w w 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
Analysis
of
drug
resistance
mutations
in
pulmonary
Mycobacterium
tuberculosis
isolates
in
the
Southern
coastal
region
of
Andhra
Pradesh,
India
Polu
Giri
Prasad
a,
Mohammad
Shaik
Jasmine
b,
Kota
Neela
Mani
kanta
b,
Karumanchi
Deepthi
c,
Uday
Sankar
Allam
b,∗aDamienFoundationUrbanLeprosy&TBResearchCentre,AndhraPradesh,India bVikramaSimhapuriUniversity,AndhraPradesh,India
cKrishnaInstituteofMedicalSciences(KIMS),AndhraPradesh,India
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received1February2019 Accepted10July2019
Availableonline14August2019
Keywords: Multidrugresistance Mycobacteriumtuberculosis MTBDRplusassay Mutations Moleculardetection
a
b
s
t
r
a
c
t
Purposeandobjectives:Detectionofdrugresistanceplaysacrucialroleintuberculosis(TB) treatmentandpreventionofMycobacteriumtuberculosis(MTB)transmission.Theaimofthis studywastodeterminethelevelsandpatternsofresistanceofMTBisolatestotwokey anti-TBdrugs(rifampicin,RIFandisoniazid,INH)andthetypeofmutationsindrugresistance genes(rpoB,katGandinhA)oftheisolatesatthesoutherncoastalregionofAndhraPradesh, India,usingcommerciallyavailableGenoTypeMTBDRplusassayundertheRevisedNational TBControlProgram.
Methods:GenoTypeMTBDRplusassaywasperformedon2859sputumsmear-positive sam-plesandthemutationsinthegenesresponsibleforresistance(rpoB,katGandinhA)were analyzed.
Results:Amongthelineprobeassay(LPA)validisolates(2894),1990(68.76%)weredrug sus-ceptible,437(15.13%)wereINHmonoresistant,104(3.59%)wereRIFmonoresistant,and363 (12.54%)weremultidrugresistant.Codon531ofrpoBgeneandcodon315ofkatGgenewere foundtohavethehighestmutationfrequencyforRIFresistance(270/467;57.81%)andINH resistance(501/800;62.62%),respectively.TheRIFresistantrpoBmutationsobservedinthe sampleswereS531L(57.81%),H526Y(8.56%),D516V(6.42%),andH526D(6.20%).Mutations ininhApromoterwerefoundin24.75%INHresistantisolateswithC15Tbeingthemost common(85.85%).TheturnaroundtimesoftheLPAtestwerefrom48to72h.
∗ Correspondingauthorat:DepartmentofBiotechnology,VikramaSimhapuriUniversity,Nellore524320,India.
E-mailaddress:[email protected](U.S.Allam). https://doi.org/10.1016/j.bjid.2019.07.002
1413-8670/©2019PublishedbyElsevierEspa ˜na,S.L.U.onbehalfofSociedadeBrasileiradeInfectologia.Thisisanopenaccessarticle undertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Conclusion: ThefrequencyofmutationsinMTBinthecoastalregionofAndhraPradesh, India,issimilartothatinretreatmentcasesfrommostsettings,withcloseto80%inrpoB
codon516,526,and531,andover80%incodonskatG315and/orinhApromoter.Theincrease inINHmonoresistanceunderlinestheneedforgreaterenforcementofnationalTBcontrol programs.
©2019PublishedbyElsevierEspa ˜na,S.L.U.onbehalfofSociedadeBrasileirade Infectologia.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http:// creativecommons.org/licenses/by-nc-nd/4.0/).
Introduction
Tuberculosis(TB),causedbyMycobacteriumtuberculosis(MTB), isoneofthe leadingcauses ofdeathworldwide.Themost recentreportsofWorldHealthOrganization(WHO)showthat therewere9.0millionnewcasesofTBand1.5milliondeaths due toTB.1 Despite the evidence that TBis slowly
declin-ing,worldwideemergenceandspreadofmultidrugresistance (MDR;resistant toatleast oneofthekey first-line anti-TB drugs(isoniazid(INH)andrifampicin(RIF))andextensivedrug resistance(XDR)(MDRplusadditionalresistancetoa fluoro-quinoloneandanysecond-lineinjectabledrug)inMTBstrains havebecomeamajorobstacleforTBcontrol.2
TheemergenceofMDRandXDRTBhasleftclinicianswith hardlyany treatmentoptions, andtheirspreadhascreated aserious threattopublic health.MDR and XDRTBare on therise mainlydue toinappropriatedrugregimen,patient defaulting,previousanti-TBtreatment,delayindiagnosisand initiationofeffectivetreatment,andprimaryinfectionwith MDRTBstrains.PatientswithMDRandXDRTB(thosewhodo notrespondtotreatment)canbeaconstantsourceof trans-missionofdrug-resistantMTB.Accurateandrapiddetection ofMDRTBiscriticalfordecreasingthetransmissionofTBand reducingtheamplificationofdrugresistance.3
AcquisitionofdrugresistanceinMTBdoesnotoccurasa resultofhorizontaltransferofresistance-determininggenes orregion,butresultsfrommutations(causedbysubstitutions ordeletionsorinsertionsofnucleotidesequences)inspecific resistance-determining regionsofthe gene targets ortheir promotersoractivatingenzymesofanti-TBchemotherapeutic agents.MutationshavebeenreportedinthegeneskatG,inhA
and ahpC (resistance against INH); rpoB(resistance against Rifampicin,RIF);andinhA(resistanceagainstEthionamide).4
Conventionalcultureanddrugsusceptibilitytesting(DST) forMTBonsolid/automatedliquidculturesystemsisa time-consumingprocedure(takesupto8–12weeks),suffersfrom highercontaminationrates,andrequiresconsiderable equip-ment,media,andtechnicalexpertiseorhumanresources.5
