1

Probingthedissonanceamongthediagnosticoutputsofmultipleapproachesusedfor1

detectionofMethicillin-resistantStaphylococcusaureus(MRSA)2

UjjwalRanjanDahiya,aArnabSikidar,aPriyankaSharma,bChitraRawat,cBenuDhawan,bArti3

Kapil,bRavikrishnanElangovan,dDineshKalyanasundarama,e*4

aCentreforBiomedicalEngineering,IndianInstituteofTechnologyDelhi,NewDelhi1100165 bDepartmentofMicrobiology,AllIndiaInstituteofMedicalSciences,NewDelhi1100296 cCouncilofScientificandIndustrialResearch(CSIR)-InstituteforGenomicsandIntegrative7 Biology(IGIB),NewDelhi1100208 dDepartmentofBiochemicalEngineeringandBiotechnology,IndianInstituteofTechnology9 Delhi,NewDelhi11001610 eDepartmentofBiomedicalEngineering,AllIndiaInstituteofMedicalSciences,NewDelhi11 11002912 *Correspondingauthor:dineshk@cbme.iitd.ac.in,dineshk.iitdelhi@gmail.com13 14

15

Thisistodeclarethatthefollowingauthorsdonothaveanyconflictofinterest:16

UjjwalRanjanDahiya:d.ujjwal.iitd@gmail.com17

ArnabSikidar:arnab.sikidar@gmail.com18

PriyankaSharma:priyankap2828@gmail.com19

ChitraRawat:chitrarawat11@gmail.com20

BenuDhawan:dhawanb@gmail.com21

ArtiKapil:akapilmicro@gmail.com22

RavikrishnanElangovan:elangovan@dbeb.iitd.ac.in23

DineshKalyanasundaram:dineshk.iitdelhi@gmail.com,dineshk@cbme.iitd.ac.in24

25

26

27

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NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice.

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2

Abstract28

Methicillin resistant staphylococcus aureus (MRSA) is an extremely infectious hospital29

acquiredbacterialpathogenoftenfoundinpost-surgicalpatientsglobally.Earlydetectionof30

such pathogens is a critical requirement to eliminate or reduce the incidence of anti-31

microbial resistance as well as for effective management of the disease. Despite the32

development of multiple biochemical, microbiological and nucleic acid amplifications33

techniques(NAATs),conventionalculturemethodsarewidelyusedclinicallyowingtohigh34

variabilitybetweenthemethods,technicalskillsandinfrastructuralneeds.Further,multiple35

reportssuggestsignificantvariationamongdiagnosticoutputforMRSAdetection.Thiswork36

attempts toprobe thediscordanceamong thediagnosticoutputof threecommonlyused37

methods,whiletryingtounderstandtheunderlyingcauseofvariability.MRSAdetectionon38

217clinicalpusisolateswascarriedoutusingthreedifferentmethodsnamely,conventional39

culturemethod,qPCR-basedamplificationandamodernLAMPbaseddetectionapproach.40

Also, to confirm the presence of MRSA and distinguish from coagulase-negative41

staphylococci(CoNS),aswellastoinvestigatetheobserveddifferencesbetweenqPCRand42

LAMP outputs, melt curve analysis was performed on discordant samples. LAMP based43

MRSAdetectionwasfoundtobetheoptimummethod.Insummary,thisstudyevaluatesthe44

diagnostic efficiency of the different detection methods, while probing for possible45

explanationsfortheobserveddifferences.46

Keywords:MRSA,LAMP,rapiddiagnosis,cross-reactivity,meltcurveanalysis(MCA),qPCR,47

Nucleicacidamplificationtechniques(NAATs) 48

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1.Introduction49

Globally,methicillin-resistantStaphylococcusaureus(MRSA)isassociatedwithnosocomial50

andcommunity-acquiredhealthcareinfections(1,2).Duetotheextensiveuseofantibiotics51

,anincreasingnumberofantibiotic-resistant(ABR)bacterialstrainssuchasMRSAiscausing52

enormoushealthcarechallengetomankind(3,4).Thepathogen iscapableofproducinga53

diverserangeoftoxinsandvirulencefactors,includingtoxicshocksyndrometoxin(TSST)54

(5). A higher mortality rate of about twenty-two percent is observed in MRSA infected55

patientsincomparisontofivepercentamongstnon-MRSApatients(6–8).Theresistanceof56

MRSAtowardsβ-lactambasedantibioticsincludingmethicilliniscausedbythepresenceof57

inherentβ-lactamaseaswellastheexpressionofmecAgeneresultingintheproductionof58

penicillin-bindingproteins (PBP,PBP2,andPBP2a) that shows loweraffinity toβ-lactam59

