Guidance

Referral of influenza samples to RVU, UKHSA Colindale, 2023 to 2024

Updated 13 November 2023

Background

General principles

National surveillance of influenza depends on accurate and timely virological information. It will indicate:

• which types of influenza viruses are circulating (influenza A or B)
• which subtypes of influenza A ((H1N1)pdm09 and H3N2) or influenza B (B/Victoria and B/Yamagata)
• how closely related these are to seasonal influenza vaccine components

Influenza type classification is dependent on the characteristics of the internal genes of the segmented genome, (usually NP, M or NS1 gene) whereas influenza subtyping is dependent on the characteristics of the genes coding for the highly variable viral surface protein haemagglutinin (HA). Typically, most commercial assays for influenza detection provide influenza type information, but do not provide subtyping information. Hence the majority of influenza test results performed in a clinical laboratory setting will not provide detailed virus strain information as needed for public health surveillance purposes.

The UK Health Security Agency’s (UKHSA) national respiratory virus unit (RVU) is also the World Health Organization (WHO) designated National Influenza Centre (NIC) for England. Detailed data on the virological characterisation of influenza viruses is reported by UKHSA’s RVU to WHO on a weekly basis.

In-depth virological surveillance is achieved through the detailed analysis of a proportion of influenza positive samples which are taken as part of clinical illness diagnosis within the NHS, hospital laboratory system, and other samples taken specifically for surveillance purposes, via UKHSA laboratories.

Samples taken specifically for surveillance purposes include those taken through community-based surveillance schemes and outbreak investigations. Comparison is made between virus strains circulating in the community, causing milder illness, and those strains associated with more severe illness requiring hospitalisation, and the degree of correspondence of circulating strains to those included in the national vaccine programmes, which allows tracking of disease burden due to influenza and effectiveness of national vaccine programmes.

Sample referral pathways

This guidance focuses on the sample flow for samples from individuals infected with seasonal influenza.

Over 150 laboratories provide clinical diagnostic testing services for influenza, usually as part of wider syndromic illness testing. Testing may be delivered through point of care (PoC) testing devices, which provide information about influenza type (A or B), but no information about influenza subtype, or through batch-based laboratory polymerase chain reaction (PCR) testing, using a variety of different commercial platforms, a relatively small proportion of which provide influenza subtype information following detection of a positive sample.

It is essential that each influenza testing site has a strategy in place through the course of the epidemic season to either test or refer a proportion of samples which test positive for influenza A or B to provide subtyping and/or more detailed virological analysis. All positive samples outside of the epidemic period should be referred.

Sample referral strategies are particularly important when individual hospitals are heavily reliant on testing provided by PoC devices, which do not leave residual materials, that can be used for virological analysis. In such situations, additional samples taken from cases that test positive may be necessary to provide materials for virological characterisation (see Figure 1). All test results (positive or negative) should be reported to SGSS through electronic reporting from laboratory information systems.

Clinical NHS laboratories can refer samples to the local UKHSA public health laboratory (PHL) for influenza A subtyping, aligned to locally agreed strategies and where appropriate (as indicated in Figure 1).

All influenza positive samples from individuals with severe influenza who require treatment in Intensive Therapy Unit - ITU/High Dependency Unit - HDU/Extra Corporeal Membrane Oxygenation (ECMO) should be subtyped. This should be done locally, if testing platforms are available, or referred to the local UKHSA PHL.

UKHSA PHLs will not charge for subtyping of influenza A positive samples from severe or fatal infections if this subtyping has not already been performed by NHS laboratories.

Figure 1. An overview of the sample testing and referral system in England

UKHSA PHLs will refer influenza positive samples to the RVU reference laboratory at Colindale in accordance with nationally agreed surveillance priorities (Figure 1). NHS laboratories should not routinely refer samples directly to the RVU for influenza diagnosis.

If samples have been collected from individuals with suspected exposure to non-seasonal influenza, specific guidelines in relation to suspected exposure must be followed (See guidance on the diagnosis and management of avian influenza).

