0095-1137/89/061192-05$02.00/0
Copyright C 1989, American Society forMicrobiology
Etiology of Childhood Diarrhea
in
Korea
KYUNG-HEE KIM,* INN-SOO SUH, JUNG MOGG KIM, CHOON WON KIM,AND YANG-JACHO
Departments of Microbiology and Clinical Pathology, Hanyang University College of Medicine, Seoul 133, Korea Received 11 November1988/Accepted3 March 1989
Toassesstheroleof recentlyrecognized enteropathogensinchildhooddiarrhea inKorea, 231 childrenwith
diarrhea admitted to and 104 children without diarrhea seen at the well-baby clinic or the outpatient
department of HanyangUniversity HospitalinSeoul, Korea,wereevaluatedduringa14-month period.Stools were cultured for bacterial pathogens, including enterotoxigenic (heat-labile and heat-stable
enterotoxin-producing) and enteroadherent organisms. Only those stools obtained from patients with diarrhea were
examined for rotavirus. Ali Escherichia coli isolates were screened for Shiga-like toxin (SLT) 1, SLT-II,
enterohemorrhagicE.colifimbriae, andenteroinvasiveness bycolony hybridization. Oneor morepathogens wereidentifiedin75.8% of the childrenwithdiarrhea. Rotaviruswasthemostfrequently identified pathogen, accounting for47% ofthecases.Other major enteropathogenswereenterotoxigenicE. coli (22%), Clostridium
difficile
(16%),enteroadherent E. coli(15%),andenteropathogenicE. coli(6%). Shigellaspp.,Campylobacterjejuni, Salmonellaspp.,SLT-I-andenterohemorrhagic-E.coli-fimbria-probe-positiveE.coliserotype026:H11
andenteroinvasiveE. coliwereisolated from onlyafewpatients.Aeromonashydrophila andE.coli 0157were
notisolated. Compared with those of thecontrols,theisolationratesofheat-stable-enterotoxin-producing E.
coli (P < 0.05), C. difficile(P < 0.025), and enteroadherentE. coli (P < 0.05)weresignificantly higherinthe
patients with diarrhea. Thegreatest numberofrotavirus, enterotoxigenicE. coli, and C. difficilecases were
identified duringthe cool, dry monthsofOctoberand November.
Acute diarrhea, especially in children, isan
extraordinar-ily common disease with worldwide distribution and a
sig-nificant public health impact (10, 12, 17, 36). In Korea, infant mortality, highly correlated with diarrheal disease mortality, declined from 53 per 1,000 live births to 14 per 1,000 live
births in the last 20years(13, 23). Nevertheless, amongall
children admitted to hospitals, diarrhea has been second in frequency (10 to 18%) only to acute respiratory disease throughout the same period (13). However, the etiologic
agents that causeddiarrheal infections in children 10 or20
years ago may be different from the agents which cause
diarrhea today.
Few studies have beenconducted in Koreato determine
the incidences of the different enteric pathogens associated
withchildhood diarrhea. In addition tothe classical salmo-nellae,shigellae, enteropathogenicEscherichia coli (EPEC), enterotoxigenic E. coli (ETEC), and enteroinvasive E. coli (EIEC), the following recently recognized agents are of
interest: Campylobacterjejuni, Clostridium difficile,
Aero-monas hydrophila, enteroadherent E. coli (EAEC),
entero-hemorrhagic E. coli (EHEC), androtavirus.
Westudied pediatric patients duringa14-monthperiodat
Hanyang University Hospital inSeoul, Korea,toassessthe role of newly recognized as well as established agents of
diarrhea inKoreaninfantsandchildren andtodeterminethe clinicalsymptomsandtheseasonalityofdiarrheaassociated with themostfrequently isolated enteric pathogens.
MATERIALSANDMETHODS
Study subjects. Between February 1984 and March 1985,
231 children under 15 years ofage, admitted to Hanyang University Hospitalwithacutediarrhea,werestudiedfor the
presence of bacterial and viral enteropathogens in stool specimens collected atthetime ofadmission.
Diarrheawasdefinedasthepassageofunformed (looseor
* Corresponding author.
watery) stools with twice the usual daily frequency,
accom-panied by fever, nausea, or vomiting. During this study period, 104age-matched children without diarrhea, seenat
the well-baby clinic or the outpatient department of the hospital, were included in the study as controls. Neither patients with diarrhea nor control children had received antibiotics within 2 weeks ofspecimen collection. A clinical
history of each child was collected by a physician. Stool samples from children with diarrhea and control children wereobtained bya nurseandweretransportedinstoolcups
tothelaboratory for microbiological examination.
