Evaluating the effectiveness of anti-tuberculosis treatment by detecting Mycobacterium tuberculosis 85B messenger RNA expression in sputum

ContentslistsavailableatScienceDirect

Journal

of

Infection

and

Public

Health

jo u r n al ho me p ag e :h t t p : / /w w w . e l s e v i e r . c o m / l o c a t e / j i p h

Original

Article

Evaluating

the

effectiveness

of

anti-tuberculosis

treatment

by

detecting

Mycobacterium

tuberculosis

85B

messenger

RNA

expression

in

sputum

Ersan

Atahan

_,

_Suat

_Saribas

_,

_Mehmet

_Demirci

_,

_Aylin

_Babalık

_,

_Seher

_Akkus

_,

Ahmet

Balıkcı

_,

_Dilek

_Satana

_,

_Tevhide

_Ziver

_,

_Harika

_Oyku

_Dinc

_,

_Melike

_Keskin

_,

Dogukan

Ozbey

_,

_Banu

_Tufan

_Kocak

_,

_Nesrin

_Gareayaghi

_,

_Sahra

_Kirmusaoglu

_,

Hrisi

Bahar

Tokman

_,

_Bekir

_Kocazeybek

a_{IstanbulUniversity-Cerrahpasa,CerrahpasaFacultyofMedicine,DepartmentofPulmonaryDiseases,Istanbul,Turkey} b_{IstanbulUniversity-Cerrahpasa,CerrahpasaFacultyofMedicine,DepartmentofMedicalMicrobiology,Istanbul,Turkey} c_{BeykentUniversityMedicalFaculty,DepartmentofMedicalMicrobiology,Istanbul,Turkey}

d_{ClinicofChestDiseases,UniversityofHealthSciences,SureyyapasaChestDiseasesandChestSurgeryTrainingandResearchHospital,Istanbul,Turkey} e_{IstanbulUniversity,IstanbulFacultyofMedicine,DepartmentofMedicalMicrobiology,Istanbul,Turkey}

f_{EasternMediterraneanUniversity,FacultyofHealthSciences,NutritionandDieteticDepartment,Famagusta,Cyprus} g_{OkanUniversity,MedicalFaculty,DepartmentofMedicalMicrobiology,Istanbul,Turkey}

h_{T.C.HealthMinistryErenkoyMentalHealthandNeurologyTrainingandResearchHospital,Istanbul,Turkey}

i_{IstanbulSisliHamidiyeEtfalTrainingandResearchHospital,BloodCenter,UniversityofHealthSciences,Istanbul,Turkey} j_{T.C.Halic¸University,FacultyofArts&Sciences,DepartmentofMolecularBiologyandGenetics,Istanbul,Turkey}

a

r

t

i

c

l

e

i

n

f

o

Received7February2020

Receivedinrevisedform8May2020 Accepted19May2020 Keywords: 85BmRNA Mycobacteriumtuberculosis Anti-tuberculosistreatment

a

b

s

t

r

a

c

t

Background:Theantigen85complex(85B)issecretedinlargequantitiesfromgrowingmycobacteriaand

thepresenceofbacterialmRNAisanindicatorofcellviability.Thequantitativedetectionof85BmRNA

expressionlevelscanbeusedtoassessthesuccessofanti-tuberculosistreatmentoutcomestodetect

viablemycobacteriacells.Therefore,weevaluatedthelevelsof85BmRNAofMycobacteriumtuberculosis

strainsinpatientswithpulmonarytuberculosis.

Methods:Thirtypatientswithprimarytuberculosiswereincludedinthisstudy.Thesputumspecimensof

patientswerecollectedondays0,15,and30daysandwereculturedandevaluatedby85BmRNA-based

RT-qPCR.

Results:Overall,23ofthestudiedtuberculosisstrainsweresusceptibletotheprimaryanti-tuberculosis

antibioticsusedinthisstudy,7wereresistant.Bythe30thdayoftreatment,85BmRNAwasdetectedin

onlyoneofthesusceptiblestrains,butinall7oftheresistantstrains,thoughtherelativegeneexpression

variedbetweenthestrains.Thisdifferencebetweenthesusceptibleandresistantstrainsatday30was

statisticallysignificant(p<0.05).

Conclusion:85BmRNAexpressionlevelscouldbeusedtofollowuponprimarytuberculosiscases.85B

mRNAseemstobeagooddiagnosticmarkerformonitoringanti-tuberculosistreatmentoutcomes.

