Abstract
Background: Antimalarial drug resistance is a major challenge hampering malaria control and elimination. Plasmodium falciparum, the leading causative parasite species, has developed resistance to basically all antimalarials. Continued surveillance of drug resistance using genetic markers provides important molecular data for treatment policies. This study sought to verify the genetic mechanism of resistance to sulfadoxine-pyrimethamine and assess the occurrence of point mutations associated with artemisinin resistance in P. falciparum clinical isolates from Eritrea. Methods: Nineteen dried blood spot samples were collected from patients visiting Adi Quala, Keren and Gash Barka Hospitals, Eritrea. The patients were followed up after receiving treatment with first line artesunate-amodiaquine. Nested polymerase chain reaction and Sanger sequencing techniques were employed to genotype point mutations in the P. falciparum bifunctional dihydrofolate reductase-thymidylate synthase (Pfdhfr, PF3D7_0417200), dihydropteorate synthase (Pfdhps, PF3D7_0810800) and kelch 13 (PfK13, PF3D7_1343700) genes. Results: Eight of nineteen (42%) of the dried blood spot samples were successful for PCR-amplification. Data analyses of the PCR-positive isolates revealed the following point mutations: Pfdhfr N51I in four isolates, C59R in one isolate, S108N in four isolates, a rare non-synonymous substitution V45A in four isolates and Pfdhps K540E in four isolates. No PfK13 point mutations were reported. Conclusions: Pfdhfr C59R and Pfdhps K540E point mutations are reliable markers for the sulfadoxine-pyrimethamine quintuple mutant haplotype combination. These findings highlight first reports in Eritrea, which verify the underlying genetic mechanism of antifolate resistance. Continuous monitoring of the PfK13 marker is recommended.
Highlights
Malaria is a major vector-borne disease, endemic in 87 tropical and sub-tropical countries, causing over 400,000 deaths yearly (WHO World Malaria Report 2020)
This reduction, according to the Ministry of Health (MOH) reports, is mainly due to extensive interventions employed towards the control of malaria since the establishment of the Eritrea National Malaria Control Program (NMCP) in 1995.1 Working hand-in-hand with Roll Back Malaria (RBM) collaborators and stakeholders,[2] NMCP set up a combination of strategies including integrated vector management (IVM), early diagnosis and prompt treatment[3] leading to a remarkable decrease in incidence and mortality rates, following the gruesome 1998 malaria epidemic in the country.[4]
Sample characteristics After consent was given for participation and follow up, 19 dried blood spot (DBS) samples were successfully collected from a total of 131 patients who visited the three hospitals during the study period,[22 10] samples from Adi Quala Hospital (AQH = 10), three samples from Keren Hospital (KH = 3) and six samples from Gash Barka Referral Hospital (GBH = 6)
Summary
Malaria is a major vector-borne disease, endemic in 87 tropical and sub-tropical countries, causing over 400,000 deaths yearly (WHO World Malaria Report 2020). Eritrea, which is situated in the Horn of Africa, has experienced a significant decline in deaths and cases of malaria over the past 20 years (WHO World Malaria Report 2019) This reduction, according to the Ministry of Health (MOH) reports, is mainly due to extensive interventions employed towards the control of malaria since the establishment of the Eritrea National Malaria Control Program (NMCP) in 1995.1 Working hand-in-hand with Roll Back Malaria (RBM) collaborators and stakeholders,[2] NMCP set up a combination of strategies including integrated vector management (IVM), early diagnosis and prompt treatment[3] leading to a remarkable decrease in incidence and mortality rates, following the gruesome 1998 malaria epidemic in the country.[4] The disease is generally endemic in the Western lowlands of Gash Barka, Anseba, Debub and Semenawi Keih Bahri (Northern Red Sea) zobas (regions) whereas the Central highlands and Eastern lowlands of Maekel and Debubawi Keih Bahri (Southern Red Sea) zobas respectively have unstable, seasonal transmission. Conclusions: Pfdhfr C59R and Pfdhps K540E point mutations are reliable markers for the sulfadoxine-pyrimethamine quintuple mutant haplotype combination These findings highlight first reports in Eritrea, which verify the underlying genetic mechanism of antifolate
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