Microbiological methods. FreshstoolswereculturedforE. coli and a number of other Enterobacteriaceae, including Shigella and Salmonella spp., C. difficile, Campylobacter
jejuni, and A. hydrophila, by standard methods (3, 9, 20,39).
A portionof eachspecimenwasmade intoa10%suspension in phosphate-buffered saline and was stored at -20°C for later rotavirus assay with the use of an enzyme-linked immunosorbent assay, the rotavirus ELISA kit (K220; Dako, Copenhagen, Denmark). This assay kitwas selected asthetestmethod since previous studies havedocumented that it is equivalent to other standard reference methods, such aselectron microscopy (15, 16).
From the primary MacConkey agar (DifcoLaboratories, Detroit, Mich.) plate, five colonies of E. coli and other
membersof theEnterobacteriaceaewere pickedand inocu-latedindividually onto apeptoneagar slant. Ofthesestock cultures, each set of the five isolates was pooled (29) and tested for biochemical reactions (API 20E strip; API
Sys-tems,S.A.,LaBalmeLesGrottes, France)andenterotoxin.
Individual isolates were tested for serogroup, adhesion to
HEp-2 cells (CCL 23; American Type Culture Collection, Rockville, Md.), and DNA hybridization with probes for Shiga-like toxin (SLT) I, SLT-II, EHEC fimbriae, and
en-teroinvasiveness. Colonies confirmedasE. coliwere trans-ferred to MacConkey agar base containing 1% sorbitol
(Difco). Sorbitol-negativeE. colicolonieswere serogrouped
withspecificantiseratoserotype0157(provided byChikH.
1192
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CHILDHOOD DIARRHEA 1193
Pai, University of Calgary, Calgary, Alberta, Canada) by
standard methods (9, 26).
Heat-labile enterotoxinproductionwasdeterminedby the Yl adrenal tumor cell (CCL 79; American Type Culture Collection) tissue culture technique (34). For detection of
heat-stable enterotoxin (ST), the infant mouse model de-scribed by Dean et al. (7) was employed. The E. coli stock colonies were serogrouped with commercially prepared an-tisera (Difco). E. coli giving positive agglutination withany
of the three polyvalent EPEC antisera (poly A, B, and C)
weresubsequently groupedwith theconstituentmonovalent antiseraby slideagglutination. Thefollowing 170and O:K serogroupswerealsosought:01aOlb, 02aO2b, 04, 06, 07, 08, 09, 015, 025, 044, 075, 078, 0113, 0114:K90, 0125:
K70(B15), 0128abO128ac,and0136:K78. Strainswerethen
confirmedbytubeagglutination byusingaboiledsuspension
of the organism (9). Among strains in the enteropathogenic
serogroups, those showing enterotoxicity in a suckling
mouse model wereconsidered ETEC. The HEp-2-adhesion
assay was performed by the method of Mathewson et al. (28). A strain was considered to be adherent ifE. coliwas
observed to adhere to 50% ofthe HEp-2 cells. The adher-ence pattern(localized ordiffuse)was noted foreach adher-entstrain. In localized adherence, bacteria bindtolocalized
areas of the cells, in which they form microcolonies. In
diffuse adherence, bacteria cover the whole surface of the
cell. Non-EPEC, non-ETEC strains that adhered to HEp-2 cells were called EAEC.
Five E. coli colonies from each child were blotted onto fourseparatepieces of Whatman 541 filterpaper(Whatman, Inc., Clifton, N.J.), each of which was hybridized with the
probesforSLT-I(BamHI 1,142-base fragmentofpJN37-19),
SLT-II (SmnaI-PstI 842-base fragment of pNN110-18),
EHEC fimbriae (3.4-kilobase HindIII fragment of
pCVD419),and EIEC(17-kilobaseEcoRIdigestion fragment
ofpRM17) (24, 25, 30, 38).Colonies thathybridizedwiththe SLT and/orthe EHEC fimbria probe were tested for
cyto-toxicity to Vero and HeLa cells (2, 22), and those that
hybridized with the EIECprobes were tested inthe Sereny
test (35).
Fibroblastcell monolayercultureswereusedtodetect the cytopathic toxins of C. difficile in stools or supernatants of brain-heart infusion broth culturesbythe method of Kimet al. (20). Specificity of thetoxin was determined by
demon-stratingneutralization ofcytopathiceffects ofpositive
spec-imens by using a 1:32 dilution of antiserum to C. difficile
toxins (Anaerobe Laboratory, Virginia Polytechnic Insti-tute, Blacksburg). Specimens yielding C. difficile plus free fecal C. difficile toxinsor yieldingonly freefecal C. difficile
toxins were considered C. difficile positive.
