Enhanced Etiological Diagnosis Of Respiratory Virus Outbreaks Using Nucleic Acid Amplification Testing Against An Expanded Range Of Targets Sallene Wong 1, Bonita E. Lee 2,3, Kanti Pabbaraju 1, Kara L. Tokaryk 1, Anita Wong 1, Kevin Ho 1 and Julie D. Fox 1,4 1 Provincial Laboratory for Public Health (ProvLab), Calgary, 2 ProvLab, Edmonton, 3 Department of Pediatrics, University of Alberta, Edmonton, 4 Microbiology & Infectious Diseases, University of Calgary, Alberta, Canada STUDY BACKGROUND AND AIMS Use of nucleic acid amplification tests (NATs) has enhanced our ability to provide an etiological diagnosis in respiratory virus outbreaks. The aim of this study was to evaluate the performance of NATs on epidemiologically linked cases of respiratory infection and to assess the utility of the Luminex xTag Respiratory Viral Panel (RVP) assay to enhance outbreak investigations. Virology and molecular diagnostic laboratory technologists and assistants in PLPH (Calgary and Edmonton) undertook routine specimen extraction and testing by NASBA and RT-PCR. PASCV 2008, M-38 Samples Nasopharyngeal (NP) and throat swab specimens from 243 suspected respiratory virus outbreaks were submitted to the ProvLab for diagnostic investigation in 2006/2007 (sample n=1093). Diagnostic algorithm In our routine testing algorithm NP samples are first subjected to direct fluorescent antigen (DFA) testing for influenza virus (IFV)A, IFVB, parainfluenza (PIV)1-3 and respiratory syncytial virus (RSV). If DFA positive results are obtained no further testing of the outbreak is undertaken. DFA-negative NP samples from undiagnosed outbreaks and all throat swab specimens from outbreaks are then screened for a panel of viruses by NATs (1). Nucleic acid extraction and NATs Nucleic acid was extracted using the easyMAG® automated extractor and reagents (bioMérieux). Individual real-time NATs were directed against IFVA, IFVB, (PIV) 1-4, RSV, human metapneumovirus (hMPV) and respiratory adenoviruses (ADVs) (2). Luminex xTag RVP assay (Luminex Molecular Diagnostic Inc.) was performed according to the manufacturers instructions (3) on samples for which no positive results had been obtained by DFA/in house NATs. Sequencing of picornavirus positive samples Primers in the VP1 and 5NCR regions of picornaviruses (4) were used for amplification of positive samples. Products were purified and sequenced using the BigDye Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems). Sequences were analyzed using Sequencing Analysis Software v5.1.1 (ABI) and aligned using ClustalW. Phylogenetic trees were constructed using the MegAlign module from Lasergene 6 (DNAstar). Undiagnosed outbreak investigation by RVP Phylogenetic analysis of picornaviruses Outbreak investigations by DFA/NATs ACKNOWLEDGEMENTS REFERENCES CONCLUSIONS 2006/2007 OUTBREAK DIAGNOSIS SUMMARY MATERIALS AND METHODS Etiology of outbreaks by DFA/NATs 1st January st December 2007 Total number of outbreaks tested= 243 1st January st December 2007 Total number of outbreaks tested= 63 Etiology of outbreaks by RVP The Luminex xTag RVP assay allows for efficient, multiplex detection of a broader range of respiratory viral targets than our routine NAT panel. The added identification of picornaviruses and coronaviruses, which are not included in our current algorithm, will greatly improve our etiological diagnosis of respiratory virus outbreaks. The serotyping of rhinoviruses based on VP1 and 5NCR was identical where both sequences were available. The same serotypes of human rhinovirus belong to individual outbreaks were observed. A total of 195 outbreaks in 2006/2007 had an etiological diagnosis using DFA/in house NATs. The detection rate increased from 67% to 80% after addition of RVP testing. YearOutbreaks with etiological diagnosis Outbreaks without etiological diagnosis Total outbreaks tested % positive % % Total % YearOutbreaks with etiological diagnosis Outbreaks without etiological diagnosis Total outbreaks tested % positive % % Total % Low outbreaks activity during the summer months in An improvement from 83% to 91% in diagnosis using RVP in addition to routine testing. HKU1 and OC43 detected by RVP in Feb-06. Picornavirus detected in 9 outbreaks by RVP. 1.Fox, J. D Respiratory virus surveillance and outbreak investigation. J.Clin.Virol. 40 Suppl 1:S24-S30. 2.Lee, B. E., J. L. Robinson, V. Khurana, X. L. Pang, J. K. Preiksaitis, and J. D. Fox Enhanced identification of viral and atypical bacterial pathogens in lower respiratory tract samples with nucleic acid amplification tests. J.Med.Virol. 78: Krunic, N., T. D. Yager, D. Himsworth, F. Merante, S. Yaghoubian, and R. Janeczko xTAG RVP assay: analytical and clinical performance. J.Clin.Virol. 40 Suppl 1:S39-S46. 4.Lee, W. M., C. Kiesner, T. Pappas, I. Lee, K. Grindle, T. Jartti, B. Jakiela, R. F. Lemanske, P. A. Shult, and J. E. Gern A diverse group of previously unrecognized human rhinoviruses are common causes of respiratory illnesses in infants. PLoS.ONE. 2:e966. Positive targetsNumber of samples tested positive IFVA2 PIV42 PIV4 + OC431 OC433 NL635 HKU11 picornavirus49 Comparison of the partial 5NCR sequences for rhinoviruses from respiratory samples. Prototype sequences from GenBank are also included. A A Unclassified B A Month-year RESULTS Month-year 2006 Number of outbreaks Outbreaks without an etiological diagnosis occurred predominantly outside of the main respiratory virus season (especially summer and autumn of 2007). An improvement from 37% to 60% in diagnosis using RVP in addition to routine testing. Picornavirus (3), OC43 (2) and NL63 (2) detected by RVP in outbreaks during the period of Jan-07 to Mar-07. During the months of May to October, picornavirus outbreaks were detected by RVP. Human rhinovirus serotypes circulating during in Alberta were typed by sequencing. Sequences with greater than 95% identity to published sequences are included. Human rhinoviruses detected included: Group A: 1B, 12, 23, 29, 41, 43, 51, 58, 59 and 94. Group B: 6. Unclassified: W10, W13, W21, W23, W28, QPM, and Antwerp rhinovirus 98/99 isolate Identical rhinovirus serotypes were detected in all samples from an outbreak except 2 different serotypes were found in 2 individual outbreaks. Etiological agents detected by RVP in outbreaks without a diagnosis by DFA/in house NAT Etiological agents detected by DFA/NATs Outbreaks without diagnosis Outbreaks without a diagnosis by DFA/in house NATs for which etiological diagnosis was provided by RVP Outbreaks with etiological diagnosis by DFA/NATs 2007