Etiology of Diarrhea in Young Children in Denmark: a Case-Control ...

12 downloads 0 Views 88KB Size Report
Dec 17, 2004 - Wilhelmi, I., E. Roman, and A. Sanchez-Fauquier. 2003. Viruses causing gastroenteritis. Clin. Microbiol. Infect. 9:247–262. 44. Willshaw, G. A. ...
JOURNAL OF CLINICAL MICROBIOLOGY, Aug. 2005, p. 3636–3641 0095-1137/05/$08.00⫹0 doi:10.1128/JCM.43.8.3636–3641.2005 Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Vol. 43, No. 8

Etiology of Diarrhea in Young Children in Denmark: a Case-Control Study Bente Olesen,1,2* Jacob Neimann,3 Blenda Bo ¨ttiger,1 Steen Ethelberg,1 Peter Schiellerup,1 1 1 Charlotte Jensen, Morten Helms, Flemming Scheutz,1 Katharina E. P. Olsen,1 Karen Krogfelt,1 Eskild Petersen,1 Kåre Mølbak,1 and Peter Gerner-Smidt1 Department of Bacteriology, Mycology, and Parasitology, Department of Virology, and Department of Epidemiology, Statens Serum Institut, Artillerivej 5, DK-2300 Copenhagen S, Denmark1; Department of Clinical Microbiology, Hillerød Hospital, Helsevej 2, DK-3400 Hillerød, Denmark2; and Danish Zoonosis Centre, Danish Institute for Food and Veterinary Research, Mørkhøj Bygade 19, DK-2860 Søborg, Denmark3 Received 17 December 2004/Returned for modification 1 April 2005/Accepted 14 April 2005

Infectious gastroenteritis is one of the most common diseases in young children. To clarify the infectious etiology of diarrhea in Danish children less than 5 years of age, we conducted a 2-year prospective case-control study. Stools from 424 children with diarrhea and 870 asymptomatic age-matched controls were examined, and their parents were interviewed concerning symptoms. Rotavirus, adenovirus, and astrovirus were detected by enzyme-linked immunosorbent assay, and norovirus and sapovirus were detected by PCR. Salmonella, thermotolerant Campylobacter, Yersinia, Shigella, and Vibrio spp. were detected by standard methods. Shiga toxinproducing (STEC), attaching-and-effacing (A/EEC), enteropathogenic (EPEC), enterotoxigenic, enteroinvasive, and enteroaggregative Escherichia coli were detected by using colony hybridization with virulence gene probes and serotyping. Parasites were detected by microscopy. Overall, a potential pathogen was found in 54% of cases. More cases than controls were infected with rotavirus, Salmonella, norovirus, adenovirus, Campylobacter, sapovirus, STEC, classical EPEC, Yersinia, and Cryptosporidium strains, whereas A/EEC, although common, was not associated with illness. The single most important cause of diarrhea was rotavirus, which points toward the need for a childhood vaccine for this pathogen, but norovirus, adenovirus, and sapovirus were also major etiologies. Salmonella sp. was the most common bacterial pathogen, followed by Campylobacter, STEC, Yersinia, and classical EPEC strains. A/EEC not belonging to the classical EPEC serotypes was not associated with diarrhea, underscoring the importance of serotyping for the definition of EPEC. fectious etiologies of diarrhea in Danish children less than 5 years of age.

Infectious gastroenteritis is one of the most common diseases in humans, with particularly high morbidity in children younger than 5 years of age (3). In industrialized countries, such as Denmark, the associated mortality is low, but the social burden and economic costs due to care of ill children and parents’ absence from work are substantial because of the high incidence. Rotavirus is known to be the most common cause of severe acute, watery diarrhea in children under 5 years of age in industrialized and developing parts of the world (14, 43). In recent decades other new etiologies of diarrhea have been recognized, including noro- and sapovirus, Shiga toxin-producing Escherichia coli (STEC), and enteroaggregative E. coli (EAggEC). Furthermore, the incidence of food-borne Campylobacter and Salmonella infections has increased in many industrialized countries. Several case-control or cohort studies of enteropathogens associated with childhood diarrhea have been conducted in developing countries, but only a few analytical studies covering a broad range of newly discovered diarrheal agents have been undertaken in Europe (5, 8, 25, 35). Many studies have focused on either bacterial or viral etiologies of diarrhea (7, 27, 38). The present study, comprising examinations for bacteria, virus, and parasites, was conducted to clarify the most common in-

