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~CaseReport
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CASEREPORT
Year:2011|Volume:29|Issue:4|Page:434437

SeverepigmentedkeratitiscausedbyCladorrhinumbulbillosum
DUGajjar1,AKPal2,JMSantos3,BKGhodadra4,ARVasavada4
1Scientist,IladeviCataractandIOLResearchCentre,Ahmedabad,India
2SeniorResearchFellow,IladeviCataractandIOLResearchCentre,
Ahmedabad,India
3ResearchFellow,UnidadedeParasitologiaMolecular,Institutode
MedicinaMolecular,Av.Prof.EgasMoniz,1649028Lisboa,Portugal
4CorneaConsultant,IladeviCataractandIOLResearchCentre,
Ahmedabad,India
Dateof
Submission
Dateof
Acceptance
DateofWeb
Publication

19Aug
2011
26Sep
2011
24Nov
2011

CorrespondenceAddress:
DUGajjar
Scientist,IladeviCataractandIOLResearchCentre,Ahmedabad
India
SourceofSupport:None,ConflictofInterest:None

DOI:10.4103/02550857.90191

~Abstract

Wereportacaseofseverepigmentedkeratitiswithpoorprognosis,caused
byCladorrhinumbulbillosum.Antifungaltreatmentwithtopicalnatamycin
andfluconazoleeyedropsandoraltabletfluconazolefailedtohealtheulcer

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andresultedinperforation.Thecausativefungus,C.bulbillosum,was
identifiedonthebasisofitstypicalmicroscopicfeaturesand98%sequence
homologytoextypeisolateCBS304.90(accessionno.FM955448).The
resultsofaninvitroantifungalsusceptibilitytestindicatedthattheisolate
wassusceptibletonatamycin,amphotericinB,fluconazoleanditraconazole.
Thepresentcaseisthethirdcaseofkeratitisandthesecondcaseofhuman
keratitis.Compromisedimmunityduetolivercirrhosiscouldleadtoafailed
prognosisevenwhenthefungalisolateishighlysusceptibletoantifungal
treatment.

Keywords:Cladorrhinumbulbillosum,pigmentedkeratitis,livercirrhosis
Howtocitethisarticle:
GajjarDU,PalAK,SantosJM,GhodadraBK,VasavadaAR.Severe
pigmentedkeratitiscausedbyCladorrhinumbulbillosum.IndianJMed
Microbiol201129:4347
HowtocitethisURL:
GajjarDU,PalAK,SantosJM,GhodadraBK,VasavadaAR.Severe
pigmentedkeratitiscausedbyCladorrhinumbulbillosum.IndianJMed
Microbiol[serialonline]2011[cited2016Dec8]29:4347.Available
from:http://www.ijmm.org/text.asp?2011/29/4/434/90191

~Introduction

Pigmenteddematiaceousfungiarethethirdmostcommoncauseofmycotic
keratitis.[1],[2]Curvulariaspp.arethemostcommonfungalspeciesisolated
frompigmentedkeratitisandotherspecieslikeBipolaris,Exserohilum,
Cladosporium,Lasiodiplodia,Alternaria,Torula,Aureobasidium,
NigrosporaandEpicoccumhavealsobeenreported.[2],[3]Here,wereporta
typicalseverecaseofdematiaceousfungalkeratitiscausedbyCladorrhinum
bulbillosum.
~CaseReport

