RapidDiagnosis ofEnterovirus Infection byMagneticBead Extraction andPolymerase ChainReaction Detection of Enterovirus RNA inClinical Specimens

We describe a rapid methodforextraction anddetection ofenterovirus RNA inclinical samples. Byusing magnetic beadtechnology, enterovirus RNA was efficiently andrapidly extracted fromcerebrospinal fluid, stool, saliva, blood, pericardial fluid, urine, andcryopreserved orformalin-fixed solid tissue. Enterovirus RNA was thendetected byreversetranscription followed bypolymerase chainreaction amplification withprimers designed toallow detection ofmostenterovirus serotypes. Fordetection ofenteroviruses inspecimens from patients withacuteenteroviral disease, theoverallsensitivity ofenzymatic RNA amplification was greater than thatofcellculture isolation, especially inbloodspecimens andinstool specimens frompatients withacute cardiac disease. Enterovirus RNA was also detected incryopreserved andarchival formalin-fixed myocardial tissue frompatients withacutemyocarditis andchronic dilated cardiomyopathy. Theability tostudy archival specimens isofparticular value inconducting retrospective investigation. TheRNA extraction procedure used was considerably faster thanextraction methods using organic reagents, usedless hazardous reagents, andwas ofsimilar sensitivity. Thisdetection protocol may therefore beuseful bothforthediagnosis ofenterovirus infection andinstudying thepathogenesis ofacuteandchronic enterovirus-induced disease. Enteroviruses areimportant humanpathogens; atleast 70 serotypes whichinfect humanshavebeenidentified, includingthepolioviruses, echoviruses, andgroup A andB coxsackieviruses (CVAandCVB).Acuteenterovirus infection may beassociated with abroad spectrumofclinical features (reviewed byGrist andReid[12]), asymptomatic infection beingthemostcommon outcome.Thereisconsiderable evidence whichsuggests thatenteroviruses may also cause persistent infection associated withsuchchronic diseases as dilated cardiomyopathy (DCM)andchronic relapsing pericarditis (reviewed byMuir[23]), thechronic fatigue syndrome(1,11,34), andmore recently thepostpolio syndrome (30). Thelaboratory diagnosis ofenterovirus infection may be achieved by isolation ofvirusfromcerebrospinal fluid (CSF), blood, orheart orother affected organs.Isolation of virus fromstool orthroat alsoprovides evidence ofinfection, butthis isless significant, asasymptomatic excretion of enteroviruses isrelatively common, particularly during the summer andautumnintemperate climates. However, attemptstoisolate enteroviruses arefrequently unsuccessful foranumberofreasons.Someserotypes growpoorly incell culture. Inmany casesinfectious virus may no longer be excreted whenpatients present withsymptoms, particularly inpatients withcardiac symptoms,whicharetypically a postacute orchronic manifestation ofenterovirus infection. Serological diagnosis ofenterovirus infection iscomplicated bythelarge numberofserotypes, thedifficulty indemonstrating rising antibody titers inmany patients, andthehigh prevalence ofneutralizing antibody inthegeneral population. Themostuseful serological markeristhepresenceof enterovirus-specific immunoglobulin M,whichindicates a