Evaluation of the Xpert MTB/RIF assay for the diagnosis of smear-negative pulmonary and extrapulmonary tuberculosis in Madagascar

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Evaluation of the Xpert MTB/RIF assay for the diagnosis of smear-negative pulmonary and extrapulmonary tuberculosis in Madagascar

R. Rakotoarivelo, J. Ambrosioni, V. Rasolofo, M. Raberahona, N.

Rakotosamimanana, R. Andrianasolo, R. Ramanampamonjy, M. Tiaray, J.

Razafimahefa, J. Rakotoson, et al.

To cite this version:

R. Rakotoarivelo, J. Ambrosioni, V. Rasolofo, M. Raberahona, N. Rakotosamimanana, et al.. Evalua-

tion of the Xpert MTB/RIF assay for the diagnosis of smear-negative pulmonary and extrapulmonary

tuberculosis in Madagascar. International Journal of Infectious Diseases, Elsevier, 2018, 69, pp.20 -

25. �10.1016/j.ijid.2018.01.017�. �pasteur-01911249�

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Evaluation of the Xpert MTB/RIF assay for the diagnosis of smear-negative pulmonary and extrapulmonary

tuberculosis in Madagascar

R. Rakotoarivelo

^a,b,1

, J. Ambrosioni

^c,d,1,

*, V. Rasolofo

^e,1

, M. Raberahona

^a

, N. Rakotosamimanana

^e

, R. Andrianasolo

^a

, R. Ramanampamonjy

^a

, M. Tiaray

^a

, J. Raza ﬁ mahefa

^a

, J. Rakotoson

^a

, M. Randria

^a,2

, F. Bonnet

^f,2

, A. Calmy

^c,2

, the MadaXpert Study Group

³

aJosephRasetaBefelatananaUniversityHospital,Antananarivo,Madagascar

bTambohobeUniversityHospital,Fianarantsoa,Madagascar

cUniversityHospitalsofGeneva,Geneva,Switzerland

dHospitalClinic-IDIBAPS,Barcelona,Spain

eInstitutPasteurdeMadagascar,Antananarivo,Madagascar

fUniversityHospitalofBordeaux,Bordeaux,France

ARTICLE INFO

Articlehistory:

Received18October2017

Receivedinrevisedform25December2017 Accepted21January2018

CorrespondingEditor:EskildPetersen,Aar- hus,Denmark

Keywords:

Extrapulmonarytuberculosis

Smear-negativepulmonarytuberculosis HIV

Moleculardiagnostictest Lowresourcecountries XpertMTB/RIF

ABSTRACT

Objective:Toevaluatethefeasibilityoftheimplementationofacommercialrapidmoleculardiagnostic test(XpertMTB/RIF)fortheroutinediagnosisofsmear-negativeorextrapulmonarytuberculosis(TB)and itsdiagnosticaccuracy,andtoassessHIVprevalenceinareal-lifesettinginMadagascar.Thisstudywas setinatertiarycarehospitalinMadagascar.

Methods:Aprospectivecohortstudywasconductedofallconsecutivecaseswithsuspectedsmear- negativeand/orextrapulmonaryTBovera2-yearperiod.Caseswereclassiﬁedasproven,probable,or possibleTBcases,orashavinganalternativediagnosis.

Results:Ofthe363patientsincluded,183(50.4%)hadsuspectedsmear-negativepulmonaryTBand180 (49.6%)hadsuspectedextrapulmonaryTB.Forprovencases,thesensitivity,speciﬁcity,positiveand negativepredictivevaluesofXpertMTB/RIFwere82.4%,98.8%,98.3%,and86.6%,respectively;forproven andprobablecasesgroupedtogether,thesevalueswere65%,98.8%,98.5%,and64%,respectively.The diagnosticaccuracywasslightlylowerforextrapulmonaryTBcomparedtosmear-negativepulmonary TB.TheprevalenceofHIVinfectionwas12.1%,butalmosthalfofthesecasesdidnothaveTB(alternative diagnosisgroup).

Conclusions:TheimplementationofarapiddiagnosisprogrammeforTBinaresource-poorsettingis feasible.TheperformanceoftheXpert-MTB/RIFwasremarkableinthisdifﬁcult-to-diagnosepopulation.

