h tt p : / / w w w . b j m i c r o b i o l . c o m . b r /
Medical
Microbiology
Combination
of
commercially
available
molecular
assays
and
culture
based
methods
in
diagnosis
of
tuberculosis
and
drug
resistant
tuberculosis
Lamprini
Gkaravela
a,
Matthaios
Papadimitriou-Olivgeris
b,1,
Antigoni
Foka
a,
Fevronia
Kolonitsiou
a,
Anastasia
Spiliopoulou
a,
Nikolaos
Charokopos
c,
Apostolos
Voulgaridis
d,
Maria
Tsiamita
d,
Markos
Marangos
b,
Evangelos
D.
Anastassiou
a,
Iris
Spiliopoulou
a,∗aDepartmentofMicrobiology,SchoolofMedicine,UniversityofPatras,Rion,Patras,Greece bDivisionofInfectiousDiseases,SchoolofMedicine,UniversityofPatras,Rion,Patras,Greece cDepartmentofPulmonology,RegionalGeneralHospitalofPirgos,Pirgos,Greece
dDepartmentofPulmonology,UniversityGeneralHospitalofPatras,Rion,Patras,Greece
a
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t
i
c
l
e
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o
Articlehistory: Received31July2016 Accepted27April2017 Availableonline24June2017 AssociateEditor:JorgeSampaio
Keywords:
Mycobacteriumtuberculosis PCR
GenoTypeMTBDRplus
a
b
s
t
r
a
c
t
Earlydiagnosisoftuberculosisisofmajorclinicalimportance.Among4733clinical speci-menscollectedfrom3363patientsandsubjectedtoZiehl–Neelsenmicroscopy,4109were inoculatedontoLöwenstein–Jensenslantsand3139inBactec/9000MB.PolymeraseChain Reaction(PCR)wasperformedin3139specimens,whereas,agenotypicassaywasdirectly appliedin93MycobacteriumtuberculosiscomplexPCR-positiveforisoniazidandrifampicin resistancedetectionspecimens(GenoTypeMTBDRplus).RecoveredM.tuberculosisisolates (64)aswellas,21moresentfromRegionalHospitalsweretestedforantimycobacterial resis-tancewithaphenotypic(manualMGIT-SIRE)andagenotypicassay(GenoTypeMTBDRplus). PCRintheclinicalspecimensshowedexcellentspecificity(97.4%)andaccuracy(96.8%),good sensitivity(70.4%),butlowpositivepredictivevalue(40.3%).MGIT-SIREperformedtoM. tuber-culosisdidnotconferareliableresultin16isolates.Oftheremaining69isolates,15were resistanttostreptomycin,seventoisoniazid,seventoethambutolandfivetorifampicin. GenoTypeMTBDRpluscorrectlydetectedisoniazid(seven)andrifampicin-resistantM. tuber-culosisstrains(five),showinganexcellentperformanceoverall(100%).Susceptibilityresults bythemolecularassayapplieddirectlytoclinicalspecimenswereidenticaltothoseobtained fromrecoveredisolatesofthecorrespondingpatients.Combiningmolecularand conven-tionalmethodsgreatlycontributetoearlydiagnosisandaccuratesusceptibilitytestingof tuberculosis.
©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/ licenses/by-nc-nd/4.0/).
∗ Correspondingauthorat:DepartmentofMicrobiology,SchoolofMedicine,UniversityofPatras,UniversityCampus,Asklipeiou1,Rion
26504,Patras,Greece.
E-mail:[email protected](I.Spiliopoulou).
1 Currentaddress:DepartmentofInternalMedicine,HôpitalduJura,2800Delémont,Switzerland. http://dx.doi.org/10.1016/j.bjm.2017.04.001
1517-8382/©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Introduction
DespitethedecliningincidenceoftuberculosisinEurope,it stillremainsanimportanthealthissue.1,2Thenotificationrate
oftuberculosissteadilydecreases inGreeceduringthelast decade,however,thereare certainconcerns sincethe inci-denceissignificantlyhighmainlyduetounderreporting.1–3
The prompt diagnosis of patients with active disease ischallenging, evenin developed countries with sufficient resources.4Tuberculosisdiagnosisismainlybasedon
culture-basedtechniquescharacterizedbyalongturn-aroundtime.5
Today,in order toreduce the time ofdiagnosis,molecular assayscombinedwithconventionalmethodsareused,either directlyinclinicalspecimensorinisolatesrecoveredfrom cul-tures.Therefore,sinceacid-fastbacillismearslacksensitivity andMycobacteriumtuberculosis(MTB)treatmentis accompa-niedbycomplications, PCRbasedmethodshavebeen used foritsidentificationdirectlyinclinicalspecimensinorderto rapidlyaidclinicians.5,6 Nowadays,clinicalspecimens
orig-inatingfrompatientswithsuspicionoftuberculosismustbe subjectedtosmearmicroscopy,culture,andMTB-PCRassays.5
Thedelayinmicrobiologicconfirmationofthedisease com-bined with underreporting may provoke a delay in public healthmeasurestoidentifyandcureinfectedpatientsand households.
