A snapshot of the prevalence of dihydropteroate synthase-431V mutation and other sulfadoxine-pyrimethamine resistance markers in Plasmodium falciparum isolates in Nigeria

The threat of malaria continues to pose a major public health issue across the globe, specifically in sub-Saharan African countries, and especially among pregnant women and children under the age of five. In 2021 alone, the World Health Organization (WHO) reported that an estimated 247 million cases of malaria occurred worldwide, while the estimated number of deaths from malaria stood at 619,000 [1]. According to the report, approximately 27% of global malaria morbidity and 31.9% of mortality are attributed to Nigeria [1]. Currently, the WHO recommends chemopreventive strategies, including the use of sulfadoxine-pyrimethamine (SP) as intermittent preventive treatment during pregnancy (SP-IPTp), the use of SP (or other anti-malarials) as perennial malaria chemoprevention (PMC) and seasonal malaria chemoprevention (SMC) with amodiaquine and SP [2].

The SP-IPTp strategy, administered at fixed time points, at four-week intervals, during antenatal visits, is currently implemented in 33 sub-Saharan African countries [1]. It has been documented to ameliorate malarial illness and its associated risks both in maternal and fetal health outcomes in regions with medium to high malaria transmission [3]. The strategy is also considered one of the cost-effective malaria control tools available [4]. The effectiveness of SP-IPTp is measured by using metrics such as reduction in the incidence of maternal malaria infection, reduction in low birth weight (LBW), decrease in preterm delivery cases, and improved haematological parameters [4]. In Nigeria, the provision of SP-IPTp is recommended as part of antenatal care of all pregnant women [5]. The dosing schedule is typically set from the second trimester, and at least three doses are given before delivery, each at least one month apart.

The combination of sulfadoxine and pyrimethamine works synergistically, and preferentially, to target dihydropteroate synthetase (PfDHPS) and dihydrofolate reductase (PfDHFR), both enzymes involved in the folate biosynthesis pathway. Molecular mechanisms underpinning SP resistance are caused by single nucleotide polymorphisms (SNPs) in two distinct genes that codes for PfDHPS and PfDHFR, that is Pfdhfr and Pfdhps genes. Polymorphisms in codons of Pfdhps causing amino acid substitutions in PfDHPS at S/A436F, A437G, K540E, and A613S have been found to be associated with sulfadoxine resistance while SNPs in Pfdhfr causing amino acid changes at position 51, 59, 108 and 164 of PfDHFR confer pyrimethamine resistance [6, 7]. Pfdhfr triple mutant alleles resulting in changes N51I, C59R, S108N (IRN) and in Pfdhps gene, the A437G mutation is prevalent in West and Central African countries [8, 9]. In contrast, a quintuple mutant that combines the Pfdhfr triple (IRN) haplotype and the Pfdhps double mutant at A437G and K540E is highly prevalent in East Africa [6] while the Pfdhps K540E mutation is rarely found in West and Central Africa [10].

However, the narrative in East Africa has escalated in the last decade with the emergence of a SNP in Pfdhps causing an alanine to glycine mutation at position 581 of PfDHPS [4, 11]. This results in the ‘super resistant’ sextuple haplotype of Pfdhfr/Pfdhps (mainly the constructed haplotype: CIRNI-SGEGA). There is evidence that the CIRNI-SGEGA haplotype signals a loss of sensitivity of Plasmodium falciparum to SP and compromise the effectiveness of the SP-IPTp strategy including a reduction in its ability to prevent low birthweights [5, 6, 12, 13]. In contrast, in West Africa, the absence of 540 and 581 genotypes was reassuring for the continuation of SP-IPTp efficacy. However, recent reports have shown that the molecular repertoire of P. falciparum in West Africa may be changing. For instance, a SNP resulting in an isoleucine to valine change (Pfdhps I431V) has emerged in Nigeria and other parts of West Africa [14]. The mutant was first reported in the UK from a malaria infection imported from Nigeria [15] and it also appeared in other surveys conducted in Nigeria, and subsequently in Cameroon [16‐18]. More recently, polymorphisms in Pfdhps resulting in amino acid substitutions at 431, 581 and 613 were reported in P. falciparum isolates from Ghana [19]. It has been suggested that the new mutant might disrupt sulfadoxine binding to its active site, thus reducing the susceptibility of P. falciparum to the drug [14]. Considering the huge burden of malaria in Nigeria, particularly among pregnant women, and the lack of recent data on the extent of the spread of the Pfdhps 431V/581G mutation in the country and the region as a whole, there is an urgent need to evaluate the current prevalence of the Pfdhps 431V, Pfdhps 431V/-581G and Pfdhps 581G/-613S bearing parasites and, subsequently, the impact of these haplotypes on the protective efficacy of IPTp.

The present study assesses the prevalence of SNPs in Pfdhps in southwest Nigeria where the prevalence Pfdhps 431V/581G mutations are suspected to be increasing.

The WHO encourages the use of SP-IPTp as part of antenatal care in malaria-endemic settings in Africa. In this study, asymptomatic falciparum malaria was found in 19% of pregnant women. Previous reports on asymptomatic malaria in pregnancy, have shown asymptomatic malaria in pregnancy is prevalent across sub-Saharan African (SSA) countries, and this carries an increased risk of maternal anaemia compared to non-infected pregnant women [48, 49]. It has been well established that asymptomatic malaria infection is associated with poor maternal or fetal health [48]. SP resistance is known to modify the outcome of SP-IPTp strategy. The occurrence of maternal and placental malaria among pregnant women who received SP-IPT may be linked to reduced drug susceptibility of the malaria parasites due to SP resistance as a result of emergence of SNPs in the Pfdhfr/Pfdhps genes of P. falciparum [4, 11, 50‐52].