HIVprevalenceinthisstudywasmuchhigherthantheprevalencereportedinthegeneralpopulationin Madagascar,inpatientswithTBandpatientswithconditionsotherthanTB.

ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by- nc-nd/4.0/).

1.Introduction

Tuberculosis(TB)isamajorpublichealththreatinMadagascar (Rakotonirinaetal.,2016),withanestimatedprevalenceof442 per 100 000 inhabitants(95% conﬁdence interval(CI) 22–735) andanestimatedincidenceof234per100000inhabitants(95%

CI 193–280), which are among the highest rates worldwide (WorldHealthOrganization,2014a).Approximatelyone-thirdof all TBcases are extrapulmonary andanother third are smear- negative.AntananarivoisthemostpopulouscityinMadagascar

*Corresponding author. Infectious DiseasesService, HospitalClinic-IDIBAPS, Villarroel170,Barcelona08032,Spain.Tel.:+34640236670.

E-mailaddress:jambrosioni@intramed.net(J. Ambrosioni).

1R.Rakotoarivelo,J.Ambrosioni,andV.Rasolofocontributedequally.

2M.Randria,F.Bonnet,andA.Calmycontributedequally.

3MadaXpertStudyGroupmembersare:JuanAmbrosioni,RadoAndrianasolo, Fabrice Bonnet, Alexandra Calmy, Judit Peñaﬁel, Mihaja Raberahona, Rivo Rakotoarivelo,AndrianantenainaRakotoson,JoelsonRakotoson,NiainaRakotosa- mimanana,RadoRamanampamonjy,MamyRandria,Voahangy Rasolofo,Julien Razaﬁmahefa,andMichelTiaray.

https://doi.org/10.1016/j.ijid.2018.01.017

1201-9712/©2018TheAuthor(s).PublishedbyElsevierLtdonbehalfofInternationalSocietyforInfectiousDiseases.ThisisanopenaccessarticleundertheCCBY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/4.0/).

ContentslistsavailableatScienceDirect

International Journal of Infectious Diseases

j o u r n a lh o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / i j i d

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andTBis alsoa majorhealthconcernhere(Rakotosamimanana etal.,2014).

The diagnosis of smear-negative and extrapulmonary TB remainschallenginginresource-limitedsettings.InMadagascar, therearealsodifficultiesrelatedtothestartof anti-TBtherapy, whichisgivenasaprioritytocaseswithprovenpulmonaryTB (smear-positivesputum)duetoepidemiologicalissuesinorderto decrease transmission. However, the confirmation of smear- negative and extrapulmonary TB cases requires a positive Mycobacterium tuberculosis culture, which may take up to 8 weeks. Patientswith themost serious clinicalpresentations of extrapulmonaryTB(suchasthosewithcentralnervoussystemor digestivesystem involvement) arefrequently tooill towait for diagnostic confirmation. In this context, rapid tests for the diagnosisof TB havean impactonclinicaloutcomes.Although recentdataarelacking,multidrugresistanceisconsideredlowin Madagascarandisreportedtobeapproximately0.5%forpatients withanewdiagnosisofTBand3%forpreviouslytreatedpatients (Ramarokotoetal.,2010).

IncontrasttootherAfrican countries,theprevalence ofHIV infectionisestimatedtobelessthan1%inMadagascar(Frickmann etal.,2013;Leutscheretal.,2005),representingapproximately50 000to60 000HIV-infectedindividuals.However,bytheendof 2015,onlyaround2000peoplewerediagnosedandlessthan1000 werereceivingantiretroviraltherapy.Asigniﬁcantproportionof newdiagnosesarelatepresenterswithopportunisticinfectionsat the time of HIV diagnosis, such as cryptococcal meningitis or disseminatedTB.

Toimproveandacceleratethediagnosisofsmear-negativeand extrapulmonary TB, molecular-based techniques, suchas Xpert MTB/RIF (Cepheid, Sunnyvale, CA,USA), are useful tools.Xpert MTB/RIFhasbeenvalidatedinaseriesofsmallstudiesforboth smear-negativeandextrapulmonaryTB,showingawiderangeof sensitivitiesandspeciﬁcitiesaccordingtothesampleevaluated, butwithanoverallgoodperformance(Maynard-Smithetal.,2014;

Piatek et al., 2013). However, studiesvalidating Xpert MTB/RIF accordingtostrictclinicalcasedeﬁnitionsandinareal-lifesetting arescarce.

