What should be taken into consideration when establishing antiviral resistance surveillance? Aeron Hurt WHO Collaborating Centre for Reference and Research on Influenza, Melbourne, Australia New and improved!!! Please have a look!!

Some questions to consider when establishing resistance testing? Five broad considerations Specific considerations when establishing testing adamantane resistance testing neuraminidase inhibitor resistance testing Details regarding choice of neuraminidase inhibitor resistance assays Overview

Which drugs are being used in your country / region? Do you have a pandemic stockpile of antiviral drugs? What sort of samples will you be testing? What is the current level of resistance in circulating strains? Will other labs be testing strains from your region for resistance? Questions to consider when establishing resistance testing?

Which drugs are being used in your country / region? Do you have a pandemic stockpile of antiviral drugs? What sort of samples will you be testing? What is the current level of resistance in circulating strains? Will other labs be testing strains from your region for resistance? Questions to consider when establishing resistance testing?

-If drugs are being used in your country then resistance testing can help - guide choice of antiviral for patient management or - monitor that the current antiviral recommendations are appropriate -If little or no drugs are being used in your country/region is there still a need to test for resistance? - testing is expensive, requires expertise, etc (more later) - your data will be of use by WHO to monitor global resistance levels. - your MoH may be asked about resistance levels in your country if a neighbouring country begins reporting the detection of resistant strains - reminder that oseltamivir resistant seasonal A(H1N1) were first reported in high levels in Norway and other parts of Europe, where relatively little oseltamivir was used Which drugs are being used in your country / region?

Do you have a pandemic stockpile of antiviral drugs? What sort of samples will you be testing? What is the current level of resistance in circulating strains? Will other labs be testing strains from your region for resistance? Questions to consider when establishing resistance testing?

- if so it may be important to be able to competently conduct resistance testing - testing needs to be established BEFORE a pandemic - So if you plan to do testing in a pandemic situation it needs to be established in a interpandemic period Do you have a pandemic stockpile of antiviral drugs?

Which drugs are being used in your country / region? Do you have a pandemic stockpile of antiviral drugs? What sort of samples will you be testing? What is the current level of resistance in circulating strains? Will other labs be testing strains from your region for resistance? Questions to consider when establishing resistance testing?

-Surveillance samples - mostly from untreated patients - Hospital samples - may be from patients under treatment - immunocompromised patients under long term treatment with antivirals - Frequency of resistance is going to be different between the two sets of samples - Will cultured isolates or clinical specimens be available for testing? - How many samples need to be tested? - Will the samples need to be tested in a short time frame (eg for patient management)? What sort of samples will you be testing?

Which drugs are being used in your country / region? Do you have a pandemic stockpile of antiviral drugs? What sort of samples will you be testing? What is the current level of resistance in circulating strains? Will other labs be testing strains from your region for resistance? Questions to consider when establishing resistance testing?

- It is important to understand the levels of resistance in circulating strains - For example if there is 100% resistance in circulating strains and advice to clinicians is to not use these drugs…. is there really any need to conduct resistance testing?? -Similarly if resistance is historically extremely rare then will resistance testing result in a lot of work with very little outcome. What is the current level of resistance in circulating strains?

Which drugs are being used in your country / region? Do you have a pandemic stockpile of antiviral drugs? What sort of samples will you be testing? What is the current level of resistance in circulating strains? Will other labs be testing strains from your region for resistance? Questions to consider when establishing resistance testing?

Will other labs be testing strains from our region for resistance? -If labs decide to not conduct testing then submission of samples to a testing laboratory, such as a WHO CC, is important - Most WHO CC’s now test the majority (if not all) samples received from NICs - Therefore would mean that at least a selection of strains from that region would be tested and any major trends in susceptibility would be detected.

So after considering those questions……………..…… ….you decide you want to establish antiviral testing !!! Great… so here are some specific considerations when establishing….. adamantane resistance testing neuraminidase inhibitor resistance testing

So after considering those questions……………..…… ….you decide you want to establish antiviral testing !!! Great… so here are some specific considerations when establishing….. adamantane resistance testing neuraminidase inhibitor resistance testing

Adamantanes Inhibit M2 channel protein Amantadine and rimantadine (Symmetrel ™ and Flumadine ™) Adamantanes Full cross resistance between the two drugs

Adamantane resistance Australasia and South East Asia 100% resistance for the two currently circulating influenza A strains over the last two years All of the current resistance in both subtypes is due to the S31N mutation in M2 gene H3 H1 H1pdm 2009

Adamantane resistance All current resistance due to S31N mutation, makes adamantane resistance testing ideal for genotypic assays (four other mutations at positions 26, 27, 30, 34) Plus there isn’t an easy phenotypic assay So which genotypic assay to use??? Real-time PCR Pyrosequencing Sanger sequencing - If you have current expertise and equipment in one of these then that may be the easiest way to go - Real-time and pyrosequencing are more high throughput – but this is only relevant if you are planning on testing large number of samples - Analysis of samples that you have already extracted RNA from will also make the process cheaper

