Plasmodium falciparum isolates from southern Ghana exhibit polymorphisms in the SERCA-type PfATPase6 though sensitive to artesunate in vitro

Abstract

BackgroundIn 2005, Ghana replaced chloroquine with artemisinin-based combination therapy as the first-line treatment for uncomplicated malaria. The aim of this work was to determine for the first time, polymorphisms in the putative pfATPase6 and pftctp, pfmdr1, pfcrt genes in Ghanaian isolates, particularly at a time when there is no report on artemisinin resistance in malaria parasites from Ghana. The sensitivity of parasite isolates to anti-malaria drugs were also evaluated for a possible association with polymorphisms in these genes.MethodsThe prevalence of point mutations in the above Plasmodium falciparum genes were assessed from filter-paper blood blot samples by DNA sequencing. In vitro drug sensitivity test was carried out on some of the blood samples from volunteers visiting hospitals/clinics in southern Ghana using a modified version of the standard WHO Mark III micro-test.ResultsAll successfully tested parasite isolates were sensitive to artesunate; while 19.4%, 29.0% and 51.6% were resistant to quinine, amodiaquine and chloroquine respectively. The geometric mean of IC50 value for artesunate was 0.73 nM (95% CI, 0.38-1.08), amodiaquine 30.69 nM (95% CI, 14.18-47.20) and chloroquine 58.73 nM (95% CI, 38.08-79.38). Twenty point mutations were observed in pfATPase6 gene, with no L263E and S769N. All mutations found were low in frequency, except D639G which was observed in about half of the isolates but was not associated with artesunate response (p = 0.42). The pftctp gene is highly conserved as no mutation was observed, while CVIET which is chloroquine-resistant genotype at codon 72-76 of the pfcrt gene was identified in about half of the isolates; this was consistent with chloroquine IC50 values (p = 0.001). Mutations were present in pfmdr1 gene but were not associated with artemisinin response (p = 1.00).ConclusionThe pfATPase6 gene is highly polymorphic with D639G appearing to be fixed in Ghanaian isolates. These may just be spontaneous mutations as all parasite isolates that were tested displayed satisfactory in vitro response to artesunate. However, there is no improvement in susceptibility of the parasites to chloroquine five years after its proscription.

Monitoring artemisinin resistance in Plasmodium falciparum: comparison of parasite clearance time by microscopy and real-time PCR and evaluation of mutations in Pfatpase6 gene in Odisha state of India

“…The four important SNPs in Pfatpase6 gene reported to be associated with artemisinin resistance in different studies are L263E, E431K, A623E, and S769N (Tahar et al 2009;Jambou et al 2010;Krishna et al 2010;Zakeri et al 2012). However, L263E was not observed in any of the field isolates in all the studies conducted so far (Kwansa-Bentum et al 2011;Tanabe et al 2011) but only detected in vitro (Uhlemann et al 2005). The studies on P. falciparum isolates from Suriname too revealed L263, A623, and S769 codons in Pfatpase6 gene to be wild type (Adhin et al 2012).…”

“…Also, in Southern-Iran, L263 and A623 were reported to be wild type (Zakeri et al 2012). The presence of S769N also made occasional impacts from few studies (Jambou et al 2005;Pillai et al 2012;Zakeri et al 2012) like that of the French Guiana (Legrand et al 2008) while there are reports which observed its absence in the P. falciparum isolates that were analyzed (Mugittu et al 2006;Sisowath et al 2007;Zhang et al 2008;Ferreira et al 2008;Menegon et al 2009;Wangai et al 2011;Kwansa-Bentum et al 2011;Tanabe et al 2011;Brasil et al 2012;Saha et al 2013) or even denied the association of this SNP with artemisinin resistance (Phompradit et al 2011;Cui et al 2012). Another study from Yaounde, Cameroon, where all these four mutations were tested, high prevalence of E431K was warranted and alarmed (Tahar et al 2009).…”

Genetic Diversity and Lack of Artemisinin Selection Signature on the Plasmodium falciparum ATP6 in the Greater Mekong Subregion

“…The 68 pfatp6 sequences recently obtained in southern Ghanaian samples were partial sequences and were excluded in our analysis [54]. Comparison of all pfatp6 sequences identified a total of 71 SNPs, including 30 synonymous and 41 non-synonymous.…”

Comparative studies of serum-free media and detection techniques for <i>in vitro</i> drug sensitivity assessment of <i>Plasmodium falciparum</i>

“…Concerns about artemisinin resistance have been reported recently [22], and all malaria-endemic countries have been warned to be more vigilant in monitoring antimalarial drug efficacy, to allow the early detection of artemisinin resistance and aid global malaria control [23][24][25]. Furthermore, most rapid diagnostic test kits currently available on the market are based on HRPII and pLDH (as a biomolecule).…”