1

The use of PCR in the surveillance, characterization and

diagnosis of influenza

Report of the 10th meeting of the WHO Working Group for the Molecular

Detection and Subtyping of Influenza Viruses and the use of Next Generation

Sequencing (NGS) in GISRS

Saint Petersburg, Russian Federation, 21–22 August 2018

2

ISBN 978-92-4-000069-8

© World Health Organization 2020

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This publication contains the report of the use of polymerase chain reaction (PCR) in the surveillance, characterization and diagnosis of influenza: report of the 10th meeting of the WHO Working Group for the Molecular Detection and Subtyping of Influenza Viruses and the use of Next Generation Sequencing (NGS) in GISRS and does not necessarily represent the decisions or policies of WHO.

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Abbreviations and acronyms

AAHL Australian Animal Health Laboratory

AI avian influenza

CC collaborating centre

CDC Centers for Disease Control and Prevention (US)

CEIRS Centers of Excellence for Influenza Research and Surveillance

CHP Centre for Health Protection (Hong Kong SAR, China)

CNIC Chinese National Influenza Center (Beijing, China)

CRH Children’s Research Hospital

CSIRO Commonwealth Scientific and Industrial Research Organisation (Australia)

EEIQAP European external influenza quality assessment programme

EIQAP external influenza quality assessment programme

EQAP External Quality Assessment Programme

EU European Union

EURO WHO regional office for Europe

FAO Food and Agriculture Organization of the United Nations

FDA Food and Drug Administration (US)

GISAID Global Initiative on Sharing All Influenza Data

GISRS Global Influenza Surveillance and Response System (WHO)

HA haemagglutinin

HAI haemagglutination inhibition

HPAI highly pathogenic avian influenza

IAV influenza A virus

IBV influenza B virus

ICV influenza C virus

LPAI low pathogenicity avian influenza

M matrix

NA neuraminidase

NGS next-generation sequencing

NIC national influenza centre

NIID National Institute of Infectious Diseases (Japan)

NIRC national influenza reference centre (US)

OFFLU OIE/FAO Network of Expertise on Animal Influenza

OIE World Organisation for Animal Health

PHE Public Health England (the United Kingdom)

PT proficiency testing

QA quality assurance

RNA ribonucleic acid

rRT-PCR real-time reverse-transcription polymerase chain reaction

5

RSV respiratory syncytial virus

RT-PCR reverse-transcription polymerase chain reaction

SAR Special Administrative Region

ToR terms of reference

UKAS the United Kingdom Accreditation Service

US United States

USA United States of America

VCM vaccine composition meeting

VE vaccine effectiveness

VIDRL Victorian Infectious Diseases Reference Laboratory

WER Weekly Epidemiological Record

WGS whole genome sequencing

WHO World Health Organization

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Contents

Abbreviations and acronyms ........................................................................................................................ 2

1. Introduction .............................................................................................................................................. 8

1.1. Background – PCR and the WHO PCR Working Group ................................................................. 8

1.2. Meetings of the PCR Working Group ............................................................................................ 8

2. Updates from WHO collaborating centres, H5 reference laboratories, NICs and OFFLU .................... 9

2.1. Dr Rodney Daniels, WHO CC for Reference and Research on Influenza (the Francis Crick

Institute), London, United Kingdom of Great Britain and Northern Ireland (the United Kingdom) ......... 9

2.2. Dr Yi-Mo Deng, WHO CC for Reference and Research on Influenza (Victorian Infectious

Diseases Reference Laboratory [VIDRL]), Melbourne, Australia .............................................................. 9

2.3. Mr John Franks, WHO CC for Studies on the Ecology of Influenza in Animals (St Jude Children’s

Research Hospital [CRH]), Memphis, United States of America ............................................................ 10

2.4. Dr Tsutomu Kageyama, National Institute of Infectious Diseases (NIID), Tokyo, Japan ............. 10

2.5. Dr Hui-Ling Yen, WHO H5 Reference Laboratory, School of Public Health, The University of

Hong Kong, China, Hong Kong SAR ......................................................................................................... 11

2.6. Dr Frank Wong, Commonwealth Scientific and Industrial Research Organisation (CSIRO),

Australian Animal Health Laboratory (AAHL), Geelong, Victoria, Australia............................................ 11

2.7. Dr Herman Tse, Centre for Health Protection (CHP), China, Hong Kong SAR ............................. 12

2.8. Dr Joanna Ellis, NIC, Public Health England (PHE), London, the United Kingdom ...................... 12

2.9. Dr Steve Lindstrom and Dr John Barnes, US CDC ....................................................................... 12

3. PCR-related activities in OFFLU ........................................................................................................... 13

4. Guidance for the use of NGS ............................................................................................................... 14

4.1. Overview of the latest NGS developments ................................................................................. 14

4.2. Use of NGS in individual institutions ........................................................................................... 14

4.2.1. US CDC................................................................................................................................. 14

4.2.2. Chinese National Influenza Center (CNIC), Beijing, China ................................................... 15

4.2.3. WHO CC for Reference and Research on Influenza (the Francis Crick Institute), London,

the United Kingdom ............................................................................................................................ 15

4.2.4. WHO CC for Reference and Research on Influenza (VIDRL), Melbourne, Australia ........... 15

4.2.5. WHO CC for Studies on the Ecology of Influenza in Animals (St Jude CRH), Memphis,

Tennessee, USA ................................................................................................................................... 15

4.2.6. NIID, Tokyo, Japan ............................................................................................................... 16

4.2.7. WHO H5 Reference Laboratory, School of Public Health, The University of Hong Kong,

Hong Kong SAR, China ......................................................................................................................... 16

4.2.8. OFFLU .................................................................................................................................. 16

7

4.2.9. CHP, Hong Kong SAR, China ................................................................................................ 16

4.2.10. PHE, London, the United Kingdom ...................................................................................... 17

4.2.11. Smorodintsev Research Institute of Influenza, Saint Petersburg, Russian Federation ....... 17

4.2.12. D.I. Ivanovsky Research Institute of Virology, Moscow, Russian Federation ..................... 17

4.2.13. State Research Centre of Virology and Biotechnology VECTOR, Koltsovo, ........................ 17

4.3. Discussion on the use of NGS in GISRS ....................................................................................... 18

4.4. Guidance for NICs on the use of NGS ......................................................................................... 18

5. PCR protocols for GISRS ...................................................................................................................... 19

5.1. Overview of current PCR protocols for GISRS ............................................................................. 19

5.2. Gaps and actions ......................................................................................................................... 20

6. Quality Assurance ............................................................................................................................... 20

6.1. EQAP: observations on progress made and future plans ........................................................... 20

6.2. WHO Regional Office for Europe (EURO) EEIQAP: experiences and lessons learned................. 21

6.3. OFFLU strategy on Proficiency Testing panel (PT) for PCR ......................................................... 22

6.4. Experiences and lessons learned from the USA CDC .................................................................. 22

6.5. Discussion on the WHO EQAP ..................................................................................................... 22

7. Way forward ....................................................................................................................................... 23

7.1. Strategy for GISRS surveillance ................................................................................................... 23

7.2. GISRS capacity: gaps and priority actions ................................................................................... 24

7.3. New technology and rapid influenza diagnostics ....................................................................... 24

7.4. Updating the WHO laboratory manual ....................................................................................... 24

7.5. Possible publication in a peer-reviewed journal ......................................................................... 24

