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10.1128/JCM.43.8.3636-3641.2005.

2005, 43(8):3636. DOI:J. Clin. Microbiol.Gerner-SmidtKrogfelt, Eskild Petersen, Kre Mlbak and PeterHelms, Flemming Scheutz, Katharina E. P. Olsen, KarenEthelberg, Peter Schiellerup, Charlotte Jensen, MortenBente Olesen, Jacob Neimann, Blenda Bttiger, SteenDenmark: a Case-Control StudyEtiology of Diarrhea in Young Children in

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JOURNAL OF CLINICAL MICROBIOLOGY, Aug. 2005, p. 36363641 Vol. 43, No. 80095-1137/05/$08.000 doi:10.1128/JCM.43.8.36363641.2005Copyright 2005, American Society for Microbiology. All Rights Reserved.

Etiology of Diarrhea in Young Children in Denmark: aCase-Control Study

Bente Olesen,1,2* Jacob Neimann,3 Blenda Bottiger,1 Steen Ethelberg,1 Peter Schiellerup,1

Charlotte Jensen,1 Morten Helms,1 Flemming Scheutz,1 Katharina E. P. Olsen,1

Karen Krogfelt,1 Eskild Petersen,1 Kre Mlbak,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,

Hillerd Hospital, Helsevej 2, DK-3400 Hillerd, Denmark2; and Danish Zoonosis Centre, Danish Institutefor Food and Veterinary Research, Mrkhj Bygade 19, DK-2860 Sborg, 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 infectiousetiology of diarrhea in Danish children less than 5 years of age, we conducted a 2-year prospective case-controlstudy. Stools from 424 children with diarrhea and 870 asymptomatic age-matched controls were examined, andtheir parents were interviewed concerning symptoms. Rotavirus, adenovirus, and astrovirus were detected byenzyme-linked immunosorbent assay, and norovirus and sapovirus were detected by PCR. Salmonella, ther-motolerant Campylobacter, Yersinia, Shigella, and Vibrio spp. were detected by standard methods. Shiga toxin-producing (STEC), attaching-and-effacing (A/EEC), enteropathogenic (EPEC), enterotoxigenic, enteroinva-sive, and enteroaggregative Escherichia coli were detected by using colony hybridization with virulence geneprobes 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, Campy-

lobacter, sapovirus, STEC, classical EPEC, Yersinia, and Cryptosporidium strains, whereas A/EEC, althoughcommon, was not associated with illness. The single most important cause of diarrhea was rotavirus, whichpoints toward the need for a childhood vaccine for this pathogen, but norovirus, adenovirus, and sapoviruswere 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 notassociated with diarrhea, underscoring the importance of serotyping for the definition of EPEC.

Infectious gastroenteritis is one of the most common dis-

eases in humans, with particularly high morbidity in childrenyounger than 5 years of age (3). In industrialized countries,such as Denmark, the associated mortality is low, but the socialburden and economic costs due to care of ill children andparents absence from work are substantial because of the highincidence. Rotavirus is known to be the most common cause ofsevere acute, watery diarrhea in children under 5 years of agein industrialized and developing parts of the world (14, 43). Inrecent decades other new etiologies of diarrhea have beenrecognized, including noro- and sapovirus, Shiga toxin-produc-ing Escherichia coli (STEC), and enteroaggregative E. coli(EAggEC). Furthermore, the incidence of food-borne Campy-lobacter and Salmonella infections has increased in many in-dustrialized countries.

Several case-control or cohort studies of enteropathogensassociated with childhood diarrhea have been conducted indeveloping countries, but only a few analytical studies coveringa broad range of newly discovered diarrheal agents have beenundertaken in Europe (5, 8, 25, 35). Many studies have focusedon 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-

fectious etiologies of diarrhea in Danish children less than 5

years of age.

MATERIALS AND METHODS

Study design. The present study was designed as a prospective case-controlstudy and was conducted from March 2000 to December 2001. Cases were

consecutively collected among children less than 5 years of age with stool samplessubmitted for the diagnosis of infectious diarrhea to Statens Serum Institut that

covered 8 of the 16 Danish counties, corresponding to ca. 45% of the Danishpopulation. Verbal agreement to participate in the study was initially receivedfrom patients parents through their physician. Parents who agreed to participate

were mailed additional information about the study, a consent form, and aquestionnaire. The parents were requested to review the questionnaire immedi-

ately and sign and return the consent form by mail. Patients were excluded fromthe study if the parents were unable to speak Danish or if the patient had anunderlying malignant illness, did not have diarrhea, had siblings already enrolled,

or were part of recognized general outbreaks. Controls were selected from theDanish Civil Registry, which is a complete and continuously updated populationregister. Controls were individually matched with cases on gender, week of birth

and county of residence. Eligible controls were mailed a consent form and aquestionnaire. The aim was to include two controls per case. If the controls did

not respond after approximately 10 days, new controls were chosen. Controlswere excluded from the study if they had had diarrhea or abdominal pain withfever during the preceding month or if the parents did not speak Danish. The

study was approved by the Danish Scientific Ethical Committee (KF 01-235/95and 01-148/96).

