Single Nucleotide Polymorphisms of Pfdhfr and Pfdhps Genes: Implications for Malaria Prophylactic Strategies in Maiduguri, Northeast Nigeria

Background . The success of Intermittent Preventive Treatment in Pregnancy (IPTp), Intermittent Preventive Treatment in Infancy (IPTi), and Seasonal Malaria Chemoprevention (SMC) depends on sulfadoxine-pyrimethamine (SP) eﬃcacy. Objective . The study determined Single Nucleotide Polymorphisms (SNPs) of Plasmodium falciparum dihydrofolate reductase ( Pfdhfr ) and dihydropteroate synthase ( Pfdhps ) in Maiduguri, Northeast Nigeria. Materials and Methods . Giemsa-stained blood smears, capillary blood, and dried blood spot samples were collected from 63 subjects with uncomplicated malaria in Maiduguri between May and October 2018. Plasmodium species was determined and parasite density (PD) was estimated using the smears. Genomic DNA (gDNA) of P . falciparum was extracted from the dried blood spot samples using QIAamp DNA Mini Kit. The gDNA was subjected to nested PCR followed by restriction fragment length polymorphism (RFLP) to determine SNPs at Pfdhfr codons N51I, C59R, and S108N and Pfdhps codons S436A/F, A437G, and K540E. Results . The subjects’ mean age ± standard deviation was 23.6 ± 8.7 (2.0–67.0) years with a geometric mean PD of 8,948 (2,100–13,400) asexual parasites/ µ l blood. SNPs prevalence at any of the six Pfdhfr and Pfdhps codons was 85.7% (54/63); the prevalence was higher ( p < 0 . 05) in Pfdhfr (82.5%; 52/63) than Pfdhps (58.7%; 37/ 63). Pfdhfr allele 108N (82.5%; 52/63) was the highest ( p < 0 . 05) mutant when compared with alleles 51I (60.3%; 38/63) and 59R (66.7%; 42/63). Triple Pfdhfr mutation was observed in 60.3% (38/63) of the isolates and was higher ( p < 0 . 05) among female subjects and SP recipients. Prevalence of Pfdhps allele 436A (28.6%; 18/63) was similar ( p > 0 . 05) to allele 437G (34.9%; 22/63), with double mutation recorded in 4.8% (3/63). K540E mutation was not observed. Conclusion . Pfdhfr and Pfdhps mutations observed in Maiduguri are suggestive of SP resistance level, and this could constitute a setback to malaria prophylactic strategies in the region if unchecked. Thus, there is a need to investigate the clinical eﬃcacy of SP.


Introduction
e scourge of malaria persisted in most countries of sub-Saharan Africa [1][2][3][4] despite the progressive reduction in global burden [5]. In 2018, the World Health Organization (WHO) reported 228 million cases of malaria worldwide, with the African Region accounting for 213 million cases amounting to 93% of the global burden. With 25% of the global morbidity and 24% of mortality, Nigeria remains the worst hit country in the world [5]. Similarly, malaria is still of public health concern in Northeast Nigeria [4,6], compounding other health, security, and social challenges in the region. However, the recent WHO World Malaria Report indicated a decline in the global burden when compared with previous years. Specifically, the global incidence rate progressively decreased from 71 cases per 1000 individuals in 2010 to 57 cases per 1000 individuals in 2018. Within the same period, the incidence rate decreased by 22% in the African Region. Plasmodium falciparum is the dominant species of the malaria parasite in Africa, accounting for 99.7% of the estimated cases in 2018 [5].
Malaria disproportionately affects all ages and sexes, with pregnant women and children below five years at the highest risk [7]. In 2018, about 24 and 11 million cases were recorded among children and pregnant women in sub-Saharan Africa, respectively; and 67% of the global mortality occurred in children below five years [5].
us, most prophylactic strategies, such as Intermittent Preventive Treatment in Pregnancy (IPTp), Intermittent Preventive Treatment in Infancy (IPTi), and Seasonal Malaria Chemoprevention (SMC), target these vulnerable cohorts. IPTp involves the administration of full doses of sulfadoxine-pyrimethamine (SP) to pregnant women starting early in the second trimester and continues monthly until delivery [8]. IPTi is the administration of full doses of SP to infants (<12 months) in regions with moderate to high malaria transmission at 10 weeks, 14 weeks, and nine months of age [9]. SMC is a complete treatment course with a fixed dose of amodiaquine and SP given monthly to children aged 3-59 months, beginning at the start of the malaria transmission season in highly seasonal transmission areas of sub-Sahara Africa [10]. Previous reports have demonstrated the efficacy of these prophylactic strategies on malaria transmission [11][12][13][14][15][16].
In Maiduguri, Northeast Nigeria, there is intense malaria transmission during the 4-6 months of the rainy season [30] with an annual average prevalence of 22.6%, lowest in April (7.5%) and highest in September (84.2%) [4]. Malaria indicators are poor in the region, only 26% of pregnant women received at least three doses of SP during the last pregnancy and 45.4% of the population used insecticide-treated nets (ITNs) [30]. IPTi and SMC are being considered for routine use in Northeast Nigeria, and a recent study has demonstrated the impact of SMC in Borno State [31]. Despite that SP is widely used, there is a dearth of molecular evidence of its resistance in the region. us, the present study determined the SNPs of Pfdhfr and Pfdhps in P. falciparum isolated in Maiduguri, Northeast Nigeria and discussed the implications on malaria prophylactic strategies.

