However,at initialmedical examination,thepatients exhibited bilateral, symmetricalflaccid weaknessaffecting thefacial and ocularmuscles,followedbydysarthriaprogressingto diaphrag-matic pa
Trang 1Case Report
c
d
e
1 Introduction
Botulism isrecognizedasone ofthemostseriousinfectious
hazards.Foodborne botulism, a notifiabledisease in Japan, the
outbreakisrecentlyveryrareforseveraldecades.Herewereporta
foodbornebotulismcase occurredinTottoriprefecture,Japanin
March,2012
2 Casepresentations
EarlyinthemorningofMarch24,2012,twomarriedpatients,a
69-year-oldmale(patient1)anda69-year-oldfemale(patient2),
wereadmittedtothehospitalinYonagoCity,TottoriPrefecture,in
Japan Both patients were able to communicate upon arrival
However,at initialmedical examination,thepatients exhibited
bilateral, symmetricalflaccid weaknessaffecting thefacial and
ocularmuscles,followedbydysarthriaprogressingto
diaphrag-matic paralysis,which ledto respiratory arrest (Figure1 The
patients were placed on respiratory support and developed
autonomic features, such as paralytic ileus and labile blood
pressure A blood test showed no abnormal signs (normal leukocyteanderythrocytecountsandnormalliverfunctiontests;
abnormalities The patients weresubsequently hospitalizedfor over1year
3 Laboratoryinvestigations
3.1 Clinicalsamples
Fecal and serum samples were taken at admission and transportedtoourlaboratories.Thesewereimmediatelycultured anaerobically for the isolation of Clostridium botulinum The samples were then homogenized in 0.2% gelatin/phosphate bufferedsaline(PBS)andfiltratedwitha0.45-mmsyringefilter Afterincubationwithtrypsin(0.2mg/ml)for1hat378C,a0.5-ml portion of the filtrate and serial dilutions of the filtrate were injected intraperitoneally (IP) into BALB/c mice (15–20g body weight) to determine the presence of botulinum neurotoxins (BoNTs),asdescribedbyKondoetal.1andinaccordancewiththe ManualforLaboratoryDiagnosticsofPathogens:Botulism(http://
Japanese) An antibody neutralization assay was conducted simultaneouslytodeterminethetoxintypeusingequalamounts
Keywords:
Japan
InMarch2012,twopatientsweretransportedurgentlyto thehospitalinTottoriPrefecture,Japan, becauseofsymptomssuggestiveofbotulism.BotulinumneurotoxintypeAwasdetectedintheclinical specimensandthefoodconsumedbythetwopatients(vacuumpackedadzuki-batto,asweetadzuki beansoupcontainingnoodles).Wewereabletomakeapromptdiagnosisoffoodbotulismassociated withtheconsumptionofadzuki-batto,fromwhichthecausativepathogenClostridiumbotulinumAbwas cultured
ß2014TheAuthors.PublishedbyElsevierLtdonbehalfofInternationalSocietyforInfectiousDiseases ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(
http://creativecommons.org/licenses/by-nc-nd/3.0/)
ContentslistsavailableatScienceDirect
j o urn a l hom e pa ge : ww w e l s e v i e r c om/ l o ca t e / i j i d
http://dx.doi.org/10.1016/j.ijid.2014.01.014
Trang 2producedin-house),asdescribedpreviously.1Theanimal
experi-mentswereperformedundertheguidelinesforanimalcareand
useofourinstitutes.Finally,BoNT/Awasdetectedinbothsamples
and BoNT/A-producing C botulinum was isolated from fecal
samples.The two patients were administered multivalent sera
againstBoNT/A,B, andFatthehospital.Consequently,no toxin
activity was observed in the serum samples of either patient
thereafter.Nonetheless,2monthslater(May2012),C.botulinum wasstillpresentinthefecalsamplesofbothpatientsandBoNT/A was detected in a fecal sample from one of the two patients (patient2)
3.2 Foodsamples
Suspectedcausativefoodswereexaminedsimultaneouslyfor thedetectionofBoNTsandC.botulinum,includingadzuki-batto, stew,andcurry,whichweretheleftoversatthepatients’home.No pathogenic bacteria were isolated from the stew or curry However, both BoNT/Aneurotoxin and theorganismproducing