Findings from this study showed that the SNPs in Pfdhfr N51I, -C59R, -S108N (Pfdhfr haplotype CIRNI) and Pfdhps-A437G are widespread in the studied population, reaching a prevalence of beyond 95%. Similar findings have been reported in Nigeria and other countries across SSA [17, 53]. This suggests that the quadruple mutations are already fixed in these populations [18, 32, 54]. Indications from other studies however revealed that the protective effect of SP-IPTp remains intact, regardless of the high prevalence of this quadruple Pfdhfr-Pfdhps mutant [2, 55].

However, the emerging of certain Pfdhps mutant haplotypes in the West African region raise concerns about the continuing effectiveness of SP-IPTp. The present study has substantiated findings from previous studies that suggested the emergence of other Pfdhfr/Pfdhps mutant alleles in Nigeria and the West African region. The present study showed that some P. falciparum isolates were carrying additional mutations at codons I431V, A581G and A613S particularly in the South. However, the frequency of these new mutations is not fixed and furthermore it is not clear how far the new mutations will influence the efficacy of SP-IPTp (and possibly other preventive interventions using SP alone; e.g. the newly revised Perennial Malaria Chemoprevention of infants). Before the advent of the Pfdhps 431V mutation in Nigeria, East Africa witnessed a similar scenario with the emergence of the quintuple (IRN + 437G + 540E) and sextuple (IRN + 437G + 540E + 581G) mutant haplotypes. These were associated with a reduction in the mean birth weight and other metrics of SP-IPTp failure in Tanzania [13, 56]. Although no 540E mutation was observed in the present study, it is possible that the emerging Pfdhps 431V and its co-occurrence with 581G and 613S could lead to a higher order mutant haplotype, and this alternative sextuple mutant might compromise the chemopreventive impact of SP-IPTp in West Africa.

Previous studies indicated that the prevalence of Pfdhps 431V varies across regions in Nigeria in clinical isolates collected at various respective study periods [13‐15]. For instance, a low prevalence of Pfdhps 431V mutation was reported in Southwest Nigeria, whereas the prevalence in the parasite population is widespread in the Southeast and Northeast Nigeria among the samples collected about a decade ago [13]. Figures 2 and 3 give a view of the spatial distribution of Pfdhps-431V and -581G in Nigeria and neighbouring countries, using data reported in surveys conducted between 2007 and 2020. This study was the first survey in Osun State and it revealed a high prevalence of the Pfdhps-431V (45.9%) and -581G (31.1%) mutations. The Pfdhps-431V mutation is found to be higher than compared to the neighbouring states, both Oyo and Kwara. Considering the upsurge of mutation in the present study, it would be necessary to bear in mind that the data for Oyo/Kwara are mostly based on older surveys. Oguike et al. [14] provided data in a study site located in Ibadan, approximately 100 km from the present study site, with a frequency of 0 and then,6.5% from a sequential sampling design between 2003 and 2008. There is a high possibility that the level of Pfdhps mutant alleles might have also increased in all the surrounding states. A more recent report by Oboh et al. [16] in Lagos and Edo States revealed a prevalence of 28.5% and 51.4%, respectively, in clinical samples collected between 2016 and 2017 [16]. This is in support that the prevalence of Pfdhps 431V mutation is increasing across the Nigerian population. This present data revealed a frequency of 31.1% for the dhps-581G bearing parasites. This also suggests an upsurge in the circulating 581G mutant allele in Southwest Nigeria similar to previous findings from Lagos and Ogun States [14, 16]. The SNPs A581G and I431V co-occur in 21.3% of the parasite population in this study. This was suggested to render the parasite less susceptible to sulfadoxine [17]. However, the paradigm is already changing to a higher order mutant allele combination consisting the Nigerian sextuple or septuple mutant haplotypes.

It is worth noting that eight distinct haplotypes were detected on the Pfdhps gene starting from ISGKAA, a single mutation, to VAGKGS with 4 mutant alleles. Other haplotypes were IAGKAA, IAGKAS, ISGKGS, VAGKAA, IAGKGS and VAGKAS. The wild haplotype ISAKAA was not found in this study in contrast to the findings reported by Oboh et al. [16]. Most haplotypes were present among the study populations except ISGKGS and IAGKGS which were only present in the group seeking healthcare at the Outpatient Unit. An important observation from this study is a relatively high prevalence of sextuple and septuple haplotypes when the SNPs from both Pfdhfr and Pfdhps genes were combined. Both haplotypes contributed approximately 25.0% of the P. falciparum isolates in the present study, and their presence might pose a high probability of the malaria parasite becoming extensively resistant to SP in Nigeria.

Despite the limitations of the study, such as the small sample size and the use of only one study site, the present data provides information about the current prevalence of molecular markers of SP resistance in Southwest Nigeria. The study revealed that a high frequency of the emerging mutations now exists within the region. In previous studies, other regions in Nigeria depicted a much higher frequency of Pfdhps 431V. Thus, the results of the present study, conducted in the Southwest region of the country, suggest a need to reassess the current prevalence of Pfdhps mutation in other regions as the current prevalence in those regions might have reached an alarming proportion. However, the exact implication of an increase in the frequency of mutation has not been established. Therefore, further work is suggested to examine in vivo susceptibility of the mutant malaria parasites to SP and evaluate the efficacy of SP-IPTp in the context of the emerging mutations.