who/bs/2016.2298 rev.1 english only expert committee …

WHO/BS/2016.2298 Rev.1

ENGLISH ONLY

EXPERT COMMITTEE ON BIOLOGICAL STANDARDIZATION

Geneva, 17 to 21 October 2016

Requests to initiate new WHO reference material projects for in vitro diagnostic

devices and reagents

Document prepared by the WHO Secretariat, based on inputs from WHO Collaborating Centres

supporting biological standardization activities. Note:

This document has been prepared for the purpose of inviting comments and suggestions on the

proposals contained therein, each of which will be considered by the Expert Committee on

Biological Standardization. The proposals have not yet been endorsed by the Expert Committee.

Comments on the proposals MUST be received by 3 October 2016 and should be addressed to

the World Health Organization, 1211 Geneva 27, Switzerland, attention: Technologies, Standards

and Norms (TSN). Comments may also be submitted electronically to the Responsible Officer:

Dr David Wood ([email protected]). The outcome of the deliberations of the Expert Committee

will be published in the WHO Technical Report Series.

© World Health Organization 2016

All rights reserved. Publications of the World Health Organization can be obtained from WHO Press, World Health

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The mention of specific companies or of certain manufacturers’ products does not imply that they are endorsed or

recommended by the World Health Organization in preference to others of a similar nature that are not mentioned. Errors and

omissions excepted, the names of proprietary products are distinguished by initial capital letters.

All reasonable precautions have been taken by the World Health Organization to verify the information contained in this

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be liable for damages arising from its use.

The named authors [or editors as appropriate] alone are responsible for the views expressed in this publication.

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WHO/BS/2016.2298 Rev.1

Introduction

The provision of global measurement standards is an important normative activity of WHO.

Biological reference preparations that are accepted internationally enable the efficacy, quality, purity

and safety of very many biological medicines, used in the prevention, treatment or diagnosis of

disease or conditions, to be stated in a common language worldwide. International biological

reference standards support the use of many biological and immunological assays for the quality

control of a wide range of biologicals including therapeutics, blood-derived products, vaccines and

immunological products of traditional types as well as those derived from modern biotechnological

approaches. They also have important applications in the standardization of materials and

approaches used in medical diagnostics such as diagnosing disease, monitoring therapy, blood safety,

and public health applications (e.g. monitoring immune status, screening for disease or susceptibility)

or otherwise characterizing biological material from individuals.

WHO biological reference standards are widely used in the development, evaluation, standardization

and control of products by industry; by regulatory authorities; and also in biological research in

academia and scientific organizations. They play a vital role in facilitating the transfer of laboratory

science into worldwide clinical practice and the development of safe and effective biologicals.

The timely development of new reference materials and standards is critically important to harness

scientific developments for new biologicals. At the same time, the active management of the

existing inventory of reference preparations requires a carefully planned programme of work to

replace established materials before the stock of containers, which comprises the standard, is

exhausted.

Considerations for assignment of priorities to development of WHO International Biological

Measurement Standards or Reference Reagents have been published (WHO TRS 932, Annex 2,

Appendix 1, 2005). These considerations are used as guiding principles by the Secretariat and the

WHO Collaborating Centres to develop a proposed programme of future work. To facilitate and to

improve transparency in the priority setting process, a simple tool has been developed which

describes the salient features of each new project proposal.

This document provides a means for the Committee and other stakeholders to review and comment

on new proposals that are under consideration. The proposals in this document

(WHO/BS/2016.2298) cover requests to initiate new projects in the areas of in vitro diagnostic

devices and reagents (Appendix 1).

WHO/BS/2016.2298 Rev.1

Appendix 1 In vitro diagnostic devices and reagents

Proposed new projects

1) BRAF V600E (proposed WHO 1st International Genomic Reference Panel) (page 4)

2) Human epidermal growth factor receptor 2 (HER2, official gene symbol ERBB2) (proposed

WHO 1st International Genomic Reference Panel) (page 6)

3) Syphilitic plasma IgG and IgM (proposed WHO 2nd International Standard) (page 8)

4) Syphilitic plasma IgG (proposed WHO 2nd International Standard) (page 8)

5) Respiratory Syncytial Virus (RSV) RNA for NAT (proposed 1st WHO International

Standard) (page 9)

6) Influenza virus types A and B RNA for NAT (proposed 1st WHO International Standard)

