In vitro amodiaquine resistance and its association with mutations in pfcrt and pfmdr1 genes of Plasmodium falciparum isolates from Nigeria

Abstract

Amodiaquine (AQ) is currently being used as a partner drug in combination with artesunate for treatment of uncomplicated malaria in most endemic countries of Africa. In the absence of molecular markers of artemisinin resistance, molecular markers of resistance to AQ may be useful for monitoring the development and spread of parasites resistance to Artesunate-Amodiaquine combination. This study was designed to assess the potential role of polymorphisms on pfcrt and pfmdr1 genes and parasite in vitro susceptibility for epidemiological surveillance of amodiaquine resistance in Plasmodium falciparum. The modified schizont inhibition assay was used to determine in vitro susceptibility profiles of 98 patients' isolates of Plasmodium falciparum to amodiaquine. Polymorphisms on parasites pfcrt and pfmdr1 genes were determined with nested PCR followed by sequencing. The geometric mean (GM) of AQ 50% inhibitory concentration (IC-50) in the 97 P. falciparum isolates was 20.48nM (95% CI 16.53–25.36nM). Based on the cut-off value for AQ in vitro susceptibility, 87% (84) of the P. falciparum isolates were sensitive to AQ (GM IC-50= 16.32nM; 95%CI 13.3–20.04nM) while 13% were resistant to AQ in vitro (GM IC-50= 88.73nM; 95%CI 69.67–113.0nM). Molecular analysis showed presence of mutant CVIET pfcrt haplotype, mutant pfmdr1Tyr86 allele and the double mutant CVIET pfcrt haplotype+pfmdr1Tyr86 in 72%, 49% and 35% respectively. The GM IC-50 of isolates harboring the wild-type pfcrt CVMNK haplotype+ pfmdr1Asn86 allele (3.93nM; 95%CI 1.82–8.46) was significantly lower (p=0.001) than those isolates harboring the double mutant pfcrtCVIET haplotype+pfmdr1Tyr86 allele (50.40nM; 95%CI 40.17–63.24). Results from this study suggest that polymorphisms in pfcrt and pfmdr1 genes are important for AQ resistance and therefore may be useful for epidemiological surveillance of P. falciparum resistance to AQ.

Ex Vivo Responses of Plasmodium falciparum Clinical Isolates to Conventional and New Antimalarial Drugs in Niger

“…This finding is of particular concern, because the use of AQ-containing combinations has been shown to select for infections that are less responsive to AQ in Uganda (24). However, no clear relationship was observed between the treatment response and the in vitro IC 50 of AQ in Gabon, and selection of this type does not seem to be occurring in Niger, while resistances to AQ have already been reported in some other African countries (25,26). Similarly, no sign of a decrease in susceptibility to LUM has been detected.…”

Role of Pfmdr1 in In Vitro Plasmodium falciparum Susceptibility to Chloroquine, Quinine, Monodesethylamodiaquine, Mefloquine, Lumefantrine, and Dihydroartemisinin

“…There was no difference in MDAQ IC 50 between the two haplotypes N86 and 86Y in isolates from Benin (43). In other works, the mutant Pfmdr1 86Y allele showed an increased MDAQ IC 50 in isolates from Nigeria (48). The Pfmdr1 86Y mutation has been shown to be associated with treatment failure after monotherapy with amodiaquine (36,49) or after combination therapy with artesunate- c n ϭ number of tested isolates for each drug.…”

Characterising temporal trends in asymptomatic Plasmodium infections and transporter polymorphisms during transition from high to low transmission in Zanzibar, 2005–2013

“…As resistance to ACT partner drugs has historically manifested before that of artemisinins (Venkatesan et al, 2014), molecular markers associated with amodiaquine resistance may serve as an important tool for surveillance of antimalarial drug resistance in Zanzibar (Froberg et al, 2012). Single nucleotide polymorphisms in the P. falciparum chloroquine resistance transporter (pfcrt) and P. falciparum multidrug resistance 1 (pfmdr1) transporter genes have been associated, both in vitro and in vivo, with resistance to amodiaquine (Echeverry et al, 2007;Folarin et al, 2011;Picot et al, 2009). Selection of pfcrt 76T and pfmdr1 86Y alleles, as well as pfmdr1 1246Y and the pfmdr1 (a.a.86,184,1246) YYY haplotype has been shown in recurrent infections after treatment with artesunateamodiaquine or amodiaquine alone (Djimde et al, 2008;Duraisingh et al, 1997;Holmgren et al, 2006Holmgren et al, , 2007Humphreys et al, 2007;Nsobya et al, 2007;Venkatesan et al, 2014).…”