So after considering those questions………………………………… ……………….you decide you want to establish antiviral testing !!! What should be considered when establishing….. adamantane resistance testing neuraminidase inhibitor resistance testing

Zanamivir and Oseltamivir (Relenza™ and Tamiflu ™) Inhibit neuraminidase + Peramivir (IV) + Laninamivir (approved in Japan in Jan 2010) NA inhibitors Generally no cross resistance between zanamivir and oseltamivir

- To put your testing in context it is important to consider current/recent levels of resistance - Resistance to either oseltamivir or zanamivir in influenza A(H3N2) or B viruses is very rare - Since conducting testing in our lab we have identified: - 1 out >1800 A(H3N2) viruses resistant to oseltamivir (<0.1%) - E119V mutation - 1 out >1600 influenza B viruses to resistant (<0.1%) - D197E mutation - It’s a different story with H1N1 viruses……. NA inhibitor resistance

Pre-pandemic seasonal A(H1N1) - Saw widespread oseltamivir resistance due to the H275Y mutation in 2007/08 - Resistance was 100% in this subtype until the pandemic Pandemic H1N So how much resistance are we seeing in the H1N subtype? - Lots of reports detecting resistance are being published….. so it’s easy to think there is a lot of resistance out there! - Oseltamivir resistance (H275Y) in pdm A(H1N1) 2009: - From our lab detected 25 out of >3000 viruses (< 1%) - Globally 383 detected out of >27,000 viruses (  1%) -Other mutations have been reported to cause mild resistance - I223R, I223K, I223V, Q313R+I427T - but these have so far been very rare !!!! - 0 of >3000 viruses tested in our laboratory NA inhibitor resistance in A(H1N1)

So now knowing the current levels of resistance, and which mutations are causing resistance… which assay should you choose? Genotypic Advantages -Rapid, familiar technology for a lot of labs, staff already skilled, -Current resistance levels suggest really need to focus only on one mutation (H275Y) - Don’t need to culture virus Disadvantages -May miss a resistant strain that has a mutation that is not H275Y (but we have just heard how common those are …….) Phenotypic (NA inhibition assays) Advantages -Will detect any change in sensitivity regardless of where the mutation is Disadvantages -More expensive, more time consuming, specialist training necessary, need to culture virus

So you if you decide to do phenotypic testing…. …there are still a few more decisions to make!!! NA-Fluor™ Kit (ABI) NA-XTD™ Kit (ABI) NA-Star® Kit (ABI) No kit Purchase components separately Fluorescence-based assayChemiluminescence-based Purchased as a complete kit - Applied Biosystems -Reagents, plates, etc -Standard protocol Not in kit form -Substrate (MUNANA) from Sigma -Plates from another supplier -Make up your own buffers -Various protocols to choose from OR

So you if you decide to do phenotypic testing…. …there are still a few more decisions to make!!! NA-Fluor™ Kit (ABI) NA-XTD™ Kit (ABI) NA-Star® Kit (ABI) No kit Purchase components separately Fluorescence-based assayChemiluminescence-based Cost (in US$) to test one virus against one drug $4.06 $6.00 $15.60 $14.80 Higher IC 50 values for virusesLower IC 50 values for viruses Similar number of steps and time to run an assay OR

Which of the NA inhibitors do you want to use in your testing? - Remember that the more drugs you include the higher the costs of testing. - Oseltamivir - most widely used influenza antiviral and history shows us that resistance to this drug can occur and spread widely - Zanamivir – used less than oseltamivir and appears to be less prone to selecting for resistance – very few resistant strains have been detected to date -Peramivir and laninamivir – at this stage only used in Japan (and S.Korea), - Peramivir has a resistance profile that is typically in between oseltamivir and zanamivir -e.g. H275Y mutant = high osel resistance, low zanamivir, moderate peramivir resistance - Laninamivir – is structurally similar to zanamivir and therefore has a resistance profile similar to zanamivir

The neuraminidase inhibition assays require training and good quality control. -Training workshops have been run in the past and future courses can be organised - WPRO and WHO CC Melbourne organised and ran an antiviral testing workshop in Not just about learning the methodology, but there is a need to be able to interpret the data correctly, detect when things have gone wrong… not just take an IC 50 value and use it. - Useful to team up with a lab that currently conducts NAI testing, like a WHO CC. This enables strains to be shared and comparison of IC 50 values. - Currently no QAP for phenotypic NAI testing

Summary Before you establish antiviral resistance testing you have to ask yourself a lot of questions !!! The most appropriate methodology for antiviral susceptibility testing will differ for different labs, Your choice of methodology will be based on various factors such as whether you want to do adamantane or NAI resistance testing whether testing treated patients or community samples resources, budget, staff expertise Regardless of the methodology chosen, it is essential that you understand the limitations of the assay and interpret the data appropriately

Thank you to all WHO National Influenza Centres and other submitting laboratories for the provision of influenza isolates and epidemiological data The WHO Collaborating Centre for Reference and Research on Influenza, Melbourne is supported by the Australian Government Department of Health and Ageing Acknowledgements