Diagnostic testing for avian influenza A(H5) and other zoonotic influenza viruses
Information on taking, submitting and processing clinical samples from patients suspected of having zoonotic (acquired from birds or mammals) influenza A of any subtype.
This document contains information on taking, submitting and processing clinical samples from patients suspected of having zoonotic (acquired from birds, mammals or other animals) influenza A of any subtype.
This guidance is aimed at clinicians and healthcare workers, health protection teams and clinical diagnostic laboratories both in the public and private sectors.
This page supersedes:
- Avian influenza A(H7) and influenza A(H5): laboratory investigations
- Avian influenza A(H7) and influenza A(H5): referral of samples
There is a wide range of influenza virus subtypes A circulating in wild birds globally. Currently, the most commonly detected avian influenza subtype with zoonotic potential is A(H5). The current UK avian influenza situation in birds and mammals can be found on the Animal and Plant Health Agency website. Influenza viruses can sporadically infect a range of mammalian species, and are endemic in swine, therefore the relevant exposure history of a suspected human case extends beyond birds.
Diagnostic testing for the A(H5) subtype is available at the UK Health Security Agency (UKHSA) Clinical Network Laboratories (CNLs), with confirmatory testing and characterisation performed in the National Influenza Reference Laboratory in the Respiratory Virus Unit, Colindale.
Information is available on when to suspect a case of zoonotic influenza, and the classification of contacts of zoonotic influenza cases and follow-up advice.
Who to inform testing is taking place
Clinicians identifying patients meeting the case definition should contact the local NHS infection or microbiology or virology services and the local health protection team (HPT) in the first instance. They will provide advice on next steps including testing and management of the case.
The infection specialist or health protection team managing the person with suspected zoonotic influenza should contact the relevant UKHSA Clinical Network Laboratory to discuss whether testing is required, and if testing is recommended, they should liaise with clinicians to ensure that an appropriate minimum diagnostic sample set is obtained.
HPTs should inform the UKHSA Acute Respiratory Infections team when a zoonotic influenza infection is suspected (via acute.respiratory@ukhsa.gov.uk) or, if out of hours, phone the Emerging and Epidemic Infections directorate (EEI) duty consultant on-call.
Testing strategy for suspected zoonotic influenza
Diagnostic testing for zoonotic influenza should be undertaken for symptomatic individuals who may have been exposed to zoonotic influenza in birds or mammals in the UK or overseas, according to the criteria described in the Managing human zoonotic influenza spillover cases and their contacts in England guidance.
Primary diagnostic testing of samples from patients with suspected avian influenza A(H5) or other zoonotic influenza infection should be via the UKHSA Clinical Network Laboratories (CNLs) (see list of CNLs, below). The CNLs undertake influenza A typing and subtyping nucleic acid amplification tests (NAATs). Influenza A viruses evolve continuously therefore CNL tests are regularly assured for diagnostic capability. A negative local influenza A test alone may not exclude zoonotic influenza in a suspected case with relevant exposure history. Clinical network laboratories will only accept samples for testing where the patient meets the case definition or after discussion with the local clinician explaining the need for testing. If initial tests are negative for zoonotic influenza and the patient develops new relevant symptoms within the 14 days following exposure, repeat the sampling and testing process.
Sampling for diagnostic testing of zoonotic influenza
When taking samples ensure that you are wearing the minimum appropriate personal protective equipment (PPE) and that all samples are labelled with the patient’s name and date of birth, the date and anatomical site of the sample. Unlabelled samples cannot be processed.
After consultation with the CNL microbiologist or virologist, the clinical team should take the following samples specifically for avian influenza A(H5) and other zoonotic influenza infection testing.
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All patients should have 2 upper respiratory tract samples (combined nose and throat or nasopharyngeal viral swabs are preferable, or nasopharyngeal aspirate if nose and throat swabs are unobtainable). Place the swabs into virus transport medium (VTM) after taking the sample. Do not use a medium containing an inactivating agent.
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If obtainable and the patient’s symptoms or clinical assessment deem it appropriate, take a lower respiratory tract sample, two if feasible (sputum, or an endotracheal tube aspirate if intubated), in addition to the upper respiratory tract samples. Do not use a medium containing an inactivating agent.
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If the patient has conjunctivitis: 2 conjunctival swabs should be taken, in addition to the upper respiratory tract samples. If symptoms affect both eyes, 2 swabs should be taken from each eye. Conjunctival swabs should be placed into VTM after they have been taken. Do not use a medium containing an inactivating agent.
All samples should be sent to the CNL. Do not send samples directly to RVU.
The CNL will use one sample for testing, and store the second. The CNL will forward the stored sample to RVU if confirmatory testing is needed.
Additional samples should be collected if local testing is needed.
