Realtime PCR Is More Sensitive than Multiplex PCR for Diagnosis and Serotyping in Children with Culture Negative Pneumococcal Invasive Disease 英文参考文献.docVIP

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Realtime PCR Is More Sensitive than Multiplex PCR for Diagnosis and Serotyping in Children with Culture Negative Pneumococcal Invasive Disease 英文参考文献.doc

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Realtime PCR Is More Sensitive than Multiplex PCR for Diagnosis and Serotyping in Children with Culture Negative Pneumococcal Invasive Disease 英文参考文献

RealtimePCRIsMoreSensitivethanMultiplexPCRfor DiagnosisandSerotypinginChildrenwithCulture NegativePneumococcalInvasiveDisease ChiaraAzzari*,MariaMoriondo,GiuseppeIndolfi,MartinaCortimiglia,ClementinaCanessa,Laura Becciolini,FrancescaLippi,MauriziodeMartino,MassimoResti DepartmentofPediatrics,AnnaMeyerChildren’sHospitalandUniversityofFlorence,Florence,Italy Abstract Background: Pneumococcal serotyping is usually performed by Quellung reaction, considered the gold standard test. Howeverthemethodcannotbeusedonculture-negativesamples.Molecularmethodscanbeausefulalternative.Theaim ofthestudywastoevaluatetheuseofMultiplex-sequential-PCR(MS-PCR)orRealtime-PCRonbloodsamplesfordiagnosis andserotypingofinvasivepneumococcaldisease(IPD)inapediatricclinicalsetting. Methodology/PrincipalFindings:SensitivityandspecificityofMS-PCRandRealtime-PCRhavebeenevaluatedbothon46 wellcharacterizedpneumococcalisolatesandon67clinicalsamplesfromchildrenwithculture-negativeIPD.Nodifference insensitivityandspecificitybetweenMS-PCRandRealtimePCRwasfoundwhenthemethodswereusedonisolates:both methodscould type100%isolatesandtheresultswerealwaysconsistentwithculture-based methods.Onthecontrary, whenusedonclinicalsamples43/67(64.2%)weretypeablebyMS-PCRand61/67(91.0%)byRealtime-PCR(p=0.0004,K Cohen0.3,McNemar’sp,0.001).Non-typeabilitybyMS-PCRwasassociatedin18/20cases(90.0%)withlowbacterialload. Thedifferencebetweenthetwomethodswaspresentbothwhentheywereusedonnormallysterilefluids(respectively31/ 33 (93.9%) typeable samples for Realtime-PCR and 24/33 (72.7%) for MS-PCR, p=0.047, 95%CL 0.03–0.98; K Cohen 0.3; McNemar’sp=0.0016)andwhentheywereusedonnasopharyngealswabs(respectively30/34(88.2%)typeablesamples for Realtime-PCR and 19/34 (55.9%) for MS-PCR, p=0.007, 95%CL 0.04–0.66); the presence of multiple pneumococcal serotypesinnasopharyngealswabswasfoundmorefrequentlybyRealtimePCR(19/30;63.3%)thanbyMultiplex-sequential PCR(3/19;15.8%;p=0.003;95%CL1.87–39.97). Conclusions/Significance:Inconclusion,