MATERIALS AND METHODS Study design. The present study was designed as a prospective case-control study and was conducted from March 2000 to December 2001. Cases were consecutively collected among children less than 5 years of age with stool samples submitted for the diagnosis of infectious diarrhea to Statens Serum Institut that covered 8 of the 16 Danish counties, corresponding to ca. 45% of the Danish population. Verbal agreement to participate in the study was initially received from patient’s parents through their physician. Parents who agreed to participate were mailed additional information about the study, a consent form, and a questionnaire. The parents were requested to review the questionnaire immediately and sign and return the consent form by mail. Patients were excluded from the study if the parents were unable to speak Danish or if the patient had an underlying malignant illness, did not have diarrhea, had siblings already enrolled, or were part of recognized general outbreaks. Controls were selected from the Danish Civil Registry, which is a complete and continuously updated population register. Controls were individually matched with cases on gender, week of birth and county of residence. Eligible controls were mailed a consent form and a questionnaire. The aim was to include two controls per case. If the controls did not respond after approximately 10 days, new controls were chosen. Controls were excluded from the study if they had had diarrhea or abdominal pain with fever during the preceding month or if the parents did not speak Danish. The study was approved by the Danish Scientific Ethical Committee (KF 01-235/95 and 01-148/96). Stool investigations. Stools were examined for bacteria, virus, and parasites. If more than one stool sample was submitted from a case, only the result from the index sample was included in the prevalence calculations in order not to introduce a bias in the comparison with the controls. Rotavirus, adenovirus, and astrovirus. A 10% stool suspension in Parker 199 medium was prepared, and the suspensions were stored at ⫺20°C. The testing for

* Corresponding author. Mailing address: Department of Clinical Microbiology, Hillerød Hospital, Helsevej 2, DK-3400 Hillerød, Denmark. Phone: 45-48294379. Fax: 45-48294384. E-mail: [email protected]. 3636

VOL. 43, 2005

DIARRHEA IN DANISH CHILDREN

3637

TABLE 1. Prevalence and adjusted ORs for diarrheal pathogenicity for potential enteropathogens acquired in Denmark and abroad in children younger than 5 years olda No. positive/no. tested (%) Organism

Rotavirus Norovirus Adenovirus Sapovirus Astrovirus Salmonella sp. Campylobacter sp. Y. enterocolitica STEC EPEC ETEC EAggEC EIEC A/EEC G. lamblia Cryptosporidium B. hominis Ascaris sp.

Acquired in Denmark

Acquired through foreign travel

Cases

Controls

Cases

Controls

50/357 (14) 19/349 (5) 16/357 (5) 11/340 (3) 3/357 (⬍1) 19/396 (5) 14/396 (4) 10/396 (3) 11/396 (3) 7/396 (2) 0/396 (0) 7/396 (2) 1/396 (⬍1) 44/396 (11) 2/344 (⬍1) 4/327 (1) 2/344 (⬍1) 1/344 (⬍1)

1/635 (⬍1) 9/629 (1) 4/633 (⬍1) 8/619 (1) 1/635 (⬍1) 4/712 (⬍1) 3/712 (⬍1) 1/712 (⬍1) 5/712 (⬍1) 5/712 (⬍1) 1/708 (⬍1) 8/712 (1) 0/712 (0) 91/714 (13) 4/585 (⬍1) 0/561 (0) 6/585 (1) 0/585 (0)

1/26 (4) 1/25 (4) 0/26 (0) 0/25 (0) 0/26 (0) 3/28 (11) 0/28 (0) 0/28 (0) 0/28 (0) 3/28 (11) 2/29 (7) 4/28 (14) 0/28 (0) 4/28 (14) 1/25 (4) 2/24 (8) 1/25 (4) 0/25 (0)

0/14 (0) 1/14 (7) 0/14 (0) 0/14 (0) 0/14 (0) 0/14 (0) 0/14 (0) 0/14 (0) 0/14 (0) 0/14 (0) 0/14 (0) 0/14 (0) 0/14 (0) 0/14 (0) 0/10 (0) 0/10 (0) 2/10 (20) 0/10 (0)

OR (95% CI)b

96 (13.1–700) 3.7 (1.7–8.1) 7.7 (2.5–23) 3.0 (1.2–7.6) 4.7 (0.5–47) 9.3 (3.1–28) 8.8 (2.5–31) 19 (2.4–150) 4.3 (1.5–13) 3.1 (1.0–9.4) 2.0 (0.1–28) 2.0 (0.8–5.1) –c 1.0 (0.7–1.4) 1.1 (0.2–5.3) – 0.5 (0.1–2.0) –

a

Mixed infections are included in this table. ORs were calculated on the basis of both domestic and imported infections and adjusted for gender, age, travel, and season. 95% CI, 95% confidence interval. c –, undefined. b