(page 11)

7) Anti-Zika virus antibody (IgG/IgM) (proposed WHO 1st International Standard) (page 13)

8) Anti-Chikungunya virus antibody (IgG/IgM) (proposed WHO 1st International Standard)

(page 15)

9) Ebola virus antigen (proposed 1st International Reference Reagent) (page 18)

10) Human Immunodeficiency Virus type 2 (proposed 2nd International Standard for HIV-2

NAT) (page 20)

WHO/BS/2016.2298 Rev.1

Proposal (title) Proposed 1st WHO International Genomic Reference Panel for BRAF V600E

Proposer (name of Institution)

NIBSC Principal contact Jennifer Boyle

Rationale BRAF mutations are present in approximately 37-50% of malignant melanomas (plus many other solid tumours including colorectal cancer, papillary thyroid carcinoma and non-small-cell lung carcinoma). Activating mutations induce stimulation of the MAP kinase pathway with subsequently increased cellular proliferation and survival. Of all BRAF mutations, 80-90% are BRAF V600E.

The BRAF V600 mutated protein is an obvious candidate for drug-targeting with inhibitor drugs in ongoing development; vemurafenib and dabrafenib are approved for metastatic melanoma treatment in patients with BRAF V600 mutations, with BRAF V600 molecular genotyping mandatory.

It is therefore important to standardise the molecular diagnosis of BRAF V600E-positive patients and monitor treatment response by quantification of BRAF V600E by the provision of a WHO international genomic reference panel to enable accurate and sensitive diagnostic testing.

Anticipated uses and users

The panel would be used by manufacturers for the calibration of diagnostic kits and by clinical & reference laboratories in the calibration of secondary standards used in multiple routine diagnostic assays for BRAF V600E detection.

Standardisation could be applied to assays utilizing both traditional formalin-fixed, paraffin-embedded tissue and ‘liquid biopsy’ circulating tumour DNA, the latter requiring highly sensitive detection and timely standardisation as a rapidly emerging technology.

Source/type of materials Two purified genomic DNAs extracted from cell lines; one BRAF wild-type, one BRAF V600E. Freeze-dried at approximately 2500 ampoules per material.

Outline of proposed collaborative study

The collaborative study would evaluate the materials using a variety of diagnostic genotyping techniques. Quantitative data derived from the collaborative study would be used to establish a consensus value for each of the materials.

Issues raised by the proposal

Feedback from potential end-users and resource availability will determine if the panel should be provided at a range of BRAF V600E levels as ‘ready-to-use’ standards, or as a single BRAF V600E standard for end-user’s dilution (along with the BRAF wild-type material).

V600E is the predominant BRAF mutation in metastatic melanoma; other, much rarer mutations would not be covered by the panel (V600E complex (V600Ec), V600D, V600K and V600R).

WHO/BS/2016.2298 Rev.1

Action required ECBS to endorse proposal

Proposer's project reference

TDI-00006 Date proposed: 1st July 2016

CONSIDERATIONS FOR ASSIGNMENT OF PRIORITIES (TRS932)

Approval status of medicine or in vitro diagnostic method

The approval of vemurafenib and dabrafinib, two BRAF inhibitor drugs, have made BRAF V600 molecular genotyping mandatory in selecting patients who will benefit from these therapies. Companion diagnostic assays have been approved for both drugs, but their use is not obligatory. The high cost of these genotyping systems means that a variety of unregulated tests of variable quality continue to be used.

Number of products or methods

Materials to enable standardisation of all commonly used PCR-based assays for BRAF V600, as well as next-generation sequencing.

Public health importance

The incidence and mortality rates of melanoma have risen sharply throughout the world over the past few decades. With over 130,000 new case of melanoma diagnosed globally each year, there is a strong need to improve the quality of diagnosis and treatment.

Global importance

Melanoma occurs in all countries, with Caucasians living in low and middle latitude countries at the highest risk.

Global need from regulatory & scientific considerations

Accurate genotyping is required to ensure patients are matched to the optimal drug treatment. Tumour biopsies from which DNA is extracted contain variable levels of tumour cells. Even commercial assays have been demonstrated to lack sufficient analytical sensitivity to detect mutations in small tumours, unless macrodissections are performed before DNA is extracted. Without sensitivity standards and calibrants, satisfactory assay performance cannot be guaranteed.

