1. © The Children's Mercy Hospital, 2014. 03/14 Clinical Laboratory Testing for Detection of Influenza. Rangaraj Selvarangan. BVSc, PhD, D(ABMM). Associate Professor, UMKC-SOM Director, Microbiology Laboratory Director, Laboratory Medicine Research Affairs Children's Mercy Hospitals and Clinics Kansas City, MO 64108 International Conference on Flu, June 8-10, 2015 Chicago, USA

2. © The Children's Mercy Hospital, 2014. 03/14 2 Objectives Describe laboratory tests available for detection of influenza infection Identify strengths and weaknesses of traditional and molecular laboratory tests for Influenza Discuss FDA ruling on Rapid antigen tests Describe the potential clinical impact of rapid molecular testing for influenza

3. © The Children's Mercy Hospital, 2014. 03/14 3 Introduction Respiratory viral illness is one of the most common infection in children and adults Influenza and RSV cause seasonal outbreaks; early diagnosis improves care. Non-influenza respiratory illness are also common; estimated at 500 million/year, economic impact of $40 billion/year 1 Several studies show that a substantial percentage of both out patient (>70%) 2 and in-patient antibiotic use (up to 50%) is either unnecessary or inappropriate. In 2008, there were 142,000 visits to emergency departments for adverse events attributed to antibiotics. 3 1 Fendrick et al Arch Intern Med 2003, 2 Hersch et al Ped 2011, 3 Shehab et al Clin Inf Dis 2008

4. © The Children's Mercy Hospital, 2014. 03/14 4 Influenza Epidemics Member of the Orthomyxovirus, Influenza type A and B cause seasonal epidemics in humans. Transmission via large-particle droplets. Incubation 1-4 days. Every year in the United States, on average: 5% to 20% of the population gets the flu more than 200,000 people are hospitalized from flu-related complications about 36,000 people die from flu-related causes Shedding: Adults 5-10 days, Children >10 days, immunocompromised weeks or months.

5. © The Children's Mercy Hospital, 2014. 03/14 5 Influenza in Children Children aged 0–4 years – 100/100,000 children for those without high-risk medical conditions. – 500/100,000 children for those with high-risk medical conditions – Highest among children aged 0–1 years and are comparable to rates reported among persons aged > 65 years. Signs: Abrupt onset of constitutional and respiratory signs (fever, myalgia, headache, sore throat, cough). Children- otitis media, nausea and vomiting. Hospitalization: 4%-11% ICU, 3% mechanical ventilation. Complications: Otitis media, Bacterial Pneumonia, Encephalitis, encephalopathy, Myocarditis, Myositis PPV for clinical definition in children 79% to 88%.

6. © The Children's Mercy Hospital, 2014. 03/14 6 Influenza Epidemics Prophylaxis Diagnosis Treatment

7. © The Children's Mercy Hospital, 2014. 03/14 7 Influenza Culture Tube culture- PMK, MDCK (trypsin in medium), ML Co-cultured cells: R-Mix (DHI)- A549+ML, R-Mix Too- A549+MDCK. Rapid results >80% of flu specimens are positive on Day 1. R-Mix Too- may be better in FluB recovery and it does not support SARS-CoV. Dunn et al JCM 2004. Total 3803 respiratory specimens; compared tube culture to R-Mix SV. Flu A 238/241 (99%), Flu B 36/38 (95%) and RSV 52/60 (87%). Cryopreserved R-Mix Ready cells: Kim et al JCV 2008. Higher and early recovery of viruses in R- Mix ready cells Vs tube culture. Option for small volume laboratories and surplus inventory during peak winter season.

8. © The Children's Mercy Hospital, 2014. 03/14 8 Influenza DFA DFA for influenza is a highly sensitive and specific test. Technical expertise needed, TAT few hours Sensitivity is affected by sample quality- flocked swabs provide good cell recovery for DFA. Screening antibodies for 7 viruses are available from several vendors. Identification is by mAb. Cytospin improves DFA performance on slide preparations. Combination antibodies FluA/respiratory virus help for quick screening during peak influenza season. D3 Duet (DHI) SimulFluor (Millipore).

