DEVELOPMENT AND USE OF NESTED POLYMERASE-CHAIN-REACTION (PCR) FOR THEDETECTION OF ADENOVIRUS FROM CONJUNCTIVITIS SPECIMENS

Background: The standard virus isolation method for detecting adenovir us is time consuming and direct detection of viral antigens in smears has its limitations. Therefore a rapid and a reliable method to identi fy virus in clinical specimens is desirable. Objective: To develop and evaluate nested PCR as a tool for detecting adenovirus from conjuncti val swabs of patients with acute conjunctivitis during an epidemic. St udy design: A total of 201 patients with acute conjunctivitis were see n between August and November 1996. Conjunctival swabs from the most r ecently affected eyes were collected from 20 random patients and proce ssed for antigen detection in direct smears, for adenovirus, enterovir us (EV70) and coxsackievirus A24 variant and adenovirus isolation by c ulture. Nested PCR was performed using oligonucleotides to amplify 100 4 basepair (bp) and 956 bp fragments of DNA coding for adenovirus hexo n protein. The neutralisation test, to type the adenovirus, was done o n four isolates selected at random. Results: The PCR could detect 0.00 32 fg of adenovirus DNA (corresponding to 8.3 x 10(-3) adenovirus part icles). The EV70 and coxsackievirus A24 antigens were not detected. Th e specimens were positive for adenovirus by all three techniques in se ven patients: (a) by direct smear and PCR in 2; (b) by virus isolation and PCR in 2; and (c) by PCR alone in five patients. In one patient t he direct smear alone was positive. The PCR required 3 days to detect the virus, antigen detection provided diagnosis the same day and virus isolation required 8-27 days. A total of four isolates selected at ra ndom were identified as serotype 7a. Conclusion: The nested PCR is a r eliable and rapid technique for detection of adenovirus from conjuncti val swabs. The adenovirus serotype 7a was the likely causative agent o f this epidemic conjunctivitis. (C) 1998 Elsevier Science B.V. All rig hts reserved.