Previous Article | Next Article 
Journal of Clinical Microbiology, September 2009, p. 3055-3056, Vol. 47, No. 9
0095-1137/09/$08.00+0 doi:10.1128/JCM.01388-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
| FAST-TRACK COMMUNICATION |
Influenza A Virus Subtyping: Paradigm Shift in Influenza Diagnosis
Received 15 July 2009/
Accepted 23 July 2009
 Top INTRODUCTION REFERENCES |
|---|
Seasonal influenza causes substantial mortality and morbidity in the United States, with approximately 365,000 hospitalizations and 50,000 deaths per year (8, 9). Multiple influenza virus types and subtypes cause human infection, and currently circulating subtypes of influenza A virus include H1N1, H1N2, and H3N2. On 17 April 2009, a novel triple-reassortant swine-origin influenza A/H1N1 virus was identified by the Centers for Disease Control and Prevention (CDC) in two children living in adjacent counties in California (2). By 12 June 2009, pandemic A/H1N1 (previously referred to as novel influenza A/H1N1 or swine-origin influenza A/H1N1) had caused an estimated 29,669 cases of infection in 74 countries, including 145 deaths worldwide (10).
Respiratory illness caused by influenza virus is difficult to distinguish from other circulating viruses, and making a diagnosis of influenza based on clinical presentation alone is difficult, with reported sensitivity ranging from 38% to 79% (6, 7). In addition, during the 2008 to 2009 influenza season in the United States, testing at the CDC revealed that 99.5% of seasonal influenza A/H1N1 viruses were oseltamivir resistant but retained susceptibility to zanamivir (100%) and the adamantanes (99.4%) (1). Influenza A/H3N2 viruses were susceptible to both neuraminidase inhibitors (100%) but were resistant to the adamantanes (100%). As a result, the CDC recommended empirical treatment with either zanamivir alone or a combination of oseltamivir plus an adamantine, unless the results of influenza A virus subtyping are available (1). This challenging treatment algorithm is further complicated by the need for early initiation of antiviral therapy within 48 h of the onset of symptoms. The emergence of pandemic influenza A/H1N1 virus while cocirculating with seasonal influenza A/H1N1, A/H3N2, and B viruses of variable susceptibilities combined with the need for rapid and sensitive testing results requires a new paradigm for influenza diagnosis.
We introduced influenza virus reverse transcription-PCR (RT-PCR) testing in our laboratory using the Luminex xTAG respiratory viral panel (RVP) (Luminex, Houston, TX) during the 2007 to 2008 season, but, following reports of oseltamivir resistance, we started reporting both influenza type A virus and subtypes H1 and H3 to allow for more accurate selection of antiviral therapy. At the end of April 2009, we identified our first cases of pandemic A/H1N1 influenza virus, which typed as influenza A but were unsubtypeable (negative for subtype H1 or H3) using RVP and hence allowed easy discrimination from seasonal influenza A/H1N1 and A/H3N2 viruses. Herein we describe the results of influenza A virus subtype identification using the RVP during the 2007 to 2008 and 2008 to 2009 influenza seasons.
The RVP is a multiplex RT-PCR assay that allows for the simultaneous detection of 17 respiratory virus types/subtypes, including metapneumovirus; coronavirus OC43, 229E, NL63, and HKU1; influenza A virus subtypes H1 and H3; influenza B virus; parainfluenza virus types 1 through 4; respiratory syncytial virus types A and B; adenovirus; and rhinovirus (5). The RVP targets the following influenza virus genes: the matrix gene of influenza A virus, the two hemagglutinin genes of influenza A virus subtypes H1 and H3, and the prehemagglutinin gene of influenza B virus. All influenza A and B virus-positive samples were sent to the Illinois Department of Public Health (IDPH) for confirmation of influenza virus types and subtypes using the CDC-developed human influenza virus real-time RT-PCR detection and characterization panel (CDC PCR). Beginning in April 2009, the CDC-developed RT-PCR for pandemic H1N1 was also performed by the IDPH on all positive samples. Discordant samples were repeated by using both the RVP and a second RT-PCR, the respiratory MultiCode-PLx assay (EraGen Biosciences, Madison, WI). The RVP median fluorescence intensity (MFI) units were recorded, and data analysis was carried out using STATA 10 (StataCorp, College Station, Texas). We calculated the sensitivity and positive predictive values (PPV) of the RVP for subtyping influenza A viruses. This study was approved by the institutional review board of Rush University Medical Center.