rotavirus, adenovirus, and astrovirus antigens was performed by using commercially available antigen capture tests from Dako (Taastrup, Denmark): IDEA Rotavirus (K6020), IDEA Adenovirus (K6021), and Amplified IDEA Astrovirus (K6042). The instructions from the manufacturer were followed, except that Parker 199 was used instead of the supplied sample buffer. Norovirus and sapovirus. The stool suspensions described above were used. RNA was extracted by QIAamp Viral RNA minikit (QIAGEN GmbH, Hilden, Germany) according to the manufacturer⬘s instructions. The purified RNA was kept at ⫺80°C until testing. Reverse transcription-PCR (RT-PCR) for the detection of noroviruses was performed by using slightly modified JV12/JV13 primers (JV12Y, ATACCACTATGATGCAGAYTA; JV13I, TCATCATCACC ATAGAAIGAG) (40) and a One-Step RT-PCR kit (QIAGEN). Viral RNA was reverse transcribed for 30 min at 50°C. The thermocycling consisted of 40 cycles of 30 s at 94°C, 30 s at 37°C, and 30 s at 72°C, with a final extension at 72°C for 10 min. RT-PCR for sapoviruses was performed with primers JV33 and SR80 (39) using the same procedure as for noroviruses but with an annealing temperature of 49°C. Bacteria. The SSI enteric medium was used for isolation of Shigella spp., Salmonella spp., Yersinia enterocolitica, and Vibrio spp. (4). For the isolation of Campylobacter coli and C. jejuni, the modified charcoal cefoperazone deoxycholate agar was used (11). Stool samples were examined for diarrheagenic E. coli by colony dot blot hybridization of colonies from the primary plate of the SSI enteric medium using DNA probes detecting stx1 and stx2 of STEC (34, 44); eae of enteropathogenic E. coli (EPEC), attaching-and-effacing E. coli (A/EEC), and some STEC strains (13); ipaH of enteroinvasive E. coli (EIEC) and Shigella strains (37); and elt, estA, and estB of enterotoxigenic E. coli (ETEC) (33) and pCVD432 for detection of EAggEC (2). Single colonies reacting positively with the DNA probes were isolated and characterized further. The strains were confirmed as being E. coli by using the Minibact E kit (SSI) (15) and a test for ␤-glucuronidase production on PGUA plates (SSI) (19). stx1- or stx2-positive strains were confirmed by using the Vero cell assay (18). O:H serotyping was performed according to the method of Ørskov and Ørskov (24). Non-STEC E. coli strains reacting with the eae probe belonging to one of the classical EPEC serotypes (O26:H⫺, O26:H11, O26:H34, O55:H⫺, O55:H6, O55:H7, O86:H⫺, O86:H34, O111:H⫺, O111:H2, O111:H25, O114:H⫺, O114:H2, O119:H⫺, O119:H2, O119:H6, O125ac:H⫺, O125ac:H6, O125ac:H21, O126:H-, O126:H2, O126:H21, O126:H27, O127:H⫺, O127:H6, O126:H21, O128ab:H⫺, O128ab: H2, O128ab:H7, O128ab:H12, O142:H⫺, O142:H6, O158:H⫺, and O158:H23) (6, 9, 10, 16, 17, 20, 21, 24, 28–32) were classified as EPEC; similar strains not belonging to these serotypes were classified as A/EEC.

Parasites. Formalin (10%) was added to the specimen to a total volume of 20 ml. The specimen was then sifted through a gauze mesh, diluted 2:1 with phosphate-buffered saline (pH 7.2), and spun at 1,600 rpm for 2 min. The pellet was resuspended in 7 ml of phosphate-buffered saline plus 3 ml of ethyl acetate and centrifuged for 2 min at 1,600 rpm. Two drops were placed on a glass slide and covered before being examined with a ⫻10 objective lens for ova and a ⫻20 objective lens for protozoan cysts after staining with a drop of 1% iodine in 2% potassium iodide. A drop from the pellet was spread on a slide, dried, and Ziehl-Neelsen acid fast stained for Cryptosporidium and Cyclospora spp. Interviews. The cases were seen initially by a physician, and clinical information was subsequently collected through telephone interviews with a parent without the assistance of a physician. The parents of cases were questioned about clinical symptoms, treatment, and other relevant clinical information by using the standard questionnaire sent out when the child was enrolled in the study. In order to facilitate data collection and reduce interviewer bias, all interviews were conducted by using a computer-aided telephone interviewing system. For use of medication, the period in question was 4 weeks, and for foreign travel it was 1 week before the onset of symptoms. Statistical methods. Pathogenicity was expressed as an odds ratio (OR), i.e., the odds of a pathogen-positive specimen being collected from a child with diarrhea divided by the odds of a pathogen-positive specimen being collected from a control. ORs were calculated by multivariate logistic regression, adjusting for the matching variables gender, age group, and season, as well as for foreign travel. Three age groups (children ⬍1 year old, 1 year old, and ⬎1 year old) and four seasons (February to April, May to July, August to October, and November to January) were used. Percent population attributable risks (PAR%) were estimated by using the formula by Miettinen (22).

RESULTS A total of 424 cases and 866 controls were enrolled in the study. Samples were submitted from hospitals for 16% and from general practitioners for 84% of cases. Stools from all cases and 726 of the controls were examined for bacterial pathogens, but some samples did not contain enough material for viral and parasitic analysis (Table 1). Overall, a potential pathogen was found in 54% of cases and 22% of controls, when only samples examined for all pathogens were considered.

3638

OLESEN ET AL.

J. CLIN. MICROBIOL.

TABLE 2. Age distribution of Danish children with gastroenteritis caused by the most common pathogensa No. (%) Age (yr)

All patients

Salmonella sp.

Campylobacter sp.