©2020TheAuthor(s).PublishedbyElsevierLtdonbehalfofKingSaudBinAbdulazizUniversityfor

HealthSciences.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.

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

Introduction

Mycobacterium tuberculosis (M. tuberculosis) is an obligate

humanpathogenthatcausestuberculosis(TB),whichisstilla

mor-∗ Correspondingauthorat:IstanbulUniversity,CerrahpasaFacultyofMedicine, DepartmentofMedicalMicrobiology,CerrahpasaStreet,34098Istanbul,Turkey.

E-mailaddress:suatsaribas@gmail.com(S.Saribas).

talpublichealthproblem[1].IntheWHO’sglobalTBreport(2019),

approximately10.0million(range:9.0–11.1million)peoplehadTB

in2018andtherewereanestimated1.2millionTBdeathsamong

HIV-negativepeople[2].Currentlaboratorymethodsforthe

diag-nosisofTBandfollow-upofanti-TBtreatmenttakealongtimedue

totheslowgrowthrateofM.tuberculosis[3].Thesuccessofan

anti-TBtreatmentisusuallycorrelatedwiththeconversionofasputum

culturefrompositivetonegative[4].Usually,thefirst-line

anti-TBregimenincludesfiveantimicrobialagents(isoniazid,rifampin,

https://doi.org/10.1016/j.jiph.2020.05.016

1876-0341/©2020TheAuthor(s).PublishedbyElsevierLtdonbehalfofKingSaudBinAbdulazizUniversityforHealthSciences.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

ethambutol,streptomycinandpyrazinamide),andthetreatment

choicemaybechangedafterobtainingtheresultsof

antimicro-bialsusceptibilitytests[3].Nucleicacidamplificationtests(NAATs)

ofmycobacterialDNA/rRNAhave proventobefastandreliable

diagnostictestsforTBdiagnosisandfollow-uponthesuccessof

anti-TBtherapy[5].ManyNAATmethods,includingthosebasedon

IS6110,65kDaheatshockprotein,and16SrRNAdetection,areused

eitherinTBdetectionorfollow-upaftertreatment[6–10].

Unfor-tunately,thesemethodscannotdifferentiatebetweenviableand

non-viableM.tuberculosis[11].Theantigen85complexofM.

tuber-culosisandMycobacteriumbovisBCGgeneratesahumoralresponse

inTBpatientsthatisspecificforgrowingmycobacteria[12].The

antigen85complex includesthree30–32-kDaproteinsthatare

locatedintheextracellularspaceandsecretedduringthegrowthof

M.tuberculosisbyanenergy-dependentprocess[12].Thehalf-lifeof

bacterialmRNAsareveryshort,comparedtorRNAorgenomicDNA,

andthereforemycobacterialmRNAmaybeabettertargetforthe

detectionofmycobacterialviability[13].Also,thecharacteristics

ofmRNA-basedassayssuggestthattheseassaysmaydistinguish

betweenviableandnon-viableorganisms.Theseassays maybe

usefulformonitoringtheefficacyofanti-TBtherapy[14].

WeaimedtodetectM.tuberculosis85BmRNAfromsputum

specimensandevaluatetheperformanceofusingmRNAto

mon-itortheresponse toanti-TBtherapyusing reverse-transcription

quantitativePCR(RT-qPCR)insputumspecimensfromTBpatients

receivingfirst-lineanti-TBtreatment.

Materialsandmethods

Studyareaandgroups

ThisprospectivestudytookplacebetweenOctober2017and

October2018andinvolvedthefollowingparticipants:

1.MedicalMicrobiologyDepartment,MedicalFacultyofBeykent

University.

2.ChestDiseasesClinicsofIstanbulSureyyapasaChestDiseases

andThoracicSurgerytrainingandresearchhospital.

3.ChestDiseasesClinicsofCerrahpasaMedicalFaculty,Istanbul

University-Cerrahpasa.

Fifty-fivesputumsampleswereobtainedwithhighlysuspicious

clinical/radiologicalandhistopathologicalevidenceofpulmonary

TBfrompatientswhowerereferredtotheoutpatientclinicsof

IstanbulSureyyapasaChestDiseasesandThoracicSurgery

train-ingand researchhospital. A total of30 patientswereincluded

afterTB wasconfirmedinthesputumsamplesbybothpositive

Lowenstein–Jensen(LJ)testsandtheBACTECMGIT960method

(BDDiagnostics,Sparks,MD) onday0.Thesputum samplesof

TB-positivepatientsweretakenondays0,15,and30.Thesex

distri-bution(M/F)andmeanageofthepatients,respectively,were16/14

and38.63±16.33years(range21–76years).Allparticipantssigned

awritteninformedconsentformthatwasapprovedbytheClinical

ResearchEthicsBoardofIstanbulUniversity,CerrahpasaFaculty

ofMedicine(No:83045809/604.01,Date:08.10.2015).Thesame

InstitutionalEthicsBoardalsoapprovedthisstudy.TotalRNA

iso-lationand85BmRNAstudieswereperformedintheMicrobiology

laboratoryofBeykentUniversity.