8. Proposed action points ....................................................................................................................... 25

References .................................................................................................................................................. 25

Annex 1: List of participants ....................................................................................................................... 26

Annex 2: Declarations of interest ............................................................................................................... 28

Annex 3: Meeting agenda ........................................................................................................................... 29

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1. Introduction

1.1. Background – PCR and the WHO PCR Working Group

Molecular detection methods enable the rapid and accurate detection of influenza viruses; an

example of such a method is the real-time reverse-transcription polymerase chain reaction (rRT-

PCR) assay. Although such methods are widely used in routine surveillance of seasonal influenza

viruses, the emergence of A(H7N9) and the various A(H5) reassortants (e.g. H5N1 and H5N6) in

the human population demonstrates the importance of accurately detecting and subtyping non-

seasonal viruses. The accumulation of large volumes of full genome molecular data with high-

throughput next-generation sequencing (NGS) allows for the timely tracking of influenza virus

evolution, to inform decision-making in vaccine development, use of antiviral drugs and

pandemic response strategies. Therefore, unified standards and protocols are required for the

World Health Organization (WHO) Global Influenza Surveillance and Response System (GISRS)

network of laboratories, to maintain sensitivity and precision in influenza virus detection and

screening. The WHO Working Group for the Molecular Detection and Subtyping of Influenza

Viruses and the use of NGS in GISRS (the PCR Working Group)1 acts as an expert technical group

to advise GISRS on developments in molecular technologies, to ensure that standards and

protocols are maintained and updated.

1.2. Meetings of the PCR Working Group

The PCR Working Group was initially established after the outbreak of highly pathogenic avian

influenza (HPAI) A(H5N1) to provide advice on the use of rRT-PCR in the detection and subtyping

of influenza viruses. The 2017 meeting was held in Hong Kong Special Administrative Region

(SAR), China, on 12–13 April; since the meeting, the following actions have been taken:

• PCR protocols have been updated;2

• an executive summary was published in the Weekly Epidemiological Record (WER) and a

full meeting report was produced; and

• a guidance document on NGS for the GISRS network was drafted.

The objectives of the 2018 meetings were to:

• review work performed since the previous meeting;

• review the PCR-related activities and quality assurance (QA) in the World Organisation for

Animal Health/Food and Agriculture Organization of the United Nations (OIE/FAO)

Network of Expertise on Animal Influenza (OFFLU);

• update the currently published PCR protocols, and identify gaps and follow-up actions;

1 http://www.who.int/influenza/gisrs_laboratory/pcr_working_group/en/ 2 http://www.who.int/influenza/gisrs_laboratory/molecular_diagnosis/en/

http://www.who.int/influenza/gisrs_laboratory/pcr_working_group/en/http://www.who.int/influenza/gisrs_laboratory/molecular_diagnosis/en/

9

• review the guidance for national influenza centres (NICs) on the use of NGS;

• discuss the results and plans for QA for GISRS; and

• discuss the strategy for GISRS surveillance, gaps and priority actions.

The expected outcomes of this meeting were to:

• update PCR protocols and guidance on the WHO website;

• finalize a consensus guidance document on the use of NGS for NICs;

• finalize the terms of reference (ToR) document for the working group;

• update the way forward for the WHO External Quality Assessment Project (EQAP), if

required to meet the changing needs of NICs and influenza laboratories;

• identify actions and ways forward for the working group; and

• publish a meeting report and executive summary in the WER.

2. Updates from WHO collaborating centres, H5 reference laboratories,

NICs and OFFLU

Representatives from the WHO collaborating centres (CCs), H5 reference laboratories, NICs and

OFFLU provided general updates on their activities over the previous year. A summary of their

presentations is outlined below.

2.1. Dr Rodney Daniels, WHO CC for Reference and Research on Influenza (the Francis

Crick Institute), London, United Kingdom of Great Britain and Northern Ireland (the

United Kingdom)

• The United Kingdom CC has moved to using MiSeq NGS for all surveillance work. The CC

performs everything up to the library preparation stage; the sample is then passed to the

advanced sequencing facility at the Francis Crick Institute.

• Training of NICs on NGS was conducted using the Illumina MiSeq platform. A few NICs

have followed their NGS protocol and used DNASTAR’s laser gene suite of programs for

data analysis.

• Most of the NICs around the world have rRT-PCR assays in place, and the frequency of

virus typing or subtyping misdesignations has dropped considerably. Occasional

misdesignations are detected, but these are from poorly resourced laboratories.

2.2. Dr Yi-Mo Deng, WHO CC for Reference and Research on Influenza (Victorian

Infectious Diseases Reference Laboratory [VIDRL]), Melbourne, Australia

• A record high number of influenza virus samples were received at the CC in the 2017

influenza season. Most of the viruses isolated were A(H3) (52.6%), followed by

B/Yamagata-lineage (26.4%).

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• The amount of NGS work continued to increase in 2017, with 1400 haemagglutinin (HA),

neuraminidase (NA) and matrix (M) genes, and 109 whole viral genomes sequenced, all

performed in-house using the Ion Torrent PGM platform. A total of 1590 virus genes were

submitted to the Global Initiative on Sharing All Influenza Data (GISAID).

• The CC participated in WHO’s global pilot study for respiratory syncytial virus (RSV)

surveillance. An in-house rRT-PCR assay was established to distinguish between RSV-A/B

in a single test; a whole genome sequencing (WGS) protocol with NGS for RSV was also

optimized.

• In 2017, 2.9% of Australian A(H3N2) viruses sequenced were sensitive to adamantanes

(Hurt et al., 2017). Full genome sequencing identified these viruses as being closely

related genetically, and likely to have been spread from a single source. No sensitive

viruses were identified after September 2017.

• Vaccine effectiveness (VE) in Australia for 2017 was 33% (Sullivan et al., 2017). The VE for

A(H1N1)pdm09 viruses and influenza B viruses (IBVs) was 50%, whereas for A(H3) viruses

it was only 10%.

2.3. Mr John Franks, WHO CC for Studies on the Ecology of Influenza in Animals (St Jude

Children’s Research Hospital [CRH]), Memphis, United States of America

• RT-PCR screening with the United States (US) Centers for Disease Control and Prevention

(CDC) influenza A virus (IAV) primers is performed on all animal swabs before isolation.

Screening is performed in-house because most surveillance sites have no capacity for PCR

screening or isolation. The turnaround time for full genome sequencing is currently

hindered by the time taken for data analysis, because no dedicated team is available.

• Sequence data from the Centers of Excellence for Influenza Research and Surveillance

(CEIRS) network are routinely uploaded to the Influenza Research Database (IRD) through

the Data Processing and Coordinating Center (DPCC), which completes the annotation

and submits the final sequences through the National Center for Biotechnology

Information (NCBI), not through GISAID. All sequences that enter this pipeline must be

made publicly available within 30 days.

2.4. Dr Tsutomu Kageyama, National Institute of Infectious Diseases (NIID), Tokyo,

Japan

• Mutations were identified in B/Yamagata-lineage viruses, resulting in a mismatch with

current probes used at the NIID. Redesigned probes gave improved detection and

identification of these viruses. It is not known whether the CDC primer/probe sets have

issues with detecting these mutated B/Yamagata-lineage viruses.

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• In 2017–2018, three cases of A(H5N6) were detected in poultry and wild birds in Japan.

