Low prevalence of Plasmodium falciparum parasites lacking pfhrp2/3 genes among asymptomatic and symptomatic school-age children in Kinshasa, Democratic Republic of Congo

Background: Loss of efficacy of diagnostic tests may lead to untreated or mistreated malaria cases, compromising case management and control. There is an increasing reliance on rapid diagnostic tests (RDTs) for malaria diagnosis, with the most widely used of these targeting the Plasmodium falciparum histidine-rich protein 2 (PfHRP2). There are numerous reports of the deletion of this gene in P. falciparum parasites in some populations, rendering them undetectable by PfHRP2 RDTs. The aim of this study was to identify P. falciparum parasites lacking the P. falciparum histidine rich protein 2 and 3 genes (pfhrp2/3) isolated from asymptomatic and symptomatic school-age children in Kinshasa, Democratic Republic of Congo.

Methods: The performance of PfHRP2-based RDTs in comparison to microscopy and PCR was assessed using blood samples collected and spotted on Whatman 903™ filter papers between October and November 2019 from school-age children aged 6-14 years. PCR was then used to identify parasite isolates lacking pfhrp2/3 genes.

Results: Among asymptomatic malaria carriers (N = 266), 49%, 65%, and 70% were microscopy, PfHRP2_RDT, and pfldh-qPCR positive, respectively. The sensitivity and specificity of RDTs compared to PCR were 80% and 70% while the sensitivity and specificity of RDTs compared to microscopy were 92% and 60%, respectively. Among symptomatic malaria carriers (N = 196), 62%, 67%, and 87% were microscopy, PfHRP2-based RDT, pfldh-qPCR and positive, respectively. The sensitivity and specificity of RDTs compared to PCR were 75% and 88%, whereas the sensitivity and specificity of RDTs compared to microscopy were 93% and 77%, respectively. Of 173 samples with sufficient DNA for PCR amplification of pfhrp2/3, deletions of pfhrp2 and pfhrp3 were identified in 2% and 1%, respectively. Three (4%) of samples harboured deletions of the pfhrp2 gene in asymptomatic parasite carriers and one (1%) isolate lacked the pfhrp3 gene among symptomatic parasite carriers in the RDT positive subgroup. No parasites lacking the pfhrp2/3 genes were found in the RDT negative subgroup.

Conclusion: Plasmodium falciparum histidine-rich protein 2/3 gene deletions are uncommon in the surveyed population, and do not result in diagnostic failure. The use of rigorous PCR methods to identify pfhrp2/3 gene deletions is encouraged in order to minimize the overestimation of their prevalence.