Advocating for PCR-RFLP as molecular tool within malaria programs in low endemic areas and low resource settings

Abstract

The road to malaria elimination for low- and middle-income countries is paved with obstacles, including the complexity and high costs of advanced molecular methods for genomic analysis. The usefulness of PCR-RFLP as less complex and affordable molecular surveillance tool in low-endemic malaria regions was assessed in a cross-sectional study conducted in Suriname, currently striving for malaria elimination, but plagued by recent P. vivax outbreaks. Molecular analysis of two highly polymorphic genes Pvmsp-1 F2 and Pvmsp-3α was performed for 49 samples, collected during October 2019 through September 2021 from four different regions with varying malaria transmission risks. RFLP-profiling revealed that outbreak samples from three indigenous villages, almost exclusively, harbored a single clonal type, matching the "Palumeu" lineage previously described in 2019, despite multiple relapses and drug pressure exerted by mass drug administration events, suggesting a limited P. vivax hypnozoite reservoir in Suriname. In contrast, isolates originating from Sophie, a mining area in neighboring French Guiana displayed a highly heterogeneous parasite population consistent with its endemic malaria status, demonstrating the differentiating capacity and thus the usefulness of PCR-RFLP for P. vivax genetic diversity studies. Outbreak reconstruction emphasized the impact of undetected human movement and relapses on reintroduction and resurgence of P. vivax malaria and PCR-RFLP monitoring of circulating parasites guided the roll-out of targeted interventions. PCR-RFLP seems a suitable molecular alternative in low-endemic areas with restricted resources for outbreak analysis, for monitoring the spread or containment of circulating strains and for identification of imported cases or potential foci.

Fig 1. Map of Suriname with study sites.

The study sites are represented by colored circles. Paramaribo, the capital of Suriname is not a study site, but is shown in the picture for reference purposes. This figure was created using a base map made with Natural Earth, free vector and raster map data @ naturalearthdata.com in QGIS version version 3.32.2-Lima and was retraced and edited using Corel Draw X7.

Fig 2. RFLP Profile Distribution for Pvmsp-1 F2 and Pvmsp-3α per area.

Trend lines for Palumeu, Apetina and Pelele Tepoe represent the total number of cases per region, diagnosed in Suriname in the period January 2019 to October 2021. The gray bar on the X-axis corresponds with the study period. Parasite isolates investigated within the study period are depicted as circles divided in two halves. Circle size in Sophie was increased just to achieve a better visual representation. Data from earlier molecular characterizations on parasite isolates outside the study period are symbolized by divided squares. Different allelic profiles for each of the genes Pvmsp-1 F2 and Pvmsp-3α are linked to a different color or different pattern. Identical RFLP-profiles within a gene are presented with the same color. The color or pattern on the left side of each circle represents the corresponding Pvmsp-1 F2 RFLP profile, while the color on the right side represents the corresponding Pvmsp-3α gene profile. Letters A and B mark two pregnant/breastfeeding participants from Pelele Tepoe with recurring malaria. Letters C and D denote two participants from Sophie, who experienced previous malaria episodes. Letters x, y and z denote participants from Pelele Tepoe with diverging allelic variants. An additional sample from Taluen, French Guiana collected in June 2021 is depicted as a triangle in the Sophie panel.