Study area and field samples
RDTs were collected as part of malaria surveillance in northern Senegal where the incidence of malaria is low (<5 ‰). Febrile patients who went to public or private health facilities were tested by RDTs and treated. All RDTs used (negative and positive) were stored in plastic bags at room temperature (Fig. 1A).
In Senegal, malaria transmission is closely related to the rate of rainfall and generally increases during the rainy season. The density of vector populations is dependent on rainfall.
For this study, two (2) health districts of two (2) regions were concerned, including that of Saint-Louis in the Saint-Louis region and that of Ranérou in the Matam region. The large shares of malaria cases recorded in the two regions were reported in these two districts. In recent years, the district of Saint-Louis had recorded the majority of malaria cases in the Region. As part of malaria elimination, the programme of Senegal began in 2018 to use primaquine combined with the usual treatment of malaria cases in this area to reduce gametocyte carriage and malaria transmission. The 2018 epidemiological data showed that the number of malaria cases in this district had doubled compared to 2017 despite the intensification of control strategies. In order to better appreciate the impact of primaquine on gametocyte carriage, it is necessary to study the gametocyte prevalence in 2018 in the district of Saint-Louis, which will be compared with the prevalence in 2017 before using primaquine and the prevalence in 2018 in the district of Ranérou where primaquine is not used. At the time of testing, patients were informed about the protocol and their consent was obtained.
A total of 303 positive RDTs were randomly selected from different time periods and sites. In Saint-Louis, we chose 101 positive TDRs completed in 2017 and 101 completed in 2018. In Ranérou 101 positive TDRs of 2018 were chosen.
RDT devices
The RDTs used in the northern part of the country for malaria surveillance were SD-Bioline Malaria Ag Pf for the detection of P. falciparum specific Histidine-Rich Protein II (HRP-II) antigen. DNA extraction was done by opening the TDR cassettes (Fig. 1C).
RDT-DNA extraction methods
Several methods of P. falciparum DNA extraction have been described [11,14]. This study used the DNA extraction protocol with Chelex-100 described by Wooden et al. which is the result of a combination of several techniques taking into account a variable number of strains of the parasite [17]. The RDTs were opened using a metal spatula to access the nitrocellulose strip. This band was taken using forceps and the blood deposition and absorption filters were cut into small pieces in labelled Eppendorf tubes (Fig. 1C). The migration zone of the blood sample on the nitrocellulose membrane was carefully scraped with a scalpel to collect the maximum amount of DNA from the devices (Fig. 1D). For each sample the material used was cleaned in 96% ethanol, then rinsed with water and dried on clean tissue paper, to minimize cross-contamination during sample preparation.
In each tube, 800 μl of 0.5% saponin solution containing 0.5 g of saponin and 100 ml of PBS diluted to 1x were added. The tubes were then centrifuged for 10 minutes at 150 rpm and incubated at room temperature overnight. The liquid was completely aspirated from the tubes by using a micropipette and the samples were then washed twice by adding 800 μl of 1xPBS for 5 minutes. Then 150 μL of sterile water and 75 μL of 20% Chelex solution were added into the tubes, which were tightly closed and vortexed for 15 seconds and incubated with a dry-bath incubator for 8 minutes. After centrifugation at maximum speed for 5 minutes, the supernatant containing the DNA was aspirated with a micropipette and then transferred to sterile tubes leaving the Chelex at the bottom of the tube [17].
qPCR assay
The validation of the technique was done using 6 different samples. Two (2) different RDT samples (Paracheck ™ -Pf and SD Bioline Malaria Ag-Pf) were made by depositing 5 μL of 0.5% and 1% diluted gametocytes cultures in the RDT wells. From the 1% diluted culture 50 μL were deposited on a filter paper. To these samples, two more positive RDTs were added whose gametocytes with different densities were identified by microscopy and another sample of filter paper whose PCR was positive for the detection of gametocytes as a positive control. qPCR-steps were done in duplicate for samples. The SYBR Green Master Mix has been standardized to detect and quantify Pfs25 gene transcripts using the BIO-RAD CFX96™ tool which showed positive results.
For the Pfs25 assay we used a specific female gametocytes primers tested by Schneider et al. [18]. The Forward (5’-CCATGTGGAGATTTTTCCAAATGTA, location: 253852–1253828) and Reverse (5’-catttaccgttaccacaagttaCATTC, location: 1253710–1253736) primers were used for amplification. For these primers, Schneider et al. had shown that the DNA sequences of the target regions (DNA produced in vitro: ivDNA) and the cDNA were identical. The Pfs25 primers used are specific to mature gametocytes and can be considered to quantify only female gametocytes. Schneider et al. had also shown that no significant nucleotide polymorphism were detected in the primer regions among almost 2000 samples of P. falciparum collected worldwide [18].
Field sample analysis
The real time-PCR was performed in a 20 µL reaction. The reaction was prepared with SYBR Green Master Mix. DNA template and Nuclease-Free Water were used. The Pfs25 primers used are specific for female gametocytes and showed limited polymorphism [18]. Each 20 μL reaction mixture contained 2 μL of 2 μL of DNA sample, 10 μL of 2x SYBR Green master mixtures, 2.25 µL of primers at a final concentration of 300 nM and 5.75 µL of Nuclease-Free Water. Amplification included a template denaturation step at 95°C (10 min) followed by 45 cycles of 15 seconds at 95°C, and 1 minute at 60°C, with fluorescence acquisition at the end of each extension step. Reactions were run in 96-well PCR plates on a Bio-Rad CFX Connect real time PCR Detection System. For the study 4 plates were used for 303 samples.
Data analysis
The Bio-Rad CFX Manager 3.0 software was used for the post-amplification data analysis.
Quantification cycle (Cq) or cycle threshold (Ct) determination mode was set to single threshold with baseline-subtracted curve fit and a user defined threshold of 50 relative fluorescence units (RFUs) for analysis of parasite. A cycle threshold value (Ct) <35 was chosen for positive PCRs. Samples with a cycle value greater than 35 Ct were considered negative for PCR. The data was also exported to Excel and analysed with the Epi Info software.