Theaimofthisstudywastoevaluatetheoperationalfeasibility of theimplementationofa routine molecularrapidtest (Xpert MTB/RIF)forthediagnosisofTBanditsperformanceforsuspected cases of smear-negative and extrapulmonary TB in a real-life setting in Madagascar. It was also aimed to estimate HIV prevalenceinthisat-riskpopulation.

2.Methods

TheJosephRasetaBefelatananaHospital(HUJRB)isa350-bed tertiaryreferraluniversityhospitallocatedin Antananarivo,the capitalcityofMadagascar.Itincludesallmedicalspecialtiesandan intensivecaremedicinedepartment.

2.1.Patientpopulation

Allpatientswithsuspected smear-negative and/orextrapul- monary TB (adult patients with a febrile illness and chronic respiratorysymptoms,pleuraleffusion,chronicabdominalpainor ascites, chronic meningitis, or other symptoms suggestive of extrapulmonary TB) presenting or referred to the infectious diseases, pulmonology, gastroenterology, or neurology services ofHUJRB wereeligiblefor inclusionin thestudy. Patientswho contributedboth pulmonaryandextrapulmonary sampleswere consideredasextrapulmonarycases.

AllsuspectedTBcasesatinclusion werere-evaluatedattheendof thestudybyan internationalpanel of three infectiousdiseases specialists(validationteam)notdirectlyinvolvedintheinclusion

processorthefollow-upofcases.Followingthisevaluation,patients wereclassiﬁedintothefollowingfourclinicalcategories:(Rakoto- nirinaetal.,2016)provenTB:culture-positiveforM.tuberculosisin any sample; (World Health Organization, 2014a) probable TB:

culture-negative,butwithclinicaldatacompatiblewithTBanda clinicalresponsetoanti-TBspeciﬁctreatmentintheabsenceof other antimicrobial therapy; (Rakotosamimanana et al., 2014) possible TB: culture-negative, but clinical and biological data compatible with TB; clinical evolution on anti-TB therapy not available,butnootheralternativediagnosis;(Ramarokotoetal., 2010)alternativediagnosis:anotherclinicalconditionwascon- ﬁrmedandretainedandtherewasnolaterevidencesuggestingTB.

The criteria of Marais et al. were used for the diagnosis of tuberculousmeningitis(Maraisetal.,2010).

Thisclassiﬁcationandstratiﬁcationofcaseswasindependentof XpertMTB/RIFandthepanelwasblindedtotheseresults.

2.2.Diagnostictests

For suspected pulmonary TB patients, direct microscopic examinationofsputumsmearsafterZiehl–Neelsenstainingwas performedatHUJRB.Allnegativesputumacid-fastbacillus(AFB) smears from pulmonary TB patients and specimens from extrapulmonary TB patients were sent to the Mycobacteria Laboratory at the Institut Pasteurde Madagascar for a second smearexamination,culture,andXpertMTB/RIFscreening.

Forallbiologicalfluids(sputum,gastricaspirate,urine,cerebrospinalfluid,pleuraleffusion,peritonealfluid), 0.5ml wasused for the Xpert MTB/RIF test, which was performed according to the manufacturer's instructions.Theremainingsamplesweredecon- taminatedbysodiumlaurylsulphatemethod(TacquetandTison, 1961).Biopsiesweregroundanddilutedindistilledwater,and0.5ml wasusedfortheXpertMTB/RIFassay.Theremainingsampleswere decontaminatedwithH2SO4(Davidetal.,1989).Decontaminated specimenswereexaminedunderafluorescencemicroscopeandthe remainingsampleculturedonstandardLöwenstein–Jensenmedi- um.Mycobacterialisolateswereidentifiedaccordingtogrowthon themedium,colonymorphology,andtheSDBiolineTBAgMPT64 test.Drugsusceptibilitytestswereperformedusingtheindirect proportionmethod(Canettietal.,1963).

