WHO external quality assurance scheme for malaria nucleic acid amplification testing (NAAT EQA)

The purpose of the WHO malaria NAAT EQA scheme is to offer an independent and periodic means for clinical, reference and research laboratories to verify the quality of their nucleic acid amplification (NAA)-based malaria diagnostic methods and to monitor performance over time. This is achieved through the provision of well-characterized and quality-controlled panels consisting of a blinded mix of Plasmodium-positive and -negative samples. Participants are then issued an EQA report upon submission of their analysis results. Participation in the scheme is voluntary, and results are confidential.

The WHO malaria NAAT EQA scheme is coordinated by the WHO's Global Malaria Programme and distributed by the United Kingdom National External Quality Assessment Service (UK NEQAS), with a priority given to not-for-profit laboratories based in developing countries. Participation so far is free of charge.

The scheme will enable laboratories to obtain an independent assessment of their NAA-based diagnostic methods in order to determine if they meet the minimum level of quality required. If any laboratory does not meet this requirement, some level of remote technical support will be offered. The laboratory will have the opportunity work on identifying and addressing any source of error in order to improve the quality and reliability of its methods.

Any laboratory using NAA-based methods such as nested PCR, RT-PCR, LAMP or NASBA to detect malaria parasites is eligible for enrolment in the WHO NAAT EQA scheme. However, given that the scheme is currently run with limited funding, priority is given on a first come, first serve basis to not-for-profit laboratories based in developing countries, with the aim to generate a balance of laboratories according to testing indication, i.e. research, surveillance/surveys and geography. Laboratories wishing to participate should send a message to MalNAATEQA@who.int. In addition, calls for interest in participation are sent out to potentially interested laboratories, and enrolment is complete once favourable responses are received and letters of agreement between WHO and the participating laboratories are signed.

Besides offering the free WHO NAAT EQA scheme coordinated by WHO, UK NEQAS also coordinates its own malaria molecular scheme that is subject to fees for participation ( http://www.ukneqasmicro.org.uk/parasitology/index.php/molecular-detection-of-malaria). Laboratories should participate in either the WHO scheme or the UK NEQAS scheme, but not both at the same time.

The WHO NAAT EQA scheme distributes EQA panels twice a year, usually once at the end of January and once at the end of July. Exact dates depend on the practical organization of the scheme, i.e. the readiness of panels and capacity at UK NEQAS. Dates are confirmed by e-mail communication when calls for interest in participation are sent out, usually about 4–8 weeks ahead of each round of panel distributions.

The WHO malaria NAAT EQA scheme is coordinated by WHO, in collaboration with Public Health England (PHE) through UK NEQAS, with support from the Foundation for Innovative New Diagnostics (FIND). WHO is responsible for the overall coordination and promotion of the scheme, while UK NEQAS holds the central repository of EQA materials and manages the scheme operations. This includes the preparation, characterization, storage and shipping of EQA panels; the issuance of EQA reports to the participating laboratories; and the handling of any logistical queries. The EQA panels undergo independent testing by referee laboratories before being distributed to the laboratories participating in the scheme.

Currently, FIND provides financial support to the overall scheme and remote technical support to the participating laboratories, if required. WHO also organizes regular meetings (at least yearly) of an Advisory Group to review the scheme procedures, provide technical expertise for potential modifications to procedures and principles, review overall results of the scheme, including complaints, and discuss actions required to address major issues, if any.

Interest in participation can be communicated by e-mail at any time to the WHO/GMP contact at MalNAATEQA@who.int. Calls for interest in participation are also sent out by e-mail to potentially interested laboratories about 4–8 weeks before each round of panel distributions. A Letter of Agreement (LOA) and laboratory profile template are issued to the interested institution or organization.

The laboratory profile template requires participants to provide contact and shipping information (including special requirements such as permits for importation of biological samples), and to describe the laboratory’s capacity for identifying Plasmodium spp. It, along with the LOA (between the participating laboratory and WHO), needs to be completed and sent back to MalNAATEQA@who.int. Once the LOA has been signed by both parties and the laboratory profile is complete, the laboratory is registered in the scheme for an unlimited period of time. The laboratory will receive login details to connect to the UK NEQAS webportal for the WHO NAAT EQA scheme, and will be contacted ahead of each round of panel distributions to organize shipments.

The laboratory profile is a template that contains a series of questions to be completed before a laboratory can enrol in the WHO NAAT EQA scheme. Its purpose is to clarify which types of malaria diagnosis will be done on the EQA panels at the laboratory, i.e. which nucleic acid extraction and amplification methods will be used, and most importantly, the laboratory’s capacity in terms of diagnostic outputs, e.g. detection of Plasmodium spp. only or additional identification of species, including P. knowlesi or not.

The scheme organizers need this critical information in order to accurately calculate success scores when laboratories submit their EQA testing results. Correct completion of the laboratory profile is mandatory for enrolment in the WHO NAAT EQA scheme.

Participation in the scheme is currently free of charge; the laboratory simply has to cover any costs associated with the reception of the panels (e.g. customs charges, travel to nearest airport) and the testing itself.

However, to ensure the long-term sustainability of the scheme, the implementation of a participation fee is planned for the near future to cover at least part of the running costs of the scheme. The fee will be determined based on a dedicated cost estimation model.