The A(H5N6) viruses were similar to European, Middle Eastern and African A(H5N8)

viruses.

• Japanese animal quarantine authorities detected three A(H7N9) viruses in duck meat of

origin from China: one low pathogenicity avian influenza (LPAI) virus and two HPAI

viruses.

2.5. Dr Hui-Ling Yen, WHO H5 Reference Laboratory, School of Public Health, The

University of Hong Kong, China, Hong Kong SAR

• The centre hosted Croucher summer courses in June–July 2018 as part of its training

programme. The courses were for vaccinology for public health and clinical practice, and

for emerging virus infections.

• During the wild bird and wet market surveillance in 2017–2018 10 HPAI A(H5N6) isolates

were detected.

• HPAI A(H7N9) was detected in environmental samples from wet market surveillance in

2016; however, no A(H7N9) was identified in wild birds or poultry samples in 2017–2018.

• An A(H5N8) outbreak occurred in domestic birds in Saudi Arabia, and about 9 million birds

were depopulated. Sequence identity of the samples was over 99%, which suggested a

single-introduction outbreak.

• In 2017, 5421 samples were collected for swine surveillance in China, and 106 influenza

type A viruses were identified (47 H1N1, 10 H1N2, 48 H3N2 and 1 H3N1). Genetic analysis

demonstrated extensive reassortment with A(H1N1)pdm09.

• The A(H5) primers and probes used at the laboratory were updated and optimized for

clade 2.3.4.4.

2.6. Dr Frank Wong, Commonwealth Scientific and Industrial Research Organisation

(CSIRO), Australian Animal Health Laboratory (AAHL), Geelong, Victoria, Australia

• Many OIE/FAO national and regional reference laboratories typically use rRT-PCR for

front-line testing in animal influenza outbreak investigations and some surveillance

programmes.

• Since the evolution of A(H5), laboratories in different regions have developed their own

modifications to pan-IAV M gene primer/probe sets, many based on the Spackman et al.

(2002) TaqMan RT-PCR assay from the US Department of Agriculture (USDA). Animal

health laboratories also use a mix of H5 RT-PCR assays.

• The European Union (EU) reference laboratory for avian influenza is now the Instituto

Zooprofilattico Sperimentale delle Venezie (IZSVe), Padua, Italy.

12

• AAHL is tasked with managing OIE and FAO proficiency testing (PT) programmes for the

South-East Asia, South Asia and East Asia regions. Similarly, the Animal and Plant Health

Agency, Weybridge, the United Kingdom – in its capacity as the EU Avian Influenza

Reference Laboratory up to 2018 – has managed PT programmes for northern

hemisphere national and regional animal health diagnostic laboratories.

• Many regions require written approval before influenza genome sequence data can be

uploaded and shared publicly. Failure to obtain written approval or acknowledgement

can result in national laboratories no longer submitting animal sector samples to

international avian influenza reference centres for analysis.

2.7. Dr Herman Tse, Centre for Health Protection (CHP), China, Hong Kong SAR

• A large number of specimens were submitted in 2017, with about 7000 samples

processed each week during the peak of the season. More than a third of the samples

were A(H3). An increase in IBV cases, predominantly from the B/Yamagata-lineage, was

seen at the end of 2017.

• Four triple deletion B/Victoria-lineage mutants were detected at the end of 2017, and by

March 2018, only B/Victoria-lineage double deletion mutants were present.

• Influenza C virus (ICV) has been included in surveillance since 2014, when the rRT-PCR

protocols were first introduced. Most ICV cases were mild with no reported deaths.

2.8. Dr Joanna Ellis, NIC, Public Health England (PHE), London, the United Kingdom

• The subtyping rRT-PCR is currently being updated due to the suboptimal detection of the

A(H3N2) 3C.2a2 subgroup.

• A number of rRT-PCR assays for avian influenza (AI) have been rolled out to regional

laboratories for detection of A(H5) and A(H7). Positive samples are referred to PHE for

confirmation with additional A(H5), A(H7) and A(H9) assays and WGS.

• The NIC participated as a WHO RSV reference laboratory in the WHO RSV surveillance

global pilot study. RSV detection data from both sentinel and non-sentinel surveillance

pertinent to the study were reported to WHO, with data being published on the website.

2.9. Dr Steve Lindstrom and Dr John Barnes, US CDC

• The portal to access the US CDC Laboratory Support for Influenza Surveillance (CLSIS) has

changed, and users need to make a profile to be added before 2019, when the old site

will no longer be available. Thus far, there are 317 registered users in 96 countries.

• The US CDC rRT-PCR influenza kits have been distributed to 132 countries globally, with

an expected 900 kits to be consumed by the end of 2018.

• About 400 human cases of swine A(H3N2) variant (H3N2v) viruses were detected in the

American north-west in 2016; almost all cases had been in direct contact with swine. In

13

2017, only 67 cases were identified; however, they were more widespread. All cases have

been associated with state fairs and all sequences are available on GISAID.

• The genomes of all specimens submitted to the US CDC are sequenced using NGS

(~6000/year).

3. PCR-related activities in OFFLU

Over the 2017–2018 season there has been a continued dispersal of clade 2.3.4.4 A(H5N8) viruses

in the northern hemisphere. This panzootic virus is causing concern because it has spread to

western Europe and south of the equator in Africa; it has been detected in wild birds and

subsequently in domestic poultry as far south as South Africa. Reassorted A(H5N6) viruses with

the HA related to Group B A(H5N8) viruses also emerged in 2017. A(H5N6) viruses have

diversified greatly into multiple 2.3.4.4 sublineages, and numerous reassortant genotypes have

been detected. These A(H5N6) viruses have caused sporadic poultry-to-human spillover

infections, with 20 confirmed human cases since 2014. There is concern that these viruses could

pose a greater zoonotic risk owing to their dynamic reassortment. A(H5N6) has also replaced

A(H5N1) as the dominant HPAI virus in southern China, although A(H5N1) is still endemic in

several countries, notably in Egypt (clade 2.2.1), South-East Asia (clade 2.3.2.1C) and West Africa

(clade 2.3.2.1C). Notifications of AI due to A(H7N9) have been limited to China, with no poultry

detections in neighbouring countries.

An overview of capacity-building and PT in Asia was also provided. The main intention of the

OIE/FAO has been to strengthen diagnostic capacities through the development of regional

veterinary laboratory networks. PT in influenza detection and diagnosis for veterinary

laboratories has been performed in the region since 2009. This PT programme has included an

avian diseases PCR panel with IAV subtypes that are relevant and circulating: H5, H7 and H9,

Newcastle disease virus class II, and AI HxNx that covered the different clades of A(H5) HPAI

viruses including A(H5N1), A(H5N8) and A(H5N6). Eighteen laboratories participated in the 2017–

2018 PT, with most returning the correct results against A(H7) and A(H9); however, detection

sensitivities against the A(H5) samples were variable. Currently, outside of the United States of

America (USA), surveillance activities for swine influenza at the animal–human interface is low

for the animal health sector; however, the FAO Regional Office for Asia and the Pacific is

undertaking a regional distribution of a swine diseases panel that includes influenza.

14

4. Guidance for the use of NGS

4.1. Overview of the latest NGS developments

The following information on the latest instrument and software developments for NGS was

presented by the expert from CDC:

• Illumina’s new iSeq and MiniSeq instruments offer a lot of sequencing capacity at an

affordable price. These instruments are likely to be popular with NICs because they should

provide plenty of coverage and have shorter running requirements.