An international reference panel will enable global standardisation of BRAF V600 genomic diagnostics to ensure a consistent, accurate and safe approach.

ECBS outcome [BLANK]

WHO/BS/2016.2298 Rev.1

Proposal (title) Proposed 1st WHO International Genomic Reference Panel for HER2


NIBSC Principal contact Jennifer Boyle

Rationale Human epidermal growth factor receptor 2 (HER2, official gene symbol ERBB2) is a member of the epidermal growth factor receptor family of receptor tyrosine kinases. ERBB2 is amplified (and thus HER2 protein is overexpressed) in 18-20% breast cancers, and is associated with increased tumour aggressiveness. HER2 overexpression is also associated with more aggressive forms of ovarian, stomach and uterine cancer.

Detection of HER2 overexpression is clinically actionable, with HER2-positive tumours treatable with herceptin (trastuzumab). Nucleic-acid based ERBB2 quantification methods are of increasing diagnostic use (particularly RNA). It is therefore important to facilitate timely standardisation of the molecular diagnosis of HER2 overexpression-positive patients and monitoring of treatment response by quantification of ERBB2 by the provision of a WHO international genomic reference panel to enable accurate and sensitive diagnostic testing.


The panel would be used by manufacturers for the calibration of diagnostic kits and by clinical and reference laboratories in the calibration of secondary standards used in multiple routine diagnostic assays for ERBB2 characterisation.

Standardisation could be applied to assays utilising both formalin-fixed, paraffin-embedded tissue and fresh-frozen solid tumour biopsies.

Source/type of materials The panel would comprise freeze-dried cells for DNA or RNA extraction at approximately 2500 ampoules per material.

One cell line with confirmed both high ERBB2 expression and copy number would be chosen from several in-place candidates. Similarly, ERBB2 status of a likely wild-type cell line would be confirmed. A dilution series of the high ERBB2 mRNA expression and copy number cell line in the ERBB2 wild-type cell line would generate four materials providing low, low medium, high medium and high values for both mRNA expression and DNA copy number.


The collaborative study would evaluate the materials using a variety of diagnostic genotyping techniques. Quantitative data derived from the collaborative study would be used to establish a consensus value for each of the materials.


The National Institute for Standards and Technology (NIST) has produced a standard reference material for ERBB2. However, this is of genomic DNA suitable only for the measurement of DNA copy number, and not mRNA expression which is much more commonly used in diagnostics.


WHO/BS/2016.2298 Rev.1


TDI-00027 Date proposed: 1st July 2016



Herceptin (trastuzumab) is approved for breast cancer treatment in patients with HER2 overexpression. The European patent expired in 2014, and with the USA patent expiring in 2019, biosimilars are already emerging.

Laboratory diagnostics by immunohistochemistry for HER2 protein overexpression and fluorescence in situ hybridization for ERBB2 gene amplification are known to be inaccurate; nucleic-acid based quantification methods are of increasing use (particularly for mRNA).


Materials to enable standardisation of all commonly used DNA- and RNA-based quantitative PCR-based assays for ERBB2.


There are 1.6 million new breast cancer cases globally each year, of which approximately 20% are HER2 overexpression-positive.

Herceptin biosimilars may vary in quality, efficacy and safety. Provision of an international reference panel to calibrate diagnostic assays will ensure biosimilar products are safe and effective.

Global importance

Just over half of breast cancer cases (including HER2 overexpression-positive cases) are from less developed countries (data from World Cancer Research Fund International).


Accurate genotyping is required to ensure patients are matched to the optimal drug treatment. The release of Herceptin biosimilars will bring effective treatments to many more patients than previously.

No standards currently exist for the measurement of RNA-based HER2/ERBB2 over-expression assays, an increasingly typical diagnostic approach.

An international reference panel will enable global standardisation of HER2/ERBB2 genomic diagnostics to ensure a consistent, accurate and safe approach.


WHO/BS/2016.2298 Rev.1

Proposal (title) The second International Standard for Syphilitic plasma IgG and IgM & second International Standard for Syphilitic plasma IgG


e.g. NIBSC Principal contact David Padley

Rationale Based on current uptake the Syphilitic plasma IgG and IgM (human)(1st International Standard) 05/132 & the Syphilitic plasma IgG (human)(1st International Standard) 05/122 will run out in 2-3 years


Reference labs, hospital labs, GUM/STI clinics, manufacturers

Source/type of materials Syphilitic human plasma


Collaborative study with candidate samples freeze-dried, frozen bulks and clinical samples for commutability. 15- 20 laboratories hopefully participating.


