127© 2018 Journal of Natural Science, Biology and Medicine | Published by Wolters Kluwer - Medknow 127

Original Article

IntroductIon

Hookworm infection is one of the important causes ofiron deficiency anemia among children worldwide,[1]which is primarily caused by two hookworm species,namely,Ancylostomaduodenale(835million)andNecatoramericanus (135 million)[2] A. duodenale is mainlydistributed inMiddleEast,NorthAfrica, India,Australia,andEurope,whereasN.americanus ismore common intheWesternHemisphere, Sub‑Saharan, EasternAsia, andSoutheastAsia.[3] In India,N.americanus is predominantinSouth IndiaandA.duodenale inpredominant inNorthIndia regions.[4]Approximately 400 million childrenworldwide are infectedwith intestinal parasites, whichresultinanemia,growthretardation,cognitiveimpairment,increasedsusceptibilitytootherinfection,andseveralacutecomplications.[4,5] Different species of hookworm differ

in theirmorphology, pathogenesis, life cycle, and clinicalfeatures.Therefore,specificidentificationanddifferentiationofhookwormspeciesisimportantpublichealthmeasureformonitoringtheseverityofillnessandtheefficacyofmassandeffectivetreatment.Althoughthedrugofchoiceissimilarforbothspeciesofhookworm,theseverityofanemiadiffersdepending on the different species andworm load in theintestine.[6,7]Further,theseverityofclinicalmanifestationsalsodependsontheeggintensityandpositivecorrelationisobservedbetweentheinfectinghookwormspeciesandtheseverityofanemia.[8,9]

Comparative Evaluation of Harada–Mori and Agar Plate Culture for the Identification of Hookworm Species under Limited

ResourcesT. Shantikumar Singh, Nongmaithem Onila Chanu, Sudip Dutta1

Departments of Microbiology and 1Pediatrics, Sikkim Manipal Institute of Medical Science, Gangtok, Sikkim, India

Background:Humanhookworminfectionhaswidespreadsocioeconomicandpublichealthimplications.Severalcoproculturetechniqueshavebeendevelopedformorphologicalidentificationofhookwormlarvaeunderlimitedresourceavailability.TheobjectiveofthisstudywastocomparetheperformancesofHarada–Moriculture(HMC),agarplateculture(APC),andmodifiedAPC(MAPC)ofhookwormpositivestoolspecimenforidentificationofhookwormspeciesoccurringinEastSikkim,India.Materials and Methods:Thisprospectivecohortstudywasperformedusing180stoolspecimencollectedfromchildrenwhoattendedCentralReferralHospitalandSirThodupNamgyalMemorialHospital,withthecomplainedofgastrointestinalsymptoms.Bloodsampleswerealsocollectedtocorrelatewiththecompletebloodcount(CBC).ThehookwormpositivestoolspecimenevaluatedbymicroscopywassubjectedtoHMC,APC,andMAPCtechniquestoharvesthookwormlarvae.Stoll’sdilutioneggcountfordeterminingeggintensityandCBCwerealsoperformedforthechildrenwhowerepositiveforhookworm’seggsintheirstoolsample.Results:ThisstudyobservedapredominanceofNecatoramericanus(75%)overAncylostomaduodenale(25%).CBCresultsshowedhighpackedcellvolumevaluesin9/12,lowhemoglobin9/12,andhigheosinophilcountinallthepositivechildren.Stoll’sdilutioneggcountshowedmoderateinfectionin66.6%,lightandheavyinfectionsin16.7%ofchildren’s.APCmethodwassuperiortoHMCandMAPCinculturingandidentifyinghookwormspecies.Conclusions:APCwasobservedtoyieldbetterresultsandwaseasiertoperforminlimitedresourcelaboratorysettingcomparetoMAPCorHarada–Moriculturetechniques.