it,C.botulinum,weredetectedintheadzuki-batto,asweetadzuki beansoupcontainingflatwheatnoodlesmadeinMiyako,Iwate Prefecture in Japan Medical interview revealed that the two patientshadeatenthisdishforlunchonMarch23,2013.Thetoxin titerintheadzuki-battowasestimatedtobe75000IPLD50/gofthe foodsampleusingmouseassays.1
3.3 Characterizationoftheisolates
Theinvivoantibodyneutralizationassaysusinganti-BoNT/A,B,
E,andFantiserumrevealedthattheorganismsisolatedfromboth the patients and thefood produced onlyBoNT/A, whereas PCR assaysforthedetectionandcharacterizationofBoNTgenes(BoNT/ A–G)usingmultiplexprimersets2incombinationwiththeprimer setsfromTakaraBio,Shiga,Japan(CodeNo.S021-S027),showed that they were positive for both BoNT/A and BoNT/B genes Correspondingly,thebacterialisolateswerealsopositiveforthe
Patient 1
March 23rd March 24th May 21st, 23rd June 26th
Evening 2:30 3:00 7:00 Evening
Patient 2
Emergency call Arrival at the hospital
Signs: slurred speech, difficulty swallowing Symptoms: flaccid weakness affecting facial/ocular muscles, dyspnea,
diaphragmatic paralysis, respiratory arrest, paralysis in skeletal muscle
Intervention: artificial respiration, enema, anti-BoNT antiserum
Signs: nausea, slurred speech, difficulty swallowing, abdominal
distension Symptoms: flaccid weakness affecting facial/ocular muscles, dyspnea,
diaphragmatic paralysis, respiratory arrest Intervention: artificial respiration, enema, anti-BoNT antiserum
Artificial respiration
Signs: drooping
eyelids, nausea
Sign: drooping
eyelids
Injection of anti-BoNT antisera
Feces: BoNT/A detected,
C botulinum A(b) isolated
Sera: BoNT/A detected
Feces: BoNT/A detected (1 of 2 patients),
C botulinum A(b) isolated (2 of 2) Sera: BoNT/A not detected
Adzuki-batto
Stew
Curry
Consumed foods
BoNT/A detected
C botulinum Ab isolated
Reversed Conscious, with mobility in toes, malar arch,
temporomandibular joint (TMJ), fingers, and neck
Unconscious
/l
Trang 3ha33 and p47 genes,which are representativemarkers for the
BoNT/AandBoNT/Bgeneclusters,respectively.3 These
observa-tionsconfirmedthatthecasesofinfectionwereassociatedwiththe
intakeofadzuki-battocontaminatedwithC.botulinumAb
4 Discussion
Sincethefirstcaseoffood-bornebotulismwasreportedin1951
inrelationtotheconsumptionof‘Izushi’,afishfermentedinrice
servedwithmaltedriceandvegetables,severalfood-bornecases
havebeenreportedinJapan.4Themaincauseofthepathogenicity
ofC.botulinuminfectionisBoNT,whichislargelyclassifiedinto
typesAtoG.5WhiletypeEbotulismwasdominantuntilthe1980s,
thenumbersofcasesoftypeAandtypeBbotulismhaveincreased
inrecentyears.6Ourrapidresponsetothisemergencycasethus
provideddatasupportingclinicaltreatment withthe
epidemio-logicalconclusion
Thesecasesshowedanunusualclinicalaspect:C.botulinumand
BoNT/Awerecontinuouslydetectedin thefecesofthepatients
Onepossibleexplanationisthat thepatients consumeda large
amount of the causative food, since neither patient had an
underlyingimmunosuppressionorgutabnormalities; also,high
titersofBoNT/A(75000IPLD50/g)weredetectedinthecausative
food It is considered that the BoNT-dependent paralytic ileus
furtherprolongedtheaccumulationofC.botulinumanditstoxin
Vacuum packing provides an atmosphere that allows the
growth of anaerobes but not Enterobacteriaceae,7 which poses
further potential risks for infection with Clostridium bacteria
throughtheconsumptionofthesefoodtypes.Weconcludedthat
these cases occurred in association with the consumption of vacuum-packed adzuki-battoaccidentally contaminated withC botulinumAb,whichovergrewandproducedtheneurotoxin
Acknowledgements
Thisworkwassupportedfinanciallyinpartbygrantsfromthe Ministry of Health, Labour and Welfare, Japan (H25-shokuhin-ippan-010,Shokkenpi-H24)
Conflictofinterest:Noconflictofinteresttodeclare
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