Positive/external control of rapid diagnostic tests: A step forward in malaria screening.

Sir,

Malaria is a mosquito-borne disease caused by a parasite and is a major global health issue. The high morbidity and mortality are attributed to the development of resistance of the parasite to antimalarial drugs and to currently available insecticides. Various techniques are available for malaria diagnosis. Methods used routinely for the diagnosis of malaria are microscopy and immunochromatographic rapid diagnostic tests (RDTs). Microscopy is regarded as the “gold standard” for malaria diagnosis (WHO, 1999).[1] However, the lack of skilled technologists in medical facilities in affected areas often leads to a poor interpretation of result so RDTs are invariably used to diagnose malaria. Procuring good-quality RDTs, however, does not necessarily guarantee good field performance. Improper storage, transport, and handling of malaria RDTs may affect the performance of RDTs leading to false-negative results. ISO 15189 standards also require any laboratory to have a procedure for the reception, storage, acceptance testing of reagents, and consumables before putting them to use. Here, we present a simple and stable methodology of preparing and preserving malaria parasite-infected blood as samples as external quality control for the validation of every new lot RDTs.

We selected two different RDT kits of the single manufacturer (Sure Test). Details are given in Table 1.

Table 1

Rapid diagnostic test type and their antigen specificity

	RDT type	Parasite antigen specificity	Number of bands (including controls)
RDT 1	PF/PV	HRP2/pLDH	3
RDT 2	Pan	LDH	2

HRP2 = Histidine-rich protein 2, pLDH = Parasite lactate dehydrogenase, LDH = Lactate dehydrogenase, RDT = Rapid diagnostic test, PF/PV = Plasmodium falciparum/ plasmodium vivax

Dried Plasmodium falciparum-infected blood preparation – Parasite used in the study was Plasmodium falciparum. Sample was collected from the patient who was found to be positive for malarial parasite Plasmodium falciparum. At the time of diagnosis, 90% or more parasites were at gametocyte stage, and the parasitic load was 200 parasites/μl. Figure 1 illustrates the preparation of dried Plasmodium falciparum-infected blood and how it is reconstituted.

Figure 1

Preparation of dried Plasmodium falciparum-infected blood

Of all the aliquots, one representative sample was taken and was rehydrated with PBS-Tween 20 solution and tested on both RDTs for 10 consecutive days. The results were positive on all occasions. For lot-to-lot verification, we are using this as a positive control sample.

Figures 2 and 3 show new lots of RDTs that has been verified. After a period of 14 months of storage at 4°C–6°C, no loss in reactivity compared to baseline was observed suggesting the stability of parasite in dried form at 4°C–6°C. However, one of the limitations of our study was that we have prepared only Plasmodium falciparum dried sample and have not included Plasmodium vivax which should have been done. Another limitation is that we have included only two RDTs and that too of the same manufacturing company.

Figure 2

Reconstituted positive control on rapid diagnostic test 2

Figure 3

Reconstituted positive control on rapid diagnostic test 1

Although positive control wells (PCWs) have been proposed as point-of-care quality-control tools, as the third component of a tiered quality assurance program;[234] how readily they are available at an affordable price is the issue. Use of dried Plasmodium falciparum-infected blood as positive control of RDTs appeared to be appropriate, affordable, and convenient method of quality check of RDTs.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.