Y. enterocolitica

STEC

EPEC

Rotavirus

Norovirus

Adenovirus

Sapovirus

0 1 2 3 4

116 (27) 173 (41) 85 (20) 27 (6) 23 (5)

9 (8) 7 (4) 4 (5) 1 (4) 1 (4)

1 (⬍1) 6 (4) 2 (2) 2 (7) 3 (13)

2 (2) 3 (2) 3 (4) 2 (7) 0 (0)

3 (3) 5 (3) 2 (2) 0 (0) 1 (4)

5 (4) 5 (3) 0 (0) 0 (0) 0 (0)

12/103 (12) 20/159 (13) 16/76 (21) 1/24 (4) 2/21 (10)

3/98 (3) 14/158 (9) 3/76 (4) 0/76 (0) 0/19 (0)

6/103 (6) 8/159 (5) 1/75 (1) 0 (0) 1/20 (⬍1)

4/112 (4) 5/155 (3) 2/70 (3) 0/23 (0) 0/20 (0)

a

Percentages are shown in parentheses. If nothing else is noted, the denominator is identical to the number of patients. Mixed infections are included in this table.

Among all samples tested, a virus was found in 26% of cases and 3% of controls; a bacterial pathogen was found in 17% of cases and 4% of controls excluding A/EEC; and a parasite was found in in 4% of cases and 3% of controls. In order of observed frequency, rotavirus, Salmonella, norovirus, adenovirus, thermotolerant Campylobacter, STEC, EPEC, sapovirus, Y. enterocolitica, and Cryptosporidium spp. were found at higher prevalences in patients than in controls (Table 1). A/EEC was equally common among cases (11%) and controls (13%) and thus not associated with diarrhea. A number of microorganisms were found only rarely, including astrovirus, ETEC, EIEC, Ascaris spp., Blastocystis hominis, and Giardia lamblia. No Shigella, Vibrio, or Cyclospora spp. were isolated. The majority of children infected by virus, EPEC or STEC were under 3 years of age (Table 2). Rotavirus was by far the most common pathogen detected and had the strongest association with disease (OR ⫽ 96). Consequentially, a higher proportion of disease (PAR%) was attributed to this pathogen (13.2%) than to any other microorganism (Table 3). The most frequently isolated bacterial pathogen was zoonotic Salmonella. Five serotypes were seen: serovars Typhimurium, Enteritidis, Agona, Hadar, and Bovismorbificans in 9, 7, 3, 2, and 1 patient, respectively. The serotypes Typhimurium, Agona, Derby, and Braenderup were found in each of four controls. Eleven patients and five controls were infected with STEC. Five patients were infected with different serotypes, and two each were infected with STEC O157:[H7], O26:H⫺, and O103:H2. Three healthy children also carried these three serotypes. All STEC isolates were eae positive except one serotype O126:H20 strain. Ten and five EPEC of classical O:H serotypes were isolated from cases and controls, respectively. The most common serotype was O55:

TABLE 3. Proportion of disease of enteropathogens with adjusted ORs larger than unity in Danish cases of childhood diarrheaa Organism

PAR%

Rotavirus..............................................................................................13.2 Salmonella sp....................................................................................... 4.6 Norovirus ............................................................................................. 3.9 Adenovirus........................................................................................... 3.6 Campylobacter sp................................................................................. 2.9 Y. enterocolitica.................................................................................... 2.2 Sapovirus.............................................................................................. 2.0 STEC .................................................................................................... 2.0 EPEC.................................................................................................... 1.6 EAggEC ............................................................................................... 1.3 a

Mixed infections are included in this table.

[H7], which was found in 6 of the 10 EPEC patients and in none of the controls. EPEC was only isolated from cases below 2 years of age (Table 2), and was the second most common (4%) bacterial cause of diarrhea in that age group. A total of 2% of cases had mixed infections with pathogens associated with diarrhea. Two patients were infected with both Salmonella and norovirus and single patients with the following combinations: rota- and adenovirus, noro- and adenovirus, rota- and norovirus, EPEC and adenovirus, EPEC and astrovirus, and EPEC and ETEC. The seasonal prevalence was most prominent for rotavirus, with most infections seen in the period from January to May. The bacterial infections tended to occur during summer and fall (Fig. 1). Clinical data. Selected clinical data on the patients are given in Table 4. Overall, the viral infections were characterized by vomiting. Bloody diarrhea was characteristic for Campylobacter infections, but this symptom was also seen among Salmonellainfected patients, whereas only two of nine patients infected with STEC had bloody diarrhea. None developed hemolyticuremic syndrome. Both Salmonella and rotavirus infections were characterized by loss of weight, fever, and high hospitalization rates. Antibiotic treatment was given to 21% of children infected with Salmonella spp. in contrast to 5% of children (P ⫽ 0.061) with no pathogen identified and 4% of children (P ⫽ 0.1) with viral infections. All children survived their infections. DISCUSSION A potential pathogen was detected for 54% of the patients, and an infectious agent associated with diarrhea was established for 45% of the patients in the present study. In spite of its seasonal occurrence, rotavirus was the most frequently isolated pathogen. The severity of the rotavirus infections was comparable to those caused by Salmonella sp. Norovirus, adenovirus, and sapovirus were also important pathogens of childhood diarrhea. Noro- and sapoviruses have up till now generally not been tested for in microbiology laboratories, and the incidence of these pathogens is therefore probably underestimated. Astrovirus, a well-known diarrheagenic pathogen (41), was only identified in a few children in the present study, which may explain why it was not significantly linked statistically to diarrhea. Large case-control studies and cohorts on diarrhea have been conducted in The Netherlands (8), Italy (5), and the United Kingdom (35, 42), and in these studies viruses were