Anotherissueofincreasingimportanceworldwideisthe resistanceofM.tuberculosistofirst-linedrugs.1,2Thismainly
arises from non-adherence toantimycobacterial treatment regimens,orthemigrationflowfromregionswithhighrates of multi-drug resistant tuberculosis (MDR-TB).1,2,7 Strains
exhibitingresistancetobothisoniazid(INH)andrifampicin (RMP)are characterizedasMDR-TB.1 Identificationof
resis-tancetofirst-linedrugsisimportanttobeposedassoonas possiblesinceinfectionbysuchstrainsneedsprolonged treat-mentanddisplayslowercurerates.6 Nowadays,inaddition
totraditionalmethodology, DNA strip assaysthat can reli-ablydetectthemostcommonmutationsassociatedwithrpoB (RMPresistance)andkatG/inhA(INHresistance)genes,suchas GenoTypeMTBDRplus,canbeappliedintheDiagnostic Labo-ratoryeitherattheDNAextractsofisolatedstrains,ordirectly totheclinicalspecimen.5,8
Theaimofthepresentstudywastoevaluatethe combi-nationofmolecularassaysandconventionalmethodsforthe diagnosisoftuberculosisandcharacterizationof antimicro-bialresistanceamongM.tuberculosisisolates.
Materials
and
methods
Studypopulation/samplecollection
Atotalof4733clinicalspecimenswerecollected from3363 patientsattendingtheUniversityGeneralHospitalofPatras (UGHP)and theRegional GeneralHospitalofPirgos(RGHP), both located in Southwestern Greece, during a three-year period(January2009toDecember2011).Frommanypatients more than one sample was collected based on clinical grounds.Thestudywas approvedbytheEthics Committee oftheUGHP.
The clinical specimens included: sputa (n=1233), sputa after bronchoscopy (SAB, n=360), bronchial lavages (BL, n=1087),gastricaspirates(n=117),urine(n=490),pericardial (n=19),peritoneal(n=10),cerebrospinal(CSF,n=346),synovial (n=20),ascitic(n=201)andpleuralfluids(n=688),aswellas, tissuespecimens(n=162). Sampleswere assignedinto two groups:pulmonaryspecimens(n=2680)andextrapulmonary specimens(n=2053).
DirectdetectionofM.tuberculosiscomplexinclinical specimens
Ziehl–Neelsen(ZN)stainingwasperformedtoallclinical spec-imens(4733).Inaddition,molecularmethodswereperformed among3139clinicalspecimens,accordingtomanufacturer’s instructions.Testingwasperformedwheneverrequestedby theclinicians.COBASAMPLICORMTBPCR(RocheDiagnostics, Rotkreuz,Switzerland)wasusedin814specimensobtained duringJanuarytoSeptember of2009,whereas,COBAS Taq-ManMTB(RocheDiagnostics)wasappliedafterwardsin2325 specimens,thereafter(October2009–December2011).
IsolationofM.tuberculosisstrains
Specimens collected from non-sterile sites were con-centrated and decontaminated by the N-acetyl-cystein (NALC)–NaOH method (BectonDickinson, LePont-De-Claix, France). Fig. 1 shows the processing of clinical speci-mens.Inoculationof4109specimensontoLöwenstein–Jensen slants (LJ,bioMérieux, Marcyl’Etoile, France)and 3139into Bactec/9000MBculturevials (BectonDickinson)was carried out.Intobothmedia,2515specimenswereinoculated. Acid-fastbacteria recoveredfromsolid andliquidculturemedia wereidentifiedtospecieslevelbyareverselineblot hybridiza-tion (RLBH) assay (GenoType MycobacteriumCM/AS; Hain LifescienceGmbH,Nehren,Germany).
Susceptibilitytesting Molecularbasedmethod
IncaseofapositivePCRresultforthepresenceofM. tuberculo-siscomplexintheclinicalspecimens,GenoTypeMTBDRplus (Hain Lifescience GmbH) was directly applied in the DNA extract,inordertorapidlydetectthepresenceofmutations conferringresistancetoINHandRMP.
