Drug-resistant H7N9 strains to change treatment: researcher

[tetano]

TAIPEI--The discovery that some H7N9 bird flu virus strains have developed drug resistance will affect the strategies for dealing with future cases, a researcher said Saturday.

Some H7N9 strains found in a Taiwanese businessmen who became the first and only confirmed case outside China in late April after returning from there, were resistant to Tamiflu, a drug used to prevent and treat flu, said Shih Shin-ru, director of Chang Gung University's Research Center for Emerging Viral Infections.

Since there have also been H7N9 virus strains with the same drug-resistant gene found in a case in Shanghai, Shih said, Lee was probably exposed to large amounts of the virus in China.

..

http://www.chinapost.com.tw/taiwan/national/national-news/2013/05/27/379652/Drug-resistant-H7N9.htm

[Giuseppe]

Re: Drug-resistant H7N9 strains to change treatment: researcher

For the sake of clarity, these findings were also cited in the recent WHO/China Joint Mission assessment paper: http://www.who.int/influenza/human_a...eport2013u.pdf


(...)

The first three isolates contained a number of genetic signatures previously associated in other subtypes with low pathogenicity in poultry, enhanced capacity for mammalian infection, resistance to the adamantane class of antiviral drugs and sensitivity to the neuraminidase inhibitors oseltamivir and zanamivir. These signatures include the following:

? A single arginine at the HA cleavage site, consistent with low pathogenicity in poultry;
? Deletion of five amino acids in the NA stalk, associated with adaptation to poultry;
? Q226L substitution in the HA, associated with enhanced binding to the α-2,6-linked sialylated receptors found in the mammalian respiratory tract;
? E627K in the PB2 protein, associated with viral replication at the lower temperature of the mammalian respiratory tract;
? S31N in the M2 protein, conferring resistance to adamantanes;
? Absence of the H275Y substitution in the NA, associated with resistance to the oseltamivir in H1N1 viruses;
? R292K in one virus (A/Shanghai/1/2013), associated with markedly reduced sensitivity to oseltamivir and modestly reduced sensitivity to zanamivir.

In vitro analyses confirmed that all three viruses bound both α -2,3- and α -2,6 linked sialic acids, suggesting an ability to bind to both avian and mammalian cells.

In functional assays, all three isolates were sensitive to both NAIs. In the case of A/Shanghai/1/2013, this result appears to reflect the presence of a mixture of viruses with R or K at position 292 of the NA.

(...)

[Biological]

Re: Drug-resistant H7N9 strains to change treatment: researcher

Just to note:
53(M) Lee (Taiwan (imported via Suzhou, Jiangsu)

16-Apr-13 recieved 75mg Tamiflu PO (Taipei Medical University Hospital)
20-Apr-13 recieved 150mg Tamiflu PO and on 24-Apr-13 recieved peramivir IV (National Taiwan University Hospital (NTUH)

Hospital releases avian flu patient after 35 days - Taipei Times

http://www.taipeitimes.com/News/taiwan/archives/2013/05/25/2003563136

Chang et al. 2013 Lancet (75mg/150mg)

http://download.thelancet.com/flatcontentassets/pdfs/S0140673613609435.pdf

[NS1]

Re: Drug-resistant H7N9 strains to change treatment: researcher

The original deposit of the Taiwan E1 passage does not show the TamiFlu Resistance marker, but 2 isolates of ostensibly (no metadata) alternate tissue samples from a subsequent Taiwan deposit show the same change as the emergent H7N9 index case from Shanghai and two other May samplings from the Pasteur Shanghai release.

At this time, 5 of the emergent H7N9 human sequences indicate genotypic TamiFlu Resistance.

[NS1]

Re: Drug-resistant H7N9 strains to change treatment: researcher

Fatalities
Associated With
TamiFlu Resistance
via
NA 292K

Of the 5 sequences representing 4 human cases that carry the NA 292K polymorphism, 2 fatalities have occurred, both in Shanghai. At least 3 of the 4 cases required treatment involving in extremis measures such as ECMO.