A42yearoldmalefarmerpresentedintheoutpatientdepartmentofthe
ophthalmologyclinicwithahistoryofseverepainfultearing,rednessand
dimnessofvisionintherighteyesince10days.Althoughthepatientwasa
farmer,hedidnotrecallanyincidentofintrusionofaforeignbodyinthe
eyeoranyoculartrauma.Thepatientwasonmedicationforlivercirrhosis
sincethelast6monthsandwasalcoholicsincethepast20years.Thedetails
ofthemedicationusedforlivercirrhosiswerenotavailable.
Onexamination,therighteyeshowedperceptionoflightintheupperand
nasalquadrantsonly.Thelidsshowedhighoedemaandtheconjunctiva
showedhighcircumciliarycongestion.Aphotoslitlampexamination
revealedthattheulcerwascoveringtheentirecorneaexceptthelimbus
[Figure1]a.Thesizeoftheulcerwasaround89mmverticallyand9mm
horizontallywithirregularmargins.Theupperhalfofthelesionsshoweda
reddishbrownpigmentwithcompletestromalinfiltrationreachinguptothe
endothelium.Thickandcheesyhypopyonwasalsopresentatadepthof

about2mm.Thedetailsoftheanteriorchamberwerenotvisibleasthe
cornealabscesswascoveringtheentirecornea.Tensionwasobservedtobe
digitallyhigh.Thelefteyewasnormal.
Figure1:(a)Slitlampphotographshowing
apigmentedplaquelikecornealulcer
coveringtheentirecornea(89mm
verticallyand9mmhorizontally)with
irregularmargins.(b)10%KOHwet
mountofthescrappingmaterialshowing
severalseptatefungalhyphae,
magnification400.(c)Growthofcase
isolateCladorrhinumbulbillosum(ICIRC
CC93)onriceagarshowingaflat,
spreadingcolonywithdarkbrown,sparse,
aerialmyceliumafter10daysofgrowth.
(d)Severalmicrosclerotiatakeninnormal
saline,teasedandseenatalower
magnification,100.(e)Microsclerotia
(measuringupto160m)seenathigher
magnificationshowingcompact
aggregationsofhyphaecomposedof
swollen,pigmentedcellsstainedwith
lactophenolcottonblue,magnification
400.(f)Flaskshapedconidiogenouscells
measuringupto11.57m,magnification
1000
Clickheretoview

Cornealscrapingsweretakenfromtherighteyeandthemediawere
inoculatedforbacterialculture,fungalculture,KOHmountandGram
staining.The10%KOHmountshowedplentyofpigmentedfungal
filamentsthatwerehighlyseptatewithirregularmargins[Figure1]b.Gram
stainingshowedprominentseptate,branchedfungalhyphaeandabsenceof
bacteria.Aprovisionaldiagnosisoffungalulcerwasmadeandantifungal
treatmentwasinitiatedwithtopicalnatamycineyedrops(Natamet5%
suspension,SunPharmaceuticalsInd.Ltd,Halol,India),vigamoxeyedrops
(Moxifloxacin0.5%suspension,AlconLaboratoriesInc.,Texas,USA),
fluconazoleeyedrops(Zocon0.3%suspension,FDCLtd,Aurangabad,
India)hourly,andatropineeyedrops(Atropine,0.1%suspension,Taj
PharmaLtd,Mumbai,India)threetimesaday.Thepatientwasalso
prescribedtabletIopar(250mgtoreduceIOP)andanantifungaltablet
fluconazole(150mg)onceaday.
Thenutrientagarplateandbloodagarplateinoculatedwithscraping
materialshowedgrowthofseveralcoloniesofasinglefunguswithin2days.
Thefollowingweek,thepatientwasrelativelycomfortablebutnochange
wasnotedinthesizeoftheinfiltrate.Thetherapywascontinued.
Unfortunately,therewasnofurtherimprovementandhiscompliance
continuedtobepoorforanother2months.Ateachpresentation,hewas
placedonatopicalandsystemicantifungalregime.Theverypoorprognosis
andtheneedfortherapeutickeratoplastywereexplainedtothepatient.
However,thepatientdeclinedkeratoplasty.After2.5months,thepatient
complainedofseverepain.Anexaminationrevealedcornealperforationand
aflatanteriorchamber.Thepatientwasreferredtoagovernmenthospital
duetofinancialreasonsandsincethenlosttofollowup.
Mycologicalstudies

