Routine parallel diagnosis of malaria using microscopy and the malaria rapid diagnostic test SD 05FK60: the experience of Médecins Sans Frontières in Myanmar

Microscopy is the reference standard for diagnosing malaria but is time consuming and requires considerable training and experience. It is ill suited as a diagnostic tool in settings where the case load is high and resources limited [1, 2]. Lateral-flow immunoassays (LFIs), often called rapid diagnostic tests (RDTs), are an attractive alternative as they are quick and easy to carry out [1‐7]. In practice however, malaria is often diagnosed solely on the basis of clinical symptoms, without using either method; this results in over-treatment of malaria and the risk of missing true underlying pathologies. To improve the accuracy of malaria diagnosis and avoid unnecessary treatment, the World Health Organization (WHO) recommends parasitological confirmation of malaria whenever possible, by RDT or by microscopy [1, 8‐11].

Among the newer generation of malaria RDTs are three-band tests that detect both the Plasmodium falciparum-specific antigen histidine-rich protein 2 (HRP2) and the pan-Plasmodium-specific lactate dehydrogenase (pLDH), expressed in the four most common malaria parasite species [P. falciparum, Plasmodium ovale, Plasmodium malariae and Plasmodium vivax[6, 12‐16].

In 2009, Médecins Sans Frontières (MSF) introduced routine use of HRP2/pan-pLDH RDTs into its programmes at sites where both P. falciparum and non-falciparum malaria are present (i e, Southeast Asia, the Americas and the Ethiopian highlands). In Myanmar, which has an enormous malaria burden, accounting for 7% of all malaria cases in Southeast Asia and India [7], P. vivax is second only to P. falciparum as the cause of malaria, so the test chosen was the SD Bioline 05FK60 Malaria Ag P.f/ Pan (Standard Diagnostics, Kyonggi, Republic of Korea), hereafter referred to as SD 05FK60.

The choice was made for the SD 05FK60 because it was one of the best performing assays in the WHO/TDR (Special Programme for Research and Training in Tropical Diseases)/FIND (Foundation for Innovative New Diagnostics)/CDC (Centers for Disease Control) evaluation in Round 1 and because continuous supply and importation to Myanmar could be guaranteed. However, since 2007, evaluations of the SD 05FK60 were conducted in returned travellers or as mentioned before by the WHO/TDR/FIND/CDC programme but not on a field site. In these evaluations, the SD 05FK60 showed good performance for detection of malaria caused by P. falciparum, with varying results for P. vivax detection [17‐22].

However, in spite of the generally good performance of malaria RDTs, their use on the ground can be problematic: medical staff frequently mistrust the results and problems with the products (e g, poor migration of blood and buffer, faint lines, incorrect placement of the lateral-flow strip in the cassette) are encountered regularly [23‐25]. This often results in low coverage of RDT implementation and poor adherence to RDT results (as the basis for treatment decisions) in clinical practice [26]. In order to monitor the performance of this newly introduced RDT in MSF’s programmes in Myanmar and to respond to clinicians’ concerns of poor RDT performance, it was decided to test patients in parallel with microscopy and RDT until the end of 2011 in our programmes in Rakhine State. In Myanmar, approximately 68% of the population of over 60 million total, is thought to be at risk for malaria with the high risk areas concentrated near international borders, including Rakhine state. The yearly burden in the entire country is to be over two million cases [16].

Commercially available HRP-2/pLDH tests have been evaluated previously in Myanmar [29, 30] but evaluations of the SD 05FK60 are available only from other areas and were conducted before 2007 [17‐22]. The study showed overall good performance for the SD 05FK60 RDT in the identification of malaria infections caused by P. falciparum and satisfactory results for non-falciparum infections. The poorer detection and identification of P. vivax/P. malariae than that of P. falciparum is consistent with other evaluations of the SD 05FK60 and pan-pLDH tests in general [17‐22].

However, it should be pointed out that in Round 1 of the WHO/TDR/FIND/CDC evaluation, the panel detection score (PDS) of the SD 05FK60 reported at parasitaemia levels of 200 parasites/μl was 96.2% for P. falciparum and 35.0% for P. vivax[18]. The same product was retested in Round 3 of this evaluation and showed a slightly decreased PDS of 92.9% for P. falciparum along with an improved detection score of 97.1% for P. vivax[19]. It is unclear which version of the product was received in the programme - the catalogue number always remained the same – and this inconsistency is a commonly encountered problem by MSF when an in vitro diagnostic product is changed.

The sensitivity of the SD 05FK60 improves with increasing parasitaemia. This is a well-known limitation of RDTs. Less expected were the findings that 25 P. falciparum infections and 12 P. vivax/P. malariae infections with +++ or ++++ parasitaemia were undetected by the SD 05FK60. The non-detection of P. falciparum infections at high parasitaemia levels could be explained by the prozone effect [31‐33] or by deletion of the histidine-rich repeat region in the HRP-2 gene [34]. It was not possible to conduct further molecular analysis of the Plasmodium cohort in the area to undermine this argument. However, this does not explain the false negative pan-pLDH band.

Translating these results into the impact on patients, 52 (10.2%) of a total 531 P. falciparum mono-infections would have been missed by using the RDT without microscopy. Although the specificity has been high for both P. falciparum and non-falciparum infections: 98.5% and 98.7% respectively, thirteen infections were misidentified as P. vivax/P. malariae malaria and as a result would have been treated with chloroquine, a drug to which P. falciparum is unlikely to be susceptible [35]. Considering the P. vivax/P. malariae cases, 121 out of 587 (20.6%) individuals would have not received any malaria treatment despite being infected. Only 31 individuals out of a total of 1,337 (2.3%) would have been unnecessarily treated with anti-malarials on the basis of RDT.

Although the results of the performance of the SD 05FK60 are satisfactory, MSF decided to opt for microscopy as the first choice for malaria diagnosis in its programmes in Rakhine State, Myanmar. This was a pragmatic decision based on the clinicians’ greater trust in microscopy, particularly in light of the cases with high parasitaemia that would have been missed if microscopy had not been used in addition to the RDT. MSF reserves RDTs for use in decentralized settings and where a high number of patients precludes microscopy. Therefore MSF decided to continue using microscopy in the two clinics in Rakhine State, Myanmar for the routine diagnosis of malaria, although RDTs have been found to be more cost-effective and more feasible in other settings [36‐38].

This study has several limitations. The data were collected as part of routine programmatic activities and were unlinked to patient records. Thus, it was impossible to collect clinical information, such as recent intake of anti-malarials or the presence of other factors, which may have influenced the results. Another limitations is that the + system has been used to grade parasitaemia instead of a parasite count. This less accurate estimation may have influenced the results on sensitivity especially in the stratified analysis of parasitaemia levels.

It is important to note that the described data were obtained in true field conditions, i e, conditions that were suboptimal in terms of RDT storage and handling and staff expertise, and non-standardized specimens may have been present. It is known that such factors influence RDT performance [39, 40], but it is difficult to optimize all these variables under difficult real-life conditions. However, this is also a strength of the study, as it represents test performance under field conditions.

MSF is able to train and maintain good malaria microscopy in its field settings. However, this may not be possible in a wider public health arena. In 2006 the WHO estimated that, if the recommendation to obtain parasitological confirmation of malaria before prescribing treatment were to be followed, screening would need to be scaled up from 122 million to 780 million tests each year in Southeast Asia alone, a 600% increase. Such scaling up would undoubtedly be difficult to achieve without the use of simple, reliable RDTs [7].