basedantibiotics(9).RapididentificationandtimelyisolationofMRSAinfectedsubjectsare60

crucialtoavoidcomplications(10,11).ConventionalmethodsforthedetectionofMRSAcan61

takeupto48hoursormoretimeduetotime-consumingprotocolsincludingculture,colony62

morphology,andanti-microbialsusceptibilitytesting(12,13).Lately,healthinstitutionsare63

givinghigh importance to rapid identificationof bacterial isolates and screeningof their64

antimicrobialsusceptibility,especiallyinpositivebloodcultureisolates(14,15).Thisledto65

deploymentofmultiplenucleicacidamplificationtechniques(NAAT)basedapproachesfor66

fast detection of MRSA and other pathogens (16). Many commercial assays based on67

polymerase chain reaction (PCR) such as FluoroType® MRSA system (Hain-life science68

GmbH, Nehren, Germany) have been developed. Although these assays are costly and69

requireshightechnicalskills(17,18).70

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4

Recently,thenextsteptakentowardsmakingMRSAdetectionfastandaffordablewithhigh71

sensitivity and selectivity is the use of isothermal amplification approaches (19,20). The72

various isothermal amplification methods are loop-mediated isothermal amplification73

(LAMP),primer-generationrollingcircleamplification(PG-RCA),recombinasepolymerase74

amplification (RPA), nucleic acid sequence-based amplification (NASBA), helicase-75

dependent amplification (HDA), exponential amplification reaction (EXPAR), and whole76

genome amplification (WGA). Among these, LAMP is a popular, well-studied, and77

standardizednucleicacidamplificationtechniquewithhighspecificityandsensitivity(21).78

LAMP utilizes polymerases such as Bst, capable of auto cycling strand-displacement79

mediatedamplification.Asetof4or6specificprimersalongwithdNTPsarenecessaryfor80

target sequence amplification (19).Amplification throughLAMPmethod canbedetected81

throughmultiplemethodsviz.turbidimetric, fluorescenceandpHchangesinthereaction82

mixture(22,23).RecentworkfromourgroupreportedaportablesystemSMOLfortherapid83

diagnosisofSalmonellaTyphiandSalmonellaParatyphiA(14).84

Whileworking towards designing a LAMP-based assay forMRSA detection, a significant85

discordanceindiagnosticresultswasoftenobservedincomparisontoresultsofdetection86

through culture method and qPCR. Perplexed with the observation, we looked in the87

literature and foundmultiple reports suggesting the differences in diagnostic results in88

MRSA detection (24–27). In the presentwork, we aim to understand the degree of and89

underlying factorsbehindsuchdiscordance.Toachievethis,wecomparedthediagnostic90

results in 217 mixed flora clinical samples, obtained by three different approaches viz.91

LAMP-assay, qPCR method and culture method. The performance of these diagnostic92

methods is comprehensively compared with each other in terms of sensitivity and93

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robustness.Availableliteraturesuggestspresenceofmultiplepathogens,inhibitoryprotein94

andcoagulase-negativestaphylococci(CoNS)contaminationassomeoftheputativesource95

oferrorinMRSAdetection(28).ToassesstheroleofCoNScontamination,discordantresults96

between NAAT methods were subjected to melt curve analysis (MCA). Mass97

spectrophotometrybaseddetectionofrandomsampleswastriedtosubstantiatethefinding.98

The study designed is comprehensively illustrated in Figure 1. The manuscript while99

comprehensivelyassessing theeffectivenessandrobustnessof thesediagnosticmethods,100

alsohighlightanddiscussthecriticalfactorstobeconsideredwhiledevelopingarapidMRSA101

detectionassay.102

103

2.Materialsandmethods104

2.1Instituteethicalapprovalandcollectionofclinicalsamples105

ThestudywasethicallyapprovedbyInstitutionalethicscommittee(documentnumberIEC-106

569/02dated02.11.2018).Twohundredandseventeenclinicalhumanpussampleswere107

collected from203patientswithclinical symptomsofStaphylococcusaureus infection. In108

fourteenpatients,twosampleswerecollectedondifferentdates.Thesampleswerecollected109

after taking consents from the volunteering patients. The pus sampleswere sent to the110

departmentofmicrobiologyfromwardsandout-patientdepartmentofAllIndiaInstituteof111