Processingofspecimens

The analysis was performed at the Microbiology

Labora-toryof BeykentUniversity.Sputumsampleswereprocessedfor

microscopy(smearexamination)andculturedintheBACTECMGIT

960andLJsystems.Samplesweredecontaminatedwith

N-acetyl-l-cysteineand 2%sodiumhydroxide (NALC–NaOH)and 500-␮L

aliquots of decontaminated sample were stored at −70 ◦_{C for}

RT-qPCR.Themycobacterialisolatesobtainedinculturewere

sub-jectedtolimitedbiochemicaltestingforspeciescharacterization

usingtheBACTECNAPTBDifferentiationTestKit(Becton

Dickin-son,Sparks,MD,USA).

MycobacteriagrowthIndicatortubedrugsusceptibilitytesting

Mycobacteriagrowthindicatortubedrugsusceptibilitytesting

(MGIT-DST)wasperformedusingtheBACTECMGIT960SIREkit

accordingtothemanufacturer’sinstructions[15].Inthetest,800

␮LofOADCsupplementand100␮Lofdrugswereaddedtoeach

MGITtube.AfewcoloniesontheLJmediumweresuspendedin

PBSandvortexedtoprepareasuspension.A1:5dilutionofthis

suspensionwaspreparedusingPBSand500-␮Lsampleswere

inoc-ulatedintoeachdrug-containingtubeandagrowthcontroltube.

Thefinaldrugconcentrationswere1.0␮g/mLforstreptomycin,

0.1␮g/mLforisoniazid,1.0␮g/mLforrifampin,and5.0␮g/mLfor

ethambutol.Drugsusceptibilitytestingofpyrazinamidewas

per-formedinadifferentsetofsamplesbymonitoringthegrowthof

M.tuberculosis,inliquidmediumatpH5.9containing0.1␮g/mLof

pyrazinamide,bytheBACTECMGIT960system.Agrowthcontrol

tubewithgrowthsupplementbutwithoutdrugwasalsoincluded

andtherelativegrowthratiosbetweenthedrug-containingtube

anddrug-freegrowthcontroltubeweredeterminedbythe

sys-tem’ssoftwarealgorithm.Thesusceptibilityresultswerereported

bytheMGIT960system.

Molecularmethods

85BmRNAdetectionwithRT-qPCR

85B mRNA from M. tuberculosis was extracted from

decon-taminated samples using TRIzol (Invitrogen, Carlsbad, CA,

USA) according to the manufacturer’s instructions, including a

previously-described modification [16]. The RNA concentration

andpurityratios(OD260/280,OD260/230)weremeasuredusing

theNanoDropND-1000spectrophotometer(ThermoFisher

Scien-tific,Wilmington, DE,USA). PurifiedRNAwasstored at −70◦_C

untilanalysis.Theprimersandprobesusedwerespecifictothe

85Bsequence andamplifya130-bp region(GenBankAccession

number:X62398).

Theprimersandprobeswerespecificto85Bsequenceand

mul-tipliedaspecific130bpregionof85BmRNA(GenBankAccess

num-berı:X62398);85BmRNAforward(5-TCAGGGGATGGGGCCTAG-3),

85B mRNA reverse (5-GCTTGGGGATCTGCTGCGTA-3) and 85B

mRNAprobe(5-FAM-TCGAGTGACCCGGCATGGGAGCGT-Tamra-3)

[16,17]. Mycobacterial 16S reference gene was used for

nor-malization of 85B mRNA gene expression levels. For the 16S

reference gene; RT primer (5-CCCAGTAATTCC-3), 16S Reverse

primer (5-CGCTCGCACCCTACGTATTAC-3), 16S Forward primer

(5-TTCTCTCGGATTGACGGTAGGT-3) and 16S Probe

(5-6FAM-AGCACCGGCCAACTACGTGCCAG-TAMRA-3)sequenceswereused

[18]. RT-qPCR was performed using a LightCycler 480 II

Real-TimePCRSystem(RocheDiagnosticsGmBH)usingthe2XOneStep

qRT-PCR Mastermix Kit (PrimerDesign Ltd., Southampton, UK)

accordingtothemanufacturer’sinstructions. TheqPCRreaction

wasperformedinatotalvolumeof20␮Lconsistingof5␮Loftotal

RNA(40ng/␮L),10␮Lof2XqPCRMastermixKit(PrimerDesignLtd.