• DNA Electronics has a new device – LiDia – which is not a full genomics instrument but

targets small pieces of the genome to provide a diagnostic result. The instrument already

detects enterobacteria and bloodborne pathogens, and should be able to be used in work

on influenza in the future.

• Pacific Biosciences have a new sequencing instrument – Sequel – which can generate an

accurate consensus of a single molecule, using a circular consensus sequencing method.

The method involves re-sequencing the target many times to provide high-quality scores.

• The Oxford Nanopore MinION has released:

o MinIT – a graphic processing unit that facilitates base calling (the base calling

fidelity still needs significant improvement, but the machine is capable of

sequencing RNA directly); and

o Flongle – a MinION flow cell dongle that dissociates from the electronics, thus

reducing cost.

Participants also briefly discussed the following techniques:

• comprehensive virus enrichment;

• Ion Torrent for the detection of multiple viral pathogens;

• direct sequencing of viral mRNA using MinION;

• flow cytometry and nano-fluidics for single-cell transcriptomics;

• multiplex surveillance of influenza A and B viruses; and

• the iterative refinement meta-assembler (IRMA) analysis pipeline.

4.2. Use of NGS in individual institutions

4.2.1. US CDC

The development of NGS has moved the focus of the US CDC from antigenic testing to WGS.

Currently, national influenza reference centres (NIRCs) perform sequencing and virus

isolation for the CDC on clinical specimens that are rRT-PCR positive for influenza viruses.

Data are analysed and curated by each NIRC, and shared via the cloud with the CDC. In

15

addition, the CDC is working with Oxford Nanopore in the development of the MinION for

field-based work; data have been published on the use of MinION for direct RNA sequencing

(Keller et al., 2018).

4.2.2. Chinese National Influenza Center (CNIC), Beijing, China

The CNIC provided information on a TaqMan low-density array (TLDA) platform for the

simultaneous detection of multiple pathogens from a single sample. This process provides

rapid and high-throughput detection with fewer nucleic acids. The CNIC also performed

studies to compare the Nanopore MinION Q10 and the Illumina MiSeq Q30. Although the

MinION allows for longer sequence reads, the high error rate makes it unsuitable for single

nucleotide polymorphism (SNP) analysis.

4.2.3. WHO CC for Reference and Research on Influenza (the Francis Crick Institute),

London, the United Kingdom

The United Kingdom CC uses Illumina technology for NGS, and is currently trying to improve

pipelines. A new NGS eight-primer set has been developed for IAV for use at the CC, and this

new set works better than a published three primer set. The published IBV NGS 13-primer set

is still in use at the CC. A primer set for ICVs has also been developed, to investigate samples

from hospitalized children in Cameroon.

4.2.4. WHO CC for Reference and Research on Influenza (VIDRL), Melbourne, Australia

An in-house Ion Torrent PGM platform together with an in-house semi-automated NGS

analysis pipeline (FluLINE) has been in routine use at the Australian CC since 2014. The CC still

uses an isolation-first approach followed by sequencing of the HA, NA and M genes. In 2017,

109 full virus genome sequences were submitted to GISAID. The CC also participates in AI

surveillance in Cambodian wet markets, in collaboration with Institut Pasteur in Cambodia.

In 2017, an outbreak of LPAI A(H7N3) occurred with a very high mortality rate in ducks; WGS

did not provide any insight into why this LPAI had such a high fatality rate in ducks. Also,

several cases of reassortment between A(H5) and A(H9) viruses were found in wet market

specimens through the use of WGS.

4.2.5. WHO CC for Studies on the Ecology of Influenza in Animals (St Jude CRH),

Memphis, Tennessee, USA

The Memphis CC is a member of the CEIRS network, which allows all institutions and smaller

contractors within the network to access NGS services from other members of the network.

These requests can be made when resources are lacking, or when the in-house NGS is

overloaded. This service is not restricted to CEIRS network members but is at the discretion

16

of individual institutions, based on available funds and with priority given to members of the

network.

4.2.6. NIID, Tokyo, Japan

The NIID uses Illumina MiSeq platforms for NGS. The system has not been automated and,

currently, amplification of viral RNA by RT-PCR and DNA fragmentation and adaptor ligation

are all performed manually. Comparisons between RT-PCR amplified RNA and non-amplified

RNA found that coverage is lower in the non-amplified samples from clinical specimens. From

2017, NGS was mainly used for the sequencing of isolates; however, Sanger sequencing is still

used for obtaining rapid results.

4.2.7. WHO H5 Reference Laboratory, School of Public Health, The University of Hong

Kong, Hong Kong SAR, China

The use of NGS varies throughout the laboratory, depending on access to an Illumina MiSeq

platform. Those without direct access to a MiSeq use the core facility, which has a 3-week

turnaround time. Although some laboratories rely solely on NGS for surveillance, others work

partially with NGS and partially with Sanger sequencing. Generally, when sequencing only a

few isolates, Sanger sequencing is used to obtain rapid results.

4.2.8. OFFLU

NGS is available in most of the OIE/FAO reference laboratories for AI. Some laboratories

possess more than one NGS platform, although the Illumina MiSeq is the most commonly

used platform. NGS technology is used in several laboratories for routine IAV genome

characterization and detection, but is not used as a primary diagnostic tool in outbreak

situations because of the turnaround time. Another issue with the use of NGS in the animal

health sector is that diagnostic laboratories do not necessarily target IAV exclusively; hence,

data outputs and reporting need to consider possible impacts on trade and livestock

quarantine. Further evaluation is needed to identify more standardized NGS workflows for

the OFFLU network.

4.2.9. CHP, Hong Kong SAR, China

NGS is not used on a routine basis at the CHP but some evaluation work was completed in

the past year using the Illumina MiSeq platform. Full coverage of IAV and IBV genomes are

generally achieved. The read depth is inversely correlated with segment length for IAV, and

this could be mitigated by using a segment-specific primer strategy, as for the IBV primer set.

NGS is currently only economical when processing a large number of samples, which may not

be practical in influenza-related public health situations. CHP is looking to explore automated

17

tools for NGS data analysis and to further evaluate NGS on clinical specimens with varying

virus concentrations.

4.2.10. PHE, London, the United Kingdom

The United Kingdom Accreditation Service (UKAS) International Organization for

Standardization (ISO) 15189 has recently accredited the PHE NIC. The Illumina MiSeq platform

is used for NGS, which has replaced Sanger sequencing for peak seasonal influenza

surveillance. The NGS workflow is either semi-automated or fully automated, with some of

the workflow being completed by an onsite sequencing service and data being analysed using

a suite of programs and the BioNumerics software platform. During the 2017–2018 season,

1641 samples were processed for sequencing compared with 621 samples in the 2016–2017

season. Use of WGS during a hospital outbreak in 2016–2017 identified a problem with

infection control and movement of patients from the emergency department of a regional

hospital. For the 2018–2019 season, PHE is moving to a sequence-first approach.

4.2.11. Smorodintsev Research Institute of Influenza, Saint Petersburg, Russian

Federation

NGS has been in routine use at the institute since 2015 with the Illumina MiSeq platform.

