Rev. Med. Virol. 2002; 12: 375–389. Published online in Wiley InterScience (www.interscience.wiley.com). Reviews in Medical Virology DOI: 10.1002/rmv.370 Molecular diagnosis of influenza Joanna S. Ellis* and Maria C. Zambon Respiratory Virus Unit, Enteric, Respiratory and Neurological Virus Laboratory, Public Health Laboratory Service, Central Public Health Laboratory, 61 Colindale Avenue, Colindale, London NW9 5HT, UK SUMMARY The past decade has seen tremendous developments in molecular diagnostic techniques. In particular, the development of PCR technology has enabled rapid and sensitive viral diagnostic tests to influence patient management. Molecular methods used directly on clinical material have an important role to play in the diagnosis and surveillance of influenza viruses. Molecular diagnostic tests that allow timely and accurate detection of influenza are already implemented in many laboratories. The combination of automated purification of nucleic acids with real-time PCR should enable even more rapid identification of viral pathogens such as influenza viruses in clinical material. The recent development of DNA microarrays to identify either multiple gene targets from a single pathogen, or multiple pathogens in a single sample has the capacity to transform influenza diagnosis. While molecular methods will not replace cell culture for the provision of virus isolates for antigenic characterisation, they remain invaluable in assisting our understanding of the epidemiology of influenza viruses. Copyright # 2002 John Wiley & Sons, Ltd. Accepted: 7 August 2002 INTRODUCTION Influenza viruses continue to be a major cause of respiratory tract infection, resulting in significant morbidity, mortality and financial burden. Each year, increased hospitalisation rates and excess deaths are attributable to influenza infections. Although influenza for most people is a mild ill- ness, which is resolved in 1–2 weeks, complications associated with influenza infection can occur in both the upper and lower respiratory tract, some of which may be fatal [1]. The potential for devel- oping complications is higher in certain risk groups, such as the elderly and individuals with chronic medical conditions. An accurate diagnosis of influenza by a physician is difficult since several different pathogens can produce respiratory ill- nesses with similar clinical symptoms. Conse- quently, there is a requirement for sensitive and rapid diagnostic techniques to verify the clinical diagnosis of influenza and improve the quality of surveillance systems. Moreover, the development of specific anti-influenza neuraminidase inhibitors has increased the potential for rapid and accurate diagnostic tests for influenza viruses to contribute to the management of patients. A number of laboratory methods for the diagnosis of influenza are currently available (Table 1). Each of these methods has advantages and disadvantages, and some, or all, of these factors may influence the method of choice. Although molecular technology has transformed the diagnosis of a number of dis- eases caused by RNA viruses, for example HIV [2], the application of molecular methods to the detec- tion of respiratory pathogens is still comparatively new and expanding rapidly. MOLECULAR METHODS FOR THE DETECTION OF INFLUENZA VIRUSES Choice of molecular assay A number of molecular methods can be employed for the detection of influenza viruses, the majority of which are based on PCR methodology (Table 2). When selecting which assay to use, there are a R RE EVI IEW Copyright # 2002 John Wiley & Sons, Ltd. *Corresponding author: Dr J. S. Ellis, Respiratory Virus Unit, Enteric, Respiratory and Neurological Virus Laboratory, Public Health Laboratory Service, Central Public Health Laboratory, 61 Colindale Avenue, Colindale, London NW9 5HT, UK. E-mail: jellis@phls.org.uk Abbreviations used BDNA, branched chain DNA; EIA, enzyme immunoassay; HI, hae- magglutination inhibition; HMA, heteroduplex mobility assay; IF, immunofluorescence; ILI, influenza-like illness; LCR, ligase chain reaction; M, matrix; NASBA, nucleic acid sequence-based amplifica- tion; NP, nucleoprotein; NS, non-structural; PIV, parainfluenza; RFLP, restriction fragment length polymorphism; WHO, World Health Organisation.