Direct identification from positive blood broth culture by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS)

brazjinfectdis2017;21(3):339–342

w w w . e l s e v i e r . c o m / l o c a t e / b j i d

The

Brazilian

Journal

of

INFECTIOUS

DISEASES

Brief

communication

Direct

identification

from

positive

blood

broth

culture

by

matrix-assisted

laser

desorption-ionization

time-of-flight

mass

spectrometry

(MALDI-TOF

MS)

Maria

Goreth

Barberino,

Marcio

de

Oliveira

Silva

,

Ana

Carolina

Palmeiras

Arraes,

Luís

Cláudio

Correia,

Ana

Verena

Mendes

HospitalSãoRafael,Salvador,BA,Brazil

a

r

t

i

c

l

e

i

n

f

o

Received23June2016 Accepted14March2017 Availableonline8April2017

Keywords:

MALDI-TOF Bloodculture

Bacterialidentification

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b

s

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t

Bloodstreaminfections(BSIs)areamongthemostconcerningbacterialinfections.Theyare oneoftheleadingcausesofmorbidityandmortality,andoccurin30–70%ofcriticalcare patients.Thepromptidentificationofthecausativemicroorganismcanhelpchoosingthe appropriateantimicrobialtherapythatwillleadtobetterclinicaloutcomes.Bloodculture isoneofthemostrelevanttestsformicrobiologicaldiagnosisofbacterialinfections.The introductionoftheMALDI-TOFmicrobiologicaldiagnosissignificantlydecreasedthetime ofidentifyingmicroorganisms.However,itdependsonthegrowthonsolidculturemedium. Inthisstudy,538bottlesofpositivebloodcultureswereevaluatedtotesttheaccuracyof aninhousemodifiedprotocol.Thestudysampleconsistedof198Gram-negativeand350 Gram-positivebacteria.Inall,460(83.94%)specieswereidentifiedbasedonthedirectplate findings.Theprotocolallowedtheidentificationof185/198(93.43%)oftheGram-negative bacteria,includingaerobes,anaerobes,andnon-fermenters,and275/350(78.85%)ofthe Gram-positivebacteria.Theproposedmethodhasthepotentialtoprovideaccurateresults incomparisontothetraditionalmethodwiththepotentialtoreducetheturnaroundtime fortheresultsandoptimizeantimicrobialtherapyinBSI.

©2017SociedadeBrasileiradeInfectologia.PublishedbyElsevierEditoraLtda.Thisisan openaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/ by-nc-nd/4.0/).

Bloodstreaminfections(BSIs) areoneofthe leadingcauses ofadmittancetointensivecareunits,1_{afflictingupto2%of} inpatients.2_{Asmanyas19millioncasesarereportedyearly} worldwide,3 _{whichmakesBSIsoneoftheleadingcausesof}

∗ _{Correspondingauthor.}

E-mailaddress:oliveirasm@yahoo.com.br(M.O.Silva).

morbidity andmortality. Itoccurs in30–70%among inten-sivecarepatients,especiallywhenanantimicrobialeffective therapyisnotreadilyinstituted.4–6_{Sepsisiscausedbya} vari-ety ofmicroorganisms, and timely diagnosis isofextreme

http://dx.doi.org/10.1016/j.bjid.2017.03.007

1413-8670/©2017SociedadeBrasileiradeInfectologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

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importance due to the severity of the patient’s condi-tion,whichdemandspromptandappropriateantimicrobial therapy.7_{Inmostcases,immediateempiricaltreatmentis} ini-tiated,whichmustbetailoredtothemicroorganismrecovered fromblood culturesand onantibioticsusceptibilitytesting. Althoughtheintroductionofautomatedsystemsinthe rou-tine of microbiology laboratories has led to a decrease in turnaroundtime,thedelay intheidentification of microor-ganisms implicated in BSIs is still considerable, given the severityofthepatient’sstatus.Themicrobiologicaldiagnosis ofbacteremiaconsistsofperformingbloodtestpositivation after the sample is Gram stained. It is then followed by subcultivationofasolidmediumandtestingofantibiotic sus-ceptibilitythat takes 12–48h on average dependingon the microorganism.8_{Inviewofthedelayobservedbetween} sam-plecollection and availabilityofblood cultureresults, new methodsbased on molecular biology have been developed andincorporatedintolaboratoryroutines,buttheyare consid-eredlimitedandexpensive.9_{‘Matrix-assistedlaserdesorption} ionizationtime-of-flightmassspectrometry(MALDI-TOFMS)’ introducedinmicrobiologysince1995,isregardedasa revolu-tionarymethodinclinicalmicrobiology,becauseitidentifies microorganisms quickly and accurately, including bacteria andfungi.8,10_{ThemainuseofMALDI-TOFisinthe} identifi-cationofmicroorganismsgrowinginculturemedium,which takes 18–24h. This is a classical method diffused in most microbiology centers. In order to accelerate the identifica-tionofmicroorganisms,variousresearchprotocolsforyeast fungiandbacteriahavebeen developeddirectly from posi-tivebloodculturevialswithintwotofivehours,depending on the technique used. This study sought to improve the in house protocol through the experience of other stud-ies,developingamethodcapableofidentifyingbacteriaand yeast fungifrom positive blood cultures using mass spec-trometry (MALDI TOF), obtaining results in less than two hours. Thus, this protocol significantly reduces the time requiredforidentificationoftheetiologicagent,reducesthe costsofthetest,andoptimizestheearlyuseofappropriate antimicrobials.5–8

