1. Introduction
Malaria remains a global health problem despite progress over the last decade. In 2015, there were an estimated 212 million new malaria cases including 429 000 deaths.1
Still in 2015, 90% of malaria cases and 92% of malaria deaths occurred in Africa. Three quarters of these cases and deaths were estimated to have occurred in fewer than 15 countries, with Nigeria and Democratic Republic of the Congo accounting for more than a third.1
In DRCongo, Malaria is the main cause of morbidity and mortality, accounting for more than 40% of all outpatient visits and for 19% of deaths among under five years children.2
Furthermore, it has been estimated that the DRC accounts for 11% of all malaria cases in sub-Saharan Africa, given that nearly the entire population (97%) lives in high transmission zones (equatorial and tropical facies) where the most common vector encountered is Anopheles gambiae (an excellent malaria plasmodium vector) and the Plasmodium falciparum is the most frequent species responsible for the majority of severe forms. 2,3
Effective Malaria control and elimination relies on appropriate malaria case management based on early and accurate diagnosis. Accurate diagnosis facilitates appropriate and prompt treatment, reduces drug misuse, minimizes the risk of developing drug resistance and thus contribute to reducing malaria morbidity and mortality.
The WHO recommends the Malaria diagnosis to rely on the microscopic examination as gold standard. However, in rural and semi urban settings where lack of equipment, reagents, trained and skilled personnel and electricity prevents this essential diagnosis, the use of Rapid Diagnostic Tests (RDTs) offers a great potential for quick and accurate diagnostic reducing the economic burden of the disease. 4,5
In the DRCongo and throughout Malaria endemic areas, RDTs have become a primary and critical tool for Malaria diagnosis. 6 In fact, they accounted for 74% of diagnostic testing among suspected cases in Africa in 2015. 1
Malaria RDT kits are designed to detect either Plasmodium falciparum specifically or discriminately detect both P. falciparum in addition to another human malaria parasite or indiscriminately detect all human malaria parasites. The main antigens that malaria RDT kits detect are PfHRP2, parasite lactate dehydrogenase (pLDH), and parasite aldolase (pAldo). 4
PfHRP-2 is a P. falciparum specific antigen with the advantage of being highly abundant and heat stable. HRP2-based RDTs are known to generate false-positive results in the setting of persistent circulating HRP2 antigen after antimalarial treatment and false-negative results in individuals with low levels of parasitemia beneath the assay’s threshold for detection, typically around 200 parasites/µL. 7
From 2010 onwards, false-negative results have been reported in individuals infected with P. falciparum parasites harboring a deletion of the pfhrp2 gene. Several of these reports also identified co-existing deletions of the P. falciparum histidine-rich protein 3 (pfhrp3) gene, which produces an antigen that exhibits some cross-reactivity with HRP2. 7,8,9,10,11,12,13
While the WHO recommends not to initiate antimalarial treatment without biological evidence, evolutionary selection of hrp2 deleted parasites could occur since only positive tested patients will be taken care of. The non-targeted patients harboring hrp2 deleted parasites will facilitate spread of hrp-2 deleted strains, jeopardizing progress towards control and/or elimination in low setting countries.
To date, only one study has investigated pfhrp2-deleted mutant parasites in DRC. The study was jointly conducted by the University of North Carolina, the Kinshasa School of Public Health and the National Malaria Control Program. It has reported a country-wide prevalence of 6.4% among under five children, with highest prevalence reported in Kinshasa and Kivu. The survey also provided spatial distribution and population genetics of these deletions.7
However, this nationwide study couldn’t explore clinical differences between pfhrp2-deleted and wild type Pf malaria due to limited clinical data and population study (majority being asymptomatic), nor didn’t it allow to draw conclusion about the relative virulence of pfhrp2-deleted parasites.
Our research is a part of the response to the aforementioned limitations and a contribution to ongoing studies aiming at better characterizing this phenomenon and developing novel diagnostic methods and tools, affordable in low income countries.
2. Objectives
The purpose of this research is to determine epidemiological, clinical and genetic features of hrp2 deleted P. falciparum infection among under five children in a highly prevalent area.
The study aims to provide evidence to leverage decision making in terms of tailored RDT procurement and provision and the need to establish surveillance system of pfhrp2-deleted mutants. Also, useful data will be generated and used for the development of novel reliable and affordable diagnostic methods and tools in low income countries.
3. Methods
We will conduct a cross sectional study targeting 3 Malaria Sentinel sites in Kinshasa (Centre Mamakoko, Centre des soeurs de Kingasani, Hôpital Pédiatrique de Kalembelembe). Samples will be collected from at least 98 under five children (prevalence of hrp2 deletion being 6.4%7, a desired precision set at 5% and confidence level of 95%) both in the community for asymptomatic and in the 3 targeted health facilities for symptomatic children. Heel or finger-prick blood from each child will be collected to perform light microscopy and then applied for hrp2-RDT and Dried Blood Spots using filter paper. Membrane of spent cassettes as well as DBS will be processed for genomic DNA extraction. PCR will be used to identify P. falciparum infection as well as to detect hrp2/3 gene deletions and genomic variations for RDT-/PCR+ samples as previously described by Cheng Qin et al.14
Statistical Data analysis Method to be determined.
Methods to learn in Japan: PCR techniques, DNA extraction and amplification, parasite genome sequencing and characterization, genomic epidemiology, novel diagnostic tools development and other molecular parasitology methods
4. Expected outcomes and conclusion:
Kinshasa is one of the area where the prevalence of hrp2 deletion has been the highest in a previous survey conducted by the University of North Carolina using 2013-2014 DHS nationwide data.7
Thanks to the research we will conduct, updated and specific prevalence will be known as well as clinical patterns and genetic diversity associated with hrp2/3 deletion in Kinshasa. Evidence generated will be used to develop tailored community surveillance strategies and novel diagnostic tools for effective disease control and progress towards disease elimination. Results of this study will also be used to make advocacy for scaling up such studies so as to have specific data for each province and implement tailored interventions taking into account genetic and geographical variations within provinces of the DR Congo.

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