9. © The Children's Mercy Hospital, 2014. 03/14 9 Influenza Rapid Antigen Test Rapid antigen test for influenza have moderate sensitivity for seasonal Flu A/B (50-80%) and poor sensitivity (10% to 40%) for 2009 H1N1 virus. Specificity usually >90%. Influenza antigen negative results need confirmatory test performed; Culture, DFA, RT-PCR. Benefit of Influenza rapid antigen test in the OP setting – patient triage, infection control, reduced antibiotic usage, timely antiviral treatment, reduce unnecessary diagnostic workups, overall reduction in medical care cost.

10. © The Children's Mercy Hospital, 2014. 03/14 10

11. © The Children's Mercy Hospital, 2014. 03/14 11 Rapid Antigen Test-Flu A/B Host Factors: –Age –Duration of illness –Specimen type –Specimen transport

12. © The Children's Mercy Hospital, 2014. 03/14 12 Host factors Age: Children excrete high viral loads during influenza infections Duration of illness: Viral load in respiratory secretions decrease with duration of illness. Testing within 3 days of illness onset improves detection Specimen Type: Nasopharyngeal aspirates, nasopharyngeal washes, nasopharyngeal swabs, mid turbinate swabs are preferred specimens. Nasal and throat swabs results in sub-optimal yield Specimen Transport: Collection in viral transport medium and rapid transport on ice improves yield Cheng et al., 2009; Esposito et al., 2011; Loeb et al., 2012; Talbot et al., 2010

13. © The Children's Mercy Hospital, 2014. 03/14 13 Specimen collection Nasopharyngeal aspirates and nasal washes: High viral load, diluted, mucous can interfere with EIA/DFA performance. Cell quantity may vary between collection. Suitable for culture, PCR and later flow tests. Recent experience indicates that a combination of NP swab and throat swab in VTM may improve diagnostic yield. Flocked swabs: More cells collected, less mucous, well suited for DFA. Nasopharyngeal and mid- turbinate swabs available.

14. © The Children's Mercy Hospital, 2014. 03/14 Tilt patient head 70 degree angle and against wall Insert swab straight back (not upward) until resistance is met Rotate the swab 5-10 times to loosen cells Remove swab and inoculate VTM

15. © The Children's Mercy Hospital, 2014. 03/14 15 Swab studies Abu-Diab et al JCM 2007; NPA Vs NP swab. 455 children. Sensitivity of NP swab 98.5%. Allen et al PASCV 2008 poster # M28; MTS Vs NP swab. 203 children, PCR gold standard. NPS =87.5%, MTS =79%. 8% of both NPS and MTS insufficient for DFA. Nurse prefer MTS. Selvarangan et al PASCV 2009 poster # M51. 200 children. NPA Vs MTS. RSV antigen and SV culture. RSV antigen sensitivity 66% for MTS and 70% for NPA. Nurses prefer MTS over NPA.

16. © The Children's Mercy Hospital, 2014. 03/14 16 Rapid Antigen Test-Flu A/B Viral Factors: –Influenza activity –Viral subtype

17. © The Children's Mercy Hospital, 2014. 03/14 17 Test Performance Sensitivity : proportion of actual positives which are correctly identified as such (i.e. the percentage of sick people who are identified as having the condition) Specificity : proportion of negatives which are correctly identified (i.e. the percentage of well people who are identified as not having the condition). The probability of the presence or absence of disease given the results of a test. Positive predictive value (PPV) : proportion of patients with positive test results who are correctly diagnosed. Negative predictive value (NPV): proportion of patients with negative test results who are correctly diagnosed. Wikipedia.org

18. © The Children's Mercy Hospital, 2014. 03/14 Beginning and end of the season- It is advisable to confirm all positives by culture due to low PPV Test Result

19. © The Children's Mercy Hospital, 2014. 03/14 RADT RVP RADT, RVP ProFlu PCR LDT-PCR FluA/B Cx Influneza pandemic 2009

20. © The Children's Mercy Hospital, 2014. 03/14 Rapid AntigenPCR Pre pH1N1Post pH1N1Pre pH1N1Post pH1N1 NPO (n=22)19 (86.4%)14 (63.6%)01 (4.5%) Hosp Ntwrk (n=31)24 (77.4%)19 (61.3%)4 (12.9%)8 (25.8%) Comm Hosp (n=43)40 (93%)36 (83.7%)00 Academic Inst (n=33)18 (55%)8 (24%)*5 (15%)14 (42%)* Survey of Lab Methods Selvarangan et al ASM conference 2010 Use of molecular methods for detection of respiratory viruses 2009 = 18% (Selvarangan, ClinMicronet Survey) 2011= 36% (Miller, ClinMicronet Survey) 2013 = 63% (Miller, ClinMicronet Survey)

21. © The Children's Mercy Hospital, 2014. 03/14 The updated VE estimate against influenza A H3N2 viruses was 18% (95% confidence interval (CI): 6%-29%). The VE estimate against influenza B viruses this season was 45% (95% CI: 14% – 65%).