Keywords:Coproculture,diagnosis,gastrointestinal,hookworm

Address for correspondence: Ms. Nongmaithem Onila Chanu, Department of Microbiology, Sikkim Manipal Institute of Medical Science,

Gangtok ‑ 737 102, Sikkim, India. E‑mail: nongmaithem2008@gmail.com

Access this article online

Quick Response Code:Website:www.jnsbm.org

DOI:10.4103/jnsbm.JNSBM_234_17

Abstract

This is an open access journal, and articles are distributed under the terms of the Creative Commons Attribution‑NonCommercial‑ShareAlike 4.0 License, which allows others to remix, tweak, and build upon the work non‑commercially, as long as appropriate credit is given and the new creations are licensed under the identical terms.

For reprints contact: reprints@medknow.com

How to cite this article: SinghTS,ChanuNO,DuttaS.Comparativeevaluation ofHarada–Mori and agar plate culture for the identificationof hookworm species under limited resources. J Nat Sc BiolMed2018;9:127‑31.

Singh, et al.: Culture techniques for hookworm species identification

Journal of Natural Science, Biology and Medicine ¦ Volume 9 ¦ Issue 2 ¦ July-December 2018128

Thefollowingcharacteristicdifferencebetweenfilariform(L3)larva ofA.duodenale andN.americanus can be used todifferentiatethespeciesbycoproculture(1)size(A.duodenale720µm,whereasN.americanus is660µm); (2)cuticle incaseofA.duodenale is shorter (10µm), lumen larger,andboundedbytwothinchitinouswall,andinN.americanus,itislarger(15µm),lumenisshort,andboundedbytwothickchitinouswall;(3)thereisnogapbetweentheesophagusandintestineinA.duodenale,incaseofN.americanus,thereisagapduetoprominentanteriordilatationoftheintestinallumen;and(4)posteriorendoftheintestinehasarefractilebodyinA.duodenale;however,thisisabsentinN.americanus.[4]

Directmicroscopyexaminationof stool sample is a simpleand rapid test to diagnose hookworm infection; however,microscopyalonecannotdifferentiatebetweenthehookwormspecies and other similar species such as Strongyloidesnematodes due tomorphological similarities of the egg.[10]Hence,variouscoproculturetechniquesareemployedforthemorphologicalcharacterizationofA.duodenale,N.americanus,andStrongyloides.Althoughhookworminfectionisoneoftheimportantcausesofanemiaamongchildren,studiesexaminingtheprevalenceofhookwormspeciesinSikkimregionofIndiaare lacking.Hence, this study aimed to detect hookworminfectionandhookwormspeciesprevalent inSikkimregionofIndia.ThisstudyalsocomparedHarada–Moriculture,agarplateculture(APC),andmodifiedAPC(MAPC)techniquesin the recovery of hookworm larvae andmorphologicalcharacterizationofhookwormspecies.Inaddition,theburdenofeggintensitybyStoll’sdilutioneggcountwasalsostudied.

mAterIAls And methods

Thisprospectivestudywascarriedoutin180childrenwhoattendedSirThodupNamgyalMemorialHospitalandCentralReferralHospitallocatedinEastSikkim,India,duringMay2015 toMay 2016.Children in the age group 0–15whopresentedwithgastrointestinalsymptomswereinvestigatedforintestinalparasiticinfection.Allstoolspecimenswerecollectedbeforeradiologicalstudiesusingbariumortheadministrationofbismuth,mineraloil,andantidiarrhealmedicationthatcouldinterferewith the detection and identification of intestinalparasites. Children above 15 years and children below15yearswithout gastrointestinal symptomswere excludedfromthisstudy.Writteninformedconsentwasobtainedfromparents/guardiansofthechildren.ThisstudywasapprovedbyInstitutionEthicsCommittee.(SikkimManipalUniversity).

