Date Published: December 3, 2018
Publisher: F1000 Research Limited
Author(s): James Abugri, Felix Ansah, Kwaku P. Asante, Comfort N. Opoku, Lucas A. Amenga-Etego, Gordon A. Awandare.
Background: The emergence and spread of resistance in
Plasmodium falciparum to chloroquine (CQ) necessitated the change from CQ to artemisinin-based combination therapies (ACTs) as first-line drug for the management of uncomplicated malaria in Ghana in 2005. Sulphadoxine-pyrimethamine (SP) which was the second line antimalarial drug in Ghana, was now adopted for intermittent preventive treatment of malaria in pregnancy (IPTp).
Malaria remains a major global health concern especially in sub Saharan Africa.
P. falciparum malaria is considered the most severe and also the leading cause of morbidity and mortality, especially among children under five years (
Schumacher & Spinelli, 2012). In 2016 a global estimate of 216 million malaria cases was reported, which led to about 445,000 deaths (
WHO, 2017). The global malaria mortality rate, however, has reduced by 29% since the year 2010, as a result of increased preventive and control measures (
The resulting nested PCR products for each of the four genes containing the SNP alleles of interest were analyzed by restriction fragment length polymorphism (RFLP). Each of the restriction digestion reactions was set at a final volume of 15 µL containing 5 µL of the nested PCR product, 1X FastDigest Green buffer and 0.3 µL of the appropriate restriction enzyme (Thermo Scientific). The restriction enzymes used, incubation temperature, incubation time as well as the expected band sizes for the wild-type and the mutant alleles of the point mutations were as reported in previous studies (
et al., 2001a;
et al., 1998). Ten microlitres of the restriction digestion fragments were resolved on 2 % agarose gel stained with ethidium bromide and the resulting image resolved with the Amersham Imager 600 (GE, USA). Purified DNA obtained from laboratory strains of
P. falciparum (Dd2, 3D7, FCR3, K1, 7G8 and W2) were used as controls for the sensitive and resistant alleles for each gene.
P. falciparum resistance to antimalarial drugs remains one of the biggest threats to the control and elimination of malaria globally. In Ghana, a change in the use of CQ to ACTs was implemented in 2005 as a result of high rate of malaria treatment failure (
et al., 2007). In this study, we determined the prevalence of alleles associated with CQ and antifolate resistance using clinical isolates from three malaria endemic regions with varying transmission intensities in Ghana. We observed a decreasing prevalence of CQ resistance-associated alleles but an increasing prevalence of SP resistance-associated alleles. The distribution of the alleles across the three study sites were not significant, except for
pfdhps 437G which was significantly higher in Accra compared to Navrongo and Kintampo. The frequency of
pfdhfr/pfdhps haplotypes in 2012–2013 and 2016–2017 were not significantly different across the three study sites. Both
in vitro and molecular surveillance studies have associated CQ resistance mainly with the
pfcrt 76T allele, but also with
pfmdr1 86Y and 184F alleles.
and pfmdr1 86Y mutant alleles have also been reported to decrease
susceptibility to amodiaquine but increase parasite sensitivity to dihydroartemisinin, lumefantrine and mefloquine (
et al., 2014;
et al., 2016). Despite the use of ACTs (artemether-lumefantrine, artesunate-amodiaquine, and dihydroartemisinin-piperaquine) in Ghana since 2005, decreasing prevalence of
pfcrt 76T and
pfmdr1 86Y mutant alleles were observed in this study when compared to study by Duah and colleagues in 2013 (
et al., 2013). This shows a gradual decline in the frequencies of these alleles since the discontinuation of CQ as an antimalarial in Ghana, this observation is consistent with findings in other malaria endemic populations in east Africa such as Tanzania, Malawi , Kenya and Zambia where artemether lumefantrine is the first-line drug for uncomplicated malaria (
et al., 2013;
et al., 2009;
et al., 2016). A study in Kenya posits that the K76 is preferential selection by Artemeter Lumefantrine(AL) (
et al., 2015) The fitness cost of harbouring the mutant alleles is thought to select against them in favour of the non-resistant background alleles (
et al., 2009;
et al., 2015). Unlike
pfcrt 76T and
pfmdr1 86Y, the prevalence of
pfmdr1 184F mutant allele (65%) appears to have not varied so much from 2005 to 2017 when compared to the 43% to 69% prevalence reported from 2005 to 2010 (
et al., 2013). Contrary to this observation, a study in Tanzania reported an increasing prevalence of
pfmdr1 N86 and 184F following the introduction of artemether-lumefantrine (
et al., 2011). Notably, parasites that have a combination of
pfmdr1 mutant alleles (N86, 184F and D1246) are reported to have reduced sensitivity to artemether-lumefantrine treatment (
Baliraine & Rosenthal, 2011;
et al., 2009;
et al., 2014). Other studies have also linked duplication of
pfmdr1 to resistance to partner drugs of ACTs (Rodrigues, Henriques et (
et al., 2011;
et al., 2010).
This study reports an increasing prevalence of CQ sensitive clinical isolates after 12 years of CQ withdrawal at three different study sites that capture the eco-epidemiology of malaria in Ghana. The prevalence of the antifolate drug resistant alleles remain relatively high across the study sites. Besides, there is an increasing trend in the frequency of SP-resistance associated alleles at all sites. Taken together, these observations point to the need for a robust antimalarial drug discovery strategy to provide a vast array of alternatives for chemotherapy in readiness for the likelihood of future poor parasite response to the use of SP for prevention of malaria in pregnant women and for SMC in children. However, it is pre-mature to recommend the discontinuation of SP use due to the high prevalence of antifolate drug resistance alleles since the drug can be efficacious where there is fixation of these alleles.
The data supporting this article is available online at Open Science Framework: Dataset 1. Prevalence of chloroquine and antifolate drug resistance alleles in
Plasmodium falciparum clinical isolates from three areas in Ghana.
et al., 2018) under a CC0 1.0 Universal licence.