Absence of Association between Methylene Blue Reduced Susceptibility and Polymorphisms in 12 Genes Involved in Antimalarial Drug Resistance in African Plasmodium falciparum.

Joel Mosnier,Bruno Pradines,Isabelle Fonta,Nicolas Benoit,Rémy Amalvict,Océane Delandre,on Group,Marylin Madamet,Marie Robert,Mathieu Gendrot,Francis Foguim

doi:10.3390/ph14040351

Abstract

Half the human population is exposed to malaria. Plasmodium falciparum antimalarial drug resistance monitoring and development of new drugs are major issues related to the control of malaria. Methylene blue (MB), the oldest synthetic antimalarial, is again a promising drug after the break of its use as an antimalarial drug for more than 80 years and a potential partner for triple combination. Very few data are available on the involvement of polymorphisms on genes known to be associated with standard antimalarial drugs and parasite in vitro susceptibility to MB (cross-resistance). In this context, MB susceptibility was evaluated against 482 isolates of imported malaria from Africa by HRP2-based ELISA chemosusceptibility assay. A total of 12 genes involved in antimalarial drug resistance (Pfcrt, Pfdhfr, Pfmdr1, Pfmdr5, Pfmdr6, PfK13, Pfubq, Pfcarl, Pfugt, Pfact, Pfcoronin, and copy number of Pfpm2) were sequenced by Sanger method and quantitative PCR. On the Pfmdr1 gene, the mutation 86Y combined with 184F led to more susceptible isolates to MB (8.0 nM vs. 11.6 nM, p = 0.03). Concerning Pfmdr6, the isolates bearing 12 Asn repetitions were more susceptible to MB (4.6 nM vs. 11.6 nM, p = 0.005). None of the polymorphisms previously described as involved in antimalarial drug resistance was shown to be associated with reduced susceptibility to MB. Some genes (particularly PfK13, Pfugt, Pfact, Pfpm2) did not present enough genetic variability to draw conclusions about their involvement in reduced susceptibility to MB. None of the polymorphisms analyzed by multiple correspondence analysis (MCA) had an impact on the MB susceptibility of the samples successfully included in the analysis. It seems that there is no in vitro cross-resistance between MB and commonly used antimalarial drugs.

Highlights

According to the World Health Organization (WHO) 2020 report [1], half of humanity is exposed to malaria in 87 countries in 2019 with 229 million malaria cases and409,000 deaths (97% were African and 67% were children under five years old)
Very few data are available on the involvement of polymorphisms on genes known to be associated with standard antimalarial drugs and parasite in vitro susceptibility to Methylene blue (MB)
We detected in the present study a higher prevalence of parasites with reduced in vitro susceptibility to chloroquine than parasites harboring mutated haplotype CVIET, suggesting that additional factors may be involved in chloroquine resistance

Summary

Introduction

According to the World Health Organization (WHO) 2020 report [1], half of humanity is exposed to malaria in 87 countries in 2019 with 229 million malaria cases and409,000 deaths (97% were African and 67% were children under five years old). Pharmaceuticals 2021, 14, 351 phenomenon involving the deletion of the pfhrp (P. falciparum histidin-rich protein 2) gene that codes the main protein used in malaria rapid diagnostic tests for first line diagnosis in Africa [2]. P. falciparum drug resistance to most antimalarial compounds has emerged in Southeast Asia and spread to Africa [3,4]. Resistance to artemisinin-based combination therapy (ACT), the last WHO recommended antimalarial drugs as first-line treatment for uncomplicated malaria, emerged in Western Cambodia, Myanmar, Thailand, and throughout Southeast Asia [5,6]. In this context, the development of new antimalarial drugs and new ACT partners became an emergency. It is mandatory to identify mutated genes linked to resistance in order to avoid cross-resistance between drugs and choose good partners for new ACT therapies

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