Prevalence of mutations in the Plasmodium falciparum chloroquine resistance transporter, PfCRT, and association with ex vivo susceptibility to common anti-malarial drugs against African Plasmodium falciparum isolates
Authors: Foguim Francis Tsombeng , Bogreau Hervé , Gendrot Mathieu , Mosnier Joel , Fonta Isabelle , Benoit Nicolas , Amalvict Rémy , Madamet Marylin , Wein Sharon , Pradines Bruno ,   The French National Reference Centre for Imported Malaria Study Group, Augis V. , Bastien P. , Benoit-Vical F. , Berry A. , Brouqui P. , Chauvin P. , Cividin M. , Courtier F. , Delaunay P. , Delhaes L. , Drancourt M. , Dubosc N. , Gaillard T. , Genin A. , Garnotel E. , Javelle E. , L’Ollivier C. , Lagier J. C. , Ledault E. , Leveque M. , Malvy D. , Marty P. , Ménard G. , Menu E. , Millet P , Minodier P , Parola P. , Picot S , Pomares-Estran C. , Ranque S. , Receveur M. C. , Robin A. , Sappa E. , Savini H. , Sevestre J. , Simon F. , Sterkers Y. , Surcouf C. , Varlet E. , Wolff A. 
Published: June 2020

Abstract
Background
The Plasmodium falciparum chloroquine transporter gene (pfcrt) is known to be involved in chloroquine and amodiaquine resistance, and more particularly the mutations on the loci 72 to 76 localized within the second exon. Additionally, new mutations (T93S, H97Y, C101F, F145I, M343L, C350R and G353V) were recently shown to be associated with in vitro reduced susceptibility to piperaquine in Asian or South American P. falciparum strains. However, very few data are available on the prevalence of these mutations and their effect on parasite susceptibility to anti-malarial drugs, and more particularly piperaquine in Africa.

Methods
A molecular investigation of these mutations was performed in 602 African P. falciparum parasites collected between 2017 and 2018 on malaria patients hospitalized in France after a travel in African countries. Associations between genotypes and in vitro susceptibilities to piperaquine and standard antimalarial drugs were assessed.

Results
None of the mutations, previously described as associated with piperaquine resistance, was found in the 602 P. falciparum African isolates. The K76T mutation is associated with resistance to chloroquine (p < 0.0002) and desethylamodiaquine (p < 0.002) in Africa. The K76T mutation is not associated with in vitro reduced susceptibility to piperaquine. The mutation I356T, identified in 54.7% (n = 326) of the African isolates, was significantly associated with reduced susceptibility to quinine (p < 0.02) and increased susceptibility to mefloquine (p < 0.04). The K76T and I356T mutations were significantly associated in West African isolates (p = 0.008).

Conclusion
None of the mutations in pfcrt found to be associated with piperaquine reduced susceptibility in Asia or South America (T93S, H97Y, C101F, F145I, M343L C350R and G353V) were found in the 602 African isolates including the three isolates with reduced susceptibility to piperaquine. The K76T mutation, involved in resistance to chloroquine and amodiaquine, and the I356T mutation were not associated with in vitro reduced susceptibility to piperaquine. Differences in mefloquine susceptibility between I356 and 356T isolates were, while statistically different, minimal. Further analyses are needed with a more important sample size from the same geographic area to confirm the role of the I356T mutation on quinine susceptibility.