Etiology of Acute Infectious Diarrhea in a Highly Industrialized Area of Switzerland

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1 GASTROENTEROLOGY 1985;88:75-9 Etiology of Acute Infectious Diarrhea in a Highly Industrialized Area of Switzerland J. LOOSLI, K. GYR, H. STALDER, G. A. STALDER, W. VISCHER, J. VOEGTLIN, M. GASSER, and B. REICHLIN Division of Gastroenterology, University Hospital. Basel; Department of Medicine, Kantonsspital, Liestal; Microbiological Laboratory, Ciba-Geigy Ltd" Basel; and Institute of Hygiene and Microbiology, University of Basel, Switzerland During an 18-mo period between 1981 and 1982, a prospective study was conducted in 119 adult patients with acute diarrhea. A diarrhea-inducing microorganism or toxin could be identified in 38.7% of the patients. Salmonella sp and Campylobacter jejuni were the leading agents that caused diarrheal illness in 25% of the investigated population, Clostridium difficile was found in 6%, mainly after previous antibiotic therapy, Rotavirus was rarely isolated and enterotoxigenic Escherichia coli were not found, Clinical features in patients in whom an invasive agent was isolated did not differ from those in patients in whom no enteropathogens were found, although the occurrence of fecal leukocytes and positive hem occult tests in the former group was significantly more frequent. More than 30% of the patients with negative stool cultures, however, showed fecal leukocytes and positive occult blood, which is suggestive of the existence of one or more invasive agent(s) so far unknown or not recognized, Acute infectious diarrhea is one of the principal causes of illness in the world (1), In many developing countries it represents the most important single cause of disease, malnutrition, and death among children below the age of 5 yr. In industrialized nations, infectious diarrhea is the second most common cause of illness after upper respiratory infections (2). It usually takes a milder course and rarely leads to death (3), Received April 24, 1984, Accepted July 20, 1984, Address requests for reprints to: Dr. J. Loosli, Division of Gastroenterology, University Hospital. CH-4031 Basel, Switzerland. The authors thank Mr. Wolfgang Weber for data processing, Dr. May ZimmerIi for reading the manuscript, and Mrs. Carita Frei and Mrs. Verena Moser for secretarial assistance. They also thank Dr. N. R. Blacklow of the University of Massachusetts Medical School for performing the Norwalk assays in his laboratory by the American Gastroenterological Association /85/$3.30 Epidemiologic data on acute infectious diarrhea have become available only very recently from countries of the Third World, from children in industrialized nations (4), and from travelers abroad (5-9). Most reports dealt with single outbreaks or with diarrhea caused by special pathogens (10,11). Little is known of the etiology and the epidemiology of infectious diarrhea in adults in the industrialized world. A prospective study on infectious diarrhea in adults has so far been reported only from the United Kingdom (12). The present prospective study was undertaken to determine the cause and epidemiolbgy of acute diarrhea in adults in a highly industrialized area of northwestern Switzerland. Special interest was given to newly identified gastrointestinal pathogens. Methods Patients Between July 1981 and December 1982 all patients above the age of 15 yr seeking medical attention for acute infectious diarrhea at the Kantonsspital in Basel and in Liestal were admitted to a prospective study if the following criteria were met: at least three unformed stools daily for not more than 3 days preceding the medical consultation, accompanied by one or more of the following additional symptoms: fever, abdominal cramps or pain, nausea, or vomiting. During the same period, patients admitted for acute diseases other than gastrointestinal ones were chosen as controls. It was intended to include 1 control patient per week during the period when diarrheal patients were entering the hospital. The two hospitals serve a highly industrialized area with roughly 300,000 inhabitants and little agricultural activities. On entry to the study, a careful history was taken from each patient including information on recent foreign travel, previous antibiotic therapy, or consumption of clams,