What tests will be performed
The CNL will use nucleic acid amplification tests for influenza A, including specific subtype testing for H5, seasonal H1 and H3. Testing for the H5 subtype must be undertaken, even if influenza A is negative.
Additional testing for other common viral causes of respiratory tract infection may also be undertaken. If any additional tests are required, please discuss with the CNL at the time of referral. If alternate diagnoses are under consideration and samples for investigation of other infections are being sent, these must be packaged with separate request forms for testing. Always inform each laboratory of the risk of infection with HG3/4 pathogens.
If an NHS referring laboratory chooses to perform its own respiratory virus panel (in addition to requesting zoonotic influenza testing in a CNL), a local risk assessment should be performed for handling a suspected CL3 organism, and appropriate health and safety measures followed.
Non-inactivated samples must be handled at CL3. Aliquots of the original sample should be separated and inactivated by lysis or other validated method.
Following inactivation, sample aliquots may be handled at CL2 for molecular testing.
Specific subtype testing for zoonotic influenza viruses (including avian and mammalian sources) other than influenza A(H5) is not available in CNLs. If non-A(H5) zoonotic influenza infection is suspected, follow the same sampling and testing guidance ‒ samples should be sent to CNLs, and the duty UKHSA CNL microbiologist or virologist will liaise with the Respiratory Virus Unit (RVU) Colindale, so that appropriate diagnostics can be organized.
Transport of samples to Clinical Network Laboratories for zoonotic influenza diagnostic testing
Details of the address of each of the laboratories laboratories that can test for zoonotic influenza (Clinical Network Laboratories or NHS Collaborating Laboratories) are listed in Table 1 and on the public health laboratories website.
Send all samples for influenza testing to the CNL. Do not send any samples directly to RVU. The CNL will forward samples to the RVU if required.
Highly pathogenic avian influenza A(H5) and some other zoonotic influenza viruses are Advisory Committee on Dangerous Pathogens (ACDP) Hazard Group 3 pathogens. Diagnostic samples should be sent by Category B transport, (UN3373, packing instruction P650) clearly indicating that the samples contain a suspected HG3 pathogen on any referral form to ensure safe handling on receipt.
Provide the contact details for your clinical laboratory for the purposes of telephone and hard copy reporting. This must include an out-of-hours telephone number.
Send samples by DX transfer or courier services according to urgency and timing.
The referring laboratory is responsible for booking and paying for the transport.
Influenza test result communication
Results from the CNL should be provided within 24 hours of receipt of the sample at the testing laboratory.
CNL staff are responsible for informing the referring clinical centre, health protection team and UKHSA acute respiratory infections team of the results including via urgent phone communication to the referring clinical centre.
The relevant clinical team which requested the tests will need to be available to receive the results.
The CNL testing process may generate negative, positive, or inconclusive results for the presence of influenza A of zoonotic origin, as well as for the presence of other respiratory virus pathogens.
In the event of a presumptive positive result for zoonotic influenza, the NHS trust must notify the NHS England Emergency Preparedness Resilience and Response (EPRR) Duty Officer to discuss activating the HCID network immediately. For influenza which is novel or classified as HCID, the network will move the individual or individuals to specialist facilities where possible or support their continued local management, where appropriate.
The clinical and infection control management of the person tested and public health actions relevant to each result will be discussed with the referring infection specialist or other clinical team. This will include antiviral treatment and whether additional testing is required.
The UKHSA CNL should contact RVU to arrange forwarding of samples for confirmatory testing in the event of an avian influenza A(H5) presumptive positive result, or if zoonotic influenza testing is inconclusive, following the guidance for CNLs referring to the RVU.
Referral of presumptive positive avian influenza A(H5) or suspected other zoonotic influenza human samples to the Respiratory Virus Unit: advice for UKHSA Clinical Network Laboratories
All presumptive positive avian influenza A(H5) or other zoonotic influenza A positive samples must be notified to RVU by telephone and arrangements made to forward urgently for confirmatory testing and virus characterization including whole genome sequencing.
For cases where zoonotic influenza testing was inconclusive, or where influenza A(H5) negative samples have been obtained, but clinical suspicion remains, or there is suspicion of another zoonotic influenza (non-A(H5)) exposure, CNLs should contact the RVU to discuss appropriate testing and sample referral.
Contact RVU in working hours Monday to Friday, 09:00 to 17:00, on:
- 020 8327 7125
- 020 8327 7002
- 020 8327 6017
If managing out-of-hours, contact the Epidemic and Emerging Infections (EEI) duty consultant for advice.
After discussion with RVU, samples should be sent by category B transport, packaged accordingly, with clear labelling to indicate an urgent sample with suspected Hazard Group 3 pathogen. Routine daily DX transfer can be used or specific courier services if required out of hours.
The completed E73 referral form must be included in the package.
