Diagnosis ofEnteroviral Meningitis byUsingPCR witha Colorimetric Microwell Detection Assayt
Аннотация
A 5-h,user-friendly PCR assayforthediagnosis ofenteroviral meningitis was developed. Reverse transcription andamplification were performed ina one-step reaction using rTthpolymerase. Carryover contamination was prevented withdUTPanduracil N-glycosylase. Detection wasperformed colorimetrically on amicrowell titer plate. Sensitivity, specificity, positive predictive value, andnegative predictive value were94.7, 97.4, 94.7, and97.4%, respectively. Theenteroviruses (EVs)comprise 68distinct serotypes of humanpathogens, andarecollectively responsible for10to15 million symptomatic infections intheUnitedStates eachyear (10). EVscause .85%ofthecasesofaseptic meningitis (1,9) andmay bedifficult todistinguish frommeningitis dueto bacteria andherpes simplex virus, particularly inyounginfants andchildren. Thisresults inunnecessaryhospitalization and treatment, withantibiotics and/or antiherpesvirus medications, ofthousands ofchildren eachyear.Cellcultures ofcerebrospinal fluid (CSF)forEVstakean averageof6or7daysfor identification ofgrowth (2), aretypically observed for2 or3 weeksbefore being reported asnegative, andmaybeonly 65to 75%sensitive. Evenwiththosedrawbacks, cellculture hasa significant impact on themanagement ofpatients withEV meningitis (2), anda more rapid andmore sensitive technique wouldbeexpected toproduce an evengreater impact. Usingseparate reversetranscription (RT)andPCR steps andradioactive or chemiluminescent detection systems, severalinvestigators havereported successful detection ofEVsin CSF(6,8,9).Whilethese assayshavebeenmore rapid and more sensitive thancellculture, theyrequire opening the reaction tubes andadding new reagents between theRT and PCR steps, increasing theriskofcarryovercontamination. Furthermore, only one oftheearlier studies (9)incorporated uracil N-glycosylase topreventcarryovercontamination, a recentimprovement inPCRquality control (5). Finally, studies todatehaveusedcumbersome, noncolorimetric detection schemes. Thisreportdescribes theapplication ofa new PCR assaythatutilizes a single enzyme forbothRT andPCR, incorporates uracil N-glycosylase, anddetects theamplified product ina simple, microwell colorimetric assay. Twenty-seven EV serotypes (coxsackieviruses A3,A7,A9,