Surveillance

Influenza strain surveillance informs the global vaccination programme and provides information for empirical antiviral choice, as well as pandemic early warning systems.

Influenza strain surveillance includes:

• UKHSA national virological data reported weekly, both nationally and internationally
• aggregate genetic and antigenic data submitted to WHO at the end of January and early September, as the UK evidence to guide the annual formulation of the influenza vaccine
• antiviral susceptibility surveillance undertaken at national level based on whole genome data from community samples, in addition to data and samples received from our regional UKHSA public health laboratories, and reported weekly to provide an estimate of the incidence of antiviral resistance

Figure 2. Epidemic sample referral periods

¹ Early and late in season; send all influenza positive samples to RVU
² Mid-season; send 10% influenza positive samples (maximum 50 per week)

Influenza positive samples should be submitted regularly throughout the season (see Appendix 1) to ensure virological data is available for accurate weekly reporting. Early and late in the season, all positive samples should be referred. During the peak weeks of the season, a proportion of representative positive samples will be referred (Figure 2).

All clinical sample contributions are very valuable and boost the overall national picture of surveillance. UK national virological surveillance data is regarded as official national statistics with some of the highest viewing figures of content on the government website, GOV.UK.

2023 to 2024 season

In the 2023 to 2024 season, influenza positive samples received at the national reference laboratory will be selected for detailed analysis using influenza whole genome sequencing (WGS), subject to technical qualification of having sufficient material to complete full characterisation, typically by ensuring samples are used with a Cycle threshold (Ct) value and adequate volume (minimum of 600μl).

Antigenic (phenotypic) characterisation is performed on a subset of viruses which are selected for virus isolation in cell culture. Both genotyping and phenotyping pathways have stringent requirements for clinical sample quality to increase chances for completed full analysis. Hence, selection for further characterisation beyond subtype classification will be based primarily on the PCR Ct value, with exemption for samples associated with severe illness or death (see Appendix 1), and those with unusual testing profile. Examples of the latter would be samples with unexpected discrepancies between assays (typing versus subtyping or when analysed on different platforms), as this pattern of results could indicate unusual viruses or performance issues with individual assay platforms.

Good completion of the questionnaire on the typing of influenza strains request form (E3), especially for these exemption samples, is crucial to prevent them from being inadvertently excluded based on Ct.

Reporting of results from national reference laboratory during the 2023 to 2024 season

The information obtained from each referred sample is an important component of the UK influenza virological surveillance. Characterisation data is used to compare how similar the currently circulating influenza viruses are to the strains included in seasonal influenza vaccines, and to monitor for changes in circulating influenza viruses.

For typical influenza positive samples, referring laboratories will receive a standard report, without further sample specific results, together with a link to the aggregated characterisation data provided in the weekly national influenza report.

The data will be reported in the weekly national joint flu and COVID-19 reports.

Reporting of results of genome sequencing, virus isolation, antigenic typing or genomic typing for antiviral susceptibility markers of influenza virus to referring laboratories will be performed only for those samples where a specific request for characterisation has been made for patient management or epidemiological purposes, for example as part of an outbreak. Reports to referring laboratories will also be issued if clinical or epidemiological follow-up is required during surveillance testing, for example, if antiviral resistance is suspected.

Surveillance samples from UKHSA public health laboratories

UKHSA Colindale (RVU) requests the influenza referrals detailed below and summarised in Figure 2 and Appendix 1, from regional UKHSA public health laboratories for surveillance purposes. No charge is made for processing these. RVU will notify laboratories about changes in the sample referral periods.

Out of season and early in influenza season

All influenza positive samples from hospital and community source should be referred. Samples from returning travellers must be sent together with details of travel history, as these out of season or early season travel associated influenza strains are of particular interest for surveillance purposes.

Mid and late influenza season

Refer approximately 10% of influenza positive samples up to a maximum of 50 samples per week. In these referred samples, include all influenza positive samples from referral categories A to G shown below and in Appendix 1, and complete the batch with samples randomly selected from category H.

