BF所致儿童腹泻

Enterotoxigenic Bacteroides fragilis–Associated Diarrhea in Children0–2Y ears of Agein Rural BangladeshPreeti Pathela,1K.Zahid Hasan,3Eliza Roy,3Korshed Alam,3Fazlul Huq,3A.Kasem Siddique,3and R.Bradley Sack2 Departments of1Epidemiology and2International Health,Johns Hopkins Bloomberg School of Public Health,Baltimore,Maryland;3International Centre for Diarrhoeal Disease Research,Bangladesh,Dhaka,BangladeshThe burden of enterotoxigenic Bacteroides fragilis(ETBF)–related diarrhea was determined in a birth cohort of252children in rural Bangladesh.Isolation rates of ETBF in stool and risk factors for acquisition of ETBF and disease were established.Of382B.fragilis–positive specimens,14.4%of the strains found in them produced enterotoxin,as determined by a tissue-culture assay.The overall isolation rate of ETBF was2.3%(40/1750) from diarrheal specimens and0.3%(15/5679)from nondiarrheal specimens collected throughout the2years of the study().ETBF was isolated from20.3%(40/197)of the B.fragilis–positive diarrheal specimens P!.001and from8.1%(15/185)of the B.fragilis–positive nondiarrheal specimens()and was significantlyP!.001associated with acute diarrheal disease in childrenу1year of age().The diarrheal illness was mildP p.0001in nature.In conditional multivariate analyses that examined environmental and host risk factors,the presence of livestock in the household area was linked to the acquisition of ETBF(chickens,;cows,).P!.05P p.06 ETBF was found to be a small but significant contributor to diarrheal disease in this rural community.Improved management of livestock may be useful for the prevention of ETBF infection.Acute diarrheal disease remains a problem for millions of children worldwide.In developing countries,it is1 of the2main causes of death and contributes greatly to the severity of malnutrition[1].In the preceding2 decades,significant advances have been made in the identification of enteropathogens and in the under-standing of pathogenic mechanisms.Yet,some acute diarrheal illnesses still have unknown etiologies.Iden-tifying new enteropathogens and establishing the path-ogenicity of recently recognized ones remain important goals for understanding childhood diarrhea. Bacteroides fragilis(BF)bacteria are gram-negative,Received12July2004;accepted19November2004;electronically published 14March2005.Presented in part:The analysis and manuscript were completed as a part of a doctoral thesis project(P.P.).Financial support:US Agency for International Development(grant HRN-A-00-96-90005-02).Reprints or correspondence:Dr.R.Bradley Sack,Dept.of International Health, Johns Hopkins Bloomberg School of Public Health,615N.Wolfe St.,Rm.5035, Baltimore,MD21205(rsack@).The Journal of Infectious Diseases2005;191:1245–52ᮊ2005by the Infectious Diseases Society of America.All rights reserved. 0022-1899/2005/19108-0008$15.00anaerobic rods that are normal inhabitants of the largebowel of mammals.A subgroup of this species has beenfound to produce an enterotoxin.Enterotoxigenic BF(ETBF)secretes an extracellular20-kDa metallopro-tease toxin(termed BF toxin[BFT])that causes swellingof human colonic epithelial cells without cell injury andalters the distribution of actinfilaments in intestinalepithelial cells.BFT acts by causing a“leaky epithelium”and stimulating active chloride secretion by cells,whichmay contribute to the pathogenesis of diarrhea in hu-mans[2].ETBF strains werefirst described in1984,when My-ers et al.[3]found that they produced a secretory re-sponse in ligated ileal loops of calves and lambs.Shortly thereafter,this novel enteropathogen was proven to beassociated with diarrheal disease in a variety of youngdomestic animals[4–6].In1987,isolation of ETBF from human adults andchildren with acute and chronic diarrhea was described[7].Thefirst controlled study of ETBF disease in Apachechildren with and without diarrhea who were attendingan Arizona clinic found a strong association betweenacute diarrhea and ETBF in children11year of ageETBF-Associated Diarrhea in Infants•JID2005:191(15April)•1245[8].To assess the significance of ETBF infections in the de-veloping world,a subsequent controlled study[9]of children !5years of age who were admitted to the hospital of the In-ternational Centre for Diarrhoeal Disease Research,Bangladesh (ICDDR,B)in Dhaka was performed.Again,ETBF was sig-nificantly associated with diarrhea in children11year