Malaria in the 21st Century continues to pose a significant challenge to the health and well-being of populations living in malaria-endemic regions worldwide. As per the latest estimates, 198 million cases of malaria occurred globally in 2013, leading to 584,000 preventable deaths [
1]. The burden exerted by this disease is heaviest on the African continent, where 90 % of the deaths due to malaria globally occurred [
2,
3]. Of these, 78 % were children under the age of 5 years [
1,
4]. In Kenya, malaria accounted for 8.82 million cases out of the 41.8 million of outpatient cases reported across Kenyan healthcare facilities in the year 2013 [
5]. Pregnant women and young children make up the most vulnerable group to the disease, with 3 % of all deaths in children ≤5 years of age attributed to malaria [
5,
6].
Accurate diagnosis followed by prompt treatment with efficacious anti-malarial drugs remains an important cornerstone in the fight against the disease [
7]. Microscopy is recommended as the standard tool for parasitological confirmation of malaria as it is highly adaptable to the poor and marginalized setting in most malaria-endemic countries where the majority of cases occur [
1,
7]. Rapid diagnostic test (RDT) strips are also employed in malaria diagnosis and are based on detection of specific parasite antigens in a patient’s blood sample. With proper training, RDTs are easy to use and sensitive in detecting low parasitaemia [
8,
9]. Efforts to fight back the severe socio-economic impact of the disease have in recent decades been hampered by emergence of strains of
Plasmodium falciparum- and
Plasmodium vivax-resistant to past and present anti-malarial drugs [
10,
11]. Developed in the 1930s as an effective replacement for quinine, chloroquine (CQ) was quickly adopted as the drug of choice against malaria in all malaria-endemic regions globally [
12]. Its key selling points were its low cost, low toxicity and high efficacy in clinical cases, which in the decades following its development made the greatest impact in sub-Saharan Africa where morbidity and mortality due to malaria was drastically reduced [
13]. Extensive use and availability of CQ as a monotherapy for malaria prophylaxis led to the emergence of CQ-resistant (CQR)
P. falciparum and
P. vivax strains [
12,
14]. First noted in Southeast Asia and South America in the late 1950s, CQR
P. falciparum spread out from these foci to cover all malaria-endemic regions globally, emerging in Africa in the 1970s [
14].
Plasmodium vivax resistance to CQ emerged much later, pointing to a different genetic mechanism in acquisition of the CQR phenotype [
15]. CQ targets the parasite haematin detoxification pathway in the parasite digestive vacuole (DV) in a two-pronged attack where it adsorbs onto growing hemozoin polymers and also binds to toxic haematin molecules generated as the parasite digests host haemoglobin resulting in accumulation of toxic haematin in the DV [
16,
17]. Genetic cross studies between CQR and CQ-sensitive (CQS) laboratory strains of
P. falciparum identified a 13 exon gene,
pfcrt, on chromosome 7 as the candidate gene for the CQR phenotype [
18]. The translated product of
pfcrt is a multi-domain transmembrane protein localized on the parasite DV. It is thought to play a role in maintenance of osmotic balance by shuttling one or more essential osmolytes across the DV membrane [
18‐
20]. To counter CQ,
P. falciparum is believed to primarily employ a mutant
pfcrt product to increase efflux of CQ from the DV [
20]. This phenotype is the result of complex genetic changes involving point mutations on ten or more codons of the
pfcrt gene [
18,
21,
22]. Numerous studies have identified the K76T mutation as the central factor in CQR development [
18,
21,
23]. This mutation is present in all CQR laboratory lines and field isolates from various malaria-endemic regions and can reliably be used as a CQR marker alone [
23] or in concert with other
pfcrt mutations. Other point mutations on
pfcrt aside from K76T seem only to modulate CQR as determined by the change on codon 76 [
23]. The Pgh-1 (P-glycoprotein homologue 1) encoded by the
P. falciparum pfmdr-
1 gene bearing specific mutations, particularly the N86Y substitution, has been mentioned as a CQR modulator [
24,
25]. Statistical associations between N86Y
pfmdr-1 and CQR were drawn initially but this gene was later shown to bear no independent effect on CQR and neither does it strengthen K76T based CQR [
23]. Its role in CQR may however relate to parasite fitness adaptation in response to physiological changes from
pfcrt point mutations [
12].
Therapeutic efficacy studies are critical in guiding drug policy and are recommended every 2 years where possible [
1]. In light of emerging resistance to current first line anti-malarial drugs, namely artemisinin, the importance of reconsidering the efficacy of CQ cannot be understated, given the time interval since its withdrawal. Malawi was the first country in Africa to cease administration of CQ in malaria chemotherapy in 1993 following widespread treatment failure [
24]. Kenya followed suit in 1999 as did many sub-Saharan countries on the continent [
26]. Sulfadoxine-pyrimethamine (SP) replaced CQ as the first-line anti-malarial drug in these countries but was discontinued in 2004 following rapid development of parasite resistance to these drugs and replaced with artemisinin-based combination therapy (ACT) [
7]. Where CQ use was effectively discontinued through concerted public awareness efforts, studies have shown that in absence of drug pressure CQR strains of
P. falciparum with an associated loss in fitness have steadily been replaced by CQ-sensitive (CQS isolates). As has been the case especially in Malawi, where the withdrawal of CQ was supported by an intensive national sensitization programme, the population frequencies of mutations associated with CQR in
pfcrt have reduced from 85 % in 1992 to 13 % in 2000 [
24]. Patterns in CQR reversal heavily depend on national drug policies and public awareness campaigns as evidenced by persistence of CQR strains of
P. falciparum in certain regions where CQ withdrawal was inconclusive in contrast with regions that efficiently removed CQ out of national circulation [
24]. Although the trend has not been as drastic as in Malawi, other reports of significant rises in prevalence of CQS
P. falciparum isolates have emerged [
27]. This reversal to CQS presents an interesting scenario to drug policy makers in these regions where CQ can be re-introduced as malaria chemotherapy, although current knowledge would advocate for CQ administration in concert with another anti-malarial drug that differs in mechanism of action to slow down development of anti-malarial drug resistance [
1,
7,
24]. Sixteen years have passed since Kenya withdrew CQ as the first line anti-malarial drug in 1999. A retrospective study done in Kilifi, Kenya [
27] reported a
pfcrt 76T prevalence of 63 %, down from 94 %, in the 13 years that had passed since CQ withdrawal. The current study thus sought to survey the current sensitivity levels of
P. falciparum to CQ in Msambweni area, coastal Kenya, 16 years after official withdrawal of CQ as the first line of treatment for uncomplicated malaria.