African isolates show a high proportion of multiple copies of the Plasmodium falciparum plasmepsin-2 gene, a piperaquine resistance marker

Didier Leroy, Fiona Macintyre, Yeka Adoke, Serge Ouoba, Aissata Barry, Ghyslain Mombo-Ngoma, Jacques Mari Ndong Ngomo, Rosauro Varo, Yannelle Dossou, Antoinette Kitoto Tshefu, Tran Thanh Duong, Bui Quang Phuc, Bart Laurijssens, Roland Klopper, Nimol Khim, Eric Legrand, Didier Ménard, Didier Leroy, Fiona Macintyre, Yeka Adoke, Serge Ouoba, Aissata Barry, Ghyslain Mombo-Ngoma, Jacques Mari Ndong Ngomo, Rosauro Varo, Yannelle Dossou, Antoinette Kitoto Tshefu, Tran Thanh Duong, Bui Quang Phuc, Bart Laurijssens, Roland Klopper, Nimol Khim, Eric Legrand, Didier Ménard

Abstract

Background: Today, the development of new and well-tolerated anti-malarial drugs is strongly justified by the emergence of Plasmodium falciparum resistance. In 2014-2015, a phase 2b clinical study was conducted to evaluate the efficacy of a single oral dose of Artefenomel (OZ439)-piperaquine (PPQ) in Asian and African patients presenting with uncomplicated falciparum malaria.

Methods: Blood samples collected before treatment offered the opportunity to investigate the proportion of multidrug resistant parasite genotypes, including P. falciparum kelch13 mutations and copy number variation of both P. falciparum plasmepsin 2 (Pfpm2) and P. falciparum multidrug resistance 1 (Pfmdr1) genes.

Results: Validated kelch13 resistance mutations including C580Y, I543T, P553L and V568G were only detected in parasites from Vietnamese patients. In Africa, isolates with multiple copies of the Pfmdr1 gene were shown to be more frequent than previously reported (21.1%, range from 12.4% in Burkina Faso to 27.4% in Uganda). More strikingly, high proportions of isolates with multiple copies of the Pfpm2 gene, associated with piperaquine (PPQ) resistance, were frequently observed in the African sites, especially in Burkina Faso and Uganda (> 30%).

Conclusions: These findings were considered to sharply contrast with the recent description of increased sensitivity to PPQ of Ugandan parasite isolates. This emphasizes the necessity to investigate in vitro susceptibility profiles to PPQ of African isolates with multiple copies of the Pfpm2 gene and estimate the risk of development of PPQ resistance in Africa. Trial registration Clinicaltrials.gov reference: NCT02083380. Study title: Phase II efficacy study of artefenomel and piperaquine in adults and children with P. falciparum malaria. https://ichgcp.net/clinical-trials-registry/NCT02083380&cntry=&state=&city=&dist= . FSFV: 23-Jul-2014; LSLV: 09-Oct-2015.

Conflict of interest statement

FM and DL are employees of Medicines for Malaria Venture. None of the other authors declare competing interests.

Figures

Fig. 1
Fig. 1
Pfmdr1 and Pfpm2 gene copy numbers of Plasmodium falciparum isolates collected from Southeast Asia (in red) and from Africa (in black). Proportion of the isolates from Southeast Asia and African are given for each group: Pfpm2 single copy/Pfmdr1 single copy (lower-left quadrant), Pfpm2 single copy/Pfmdr1 multiple copies (upper-left), Pfpm2 multiple copies/Pfmdr1 single copy (lower-right) and Pfpm2 multiple copies/Pfmdr1 multiple copies (upper-right)
Fig. 2
Fig. 2
Kelch13 mutations, Pfmdr1 and Pfpm2 gene copy numbers of Plasmodium falciparum isolates collected from four sites in Southeast Asia (a) and from nine sites in Africa (b). Each of the kelch13 mutations are presented with different symbols and colours. Open triangle represents isolates with unavailable kelch13 data. The four quadrants in both panels present isolates with Pfpm2 single copy/Pfmdr1 single copy (lower-left quandrant), Pfpm2 single copy/Pfmdr1 multiple copies (upper-left), Pfpm2 multiple copies/Pfmdr1 single copy (lower-right) and Pfpm2 multiple copies/Pfmdr1 multiple copies (upper-right)

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