RESULTS
Etiologic agents. During the 14monthsofthestudy, fecal
samplesfromthe 231patientswithdiarrhealillness admitted tothehospital and from the 104control subjectsweretested for enteric pathogens. One or more recognized etiologic agentswerefound in76% ofpatientswithdiarrhea(Table 1). Multiple infections occurred commonly in patients with C. difficile (78%), EAEC (71%), ETEC (61%), rotavirus(44%),
or EPEC (41%) infection. Only 111 patients (48%) were
infected with a single pathogen.
Rotavirus was the most frequently identified pathogen, accountingfor 47% ofthecases, followed by ETEC (23%),
C. difficile (16%), EAEC (15%), and EPEC (7%). Shigella species, Salmonella species, Caînpvlob((ter jejini, EIEC,
TABLE 1. Prevalence ofenteropathogens inKoreanchildren withand without diarrhea
No.(%i) with identified enteropathogen" Enteropathogen Diarrheal Control
group group value" (n =231) (ni = 104) vau'
Rotavirus 107(46.8) ND
ETEC 52(22.5) 14(13.5)
STproducing(ST+) 45(19.5) 11(10.6) <0.05 Heat-labile-enterotoxin 6(2.6) 3(2.9)
producing(LT+)
ST+/LT+ 1 (0.4) 0
OtherST+ Eniteroba(uteria(ce'e 9 (4.8) 1(1.0)
C.difficile 36(15.6) 7(6.7) <0.025
EAEC 34(14.7) 8(7.7) <0.05
HEp-2 cell-adherent
K. pneunzoliiae 3 (1.3) 0
EPEC 15 (6.5) 5(4.8)
SLT-I+/EHECfimbria"+' 2 (1.3) 0
Shigella spp. 4 (1.7) 0
Salnmonella spp. 1(0.4) 0
Camnpyloba(cerjejini 1(0.4) 0
EIEC 1(0.4) 0
`Multiplepathogens isolatedfrom the samepatientarelisted separately.
Nopathogens wereidentified in 56(24.2%)of thepatientswithdiarrheaand in52(52.9%)ofthe controls. ND,Notdone.
"By thechi-squaretest.
OtherST-positive(ST+) Enierobocteriaceoewere fiveKlebsiella
pneu-mionlae. twoCitrobacter dii'ersu.s,one K. oxstoca, and oneAcinelobacter calcoaceticus subsp.onitr(aiisisolate in the children with diarrhea and one K.
pnetnonioae isolate in the controlgroup.
"1SLT-IP. SLT-I probe positive; EHEC fimbria+, EHEC fimbria probe positive.
and SLT-I-probe-positive E. coli 026:H11 were isolated from only a few patients. A. hydrophila and E. coli 0157 were not isolated. All three SLT-I-probe-positive E. coli 026:H11 strains hybridized with the EHEC fimbria probes and produced cytotoxin that was neutralized by anti-Shiga toxin antiserum. Two of the three SLT-I- and EHEC fim-bria-probe positive strains were isolated from a child with
intussusception andwere verotoxin positive (oneat a 1:625 dilution and the other at 1:3,125). Stool cultures from this child were negative for other bacterial pathogens and rota-virus. Most(86%)of the ETEC produced only ST. The most
frequent serogroups of E. coli were as follows: of ST-producingE. coli,015 and025; ofEPEC, 086:K61; and of EAEC, 06 and 025.
A number of asymptomatic control subjects were also found to be infected with potential pathogens (Table 1). Rotavirus was not evaluated in the controls. Most of the ETEC produced only ST, and none produced both ST and heat-labile enterotoxin. Compared with those of isolates from the patients with diarrhea, the isolation rates of ST-positive E. coli (P < 0.05), C. difficile (P < 0.025), and EAEC (P < 0.05) were significantly lower in the control group. Although EPEC was isolated more frequently from the patientswith diarrhea than from the controls, the
differ-ence was not statistically significant.
On the basis of age groupof thepatients (Fig. 1),infecting
agentswere found in 157(82%) of192children less than 18 months old with diarrhea, compared with 22 (56%) of 39 children 18months through 15years old with diarrhea (P<
0.05). Rotaviruswas isolatedmorefrequentlyfrom children aged 12 to <18monthsthanfrom those of other age groups.
For other enteropathogens, the isolation rates were not
significantly different among different age groups.
