Skip to main content
PMC home page
Wiley - PMC COVID-19 Collection logoLink to Wiley - PMC COVID-19 Collection
. 2008 Nov 21;81(1):167–175. doi: 10.1002/jmv.21368

Development and implementation of a molecular diagnostic platform for daily rapid detection of 15 respiratory viruses

Annika Tiveljung‐Lindell 1,2,✉, Maria Rotzén‐Östlund 1,2, Shawon Gupta 1,2, Richard Ullstrand 1,2, Lena Grillner 1,2, Benita Zweygberg‐Wirgart 1,2, Tobias Allander 1,2
PMCID: PMC7167155  PMID: 19031448

Abstract

Acute respiratory tract infections are caused by a large number of viruses. Diagnostic methods have until recently been available only for a limited number of these viruses. With the objective to achieve sensitive assays for all respiratory viruses, a rational workflow in the laboratory, and a short turn‐around time, a real‐time PCR diagnostic platform for daily rapid detection of 15 respiratory viruses was developed. The system was evaluated on 585 stored nasopharyngeal aspirates from hospitalized children. Previous analysis by immunofluorescence and virus isolation identified viruses in 37% of the samples while the new PCR diagnostic panel detected 57% virus positive samples. The new platform was introduced in the laboratory in October 2007 and has then fully replaced the standard immunofluorescence assay for rapid detection of viruses and virus isolation. J. Med. Virol. 81:167–175, 2009. © 2008 Wiley‐Liss, Inc.

Keywords: real‐time PCR, respiratory tract infection, influenza, respiratory syncytial virus, human bocavirus

