Geographical emergence of sulfadoxine-pyrimethamine drug resistance-associated P. falciparum and P. malariae alleles in co-existing Anopheles mosquito and asymptomatic human populations across Cameroon

ABSTRACT Malaria molecular surveillance remains critical in detecting and tracking emerging parasite resistance to anti-malarial drugs. The current study employed molecular techniques to determine Plasmodium species prevalence and characterize the genetic diversity of Plasmodium falciparum and Plasmodium malariae molecular markers of sulfadoxine-pyrimethamine resistance in humans and wild Anopheles mosquito populations in Cameroon. Anopheles mosquito collections and parasitological survey were conducted in villages to determine Plasmodium species infection, and genomic phenotyping of anti-folate resistance was accomplished by sequencing the dihydrofolate-reductase (dhfr) and dihydropteroate-synthase (dhps) genes of naturally circulating P. falciparum and P. malariae isolates. The malaria prevalence in Elende was 73.5% with the 5–15 years age group harboring significant P. falciparum (27%) and P. falciparum + P. malariae (19%) infections. The polymorphism breadth of the pyrimethamine-associated Pfdhfr marker revealed a near fixation (94%) of the triple-mutant -A16I51R59N108I164. The Pfdhps backbone mediating sulfadoxine resistance reveals a high frequency of the V431A436G437K540A581A613 alleles (20.8%). Similarly, the Pmdhfr N50K55L57R58S59S114F168I170 haplotype (78.4%) was predominantly detected in the asexual blood stage. In contrast, the Pmdhps– S436A437occured at 37.2% frequency. The combined quadruple N50K55L57R58S59S114F168I170_ S436G437K540A581A613 (31.9%) was the major circulating haplotype with similar frequency in humans and mosquitoes. This study highlights the increasing frequency of the P. malariae parasite mostly common in asymptomatic individuals with apparent P. falciparum infection. Interventions directed at reducing malaria transmission such as the scaling-up of SP are favoring the emergence and spread of multiple drug-resistant alleles between the human and mosquito host systems.

M alaria in Africa is principally caused by Plasmodium falciparum with Plasmodium malariae, Plasmodium ovale, and Plasmodium vivax considered as minority species.Efforts to control and eliminate malaria have been accelerated by anti-malarial drugs with control interventions such as intermittent preventive treatment in pregnancy (IPTp), perennial malaria chemoprevention (PMC), and seasonal malaria chemoprevention (SMC) employing anti-folates notably sulfadoxine-pyrimethamine (SP) as a chemopro phylaxis regimen for P. falciparum infections in many areas of Africa (1).Chemopreven tion is known to be a generally safe and cost-effective strategy for targeting the most vulnerable populations to malaria.This strategy has yielded striking success in shrinking the global malaria mortality curve, particularly for pregnant women and infants (2,3).However, the overreliance and indiscriminate use of the drug have been major multiplying factors for drug resistance which present an obstacle in malaria control where it has contributed to the rebound in malaria cases in Africa from 211,000,000 cases and 577,000 deaths in 2015 to 234,000,000 cases and 609,000 deaths in 2022 (1).Recent reports by the World Health Organization (WHO) indicate that the emergence and spread of Plasmodium parasite resistance by Anopheles mosquitoes threatens to reverse the gains achieved in malaria control over the past decade (1,4).The increasing occurrence and spread of drug resistant alleles render a threat to malaria treatment efficacy in pregnancy and SMC in which SP remains the frontline intervention in Africa, thus emphasizing the need for continuous monitoring and surveillance (5).
The anti-folate SP is a slow-acting combination drug with a prolonged elimination half-life of up to 14 days in blood, usually employed as a chemoprophylaxis strategy against malaria.The drug inhibits two key enzymes in the P. falciparum folate metabolic pathway notably dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) (6).Failure of SP therapy is associated with the accumulation of point mutations in these two parasite genes, and mutations in the genes encoding these enzymes have been validated as causal variants of SP drug resistance in field parasite isolates (7).The landscape of P. falciparum resistance to pyrimethamine is mediated by a stepwise progressive selection of a series of non-synonymous point mutations on codons 50, 51, 59, and 164 originating from the ancestral S108N mutant allele.Parasites harboring these N 51 I-C 59 R-S 108 N triple-mutant alleles often exhibit marked reduction to pyrimethamine susceptibility in vitro with a higher risk of SP treatment failure in clinical surveys (8).Moreover, the presence of an additional I164L quadruple mutant is known to confer complete SP clinical failure as observed in various studies across Africa, Southeast Asia, and Latin America (9).Similarly, P. falciparum resistance to sulfadoxine is primarily driven by the presence of the A437G and K540E codon mutations in the dhps backbone.This becomes programmatically relevant for malaria control as the increasing frequency of the quintuple mutation (Pfdhfr IRN + Pfdhps GE) in local parasite populations is predictive of complete SP clinical tolerance (10).Key mutations in the dhfr and dhps genes have been exploited as molecular markers for mapping, inferring, and tracking the frequency of circulating drug-resistant parasite haplotypes in real time and geography (11).Based on this, the WHO recommends that the use of IPTp and PMC with SP be implemented based on a >50% frequency threshold of the dhps 437G + 540E allele (5,12), further emphasizing the need for routine geographical surveillance to guide malaria interven tion policy decisions.
