Comparison of GeneXpert MTB/RIF assay and LED-FM microscopy for the diagnosis of extra pulmonary tuberculosis in Khyber Pakhtunkhwa, Pakistan

h tt p : / / w w w . b j m i c r o b i o l . c o m . b r /

Clinical

Microbiology

Comparison

of

GeneXpert

MTB/RIF

assay

and

LED-FM

microscopy

for

the

diagnosis

of

extra

pulmonary

tuberculosis

in

Khyber

Pakhtunkhwa,

Pakistan

Anwar

Sheed

Khan

_,

_Sajid

_Ali

_,

_Muhammad

_Tahir

_Khan

_,

_Sajjad

_Ahmed

_,

Yasir

Khattak

_,

_Abduljabbar

_,

_Muhammad

_Irfan

_,

_Wasim

_Sajjad

a_{KhyberMedicalUniversityPeshawar,Peshawar,Pakistan} b_{ProvincialTBReferenceLaboratory,Peshawar,Pakistan}

c_{UniversityofPeshawar,CentreforBiotechnologyandMicrobiology,Peshawar,Pakistan} d_{CapitalUniversityofScience&Technology,Islamabad,Pakistan}

e_{DepartmentofBiogenetics,NationalUniversityofMedicalSciences,Rawalpindi,Punjab,Pakistan} f_{UniversityofHaripur,DepartmentofMedicalLabTechnology,Haripur,Pakistan}

g_{SarhadUniversityofScienceandInformationTechnology,InstituteofBiologicalSciences,Peshawar,Pakistan}

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Articlehistory:

Received4April2017 Accepted28February2018 Availableonline27April2018 AssociateEditor:AfonsoBarth

Keywords: GeneXpert LED-FMmicroscopy Sensitivity Specificity

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GeneXpertisoneoftherecenttechnologicalinstrumentsusedtodiagnosetuberculosisin ashortspanoftime.Inthisstudy,theperformanceofGeneXpertMTB/RIFassayforthe diagnosisofextrapulmonarytuberculosis(EPTB)wascomparedwithlight-emittingdiode FluorescentMicroscopy(LED-FM)inKhyberPakhtunkhwa,Pakistan.Atotalof737EPTB sam-pleswerecollectedfromtuberculosis(TB)suspectedpatients.Outofthesesamples,male tofemaleratiowas53%(n=390)to47%(n=347)respectively.Thesensitivityandspecificity was73%and100%forGeneXpert,while40%and100%forLED-FMmicroscopy.Thisshows thatthesensitivityofGeneXpertis40–50%,higherthanLED-FMmicroscopy.GeneXpertalso detectedlownumberofbacilliascomparedtoLED-FMmicroscopy.

©2018SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

licenses/by-nc-nd/4.0/).

∗ _{Correspondingauthorat:ProvincialTBReferenceLaboratory,Peshawar,KhyberPakhtunkhwa,Pakistan.}

E-mail:Anwar786kp@hotmail.com(A.S.Khan).

https://doi.org/10.1016/j.bjm.2018.02.011

1517-8382/©2018SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Mycobacterium tuberculosis complex (MTBC) is the causative agentoftuberculosis(TB).TBisthesecondleadingcauseof deathafterHIVworldwide.Itisestimatedthat9millionpeople developTBandamongthem1.5milliondieeachyearoutof which360,000peopleareHIVpositive.1_{InPakistan350cases}

ofTBper100,000populationarereported.Pakistanisranked 5thamong22highburdenTBcountries.2_{MTBCmainlyinfect}

lungs(pulmonary)andseldomlyaffectsotherpartsofthebody knownasextrapulmonarytuberculosis(EPTB).3,4_Worldwide,