of age. To our knowledge,to date,no community surveillance stud-ies of ETBF infections have been reported.The present study was conducted to describe the prevalence of ETBF organisms and the epidemiological and clinical characteristics of ETBF infections in children!2years of age in rural Bangladesh.SUBJECTS,MATERIALS,AND METHODSStudy population.A household surveillance study was carried out in Mirzapur,Bangladesh,from1993to1996.Thefield team was composed of12trained female community health workers (CHWs)and a physician.From July to August1993,CHWs conducted a door-to-door census in10villages,identified preg-nant and married women of childbearing age,and invited them to participate in the study.From August1993to October1994, 288newborns were enrolled into the study.A household baseline survey that collected socioeconomic and environmental information on families of enrolled newborns was conducted.Two hundredfifty-two children were followed for diarrheal morbidity from birth until2years of age.CHWs visited the participants’homes twice weekly to record occurrences of diarrhea and respiratory tract infections and clinical features of illnesses.Stool specimens or rectal swabs were collected from children when they had diarrhea,as well as on a monthly basis. Diarrhea was defined asу3loose stools,or any number of stools containing blood,within a24-h period.Diarrheal episodes were separated by at least3diarrhea-free days.Informed consent was obtained from a parent or guardi-an for inclusion of each child in the study.During the study, children were provided with health care by a pediatrician in the home or at Kumudini Hospital,located4–9km from each study village.If a child had diarrheal illness,oral rehydration therapy was provided,and the physician administered antibi-otics if indicated.The protocol was approved by the Ethical Review Committee of ICDDR,B,and the analysis plan was ap-proved by the Committee on Human Research of the Johns Hopkins Bloomberg School of Public Health. Microbiological assessment.Stool specimens and rectal swabs were collected in the morning,kept in an ice cooler,and trans-ported to the laboratory for processing that afternoon.The specimens were tested for recognized bacterial,viral,and par-asitic enteropathogens—such as Escherichia coli,Campylobacter jejuni,Shigella species,Salmonella species,Vibrio cholerae,Cryp-tosporidium species,Entamoeba histolytica,rotavirus,and as-trovirus—using methods that are routine at ICDDR,B[10].Isolation of BF and assays for toxin production.All stool specimens were tested for the presence of BF.Although earlier investigations of ETBF used PINN medium,which contains polymyxin B,irgasan,nalidixic acid,and novobiocin[8,9,11], it has been reported to be less than satisfactory for growing BF [9].Thus,alternative media were used.Specimens collected between1October1993and31March1995were cultured for BF by streaking them onto Bacteroides bile esculin(BBE)agar for colony isolation.This medium allows the growth of several species of Bacteroides and has been shown to have the selectiv-ity necessary for the isolation of BF[12].Because information suggested that another medium was superior to BBE for grow-ing BF,from1April1995to the end of the study(30September 1996),colonies were grown on Reuben BF media(RBF),which was specifically formulated for the primary isolation of BF from stool specimens.The novel medium consisted of3.7%Difco brain-heart infusion base,1%yeast extract,1%casamino acids, vitamin K,hemin additives,∼20%ox bile,4m g/mL gentamicin, 1m g/mL pefloxacin,and100m g/mL kanamycin.When enriched with5%lysed sheep blood,this medium provided good growth of isolates while inhibiting normalflora in stool[13].Colonies were subcultured on blood agar,and BF colonies were identi-fied by their characteristic mottled appearance.In all instances, plates were incubated anaerobically for48h at37ЊC.Specimens were stored in chopped meat medium.When diarrheal specimens tested positive for BF,colonies were tested for enterotoxin production.Additionally,1of every 5of the monthly stool specimens was tested for bo-ratory workers were not aware of the source of the BF cultures. The cultures were coded only with numbers,handled in the order in which they were received,and decoded later.A tissue-culture assay using the cloned human colonic-epithelial-cellline HT29/C1was used to detect ETBF in BF-positive