Update of Antiviral Resistance in Seasonal and Pandemic Influenza Viruses 3 rd NIC Meeting, Beijing,...

Update of Antiviral Resistance in Seasonal and Pandemic

Influenza Viruses3rd NIC Meeting, Beijing, China

August 18-20th 2009

Aeron HurtAeron Hurt

WHO Collaborating Centre for Reference and Research on Influenza, WHO Collaborating Centre for Reference and Research on Influenza, MelbourneMelbourne

OverviewOverview

Neuraminidase InhibitorsNeuraminidase Inhibitors– Emergence of oseltamivir resistance in A(H1N1) in Emergence of oseltamivir resistance in A(H1N1) in

20082008– Resistance monitoring of pandemic H1N1 2009 Resistance monitoring of pandemic H1N1 2009

virusesviruses

Adamantanes Adamantanes – Current levels of resistance in seasonal and Current levels of resistance in seasonal and

pandemic virusespandemic viruses

SummarySummary

Influenza antiviral drugsInfluenza antiviral drugs

Neuraminidase (NA) inhibitors

Zanamivir and Oseltamivir(Relenza™ and Tamiflu ™)

Used since 1999

Large volumes stockpiled for pandemic use

Effective for influenza A and B

Prior to 2007, resistance was uncommon

Inhibit neuraminidase

Emergence of oseltamivir resistanceEmergence of oseltamivir resistance

European 2007/2008 influenza seasonEuropean 2007/2008 influenza season

– Oseltamivir resistant isolates first detected in France, UK and Norway in late Oseltamivir resistant isolates first detected in France, UK and Norway in late 20072007

• H274Y mutation in NA responsible for the resistanceH274Y mutation in NA responsible for the resistance



– Oseltamivir resistant isolates first detected in France, UK and Norway in late Oseltamivir resistant isolates first detected in France, UK and Norway in late 20072007

• H274Y mutation in NA responsible for the resistanceH274Y mutation in NA responsible for the resistance

Change in ICChange in IC5050

OseltamivirOseltamivir 1500-fold1500-fold

ZanamivirZanamivir No changeNo change

What impact does the H274Y have on resistance?What impact does the H274Y have on resistance?



– Oseltamivir resistant isolates first detected in France, UK and Norway in late 2007Oseltamivir resistant isolates first detected in France, UK and Norway in late 2007• H274Y mutation in NA responsible for the resistanceH274Y mutation in NA responsible for the resistance

– From untreated patients - low oseltamivir usage in EuropeFrom untreated patients - low oseltamivir usage in Europe

0

1

2

3

4

5

6

Japan USA Europe

No

. of

pre

scri

pti

on

s (m

illio

ns)

Oseltamivir prescriptions in 2007

Source : IMS Rx data

Germany: 49 % Germany: 49 % France: 38 %France: 38 %Greece: 1 %Greece: 1 %Finland: 3 %Finland: 3 %Belgium: 7 %Belgium: 7 %Austria: 2 %Austria: 2 %

Other countries with Other countries with negligible usenegligible use





– Subsequent testing revealed spread of mutant throughout EuropeSubsequent testing revealed spread of mutant throughout Europe


Meijer et. al., EID, 15 (4), 2009






– Variable resistance seen in different countriesVariable resistance seen in different countries


47 %47 %

1 %1 %

Compared with < 1% Compared with < 1% resistance in previous resistance in previous

seasons across all countries!!seasons across all countries!!

67 %67 %

Meijer et. al., EID, 15 (4), 2009






– Variable resistance seen in different countriesVariable resistance seen in different countries

– ““Fit” oseltamivir resistant virus circulating not driven by drug usageFit” oseltamivir resistant virus circulating not driven by drug usage USA 2007/2008 influenza seasonUSA 2007/2008 influenza season

– Oseltamivir resistant viruses being detectedOseltamivir resistant viruses being detected

25%25%16%16%

??

25%25%16%16%

WHO CC Melbourne received H1 viruses from WHO CC Melbourne received H1 viruses from these countries during 2008 seasonthese countries during 2008 season

% Resistance in A(H1N1) viruses in % Resistance in A(H1N1) viruses in 20082008

0%0%(n=5)(n=5)

6%6%(n=34)(n=34)

30%30%(n=10)(n=10)

32%32%(n=22)(n=22)

44%44%(n=34)(n=34)

100%100%(n=26)(n=26)

86%86%(n=111)(n=111)

100%100%(n=7)(n=7)

91%91%(n=11)(n=11)

75%75%(n=4)(n=4)

Emergence of H274Y mutant viruses in different countries.