Collection and processingThe children/guardianswere given a labeled, leak‑proofcontainerwithaplasticscoop(Hi‑Media)tocollectthesampleasperthestandardprocedureoutlinedbytheWorldHealthOrganizationandwasprocessedwithin4hofcollection.Then,thesampleswereexaminedmicroscopicallybeforesmearsandafterformal‑etherconcentration,techniqueusingsalineandiodinewetmountcoverslippreparation.Positivesamplesforhookwormeggswerefurthersubjectedtothreecoproculture

methods to obtained rabditiform larvae formorphologicalspeciation.Stoll’sdilutioneggcountmethod(numberofeggpergramofstool)toidentifythewormburdenandintensityoftheinfectionwasconductedinallthepositivespecimens.Complete blood counts (CBCs) were performed usingAcTTM5diffCapPierceHematologyAnalyzer.

Stool cultureAbout0.5–1goffreshsamplesthatcontainedhookwormeggswereculturedtotherabditiformlarvaat25°C–28°CbytheHarada–Moriculture(HMC)technique.[6]Thetubewaskeptfor 7–10days and checkeddaily.ForAPC, approximately2gramsof samplewereusedand incubatedat26°C–33°Cfor2days.[11]ForMAPC,acanalof1cmwidewasmadeandapproximately2gramsofsamplesweresmearedontheagarandincubatedat26°C–33°Cfor2–3daysaspreviouslydescribed.[12‑14] Quantitative hookworm egg countswereobtained byStoll’s dilution egg count[11], and resultswereexpressedas100–500eggsinfeces.

Statistical analysisThestatisticalanalysiswasperformedusingMicrosoftExcelsheet.Chi‑square testwas used tomeasure the probabilityofassociationbetweenthethreemethods,prevalencerateofinfectionamongtheageandgendergroup.Differentvariablesweresummarizedusingfrequencytables.

results

Theoverallintestinalparasiticinfectionrateobservedinthisstudywas 32.2% (58/180) inwhich hookworm infectionratewas 6.6% (12/180) [Table 1]. Hookworm infectionassociatedwith orwithout other parasiteswas 41.6% and58.3%,respectively.TheincidencerateofN.americanusandA.duodenaleinthisstudywas75%and25%,respectively.Otherpredominantintestinalparasitesobservedinthisstudyweregiardiacyst(7.7%),Entamoeba histolytica/Entamoebadispar(5.5%),andTaeniaeggs(5%).Infectionratewasloweramongfemalethenmalechildrenandsignificantly(P<0.05)higherprevalenceofhookwormwasobservedintheagegroupof6–10[Table2].Diarrheaaccompaniedwithdehydration,weakness, fever, and bloatingwas themost prominentsymptomsatpresentation(66%),andothersymptomsincludeddiarrhea alone (15%), loss ofweight and appetite (8%),abdominalpain(7%),dysentery(3%),andconstipation(1%).CBCresultsshowedpackedcellvolumevalueshighin5%of children’s due to dehydration, 75%of affected childrenwereanemicandalltheaffectedchildrenhadhigheosinophilcount[Table4].

Coproculture positivity was observed in 4/12 (33.3%),12/12(100%),and8/12(66%) inHarada–Morifilterpapermethod,APC,andMAPC,respectively[Table3].Filariformlarvae ofN. americanus varied from 500 to 620µm,whileA. duodenale varied from 690 to 750µm. Stoll’sdilutioneggcountshowedlight(100–500)in2/12(16.7%),moderate (eggs = 600–1000) in 8/12 (66.6%), and heavyinfections(>1000)in2/12(16.7%)children.