Atotalof538 positivebottlesMonitored byAutomating BacT/ALERT® 3D (bioMérieux, Marcy l’Etoile, France) were analyzedatSãoRafaelHospital’sMicrobiologyLaboratoryin Salvador, Bahia, Brazil, between May and November 2014. Identifyingmicroorganismsdirectlyfromapositiveblood cul-turebottlewascomparedtotheidentificationaftergrowth inculturemediumplatesusingtheMALDI-TOFmethod.The techniqueconsistedofadding1mLofthepositiveblood sam-pletoEppendorftubewith200␮Lof2%saponin,followedby vortexingforaperiodof30sandincubationfor5minatroom temperature.Thesamplewasthencentrifugedat14,500×g

(1min) and thesupernatant wasdiscarded. Thepelletwas homogenizedin 1mL ofsteriledistilled water, vortexed to completedissolution,andcentrifugedat14,500×gfor1min. Washingofthe pelletindistilled waterwasrepeated twice inaccordancewiththeadaptedprotocol.11Subsequently,the supernatant was removed and spots were made from the homogenized pellet. After air-dryingat room temperature, eachspotwasoverlaidwith0.5␮Lofformicacid(bioMérieux) and1␮Lofthematrixsolution(␣-cyano-4-hydroxycinnamic acid) (bioMérieux)was deposited onto the spots. Afterthe

spots were air-dried, the target slide was analyzed by the VITEK®MSsystem.

Finally, the results were compared with those obtained fromthegrowthofthemicroorganismintheculturemedium identifiedbyMALDI-TOF(VITEK®MS,bioMérieux).Theresults usingtheprotocolofidentificationdirectlyfromthepositive bloodculturebottleswerecomparedtothoseobtainedwith theidentificationofthesamemicroorganismisolatedfroma solidmediumaftersubcultivationofthepositivebottleusing MALDI-TOF.Qualitycontrol wasperformedacross readings usingthereferenceE.coliATCC8739strain.

Theagreementratewascalculatedbasedonthe propor-tionofidenticalresultsobtainedusingtheprotocolanddirect identificationofculturesforeachspecimen.Partialagreement wasconsideredwhentheprotocolidentifiedatleastoneofthe microorganismsdetectedbythedirectmethodoftheculture plate.Sensitivityandspecificitywerecalculatedby consider-ingthecultureasgold-standardfortruepositiveandnegative findingsforeachspecies.Theinter-methodagreementbeyond chancewasassessedbycalculatingFleiss’sKappaCoefficient. Theproportionofcorrectidentificationperspeciesandgroup (Gram-positiveandGram-negative)wasalsodetermined.The analyseswereperformedusingtheRprogramminglanguage andenvironment.

Duringthestudyperiod,538positivebloodcultureswere analyzedusingbothmicroorganismidentificationmethods. Thesamplewaspredominantly(90.9%)monomicrobial,with asmallerproportion(2.1%)ofpolymicrobialcultures.Only7% ofthebottles werenegativeandshowed nogrowthon the culturemedium.Thesewereconsideredfalsepositiveresults. Most(63.7%)ofthe460microorganismsidentified(83.9%of the total)were Gram-positive with all the sampleswith a scorevalue>90%.Outof88incongruoussamples,60(68.2%) were not detected and 28 (31.8%) were misidentified. The highestpercentageofcorrectlyidentifiedmicroorganisms,in terms ofgenera and species levels, was found among the Gram-negativebacteria,with186/199(93.5%)versus274/349 (78.5%)ofGram-positivebacteria(Table1).Ahigher sensitiv-ity(84.7%;95%CI:81.6%–87.8%)thanspecificity(77.5%;95%CI: 64.6%–90.4%)wasobserved,althoughtheconfidenceinterval ofthelatterwaslargerduetothesmallproportionofnegative culturesinthestudysample.Agreementanalysisshowedan overallagreementof81.4%(95%CI:78.1%–84.7%),withakappa of0.798(95%CI:0.762–0.834).Agreementincreasedto84.2% (95%CI:81.1%–87.3%).Partialidentificationswereobservedin 11 samples.Innineofthem,the cultureshowed one addi-tionalspecieswhereasintwosamplestheMALDI-TOFmethod identifiedoneadditionalspecies.