Culturebasedmethod
SixtyfourMTB(oneperpatient)isolatedfrom3363patients, andanother21isolatesobtainedfrompatientshospitalized inotherRegionalHospitalsofSouthwesternGreecethatwere sent to our Microbiology Laboratory, were tested for sus-ceptibilitytoantimycobacterialagents.Susceptibilitytesting was performedbyaphenotypic method(manual Mycobac-teria Growth Indicator Tube, MGIT-SIRE; Becton Dickinson) forstreptomycin,INH,RMPandethambutol(concentrations tested:0.1mg/L,1.0mg/L,1.0mg/Land5.0mg/L,respectively). Inordertoidentifythemostcommonmutationsconferring resistancetoINHand RMPtheGenoTypeMTBDRplus(Hain
Ziehl-Neelsen in 4733 samples 4109 specimens were inoculated in LJ 1594 specimens were inoculated only in LJ 20 MTB 5 NTM 61 MTB 7 NTM 4 MTB 3 NTM 100 mycobacterium spp from 78 patients 64 patients positive for MTB
85 MTB (one per patient)
MGIT-SIRE GenoType MTBDRplus 21 patients positive for MTB
from other hospitals No PCR was performed in 1594 specimens PCR was performed in 3139 specimens COBAS AMPLICOR MTB was applied in 814 specimens COBAS TaqMan MTB was applied in 2325 specimens
In case of positive PCR, GenoType MTBDRplus was applied directly in the
specimen (one per patient)
2515 specimens were inoculated only in both media
624 specimens were inoculated only in BACTEC 3139 specimens were
inoculated in BACTEC
Fig.1–Clinicalspecimens’flowchart.ZN,Ziehl–Neelsen;LJ,Löwenstein–Jensen;MTB,M.tuberculosis;NTM, non-tuberculousmycobacteria;MGIT-SIRE,MycobacteriaGrowthIndicatorTubeforsusceptibilitytesting.
LifescienceGmbH)wasappliedaccordingtotheinstructions ofthemanufacturer.
Statisticalanalysis
SPSSversion19.0(SPSS,Chicago,IL)softwarewasusedfordata analysis.Specificity,sensitivity,positiveandnegative predic-tivevalues(PPV,NPV)werecalculatedinordertoassessthe diagnosticperformanceofZNandPCR,aswellas,the perfor-manceofGenoTypeMTBDRplustoINHandRMPresistance. Thegold standard comparator forM. tuberculosis detection waspositivityofliquidand/orLJcultures,whereas,forINH andRMPresistanceitwasthemanualMGIT-SIREtesting.The accuracyofaforementionedmethodswasinvestigatedusing receiveroperatingcharacteristic(ROC)analysis.
Results
From a total of 4733 clinical specimens, 100 specimens collected from 78 patients (2.1%) were culture-positive for mycobacteria byeither method. Ninety-three (2.3%) out of 4109specimensinoculatedontoLJwerepositive,whereas,75 outof3139(2.4%)werepositivefromBactecvials.ZNwas posi-tivein72(1.5%)samples,64ofwhichwerepulmonary(2.4%vs 0.4%,p<0.001).EventhoughZNshowedhighspecificity,NPV, andaccuracyforpulmonaryandextrapulmonaryspecimens,
itssensitivityandPPVwerelow(Table1).BLandSAB sam-plesshowedhigherPPVascomparedtosputa.Samplesfrom whicha“non-tuberculousmycobacteria”(NTM)wereisolated didnotdemonstrateapositiveZNstain.
Intotal,78patientshadpositiveculturebyeithermedia: 64M.tuberculosis,threeM.kansasii,threeM.fortuitum,twoM. simiaeand oneofeachM. avium,M. malmoenseand M. gor-donae.Itwasnotpossibletoidentifystrainstospecieslevel fromthreepatients,thus,theywereclassifiedasNTM (non-tuberculousmycobacteria).