However,basedonthe knowledgeofreportedmutationsof theanti-TBdrugresistancegenes,rapidmoleculardiagnostic lineprobeassay(LPA)hasbeendevelopedforthedetection of drug resistance in MTB. INNO-LiPA RifTB (Innogenetics N.V,Ghent,Belgium),GenoType®MTBDRplus(fordetectionof INHandRIFresistance),andGenoType®MTBDRsl(for detec-tionofresistanceagainstethambutol,fluoroquinolonesand aminoglycosides) (Hain Lifescience, Nehren, Germany) are commerciallyavailabletypesofLPA thatare beingused to detect simultaneously the reported mutations in the drug
resistancegenesofMTB.In2008,genotypeMTBDRplus LPA hasbeenrecommendedforrapiddetectionofdrugresistance ofMTBbyWHO.1
Manystudieshaveshownthatthespecificityand sensitiv-ityofGenoTypeMTBDRplusassayforRIFandINHresistance arefoundtobecomparabletoconventionalphenotypicdrug susceptibilitytesting(DST).6–8Ameta-analysisstudycarried
out in2008foundthattheGenoTypeMTBDRplusassayhas asensitivityof98%fordetectingRIFresistanceand89%for detectingINHresistance,andspecificityof99%forbothRIF andINH.9
Globally,manyreportshavebeenpublishedondetection and diagnosis that are based on rapid advanced molecu-lar assays. However,owingtothe geographicaldiversityof MTBclinicalisolatesacrosstheworld,thereisapossibility ofvaryingefficacyofmoleculargenotypingmethods,which mayhavediagnosticimplications.Inaddition,themutations responsibleforresistancecoulddifferwithregionand thus provideaninsightintotheepidemiologyofthedisease. How-ever,themagnitudeofdrugresistantMTBisolatesandpattern ofdrugresistancemutationsisnotwellknowninthesouth coastalregionofAndhraPradesh,India.Therefore,thepresent study wasundertakentodeterminetheanti-TBdrug resis-tanceandassociatedpatternsofmutationsinrpoB,katGand
inhAgenesofMTBisolatesfrompatientsofthisregionusing commerciallyavailableGenoTypeMTBDRplusassayunderthe RevisedNationalTBControlProgram(RNTCP)ofIndia,andto evaluatethepossibleriskfactorsassociatedwith mycobacte-rialresistanceinthepatientsofthisregion.
Materials
and
methods
Mycobacteriumtuberculosisisolates,studysites,and sampleprocessing
Thisstudywaspartofaprogrammaticmanagementofdrug resistant TB,conductedbyDamien FoundationIndiaTrust, with thesupport oftheDistrict TBControlOfficer (DTCO), Nellore,AndhraPradesh,India.ThiscenterhasaTBculture anddrugsusceptibilitytesting(C&DST)referrallaboratoryfor RNTCP,accreditedbytheNationalMycobacteriology Accred-itation System of Central TB Division Ministry of Health, GovernmentofIndia.Datainthisstudyarefrompatientswho hadregisteredduringaperiodofoneyear.
Five thousand and eighty-seven non-duplicate(one iso-late per patient) clinical isolates of MTB were obtained from the clinical samples of pulmonary TB patients from microscopycentersoperatingundertheaegisoftheRNTCP,
locatedin Southern districtsofAndhra Pradesh (Anantha-pur,Chittoor,Prakasam,Kadapa,Kurnool,andNellore),India. Standard microbiological techniques were used for isola-tionand identificationofthe isolates.Allthe chosencases werepulmonaryTBpatientswithsymptomsofweightloss, chestpain, night fever,blood mixedsputum, andso forth. Informationabout patients was obtained byphysicianson astandardformthatisusedforpatientssuspectedtohave TB. This study received ethical clearance from the ethics committeeofKrishnaInstitute ofMedicalSciences (KIMS), Nellore.Patients wereidentifiedand managedaccording to RNTCP guidelines (RNTCP, 2009 guidelines).Informed con-sentwasobtainedfromallparticipantsincludedinthestudy. AccordingtoRNTCPguidelines,occurrencesofTBinpatients who underwentcategory IItreatment(2(SHRZE)3+1(HRZE)3
5(HRE)3) with four-month follow up were considered as
previouslytreated casesand those inpatients who under-wentcategoryItreatment(2(HRZE)34(HR)3)wereconsidered
asnewcases(H=isoniazid,R=rifampicin,Z=pyrazinamide, E=ethambutol,S=streptomycin).
Isolation and identification of MTB was carried out in acertifiedmicrobiologylaboratoryusingZiehl–Neelsen(ZN) stainingor acid-fastbacilli (AFB)stainingtechnique.10 The
smearsweregradedaccordingtothenumberofbacilliseen on the slide, as per RNTCP guidelines. All smear positive (>1+AFB)sputumsamplesreceivedwithin48–72hofsample collectionincoldchainweresubjectedtoonlyLPA.Smear neg-ativeandscantyacidfastbacterialsamplesweresubjected tocultureonLowenstein–Jensen(L–J)solidmediaforgrowth ofMTB.Theculture-positivesamplesforMTBwere further subjectedtoLPAanalysis.Thesampleswereprocessedby N-acetyl-L-cysteine-sodiumhydroxide(NALC-NaOH)methodof digestionanddecontamination.11
Lineprobeassay/GenotypeMTBDRplusassay
The GenoType MTBDRplus assay version 2.0 (Hain Life Sciences, Nehran, Germany) was performed in accordance with the manufacturer’s instructions (http://www.hainlifescience.de). The LPA is a multiplex polymerase chainreaction (PCR) based genotypictest that identifies MTB complex and simultaneously detects the mutationsthatconferresistancetoRIF andINH.Theassay has an additional advantage over other LPAs because the GenoTypeMTBDRplusassayidentifiesmutationsintherpoB
gene(coding forthe ß-subunit oftheRNA polymerase)for detectionofRIFresistance,mutationsinthekatGgene(coding forthecatalaseperoxidase)forhigh-levelINHresistance,and mutationsinthepromoterregionofinhAgene(codingforthe NADHenoylACPreductase)forlow-levelINHresistance.H37 RVwasusedasthepositivecontrol.
