ww 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
Original
Article
Isoniazid
and
rifampin
drug
susceptibility
testing:
application
of
2,3,5-triphenyl
tetrazolium
chloride
assay
and
microscopic-observation
drug-susceptibility
assay
directly
on
Ziehl-Neelsen
smear
positive
sputum
specimens
Pottathil
Shinu
a,∗,
Varsha
Singh
a,
Anroop
Nair
baDepartmentofMicrobiology,M.M.I.M.S.R.,MMUniversity,Mullana,Ambala133207,India
bDepartmentofPharmaceuticalSciences,CollegeofClinicalPharmacy,KingFaisalUniversity,Al-Ahsa,SaudiArabia
a
r
t
i
c
l
e
i
n
f
o
Articlehistory: Received19June2015 Accepted22September2015 Availableonline27November2015 Keywords: Tuberculosis Rapid Evaluation Löwenstein–Jensen
a
b
s
t
r
a
c
t
Thecurrentstudywasaimedtoevaluatetheperformanceofdirect2,3,5-triphenyl tetra-zoliumchlorideassayanddirectmicroscopicobservationdrugsusceptibilityassaywith indirectLöwenstein-JensenproportionmethoddirectlyonZiehl-Neelsensmearpositive sputumspecimens.
Methods:Directacidfastbacillismearpositivesputumspecimens(n=264)weresubjected toisoniazidandrifampicindrugsusceptibilitytestingbydirect2,3,5-triphenyltetrazolium chlorideassay,directmicroscopicobservationdrugsusceptibilityassay,andthe perform-anceswerecomparedwithindirectLöwenstein-Jensenproportionmethod.
Results:Thedirect2,3,5-triphenyltetrazoliumchlorideassaydemonstratedanoverall sen-sitivity,specificity,positivepredictivevalue,andnegativepredictivevalueof99.2%,82.4%, 99.2%, and 88.5%, respectively, for the detection of isoniazid and rifampicin resistant MycobacteriumtuberculosisisolateswhencomparedtoindirectLöwenstein-Jensen pro-portionmethod.Likewise,theoverallsensitivity,specificity,positivepredictivevalueand negativepredictivevalueofdirectmicroscopicobservationdrugsusceptibilityassaywere 98.8%,82.4%,99.2%,and78.2%,respectively.
Conclusion: Thedirect2,3,5-triphenyltetrazoliumchlorideassaywasfoundtobean eco-nomicalalternativemethodfortherapidandaccuratedetectionofisoniazidandrifampicin resistancefromdirectacidfastbacillismearpositivesputumspecimens.
©2015ElsevierEditoraLtda.Allrightsreserved.
∗
Correspondingauthor.
E-mailaddress:shinup1983@gmail.com(P.Shinu). http://dx.doi.org/10.1016/j.bjid.2015.09.006
Introduction
Tuberculosis(TB)continuestobealeadingpublichealth prob-lem across the world particularly in developing countries (SoutheastAsiaaccountsfornearly40%ofglobalTBcases).1 TBisfurthercomplicatedbytherisingincidenceofmultidrug resistant (MDR) strains of tubercle bacilli and extensively drug-resistant TB,challenging TBcontrol efforts inseveral low-andmiddle-incomecountries.2,3 InIndia, theMDRTB is distributed among 2.9% and 15.3% of newly and previ-ouslytreatedTBcases,respectively,aspublishedbyRevised NationalTuberculosisControlProgram.4However,therising incidence and rate of transmission of MDR and extensive drug-resistant TB may be minimized by timely detection of the cases.5 Currently available drug susceptibility test-ing for mycobacterial isolates use an egg or agar based medium.Thesetraditionalculturemethodsdetect Mycobac-teriumtuberculosis(MTB)growth afteranaverageofthree weeksunderoptimalconditions,andthedrugsusceptibility testingtakesadditionalthreetofourweeks.6,7However, auto-matedsystems(brothbased)considerablyreducedthetime todetectionofdrugresistanceofmycobacterialisolates,but requireexpensiveinstrumentationandaretechnically com-plicated,limitingtheirapplicationinlowresourcesettings.8,9 Furthermore,molecularmethodsusedforthe characteriza-tionofgenesthatconferresistancetofirst-lineantimicrobial agentssuchasIsoniazid(INH)andRifampicin(RIF)are avail-able;however,thehighcostofinstrumentsandspecialized expertisetoperformthesemethodslimittheirwidespread use indeveloping countries.10 Furthermore, various colori-metric and non-colorimetric assays have been developed recently.However,all