Although the institute can perform a large amount of NGS, funding restrictions meant that a

relatively small number of specimens (347) were sequenced from 2015 to 2017. During the

2017–2018 season, 243 influenza viruses from 40 regions of the Russian Federation were

sequenced. The criteria for selection include geographical location, vaccination history and

hospitalization. Due to specimen batching there is a significant time lag between sample

collection and sequencing. NGS is mainly used during the peak and the end of the influenza

season. Early and urgent specimens are still sequenced using Sanger technology. NGS is

mainly used for human seasonal viruses but is also used for surveillance of swine influenza

viruses from pig farms in the Saint Petersburg and neighbouring regions. The institute is also

in the process of developing an NGS protocol for virus detection and characterization.

4.2.12. D.I. Ivanovsky Research Institute of Virology, Moscow, Russian Federation

The institute has two laboratories that focus on HA and NA sequencing, and a recent

restructure of the institute has provided limited access to an NGS platform. Currently, certain

surveillance specimens, laboratory-generated reassortants and escape mutants are sent for

NGS.

4.2.13. State Research Centre of Virology and Biotechnology VECTOR, Koltsovo, Russian

Federation

18

The centre uses an Illumina MiSeq platform for NGS. In 2017–2018, the centre obtained

experience in sequencing primary clinical samples and viral isolates. The centre is involved in AI

surveillance from across 50 regions of the Russian Federation, and swine surveillance is

underway. In 2017–2018, several hundred human and avian viruses were sequenced.

4.3. Discussion on the use of NGS in GISRS

NGS and WGS have become an important tool for global pandemic preparedness; however, in a

public health situation, rRT-PCR and Sanger sequencing may provide results in a more timely

manner. In epidemic or pandemic events, these techniques combined could offer a

comprehensive overview, allowing a rapid initial response to an outbreak, with a more detailed

understanding once WGS data become available.

A general issue identified among the centres was the need for pathogen-specific primers to

ensure good sequence coverage with NGS. This problem is evident in the animal health sector,

where random or non-targeted libraries that are used to cover a broad range of pathogens

provide lower coverage of IAV. In addition, comparisons between the universal IAV primers and

segment-specific IBV primers further demonstrate the benefits of using targeted primers to

obtain good sequence coverage.

The use of NGS during a respiratory disease outbreak of unknown cause was also raised. Most

centres have limited experience in this area and generally receive specimens containing

pathogens predetermined by sentinel partners. The CHP in Hong Kong has a comprehensive

multiplex PCR that can detect up to 16 pathogens, and it is aiming to expand the multiplex to 38

pathogens; however, the turnaround time is quite slow and the process has not been

incorporated as part of routine services. The continued improvements to NGS technology may

improve this situation in the future. The Oxford Nanopore MinION technology is an excellent

example of this; the fidelity of the technology needs to be improved, but it could provide a

consensus sequence in real time that is accurate enough to identify the pathogen, and thus could

improve understanding of the virus emerging in an outbreak situation. The MinION is currently

the fastest instrument on the market for outbreak investigations, and it has already been used

with Ebola and Zika.

4.4. Guidance for NICs on the use of NGS

During the 2017 PCR Working Group meeting, it was established that a guidance document was

needed for NICs on the implementation of NGS for influenza virus surveillance. A draft document

was submitted to the 2018 PCR Working Group members for review, and the following were

identified as key points of consideration for NICs:

• There are high startup costs for NGS (e.g. for purchasing equipment and reagents, and for

creating space and housing). The CCs in London and Memphis were able to access NGS

19

technology because the equipment was available in a shared core facility; without such

availability, it is unlikely that these CCs would have been able to access NGS technology.

• There are ongoing costs associated with NGS. Servicing of instruments, additional

computational equipment and having onsite representatives to help troubleshoot are all

costs that need to be considered.

• Supportive resources are essential for NGS; in particular, there is a need for bioinformatics

for sequence assembly and data analysis. Data storage adds another complication;

typically, extensive networks are required to house all the information.

• NICs who wish to implement NGS need to consider the economy of scale. When a large

number of samples require sequencing, NGS is a fiscally suitable option. However, if this

is not a frequent occurrence, then an expensive (although costs are coming down)

machine may sit unused; this lack of use may be detrimental to the machine’s functioning.

In addition, if sequencing needs to be put on hold until enough samples have been

accumulated, the turnaround time may become problematic, especially in a public health

situation. The shelf life of reagents also needs to be considered. Overall, unless there is a

need for high-throughput sequencing on a relatively frequent basis, NGS may not be a

viable option.

Generally, if a NIC believes that NGS can help them achieve their goals and the NIC is equipped

with resources and finances, then the NIC should be encouraged to implement NGS. Due to the

seasonality of influenza in most countries, integration with other groups within the same institute

(or developing an NGS network similar to that seen within the CEIRS network) may help to reduce

costs by burden sharing and ensure better turnaround time. The generation of a questionnaire

or spreadsheet for NICs that would allow calculation of cost–effectiveness and provide support

for long-term investment in NGS was suggested.

An emphasis was also placed on the continual submission of sequence data to GISAID, whether

generated through NGS or Sanger sequencing. Guidance needs to be given on the quality of data

submitted to GISAID, as well as the minimum requirements (e.g. full-length sequences of HA, NA

and M gene segments). In addition, continued virus isolation needs to be encouraged in all NICs

for biological characterization purposes.

5. PCR protocols for GISRS

5.1. Overview of current PCR protocols for GISRS

The 2017 update of the WHO information for molecular diagnosis of influenza virus was

discussed. This document covers conventional RT-PCR protocols, rRT-PCR protocols and

sequencing protocols for the molecular diagnostics and detection of influenza viruses. It also

provides some general guidelines for PCR, covering topics such as:

20

• interpretation of RT-PCR results;

• referral of samples for further characterization;

• validation;

• training of personnel; and

• equipment.

5.2. Gaps and actions

As in previous years, participants noted that protocols and primer/probe sequences should be

reviewed and updated promptly. It was agreed that the addition of a table of contents to the

document in 2017 significantly improved navigation and ease of use. The following actions were

also discussed:

• Validation of protocols – A list of clades and viruses that a protocol has been validated

against should be included, to allow users to decide which protocol is best suited for their

needs and resources. Updates to the A(H5) table may be needed, and a similar table for

A(H7) should be generated.

• NGS protocols and primer sets – Due to the increased demand for NGS, protocols and

primer sets used by individual centres and institutions should be included in the

document. The protocols should only include the steps before the library preparation

stage. The NGS guideline document should also be referenced once completed.

• Updates to the A(H7) guidelines – PCR protocols for H7 viruses are currently provided by

the Department of Virology, Erasmus MC Rotterdam, Netherlands. A comparative study

should be completed between the other A(H7) protocols available and those from

Erasmus MC.

• Potential updates for the IBV primer/probes – The mutated B/Yamagata-lineage viruses

identified at the NIID require testing with the published CDC primer/probe sets to

determine whether the CDC primer/probe sets need to be updated.

• A(H10) protocols – There has been no persistent detection of A(H10N8) since the zoonotic

spill over; however, because the protocol is already available, it will remain in the

document and there is no current need to update it.

6. Quality Assurance

6.1. EQAP: observations on progress made and future plans

The main objectives of the WHO EQAP are to:

• ensure maintenance of the detection of IAV subtypes and type B viruses by RT-PCR among

participants, by monitoring quality and standards of performance;

21

• continue the option of voluntary testing of viruses with reduced susceptibility to NAIs;

and

• promote good laboratory practices.