MedicalSciences,NewDelhi.112

113

2.2Culture-baseddetectionofMRSA114

Thedetailedprotocolfortheconventionalculture-baseddiagnosticapproachforMRSAis115

giveninthesupplementarysectionandinvolvedculturingoftheclinicalsamplesonblood116

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andMacConkeyAgarplatesfollowedbybiochemicaltests.Clinicalpusaspirateswereplated117

on blood Agar plate in carbon dioxide enriched atmosphere (5% CO2 incubator) for118

performingaβ-hemolysistest.Thepresenceofyellowtocreamwhitecoloniessignifiedthe119

presenceofS.aureusorStreptrococcuspyrogens.Fordifferentiatingbetweentwopossible120

pathogens,catalasetestswereperformedbymixingasmallvolumeofinoculumfromeach121

sampletoa3%hydrogenperoxidesolution.Thereleaseofbubblesconfirmsthepresence122

ofMRSAinclinicalisolates.Further,coagulasetestswereperformedbyincubatingthepus123

sampleswithcoagulaseplasma(HIMEDIA®,India)forobservationofbloodclotsonaglass124

slide.Cefoxitinwasusedasamarkertodetectmethicillinresistancethroughantibioticdisc125

diffusiontestsandtheresultswereanalyzedbycurrentCLSIguidelines.(29)Tounderstand126

thelimitofdetection,serialdilutionsofMRSAcellsspikedbloodculturemediawereused127

forstandardizationsforconcentrationrangeof5to500CFU/mL(supplementarysection).128

Theconventionalculturemethodtakesabout72hours.Alltheculture-baseddetectionof129

clinicalpussampleswerecarriedoutattheDepartmentofMicrobiologyoftheassociated130

hospital.131

132

2.3SampleprocessingforNAATbaseddetection133

Elutionofsamplesinbufferwasperformedpost4hoursofincubationofthepussamples,134

followedbylysisat95°Cfor10minutesinheatedwaterbath.Pussamplesupernatantthus135

preparedwasstoredat4°Cuntilrequiredfurther.Supernatantfromeachclinicalsample136

wasthenusedforsettingupLAMPorqPCRreactions.137

138

2.4LAMPbaseddetectionofMRSA139

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LAMP reagents master mix were purchased from Optigene Private Limited, India, a140

subsidiaryofAmpligene,UK.TheprimersformecAgenewerecustomdesignedbyourgroup141

andsynthesizedbyIntegratedDNATechnologies,USA,forLAMPexperiments.LAMPbased142

nucleicacidamplificationusedinthisstudywasbasedon4primers;forwardouterprimer143

(F3),backwardouterprimer(B3),forwardinnerprimer(FIP),andbackwardinnerprimer144

(BIP).TheFIP,BIP,F3,andB3primersforthemecAgeneweredesignedinPrimerExplorer®145

version5(Table1).BLAST®programwasusedforverificationofprimerspecificity,prior146

toexperimentation.TheLAMPmethodhasbeenexplainedelsewhereindetail.(30)Before147

settingupthereactions,solutionsforprimermixandLAMPreagentsmixwerepreparedto148

enable faster, convenient and precise experimentation. For preparing primermix, 20 µL149

from100µMofFIPandBIPeach,5µLfrom100µMofF3andB3each,and30µLofnuclease150

freewaterweremixed.FortheLAMPassay,5µLofprimermix,10µLofLAMPreagentmix,151

5µLofclinicalsamplelysate(templateDNA)and5µLofnucleasefreewaterwasusedto152

setupa25µLreaction.Allthesampleswereplacedinavialandsealedwithparafilmbefore153

thetreatmentat65°CforLAMP.AmplifiedLAMPreactionsweretestedafteradding2µLof154

1000XSYBRgreenineachtube,followedbyvisualdetectionofpositivesamples.155

156

2.5Cross-reactivityandspecificityofLAMPassay157

Several common pathogenic bacterial species such as S. Havana, S. Paratyphi B, S.158

Typhimurium,S.Typhi,Escherichiacoli,Pseudomonasaeruginosa,Acinetobacterbaumanii,159

Enterococcus fecalis, Klebsiella pneumonia, and Shigella flexneri at a concentration of 106160

CFU/mLwere tested individually to evaluate the cross-reactivity of the primers. Similar161

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protocol of DNA isolation and LAMP amplifications were followed as described in the162

precedingsections.163

164

2.6Quantitativepolymerasechainreaction(qPCR)165

KAPA SYBR master mix 2X was procured from Sigma Aldrich, USA, for qPCR-based166

amplifications.Allotherreagentsincludingbuffers,broth,albumin,etc.,wereobtainedfrom167

