A food-borne outbreak of gastroenteritis caused by norovirus GII in a university located in Xiamen City, China

A

food-borne

outbreak

of

gastroenteritis

caused

by

norovirus

GII

in

a

university

located

in

Xiamen

City,

China

Zhinan

Guo

,

Jianwei

Huang

,

Guoqing

Shi

,

Cheng-hao

Su

,

Jian

Jun

Niu

*

a_{XiamenCenterforDiseaseControlandPrevention,Fujian,People’sRepublicofChina} b

ChineseFieldEpidemiologyTrainingProgram,ChineseCenterforDiseaseControlandPrevention,Beijing,People’sRepublicofChina c

ZhongshanHospital,XiamenUniversity,Xiamen,Fujian,People’sRepublicofChina

1. Introduction

Noroviruses(NoVs)aremembersoftheCaliciviridaefamilyand arerecognizedas acause ofacute non-bacterialgastroenteritis worldwide.1,2_{NoVscanbedividedintofivegenogroups,}

designat-edGI–GV.3_{ThemajorityofhumanNoVscanbeclassifiedintothree}

genogroups, GI, GII, and GIV, which are subdivided into the followinggenotypes:atleasteightGI,21GII,andtwoGIV.3,4_GII.4

has been associated with the majority of viral gastroenteritis outbreaksworldwideinrecentyears.5

Foods may be contaminated by contact with human fecal matteratthesource6 _{orby unhygienicmanipulation bya food}

handler excreting thevirus.2 _{The virus can be excreted in the}

absenceofsymptoms,7_{soimpropermanipulationbyan}

asymp-tomaticfoodhandlercanleadtoanoutbreak.8,9

OnDecember19,2012,itwasreportedthat30peoplefromthe university in Jimei District, Xiamen City, China had developed

acutediarrheaandfever.Rectalswabsamplesfromsevenofthe patients were found tobe positive for NoV GII using reverse-transcriptasePCR(RT-PCR).In ordertoprovideeffectivecontrol measures,wesurveyedtheoutbreaktoverifyadditionalcasesand the source of infection, vehicle for infection, and mode of transmission.

2. Methods 2.1. Studydesign

The outbreak was investigated immediately after it was reportedonDecember19,2012.Theinvestigationwasexempted fromethicalapprovalandanyrequirementforinformedconsent becauseitwasinresponsetoanacutepublichealthemergency. Despitethiscontingency,westillinformedeachsubjectverbally beforetheinvestigationandasignedquestionnairewasobtained. The whole investigation was recorded comprehensively by an investigator and all of the relevant documents were filed appropriately.

ARTICLE INFO

Articlehistory:

Received6November2013 Receivedinrevisedform30June2014 Accepted30June2014

CorrespondingEditor:EskildPetersen, Aarhus,Denmark Keywords: Food-borne Gastroenteritis Norovirus Case–controlstudy SUMMARY

Objectives:WeinvestigatedadiarrheaoutbreakthatoccurredatauniversityinChinatoidentifythe etiologicalagentoftheoutbreak,sourceofinfection,modeoftransmission,andriskfactors. Methods:Inthiscase–controlstudy,wecomparedthefoodsourcesandexaminedthefoodandwater itemsconsumedbetweentheprobableandconfirmedcasesandtheasymptomaticcontrolstudents, whowereselectedrandomlyandfrequency-matchedbyclassandageataratioof1:2.

Results:Outof7141students(excludingteachers),87(1.2%)developedanillness.Thirty-threeof44 (75%)casesand11of88(13%)controlstudentshadconsumedbreadproductssuppliedbyanunlicensed smallbakery(oddsratio21,95%confidenceinterval8–60).NorovirusGIIwasdetectedinsevenpatients andinafoodhandleratthebreadworkshopandhis8-month-oldson.

Conclusions: Theoutbreakofgastroenteritiswascausedmainlybybreadproductscontaminatedwith norovirusGII.AfoodhandlerwithanasymptomaticnorovirusGIIinfectionwasthepossiblesourceof infection.

ß2014TheAuthors.PublishedbyElsevierLtdonbehalfofInternationalSocietyforInfectiousDiseases. ThisisanopenaccessarticleundertheCCBY-NC-NDlicense( http://creativecommons.org/licenses/by-nc-nd/3.0/).

* Correspondingauthor.

E-mailaddress:[email protected](J.J.Niu).