This study analyzed positive blood cultures of patients withbacteremiaincludingGram-positiveandGram-negative bacteria. It compared direct microorganism identification from positive bottles to growth on solid medium using MALDI-TOFMS.Thelatterreliesonthedetectionof riboso-malproteinsandisconsideredafastandaccuratemethodfor microbiologicaldiagnosis.Itisdescribedinagreatnumberof studiesasanimportanttooltoaidphysiciansinchoosingthe appropriateantimicrobialtherapy,therebyreducingpotential adverse events in patientswith BSI.1 _{Our protocol showed} agreementintheidentificationofthemicroorganismgrown on a solid medium. In relation to established protocols,

brazj infect dis.2017;21(3):339–342

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Table1–Proportionofcorrectidentificationperspeciesandgroup.

Species n Correctidentification Species n Correctidentification

Gram-negative Gram-positive A.baumannii 8 8 BGP-coryne 4 2 B.fragilis 2 2 E.faecium 8 5 C.indologenes 1 1 E.faecalis 27 26 C.koseri 2 2 S.hominis 37 32 E.cloacae 8 8 L.monocytogenes 1 1 E.coli 56 52 Leuconostoc 1 0 E.aerogenes 2 1 M.luteus 2 2 M.osloensis 1 1 R.mannitolytica 2 0 Kpneumoniae 74 72 S.agalactiae 2 2 E.meningoseptica 4 4 S.aureus 31 27 K.oxytoca 1 0 S.capitis 22 19 P.aeruginosa 9 8 S.epidermidis 98 64 P.mirabilis 9 9 S.haemolyticus 49 39 P.putida 3 3 S.hominis 37 32 P.vulgaris 3 3 S.infantarius 1 1 P.rettgeri 1 1 S.pneumoniae 4 4 S.marcescens 11 8 S.saprophyticus 5 5 S.maltophilia 1 1 S.pneumoniae 4 4 Salmonellaspp. 3 2 S.vestibularis 1 0 – – – S.warneri 8 5 – – – S.viridans 1 0 – – – Strep.constellatus 1 1 – – – S.mitis/oralis 3 3 Total(%) 199(100.00) 186(93.46) Total(%) 349(100.00) 274(78.51) Total 548*(100.00) 460(83.94)

Totalnumberofbacteria(548)isbiggerthanthetotalnumberofsampleduetopolymicrobialcultures.

similarorsuperioragreementwasobtained,regardlessofthe processingperiodssuchastheoneinastudyconductedby Martinyetal.,2012.11_{Usinganin-housemethod,theyfound} 73.7%ofcorrectidentificationsoverall,with86.4%correctly identified Gram-negative bacteria and 73.7% Gram-positive generaand species.Moussaouilet al.,2010,showedcorrect identification of 89.7% of the bottles12_{; Drancourt, 2010,} found94.0%forGram-negativeand67.0%forGram-positive bacteria13_{;andLaScola&Raoult,2009,obtained88.0%} cor-rectlyidentifiedGram-negativesand67.0%Gram-positives.8

Thepresent study yielded84% ofcorrectidentifications at the species level, with 93.5% being Gram-negative and 78.5%Gram-positive bacteria. Sensitivity(77.5%)and speci-ficity(84.7%)valueswereconsistentwiththosewidelyfound inmedicalliterature,andcanassistmicrobiologistsintheir decision-makingregardingtheidentificationmethodtouse. InspiteofthecontroversiesaroundKappameasurementof agreementclassifications,thestudyshowedakappaof0.798 –whichreflectssubstantialagreementaccordingtomostof theliterature.14,15

Our study showed reliable and accurate results when usingMALDI-TOFcomparedtoconventionalmethods.Ourin houseprotocolisconsideredtobeanefficientandsuccessful methodforroutineidentificationofbacterialisolatesin clin-icalmicrobiology;unlikeotherstandardizedprotocols,which are described aslengthy and asdifficultto bedeployed in routinemicrobiology.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

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