Identificationofnon-tuberculosismycobacteria(NTM) Identification ofnon-tuberculosis mycobacteria (NTM) was donebasedoncolonymorphology,pigmentation,growthrate onconventionalsolid,andmediacontainingp-nitrobenzoic
acid(PNB).Inaddition,biochemicaltestssuchasniacintest andheat-stablecatalasetest(pH6.8,68◦C)wereperformedas
Suspected MDR-TB cases (5087) AFB staining Smear negative (2200) Smear positive (2859) Rejected (28) Culture positive (204) Scanty (380) +3 (852) +2 (788) +1 (839) Genotypic MDR plus/LPA LPA invalid (166) Biochemical analysis NTM (15) RIF (104 (3.59%) INH (437 (15.10%) MDR (363 (12.54%) Drugs sensitive 1990 (68.76%) LPA valid (2894) Drug resistant (904 (31.23%)
Fig.1–Flowdiagramofoverallmethodologyfollowedin thestudyandpatternofdrugresistanceofMTBisolates. Figuresinbracketaresamplenumberandtheirrespective percentageoftotal.
pertheprotocolsinTrainingManualforMycobacterium tuber-culosisCulture&Drugsusceptibilitytesting.10
Turnaroundtime(TAT)
Turnaroundtimewascalculatedasthetimebetweenthedate ofreceivingsputumsampleattheprimaryhealthcenterand dateofMTBDRplustestresults.
Statisticalmethods
SPSSsoftware20.0wasusedfordataanalysis.Thestatistical analysiswas performedusingSPSSsoftware(Version 17.0). Allvaluesareexpressedintheformofpercentagesandthe Chi-square testwas appliedwherevernecessary. Statistical significancewassetatp≤0.05.
Results
A total of 5087 sputum samples were obtained from pri-maryhealthcentersofvarioussoutherndistrictsofAndhra Pradesh,India,fromDecember2013toDecember2014 (one-yearperiod).Amongthestudyisolates,2859(56.14%)samples were AFB smearpositive and 2200 (43.24%) samples were smearnegative.Thesmear-positivesamplesweregradedon ascalefrom0to3+.Threehundredandeighty(13.09%) sam-plesgavescantypositiveresultsand30sampleswererejected. Atotalof839(29.34%)sampleshadanAFBcountofoneto 10per100fields(smear1+),788(27.56%)sampleshadoneto ninebacilliperfield(smear2+),and852(29.80%)sampleshad morethanninebacilliperfield(smear3+)(Fig.1).Ofthe smear-negativespecimens,204/2200(9.27%)wereculturepositivefor AFBand were includedforthefinalLPA analysis,ofwhich 204/204(100%)confirmedtobeMTBcomplex(i.e.,presenceof TUBband(Fig.2)).
Fig.2–RepresentativeDNAstrippatternsofGenoTypeMTBDR-plusstrip.Lane1,M.tuberculosiscomplexH37Rvlaboratory controlstrain(rpoB,katG,inhAWT);Lane3,4,6,9&10sensitivetoRifampicin(RIF)andIsoniazid(INH);Lane2,INH monoresistant(katGS315T1mutation)Lane5,MDR-TB(rpoBS531LandkatGS315T1mutation);Lane7,RIFmonoresistant (rpoBunknownmutation);Lane8,TUBbandabsent(LPAinvalid)Lane11,DNAnegativecontrol.
Table1–Distributionofhostandmicrobialcharacteristicsamongdrug-sensitive,drug-resistant,andMDRTBcases amongpatientsofsoutherncoastalregionofAndhraPradesh,India.
S.No Characteristic Value Invalid Drugsensitive INHmonoresistant RIFmonoresistant MDR
Sex 1 Male 2481(81.10%) 119 1645(66.30%) 376(15.19%) 78(3.14%) 263(10.56%) 2 Female 579(18.92%) 47 345(59.58%) 61(10.53%) 26(4.49%) 100(17.27%) Age(Range) 1 <20 160(5.22%) 9 99(61.87%) 14(8.75%) 10(6.25%) 29(18.12%) 2 21–40 1410(46.07%) 66 901(63.90%) 208(14.75%) 48(3.40%) 187(13.26%) 3 41–60 1267(41.47%) 77 842(66.45%) 184(14.82%) 39(3.07%) 125(9.78%) 4 ≥61 223(7.28%) 14 148(66.364%) 32(14.34%) 7(2.13%) 22(9.86%) HIV-Co-infection 1 +ve 227(7.41%) 6 178(78.41%) 25(11.01%) 4(1.76%) 14(6.16%) 2 −ve 2833(92.58%) 160 1812(63.96%) 412(14.54%) 100(3.52%) 349(12.31%) TBhistory 1 New 243(7.94%) 6 184(75.72%) 29(11.93%) 5(2.05%) 19(7.81%) 2 Treated 2817(92.05%) 160 1806(64.11%) 408(14.48%) 99(3.51%) 344(12.21%)
INH,Isoniazid;RIF,Rifampicin;MDR,MultidrugResistant;Invalid-Sampleswereeithernegativeorresultswerenotclear.
Ofthefinal3060sputumsamples,2481(81.10%)werefrom malepatientsand579(18.92%)fromfemalepatients.Majority ofthepatientswereinthe21–40(1410/3060;46.07%; econom-icallyproductivegroup)and41–60(1267;41.47%)agegroups. Outof3060patients,227(7.41%)werecoinfectedwithHIVand MTB(Table1).Themeanageofthestudypatientswas41.5 years.Amongthe3060cases,243(7.94%)werenewand2817 (92.05%)includedpatientswhowereundergoingTBtreatment orpreviouslytreated(i.e.,DOTSpluscategoryIItreatment).