Stool investigations. Stools were examined for bacteria, virus, and parasites. Ifmore than one stool sample was submitted from a case, only the result from theindex sample was included in the prevalence calculations in order not to intro-

duce 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 20C. The testing for

* Corresponding author. Mailing address: Department of ClinicalMicrobiology, Hillerd Hospital, Helsevej 2, DK-3400 Hillerd, Den-mark. Phone: 45-48294379. Fax: 45-48294384. E-mail: [email protected].

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rotavirus, adenovirus, and astrovirus antigens was performed by using commer-cially 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 thatParker 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 manufacturers instructions. The purified RNA was

kept at 80C until testing. Reverse transcription-PCR (RT-PCR) for the de-

tection of noroviruses was performed by using slightly modified JV12/JV13primers (JV12Y, ATACCACTATGATGCAGAYTA; JV13I, TCATCATCACC

ATAGAAIGAG) (40) and a One-Step RT-PCR kit (QIAGEN). Viral RNA was

reverse transcribed for 30 min at 50C. The thermocycling consisted of 40 cyclesof 30 s at 94C, 30 s at 37C, and 30 s at 72C, with a final extension at 72C 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 temper-ature of 49C.

Bacteria. The SSI enteric medium was used for isolation of Shigella spp.,

Salmonella spp., Yersinia enterocolitica, and Vibrio spp. (4). For the isolation ofCampylobacter coli and C. jejuni, the modified charcoal cefoperazone deoxy-

cholate 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 Shigellastrains (37); and elt, estA, and estB of enterotoxigenic E. coli (ETEC) (33) and

pCVD432 for detection of EAggEC (2). Single colonies reacting positively withthe 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-positivestrains were confirmed by using the Vero cell assay (18). O:H serotyping was

performed according to the method of rskov and rskov (24). Non-STEC E.

coli strains reacting with the eae probe belonging to one of the classical EPECserotypes (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, 2832) were classified as EPEC; similar strains notbelonging to these serotypes were classified as A/EEC.

Parasites. Formalin (10%) was added to the specimen to a total volume of 20ml. The specimen was then sifted through a gauze mesh, diluted 2:1 with phos-

phate-buffered saline (pH 7.2), and spun at 1,600 rpm for 2 min. The pellet wasresuspended in 7 ml of phosphate-buffered saline plus 3 ml of ethyl acetate andcentrifuged 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 20objective 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, andZiehl-Neelsen acid fast stained for Cryptosporidium and Cyclospora spp.

Interviews. The cases were seen initially by a physician, and clinical informa-

tion was subsequently collected through telephone interviews with a parentwithout the assistance of a physician. The parents of cases were questioned aboutclinical symptoms, treatment, and other relevant clinical information by using the

standard questionnaire sent out when the child was enrolled in the study. Inorder to facilitate data collection and reduce interviewer bias, all interviews were

conducted by using a computer-aided telephone interviewing system. For use ofmedication, the period in question was 4 weeks, and for foreign travel it was 1week 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 collectedfrom a control. ORs were calculated by multivariate logistic regression, adjustingfor 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 Novemberto 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 thestudy. Samples were submitted from hospitals for 16% andfrom general practitioners for 84% of cases. Stools from allcases and 726 of the controls were examined for bacterialpathogens, but some samples did not contain enough materialfor viral and parasitic analysis (Table 1). Overall, a potentialpathogen was found in 54% of cases and 22% of controls, whenonly samples examined for all pathogens were considered.

