Yellow Fever Tools and Guidance

Jean-François (Jeff) Lemaire

yellow fever tools guidanceTools and guidance contents 

This page contains information on: 

Yellow fever case definition

The complete case definition for YF including suspected, probable, confirmed and discarded cases can be found on page 7 of YF: Vaccine Preventable Diseases Surveillance Standards are available for download, in addition to the YF outbreak toolbox.

What to do in case of suspected YF

When a case of YF is suspected, a number of steps must be undertaken to obtain the best possible YF diagnostic results, and these are primarily under the responsibilities of the clinic or physician. These steps include a thorough completion of a specimen submission form, careful collection of a specimen volume adequate for testing and timely processing, and appropriate storage of the specimens prior to shipment to ensure good specimen quality and integrity upon receipt. The correct use of packaging and attention to shipping details and regulations will help guarantee the safe arrival of the specimen at the laboratory. The importance of effective communication with the testing laboratory, transportation company, and everyone involved at all stages underpins the entire process and cannot be emphasized enough.

A Flowchart for YF suspected case is provided as a clinicians guide to collection, packaging and transportation of specimens from suspected YF cases. The same guidance can be used by investigative surveillance teams. Additional information can be found in YF Manual Chapter 3.

Additional sample acquisition, packaging and specimen transportation guidance within the country of origin can be found in the WHO District guidelines for yellow fever surveillance and in job aids.

International shipping guidance

Guidance for shipping of YF specimens from NL’s to RRL’s including the booking form are in this SOP, and in a training video on international shipping of YF samples. 

Laboratory testing for yellow fever

Laboratory testing of specimens from suspected YF cases can change the case classification to discarded, probable, or confirmed YF.

The process for YF diagnostic testing starts with the availability of an adequate specimen volume for use in all assays that may be required, in addition to relevant patient information. In particular this includes history of YF vaccination, place of residence and recent travel history, the date of onset of symptoms and the collection date of the specimen. The most usual sample type for YF testing is serum, and efforts to prevent haemolysis of the blood before the serum is separated out is critical to good results. If specimens arrive without the required information, efforts should be made to obtain them. Communication of these critical aspects by YF testing laboratories to clinics and physicians is integral to ensuring good quality samples are received and that results can be properly interpreted. This communication is the responsibility of the YF laboratory. Failure to communicate these preparative steps prior to testing will compromise results.

Laboratory YF diagnostic testing for living suspected YF cases falls into two main categories: molecular and serologic.

 Molecular testing using YF RT-qPCR

A positive YF RNA result obtained from RT-qPCR is confirmatory for the diagnosis of YF and no further testing is needed. A negative result does not confirm the absence of a YF infection and serological testing is required. YF RT-qPCR is used when the method is available and validated for use in the NL and the specimen was obtained no more than 14 days after the onset of symptoms. If these conditions are not met, serological YF diagnostic testing methods should be used.

A laboratory-developed YF RT-qPCR test is well established for use in most of the region of the Americas, and recently, a commercially-produced kit, the altona Diagnostics’ RealStar Yellow Fever Virus RT PCR Kit 1.0 was approved for use in the GYFLaN by WHO, facilitating the expansion of molecular testing in African and Eastern Mediterranean Regions. Both are based on the YF diagnostic testing method of Domingo et al. (2012). Additional guidance on the use of molecular assays for YF and on verification of assays prior to adoption by the laboratory is available. These assays do not distinguish between naturally-acquired infections and rare adverse reactions to the YF vaccine, underlining the importance of obtaining vaccination information. A specialized method for differentiating between natural and vaccine-related adverse events was developed by Hughes et al. (2018), and is used under special circumstances within the GYFLaN.

Serological testing using YF immunoglobulin M (IgM) methods

Serological testing for YF immunoglobulin M (IgM) is currently the most common front-line tool employed in the Global Yellow Fever Laboratory Network (GYFLaN) because of its availability in African YF laboratories, and because it is useful for specimens obtained during a wide range after onset of symptoms.  Methods include enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay (IFA), and lateral flow assay (LFA). The CDC YF MAC-ELISA based upon the publication of Martin et al. (2000), is a widely-used laboratory-developed test (LDT), but many laboratories especially in the region of the Americas have developed their own LDT’s. Recently, the YF MAC-HD ELISA kit from ATCC® and the Standard Q Yellow Fever IgM LFA from SD Biosensor have been evaluated and approved for use in the GYFLaN by the EYE-LTWG according to the Operational guidance on the use of yellow fever assays in the context of surveillance. A job aid is available to help in the verification of assays prior to routine use in the laboratory. The Euroimmun Biochip YF IFA is commercially-available.  Regardless of the assays used, the occurrence of false-negative results from specimens taken within the first 7 days following onset of symptoms may sometimes necessitate the acquisition of follow-up specimens. YF IgM positive results must be confirmed by the use of a plaque-reduction neutralization test (PRNT), to eliminate false-positive results.

YF testing algorithm for the GYFLaN

Laboratory testing for YF takes place at Sub-National (SNL), National (NL), Regional Reference (RRL)/WHO Collaborating Centers (WHO-CC) and Global Specialized (GSL) Laboratories within the GYFlaN. The types of testing performed is dependent on role and capacity of the laboratories, but submitted specimens are generally tested in SNL/NL first, and referred to RRL/WHO-CC for confirmation if needed, with rare instances of referral to the GSL.

The EYE-LTWG has developed YF testing algorithms for use in the GYFLaN that encompass testing methods and use-case scenarios encountered in all YF-endemic regions.

These include comprehensive YF testing algorithms for NL’s that show the workflow and decision-making sequences for routine surveillance and for active YF outbreaks including fatal cases. A distinct algorithm for confirmation of IgM-positive specimens performed at Regional Reference Laboratories is available. It includes guidance on the interpretation of PRNT results in the context of differential testing. Finally, a simplified YF testing algorithm for countries with no access to YF RT-qPCR, ELISA and PRNT was also developed and is to be used exclusively in coordination with their WHO regional YF laboratory network coordinator. Video presentations for the use of YF algorithms in routine surveillance/outbreaks and confirmatory testing are available. 

YF testing guidance and resources for non-endemic regions can be accessed using these links:

USA: Testing for travellers returning to the USA with suspected YF is available via the US CDC.

Europe: European laboratories with YF testing capabilities are affiliated with the European Centre for Disease Control and Prevention Emerging Viral Diseases-Expert Laboratory Network (EVD-LabNet). This is a list of affiliate laboratories listed by country. 

Biosafety for the YF laboratory 

Yellow fever virus is classed as a BSL-3 virus, as the virus can be fatal in unvaccinated persons. Risk assessments should be performed for all YF diagnostic laboratories, and safety procedures should be adjusted according to the assessments. The WHO Biosafety Manual is available for download. 

Basic laboratory safety practices for use with YF diagnostic specimens include:

  • Use of HEPA-filtered biocontainment (BSL-2) when removing packaging materials from shipments and when manipulating specimens
  • YF-vaccination of laboratory personnel
  • Use of personal protective equipment such as gloves, lab coats and safety glasses

Note that YF virus isolates should be manipulated using BSL-3 biocontainment or according to local biosafety regulations.

Additional biosafety resources

YF lab manual Ch 3 contains biosafety information.

https://www.who.int/activities/safeguarding-biosafety-and-biosecurity-in-laboratories

https://www.who.int/news/item/14-01-2021-who-publishes-latest-manual-on-biosafety-in-laboratories

Biosafety in Microbiological & Biomedical Laboratories (BMBL) sixth edition)