ContentslistsavailableatScienceDirect

International

Journal

of

Infectious

Diseases

http://dx.doi.org/10.1016/j.ijid.2014.06.022

1201-9712/ß2014TheAuthors.PublishedbyElsevierLtdonbehalfofInternationalSocietyforInfectiousDiseases.ThisisanopenaccessarticleundertheCCBY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/3.0/).

2.2. Casedefinitions

NoVcaseswereidentifiedbytheonsetofvomitingordiarrhea (3times/day)inanypersonattheuniversityduringDecember9– 23,2012.Probablecasesweresuspectedcaseswithatleasttwoof the following symptoms: fever, abdominal pain, nausea, and headache. Confirmed cases weresuspected and probablecases withNoVGII-positivestoolspecimens,rectalswabs,orvomit.

2.3. Casefinding

Electronic medical records collected from Jimei District Hospitalandtheuniversityinfirmarieswerereviewed systemati-cally,especially forpatientswithvomiting,diarrhea,abdominal pain,and fever. Casedefinitions wereapplied tothereview of medical records toidentify cases. The teachers and staff were requestedto verbally reportcases of illness in anystudent by telephonefromDecember11to23,2012.

2.4. Case–controlstudy

All of the probable and confirmed cases with onset from December12to19,2012wereenrolledinthecase–controlstudy. Thecontrolstudentswereselectedrandomlyfromasymptomatic studentsataratioof1:2,frequencymatchedbyclassandage.A questionnairesurveywasconductedbyface-to-faceinterviewto collect the following data: demographic information, date of illness onset, symptoms, duration of illness, and history of exposure to suspected food and water. We also compared the refectoryrecordsbetweencasesandcontrolstudents.

2.5. Laboratoryinvestigation

In order to identify the possible pathogens, including NoV, rotavirus,Vibriocholerae,Shigella, andSalmonella,we collected rectalswabs,stoolsamples,vomit,andhandswabspecimensfrom cases,refectory employees in theuniversity, and employees of unlicensedbakeries.Thetotalbacterialcountwasobtainedfrom watersamplestoidentifypossiblebacterialcontamination,anda fecal coliform count was also obtained to determine fecal contamination.Allbacterialtestswereperformedinaccordance withstandard protocols. Viral nucleic acidwas extractedfrom rectalswabsandvomit.Suspensionsofrectalswabswereprepared byadding2mlphosphate-bufferedsalinefollowingbrief vortex-ing.Intheextractionstep,analiquotof1mlliquidfeceswasspun at 1000g for 5min, and total RNA wasextracted from 200

m

Laboratorytestswereconductedinaccordancewithstandard protocols.ThespecimensweretestedintheLaboratoryofXiamen CenterforDiseaseControlandPreventionforbacterialpathogens usingconventionalmicrobiologicalmethodsandforNoVbyRT-PCR.

2.6. GenotypeanalysisofNoV

WemanagedtoidentifyNoVRNAinninesamplesusing real-timePCR;theRNAoftheseninepositivesampleswasisolatedusing a furtherlaboratoryassay.The Qiagen OneStep RT-PCR Kit was used to amplify the RNA-dependent RNA polymerase (RdRp) fragmentofNoV.TheprimersequenceswereNVRPf50

-ATACCAC-TATGATGCAGATTA-30_andNVRPr50

-TCATCATCACCATAGAAAGAG-30_{.Agarosegelelectrophoresiswasutilizedtoretrieveamplification}

products of 327bp, and the products were sent to Invitrogen (Shanghai, China) for sequencing after purification. Multiple genotype sequences were downloaded from GenBank, and the clusterand neighbor-joiningmethod was performed toidentify

thegeneticrelationshipbetweenlocalNoVandotherstrainsfrom homeandabroad.

2.7. Environmentalinvestigation

Toidentifyinfectionriskfactors,weimplementedan environ-mentalsurvey,includingtheworkingconditionsinthebakery,the bread-makingprocess,andthebreadsaleschannels.

2.8. Statisticalanalysis

Univariateanalysis(Student’st-test,PearsonChi-squaretest) wasusedtocomparethedistributionsofageandsexbetweenthe casesandcontrols.TheMantel–HaenszelChi-squaretestwasused toassessrisk factors.Statisticalanalyseswereperformed using SPSSversion15.0(SPSSInc.,Chicago,IL,USA).p-Valueswere two-sidedandp<0.05wasconsideredstatisticallysignificant.

