Background
The malaria parasite
Plasmodium falciparum is one of the major causes of morbidity and mortality in sub-Saharan African countries, especially in children and pregnant women. Many factors have contributed to the development and spread of drug resistance, including gene mutations and drug pressure [
1]. Resistance to chloroquine (CQ), the most widely used and affordable antimalarial drug, has contributed to the increased in mortality and morbidity caused by
P. falciparum infections in endemic areas [
2]. Resistance to chloroquine, the cheapest and most widely available anti-malarial, has reached significantly high levels leading to replacement with artemisinin-based combination therapy (ACT) in many malaria-endemic countries [
3].
Molecular genotyping and characterization of mutations for single nucleotide polymorphism (SNPs) have been used for drug resistance monitoring and could predict emerging or existing drug resistance patterns. Genetically, chloroquine and amodiaquine resistance has been linked to
P. falciparum chloroquine resistance transporter (
Pfcrt) gene from different parts of the world [
4-
7], as well as mutations in
P. falciparum multidrug resistance 1 (
Pfmdr1) [
8,
9].
Polymorphisms in the
Pfmdr1 gene have been shown by transfection to modulate higher levels of chloroquine resistance and to affect mefloquine, halofantrine, and quinine resistance [
10-
12]. The role played by
Pfmdr1 mutations (N86Y, Y184F, S1034C and D1246Y) in mediating
in vivo and
in vitro chloroquine resistance has received a lot of research interest [
13-
15]. It has been reported that mutations in the region of
Pfcrt encompassing codons 72 – 76 is a key marker of
P. falciparum chloroquine resistance [
16]. Substitutions in the wild type allele, encoding CVMNK, give rise to several resistant variants, of which the most common are CVIET in South-East Asia and Africa and SVMNT, which has been reported in South America [
4] and Asia [
17], but rarely in Africa [
18]. The change in single codon of
Pfcrt gene from Lys (K) to Thr (T) at position 76 (K76T) thus is reported to play a decisive role in conferring resistance to chloroquine [
4]. Prolonged use of chloroquine monotherapy has imposed high selection pressure, leading to a substantial increase in the prevalence of this marker in parasite populations worldwide.
In Nigeria, despite the change in National malaria drug policy to ACT because of the widespread and high-level clinical failure rate of chloroquine, CQ is still widely used in the treatment of malaria in the country [
19], because it is accessible and affordable. Previous studies have reported resistance to chloroquine by a change at position 76 (K76T) in children treated with chloroquine in Lagos, just before the change in policy [
20], in Ibadan [
21] and in Osogbo [
22]. However, in the above studies, CQ was still the first-line antimalarial medicine before a change in policy to ACTs and the children were treated with chloroquine. The studies did not also provide expanded haplotype information on single nucleotide polymorphisms (SNPs). Therefore, this study was carried out to elucidate single nucleotide polymorphisms in
Pfcrt and the point mutations in
Pfmdr1 genes with the aim of determining the status of CQ-resistant
Plasmodium falciparum genes in a diverse and highly populated setting in Lagos, Nigeria, in the light of reports on return of CQ sensitive (wild type) parasites in Malawi and Kenya [
23,
24] after the total removal of CQ from the population.
Discussion
Self-treatment with chloroquine and other monotherapies are still high in the population because of their affordability, accessibility, and low implementation of the malaria treatment guidelines that recommends the use of ACTs. The access to, and high usage of CQ and other monotherapies in the treatment of malaria makes the determination of chloroquine resistant Plasmodium falciparum in Lagos imperative several years after the change in malaria treatment policy. CQ is still sold especially in the informal private sector among the Private Propriety Medicine Vendors (PPMVs), otherwise known as medicine retailers, a group that provide malaria treatment to over 60% of fever patients in the country. Further, the PPMVs sell antimalarial medicines on the basis of their clients’ complaints and are not permitted to do a blood based test due to policy restrictions.
Mutations in the region of
Pfcrt codons 72 – 76 is said to be a key marker of
P. falciparum chloroquine resistance [
16]. This study showed high prevalence of
Pfcrt CVIET haplotype (72–76). This result is consistent with previous study where majority (98%) of the isolates genotyped carried the chloroquine resistant CVIET haplotype in Uganda [
31] and in Swaziland [
32]. Persistence of high prevalence of CVIET was also reported in Ethiopia due the continuous use of chloroquine for the treatment of
P. vivax [
33]. This study showed that the SVMNT haplotype does not occur in Lagos, Nigeria. The absence of the SVMNT haplotype which is known to occur majorly in South America is consistent with many other reports in Africa [
16,
32] except studies in part of East Africa (Tanzania and Angola)that reported SVMNT [
18,
34]. This was presumed to be as a result of
P. falciparum resistance to amodiaquine or its metabolite desethyl-amodiaquine following the use of amodiaquine as monotherapy.