During the influenza seasons of 2007 to 2009, a total of 295 influenza A virus-positive specimens from 289 patients underwent subtype identification by both RVP and CDC PCR (Table 1). Six duplicate samples were excluded from analysis. The median age of patients was 22 years old (range, 1 month to 90 years), and 47% of the patients were less than 21 years old. The majority of specimens, 92%, were from nasopharyngeal swabs collected in M4RT viral transport medium (Remel, Lenexa, KS).
|
View this table: [in a new window] |
TABLE 1. Summary of subtyping results for all influenza A virus-positive specimens collected from 2007 to 2009
|
The dominant influenza A virus subtype during the 2008 to 2009 influenza season was influenza A/H1N1. The RVP assay subtyped 46 of 46 specimens as being seasonal influenza A/H1N1 virus (7 from 2007 to 2008 and 39 from 2008 to 2009). One influenza A/H1N1 specimen could not be subtyped by CDC PCR, and two influenza A/H1N1 specimens were found to be negative by CDC PCR. All three specimens were determined to be repeat positive by both RVP and the respiratory MultiCode-PLx assay. The overall sensitivity of RVP for subtyping seasonal influenza A/H1N1 virus was 100%.
Between 27 April and 17 June 2009, 176 unsubtypeable influenza A viruses (negative for H1 or H3 by RVP) were tested by the CDC PCR for pandemic A/H1N1 virus. A total of 175 of 176 specimens were confirmed to be pandemic A/H1N1, and 1 specimen was reported as indeterminate. Five of the 176 specimens (repeated twice) gave an indeterminate result by the RVP (MFI range, 150 to 293 U) but were confirmed to be pandemic A/H1N1 by the CDC PCR. The RVP had a sensitivity and PPV of 97.1% and 99.4%, respectively, for pandemic A/H1N1. The overall PPV of the RVP for pandemic A/H1N1 over the two influenza seasons for all unsubtypeable specimens (including eight negative H3 subtypes) was 95.5%.
The 2008 to 2009 influenza season has been an especially challenging one for clinical laboratories and physicians. The need for rapid and sensitive typing and subtyping of influenza viruses has proven to be essential for accurate diagnosis, treatment, and postexposure prophylaxis. The variable resistance patterns of influenza viruses and the need for accurate epidemiologic studies mean that it is no longer sufficient to merely report the presence of influenza A or B virus types. In response to this changing paradigm in influenza diagnosis and treatment, nucleic amplification tests are preferred because of their speed, sensitivity, and ability to subtype influenza viruses directly on clinical specimens (3).
The results of our study show that the RVP assay was able to accurately classify the subtypes of 276 of 289 (95.5%) influenza A viruses, including 171 of 176 pandemic A/H1N1 viruses. Our results are consistent with the study by Ginocchio et al., in which 99 of 101 influenza A viruses unsubtypeable by RVP were confirmed to be pandemic A/H1N1 (4). However, our findings suggest that while the majority of unsubtypeable influenza A viruses can be reported as pandemic A/H1N1, this level of confidence may diminish if A/H3N2 is the dominant cocirculating seasonal influenza virus, as eight A/H3N2 specimens had negative H3 subtypes as determined by using the RVP. Ginocchio et al. reported that the RVP assay was able to subtype 100% of influenza A/H3N2 viruses (4). The difference in our results may be due to methodologic errors, but we think this is unlikely since all unsubtypeable specimens were repeated twice using RVP. In addition, during the 2006 to 2007 influenza season, we submitted 51 influenza virus-positive specimens to IDPH for subtyping, including 7 specimens that failed to be subtyped by RVP (data not shown). Another possible explanation for the failure to subtype H3 may be that these were low-positive specimens. On a practical level, failure of H3 subtyping is currently unlikely to be of clinical significance as both seasonal A/H3N2 and pandemic A/H1N1 viruses remain sensitive to oseltamivir. However, the ability to distinguish A/H3N2 and pandemic A/H1N1 is clearly of epidemiologic importance.
