Development and use of nested polymerase chain reaction (PCR) for the detection of adenovirus from conjunctivitis specimens.

Abstract

The standard virus isolation method for detecting adenovirus is time consuming and direct detection of viral antigens in smears has its limitations. Therefore a rapid and a reliable method to identify virus in clinical specimens is desirable. To develop and evaluate nested PCR as a tool for detecting adenovirus from conjunctival swabs of patients with acute conjunctivitis during an epidemic. A total of 201 patients with acute conjunctivitis were seen between August and November 1996. Conjunctival swabs from the most recently affected eyes were collected from 20 random patients and processed for antigen detection in direct smears, for adenovirus, enterovirus (EV70) and coxsackievirus A24 variant and adenovirus isolation by culture. Nested PCR was performed using oligonucleotides to amplify 1004 basepair (bp) and 956 bp fragments of DNA coding for adenovirus hexon protein. The neutralisation test, to type the adenovirus, was done on four isolates selected at random. The PCR could detect 0.0032 fg of adenovirus DNA (corresponding to 8.3 x 10(-3) adenovirus particles). The EV70 and coxsackievirus A24 antigens were not detected. The specimens were positive for adenovirus by all three techniques in seven 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 the 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 random were identified as serotype 7a. The nested PCR is a reliable and rapid technique for detection of adenovirus from conjunctival swabs. The adenovirus serotype 7a was the likely causative agent of this epidemic conjunctivitis.