Fromthe3139clinicalspecimenstestedbycultureandPCR (COBASAMPLICORMTBPCRorCOBASTaqManMTB),results werevalidin2958specimens.Ofthese,71specimens(2.4%) wereculture-positiveand124(4.2%)PCR-positiveforM. tuber-culosiscomplex(Table2).Ontheotherhand,invalidresults werefoundin181specimens(5.8%)(negativePCRresultfor theinternalcontrol),ofwhich125werepulmonary(125/2122, 5.9%)and56extrapulmonary(56/1017,5.5%).PCRshowedhigh specificity,NPV,andaccuracy,goodsensitivityandpoorPPV, especiallyforextrapulmonaryfluids(gastricaspirates,urine, pericardial,peritoneal,CSF,synovial,ascitic,andpleural),as shown in Table 2. No statistically significant difference in theperformanceofCOBASAMPLICORMTBPCRascompared toCOBASTaqManMTBwasobserved.Fromthe 85totalM. tuberculosis(64beingoursplus21fromothersites,Fig.1), phe-notypic susceptibilitytesting bymanual MGIT-SIREdidnot conferareliableresultin16isolates(18.8%),duetomedium
Table1–PerformanceofZiehl–Neelsenintheidentificationofculture-positiveclinicalspecimensformycobacteria. Clinicalspecimens Number Culturepositive Ziehl–Neelsen
TP FP TN FN Sensitivity Specificity PPV NPV Accuracy
Pulmonaryspecimens 2680 81 22 42 2557 59 27.2% 98.4% 34.4% 97.7% 96.2% BL 1087 31 10 4 1052 21 32.3% 99.6% 71.4% 98.0% 97.7% Sputa 1233 34 7 35 1164 27 20.6% 97.1% 16.7% 97.7% 95.0% SAB 360 16 5 3 341 11 33.3% 99.1% 62.5% 96.9% 96.1% Tissue 162 10 2 0 152 8 20.0% 100% 100% 95.0% 95.1% Otherfluidsa 1891 9 0 6 1876 9 0.0% 99.7% 0.0% 99.5% 99.2% Allspecimens 4733 100b 24 48 4585 76 24.0% 99.0% 33.3% 98.4% 97.4%
TP,truepositive;FP,falsepositive;TN,truenegative;FN,falsenegative;PPV:positivepredictivevalue;NPV,negativepredictivevalue;BL,
bronchiallavages;SAB,sputaafterbronchoscopy.
a Gastricaspirates,urine,CSF,pericardial,peritoneal,synovial,ascitic,andpleuralfluids.
b Included85M.tuberculosisand15“non-tuberculousmycobacteria”(NTM)strains.
contaminationbyotherbacteria.Fromtheremaining69 iso-lates,15(21.7%)wereresistanttostreptomycin,seven(10.1%) toINH,seven(10.1%)toethambutolandfive(7.2%)toRMP.
TheGenoTypeMTBDRplusmethod appliedtoall 85 iso-latescorrelatedverywellwiththeconventionalsusceptibility method.Basedonthemutationsfound,sevenisolateswere identifiedasresistanttoINH(8.2%)andfivetoRMP(5.9%).The mostcommon mutationpatterns identifiedbythis method aredepictedinTable3.Onlyonemulti-drugresistantisolate wasdetectedbyphenotypicand/orgenotypicmethodologies. Three isolates (one MDR and two INH-resistant) exhibited a subpopulation of susceptible cells (additional hybridiza-tion with the wild typeprobe) originating from previously treatedpatients. TheperformanceofGenoTypeMTBDRplus ascomparedtoMGIT-SIREamongthe69isolatesforwhich bothmethodsconferred aresult,excellent (100%) sensitiv-ity,specificity,PPV,NPVandaccuracywererecordedforthe identificationofresistancetoINHandRMP.
In addition, the application of GenoType MTBDRplus methoddirectlyto93 PCR-positiveclinicalspecimensfrom differentpatients(48culture-negativespecimensincluded), showed excellent performancefor 72 sampleswith anOD >0.350 byCOBAS AMPLICOR MTBPCR ora thresholdcycle
Ct≤36byCOBASTaqManMTB.Nodifferencesweredetected amongthesusceptibilityresultofGenoTypeMTBDRpluswhen applieddirectlytospecimensortheisolaterecoveredfromthe samepatient(45patients).
Discussion
DespitethedecliningtuberculosisincidenceinEurope,delay inthediagnosisbytraditionalculture-basedmethodshasas aconsequenceseriousadverseeffectsontreatment,andan increase ofM. tuberculosisresistancerates toprimary anti-tuberculousagents.SincesensitivityandPPVofZNarepoor, the applicationofnewmolecularmethodsisnecessaryfor rapid,directandreliablediagnosisoftuberculosis,aswellas, identificationofresistance.1,6
PCR(COBASAMPLICORMTBPCRorCOBASTaqManMTB) showedhighspecificity,NPVandaccuracyforthedetection ofM.tuberculosis complexfrom bothpulmonaryand extra-pumonarysamples.PCR’ssensitivitywashigherwhenapplied inpulmonarysamples,ascomparedtoextrapulmonaryones (71.4%vs62.5%),probablyduetothefactthatthetwoPCR kitsusedinthisstudyweredesignedforpulmonarysamples
Table2–PerformanceofPCRintheidentificationofculture-positiveclinicalspecimensforM.tuberculosis(excluding181 samplesforwhichPCRresultwasinvalid).