thesemethodswererestrictedor not evaluatedunderfieldconditions,suggestingtheprerequisite of an accurate, rapid, inexpensive, and technically simple method for the detection of MDR TB in resource-limited settings.11–18 In India, most of the mycobacterial clinical laboratoriesareexperiencingintricacyinobtainingdrug sus-ceptibilityinformationforMTB isolatesdueto financialor technicalreasons.19 Moreover, treatmentofTBunaware of susceptibilityinformationmayincreasetheriskoftreatment failure,thespreadofresistantstrains,andthedevelopmentof resistancetoadditionaldrugsaswell.20Inviewoftheabove issues,thecurrentstudywasdesignedtoevaluatethe perfor-manceofmicroscopicobservationdrugsusceptibility(MODS) assay and 2,3,5-triphenyl tetrazolium chloride (TTC) assay with indirect proportion method (using Löwenstein-Jensen (LJ)media)directlyonZiehl-Neelsen(ZN)smearpositive spu-tumspecimens.Moreover,toourknowledge,thisisthefirst report to investigate the performance of direct TTC assay and direct MODS directly on ZN smear positive sputum specimens.
Materials
and
methods
Studysettings
Thestudy was conductedin the Department of Microbiol-ogy,MMInstituteofMedicalSciencesandResearch,Mullana,
Ambala, India, a tertiary care hospital (1050 bedded) that alsofunctionsasaperipheralcenterfortheRevisedNational TuberculosisControlProgram.
Patientscreeningandrecruitment
Atotalof283patients,whowerepositivefordirectacidfast bacilli sputum smearmicroscopy (stained using ZN stain-ingmethod)andmeetinganyofthefollowingcriteriawere recruited: receiving anti tuberculosis treatment, suspected TB treatment failure, or suspected TB relapse cases were included.However,smearnegativecasesofpulmonary tuber-culosisandsalivarysputumsampleswereexcluded.
For the present study,atotaloftwosputumspecimens (spotsampleatclinicandmorningsampleathome)fromeach patientwerecollectedandallthespecimensweretransported immediately tothemicrobiologylaboratory (Departmentof Microbiology,M.M.I.M.S.R)andrefrigerated(4–8◦
C)overnight. Uponreceiptofthesecondsputumspecimen,bothspotand earlymorningsputumsampleswerestainedusingZN stain-ingtechniqueandreportswereissuedtothepatients[asall the sputumsamples were collected forroutinedirect Acid fastbacilli(AFB)smearexamination].Forthisstudy,thespot sampleatclinicand morning sampleathome(bothdirect AFBsmearpositivesputumsamples)werecollectedandthen mixedirrespectiveofthesmeargradesobtainedinthe rou-tine direct AFB smearexaminationby pouringthe sample fromonebottletoanothercontainingundrilledglassbeads (1–2mm,Himedia,Mumbai,India).Aftermixing,thesputum specimenswerehomogenized(usingaPasteurpipetteforone min) and vortexedfortwomintoassureuniform distribu-tionofbacilli.Afterhomogenization,thesputumspecimens were divided into two aliquots. Thefirst aliquot was sub-jected to directAFB smear examinationand culture on LJ media,directMODSassayanddirectTTCassay.Thesecond aliquotwasstoredinadeepfreezer(REMI;UDFV-185,Mumbai, India)at−80◦Cforthreemonthsortillthedrug susceptibil-ity test resultswere available.If the DST resultsusing the firstaliquotofsputumwerenotinterpretable,thenthe sec-ondaliquotwasusedtorepeattheindirectLöwenstein-Jensen proportionmethod(LJPM),directTTCassayanddirectMODS assay.
Laboratory
methods
Specimenprocessingandinoculumpreparation
Thefirst aliquot ofthe sputumspecimen[2–3mLin14mL BD FalconTM centrifuge tube (Becton Dickinson, Franklin Lakes, NJ, USA)]was subjected todirect AFBsmear exam-ination and N-acetyl l cystiene (NALC)–sodium hydroxide (NaOH)decontaminationmethod(finalNaOHconcentration 1%).6 AfterNALC–NaOHdecontaminationmethod,the sed-iment in each tube was suspended again in phosphate buffered saline to2mLand mixed well.This resuspended sedimentwasusedasacommoninoculumsourcefordirect TTC assay, direct MODS assay, and indirect LJ proportion method.