Australia

Macau

Malaysia

New Zealand

New Caledonia

Philippines

Singapore

South Africa

Taiwan

Thailand

2007 2008

Oct Nov Dec Jan Feb Mar Apr May Jun Jul Aug Sep Oct

Oseltamivir resistant H274Y virus

Oseltamivir sensitive virus

Emergence of H274Y mutant viruses in different countries.

Australia

Macau

Malaysia

New Zealand

New Caledonia

Philippines

Singapore

South Africa

Taiwan

Thailand

2007 2008

Oct Nov Dec Jan Feb Mar Apr May Jun Jul Aug Sep Oct

Predominantly H274Y mutants

Predominantly H274Y mutants

Meijer et. al., EID, 15 (4), 2009Meijer et. al., EID, 15 (4), 2009

20 24 28 32 36 40 44 48 52 20 24 28 32 36 40 44 48 52

9090

100100

= prevalence of oseltamivir-

resistant A(H1N1) H274Y mutants

in Oceania, South East Asia and South Africa

Speed of H274Y global spreadSpeed of H274Y global spread

From 0 to 100% in one year!From 0 to 100% in one year!

..and now in 2009…..and now in 2009…

AustraliaAustralia 97% (n=33)97% (n=33)

ThailandThailand 100% (n=18)100% (n=18)

SingaporeSingapore 100% (n=17)100% (n=17)

PhilippinesPhilippines 100% (n=9)100% (n=9)

FijiFiji 100% (n=7)100% (n=7)

New ZealandNew Zealand 100% (n=6)100% (n=6)

TahitiTahiti 100% (n=1)100% (n=1)

Virtually 100% of seasonal A(H1N1) viruses are Virtually 100% of seasonal A(H1N1) viruses are oseltamivir resistantoseltamivir resistant

Regional publication – Regional publication – WHO CC and NIC collaborationWHO CC and NIC collaboration

It was great It was great opportunity for an NIC / opportunity for an NIC /

CC collaboration and CC collaboration and co-publication –co-publication –

27 authors!!!27 authors!!!

M N P N Q K…………………N A P N F H Y EM N P N Q K…………………N A P N F H Y E

1 2 3 4 5 6 ………………………270 271 272 273 274 275 276 277 1 2 3 4 5 6 ………………………270 271 272 273 274 275 276 277

N1 amino acid sequenceN1 amino acid sequence

H = Oseltamivir SensitiveH = Oseltamivir SensitiveY = Oseltamivir ResistantY = Oseltamivir Resistant

QuestionQuestion: If this is residue 275 why do : If this is residue 275 why do people refer to the mutation as H274Y?people refer to the mutation as H274Y?

AnswerAnswer: Because they are referring to the : Because they are referring to the residue based on N2 numberingresidue based on N2 numbering

H274Y or H275Y ?H274Y or H275Y ?

M N P N Q K…………………N A P N F H Y EM N P N Q K…………………N A P N F H Y E

1 2 3 4 5 6 ………………………270 271 272 273 274 275 276 277 1 2 3 4 5 6 ………………………270 271 272 273 274 275 276 277


M N P N Q K………………….... G S A Q H V EM N P N Q K………………….... G S A Q H V E

1 2 3 4 5 6 ………………………….. 270 271 272 273 274 275 276 1 2 3 4 5 6 ………………………….. 270 271 272 273 274 275 276


Equivalent Histidine residueEquivalent Histidine residue

H274Y or H275Y ?H274Y or H275Y ?

Traditionally N1 residues have been numbered Traditionally N1 residues have been numbered based on the equivalent residue in the N2 based on the equivalent residue in the N2 neuraminidaseneuraminidase

However, either However, either H274YH274Y or or H275YH275Y are are acceptable for reporting, publication, etcacceptable for reporting, publication, etc

Important that:Important that:– State which numbering system you are usingState which numbering system you are using

• eg H274Y (based on N2 numbering) eg H274Y (based on N2 numbering)

– Ensure that you are looking at the correct Ensure that you are looking at the correct residue!!!residue!!!

H274Y or H275Y ?H274Y or H275Y ?