All available original clinical sample or samples collected for testing must be sent (minimum 400 microlitres (µl), ideally 600 µl or more in VTM, not lysed or inactivated). In the event there is a lower volume, or no clinical material remains, contact RVU for further advice.
For influenza A unsubtypeable results, where the patient does not meet the criteria for clinical suspicion of zoonotic influenza infection refer to the RVU seasonal influenza referral guidelines, and use the seasonal influenza referral form.
Reporting of confirmatory avian influenza A(H5) or other zoonotic influenza results
RVU will report all results of confirmatory testing, positive and negative, by phone to the referring laboratory (for example, the CNL testing laboratory).
The Acute Respiratory Infections Team should be notified by phone (followed up by email) in hours. Out of hours, notify the EEI duty consultant by phone. The local HPT will be updated by the ARI team if results are obtained during working hours, or by the EEI consultant outside of working hours.
Key contacts
Find contact Information for local health protection teams.
Table 1. UKHSA and Collaborating NHS Clinical Network Laboratories
| Region/HPT | Primary contact laboratory | Secondary contact laboratory |
|---|---|---|
| Midlands | Midlands Regional Public Health Laboratory (UKHSA Birmingham) | n/a |
| South East | South East Public Health Laboratory (Southampton) | South West Regional Public Health Laboratory (UKHSA Bristol) |
| South West | South West Regional Public Health Laboratory (UKHSA Bristol) | n/a |
| East of England | East of England Regional Public Health Laboratory (UKHSA Cambridge) | n/a |
| London | East of England Regional Public Health Laboratory (UKHSA Cambridge) | n/a |
| North East | North East Public Health Laboratory (Newcastle) | North West Regional Public Health Laboratory (UKHSA Manchester) |
| North West | North West Regional Public Health Laboratory (UKHSA Manchester) | n/a |
| Yorkshire and the Humber | Yorkshire and the Humber Public Health Laboratory (Leeds) | North West Regional Public Health Laboratory (UKHSA Manchester) |
Table 2. UKHSA national teams
Respiratory Virus Unit, Colindale
Email: rvu.incidentresponse@ukhsa.gov.uk
Telephone:
- 020 8327 7125
- 020 8327 7002
- 020 8327 6017
Acute Respiratory Infections Unit
Email: acute.respiratory@ukhsa.gov.uk
Telephone: 020 8200 4400 (ask to speak with ARI duty senior)
Epidemic and emerging infections duty consultant
No email
Telephone: 020 7123 0333
Results: definitions and actions for UKHSA Clinical National Laboratories
Avian influenza A(H5) positive
If the CNL obtains a presumptive positive result for avian influenza A(H5) regardless of any other influenza A typing or subtyping result and regardless of any other respiratory pathogen detection rapid communication is essential, so that public health control measures can be implemented. An incident management team will be required to plan the management of presumptive positive cases. See section on referral of presumptive positives to RVU for guidance on sample referral.
Avian influenza A(H5) negative
If the test results show that the sample was influenza A negative AND avian influenza A(H5) subtyping negative, then the patient should be managed according to any other relevant test results. If the test results are influenza A positive and subtyped as seasonal A(H3) or A(H1) (without any Ct mismatch), AND A(H5) subtyping is negative then the patient should be managed as for seasonal influenza and any other relevant test results.
Note: samples from seasonal H3 or H1 positive individuals who were exposed to avian influenza should still be referred to RVU for whole genome sequencing, as per the routine seasonal referral guidance.
If appropriate samples were obtained and an alternative diagnosis is likely, then avian influenza A(H5) may be considered excluded. If clinical suspicion remains that the patient has avian or other zoonotic influenza, including the potential for a co-infection, the UKHSA CNL duty microbiologist or virologist should contact the RVU to discuss the results and the options for further testing.
Avian influenza testing result inconclusive
Inconclusive results can be any of the following:
- influenza A positive but seasonal A(H1)/A(H3) and A(H5) subtyping negative (influenza A unsubtypeable)
- influenza A negative and seasonal A(H1)/A(H3) subtyping positive.
- influenza A positive and seasonal A(H1)/A(H3) subtyping positive, but with a significant PCR cycle threshold (Ct) mismatch (>5 Ct difference between typing and subtyping).
Influenza A unsubtypeable samples, or samples with discrepant results between influenza A detection and subtyping results can be an indication of zoonotic influenza viruses, requiring urgent investigation. These results can also be an indication of assay performance issues with seasonal influenza viruses that require further investigation.
Where inconclusive results are obtained, these should be discussed with the RVU for further investigation as outlined in Referral of influenza samples to RVU, UKHSA Colindale. The patient should continue to be managed as a suspected avian or zoonotic influenza case, until a definitive result is obtained unless a clear alternative diagnosis has been made and dual pathology excluded.