DirectTTCassay
The TTC assay was carried out as described by Moham-madzadehetal., withfewmodifications.17 Briefly,foreach sputumspecimen panelsof four screwcapped round bot-tomborosilicateglasstubes (Himedia,Mumbai)were used. Ofthefour screwcapped tubes,twotubeswerecontaining 1.6mLofMiddlebrook7H9broth,300LofPANTA(PANTA;BBL MGITPANTAAntibioticMixture;BD),300LofsolutionsofINH 1g/mLor RIF 10g/mL,300Lofthe processedspecimen and500LofTTCsolution(30g/mL),givingafinalvolume of3mLpertube,andthecriticalconcentrationsof0.1g/mL INH,1g/mLRIF and5g/mLTTC, respectively.Oneofthe othertwotubescontainingMiddlebrook7H9brothandPANTA mixtureservedasasterilecontroltubeand theothertube asagrowth controltubewheretheinoculumofeach sam-plewasincluded.Allthetubeswereincubatedat37◦Cand after72hofincubationthesetubeswereinspectedforcolor change(growthwasindicatedbytheformationofaredringor thereddishdiscolorationoftheMiddlebrook7H9broth).Inthe absenceofacolorchange,theincubationwascontinuedand thetubeswereinspectedondays3,5,7,9,11,13,15andupto 21daysforachangeincolor.Drugsusceptibilityresultswere interpretedonthesamedaythatthereddishdiscolorationor redringappearedingrowthcontroltube.Asamplewas con-sideredsusceptibleifredringorredcolorwasproducedinthe growthcontroltube(thedrugfreetube)butnotinthe drug-containingtube.Asamplewasconsidered resistantif both thedrug-freeandthedrug-containingtubesshowedredring orredcolorformation.
DirectMODSassay
ThedirectMODSassaywasperformedasdescribedbyBwanga etal.withminormodifications21 inthe readingtime inter-valoftheassay.Forthisassay,a24-welltissuecultureplate (Himedia, Mumbai, India) was used. To each well 700L ofMiddlebrook 7H9broth, 100L mixture ofpolymyxin B, amphotericinB,nalidixicacid,trimethoprim,andAzlocillin (PANTA;BBLMGITPANTAAntibioticMixture;BD),100Lof solutionsofINH1g/mLorRIF10g/mLand 100Lofthe processedspecimenwasadded,thusobtainingafinalvolume of1mLperwellandcriticalconcentrationsof0.1g/mLINH and1g/mLRIF.Agrowth controlwellwithMiddlebrook7 H9broth–PANTAplustheinoculumandthesterilecontrol wellwithMiddlebrook7H9broth-PANTAwereincludedfor eachsample.Allthecultureplatesweresealedusinga cello-phanetape,keptinziplockbagsandincubatedat37◦C.The drug-freecontrolwellswereobservedondays3,5,7,9,11, 13,15,andupto21daysusinganinverted-lightmicroscope (Zeiss,Axiovert25,Germany)at40×magnifications.After15 daysof incubation, the observation was restricted toonce ortwiceand continuedup to21days. Growthwas defined astheemergence ofvisually detectableserpentineclusters ofbacteria(cordformation).Drugsusceptibilityresultswere interpreted on the same day that distinct growth visual-izedincontrolwells.Asamplewasconsideredsusceptibleif growthwasvisibleinthedrug-freewellbutnotinthe drug-containingwell.Asamplewas consideredresistant if both
the drug-freeand thedrug-containingwellsshowedvisible growth.
IndirectLJproportionmethod
Theproportionmethod wasperformedaccording to estab-lishedproceduresonLJmediumwithcriticalconcentrations of0.2gmLforINHand40gmLforRIF.Astrainwas classi-fiedassusceptibletotheparticulardrugonlyifthenumber ofcoloniesthatgrewonthedrug-containingmediumis<1% ofthenumberofcoloniesthatgrewonthedrugfreecontrol tubeandresistantifthenumberofcoloniesis>1%.4
Identification
of
Mycobacterium
isolates
Theidentificationofmycobacterialisolateswasachievedby positive AFB smear, growth rate, colony pigmentation and morphology,nitratetest,catalasetest(at68◦C),growthonLJ mediacontainingp-nitrobenzoicacid.22Thequalitycontrolof eachnewlotofLJmediaandMiddlebrook7H9brothwas per-formedusingreferencestrains[MTBH37Ra(ATCC25177)and Mycobacteriumkansasii(ATCC12478)].Further,thenewlotof LJmediaandMiddlebrook7H9brothcontainingINHandRIF werecarriedoutusingH37RvMycobacteriumtuberculosisstrain ATCC27294(susceptiblecontrol),M.tuberculosisINHresistant (ATCC35822) andRIF resistant M.tuberculosis (ATCC35838) strains,respectively.