Study Area and Study Population.
e study was conducted in Maiduguri, Northeast Nigeria, a region with seasonal malaria transmission that peaks in the rainy months of August and September [4]. Maiduguri is the largest city in the region and the capital of Borno State, which shares international borders with republics of Cameroon, Chad, and Niger. It is located on latitude 11°40′N-11°44′N and longitude 13°05′E-13°14′E with an average annual rainfall of 613 mm, an average annual temperature of 25.8°C (11.9-44.0°C), and relative humidity of 15-72% [32]. e weather is categorized into harmattan (October-February), hot (March-May/June), and rainy seasons (June/July-September) [33]. e estimated population is over one million people [34] who are predominantly Kanuri. However, other indigenous ethnic groups and non-Nigerians are also present. ey mainly engage in farming, livestock keeping, trading, fishing, artisanship, civil service, and nongovernmental organizations.

Subject Enrolment and Sample Collection.
Between May and October 2018, 235 individuals were screened for malaria by light microscopy at the University of Maiduguri Teaching Hospital (UMTH), Muhammed Shuwa Memorial Hospital (MSMH), and Teachers' Village (TV), Maiduguri. Of the 128 individuals with malaria, 106 subjects who met the inclusion criteria and provided informed consent were enrolled. e demographic and medical data were obtained following a comprehensive physical examination. Giemsa-stained thick and thin smears, capillary blood, and dried blood spot samples were prepared for all subjects [35]. in and thick smears were used for species identification and parasitaemia quantification, respectively, while capillary samples were used to determine haematocrit values [36]. e dried blood spot samples were labelled, placed in an air-tight zip bag with desiccants, and stored at 4°C until use. e subjects were treated with artemether-lumefantrine (AL) and monitored for 14-day posttreatment. e study protocol was reviewed and approved by the Research Ethics Committee, UMTH.

Statistical Analysis.
e data were analyzed with SPSS version 21.0 for Windows (IBM Corporation, Armonk, NY, USA) and presented as means ± standard deviation, range, and percentages. Categorical variables were compared using Pearson Chi-square or Fisher's exact test, while Student's ttest and analysis of variance (ANOVA) were used for continuous variables. Significance difference was inferred at p < 0.05.

Baseline Characteristics of the Subjects at Enrolment.
e baseline characteristics of the 63 subjects whose samples were analyzed are presented in Table 1. e overall mean age ± standard deviation (range) of the subjects was 23.6 ± 8.7 (2.0-67.0) years with a mean duration of illness of 3.1 ± 1.6 days and anaemia prevalence of 27.0% (17/63). Headache and fever accounted for the highest (p < 0.05) proportion of symptoms presented by the subjects at enrolment. e proportion of subjects who consumed SP in the last 12 months (69.8%; 44/63) was higher (p < 0.05) than those who did not (30.2%; 19/63). e geometric mean parasite density (range) was 8,948 (2,100-13,400) asexual parasites/μl blood. All subjects responded adequately to AL treatment and the outcomes presented elsewhere.