specimens. HT29/C1cells that are treated with the supernatants of ETBF strains develop morphological changes,including loss of cell-to-cell attachments,rounding,and swelling within as little as 1h after treatment.Morphological changes progress over at least thefirst24h after treatment with supernatants of ETBF strains.This assay was proven to be89%sensitive and100% specific in detecting ETBF strains as defined by the previously used lamb–ligated-intestinal-loop assay[14].Statistical analyses.T o assess the pathogenicity of ETBF, children with ETBF-positive diarrheal specimens were compared with children with ETBF-positive nondiarrheal specimens.A sim-ilar comparison was done for children with BF-positive speci-mens.x2and Fisher exact tests were used to compare differences between groups.To determine if household and biological factors were as-sociated with acquisition of ETBF,a nested matched case-con-trol study was conducted.Case patients were defined as all ETBF-positive children,and control subjects were defined as ETBF-negative children matched to case patients by age(within1246•JID2005:191(15April)•Pathela et al.Table1.Isolation rates of enterotoxigenic Bacteroides fragilis(ETBF),distributed by age group.Age groupDiarrheal specimens a Nondiarrheal specimens bOR(95%CI)P BFpositiveETBFpositive Rate,%BFpositiveETBFpositive Rate,%0–5months18316.7251 4.0 4.80(0.34–262.2).29 6–11months49510.23837.9 1.33(0.24–9.10).71 12–17months571628.144715.9 2.06(0.70–6.58).15 18–23months731621.9784 5.1 5.19(1.55–22.28).003 Total1974020.3185158.1 2.89(1.49–5.84)!.001NOTE.CI,confidence interval;OR,odds ratio.a89.1%(197/221)of BF organisms isolated from diarrheal specimens were tested for enterotoxin production.b21.5%(185/862)of BF organisms isolated from nondiarrheal specimens were tested for enterotoxin production.3months)and season of specimen collection.A second analysis to determine risk factors for diarrhea-associated ETBF infection was performed.ETBF-positive children with diarrheal speci-mens were matched by age and season of specimen collection to ETBF-negative children with nondiarrheal specimens.In-dependent variables in the regression models included factors recorded during household surveys(i.e.,water sources,pres-ence of livestock in the household area,sex,birth weight,and duration of exclusive breast-feeding).Indicators of nutritional status(normal versus low z scores of weight-for-age,height-for-age,and weight-for-height)were also studied in this analy-sis.Significant variables in the univariate analyses(at an a level of0.05)and potential confounders were included in conditional multivariate logistic regression models.Statistical analyses were performed using Stata(version7.0;StataCorp).RESULTSA total of7429specimens(1750diarrheal and5679nondi-arrheal)were collected from the252children included in the present study.The overall isolation rate of BF,which included ETBF and non-ETBF strains,was14.6%(1083/7429specimens). From October1993to March1995,a total of3740specimens were tested for BF using BBE medium,and from April1995 to September1996,a total of3689specimens were tested for BF using RBF medium.The overall distributions of diarrheal and nondiarrheal specimens were similar for the2culturing periods.From October1993through March1995,there were 786(21.0%)diarrheal specimens and2954(79.0%)nondiar-rheal specimens.From April1995through September1996, there were964(26.1%)diarrheal specimens and2725(73.9%) nondiarrheal specimens.The isolation rate for BF when BBE medium was used was11.1%(417/3740),which was signifi-cantly less than the18.1%(666/3689)isolation rate when RBF medium was used().P!.001BF was not isolated more frequently in children with diar-rhea;actually,the reverse was true.BF was isolated in221 (12.6%)of1750diarrheal specimens and in862(15.2%)of 5679nondiarrheal specimens().P p.008A total of382BF-positive specimens were tested for entero-toxin production.These were composed of89.1%of the BF isolates from the diarrheal specimens(some cultures were lost in storage)and a random sample of21.5%of the BF isolates from the nondiarrheal specimens.The overall isolation rate of ETBF was2.3%(40/1750)from diarrheal specimens and0.3% (15/5679)from nondiarrheal specimens().P!.001Table1shows the isolation rates of ETBF in the382BF-positive specimens.A total of14.4%(55/382)of the BF isolates were enterotoxin producers.ETBF was identified