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. 2022 Jun 16;2(2):mtsi.v2i2.2022.198.
doi: 10.48327/mtsi.v2i2.2022.198. eCollection 2022 Jun 30.

[Pcr-rflp genotyping of pfcrt and pfmdr1 in plasmodium falciparum isolates from children in Vatomandry, Madagascar]

[Article in French]
Élisabeth Ravaoarisoa  1   2 , Voahangy Hanitriniaina Isabelle Andrianaranjaka  1   2 , Aina David Ramanantsahala  1 , Tovonahary Angelo Rakotomanga  1   2 , Fanomezantsoa Ralinoro  1   2 , Rianasoambolanoro Rakotosaona  3 , Ranjàna Hanitra Randrianarivo  1 , Danielle Aurore Doll Rakoto  1 , Victor Jeannoda  1 , Arsène Ratsimbasoa  1   4
Affiliations

[Pcr-rflp genotyping of pfcrt and pfmdr1 in plasmodium falciparum isolates from children in Vatomandry, Madagascar]

[Article in French]
Élisabeth Ravaoarisoa et al. Med Trop Sante Int. .

Abstract
in English, French

Background: Malaria is a parasitic disease caused by a hematozoan of the genus Plasmodium. Early diagnosis followed by effective treatment is one of the keys to control this disease. In Madagascar, after more than 60 years of use for the treatment of uncomplicated malaria, chloroquine (CQ) was abandoned in favor of artesunate + amodiaquine (ASAQ) combination because of high prevalence of CQ treatment failure. Surveillance based on the assessment of therapeutic efficacy and genetic markers of resistance to antimalarials is therefore essential in order to detect the emergence of potentially resistant parasites as early as possible. In this context, our study aimed to genotype the Plasmodium falciparum chloroquine resistance transporter gene or Pfcrt and Plasmodium falciparum multidrug resistance gene 1 or Pfmdr1 in isolates collected from children in the district of Vatomandry.

Methods: A total of 142 P. falciparum isolates collected during active case detection of malaria in children under 15 years old, between February and March of 2016 and 2017 in Vatomandry district, were analyzed. Pfcrt (K76T codon) and Pfmdr1 (N86Y codon) genotyping was carried out by polymerase chain reaction followed by enzymatic digestion (restriction fragment length polymorphism) or PCR-RFLP.

Results: The successful rates of amplification of Pfcrt and Pfmdr1 genes were low, around 27% and 39% respectively. The prevalence of isolates carrying the mutant Pfcrt K76T codon and the mutant Pfmdr1 N86Y codon was 2.6% [95% confidence interval (95% CI): 0.1 - 15.0%] and 36% [95% CI: 23.7 - 49.7%] respectively.

Conclusion: Despite the limited number of samples analyzed, our study highlighted the circulation of isolates carrying both the mutant Pfcrt K76T and Pfmdr1 N86Y alleles. Although the prevalence of mutations in Pfcrt and Pfmdr1 genes that we observed was low, other studies should be carried out in order to follow the evolution of these markers in time and space. The use of more sensitive methods will better characterize P. falciparum strains circulating in Madagascar. Artesunate-amodiaquine is used as a first-line treatment for uncomplicated malaria in the country; it is also crucial to monitor the other codons, i.e. 184 and 1246 of the Pfmdr1 gene, implicated in the resistance of P. falciparum to amodiaquine in Africa.

Contexte: La surveillance de l'efficacité thérapeutique et des marqueurs génétiques de la résistance aux antipaludiques est primordiale afin de détecter le plus tôt possible l’émergence des parasites potentiellement résistants. Dans ce contexte, notre étude a pour objectif de réaliser le typage du gène Plasmodium falciparum chloroquine resistance transporter ou Pfcrt et Plasmodium falciparum multidrug resistant gene 1 ou Pfmdr1 sur des isolats provenant des enfants du district de Vatomandry.

Méthodologies: Au total, 142 isolats de P. falciparum collectés lors d'un dépistage actif du paludisme chez des enfants âgés de moins de 15 ans, entre février et mars des années 2016 et 2017 à Vatomandry, ont été analysés. Le typage du codon K76T du gène Pfcrt et du codon N86Y du gène Pfmdr1 a été ensuite effectué par la technique de polymérisation en chaîne suivie de digestion enzymatique (restriction fragment length polymorphism) ou PCR-RFLP.

Résultats: Le taux de succès d'amplification des gènes Pfcrt et Pfmdr1 était faible, de l'ordre de 27 % et 39 % respectivement. La prévalence des isolats mutants pour le codon K76T de Pfcrt était de 2,6 % [intervalle de confiance à 95 % (IC95%) : 0,1 - 15,0 %] et de 36 % [IC95% : 23,7 - 49,7 %] pour le codon N86Y du gène Pfmdr1.

Conclusion: Notre étude a mis en évidence la présence d'isolats portant à la fois la mutation au niveau du codon K76T de Pfcrt et N86Y de Pfmdr1. Bien que le taux de mutation que nous avons observé soit faible, d'autres études méritent d’être effectuées afin de suivre l’évolution de ces marqueurs dans le temps et dans l'espace à Madagascar.

Keywords: Antimalarial; Children; Indian Ocean; Madagascar; Pfcrt; Pfmdr1; Plasmodium falciparum; Resistance; Vatomandry.

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Figures

Figure 1
Figure 1
Site d’étude (Programme national de lutte contre le paludisme, Madagascar) Study site (National Malaria Control Program, Madagascar)
Figure 2
Figure 2
Electrophorèse des produits de digestion de Pfcrt avec l'enzyme ApoI Electrophoresis of restriction fragments of Pfcrt after digestion with ApoI
Figure 3
Figure 3
Electrophorèse des produits de digestion de Pfmdr1 avec l'enzyme AflIII Electrophoresis of restriction fragments of Pfmdr1 after digestion with AfIII
See this image and copyright information in PMC

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