VOL. 43, 2005

DIARRHEA IN DANISH CHILDREN

3639

FIG. 1. Bubble diagram showing the relative prevalence per month of the most frequently occurring pathogens among cases.

also found to be the leading cause of diarrhea. A number of studies have shown rotavirus to be a leading cause of gastroenteritis in children (14, 43). However, English (35), Dutch (8), and Finnish (26) community studies found the percentage attributable to norovirus to be clearly higher than in our study, and this is also true for sapovirus (8). The Finnish study showed that norovirus caused milder symptoms than rotavirus, and thus parents of patients with norovirus infections will be less likely to seek medical attention. Community studies are therefore expected to disclose a higher proportion of norovirus than studies involving patients admitted to hospitals or seen by a physician. The fact that the stool suspensions in our study had been frozen and thawed several times before being tested

by RT-PCR may also have diminished the sensitivity of the norovirus and sapovirus detection. Similarly, astrovirus causes milder symptoms than other viruses tested for in the Finnish study (26) and are therefore less likely to be detected among samples from medical facilities. In addition, it has been shown that the sensitivity of the enzyme-linked immunosorbent assay for astrovirus used here is only 50 to 60% compared to PCR tests used in other studies (41). With regard to bacterial causes, our findings are in general agreement with previous case-control studies. However, in most studies (5, 8, 25, 35), STEC was isolated in ⬍1% of cases versus 3% in the present study; in Italy (5), 19% of stools from cases younger than 10 years old were positive for Salmonella versus 5% in the present study.

TABLE 4. Selected clinical data of the most important causes of gastroenteritis in Danish children under 5 years of agea Organism

Rotavirus Adenovirus Norovirus Sapovirus Salmonella sp. Campylobacter sp. Y. enterocolitica STEC EPEC No pathogen

No. of patients (%) with:

No. of patients

Vomiting

Bloody diarrhea

Fever

Hospitalization

Dehydrationb

Loss of wt

45 12 10 10 19 12 10 9 7 183

42 (93) 8 (67) 8 (80) 6 (60) 9 (47) 2 (17) 3 (30) 3 (33) 2 (29) 71/181 (39)

2 (4) 0 (0) 1 (10) 0 (0) 5 (26) 11 (92) 2 (20) 2 (22) 1 (14) 11/181 (6)

41 (91) 7 (58) 7 (70) 5/9 (56) 17 (90) 11 (92) 8 (80) 4 (44) 4 (57) 99/177 (56)

13 (29) 0 (0) 2 (20) 2 (20) 5 (26) 2 (17) 1 (10) 3 (33) 3 (43) 23/182 (13)

11 (24) 1 (8) 2 (20) 1 (10) 5 (26) 2 (17) 0 (0) 2 (22) 2 (29) 23 (13)

33 (73) 5/9 (56) 6 (60) 6/9 (67) 14/18 (78) 4/9 (44) 5/9 (56) 2 (22) 2/5 (20) 67/158 (42)

a Percentages are shown in parentheses. Mixed and potentially mixed infections (cases with a secondary bacterial pathogen isolated within 30 days before and after the index pathogen) were excluded. If nothing else is noted, the denominator is identical to the number of patients. b Patients who received treatment with oral or intravenous fluid.

3640

OLESEN ET AL.