2 76 LOOSLI ET AL. GASTROENTEROLOGY Vol. 88, No.1, Part 1 other shell fish, chicken, or raw meat. A thorough clinical examination was then performed. Biochemical Tests and Clinical Investigations Blood samples were collected on admission and when possible, a second blood sample was taken 8-14 days after entry into the study. Total and differential blood count and serum electrolytes were measured. Liver enzymes and blood cultures were obtained in severely ill patients only. Blood samples were taken for viral studies. Proctosigmoidoscopy with or without rectal biopsy was performed when pseudomembraneous colitis or other severe mucosal damage was suspected. Collection, Transport, and Storage of Fecal Specimens Fecal specimens were collected as soon as the patient was admitted to the study. Stool samples were recorded as watery, bloody, or loose, containing mucus or pus. Specimens from each sample were placed into four tubes, one containing Cary-Blair transport medium, two with no additive, and one containing merthiolate-iodineformalin solution. Duplicate smears were made on slides for examination of leukocytes, and a modified guaiac test (Hemoccult, Smith Kline Diagnostics, Sunnyvale, Calif.) was performed. The two specimens without additives were kept at - 20 C until examination. Bacteriologic Examination of Feces In stool samples the following bacteria, parasites, toxins, and viruses were searched for: Salmonella sp, Shigella sp, Campylobacter jejuni, Clostridium difficile (bacteria and toxin), Yersinia enterocolitica, Vibrio cholerae, Vibrio parahaemolyticus, protozoa (Entamoeba histolytica, Giardia lamblia), helminths, enterotoxigenic Escherichia coli (thermolabile and thermostable toxins), Aeromonas hydrophila, Edwardsiella sp, rotavirus, and Norwalk virus. Each stool specimen was examined under the microscope in the native state and after gram staining. Salmonella and Shigella were searched for by conventional methods. * Yersinia enterocolitica was grown on Salmonella Shigella agar (Difco Laboratories Inc., Detroit, Mich.) Supplemented with 2% deoxycholate as well as on cefsulodin Irgasan Novobiocin agar (13). Both were incubated at 28 C for 48 h. At the same time, cold enrichment was also performed in phosphate-buffered saline solution at 4 C for 3 wk. Suspect colonies were identified biochemically. Campylobacter jejuni was searched for on Butzler's medium (14). The plates were incubated for 48 h at 43 C using an anaerobic gaspak system without catalyst. Clostridium difficile was cultured on cydoserin cefoxitin fructose (egg yolk agar) medium (15). The toxin was dembnstrated by * All aerobic, gram-negative organisms were worked up and identified. the method published by Ryan et al. (16). For the demonstration of ertterotoxins of Escherichia coli, subcultures of three individual colonies each of Escherichia coli and a pool of 10 colonies were made from each fecal specimen and kept at -20 C until examination for enterotoxin formation. Thermostable enterotoxin was demonstrated by intragastric injection of crude filtrate into 2-4-day-old suckling mice (17) and thermolabile enterotoxin by tests for cytopathogenicity in cultures of Y-1 cells (18). The presence of Staphylococcus aureus was noted if >30 colonies grew from one loopful of fecal suspension on mannitol-salt-phenol red agar. Other bacteria were identified and noted if they made up the predominant aerobic flora or if no Escherichia coli were present. Examination for protozoa and helminths was made in fecal samples from the specimen tubes containing merthiolate-iodineformalin solution (19). Rotaviruses were demonstrated by means of an enzymelinked immunosorbent assay technique (Rotazyme, Abbott Laboratories, Chicago, Ill.). In 30 patients who showed negative stool cultures, investigations for Norwalk antigens in stool samples and for seroconversions to Norwalk virus were performed as described elsewhere (20). Statistical Tests All data were analyzed using the X Z test. Results During the 18-mo study period, 140 patients with acute infectious diarrhea were admitted to the two hospitals. After final evaluation, 21 patients were excluded from the study because their diarrhea lasted >3 days before admission. Of the 28 control patients, 1 patient was later found to have carcinoma of the colon and was therefore excluded. The control patients, most of whom had cardiovascular disease, were significantly older than the diarrheal patients. Forty-nine enteric pathogens were isolated from 46 (38.7%) of the 119 patients with diarrhea (Table 1). In 3 cases, two pathogens were found; these were Salmonella sp associated with Clostridium difficile, Trichuris trichiura, or rotavirus. In 16 of the 73 patients in whom no enteropathogens were found, one or more of the following agents not considered as pathogenic were isolated: Staphylococcus auteus, Pseumononas aeruginosa, Klebsiella sp, Entamoeba coli, Endolimax nana, and Hafnia alvei. Not detected were Yersinia enterocolitica, enterotoxigenic Escherichia coli, Vibrio cholerae, Vibrio parahaemolyticus, Entamoeba histolytica, Aeromonas hydrophila, and Edwardsiella sp. Rotaviruses were found in 4 patients. Thirty stool samples in which no pathogens were found were investigated for Norwalk virus antigen and all were negative. Twenty-one convalescent sera from the 30 patients (65%) possessed antibody to