None.



Date proposed: 05-07-2016





Global importance


WHO/BS/2016.2298 Rev.1

Proposal (title) 1st WHO International Standard for Respiratory Syncytial Virus (RSV) RNA

for NAT


NIBSC Principal contact Clare Morris

Rationale Respiratory tract infections can be caused by a plethora of pathogens, all with varying degrees of severity. RSV is a seasonal infection during winter months and is major cause of lower respiratory tract infections often causing severe disease in the elderly or very young; in many cases hospitalisation is required. Early diagnosis is therefore critical for correct diagnosis and therefore early application of treatment. There is no vaccine for RSV.

Laboratories are moving away from serological screening towards more sensitive molecular methods. However data returned through EQA schemes indicates that there is a large degree of variation across all assays. Assays are typically reporting in qualitative format, however the large variation in results reported on the same sample suggests the incidence of incorrect reporting both as false negative and false positive occurs. Without the availability of an international standard it is also very difficult to accurately determine LOD and LOQ. RSV can be divided into two main subtypes, A and B. It is unknown whether current molecular assays detect both strains with equivalent sensitivity


Manufactures of RSV assays

Laboratories with in house assays

Providers of secondary reference materials

Source/type of materials Literature reports indicate that RSV can be grown to high titre in HEp-2 and MA-160 cells in tissue culture and therefore it is anticipated that candidate material for RSV A and RSV B will be produced in this way.

Laboratories will typically screen for RSV in a range of respiratory samples – sputum, throat swabs, BAL. In collaboration with clinical laboratories, such samples will be sought for inclusion in the study to contribute towards commutability assessment.


Study to include up to 30 international laboratories running a variety of commercial and in house assays


Availability of clinical material to assess commutability. Availability of clinical material to assess commutability may be limited. Typically a high volume (>500ml) of individual patient material is needed to aliquot amongst study participants in order for identical un manipulated clinical material to be assessed as would mimic the situation in diagnostic laboratories.


WHO/BS/2016.2298 Rev.1


Date proposed: 17 June 2016



Diagnostic methods are CE marked where they are commercial, however status of in house methods is unknown


4 commercial assays known for single detection of RSV, multiple assays >6 for the multiplex detection of RSV. Unknown number of in house methods


PHE figures indicate that typically 1000-1200 cases are reported in the UK during the winter season.

Global importance

WHO data indicates that RSV is responsible for in the order of 30 million global respiratory tract infections annually


Lack of vaccine material and the high number of hopitailsations resulting from RSV infections in the young and elderly lead to an urgent need for accurate reporting.


WHO/BS/2016.2298 Rev.1

Proposal (title) 1st WHO International Standard for Influenza types A and B for NAT


NIBSC Principal contact Clare Morris

Rationale Respiratory tract infections can be caused by a plethora of pathogens, all with varying degrees of severity. Influenza is a major cause of respiratory tract infections. Influenza has many subtypes determined by the combination

of hemagglutinin (HA) and neuraminidase (NA), two proteins on the surface of

the virus: The severity of disease can be dependent on the combination of HA and NA. Influenza has been devised into 3 types, A, B and C. Types A and B are known to be capable of causing the most severe form of illness. Assays are reported to be capable of detecting both type A and B with equivalent sensitivity. However data returned through EQA schemes indicates that there is a large degree of variation across all assays, it is unknown to what degree this is caused by variant or type. The use of molecular assays is becoming increasing more frequently in the determination of a respiratory illness being caused by influenza. In particular the use of rapid assays.


Manufactures of Influenza A/B assays

Laboratories with in house assays

Providers of secondary reference materials

Source/type of materials Literature reports indicate that both Influenza A and B can be grown to high titre in tissue culture and therefore it is anticipated that candidate material for A and B will be produced in this way.

The selection of appropriate strain is addressed below in issues raised.

Laboratories will typically screen for influenza in a range of respiratory samples – sputum, throat swabs, BAL. In collaboration with clinical laboratories, such samples will be sought for inclusion in the study to contribute towards commutability assessment.


