Quality assurance of malaria rapid diagnostic tests: An aid in malaria elimination

Diagnosis is the mainstay of effective management of any disease. Malaria is a tropical infectious disease with 241 million cases occurring in 20201. In this year, the World Health Organization-South East Asia Region (WHO-SEARO) accounted for about two per cent of the total malaria burden. Diagnosis not only plays a pivotal role in treating a disease but also in its overall management2. Hence, the importance of appropriate diagnosis is well recognized in malaria control programme. There is a potential to save 100,000 lives annually and to deter 400 million unnecessary treatments when the diagnosis for malaria remains accurate and follows practical algorithm3. Clinical symptoms of fever, chills and rigour are primarily recognized due to malaria4, leading to false diagnosis because non-specific symptoms mimic many other diseases including many viral infections. Clinical diagnosis alone does not suffice disease recognition, and laboratory detection plays a critically important role. The ‘gold standard’ method for diagnosing malaria is examining thick and thin blood smears under a microscope5. This method is widely used but has certain limitations, such as the need for an expert microscopist for species identification and its limited threshold concentration of detection of parasites per microliter of blood compared to that of other techniques6. The other commonly used methods are polymerase chain reaction (PCR) and rapid diagnostic test (RDT) kits. The PCR is much more sensitive compared to microscopy. Still, its high cost, the requirement of a laboratory setup having equipment and skilled human resources make its use unrealistic for resource-limited settings7. Rapid diagnostic test kits (RDTs) are immunochromatographic strips used extensively for malaria detection worldwide, which were developed in the mid-1990s8,9. These kits require one drop of blood for malaria detection based on the malaria-specific antigen (Ag). As the name suggests, RDTs can be quickly performed by individuals receiving a short training. Furthermore, it does not require skilled persons as it is a pre-requisite for microscopic diagnosis and does not require electricity or any equipment10.

Since 2008, the Foundation for Innovative New Diagnostics (FIND) in collaboration with the WHO has established an evaluation system for quality assurance (QA) of malaria RDTs, which help improving their sensitivity and specificity11. The accuracy of RDT remains dependent on many factors such as the quality control (QC) in manufacturing RDTs, their field performance, stability at high storage temperature, during transport, storage and the method of use12. Numerous factors determine the sensitivity of malaria RDTs, such as the rate with which blood reaches the nitrocellulose strip, how the antibody (Ab) adheres to the strip, interaction between Ab and Ag and the accuracy of the Ab-dye conjugate. Further, these are all subject to deterioration in poor storage and carriage conditions13. The National Centre for Vector Borne Disease Control (NCVBDC), formerly known as NVBDCP (National Vector Borne Disease Control Programme), New Delhi, introduced RDTs for the diagnosis of malaria during 2004-2005 as the load of negative and all positive slides for malaria was about five million and 1.5 million, respectively, for cross-checking its quality14. Initially, it was introduced in areas with high transmission where reaching out was difficult or where microscopic facilities were non-functional due to operational issues. With an existing method for QA of microscopy, in the year 2007, a standard operating procedure (SOP) manual was put in place by NCVBDC for QA and adequate monitoring of laboratory services performing malaria RDT testing at the peripheral level13. These RDTs are accessible, used by accredited social health activist (ASHA) workers at the village levels in areas where malaria prevalence is moderate to high and hence are considered point of care.

The Indian Council of Medical Research-National Institute of Malaria Research (ICMR-NIMR), New Delhi, and NCVBDC jointly initiated a structured programme for QA of malaria RDT in August 2009 to assess the quality of RDTs purchased and provided by the National Programme. The NCVBDC was identified as a nodal centre and ICMR-NIMR as a National Referral Laboratory for this programme.

The overall goal of a QA process for malaria RDT is to ensure that quality RDTs reach in the hands of end-users through monitoring of technical standards of them and decreasing the costs brought about by misdiagnosis15. Each stage of RDT workflow is examined to ensure that the best quality of malaria RDT passes the QC16. The key components of the QA of RDTs for malaria include preparation of QC samples, pre-dispatch QC (testing of malaria RDT procured by the National Programme or directly by manufacturers), post-dispatch QC (testing of RDT sent by the National Programme officers which are used in the field), External Quality Assurance Scheme (EQAS) and internal QC. Panels of known parasitaemia of both species, i.e. Plasmodium falciparum and Plasmodium vivax, and also negative control panels are prepared.

The ICMR-NIMR was certified for its quality practice by the WHO-FIND in the year 2016 for the first time due to successful completion of an external QA (EQA) assessment and fulfilment of all the requirements for laboratory quality of the WHO-FIND malaria rapid diagnostic test QA programme. Since then, the laboratory has undergone EQA assessments to maintain its quality standards.

