1. 12/2/20041 Modern Methods for Influenza Detection and Sub-typing Ronald Cheshier

2. 12/2/20042 Introduction Training provided by the CDC, Association of Public Health Laboratories (APHL), and the National Laboratory Training Network (NLTN) October 25, 2004 to October 29, 2004 at the Georgia State Laboratory This course provided up to date information on Influenza epidemiology, surveillance, and laboratory testing protocols. One of the primary goals was to encourage the State Laboratories to develop the tools necessary for rapid response to a Pandemic Influenza A scenario.

3. 12/2/20043 Influenza Virus review Family Orthomyxoviridae: three types  Influenza A  Influenza B  Influenza C (not considered of critical importance)  Segmented (8) ssRNA genome with lipid envelop

4. 12/2/20044 Influenza A Further classified by Hemagglutinin (H) and Neuraminidase (N) sub-types Current circulating strains are H1N1 and H3N2 Human subtypes include H1N1, H3N2, H1N2, and H2N2 Avian subtypes include H1 to H15 and N1 to N9 Bird  human H5N1, H9N2, H7N7, H7N2, H7N3

5. 12/2/20045 Influenza B Produces less serious disease than does Influenza type A Not categorized as by H or N type as Influenza A is

6. 12/2/20046 Influenza C First isolated in 1949 Not known to be responsible for epidemics

7. 12/2/20047 Anticipated circulating strains for 2004/05 Northern hemisphere  A/Fujian/411/2002(H3N2)-like  A/New Caledonia/20/99 (H1N1)-like  B/Shanghai/361/2002-like

8. 12/2/20048 Influenza as a public health threat Influenza Viruses are the respiratory viruses of greatest public health importance, particularly Influenza A

9. 12/2/20049 Epidemiology, Prevention, and Control of Influenza; esp. Influenza A Why is Influenza A such a public health threat?  antigen drift (variation within the HN sub-type) or antigenic shift (variation between different HN sub-types) makes large portions of the population immunologically naïve on a regular basis  Influenza epidemics can be characterized as inter- pandemic or pandemic  Annual average US winter epidemics affect 5% to 20% of the population with approximately 200,000 influenza-related hospitalizations during the 1990’s and 36,000 influenza related deaths.  At one time it was thought that new HN variants were due to re-assortment of genetic material from an avian strain and a human strain in a third animal, like a pig. Modern evidence suggests that humans may act as this mixing vessel

10. 12/2/200410 For example A pig or person is infected with an avian influenza like an H5N4 at the same time the pig or person is infected with a human strain like H1N1; During the infections the two genomes re-assort to an H5N1 (with the avian H and the human N)

11. 12/2/200411 Global and US surveillance  Avian influenza: H5N1: HIGHLY PATHOGENIC  Present in Asia since 1996  Extent/distribution not firmly established  Threat level is high  In 1997 there were 18 confirmed cases of H5N1 with 6 deaths  Other avian strains being watched include H7N7 and H9N2

12. 12/2/200412 Pandemic Influenza  Recipe for a human pandemic  Emergence of a novel sub-type of influenza to which the population is immunologically naïve  Replication in humans  disease  Efficient human-to-human transmission  Note: H5N1 has meet all criteria except the third one.

13. 12/2/200413 Pandemic planning  An influenza pandemic will be unlike other public health emergencies or common disasters.  Inevitable  Will arrive with very little warning  Locally explosive epidemics  Widespread, not focused like a bio- terrorism event  Will put an extraordinary strain on human and material resources  Effect will be relatively prolonged –weeks to months

14. 12/2/200414 Laboratory issues Laboratory safety Tissue culture techniques Rapid test kits HA/HI sub-typing Immuno-fluorescent testing Real time PCR analysis  Molecular typing and sub-typing

15. 12/2/200415 Laboratory/Epidemiology Topics of Discussion Reviewing current Influenza surveillance testing algorithm Brief comments on alternative Influenza surveillance algorithms and the ability of this laboratory to support them.