If a laboratory has more than 50 samples per week in referral categories A to G, please make sure that the 50 samples referred include all those in categories C and D below. Referral categories A to G can be any Ct value.

Referral categories

A. Patients with complicated influenza [note 1], including patients admitted to any area of the hospital. All ITU/HDU/ECMO and fatal cases should be included.

B. All influenza positive samples in which the oseltamivir resistance mutation 275Y has been detected.

C. All influenza A positive samples that cannot be subtyped as (H1N1)pdm09, H3 or H5 [note 2], H7 [note 2]. Refer to Influenza A positive samples and subtyping on this category. All Influenza B positives are requested for lineage determination.

D. Influenza positive samples that have an unusual or unexpected PCR profile/Ct values for generic influenza A or specific subtyping.

E. Influenza positive samples where shedding of live attenuated influenza virus (LAIV) is suspected from the patient’s vaccination history or laboratory results [note 3].

F. Influenza positive samples from influenza only co-infections, or influenza and SARS-CoV-2 co-infections, with influenza PCR Ct values ≤31.

G. Influenza positive samples from all individuals with a vaccination history for current season.

H. Samples positive for influenza B, A (H3N2), or A (H1N1)pdm09, with PCR Ct values ≤31.

[note 1] Complicated influenza: Influenza requiring hospital admission and/or with symptoms and signs of lower respiratory tract infection (hypoxaemia, dyspnoea, lung infiltrate), central nervous system involvement and/or a significant exacerbation of an underlying medical condition.

[note 2] Please make sure you urgently inform RVU of any H5/H7 positive samples or unsubtypable samples from individuals with suspected exposure to avian influenza before sending for confirmation due to the public health significance of these results. See guidance on the diagnosis and management of avian influenza.

[note 3] Patients who have any positive result and a recent history of vaccination (within a 2-week period) or have been in contact with an individual who has recent history of vaccination with LAIV.

It is recommended to send samples in regular batches if possible, except for those in categories A to E, which should be sent rapidly to RVU for investigation (see acceptable referred samples), and with the reason for sending, for example ECMO patient, clearly stated on the typing of influenza strains request form (E3).

Influenza A positive samples and subtyping categories

Influenza A detections may fall into one of several categories as far as subtyping information is concerned; either:

A. Influenza A subtyping performed, and subtype successfully allocated.

B. Influenza A subtyping not performed and subtype unknown.

C. Influenza A subtyping performed, but no subtype determined because the samples have failed subtyping assays for seasonal influenza (H1N1)pdm09 or H3N2. These are described as unsubtypable samples.

Note, the subtyping category unsubtypable (meaning non-(H1N1)pdm09, non-H3, non-H5 or non-H7/H9) is different from not subtyped (in other words subtyping not attempted), which have separate tick-boxes on the typing of influenza strains request form (E3).

The RVU reference laboratory does not provide a routine subtyping service for influenza A positives. This should be performed locally through NHS testing and/or through the regional UKHSA PHLs, with locally agreed strategies for sample referral. A plausible technical explanation for failure to subtype includes a low viral load, where subtyping assays may have a lower sensitivity compared to the generic detection assay.

Note, it is unusual to see Ct differential ≥5 between typing and subtyping assays. Therefore, a sample for providing a Ct ≤30 in a generic influenza A assay should subtype.

All category C samples for which subtyping attempts fail must be forwarded to the UKHSA reference laboratory.

In some influenza seasons, influenza A samples are classified as unsubtypable due to minor virus strain sequence variations affecting performance of commercial platforms or assays. Such variation can reflect very recent in-season virus evolution and should be forwarded together with information on the assay or manufacturer combination used for detection by completing the relevant sections in the typing of influenza strains request form (E3) for further investigation.

Unsubtypable results can also arise if the sample contains non-seasonal influenza. This possibility is important to consider especially in cases of severe illness [note 4] particularly if there is a history of travel or unusual zoonotic exposures, especially considering the ongoing worldwide epizootic of H5N1. Where available, H5 specific assays should be urgently performed locally or in the appropriate UKHSA or NHS collaborating laboratories  and the sample forwarded to the Reference laboratory - for confirmatory testing (following specific guidance on diagnosis and management and using the E73 request form) or further characterisation.