3 January 1985 INFECTIOUS DIARRHEA IN SWITZERLAND 77 Table 1. Agents Identified in 119 Patients With Acute Diarrhea n % Patients with identified pathogens Pathogens Invasive Salmonella sp 17 a 14.3 Campylobacter jejuni Shigella sp Yersinia enterocolitica 0 0 Noninvasive Clostridium difficile Vibrio sp 0 0 Enterotoxigenic Escherichia coli 0 0 Viruses Rotavirus Norwalk virus 0 0 Protozoa Giardia lamblia Entamoeba histolytica 0 0 Helminths Trichuris trichiura a Mixed infection in 3 patients: Salmonella sp + Clostridium difficile. Salmonella sp + rotavirus. Salmonella sp + Trichuris trichiura. Norwalk virus but serologic titers were all less than fourfold. In the control group (n = 27), no enteric pathogens were isolated except in 1 patient who excreted Clostridium difficile (toxin negative) together with Pseudomonas fluorescens, a nonenteropathogenic agent. These patients showed no symptoms of gastrointestinal disease. Staphylococcus aureus was isolated from two stool samples of the control group. No patient showed fecal leukocytes; 1 patient had a positive hemoccult test, but no agent was found in the stool. Table 2 summarizes anamnestic, clinical, and laboratory data in the 119 patients with acute diarrhea. Patients with diarrhea due to an invasive pathogen showed no statistically significant different clinical features in the incidence of abdominal pain, vomiting, fever, stool frequency, or the occurrence offrank blood in stools when compared with those patients in whom no pathogens or no enteropathogenic agents were isolated. However, specific food exposure and foreign travel were significantly more frequent in the former group. In patients with diarrhea induced by invasive agents, fecal leukocytes were found in 85.7% and a positive modified guaiac test in 80%. This is significantly different from the group in which no pathogens were found (32.9% and 34.2%, respectively). White blood cell count did not differ in the two groups. In the Campylobacter group (15 patients), 7 patients (47%) noted previous consumption of poultry; 5 of these patients gave an additional history of ingestion of clams or raw meat. In the group with Salmonella infections (17 patients), 5 (29%) had eaten poultry or clams, or both. There is no statistical difference in food exposure between these two groups. All 3 patients with Shigella infection, 6 of 15 patients with Campylobacter infection, and 5 of 17 patients with Salmonella infection had been abroad during the 3 wk preceding their diarrheal illness. Clinical features, the occurrence of fecal leukocytes, and occult blood were similar in all groups with invasive pathogens. Clostridium difficile was found in 7 patients with acute diarrhea (1 patient had a mixed infection with Salmonella sp). The toxin of Clostridium difficile was identified in only 4 of the 7 patients. Five patients had previous antibiotic therapy, 3 with a single antibiotic (amoxycillin, clindamycin, or cefur- Table 2. Anamnestic, Clinical, and Laboratory Findings in 119 Patients With Acute Diarrhea Due to Different Etiologic Agents "Invasive" (n = 35) Salmonella spa Clostridium Shigella sp No pathogens difficile Rotavirus Campylobacter jejuni (n = 7) (n = 4) (n = 73) n % n n n % Significance b Specific food exposure 13/ /6 7/ S Foreign travel 14/ /7 0 12/ S Previous antibiotic use 2/ /7 18/ NS Temperature >38 C at admisison 10/ /6 0/3 13/ NS >12 stools/day last 24 h 13/ /7 3/4 13/ NS Frank blood in stool 5/ /7 0/4 6/ NS Severe abdominal pain 28/ /7 3/4 49/ NS Vomiting 16/ /7 4/4 33/ NS White blood cell count> 10,000/1L1 5/ /5 1/3 13/ NS Fecal leukocytes 30/ /6 3/4 24/ S Occult fecal blood 28/ /6 3/4 25/ S a S, significant; NS, not significant. Salmonella sp associated with: Clostridium difficile 1 x, rotavirus 1 x, Trichuris trichiura 1 X. b Statistical comparison was made between the invasive group and the group with no pathogens.