22. © The Children's Mercy Hospital, 2014. 03/14 22 Rapid Antigen Test-Flu A/B Analytical Factors –Test characteristics –Result interpretation Post Analytical Factors –Reporting Time –Clinical interpretation

23. © The Children's Mercy Hospital, 2014. 03/14 Next –Generation Influenza RADT 3M Influenza BD Veritor Quidel Sofia

24. © The Children's Mercy Hospital, 2014. 03/14 24 Rapid Antigen Tests- Instrument Reader Hassan et al JCM 2014

25. © The Children's Mercy Hospital, 2014. 03/14 25 Rapid Antigen Tests- Instrument Reader Dunn et al DMID 2014

26. © The Children's Mercy Hospital, 2014. 03/14

27. Manufacturer 1: ref method Culture; 79% sensitivity (75-83%)

28. © The Children's Mercy Hospital, 2014. 03/14 FDA Ruling May 2014-RIDT Reference Method: Viral culture Sensitivity Flu A Point estimate of 90% (95% C.I. >80%) Flu B Point estimate of 80% (95% C.I. >70%) Specificity lower bound of the 95% CI exceeding 90% for both, Flu A and Flu B. Reference Method: Molecular method Sensitivity Flu A Point estimate of 80% (95% C.I. >70%) Flu B Point estimate of 80% (95% C.I. >70%) Specificity lower bound of the 95% CI exceeding 90% for both, influenza A and influenza B.

29. © The Children's Mercy Hospital, 2014. 03/14 FDA Ruling May 2014-RIDT Laboratory Tests: Influenza viral antigens or influenza viral gene segments (protein or nucleic acid), either in single unit test formats or multi-test formats Monitoring Performance: Conduct annual analytical testing of their device with contemporary strains Standardized panel of viruses selected in coordination with FDA. Dilutions at 10e2 and 10e5 TCID50/mL in triplicate. Detection of all replicates at least at one dilution. Standardized panels of well characterized viral stocks could possibly be available from CDC or commercial vendors. The testing could be conducted in-house or at a contract laboratory. Absence of analytical reactivity would be reflected in labeling as a limitation. Emerging Influenza strains: Provide analytical reactivity report to FDA within 60 days of emerged virus strain availability.

30. © The Children's Mercy Hospital, 2014. 03/14 30 Diagnostic Challenges Problem1: Lack of a rapid and highly sensitive method for detection of Influenza at POC setting. Problem 2: Lack of sensitive method for extensive detection of respiratory viruses in hospitalized patients –Impact: Improper use of antivirals, overuse of antibiotics, additional diagnostic workup, improper infection control measures, over all increase in health care cost.

31. © The Children's Mercy Hospital, 2014. 03/14 31 Diagnostic Challenge-Solution Problem 1: Lack of a rapid and highly sensitive method for detection of Influenza at POC setting. Solution- RADT-Instrument read, Rapid multiplex RP- NAAT- Alere™ i Influenza A & B, iQuum Liat Influenza A/B, Focus Simplexa™ FluA/B & RSV Direct, Cepheid Xpert ® Flu, and GenXpert and BioFire Filmarray™ RP Considerations: Clinical performance data limited, Cost, clinical experience limited, Lack of complete understanding of the hierarchy among these tests

32. © The Children's Mercy Hospital, 2014. 03/14 32 Molecular Nucleic Acid Amplification Methods for Respiratory Virus Detection

33. © The Children's Mercy Hospital, 2014. 03/14 33 Multiplex RP-NAAT: Rapid BioFire Filmarray™ RP (~60 min) Cepheid Xpert ® Flu(75 min) Focus Simplexa™ FluA/B & RSV Direct (~60 min) iQuum Liat Influenza A/B (20 min ) Alere™ i Influenza A & B (15 min )