Sigma-Aldrich,USA.qPCRreactionswerecarriedoutforvalidationoftheresultsofclinical168

samples.Inadditiontotheabove,knowndilutionofMRSAculturewasusedtogeneratethe169

standardcurvebetweenthemecAcopynumbers(consideringonecopyofthegeneperCFU)170

andCtvalues(obtainedintheqPCRreactions).TheouterprimersF3andB3wereusedin171

qPCRreactionstoamplifythemecAgene.ThestandardcurveofCtvs.copynumberwasused172

forestimatingthecopynumberintheclinicalpussamples.Thelysateprepared(described173

intheearliersections)wasusedfortheqPCRreactions.Forboththeclinicalsamplesandfor174

thestandardcurveexperiments,7.5µLofKAPASYBR2X(Promega,USA)mastermix,2µL175

ofclinicalsamplelysateand3µLofpre-mixedprimers(10µMofF3andB3each)wereused.176

Thereactionswerecarriedoutat95°Cfor30sforinitialdenaturationfollowedby40cycles177

ofdenaturationat95°Cfor10s,andextensionat57°Cfor30s.Thereactionswerecarried178

outinrealtimeqPCRmachineLightCycler®480(RochemolecularsystemsInc.,USA).179

180

2.7MeltCurveAnalysisfordifferentiationMRSAfromCoNS181

To further probe the differences observed between the diagnostic methods, melt curve182

analysis (MCA)was performed. Primer sequences (Table 2) specific to the conservative183

domainsofStaphylococcusgenus(16S),S.aureus (ITS),andmecAgenewereused for the184

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assay.(31)ForMCAassay,qPCRreactionsweresetuptocheckamplificationof16S,ITS,and185

mecA genes in the samples with discordant results between the two NAAT diagnostic186

methods. The reactions were carried out in real-time qPCR machine CFX96® (BioRad187

Technologies, USA). Samples in which all three gene sequences were amplified were188

designatedasMRSA,whilesampleswithnoamplificationofITSampliconweredesignated189

asCoNSpositive.190

191

2.8Statisticalanalysis192

Theeffectivenessandefficiencyofthethreediagnosticmethodswasestimatedbasedonthe193

diagnostic outputs, by calculating the clinical sensitivity and negative predictive value194

(NPV).(32) Power analysiswas also performed on the results. For the sake of statistical195

analysis, thesampleswithnegative results inall threemethods (absenceofMRSA)were196

takenasreferenceforestimatingNPVandsensitivity.197

198

3.0Results199

3.1Cross-reactivitytestsfortheprimersagainstotherpathogens200

Thecross-reactivitywastestedforthemecAprimersequencesusedforMRSAdetectionin201

thestudy.LAMPwasperformedagainstnucleicacidsofthefollowingbacterialpathogens:S.202

Havana,S.ParatyphiB,S.Typhimurium,S.Typhi,Escherichiacoli,Pseudomonasaeruginosa,203

Acinetobacter baumanii, Enterococcus fecalis, Klebsiella pneumonia, and Shigella flexneri.204

AmplificationinallsampleswasobservedvisuallypostadditionofSYBRgreen.Theprimer205

sequenceswerefoundtobespecificandnoamplificationwasobservedwithDNAofabove-206

mentionedorganisms(Figure2).207

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3.2DiagnosticresultsfordifferentapproachesusedforMRSAdetection208

MRSA detection on all 217 clinical samples was performed using threes diagnostic209

approaches as mentioned above and the diagnostic outputs were tabulated (Table 3).210

Conventionalculture-basedMRSAdetectionon217clinicalsamplesresultedin30positive211

samplesand187negatives (Figure3).The isothermalamplificationbasedLAMPmethod212

wasconfirmedusingvisualdetectionafteraddingSYBRgreendyeandshowed104positive213

samplesand113negatives.214

qPCRbaseddiagnosisaswellasquantificationofmecAcopynumberinclinicalpussamples215

was performed after preparing a standard curve between Ct vs mecA copy numbers216

(supplementary section). qPCR based estimation ofmecA copy number showed distinct217

variationamongthetestedsamples(supplementarysection).Basedonthestandardcurve,218

a cut off valueof Ctequal to23.65wasused fordistinguishingbetween thepositive and219

negative MRSA detection and further statistical analysis. qPCR based MRSA detection220

resulted in 67 positive samples and 150 negative samples (Figure 2). On comparing the221

diagnosticoutputs,atotalof13sampleswerefoundpositiveinallthreemethodswhile95222

sampleswerefoundnegativeacrossthemethods(Figure3).Sample-wisedetailedresults223

forallthreediagnosticapproachesareincludedinthesupplementarytables.224

225

3.3Statisticalsignificanceofdifferentapproaches226

ThesensitivityandNetpredictivevalues(NPV)forMRSAdetectioninthreemethodswere227

estimatedusingstatisticalanalysisbasedonthediagnosticoutputs(Table3).Atotalof95228

samples showedMRSA absence in all three approaches, and thus used as reference for229

estimatingsensitivityandnegativepredictivevalue(NPV).CulturebasedMRSAdetection230

