What should be taken into
consideration when establishing
antiviral resistance surveillance?
Aeron Hurt
WHO Collaborating Centre for Reference and
Research on Influenza, Melbourne, Australia
www.influenzacentre.org
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Overview
• 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
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?
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?
Which drugs are being used in your country / region?
-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
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?
Do you have a pandemic stockpile of antiviral drugs?
- 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
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?
What sort of samples will you be 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)?
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?
What is the current level of resistance in circulating strains?
- 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.
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?
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
Adamantanes
Inhibit M2 channel protein
Amantadine and rimantadine
(Symmetrel ™ and Flumadine ™)
Full cross resistance between
the two drugs
Adamantane resistance
100% resistance for the two
currently circulating
influenza A strains over the
last two years
Australasia and
South East Asia
100
H3
% adamanatne resistant
90
H1pdm 2009
80
70
60
H1
50
H3
40
pdmH1 2009
30
H1
20
10
0
2005
2006
2007
2008
2009
2010
2011
Sam ple date (Year)
All of the current resistance in
both subtypes is due to the
S31N mutation in M2 gene
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
Sanger sequencing
Pyrosequencing
- 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
NA inhibitors
Inhibit neuraminidase
Zanamivir and Oseltamivir + Peramivir (IV) + Laninamivir
(Relenza™ and Tamiflu ™) (approved in Japan in Jan 2010)
Generally no cross resistance
between zanamivir and oseltamivir
NA inhibitor resistance
- 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 in A(H1N1)
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 H1N1 2009
- So how much resistance are we seeing in the H1N1 2009 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
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!!!
Fluorescence-based assay
No kit
Purchase components
separately
Not in kit form
- Substrate
(MUNANA)
from Sigma
- Plates from
another
supplier
- Make up your
own buffers
- Various
protocols to
choose from
NA-Fluor™ Kit
(ABI)
OR
Chemiluminescence-based
NA-Star® Kit
(ABI)
Purchased as a complete kit
- Applied Biosystems
- Reagents, plates, etc
- Standard protocol
NA-XTD™ Kit
(ABI)
So you if you decide to do phenotypic testing….
…there are still a few more decisions to make!!!
Fluorescence-based assay
No kit
Purchase components
separately
NA-Fluor™ Kit
(ABI)
OR
Chemiluminescence-based
NA-Star® Kit
(ABI)
NA-XTD™ Kit
(ABI)
Cost (in US$) to test one virus against one drug
$4.06
$6.00
Higher IC50 values for viruses
$15.60
$14.80
Lower IC50 values for viruses
Similar number of steps and time to run an assay
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 2009
- 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 IC50 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 IC50 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
Acknowledgements
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
www.influenzacentre.org