TheMTBDRplustestwasdonefor3060TBsuspected sam-plesandamongthese,validresultswere obtainedfor2894 (94.57%) samples.Theremaining166 (5.42%)sampleswere eithernegativeor resultswere not clear;hence,these iso-lateswere notincludedforfurtherMTBDRplusstudies.LPA invalidsamples were furthersubjected tobacterial culture andbiochemicalmethodstocharacterizethesmear-positive and LPA-negative isolates.Out of166 acid fastisolates, 15 (0.5%)wereidentifiedasNTMbasedontheirgrowthonmedia containingPNB, negativeresponse toniacintest, and pos-itive responsetocatalase activity at68◦C and pH6.8. The MTBDRplusLPAinvalidrateincreasedas13/852(1.5%),78/839 (9.3%),14/788(1.8%),and61/380(16.0%);andtheAFBsmear
gradingdecreasedas3+,2+,1+,andscanty.Thiscorrelation wasstatisticallysignificant(p<0.005).
DirectMTBDRplus testresultsshowedthat2894sputum sampleswere MTBpositive,that is,presenceofTUBband, whichboundtheampliconstotheMTBcomplex.Drug resis-tancetooneormoreanti-TBdrugswasfoundin904(31.23%) MTBisolatesof2894patients (Fig. 1). AsshowninTable1, among MTBDRplus performed, 1990 (68.76%) isolates were susceptibletobothRIFandINH,104(03.59%)wereRIF monore-sistant,437(15.10%)wereINHmonoresistant.and363(12.54%) wereMDR(resistanttoboththefirstlineofdrugs:RIFandINH) (Table1andFig. 1). Anaverageturnaroundtime, including specimentransportationtimeforLPAperformedfrom differ-entregions,wasfivedays.
TheGenotypeMTBDRplusdetectedmutations, responsi-bleforRIFandINHresistance,aredisplayedinTables2and3, respectively.Amongall467RIFresistantisolates (363-MDR-TB strains and 104-RIF monoresistant), missing wild type (WT) bandalongwiththe presenceofknownmutantband wasdetectedin369(79.01%) andmissingWTwithnogain in mutant band was found in 98 (20.98%) strains (i.e., no hybridization totherpoBWTnortoeitherofthemutation
Table2–GenemutationpatternasdetectedbyGenoTypeMTBDRplusassayinRifampicinresistantMTBisolates.
Gene Bandmissing Generegion Mutationspresent MDR ’RIF’Monoresistant Total
+ H526Y 5 3 8 rpoB + S531L 6 6 12 + D516V 1 3 4 + D516V,S531L 1 0 1 + H526D 3 2 5 WT1,WT2 506-509,510-513 UK 0 1 1 WT1,WT7 506-509,526-529 H526Y 1 0 1 WT2 510-513 UK 12 6 18 WT2,WT3 510-513,513-517 UK 3 1 4 WT2,WT3 510-513,513-517 H526D 0 1 1 WT2,WT3,WT4 510-513,513-517,516-519 UK 5 0 5 WT2,WT3,WT8 510-513,513-517,530-533 UK 2 1 3 WT2,WT7 510-513,526-529 UK 1 0 1 WT2,WT8 510-513,530-533 UK 5 1 6 WT3 513-517 UK 3 1 4 WT3 513-517 D516V 1 0 1 WT3,WT4 513-517,516-519 D516V 18 5 23 WT3,WT4 513-517,516-519 UK 13 4 17 WT3,WT4,WT7 513-517,516-519,526-529 UK 1 0 1 WT3,WT4,WT8 513-517,516-519,530-533 UK 3 1 4 WT3,WT4,WT8 513-517,516-519,530-533 D516V 1 0 1 WT4,WT5 516-519,518-522 UK 2 2 4 WT4,WT5,WT8 516-519,518-522,530-533 UK 1 0 1 WT5,WT6,WT8 518-522,521-525,530-533 UK 1 0 1 WT5.WT6 518-522,521-525 UK 1 0 1 WT5,WT8 518-522,530-533 UK 0 1 1 WT6 521-525 UK 1 0 1 WT6,WT7 521-525,526-529 UK 1 0 1 WT7 526-529 H526Y 22 1 23 WT7 526-529 H526D 19 3 22 WT7 526-529 H526Y,H526D 2 0 2 WT7 526-529 UK 17 2 19 WT7,WT8 526-529,530-533 UK 1 0 1 WT7,WT8 526-529,530-533 H526D 1 0 1 WT7,WT8 526-529,530-533 H526Y 1 0 1 WT8 530-533 S531L 203 55 258 WT8 530-533 UK 0 4 4 WT8 530-533 H526Y 5 0 5 Total 363 104 467
MDR,Multidrugresistant;RIF,Rifampicin;WT,Wildtype;MUT,Mutant;UK,Unknown;+,Heteroresistant.
Table3–PatternofgenemutationsdetectedbyGenoTypeMTBDRplusassayinIsoniaziddrug-resistantMTBisolates.
