Rapid and sensitive diagnosis of human adenovirus infections by a generic polymerase chain reaction
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.
Section snippets
Viral strains
Reference strains of adenovirus types 1 to 47 were kindly supplied by Dr. Dean Erdman from the Centre of Disease Control and Prevention (Atlanta, GA). Wild adenovirus types 1, 3, 5 and 19, influeza B virus, parainfluenza 1 virus, herpes, simplex 1 virus, varicella zoster virus, syncytial respiratory virus and parvovirus B19 strains were obtained from the Centro Nacional de Microbiologia (Majadahonda, Spain) archives.
Clinical samples
135 clinical samples distributed as follows: Group 1 included 43 conjunctival
Viral isolates
PCR products of the expected size were obtained for all 47 adenovirus serotype reference strains as well as for all wild isolates. Sensitivity showed optimal in a wide range of chemical conditions: Cl2Mg concentration between 2 and 6 mM, dnTp from 200 to 1000 μM and primers from 0.2 to 1 μM. Denaturation temperatures showed optimal from 92 to 95°C and annealing temperatures between 50 and 60°C. No amplification was obtained with other viruses. Serial dilution experiments with a type 1 wild
Discussion
The nested PCR described above was shown to be able to detect all 47 adenovirus serotypes with high sensitivity. Only three of the published PCR techniques (Hierholzer et al., 1993, Kidd et al., 1996, Puig et al., 1994) have been shown to detect all 47 serotypes with different sensitivity depending on the serotype. The sensitivity of the method was calculated only for adenovirus 1, which is the one showing more mismatches with primer sequences (Fig. 1). Consequently, a higher sensitivity should
Acknowledgements
The authors thank Dr Dean Erdman from the Centres for Disease Control and Prevention (Atlanta, GA) for supplying adenovirus reference strains and for serotyping of wild adenovirus isolates. The authors also thank technicians Ana Castellanos, Soledad Carlos, Nieves Cruz, Domingo Estévez, Concepcion Hoyas, Manuela López, Paloma Lucas, lnés Parera and Manuel Vera for their work on routine diagnosis, which has been the source of the clinical samples selected for this work. This work was developed
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