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showeda low sensitivity of 24.6%andNPVof 50.8%. LAMPbaseddetection resulted in231

85.2%sensitivityand84.1%NPV.SensitivityandNPVof54.9%and63.3%respectivelywas232

observedinthecaseofqPCR-basedMRSAestimation.233

234

3.4Estimatingthediscordanceindiagnosticoutputs235

Observed MRSA detection result using three different methods showed significant236

differences.Toassessthedegreeofvariabilitybetweenthediagnosticoutputs,weestimated237

percentagediscordance(non-concordantasapercentageof total)within theresults.The238

diagnosticoutputswerecategorized in8differentgroupbasedontheresults (Table4a).239

While considering results from all three methods together, a total of 108 samples (13240

positive and95negative) showed concordant results, resulting in overall discordance of241

50.2%.Next,weestimatedthepairwisediscordancebetweentheapproaches(Table4b).In242

pairwisecomparison,highestdiscordanceof36.9%wasobservedbetween theresultsof243

culture and LAMP methods. This was followed by an estimated discordance of 31.8%244

betweencultureandqPCRresults.Finally,betweenthetwoNAATmethodstheestimated245

discordancewasfoundtobe31.8%.246

247

3.5RoleofCoNSpresenceinthemixedculture(MeltcurveAnalysis)248

SincebothqPCRandLAMPbaseddetectionofMRSAinthestudywasbasedonmecAgene,249

a significant discordance of 31.8% was poorly understood. To assess the role of CoNS250

contaminationintheobserveddiscordanceweperformedMCA.The69discordantsamples251

betweenLAMPandqPCRmethodsweresubjectedtomeltcurveanalysis(MCA)tofindthe252

presenceofCoNS in thesamples.Sampleswere tested for specificamplificationpeaksof253

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mecA(79.5°C),ITS(86.5°C)and16srRNA(83.5°C)inmeltcurves.AmplificationofITSgene254

alongwiththemecAgeneand16SrRNAgeneconfirmsthepresenceofMRSA,whileabsence255

ofITSpeakinmeltcurvesignifiedCoNScontamination.Meltcurveanalysisconfirmedthe256

presence of CoNS in 17 out of the 69 tested samples,while restwere found to be CoNS257

negative(Figure4,Table5).258

259

4.Discussion260

InfectionbyMRSAisawidelyacknowledgedpublichealthchallengeasoneofthehospital-261

acquiredinfections.(33)Lowaffinitypenicillin-bindingprotein(PBP2),analteredprotein262

encodedbythemecAgenepresentinthechromosomeStaphylococcalcassettechromosome263

mec (SCCmec) is responsible for manifesting methicillin resistance in S. aureus.(34,35)264

TreatmentofMRSAisoftenchallengingduetoitsassociationwithmultipleantimicrobial265

resistances.ThusarapidandaccuratedetectionofMRSAinfectionscanplayacriticalrolein266

effectivetreatmentandmanagementofpatients(36).267

In conventional culture-based detection of MRSA, samples showing bacterial growth on268

primary plates are further processed for strain identification tests like coagulase and269

catalaseandthenfinallyforanti-microbialsusceptibilityusingdiscdiffusionassay.Culture270

based detection of MRSA face inherent challenges including but not limited to longer271

processingtimeandlimitedrangeofdetectioninlowinoculumculture(12).Thus,theneed272

ofarapidandrobustdetectionofMRSAhasledtothedevelopmentofmultipledetection273

approaches,majorlybasedonnucleicacidamplificationofaspecificgeneinMRSAgenome274

(37,38).Despitethelargenumberofefforts,aplethoraofliteratureisavailableshowcasing275

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theshortcomingsoftheavailabledetectionmethodsandtheresultantvariationamongthe276

diagnosticmethods(25,39).277

MRSA detection can be performed on clinical isolates from several anatomical sites like278

woundexudates(pus),nasalswabs,skinswabs,andbloodsampleswithnasalswabsbeing279

preferred by clinical test kits (40,41). However, the relevance of pus culture over other280

anatomic samples, especially blood, is recently reported in the literaturewith new tests281

beingdevelopedbasedonpuscultures(14,42,43).282

ThepresentstudycomparedthediagnosticoutputsofMRSAdetectionbythreecommonly283

used approaches namely, culture-based method, qPCR, and loop-mediated isothermal284

amplification. On comparing the diagnostic outputs of these methods, 95 samples were285

foundtobenegativeacrossthethreemethodsandthustakenasnegativecontrolsforthe286

purpose of statistical analysis. A significant difference between the sensitivities of these287

methodswasseenwiththeculture-basedmethoddetecting30MRSApositivesampleswhile288

qPCRbasedassaydetected67positivesamples.HighestMRSAdetectionof104sampleswas289

observedinLAMP-basedassay.ItwouldbeimperativetomentionthatMRSApresencein39290

sampleswereexclusivelyidentifiedbyLAMPmethod(Table4a).291

Both overall and pairwise discordance were estimated and suggested considerable292

differencesinthediagnosticsoutputs(Figure4a,4b).DiagnosticresultsforNAATsmethods293

