Influenza

Globally, influenza detections remained low in week 41 with influenza A virus detections predominant.

In the northern hemisphere, influenza percent positivity was elevated (above 10%) in some countries in Central America and the Caribbean, Western Africa, Southern, South-East and Eastern Asia and a single country in Tropical South America and Northern Europe. Percent positivity was over 30% in some countries in Western, Southern and South-East Asia and in single countries in Western and Eastern Africa. Increases in activity were observed in some countries in Central America and the Caribbean, Western Africa, Western, Southern and South-East Asia and in single countries in South West Europe, Eastern Africa and Eastern Asia.

In the southern hemisphere, influenza percent positivity was elevated (above 10%) in some countries in Tropical South America and in single countries in Temperate South America, Eastern Africa and Oceania. Percent positivity was over 30% in a single country in South-East Asia. A small increase in activity was reported in a single country in Tropical South America.

In the zones with elevated positivity, influenza A(H1N1)pdm09 was predominant in Northern Europe, Eastern Africa, Western, Southern and South-East Asia and Oceania and influenza A(H3N2) was predominant in Eastern Asia. Influenza B was predominant in Central America and the Caribbean, Tropical and Temperate South America and in Western Africa.

SARS-CoV-2

Globally, SARS-CoV-2 positivity remained stable and low across most reporting countries, with elevated positivity (above 10%) reported in some countries in Central America and the Caribbean, Northern and South West Europe, and in single countries in Northern Africa, Western, South-East and Eastern Asia. Increases in activity were observed in two countries in Southern Asia and in single countries in Central America and the Caribbean, Northern Europe, Western, South-East and Eastern Asia.

Respiratory syncytial virus (RSV)

RSV positivity was elevated (above 10%) in a few countries in Central America and the Caribbean and Temperate South America and in single countries in Western Africa and Eastern Asia. Percent positivity was over 30% in a single country in Southern Asia. Increases in activity were observed in single countries in Central America and the Caribbean, Western and Eastern Africa and Southern Asia. RSV and influenza activity were both elevated in countries in Central America and the Caribbean, Temperate South America, Western Africa, Southern and Eastern Asia.

Severity assessment

The severity assessments here are reported from countries, areas and territories. Assessments for transmissibility can be reported based on syndromic parameters and/or influenza-specific parameters. In the northern hemisphere temperate and subtropical areas, influenza-specific transmissibility was reported as below seasonal threshold in 16 countries, low in two countries and moderate in a single country; transmissibility using syndromic data was reported as below seasonal threshold in nine countries and low in two countries. In the southern hemisphere temperate and subtropical areas, influenza-specific transmissibility was reported as below seasonal threshold in one country. Influenza-specific transmissibility was reported as below seasonal threshold in a single country in the tropical areas.

Molecular detection protocols and reagents

The PCR/NGS WG considered the inclusion of two multiplex polymerase chain reaction (PCR) protocols in the compendium of influenza virus molecular detection, one from the NIC in Pune, India (verified by WHO Collaborating Centre for Reference and Research on Influenza in Melbourne, Australia) and one from the NIC in China, Hong Kong Special Administrative Region. Broadening the range of validated options would provide NICs and national influenza laboratories with alternatives to the existing United States Centers for Disease Control and Prevention (CDC) multiplex protocol, particularly for laboratories unable to access CDC International Reagent Resource (IRR) reagents. The PCR/NGS WG encouraged the originating laboratories to provide further validation data and feedback before formal inclusion.

The PCR/NGS WG observed that there is currently no comprehensive list of commercially available PCR assays systematically validated for NIC use. Concerns were raised about variable performance, limited transparency on assay design, infrequent updates and the restriction of some commercial assays to specific thermocycler platforms. By contrast, assays developed by CDC were noted to benefit from established performance monitoring and periodic updates in response to viral evolution. Members emphasized the need for clearer, standardized guidance on verifying new or alternative commercial assays, which could be supported through a dedicated workshop. The WHO RSV reference laboratories indicated plans to develop a WHO training course on PCR assay verification for NICs, which could be extended to influenza.

Laboratory testing algorithms and integrated surveillance

The PCR/NGS WG reviewed the GISRS laboratory testing algorithms. Members agreed that algorithms should remain flexible and be adapted to the prevailing epidemiological situation, with NICs determining the most appropriate approach. NICs with the capacity for routine RSV testing were encouraged to test all specimens, since limiting RSV testing to influenza-negative and SARS-CoV-2-negative specimens may miss coinfections.

The PCR/NGS WG recommended that influenza B lineage detection continue, particularly where not all specimens are sequenced and reagents remain available, while recognizing that no B/Yamagata-lineage viruses have been detected in recent years and that initial lineage testing may be less critical where all surveillance specimens are systematically sequenced. In view of increased influenza A(H5) activity, the need to update primers and probes for H5 detection was emphasized; several NICs have already revised their H5 assays, and the IRR provides H5 synthetic controls to support assay validation and routine testing. Existing WHO guidance recommends systematic testing of at least 50 specimens per week from influenza-like illness, acute respiratory infection and severe acute respiratory infection surveillance, with subtyping and lineage determination expected (WHO, 2022). The PCR/NGS WG discussed introducing guidance on a minimum subtyping threshold and encouraged resource-limited NICs to prioritize subtyping for severe cases, unusual events and outbreak investigations.