Withregardtotheestablishment ofa phylogenetictree, we downloadedthe17sequencesofNoVGIandGIIfromGenBankas referencesequencesforalignmentanalyses.Thesesequenceswere alignedbyClustalW2.0,thenaphylogenetictreewasconstructed usingtheneighbor-joiningmethodofMEGAversion6.0.

3. Results

Weidentified87casesincluding38suspectedcases,42possible cases,and sevenconfirmedcases thatoccurred fromDecember 11to22,2012.Allcaseswerestudents.Theattackratewas1.2% (87/7141). No cases were identified among faculty or staff members.Also, no case wasfoundamong thefamilymembers oftheteachersandstaffortheurbanresidentslivingaroundthe university.Amongthe87 cases,62(71%) experiencedvomiting andothercommon symptoms,includingnausea(66%), diarrhea (60%), abdominal pain (46%), fever (25%), headache (5%), and dizziness(2%).Nonewerehospitalized.Sixty-onepercent(53/87) weremale,andthemedianagewas20years(range18–23years). Theoutbreaklastedfor12days,startingat08:00honDecember 11.AccordingtotheincubationperiodofNoV,theepidemiccurve inaffectedclassesappearedtobethatofa continuouscommon source pattern with seven peaks (Figure 1). The time interval betweenadjacentpeakswas24h(exceptbetweenpeaks5and6, whichwas72h).

Theattack raterangedfrom0.6%to1.6%, and therewasno significant difference between the different grades of the university (Chi-square=7.12, p=0.07). The attack rate of the 31%(61/194)affectedclassesrangedfrom2.0%to13%.Theattack rate in male students was 3.6%, compared to 4.0% in female students; the difference was not significant (Chi-square=0.24,

p=0.6).

Teachers and students all drank barreled water that was providedbyXiamenWahahaFoodCo.Ltd,andhadmealsinthe universityrefectories.Among87cases,63(72%)hadeatenbread products2daysbeforetheonsetofillness,comparedto41(84%) among the 49 probable and confirmedcases. According to the epidemiccurve,weobservedatotalofsevenincidencepeaks,and thebreadconsumptionpeakoccurredat24hearlierthanevery incidencepeak(Figure1).

Forty-four probable and confirmed cases and 88 control studentswereenrolledinthecase–controlstudy.Thirty-eightof 44(86%)casesand14/88(16%)controlstudentsatebreadproducts (odds ratio (OR) 33.5, 95% confidence interval (CI) 11–111)). Thirty-threeof44(75%)casesand11/88(13%)controlstudentsate breadproductssuppliedbyanunlicensedsmallbakery(OR21,95% CI8–60)(Table1).

Theenvironmentalinvestigationdemonstratedthatthebakery workshoplocatedinBaishiVillagewasonly15m2_.Thedrinking

waterintheworkshopwasderivedfromamunicipalwatersupply. Theshopkeeperofthebakeryservedastheonlybaker.Therewere fivepeoplein hisfamilyand allofthemwereingoodphysical health,exceptfor hisson,whowasreported tohavefeverand influenzaonDecember11, 2012.According totheshopkeeper’s statement,thestandard protocolforbakingbreadmaintained a temperatureof 180–2508C for atleast 20min. However, there wereno records of bakingtemperatures ortimes. Thefinished productsweredeliveredtothe‘newandbetterpastryboutique’in the1FcanteenoftheuniversityonMondaytoFridayeveryweek. NoVGIIwasdetectedinsevencases,thefoodhandlerofthe bakery,andhis8-month-oldson(Table2).Otherenvironmental samplescollectedfromthebakery,employeesof theuniversity refectories, and thefood handler’sotherthree familymembers

testednegativeforNoV,Shigella,V.cholerae,andStaphylococcus aureus.TotalbacterialandcoliformcountsinfiveWahahabarreled waterand19tapwatersampleswerenormal.

PhylogeneticanalysisindicatedthattheNoVstrainsdetectedin ninespecimens(sevenconfirmedoutbreakcases,thefoodhandler, andhis8-montholdson)wereallNoVGII.4referencestrains.The homogeneitywas>97%over329bpoftheRdRpregion.Further analysisrevealedthatthevirusthatcausedthisoutbreakwasthe samestraindetectedduringNoVsurveillanceinXiamenCityin 2010 (HM195200jNoV Hu/Xiamen/2010/GII.4). Moreover, we foundthatthestrainwashighlyhomologouswithEU366113jNoV NoVHu/Beijing/2006andAB294793jNoVHu/JP/2006/GII.4,which showedthatNoVGII.4wasthedominantgenotype,withawide geographicdistribution(Figure2).