in40(20.3%) of197BF-positive diarrheal specimens and in15(8.1%)of 185BF-positive nondiarrheal specimens().ETBF wasP!.001isolated more frequently from the stool specimens of children у1year of age than from the stool specimens of children in other age groups.The difference between the isolation rates of ETBF in BF-positive children attained statistical significance in the oldest age group(18–23months).The numbers of ETBF-positive specimens in the younger age groups were low.The data,analyzed according to whether the specimens were from children!1year of age orу1year of age,showed that isolation rates of ETBF in diarrheal specimens versus those in nondi-arrheal specimens for those!1year of age were not statistically significantly different(11.9%vs.6.4%;odds ratio[OR],2.00; 95%confidence interval[CI],0.50–9.53),but were statistically significantly different for childrenу1year of age(24.6%vs.9.0%;OR,2.73;95%CI,1.51–7.62).Both non-ETBF strains and ETBF were isolated in diarrheal and nondiarrheal specimens throughout the year.In Bangla-desh,the dry,hot spring occurs from March to May;the wet, hot summer occurs from June to October;and the winter oc-curs from November to February.BF was significantly more prevalent in nondiarrheal specimens than in diarrheal speci-mens during all seasons(data not shown).The seasonal dis-tribution of isolation of ETBF(figure1)was also examined. When the isolation rates of ETBF in BF-positive nondiarrheal specimens were compared with those in diarrheal specimens, the diarrheal specimens had higher isolation rates of ETBF dur-ing each season,but they were statistically significantly higherETBF-Associated Diarrhea in Infants•JID2005:191(15April)•12471248•JID 2005:191(15April)•Pathela etal.Figure 1.Seasonal isolation rates of enterotoxigenic Bacteroidesfragilis(ETBF)in diarrheal specimens (D;black bars )and in nondiarrheal specimens(ND;gray bars ).a,,comparing isolation rates of ETBF in diarrhealP p .03specimens taken during March–May and November–February with isolationrates of ETBF in diarrheal specimens taken during June–October.b,P p,comparing isolation rates of ETBF in diarrheal specimens and non-.001diarrheal specimens taken during June–October.Table 2.Clinical profile of 18diarrheal episodes in which en-terotoxigenic Bacteroides fragilis (ETBF)was isolated.Characteristic No.Loose stool episodes 15Mucoid stool episodes 9Bloody stool episodes 4Vomiting episodes 1Fever episodes 1Dehydration episodes 1Maximum stools passed per day,median (range)5(3–20)Duration (in days)of ETBF episode,median (range)6(2–39)NOTE.The 18diarrheal episodes were in 15children.At the time of the isolation of ETBF ,2children were 0–5months of age,3children were 6–11months of age,8children were 12–17months of age,and 5children were18–23months of age.during the summer (30.8%in diarrheal specimens vs.8.6%innondiarrheal specimens)().In the nondiarrheal spec-P p .001imens,the isolation rates of ETBF did not change significantlyfrom season to season.In the diarrheal specimens only,the peakin the isolation rate of ETBF (30.8%)in the summer differedsignificantly ()from the isolation rates of ETBF in di-P p .03arrheal specimens in the spring and winter (15.1%and 15.3%,respectively).Of the 55specimens from which ETBF was isolated,14spec-imens were from children who already had tested positive forETBF;therefore,41children (16.3%of all study children)hadat least 1ETBF-positive specimen.Repeat isolations of ETBFfrom diarrheal specimens came from separate diarrheal episodes,which indicates that these were sequential infections rather thanpersistent ETBF-related symptoms.The children who tested pos-itive for ETBF more than once during the study had 2–4ETBFepisodes each.The repeat isolations of ETBF were separated bydiarrheal episodes that were caused by other pathogens.The data were further examined to determine whether spec-imens harboring ETBF had other enteropathogens.Fifteen of55ETBF isolates were not accompanied by diarrheal symptoms.There were a total of 40ETBF-positive diarrheal episodes in33children.Of these 40episodes,22(55%)were accompaniedby other bacterial and/or parasitic agents (data not shown).The clinical data were analyzed after exclusion of the ETBF-positive diarrheal episodes in which multiple enteropathogenswere identified,which left for analysis 18separate diarrheal ep-isodes in 15children.As is shown in table 2,there was a strikingdifference