Comparison of the IMDx Influenza A, Influenza B, and Respiratory Syncytial Virus A/B
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1 JCM Accepts, published online ahead of print on 1 October 2014 J. Clin. Microbiol. doi: /jcm Copyright 2014, American Society for Microbiology. All Rights Reserved. 1 2 Comparison of the IMDx Influenza A, Influenza B, and Respiratory Syncytial Virus A/B assay on the m2000 platform with Real-time Reverse Transcriptase PCR assays Dena Adachi, 1, Julian W Tang, 1,2,3, Roberta Lundeberg 1, Graham Tipples 1,2, Carmen L. Charlton 1,4 and Steven J. Drews 1,5* Alberta Provincial Laboratory for Public Health, Alberta, Canada; Departments of 2 Medical Microbiology and Immunology; 3 Medical Genetics, and 4 Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta, Canada; 5 Department of Medical Microbiology and Immunology, University of Calgary, Calgary, Alberta, Canada *Corresponding Author: Steven Drews Alberta Provincial Laboratory for Public Health University of Alberta Hospital, St, 2B1.03, Edmonton AB T6G 2J2. Canada. Tel: Fax:
2 The increasing demand for timely respiratory virus testing for both diagnostic and surveillance purposes emphasizes the need to strike a sustainable approach for testing clinical specimens. Streamlined automated approaches allow for clinically relevant diagnosis while avoiding pitfalls (e.g. subjective interpretation and re-test algorithms) that impact on laboratory workflow and test turn-around-times. The IMDx influenza A/B and respiratory syncytial virus (RSV) assay (Abbott Molecular, Abbott Park, Illinois, U.S.A) is an FDA approved molecular assay for influenza A, influenza B and combined RSV A/B testing on the Abbott m2000 platform. The assay requires minimal operator manipulation; total nucleic acid extraction and molecular detection are performed on the same platform. A MaxRatio algorithm, an automated approach utilizing several variables including cycle number, curve shapes and relative measures of amplification efficiency, is used to determine test positivity to avoid subjective interpretations. The assay also contains an internal MS2 control to monitor for inhibition (7). This evaluation study compares detection of influenza A/B and RSV by the IMDx assay against: our modified influenza A (CDC-M) and influenza B (NS1) single-plex real-time reverse-transcriptase PCR (rrt-pcr) assays, or laboratory-developed tests (LDTs) (3,6), a RSV A/B rrt- PCR LDT (8), and the Luminex respiratory virus panel (RVP) Classic assay (6). The MagMax total-nucleic-acid-extraction system (Life Technologies Corp, Burlington, ON, Canada) was used for all the assays except the IMDx. Our analysis utilized nasopharyngeal swab specimens of convenience collected in Alberta, Canada in the respiratory season. Selected specimens (n=371) were blinded and evaluated. To assess accuracy, specimens were run in parallel by aforementioned assays with commercial tests run as per the package inserts. Specimens 2
3 positive for other respiratory virus targets (Luminex RVP Classic) were run in parallel for each assay to determine specificity. No cross reactivity was observed for specimens positive for: adenovirus (n=8); human coronavirus 229E (n=4), HKU1 (n= 1), OC43 (n=5); parainfluenza (PIV) 1 (n=6), PIV2 (n=5), PIV3 (n=5), or PIV4 (n=3), rhino/enterovirus (n=10), and human metapneumovirus (n=14). Relative limits of detection (LOD), determined to be 50% of replicates positive (2) were calculated. LOD panels used Influenza A pdm09 and seasonal influenza H3 collected in 2014, one influenza A, one influenza B, one RSVA and one RSVB specimen diluted in viral transport media in serial dilutions from 10-1 to The vast majority of these H3 isolates in Canada in the respiratory season belonged to A/Texas/50/2012 strain ( The relative LODs are identified in Table 2. For Luminex RVP assays, values are shown when greyzones are reported either as positive (normal font) or negative (italicized). Two approaches were used to calculate sensitivity and specificity: comparison of IMDx and Luminex RVP Classic assay against rrt-pcr, and comparison of each result against an aggregate gold standard (Aggstd: each specimen defined as positive if >2 independent tests scored the result as positive) with key data bolded in Table 2 (1). Sensitivity, specificity and 95% confidence intervals calculations were determined (Vassarstats Clinical Calculator 1, 2014). When an LDT rrt-pcr was used as the gold standard, the results were influenza A (positive n=94, negative n=129), influenza B (positive n=69, negative n=99) and RSV A/B (positive n=76, negative n=94). When the Aggstd was used as the gold standard the results were influenza A (positive n=92, negative n=131), influenza B (positive n=66, negative n=102) and RSV A/B (positive 3