(qPCRandLAMP)showedsignificantdiscordancewithculturemethod,andwereable to294

detect more number of MRSA bearing samples. The observed discordance between the295

diagnosticoutputsofcultureandmolecularmethodhighlightsthelimitationoftraditional296

methodgiventhetimeconsumingprotocolsprocessandothervariablesincludingtheslow297

rateofgrowthofbacteria,reducedsensitivityduetopresenceofotherpathogens,andpus298

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beingaheterogeneousbodyfluidbearinghighconcentrationofimmunecellsthathampered299

theviabilityoftheMRSApathogen(44).ResultsofNAATmethodsincludingqPCRandLAMP300

werefoundtobemorelargelysimilarincomparisontoculturebasedassay.Amongthe69301

samples found discordant between theNAATsmethods, 53were exclusively detected in302

LAMPwhile16weredetectedMRSApositiveinqPCRmethod.SincebothLAMPandqPCR303

assayarebasedonmecAgeneamplification,observeddiscordancebetweenthetwoisnot304

well-understood.Although,itcanbeexplainedtosomeextentconsideringhighsensitivity305

androbustnessreportedforLAMPbasedassaysinmultiplereports(32,45).306

Inaddition to this, thepresenceofanotherpathogenCoNSpossessingmecAgeneand its307

influence onNAATmethods based detection cannot be overlooked (31). Themethicillin308

resistancetargetgenemecAisoftenfoundintwoorganismsnamely,CoNSandS.aureus,(28)309

and hence merely detecting mecA is not sufficient to distinguish between MRSA and310

methicillinresistantCoNS.Kahánkováetal.employedamulti-locusPCRbasedapproachto311

amplify multiple DNA segments of both species simultaneously (46). However, the312

performanceofmulti-locusPCRhasyieldedlimitedsuccessasreportedbyotherresearch313

groups (47,48).Hence,melt curveanalysiswasperformed in the69clinicalpussamples314

exhibitingdiscordant results to rule out thedetectionof falsepositives anddifferentiate315

betweenMRSA andCoNS (Table 5). Seventeen sampleswere found to be CoNSpositive,316

while therest52sampleswere found tobeMRSApositive.Statisticalanalysiswasagain317

performedforNAATsmethodswhileconsideringtheCoNSidentifiedsamplesasnegative318

(Table6).Theresultsshowthatisothermalamplification(LAMP)baseddetectionofMRSA319

wasfoundtobetherobustapproachamongthethreemethods,withsensitivityof85.7and320

NPVof86.7%(Table6).321

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15

LAMPiscomparativelythelatestdetectionassayamongstthethreemethodsandcouldbe322

employed inhospitalorcommercial laboratorysettings forrapiddetection,given its low323

limit of detection and the capability todetect cell viability. Perhaps thebest strategy for324

MRSAdetectionwouldbetouseLAMPbasedassayforquickscreeningofclinicalsamples325

andthenperformmultiplexPCRorbiochemicalassaytore-confirmthepresenceofMRSAif326

sodesiredby theclinician.Suchapproachwillhelp insaving theprecious time lostwith327

processingofnegativesamplesinconventionalmethods,whilereducingthecosttoo.328

329

5.Conclusions330

In conclusion, our study highlights significant discordance in outputs of the diagnostic331

performancesandefficiencybetweenthreedifferentmethodsusedforMRSAdetectionin332

clinicalpusisolates.Significantdiscordancewasseenamongthediagnosticresultsofthree333

methods,withLAMPbasedmethoddetectinghighestnumberofMRSAinfections.Withinthe334

nucleicacidamplification-basedmethods,case-wisecomparisonwasmadetoidentifythe335

non-concordantsamples.Ameltcurveanalysis(MCA)wasusedtoidentifythepresenceof336

CoNS for non-concordant samples while other reasons leading to discrepancies are337

elaborately discussed. The study highlights the robustness and loopholes of different338

approaches used for MRSAmethod and the factors which should be considered for the339

developmentofanadvancedandmorespecificdetectionmethod.Oneofthebeststrategies340

forMRSAdetectionwouldbetouseLAMPbasedassayforquickscreeningofclinicalsamples341

andthenperformmultiplexPCRorbiochemicalassaytore-confirmtheMRSApresenceif342

desiredbytheclinician.343

344 345

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16

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medicalscience :GMSe-journal.2009.483 48. FrenchGL.Methodsforscreeningformethicillin-resistantStaphylococcusaureus484