While an enormous data set exists for P. falciparum, there is a paucity of informa tion on the drug resistance polymorphism profile of P. malariae to conventional SP drugs.This becomes very important as increasing epidemiological evidence is bringing to prominence the occurrence of P. malariae as an important parasite species often co-niching with P. falciparum in high-malaria transmission settings such as Cameroon (11).Unfortunately, research on P. malariae biology remains largely neglected and lagging because it is less frequently recognized to account for malaria cases although PCR detection has identified this parasite species to be more common and persistent in recent years than previously thought (13).Consequently, it is empirical to determine whether SP pressure has selected dhfr and dhps genetic variants in circulating P. malariae populations since chronic, low-density asymptomatic infection is a successful biological adaptation unique to this species (14).Although P. malariae infections are not usually treated directly with SP, the high frequency of mixed infections with P. falciparum and non-falciparum species common in malaria-endemic settings implies that a large genome pool of P. malariae parasites has inevitably been exposed to indiscriminate SP therapy.This ultimately creates an enabling environment for the emergence of drugresistant alleles that may compromise treatment success.
In the quest of mapping resistance epidemiology, Plasmodium drug resistance typing has traditionally relied on the blood stage of the parasite in the human host without much attention to the mosquito vector (15,16).Limited data exist on Plasmodium midgut and salivary gland life cycle stages in the mosquito which otherwise constitutes a key developmental phase responsible for sexual fertilization and parasite genetic recombination.Moreover, allele frequency may differ between the two host systems due to super-infection, multiplicity of infection, and immune clearance in humans, as well as bottleneck checkpoints, genetic recombination, and fitness cost in mosquitoes which could impact the disparate transmissibility of mutant parasite variants.Thus, this study characterizes the genetic determinants of the dhfr and dhps backbones involved in SP resistance in P. falciparum and P. malariae parasites in the Anopheles and human host systems.

Comparison of Dhfr and Dhps Genes between P. malariae and P. falciparum
Nucleotide alignment of indigenous Cameroonian Pmdhfr and Pmdhps isolates with the equivalent P. falciparum dhfr and dhps GenBank reference sequences reveals a sequence homology score of 72% and 80%, respectively.Similarly, the A/T nucleotide base content for both parasite species is 75% for Pfdhfr, 75% for Pfdhps, 73% for Pmdhfr, and 75% for Pmdhps, suggesting a high-mutation plasticity rate.

Polymorphism profile and genetic diversity statistics of Pfdhfr and Pfdhps gene sequences from P. falciparum-infected Anopheles mosquitoes and human populations
A 635-bp fragment of the dhfr gene (spanning nucleotide 40 to 633) was amplified from the P. falciparum parasite stages in the Anopheles mosquito vector and the human host.A total of 234 samples (70 H/T isolates + 89 midgut isolates + 17 mixed mos quito stages + 58 human blood stage) were sequenced (Table S1).Combined multiple sequence alignments with the PlasmoDB reference (PF3D7_0417200) reveal varying near-fixation frequency of singleton mutant alleles in drug resistance-associated loci [N51I (93.2%);C59R (93.6%);S108N (98.3%)] in both the human and mosquito host systems (Fig. 2).Similarly, the frequencies of the double-mutant alleles -A 16 I 51 C 59 N 108 I 164 and -A 16 N 51 R 59 N 108 I 164 -were 2.9% (n = 7) and 1.3% (n = 3), respectively; and a triple AIRNI allele frequency of 94.4% (n = 221) as shown in Table S1 to S5.The frequency of the triple-mutant alleles was similar between the human (94.8%) and mosquito stages (H/T: 91.4%, midgut: 95.5%).The wild-type allele (ANCSI) circulated at a generally low prevalence of 3.4% predominantly in the midgut (n = 3/89) as outlined in Table S5.
No mutant alleles were detected at codons 16 and 164 in the Pfdhfr gene.Genetic diversity statistics of aggregated Pfdhfr stage-specific population reveals five haplotypes, dominated by the mutant parasite cluster (H2) with a reduction in diversity (Hd = 0.115) and a negative Tajima value (D = −0.96879)(P > 0.10) as shown in Table S6.Maximumlikelihood phylogeny employing the Hasegawa-Kishino-Yano (HKY) model shows five clades (Fig. 3) principally sized by the mutant sequence types (H2: n = 221) followed by the wild (H1: n = 4) and minority isolates (H3 to H5) as seen in Fig. 4.There was no marginal difference (P = 0.83) in the haplotype diversity between distinct mosquito stages and human asexual forms.