[note 4] Please note that it is a requirement to subtype all influenza detections from individuals hospitalised in critical care.

Viral mutations can affect pan-influenza typing assays targeting the internal genes with loss of sensitivity compared with subtyping results, as evidenced by higher-than-expected Ct values. This may lead to false negative results and/or loss of sensitivity of the surveillance. Such discrepant results can usually only be detected by laboratories that perform multiple assays and observe an unusual or discordant pattern of reactivity – please forward any affected samples together with details of assay used for detection.

RVU will further investigate subtyped samples for which characterisation is required for an outbreak investigation. Subtyped samples from localised or unusual outbreaks should always be submitted and will be subject to enhanced testing including sequencing. There is no PCR Ct value cut-off for referring influenza samples from outbreaks (see acceptable referred samples). Please make sure the samples are clearly marked as from an outbreak, with details of the outbreak setting name or location and type, and the HPZone reference if known, using the typing of influenza strains request form (E3).

RVU will also perform enhanced analysis on influenza positive samples from children aged 2 to 17 years. Where influenza vaccination history is known, please include information on date of vaccination and type (inactivated or LAIV) on the referral form. There is no PCR Ct value cut-off for referring influenza samples from vaccinated children in this age group (see acceptable referred samples below).

Please use the current version of the typing of influenza strains request form (E3). The form collects information such as the SARS-CoV-2 status of the referred sample, to guide the processing pathway of the sample for virus characterisation as well as information on detection and any subtyping assays used in the sender’s laboratory, which can help identify trends in assay performance including assay failures and is thus an important piece of information for the Reference laboratory to fulfil its national advisory role. It is vital that the requested information – mainly through tick boxes – is completed as best as possible to support this function.

Surveillance samples from the NHS

NHS laboratories normally contribute to influenza surveillance through forwarding influenza samples to their regional PHLs who perform influenza subtyping and where required, H275Y detection (in A(H1N1)pdm09 viruses) and chose suitable materials from categories A to G (see categories and Appendix 1) for referral to the RVU.

NHS laboratories which perform their own subtyping and H275Y detection (in A(H1N1)pdm09 viruses) do not need to send their samples to their regional UKHSA public health laboratory for surveillance purposes. These laboratories could send their characterised materials, with subtype and Ct (or similar information) fitting into categories A to G, directly to RVU.

Influenza A positive samples from fatal cases and ITU/HDU/ECMO cases should be sent directly to RVU – ideally after subtyping and where applicable H275Y testing have been performed. It is important that the current version of the typing of influenza strains request form (E3) is used to capture information on the SARS-CoV-2 status of the referred sample to help guide the processing pathway of the sample for virus characterisation.

NHS laboratories which do not subtype and/or test for H275Y mediated resistance (in A(H1N1)pdm09 viruses) need to send their materials to regional UKHSA PHLs or NHS collaborating laboratories if they require this information for clinical reasons (see Table 1 and Table 2 for services provided).

Table 1. Services provided by UKHSA laboratories (performed locally)

UKHSA laboratories	Flu subtyping, A(H1N1)pdm09/H3	Avian subtyping, H5	Avian subtyping, H7	A(H1N1)pdm09, H275Y (Oseltamivir sens/res)
Birmingham	Yes	Yes	Yes	Yes
Bristol	Yes	Yes	No	No
Cambridge	Yes	Yes	No	Yes
Manchester	Yes	Yes	No	Yes

Table 2. Services provided by NHS collaborating laboratories (performed locally)

NHS collaborating laboratories	Flu subtyping, A(H1N1)pdm09/H3	Avian subtyping, H5	Avian subtyping, H7	A(H1N1)pdm09, H275Y (Oseltamivir sens/res)
Brighton	Yes	No	No	No
Leeds	Yes	No	No	No
Newcastle	Yes	No	No	No

Testing to inform patient management

Please note RVU will charge for any service that is for immediate patient management. To support clinical diagnosis and management, RVU can receive the following samples.