OverviewOverview



virusesviruses

Adamantanes Adamantanes – Current levels of resistance in seasonal and Current levels of resistance in seasonal and


SummarySummary

•As of 31 July, 162,000 cases confirmedAs of 31 July, 162,000 cases confirmed

•Unprecedented volumes of oseltamivir (and Unprecedented volumes of oseltamivir (and zanamivir) used to treat infected patients and zanamivir) used to treat infected patients and

prophylax contactsprophylax contacts

•Only eight reported cases of oseltamivir resistanceOnly eight reported cases of oseltamivir resistance• Denmark, 4 x Japan, Canada, Hong Kong, Denmark, 4 x Japan, Canada, Hong Kong,

SingaporeSingapore

•All resistant viruses contained the H274Y All resistant viruses contained the H274Y mutation mutation

Pandemic H1N1 2009Pandemic H1N1 2009

•Only eight reported cases of oseltamivir resistanceOnly eight reported cases of oseltamivir resistance• Denmark, 4 x Japan, Canada, Singapore, Hong KongDenmark, 4 x Japan, Canada, Singapore, Hong Kong

Patients under Patients under oseltamivir oseltamivir prophylaxisprophylaxis

No evidence of No evidence of transmission of resistant transmission of resistant

virus to contactsvirus to contacts

Patient did not Patient did not receive oseltamivirreceive oseltamivir

Of more concern if Of more concern if resistant viruses are resistant viruses are

occurring in the absence occurring in the absence of drug selective of drug selective

pressurepressure

Oseltamivir resistant casesOseltamivir resistant cases

Already infected?Already infected?Suboptimal dose?Suboptimal dose?

Patient Patient received full received full oseltamivir oseltamivir

dosedose

Current resistance testing Current resistance testing at WHO CC, Melbourneat WHO CC, Melbourne

Clinical specimens Clinical specimens (that have not been (that have not been

cultured)cultured)

Cultured Cultured isolates isolates

NA enzyme NA enzyme inhibition assayinhibition assay(Fluorescence-(Fluorescence-

based) based)

Pyrosequencing Pyrosequencing or conventional or conventional

sequencingsequencing

NA enzyme inhibition assayNA enzyme inhibition assay

Neuraminidase enzyme inhibition assay (fluoresence-Neuraminidase enzyme inhibition assay (fluoresence-based) testing of cultured isolatesbased) testing of cultured isolates

April May June July Aug TOTAL

New Zealand 4 0 22 1 0 27

Australia 0 15 69 27 0 111

Philippines 0 1 0 0 0 1

Singapore 0 4 7 0 0 11

Fiji 0 0 3 0 0 3

TOTAL 4 20 101 28 0 153

None of the isolates tested have demonstrated resistance None of the isolates tested have demonstrated resistance to either oseltamivir or zanamivirto either oseltamivir or zanamivir

Genetic analysisGenetic analysis

Conventional sequencingConventional sequencing Pyrosequencing Pyrosequencing

– Mutation detectionMutation detection• H274YH274Y

– Rapid: following PCR, can analyse 96 samples in Rapid: following PCR, can analyse 96 samples in less than one hourless than one hour

– Dr Yi-Mo Deng, WHO CC, MelbDr Yi-Mo Deng, WHO CC, Melb

None of the clinical specimens tested have contained the None of the clinical specimens tested have contained the H274Y mutationH274Y mutation

Monitoring for H274Y mutationMonitoring for H274Y mutation

Advantages Advantages – Most likely resistance mutation to arise in N1 under Most likely resistance mutation to arise in N1 under

oseltamivir pressureoseltamivir pressure– Quick, most labs have PCR / sequencing expertiseQuick, most labs have PCR / sequencing expertise

Disadvantages Disadvantages – Other oseltamivir or zanamivir mutations which can Other oseltamivir or zanamivir mutations which can

confer resistance may be missedconfer resistance may be missed• Just because it has a H @ 274, doesn’t mean that it is Just because it has a H @ 274, doesn’t mean that it is

sensitive to oseltamivir!sensitive to oseltamivir!

– New strain unsure of which other resistance New strain unsure of which other resistance mutations may occur (eg N294S in H5N1)mutations may occur (eg N294S in H5N1)

OverviewOverview



virusesviruses

AdamantanesAdamantanes– Current levels of resistance in seasonal and Current levels of resistance in seasonal and


SummarySummary


Adamantanes

Inhibit M2 channel protein

Amantadine and rimantadine(Symmetrel ™ and Flumadine ™)

Used since 1967


Adamantanes

Inhibit M2 channel protein

Amantadine and rimantadine(Symmetrel ™ and Flumadine ™)

Used since 1967

Effective only for influenza A

Rapidly select for resistance

Adamantane resistanceAdamantane resistance

0

10

20

30

40

50

60

70

80

90

100

2005 2006 2007 2008 2009

Sample date (Year)

% a

dam

anat

ne

resi

stan

t

A(H3N2)A(H3N2)