in ETBF-positive case patients by sex.With the ex-ception of 4diarrheal episodes that were accompanied by bloodin the stool and 3episodes that were persistent (lasting у14days),the illnesses were generally found to be mild and secre-tory in nature.Case-control studies.A nested case-control study of 40ETBF-positive children and 78matched ETBF-negative control subjects was performed to assess the association of risk factors with the acquisition of ETBF.For 1ETBF-positive child,a matched control subject was not identified.The household and host characteristics,as well as distributions of other entero-pathogens,were similar in case patients and control subjects.In the analysis to determine risk factors for diarrhea-associated ETBF infections,32of 33children who had diarrhea-associated ETBF-positive episodes were matched by age and season of spec-imen collection to 55ETBF-negative control subjects who had nondiarrheal specimens.One ETBF-positive child was not in-cluded in this analysis because a matched control subject was not identified.There were no significant differences between case patients and control subjects with regard to household charac-teristics,nutritional indicators,and other enteropathogens.Univariate conditional logistic regressions for both case-con-trol analyses are shown in table 3.No potential risk factors showed a significant association with ETBF-positive diarrheal specimens,but the presence of chickens or cows in the house-hold area was statistically significantly associated with the ac-quisition of ETBF.As is shown in table 4,only the adjusted OR for the presence of chickens in the household area remained significantly associated with the acquisition of ETBF.The pres-ence of cows in the household area had an association that approached statistical significance ().P p .06DISCUSSION Although numerous enteropathogens are known to cause di-arrheal infections,the etiologies of a substantial number of cases of diarrhea remain unrecognized,and this suggests that addi-tional,unidentified pathogens may be responsible for disease.Our results confirm that the isolation of ETBF is low but isTable3.Crude odds ratios(95%confidence intervals)for acquisition of en-terotoxigenic Bacteroides fragilis(ETBF)and disease,by variable.Variable Acquisition of ETBF a ETBF-associated disease bSexMale 1.00 1.00Female0.80(0.39–1.64)0.47(0.17–1.28) Exclusive breast-feedingу3months 1.00 1.00!3months0.72(0.29–1.79)0.82(0.32–2.11) First born in familyY es 1.00 1.00No0.48(0.20–1.16)0.31(0.10–0.99)c Low birth weightY es0.62(0.26–1.46)0.36(0.09–1.37)No 1.00 1.00Mother’s education15years of schooling 1.00 1.00р5years of schooling 1.09(0.41–2.88)0.89(0.34–2.28) Father’s education15years of schooling 1.00 1.00р5years of schooling0.71(0.31–1.61)0.33(0.10–1.06) Household monthly salaryу3000Taka 1.00 1.00!3000Taka0.79(0.38–1.65) 1.53(0.61–3.81) Family sizeр6members 1.00 1.0016members 1.40(0.66–2.98) 1.23(0.54–2.81) Source of drinking waterTube well 1.00 1.00Surface 1.41(0.08–23.57)0.78(0.07–8.88) Source of washing waterTube well 1.00 1.00Surface0.78(0.19–3.22)0.20(0.02–1.79) Type of latrine used by motherSanitary 1.00 1.00Open 2.28(0.65–8.04) 1.76(0.53–5.83) Type of latrine used by childSanitary 1.00 1.00Open 1.00(0.23–4.35) 1.28(0.23–7.14) Chickens in household areaY es 5.89(1.31–26.49)d 3.36(0.71–15.77) No 1.00 1.00Cows in household areaY es 2.17(1.02–4.62)d 1.32(0.56–3.08)No 1.00 1.00Site of cookingDry seasonInside house 1.00 1.00Outside house 1.37(0.63–2.99)0.79(0.32–1.92) Wet seasonInside house 1.00 1.00Outside house 1.10(0.39–3.06)0.70(0.18–2.65) Own tube wellY es 1.00 1.00No0.95(0.44–2.06) 1.16(0.38–3.38) Store waterY es0.97(0.40–2.33) 1.00(0.32–3.13)No 1.00 1.00a For the analysis,40ETBF-positive children were matched to78ETBF-negative control subjects.For1ETBF-positive child,a suitable control subject was not identified.b For the analysis,32ETBF-positive children with diarrheal specimens were matched to55 ETBF-negative control subjects.c.P p.05d.P!.05Table4.Adjusted odds ratios(95%confidenceintervals)for the acquisition of enterotoxigenicBacteroides fragilis(ETBF),by selected variables.Variable Acquisition of ETBFSexMale 1.00Female0.62(0.28–1.37)Chickens in household areaY es 5.41(1.09–26.91)aNo 1.00Cows in household areaY es 2.30(0.97–5.44)No 1.00NOTE.For the analysis,40ETBF-positive childrenwere matched to78ETBF-negative control subjects.For1ETBF-positive