Time
to
results
for
the
drug
susceptibility
assays
Timeto resultswascalculated (in terms ofdays)from the datesofDSTinoculationandtillvalid(interpretable)results wereobtained.Theseresultswereinterpretedaseither ‘resis-tant’or‘susceptible’whereasinvalidresultswerereferredas ‘nogrowth’or‘contaminatedtube/well’evenafterrepeated testing.
Statistical
analysis
The performanceofthe direct MODS assay and the direct TTC assay was compared with the indirect LJ PM and results were interpreted in terms of sensitivity, speci-ficity,positivepredictivevalue(PPV),andnegativepredictive value (NPV). The following formulae were used for calcu-lations; sensitivity was true positives/(true positives+false negatives)×100; specificity was true negatives/(true neg-atives+false positives)×100; PPV was true positives/(true positives+false positives)×100; and NPV was true nega-tives/(true negatives+false negatives)×100. Themean and standarddeviations(SD)werecalculatedforageandgender aswell.Thedifferencesbetweencontaminationratesofdirect TTCassayanddirectMODSassaywerecomparedwithindirect LJPMandthesignificancewasdeterminedbyFisher’sexact testusingGraphpadprismonlineversionsoftware.
Ethic
statement
Maharishi Markendeshwar University Ethical Committee offeredawaiverofinformedconsentforthisstudybecausethe specimensusedwerecollectedforuseintheapprovedroutine directAFBsmearexaminations(Let.No.IEC/MMIMSR/13/47). The anti-tubercular drug susceptibility testing (using Indi-rectLJproportionmethod,directTTCassayanddirectMODS assay) were carried out in parallel with direct AFB smear positivesputumspecimensobtainedfromroutinedirectAFB smearexaminations.
Results
ThestudywascarriedoutfromMarch2013toApril2014and thestudypopulationincludedbothmale(n=177)andfemale (n=87)patientswithameanageof42.2years(SD14.4).Ofthe 264sputumspecimensprocessed,atotalof224(84.9%)valid resultswereobtainedinalltheassaysinvestigated.Ofthetotal validresults(n=224)obtainedinalltheassaysstudied,81.4% ofthedirectTTCassay(validresults,n=215)and82.8%direct MODS assay (valid results, n=217)were interpretable with indirectLJproportionmethod(validresults,n=221)andwere consideredforstatisticalanalysis(Fig.1).Table1showsvalid andinvaliddrugsusceptibilitytestresultsobtainedbyallthe assaysinvestigated.WithdirectTTCassay,74.6%(n=197)of the264resultswerevalidatinitialtesting.However,repeated directTTCassaycouldprovidevalidresultsin4.6%(n=12/264) ofthecontaminatedcultures,3.8%(n=10/264)indeterminate results(lackofgrowthingrowthcontrolwells),respectively, accomplishingatotalpercentageof82.5%(219/264).Similarly, indirectMODSassay76.1%(n=201)ofthe264resultswere validatinitialtesting.However,repeateddirectMODSassay added1.5%(n=4/264)specimenswith lackofgrowth,4.6%
(n=12/264)ofthecontaminatedcultures,and0.8%(n=2/264) ofdriedwells, givingatotal of83% (219/264)validresults. Furthermore,insufficientgrowthofMTB,contaminated cul-tures and growth of non-tuberculous mycobacteria (NTM) resultedin16.9%ofinvalidresultsinalltheassays investi-gated(Table1).
Table2demonstratesturnaroundtimesofdirectTTCassay and directMODS assays. The mediantime to detectionof directTTCassaywasninedays(range5–21days).Incontrast, themediantimetodetectionofdirectMODSassayand indi-rectLJPMMethodwassevendays(range3–21days)and68 days(range42–120days),respectively.Amongthevalidresults obtained withthe directTTC assay(n=215) andthe direct MODS assay (n=217),boththe directTTC assayand direct MODS assaycouldprovide interpretableresultsin94% and 93.6% ofthesputumspecimenswithin5–15days and3–15 days,respectively.