Material & Methods

Panels for quality control (QC) were prepared at selected field sites having laboratories of ICMR-NIMR fulfilling laboratory infrastructure requirements. QC-samples were evaluated by diagnostic PCR and serological test, which included HIV, HCV and HbsAg test and Ag (pldh, pfhrp2, paldolase and pvldh) characterization by enzyme-linked immunosorbent assay (ELISA). The lot-testing laboratory followed NCVBDC SOPs from 2009 to 2013 for QA of RDTs13. Thereafter, from the Year 2014 Methods Manual for Laboratory QC Testing of Malaria RDT Ver. 7 June 2014 and Ver. 8 June 2016 and updated versions were used for testing the RDTs17.

Rapid diagnostic test (RDT) quality control (QC) sample preparation:

Microscopy assessment: The first step for preparing a QC panel was its assessment using microscopy. Parasitological assessment during panel preparation was conducted by two level 1 WHO-certified microscopists. The results of their assessment were blinded for both the microscopists.

Parasitized blood was diluted with O^+ve blood to maintain the haematocrit level and with AB^+ve fresh frozen plasma to attain uniform parasite density of 200/μl (low positive) and 2000/μl (high positive). On the same lines, negative control panels were also prepared. Panels were transported in liquid nitrogen containers/dry ice and were stored at −80°C deep freezer. Table I shows the details of the total panel prepared at ICMR-NIMR, New Delhi, and at other field units over the years.

Validation of rapid diagnostic test (RDT) panels: Dried blood spots on Whatman 3 mm chromatography filter paper were prepared from patient’s blood, and PCR for all five species of malaria parasite infecting humans was performed for confirmation of species for mono-infection. ELISA for HBsAg, HCV and HIV antibody were performed. Characterization of the prepared QC panel was performed to detect the Ag HRP2, LDH and aldolase levels expressed in a blood sample. External characterization of Ag was performed at Hospital for Tropical Diseases, London, till 2016, and subsequently in-house characterization for HRP2 and PLDH Ag levels were being carried out for standardization.

PCR proficiency: The QA RDT laboratory at ICMR-NIMR is enrolled under the United Kingdom National External Quality Assurance Services for PCR proficiency. The assessment was done biannually under the WHO Malaria Nucleic Acid Amplification Test (NAAT) EQA Scheme. A set of ten samples with masked identities was received from the WHO malaria NAAT scheme, and after performing the analysis, the results were uploaded on the website for their assessment. This laboratory has completed eight rounds of distributions so far with twice 100 per cent scores.

Pre-dispatch lot testing: Various lots of different brands of malaria RDTs were received for lot testing through government central procurement agencies such as the United Nations Office for Project Services, Rail India Technical and Economic Service, Central Medical Services Society, regulatory body such as Drugs Controller General of India and directly from manufacturers. The requests received for testing RDTs were of different targeted Ags and their combinations available in India like Pf/Pv/Pan, Pf/Pv, Pan and Pf. The turn around time for reporting of lot test was five working days.

RDT collection: Primary health centres (PHCs)/sub-centres/ASHA workers were trained to send representative RDT samples for their quality testing. The malaria officers from the National Programme collected RDT samples from various PHCs and districts and sent those to the referral laboratory every quarter. From each district, one PHC was selected every three months. One RDT was picked up from the PHC, two from two sub-centres and four RDTs from ASHAs. In the next quarter, another PHC was selected and the procedure was repeated. RDTs were collected from different states of India (Fig. 1; map prepared using ESRI India’s ArcGIS ArcMap 10.5 software purchased by ICMR-NIMR).

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Fig. 1

A spot map showing States of India depicting the spread of centres from where RDTs were collected for quality assurance. Source: ESRI India’s ArcGIS ArcMap 10.5 software. RDT, rapid diagnostic test.

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Testing of RDTs using panels: It is important to assess the quality of the RDTs with known low and high positive samples before they are supplied to the testing laboratory to confirm that they are free from any manufacturing defects and have desired sensitivity and specificity. This was achieved by lot testing of the kits using the characterized panel having a parasite density of 200 p/µl with Ag concentration within the range as per the WHO guideline. From each RDT lot, 58 RDTs were drawn and tested using the WHO protocol18. Four sets of positive QC panels for P. vivax and P. falciparum and ten negative panels were used to test RDTs. Thus, a total of 58 RDTs (24 Pf panels, 24 Pv panels and 10 negative panels) were tested. For long-term RDT testing, i.e. six months before expiry, total 10 RDTs (4 Pf panels, 4 Pv panels and 2 negative panels) were tested from the year 2015 till date.