The number of laboratories participating in the WHO EQAP increased from 54 in 2007 to 160 in

2017. Correct identification of all specimens in the panel increased from 67% in 2007 to 87% in

2017, with 93% of laboratories correctly identifying all A(H5) samples in 2017. Generally,

participation in the WHO EQAP has been increasing in each region, and all the laboratories from

the WHO Eastern Mediterranean Region who participated in the 2017 EQAP (panel 16) correctly

identified all samples – a first for any region.

In 2017 (panel 16), 179 laboratories were invited to participate; of these laboratories, 160

reported results. Panel 16 comprised four A(H5) samples, a clade 2.3.2.1 and a clade 2.3.4.4 virus

at two different concentrations, an A(H7) virus, an A(H1N1)pdm09 virus, an A(H3N2) virus, a

B/Yamagata-lineage virus, a B/Victoria-lineage virus and a negative control. Of the 160 reporting

laboratories, more than 94% were able to identify each of the viruses in the panel. Ten

laboratories returned incorrect type or subtype results, and no apparent cause was identified.

Also, five participants using the same protocol could not subtype one or both samples of

influenza A(H5) clade 2.3.4.4, and three false positives were reported.

6.2. WHO Regional Office for Europe (EURO) EEIQAP: experiences and lessons learned

The results from the 2017–2018 WHO EURO Regional External Influenza Quality Assessment

Programme (EEIQAP) were presented. The main objectives of the EURO EEIQAP are to assess

laboratory performance in the following areas:

• molecular detection of type, IAV subtype and IBV lineage;

• virus isolation;

• antigenic characterization of isolated viruses; and

• genetic characterization of clinical specimens or isolated viruses.

The panel was designed by the National Institute for Public Health and the Environment (RIVM)

in the Netherlands, and pre-testing was performed by the NIC in Rotterdam, Netherlands, and

the NIC in Lyon, France. The panel contained eight samples of influenza type A or B viruses of

various concentrations, and one negative sample. Molecular detection was performed by

55 laboratories, more than 96% of which correctly identified the type and HA subtype or lineage.

Virus isolation was completed by 44 laboratories; isolation of B/Victoria-lineage virus was the

least successful. This reduced success was attributed to negative molecular testing results and to

laboratories attempting isolation from virus-positive specimens only. Preliminary

characterization results suggest that genetic characterization was accurate for most specimens.

22

Based on these results, a regional corrective action plan will be developed and implemented, to

provide group and individual laboratory training before the next EEIQAP.

6.3. OFFLU strategy on Proficiency Testing panel (PT) for PCR

The 2017 OFFLU global PT was organized by the AAHL and was limited to OIE/FAO reference

laboratories and major OFFLU contributors. The OFFLU PT aimed to assess detection of AI viruses

of current concern to avian health and detection of zoonoses. The panel comprised 15 samples

with 13 AI viruses (nine H5, three H7 and one H9N2), an avian paramyxovirus-1 (APMV-1) and a

negative control. The aims for this panel were to assess M detection and HA subtyping, and assay

repeatability, analytical sensitivity and specificity.

For M detection, five of eight laboratories correctly identified all isolates. For detection of A(H5),

there were issues with reproducibility because some centres from the northern hemisphere

struggled with the A(H5N6) clade 2.3.4.4 sublineages from South-East Asia and China. Three

laboratories did not correctly identify an A(H7N2) virus from the southern hemisphere; therefore,

more A(H7) samples will be included in future panels. Overall, the results suggest that the OFFLU

global PT programme is valuable and necessary. The possibility of expanding the OFFLU PT

programme to laboratories within the WHO network and some public health laboratories was

discussed. The inclusion of other animal viruses in the panel, or the generation of an additional

panel for non-avian viruses was also raised.

6.4. Experiences and lessons learned from the USA CDC

The US CDC has recently updated the B/Victoria-lineage panel to include the detection of the

double deletion variant. This virus was found in the USA in 2016–2017 and has been detected in

many South American and European countries. The current IBV lineage (B/Victoria and

B/Yamagata) assays are not affected. The recent B/Victoria-lineage triple deletion virus detected

in China, Hong Kong SAR, other parts of Asia, some countries in Africa and the USA will be non-

reactive in the B/Yamagata-lineage and B/Victoria-lineage double deletion assays. A new IBV

genotyping panel will be available in the autumn of 2018.

The performance evaluation of 2017 was completed in conjunction with the Pan American Health

Organization (PAHO). The exercise was completed with 17 countries, 15 of which scored 100%.

Only one laboratory did not identify the samples correctly, despite having the capacity to do so.

6.5. Discussion on the WHO EQAP

The general purpose of the WHO EQAP was discussed. NICs are highly encouraged to participate

in the EQAP. The results of the EQAP for a NIC are often used to promote the NIC to a country’s

ministry of health, which may have economic implications. It was therefore suggested that panel

composition and isolate concentrations need to be stabilized. The use of challenging samples was

23

still deemed to be important in identifying gaps and areas for improvement. Demonstration of

improvement in future panels and changes to assays may need to be emphasized in future

reports. The impact of the inactivation methods used on viruses included in the panel was also

discussed. Laboratories have commented on the reduced quality of the viral RNA for Sanger

sequencing when samples are Triton-X100 inactivated and vacuum dried. Heat inactivation was

suggested as a better way to retain viral RNA of a quality suitable for Sanger sequencing.

The use of PT programmes by NICs for their network of within-country laboratories was

discussed. Currently, NICs in the United Kingdom and the Netherlands have an established PT

programme for their network laboratories. For PHE in the United Kingdom, and for AAHL and the

Melbourne CC in Australia, panel generation was moved to an independent accredited panel

provider. Requests were made for training on the generation and establishment of PT

programmes by NICs in central Asian countries, and a similar request has been made by

laboratories in the OFFLU network. At AAHL, a number of focal point national animal health

laboratories are being trained on the provision of PT within their countries, and this continues to

be an ongoing activity supported by the OIE and FAO in South-East Asia and the wider Asia region.

The possibility of WHO providing funding for a PT training programme was raised.

7. Way forward

7.1. Strategy for GISRS surveillance

For seasonal influenza surveillance, rRT-PCR is the method of choice for virus detection and

diagnosis; generally, NICs are proficient in rRT-PCR and should be encouraged to maintain this

capacity. Sequencing of specimens by some NICs and the CCs is important because it provides

information on circulating genetic groups, and it is particularly helpful for the A(H3) viruses.

Although almost 90% of A(H3)-positive clinical samples can be isolated, haemagglutination

inhibition (HAI) assays can only be performed on about 25% of these samples. A plaque reduction

or focus reduction neutralization assay was developed to combat the HAI issue for A(H3) viruses.

However, these assays are time consuming and are not amenable to automation, meaning that

fewer viruses can be processed compared with antigenic characterization by HAI. NICs that ship

samples for seasonal surveillance to the CCs should endeavour to submit only samples from the

most recent relevant periods to inform WHO consultation meetings on influenza vaccines

composition (VCMs); that is, samples with collection dates from September to January should be

shipped for the northern hemisphere VCM (held in February) and those with collection dates

from February to August should be shipped for the southern hemisphere VCM (held in

September). The usefulness of data from samples collected before the time periods indicated is

limited for recommendations being made at the VCMs. Where zoonotic virus infection is

24

expected, NICs are encouraged to send specimens to a CC or an H5 reference laboratory as soon

as possible, particularly if the NIC cannot detect and identify the virus subtype, and lacks the

necessary containment facilities to attempt virus isolation.

