This page includes information on the YF Laboratory Manual and YF diagnostic testing assay evaluation reports.
Yellow Fever Laboratory Manual
A technical manual for laboratory diagnosis of YF has been developed as a comprehensive reference for all aspects related to YF diagnostic testing in the Global Yellow Fever Laboratory Network (GYFLaN). The entire YF Manual can be downloaded as a PDF.
The Laboratory manual for yellow fever consists of 9 chapters and annexes that include an overview of YF, the role of the GYFLaN in the EYE strategy, the use of clinical specimens for YF diagnostic testing, testing algorithms, antibody and molecular detections methods, confirmatory testing, data management and reporting of laboratory results, and quality assurance through assessment and accreditation.
Topics within these chapters are described below.
Chapter 1: Yellow fever: An overview. The structure and biology of yellow fever virus, transmission cycles and geographical distribution are discussed. Epidemiological aspects of human disease, modes of YF virus infection and immune response, and the roles of surveillance to track disease are described. The clinical description of the disease, the case definition of YF, and laboratory diagnosis are introduced, with more detailed descriptions of diagnostic methodology in later chapters.
Chapter 2: The Global Yellow Fever Laboratory Network and the role of the yellow fever diagnostic laboratory network in the Eliminate Yellow Fever Epidemics (EYE) strategy. The history, objectives and organization of the GYFLaN are described, including how the tiered laboratory network coordination works in undertaking the roles and major activities of the network laboratories. The EYE strategy’s commitment to high-quality YF diagnostic testing services through laboratory strengthening is discussed.
Chapter 3: Clinical specimens for the laboratory confirmation of yellow fever. The best practices for collection, processing and storage of serum specimens are detailed, including how best to time the collection of blood for YF testing. Collection of specimens for confirmation of YF in fatal human cases and collection from non-human primates require special considerations which are discussed, along with general guidelines for the preparation and transport of clinical specimens. Safety procedures for incoming clinical specimens are described, as is the use of alternative specimens for YF diagnostic testing.
Chapter 4: The algorithm for laboratory confirmation of yellow fever cases. An algorithm for YF diagnostic testing is based on the timeline of clinical symptoms and immune response following YF virus infection. Chapter 4 discusses the GYFLaN testing algorithm in this context and details appropriate specimen types and timing of specimen collection for YF testing. The YF assays used in the NLs for diagnosis of YF and under what circumstances they are appropriate for use are described, including the interpretation of test results and referral of samples to the RRLs.
Chapter 5: Antibody Detection Methods for Yellow Fever diagnosis. The kinetics of the immune response against YF virus infection and how this relates to the IgM immunoassay is described, in addition to the selection and comparison of YF IgM detection assays. Details on the setting up of the YF MAC-ELISA in the laboratory, how calculations are performed, and test interpretation are detailed. Alternative specimens for YF IgM detection and other serological techniques and methodologies are described.
Chapter 6: Detection of viral RNA by RT-PCR for the confirmation of YF virus infection. This chapter describes the best practices for the collection and processing of clinical specimens for molecular detection of YF virus and for extraction of RNA. Considerations for the use of molecular diagnostic methods and YF RNA detection methods by RT-qPCR are discussed, including methods for vaccine-specific identification. Quality assurance and quality control aspects, test validity, data interpretation, and assay limitations are detailed.
Chapter 7: Yellow fever confirmatory and reference testing at the Regional Reference Laboratory. The RRL YF confirmatory testing algorithm is presented including the differential diagnostic testing by flavivirus MAC-ELISAs and PRNT. The histopathologic examination of specimens from fatal cases is described as well as specialized testing such as viral isolation. The reporting of results of confirmatory testing is discussed.
Chapter 8: Data management and reporting of laboratory results. Data management goals for the GYFLaN are discussed in this chapter. Specimen accessioning and the recording of laboratory results and their accompanying data is described, as is the reporting to Expanded Programme on Immunization (EPI) teams and to WHO. Data management and results reporting for specimens sent to the RRLs for confirmatory testing is also included.
Chapter 9: Quality assurance, quality control, and assessment of laboratory capacity and performance. This chapter describes the establishment and benefits of a quality management system and the technical elements therein. The key objectives of laboratory quality assessment and assurance, details on monitoring and standardization of assay performance, the WHO external quality assurance programme, and the process of WHO assessment and accreditation are presented.
YF diagnostic assay evaluation reports
WHO issued expressions of interest (EOI) requests for commercial YF diagnostic assays to be evaluated for use in the GYFLaN. The evaluation process begins with an applicant dossier screening, after which successful applications undergo a complete documentation dossier review, a quality management system audit, and an independent YF diagnostic assay performance evaluation. The combined findings are used to inform the decision-making process as to whether WHO, advised by the EYE-LTWG, recommend assays for use within the GYFLaN. So far, one EOI request for YF molecular assays and one for YF IgM serology assays were published in July 2020 and January 2021, respectively.
In the first EOI round for YF molecular assays, one manufacturer (altona Diagnostics, Hamburg, Germany) submitted RealStar® Yellow Fever Virus RT-PCR kits for independent evaluation by EYE-LABS. The assay was found to be suitable for use in the GYFLaN as described in this report.
A second EOI request for YF molecular assays was published in December 2022.
The EOI for YF serological assays received two submissions: ATCC® (Manassas, Virginia, USA), submitted the YF MAC-HD ELISA kit, and SD Biosensor (Gyeongii-do, Republic of Korea) submitted the Standard Q Yellow Fever IgM lateral flow assay for evaluation. Both assays were found to be appropriate for use in the GYFLaN. Use these links to access the ATCC YF MAC-HD and the SD Biosensor Standard Q Yellow Fever IgM assay evaluation reports.
Practical information relating to the use of commercial YF assays can be found in Operational guidance on the use of yellow fever assays in the context of surveillance.