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Table1.SequenceofprimersusedforLAMPbasedamplificationofmecAgene.488

Primername Primersequence

F3 5’-TGATGCTAAAGTTCAAAAGAGT-3’

B3 5’-GTAATCTGGAACTTGTTGAGC-3’

FIP 5’-TGAAGGTGTGCTTACAAGTGCTAAT-CAACATGAAAAATGATTATGGCTC-3’

BIP 5’-TGACGTCTATCCATTTATGTATGGC-AGGTTCTTTTTTATCTTCGGTTA-3’

489 Table2.PrimersequenceusedforMeltCurveAnalysis(MCA)baseddistinctionbetweenMRSAandCoNS.490 491

Primername Primersequence Target

genePresenceofgeneinorganism

MRSAconfirmation

CoNSconfirmation

F-16S 5’-ACTTCGGGAAACCGGAGC-3’ 16SrRNA

GenespecifictoStaphylococcus

(MRSA,CoNSetc.)

Amplificationofallthreetargets

Amplificationofonly16SrRNAandmecAgenebutnotITS

B-16S 5’-ACCGTGTCTCAGTTCCAG-3’

F-ITS 5’-GTTAGAGCGCACGCCTGATA-3’ITS

GenespecifictoS.aureusbutnotdoesnotconfirm

methicillinresistanceB-ITS 5’-AATGGTGGAGACTAGCGGGA-3’

F-mecA 5’-TGATGCTAAAGTTCAAAAGAGT-3’ mecAgene

GenespecifictobothMRSAand

CoNSB-mecA 5’-GTAATCTGGAACTTGTTGAGC-3’

492

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Table3.Cultureresultsofthe225clinicalpussamplestestedforMRSApresenceusingthreedifferentdiagnosticmethods.493 StatisticalAnalysisofdiagnosticperformanceofthemethodsisincludedinthetable.494

495

DiagnosticMethod

Detection,n(%) ObservationSensitivity*

(%)

NegativePredictiveValue(NPV)**(%)

Positive NegativePositiveinatleastonemethod

Positiveinallthree

methods

Negativeinallthreemethods

Culturemethod 30 187

122 13 95

24.6 50.8

qPCR 67 150 54.9 63.3

LAMPassay 104 113 85.2 84.1

496 n:numberofsamples;NPV:negativepredictivevalue;LAMP:loop-mediatedisothermalamplification;497 qPCR:quantitativepolymerasechainreaction.Formulasusedforstatisticalcalculationsareincludedinthesupplementarysection.498 *Sensitivity=Positiveinthecorrespondingmethod/Positiveinatleastoneofthemethods499 **NPV=Negativeinallthemethods/Negativeinthecorrespondingmethod500

501 502

503 504 505 506 507 508 509 510 511 512 513

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Table4a.EstimatingthediscordancebetweentheMRSAdetectionresultsthroughthreedifferentapproaches.Diagnostic514 outputof217clinicalsampleswascategorizedin8groups.515

516

Concordance LAMPassay qPCR Culturemethod n(%)

$Concordant + + + 13Discordant + + - 38Discordant + - + 14Discordant - + + 1Discordant - - + 2Discordant - + - 15Discordant + - - 39*Concordant - - - 95

Total 217 %Discordance=[(Total–Concordant)/Total]*100 50.2

n:numberofsamples;NPV:negativepredictivevalue;LAMP:loop-mediatedisothermalamplification;517

qPCR:quantitativepolymerasechainreaction518

*Truenegative=>Negativeinallmethods=95519 $Truepositive=>Positiveinallmethods=13520 521

522

523

524

525

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Table4b.PairwisecomparisonandestimationofdiscordanceinMRSAdetectionresultsusingthreedifferentmethods.526 527

Concordance FindingsCulturevs.qPCRn(%)

Culturevs.LAMPn(%)

qPCRvs.LAMPn(%)

Concordant (+),(+) 14 27 51

Concordant (-),(-) 134 110 97

Discordant (+),(-) 16 3 16

Discordant (-),(+) 53 77 53

Total 217 217 217

%Discordance=[(Total–Concordant)/Total]*100

31.8 36.9 31.8

528 n:numberofsamples;LAMP:loop-mediatedisothermalamplification;qPCR:quantitativepolymerasechainreaction529

530 531

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Table5.Summarizedresultsformeltcurveanalysisfor69discordantsamples.532

OrganismNoof

samples(%)