10%ofextrapulmonarycasesarereportedeveryyear.5_More

than 50% ofTB casesare reportedwith HIV co-infection.6

ThereasonforincreasedEPTBincidencesinPakistanisstill vagueduetolackofproperdiagnosis.Microscopic observa-tion ofM. tuberculosis in sputum smears still remains the pillaroftuberculosis(TB)diagnosisindevelopingcountries, despiteitspoorsensitivity.7_{Fluorescencemicroscopy(FM)of}

smearshavebeenstudiedasanalternativetoconventional lightmicroscopy withZiehlNeelsen staining(ZN).8 _Onthe

basisofthese findings, World Health Organizationin 2011 replacedconventionalFMbyLED-FMandphaseinLED-FMas analternativetoZNmicroscopy.9

Thereareanumberoftestsavailableforthediagnosisof tuberculosiseachhavingtheirownlimitations.Conventional microscopy haslow sensitivityand culturerequires longer time for positivity. Thecommercially available automated, liquidMGIT(MycobacteriumGrowth Indicatortube)culture systemistime-consumingandrequiresspecialized laborato-ries.Ontheotherside,nucleicacidamplificationtechniques notonlyprovidethe advantageofrapidityofdiagnosis but alsodetectevenlowgenomiccopiesinvariousspecimensand curtailthetransmissionofthedisease.10

TheWorld HealthOrganization(WHO)hasendorsedthe implementation of GeneXpert MTB/RIF assay for national tuberculosisprogramsindevelopingcountries.11 _TheXpert

MTB/RIF (Cepheid Inc.) isan automated, user friendly and rapidtestbasedonnestedreal-timePCRassayand molecu-larbeacontechnologyforMTBdetectionandRIFresistance.12

Theresultsare obtainedwithinashortperiodoftime(2h). Moreoverthistechniqueisnotpronetocross-contamination, requiresminimalBiosafetyfacilitiesandhasahighsensitivity insmear-negativepulmonaryTB.13_{ThediagnosisofEPTBis}

oftendifficulttoestablish,consideringthatnumberof bacte-riainspecimensisoftenverylow,acollectionoftenrequires invasiveprocedures,anditisnoteasytoobtainmultiple sam-ples.InthisscenarioGeneXpertisapotentiallyusefultoolfor extrapulmonaryspecimens.14

Thecurrentstudyaimstoinvestigatethediagnostic accu-racyofGeneXpert forEPTBspecimens andcompared with LED-FMmicroscopy.Thepercentageofenhancedcase detec-tionrateinEPTBsampleswasalsostudied.

Materials

and

methods

Thisstudy was conductedin Provincial TBReference Lab-oratory Peshawar KhyberPakhtunkhwa, from January 2013 to January 2015 in which two different techniques that is

GeneXpert MTB/RIF assay and LED-FM microscopy were compared. The result of both tests was evaluated against referencestandardculturemethod.

Area/patientsselection

Atotalof737samplesofEPTBonthebasisofclinical presenta-tion,radiologicalfindingandhistopathologicalevidencewere collectedfromTBsuspectsfromthreetertiarycarehospital, 332samplesfromLadyReadingHospital(LRH),149and256 samplesfromKhyberTeachingHospital(KTH)andHayatabad MedicalComplex(HMC)respectively.Sampleswereaspirated fromdifferentbodypartsi.e.,n=259Pleuralfluids,n=25 bio-logicalalveolarlavage(BAL),n=59cerebrospinalfluid(CSF),

n=82pus,n=125asceticfluid,n=13synovialfluid,n=47urine,

n=56pericardialfluids,n=36bonemarrow,n=35softtissues.

