (62.1%), fever (55%) and abdominal cramp (80%). The frequency of resistance to conventional antimicrobial agents such as ampicillin, trimethoprim-sulfamethoxazole, chloramphenicol and tetracycline was high throughout this study. Multiple antibiotic resistances to three or
Table 2 Isolation and age distribution of enterotoxigenic Escherichia coli (ETEC) in hospitalized diarrhea patients in Denpasar, Bali, Indonesia ETEC Positive Isolation 0-1 years (n ⫽ 167) ST LT ST/LT All
6 7 0 13 (7.8%)
ST: heat-stable toxin, LT: heat-labile toxin.
1-5 years (n ⫽ 147) 25 4 1 30 (20.4%)
5-15 years (n ⫽ 53) 7 4 0 11 (20.8%)
⬎15 years (n ⫽ 122)
All age
13 6 0
51 21 1
19 (15.6%)
73 (14.9%)
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Table 3 Occurrence and association of Toxins and Colonization Factor Antigens (CFAs) in enterotoxigenic Escherichia coli (ETEC) isolates
Table 5 The percentage of antimicrobial resistance patterns of enterotoxigenic Escherichia coli (ETEC) isolated from Denpasar, Bali, Indonesia
Toxin
No. of isolates
CFA types
No. of isolates (%)
ETEC
ST
51
CFA/I CS1 ⫹ CS3 CS5 ⫹ CS6 CS6 CS17 CS14 None CFA/I CS5 None None
6 (11.8) 3 (5.9) 1 (2.0) 6 (11.8) 1 (2.0) 1 (2.0) 33 (64.7) 2 (9.5) 1 (4.8) 18 (85.7) 1 (100)
LT
21
ST and LT
1
LT (n⫽21) ST (n⫽46)
% of Isolates resistant toa AM
SXT
C
Te
CF
NA
NOR
CIP
CRO
67
48
62
95
86
0
0
0
0
83
70
61
85
87
0
0
0
0
a
Abbreviations: ETEC: enterotoxigenic Escherichia coli; LT: the heatlabile toxin; ST: heat-stable toxin; AM: Ampicillin; SXT: Trimethoprim/ sulfamethoxazole; C: Chloramphenicol; Te: Tetracycline; CF: Cephalothin; NA: Nalidixic Acid; NOR: Norfloxacin; CIP: Ciprofloxacin; CRO: Ceftriaxone.
ST: heat-stable toxin, LT: heat-labile toxin.
more antimicrobial agents were observed (Table 5). Of interest, is that the only female patient who expressed ST/LT toxin, displayed resistance to ampicillin, trimethoprim-sulfamethoxazole, chloramphenicol, tetracycline and cephalothin. None of the ETEC isolates were found to be resistant to norfloxacin, ciprofloxacin or nalidixic acid (Table 5). All enterotoxigenic Escherichia coli isolates were susceptible to ceftriaxone by MIC test. The MIC was ⬍ 0.5 g/ml. The MIC50 was 0.2 g/ml with a range of 0.125-0.25 g/ml. The MIC90 was 0.4 g/ml with a range of 0.125-0.5 g/ml. MIC result showed that ETEC isolates are susceptible to ceftriaxone. A review of seasonal patterns revealed that ETEC diarrhea occurred all year-round in patients. The isolation rate of ETEC increased during the start of the rainy season in October (Fig. 1). The distribution of ETEC shows that the isolation rate of ETEC increased during the months of October through March (Fig. 1). All the patients who participating on this study came from urban area, no travelers among them and no underlying diseases accompanied the diarrhea.
4. Discussion This study was conducted in the city of Denpasar, Bali, Indonesia, which is frequently visited by travelers. The aim of the study was to determine the prevalence of ETEC and Table 4 Distribution of enterotoxigenic Escherichia coli (ETEC) among sex group in hospitalized diarrhea patients in Denpasar, Bali, Indonesia Sex group
Female Male
No. of Positive Patients
No. of ETEC Positive Patients ST
LT
ST/LT
Total (ETEC)
228 261
29 (12.7%) 22 (8.4%)
13 (5.7%) 8 (3.1%)
1 (0.4%) 0
43 (18.9%) 30 (11.5%)
ST: heat-stable toxin, LT: heat-labile toxin.