Currently, the panel includes 10 lyophilized blood (LB) and 10 dried blood spot (DBS) samples. Malaria-containing samples have varying parasite densities, ranging from 50 to 2 000 000 parasites per microliter of blood (p/mL). Parasite-positive samples can be from any of the five species known to infect humans, i.e. P. falciparum, P. vivax, P. malariae, P. ovale or P. knowlesi, depending on availability. The lyophilized and DBS samples do not share the same composition, i.e. different species and/or parasite densities can be included.

Briefly, parasites are obtained either through in vitro culture of laboratory strains – in the case of P. falciparum and P. knowlesi – or by using leftover blood from clinical samples. Parasite-negative blood for negative control samples and for the dilution of parasites is obtained from accredited blood banks and pre-screened by PCR to confirm the absence of Plasmodium parasites.

After determining the densities of the cultured or clinical parasite samples by expert microscopy, they are diluted with parasite-negative blood to parasite densities ranging from 50 to 2 000 000 p/mL. Samples are mixed thoroughly at each dilution step. To prepare LB samples, dilutions are then aliquoted in 500 µL volumes and lyophilized. To prepare DBS samples, 50 µL volumes are deposited on Protein Saver 903 DBS cards and air-dried for 3–4 hours. All samples are stored at -80°C for long-term storage, but can be shipped and transported at ambient temperature.

A more detailed description of the preparation procedures can be found in the Operational Manual of the WHO NAAT EQA scheme, version 1.0.

Each laboratory should test the panels using the extraction and amplification methods that it routinely uses for testing clinical or research study samples. Laboratories are permitted to submit only one result per sample. Laboratories are requested to provide information on the methods used, such as the test’s name or kit details, along with their final result in the online results submission form. Repeat testing should only be done in the case of indeterminate results or suspicion of methodological errors, just as would be done routinely for any clinical or research study sample.

An incident review form is available online from the UK NEQAS website. Laboratories receiving substandard results should complete this form and send it to the scheme manager at parasit@ukneqas.org.uk, with copy to the WHO contact at MalNAATEQA@who.int. Laboratories can also request a repeat panel to be shipped for re-testing and further investigation, at the same e-mail addresses. Additional remote technical support is currently provided by FIND (until 30 June 2018) and by members of the WHO Advisory Group and/or representatives of the referee laboratories if their time and workload allow. It is the participating laboratory’s right to decide if its EQA results can be shared with these third-party sources of technical support, as well as other information such as instruments or reagents used and results from any repeat or troubleshooting experiments generated through this investigational process.

It is the participating laboratory’s right to decide if its EQA results can be shared with these third-party sources of technical support, as well as other information such as instruments or reagents used and results from any repeat or troubleshooting experiments generated through this investigational process.

From time to time, webinars will be organized to give registered laboratories the opportunity to ask questions from a panel of experts in the field of molecular-based malaria diagnostics.

Ultimately, it is the laboratory’s responsibility to investigate and address any problems. Sponsors of the laboratory should consider allocating appropriate resources to improve the quality management system (QMS) of the laboratory, as required.

Yes. All testing results are kept strictly confidential; only the laboratory itself can access them through the UK NEQAS webportal using its specific login and password. Otherwise, only staff from the scheme’s organizing team can access individual results for the purposes of data analysis and reporting. Only anonymized and aggregated results will be used for publication or communication to the scheme’s Advisory Group.

If substandard results require any remedial action or technical support, the scheme organizers will first seek the laboratory’s interest and approval to involve any third parties such as technical staff from FIND or members of the Advisory Group. If the laboratory agrees, test results will then be communicated to facilitate troubleshooting and remediation.

Each laboratory is free to communicate part or all of its testing results to any third party without the need to inform WHO or UK NEQAS. Communication of the scheme results is encouraged in order to confirm the laboratory’s performance level for potential collaborators, donors, regulatory authorities and others. Communication of substandard results is also encouraged in order to seek assistance or funding for training workshops, and to work on improving the laboratory’s performance.

Participants can send an e-mail to the UK NEQAS scheme manager at parasit@ukneqas.org.uk, with a description of the problem, screenshots or other useful information.

As described under the question ‘what if I get substandard results’, the laboratory can request a repeat panel to be shipped for re-testing and further investigation of the reasons behind the bad results. Repeat panels can be requested by contacting the scheme manager at parasit@ukneqas.org.uk, with copy to the WHO contact at MalNAATEQA@who.int. Remote technical support can also be offered by FIND (until 30 June 2018) and/or staff from the referee laboratories of the scheme and the WHO Advisory Group, depending on availability.

It is nevertheless the laboratory’s responsibility to work on improving its performance and solving any quality issues. Therefore, other options should also be considered, including participating in training workshops, working with collaborating laboratories, investing in an improved QMS, and so on.

If a specimen vial from the EQA panel set gets damaged, dropped or lost for some reason, the scheme can send repeat specimens. For this, the laboratory can simply contact the scheme manager at parasit@ukneqas.org.uk, with copy to the WHO contact at MalNAATEQA@who.int, explaining the reason for the request and specifying how many vials of which specimen are required. As the scheme is run with limited funding, this offer of repeat specimens should be used sparingly, only in the case of real need.