Sampleprocessing

Cultureprocessingprocedure

All samples were subjected to decontamination process according to standard protocol except bone marrow and

CSF.15,16 _{Some specimens should not be decontaminated,}

becauseeithertheyaresterilesourcei.e.CSFor decontamina-tionprocessisaroughmethodandthereisariskofkillingTB bacilli.17_{Afterdigestionanddecontamination,sampleswere}

dilutedwith50mLsterilephosphatebuffer,pH6.8,to mini-mizethecontinuingactionofNaOHandtolowerthepHand specificgravityofthespecimenbeforecentrifugation. Follow-ingrefrigeratedcentrifugation,thefalcontubeswerekeptfor 5mintoallowaerosolssettledown.Oncethesupernatantwas decanted,1mLofbufferwasaddedandvortexedto homog-enized the pallets.200ulofthe samplewas added to7mL MGITtubesupplementedalreadywithPANTA.Allthetubes wereincubatedinMGIT(Bactec960,BD,USA)semi-automated liquidculturesystemmachinefor42daysat35–38◦C. PostMGITidentification

The identification of MTBC was done by two different methods, including Ziehl Nelson staining (ZN) Microscopy and TBC(Becton, Dickinsonand Company)device method. StainingusingZNwascarriedoutaccordingtostandard pub-lishedprocedure,andslideswereexaminedwithbright-field microscopy (OlympusCX21)at1000×magnification. TBcis arapidimmuno-chromatographicdevicewhichwasusedto determineclinicalisolatestothespecieslevel.

GeneXpertMTB/RIFassayandLED-FMmicroscopy

SmearforLEDfluorescentmicroscopywereprepared accord-ing tostandard procedures.11 _{Smearsfor FMwere stained}

usingauraminO.Briefly,smearswerefloodedwithAuramin Ofor10min,destainedwithacidalcoholfor2min,andthen counterstained with methylene blue for oneminute. With auramineOstaining,Mycobacteriaappearasbrightyellow flu-orescentrodsonadarkbackground.Theslideswereexamined withZEISSEXTARO300at20×magnification.Thepresenceor absenceofAFBwasreportedusingWHO/IUATLDguideline.18

The sediment samples by cytocentrifugation were processed for Xpert MTB/RIF assay. Using a fresh trans-ferpipette,2mLoftheprocessedsamplewastransferredto

Table1–SamplewisecomparisonofLED-FM,GeneXpertandculture.

Types Totalsample2015 2014 Totalsample Totalnumberof

processedsample

Positive

LEDFM GeneXpert Culture

Pleuralfluid 85 45 129 259 22(8%) 30(12%) 52(20%) BAL 1 1 23 25 3(12%) 7(28%) 8(32%) CSF 18 15 26 59 5(8%) 10(17%) 12(20%) Pus 31 20 31 82 4(5%) 10(12%) 12(15%) Acseticfluid 48 20 57 125 8(6%) 14(11%) 14(11%) Synovailfluid 1 1 11 13 2(15%) 6(46%) 7(54%) Urine 15 10 22 47 4(9%) 4(9%) 7(15%) Pericardialfluid 17 10 29 56 2(4%) 6(11%) 7(12%) Bonemarrow 20 15 1 36 0 3(8%) 6(17%) Tissues 16 8 11 35 2(8%) 5(14%) 5(14%) Total 252 145 340 737 52(7%) 95(13%) 130(18%) M=390 53% F=347 47%

theXpertMTB/RIF cartridge10.Load thecartridgeintothe GeneXpert instrument as per manufacturer’s instructions accordingtostandardprotocol.14,19_{TheMTB/RIFassay}

inter-pretationissoftwarebased and notuserdependent.20 _The

sensitivity and specificity of each sample were calculated accordingtothefollowingformula

Sensitivity: Truepositive

Truepositive+Falsenegative×100 Specificity:

Truenegative

Truenegative+Falsepositive×100

Results

Ourstudywasthefirsttoshowthesuccessful implementa-tionofLED-FMstainingandXpertMTB/RIFservicesinroutine programmaticconditionsinKhyberPakhtunkhwa,Pakistan.A totalof737sampleswereprocessed,53%(n=390)weremale and47%(n=347)werefemale.

DetectionofM.tuberculosisbyXpertMTB/RIFassay

TotalpositiveonXpert MTB/RIFassaydeclared13%(n=95).