Discussion
e present study reports the prevalence of Pfdhfr SNPs at codons N51I, C59R, and S108N and codons S436A/F, A437G, and K540E of Pfdhps and discusses the implications on malaria prophylactic strategies in Maiduguri, Northeast Nigeria. Pfdhfr and Pfdhps genes have been widely used as biomarkers for monitoring pyrimethamine and sulfadoxine resistance, respectively [23,24,26,27,36]. e detection of Pfdhfr mutations in the present study is an indication of circulating pyrimethamine-resistant P. falciparum strains in the region. is finding is in accordance with studies that have previously established pyrimethamine resistance in Nigeria [20][21][22] and other African countries [17,24,25]. Among the codons of Pfdhfr evaluated, codon S108N accounted for the highest mutation, with all 52 mutant strains carrying the mutant allele 108N. is is similar to previous reports of a preponderance of the mutant allele 108N in populations where SP is deployed [21,37]. Although the mutant allele 108N is said to confer low pyrimethamine resistance [21,23], the presence of mutant alleles 51I and/or 59R could have reinforced the level of pyrimethamine resistance in the region. High pyrimethamine resistance is associated with triple Pfdhfr mutants (51I, 59R, and 108N) in West Africa [28,40]. e high proportion (60.3%) of triple mutant alleles observed in the present study points to the extent of pyrimethamine resistance in Maiduguri. e present value is significantly lower than 81.3% reported in Damboa, another town in the region [22]. Oguike et al. [22] studied pregnant women who might have been exposed to pyrimethamine pressure from IPTp, resulting in a higher resistance level. Similarly, the present value is lower than the values (92.9-98.7%) reported from Southern Nigeria [15,22]. is disparity could be largely attributed to malaria transmission intensity and higher IPTp coverage in Southern Nigeria [30]. Furthermore, the significantly higher prevalence of triple Pfdhfr mutations in P. falciparum isolated from female subjects and SP recipients is clearly suggestive of the influence of routine IPTp. e majority of the female subjects who participated in the study admitted previous consumption of SP within the last 12 months prior to the study.
Pyrimethamine is mainly coformulated with sulfadoxine and is coadministered for malaria prevention in pregnant women and children [8][9][10]. us, the present study also examined point mutations in Pfdhps, a gene associated with sulfadoxine resistance. Two mutant alleles 436A/F and 437G were observed among the parasite population, and the prevalence is similar to the report from other parts of the region [22]. It is worthy to note that the lack of K540E mutation in the present study is similar to previous studies that reported the rare mutant allele 540E in West Africa [15,21,22]. However, this does not undermine the possibility of sulfadoxine resistance among the population since the majority of the isolates with the mutant alleles 436A and 437G also have triple Pfdhfr mutant alleles indicating a high level of SP resistance.
Sulfadoxine and pyrimethamine are essential components of the WHO prescribed malaria prophylactic strategies such as IPTp, IPTi, and SMC [8][9][10]. us, the success of these strategies primarily depends on the clinical efficacy of both drugs. e present study revealed the presence of sulfadoxine-and pyrimethamine-resistant P. falciparum strains in Maiduguri as implied by the detection of Pfdhfr and Pfdhps mutant alleles. IPTp is routinely used in Nigeria to prevent malaria in pregnancy [30]. e poor coverage of this strategy in Northeast Nigeria [30] is often implicated as the cause of persistent malaria in pregnancy in the region. Besides, the current evidence of SP resistance in the region provides an additional explanation as to why malaria   [41]. is is in agreement with previous studies that reported SMC efficacy in areas with high SP resistance and low amodiaquine resistance [42].

Conclusion
e detection of Pfdhfr and Pfdhps mutant alleles in Maiduguri is an indication of compromised SP clinical efficacy in Northeast Nigeria and could adversely affect malaria prophylactic strategies if unchecked. However, the implications can be minimized by improved IPTp coverage, systematic IPTi deployment, sustained amodiaquine efficacy, and the use of other malaria preventive measures such as ITNs.
Data Availability e data are available with STB and can be accessed by readers following a convincing request clearly stating while access to the data is required. Acknowledgments e authors acknowledged the technical support provided by Mr. Rotimi Olatunde of the Department of Pharmacology and erapeutics, University of Ibadan, Nigeria. e University of Maiduguri, Nigeria, is highly appreciated for providing an enabling platform. is study was supported by Tertiary Education Trust Fund (TETFUND), Nigeria, through an Institutional-based Research Grant jointly 162 bp 148 bp Ladder S8 S11 S15 S22 S29 S33 S51 S59  Journal of Tropical Medicine 5 awarded to STB (PI), UKS, and OAS. However, the publication was funded by the authors' personal funds.

Supplementary Materials
Supplementary