VOL. 27,1989
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FIG. 1. Isolationrateofrotavirus, ST-positive (ST+)E. coli, C.
difficile, EAEC, and EPEC on the basis of age group of patients. Multipleisolations of two ormore pathogens from the same patient are separated into components. Abbreviations: m, months; y, years.
Clinical patterns of diarrheal illness.Clinical datafrom the 107patients (under 2 yearsold) with single-agent infections due torotavirus, ETEC, EPEC, EAEC, and C.
difficile
wereexamined(Table 2). The clinical manifestations seen in the
five groups of patients were not significantly different from eachother, although vomiting and dehydration tended to be morefrequent inrotavirusinfectionandfeverappeared to be more common in C. difficile infection. The mean ages of patients infected with rotavirus, ST-positive E. coli, C.
difficile, and EAEC were 12, 9.5, 9, and 11.5 months,
respectively.
Seasonal patterns of diarrheal illness. A seasonal
fluctua-tion in the incidence of the three most frequently isolated
pathogens, rotavirus, ST-positive E. coli, and C.
difficile,
was observed (Fig. 2). Although diarrheal infections oc-curred throughout the year, infections due to these three agents peaked during thecool, dry months ofOctober andNovember, overlapping the season ofpeak hospital
admis-sion of diarrhea patients. Epidemiologic studies covering
1985through 1987 have revealed that thehospital admission
rateofpatients with diarrheapeaked inOctoberand Novem-ber.
DISCUSSION
This is the first and the most comprehensive study in Koreaand in NortheastAsiaof thenewlyrecognizedaswell
as established agents associated with diarrhea in children.
Rotavirus, ETEC, C.
difficile,
and EAEC were commonlyfound in the hospitalized diarrhea patients. Rotavirus was
TABLE 2. Major clinicalsymptoms associated with the most frequently isolatedenteric pathogensin 107patients (2 years old
oryounger) withsingle-agent infections
Enteropathogen %of children with the followingsymptoms":
(no.ofpatients) Vomiting Fever Dehydration
Rotaviruses(60) 83.3 53.3 85.0
ETEC (22) 63.6 54.5 63.6
EPEC(10) 60.0 20.0 30.0
EAEC(9) 44.4 77.8 66.7
C.difficile(6) 50.0 100.0 50.5
"Bloodystool was not a symptom in any of these 107 patients.
F M A M J J A_ _ N D J F M
1984 1985
4 6 11 14 9 13 10 9 17 64 15 11 8 17
No.ofpatients studied each month
FIG. 2. Seasonal fluctuation in the incidence ofrotavirus, ST-positive (ST+) E. coli, and C. difficile infections in 207 children under 2 yearsofageinSeoul, from February1984through March 1985. Multiple pathogens isolated from the patientarelisted sepa-rately.
mostfrequentlydemonstrated agentoverall,and itpeakedin incidence in children 12to<18months old. Itsisolationrate
washigherthan those revealed in 1981 and 1983 (26 to31%)
in hospitalized children with diarrheaatHanyangUniversity Hospital (15, 16). It was higher than those in northeastern Brazil (19.4%), the Central African Republic (17.6%), Paris
(36%), and CostaRica(45.3%) butlower than thatinJapan
(63%) (4, 10, 12, 21, 27). However, since asymptomatic as
well as diarrheagenic rotavirus infections occur in young children (1, 4), it would be useful toknow thefrequencyof
asymptomatic rotavirus infections in healthy children in Korea.
ETEC were found in 23% of cases; virtually all isolates produced STonly. The isolationrate ofST-positiveE. coli wassignificantly higherin thepatients with diarrhea than in the controlpatients(P<0.05). InaCostaRican studyalso,
almost allETEC isolates wereSTproducers(27). Although
the early study identified heat-labile-enterotoxin-producing
bacteria as pathogens (33), they have been found as fre-quently in control subjects as in patients with diarrhea in MexicoCity (8) and in Seoul (this study).
C.
difficile
wasthethirdmostfrequently isolated pathogenfound in children withdiarrhea. Althoughsomecontroversy surrounds the role of C.
difficile
in diarrheal disease ofinfants,this revealedasignificant association ofthisspecies
with diarrhea (P<0.025). Themeanageofthepatientswho excreted C.
difficile
in their stools was ca. 9 months.Colonization of infants with C.
difficile
seemed toincreaseduringweaning, probably because ofothercarriers orfood
or environmental sources. The babies, in turn, may be an
exogenoussourceof C.difficilefor adults whoare
receiving
antibiotics.