The present study showed prevalence of 62.2% and 69.0% for
Pfmdr1 Y86 and F184 (mutant-type) respectively. The role of
Pfmdr1 gene mutations in anti-malarial drugs resistance is still controversial. An
in vivo study where a chloroquine-resistant infection was reported showed absence of mutations at codons
Pfmdr1 86 and 1246 in the [
35,
36]. Similarly, a study in South-Eastern Iran reported a strong association between
pfcrt K76T, but not
pfmdr1 N86Y mutation and
in vivo chloroquine resistance [
37]. A study in Haiti reported mutation in F184 only in the
Pfmdr1 gene and no mutation was seen in
Pfcrt gene codon 72–76 [
38]. In contrast, Y86 have been reported to be responsible for chloroquine resistance in combination with
Pfcrt 76 T [
13,
21,
39] and another study from Madagascar reported an association between
Pfmdr1 Y86 mutant alleles and chloroquine clinical resistance with no such association with
Pfcrtgene [
40]. Polymorphisms in the
Pfmdr1 gene have been said to be under artemether-lumenfantrine selection pressure [
41]. Selection of
Pfmdr1 Y86by amodiaquine and chloroquine were reported previously in the Gambia [
42], and in Kenya [
43]. Thus, the success of treatment with ACTs may largely depend on the parasite’s existing level of tolerance to the partner drugs.
Some earlier studies in the pre-ACT days in South-West Nigeria reported high chloroquine-resistant parasites in children treated with CQ when CQ was the drug of choice for the treatment of malaria and they only determined mutation at position 76 (K76T). Mutation at 76 (K76T) had been reported to play a decisive role in conferring resistance to CQ [
4]. In a semi-urban area of Lagos, South-West Nigeria, the prevalence of T76 mutation was 74.6% [
20], while another study In Ibadan; South-West Nigeria reported 62% and 29% for T76 and Y86 mutations respectively [
21]. Prevalence of 74%, 29% and 64% were reported for mutations at T76, Y86 and F184 respectively in children with
P. falciparum even before they were treated in Osogbo, South-West Nigeria while in another study in Ibadan (south-West Nigeria), a prevalence of 60%, 33% and 14% mutations at T76, Y86 and F184 respectively was reported in children whose age ranged from 6 month - 12 years [
44]. Also another study in Ogun State, still in the South-West zone of Nigeria, reported a prevalence of 96.9% at K76T among children under the age of five years [
45].
Our study in Lagos, South-West Nigeria, has showed the persistence of chloroquine-resistant parasites circulating in children four years after the change in policy for the treatment of uncomplicated malaria from CQ to ACTs. It is therefore important that apart from the change in policy to ACTs, there is an urgent need to restrict the use of chloroquine in the general population by the regulatory agency for drugs. Since resistant phenotypes often have fitness costs [
46], their prevalence is likely to decline after removal of the selective pressure. In countries where the change in policy from chloroquine to ACT was strictly enforced, marked decrease in chloroquine-resistant parasites in the population was recorded. In a recent surveillance study in Honduras, Central America, where CQ is still used for the management of uncomplicated malaria, all the samples tested showed CQ susceptibility in the
Pfcrt “CVMNK” genotype in codons 72–76 [
47].
There was a decrease in the frequency of
Pfcrt76T mutation when CQ was abolished in the treatment of
P. falciparum malaria in the People’s Republic of China [
48]; prevalence of mutant alleles of
Pfcrt76T decreased from 64.5% in 2002 to 16% in 2004 and that of the mutant
Pfmdr1 86Y alleles decreased from 46.6% to 2.7% two and half years after successful withdrawal of CQ in coastal Tanzania [
49]. It was also reported that the prevalence of the CQ-resistant
Pfcrt76T genotype decreased from 85% in 1992 to 13% in 2000 in Malawi [
49]. In 2001, CQ cleared 100% of 63 asymptomatic
P. falciparum infections as no isolates were resistant to CQ
in vitro, and no infections with the CQ resistant
Pfcrt76T genotype were detected [
50]. Similarly, it was shown that CQ was again an efficacious treatment for malaria, 12 years after it was successfully withdrawn from use in Malawi [
23]. Similar result was also reported in Kenya where the frequency of the
Pfcrt-76 mutant significantly decreased from around 95% to 60%, though, the frequency of
Pfmdr1-86 did not decline substantially [
24]. In Tanzania, where chloroquine is no longer in use, the frequency of the wild type CVMNK haplotype increased from 6% in 2003 to 30% in 2007. These findings may reflect decreasing drug pressure of chloroquine on the parasite populations in these areas.
Surveillance for antimalarial drug resistance, using the platform provided by the National Malaria control Programme in Nigeria should be supported to undertake regular and robust monitoring of malaria parasite resistance genes for trends. The use of amodiaquine has been associated to a certain extent with
Pfcrt76T and
Pfmdr1 86Y mutations [
21,
43]. Furthermore, the partner drugs to ACT are also threatened by the development of resistance if treatment of malaria with antimalarial monotherapy is not abolished [
51,
52]. Importantly, the implementation of the current malaria treatment policy using the ACTs should be strengthened, vigorously promoted, through regular training, supervision among health workers; and the institution of best procurement practices for malaria medicines at all levels based on National Policy recommendations. The regulatory agency, National Agency for Food Drug Administration and Control (NAFDAC) should regulate access to chloroquine in Nigeria while the general public should be enlightened on the high levels of circulating resistant-malaria parasite genes to chloroquine, its low efficacy and to discourage its continuous use for the for the treatment of uncomplicated malaria. Access to ACTs should be expanded and made affordable especially in the private sector where a high number of persons with fever are treated.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
OO participated in sample collection and processing, carried out the molecular genetic analyses and drafted the manuscript. OW conceived the study, participated in the design of the study and coordination and helped to draft the manuscript. CJ provided the platform for molecular analysis, the reagents used and contributed immensely to the molecular genetic studies and in drafting the paper. All the authors read and approved the final manuscript.