DISCUSSION
Accurate knowledge of drug resistance epidemiology is fundamental for malaria control and elimination.This becomes important as efforts to control malaria are encountering hurdles arising from the silent burden of asymptomatic infection (17) and the emergence of drug resistance (18).Molecular detection methods such as PCR have illuminated fine insights into parasite species composition, revealing the hidden burden of P. malariae which often presents as asymptomatic sub-microscopic parasitemia (19).Here, we report evidence of the increasing circulation of P. malariae as a significant cause of malaria in Cameroon often in mixed infection with P. falciparum, consistent with previous data documenting a high proportion of P. malariae infection in mosquitoes (11).This high P. malariae prevalence can be attributed to expanded access to anti-malarial interventions mainly directed to the treatment of P. falciparum infection while indirectly favoring the silent rise of the former and selecting for mutations in drug-resistant backbones.Indeed, the adaptive survival of P. malariae is sustained by a 72-hour life cycle charac terized by waves of slow multiplication in infected erythrocytes before eventual lysis.Despite the high Pf/Pm co-infection prevalence, there was a reduction in P. malariae parasite density compared with P. falciparum.Inter-parasite species competition could be responsible for the comparatively low P. malariae parasitemic biomass as observed in sympatric infection.The superiority of P. falciparum in Cameroon still poses a challenge to the national malaria control program as the deployment of therapeutic interventions is in parallel favoring the emergence and spread of drug resistance.In addition, the high frequency of PCR-identified P. falciparum not detected by the conventional RDT poses a major concern particularly with the paucity of data on histidine-rich protein-2 gene deletion in Cameroon.Such P. falciparum diagnostic "refugia" phenotypes may compromise the performance and shelf-life of RDTs in malaria detection accounting for the substantial false negatives.
On the other hand, the malaria cycle involves Plasmodium parasites encountering dramatic environmental changes during transition between the mosquito and human hosts, which could impact fitness, population size, allele composition, and differential transmissibility of variants (20).Indeed, anti-malarial drugs and immune response in humans as well as population bottlenecks during ookinete migration across the midgut barrier and sporozoite invasion of the salivary gland in the mosquito definitive host can shape the overall landscape of parasite diversity and drug resistance allele frequency.In this regard, data generated from this study show the presence of mutations in the Pfdhfr/Pfdhps and Pmdhfr/Pmdhps gene backbones suggesting a directional selection of resistant parasite sub-populations by continuous SP drug pressure in the human host that are transmitted by mosquitoes (21).These mutations drive the evolution of SP resistance that led to the withdrawal of the drug as a monotherapy option against malaria (22).While the current SP formulation combines a partner drug, the persistent drug pressure still selects for drug resistance phenotypes that may culminate in the loss of drug efficacy for malaria control.This is supported by the saturation of the Pfdhfr IRN triple-mutant alleles in synergy with a high frequency of the Pfdhps A437G mutation in both host systems suggesting that resistance alleles to pyrimethamine and sulfadoxine are increasingly selected in the field (23,24).This is of major concern since the Pfdhps 437G marker is associated with partial parasite resistance to sulfa drugs.The Pfdhps K540E mutation linked with full resistance was identified only in a single isolate, confirming the low prevalence of this allele in Cameroon similar to previous studies (23,24).
Moreover, the novel Pfdhps I 431 V allele previously detected in Nigeria and observed to be spreading across Central Africa (25,26) including Chad and the Democratic Republic of Congo was identified in this study at a frequency of 27.1%.This allele exhibited similar frequency (30.8%) in the H/T and human stages although lower in the midgut stage (18.1%),possibly because of sporozoite population expansion in the salivary gland from ruptured oocyst that successfully traversed the midgut barrier (20).Molecular docking evidence from Oguike et al. (27) showed that the I 431 V mutation enhances resistance to SP by lowering the binding affinity of the drug to the dhps enzyme target.Although the effect of the Pfdhps-431V mutation on SP resistance is not yet known, the progressive increase of this mutant allele is of prime concern to regularly monitor its impact on the effectiveness of SP-based IPT programs in Cameroon.Hence, the increasing frequency of this I 431 V co-existing with the A437G among heterogeneous parasite populations raises an alarm on the long-term therapeutic longevity of SP in Cameroon.
Despite this observation, the extensive use of antibiotics such as Cotrimoxazole (containing trimethoprim and sulfamethoxazole ingredients) known to select for dhfr/dhps mutant alleles could also be directly contributing to resistance expan sion.Nonetheless, molecular surveillance data from this study highlight that routine implementation of SP as prophylaxis to prevent P. falciparum infection in pregnancy or in combination with amodiaquine (SP + AQ) for SMC programs in children in Cameroon is still effective despite the presence of mutations.