34. © The Children's Mercy Hospital, 2014. 03/14 34 Alere i Influenza Vs Culture Specimen DetectionTPFPTNFNTotal % Sensitivity (95% CI) % Specificity (95% CI) PPV (95% CI) NPV (95% CI) Flu A Children12853201454 99.298.496.299.6 (95.1-99.9) (96.2-99.4) (91.0-98.6)(98.0-99.9) Adults17256075 10096.589.4100 (77.0-100)(87.0-99.4)(65.4-98.1)(92.0-100) Flu B Children7003792451 97.2100 99.5 (89.4-99.5) (98.7-100) (93.5-100)(97.9-99.9) Adults13062075 100 (71.6-100)(92.7-100)(71.6-100)(92.7-100) Bell et al; Journal of Clinical Virology 2014 61, 81-86

35. © The Children's Mercy Hospital, 2014. 03/14 35 Alere i Influneza Vs ProFlu PCR Alere™ i Influenza A&B vs. real-time RT-PCR TPFPTNFNTotal % Sensitivity (95% CI) % Specificity (95% CI) PPV (95% CI) NPV (95% CI) Influenza A MTS761709156 89.498.698.788.6 (80.4-94.7)(91.3-99.9)(92.0-99.9)(79.0-94.3) NPW/A27145477 87.197.896.491.8 (69.2-95.8)(87.0-99.9)(79.8-99.8)(79.5-97.4) Total103211513233 88.898.398.189.8 (81.3-93.7)(93.3-99.7)(92.6-99.7)(82.9-94.3) Influenza B MTS31−124−155 100 (86.3-100)(96.3-100)(86.3-100)(96.3-100) NPW/A27−50−77 100 (84.5-100)(91.1-100)(84.5-100)(91.1-100) Total58−174−232 100 (92.3-100)(97.3-100)(92.3-100)(97.3-100) Bell et al; J Clin Microbiol.Bell et al; J Clin Microbiol. 2014 Nov;52(11):3992-5.

36. © The Children's Mercy Hospital, 2014. 03/14 36 Alere i Influenza Vs ProFlu PCR

37. © The Children's Mercy Hospital, 2014. 03/14 37 Cobas Liat Influenza assay FDA-cleared, rapid (<20 min) PCR assay (Roche cobas® Liat) to Focus Simplexa™ Flu A/B & RSV Direct using respiratory swabs (n=197). The cobas Liat influenza A and B assays demonstrated sensitivities of 99.2% (123/124) and 100% (23/23), respectively, while showing a specificity of 100% for both targets. Binnicker et al: J Clin Microbiol.Binnicker et al: J Clin Microbiol. 2015 Apr 29. pii: JCM.00791-15.

38. © The Children's Mercy Hospital, 2014. 03/14 38 Multiplex RP-NAAT: Extended Panel Luminex xTAG RVP (n=12) and RVP FAST (n=8) BioFire Filmarray RP (n= 20 )Nanosphere RV Plus test (n=7) GenMark eSensor RVP (n=14)

39. © The Children's Mercy Hospital, 2014. 03/14 39 CAP-ID Resp Panel-FLu 2014 2014 IDR-A summary

40. © The Children's Mercy Hospital, 2014. 03/14 Flu Antiviral Resistance Influenza viruses Antiviral2009 H1N1 Seasonal H1N1 Seasonal H3N2 Flu B AdamantanesResistant Susceptible ResistantN/A Oseltamivir Susceptible* Resistant Susceptible Zanamivir Susceptible * Few oseltamivir-resistant 2009 H1N1 strains reported CDC

41. © The Children's Mercy Hospital, 2014. 03/14 41 Cost of the test Reimbursement issues Loss of detection due to mutations* Competitive inhibition of analytes in a multiplex assay Limited clinical experience for certain infections: coronavirus, rhinovirus and co- infections. *Hawkinson et al DMID 2013 Multiplex RP NAAT Disadvantages

42. © The Children's Mercy Hospital, 2014. 03/14 42 Influenza Diagnostics- Summary Rapid antigen tests are useful for OP management. Antigenic drift and shift in Influenza strains influences test performance. Specimen collection method and VTM approved for the antigen test need to be followed Monitor QC controls and review data periodically Reflex antigen negative test result to confirmatory testing Rapid molecular tests for Influenza will improve diagnostic yield SV culture is a simple assay with Flu isolation mostly by day 1 Multiplex PCR assays improve Flu detection and other respiratory infections associated with ILI Epidemiological data or individual Flu subtype testing may be necessary to determine antiviral resistance.