Tables3and4demonstratesperformanceofdirectMODS assayand directTTCassaywithindirectLJPM.Amongthe validresultsobtainedbydirectTTCassay(n=215),5.6%,1.4%, and9.8%oftheMTBisolateswereresistanttoINH,RIFand bothINHandRIF,respectivelywhencomparedtoindirectLJ PMmethod(in indirectLJ PMmethod5.6%,1.4%,and9.8% isolates were resistant to INH, RIF, and both INH and RIF, respectively).Further,theoverallsensitivity,specificity, PPV andNPVofdirectTTCassaywere99.2%,82.4%,99.2,and88.5% forthedetectionofINHandRIFresistantMTBisolates. Sim-ilarly, ofthe interpretable resultsobtainedbydirect MODS assay (n=217), 6%,1.8%and9.7%ofthe MTBisolateswere resistanttoINH,RIFandbothINHandRIF,respectivelywhen comparedtoindirectLJPMmethod(inindirectLJPMmethod 5.1%,1.8%,and9.7%isolateswereresistanttoINH,RIF,and bothINHandRIF,respectively).Further,theoverall sensitiv-ity,specificity,PPVandNPVofdirectMODSassaywere98.8%, 82.4%,99.2%,and78.2%,respectivelyforthedetectionofINH
Total number of patients recruited, N=283
Valid results in direct TTC assay, N=218 (82.6%)
Valid results in indirect LJ PM, N= 221(83.7%) Revised included, N=264(93.3%)
Valid results in direct MODS assay, N=219 (83%)
Valid results with both indirect LJ PM and direct MODS assay N=217 (82.2%)
Valid results with both indirect LJ PM and direct TTC assay N=215 (81.4%)
Excluded;
=4(1.4%) =19(6.7%) =15(5.3%) Not submitted second sputum
Couldn’t produce sputum Total
Drug susceptibility tests were carried out by indirect LJ PM, direct TTC assay, direct MODS assay methods on 264 direct AFB smear positive sputum specimens
Table1–Validandinvaliddrugsusceptibilitytestresults(n=264). Results IndirectLJPM (%) DirectTTC assay (%) DirectMODS assay (%)
Validtestresults
SensitivetobothINHandRIF 189(71.6) 184(69.7) 183(69.3)
ResistancetoINHandRIF 21(8) 21(8) 21(8)
ResistancetoINH 9(3.4) 10(3.8) 11(4.2)
ResistancetoRIF 2(0.8) 3(1.1) 4(1.5)
Subtotal 221(83.7) 218(82.6) 219(83)
Invalidtestresults
Nogrowth 27(10.2) 24(9.1) 25(9.5)
Contaminatedcultures 13(4.9) 19(7.2) 17(6.4)
Non-tuberculousmycobacteria 3(1.1) 3(1.1) 3(1.1)
Subtotal 43(16.3) 46(17.4) 45(17.1)
INH,isoniazid;RIF,rifampicin;LJPM,Löwenstein-Jensenproportionmethod;TTCassay,2,3,5-triphenyltetrazloiumchlorideassay;directMODS assay,microscopicdrugsusceptibilityassay.
Table2–Turnaroundtimeofthedrugsusceptibility assays.
Results(no.ofdays) DirectTTCassay
n=215 (%)
DirectMODSassay
n=217 (%) 3 – 1(0.5) 5 2(0.9) 53(24.7) 7 83(38.6) 77(35.8) 9 62(28.8) 35(16.3) 11 34(15.8) 19(8.8) 13 12(5.6) 2(0.9) 15 9(4.2) 16(7.4) After15days 13(6.1) 14(6.5) DirectTTCassay,2,3,5-triphenyltetrazloiumchlorideassay;direct MODSassay,microscopicdrugsusceptibilityassay.
andRIFresistantMTBisolateswhencomparedtoindirectLJ PMmethod.