TABLE 1. Prevalence and adjusted ORs for diarrheal pathogenicity for potential enteropathogens acquired in Denmark and abroad inchildren younger than 5 years olda

Organism

No. positive/no. tested (%)

OR (95% CI)bAcquired in Denmark Acquired through foreign travel

Cases Controls Cases Controls

Rotavirus 50/357 (14) 1/635 (1) 1/26 (4) 0/14 (0) 96 (13.1700)Norovirus 19/349 (5) 9/629 (1) 1/25 (4) 1/14 (7) 3.7 (1.78.1)

Adenovirus 16/357 (5) 4/633 (1) 0/26 (0) 0/14 (0) 7.7 (2.523)Sapovirus 11/340 (3) 8/619 (1) 0/25 (0) 0/14 (0) 3.0 (1.27.6)

Astrovirus 3/357 (1) 1/635 (1) 0/26 (0) 0/14 (0) 4.7 (0.547)Salmonella sp. 19/396 (5) 4/712 (1) 3/28 (11) 0/14 (0) 9.3 (3.128)Campylobacter sp. 14/396 (4) 3/712 (1) 0/28 (0) 0/14 (0) 8.8 (2.531)Y. enterocolitica 10/396 (3) 1/712 (1) 0/28 (0) 0/14 (0) 19 (2.4150)STEC 11/396 (3) 5/712 (1) 0/28 (0) 0/14 (0) 4.3 (1.513)EPEC 7/396 (2) 5/712 (1) 3/28 (11) 0/14 (0) 3.1 (1.09.4)ETEC 0/396 (0) 1/708 (1) 2/29 (7) 0/14 (0) 2.0 (0.128)EAggEC 7/396 (2) 8/712 (1) 4/28 (14) 0/14 (0) 2.0 (0.85.1)EIEC 1/396 (1) 0/712 (0) 0/28 (0) 0/14 (0) c

A/EEC 44/396 (11) 91/714 (13) 4/28 (14) 0/14 (0) 1.0 (0.71.4)G. lamblia 2/344 (1) 4/585 (1) 1/25 (4) 0/10 (0) 1.1 (0.25.3)Cryptosporidium 4/327 (1) 0/561 (0) 2/24 (8) 0/10 (0)

B. hominis 2/344 (1) 6/585 (1) 1/25 (4) 2/10 (20) 0.5 (0.12.0)

Ascaris sp. 1/344 (1) 0/585 (0) 0/25 (0) 0/10 (0) a Mixed infections are included in this table.b 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.

VOL. 43, 2005 DIARRHEA IN DANISH CHILDREN 3637


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Among all samples tested, a virus was found in 26% of casesand 3% of controls; a bacterial pathogen was found in 17% ofcases and 4% of controls excluding A/EEC; and a parasite wasfound in in 4% of cases and 3% of controls. In order ofobserved frequency, rotavirus, Salmonella, norovirus, adenovi-rus, 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 ofmicroorganisms were found only rarely, including astrovirus,ETEC, EIEC, Ascaris spp., Blastocystis hominis, and Giardialamblia. No Shigella, Vibrio, or Cyclospora spp. were isolated.The majority of children infected by virus, EPEC or STECwere under 3 years of age (Table 2).

Rotavirus was by far the most common pathogen detectedand had the strongest association with disease (OR 96).Consequentially, a higher proportion of disease (PAR%) wasattributed to this pathogen (13.2%) than to any other micro-organism (Table 3). The most frequently isolated bacterialpathogen was zoonotic Salmonella. Five serotypes were seen:serovars Typhimurium, Enteritidis, Agona, Hadar, and Bovis-morbificans in 9, 7, 3, 2, and 1 patient, respectively. The sero-types Typhimurium, Agona, Derby, and Braenderup werefound in each of four controls. Eleven patients and five con-trols were infected with STEC. Five patients were infected withdifferent serotypes, and two each were infected with STECO157:[H7], O26:H, and O103:H2. Three healthy childrenalso carried these three serotypes. All STEC isolates were eaepositive except one serotype O126:H20 strain. Ten and fiveEPEC of classical O:H serotypes were isolated from cases andcontrols, respectively. The most common serotype was O55:

[H7], which was found in 6 of the 10 EPEC patients and innone of the controls. EPEC was only isolated from cases below2 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 pathogensassociated with diarrhea. Two patients were infected with bothSalmonella and norovirus and single patients with the following

combinations: rota- and adenovirus, noro- and adenovirus, ro-ta- and norovirus, EPEC and adenovirus, EPEC and astrovi-rus, 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 andfall (Fig. 1).

Clinical data. Selected clinical data on the patients are givenin Table 4. Overall, the viral infections were characterized byvomiting. Bloody diarrhea was characteristic for Campylobacterinfections, but this symptom was also seen among Salmonella-infected patients, whereas only two of nine patients infectedwith STEC had bloody diarrhea. None developed hemolytic-uremic syndrome. Both Salmonella and rotavirus infectionswere characterized by loss of weight, fever, and high hospital-ization rates. Antibiotic treatment was given to 21% of chil-dren infected with Salmonella spp. in contrast to 5% of chil-dren (P 0.061) with no pathogen identified and 4% ofchildren (P 0.1) with viral infections. All children survivedtheir infections.