Gene Bandmissing Generegion Mutationspresent MDR ’INH’monoresistant Total
katG WT 315 S315T1 247 250 497 WT 315 S315T2 2 2 4 WT 315 S315T1,S315T2 0 4 4 WT 315 UK 31 47 78 + S315T1 3 16 19 Total 283 319 602 inhA WT1 15/16 C15T 62 92 154 WT1 15/16 UK 2 2 4 WT2 −8 T8C 2 6 8 WT2 −8 UK 0 1 1 WT2 −8 T8A 0 2 2 WT1,WT2 15/16,-8 C15T 9 7 16 WT1,WT2 15/16,-8 UK 2 4 6 + T8C 1 1 + C15T 2 4 6 Total 80 118 198
probes). These isolates with the absence of both WT and mutantband were consideredas unknown(UK). Themost prominentknowngeneticmutationconferringRIFresistance wasincodonS531LofrpoBgene(271/467;58.8%)diagnosed bylossofthe WT8 band and presenceofMUT3band, fol-lowedbyH526Y mutation(40/467;8.56%), D516Vmutation (30/467; 6.42%), and H526D mutation (31/467; 6.63%). The mutationS531L was foundin 210/363 (57.85%) and 61/104 (58.65%)ofMDRstrainsand mono-RIF strains,respectively. ThisdifferenceofrpoBS531LmutationsinMDR-TBisolates compared with RIF monoresistant isolates was not statis-ticallysignificant.In threeMDR-TB strains, morethan one mutation(D516V,S531L-(n=1)andH526Y,H526D-(n=2)was foundinrpoBgene(Table1)andwereabsentinRIF monore-sistantisolates.AmongtheseMDR-TBisolateswithmorethan onemutation,oneisolateshowedmutationsintwoseparate codons,thatis,516and531andtwoisolatesshowed muta-tionsinthesamecodon,thatis,526.
Out of 98 (20.98%) RIF-resistant isolates with unknown mutationsormutantbands,25wereobservedinRIF monore-sistantstrains and 73 were observed inMDR isolates. The 98isolatesincluded missingWT1/WT2 (1;1.02%),WT2 (18; 18.36%), WT2/WT3 (4; 4.08%), WT2/WT3/WT4 (5; 5.10%), WT2/WT3/WT8(3;3.80%),WT2/WT7(1;1.02%)WT2/WT8(6; 6.12%),WT3(4;4.08%),WT3/WT4(17;17.34%),WT3/WT4/WT7 (1; 1.02%), WT3/WT4/WT8 (4; 4.08%), WT4/WT5 (4; 4.08%), WT4/WT5/WT8 (1; 1.02%), WT5/WT6/WT8 (1; 1.02%), WT5/WT6 (1; 1.02%), WT5/WT8 (1; 1.02%), WT6 (1; 1.02%), WT6/WT7(1;1.02%), WT7(19;19.38%),WT7/WT8 (1;1.02%), WT8 (4; 4.08%). Mixed pattern to RIF with all WT probes presentalong withthe presenceofoneor moreadditional mutantbands was found in30/467 (6.42%), mostcommon beingS531L(13/30;43.3%), followedbyH526Y(8/30;26.6%), D516V (5/30; 16.6%), and H526D (5/30; 16.6%), while one MDR-TB isolate with D516V had an additional mutation, S531LintherpoBgene.
Out of 800 INH-resistant isolates detectedby GenoType MTBDRplus,mutationsinkatGwerefoundin602/800(75.25%) isolatesand mutationsin inhAwere foundin198 (24.75%) isolates.Outof602INH-resistantisolates,mutationsinkatG
werefoundin319(53%)INH-monoresistantisolatesand in 283(47%)MDRisolates.Knownmutationsweredetectedat S315T1(MUT1band)in520/602(86.37%)andS315T2in8/602 (1.32%) ofthe katG mutant isolates. Four INH-mono resis-tantisolatesshowedthepresenceofbothS315T1andS315T2 mutationsinkatGandwereabsentinMDR-TBisolates. Miss-ingWTswiththeabsenceofmutantbandwerefoundin78 (12.95%)ofkatGmutants,andoutofthese,31werefoundin MDRisolatesand47inINHkatG-monoresistantstrains.Mixed patterntoINHwithallWTprobespresentalongwiththe pres-enceofoneormoremutantbandswasfoundin19(3.15%) isolates,knownmutation,S315T1waspresentinallthe iso-latesofbothMDR(3/19)andINHkatGmonoresistantisolates (16/19).
MutationsininhAwerefoundin198(24.75%)INH-resistant isolates,ofwhich118were ininhAINH-monoresistant iso-lates and 80 were in MDR isolates. The most commonly knowngeneticmutationobservedininhAregulatoryregion wasC15T(176/198; 88.88%), followedby T8C(9/198;4.54%) andT8A(2/198;1.00%).MissingWTswithunknownmutations
werefoundin11(5.55%)inhAmutantisolates.MixedWTand mutantpatterntoINHwasfoundin7/198(3.5%)ofthetotal INH-resistantisolates.15/437(3.4%)oftheINHmonoresistant isolateshad mutationsinbothinhAand katGgenes. Muta-tions inC15Twere relativelyhigherinINHmono-resistant (103/118)thanMDRstrains(73/80),butthedifferencewasnot significant.MutationsinrpoBandkatGgeneaccountfor78% (283/363) ofall MDR casesandmutationsinrpoBand inhA
genesaccountfor22%(80/363)ofalltheMDRcases.NoMDR casewasfoundhavingmutationsinallthreegenes(rpoB,katG
&inhA).
Discussion
Andhra Pradesh (AP) is one of the largest south-ern states in India and it records 1, 07,293 patients, annually, enrolled under RNTCP program for TB treat-ment with ca. 8% of the suspected harboring MDR TB (http://www.tbfacts.org/tb-statistics-india/,2015).Since2006, APhasbeenimplementingthesecondlineanti-TBtreatment under the Directly Observed Treatment Short course plus program(DOTSplus) ofRNTCPfordiagnosisand treatment. In this study,we aimedtodetermine the patterns ofdrug resistanceagainsttwofirstlineanti-TBdrugs(RIFandINH). Wealsoanalyzedthe frequencyandtypeofdrug resistant mutationsin the81-bp hotspotregionofrpoBgene, codon 315ofkatGgeneandalterationsintheinhApromoterregion foundupstreamfromthemabA-inhAlocusinclinicalisolates frompulmonaryTBpatientsofsoutherndistrictsofAP,India, enrolled undertheRNTCPprogramusingthecommercially availableGenoType® MTBDRplusassay.