Discussion
TBcontinuestobealeadingcauseofmorbidityand mortal-ity acrossthe world.Globally, anestimated480,000 people developed MDR-TB in 2013.1 However, due to scarcity of
cost-effective rapid diagnostic tests, the National TB con-trol programsareunabletoroutinelycarryout surveillance forMDR-TB.19Therefore,thecurrentstudywasdesignedto evaluatetheperformanceofdirectTTCassay(anew,rapid, technically simple,cost-effective diagnostictechnique) and directMODSassayincomparisonwithindirectLJPMdirectly onZNsmearpositivesputumspecimens.Inthecurrentstudy, themale:femaleratiowas2:1andameanageof42.2years(SD 14.4).Thisdataisinagreementwithapreviousstudycarried outbyRaoetal.23Ofthe264sputumspecimensprocessedin thisstudy,atotalof84.9%ofvalidresultswereobtainedin all assaysinvestigatedconfirming previousreportswherein 89–95% of samples subjected to direct MODS assay could providevalidresults.21However,duetothelackofpublished studies,thecomparisonofdirectTTCassaywasnotpossible. Furthermore,therelativelylowerproportionofvalidresults inthecurrentstudy (84.9%validresultsobtainedinall the assaysinvestigated)maybeattributedtopatientrecruitment criteriaadoptedinthestudy.Morespecifically,inthecurrent studywehaverecruitedpatientsreceivinganti-tuberculosis treatment(ATT).ThisATTmighthavekilledtheMTBinthe sputumspecimens,andthedeadbacillimusthaveappeared inthedirectsputumsmeargivingafalse-positiveAFBsmear result.Inaddition,inthisstudy,thevalidresultsobtainedat initialtesting withdirectTTCassayanddirectMODSassay
Table3–PerformanceofdirectTTCassaywithindirectLJPM.
Typeofassay IndirectLJPM Sensitivity(%) 95%C.I. Specificity 95%C.I. PPV 95%C.I. NPV 95%C.I. DirectTTCassay S R INH S 201 2 98.5 95.4–99.6 81.8 47.8–96.8 99 96.1–99.8 75 42.8–93.3 R 3 9 RIF S 211 1 100 97.8–100 75 21.9–98.7 99.5 97–100 100 30.1–100 R 0 3 INH+RIF S 192 2 99 95.9–99.8 90.5 68.2–98.3 99 95.9–99.8 90.5 68.2–98.3 R 2 19
Table4–PerformanceofdirectMODSassaywithindirectLJPM.
Typeofassay LJPM Sensitivity(%) 95%C.I. Specificity 95%C.I. PPV 95%C.I. NPV 95%C.I. DirectMODSassay S R
INH S 202 2 98.1 94.8–99.4 81.9 47.8–97 99.0 96.1–99.8 69.2 38.9–89.6 R 4 9 RIF S 212 1 99.5 97–100 75.0 22–99 99.5 97–100 75.0 21.9–98.7 R 1 3 INH+RIF S 194 2 99.0 96–100 90.5 68.2–98.3 99.0 96–100 90.5 68.2–98.3 R 2 19
were74.6%(197/264)and76.1%(201/264),respectively.These findings indicate that theseassays are easy to perform in mycobacteriologylaboratories.Forfewsputumsamples, with-out valid results at initial testing, the main reasons were contaminationofculturemedia,lackofgrowth,anddrying ofthewells (inthedirectMODSassay).Thecontamination rateofculturemediawas4.9%,7.2%and6.4%inindirectLJPM, directTTCassay,anddirectMODSassay,respectively(Table1). TherelativelylowercontaminationrateofIndirectLJPMwhen comparedtodirectTTCassayanddirectMODSassaysmaybe attributedtotheconsistencyofculturemedia.Middlebrook 7H9broth(aliquidculturemedia)ismorepronetodevelop contaminationasreportedearlier.24Thesecondmajor prob-lemforfailingtoobtain validresultsinall assayswaslack ofgrowthintheculturemedia(Table1),whichmaybedue topatientrecruitmentcriteriaadoptedinthisstudy(patients takingATTwererecruited)ormaybeduetothetoxicitycaused byNaOH,thatmighthavekilledMTBstrains (NaOHinthe NALCNaOHdecontaminationmethod)inthesputum speci-mens.Theotherreasonfortheinvalidresultsintheassays wasthegrowthofNTM(3.1%).ThisreducedrateofNTMin thisstudyindicateseitherlowprevalenceofNTMinthis geo-graphicalareaorpropersterilization techniquesadoptedin thecurrentstudy.25