Influenza positive samples for antiviral susceptibility testing for clinical purposes

Clinical susceptibility testing is not performed in RVU, but the laboratory analyses influenza whole genomes for genetic markers that have theoretically or clinically been linked with antiviral resistance. Laboratories with genuine suspicion of antiviral resistance must contact the RVU laboratory before sending samples for sequencing with the explicit purpose of antiviral genotypic testing. The routine turnaround time for results on these samples is such that the results are unlikely to be able to impact on patient management, unless specific arrangements are made.

Antiviral (AV) genotypic testing should be considered when there is clinical concern about AV treatment failure. The greatest risk of oseltamivir resistance is currently in immunocompromised patients with influenza A(H1N1)pdm09. Further recommendations can be found in the guidance on the use of antiviral agents for the treatment and prophylaxis of seasonal influenza.

Acceptable referred samples

Reference laboratory work requires original clinical material. This is because a range of different analytical approaches may be undertaken, some of which are incompatible with diverse lysis buffers. To prepare virus isolates it is necessary to use original material.

Please do not send nucleic acid extracts or samples in lysis buffer. Extracts and samples referred in lysis buffer in referral categories F to H will be stored without testing if received, with a report sent to the referring laboratory to indicate this.

If nucleic acid extracts or samples in lysis buffer are in categories A to D, please consider re-swabbing of the patient to enable further detailed characterisation (such as virus isolation for assessment of phenotypical features and whole genome sequencing).

Please do not refer samples with Ct values over 31, unless in categories A to E (see Appendix 1). Samples with a Ct value over 31 are unlikely to be successfully sequenced or cultured.

Please do not refer samples which have been tested with an assay that does not generate PCR Ct values, unless in categories A to E (see Appendix 1).

A checklist to aid sample referral is provided in Appendix 2.

It is recommended to send samples in regular batches if possible, except for those in categories A to E, which should be sent rapidly to RVU for investigation. The current version of the typing of influenza strains request form (E3) should be used.

Appendices

Appendix 1: surveillance samples

Referrals requested from UKHSA public health laboratories to RVU.

Out of season and early season

All influenza positive samples from hospital and community sources, including influenza positive samples from returning travellers.

Mid- to late-season

Refer approximately 10% influenza positive samples, up to a maximum of 50 samples per week.

Include all influenza positive samples from referral categories A to G, and complete the batch with samples randomly selected from category H.

It is recommended to send samples in regular batches if possible, except for those in categories A to E, which should be sent rapidly to RVU for investigation. Influenza B samples (all detections requested) are non-urgent and can therefore be sent in larger batches throughout the season.

Appendix 2: checklist for referral of influenza samples to RVU

Has the relevant section in the guidance been checked for sample referral?

• see section UKHSA public health laboratories
• see section for NHS laboratories
• see section for testing to inform patient management

Is the current version of the typing of influenza strains request form (E3) being used?

Has all relevant patient information been included on the typing of influenza strains request form (E3)?

• include details of ICU/HDU/ECMO or fatal case

Is the sample an acceptable referred sample type?

• see guidance for acceptable referred samples
• include reference number, sample type, date of collection and date sent to UKHSA

Has the relevant safety information been provided on the typing of influenza strains request form (E3)?

• do you suspect that the patient is infected with a Hazard group 3 or 4 pathogen?
• is there foreign travel history?

Have the sending laboratory influenza and SARS-CoV-2 (if performed) results been provided?

• influenza type or subtype? If SARS-CoV-2 tested for, positive or negative?
• assay used and Ct values where generated must be included

If antiviral susceptibility testing requested, has this been discussed with the reference laboratory before sending sample?

• if no, call reference laboratory first to discuss

Is the referred sample from an influenza outbreak?

• if yes, provide details of the name or type of setting and HPZone reference if known, on the typing of influenza strains request form (E3)

Has the patient received the current season flu vaccine? Has the patient received antivirals in the past 14 days?

• if yes, provide details on the typing of influenza strains request form (E3)