TheoverallRIFresistanceinourstudywas51.65%(467/904) of drug resistant isolates and is considered as surrogate markerfordetectionofMDRTB.Theisolatesmonoresistantto RIFwerealsoconsideredunderMDRcategoryandthepatients were given treatment under categoryIV of DOTSplus pro-gram(treatmentgiventoMDRpatients).Similarfindingswere reportedbyJanmejaandRaj,1998,fromHaryana(49%)12and
Rawatetal.,2009,fromUttarakhand(57.22%).13However,
per-cent RIF resistanceobservedinourstudy wasmuchlesser thanthepercentRIFresistancereportedinotherstudiesby Singhaletal.,2015fromNewDelhi,India(73.9%)14andBarnard
et al., 2008from SouthAfrica(91%).15 AmongRIF-resistant
MTBisolates,58.8%hadasinglenucleotideSer-to-Leu sub-stitutionatrpoB531positionfollowedbyHis-to-Aspat526, Asp-to-Valat516,andHis-to-Tyrat526positions.Thesedata areinaccordancewiththemutationfrequenciesreported pre-viouslybyotherstudies.8
Accordingtotheresultsofmanyearlierstudies,themost commonlyobservedmutationsamongallthemutations lead-ingtoRIFresistancewasinthe531regionofrpoBgene,mainly S531Lmissensemutation(58.8%).Mostofthepointmutations observedinthisstudywereinthecodons531,516,and526. Theseresultscoincidewiththepreviousresultsreportedby others.ThefrequencyofthemajormutationS531L(serineto threoninesubstitution)(58.8%)observedinourstudyis con-sistentwiththose foundinother studiesinIndia((Singhal etal.,2015(59.0%),14Lingalaetal.,2010(53.6%),16Mauryaetal.,
et al., 2014 (50%), and South Vietnam (Huyen et al., 2010 (50%)).18,19Nonetheless,thefrequencyofS531Lwasfoundto
belowerthan someofthe otherreportsfrom India(Yadav etal.,2013(72%),Mohanetal.,2014(81.5%)andRaveendran etal.,2012(84.6%)),Germany(Hillemannetal.,2007(73.6%)), Denmark(Vijdeaetal.,(86%)),andRussia(Nikolayevskyyetal., 2010(94%)).6,20–24 In the present study,frequency ofS531L
mutationwas nearly same in both RIF monoresistant iso-lates(55/104;53%)andMDR-TBisolates(203/363;56%).This isincontrasttoother reportsfrom IndiaandSouthAfrica, wheretheS531Lmutationwassignificantlyhigherin MDR-TBthan inRIFmonoresistantstrains.14,15,25 However,these
resultswereinconcordancewiththeresultsofYadavetal., 2013andRaizadaetal.,2014.6,9
ForINHresistance,ithasbeenreportedpreviouslybymany authorsthatthemostcommonmutationobservedwasinthe 315regionofkatG,whichwaspresentin75.25%(602/800)of allINHresistantisolatesandtheS315T1mutation(497/602; 82.5%)thatledaminoacidserinetothreoninesubstitutionhas alsobeenrelatedtohighlevelofresistancetoINH.Theseare inaccordancewiththeworldwidereportedfigures(50–90%) ofall observed phenotypic INH resistance associated with thekatG315mutation.InTBendemiccountries, ithasbeen reportedthathighprevalenceofkatGmutationwasassociated withamajorityoftheINHresistantisolates.17,19,26–28
MutationsininhApromoterwerefoundin24.75%(198/800) INHresistance,whileC15Tbeingthe mostcommon muta-tion(85.85%;170/198).TheinhAmutationsaccountedfor27% (80/363)and20.88%(118/437)ofmono-INHandMDRstrains, respectively.Out of198 totalINHdrugresistant mutations, 60%(118/198)ofinhAmutationswerefoundinINH monore-sistantand40%(80/118)wereinMDRTBisolates.Mutation occurredin24.75%ofINHresistantMTBstrainsinthe reg-ulatoryregionofinhA(invariablyC15T),whichissimilarto otherstudiesbySinghaletal.,2015(13.4%),Huyenetal.,2010 (18%),andBrossieretal.,2006(17%),butconsiderablylower thanthatreportedbyBarnardetal.,2008(40%).8,14,19Lowor
intermediatelevelsofINHresistancewasshowntobe asso-ciatedwiththemutationsofregulatoryregionofinhAgene (20–35%)byvariousstudies,whichareinagreementwithour results.29
Inourstudywedidnotfindanycombinedmutationsin
katG and inhA, as compared withother studies.6,30 Unlike
RIFresistancethathasbeenshowntobeasurrogatemarker fordetectionofMDRTB,theclinicalimplicationofthelow level INH resistance needs further investigations. We did notfind any association between a particularmutation in
katGand/orinhAandtheoccurrenceofINHmonoresistance or MDR TB.In contrast to our results,previous studies by Barnardetal.,2008,andBolotinetal.,2009,reporteda sig-nificantlyhigherlevelofassociationofS315TofkatGand/or
inhAmutationsinMDR-TBisolatesthan inINH monoresis-tantTBisolates.MutationsinthekatGgeneofstrainsresistant toINHwereobservedathigherfrequencycomparedwiththe
inhApromotermutations.15,31UnlikeRIFresistancemutations
(specificto81-bpregionoftherpoBgene),mutationscausing resistancetoINHaremorecomplexandinvolvesmutations inseveral genessuch askatG, ahpC, fabG1, kasA,furA, ndh
andoxyR-ahpCintergenicregion.32,33Consideringthis,LPAtest
mighthavefailedtodetectallmutationsoutsidethekatGgene
thatis,inothergenomicregionsthatconferINHresistancein someofthedrugresistantisolates.FurthersequencingofTB strainswouldbenefittocharacterizethemutationsinthegene regionotherthankatGgeneandinhApromoter.