Standardanalyses(cytologicalandbiochemical)forbiological fluids(cerebrospinalfluid,peritonealfluid,pleuraleffusion)and the pathological examination of tissue samples (lymph nodes, cutaneous biopsies, pleural and synovial biopsies) were also performed at HUJRB. HIV serology testing was offered to all patients.HIV statuswasconfirmedusingthreedifferentconfor- mational rapid tests, according to national recommendations.

Xpert MTB/RIF and all other standard microbiological and pathologicaltestresultswereavailableinreal-timeand usedto assistthecliniciansinmakingmedicaldecisions.

2.3.Datamanagementandstatisticalanalyses

Aspecificcasereportformwascreatedforthestudyandalldata werecollectedinadedicateddatabase.TheChi-squaretest and Fisher'sexacttestwereusedasappropriatetocomparecategorical variables. Continuous variables were compared between sub- groupsusingtheMann–Whitneytest.Allp-valueswereconsidered significant at p<0.05. The statistical analyses were performed usingIBMSPSSandRsoftware.Theperformanceofthediagnostic methodswastestedusingtwogoldstandards:(Rakotonirinaetal., 2016) proven cases, and (World Health Organization, 2014a) proven and probable cases grouped together. Exact binomial confidencelimitswerecalculatedfor testsensitivity,specificity, and positive and negative predictive values (PPV and NPV).

Youden'sindexwascalculatedforXpertMTB/RIF.

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2.4.Ethics

ThestudywasapprovedbytheNationalEthicsCommitteeof theMinistryof Health of Madagascar (No.049-MSANP/CE). All patients gave informed consent before any procedure was performed.

3.Results 3.1.Patients

Between September 2013 and June 2015, 363 patients were includedandatotalof438sampleswereanalysed.Twohundredand ninety-eight (82.1%) patients contributed one sample, 56 (15.4%) contributedtwo,andnine(2.38%)contributedthreeormoresamples.

Amongthe56patientswithtwostudiedsamples,six(10.7%)hadtwo pulmonary specimens, 33 (58.9%) had one pulmonary and one extrapulmonaryspecimen,and17(30.4%)hadtwoextrapulmonary specimens.Allcaseswiththreeormorespecimenshadatleastone pulmonaryandoneextrapulmonaryspecimen.

Thebaselinecharacteristicsofthestudypatientsandthefinal classificationofsuspectedcasesareshowninTable1.Inbrief,183 (50.4%)patientshadsuspectedpulmonaryTBand180(49.6%)had suspectedextrapulmonaryTBormixedpulmonary/extrapulmonary TB. Overall,proven cases accounted for 119(32.8%) patients, probable casesfor41(11.3%),andpossiblecasesfor56(15.4%).Analternative diagnosiswasidentifiedin147cases(40.5%).Themedianageofthe patientswas38.7 years(standarddeviation 12.1years)and 232 (63.9%)weremale.Thedistributionofproven,probable,possible, and alternative diagnoses differed according to the clinical presentation(pulmonaryvs.extrapulmonary,p<0.001).

In 38 of314 cases, theHIV serologywaspositive, giving an overallprevalenceof12.1%.Sixteencases(45.7%)wereknowntobe HIV-positiveatstudyentryand22(54.3%)werediagnosedinthe setting ofthestudy. Thirteencases(34.2%) had provenTB, ﬁve cases(13.2%)hadprobableTB,andfourcases(10.5%)hadpossible TB.Sixteenof38HIVcases(42.1%)werediagnosedamongpatients for whom an alternative diagnosiswas available. The CD4 cell count was available for 34 cases (89.5%), with a median (interquartilerange)valueof108(38–212)cells/mm³.HIVserology couldnotbeperformedin32(8.8%)of363patients,anditwas refusedbythepatientin17cases(4.7%).

3.2.Samples

Of the 438 samples included, 205 (46.8%) were respiratory samples(sputumand/orgastricaspirates)and233(53.2%)were extrapulmonary samples (Table 2). A negative Ziehl–Neelsen sputumstainwasaconditionatscreeningforstudyinclusion.An auraminestainwasthenperformedonthenewresearchsample and tested positive in 32 (15.6%) respiratory specimens. Nine (3.82%)extrapulmonarysampleswerealsoauramine-positive.