Updates to the GISAID Data Science Initiative’s EpiFlu platform

The PCR/NGS WG reviewed recent enhancements to the GISAID Data Science Initiative’s EpiFlu platform, including:

• introduction of the higher-resolution views from subtype and clade levels down to individual mutations, with the ability to overlay PCR and NGS data, and enhanced data-visualization dashboards;
• an updated Primer Checker tool incorporating newly available primers, including the H5 set from WHO Collaborating Centre at the National Institute of Infectious Diseases (NIID), Japan, and PCR/NGS WG members were invited to submit a prioritized list of further primers for inclusion;
• new metadata fields and tools, including a “specimen source” field, updated H5 genotyping tools and linkage of sequences to publications; and
• updated context on genotyping standards and nomenclature for animal influenza viruses, as provided in the OFFLU Genotyping Holding Statement.

GISRS laboratories are encouraged to continue providing feedback on the utility and preferred formats of the new dashboards and features.

Reviewing quality assessment results

To track testing quality across the GISRS network, the working group reviewed the GISRS external quality assessment programme (EQAP) 2024 results. A total of 192 laboratories participated in the 2024 round of the EQAP, with improvements noted in on-time reporting. Of the participating laboratories, 83% reported all influenza detection and subtyping results correctly. Most errors occurred in the detection of nonseasonal influenza viruses, and were typically attributed to protocol or quality-control issues rather than kit limitations. In the sequencing component, 76 laboratories participated, most of which used Illumina platforms, followed by Oxford Nanopore Technologies platforms. Most laboratories returned whole-genome data, although some submitted only haemagglutinin and neuraminidase sequences. Recommendations included retesting specimens with high cycle threshold (Ct) values or inconsistent results, strengthening quality management systems to reduce errors and prevent cross-contamination and carefully reviewing results before reporting.

For future panels, the PCR/NGS WG agreed to continue including nonseasonal influenza viruses, such as A(H5), A(H7) and A(H9), annually even when not circulating locally, to maintain global detection capacity and to provide clear guidance on expected subtypes while allowing flexibility based on regional relevance. The sequencing component will remain educational until broader capacity is established, with future expansion to include zoonotic strains and key markers, such as those associated with host adaptation and antiviral resistance, and with emphasis on evaluating both wet-laboratory sensitivity and bioinformatics pipeline performance. RSV will remain an optional component given reagent and resource limitations. Efforts will continue to encourage open data sharing where administrative or technical concerns persist.

Sample collection and its impact on surveillance

Since the coronavirus disease 2019 (COVID-19) pandemic, many diagnostic laboratories have discontinued traditional viral transport medium in favour of lysis or inactivating buffers, which are compatible with combined viral and bacterial workflows and perceived as safer for laboratory staff. Reversing this shift has proven difficult, particularly where primary laboratories supply specimens to reference laboratories. The PCR/NGS WG noted important consequences for surveillance. Specimens in inactivating buffers cannot be cultured, which severely limits virus isolation and the development of candidate vaccine viruses. In addition, although such buffers are compatible with real-time reverse transcription PCR (RT-PCR), they present challenges for NGS.

Bioinformatics training and quality assessment

The GISAID Data Science Initiative offered to support bioinformatics training through in-person workshops, including one preceding the International Meeting on Respiratory Pathogens 2025 in Singapore and one in Japan around the time of WHO consultation on the composition of influenza virus vaccines in September 2025, and through virtual workshops to increase accessibility. A new bioinformatics quality assessment exercise will compare and assess the pipelines used across GISRS to assemble influenza NGS data, drawing on a mix of in-house and publicly available raw data. Outputs will be compared against expected results, focusing on correct identification of subtype and lineage; detection of in-frame insertions and deletions, mixed populations (including antiviral-resistance markers) and mixed infections; differentiation of B/Yamagata from B/Victoria; handling of zoonotic and swine-variant viruses; and performance across Illumina and Nanopore data. Recognizing that mixed infections and minor-variant detection remain difficult for many pipelines, the PCR/NGS WG emphasized the value of pipelines that at least flag possible mixed infections.

Conclusion

PCR/NGS WG members will continue to strengthen laboratory and surveillance capacity across GISRS. Updated PCR and NGS protocols will continue to be collected, consolidated and posted annually on the WHO website. A training package on molecular assay verification for NICs, covering influenza and RSV, will be developed and supported by a structured training course. Bioinformatics training will be offered through virtual and in-person workshops linked to the next round of the GISRS bioinformatics quality assessment exercise. An internal list of validated commercial molecular assay kits will be maintained and shared with regional offices and NICs on request. Nonseasonal influenza viruses, including A(H5), A(H7) and A(H9), will continue to be included in annual EQAP panels to maintain global detection capacity. Together, these actions will help GISRS sustain robust, flexible and collaborative laboratory surveillance of influenza and other respiratory viruses.