DISCUSSION

A potential pathogen was detected for 54% of the patients,and an infectious agent associated with diarrhea was estab-lished for 45% of the patients in the present study. In spite ofits seasonal occurrence, rotavirus was the most frequently iso-lated pathogen. The severity of the rotavirus infections wascomparable to those caused by Salmonella sp. Norovirus, ad-enovirus, and sapovirus were also important pathogens ofchildhood diarrhea. Noro- and sapoviruses have up till nowgenerally not been tested for in microbiology laboratories, andthe incidence of these pathogens is therefore probably under-estimated. 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 statisti-cally to diarrhea.

Large case-control studies and cohorts on diarrhea havebeen conducted in The Netherlands (8), Italy (5), and theUnited Kingdom (35, 42), and in these studies viruses were

TABLE 2. Age distribution of Danish children with gastroenteritis caused by the most common pathogensa

Age(yr)

No. (%)

All patientsSalmonella

sp.Campylobacter

sp.Y. enterocolitica STEC EPEC Rotavirus Norovirus Adenovirus Sapovirus

0 116 (27) 9 (8) 1 (1) 2 (2) 3 (3) 5 (4) 12/103 (12) 3/98 (3) 6/103 (6) 4/112 (4)1 173 (41) 7 (4) 6 (4) 3 (2) 5 (3) 5 (3) 20/159 (13) 14/158 (9) 8/159 (5) 5/155 (3)

2 85 (20) 4 (5) 2 (2) 3 (4) 2 (2) 0 (0) 16/76 (21) 3/76 (4) 1/75 (1) 2/70 (3)3 27 (6) 1 (4) 2 (7) 2 (7) 0 (0) 0 (0) 1/24 (4) 0/76 (0) 0 (0) 0/23 (0)4 23 (5) 1 (4) 3 (13) 0 (0) 1 (4) 0 (0) 2/21 (10) 0/19 (0) 1/20 (1) 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.

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

Organism PAR%

Rotavirus..............................................................................................13.2Salmonella sp....................................................................................... 4.6Norovirus ............................................................................................. 3.9

Adenovirus........................................................................................... 3.6Campylobacter sp................................................................................. 2.9Y. enterocolitica.................................................................................... 2.2Sapovirus.............................................................................................. 2.0STEC .................................................................................................... 2.0EPEC.................................................................................................... 1.6EAggEC............................................................................................... 1.3

a Mixed infections are included in this table.

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

by RT-PCR may also have diminished the sensitivity of thenorovirus and sapovirus detection. Similarly, astrovirus causesmilder symptoms than other viruses tested for in the Finnishstudy (26) and are therefore less likely to be detected amongsamples from medical facilities. In addition, it has been shownthat the sensitivity of the enzyme-linked immunosorbent assayfor astrovirus used here is only 50 to 60% compared to PCRtests used in other studies (41). With regard to bacterial causes,our findings are in general agreement with previous case-con-trol studies. However, in most studies (5, 8, 25, 35), STEC wasisolated in 1% of cases versus 3% in the present study; inItaly (5), 19% of stools from cases younger than 10 years oldwere positive for Salmonella versus 5% in the present study.

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

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

Organism No. ofpatients

No. of patients (%) with:Vomiting Bloody diarrhea Fever Hospitalization Dehydrationb Loss of wt

Rotavirus 45 42 (93) 2 (4) 41 (91) 13 (29) 11 (24) 33 (73)Adenovirus 12 8 (67) 0 (0) 7 (58) 0 (0) 1 (8) 5/9 (56)Norovirus 10 8 (80) 1 (10) 7 (70) 2 (20) 2 (20) 6 (60)Sapovirus 10 6 (60) 0 (0) 5/9 (56) 2 (20) 1 (10) 6/9 (67)Salmonella sp. 19 9 (47) 5 (26) 17 (90) 5 (26) 5 (26) 14/18 (78)Campylobacter sp. 12 2 (17) 11 (92) 11 (92) 2 (17) 2 (17) 4/9 (44)Y. enterocolitica 10 3 (30) 2 (20) 8 (80) 1 (10) 0 (0) 5/9 (56)STEC 9 3 (33) 2 (22) 4 (44) 3 (33) 2 (22) 2 (22)EPEC 7 2 (29) 1 (14) 4 (57) 3 (43) 2 (29) 2/5 (20)No pathogen 183 71/181 (39) 11/181 (6) 99/177 (56) 23/182 (13) 23 (13) 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 afterthe 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.