Southampton,UK),1␮Lofprimer/probemix(FAMreporter),and4

␮Lofnucleasefreewater.PCRconditionswereasfollows:reverse

transcriptionat55◦Cfor10minandenzymeactivationat95◦C

for2minfollowedby50cyclesofdenaturationfor10sat95◦C

andannealingfor60sat60◦C.LightCycler480software(version

Table1

ThedistrubutionofMGIT960and85BmRNA/16SrRNAresultsof30M.tuberculosis positivepatientswithpulmonarytuberculosisbefore(day0)andafterthestartof thetuberculostaticdrugs.

MGITn(%) 85BmRNA/16SrRNAn(%)

Positive Negative Positive Negative

Day0 30(100) 0(0) 30(100) 0(0)

Day15 17(56.6) 13(43.3) 17(56.6) 13(43.3)

Day30 7(23.3) 23(76.6) 8(26.67) 22(73.3)

resultswereanalysedaccordingtothedeltaCtmethodbyrelative

quantationmodüleofsoftware.

Statisticalanalyses

ThePearsoncorrelationcoefficientwasusedtoassesstheMGIT

960and85BmRNAcomparisonforday-30samples.Student’s

t-testwasusedtocomparethemeanagesandsexbetweengroups.

TheMann–WhitneyUtestwasusedtocomparethe85BmRNA

expressionbetweentheresistantandsusceptiblegroups.All

sta-tisticalanalyseswereperformedusingtheSPSSversion20(IBM

Corporation,Somers,NY,USA)andMedCalcversion13(MedCalc,

Mariakerke,Belgium)statisticalsoftwarepackages.

Results

Thesexdistribution(M/F)andmeanageofthepatients,

respec-tively,were16/14and38.63±16.33years(range21–76years).

ThedistributionoftheMGIT960and85BmRNAresultsofthe30

M.tuberculosis-positivepatientswithpulmonaryTBbefore(day0)

andafterthestartoftheanti-TBdrugsisshowninTable1.There

were100%correlationsbetweentheMGIT960and85BmRNA

lev-elsforsamplesfromdays0and15, butthePearsoncorrelation

coefficientwas0.914853fortheday-30samples.

Table2showsthedrugsusceptibilitytestingresultsofpatients

1,2,4,6,13,and16,whohadresistantandmultidrug-resistant

TB(MDR-TB)ondays0and30andonesusceptiblestrainonday

0.WhilefiveofthestrainswereMDR,theothertwostrainswere

isoniazid-andstreptomycin-resistant.Thehighestresistanceratio

wasobservedforisoniazid(23.3%),followedbystreptomycin(20%).

Onlyonesusceptiblestrainindicatedpositivity(480level)onday

30,whereastheother22M.tuberculosisstrainswerenegativefor

85BmRNAonday30.Wesuggesteditasfalsepositive.

Table 3 shows the 85B mRNA expression levels and

cul-tureresultsofantibiotic-susceptibleand-resistantM.tuberculosis

strainsondays0,15,and30.The85BmRNAlevelsofsevenMDRM.

tuberculosisstrainsondays0,15,and30areshowninFig.1.There

aresevenstrainswithMDRTB.Inthecomparisonofthe85BmRNA

levelsbetweentheresistantandsusceptibleM.tuberculosisstrains

ondays0and30,theresistantstrainshadsignificantlydifferent

85BmRNAlevelsthanthesusceptiblestrains(p<0.05[p:0.026])

(Fig.2).

Discussion

TounderstandthekillingefficacyofaTBtreatment,atleasttwo

monthsarerequiredtoobservetheconversionofasputum

cul-turefrompositivetonegative,whichhasbeenshowntocorrelate

withtherelapserate2yearsafterthefinalizationoftherapy[17].

Inthisstudy,wemeasured85BmRNAlevelsinsputumcollected

fromTBpatientsthatreceivedonemonthoftherapy.Weaimedto

evaluatetheusefulnessof85BmRNAlevelsasasurrogatemarker

ofTBtreatmenteffectiveness.Ourmainaimwastoshortenthe

durationofteststhatevaluatetheefficiencyofanti-TBtreatment

regimens.