Thecaseisolate(ICIRCCC93)wassubculturedonSabouraud'sDextrose
Agar(SDA)(HimediaLabsLtd,Mumbai,India)andriceagar(preparedin
thehouse)andinoculatedat25Cand37C.Thefluffy,purefungalgrowth
wasbrowninthemiddleandwhiteattheperiphery.Colonieswerefast
growingreachingadiameterof6880mmwithin4daysat25Candat7
daystheyreachedtheedgeofthePetridishMoreDetails.After10days,they
turneddarkbrown[Figure1]c.ThepurefungusfromtheSDAplatewas
mountedwithlactophenolcottonbluestain.Septatefilamentswere
observed,andinafewfields,microsclerotiawerealsoobserved.Thefungus
wasidentifiedasCladorrhinumspp.byitsmorphologicalandmicroscopic
features.Amicroscopicpictureofseveralmicrosclerotiaisshownin[Figure
1]d.Theaveragelengthofasinglemicrosclerotiumis160mm.
Microsclerotiaseenatahighermagnificationshowedcompactaggregations
ofhyphaecomposedofswollen,pigmentedcells[Figure1]e.Rarely,flask
shaped,lateralconidiogenouscellsmeasuringupto11.57mmwerealso
seen[Figure1]f.
Foridentificationofthefungus,sequencingoftheITS(internaltranscribed
spacer)regionwasdone.TheDNAwasextractedfromthefungususing
cetyltrimethylammoniumbromide(CTAB)buffer.SequencingoftheITS
regionwasdoneatFirstBaseLaboratoriesSdn.Bhd,Malaysia,using
primersITS1(F5'TCCGTAGGTGAACC3')andITS4(R
5'TCCTCCGCTTATTGATATGA3'),whichamplifythefollowinggenesof
thefungalgenome:Partial18SrRNAgene,completeITS1,5.8SrRNAgene
andITS2regions,andpartial28SrRNAgene.SequencingoftheLSU
region,usingtheMicroSeqD2LSUrDNAfungalidentificationkit
(AppliedBiosystems,CA,USA),wasdoneatthesequencingfacilityof
GujaratStateBiotechnologyMission(GSBTM),GovernmentofGujarat,
India.TheITSandLSUsequencesweresubmittedtotheNCBIdatabase.
TheITSsequenceofthecaseisolate(ICIRCCC93)showed98%sequence
similaritytoC.bulbillosumCBS304.90(accessionno.FM955448)witha
BLASTsearchexpectvalueofzero.The98%matchindicatedthatthe
isolatewasrepresentativeofC.bulbillosum.TheD2LSUsequenceshowed
amaximumsimilarityscoreof98%withCercophoracostaricensis
(accessionno.AY780059)withanexpectvalueof5E12.ABayesian
phylogenetictreewasbuiltusingMRBAYES(version3.0).Theadjoining
ITStreeclearlyclustersthepresentcaseisolatewiththeextypeisolateof
C.bulbillosum(CBS304.90)[Figure2].
Figure2:Phylogenetictreeinferredin
MrBayesv.3.0resultingfromaBayesian
analysisof565charactersoftheITS
nrDNAregion(includingcodedindels).
Thenewlygeneratedsequenceislistedby
itsisolatenumber(ICIRCCC93).Extype
cultureofCladorrhinumbulbillosumisin
bold.Thecaseisolate(ICIRCCC93)
clusterswithCladorrhinumbulbillosum
CBS304.90(accessionno.FM955448).
Bayesianposteriorprobabilitiesareshown
atthenodes.Thetreewasrootedtotwo
isolatesofChaetomiumglobosum
(DQ336707andHQ224669).Thescalebar
represents0.1substitutionspersite