SampleID

CoNS 17(24.3)US322, AK491, CD566, SK733, RA976, SI989, AP755, SU370, SK870,MK889,IA128,N368,V882,AK285,Sl292,SD269,NB291

MRSA 52(75.7)

RO073, NN999, DP141, PC923*, PO083, RC152, RN055, SD079, ND430,LK633, WK892, SK151, VS270, SD829, NK116, JT336, KD055, NM031,VJ269,SK515,SB228,G303,AK285,PD464,CS513,JK931,AY258,KP056,AR905,UD757,PD171,NJ590,DS969,BP368,DS858,RS224,D291,AK865,SC940, PK448, SD831, H418, MD272, MD272, JM305, RK576, SS115,VB411,DG170,BS544,SR661,PD884

Total 70 533

Table6.MRSAdetectionresultsandstatisticalanalysisforLAMPandqPCRmethodafterconsideringCoNScontamination.534

NucleicacidbasedDiagnosticMethods

FrommeltcurveanalysisSensitivity

(%)

Specificity

(%)

Positivepredictivevalue(PPV)

(%)

Negativepredictivevalue(NPV)

(%)True

PositiveTrue

NegativeFalse

positive*False

negative**

qPCR 64 109 3 41 61.0 97.3 95.5 72.7

LAMPassay 90 98 14 15 85.7 87.5 86.5 86.7

n:numberofsamples;TP:truepositive;FP:falsepositive;TN:truenegative;FN:falsenegative;PPV:positivepredictivevalue;NPV:negativepredictivevalue;535 LAMP:loop-mediatedisothermalamplification;qPCR:quantitativepolymerasechainreaction.536 *FalsePositive–SamplesdetectedasMRSApositivebutfoundcontaminatedwithCoNSthroughMCA.537 **Falsenegative–(Totalpositiveinthecorrespondingdiagnosticmethod–Truepositive)538

539

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540

541 Figure1.Schematicshowingworkflowforthecomparativestudy.542

543

Elution in peptone waterStreaking on blood agar

plates and culture

b - hemolysis test on blood agar media

Positive Negative

Catalase test

Coagulase test

Disc diffusion test for antibacterial resistance

Incubation of swab samples at 37 deg for 4 hours

Lysis by heating 95 deg C for 10 minutes

LAMP based isothermal amplification

Visual detection of amplification using SYBR

green dye

qPCR based amplification of mecA gene

Estimation of mecA copy number in pus lysates

Comparison of diagnostic results

Concordant diagnostic results

Non-concordant diagnostic results

Melt curve analysis (MCA)

Pus samples on swab

26

544 Figure 2. (a) Case wise comparison of diagnostic efficacy different molecular diagnostic545 methods used for MRSA detection. (b) Comparative performance of different diagnostic546 methodson54Clinicalpussamples(6samplesshowednegativeresultsinallthreemethods547 while48clinicalpusisolatesrecordednon-concordantresults).548 549 550

551 552 Figure3.Comparativeperformanceofdifferentdiagnosticmethodson122positiveclinical553 pus samples out of 217 total samples (95 samples showed negative results by all three554 methodsandhenceexcludedfromthediagram).555 556

0

20

40

60

80

100

120

140

Negativecontrol

Positivecontrol(500CFU/mL)

K.pneumoniae

S.typhi

P.aeroginosa

A.baumanii

E.fecalis

S.paratyphiB

S.typhimurium

S.havana

E.coli

S.flexneri

Arbitraryfrequencyunits

27

557 Figure4.Meltcurveanalysis(MCA)foridentifyingCoNScontaminationinthediscordantsamples.(a)to(c)Meltcurvesforthree558 controlsamplesand(d)to(f)threerepresentativesamples(JT336,NK116andCD566)559 560

-1000

100200300400500600

65 70 75 80 85 90 95

-d(RFU)/dT

Temperature(°C)

MRSAcontrol mecA16SrRNAITS

-10

40

90

140

190

65 70 75 80 85 90 95

-d(RFU)/dT

Temperature(°C)

NegativeControl mecA16SrRNAITS

0

10

20

30

40

50

60

65 70 75 80 85 90 95

-d(RFU)/dT

Temperature(°C)

CoNS (S.epidermadis)control

mecA16SrRNAITS

-10010203040

5060

65 70 75 80 85 90 95

-d(RFU)/dT

Temperature(°C)

(JT336) mecA16SrRNAITS

0

10

20

30

40

50

60

65 70 75 80 85 90 95

-d(RFU)/dT

Temperature(°C)

(NK116) mecA16SrRNAITS

0

10

20

30

40

50

65 70 75 80 85 90 95

-d(RFU)/dT

Temperature(°C)

(CD566) mecA16SrRNAITS

(a) (b) (c)

(d) (e) (f)