4 78 LOOSLI ET AL. GASTROENTEROLOGY Vol. 88, No, 1, Part 1 oximj, and 2 with a combination of amoxycillin and co-trimoxazol. One patient, who had no previous antibiotic therapy, had a mixed infection with Clostridium difficile and Salmonella sp. Clinical symptoms in all patients were fever, abdominal pain, or watery diarrhea. Vomiting and bloody stool were seen in only 1 patient (Table 2). Rectosigmoidoscopy was carried out in 6 patients with Clostridium difficile infection: pseudomembranous colitis was found in 4 patients and a nonspecific colitis was histologically verified in 2 patients. In the 4 patients with rotavirus infection, the predominant clinical features were vomiting, abdominal pain, and frequent and watery stools. Fever was absent. Three of the 4 patients had occult blood and fecal leukocytes in the stools. Three cases occurred in fall and winter and 1 case occurred during the summer. Discussion Although there are numerous studies on diarrhea in special population groups, e.g., travelers (5-9), only scanty data are available on the etiology and epidemiology of acute infectious diarrhea in adults in the industrialized nations. To our knowledge, the present study represents the first prospective investigation on the etiology, anamnestic, and clinical features of acute infectious diarrhea in continental Europe, in which newly recognized enteric pathogens, such as Campylobacter jejuni, enterotoxigenic Escherichia coli, Clostridium difficile, rotavirus, and Norwalk virus were searched for. Pathogenic agents were identified in 46 of 119 patients (38.7%). The most frequent pathogens were Salmonella sp, Campylobacter jejuni, and Clostridium difficile, whereas Shigella, rotavirus, and Giardia lamblia were much rarer (Table 1). Yersinia enterocolitica, Vibrio cholerae and parahaemolyticus, Entamoeba histolytica, Aeromonas hydrophila, Edwardsiella sp, or enterotoxigenic Escherichia coli were not identified as causes of acute infectious diarrhea in the patients surveyed. Other types of Escherichia coli (enteroinvasive and enteropathogenic) were not searched for; enteropathogenic Escherichia coli is reported to be rare in industrialized nations (21). We did not look for causes of food poisoning because the toxins of Staphylococcus aureus, Bacillus cereus, and Clostridium perfringens are detectable in stools only at highly specialized laboratories and analysis of suspicious food was not possible in this study group. The percentage of microorganisms detected in this study population was somewhat lower than that reported by Jewkes et al. (12) from a population in suburban northwest London. This discrepancy could well reflect different selection criteria, because in their study patients whose diarrhea had ceased on admission were excluded, whereas in our investigation all patients were enrolled when their diarrhea started within the last 72 h before admission, irrespective of whether the diarrhea continued or not on entry to the study. A much higher identification rate of pathogens has also been reported from the Third World countries such as Bangladesh (22,23). This higher rate of pathogens is most likely due to the inclusion of a large number of children in whom pathogens were found in up to 70% of the cases; rotavirus was also often found (22). When the study populations are stratified according to age groups, the identification rate observed in adults becomes very similar to that observed in the present study. In contrast to the Bangladesh study, enterotoxigenic Escherichia coli and Vibrio cholerae are virtually absent in industrialized European countries and the United States (24-26). Patients traveling abroad or those who have been exposed to certain foods were more likely to harbor invasive agents. The present study also confirms the observation that Clostridium difficile infection is associated with previous use of antibiotics. In our study population, it was not possible to distinguish between patients with diarrhea caused by invasive agents and those in whom no agent or a nonenteropathogenic agent was found based on the clinical features of fever, abdominal pain, or vomiting. However, tests for fecal leukocytes and occult blood in the stool correlated extremely well in our patients and showed a sensitivity of ~ 8 for 0 % diarrhea with invasive agents. It is our experience that one of these tests may be sufficient for routine diagnostic use; the procedure of choice in our areas is the modified guaiac test (27). The present study, however, does not allow any conclusion on the specificity of the tests as agents causing noninvasive diarrhea such as enterotoxigenic Escherichia coli and Vibrio cholerae did not occur in our adult study population and viral diarrhea was rarely identified. Although Norwalk virus-induced diarrhea was found in ~ 3 of % the British population studied (12), we did not detect the Norwalk agent in the stool nor a diagnostic rise of antibody titer in the sera of the 30 patients investigated. This difference may be explained by the fact that Norwalk agent-induced diarrhea is usually associated with mild clinical symptoms, which do not lead the patient to seek hospital care, and therefore such subjects would be excluded from the present investigation. The fact, however, that 65% of the sera were positive for Norwalk virus antibody proves that Norwalk virus infection is prevalent in the area studied (28). It is surprising to note that in only 38% of the 119 investigated patients could a diarrhea-inducing mi-

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