Rapid and sensitive diagnosis of human adenovirus infections by a generic polymerase chain reaction

Abstract

A new adenovirus specific nested polymerase chain reaction (PCR) method is described. It was designed inside the hexon protein gene of the adenovirus genome, and was able to detect DNA of all 47 human adenovirus types in a wide range of clinical samples. A sensitive internal control system able to assure proper analytical conditions for the amplification of as few as 100 molecules of a heterologous DNA was included to avoid false negative results. Sensitivity was estimated at about 10 molecules per tube of a plasmid containing an insert of the first amplification product. The method was able to detect adenovirus infection in 31/43 conjunctival scrapings from patients with acute kerato conjunctivitis 10/40 nasopharyngeal aspirates from patients admitted to hospital with acute respiratory disease and 2/26 urine samples from patients with haemorrhagic cystitis with better sensitivity than cell culture or rapid diagnosis by antigen detection by immunofluorescence (IF) in the case of respiratory specimens. Only two of 17 stools positive for a group F adenovirus specific latex immunoassay were PCR negative. The internal control system avoided a false negative result on another two stool samples. In conclusion, the method described below was shown to be useful for rapid diagnosis of adenovirus infections with higher sensitivity than antigen detection by IF.

Introduction

Human adenoviruses are a major cause of different clinical syndromes including gastroenteritis, respiratory disease, conjunctivitis, haemorrhagic cystitis and exanthema (Horwitz, 1996). They comprise 47 different types grouped in seven groups (A, B1, B2, C, D, E, F). Correlation between viral type or group and organ tropism is partial (Horwitz, 1996).

Although viral isolation is the gold standard for direct detection of adenovirus, it often results in a delay of several days or weeks before the results are available (Krisher and Menegus, 1987, Mahafzah and Landry, 1989) and, therefore, is not useful for making therapeutic decisions. Serology is also slow as a result of the requirement for convalescent sera (Thiele et al., 1989). Direct antigen detection methods based on enzyme immune assay (de Paiva et al., 1992, Grondahl et al., 1999, Nakagawa et al., 1995, Rabalais et al., 1992), immunofluorescence (IF) (de Paiva et al., 1992, Freymuth et al., 1998, Mahafzah and Landry, 1989, Rabalais et al., 1992, Shaw et al., 1995, Torres and Vicente, 1992, Trabelsi et al., 1992), or specific latex agglutination (Dahling et al., 1993, Trabelsi et al., 1992) are used widely for rapid diagnosis of adenovirus infections; however, some of these techniques are less sensitive than cell culture (Torres and Vicente, 1992).

Genome amplification techniques based on the polymerase chain reaction (PCR) provide a rapid and sensitive alternative for adenovirus detection in clinical samples. These techniques have been applied either to detect all adenovirus types (Allard et al., 1992, Dalapathy et al., 1998, Flomenberg et al., 1997, Hierholzer et al., 1993, Jackson et al., 1996, Kidd et al., 1996, Kinchington et al., 1994, Kuwano et al., 1997, Lawler et al., 1994, Matsuse et al., 1994, Morris et al., 1995, Morris et al., 1996, Puig et al., 1994, Raty et al., 1999, Saitoh et al., 1996, Turner et al., 1993), or for the specific detection of some adenoviruses (Pring Akerblom and Adrian, 1994, Pring Akerblom et al., 1997, Tiemessen and Nel, 1996). DNA polymerase inhibitors are frequent in clinical samples and can be the cause of false negative results despite the high sensitivity of PCR, specially when urine or faecal samples are involved (Chernesky et al., 1997, Glimaker et al., 1992) but also with conjunctival swabs (Dalapathy et al., 1998). This can be avoided using internal control systems to monitor the reaction conditions in each individual tube.

In the present report, a new nested PCR method is described which is able to detect all 47 adenovirus types in clinical samples with a sensitive internal control system valid to assure the quality of reaction conditions in each individual tube.