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Surveillance data indicate that thermotolerant Campy-lobacter is the most common bacterial gastrointestinal patho-gen for all ages in Denmark (1). However, in the present studyof children younger than 5 years, it was surpassed by Salmo-nella as the most common bacterial pathogen. Y. enterocoliticawas isolated from 3% of patients, indicating that this speciescontinues 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 tothe classical EPEC serotypes were associated with diarrhea.A/EEC, i.e., E. coli isolates harboring the eae gene but notproducing Shiga toxin or belonging to the classical EPEC se-rotypes, was the most commonly isolated potential pathogen inthe present study. However, A/EEC was isolated at a similarproportion from cases and controls. The presence of the geneencoding bundle-forming pilus, a recognized EPEC virulencefactor (23), was neither alone nor in combination with the eaegene associated with diarrhea (data not shown). Recent studieshave applied a definition of EPEC where the serotype of theinfecting strains is not taken into account (25, 27). Had we

defined EPEC by the presence of the eae gene in stx-negativestrains, we would not have identified EPEC as a diarrheagenicpathogen. It is, therefore, premature to define EPEC indepen-dently of the serotype. Until virulence factors that may becofactors for diarrhea in EPEC infections have been identified,we recommend including the serotype or at least the O groupfor the identification of EPEC in young children. ClassicalEPEC was only isolated from children below 2 years of age andwas the second most common bacterial pathogen in this agegroup, indicating that this pathogen is an important cause ofsporadic diarrheal illness in infants.

Eleven and five STEC strains were isolated from patientsand controls, respectively. The serotypes O26:H, O103:H3

(both stx1 and eae positive), and O157:H7 (stx2 and eae posi-tive) were found in both cases and controls. This is interestingbecause these three serotypes are not only the most commonbut also among the most virulent STEC types (36). Althoughmost patients infected with STEC stop excreting the organismshortly after the acute illness has passed, a substantial propor-tion may continue to carry the organism for several months(12, 36). This may partly explain the occurrence of these patho-gens among the control children in the present study. Very fewETEC, EAggEC, and EIEC were isolated. ETEC and EIECare well-known pathogens, but they are uncommon in childrenfrom industrialized countries. EAggEC was isolated more fre-quently from cases than controls. However, this difference wasnot statistically significant.

Parasitic infections were rare in the present study, thoughcryptosporidia were isolated from 1.7% of cases. A small num-ber of children were diagnosed with Giardia, explaining why noassociation with diarrhea was found for this pathogen. In gen-eral, infections with Giardia lamblia are a minor cause of di-arrhea in Denmark. In 2004, the rate ofG. lamblia was 1.7% insamples where examinations for parasites were requested by aphysician (unpublished data [Maiken Arendrup, Laboratory ofMycology and Parasitology, Statens Serum Institut, Copenha-gen, Denmark]).

Only few cases in the present study (6.6%) acquired theirinfections during foreign travel. Salmonella and EPEC werethe most common travel related pathogens. Not surprisingly,

Cryptosporidium, ETEC, and EAggEC were also associatedwith travel. Very few viruses were isolated from cases with atravel history. In general, there were overlapping signs andsymptoms between the different etiologic categories. Some typ-ical patterns were, however, observed. Bloody diarrhea wasreported in 92% of the Campylobactercases and 20 to 26% ofthe patients with Salmonella, Y. enterocolitica, and STEC in-fections but rarely among the patients with a viral infection. Asexpected, vomiting was a more prominent finding among casesinfected with viruses than bacteria.

In conclusion, rotavirus was confirmed as the most commonpathogen in childhood diarrhea, especially during winter andspring in Denmark. This fact, in combination with the severityof the infections, warrants consideration of a rotavirus vaccinein the childhood immunization program. The role of diarrhea-genic E. coli was confirmed for STEC and classical EPEC.A/EEC, i.e., attaching-and-effacing E. coli not belonging to theclassical EPEC serotypes, was commonly identified but was notassociated with disease. Parasites are only rarely identified inchildren with diarrhea with the possible exception of patients

who have traveled abroad. Routine examination of stool cul-tures from children less than 5 years of age should include testsfor viruses in the colder months and for STEC and classicalEPEC. Ideally, identification of STEC should be based ondetection of the Shiga toxins or their genes. As for EPEC,routine examination may be restricted to children below 2years of age but must include a combination of detection of theeae gene and the serogroup of the infecting strain.

ACKNOWLEDGMENTS

We are grateful to Carsten Struve and Andreas Munk Petersen forhaving contributed to the data collection and to Brita Bruun for criticalreading of the manuscript. We thank the staff of the laboratories thattook part in the study.

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