Clinicalspecimens Number Culturepositive PCRa
TP FP TN FN Sensitivity Specificity PPV NPV Accuracy
Pulmonaryspecimens 1997 63 45 45 1889 18 71.4% 97.7% 50.0% 99.1% 96.8% BL 998 27 22 26 945 5 81.5% 97.3% 45.8% 99.5% 96.9% Sputa 699 25 15 13 661 10 60.0% 98.1% 53.6% 98.5% 96.7% SAB 300 11 8 6 283 3 72.7% 97.9% 57.1% 99.0% 97.0% Tissue 63 3 3 4 56 0 100% 93.3% 42.9% 100% 93.7% Otherfluidsb 898 5 2 25 868 3 40.0% 97.2% 7.4% 99.7% 96.9% Allspecimens 2958 71 50 74 2813 21 70.4% 97.4% 40.3% 99.3% 96.8%
TP,truepositive;FP,falsepositive;TN,truenegative;FN,falsenegative;PPV,positivepredictivevalue;NPV,negativepredictivevalue;BL,
bronchiallavages;SAB,sputaafterbronchoscopy.
a 774samplesweretestedwithCOBASAMPLICORMTBPCRandtheremaining2184sampleswithCOBASTaqManMTB.
Table3–PatternsofGenoTypeMTBDRplusresultsascomparedtomanualMGIT-SIRE.
INH RMP
MGIT-SIRE MTBDRplus MGIT-SIRE MTBDRplus
katG inhA Result Number rpoB Result Number
R WT WT2,Mut2(A-16G) R 4 R WT2,WT3,Mut2A(H526D),Mut2B (H526Y),Mut3(S531L)
R 1
R WT WT,Mut2(A-16G) R+S 3 R WT8,Mut2B(H526Y),Mut3(S531L) R 1
S WT WT S 62 R WT8,Mut3(S531L) R 1
np WT WT S 16 R WT2,WT3,Mut3(S531L) R 1
R WT,Mut1(D516V) R+S 1
S WT S 64
np WT S 16
MGIT-SIRE,MycobacteriaGrowthIndicatorTubeforsusceptibilitytesting;INH,isoniazid;RMP,rifampicin;,deletionofhybridizationsignal withwild-typeprobes;WT,wild-type;Mut:mutation;R,resistant;S,susceptible;np,notperformedduetocontamination.
only,aspublished.9–11TheoverallreportedsensitivityofPCR
differedwidelyinpreviouspublications(61.3–96.1%).10–14 In
ourstudy,theoverallPCRsensitivitywas70.4%,sinceonly72 (1.5%)specimensweresmear-positive.Themainadvantageof PCRisspeed,and,whencombinedwithGenoTypeMTBDRplus assaydirectlyinthePCR-positivespecimencanminimizethe timedelay fromspecimen collectiontoeffectivetreatment administration.Thiscanalsofacilitatepublichealth author-itiesto trace householdcontacts, especially inthe case of resistanttuberculosisandstopfurthertransmission.
ThemaindrawbacksofPCRforthedetectionofM. tuber-culosiscomplexareinhibitionandpresenceoffalsepositive results.Inhibitionwasobservedin5.8%ofsamples,noneof whichwasculturepositive.Thepercentageisslightlyhigher than previouslyreported (1.3–4.9%).9,10,12,15,16 Therelatively
highpercentage(2.5%)ofPCRfalsepositiveresultsconstitutes anothercontroversialfact.Therearetwodifferentetiologies forsuchanobservation.Firstly,theburdenofM.tuberculosis inthesamplemaybetoolowtoyieldapositiveculture,and secondly,PCRremainspositiveforatleastfourmonthsdueto thepresenceofnon-viableM.tuberculosis,asisthecaseafter initiationoftherapy.17,18ThelatteristrueforourInstitution,
sinceitisnotclearamongcliniciansthatonlysmearand cul-ture,andnotPCR,shouldbeorderedincaseoffollow-upto assesstreatmentefficacyanderadicationofmycobacteria.