Inthepresentstudy,ofthe264samplesprocessed,atotal of82.6%and83.3%validresultswereobtainedbydirectTTC assayanddirectMODSassay,respectively.Thisdatasuggest the potential of these assays to be implemented for rou-tinedirectanti-tuberculardrugsusceptibilitytesting.Further, bothdirectTTCassay(validresults,n=215)anddirectMODS assay(validresults,n=217)couldrender39.5%and60.4%valid resultswithinthefirstsevendays.However,by15daysof inoc-ulation,bothdirectTTCassayanddirectMODSassayscould provide54.4%and33.2%susceptibilityresults.Thereby,both directTTCassayanddirectMODSassaycoulddetectequal proportionofvalidresultswithin15 daysofinoculation. It isevidentfromTable2thatthemediantimetodetectionof directTTCassayanddirectMODSassaywas 9and 7days, respectively.Thisdatasupportsthe WHOrecommendation [thattheuseofvalidatedrapid(detectresistancewithin2–3 weeks)drugresistancedetectionmethodsforthedetectionof MDRTBifmolecularmethodsarenotavailable]tousedirect TTCassayforthedetectionofdrugresistanttuberculosis.26In thecurrentstudysetting,thedirectTTCassaywasfoundto beveryaccurate,quick(mediantimetodetectionwas9days),
andeasytoperformwhencomparedtoindirectLJPM(median timetodetectionwas68days)andmightrepresenta signif-icant advancementindetectionofMDRTBfromthepublic healthandindividualpatientperspectives.
Variouscolorimetric assayshavebeen proposed.Among these,directTTCassayusestheabilityofmitochondrial dehy-drogenase enzyme of living cells to reduce 2,3,5-TTC (an yellowcoloreddye)toaninsolubleredcoloredformazan crys-tals.ThisinsolubleTTCformazancrystalappearsasaredring atthebottomofthetubeorreddishdiscolorationofthe cul-turetube.17Inthecurrentstudy,whencomparedtoindirect LJPM,directTTCassaycouldprovideanexcellentsensitivity (98.5%)andspecificity(81.8%)forINH,100%and75%forRIF, respectively,and99%and90.5%forbothINHandRIF, respec-tively.TheoverallsensitivityandspecificityofdirectTTCassay was99.2%and 82.4%,respectivelyforthe detectionofdrug resistantMTBisolates.Thesefindingsareinaccordancewith Mohammadzadehetal.(carriedoutthestudydirectlyonMTB isolates)whereinasensitivityandspecificityof100%and92%, forbothINHandRIF,respectivelywereobtained.17However, nopreviousreportswereavailableinliteraturetocomparethe performanceofdirectTTCassaywiththecurrentstudy.The directTTCassaymayberecommendedinresourcepoor sett-ings fortherapiddiagnosisofMDR-TBduetoits excellent sensitivity,specificity,andeaseofimplementation. Further-more,therelativelyhighercontaminationratesinthedirect TTCassay(7.2%)whencomparedtoIndirectLJPM(4.9%)was alsonotstatisticallysignificant (p=0.36,Fisher’s exacttest) recommendingitsuseintheroutinedrugsusceptibility test-ing.TheotheradvantagesofdirectTTCassayare(a)decreased mediantimetodetection(mediandetectiontime9days).This mediandetection timewasrelatively highwhencompared todirectMODSassay(mediandetectiontime7days). How-ever, due totheease ofimplementation,highersensitivity andspecificityofdirectTTCassay,themediantimeof9days appearstobereasonableforthedetectionofdrugresistant TB;(b)indirectTTCassay,94%ofvalidresultswereobtained within15daysafterinoculation; (c)thedirectTTCassayis technicallyeasytoperformasoncetherequiredamountof Middlebrookmediaanddrugconcentrationsarepreparedin screwcappedtesttubesitcanbestoredat4–8◦
Cforfutureuse, minimizingtheworkloadinthelaboratory;(d)easytoreadthe results[asthegrowthofMTBisindicatedwithformationof redringatthebottom(Fig.2)]orareddishdiscolorationofthe culturemedia;(e)reducedaerosolproductionduetotheuseof
Fig.2–Photographshowingredringformationinthe directTTCassay[fromlefttoright(a)mediacontroltube, (b)growthcontroltube,(c)isoniazidresistantand(d) rifampicinresistant].