Table1showsresultsofEPTBforGeneXperttechnique.The

sensitivityvaluesobtainedthroughGeneXpert rangesfrom 50to100%whileSpecificityremainshigh(100%)asshownin

Table2.

DetectionofM.tuberculosisbyculturemethod

Totalpositiveonculturewas18%(n=130)(Table1).Further, therewerenosamplesfoundthathaveculturepositiveresults andGeneXpertandLED-FMnegative.

DetectionofM.tuberculosisbyLEDfluorescence microscopy

TotalpositiveonLED-FMmicroscopictechniquedeclared7%

(n=52)(Table1).ThelowestsensitivityrecordedbyLED-FMis0

forbonemarrowwhilethehighestis57%forasceticfluidand urine samples. Specificity for LEDfluorescence microscopy remainshigh(100%)asshowninTable2.

Discussion

Inthecurrentinvestigation,thediagnosticperformancefor EPTBcasesoftwodifferenttechniqueswereevaluated.The resultswerecomparedwithreferencestandardculture tech-nique.Atotalof737EPTBsampleswereanalyzedwiththe detectionrateofLED-FM,7%(n=52),GeneXpertMTB/RIFassay 13% (n=95), whileforreferencestandardculturetechnique 18% (n=130). Thesensitivity and specificity for GeneXpert MTB/RIF assay was 73% and 100%, while that of LED-FM microscopywas40%and100%respectively.Abetterdetection rateofGeneXpertwasreportedinastudywhencomparedto smearmicroscopyandculture.21

The results revealed that the detection rate (specificity andsensitivity)ofthetestedsamplesforGeneXpertMTB/RIF assay was almost twofold to that of LED-FM microscopy

(Table2).Previousinvestigationsshowedthatthespecificity

and sensitivityofGeneXpertMTB/RIF was79.0%and 97.3% respectively.22_{Someotherstudiesshowedspecificityand}

sen-sitivityof95%and100%respectivelyin340positivesamples.23

Thishigherratiointermofsensitivityincomparisontoour resultsmaybeduetosamplesize.

Thecurrentconventionaltechniques,i.e.LED-FMand cul-turesystemnotonlyrequirestheBiosafetyLevelIII(BSLIII)and trainedpersonnelbutalsotakeseveralweekstoyieldresults. TheGeneXpertMTB/RIFisaproventechnologyinTB diagno-sis.TheGeneXpertMTB/RIFassayisindependentoftheuser’s skillsandroutinestaffwithminimaltrainingcanusethetest. Theefficacy ofGeneXpert MTB/RIFwasproved tobemuch higherthanconventionalLED-FMandcomparabletothatof culture.Weobservedasensitivityof73%and specificityof 100%, whichwas comparativelyhigher than thesensitivity observedinastudywhichconsisted52%ofEPTBsamples.24

InanotherpublishedworkcarriedoutinEPTBcases,Tortoli etal.,2012.22_{reported86.9%sensitivityand99.7%specificity}

T able 2 – Compar ison betw een LED-FM, GeneXpert sensiti vity and specificity . T otal Pleur al fluid BA L CSF Pus Ascetic fluid Syno v ail fluid Urine P ericar dial Fluid Bone Marr o w T issues. GX LED-FM GX LED-FM GX LED-FM GX LED-FM GX LED-FM GX LED-FM GX LED-FM GX LED-FM GX LED-FM GX LED-FM GX LED-FM Sensiti vity % 73 40 58 42 88 38 83 42 83 33 100 57 86 29 57 57 86 29 50 0 100 40 Specificity % 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 PPV 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 NPV 95 89 90 87 94 77 96 87 98 90 100 95 86 55 93 93 98 91 91 83 100 91 PPV , positi v e pr edicti v e v alue; NPV , ne g ati v e pr edicti v e v alue; GX, GeneXpert; CSF , cer e b rospinal fluid; LED-FM, light emitting diode fluor escent micr oscop y.