Singh, et al.: Culture techniques for hookworm species identification

Journal of Natural Science, Biology and Medicine ¦ Volume 9 ¦ Issue 2 ¦ July-December 2018 129

dIscussIon And conclusIon

Microscopic examination of direct stool samplemountshowed6.6%ofthechildrenhadhookwormeggs[Figure1].Thisfindingisconsistentwithotherstudies;however,afewstudieshavereportedhigherincidencerate(39.1%).[15‑18]Thesepreviousstudieswerebasedonsinglefecalexamination,which

Table 2: Age‑ and sex‑wise distribution in the study population

Age group Total male (%)

Positive male (%)

Total female (%)

Positive female (%)

1‑5 12(6.6) 1(0.5) 4(2.3) ‑6‑10 61(33.8) 5(2.7) 63(35) 3(1.7)11‑15 24(13.4) 2(1.2) 16(8.9) 1(0.5)Total 97(53.8) 8(4.5) 83(46.2) 4(2.3)

Table 4: Results of complete blood count for the children with hookworm infection

Parameters Normal range Males (%) Females (%)Hemoglobin(g/dl) >11 1(8.3) 2(16.6)

<11 7(58.4) 2(16.6)Hct(%) <36 3(25) 0

<54 5(41.6) 4(33.4)MCH(pg) <27 7(58.3) 3(25)

>32 1(8.3) 1(8.3)MCHC(g/dl) <31 6(50) 4(33.3)

>35 2(16.6) 0Eosinophilcount <0.04 0 0

>0.40 8(66.6) 4(33.3)MCH:Meancorpuscularhemoglobin,MCHC:Meancorpuscularhemoglobinconcentration,Hct:Hematocrit

Table 3: Comparisons of Harada‑Mori culture, Agar plate culture, and modified agar plate culture method

Characteristics HMC (M1)

APC (M2)

MAPC (M3)

Comments

Numberofpositive(n=12)(%)

4(33.3)

12(100)

8(66.6)

Pair‑wisecomparisonM1:M2P<0.05;M1:M3P<0.05;M2:M3P>0.05

Timetakentohatchlarva(days)

7‑10 2‑5 2‑3

HMC:Harada‑Moriculture,APC:Agarplateculture,MAPC:Modifiedagarplateculture

Table 1: Intestinal parasites among the children (n=58)

Types of parasites Total (%)Taeniaspecies 9(5)Ascaris lumbricoides 6(3.3)Entamoeba histolytica 10(5.5)Hymenolepis nana 2(1.2)Giardia intestinalis 14(7.7)Trichuris trichiura 3(1.7)Enterobius vermicularis 2(1.2)Hookworm 12(6.6)

possiblymayhavemissedthechancesoftheactualprevalencerate.Moreover,examinationbasedonmicroscopymayalsomissthehookwormeggsintheeventoflightinfection.TheinabilityofconventionalmicroscopytodifferentiatebetweenN.americanus,A.duodenale,andStrongyloidesisalsoamajorlimitation.Besidesmolecularmethods,coproculturemethodsarealternativemethodsforspeciesdifferentiation.InthishillyareaofNortheasternSikkim,India,ourstudyshowedthemajorprevalenceofN.americanusspecies[Figures2and3].Nomixedinfectionwasobservedamonghookwormspeciesinthisstudy.ComparedtotheHMCandMAPC,APCshowedsignificantlyhigherpositiverates.Intermsoftotaltimetakentohatchthefilariformlarva,APCandMAPCtookanalmostsimilartimeperiodofaround2–5days,insomecases,MAPCtook only 2 days.However, inHMC, it takes 7–10 days.In other similar previous studieswith 10.15%hookwormincidencerate,11.53%wereA.duodenaleand88.47%wereN.americanus. Incontrast, somestudieshave reported theequalprevalenceofA.duodenaleandN.americanus.However,allthesestudieswerebasedonHMCtechniquesonly.[12,16,19,20]Thedifferenceobservedmaybeduetogeographicalvariationsorprobablyexistenceofsubstrains.