Surveillance data indicate that thermotolerant Campylobacter is the most common bacterial gastrointestinal pathogen for all ages in Denmark (1). However, in the present study of children younger than 5 years, it was surpassed by Salmonella as the most common bacterial pathogen. Y. enterocolitica was isolated from 3% of patients, indicating that this species continues to be an important pathogen in young children. Several points of interest relate to diarrheagenic E. coli. Apart from STEC, only eae gene-positive strains belonging to the classical EPEC serotypes were associated with diarrhea. A/EEC, i.e., E. coli isolates harboring the eae gene but not producing Shiga toxin or belonging to the classical EPEC serotypes, was the most commonly isolated potential pathogen in the present study. However, A/EEC was isolated at a similar proportion from cases and controls. The presence of the gene encoding bundle-forming pilus, a recognized EPEC virulence factor (23), was neither alone nor in combination with the eae gene associated with diarrhea (data not shown). Recent studies have applied a definition of EPEC where the serotype of the infecting strains is not taken into account (25, 27). Had we defined EPEC by the presence of the eae gene in stx-negative strains, we would not have identified EPEC as a diarrheagenic pathogen. It is, therefore, premature to define EPEC independently of the serotype. Until virulence factors that may be cofactors for diarrhea in EPEC infections have been identified, we recommend including the serotype or at least the O group for the identification of EPEC in young children. Classical EPEC was only isolated from children below 2 years of age and was the second most common bacterial pathogen in this age group, indicating that this pathogen is an important cause of sporadic diarrheal illness in infants. Eleven and five STEC strains were isolated from patients and controls, respectively. The serotypes O26:H⫺, O103:H3 (both stx1 and eae positive), and O157:H7 (stx2 and eae positive) were found in both cases and controls. This is interesting because these three serotypes are not only the most common but also among the most virulent STEC types (36). Although most patients infected with STEC stop excreting the organism shortly after the acute illness has passed, a substantial proportion may continue to carry the organism for several months (12, 36). This may partly explain the occurrence of these pathogens among the control children in the present study. Very few ETEC, EAggEC, and EIEC were isolated. ETEC and EIEC are well-known pathogens, but they are uncommon in children from industrialized countries. EAggEC was isolated more frequently from cases than controls. However, this difference was not statistically significant. Parasitic infections were rare in the present study, though cryptosporidia were isolated from 1.7% of cases. A small number of children were diagnosed with Giardia, explaining why no association with diarrhea was found for this pathogen. In general, infections with Giardia lamblia are a minor cause of diarrhea in Denmark. In 2004, the rate of G. lamblia was 1.7% in samples where examinations for parasites were requested by a physician (unpublished data [Maiken Arendrup, Laboratory of Mycology and Parasitology, Statens Serum Institut, Copenhagen, Denmark]). Only few cases in the present study (6.6%) acquired their infections during foreign travel. Salmonella and EPEC were the most common travel related pathogens. Not surprisingly,

J. CLIN. MICROBIOL.

Cryptosporidium, ETEC, and EAggEC were also associated with travel. Very few viruses were isolated from cases with a travel history. In general, there were overlapping signs and symptoms between the different etiologic categories. Some typical patterns were, however, observed. Bloody diarrhea was reported in 92% of the Campylobacter cases and 20 to 26% of the patients with Salmonella, Y. enterocolitica, and STEC infections but rarely among the patients with a viral infection. As expected, vomiting was a more prominent finding among cases infected with viruses than bacteria. In conclusion, rotavirus was confirmed as the most common pathogen in childhood diarrhea, especially during winter and spring in Denmark. This fact, in combination with the severity of the infections, warrants consideration of a rotavirus vaccine in the childhood immunization program. The role of diarrheagenic E. coli was confirmed for STEC and classical EPEC. A/EEC, i.e., attaching-and-effacing E. coli not belonging to the classical EPEC serotypes, was commonly identified but was not associated with disease. Parasites are only rarely identified in children with diarrhea with the possible exception of patients who have traveled abroad. Routine examination of stool cultures from children less than 5 years of age should include tests for viruses in the colder months and for STEC and classical EPEC. Ideally, identification of STEC should be based on detection of the Shiga toxins or their genes. As for EPEC, routine examination may be restricted to children below 2 years of age but must include a combination of detection of the eae gene and the serogroup of the infecting strain. ACKNOWLEDGMENTS We are grateful to Carsten Struve and Andreas Munk Petersen for having contributed to the data collection and to Brita Bruun for critical reading of the manuscript. We thank the staff of the laboratories that took part in the study. REFERENCES 1. Anonymous. 2002. Annual Report on Zoonoses in Denmark 2001. Ministry of Food, Agriculture, and Fisheries, Copenhagen, Denmark. 2. Baudry, B., S. J. Savarino, P. Vial, J. B. Kaper, and M. M. Levine. 1990. A sensitive and specific DNA probe to identify enteroaggregative Escherichia coli, a recently discovered diarrheal pathogen. J. Infect. Dis. 161:1249–1251. 3. Bern, C., J. Z. I. de Martines, and R. I. Glass. 1992. The magnitude of the global problem of diarrhoeal disease: a ten-year update. Bull. W. H. O. 70:705–714. 4. Blom, M., A. Meyer, P. Gerner-Smidt, K. Gaarslev, and F. Espersen. 1999. Evaluation of Statens Serum Institut enteric medium for detection of enteric pathogens. J. Clin. Microbiol. 37:2312–2316. 5. Caprioli, A., C. Pezzella, R. Morelli, A. Giammanco, S. Arista, D. Crotti, M. Facchini, P. Guglielmetti, C. Piersimoni, I. Luzzi, et al. 1996. Enteropathogens associated with childhood diarrhea in Italy. Pediatr. Infect. Dis. J. 15:876–883. 6. Cravioto, A., R. J. Gross, S. M. Scotland, and B. Rowe. 1979. An adhesive factor found in strains of Escherichia coli belonging to the traditional infantile enteropathogenic serotypes. Curr. Microbiol. 3:95–99. 7. Dennehy, P. H., S. M. Nelson, S. Spangenberger, J. S. Noel, S. S. Monroe, and R. I. Glass. 2001. A prospective case-control study of the role of astrovirus in acute diarrhea among hospitalized young children. J. Infect. Dis. 184:10–15. 8. de Wit, M. A., M. P. Koopmans, L. M. Kortbeek, W. J. Wannet, J. Vinje, F. van Leusden, A. I. Bartelds, and Y. T. van Duynhoven. 2001. Sensor, a population-based cohort study on gastroenteritis in The Netherlands: incidence and etiology. Am. J. Epidemiol. 154:666–674. 9. Donnenberg, M. S. 1995. Enteropathogenic Escherichia coli, p. 709–726. In M. J. Blaser, P. D. Smith, J. I. Ravdin, H. B. Greenberg, and R. L. Guerrant (ed.), Infections of the gastrointestinal tract. Raven Press, Ltd., New York, N.Y. 10. Gomes, T. A., M. A. Vieira, I. K. Wachsmuth, P. A. Blake, and L. R. Trabulsi. 1989. Serotype-specific prevalence of Escherichia coli strains with EPEC adherence factor genes in infants with and without diarrhea in Sao Paulo, Brazil. J. Infect. Dis. 160:131–135.

