INTRODUCTION

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Millions of people travel from industrialized to developing countries each year. Traveler's diarrhea is the most common disorder encountered by these travelers during their journey and thereafter. While traveler's diarrhea generally lasts less than a week (7), approximately 3% of international travelers to high-risk areas develop persistent diarrhea. By definition, the duration of persistent diarrhea is at least 14 days (3), but it may last several months to a year. In approximately 50% of travelers with persistent diarrhea, diarrhea lasts more than 30 days (11). Besides travelers, persistent diarrhea is also a major cause of morbidity in expatriates and other long-term foreign residents in developing countries (1, 30).

The etiology of persistent diarrhea in travelers remains unknown in at least 50% of cases (11, 26). While the etiology of acute traveler's diarrhea has been studied extensively, there are only a few reports in which the etiology of persistent diarrhea in travelers and foreign residents has been studied. Although enterotoxigenic Escherichia coli (ETEC) is considered to be the most important cause of acute traveler's diarrhea (7), it has rarely been implicated as a cause of persistent diarrhea in travelers (30). In view of the possible association of enteroaggregative E. coli (EAggEC) with persistent diarrhea in children in developing countries (5, 10, 12), it has been suggested that EAggEC may also be a cause of persistent diarrhea in travelers.

We performed a case control study to determine the role of ETEC and EAggEC in acute and persistent diarrhea in returned travelers. In addition, the presence of diffuse adherent E. coli (DAEC) and enteropathogenic E. coli (EPEC) was determined.

	MATERIALS AND METHODS

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Definitions. Diarrhea was defined as at least three loose stools in 24 h, any number of watery stools, or 1 or 2 loose stools in 24 h accompanied by at least one of the following symptoms: nausea, vomiting, abdominal cramps, or fever of >38°C.

Patients and controls. Patients were included who presented with diarrhea from December 1995 to August 1996 at the Outpatient Department for Tropical Diseases at the Academic Medical Centre, Amsterdam, The Netherlands, and the Leopold Institute of Tropical Medicine, Antwerp, Belgium. Cases were defined in terms of patients who developed diarrhea during a stay in tropical areas or within the first 2 weeks after their return.

Investigation of stool samples. Stool samples from patients and controls were collected on the day of presentation at the outpatient department, immediately transported to the laboratory, stored at 4°C, and processed within 24 h.

Detection of diarrheagenic E. coli. Stool samples were inoculated onto cysteine lactose electrolyte-deficient (CLED) agar plates, and after incubation for 18 h at 37°C, a "sweep" of the complete bacterial growth on the agar was collected with a sterile cotton swab and stored in glycerol-peptone at 70°C, as described previously (28).

Detection of other enteropathogenic bacteria. All stool samples were inoculated onto Salmonella-Shigella (SS) agar, CLED agar, Hektoen enteric agar (HEA), MacConkey agar containing potassium-tellurite, and thiosulfate-citrate-bile salts-sucrose agar and incubated for 24 to 48 h at 37°C. For enrichment, Hajna GramNegative broth was inoculated, and after incubation for 24 h at 42°C, cells were subcultured onto SS agar and HEA. For selective enrichment of Salmonella species, feces was inoculated onto a semisolid selective-motility enrichment medium (14), incubated at 37°C, and subcultured onto SS agar. Yersinia species were isolated by inoculation of cefsulodin-irgasan-novobiocin (CIN) agar, which was incubated for 48 h at room temperature. In addition, glucose broth was inoculated and incubated for 1 week at 4°C and subcultured onto CIN agar. For detection of Campylobacter species, Butzler Campylobacter selective medium was inoculated, and plates were incubated for 48 h at 42°C under microaerophilic conditions. For isolation of Plesiomonas shigelloides, mannitol-MacConkey agar plates were inoculated and incubated for 18 to 36 h at 37 and 42°C. All isolates were further characterized by standard microbiological identification methods and with commercially available antisera. For detection of Aeromonas species, blood agar plates containing 10 µg of ampicillin per ml were inoculated and incubated for 24 to 48 h at 37°C and biochemically characterized as described by Kuijper et al. (20). The methods used for bacterial culture in Amsterdam and Antwerp were identical.

Parasitological examination. Parasitological investigation of stool samples was performed by direct examination of stool in eosin solution. In addition, samples were concentrated as described by Ridley (25) and examined after staining with potassium-iodine. Stool samples were examined for the presence of ova, cysts, and cyclospora. Identical protocols for identification of parasites were used in Amsterdam and Antwerp.

