Using the Full Spectral Capacity (six Channels) of a Real-time PCR Instrument Can Simplify Diagnostic Laboratory Screening and Typing Protocols for Pandemic H1N1 Influenza

Overview

Journal Influenza Other Respir Viruses

Publisher Wiley

Specialty Microbiology

Date 2011 Feb 11

PMID 21306574

Citations 5

Authors

Mark J Hopkins

Jay F Moorcroft

Jailson B Correia

Ian J Hart

Affiliations

Soon will be listed here.

Abstract

Background: Timely reporting of influenza A virus subtype affects patient management. Real-time PCR is a rapid and sensitive method routinely used to characterise viral nucleic acid, but the full spectral capability of the instruments is not employed.

Objectives: To evaluate a hexaplex real-time PCR assay (Flu-6plx assay) capable of detecting influenza A and B, hMPV, respiratory syncytial virus (RSV) and distinguishing 2008 'human' influenza A/H1 from 2009 pandemic A/H1 subtypes.

Methods: Respiratory specimens (n = 213) were tested using the Flu-6plx assay and a further four monoplex PCRs targeting hMPV, RSV, influenza A and B. The FDA-approved ProFlu ST test was used to validate the Flu-6plx PCR influenza A/H1 subtyping components. Discrepant 2009 pandemic A/H1 results were further tested using the CDC swine H1 assay. Results The Flu-6plx assay had excellent sensitivity identifying 106/106 influenza A RNA-positive samples. The ProFlu ST test was a less sensitive subtyping test, and discrepant analysis could not confirm A/H1 status for four samples resulting in Flu-6plx PCR specificities of 98% and 95% for human A/H1 and 2009 pandemic A/H1, respectively. Co-infection affected the sensitivity of the Flu-6plx PCR hMPV component whereby low-level hMPV RNA could be masked by much higher concentrations of influenza A virus RNA.

Conclusions: The Flu-6plx assay is a sensitive and specific test for the universal detection of influenza A infection and determination of A/H1 subtype. Concomitant detection of influenza B, hMPV and RSV demonstrates the utility of hexaplex real-time PCRs in viral diagnostics.

Citing Articles

An Influenza A Vaccine Based on the Extracellular Domain of Matrix 2 Protein Protects BALB/C Mice Against H1N1 and H3N2.

Ong H, Yong C, Tan W, Yeap S, Omar A, Razak M Vaccines (Basel). 2019; 7(3).

PMID: 31430965 PMC: 6789677. DOI: 10.3390/vaccines7030091.

A newly developed tetraplex real-time RT-PCR for simultaneous screening of influenza virus types A, B, C and D.

Henritzi D, Hoffmann B, Wacheck S, Pesch S, Herrler G, Beer M Influenza Other Respir Viruses. 2018; 13(1):71-82.

PMID: 30264926 PMC: 6304318. DOI: 10.1111/irv.12613.

Modulation of nasopharyngeal innate defenses by viral coinfection predisposes individuals to experimental pneumococcal carriage.

Glennie S, Gritzfeld J, Pennington S, Garner-Jones M, Coombes N, Hopkins M Mucosal Immunol. 2015; 9(1):56-67.

PMID: 25921341 PMC: 4703943. DOI: 10.1038/mi.2015.35.

The impact of the H1N1 influenza pandemic on clinical presentations and viral epidemiology of acute respiratory infection in preschool children in Brazil.

Fawkner-Corbett D, Duarte M, Rose K, Fonceca A, Bezerra P, Hopkins M Pediatr Infect Dis J. 2012; 31(6):653-5.

PMID: 22414906 PMC: 3378642. DOI: 10.1097/INF.0b013e3182501ff4.

Viral and atypical bacterial detection in acute respiratory infection in children under five years.

Bezerra P, Britto M, Correia J, Duarte M, Fonceca A, Rose K PLoS One. 2011; 6(4):e18928.

PMID: 21533115 PMC: 3078930. DOI: 10.1371/journal.pone.0018928.