SuspectedextrapulmonaryTBcasesprovidedalargevarietyof clinicalspecimens.Themostfrequentwerecerebrospinalﬂuidin 77(33%)cases,asciticﬂuidin62(26.6%),andpleuraleffusionin50 (21.5%)cases.Amongtheremaining44samples(18.9%),themost frequentwerelymphnodes (n=16),urine (n=8),subcutaneous abscesses(n=6),and pleuralbiopsies (n=6).Culture and Xpert MTB/RIFhadanoverallpositivityrateofaround40%forpulmonary TBand24%forextrapulmonaryTB(Table2).

Forthe56(15.4%)caseswithmorethanonesamplestudied,28 (50%)caseshadtwonegativesamples,15cases(26.8%)hadone Table1

BaselinecharacteristicsofthestudypatientsandﬁnalclassiﬁcationofsuspectedTBcases

Allcases SuspectedpulmonaryTBcases SuspectedextrapulmonaryTBcases^a p-Value

n=363(%) n=183(%) n=180(%)

Sex(n=363) 0.099

Female 131(36.1%) 58(31.7%) 73(40.6%)

Male 232(63.9%) 125(68.3%) 107(59.4%)

Age(n=363),median(SD)years 38.7(15.2) 40.0(15.3) 37.3(15.0) 0.082

HIVserologicalstatus(n=314) 0.206

Negative 276(87.9%) 140(85.4%) 136(90.7%)

Positive 38(12.1%) 24(14.6%) 14(9.33%)

PreviouslyknownasHIV-positive 16(42.1%) 11(45.8%) 5(35.7%)

Classiﬁcationofsuspectedcases(n=363) <0.001

ConﬁrmedTB 119(32.8%) 69(37.7%) 50(27.8%)

ProbableTB 41(11.3%) 10(5.46%) 31(17.2%)

PossibleTB 56(15.4%) 20(10.9%) 36(20.0%)

Alternativediagnosis 147(40.5%) 84(45.9%) 63(35.0%)

TB,tuberculosis;SD,standarddeviation.

aIncludingdisseminateddisease(pulmonaryandextrapulmonaryclinicalpresentation).

Table2

Overallresultsfordirectexamination,culture,andXpertMTB/RIFforthe438samplesfrom363patientswithsuspectedsmear-negativepulmonaryorextrapulmonaryTB

Allsamples Pulmonarysamples Extrapulmonarysamples p-Value

n=438(%) n=205(%) n=233(%)

Directexamination^a(n=438) <0.001

Negative 397(90.6%) 173(84.4%) 224(96.1%)

Positive 41(9.36%) 32(15.6%) 9(3.86%)

Culture^b(n=438) <0.001

Negative 299(68.3%) 122(59.5%) 177(76.0%)

Positive 139(31.7%) 83(40.5%) 56(24.0%)

XpertMTB/RIF(n=437) <0.001

Negative 298(68.2%) 121(59.3%) 177(76.0%)

Positive 139(31.8%) 83(40.7%) 56(24.0%)

TB,tuberculosis.

aZiehl-Neelsenstain

b Löwenstein–Jensenmedium.

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positiveand one negativeresult,and 13cases(23.2%) had two positive samples. In 57% of cases with a pulmonary and an extrapulmonary sample studied, at least one was positive, compared to 41% of cases with two extrapulmonary samples and33%ofcaseswithtwopulmonarysamplesanalysed(p=0.310).

Fortheninecases(2.38%)withthreeormoresamplesstudied,all sampleswerenegativeinsixcases(66.7%)andoneortwosamples werepositiveinthreecases(33.3%).

3.3.Performanceofdiagnostictests

Thesensitivity,speciﬁcity,PPV,andNPVofXpertMTB/RIFare shown in Table 3. Xpert MTB/RIF was analysed using the

microbiologygoldstandard(provencases,positiveculture),aswell as the alternate gold standard (proven/probable cases, positive culture or clinical responsewithout an alternative diagnosis or combinedtreatments).Overall,XpertMTB/RIFshowedasensitivity of0.824(95%CI0.751;0.883)andanextremelyhighspecificityof 0.988 (95% CI 0.956; 0.999). The sensitivity was lower for extrapulmonary TB than for pulmonary TB. For extrapulmonary samples,analyseswere performed for the three most prevalent types (cerebrospinalfluid,asciticfluid,and pleuraleffusion)andother samples(Table4).Theoverallrateofpositiveandnegativeresults according totheclinical category(proven, probable,possible,or alternativediagnosis)isshowninFigure1.