Overall MDR-TB rateobserved inthis study was 12.54% (363), which is slightly lower than that in other studies reported from other parts of India (Chandigarh (27.6%),34
Tamil Nadu(25%),35 Andhra Pradesh(26.7%),20 and Gujarat
(30.2%)36).In contrast,higher rates ofmultidrugresistance
wereobservedinDehradun(57.22%)13andDelhi53.6%37and
rates are inconcordance withthose inSewagram Wardha (9.2–9.6%).38
Inthisstudy,weobservedsignificantlyhighernumberof isolateswithmissingWTbandandwithoutmutantbandand were intherate of21% (n=98/467)inrpoB,13% (n=78/602) in katG,and 5.55% (n=11/198) ininhA, which indicate that the mutationsconferringdrug resistanceare notthe com-monmutationsofthegenicregionthatareincorporatedin the present MDRTBplus stripand are probablyrare muta-tions. Interestingly, such unknown mutations were found comparativelymoreinRIFmonoresistant(24.03%vs20%)and INHmonoresistantisolates(12.5%vs9.6%)thaninMDR-TB isolates.Butthedifferenceobservedwasnotstatistically sig-nificant.Similarly,highernumberofnewmutationshavebeen reported from India,14,39 Vietnam19 and Uganda.40 Further
characterizationofthesegenemutationsisnecessaryinorder to detect newand emerging drug resistance mutations. It wouldbeinterestingtoknowanydirectrelationshipbetween specificmutationsdetectedwithtreatmentoutcomesofINH monoresistantcases.
Another interestingobservationwas ahigherpercentof TB isolates from female patients that were found to be MDR(100/579;17.27%)comparedtomalepatients(263/2481; 10.56%),thoughthemajorityoftheisolateswerefrommale patients (2481/3060, 81.10%) compared to female patients (579/3060,18.92%).Thedifferencebetweenthetwowas sta-tisticallysignificant(2=21.863,p<0.005).Thesefindingswere
inlinewithotherstudiesreportedbyBaziraetal.,2010and Lomtadzeetal.,2009.Thismightberelatedtohealthseeking behavior, withprolonged delays infemale patients (proba-blyduetolackofcontroloffinancialresourcesathousehold levels)ashasbeenreportedbyBaziraetal.,2010.41,42
How-ever,reasonsfortheincreasedriskofMDRTBamongwomen patientsobservedinourregionare notknownand require furtherinvestigation.
Patients’ age-wisedistribution of MDRTB also revealed someinterestingobservations.Of2894MTBpositivepatients, 86% of the patients were in the age group of20–50years. Intriguingly,high percentofMDR-TB(29/363;18.12%)cases werereportedintheagegroupofbelow20years.Ofthe363 MDR-TB isolates,187/363(13.26%)were fromthe agegroup 21–40yearsand125/363(9.78%)werefromtheagegroup41–60 years(2=17.43,p<0.005).Asyoungadultmalesarefrom
eco-nomicallyproductivesectionofsociety,highMDR-TBburden inthemhavemanysocio-economicimplications.Wecould notgettheinformationregardingotherreportedclinicaland socio-demographic risk factors such as alcoholism, smok-ing, number of previous treatments, irregular treatments, lungcavities inchest X-ray,occupation, educationalstatus, monthly income, site of TB disease,and diabetes. Further
studiesarewarrantedtoanalyzetheriskfactorsofMDRTB inthisregion.
In India, MDR TB among notified new pulmonary TB patientsis2.2%with95%CIof1.9–2.6%andMDR-TBamong previouslytreatedpulmonaryTBcasesis15%with95%CIof 11–19%.43Globally,3.7%ofnewcasesand20%ofpreviously
treated casesare estimated tobe MDR TB. In the present study,the prevalenceof MDRTB caseswas high in previ-ouslytreated cases(12.21%;344/2817)and relativelylow in newcases(7.81%; 19/243).The differencebetween the two wasstatisticallysignificant(2=6.4,p<0.05).Theresultsofthe
presentstudy are inconcordancewiththe figuresreported byRNTCPand WHOglobalsurveys. Guptaet al.,2014,and Kauret al.,2016, had alsoobservedthe significantlyhigher numberofMDRTBinpreviouslytreatedcasescomparedto newcases.44,45 Asfoundinmany otheranti-TBdrug
resis-tancesurveillancestudies,ourstudyalsosuggeststhathistory ofanti-tuberculartreatmenthasbeenconsistentlyassociated withtheriskofMDRTB.14,44–46Similarly,primary
monoresis-tancetoRIFwasobservedinfive(2.05%)casesandacquired resistancein99 (3.51%)cases.Primary INHmonoresistance wasdetectedin29(11.93%)casesandacquiredresistancein 408(14.48%)cases.