Clickheretoview

Invitroantifungalsusceptibilitytesting
Invitroantifungalsusceptibilitytestingwasdoneagainstnatamycin
(Natamet5%suspensionSunPharmaceuticalsInd.Ltd,Halol,India),
itraconazole(Itral1%suspensionJawaPharmaceuticals,Gurgaon,India),
fluconazole(Nuflucon0.3%suspensionNuLifePharmaceuticals,Pune,
India),andamphotericinB(RM462,HimediaLabsLtd,Mumbai,India)
usingthemicrodilutionmethodandbyfollowingCLSIguidelines.All
antifungalagentsweredissolvedinDMSOandamphotericinBwas
dissolvedinwater.Theinoculumswerepreparedbycoveringthe7dayold
cultureplatewith0.85%normalsaline.Thiswasfollowedbygentleprobing
ofthecolonieswiththehelpofapipetteandadjustingthedensitiesofthe
suspension(readat530nm)toafinalinoculumof0.5McFarlandstandard.
Thefinaldrugconcentrationrangeswere0.008to32mg/mlforallthefour
antifungalagents.AlltheantifungalagentsweretestedinRPMI1640media
with2%glucoseandwithoutsodiumcarbonate.Theresultsoftheinvitro
antifungalsusceptibilitytestindicatedthattheMICwas0.064mg/mlfor
natamycinandamphotericinB,0.016mg/mlforfluconazoleand<0.016
mg/mlforitraconazole.
~Discussion

Here,wereportaseverecaseofpigmentedfungalkeratitiscausedbyan
endophyticfungiC.bulbillosum.
Cladorrhinumspp.arefoundmainlyondungandplantmaterialinthesoil
assaprobes.[4]C.bulbillosumbelongstotheLasiosphaeriaceaefamilyof
theorderSordariales.Therearealmost29generafoundinthisfamily
includingCladorrhinum.Outofthose29genera,only2arefoundtobe
pathogenictohuman,namelyCladorrhinumandArnium.[5],[6],[7]Inthe
genusCladorrhinum,C.bulbillosumistheonlyreportedpathogenicspecies
causingkeratitiswithonlytworeportedcasessofar.Thefirstcaseof
keratomycosisbyCladorrhinumspp.wasreportedina12yearold
Argentineanboywhowasworkingwithhorsesin1979(strainCBS604.75)
[7]andthesecondcasewasreportedinaPercheroncrosshorsein1997.[5]
TheearliercasewaslaterdescribedasthenewspeciesC.bulbillosumby
MouchaccaandGams.[8]Cladorrhinumspeciesweremainlydistinguished
onthebasisoftheirabilitytoproducemicrosclerotia,theconidialshape,
andmoreimportantlythetuftedaggregationoftheconidiophores.Ourcase
isolateofC.bulbillosum(ICIRCCC93)hadallthemorphological
characteristicstypicalofthisspeciesandalsoclustersalongwiththeextype
isolateofC.bulbillosumintheadjoiningphylogenetictree.The
evolutionaryrelationshipbetweenCladorrhinumspeciesandother
ascomycetesisnotveryclear.Inarecentpaperdescribingtwonewspecies
ofCladorrhinum,theauthorsveryefficientlydiscussthephylogenetic
placementofthisgenus.[4]
Sofar,noneoftheotherspeciesofCladorrhinumareassociatedwithany
humanoranimaldisease.Boththeabovementionedcasesweresuccessfully
treatedusingmiconazoleantifungalagentalthoughatherapeutic
keratoplastywasperformedonthehorse.Thepresentcaseshowedpoor
prognosis,inspiteofgoodinvitroantifungalactivityofallthefourtested
agents.Ourpatientwasonmedicationforlivercirrhosisandsothepoor
prognosismaybeattributedtothepatient'simmunocompromisedcondition.
Bothlivercirrhosisandalcoholismarewellestablishedreasonsfor
compromisedimmunity.Patientswithliverfailureareatriskofincurring