child,a suitable control subject was notidentified.a.P!.05significantly associated with acute diarrheal disease in children у1year of age in rural Bangladesh.The disease is relatively mild and is readily treated with oral rehydration therapy.The present study—which is,to our knowledge,thefirst longitu-dinal study to be conducted at the household level on rural children of a developing country—found that16.3%of all children were infected with ETBF during thefirst2years of life.To our knowledge,this study is also the second one[15] to indicate a seasonal pattern of ETBF infection and may be thefirst to demonstrate that ETBF infection is associated with environmental and household factors.An interestingfinding was that RBF medium was more sen-sitive in identifying BF organisms than was the traditional BBE medium.The isolation rate when RBF medium was used was about twice the rate seen when BBE medium was used(18.1% vs.11.1%)().In the future,studies of BF should benefit P!.001from the use of RBF medium.In the present study,BF of any type was isolated from12.6% of diarrheal specimens and from15.2%of nondiarrheal spec-imens.Thus,unlike thefinding from an Oklahoma hospital study by San Joaquin et al.[15],but in accordance with the finding from a study of Apache children by Sack et al.[8], there was no significant association between the presence of any BF and diarrhea in Mirzapur.That BF was consistently more often isolated from nondiarrheal specimens supports the idea that,like E.coli,it is a normal inhabitant of the colon. Approximately14%of BF isolates tested were enterotoxin producers.Similarly,San Joaquin et al.[15]discovered that,in Oklahoma,∼15%of BF in stool specimens(regardless of di-arrheal status,age,and season)were enterotoxin producers. After enumeration of the organisms in municipal sewage in Montana,it was found that9.3%of BF was ETBF[16].ETBF may make up a considerable portion of the BFflora of the intestinal tract,and,like diarrheogenic E.coli,BF may have additional virulence factors that are important in the patho-genesis of diarrheal disease.Recently,in epidemiological studies conducted throughout the world[8,9,15,17–21],ETBF has been explored as a human diarrheal pathogen in persons presenting at clinics or hospitals. Seven of these studies have focused on the association between ETBF and childhood diarrhea[8,9,15,17–20].A1997study by Pantosti et al.[18]in Italy showed that the rate of ETBF carriage was high and was not significantly different between children with diarrhea(17%)and healthy control subjects(12%) 1month–9years of age.A Bangladeshi hospital–based study [9]found ETBF rates in children!5years of age to be lower (6.1%of case patients and1.2%of control subjects)than those in the Italian study.The rates at this Bangladeshi hospital are therefore similar to the childhood rates found in the Oklahoma study(4.4%of case patients and3.1%of control subjects)and in the study of Apache children(12%of patients and6%of control subjects).In all3studies,differences in isolation rates of ETBF in case patients and control subjects attained statistical significance only for children11year of age.In the present study,overall isolation rates of ETBF from BF-positive diarrheal and nondiarrheal specimens were high(20.3%from diarrheal specimens and8.1%from nondiarrheal specimens).Similar to the isolation rates in the3previous studies mentioned,the isolation rates for the2groups in the present study were sig-nificantly different only for those specimens taken from chil-drenу1year of age,in which the isolation rates of ETBF rose to24.6%in diarrheal specimens but remained virtually con-stant in nondiarrheal specimens.Our isolation rates of ETBF from diarrheal specimens and from nondiarrheal specimens(2.3%and0.3%,respectively)were close to those seen for a number of other organisms detected in this cohort:Shigellaflexneri(5.4%in diarrheal specimens and 0.02%in nondiarrheal specimens),Salmonella species(1.83% in diarrheal specimens and0.2%in nondiarrheal specimens), V.cholerae O1Ogawa(0.2%in diarrheal specimens and0.0% in nondiarrheal specimens),and Cryptosporidium species(0.6% in diarrheal specimens and0.2%in nondiarrheal specimens). The burden of ETBF in our study may have even been un-derestimated because of the medium initially used to detect BF, as was indicated above.To our knowledge,this is the second study to report a sea-sonal pattern in ETBF infections.San Joaquin et al.