TB is the most common opportunistic bacterial infec-tion amongthe humanimmunodeficiency syndrome (HIV) infected patients. Furthermore, MDR-TB co-infected HIV patientsareathigherriskofdeaththantheHIVnon-infected individualsindevelopingcountrieslikeIndia.47Inourstudy, out of2894TB positivepatients, 227 patients (7.41%)were foundtobeco-infectedwithHIV.Outof227HIVco-infected patientsamples,184(81.05%)weredrugsensitive,25(11.01%) wereINHresistant,4(1.76%)wereRIFresistant,and14(6.16%) wereMDR(resistanttobothINHandRIF).MDRwasseenin only6.16%isolatesascomparedto12.31%ofisolatesfromHIV negativecases(2=7.114,p<0.05).LowerincidenceofMDRTB
inHIV-infectedpatientshasalsobeenreportedbyPraharaj etal.,2004.However,theratesofHIV/TBco-infectionin dif-ferentregionsofIndiawerereportedtobebetween0.4%and 30%.48,49
Another important observation is presence of hetero-resistant populationcharacterizedbypresenceofboth WT and the mutant probes corresponding tosensitive or sus-ceptible and resistant isolate, respectively. High rate of hetero-resistancepatternwasobservedinallthreegenes,that is,rpoB(6.42%;30/467),katG(3.15%;19/602),andinhApromoter region(3.5%;(7/198).ThemostfrequentmutationwasS531L (40.0%)inrpoBregion,S315T1(100%)inkatGgene,andC15T ininhApromoterregion(85.7%).Inourstudy,comparatively, hetero-resistantpopulationwas moreinRIF monoresistant andINHmonoresistantisolatesthaninMDR-TBcases.A sim-ilarreportofhighprevalenceofhetero-resistantTBisolates were reportedinother studiesbySingh et al.,2014,Tolani etal.,2012,andC¸AVUS¸O ˘GLUetal.,2011.30Thepresenceof
hetero-resistant TBisolates indicatesthe slowevolution of mycobacteriafromasensitivetoresistantprofileduringdrug treatmentandfutureupsurgeofMDRTBinthisregion.Italso reflectsthesuperinfectionofbothsensitiveandresistant iso-latesinthesamepatientwhomustbegivenadrugresistant TBregime.Thestudiedriskfactorssuchasage,sex,history ofTB,andHIVstatuswerenotsignificantlyassociatedwith
theobservedhetero-resistant(datanotshown).Detectionof hetero-resistantTBisolatesisanotheradvantagefortheuse ofLPA.
MajorityofLPA invalidresultsofthepresentstudy were foundinculturenegativesamplesorsputumspecimenswith lower bacillary load (1+) and their frequencyinversely cor-related tosmeargrading ashasbeen reported byprevious studies.15,39 The results of the present study also support
theunsuitabilityofLPAtestfordirectuseonsmear-negative clinical specimens and also low specificity of the smear microscopy, which has been used as a standard point of care test to diagnose MTB.6,50 Further characterization of
smear-positiveandLPA-invalidsamplesusingphenotypicand biochemicalassayssuggestedtheprevalenceofNTM(n=15) inthisregion.Usingtheseassays,wecouldidentifyNTMupto genuslevelonlyandfurtherstudiesarewarrantedtoidentify NTMspeciesspectra,soastoinitiateappropriatetreatment tofurthercurbthespreadofNTM.Theresultsofthepresent studyalsoemphasizetheneedtoisolatethecausative organ-isminpureculture,assmearmicroscopydoesnotdistinguish MTBC that causes pulmonary TB from NTMs. Surveillance orepidemiologicalstudiesshouldbedonefurthertocontrol spreadoftheseinfections.
ThoughtheGenoTypeMTBDRplushashighaccuracy cou-pledwithreducedturnaroundtime,thetestcanonlydetect drugresistancemutationsthatoriginatefromrpoB,katG,and
inhA. The assay cannot detect mutations originating from othergenomicregionsandotherresistancemechanisms.This isespeciallytrueindetectingINHresistanceandexplainsthe comparatively lowsensitivityoftheassayindetectingINH resistance.Secondly,highpossibilityofsilentmutations,that is,mutationsthatdonotleadtochangeinaminoacidand hencethestrainbeingsensitiveinphenotypicdrug suscepti-bilitystudies.Thereportedturnaroundtestingtimeincluding specimentransportationtimewasslightlyhigher(fivedays) thantheRNTCPrecommendedTAT,thatis,withinfivedays. Theoretically,LPAwascompletedwithinthreedays,however, theobservedTAT,thatis,fivedays,wasmainlyduetolagin transportationofsamplesfromprimaryhealthcentertothe testinglaboratory.ThepatientswhowerediagnosedwithMDR TBwereprovidedwithcategoryIVtreatmentofDOTSplus programwithin10daysoftestresults.
In conclusion, the present study ascertained the preva-lence ofdrug-resistant TBisolates in southern districtsof AndhraPradeshandalsothepatternofrpoB,katG,andinhA
gene mutations in the drug-resistant isolates. This study revealed that the presence of MDR TB is a major serious publichealthproblemofthisregion.Furtherstudiesare war-ranted to know the transmission dynamics of these MDR TB isolates so as to curb further community and nosoco-mial transmission ofdrug resistant TB,especially, limiting MDR-TB and XDR-TB disease progression. Our study also reported a number of unknown or uncommon mutations prevalentinthisregion,whicharenotcoveredbyroutinely usedmoleculardiagnostickits.Specificmutationsfoundby theMTBDRplusassaymayhelpinempiricchoiceofananti-TB treatment regimen. Continued surveillance through tradi-tional culture and DST methods will remain importantto individualize and customize treatment regimens for drug-resistantMTB.
Funding
Theconsumables and machines for GenoType MTBDRplus assayhavebeenprovidedfreeofcostbyDamienFoundation IndiaTrust,India,formanagementofMDR-TBpatientsunder theprogrammaticmanagementofDrugresistantTB(PMDT).
Ethical
approval
Thisstudyreceivedethicalclearancefromtheethics commit-teeofKrishnaInstituteofMedicalSciences(KIMS),Nellore.
Informed
consent
Verbalinformedconsentwasobtainedfromallparticipants includedinthisstudy.
Conflict
of
interest
TheresearchersclaimNoconflictsofinterest.
Acknowledgement
Weacknowledgethetechnicalhelpofadministrativeand lab-oratorystaffofDamienTBresearchcentre,Nellore.Wealso acknowledgethesupportextendedbyDTCO’Sofsouthern dis-trictsofAndhraPradesh.WealsothankDebasmitaN,Gopala KrishnaMandSangeethaChakraborttyforcriticalappraisalof thearticle.Dr.UdaySankarAllamacknowledgesDepartment ofScienceand Technology(DST)for theirsupportthrough EarlyCareerResearchAward(ECRA). MohammadShaik Jas-mineacknowledgesUniversityGrantsCommission(UGC)for MaulanaAzadNationalFellowship.
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