Lot testing journey of the WHO-recognized laboratory from India: Since its inception in the year 2014, 90 QC panels have been prepared, including 21 panels in 2014, 28 in 2015 and 25 in 2016, and 16 in 2018 as per the protocol following methods manual for product testing of malaria rapid diagnostic tests version 8 June 2016. The details are presented in Table I. Before testing the RDTs, panels were validated using two reference kits. The RDTs received from the field were tested for their quality using the negative panel and positive panels with 200 p/µl following the WHO protocol.

Results

Three hundred twenty three pre-dispatch RDT lots were tested according to the WHO-FIND protocol from 2014 to March 2021; 24 lots failed, and 299 passed. The long-term testing of these RDTs showed that nine failed among the 179 lots tested so far. Figure 2 shows the trend of lot testing received from different manufacturers and through national procurements from 2014 onwards. The lots were tested following the NCVBDC protocol from 2011 until 2015. From the year 2016, the WHO protocol was followed to test the lots. During 2017, due to some logistic issues, lesser number of lots were received for lot testing. The increase in the number of lots that failed in recent years was certainly a matter of concern highlighting the importance of lot testing.

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Fig. 2

Year-wise details of number of lots-tested and -failed till March 2021.

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Long-term testing of lots refers to testing the pre-lot test which are kept in the B.O.D (Bio-Oxygen Demand) chamber at 37°C and 60 per cent humidity for approximately 18 months, which is six months before its expiry. These testing provide over-the-time scale quality assessment of RDT lots at ambient temperature. The long-term testing of these RDTs showed that amongst the 179 lots tested so far, nine failed (Fig. 3), highlighting their significance. It is envisioned that these QA lots will be of high use in malaria elimination.

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Fig. 3

Year-wise details of number of lots-tested and -failed in long term testing till March 2021.

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The District Malaria Officers, from malaria-endemic states covered under the World Bank and Global Fund to Fight HIV/AIDS, Tuberculosis and Malaria projects, were oriented to the programme. Training programmes were conducted for District Programme Officers of 12 States: Assam, Meghalaya, Manipur, Mizoram, Nagaland, Arunachal Pradesh, Orissa, Jharkhand, Chhattisgarh, Karnataka, Madhya Pradesh and Gujarat during 2009. Since 2009, staff from the National Programme were picking up RDTs randomly from village levels and sending them for QA at ICMR-NIMR, New Delhi. At ICMR-NIMR, these RDTs were tested using the QC panels of known parasitaemia. As a part of post-dispatch QA, 7741 RDTs were tested in a decade’s time since the inception of the programme in 2009 till March 2019. These RDTs were received from 256 districts of 19 states. The panel detection score of these RDTs was 97.71 per cent. There were four invalid results but no false positives. The state-wise details of RDTs tested so far are detailed in Table II.

To improve compliance with post-dispatch lot testing, the Directorate of NCVBDC had advised the State Programme officers to actively participate in the programme. ICMR-NIMR also started to actively collect RDTs from different areas. The data thus generated underlined that the malaria RDTs being used in the programme were satisfactory.

Discussion

The National Framework of Malaria Elimination in India was launched in 2016 to achieve zero native cases in the country by 203019. Diagnosis of malaria has a significant role to play in its treatment, surveillance and elimination. However, emphasis should be given to the quality of RDT. Quality-assured RDTs have a major role in the context of elimination, especially in population with low parasitaemia20. A low-quality RDT may miss out malaria, thus leading to a pool of undiagnosed parasites transmitting in the same geographical area. Any lapse in detecting and interrupting such transmission will ultimately lead to an increase in the total number of malaria cases.

Establishing a lot-testing laboratory in the SEARO region of the WHO was a long journey with many key learnings as outcomes. There were many lessons learnt at each step, namely fulfilling the laboratory checklist requirement, maintaining daily records, training of staff for lot testing, biosafety training and audit systems, including internal and external audits. Keeping records of vaccination of staff (during COVID-19 pandemic), maintaining logbooks, drafting of SOPs for each laboratory procedure were other key requirements. Documentation of corrective actions and annual maintenance of equipment were critical. The other learning came from QC panel preparation like selecting sites with positive malaria cases, fulfilment of the infrastructure requirement and transportation of samples. Moreover, challenges pertaining to coordination issues with different governing bodies were encountered. The lot-testing facility should continue working for the country and support other neighbouring SEARO countries as well where no such facility presently exists.

Overall, the quality testing of malaria RDTs is critical, as a low-quality product could lead to a misdiagnosis of malaria. Quality-assured RDTs have a major role, especially in population where low parasitaemia of parasite persists. This report may help programmes and key decision-makers understand the expected usefulness of high-quality RDTs for malaria diagnosis in the context of malaria elimination. India’s lot-testing programme could serve as a learning reference for other countries that lack the resources to implement a similar programme.