Inourpreviousstudy,RT-qPCR targetingofthe85Bgene of

M.tuberculosisseemedtobeamoreusefulandrapidtechnique

thanDNA-basedmethodsfordetectingliveM.tuberculosisbacilli

fromsputumspecimens[19].ThereisgreatneedforTBsurrogate

biomarkersthatcanhelpusmonitoranti-TBtreatmentoutcomes

andpredicttreatmentsuccess.Therewerenodifferencesbetween

theMGIT960systemand85BmRNAexpressionresultsfordays0

Table2

DrugSusceptibilityTesting(DST)resultsof1,2,4,6,13and16patientswithresistantandmultidrug-resistanttuberculosis(MDR-TB)strainsand12patientwithone susceptiblestrainonday0and30.

No Gender Age Antituberculosisantibiotics(day0and30)

Isoniazid Rifampin Pyrazinamide Ethambutol Streptomyocin

1 F 50 R R S S R 2 F 22 R R S S R 4 M 16 R R S S S 5 F 23 R S S S R 6 M 57 R R R R R 12a _{E 18 S S S S S} 13 F 16 R R R R R 16 M 67 R S S S R

a_{Nogrowthwasobservedonday30.}

Table3

Quantificationofthebacillaryload(log)andcultureresultsofeightpatients(onesusceptibleandsevenresistantstrains)monitoredduring30daysofspecifictreatmentby 85BmRNA/16SrRNArelativefoldchange(log).

No Gender Age Day0 Day15 Day30

MGIT 85BmRNA/16SrRNA MGIT 85BmRNA/16SrRNA MGIT 85BmRNA/16SrRNA

1 K 50 Positive 5.12 Positive 4.13 Positive 3.51

2 K 22 Positive 5.10 Positive 4.10 Positive 3.61

4 E 16 Positive 5.06 Positive 4.15 Positive 3.98

5 K 23 Positive 4.99 Positive 4.05 Positive 3.56

6 E 57 Positive 4.94 Positive 3.99 Positive 3.45

12 E 18 Positive 5.05 Negative 2.83 Positive 2.55

13 K 16 Positive 4.95 Positive 4.18 Positive 4.04

Fig.1.(a)Thedistributionof85BmRNAgeneexpressionforantibiotic-resistantM.tuberculosisstrainsondays0,15,and30.(b)Thedistributionof85BmRNAgeneexpression forantibiotic-susceptibleM.tuberculosisstrainsondays0,15,and30.

Fig.2.Quantificationof85BmRNAgeneexpression(log)forantibiotic-susceptible andresistantM.tuberculosisstrainsondays0,15,and30.

and15,buttherewasonlyonemorepositivecaseforthe85BmRNA

expressionresultsthantheMGIT960results.Thepositivecasefor

85BmRNAexpressionwasalsoveryweak(480),butthePearson

correlationcoefficientdecreasedto0.914853forday-30samples

betweentheMGIT960and85BmRNAresults,whereasthe

corre-lationwas100%betweenthesegroupsfortheday-0andday-15

samples.Wesuggesteditasfalse-positivebutdecreasedthe

cor-relationbetweenRT-qPCRandculture.M.tuberculosisstrainsthat

wereresistanttoeitherisoniazidorrifampinshowednoreductions

in85BmRNAexpressioninthepresenceoftheanti-TBantibiotics.

Onlyonestrain,whichwasisolatedfrompatient12,showedvery

weak85BmRNAexpressionbutnogrowthintheMGIT960

sys-tem.

Therearesomerelated studiesin theliterature.Montenegro

et al.[20] evaluated52 patientswithpulmonaryTB by culture

and RT-qPCRfor a30-day treatment.Theydetected85BmRNA

inthesputumsamplesof52patientswithaconfirmeddiagnosis

ofpulmonaryTBonday0.Then,duringtreatment,theydetected

85BmRNAin13 patientsonday15 and inonlythree patients

onday30.Therefore,theyconcludedthatM.tuberculosismRNA

inthesputumsamplescanbeusedasaprognosticmarkerand

that85BmRNAquantificationisverypromisingasanearlyand

reliableindicatorformonitoringresponsestotreatment,drug

resis-tance,re-infection,andrelapse.Inanotherstudy,Hellyeretal.[10]

reportedthatexposingsensitiveM.tuberculosisstrainstoisoniazid

orrifampinfor 24hreducedthelevelsof85BmRNAto<4and

<0.01%,respectively,butnoreductionwasobservedindrug-free

controlcultures.Incontrast,thelevelsofIS6110DNAand16SrRNA

werenotreducedinthesameperiod.Inasimilarstudy,Lietal.[14]

detectedisocitratelyasemRNAinthesputumofculture-positive

TBpatientswhoreceivedarifampin-basedregimenfor1month.