screwcappedtesttubes;and(f)economical(singledirectTTC assaycosts∼20 Indianrupees,excludingthebottlecharge, instrumentcost,electricitycharge,laborcharges–datanot shown).Consideringtheseadvantages,thishighlysensitive testshouldbeusedtorapidlydetectMDRTBfromretreatment andfollowuppatientstoconfidentlyeliminateMDRdisease. Although,thedirectTTCassayhasfewlimitationsaswell;any typeofbacterial(particularly,non-tuberculousmycobacteria) orfungalgrowth,maygivecolorchangeoftheculturemedia, givingfalse-positive results. However,false-positive results duetothegrowthofnon-tuberculousmycobacteriamaybe resolvedifPNBisincorporatedinanadditionalculturetube (PNB,areagentthatinhibitsthegrowthofMTBbutnotatypical mycobacteria)andthefurtherconfirmationofculturecanbe achievedbydirectacidfaststainingandbiochemicalreactions orusingmoleculartechniques.21
DirectMODSassayutilizesthe principleofdirect micro-scopicobservationofMTBcords(serpentinearrangementof MTB),which couldbe seen inrelatively fewhours ordays beforetheproductionofformazancrystals,producingmuch fasterresultsthanthedirectTTCassay.13Inthisstudy,direct MODS assay could provide a sensitivityof 98% and speci-ficityof 87.8% (whencompared toindirect LJ PM) forINH, 99.5%and 75%forRIF, respectively,and 99% and90.5% for both INH and RIF, respectively. The overall sensitivity and specificityofdirectMODSassaywas98.8%and84.4%forthe detection ofdrug resistant MTB isolates, inline with pre-viousreportswhereinthe sensitivityand specificityranged between85% and 100%.20 Further, contaminationof direct MODSassay (6.4%)wasmoderatelyhigherwhen compared toIndirectLJPM(4.9%),notstatisticallysignificant(p=0.57, Fisher’sexacttest),underscorestheroleofMODSassayin rou-tinedrugsusceptibilitytesting.13,27Furthermore,itisevident fromTables3and4,theloweroverallperformanceofdirect MODSassaycomparedtodirectTTCassayduetothedetection offewer false-positiveresistant cases. These false-positive
results may happenif artifacts are interpreted ascords or maybeduetothegrowthofNTM.27However,false-positive results produced by the growth of NTMmay be overcome bytheadditionofawellwithaPNB,areagentthatinhibits thegrowthofMTBbutnotatypicalmycobacteria.28Theother advantagesofMODSassayarereducedincubatorspace (sin-gle24-welltissuecultureplateisadequateforfoursputum specimens)anddecreasedmediantimetodetection(median timetodetectionwassevendays).However,theuseofMODS assaymaybelimitedwhencomparedtodirectTTCassaydue toitslowertechnicalperformance,comparativelyexpensive (∼39RS,excludinginstrumentcost,electricitycharge,labor charges– datanotshown)than directTTCassay and indi-rectLJproportionmethod(∼16Indianrupeesexcludingbottle charge,electricity,andlaborcharges),requireuseofinverted microscopetointerprettheresults,andcontinuous monitor-ingofthecultureplates fortheformationofmycobacterial cords.
In conclusion, direct TTC and direct MODS assays may beusedforeffectiveandaccuratedetectionofisoniazidand rifampicin resistancedirectly from ZN smearpositive spu-tum samples.These alternativemethodsseem tohavethe potentialtoproviderapiddetectionofresistancetoisoniazid andrifampicinwhencomparedtoindirectLJPM(technically exacting, cumbersome, and time devouring) and could be implementedinlaboratorieswithlimitedresources.However, MODS assayisrelatively costlyand requireuse ofinverted microscope. On the other hand,the directTTC assay was foundtobeaneconomicalalternativemethod(easyto per-form, do not require any special instrumentation, easy to interprettheresults)fortherapidandaccuratedetectionof INHandRIFresistance(withhighersensitivityandspecificity) fromdirectAFBsmearpositivesputumspecimenswhen com-paredtodirectMODSassayandIndirectLJPM.
Conflicts
of
interest
Theauthorsdeclarenoconflictsofinterest.
Acknowledgements
WeappreciateMr.GulabhSingh,laboratorytechnician,forhis excellent technicalsupport,and thenon-technicalStafffor assistinginsputumcollectionandtransportation.
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