Data collection and analysis. Clinical data from each patient and control were collected by using a questionnaire in the Epi-info format (Amsterdam) or by direct registration in dBase (Antwerp). Data were merged into Epi-info, version 6.0 (Centers for Disease Control and Prevention, Atlanta, Ga.). Statistical analysis was performed with Stata, version 4 (Stata Corporation, College Station, Tex.).

	RESULTS

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A total of 171 patients and 109 controls were included. Of these, 63 patients and 53 controls were included in Amsterdam and 108 patients and 56 controls were included in Antwerp. Two patients and one control were excluded because of inconsistent data collection. Characteristics of patients and controls are presented in Table 1. Patients were younger than controls. The duration of stay abroad of patients was significantly shorter than that of controls, and patients presented at the outpatient department within a significantly shorter period after return than controls. In addition, patients used more imidazoles prior to presentation than controls.

Fifty-three (31%) patients presented with acute diarrhea, 73 (43%) patients had diarrhea for 14 days to 3 months, and 43 (25%) patients had diarrhea for more than 3 months.

Bacterial enteropathogens, except for diarrheagenic E. coli, were isolated from 18 patients and 3 controls. The rates of detection of these pathogens in patients with acute and persistent diarrhea are shown in Table 2. Campylobacter and Shigella species were isolated in patients with both acute and persistent diarrhea. No Plesiomonas shigelloides, Aeromonas species, or Yersinia species were isolated. Bacteriological stool investigation was incomplete in 4 patients and 11 controls.

Giardia lamblia was identified in 20 patients and 5 controls, Blastocystis hominis was identified in 13 patients and 3 controls, and cyclospora was identified in 5 patients and 0 controls (Table 2). The parasitological stool investigation was incomplete in six patients and eight controls. If stool investigations were incomplete, this was generally due to the submission of amounts of stools too small to perform all investigations.

ETEC was isolated in 18 patients and 4 controls (Table 3). EAggEC was isolated in 16 patients and 7 controls. In five patients, both ETEC and EAggEC were present. DAEC was found in 22 patients and 15 controls, while EPEC was found in 2 patients and 6 controls. In two patients and three controls, DAAA was identified (Table 3). Mixed infections with other bacteria were present in three patients with ETEC infection (one each with Salmonella sp., Shigella sp., and Campylobacter jejuni) and one control with ETEC (Campylobacter jejuni). Mixed infection with cyclospora occurred in one patient with ETEC. Mixed infections with other bacteria were present in one patient and one control with EAggEC, both with Shigella sp.

The association of infection with diarrheagenic E. coli and diarrhea was analyzed by multivariate analysis with logistic regression. Crude ORs and results of stratified analysis to assess potential confounding variables are shown in Table 4. ORs, adjusted for confounding in logistic regression models, are shown in Table 5. Infection with ETEC was significantly associated with diarrhea of less than 14 days of duration (P = 0.005; Table 5), but not with persistent diarrhea. EAggEC was found significantly more frequently in patients with acute diarrhea than in controls (P = 0.009; Table 4). However, no significant association of EAggEC infection with diarrhea remained after multivariate analysis (Table 5), which was mainly due to the fact that coinfection of EAggEC with ETEC occurred in five patients with acute diarrhea. In 9 of 15 patients with EAggEC infection and no other bacterial causes of diarrhea (except ETEC), diarrhea was acute, and in 6 patients diarrhea was persistent, lasting 14 days to 3 months in 4 of them.

Isolation of DAEC was not associated with acute or persistent diarrhea (Table 5). EPEC and DAAA were detected in too few patients and controls to permit statistical analysis.

The distributions of patients with ETEC and EAggEC among the various geographic regions visited were similar. The highest rates of detection occurred among visitors to Africa (ETEC, 21%; EAggEC, 15%) and South and Central America (ETEC, 15%; EAggEC, 27%), and the lowest detection rate occurred among visitors to Southeast Asia (ETEC, 7%; EAggEC, 5%).

	DISCUSSION

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The aim of this study was to evaluate the potential associations between diarrheagenic E. coli and diarrhea of short and prolonged duration in returned travelers.