Use of NGS by NICs was raised. Currently, sequencing of samples by this method is not

encouraged, owing to the time delays involved, although this may change as NGS technology

improves. NICs and other laboratories should be encouraged to submit samples to a CC as soon

as possible. The shift to a “sequence-first” scenario, after rRT-PCR, raises the issue of virus

isolation and loss of skill at the NICs and other reference laboratories. The capacity for virus

isolation needs to be encouraged and maintained.

7.2. GISRS capacity: gaps and priority actions

• The NGS guidance document is to be developed and completed by the middle to the end

of December 2018. An Excel spreadsheet that allows calculation of the costs of NGS for

inclusion in a decision-making tree should also be generated.

• The need to maintain virus isolation at NICs and reference laboratories was raised.

• A deadline for the end of October was recommended for CCs and participating

institutions, to inform WHO about any protocols that need to be revised.

7.3. New technology and rapid influenza diagnostics

The USA CDC has worked with the USA Food and Drug Administration (FDA) to improve on the

rapid diagnostic tests that are FDA approved. Companies that sell rapid detection tests in the USA

must now participate in annual testing and the data must be shared publicly. As a result, the

number of tests available has decreased and the compliant tests are listed on the CDC website.

7.4. Updating the WHO laboratory manual

The WHO Manual for the laboratory diagnosis and virological surveillance of influenza (WHO,

2011) was first published in 2011. Questions were raised on whether time should be invested to

update and create a printed document. It was suggested that an online manual system could be

used, with each chapter being present and with dates of updates included, similar to the OIE

Manual of diagnostic tests and vaccines for terrestrial animals 2018 (OIE, 2018).

7.5. Possible publication in a peer-reviewed journal

The possibility of publication of the efforts of the PCR Working Group in a peer-reviewed journal

was discussed. The publication would cover the roles and contributions of this group for the

guidance of NICs, with the aim of increasing the visibility of the PCR Working Group, and

advocating its importance and contribution to maintaining influenza surveillance.

25

8. Proposed action points

The outcomes of the meeting will be published in an executive summary in the WER. Proposed

action points were as follows:

• protocols on the WHO website should be reviewed to ensure that they are current;

• NGS primers and protocols should be provided;

• an NGS guidance document for NICs should be finalized;

• comparative studies on H7 protocols are to be completed;

• testing of the CDC primer/probes on the B/Yamagata-lineage mutant viruses from Japan

is to be completed; and

• the PCR Working Group ToR document needs to be reviewed.

References

Hurt A, Komadina N, Deng YM, Kaye M, Sullivan S, Subbarao K et al. (2017). Detection of adamantane-sensitive influenza A(H3N2) viruses in Australia, 2017: a cause for hope? Euro Surveill 22(47):17–00731 10.2807/1560-7917.ES.2017.22.47.17-00731 (https://www.ncbi.nlm.nih.gov/pubmed/29183552, accessed 21 January 2019).

Keller MW, Rambo-Martin BL, Wilson MM, Ridenour CA, Shepard SS, Stark TJ et al. (2018). Direct RNA sequencing of the coding complete influenza A virus genome. Sci Rep 8(1):14408 10.1038/s41598-018-32615-8 (https://www.ncbi.nlm.nih.gov/pubmed/30258076, accessed 21 January 2019).

OIE (2018). Manual of diagnostic tests and vaccines for terrestrial animals 2018. Paris, France: World Organisation for Animal Health (http://www.oie.int/standard-setting/terrestrial-manual/access-online/, accessed 21 January 2019).

Spackman E, Senne DA, Myers TJ, Bulaga LL, Garber LP, Perdue ML et al. (2002). Development of a real-time reverse transcriptase PCR assay for type A influenza virus and the avian H5 and H7 hemagglutinin subtypes. J Clin Microbiol 40(9):3256–60 (https://www.ncbi.nlm.nih.gov/pubmed/12202562, accessed 23 January 2019).

Sullivan SG, Chilver MB, Carville KS, Deng YM, Grant KA, Higgins G et al. (2017). Low interim influenza vaccine effectiveness, Australia, 1 May to 24 September 2017. Euro Surveill 22(43):17–00707 10.2807/1560-7917.ES.2017.22.43.17-00707 (https://www.ncbi.nlm.nih.gov/pubmed/29090681, accessed 21 January 2019).

WHO (2011). Manual for the laboratory diagnosis and virological surveillance of influenza,. Geneva: World Health Organization (WHO) (https://www.who.int/influenza/gisrs_laboratory/manual_diagnosis_surveillance_influenza/en/, accessed 13 January 2019).

https://www.ncbi.nlm.nih.gov/pubmed/29183552https://www.ncbi.nlm.nih.gov/pubmed/30258076http://www.oie.int/standard-setting/terrestrial-manual/access-online/http://www.oie.int/standard-setting/terrestrial-manual/access-online/https://www.ncbi.nlm.nih.gov/pubmed/12202562https://www.ncbi.nlm.nih.gov/pubmed/29090681https://www.who.int/influenza/gisrs_laboratory/manual_diagnosis_surveillance_influenza/en/https://www.who.int/influenza/gisrs_laboratory/manual_diagnosis_surveillance_influenza/en/

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Annex 1: List of participants

John Barnes Influenza Prevention and Control Team Epidemiology and Prevention Branch Influenza Division Centers for Disease Control and Prevention (CDC) Atlanta, GA United States of America

Steve Lindstrom Influenza Prevention and Control Team Epidemiology and Prevention Branch Influenza Division Centers for Disease Control and Prevention (CDC) Atlanta, GA United States of America

Rod Daniels WHO CC Crick Worldwide Influenza Centre The Francis Crick Institute 1 Midland Road London NW1 1AT United Kingdom of Great Britain and Northern Ireland

Alexander Ryzhikov Department of Zoonotic Infections and Influenza FBIR State Research Centre of Virology and Biotechnology VECTOR Koltsovo Russian Federation

Yi-Mo Deng WHO CC Victorian Infectious Diseases Reference Laboratory (VIDRL), Peter Doherty Institute for Infection and Immunity Melbourne, VIC Australia

Tatiana Timofeeva D.I. Ivanovsky Research Institute of Virology FSBI “N.F. Ganaleya FRCEM” Moscow Russian Federation

Joanna Ellis National Influenza Centre Respiratory Virus Unit Virus Reference Department Public Health England: Colindale London United Kingdom of Great Britain and Northern Ireland

Sanja Trifkovic WHO CC St Jude Children’s Research Hospital Department of Infectious Diseases Memphis, TN United States of America

John Franks WHO CC St Jude Children’s Research Hospital Department of Infectious Diseases Memphis, TN United States of America

Herman Tse Public Health Laboratory Services Branch Centre for Health Protection (CHP) Department of Health Hong Kong SAR China

Tsutomu Kageyama Laboratory of Molecular Diagnosis Influenza Virus Research Center National Institute of Infectious Diseases (NIID) Tokyo Japan

Frank Wong Agent Characterization Research Team Diagnosis, Surveillance and Response Group CSIRO Australian Animal Health Laboratory East Geelong, VIC Australia

Andrey Komissarov Laboratory for Molecular Virology Smorodintsev Research Institute of Influenza Ministry of Health of the Russian Federation Saint Petersburg Russian Federation

