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Potentialcauses of congenital infection includeToxoplasma gondii andviruses such as cytomegalovirus (CMV), enterovirus, hepatitis C virus,herpes simplex virus types 1 and 2 (HSV-1 and -2), human herpesvirustypes 6, 7, and 8, lymphocytic choriomeningitis virus, parvovirus,rubella virus, and varicella-zoster virus. Testing for each of theseagents using nucleic acid tests is time consuming and the availabilityof clinical samples such as amniotic fluid or neonatal blood is oftenlimited. The aim of this study was to develop multiplex PCRs (mPCRs)for detection of DNA and RNA agents in the investigation of congenitalinfection and an mPCR for the viruses most commonly requested in adiagnostic virology laboratory (CMV, Epstein-Barr virus, enterovirus,HSV-1, HSV-2, and varicella-zoster virus). The assays were assessedusing known pathogen-positive tissues (cultures, placentae, plasma, andamniotic fluid) and limits of detection were determined for all theagents studied using serial dilutions of plasmid targets. Nested PCRwas performed as the most sensitive assay currently available, anddetection of the amplicons using hybridization to labeled probes andenzyme-linked immunosorbent assay detection was incorporated into threeof the four assays. This allowed detection of 10 to 102 copies of each agent in the samples processed. In several patients, anunexpected infection was diagnosed, including a case of encephalitiswhere HSV was the initial clinical suspicion but CMV was detected. Inthe majority of these cases the alternative agent could be confirmedusing reference culture, serology, or fluorescence methods and was ofrelevance to clinical care of the patient. The methods described hereprovide useful techniques for diagnosing congenital infections and aparadigm for assessment of new multiplex PCRs for use in the diagnosticlaboratory.

Development of Multiplex PCRs for Detection of Common Viral Pathogens and Agents of Congenital Infections