[15]found no appreciable differences in isolation rates of ETBF from sea-son to season in Oklahoma in43case patients and18control subjects.In contrast to those results,we found a peak in di-arrhea-associated ETBF during the summer,compared with the rates in spring and winter.Isolation rates of ETBF in non-diarrheal specimens did not change seasonally.This seasonal difference suggests some as-yet-unspecified property of the di-1250•JID2005:191(15April)•Pathela et al.arrhea-associated ETBF that is lacking in those strains not as-sociated with diarrhea.In the crude analysis of risk factors for acquisition of ETBF, the presence of cows and chickens in the household area showed statistically significant,positive associations with the acquisition of ETBF.After controlling for cows in the household and sex of the child,there was still a5-fold increase in the odds of ac-quiring ETBF for children who had any chickens in the house-hold area,compared with the odds for children who had no chickens.Although it has been determined that different do-mestic animals,including lambs and calves,can have ETBF-associated diarrhea[3,11],to our knowledge there has not been a report of ETBF being isolated from the feces of chickens. It is possible that feces from chickens could yield ETBF,as has been seen in the case of Campylobacter[22].The results of the present study suggest that both cows and chickens could be sources of human infection or vehicles for the transmission of ETBF.Thisfinding points to areas of future investigation.Further surveillance studies could include col-lection of specimens from livestock with diarrhea,particularly when a case of ETBF is discovered in humans.ETBF-positive stool specimens from livestock and ill children within the same time frame would give strength to the epidemiological asso-ciation we found.Because a large proportion of families in Mirzapur keep livestock in the household area(i.e.,50%of families keep13chickens),the discovery of a definitive animal-to-human transmission of ETBF infection would have impli-cations for the prevention of this infection in this community and in comparable populations.No significant associations between ETBF-associated diar-rhea and environmental or biological risk factors could be dem-onstrated.The inhabitants of Mirzapur are a largely homo-geneous population;few disparities in living conditions and behaviors exist.Because they have access to the same water sources and exposure to the same unsanitary conditions,the children of these families had high levels of illness.For example, in this cohort,the incidence of infant diarrhea was4.25episodes per child per year(P.P.,K.Z.H.,E.R.,F.H.,A.K.S.,and R.B.S., unpublished data).Thus,because almost all children in this cohort are exposed to the same potential disease-causing con-ditions,it is difficult to determine which ones specifically are linked with diarrheal disease.Additionally,the use of ETBF-associated diarrhea as the outcome of a study could be prob-lematic,given that there is a high prevalence of mixed infections in this population and that ETBF-positive diarrheal specimens were also often found to be positive for other enteropathogens. To be able to attribute diarrhea solely to ETBF would have required omitting children who had additional enteropathogens from the study,which would have greatly reduced the numbers studied and the statistical power of the analysis.In summary,ETBF has been shown to be a small but im-portant contributor to disease burden in young children living in rural Bangladesh.Approximately16%of all children!2years of age were infected with ETBF at some time.This community study had the advantage of detecting illnesses of a mild nature; most children with ETBF-associated diarrhea would probably not be seen at a treatment center.Approximately22%of ETBF-positive diarrheal episodes were associated with bloody stool; similarfindings have been reported elsewhere[7],and,in the present study,these episodes may have been the result of un-recognized coinfections.Prevention of ETBF infection through the improved management of livestock,as well as adequate treatment of the infection with oral rehydration therapy,would help to lessen the impact of diarrheal diseases,which are a persistent problem in this part of the world. 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