bacterialandfungalinfection.Endogenousendophthalmitisafterliver
transplantationandocularsurfacedamageinpatientswithchronichepatitis
hasbeenreported.[9]Further,thefailureofthetreatmentcanalsobe
attributedtothefactthatthepatientmaynothavestrictlyfollowedthe
prescribeddrugregime.Previously,inacaseseriesonamnioticmembrane
transplants,incasesofrecurrentandperforatedcornealulcers,threecasesof
keratitiswithlivercirrhosiswereshowntobeperforatedduringtreatment.
[10]However,sincethisisthesecondreportedcaseofC.bulbillosum
keratitis,allpossibilitiesremainhypothetical.
Insummary,thiscaseillustratesthevulnerabilityofthecorneatoinfrequent
organismsandfurtherhighlightstheimportanceofthepatient'simmune
systemparalleltothetreatmentregimeincombatinginfection.
~NucleotideSequenceAccessionNumbers

TheGenBankaccessionnumbersforthecaseisolateCladorrhinum
bulbillosum(ICIRCCC93)areJF758870(ITS)andJN107807(D2LSU).
~Acknowledgments

DGthanksthefinancialsupportfromtheWOSAgrant(No.SR/WOS
A/LS1202008)ofDepartmentofScienceandTechnology,India.The
authorsthankDr.HugoMadrid,UnitatdeMicrobiologia,Facultatde
MedicinaiCinciesdelaSalut,IISPV,UniversitatRoviraiVirgili,C/Sant
Llorenc21,43201Reus,Tarragona,Spainforhishelpinidentificationof
theisolate.

~References

1. TanureMA,CohenEJ,SudeshS,RapuanoCJ,LaibsonPR.Spectrumof
fungalkeratitisatWillsEyeHospital,Philadelphia,Pennsylvania.
Cornea200019:30712.
[PUBMED][FULLTEXT]
2. GopinathanU,SharmaS,GargP,RaoGN.Reviewofepidemiological
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keratitis:Experienceofoveradecade.IndianJOphthalmol
200957:2739.
[PUBMED]

3. GargP,VemugantiGK,ChatarjeeS,GopinathanU,RaoGN.Pigmented
plaquepresentationofdematiaceousfungalkeratitis:Aclinicopathologic
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4. MadridH,CanoJ,GeneJ,GuarroJ.TwonewspeciesofCladorrhinum.
Mycologia2011103:795805.

5. ChopinJB,SiglerL,ConnoleMD,O'BoyleDA,MackayB,Goldstein
L.KeratomycosisinaPercheroncrosshorsecausedbyCladorrhinum
bulbillosum.JMedVetMycol199735:535.
[PUBMED]
6. RestrepoA,McGinnisMR,MallochD,PorrasA,GiraldoN,VillegasA,
etal.FungalendocarditiscausedbyArniumleporinumfollowing
cardiacsurgery.Sabouraudia198422:22534.
[PUBMED]

7. ZapaterRC,ScattiniF.MycotickeratitisbyCladorrhinum.Sabouraudia
197917:659.
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8. MouchaccaJandGamsW.ThehyphomycetegenusCladorrhinumand
itsteleomorphconnections.Mycotaxon199348:41540.

9. ToshikuniN,UjikeK,YanagawaT,SugaT,ShimizuT,KusudaY,etal.
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lithotripsyinapatientwithlivercirrhosis.InternMed200645:1327
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[PUBMED][FULLTEXT]
10. ChenHC,TanHY,HsiaoCH,HuangSC,LinKK,MaDH.Amniotic
membranetransplantationforpersistentcornealulcersandperforations
inacutefungalkeratitis.Cornea200625:56472.
[PUBMED][FULLTEXT]
Figures
[Figure1],[Figure2]
Thisarticlehasbeencitedby
1

Filamentousfungalinfectionsofthecornea:aglobaloverviewof
epidemiologyanddrugsensitivity
LszlKredics,VenkatapathyNarendran,CoimbatoreSubramanian
Shobana,CsabaVgvlgyi,PalanisamyManikandan
Mycoses.201558(4):243
[Pubmed]|[DOI]

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