Figure1.EpidemiccurveforreportedcasesofnorovirusIIby8-hintervalintheuniversity—Xiamen,Fujian,China,2012.

Table1

RiskofnorovirusIIbyexposurein44probableandconfirmedcasesand88controlsintheuniversity,Xiamen,Fujian,China,2012

Foodtypes/refectoryrecord Number Exposed(%) OR 95%CI Cases

(n=44)

Controls (n=88)

Cases Controls

Breadproductsprovidedbyallcompanies 38 14 86 16 33.5 11–111 Breadproductsprovidedbytheunlicensedsmallbreadcompany 33 11 75 13 21.0 8–60

Refectoryrecord89# 4 3 9 3 2.8 0.5–20 Refectoryrecord81# 13 13 30 15 2.4 0.93–6 Refectoryrecord72# 10 9 23 10 2.6 0.87–8 Refectoryrecord94# 18 27 41 31 1.6 0.7–4 Refectoryrecord92# 12 19 27 22 1.4 0.5–3 Refectoryrecord22# 12 20 27 23 1.3 0.5–3 Refectoryrecord7# 7 11 16 13 1.3 0.4–4 Refectoryrecord70# 6 13 14 15 0.9 0.3–3

Wahahabarreledwater 29 70 66 80 0.8 0.7–1.1

Refectoryrecord61# 4 11 9 13 0.7 0.1–3

Refectoryrecord90# 3 10 7 11 0.6 0.1–2

Refectoryrecord113# 2 10 5 11 0.4 0.04–2

4. Discussion

Currently,NoV isconsideredtheleadingcauseoffood-borne disease and acute non-bacterial gastroenteritis worldwide.10

InfectionswithNoV are estimatedtocause23 million casesof gastrointestinalillnessperyearintheUSA.Asymptomaticinfected individualscanstillshedvirusintheirstools.11,12_Asymptomatic

excretionofNoVisrecognizedinfacilitieswith13,14andwithout15 outbreaks.In this investigation,we documentedan outbreakof NoVGIIgastroenteritiscausedbyafoodhandlerwith asymptom-aticinfection.

In this outbreak, thestatisticalanalysis didnotidentifyany significanceamongthefoodseatenintheuniversityrefectoriesas

the vehicle of infection, because the exposure rates of cases through the refectory records were low; the epidemic curve showedacontinuouscommonsourcepatternwithsevenpeaks (Figure1),butthetimeintervalbetweenadjacentpeakswas24h (exceptbetweenpeaks5and6,whichwas72h).However,three factssuggestthatthefoodvehiclewasprobablythebreadproducts providedbythesamebakery(Table1).First,75%ofcasesand13% of control students consumed bread products supplied by the bakery (OR21, 95% CI 8–60). Second,thebreadproducts were providedonlybetweenMondayandFridayeveryweek,whichled tothe72-hintervalbetweenpeaks5and6intheepidemiccurve. Third,thesametypeofNoVGIIwasidentifiedinsevencasesand thefoodhandlerinthebakery.

Table2

Testresultsamongcasestudents,environmentalsamples,andfoodhandlersfromtheuniversitydiningroomandanunlicensedbreadworkshop

Place Type Number NorovirusII

Theunlicensedsmall breadcompany

Water 6 Negative

Plate 1 Negative

Refrigeratorsmearsamples 1 Negative

Workstation 1 Negative

Plasticbucket 1 Negative

Stainlesssteelbarrel 1 Negative

Funnel 1 Negative

Cuttingtool 1 Negative

Blender 1 Negative

Rectalswabsofthefoodhandlerfamily Thefoodhandler 1 Positive Thefoodhandler’s8-month-oldson 1 Positive

Thefoodhandler’swife 1 Negative

Thefoodhandler’sdaughter 1 Negative

Thefoodhandler’smother-in-law(sellsbreadinthenewandbetterpastryboutique) 1 Negative Theuniversityrefectories Employeerectalswabs 13 Negative

Casestudents Rectalswabs 9 7Positive

Total 41 9Positive

Thisinvestigation described anoutbreak ofNoVGII among students in the university. Illness was associated with the consumption of bread products provided by an unlicensed bakery. NoV is extremely contagious, with an estimated infectiousdoseaslowas18viralparticles.16NoVtransmission occurs through three general routes: person-to-person, food-borne, and water-borne. Food-borne transmission typically occurs by contamination from infected food handlers during preparation,processing,andservice,andinthefooddistribution system.17–19 _{The high temperature used in food processing is}

capable of eliminating viruses, however, it is likely that the contamination occurred after baking via the hands of the foodhandlerduringthepackingprocedure.We speculate that NoVGIIwasspreadasymptomaticallyamongthefoodhandler’s family, resultingin contamination ofthe bread products.This speculationis basedon thefood handlerandhis8-month-old sontestingpositiveforNoV.Thebreadproductswereprobably contaminated directly by the asymptomatically infected food handler, or indirectly via contaminated food preparation surfacesorutensils.