Combining the manual MGIT-SIRE and GenoType MTBDRplus results, resistance rates of INH (8.2%) and RMP(5.9%)arecomparabletothosepreviouslyreportedfrom theGreekNationalReferenceLaboratoryforMycobacteria.2
On the contrary, an extremely high rate of resistance to streptomycin (21.7%) was observed, which is similar to that previously reported from our Institution.19 A possible
explanationisthatMGIT-SIREshowsthehighestdiscrepancy in identifying streptomycin resistanceas compared to the performanceofseveralothermethods.20Inapreviousstudy
fromourInstitution(2001–3),anequallyhighrateof strepto-mycinresistancewasidentifiedbythemethodofproportion weused.19Anotherplausibleexplanationisdisseminationof
resistantlineagesinSouthwesternGreece.
GenoTypeMTBDRplusshowedexcellentsensitivity, speci-ficity, PPV,NPV and accuracyfor the detectionof INHand RMPresistanceamongM.tuberculosisisolates,ascomparedto
MGIT-SIRE.ThismethodisalsousefulincasesofLJslantsand BactecvialcontaminationwhereMGIT-SIREcannotprovidea result.Inourstudy,contaminationbyotherbacterialspecies wasobservedin18.8%ofthetotalsamples.Asshownina metanalysis,8sensitivityofGenoTypeMTBDRpluswashighfor
INH(95.9%)andRMP(98.9%)resistancedetection.Amongthe fiveisolatesthatwereresistanttoRMPvariousmutationsof therpoBgeneweredetected.Thetypesofmutationswere sim-ilar tothosefoundinapreviousstudy fromGreekpatients affectingmostcommonlycodons531and526.21Amongthe
sevenINH-resistantisolates,alldemonstratedthesame muta-tion in the promoter region of inhA gene (A-16G). On the contrary,adifferentinhAmutation(C-15T)was detectedin sixamong19INH-resistantM.tuberculosisisolatesfrom the islandofCrete,Greece,while,theremaininghadamutation inkatGgeneleading todifferent prevalentclonesin differ-ent geographical locations.22 The application of GenoType MTBDRplusadditionallyenabledthedetectionofamixed pop-ulationofsusceptibleandresistantcellstoINHorRMPwithin thesamesample.Allthreesamplesthatexhibited heterore-sistance originatedfrom previouslytreatedpatients, which can explainthe presenceofmixedpopulation. Apparently, the burdenoftheresistantpopulationspecifies the pheno-typicsusceptibilitypatternoftheisolate,whichwasalways resistantinourstudy.Ifitwastheotherwayaround,further studies,liketypingbymycobacterialinterspersedrepetitive unit-variable number of tandemrepeats typing, should be necessary.Moreover,theexcellentperformanceofGenoType MTBDRplusdirectlyintheclinicalspecimenwhentheCt≤36 indicatesthatwhentheinitialbacterialloadofthesampleis highenough,antimicrobialresistanceforINHandRMPcanbe identifieddirectlyfromtheclinicalspecimen,duringtheearly stepsoflaboratorydiagnosis.
Despite the high accuracy of GenoType MTBDRplus in detectingM.tuberculosisresistancetoINHandRMP,aswellas, thecontributioninidentifyingtheoccurrenceofcertain muta-tions directlyfrom thePCR-positivespecimen,thismethod cannot completely replace traditional phenotypic methods suchasMGITsinceitdetectsspecificmutations.
Molecularmethodsrepresentamajorcontributiontothe rapiddiagnosis(COBASAMPLICORMTBPCRandCOBAS Taq-ManMTB)orsusceptibilitytesting(GenoTypeMTBDRplus)of
tuberculosis,sincetheycanprovidearapidandreliableresult toclinicians,whileawaitingtheresultoftraditionalcultures (LJ and Bactec/9000MB) or susceptibility (MGIT-SIRE) meth-ods.Conventional cultureandsusceptibility testingremain thegoldstandardsfortuberculosisdiagnosis,conferringthe advantage ofisolation of strains for further epidemiologic andresistancetraitsanalyses.However,thecombinationof molecularandconventionalmethodscontributestotheearly tuberculosisdiagnosisanddrugresistancedetection.
Conflicts
of
interest
Theauthorshavenoconflictsofinteresttodeclare.
Acknowledgements
This study was supported by funds of the Department of Microbiology,University ofPatras,Patras,Greece.Wethank Prof.LoukiaZervaforcriticallyreviewingthispaper.
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