4 n=76, negative n=94). Similarly, type specific influenza A analysis for pdm09 and H3 subtypes were compared using rrt-pcr as a gold standard: pdm09 (positive n=77, negative n=96); H3 (positive n=16, negative n=94) and the Aggstd as the gold standard: pdm09 (positive n=76, negative n=97); H3 (positive n=16, negative n=94). The proportion of positives detected was not significantly different between the LDT and the IMDx for overall influenza A and influenza A pdm09 detection (Fisher s exact test, GraphPad QuickCalcs, 2014) On clinical specimens, the IMDx assay showed equivalent sensitivity and specificity for influenza A, influenza B, and RSV against both LDTs and Aggstds. Wide ranges in some 95% CI values in Table 2 (e.g. H3 xtag and IMDx) were likely due to small numbers of specimens tested. The clinical significance of low positive results and how they impact clinical management is largely unknown, and the decision to implement an assay should rest on ability to detect a target at clinically relevant levels (4,5). Overall, the IMDx assay was easier to use than the existing LDT due to its automated platform and the MaxRatio calculated interpretations, which do not require operator intervention (7). This evaluation study provides the preliminary data to support the use of the IMDx assay as an automated front-end screening assay for seasonal influenza and RSV. At our institution, clinical samples are first tested by our LDT for influenza A/B and negative samples are reflexed to a comprehensive viral panel. Replacement of our LDT with the IMDx assay to include RSV in the front line screening assay is estimated to remove approximately one-fifth of specimens from further testing, which represents a significant cost savings to the laboratory (data not shown). This analysis indicates that the IMDx 4
5 assay has similar molecular test characteristics to other molecular detection approaches for the detection of seasonal influenza A, influenza B and RSV. IMDx kits for evaluation were provided by Abbott Molecular, Abbott Park, Illinois, U.S.A. 5
6 95 Table 1. Comparable relative limits of detection for lab-developed (LDT) real-time PCRs and IMDx IMDX LDT Luminex GZ*= pos GZ=neg Influenza A H3 Influenza A Pdm Influenza B RSV A/B RSVA: 10-4 RSVA: RSVB: RSVB: *Greyzone 97 6
7 Table 2. Relative test characteristics of the IMDx influenza A/B and RSV assay. Target: pdm09 Influenza A subtype H3 Subtype not resolved All Influenza A Influenza B RSV A and B Percentage (95% CI) Percentage (95% CI) Percentage (95% CI) Percentage (95% CI) Percentage (95% CI) Percentage (95% CI) IMDx vs LDT Sensitivity 94.8% ( ) 100% ( ) 0% (0-94.5) 94.7% ( ) 95.7% ( ) 100% ( ) Specificity 99.0% ( ) 100% ( ) 100% ( ) 99.2% ( ) 100% ( ) 100% ( ) xtag RVP vs LDT Sensitivity 97.4% ( ) 93.8% ( ) 0% (0-94.5) 96.8% ( ) 89.9% ( ) 98.7% ( ) Specificity 100% ( ) 100% ( ) 100% ( ) 100% ( ) 100% ( ) 100% ( ) IMDx vs Aggstd Sensitivity 96.1% ( ) 100% ( ) NaN 96.7% ( ) 100% ( ) 100% ( ) Specificity 99.0% ( ) 100% ( ) 100% ( ) 99.2% ( ) 100% ( ) 100% ( ) LDT vs Aggstd Sensitivity 100% ( ) 100% ( ) NaN 100% ( ) 100% ( ) 100% ( ) Specificity 99.0% ( ) 100% ( ) 99.2% ( ) 98.5% ( ) 97.1% ( ) 100% ( ) xtag RVP vs Aggstd Sensitivity 98.7% ( ) 93.8% ( ) NaN 98.9% ( ) 93.9% ( ) 98.7% ( ) Specificity 100% ( ) 100% ( ) 100% ( ) 100% ( ) 100% ( ) 100% ( ) NaN: Not able to numerate. Bolded text: key test characteristics for IMDx assay. 7
8 References 1. Bolotin S, De LC, Choi KW, Lombos E, Burton L, Mazzulli T, Drews SJ Validation of the TaqMan Influenza A Detection Kit and a rapid automated total nucleic acid extraction method to detect influenza A virus in nasopharyngeal specimens. Ann. Clin. Lab Sci. 39:p doi:39/2/155 [pii]. 2. Caraguel CG, Stryhn H, Gagne N, Dohoo IR, Hammell KL Selection of a cutoff value for real-time polymerase chain reaction results to fit a diagnostic purpose: analytical and epidemiologic approaches. J. Vet. Diagn. Invest 23:p doi:23/1/2 [pii]. 3. Dawood FS, Jain S, Finelli L, Shaw MW, Lindstrom S, Garten RJ, Gubareva LV, Xu X, Bridges CB, Uyeki TM Emergence of a novel swine-origin influenza A (H1N1) virus in humans. N. Engl. J. Med. 360:p doi:nejmoa [pii]; /nejmoa [doi]. 4. Esbenshade JC, Edwards KM, Esbenshade AJ, Rodriguez VE, Talbot HK, Joseph MF, Nwosu SK, Chappell JD, Gern JE, Williams JV, Talbot TR Respiratory virus shedding in a cohort of on-duty healthcare workers undergoing prospective surveillance. Infect. Control Hosp. Epidemiol. 34:p doi: / [doi]. 5. Lau LL, Cowling BJ, Fang VJ, Chan KH, Lau EH, Lipsitch M, Cheng CK, Houck PM, Uyeki TM, Peiris JS, Leung GM Viral shedding and clinical illness in naturally acquired influenza virus infections. J. Infect. Dis. 201:p doi: / [doi]. 6. Pabbaraju K, Wong S, Tokaryk KL, Fonseca K, Drews SJ Comparison of the Luminex xtag respiratory viral panel with xtag respiratory viral panel fast for diagnosis of respiratory virus infections. J. Clin. Microbiol. 49:p doi:jcm [pii]; /jcm [doi]. 7. Shain EB, Clemens JM A new method for robust quantitative and qualitative analysis of real-time PCR. Nucleic Acids Res. 36:p e91. doi:gkn408 [pii]; /nar/gkn408 [doi]. 8. van Elden LJ, van Loon AM, van der Beek A, Hendriksen KA, Hoepelman AI, van Kraaij MG, Schipper P, Nijhuis M Applicability of a real-time quantitative PCR assay for diagnosis of respiratory syncytial virus infection in immunocompromised adults. J. Clin. Microbiol. 41:p
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