Table3

PerformanceofXpertMTB/RIFforthe438samplesfrom363patientswithsuspectedsmear-negativepulmonaryorextrapulmonaryTB;valueand95%conﬁdenceintervalfor allestimations

GoldstandardA^a GoldstandardB^b

Allsamples(n=438)

SE 0.824(0.751;0.883) 0.652(0.582;0.717)

SP 0.988(0.956;0.999) 0.988(0.956;0.999)

PPV 0.983(0.941;0.998) 0.985(0.948;0.998)

NPV 0.866(0.808;0.911) 0.694(0.63;0.753)

Youden'sindex 0.812(0.708;0.881) 0.64(0.539;0.716)

Smear-negativepulmonarysamples(n=205)

SE 0.867(0.775;0.932) 0.832(0.741;0.901)

SP 0.989(0.938;1) 0.989(0.938;1)

PPV 0.986(0.926;1) 0.988(0.932;1)

NPV 0.888(0.808;0.943) 0.845(0.76;0.909)

Youden'sindex 0.856(0.71;0.932) 0.82(0.679;0.9)

Extrapulmonarysamples(n=233)

SE 0.763(0.634;0.864) 0.495(0.398;0.593)

SP 0.987(0.928;1) 0.987(0.928;1)

PPV 0.978(0.885;0.999) 0.982(0.903;1)

NPV 0.841(0.748;0.91) 0.574(0.484;0.66)

Youden'sindex 0.749(0.562;0.863) 0.482(0.326;0.592)

TB,tuberculosis;SE,sensitivity;SP,speciﬁcity;PPV,positivepredictivevalue;NPV,negativepredictivevalue.

aProvencases(culture-positivecases).

b Provenandprobablecases(culture-positivecasesorclinicalresponsetoanti-TBtreatmentwithoutanyotherdiagnosisorothertreatment).

Table4

PerformanceofXpertMTB/RIFin233extrapulmonarysamplesfrompatientswithsuspectedextrapulmonaryTB;valueand95%conﬁdenceintervalforallestimations

GoldstandardA^a GoldstandardB^b

Cerebrospinalﬂuidsamples(n=77)

SE 0.692(0.386;0.909) 0.444(0.255;0.647)

SP 0.933(0.817;0.986) 1(0.838;1)

PPV 0.75(0.428;0.945) 1(0.64;1)

NPV 0.913(0.792;0.976) 0.674(0.52;0.805)

Youden'sindex 0.626(0.203;0.895) 0.444(0.093;0.647)

Asciticﬂuidsamples(n=62)

SE 0.643(0.351;0.872) 0.407(0.224;0.612)

SP 0.929(0.765;0.991) 1(0.698;1)

PPV 0.818(0.482;0.977) 1(0.615;1)

NPV 0.839(0.663;0.945) 0.484(0.302;0.669)

Youden'sindex 0.571(0.116;0.864) 0.407(-0.078;0.612)

Pleuraleffusionsamples(n=50)

SE 0.636(0.308;0.891) 0.241(0.103;0.435)

SP 1(0.842;1) 1(0.681;1)

PPV 1(0.473;1) 1(0.473;1)

NPV 0.889(0.739;0.969) 0.389(0.231;0.565)

Youden'sindex 0.636(0.15;0.891) 0.241(-0.217;0.435)

Othersamples(n=44)^c

SE 0.952(0.762;0.999) 0.923(0.749;0.991)

SP 0.75(0.509;0.913) 0.933(0.681;0.998)

PPV 0.8(0.593;0.932) 0.96(0.796;0.999)

NPV 0.938(0.698;0.998) 0.875(0.617;0.984)

Youden'sindex 0.702(0.27;0.912) 0.856(0.429;0.989)

TB,tuberculosis;SE,sensitivity;SP,speciﬁcity;PPV,positivepredictivevalue;NPV,negativepredictivevalue.

aProvencases(culture-positivecases).

b Provenandprobablecases(culture-positivecasesorclinicalresponsetoanti-TBtreatmentwithoutanyotherdiagnosisorothertreatment).

cIncludinglymphnodes,urine,subcutaneousabscesses,pleuralbiopsies.