Study to include up to 30 international laboratories running a variety of commercial and in house assays


Availability of clinical material to assess commutability may be limited. Typically a high volume (>500ml) of individual patient material is needed to aliquot amongst study participants in order for identical un manipulated clinical material to be assessed as would mimic the situation in diagnostic laboratories.

Selection of an appropriate strain that remains representative over time. It would be appropriate to evaluate a panel of materials and this may need to take to form of a pilot study to identify, if possible, 2/3 strains to put forward into the main study. Additional guidance needs to be sought from experts in this field. Currently strains to which assays commonly refer to as detecting are H1N1 and H3N2, these also feature widely in UK and WHO incidence reports. These viral strains are also reflected in many run control reagents.

http://www.cdc.gov/flu/images/virus/fluvirus-antigentic-characterization-large.jpg

WHO/BS/2016.2298 Rev.1

As a result these would be a good inclusion in the panel. It. Recombination events within the virus lead to frequent changes in the HA and NA combinations. Whilst commercial assays often target conserved areas of the influenza genome, it would be wise to assess the validity of the standard on an annual basis. This could be achieved by inclusion in an EQA scheme as a means of reassessing performance with a range of assays.



Flu A/B 2016 Date proposed: 17 June 2016



Diagnostic methods are CE marked where they are commercial, however status of in house methods is unknown


Commercial assays are available for single detection of Influenza A/B however many assays(>6) are on the market for the multiplex detection of Flu A/B these often fall into the rapid test field. Unknown number of in house methods


PHE figures indicate that typically 150,000 cases per week reported in the UK during the winter season.

Global importance

WHO data indicates that Flu A/B infections are responsible for 3 to 5 million cases of severe illness, and about 250 000 to 500 000 deaths annually


A vaccine is available however not adopted by all


WHO/BS/2016.2298 Rev.1

Proposal (title) The 1st International Standard for antibody (IgG/IgM) to Zika virus


NIBSC Principal contact Mark Page

Rationale In support of the WHO response to Zika virus disease, NIBSC has been requested to lead in the development of an International Standard for use in calibration and control of Zika antibody assays. Accurate diagnosis of Zika virus infection is critical for subsequent healthcare decisions particularly for pregnant women. The virus has a short viraemia and PCR based tests are only useful during this period (~9 days) after onset of symptoms (rash, fever). Diagnosis is then carried out by serology tests to measure the antibody response to the virus. Current tests are prone to high cross reactivity against other arboviruses (Dengue in particular) that are spread by the same mosquito vector. Therefore it is critical that accurate diagnosis is available and standardization of the tests is required to improve sensitivity and specificity measurements. Both IgM and IgG are currently measured to determine prior exposure where IgM would indicate a recent infection that is used in secondary testing after a negative PCR result.


Primary intended use is for calibration of assays used to measure Zika antibody IgM and IgG levels in human serum by ELISA and neutralization assays. Recommendation for this purpose will be subject to satisfactory demonstration of commutability. Primary users will be public health and other clinical laboratories, kit manufacturers and Zika vaccine manufacturers (for clinical trials).

Source/type of materials Human serum/plasma from subjects recently infected with Zika virus.

Serum from immunized transchromosomal bovines that produce fully humanized IgG.


assess the suitability of different antibody preparations to serve as the Intenational standard with an assigned unitage per mL for use in the harmonization of Zika serology assays. There is no international conventional reference measurement procedure for Zika virus antibodies and the unitage will not be traceable to the International System of Units (SI) of quantity.

characterise the antibody preparations in terms of reactivity/specificity in different assay systems.

assess each preparation’s potency i.e. readout in a range of typical assays performed in different laboratories for both IgG and IgM classes

assess commutability i.e. to establish the extent to which each preparation is suitable to serve as an interim standard for the variety of different samples and assay types.

WHO/BS/2016.2298 Rev.1


Material transfer agreements and sourcing material from embargoed countries may add to the timeline.

Action required e.g. ECBS to endorse proposal


Date proposed:




Experimental ELISA kits in development. Commercial ELISA kits are being rolled out. Neutralisation assay measured by plaque reduction.


Increasing numbers of countries are reporting cases. Zika association with microcephaly and Guillain-Barre syndrome is of high importance with potential for global impact. WHO have declared the outbreak as a Public Health Emergency of International Concern.