Thefourthmostfrequentlyisolatedpathogen,EAEC,was
found
significantly
more oftenin thepatients
with diarrhea than in the controls (P < 0.05). Localized adherenceoc-curredin 13 strains(38%),and diffuse adherenceoccurred in 18 strains (53%). Some strains (9%) showed localized and diffuse adherence in the same preparation. Cravioto et al.
(6), in the original description of HEp-2 cell adherence, reported that non-EPEC, non-ETEC strains ofE. coli that
were possible causes of diarrhea outbreaks in adults were
adherenttoHEp-2 cells. Mathewsonetal.(28)reportedthat EAEC were associated with traveler's diarrhea. Our study
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CHILDHOOD DIARRHEA 1195
suggests that EAEC may be of etiologic importance in childhood diarrhea.
The incidence of EPEC was greater in children with diarrhea than in well children, but this difference was not statistically significant. The incidences of Shigella spp., Salmonella spp., and Campylobacterjejiuni were foundtobe very low in thepatients and nonexistent in the controls. The isolation rate of these bacterial enteric pathogens was slightly lower than those that were revealed in a previous studyof children with diarrhea seen at eight pediatric clinics orhospitals located in Seoul (5). The isolation rate (1.3%) of
SLT- (or verocytotoxin-) producing E. coli 026:H11 in the present study was lower than that (2.5%) of verocytotoxin-producing E. coli 0157:H7 found in Canada (32). The frequency of SLT-producing E. coli infection has been reported to be much higher in patients with bloody diarrhea (15 to37%) and in patients with hemolytic uremic syndrome
(75%) (19, 32). None of our study subjects had bloody diarrhea. SLT-positive E. coli produces one or both of two antigenically distinct toxins, SLT-I and SLT-II, which are encoded on two distinct bacteriophages, 933J and 933W, in E. coli 933 (30, 31, 37). SLT-positive E. coli also possess
plasmid-encodedfimbriae that promote attachment to intes-tinal epithelial cells in tissue culture (18), and a DNA probe
forthefimbriae hasbeen used to detect EHEC (24). In our
study, all threeSLT-positiveE.coli026:H11 strains hybrid-ized both with SLT-I and with the EHEC fimbria probes. It isofinterest that twoof thesestrains were isolated from one
child with intussusception. This is the first study in Korea andin Northeast Asia that included EHEC. E. coli 026:H11,
anewly described enterohemorrhagic class of diarrheagenic
E. coli, maybe an uncommon cause of pediatric diarrhea in Korea. Enteroinvasiveness-probe-positive E. coli was iso-latedfrom a child with diarrhea and was found to be a lactose
fermenter. Neither A. hydrophila nor E. coli 0157:H7 was
isolated in this population. The frequent occurrence of A.
hydrophilain New YorkCity, N.Y., and in Perth, Australia (11, 14), and the large number ofsporadic cases of E. coli
0157:H7 infection reportedinCalgary,Canada(32), suggest that, like that of other enteric organisms, the incidence of these organisms may varygeographically.
Inother studies (10, 12, 21, 27), infections with ETEC had seasonal peaks in the warmer months and infections with rotavirus had seasonal peaks in the cool, dry months. In this study,infections due to rotavirus, ST-positive E. coli, and C.
difficile
all peaked in the cool, dry months. This is probablydue to thefact that hospital admission due to diarrhea also
peaked duringthat period.
Before this study, the relative frequency of the specific agents that cause severe diarrheal infections in young chil-dren in Koreawaslargely unknown.Inthisstudy, significant
differences were observed between hospitalized children withdiarrhea and control children regarding theexcretion of
ST-positive E. coli, C.difficile, and EAEC. Overall, 76% of the patients were infected with at least one potentially
pathogenic viral or bacterial enteric agent. Since this study has dealt with children admitted to a hospital, where more
severeformsof diarrheawere seen,community-based stud-ies including other viruses and parasites need to be con-ducted to determine whether the data reflect those of the general pediatric population.
ACKNOWLEDGMENTS
Wethank B. Rowe andC. Pai forproviding bacterial strains; H. LiorforserotypingE.coli026:H11;P. Echeverria and J.E. Brown for theirhelpful advice, forproviding the rabbitantibody to Shiga
toxin and the DNA probes used in this study, and for help in carrying out the cytotoxicity and DNA hybridization assays in Thailand; and J. Seriwatana (Armed Forces Research Institute of Medical Sciences, Bangkok,Thailand) for assisting with the DNA hybridization.
This study wassupported by the Korea Science and Engineering Foundationand the Mogam Biotechnology ResearchInstitute.
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