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:[email protected],[email protected] 14
15
Thisistodeclarethatthefollowingauthorsdonothaveanyconflictofinterest:16
UjjwalRanjanDahiya:[email protected]
ArnabSikidar:[email protected]
PriyankaSharma:[email protected]
ChitraRawat:[email protected]
BenuDhawan:[email protected]
ArtiKapil:[email protected]
RavikrishnanElangovan:[email protected]
DineshKalyanasundaram:[email protected],[email protected]
25
26
27
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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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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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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)