Hui-Ling Yen WHO H5 Reference Laboratory School of Public Health The University of Hong Kong Hong Kong SAR China

27

WHO Secretariat

Ehab Atia High Threat Pathogens Infectious Hazard Management WHO Health Emergencies and Communicable Diseases WHO Regional Office for Europe Copenhagen Denmark

Magdi Samaan The Global Influenza Program HQ/IPR Influenza Preparedness and Response Geneva Switzerland

Terry Besselaar The Global Influenza Program HQ/IPR Influenza Preparedness and Response Geneva Switzerland

Wenqing Zhang The Global Influenza Program HQ/IPR Influenza Preparedness and Response Geneva Switzerland

Dmitriy Pereyaslov High Threats Pathogens Health Emergency Program Division of Communicable Diseases and Health

Security WHO Regional Office for Europe Copenhagen Denmark

28

Annex 2: Declarations of interest

The 10th meeting of the WHO Working Group for the Molecular Detection and Subtyping of Influenza

Viruses and the use of Next Generation Sequencing (NGS) in GISRS [Global Influenza Surveillance and

Response System], held on 21–22 August 2018, was organized by the WHO Global Influenza Programme.

Representatives from WHO collaborating centres on influenza and WHO H5 reference laboratories of

GISRS participated. A representative from the World Organization for Animal Health/Food and Agriculture

Organization of the United Nations (OIE/FAO) Network of Expertise on Animal Influenza (OFFLU) also

participated on behalf of the veterinary sector.

In accordance with WHO policy, all participants completed the WHO form for Declaration of Interests

for WHO Experts before being invited to the meeting. These declarations were then evaluated by the

WHO Secretariat prior to the meeting. At the start of the meeting, the interests declared were disclosed

to all consultation participants.

Participants declared no personal current or recent (within the last 4 years) financial or other interests

relevant to the subject of work.

Institution Representative Personal interest

WHO CC, Atlanta Dr Stephen Lindstrom None

WHO CC, Atlanta Dr John Barnes None

WHO CC, Beijing Dr Xiang Zhao None

WHO CC, London Dr Rod Daniels None

WHO CC, Melbourne Dr Yi-Mo Deng None

WHO CC, Memphis Dr Sanja Trifkovic None

WHO CC, Memphis Mr John Franks None

WHO CC, Tokyo Dr Tsutomu Kageyama None

WHO H5 Reference Laboratory, CHP, Hong Kong SAR, China Dr Herman Tse None

National Influenza Centre, PHE, the United Kingdom Dr Joanna Ellis None

University of Hong Kong, Hong Kong SAR, China Dr Hui-Ling Yen None

CSIRO Australian Animal Health Laboratory, Geelong, Australia Dr Frank Wong None

H5 Reference Laboratory, VECTOR, Novosibirsk, Russian Federation

Dr Alexander Ryzhikov None

National Influenza Centre, Saint Petersburg, Russian Federation Dr Andrey Komissarov None

National Influenza Centre, Moscow, Russian Federation Dr Tatiana Timofeeva None

A WHO assessment concluded that none of the experts had a conflict of interest with the objectives of

the technical consultation.

29

Annex 3: Meeting agenda

Meeting of the WHO Working Group for the Molecular Detection and Subtyping

of Influenza Viruses and the use of Next Generation Sequencing (NGS) in GISRS

Research Institute of Influenza of the Ministry of Health of the Russian Federation,

Saint Petersburg, Russian Federation

21–22 August 2018

Final Agenda

Tuesday, 21 August 2018 Chair: J. Franks

09:00 – 09:30 Welcome and opening W. Zhang, Global

Influenza

Programme, WHO

D. Danilenko

Research Institute of

Influenza, St

Petersburg, Russian

Federation

Declaration of interests

Selection of chair

Appointment of rapporteur

09:30 – 09:40 Objectives and expected outcomes

M. Samaan

9:40 – 10:30 Session A: Review of WG actions since last meeting Session co-chair

T Kageyama

09:40 – 09:50 Review of actions recommended by the 2017 WG meeting M. Samaan

09:50 – 10:30 General updates from participating laboratories

(10 minutes each)

• The Francis Crick Institute, London

• VIDRL, Melbourne

• St. Judes, Memphis

Representatives from

participating labs

10:30 – 11:00 Coffee break and photo

11:00 – 11:40 General updates from participating laboratories (cont.)

• NIID, Tokyo

• HKU, Hong Kong SAR, China

• OFFLU

• CHP, Hong Kong SAR, China

• PHE, London

Representatives from

participating labs

11:40 – 11:55 Presentation PCR related activities in OFFLU (15

minutes)

F. Wong

11:55 – 12:30 Discussion All participants

12:30 – 13:30 Lunch

30

13:30 – 17:00

Session B: Guidance for NICs on the use of Next

Generation Sequencing

Session co-chair

J. Ellis

13:30 – 13:40

Overview of the latest NGS developments John Barnes

(Skype call)

13:40 – 15:40 Use of NGS in individual institutions: (10 minutes each):

• CDC, Atlanta (John Barnes, Skype call)

• CNIC, Beijing

• The Francis Crick Institute, London

• VIDRL, Melbourne

• St. Judes, Memphis

• NIID, Tokyo

• HKU, Hong Kong SAR, China

• OFFLU

• CHP, Hong Kong SAR, China

• PHE, London

Discussion: Use of NGS for risk assessment and rapid

response to in epidemics and pandemic events.

15:40 – 16:10

Coffee break

16:10 – 17:30

Discussion: Guidance for GISRS on using NGS All participants

17:30

Close of Day 1 Chair

31

Wednesday, 22 August 2018

09:00 – 10:30 Session C: PCR protocols for GISRS Session co-chair

Y. Deng

09:00 – 09:30 Overview of current PCR protocols for GISRS J. Ellis

09:30 – 10:30 Discussion on gaps and actions All participants

10:30 – 11:00 Coffee break

11:00 – 14:20 Session D: Quality assurance Session co-chair

H. Tse

11:00 – 11:30 EQAP: Observations on the progress made and future plans H. Tse

11:30 – 11:50 Experience and lessons on learnt from the EURO quality

assessment programme for PCR and future plans

D. Pereyaslov

11:50 – 12:15 OFFLU strategy on external quality assurance for PCR F. Wong

12:15 – 12:30 Discussion All participants

12:30 – 13:30 Lunch

13:30 – 17:30 Session E: Way Forward

Discussions:

1) Strategy for GISRS surveillance for:

• Seasonal viruses

• Zoonotic viruses

• Pandemic viruses

2) GISRS capacity: gaps and priority actions

3) Experience and lessons learnt from the CDC quality assurance programme for PCR and future plans (15:30 –

16:00)

4) Develop and update guidance for NICs

5) New technology and rapid influenza diagnostics

6) Updating the WHO laboratory manual

7) Possible publications in peer reviewed journal

8) Introduction of ToR for the WHO Expert Working Group on Molecular Detection and Subtyping of Influenza

Viruses for GISRS (PCR Working Group)

(Session co-chair) R.

Daniels

All participants

S. Lindstrom

(Skype call)

15:00 – 15:30 Coffee break

16:00 – 17:00 Session E: Way forward (continued)

17:00 – 17:20

Summary and expected output

Chair

17:00 – 17:20

Summary and closure W. Zhang