REFERENCES

  1. Allander T, Tammi MT, Eriksson M, Bjerkner A, Tiveljung‐Lindell A, Andersson B. 2005. Cloning of a human parvovirus by molecular screening of respiratory tract samples. Proc Natl Acad Sci USA 102: 12891–12896. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Allander T, Jartti T, Gupta S, Niesters HG, Lehtinen P, Österback R, Vuorinen T, Waris M, Bjerkner A, Tiveljung‐Lindell A, van den Hoogen BG, Hyypiä T, Ruuskanen O. 2007. Human bocavirus and acute wheezing in children. Clin Infect Dis 44: 904–910. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bellau‐Pujol S, Vabret A, Legrand L, Dina J, Gouarin S, Petitjean‐Lecherbonnier J, Pozzetto B, Ginevra C, Freymuth F. 2005. Development of three multiplex RT‐PCR assays for the detection of 12 respiratory RNA viruses. J Virol Methods 126: 53–63. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brittain‐Long R, Nord S, Olofsson S, Westin J, Anderson LM, Lindh M. 2008. Multiplex real‐time PCR for detection of respiratory tract infections. J Clin Virol 41: 53–56. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Falsey AR, Walsh EE. 2006. Viral pneumonia in older adults. Clin Infect Dis 42: 518–524. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Fry AM, Lu X, Chittaganpitch M, Peret T, Fischer J, Dowell SF, Anderson LJ, Erdman D, Olsen SJ. 2007. Human bocavirus: A novel parvovirus epidemiologically associated with pneumonia requiring hospitalization in Thailand. J Infect Dis 195: 1038–1045. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Glimåker M, Abebe A, Johansson B, Ehrnst A, Olcen P, Strannegård O. 1992. Detection of enteroviral RNA by polymerase chain reaction in faecal samples from patients with aseptic meningitis. J Med Virol 38: 54–61. [DOI] [PubMed] [Google Scholar]
  8. Glimåker M, Johansson B, Olcen P, Ehrnst A, Forsgren M. 1993. Detection of enteroviral RNA by polymerase chain reaction in cerebrospinal fluid from patients with aseptic meningitis. Scand J Infect Dis 25: 547–557. [DOI] [PubMed] [Google Scholar]
  9. Gunson RN, Collins TC, Carman WF. 2005. Real‐time RT‐PCR detection of 12 respiratory viral infections in four triplex reactions. J Clin Virol 33: 341–344. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gunson RN, Collins TC, Carman WF. 2006. Practical experience of high throughput real time PCR in the routine diagnostic virology setting. J Clin Virol 35: 355–367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Heim A, Ebnet C, Harste G, Pring‐Akerblom P. 2003. Rapid and quantitative detection of human adenovirus DNA by real‐time PCR. J Med Virol 70: 228–239. [DOI] [PubMed] [Google Scholar]
  12. Hurt AC, Alexander R, Hibbert J, Deed N, Barr IG. 2007. Performance of six influenza rapid tests in detecting human influenza in clinical specimens. J Clin Virol 39: 132–135. [DOI] [PubMed] [Google Scholar]
  13. Jartti T, Lehtinen P, Vuorinen T, Österback R, van den Hoogen B, Osterhaus AD, Ruuskanen O. 2004. Respiratory picornaviruses and respiratory syncytial virus as causative agents of acute expiratory wheezing in children. Emerg Infect Dis 10: 1095–1101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kaiser L, Aubert JD, Pache JC, Deffernez C, Rochat T, Garbino J, Wunderli W, Meylan P, Yerly S, Perrin L, Letovanec I, Nicod L, Tapparel C, Soccal PM. 2006. Chronic rhinoviral infection in lung transplant recipients. Am J Respir Crit Care Med 174: 1392–1399. [DOI] [PubMed] [Google Scholar]
  15. Kantola K, Hedman L, Allander T, Jartti T, Lehtinen P, Ruuskanen O, Hedman K, Söderlund‐Venermo M. 2008. Serodiagnosis of human bocavirus infection. Clin Infect Dis 46: 540–546. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lai KK, Cook L, Wendt S, Corey L, Jerome KR. 2003. Evaluation of real‐time PCR versus PCR with liquid‐phase hybridization for detection of enterovirus RNA in cerebrospinal fluid. J Clin Microbiol 41: 3133–3141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lee WM, Grindle K, Pappas T, Marshall DJ, Moser MJ, Beaty EL, Shult PA, Prudent JR, Gern JE. 2007. High‐throughput, sensitive, and accurate multiplex PCR‐microsphere flow cytometry system for large‐scale comprehensive detection of respiratory viruses. J Clin Microbiol 45: 2626–2634. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Ljungman P, Ward KN, Crooks BN, Parker A, Martino R, Shaw PJ, Brinch L, Brune M, De La Camara R, Dekker A, Pauksen K, Russell N, Schwarer AP, Cordonnier C. 2001. Respiratory virus infections after stem cell transplantation: A prospective study from the Infectious Diseases Working Party of the European Group for Blood and Marrow Transplantation. Bone Marrow Transplant 28: 479–484. [DOI] [PubMed] [Google Scholar]
  19. Lopez AD, Mathers CD, Ezzati M, Jamison DT, Murray CJ. 2006. Global and regional burden of disease and risk factors, 2001: Systematic analysis of population health data. Lancet 367: 1747–1757. [DOI] [PubMed] [Google Scholar]