At2months,isocitratelyasemRNAlevelsinthesputumcorrelated

morecloselywithliquidgrowththansolidculturemedium.

Desjardinetal.[17]reportedthatlevelsof85BmRNAdeclined

aftertheinitiationofanti-TBtherapyandthat90%ofpatients

con-vertednegativeafter2monthsoftreatment.Theyconcludedthat

therapiddisappearanceofM.tuberculosismRNAfromsputum

sug-geststhatitisagoodindicatorofmicrobialviabilityandauseful

markerforrapidlyassessingresponsestochemotherapy.

Mean-while,Yasiretal.[21]studied29M.tuberculosis-positivespecimens

for cultureand mRNA beforeinitiating TB chemotherapy. They

culturewhile, detecting26 (89.6%) and 28 (96.5%) as negative

bymRNA.TheyfinallyconcludedthatrapiddeclineinthemRNA

levelscorrelatedwithrapidcultureclearanceafteranti-TB

ther-apy.Mdivanietal.[3]studied65patientswithnewlydiagnosed

TBwhoweretreatedwithastandardizedfirst-lineanti-TB

treat-ment.Thesputum specimenswereevaluated atweek2and at

months1,2,and4aftertherapyinitiation.Theoverallagreement

betweenthecultureandmRNART-PCRresultswas87.1%.They

concludedthatformonitoringtheefficacyoftreatment,themRNA

RT-PCRresultsweresimilartothoseofcultureatthefollow-up

timepoints.

Inanotherstudy,Jouetal.[22]usedanIS6110-targetedDNA

PCRandalltheM.tuberculosissamples,though13daysof

isoni-azidtreatment,continuedtobepositive.Theydemonstratedthe

superiorityofanmRNAtargetinthedetectionofmycobacterial

viability;ontheotherhand,theirSTNRT-PCRassay,whichtargeted

themRNAcodingfortheubiquitous85Bantigenofmycobacteria,

couldnotdistinguishbetweenthoseculturesthatcontainedthe

antibioticandthosethatdidnot.SubculturesonLJagarconfirmed

theviabilityassessmentsoftheSTNRT-PCR.OurRT-qPCR-based

85BmRNAexpressionresultsareinlinewiththeresultsofother

studies.

InthestudyofWallisandJohnson[23],a72%decreaseinthe

85BmRNAlevelswasobservedduringtreatmentfromdays0to4,

andfollowedbya99%decreasebetweendays7and14.Inother

words,bacterialCFUcountsdeclined85%withinthefirst4days

oftreatmentandbyanadditional63%fromdays4to14.Ninety

percentofTBcasesconvertnegative,asevaluatedby85BmRNA

expression,after2monthsoftherapy.Clearanceofthe16SRNA

followedanintermediatepattern.OursevenM.tuberculosisstrains

withMDRindicated positiveresultsfor85BmRNAwithmostly

non-significantdecreases.Insuchsituations,theanti-TBtreatment

regimenshouldbeshiftedtosecond-lineanti-TBantibiotics.This

willsupplyanoptimaltreatmentregimenforpatientswithMDR

TB[23].

Inconclusion,detectingmycobacterialviabilityviaRT-qPCRof

85BrRNAtomonitortheefficacyofanti-TBtreatmentseemstobe

averypromisingwaytofollow-upTBpatientsundertheanti-TB

treatment.Theadvantageofthismethodisspeed.Ittakesweeks

towaitfortheresultsofbothsolidandliquidmycobacterial

cul-tures,whereasRT-qPCRtakesafewhours.Ontheotherhand,we

hadsomelimitations,suchaslimitedfinancialresources,which

onlyallowedustostudya2-monthperiodandpreventedusfrom

expandingthesamplesizebeyond30patients.Researchwithlarger

samplesis neededtoindicatetheadvantageof85BmRNAas a

surrogatemarkerfor theclinicaland therapeuticmonitoringof

patientswithpulmonaryTB.

Funding Nofundingsources. Competinginterests Nonedeclared. Ethicalapproval Notrequired. Acknowledgments

This work was supported by Istanbul University Scientific

ResearchProjectsCoordinationUnit,Projectnumber27154.

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