The study has been conducted in collaboration with the National Malaria Control Program (NMCP). Ten sites were selected among the 26 provinces of DRC. The study included the 3 largest cities of the country (Kinshasa, Kisangani and Lubumbashi) as well as 7 other sites that were selected based on their epidemiological facies which refers to the malaria transmission intensities . Thus, the following sites were selected: Bolenge, Karawa and Vanga for the equatorial facies characterized by high and permanent transmission; Kalima, Kamina and Fungurume for the tropical facies marked by seasonal variations with resurgences during rainy season (8 – 9 months) and Katana for the mountainous facies with low transmission. Figure 1 presents the cartography of study sites according to the selection criteria. All selected sites are NMCP’s sentinel sites for malaria surveillance, except Lubumbashi. These sites are organized within one selected Health Zone entities, per province and are part of a national network for malaria surveillance. The objectives of this network are to provide accurate and real-time data on morbidity-mortality trends, monitor the epidemiological progression for a rapid response, monitor and evaluate control interventions and progress towards the elimination of malaria. A pilot study was conducted first in Kinshasa from January to March 2017. Afterwards, the study was started in the other sites from September to December 2017.
Figure 1. Cartography of study sites across DRC.
Patients of all ages who attended one of the selected medical centers for fever and who had a positive rapid diagnostic test (RDT) for malaria or a positive thick blood smear were enrolled after informed consent.
Blood sample collection
Screening tests were performed on blood samples taken by finger prick. RDT’s were used depending of the availability of the test on site. Two RDT’s were available which detected both P. falciparum HRP2 protein: the SD Bioline malaria Ag Pf (Standard Diagnostics) assay was used to enroll patients attending all collection sites except in Kinshasa where the CareStart Malaria Pf (Access Bio) assay was used in a majority of patients. In case of lack of RDT’s, a positive thick blood smear test for malaria was performed for enrollment.
After enrollment, a blood sample was taken by finger prick and three spots were deposited on Whatman Grade GB003 filter paper (Whatma, GE Healthcare). Dried blood spots (DBS) were placed in an individual ziploc plastic bag containing silicagel desiccant and were then stored at room temperature before their transfer to the Clinical Microbiology Laboratory of the University of Liège for molecular analysis.
DNA was extracted from blood spots by using the QIAamp DNA Mini Kit (Qiagen, Germany) following the recommended protocol for DBS. The extracted DNA was stored at – 20 °C before PCR testing.
P. falciparum real-time PCR
A real-time PCR for the detection of P. falciparum was performed according to a modified previously described procedure . Briefly, the mix contained 200 nM of P. falciparum primers and probe, a volume of 2.5 µl of Double-Dye Probe/Primer for Internal Positive Control (IPC), 2.5 µl of DNA virus culture (DIA-EIC/DNA(Cy5) for IPC, 12.5 µl of 2X Taqman Universal PCR Master Mix (Applied Biosystems) and of the rest of water in a total volume of 25 µl including 5 µl of DNA template. Assays were run on a ABI 7500 Fast real-time thermocycler (Applied Biosystems).
The fragment of interest (containing codons 72 – 76) on the pfcrt gene was amplified following a previously described procedure . The PCR was run on a conventional Dyad Peltier Thermal Cycler (Bio-Rad Laboratories, CA, US). The PCR products were visualized after electrophoresis on 2% agarose gel stained with ethidium bromide.
After purification using magnetic beads AMPure XP (Beckman Coulter, CA, US), the PCR products were added to a mix of Big Dye Terminator V3.1 for the sequencing reaction. The resulting 152-bp nucleotide sequences were analyzed on an ABI 3730 DNA Analyzer automated sequencer (Applied Biosystems) using the Sanger method at the GIGA Interdisciplinary center of biomedical research of Liege University. These 152-bp nucleotide fragments of the pfcrt gene encompassing the codons at position 72-76, were aligned using GeneStudioTM Professional and compared to the reference sequence PF3D7_0709000 (https://www.ncbi.nlm.nih.gov/gene/term=PF3D7_0709000 accessed on September 11, 2018) using the on line Basic Local Alignment Search Tool (BLAST) for identifying mutations.
The protocol and the informed consent form were approved by the Ethics Committee of the Faculty of Medicine, University of Kinshasa (Approval N°: ESP MINESU 019/2016). All participants involved in the study signed an informed consent form. In case participants were young (children), the consent form was approved and signed by their parents or guardians.
Data were entered in an Excel 2010 database by an independent data clerk. Statistical analysis was performed using SPSS V. 20.0 (IBM corp, Armonk, NY). Pfcrt genotype profile was determined by the absence or presence of wild/mutant alleles. Samples for which genotype profile could not be determined were excluded from the analysis. Differences between groups were assessed using the Chi-square test for proportions and a p-value of less than 0.05 was considered statistically significant. In addition, when multiple pairwise comparison of prevalence of CQ resistance marker rates between sites was done, we used the Bonferroni correction to control the type 1 error rate.