Today's publication in The Lancet from Dr. Malik Peiris and an extensive multi-disciplinary team of Chinese organisations defines clinical progressions, treatments, outcomes and experimental findings including discussions on two TamiFlu Resistance markers, NA 292K and NA 152K. Their initial study reveals that the single 292K polymorphism increases oseltamivir resistance 100 fold.

www.thelancet.com Published online May 28, 2013 http://dx.doi.org/10.1016/S0140-6736(13)61125-3
Association between adverse clinical outcome in human disease caused by novel influenza A H7N9 virus and sustained viral shedding and emergence of antiviral resistance

We have shown that A/Shanghai/1/2013 (H7N9) virus isolate contains a mixed population of Arg/Lys at position 292 of the NA gene, and by purifying virus plaques that carry NA Arg292 and Lys292, we have noted that this mutation increases resistance to oseltamivir by 100-fold and zanamivir by 30-fold in a fluorescence-based NA inhibition assay (unpublished data).

In patient 6 (from the mechanical ventilation group), we found emergence of Arg152Lys mutation in the NA gene. This mutation was first reported from an
immunocompromised patient infected with influenza B after zanamivir treatment.13 Using the baculovirus expressed N9 NA protein, it had been shown to exhibit mild resistance to both zanamivir and oseltamivir in vitro.14

We have been concerned that the few detailed clinical progressions may not have fully reported sepsis. This paper provides insight confirming one type of clinical similarity to other zoonotics including many pH1N1 cases and H5N1 human infections:

We detected viral RNA in the serum obtained at some time during the clinical illness of 12 (86%) of the 14 patients (appendix; all three patients in the ECMO group, all four patients in the mechanical ventilation group, and five of seven patients in the pneumonia group).

[NS1]

Re: Drug-resistant H7N9 strains to change treatment: researcher

TamiFlu Resistance
via
NA 292K

Originally published on 2013-05-31-06:24 at China - H7N9 Human Isolates on Deposit at GISAID [thread].

www.thelancet.com Published online May 28, 2013 http://dx.doi.org/10.1016/S0140-6736(13)61125-3
Association between adverse clinical outcome in human disease caused by novel influenza A H7N9 virus and sustained viral shedding and emergence of antiviral resistance

Four of the fourteen Sanger Neuraminidase sequences deposited at GenBank by Fudan University today and associated with the 2013-05-28 AntiViral Resistance paper (Malik Peiris and associates, The Lancet) show NA 292K. Two patients were indicated in the paper as developing antiviral resistance at NA 292K.

We are grateful to Dr. Peiris for coordinating production of the cross-reference information with the authoring organisations concerning serialisation and patient assignment for the samplings.

NA 292K

Our preliminary groupings for the four genotypic AntiViral Resistant sequences are estimated until we receive the master cross-reference. These groups represent either patient 2 or patient 3 from the tables in the paper. ECMO was required for each. Patient 2 expired at 19 days post-onset; patient 3 remained under treatment at 46 days (as of May 18, 2013). Group 1 is very likely Patient 2 who died during ECMO treatment. At least one fatality is represented in these 2 groupings.

We have arranged Group 1 due to Hemagglutinin homology:

Group 1 with 3 Sequences
Probable as Patient 2 Fatality
Drug Resistance by Treatment Day 4

• ChinaShanghai5240T_88M_2013_04_25_TmX_f [KF028381]
• ChinaShanghai5180T_88M_2013_04_23_TmX_f [KF028380]
• ChinaShanghai5083T_88M_2013_04_20_TmX_f [KF028379]

Group 2 with 1 Sequence
Probable as Patient 3

• ChinaShanghai4842T_56M_2013_04_13_TmX_s [KF028383]

NA Polymorphism Summary

ChinaShanghai4798T_62M_2013_04_12_TmX_s [KF028387] shows 152K and is likely Patient 6 who was mechanically ventilated and successfully discharged 35 days from onset. The polymorphism developed after day 3 of oseltamivir treatment and by the sampling date on day 5 of treatment.

ChinaShanghai4821T_2013_04_12_s [KF028392] shows NA 285K [284K] with first and third base polymorphisms. Only the emergent H7N9 ChinaShanghai4664T_2013_03_05 [KC853231] and a single instance from H4N9 avian show this polymorphism in the data record.