There was one specimen in our study that was reported as indeterminate for pandemic A/H1N1 by CDC PCR due to a failure to amplify the internal extraction control Rp gene. In addition, 5 of 176 pandemic A/H1N1 specimens gave an indeterminate result for influenza A virus as determined by the RVP (mean MFI, 227 U; range, 150 to 293 U). Users of the RVP assay should therefore be aware that an indeterminate result for influenza A virus may still represent a positive result in a patient with a compatible influenza-like illness.
In summary, the emergence of oseltamivir-resistant seasonal influenza A/H1N1 and pandemic A/H1N1 influenza viruses calls for a change in the paradigm for diagnosis and treatment of influenza. In the absence of simple, accurate, and affordable assays for influenza virus susceptibility testing, clinicians will have to rely on local or regional surveillance data of circulating influenza virus subtypes to predict antiviral susceptibility and drug selection. The Luminex xTAG RVP is able to accurately subtype seasonal A/H1N1 and has a high PPV for pandemic A/H1N1.
Published ahead of print on 29 July 2009. 
|
TopINTRODUCTIONREFERENCES |
|---|
- Centers for Disease Control and Prevention. 2008-2009. FluView: 2008-2009 influenza season week 24 ending June 20, 2009. Centers for Disease Control and Prevention, Atlanta, GA. http://www.cdc.gov/flu/weekly/weeklyarchives2008-2009/weekly24.htm. Accessed 25 June 2009.
- Dawood, F. S., S. Jain, L. Finelli, M. W. Shaw, S. Lindstrom, R. J. Garten, L. V. Gubareva, X. Xu, C. B. Bridges, T. M. Uyeki, and the Novel Swine-Origin Influenza A (H1N1) Virus Investigation Team. 2009. Emergence of a novel swine-origin influenza A (H1N1) virus in humans. N. Engl. J. Med. 360:2605-2615.
[Abstract/Free Full Text] - Dwyer, D. E., D. W. Smith, M. G. Catton, and I. G. Barr. 2006. Laboratory diagnosis of human seasonal and pandemic influenza virus infection. Med. J. Aust. 185:S48-S53.[Medline]
- Ginocchio, C. C., and K. St. George. 2009. Likelihood that an unsubtypeable influenza A result in the Luminex xTAG respiratory virus panel is indicative of novel A/H1N1 (swine-like) influenza. J. Clin. Microbiol. 47:2347-2348.
[Free Full Text] - Mahony, J., S. Chong, F. Merante, S. Yaghoubian, T. Sinha, C. Lisle, and R. Janeczko. 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.
[Abstract/Free Full Text] - Monto, A. S., S. Gravenstein, M. Elliott, M. Colopy, and J. Schweinle. 2000. Clinical signs and symptoms predicting influenza infection. Arch. Intern. Med. 160:3243-3247.
[Abstract/Free Full Text] - Peltola, V., T. Reunanen, T. Ziegler, H. Silvennoinen, and T. Heikkinen. 2005. Accuracy of clinical diagnosis of influenza in outpatient children. Clin. Infect. Dis. 41:1198-1200.[CrossRef][Medline]
- Thompson, W. W., D. K. Shay, E. Weintraub, L. Brammer, N. Cox, L. J. Anderson, and K. Fukuda. 2003. Mortality associated with influenza and respiratory syncytial virus in the United States. JAMA 289:179-186.
[Abstract/Free Full Text] - Thompson, W. W., D. K. Shay, E. Weintraub, L. Brammer, C. B. Bridges, N. J. Cox, and K. Fukuda. 2004. Influenza-associated hospitalizations in the United States. JAMA 292:1333-1340.
[Abstract/Free Full Text] - World Health Organization. 2009. Influenza A(H1N1)—update 46. World Health Organization, Geneva, Switzerland. http://www.who.int/csr/don/2009_06_12/en/index.html. Accessed 12 June 2009.
|
Michael Vinikoor
Department of Medicine
Jane Stevens
John Nawrocki
Kamaljit Singh*
| ||||||
|
* Phone: (312) 942-3214, Fax: (312) 942-6787, E-mail: Kamaljit_Singh{at}rush.edu
|
Journal of Clinical Microbiology, September 2009, p. 3055-3056, Vol. 47, No. 9
0095-1137/09/$08.00+0 doi:10.1128/JCM.01388-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Copyright © 2010 by the American Society for Microbiology. For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»