Dependingon the status of host hemoglobin, a hookwormburden of 40–160 worms per individuals can lead tohemoglobinlevelof11g/dl.[21]Regardingtheintensityoftheinfection,majorityofthechildren(66.6%)inthisstudyhadmoderatefecaleggcount.Theprevalenceofanemiawashigherin females (46.8%) compare tomales (36.1%). Preschoolandschoolchildrenhavethegreatestphysiologicaldemandsforiron,andhenceareathigherriskofdevelopinganemia.Furthermore,theinfectionratewashigheramong6–10yearsoldmalechildren.Thismightbeduetomalechildrenplayingbarefootoncontaminatedsoil,closecontactwithhouseholdpets,andpoorhygiene.Thehigherintensityoffecaleggcountinthisstudycouldbeduetopoorenvironmentalsanitationandpoorhygienedespitedewormingamongthechildren.However,a largernumberofpopulationsamplingwillberequired tovalidatethisinthefuture.

Figure 1: Hookworm eggs in saline mount (×40)

Singh, et al.: Culture techniques for hookworm species identification

Journal of Natural Science, Biology and Medicine ¦ Volume 9 ¦ Issue 2 ¦ July-December 2018130

The reducedmean corpuscular hemoglobin and meancorpuscular hemoglobin concentration and increasedeosinophilcountobservedinthisstudyisareflectionofparasiticinfection.Thehookwormspeciesdiffer insusceptibility toanthelminthicdosage regimen[7,22] andhavediffering routeof infection (N.americanus infection ismainly by skinpenetrationwhileAncylostomaspp.infectionsarecommondue to ingestion of infective third‑stage larvae). Hence,speciesidentificationisparamountindesigningappropriateand effective prevention and control strategies.Anemia inchildren is aworldwide public health problem, inwhichhookworminfectionisoneofthemaincauses.A.duodenalecausesmorebloodlosscomparedtoN.americanus. IthasbeenestimatedthatasingleA.duodenaleandN.americanusworm ingests about 150µl and 30µl of blood per day,respectively.Hence,Hbconcentrationfallsastheintensityofinfectionrises.[23]Inthisstudy,9/12ofthechildrenwiththeHookworminfectionhadlowHblevel(<11g/dl),whichmayprogressivelydecreaseasthehookwormingestsblood.However,didnotobserveanysignificancedifferencebetweenthebloodcountofchildreninfectedwithN.americanusandA.duodenaleandwillprobablyneed largersamplesize tounderstandthisassociation.

Compare tomolecularmethods the cultural techniques areeasyandlessexpensive,althoughtime‑consumingbutareanidealalternativeina lessresource‑settingarealikeSikkim.Limitationofthisstudywastheuseofthesinglefecalsamplewhichmightlosethechancesofhookworminfectionsfromthesamepatients.Coprocultureisalsotime‑consumingandneedswellexperiencedstafftodifferentiatethespecies.Nevertheless,ordinaryAPCyieldedbetterresultsfortheisolationoflarvafromtheeggsinthestoolandisalsoeasiertoperforminanylaboratorywithminimalresources.

Financial support and sponsorshipNil.

Conflicts of interestTherearenoconflictsofinterest.

references1. Gezahegn A, Menkir S. Hookworm Infection and Hemoglobin

Concentration among Students in Three Selected Primary Schools,Aboboworeda,GambellaRegion,WesternEthiopia;2012.

2. HotezPJ.Oneworldhealth:Neglectedtropicaldiseasesinaflatworld.PLoSNeglTropDis2009;3:e405.

3. deSilvaNR,BrookerS,HotezPJ,MontresorA,EngelsD,SavioliL,et al.Soil‑transmittedHelminthinfections:Updatingtheglobalpicture.TrendsParasitol2003;19:547‑51.

4. SastryAS,BhatSK, editors.Small intestinalnematodes. In:MedicalParasitology. 1st ed. Pondicherry, India: Jaypee Brothers MedicalPublisher(P)Ltd.;2014.

5. World Health Organization. Control of Schistosomaisis andSoil‑Transmitted Helminths Infections. Report by The Secretariat,Executive Board. 107th Session, Provisional Agenda Item 3.3 (EB107/31).Geneva:WorldHealthOrganization;2001.