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3.4.M.tuberculosisresistancetoanti-TBdrugs

ResistancetorifampicinwasdetectedbyXpertMTB/RIFintwoof 363cases(0.55%):a62-year-oldwomanwithperitonealTBanda positiveXpertMTB/RIFinasciticﬂuid,anda28-year-oldmanwith smear-negativepulmonaryTB.Inaddition,ninecaseswerereported as undetermined (ﬁve were culture-negative and four culture- positivewithnoresistancedetectedbyconventionalmethods).All otherspecimenswerenegativeforrifampicinresistance.

4.Discussion

This studyconducted in Madagascar (the MadaXpert study) confirmed the public health problem and burden of smear- negativeandextrapulmonaryTB.Inthisresource-poorsetting,the implementationofa rapidmoleculardiagnostictest forTBin a real-lifesituationwasfeasibleandprovidedareliableandrapid diagnosisofTB.Overall,XpertMTB/RIFshowedahighsensitivity andan extremelyhighspecificity,thus confirmingits excellent performanceforthediagnosisofbothsmear-negativepulmonary TBand extrapulmonaryTB in awide rangeofspecimens when compared to culture. These results were obtained in a public referencehospitalinthecontextofacutelyillpatientspresenting withaclinicalsuspicionofTB.AnHIVseroprevalenceof12%was identifiedinthishigh-riskpopulation,whichismuchhigherthan the overall reported prevalence of around 1% for the general population.Similartopreviousreports,drug-resistantM.tubercu- losiswasveryinfrequent(Ramarokotoetal.,2010).

Sputum smear microscopy remains a common method to diagnosepulmonary TBcases,butlackssensitivity.Moreover,it

doesnotallowforthediagnosisofextrapulmonarycases,which remainsachallengingissueforclinicians(Steingartetal.,2007).

Indeed,extrapulmonary TBisfrequently underestimateddueto diagnosticdifficulties(Kulchavenya,2014),althoughitrepresentsa significantproportionofTBcasesinsomecountries(Gunaletal., 2011). Nucleic acid amplification tests have facilitated the diagnosis of some extrapulmonary TB forms, but sensitivity is poorforsomeof them(Ketataet al.,2015;Mehtaetal.,2012).

Although Xpert MTB/RIF has been largely validated for the diagnosisofpulmonaryTB(Steingartetal.,2014),extrapulmonary clinicalspecimensstillneedmoreattention(Maynard-Smithetal., 2014).

In thepresent study, XpertMTB/RIF showeda sensitivityof 76.3%forextrapulmonaryTB.Itwasabletorapidlyidentifyaround 65–70%ofconﬁrmedmeningeal,abdominal,andpleuralTBcases;

thisrepresentsahugeclinicalimpactintermsofprovidingtimely life-savingtreatment.TheoverallresultsforXpertMTB/RIFwere quitesimilartothosereportedintheliterature(BatesandZumla, 2016).Aproportionofgastricaspirateswereincludedamongthe respiratory samples. This sample type has been validated in children(Batesetal.,2013),buthasbeenpoorlystudiedinadults.

Inthepresentstudy,whenmorethanonesampleperpatientwas included,therewasatrendtowardsahigherpositivityrateincases withboth pulmonary and extrapulmonary specimens analysed, althoughthiswasnotstatisticallysigniﬁcant.

IncontrasttoneighbouringcontinentalAfricancountries,such asMozambique,theprevalenceofHIVinMadagascarisbelievedto belessthan1%inthegeneralpopulation(Frickmannetal.,2013;

Leutscheretal.,2005).However,recentlargestudiesarelacking and the possibility that a signiﬁcant proportion of cases are Figure1.PositivityrateofXpertMTB/RIFaccordingtotheclassiﬁcationofcases(proven,probable,orpossibleTBandalternativediagnoses)andtypeofextrapulmonary specimen.

CSF:cerebrospinalfluid;AF:asciticfluid;PF:pleuralfluid;others:othersampletypes(includinglymphnodes,urine,subcutaneousabscesses,pleuralbiopsies,synovialfluid, pericardialfluid,andbonemarrowaspirates.).