We found that the gastroenteritis outbreak of NoV GII was mainlyviafood-bornetransmissionintheuniversity. Person-to-persontransmissionwastestedforbutnotidentifiedasthemain riskfactorforcausingtheoutbreak.However,forthetimeperiod duringtheoutbreak,person-to-persontransmissionwasplausible. NoV outbreaks with initial food-borne transmission, further propagatedbyperson-to-persontransmission,havebeenreported, especiallyinrelativelyclosedenvironments.20,21

Thecurrentstrategytopreventfood-borneinfection recom-mendsthatfoodhandlerswithdiarrheashouldnot workuntil 48–72h after becoming asymptomatic.22,23 However, it has beendemonstratedthatshedding of NoVmaylastlonger.24,25

The virus hasbeen detectedin stoolsamples for a medianof 4weeksandforupto8weeksaftervirusinoculation.25_During

the present outbreak, transmission was person-to-person or food-borneamongthefoodhandler’sfamily10dayspreviously, because the food handler’s 8-month-old son was positive for NoV and had fever and influenza on December 11, 2012.Therefore,therecommendation forexclusion ofworkers for48–72hafterthedisappearanceofsymptomsmaynothave beensufficienttoavoidvirustransmission.Werecommendthat foodhandlersshouldonlyreturntoworkafterthreeconsecutive stool specimens collected at 1-dayintervals test negative for NoV.

The present NoV outbreak had two possible sources. One wasthatthebakerwastheinitialcasewithNoVinfection,and the food was contaminated with the virus that he carried. Meanwhile, his family memberswere infected with the same virusviaclosepersonalcontact.However,itisalsopossiblethat theinfantwastheinitialcaseandtransmittedthevirus tohis father. Infection of the food handler from his son may be attributed to poor education and lack of health knowledge and awareness. It is possible that the food handler did not thoroughlywash his handsbefore consuming food or making bread.

Amajor limitationof ourinvestigationwasourinability to analyze all bread products or their components. No bread products werecollected duringtheoutbreak. Wewere unable todeterminewhetherthebreadcomponentswereavehiclefor NoV transmission.26 A second limitation was the delay in carryingout thesurvey,1weekafter theincident,whichmay haveresultedinrecallbiasforsomefactorsandmisclassification bias.

In conclusion, this gastroenteritis outbreak emphasizes the importanceofpreventing food-bornetransmissionof NoV. This eventdemonstrates that somefood processing enterpriseslack

HazardAnalysisCriticalControlPoint(HACCP)managementand effectiveevaluationoffoodprocessingsteps.Ourresultsalsoshow thatitisnecessarytoanalyzestoolsamplesfromallfoodhandlers, especiallythosewhoareasymptomatic,duringoutbreaksofNoV gastroenteritis. Given the importance of food handlers in the preventionofNoVinfection,allmeansshouldbeusedtopromote health education,hygienic manipulation of food, cleaning, and disinfection.

Acknowledgements

WeareindebtedtoDrGeorgeConwayforhisguidanceduring theoutbreakinvestigationandforhiscommentsandsuggestions onthedataanalysisandmanuscriptpreparation.Wealsothank YidunZhangandZhiminQiufortheirvaluablecontributionstothis investigationandarticle.

Ethical approval: This study was considered tobe part of a continuing publichealth outbreak investigation bythe Xiamen MunicipalHealth Bureauand exempt frominstitutionalreview boardassessment.Alldatawerekeptconfidential,withoutpatient identifiers.

Conflictofinterest:Ourstudyreceivedafinancialgrantfromthe medical researchprogram of XiamenMunicipal Health Bureau, entitled ‘‘Spectrum study and establishment of surveillance platform of infectious diseases in Xiamen City’’ (WSK2010-01). Wedeclarethatwehavenoconflictsofinterest.

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,athttp://dx.doi.org/10.1016/j.ijid.2014.06.022.

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