16. 12/2/200416 Current testing algorithm  Inoculation of specimens into cell culture; one diploid, one Hep-2, one Viromed Rhmk and two Diagnostic Hybrid Rhmk  In the absence of CPE, “blind” Hemadsorption (HAD) at days 7 and 14.  In the absence of CPE “blind” passage of Hep2 with “blind” FA for RSV at day 14 for all patients ≤5 years old  Identification of Influenza isolates:  Immuno-fluorescent testing to identify type (A or B) followed by Hemagglutination Inhibition (HI) testing to identify sub-type

17. 12/2/200417 Alternatives to be considered The use of shell vials for more rapid isolation and identification of Influenza  Studies by Wisconsin and Iowa suggest that MDCK shell vials are more sensitive for Influenza than Rhmk cell culture  MDCK shell vials inoculated, incubated, and “blind” stained at day 5 for Influenza A and B with supernatant saved and used for HI testing (if necessary).  Things to consider: Cost: purchase shell vials commercially with goal of preparing our own shell vials once the cell culture preparation method has been established. The CDC provides the FA reagents as part of the WHO/CDC Influenza Kits received each year. In addition, commercial sources are available. Time and labor requirements; Not an issue once Arbovirus season is over.

18. 12/2/200418 Alternatives to be considered The use of Immuno-fluorescence (IFA) for Influenza A sub-typing  The CDC/WHO kit provides staining reagents for H1N1 and H3N2  Cost: We would have to purchase additional anti-mouse IgG conjugate commercially.  This procedure would decrease turn- around-time  Has the potential for allowing us to test for 2 sub-types at once by using different conjugates, I.e. FITC and Rhodamine

19. 12/2/200419 Alternatives to be considered Use of Rapid Test kits  Things to consider  Cost: These kits are very expensive and have relatively short shelf-lives  Poor positive predictive values in the absence of an outbreak (high sensitivity but low specificity)  These kits are probably more effective for use at a primary care setting during influenza season

20. 12/2/200420 Alternatives to be considered PCR: The CDC has provided the sequence data for the primers and probes for Influenza A (group) and Influenza B (group). In addition, it has provided the data for H1, H3, and H5 (avian considered as possible candidate for next pandemic).  Things to consider  Cost: While the cost of primers is probably manageable, probes are very expensive.  There will be a lag time as we will have to obtain all the probes and primers and do validation studies. This includes validation for H5; Note, we do not have any controls for that agent. The CDC has indicated they will provide a Proficiency set sometime next year.  The CDC primer sets are for H1, H3, and H5, not H1N1, H3N2, and H5N1 (no neuraminidases). The CDC is not overly concerned about this because it is the “H” (Hemagglutinin) that is related to pathogenicity but, if we choose to report based on PCR we will only be able to report on the Hemagglutinin result.

21. 12/2/200421 Suggested algorithms It is not the intent of the CDC to replace culture with PCR but rather to allow the States to have PCR testing available as a surveillance/rapid diagnostic tool Isolation will still be needed for vaccine related surveillance and production efforts Consider the purpose of this surveillance effort

22. 12/2/200422 Next Steps Laboratory Management and the State Epidemiology will have to meet to more carefully review and discuss the options available to us in terms of surveillance enhancement.

23. 12/2/200423 Items to consider It has been 20+ years since our Influenza algorithm has been changed A number of exciting new methods and tools are available to us including  The use of shell vials  Immuno-fluorescent sub-typing including multiplexing  PCR

24. 12/2/200424 Final algorithm  Should allow for the most rapid response possible  Cost considerations  Sensitivity and specificity of the tests  Validation studies, not just to satisfy CLIA requirements but also to ensure that the tests being performed are providing accurate information.

25. 12/2/200425 Presented By: Ron Cheshier, Virology Section Manager Arizona Department of Health Services - Bureau of State Laboratory Services (602) 542-6134 or cheshir@azdhs.gov