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undiagnosedcannotbeexcluded.Althoughsomecasesincludedin thisstudywerealreadyknowntobeHIV-positive,mostcaseswere diagnosedduring thestudyperiod. ThehighHIV prevalencein patientswithsuspectedTBinthisstudyisnottotallyunexpected.

However, 42.1% of HIV diagnoses were performed in patients classiﬁedbytheexpertvalidationteamashavinganalternative diagnosis. These data suggest that HIV seroprevalence in MadagascarmaybeunderestimatedandcallforlargeHIVtesting programmestobeputinplace.MostHIV casespresentedwith advanceddiseaseandamedianCD4cellcountofaround100cells/

mm³.HIVserologywaspartoftheinitialevaluationofthecasesin thestudy,butinalmost10%ofcasesitcouldnotbeperformeddue toa lackof testsduringsomeof thetime periods.In addition, approximately5%ofpatientsrefusedtobetested.

Thephysicians’perceptionoftheutilityofthisrapiddiagnostic toolwasextremelypositive;itwasconcludedthatXpertMTB/RIF representedamajoradditioninassistingclinicaldecision-making and case management. The sensitivity of Xpert MTB/RIF for extrapulmonaryTBwas76.3%inthisstudyandwascloseto59%for both conﬁrmed and probable cases when groupedtogether. In Madagascar, where TB is a major public health issue, anti-TB treatmentisprioritizedforpulmonarycasesinordertodecrease transmission. Thus, unconﬁrmed extrapulmonary cases have sometimesremained untreated for long periodsof time. Some extrapulmonaryformsofTB,suchastuberculousmeningitis,are amongthemostserious,andadelayintheinitiationoftreatment isassociatedwithapoorprognosisintermsofmortalityorserious sequelae in survivors. In these cases, the implementation of programmes allowing a rapid diagnosis has major clinical implications(Piateketal.,2013).

Thisstudyhasseveralstrengths.First,moststudiesevaluating the performance of a microbiology test have included limited clinical information. In this study, each case was thoroughly evaluated to include not only those with microbiologically confirmedTB,but alsoallcasesthatmostprobablyhadTB but remained unconfirmed. Moreover, the strict clinical follow-up allowedtheexclusionofallcaseswithadocumentedalternative diagnosis, thus providing excellent negative controls for the evaluationoftheperformanceofthetests.Second,withalmost 400patientsincluded over2 years, thesample sizeprovides a representativeoverviewoftheTBperspectiveinAntananarivoand overallinMadagascar,aswellassufficientpowertoevaluatethe diagnostictechniques. Finally,this study provided data onHIV prevalenceinahigh-riskpopulationinMadagascar.Asalimitation, theheterogeneoustypesofextrapulmonarysampleprecludedthe analysisofspecificclinicalpresentationsduetothesmallnumbers ofcasesforthedifferentTBtypes.Inaddition,itwasnotpossibleto detecttheimpactoftheearlydiagnosisofthesesevereformsofTB onmortality,asempiricaltreatmentwasavailable.

Inconclusion,thisstudy–theMadaXpertstudy–conﬁrmed thefeasibilityandusefulnessoftheimplementationofaroutine rapid molecular diagnosis programme for TB in Madagascar.

XpertMTB/RIFdemonstratedagoodperformanceforallformsof difﬁcult-to-diagnose TB, comparable to reports from previous studies.HIVprevalencewashighinthishigh-riskpopulation.M.

tuberculosisresistancetoanti-TBdrugswasverylow.Ascale-upof the Xpert MTB/RIF rapid TB diagnostic method should be encouragedin low-incomecountrieswitha highTBprevalence

(WorldHealthOrganization,2014b)givenitsabilitytoovercome operationalconcerns.

Acknowledgements

The study was funded by grants from Geneva University Hospitals (Humanitarian Commission, CGR 75 388), Bordeaux UniversityHospital,andtheAquitaineRegion.

WethankallofthetechniciansattheMycobacteriaLaboratory, InstitutPasteurdeMadagascar.

Conﬂictofinterest:RR,JA,VR,NR,MR,RA,RR,MT,JR,AA,MR,FB, AC:nonetodeclare.

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