Plasmodium falciparum from Pará state (Brazil) shows satisfactory in vitro response to artemisinin derivatives and absence of the S769N mutation in the SERCA-type PfATPase6.
OBJECTIVE
To evaluate the in vitro efficacy of artesunate (ATN) and artemether (ATH) against Plasmodium falciparum isolates from the Brazilian Amazon state of Pará and to search for mutations and/or altered copy numbers in the putative resistance-associated pfcrt, pfmdr1 and pfATPase6 genes.


METHODS
In vitro efficacy of ATN and ATH was successfully measured in 56 freshly collected P. falciparum isolates, using a conventional WHO microtest with minor modifications. Single nucleotide polymorphisms (SNPs) in the same isolates were inspected using DNA sequencing and/or PCR-RFLP. We used real-time quantitative PCR to assess gene copy numbers.


RESULTS
ATN and ATH geometric mean IC(50)s were 0.85 nm, 95% CI (0.55-1.15) and 3.0 nm, 95% CI (1.5-4.5), respectively. There was extremely limited diversity of pfcrt and pfmdr1 genotypes and three SNPs were identified in the pfATPase6 gene: one T to A synonymous mutation at nucleotide 2694 and two non-synonymous (both G to A) mutations at nucleotides 110 and 1916, causing predicted aminoacid shifts of arginine to lysine and of glycine to aspartate, respectively. The previously reported S769N mutation was not detected in any of the isolates inspected. In addition, no gene amplifications were detected in a subset of eight isolates.


CONCLUSIONS
Artemisinin derivatives display satisfactory in vitro activity locally and the pfATPase6 gene is distinct from that reported in French Guiana, suggesting that those haplotypes have not been introduced regionally.
Ferreira
Isabel D
ID
Centro de Malária e Outras Doenças Tropicais/IHMT/UEI Biologia Molecular/UEI Malária, Lisbon, Portugal.
Martinelli
Axel
A
Rodrigues
Louise A
LA
do Carmo
Ediclei L
EL
do Rosário
Virgílio E
VE
Póvoa
Marinete M
MM
Cravo
Pedro
P
D000251:Adenosine Triphosphatases / Q000235:genetics*; D000818:Animals; D000962:Antimalarials / Q000494:pharmacology*; D000077549:Artemether; D037621:Artemisinins / Q000494:pharmacology*; D000077332:Artesunate; D001938:Brazil / Q000453:epidemiology; D004351:Drug Resistance; D018628:Gene Dosage; D006801:Humans; D016778:Malaria, Falciparum / Q000453:epidemiology / Q000469:parasitology*; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics; D009154:Mutation; D021261:Parasitic Sensitivity Tests; D010963:Plasmodium falciparum / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D015800:Protozoan Proteins / Q000235:genetics; D012717:Sesquiterpenes / Q000494:pharmacology*
D000251:Adenosine Triphosphatases; D000962:Antimalarials; D000077549:Artemether; D037621:Artemisinins; D000077332:Artesunate; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D015800:Protozoan Proteins; D012717:Sesquiterpenes; C513498:Mdr1 protein, Plasmodium falciparum