HA Polymorphism Summary

Three HA sequences carry a synonymous change at the third base of aa471 producing syn471E (GAg) [syn462E (GAg)]:

Probable as Patient 2 Fatality

• ChinaShanghai5240T_88M_2013_04_25_TmX_f [KF028376]
• ChinaShanghai5180T_88M_2013_04_23_TmX_f [KF028375]
• ChinaShanghai5083T_88M_2013_04_20_TmX_f [KF028374]

All three are also TamiFlu Resistant and are from a patient that required ECMO.

This syn462E (GAg) polymorphism has been seen in non-emergent H7N9 and in 5 emergent Human H7N9 sequences, including 3 from the single Taiwan case and 2 from the Pasteur Shanghai GISAID deposit of preliminary sequences on 2013-05-23.

The revision is also found in Pandemic H1N1 2009 (pH1N1), H5N1 (wildtype), H7N3 (extensive), H7N7 (rare, passerine), H9N2 (extensive, ostrich) and H9N9. Four of the nine instances within pH1N1 (all host transition from 2009) are from China and three others are from neighboring Asian countries. Argentina shows 2 sequences from subjects aged 2 years and younger. Seven of the eight pH1N1 sequences with this polymorphism and age metadata are pediatric cases and one of those Chinese sequences was published as part of a drug resistance investigation.

Additional Reading on Emergent H7N9 Genetics

We acknowledge the authors, originating and submitting laboratories of the sequences from GenBank & from GISAID’s EpiFlu™ Database on which this research is based. An additional list is detailed in the linked PDF entitled "GISAID_Citations_H5N1_2011" at Is H7N9 Spreading from Human to Human in China? Post#164

GISAID Citations

• GeneWurx Cross Serotype Homology Analysis, Open-Access, Full-Text version
• Human Emergent H7N9 from Zhejiang Province
• Environment Emergent H7N9 from Zhejiang
• Fatal H5N1 Homology to Emergent H7N9 from Shanghai in March
• Fatal H5N1 Karo Cluster Homology to Emergent H7N9 from First Fujian Case
• Receptor Binding Site Novelty from First Taiwan H7N9 Case
• China - H7N9 Human Isolates on Deposit at GISAID, Comprehensive Genetics Discussion

[Giuseppe]

Re: Drug-resistant H7N9 strains to change treatment: researcher

Source: WHO, full PDF document: http://www.who.int/entity/influenza/...ew_31May13.pdf

[NS1]

Re: Drug-resistant H7N9 strains to change treatment: researcher

Genetic Quantification Errors

The WHO comprehensive report contains errors, including notations concerning PB2 701N, a very important mammalian adaptation. In two places (p6 and Table2), they exclude ChinaZhejiang2_64M_2013_04_03 though the sequence is clearly included in the Hemagglutinin review (Table 1).

Page 6, Section 5. Characterization of the A(H7N9) viruses (emphases mine):

The PB2 proteins from some H7N9 viruses isolated from humans have mutations at positions 627 (Glu to Lys in the human isolates from Anhui, Hangzhou and Shanghai) or 701 (Asp to Asn in A/Zhejiang/DTID-ZJU01/2013) which impart enhanced replication at temperatures similar to that of the upper airway of mammalian hosts and possibly humans as well (Hatta et al. 2007, Massin et al. 2001).

Page 26
Table 2. Markers of mammalian adaptation in internal proteins of viruses of avian origin (emphasis mine):

Table 2, PB2

701Asn one human isolate

Neuraminidase Inhibition

This supplying of Tables 3a and 3b is not relevant to this thread because no H7N9 sequence with genotypic or phenotypic drug resistance is profiled in either table. H3N2 drug resistance is managed at depth for this H7N9 review from WHO with comparatives on 5 different H3N2 viruses with 2 types of genotypic drug resistance measured using 2 different protocols. They have thoroughly measured and thoroughly reported, but not on drug resistance risk / ratings for H7N9.