ThesensitivityofGeneXpertwas40–50%higherthanthe LED-FMtechniquewhileinapreviousstudycarriedby,25_the

sensitivityofGeneXpertMTB/RIFassaywas45%higherthan microscopy technique.Ifwecompareourstudywith previ-ous study it depicts same resultsregardingsensitivity and specificity(Table2).Armandetal.,reportedthesameresults forboththetechniquesregardingspecificity.26_{Thepositivity}

of EPTB increasedfrom LED-FM to GeneXpert followed by culture gold standard. We found that the implementation ofGeneXpertservicesinTBDiagnosticcentersenhancethe yieldofEPTBpatients.Ithasashorterturnaroundtimeand simultaneouslydetectsrefamicin(RIF)resistanceinlessthan 3h.GeneXpertispreferredovertheconventionaltechniques becauseofitshighspecificity,sensitivityandwithminimal technicalexpertiserequired.Although,conventional labora-torytechniquesasLED-FMsmearmicroscopyfordiagnosisof tuberculosisarecosteffectivebutlesssensitiveascompared totheGeneXpertbecausethelargebacillaryload(105_/mL)will

berequiredforasmeartobecomepositive.

Inconclusion,specificityofGeneXpertishighinmajority ofEPTBcaseswithsmear-positivenon-respiratorysamples.In addition,ithasahighsensitivityfordetectingEPTBsamples likeascetic fluidandtissues.Thesefindingssupportrecent WHOguidelinesregardingtheuseofGeneXpertforTB diag-nosisfromEPTBspecimens.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

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e

s

1.WorldHealthOrganization.Globaltuberculosisreport2014.

Geneva:WorldHealthOrganization;2015.

2.WHO.GlobalTuberculosisReport2013.WorldHealth

Organization;2013.

3.FanningA.Tuberculosis:6.Extrapulmonarydisease.CMAJ

CanMedAssocJ.1999;160:1597.

4.SharmaSK,MohanA.Extrapulmonarytuberculosis.IndianJ

MedRes.2004;120:316.

5.RapiniRP,BologniaJL,JorizzoJL.Dermatology:2-VolumeSet.

St.Louis:Mosby;2007.

6.KumarV,AbbasAK,FaustoN,MitchellR.Chapter1.Cell

injury,celldeath,andadaptations.In:KumarV,AbbasAK,

FaustoN,MitchellR,eds.RobbinsBasicPathol.8thed.Phila:

Elsevier–Saunders;2007:13–17.

7.SteingartKR,HenryM,NgV,etal.Fluorescenceversus

conventionalsputumsmearmicroscopyfortuberculosis:a

systematicreview.LancetInfectDis.2006;6(9):570–581.

8.CorbettEL,BandasonT,CheungYB,etal.Epidemiologyof

tuberculosisinahighHIVprevalencepopulationprovided

withenhanceddiagnosisofsymptomaticdisease.PLoSMed.

2007;4(1):e22.

9.GelalchaAG,KebedeA,MamoH.Light-emittingdiode

fluorescentmicroscopyandXpertMTB/RIF®assayfor

diagnosisofpulmonarytuberculosisamongpatients

attendingAmbohospital,west-centralEthiopia.BMCInfect

Dis.2017;17:613.

10.AgrawalM,BajajA,BhatiaV,DuttS.Comparativestudyof

suspectedpulmonarytuberculosis.JClinDiagnResJCDR.

2016;10(5):DC09–DC12.

11.WorldHealthOrganization.Fluorescentlight-emittingdiode

(LED)microscopyfordiagnosisoftuberculosis:policystatement;

2011.