  20. Mackay IM, Bustin S, Andrade JM, Nissen MD, Sloots TP. 2007. Quantification of microorganisms: not human, not simple, not quick In: Mackay IM, editor. Realt‐time PCR in microbiology: From diagnosis to characterization. Norfolk: Caiaster Academic Press; p. 133–182. [Google Scholar]
  21. Mackie PL. 2003. The classification of viruses infecting the respiratory tract. Paediatr Respir Rev 4: 84–90. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Mahony J, Chong S, Merante F, Yaghoubian S, Sinha T, Lisle C, Janeczko R. 2007. Development of a respiratory virus panel test for detection of twenty human respiratory viruses by use of multiplex PCR and a fluid microbead‐based assay. J Clin Microbiol 45: 2965–2970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Nilsson AC, Alemo B, Björkman P, Dillner L, Melhus A, Nilsson B, Widell A. 2008. Around‐the‐clock, rapid diagnosis of influenza by means of membrane chromatography antigen testing confirmed by polymerase chain reaction. Infect Control Hosp Epidemiol 29: 177–179. [DOI] [PubMed] [Google Scholar]
  24. Ohm‐Smith MJ, Nassos PS, Haller BL. 2004. Evaluation of the Binax NOW, BD Directigen, and BD Directigen EZ assays for detection of respiratory syncytial virus. J Clin Microbiol 42: 2996–2999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Oosterheert JJ, van Loon AM, Schuurman R, Hoepelman AI, Hak E, Thijsen S, Nossent G, Schneider MM, Hustinx WM, Bonten MJ. 2005. Impact of rapid detection of viral and atypical bacterial pathogens by real‐time polymerase chain reaction for patients with lower respiratory tract infection. Clin Infect Dis 41: 1438–1444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Östlund MR, Wirgart BZ, Linde A, Grillner L. 2004. Respiratory virus infections in Stockholm during seven seasons: A retrospective study of laboratory diagnosis. Scand J Infect Dis 36: 460–465. [DOI] [PubMed] [Google Scholar]
  27. Ripa T, Nilsson PA. 2007. A Chlamydia trachomatis strain with a 377‐bp deletion in the cryptic plasmid causing false‐negative nucleic acid amplification tests. Sex Transm Dis 34: 255–256. [DOI] [PubMed] [Google Scholar]
  28. Rossen JW, Versluys B, Bierings M, van Ewijk E, Wolfs TF, Van Loon AM, Boelens JJ. 2006. The role of respiratory viruses in developing bronchiolitis obliterans and IPS in pedriatic HCT viruses. VIII International Symposium on Respiratory Viral Infections, Kohala Coast, Hawaii. Abstract.
  29. Schweiger B, Zadow I, Heckler R, Timm H, Pauli G. 2000. Application of a fluorogenic PCR assay for typing and subtyping of influenza viruses in respiratory samples. J Clin Microbiol 38: 1552–1558. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Shay DK, Holman RC, Newman RD, Liu LL, Stout JW, Anderson LJ. 1999. Bronchiolitis‐associated hospitalizations among US children, 1980–1996. J Am Med Assoc 282: 1440–1446. [DOI] [PubMed] [Google Scholar]
  31. Smit M, Beynon KA, Murdoch DR, Jennings LC. 2007. Comparison of the NOW Influenza A & B, NOW Flu A, NOW Flu B, and Directigen Flu A + B assays, and immunofluorescence with viral culture for the detection of influenza A and B viruses. Diagn Microbiol Infect Dis 57: 67–70. [DOI] [PubMed] [Google Scholar]
  32. Templeton KE, Scheltinga SA, Beersma MF, Kroes AC, Claas EC. 2004. Rapid and sensitive method using multiplex real‐time PCR for diagnosis of infections by influenza A and influenza B viruses, respiratory syncytial virus, and parainfluenza viruses 1, 2, 3, and 4. J Clin Microbiol 42: 1564–1569. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. van de Pol AC, van Loon AM, Wolfs TF, Jansen NJ, Nijhuis M, Breteler EK, Schuurman R, Rossen JW. 2007. Increased detection of respiratory syncytial virus, influenza viruses, parainfluenza viruses, and adenoviruses with real‐time PCR in samples from patients with respiratory symptoms. J Clin Microbiol 45: 2260–2262. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. van Elden LJ, van Loon AM, van Alphen F, Hendriksen KA, Hoepelman AI, van Kraaij MG, Oosterheert JJ, Schipper P, Schuurman R, Nijhuis M. 2004. Frequent detection of human coronaviruses in clinical specimens from patients with respiratory tract infection by use of a novel real‐time reverse‐transcriptase polymerase chain reaction. J Infect Dis 189: 652–657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Watzinger F, Suda M, Preuner S, Baumgartinger R, Ebner K, Baskova L, Niesters HG, Lawitschka A, Lion T. 2004. Real‐time quantitative PCR assays for detection and monitoring of pathogenic human viruses in immunosuppressed pediatric patients. J Clin Microbiol 42: 5189–5198. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Weinberg A, Walker ML. 2005. Evaluation of three immunoassay kits for rapid detection of influenza virus A and B. Clin Diagn Lab Immunol 12: 367–370. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Medical Virology are provided here courtesy of Wiley

RESOURCES