The experimental evaluation of a 100-fold genotypic and multi-patient severity phenotypic H7N9 sequence was, in fact, produced in the paper that is the topic of this thread.

www.thelancet.com Published online May 28, 2013 http://dx.doi.org/10.1016/S0140-6736(13)61125-3
Association between adverse clinical outcome in human disease caused by novel influenza A H7N9 virus and sustained viral shedding and emergence of antiviral resistance

We have shown that A/Shanghai/1/2013 (H7N9) virus isolate contains a mixed population of Arg/Lys at position 292 of the NA gene, and by purifying virus plaques that carry NA Arg292 and Lys292, we have noted that this mutation increases resistance to oseltamivir by 100-fold and zanamivir by 30-fold in a fluorescence-based NA inhibition assay (unpublished data).

Qualitative Errors

Two points do come to mind, however, concerning potential qualitative errors in the WHO Review:

Point A

Page 1, Section Summary (emphasis mine):

Epidemiologic studies and laboratory analyses of virus isolates have provided a vast amount of information in a very short time.

This paper was published on May 31, 2013.

Does the "vast amount of information" that is recognised by the authors on Page 1 in their tribute to transparency include the 9 human sequences with genotypic TamiFlu Resistance due to NA 292K or the single human sequence with genotypic mild TamiFlu Resistance due to NA 152K?

10 Stealth H7N9 TamiFlu Resistant Sequences

Are the authors, WHO are self-reportedly working hand-in-hand with the bench scientists, aware of the early detection during treatment at Day 2 for the very first case of emergent H7N9 who expired on March 4, 2013, more than 7 weeks prior to this paper? Was the vast information flow limited at that time? Has consideration been given to the importance of resistance that emerged at Day 4 for a case who expired after aggressive oseltamivir / peramivir combination therapy with ECMO on April 29, 2013, more than 30 days prior to publication?

• If no author or reviewer is aware, then WHO is at the helm?
• If any notion of awareness does exist, then WHY are these resistant items absent from today's report?
• At prevailing market rates, what is the probability that genetic drug resistance produces official reporting aversion?

Point B

This section may be made moot if a supplemental pdf is available at some point detailing isolation method / experimental design variations between WHO Collaborating Centers in Atlanta, Tokyo and Melbourne. A search of the WHO Influenza H7N9 topic does not show a supplement, but does prominently feature a claim that "limited information" exists for the disease.

emphasis mine:

As yet, there is limited information about the scope of the disease the virus causes and about the source of exposure.

The following evaluation cross-references data from Table 3a (Page 27) and Table 3b (Page 28) of today's "vast information" paper.

IC50 (nanoMolar) neuraminidase-inhibitor antiviral drugs

Protocol Legend

• F=fluorescent
• C=chemiluminescence

11.9 fold variance for same virus, different protocol, different lab.
Oseltamivir only

• H3N2 A/Fukui/45/2004 . . F. . 37.00 . . Atlanta
• H3N2 A/Fukui/45/2004 . . C. . 03.10 . . Tokyo

2.4 fold | 4.0 fold | 3.2 fold | 3.02 fold variance for same virus, same protocol, different lab.
Oseltamivir | Peramivir | Zanamivir | Laninamivir

• H7N9 A/Anhui/1/2013 . . F. . 0.17 | 0.06 | 0.33 | 0.46 . . Atlanta
• H7N9 A/Anhui/1/2013 . . F. . 0.41 | 0.24 | 1.06 | 1.39 . . Melbourne

Atlanta is higher on H3N2 and lower on H7N9? Are we to be concerned when drug efficiency scores are reported as highly variable with no clear annotations or explanations?

Additional Reading on Emergent H7N9 Genetics

We acknowledge the authors, originating and submitting laboratories of the sequences from GenBank & from GISAID’s EpiFlu™ Database on which this research is based. An additional list is detailed in the linked PDF entitled "GISAID_Citations_H5N1_2011" at Is H7N9 Spreading from Human to Human in China? Post#164

GISAID Citations

• GeneWurx Cross Serotype Homology Analysis, Open-Access, Full-Text version
• Human Emergent H7N9 from Zhejiang Province
• Environment Emergent H7N9 from Zhejiang
• Fatal H5N1 Homology to Emergent H7N9 from Shanghai in March
• Fatal H5N1 Karo Cluster Homology to Emergent H7N9 from First Fujian Case
• Receptor Binding Site Novelty from First Taiwan H7N9 Case
• China - H7N9 Human Isolates on Deposit at GISAID, Comprehensive Genetics Discussion