VOL. 43, 2005 11. Hutchinson, D. N., and F. J. Bolton. 1984. Improved blood free selective medium for the isolation of Campylobacter jejuni from faecal specimens. J. Clin. Pathol. 37:956–957. 12. Jensen, C., P. Schiellerup, K. E. P. Olsen, F. Scheutz, E. Petersen, P. Gerner-Smidt, and K. Molbak. Antimicrobial treatment of asymptomatic carriers of verocytotoxin-producing Escherichia coli: an empiric study. Scand. J. Infect. Dis. 37:61–63. 13. Jerse, A. E., J. Yu, B. D. Tall, and J. B. Kaper. 1990. A genetic locus of enteropathogenic Escherichia coli necessary for the production of attaching and effacing lesions on tissue culture cells. Proc. Natl. Acad. Sci. USA 87:7839–7843. 14. Kapikan, A., Y. Hoshino, and R. M. Chanock. 2001. Rotaviruses, p. 1787– 1834. In D. M. Knipe and M. J. Hudson (ed.), Fields virology. Lippincott/The Williams & Wilkins Co., Philadelphia, Pa. 15. Kjaeldgaard, P., B. Nissen, N. Lange, and H. Laursen. 1986. Evaluation of Minibact, a new system for rapid identification of Enterobacteriaceae: comparison of Minibact, Micro-ID, and API 20E with a conventional method as reference. Acta Pathol. Microbiol. Immunol. Scand. B 94:57–61. 16. Knutton, S., T. Baldwin, P. H. Williams, and A. S. McNeish. 1989. Actin accumulation at sites of bacterial adhesion to tissue culture cells: basis of a new diagnostic test for enteropathogenic and enterohemorrhagic Escherichia coli. Infect. Immun. 57:1290–1298. 17. Knutton, S., A. D. Phillips, H. R. Smith, R. J. Gross, R. Shaw, P. Watson, and E. Price. 1991. Screening for enteropathogenic Escherichia coli in infants with diarrhea by the fluorescent-actin staining test. Infect. Immun. 59:365– 371. 18. Konowalchuk, J., J. I. Speirs, and S. Stavric. 1977. Vero response to a cytotoxin of Escherichia coli. Infect. Immun. 18:775–779. 19. Lautrop, H., N. Høiby, A. Bremmelgaard, and B. Korsager. 1979. Bakteriologiske undersøgelsesmetoder. FADLs Forlag, Copenhagen, Denmark. 20. Levine, M. M., and R. Edelman. 1984. Enteropathogenic Escherichia coli of classic serotypes associated with infant diarrhea: epidemiology and pathogenesis. Epidemiol. Rev. 6:31–51. 21. Levine, M. M., J. P. Nataro, H. Karch, M. M. Baldini, J. B. Kaper, R. E. Black, M. L. Clements, and A. D. O’Brien. 1985. The diarrheal response of humans to some classic serotypes of enteropathogenic Escherichia coli is dependent on a plasmid encoding an enteroadhesiveness factor. J. Infect. Dis. 152:550–559. 22. Miettinen, O. S. 1974. Proportion of disease caused or prevented by a given exposure, trait, or intervention. Am. J. Epidemiol. 99:325–332. 23. Nataro, J. P., and J. B. Kaper. 1998. Diarrheagenic Escherichia coli. Clin. Microbiol. Rev. 11:142–201. 24. Ørskov, F., and I. Ørskov. 1984. Serotyping of Escherichia coli. Methods Microbiol. 14:43–112. 25. Pabst, W. L., M. Altwegg, C. Kind, S. Mirjanic, D. Hardegger, and D. Nadal. 2003. Prevalence of enteroaggregative Escherichia coli among children with and without diarrhea in Switzerland. J. Clin. Microbiol. 41:2289–2293. 26. Pang, X. L., S. Honma, S. Nakata, and T. Vesikari. 2000. Human caliciviruses in acute gastroenteritis of young children in the community. J. Infect. Dis. 181(Suppl. 2):S288–S294. 27. Rademaker, C. M., A. C. Fluit, M. Jansze, W. H. Jansen, J. H. Glerum, and J. Verhoef. 1993. Frequency of enterovirulent Escherichia coli in diarrhoeal disease in The Netherlands. Eur. J. Clin. Microbiol. Infect. Dis. 12:93–97. 28. Robins-Browne, R. M. 1987. Traditional enteropathogenic Escherichia coli of infantile diarrhea. Rev. Infect. Dis. 9:28–53. 29. Scaletsky, I. C., M. L. Silva, M. R. Toledo, B. R. Davis, P. A. Blake, and L. R.