6. World Health Organization. Guidelines for the Evaluation of SoilTransmitted Helminthiasis and Schistosomiasis at the CommunityLevel.SchistosomiasisanIntestinalParasiteUnit.DivisionofControlTropical Diseases. (WHO/CTD/SIP/98.1). Geneva: World HealthOrganization;1998.

7. Piekarski G. Medical Parasitology. 2th ed. Berlin, New York:Springer‑VerlagPress;1989.

8. Bundy DA, Cooper ES, Thompson DE, Didier JM, Simmons I.Effect of age and initial infection intensity on the rate ofreinfection with Trichuris trichiura after treatment. Parasitology1988;97(Pt3):469‑76.

9. BethonyJ,ChenJ,LinS,XiaoS,ZhanB,LiS,et al.Emergingpatternsofhookworminfection:InfluenceofagingontheintensityofNecatorinfection inHainanprovince,People’sRepublicofChina.ClinInfectDis2002;35:1336‑44.

10. Parija SC. Textbook of Medical Parasitology: Protozoology andHelminthology.4thed.NewDelhi:AllIndiaPublishersandDistributors;2013.

11. GarciaLS.DiagnosticMedicalParasitology.4th ed.Washington,DC:ASMPress;2001.

12. KhannaV,TilakK,PrakashPY,MukhopadhyayC.ModifiedagarplateculturemethodforcultureofStrongyloides stercoralis.TropParasitol2015;5:136‑8.

13. dosSantosNetoJG.MovementoftherhabditiformlarvaofStrongyloides stercoralis.Lancet1993;342:1310.

14. JongwutiwesS,CharoenkornM,SitthichareonchaiP,AkaraborvornP,PutaporntipC. Increasedsensitivityof routine laboratorydetectionofStrongyloides stercoralisandhookwormbyagar‑plateculture.TransRSocTropMedHyg1999;93:398‑400.

15. Ngui R, Ching LS, Kai TT, Roslan MA, Lim YA. Molecularidentification of human hookworm infections in economically

Figure 3: Filariform larvae, L3 in iodine stainFigure 2: Filariform larvae L3 in agar plate culture (×40)

Singh, et al.: Culture techniques for hookworm species identification

Journal of Natural Science, Biology and Medicine ¦ Volume 9 ¦ Issue 2 ¦ July-December 2018 131

disadvantaged communities in peninsularMalaysia.Am J TropMedHyg2012;86:837‑42.

16. Shahid SB, Wazib A, Chowdhury A. Identification of hookwormin stool by Harada Mori Culture. Bangladesh J Med Microbiol2010;4:3‑14.

17. HumphriesD,SimmsBT,DaveyD,OtchereJ,QuagraineJ,TerryahS,et al. Hookworm infection among school age children in KintampoNorth municipality, Ghana: Nutritional risk factors and response toalbendazoletreatment.AmJTropMedHyg2013;89:540‑8.

18. ChidambaramM, Parija SC, Toi PC, Mandal J, Sankaramoorthy D,GeorgeS,et al.Evaluationof theutility of conventional polymerasechain reaction for detection and species differentiation in humanhookworminfections.TropParasitol2017;7:111‑6.

19. Parija SC, Malini G, Rao RS. Prevalence of hookworm species inPondicherry,India.TropGeogrMed1992;44:378‑80.

20. Adenusi AA, Ogunyomi EO. Relative prevalence of the humanhookworm species,Necator americanus andAncylostoma duodenalein an urban community in Ogun State, Nigeria. Afr J Biotechnol2003;2:470‑3.

21. Lwambo NJ, Bundy DA,Medley GF.A new approach to morbidityriskassessmentinhookwormendemiccommunities.EpidemiolInfect1992;108:469‑81.

22. Haburchak DR. Hookworm Diseases Treatment and Management.Medscape;2016.

23. JanMS, LiuD, editors. Necator. In:MolecularDetection ofHumanParasiticPathogens.USA:CRCPress;2012.p.613.