Global importance

As above


Standardised and calibrated assays are vital for accurate diagnosis of Zika (see above)

ECBS outcome ECBS to endorse

WHO/BS/2016.2298 Rev.1

Proposal (title) Chikungunya virus reference reagent for serology

Proposer (name of

Institution)

PEI Principal

contact

Sally Baylis

Rationale The mosquito-borne Chikungunya virus (CHIKV) is a

member of the Alphavirus genus in the Togaviridae family.

Chikungunya was first identified in Tanzania in the early

1950s. The disease occurs not only in Africa but also in

Asia and the Indian subcontinent and, since 2013, has

spread to the Americas, particularly central and Southern

areas. Small outbreaks have also occurred recently in

Europe.

The diagnosis of Chikungunya requires a variety of tests

including detection of IgM and IgG antibodies. Co-

circulation of Chikungunya virus with Dengue virus (DENV)

and Zika virus (ZIKV) frequently occurs and infections

cause by these viruses share common signs and symptoms

in infected patients. Accurate diagnosis and discrimination

of CHIKV from other virus infections is important for patient

care. Analysis of IgM antibodies is particularly useful for the

confirmation of acute infection. Anti-CHIKV IgG may also be

detectable during acute infection. Anti-CHIKV IgG is also a

marker of past CHIKV infection and seroprevalence and

anti-CHIKV and indication previous exposure of different

populations to CHIKV infection.

Anti-CHIK assays vary in their performance (for example,

WHO/BS/2016.2298 Rev.1

Niedrig et al. Clin Microbiol Infect. 2009;15:880 and Prat et

al. Emerg Infect Dis. 2014;20:2129).

Anticipated uses

and users

Clinical laboratories, particularly arbovirus reference

laboratories. Research laboratories and organizations

developing CHIKV vaccines. IVD manufacturers.

Source/type of

materials

Sera to be sourced through a national and international

collaborations.

Alphavirus cross reactivity.

Outline of

proposed

collaborative study

Evaluate sera from CHIKV infected patients and potentially

blood donors. These samples will include IgM as well as

IgG reactive sera to distinguish between recent and past

infections.

Issues raised by

the proposal


Proposer's project

reference

Date

proposed:

July 2016


Approval status of

medicine or in

vitro diagnostic

A number of serological kits are available for the detection

of anti-CHIKV IgM and IgG.

WHO/BS/2016.2298 Rev.1

method

Number of

products or

methods

As above.

Public health

importance

To ensure that assays are of adequate sensitivity/specificity

for the determination of anti-CHIK IgM and IgG for

diagnostic purposes and to facilitate a better understanding

of CHIKV epidemiology globally.

Global importance The rapid spread of CHIKV in the last decade and the

similar clinical presentation with viruses such as dengue

and Zika virus require accurate diagnostic testing.

Global need from

regulatory &

scientific

considerations

As above.

ECBS outcome

WHO/BS/2016.2298 Rev.1

Proposed 1st International Reference Reagent for Ebola virus antigen

Proposal (title) Proposed 1st International Reference Reagent for Ebola virus antigen


NIBSC Principal contact Dianna Wilkinson

Rationale In support of the WHO response to the Ebola virus outbreak, NIBSC has been requested to lead in the development of reference reagent for use in control of Ebola virus antigen assays. Accurate and rapid diagnosis of Ebola virus infection is critical for subsequent healthcare decisions. Patients may present with a fever that could be confused with other fever inducing pathogens particularly malaria which is endemic in much of Africa. Rapid diagnostic kits such as those used at the point of care allow confirmation of ongoing virus infection within ~15 minutes from a simple blood prick sample. The read out is a colour reaction on a solid phase stick that gives an easily interpretable yes or no answer. A rapid response is vital to facilitate appropriate healthcare provision particularly the isolation of infected individuals. Other ELISA based assays may also be used in other settings.


Primary intended use is for a monitor for Ebola antigen assays. Recommendation for this purpose will be subject to satisfactory demonstration of commutability. Primary users will be public health and other clinical laboratories, kit manufacturers.