DIARRHEA IN DANISH CHILDREN

30.

31. 32.

33.

34.

35.

36.

37.

38.

39. 40. 41. 42.

43. 44.

3641

Trabulsi. 1985. Correlation between adherence to HeLa cells and serogroups, serotypes, and bioserotypes of Escherichia coli. Infect. Immun. 49: 528–532. Scotland, S. M., H. R. Smith, T. Cheasty, B. Said, G. A. Willshaw, N. Stokes, and B. Rowe. 1996. Use of gene probes and adhesion tests to characterise Escherichia coli belonging to enteropathogenic serogroups isolated in the United Kingdom. J. Med. Microbiol. 44:438–443. Scotland, S. M., G. A. Willshaw, T. Cheasty, and B. Rowe. 1992. Strains of Escherichia coli O157:H8 from human diarrhoea belong to attaching and effacing class of E. coli. J. Clin. Pathol. 45:1075–1078. Scotland, S. M., G. A. Willshaw, H. R. Smith, R. J. Gross, and B. Rowe. 1989. Adhesion to cells in culture and plasmid profiles of enteropathogenic Escherichia coli isolated from outbreaks and sporadic cases of infant diarrhoea. J. Infect. 19:237–249. Sommerfelt, H., K. H. Kalland, P. Raj, S. L. Moseley, M. K. Bhan, and B. Bjorvatn. 1988. Cloned polynucleotide and synthetic oligonucleotide probes used in colony hybridization are equally efficient in the identification of enterotoxigenic Escherichia coli. J. Clin. Microbiol. 26:2275–2278. Thomas, A., H. R. Smith, G. A. Willshaw, and B. Rowe. 1991. Non-radioactively labeled polynucleotide and oligonucleotide DNA probes, for selectively detecting Escherichia coli strains producing Vero cytotoxins VT1, VT2 and VT2 variant. Mol. Cell Probes 5:129–135. Tompkins, D. S., M. J. Hudson, H. R. Smith, R. P. Eglin, J. G. Wheeler, M. M. Brett, R. J. Owen, J. S. Brazier, P. Cumberland, V. King, and P. E. Cook. 1999. A study of infectious intestinal disease in England: microbiological findings in cases and controls. Commun. Dis. Public Health 2:108– 113. Tozzi, A. E., S. Goriette, and A. Caprioli. 2001. Epidemiology of human infections by Escherichia coli O157 and other verocytotoxin-producing E. coli, p. 113–148. In G. Duffy, P. Garvey, and D. A. McDowell (ed.), Verocytotoxigenic E. coli. Food & Nutrition Press, Inc., Trumbull, Conn. Venkatesan, M. M., J. M. Buysse, and D. J. Kopecko. 1989. Use of Shigella flexneri ipaC and ipaH gene sequences for the general identification of Shigella spp. and enteroinvasive Escherichia coli. J. Clin. Microbiol. 27:2687– 2691. Vila, J., A. Gene, M. Vargas, J. Gascon, C. Latorre, and M. T. Jimenez de Anta. 1998. A case-control study of diarrhoea in children caused by Escherichia coli producing heat-stable enterotoxin (EAST-1). J. Med. Microbiol. 47:889–891. Vinje, J., H. Deijl, H. R. van der, D. Lewis, K. O. Hedlund, L. Svensson, and M. P. Koopmans. 2000. Molecular detection and epidemiology of Sapporolike viruses. J. Clin. Microbiol. 38:530–536. Vinje, J., and M. P. Koopmans. 1996. Molecular detection and epidemiology of small round-structured viruses in outbreaks of gastroenteritis in The Netherlands. J. Infect. Dis. 174:610–615. Walter, J. E., and D. K. Mitchell. 2003. Astrovirus infection in children. Curr. Opin. Infect. Dis. 16:247–253. Wheeler, J. G., D. Sethi, J. M. Cowden, P. G. Wall, L. C. Rodrigues, D. S. Tompkins, M. J. Hudson, P. J. Roderick, et al. 1999. Study of infectious intestinal disease in England: rates in the community, presenting to general practice, and reported to national surveillance. BMJ 318:1046–1050. Wilhelmi, I., E. Roman, and A. Sanchez-Fauquier. 2003. Viruses causing gastroenteritis. Clin. Microbiol. Infect. 9:247–262. Willshaw, G. A., H. R. Smith, S. M. Scotland, and B. Rowe. 1985. Cloning of genes determining the production of Vero cytotoxin by Escherichia coli. J. Gen. Microbiol. 131(Pt. 11):3047–3053.