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

Abstract

Background: 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. Objective: To develop and evaluate nested PCR as a tool for detecting adenovirus from conjunctival swabs of patients with acute conjunctivitis during an epidemic. Study design: 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. Results: The PCR could detect 0.0032 fg of adenovirus DNA (corresponding to 8.3×10⁻³ 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. Conclusion: 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.

Introduction

Adenoviruses are the leading causes of acute conjunctivitis seen in clinical practice. Common clinical manifestations include epidemic keratoconjunctivitis (EKC), pharyngo-conjunctival fever and non-specific follicular conjunctivitis (Ford et al., 1987, Fitch et al., 1989). Adenoviruses are grouped into six subgenera (A–F) and 49 serotypes based on DNA homology and biological and biochemical parameters (Wadell, 1984). Epidemic keratoconjunctivitis (EKC), the most serious manifestation of adenoviral keratoconjunctivitis, is caused predominantly by serotypes 8, 19 and 37 (Darougar et al., 1977, Kemp and Hierholzer, 1986, Aoki et al., 1986). Pharyngeal-conjunctival fever, a disease commonly affecting children, is most commonly caused by serotypes 3, 4, 5 and 7 whereas serotypes 1 to 11 and 19 are agents of non-specific follicular conjunctivitis. Types 2, 3, 4, 5, 7, 10, 11, 21 and 29 have been isolated from patients with EKC (Aoki et al., 1986). The biochemical and genetic changes within the predominant serotypes have also been reported (Kemp et al., 1983, Ishii et al., 1987.). Accurate and timely aetiological diagnosis is essential to plan the most effective ways of limiting the spread of infection. The ‘gold standard’ for the aetiological diagnosis is the isolation and identification of adenovirus after growth in cell cultures (Kowalskii and Gordon, 1989). Detection of the virus directly in the conjunctival specimen by immunofluorescence (Walpita and Darougar, 1989), immunofiltration (Kowalskii and Gordon, 1989), enzyme immunoassay (Kowalskii and Gordon, 1989) and immunoelectron microscopy (Van Rij et al., 1982) have all been used with varying successes. These tests rely on antibody recognition of antigens common to all serotypes and are generally less sensitive than cultures as they require significant threshold levels of antigen for detection (Kinchington et al., 1994). Although the total diagnostic positivity rate is nearly the same between antigen detection and virus isolation technique, there are always specimens that are positive only for any one of them (Waris et al., 1990).

Polymerase chain reaction (PCR), a significant technological advancement in molecular biology, has the capacity to amplify genomic sequences from an infectious agent over a million times and yield results within several hours (Mullis et al., 1986). Recent improvements have resulted in the application of PCR as a routine technique for the diagnosis of several bacterial and viral agents (Kinchington et al., 1994).

In this report, the results are presented of a study evaluating: (1) nested PCR; (2) conventional cell culture; and (3) direct smear immunofluorescence (IF) study for the detection of adenovirus from conjunctival swabs of patients. These patients were clinically diagnosed to be suffering from acute conjunctivitis during an epidemic (August–November) in 1996 in Chennai, Tamil Nadu, South India.