Source/type of materials Full length, native, VP40 recombinant protein, expressed in E.coli. Based on 2014 Kissidougou – C15 strain

Virus-like particles developed by expression of 3 plasmids for VP40, NP and GP in HEK293T cells. Based on H. sapiens wt/GIB/2014/Kissidougou – C15 Ebola strain. VLPs are purified on 205 sucrose cushion in phosphate buffer

Soluble GP, partial nucleoprotein NP and full length VP40 produced in E.coli using Ebola Mayinga sequences, supplied in carbonate buffer by SD Biosensor Inc., S. Korea

9 coded samples will be entered into the study, formulate in thrombinised and declotted plasma. Samples will be diluted around the detection limit and aliquotted into 120ul and freeze-dried


assess the suitability of different antigen preparations to serve as the international reference reagent to monitor Ebola antigen assays.

characterise the antigen preparations in terms of reactivity and sensitivity in different assay systems.

assess each preparation’s performance i.e. readout in a range of typical assays performed in different laboratories

assess commutability i.e. to establish the extent to which each preparation is suitable to serve as an interim standard for the variety of different samples and assay types.

Issues raised by the Material transfer agreements and sourcing material from embargoed

WHO/BS/2016.2298 Rev.1

proposal countries may add to the timeline.



TBC Date proposed: 2016



Diagnostic antigen kits are under development, none licensed. Applications for WHO EUAL in progress or are listed.


Experimental antigen kits in development. Commercial kits are being rolled out.


Rapid diagnosis of Ebola is vital during an outbreak to identify infected individuals so that they can be treated quickly to prevent death and spread of the virus.

Global importance

As above


Proper monitoring of kit batches is vital for accurate diagnosis of Ebola (see above)

ECBS outcome

WHO/BS/2016.2298 Rev.1

PROPOSAL FOR A NEW / REPLACEMENT

INTERNATIONAL REFERENCE PREPARATION

Proposal 2nd International Standard for HIV-2 NAT

Proposer NIBSC Principal Contact

Clare Morris

Rationale The 1st WHO IS for HIV-2 RNA was established in 2009 with an assigned value of 1000IU/ml. Many users have contacted NIBSC stating that this value is too low for calibration purposes. It is therefore proposed to formulate a replacement materials at a higher titre (>106). Once established the remaining material for the 1st IS will be withdrawn.


This standard will used to calibrate HIV-2 NAT assays and secondary HIV-2 NAT standards. It will be used by a range of laboratories including kit manufacturers, blood fractionators, reference laboratories, diagnostics labs, EQA providers and OMCL’s.

Source/type of materials

We propose using the same HIV-2 subtype A strain as was used to manufacture the 1st HIV-2 IS. Live high titre stocks of tissue culture derived virus are already stored under vapour phase liquid nitrogen at NIBSC. Following the successful inactivation of the 1st HIV-2 IS we propose to heat inactivate stock material to formulate the 2nd standard. The viral stocks will be diluted in pooled human plasma prior to lyophilisation.


A panel of samples would be developed for evaluation in a collaborative study comprising: The current (1st) IS for HIV-2 NAT The proposed candidate 2nd HIV-2 material The frozen liquid bulk material Where possible clinical specimens of HIV-2 positive plasma (however this is expected to be very difficult to obtain) A collaborative study involving 15-20 international laboratories conducting a range of commercial and in-house assays would be invited to participate.

Issues raised by proposal

Possible sourcing of clinical material for commutability assessment

Action required Endorsement of the project

NIBSC Ref VIR-00048 ECBS Endorsement required

WHO/BS/2016.2298 Rev.1


NAT is the most widely used and most sensitive method for the detection of HIV-2 RNA in human serum and plasma.


A range of NAT-based methods are available for the detection and quantification of HIV-2 RNA, including both commercial and laboratory-developed assays.


The need to standardise NAT-based assays for HIV-2 is ongoing. NAT is routinely used to manage HIV infections and there remains a major risk of transfusion-transmitted infection due window-period donations.

Global importance

HIV-2 incidence is lower than HIV-1 and typically follows a difference course of infection, with lower viral loads and a higher percentage of longer term non progressors. Not all treatments established for HIV-1 are effective against HIV-2, therefor the accurate and timely diagnosis of HIV-2 infection is critical to ensure correct therapy can be given and to inform the individual of their infection status to prevent the risk of unknown transmissions


Whilst screening for HIV-2 RNA is not mandated in as many countries as HIV-1, due to the lower incidence and lower titre of an infected individual, accurate and sensitive assays are critical to ensure the safe blood supply in countries where HIV-2 is prevalent.


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