In 31% of patients, diarrhea lasted 14 days or less, and in these patients, a significant association of ETEC with diarrhea was found. This observation is compatible with the well established association of ETEC with classical traveler's diarrhea (7) and with findings from other studies of returned travelers (13, 18, 22). While ETEC was the pathogen most commonly detected in our patients, it should be taken into account that we used a highly sensitive method for detection of ETEC in stool samples (28). This method probably has a higher sensitivity than routine stool culture for detection of other common enteropathogens, such as Campylobacter or Shigella.

In patients with acute diarrhea, the frequency of EAggEC was similar to the frequency of ETEC. The role of EAggEC as a cause of diarrhea in travelers is not well established. Enteroadherent E. coli strains, which include both EAggEC and DAEC, have been associated with traveler's diarrhea in adults traveling to Mexico (21). However, at the time of that study, no distinction was made between localized, aggregative, and diffuse adhesion of adherent strains, and the contribution of EAggEC in this group is unknown. Increased colonization with EAggEC was observed in adult travelers to Mexico, when stool samples before and after the journey were compared, but no association with diarrhea was found (9). Finally, in a recent study of 165 returned Spanish travelers with diarrhea and their healthy travel companions, EAggEC was associated with diarrhea, which clinically resembled diarrhea caused by ETEC (13).

The detection rate of EAggEC in our study was similar to that found in the Spanish travelers (13). However, we found polymicrobial infection, particularly coinfection with ETEC, in 6 of 17 (35%) patients with EAggEC. Coinfection with ETEC and EAggEC may be a common phenomenon, considering the comparable distributions of the two categories of E. coli among the geographic regions. In addition, the uses of antibiotics by patients with EAggEC and controls were different (Table 4). Both factors account for the lack of association of EAggEC with acute diarrhea in our multivariate model (Table 5).

The majority of the patients (68%) presented with persistent diarrhea; illness lasting more than 3 months was present in 25%. Neither ETEC nor EAggEC was associated with persistent diarrhea. In a study in Nepal, ETEC was found in 19% of 37 travelers and foreign residents, who had diarrhea for more than 14 days. However, this study was performed in an area of endemicity, and no control group of individuals without diarrhea was included (30). EAggEC has been associated with persistent diarrhea in children in areas of endemicity in a number of studies (5, 10, 12), but to our knowledge, no data are available demonstrating an association of EAggEC in travelers with persistent diarrhea.

DAEC was detected at a relatively high frequency among patients and controls. The potential role of DAEC as an enteric pathogen is not well established (23). DAEC was detected in stool samples in 20% of both adult patients with ulcerative colitis and healthy controls in The Netherlands, who had not traveled during the year before the investigation (27). The latter findings suggest that DAEC strains are commonly present in stool samples from adults in The Netherlands and explain the lack of an association with diarrhea in travelers from tropical areas.

Since EPEC, similar to ETEC, is a common cause of childhood diarrhea in developing countries, several studies have investigated the role of EPEC in acute traveler's diarrhea (9, 13, 22, 24), using either serotyping or DNA probes for detection of EPEC. In none of these studies, was EPEC significantly associated with diarrhea, which is confirmed by our study. In addition, our results indicate that EPEC is also not associated with persistent diarrhea in travelers. The category of E. coli strains which hybridize with both the daaC probe and the EAggEC fragment probe and which show a mixed adherence pattern to HEp-2 cells, has been recognized before (29). We chose to evaluate the individuals with such strains separately from those infected with DAEC or EAggEC, since it is not clear to which category of diarrheagenic E. coli the strains belong and their pathogenic potential has not been evaluated.

The presence of other diarrheagenic E. coli, in particular verotoxigenic E. coli and enteroinvasive E. coli, was not investigated in the current study. However, we did not detect any of these pathogens in a pilot study among 50 returned travelers (C. Schultsz, R. van Ketel, P. Speelman, G. J. Pool, and J. Dankert, Abstr. 34th Intersci. Conf. Antimicrob. Agents Chemother., abstr. J220, p. 269, 1994), and therefore we did not expect to find any association with diarrhea in the present study.

We found a potential cause of diarrhea in less than 50% of patients. ETEC and EAggEC were the (putative) enteropathogens isolated most frequently. Twenty-eight percent of patients used antibiotics during the course of their disease, which may have influenced the detection rates of pathogens. This detection rate is, however, comparable with rates reported by others (18). Several reports indicate that persistent diarrhea, which has developed after a bout of acute diarrhea and for which no cause is found, is self-limited, although symptoms may persist for periods as long as a year (2, 11, 26). Patients with persistent diarrhea after travel, without identifiable cause, should therefore not receive recurrent antimicrobial therapy.