CFA types expressed by hospitalized patients with acute diarrhea. The proportion of ETEC isolated from the rectal swabs obtained from hospitalized patients was 14.9%. A previous study in hospitalized children less than 5 years of age in Jakarta showed that the prevalence of ETEC was 8.2% (Subekti et al., 1993). Other studies in Ujung Pandang (South Sulawesi), Kebumen (Central Java) and Jakarta, Indonesia, showed that the prevalence of ETEC were 8%, 19% and 19%, respectively (Maidin, 1983; Orndorff et al., 1996; Oyofo et al., 2001). A study in Bangladesh showed that the prevalence of ETEC was 14% (Qadri et al., 2000). Heat stable (ST) toxin was predominant in 51 (69.9%) of ETEC isolates, while 21 (28.7%) produced heat labile toxin (LT). LT and ST toxins were found in 1(1.4%) ETEC isolates. This distribution concurs with a previous study in Jakarta that showed 72% of the isolates elaborating ST, while LT alone was produced by 23% of the isolates (Oyofo et al., 2001). An examination of the age distribution among patients with ETEC showed isolation from patients of different age groups (from less than 1 month to over 60 years of age). The highest proportion was among patients 1-5 years and 5-15 years of age. This finding was in agreement with the results of a study that was conducted among patients with diarrhea in a hospital located in Dacca. The study showed that diarrhea due to ETEC is a disease of all ages, and that the organism was isolated more frequently from patients older than 1 year (Sack et al., 1977). World Health Organization (1999) also reported ETEC to be a childhood disease, due to its substantially higher incidence in early childhood than in older age groups. Surveillance of hospitalized cases of ETEC diarrhea showed that almost half of such cases occur in person aged ⬎10 years, due to the large denominator population at risk in these older groups (WHO, 1999). Sutoto et al. (1982) reported that 1.8% of all diarrheal cases needed hospitalization and that ETEC was the major cause (15.9%) of bacterial diarrhea in Jakarta, with the highest prevalence (50%) in children ⬍ 2 years old. Although, our study focused only in patients who were hospitalized for diarrheal illness, our results were in agreement with the above community-based study results obtained by Sutoto et
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403
Fig. 1.
al. (1982). We found that the prevalence of ETEC was 13.7% in children ⬍5 years old, and 20.8% in children between 5-15 years old. In a hospital-based study conducted by Khusmith et al. (1980), a similar result was reported. The high number of ETEC among children may reflect the fact that children usually receive more medical attention than adults. However, in our study, the prevalence of ETEC in adults was 15.6%, higher than that in children ⬍15 years old (14.7%), although this difference was not significant. Since ETEC was endemic in Indonesia and comprised the most prevalent cause of bacterial diarrhea, this strain posed an important role in the etiology of traveler’s diarrhea. Since 71.2% of the ETEC isolated had undetectable CFA/toxins by the existing monoclonal antibodies, it is suggested here that, continued efforts must be made to screen for CFAs for inclusion in future vaccine testing. The loss of plasmid during storage and processing may have attributed to the high number of undetectable CFAs/toxins. However, plasmid loss has been reported among ETEC isolates (Murray et al., 1983). The prevalence of CFAs has been observed to vary from one region to another (Nirdnoy et al., 1997). Previous reports showed that CFAs were identified in 59% of the ETEC isolates examined (Serichantalergs et al., 1997). In a study in Indonesia (Nirdnoy et al., 1997) showed variance in the expression of CFAs by ETEC among children less then 5 years old. Result of this study showed that 47% of the ETEC expressed CFAs. In another study conducted in Jakarta by Oyofo et al. (2001) among adults and children, result showed that CFA was expressed
in 18.2% of the ETEC isolates. In Argentinean children, 48% of the ETEC had no detectable CFAs (Binsztein et al., 1991). In a study conducted in Israel, 34.5% of the ETEC isolates had CFA, while 65.5% had no detectable CFAs (Wolk et al., 1997). In our study, ETEC disease peaked in the beginning of rainy seasons in November and decreased during the dry seasons. Contrary to our finding, a study conducted in Bangkok, Thailand, reported that ETEC was detected only in a certain period of the year (Maidin et al., 1982). The antimicrobial susceptibility test for ETEC isolates showed a high degree of resistance to ampicillin, trimethoprim-sulfamethoxazole, chloramphenicol and tetracycline (Table 5). The resistance of ETEC to trimethoprimsulfamethoxazole and tetracycline were 48% and 95% in ETEC that produced LT; 70% and 85% in ETEC that produced ST, ETEC isolates in this study had greater resistance to trimethoprim-sulfamethoxazole and tetracycline when compared to the ETEC strains that was isolated in a study that was conducted in Mexico (Jiang et al., 2000). This study showed that 31% of the ETEC strains were resistance to trimethoprim-sulfamethoxazole and 81% resistance to tetracycline (Jiang et al., 2000). The high frequency of resistance of ETEC strains to trimethoprim-sulfamethoxazole, ampicillin and tetracycline has been documented in a study among patients with traveler’s diarrhea in tropical and subtropical areas (Villa et al., 2000). The study found that ETEC strains were resistant to nalidixic acid and ciprofloxacin among patients who traveled to India.
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The current study supports the fact that ETEC is a major cause of disease in children less than 15 years of age in developing countries. The relatively high prevalence of ETEC, types of CFAs and the antimicrobial resistance patterns in this region, may be useful for future investigations in reducing morbidity attributable to ETEC infection in Southeast Asia.
Acknowledgments We thank Dr. A. M. Svennerholm for supplying the monoclonal antibodies used for the GM1 ELISA. The contributions of the Microbiology staff (NAMRU-2), the staff of Wangaya hospital and the staff of Sanglah hospital in Denpasar, Bali, Indonesia, are all appreciated. Disclaimer: The opinions or assertions expressed herein are the private views of the authors and are not to be construed as representing those of the U.S. Navy, the Department of Defense, or the Indonesian Ministry of Health. This work was supported by the Global Emerging Infections Surveillance (D0016) program of the US Department of Defense.
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