12.GuenaouiK,HarirN,OuardiA,etal.UseofGeneXpert

Mycobacteriumtuberculosis/rifampicinforrapiddetectionof rifampicinresistantMycobacteriumtuberculosisstrainsof clinicallysuspectedmulti-drugresistancetuberculosis cases.AnnTranslMed.2016;4(9),

http://dx.doi.org/10.21037/atm.2016.05.09.

13.Alvarez-UriaG,AzconaJM,MiddeM,NaikPK,ReddyS, ReddyR.RapidDiagnosisofPulmonaryandExtrapulmonary TuberculosisinHIV-InfectedPatients.ComparisonofLED FluorescentMicroscopyandtheGeneXpertMTB/RIFAssayina DistrictHospitalinIndia.TuberculosisResearchandTreatment;

2012https://www.hindawi.com/journals/trt/2012/932862/.

Accessed24.09.17.

14.VadwaiV,BoehmeC,NabetaP,ShettyA,AllandD,Rodrigues

C.XpertMTB/RIF:anewpillarindiagnosisof

extrapulmonarytuberculosis?JClinMicrobiol.

2011;49(7):2540–2545.

15.LönnrothK,CastroKG,ChakayaJM,etal.Tuberculosis

controlandelimination2010–50:cure,care,andsocial

development.Lancet.2010;375:1814–1829.

16.ParidaSK.TruefaceTsofTBdiagnosisin2012:Hypesand

realiTies.EurJMicrobiolImmunol.2012;2(4):275–281.

17.HandbookonTBlaboratorydiagnosticmethodsforthe EuropeanUnion.EuropeanCentreforDisease Preven-tionandControl.http://ecdc.europa.eu/en/publications-data/

handbook-tb-laboratory-diagnostic-methods-european-union.

Published14.03.16.Accessed24.09.17.

18.WHO.Theworldhealthreport1998–Lifeinthe21stcentury: Avisionforall.WHO.http://www.who.int/whr/1998/en/

Accessed24.09.17.

19.HelbD,JonesM,StoryE,etal.Rapiddetectionof

Mycobacteriumtuberculosisandrifampinresistancebyuseof

on-demand,near-patienttechnology.JClinMicrobiol.

2010;48:229–237.

20.BoehmeCC,NabetaP,HillemannD,etal.Rapidmolecular

detectionoftuberculosisandrifampinresistance.NEnglJ

Med.2010;363:1005–1015.

21.BatesM,O’GradyJ,MaeurerM,etal.AssessmentoftheXpert

MTB/RIFassayfordiagnosisoftuberculosiswithgastric

lavageaspiratesinchildreninsub-SaharanAfrica:a

prospectivedescriptivestudy.LancetInfectDis.

2013;13(1):36–42.

22.TortoliE,RussoC,PiersimoniC,etal.Clinicalvalidationof

XpertMTB/RIFforthediagnosisofextrapulmonary

tuberculosis.EurRespirJ.2012;40(2):442–447.

23.CausseM,RuizP,Gutiérrez-ArocaJB,CasalM.Comparisonof

twomolecularmethodsforrapiddiagnosisof

extrapulmonarytuberculosis.JClinMicrobiol.

2011;49:3065–3067.

24.ZekaAN,TasbakanS,CavusogluC.Evaluationofthe

GeneXpertMTB/RIFassayforrapiddiagnosisoftuberculosis

anddetectionofrifampinresistanceinpulmonaryand

extrapulmonaryspecimens.JClinMicrobiol.

2011;49(12):4138–4141.

25.ParsonsLM,SomosköviÁ,GutierrezC,etal.Laboratory

diagnosisoftuberculosisinresource-poorcountries:

challengesandopportunities.ClinMicrobiolRev.

2011;24(2):314–350.

26.ArmandS,VanhulsP,DelcroixG,CourcolR,LemaîtreN.

ComparisonoftheXpertMTB/RIFtestwithan

IS6110-TaqManreal-timePCRassayfordirectdetectionof

Mycobacteriumtuberculosisinrespiratoryandnonrespiratory