• Overall – Australasia and South East AsiaOverall – Australasia and South East Asia

Adamantane resistanceAdamantane resistance

0

10

20

30

40

50

60

70

80

90

100

2005 2006 2007 2008 2009

Sample date (Year)

% a

dam

anat

ne

resi

stan

t

A(H3N2)A(H3N2)

A(H1N1) seasonalA(H1N1) seasonal

A(H1N1) A(H1N1) pandemic pandemic

20092009

• Overall – Australasia and South East AsiaOverall – Australasia and South East Asia

OverviewOverview



virusesviruses

AdamantanesAdamantanes– Current levels of resistance in seasonal and Current levels of resistance in seasonal and


SummarySummary

SummarySummary

Virtually 100% of current seasonal A(H1N1) viruses are oseltamivir resistant Virtually 100% of current seasonal A(H1N1) viruses are oseltamivir resistant

However the number of seasonal A(H1N1) viruses circulating has decreased However the number of seasonal A(H1N1) viruses circulating has decreased

significantly since the emergence of pandemic A(H1N1) significantly since the emergence of pandemic A(H1N1)

– Will the seasonal A(H1N1) continue to circulate?Will the seasonal A(H1N1) continue to circulate?

SummarySummary





20 July 27 July 3 August 10 August

A(H1N1) pandemic 120 74 75 35

A(H3N2) 7 9 3 00

A(H1N1) 1 0 2 00

Influenza B 0 0 0 00

Influenza specimens analysed from Victoria, Australia, by VIDRL Influenza specimens analysed from Victoria, Australia, by VIDRL

Data kindly provided by Dr Chris Birch, VIDRL, MelbourneData kindly provided by Dr Chris Birch, VIDRL, Melbourne

SummarySummary





Large volumes of NA inhibitors have been used to treat pandemic Large volumes of NA inhibitors have been used to treat pandemic

A(H1N1) infectionsA(H1N1) infections

– Encouragingly only a few cases of resistance have been detected Encouragingly only a few cases of resistance have been detected

with no evidence of further transmissionwith no evidence of further transmission

Pandemic A(H1N1) and seasonal A(H3N2) are all resistant to Pandemic A(H1N1) and seasonal A(H3N2) are all resistant to

adamantanesadamantanes

Continued monitoring of pandemic A(H1N1) viruses is essential, Continued monitoring of pandemic A(H1N1) viruses is essential,

particularly in patients under treatment and their contactsparticularly in patients under treatment and their contacts

WPRO / SEARO Antiviral WorkshopWPRO / SEARO Antiviral Workshop

‘‘Hands-on’ laboratory based workshop Hands-on’ laboratory based workshop Melbourne WHO CC, November 9-13Melbourne WHO CC, November 9-13thth 2009 2009 Phenotypic assaysPhenotypic assays

– Fluorescence-basedFluorescence-based– Chemiluminescence-basedChemiluminescence-based

Genetic assaysGenetic assays– Real-time PCRReal-time PCR

Information to get testing establishedInformation to get testing established– SOPs, Sourcing antivirals, control viruses SOPs, Sourcing antivirals, control viruses

Submission of influenza isolates and specimensSubmission of influenza isolates and specimens

Thank you to all WHO National Influenza Centres and other submitting Thank you to all WHO National Influenza Centres and other submitting laboratories for the provision of influenza isolates and epidemiological laboratories for the provision of influenza isolates and epidemiological datadata

WHO CC Influenza, Melb staffWHO CC Influenza, Melb staff

Jo Ernest – NA inhibition assaysJo Ernest – NA inhibition assaysYi-Mo Deng – Pyrosequencing (rapid confirmation of mutations)Yi-Mo Deng – Pyrosequencing (rapid confirmation of mutations)Pina Iannello and Naomi Komadina – Conventional sequencingPina Iannello and Naomi Komadina – Conventional sequencingRobert Shaw and Helen Sjogren – Culture of virusesRobert Shaw and Helen Sjogren – Culture of viruses

The WHO Collaborating Centre for Reference and Research on Influenza, The WHO Collaborating Centre for Reference and Research on Influenza, Melbourne is supported by the Australian Government Department of Melbourne is supported by the Australian Government Department of Health and AgeingHealth and Ageing

AcknowledgementsAcknowledgements

Update of Antiviral Resistance in Seasonal and Pandemic Influenza Viruses 3 rd NIC Meeting, Beijing,...

Documents

Transcript of Update of Antiviral Resistance in Seasonal and Pandemic Influenza Viruses 3 rd NIC Meeting, Beijing,...