Uncovering hidden pfhrp2/3 deletion Plasmodium falciparum.

Deaths and transmission from malaria have declined significantly in the past decade, although the progress has somewhat stalled in recent years [1]. Accurate and timely diagnosis is essential to providing adequate treatment to malaria cases. Plasmodium falciparum histidine-rich protein 2 (PfHRP2) based rapid diagnostic test (RDT) is widely used especially in Africa, where P. falciparum accounts for 98% of estimated malaria cases [1]. Despite its importance, reports of parasites lacking pfhrp2 gene emerging across the globe are threatening the importance of this malaria diagnostic tool. Peru is the first country that reported a high prevalence of pfhrp2 and pfhrp3 deleted strains in the field and warned that PfHRP2-based RDT should be replaced with a non-PfHRP2-based RDT or quality microscopic diagnosis [2]. The threat of false-negative PfHRP2-based RDT alarmed the World Health Organization (WHO) and recommendation for accurate reporting was updated in 2014 [3], followed by multiple reports by WHO Global malaria program [4]. In the African continent, where the burden of malaria mortality and morbidity are the highest, a recent review showed that the level of pfhrp2/3 gene deletion varied among 12 countries where pfhrp2/3 deletions were tested in Africa – the highest being Eritrea (62%), and the lowest being Angola (0.4%), with a caveat that sample collection and assay varied across studies [5]. Standardized surveillance protocol is now developed by WHO to study pfhrp2/3 deletion in malaria-endemic countries [6]. Based on the protocol, suspected PfHRP2-RDT false negative individuals, who are febrile, should be screened by microscopy or a pf-pLDH RDT [6]. Being negative with PfHRP2-based RDT, but positive with other screening methods indicate potential deletion, and if suspected deletion exceeds 5%, WHO urges to switch to non-PfHRP2-based RDTs or microscopy [6]. Although molecular confirmation of pfhrp2/3 deletion is currently not required, detecting the emergence of pfhrp2/3 deletion parasite and monitoring it can assist malaria-endemic countries to make a plan accordingly. When screening for deletion, infection with multiple clones of parasites can cause difficulty to analyze the results, since the presence of pfhrp2/3 deletion parasite can be masked by the presence of pfhrp2/3 un-deleted parasites.

In an article in EBioMedicine, Grignard and colleagues [7] propose a novel multiplex qPCR assay that addresses the issue of polyclonal infection. The authors went through meticulous optimization and validation process. In the final multiplex qPCR assay, host DNA (human beta tubulin (HumTuBB)), a single-copy parasite house-keeping gene (P. falciparum lactate dehydrogenase (pfldh)), as well as pfhrp2 and pfhrp3 are simultaneously detected using specific primers and probes. They used pfldh to confirm the presence as well as quantification of parasite DNA, and used HumTuBB to normalize relative parasite densities in each sample. Using the developed and validated assay, the authors tested various field samples including; dried blood spots (DBS) from Eritrea (n = 50), whole blood collected from symptomatic individuals in Kenya (n = 150) and Tanzania (n = 149), and confirmed P. falciparum in whole blood samples collected from UK travelers (n = 113) at Public Health England Malaria Reference Library. Of those tested, 31 (62%), 0 (0%), 1 (0.7%), and 1 (3.5%) were pfhrp2/3 double deletions among samples from Eritrea, Kenya, Tanzania and UK travelers, respectively. Furthermore, when polyclonal infections were taken into account, the authors reported additional pfhrp2, pfhrp3 or pfhrp2/3 deletions among samples from Kenya, and UK travelers. The current UK recommendation for malaria diagnosis is a thick and thin smear, but when a microscopist is inexperienced or unavailable, RDT plays a back-up diagnostic role. Given the caveats reported in this paper, the authors recommend that PfHRP2-based RDT results should be interpreted cautiously if that is used when microscopy analysis is not available.

The use of pfldh quantification as normalizer without the human gene was previously reported [8]. Grignard and colleagues report that they have tried both methods (delta Ct vs delta delta Ct) and are comparable while adding a complex layer of internal control measure [7]. As it is commonly known that field samples can be collected and prepared in various ways. DBS is one of the sources of DNA the authors used in this manuscript and this could potentially contribute to a sample quality bias – partially filled or oversaturated. Incorporating an additional layer of normalization to the analysis is one of the ways to ensure the results are comparable across time and location. Detecting pfhrp2/3 deletion parasites masked by polyclonal infection may not change the result of the diagnosis, however, detecting the emergence and monitoring its spread over the course of time will enable countries and communities affected by malaria plan for the change in malaria diagnostic tools before the surge of parasite strains that are undetectable by current PfHRP2-based RDTs.

Declaration of Competing Interest

Dr. Kobayashi has nothing to disclose.