References

Helb D, Jones M, Story E, Boehme C, Wallace E, Ho K . Rapid detection of Mycobacterium tuberculosis and rifampin resistance by use of on-demand, near-patient technology. J Clin Microbiol. 2009; 48(1):229-37. PMC: 2812290. DOI: 10.1128/JCM.01463-09. View

Ieven M . Currently used nucleic acid amplification tests for the detection of viruses and atypicals in acute respiratory infections. J Clin Virol. 2007; 40(4):259-76. PMC: 7108459. DOI: 10.1016/j.jcv.2007.08.012. View

Wang R, Sheng Z, Taubenberger J . Detection of novel (swine origin) H1N1 influenza A virus by quantitative real-time reverse transcription-PCR. J Clin Microbiol. 2009; 47(8):2675-7. PMC: 2725689. DOI: 10.1128/JCM.01087-09. View

Carr M, Gunson R, MacLean A, Coughlan S, Fitzgerald M, Scully M . Development of a real-time RT-PCR for the detection of swine-lineage influenza A (H1N1) virus infections. J Clin Virol. 2009; 45(3):196-9. PMC: 7173005. DOI: 10.1016/j.jcv.2009.06.001. View

Hopkins M, Redmond C, Shaw J, Hart I, Hart C, Smyth R . Detection and characterisation of human metapneumovirus from children with acute respiratory symptoms in north-west England, UK. J Clin Virol. 2008; 42(3):273-9. DOI: 10.1016/j.jcv.2008.03.011. View

Hymas W, Hillyard D . Evaluation of Nanogen MGB Alert Detection Reagents in a multiplex real-time PCR for influenza virus types A and B and respiratory syncytial virus. J Virol Methods. 2008; 156(1-2):124-8. DOI: 10.1016/j.jviromet.2008.11.004. View

Bahrdt C, Krech A, Wurz A, Wulff D . Validation of a newly developed hexaplex real-time PCR assay for screening for presence of GMOs in food, feed and seed. Anal Bioanal Chem. 2010; 396(6):2103-12. DOI: 10.1007/s00216-009-3380-x. View

Kuypers J, Wright N, Corey L, Morrow R . Detection and quantification of human metapneumovirus in pediatric specimens by real-time RT-PCR. J Clin Virol. 2005; 33(4):299-305. PMC: 7185467. DOI: 10.1016/j.jcv.2004.11.023. View

Templeton K, Forde C, van Loon A, Claas E, Niesters H, Wallace P . A multi-centre pilot proficiency programme to assess the quality of molecular detection of respiratory viruses. J Clin Virol. 2005; 35(1):51-8. PMC: 7108443. DOI: 10.1016/j.jcv.2005.05.005. View

10.

Rolfe K, Parmar S, Mururi D, Wreghitt T, Jalal H, Zhang H . An internally controlled, one-step, real-time RT-PCR assay for norovirus detection and genogrouping. J Clin Virol. 2007; 39(4):318-21. DOI: 10.1016/j.jcv.2007.05.005. View

11.

Scriven J, McEwen R, Mistry S, Green C, Osman H, Bailey M . Swine flu: a Birmingham experience. Clin Med (Lond). 2010; 9(6):534-8. PMC: 4952289. DOI: 10.7861/clinmedicine.9-6-534. View

12.

Gunson R, MacLean A, Davies E, Bennett S, Miller R, Carman W . Development of a multiplex real-time RT-PCR that allows universal detection of influenza A viruses and simultaneous typing of influenza A/H1N1/2009 virus. J Virol Methods. 2009; 163(2):258-61. PMC: 7173015. DOI: 10.1016/j.jviromet.2009.10.006. View

13.

Brittain-Long R, Nord S, Olofsson S, Westin J, Anderson L, Lindh M . Multiplex real-time PCR for detection of respiratory tract infections. J Clin Virol. 2007; 41(1):53-6. PMC: 7172039. DOI: 10.1016/j.jcv.2007.10.029. View

14.

Gunson R, Collins T, Carman W . Real-time RT-PCR detection of 12 respiratory viral infections in four triplex reactions. J Clin Virol. 2005; 33(4):341-4. PMC: 7108440. DOI: 10.1016/j.jcv.2004.11.025. View

15.

Lassauniere R, Kresfelder T, Venter M . A novel multiplex real-time RT-PCR assay with FRET hybridization probes for the detection and quantitation of 13 respiratory viruses. J Virol Methods. 2010; 165(2):254-60. PMC: 7112774. DOI: 10.1016/j.jviromet.2010.02.005. View