Furthermore, a comparison of polymorphism frequency in the genes mediating anti-folate sensitivity shows that P. malariae has encountered a relatively lower impact of SP selection pressure compared with P. falciparum even though both parasite species have been exposed to the same drug treatment over decades and this can be linked to the multiplication cycle of the former in infected erythrocytes.The present study provides the first genetic epidemiological data of P. malariae in Cameroon by character izing the polymorphism profile in known SP drug resistance markers from the human intermediate and the mosquito definitive host systems of the parasite life cycle.The high frequency of the double L 57 R 58 -mutant haplotypes in the Pmdhfr backbone suggests a strong selection force imposed by pyrimethamine that has favored the emergence of these polymorphisms.The presence of heterozygous mixed alleles in the Pmdhfr asexual blood stage may suggest super-infection by distinct parasite genotypes arising from multiple infectious bites.Similar to P. falciparum, P. malariae is considered to acquire resistance to pyrimethamine more rapidly than sulfadoxine and this explains the reason for the high frequency of multiple polymorphisms in the dhfr than dhps backbones, consistent with previous studies (28,29).The low frequency of the Pmdhfr S 114 N polymorphism in humans and the observed distant clustering pattern of this polymorphism with Thailand isolates may suggest the local independent emergence of this allele in Cameroon.In addition, a high proportion of the Pmdhfr isolates harboring mutations at amino acids S 58 R was observed alongside a low frequency of the S 114 N that corresponds to generic codon positions C 59 R and S 108 N in Pfdhfr, respectively.Sequen tial accumulation of these mutations in the Pmdhfr backbone could have probably originated from the C 59 R ancestral residue as inferred from its high frequency.While it has been demonstrated through phenotypic studies that the 108 N and 59 R primarily confer pyrimethamine resistance in P. falciparum, no data exist on whether the equivalent mutations are linked to pyrimethamine resistance in P. malariae.
Akin to P. falciparum, the Pmdhps sequence isolates reveal a high frequency of the dominant A 437 G allele in the gene backbone.This may suggest that the absence of key mutations in the sulfadoxine resistance-conferring Pmdhps gene could be attributed to detrimental fitness costs that may impact parasite survival and transmission, particularly in mixed species infection.Phylogenetic computation of the local Pmdhfr and Pmdhps sequences separated the field mutant sequences and the Thailand isolates as distinct clades suggesting divergent parasite populations further highlighting the possibility of the de novo emergence of these mutations.
Consistent with previous studies by reference (30,31), comparative assessment of P. falciparum and P. malariae dhfr parasite populations reveals similar profiles of the mutant allele frequency and diversity between intra-(within-mosquito compartments) and inter-(between humans and mosquito) host systems.However, a contrast exists for the dhps 431V and 436A alleles with higher frequencies observed in the mosquito H/T and human stages than the midgut stage.This discrepancy could be linked to continuous pressure imposed by decades of SP use that is otherwise driving the adaptation and efficient transmission capacity of parasites harboring dominant dhfr mutant alleles with minimal fitness, thus, ensuring spread of the resistant variants across the definitive (mosquito) and intermediate (human) hosts.This study underscores the heterogeneity in transmis sion of different markers of anti-malarial drug resistance; further emphasizing that not all mutations are equal in terms of transmission-carrying capacity by local mosquito populations.
Despite the detection of mutations in these key parasite genes, current research to understand the treatment susceptibility outcome of P. malariae is limited by the difficulty in conducting in vivo therapeutic efficacy studies (TES) and the challenges of in vitro parasite culture (28).However, recently established ex vivo culture has facilitated conventional anti-malarial susceptibility evaluation (32) and the possibility of validating cross-species molecular markers which would be fundamental for future studies on this neglected parasite.Further studies employing microsatellite markers will be critical in identifying both the local geographical origin of the mutations as well as uncovering the population structure of P. malariae isolates in mixed infections and between the two host systems.The experimental infection system exploiting natural P. falciparum/P.malariae parasites in wild Anopheles vectors will shed insights on the role of mosquitoes in drug resistance genotype transmission.The limitation of the current study arises from the aspect that adult mosquitoes were randomly collected in households, thereby introducing a sampling bias since multiple blood feeding is characteristic of natural mosquito populations.Thus, parasite typing from the mosquito abdomen (midgut compartment) does not differentiate between asexual stages (trophozoites, schizonts) in the blood bolus and oocyst infection particularly over successive infectious meals.Secondly, the mosquitoes and human blood samples were not collected from the same households and sampling was done at different time periods.This hindered a real-time pair-wise comparison of the allelic frequency of molecular markers between the two host systems.Thirdly, Sanger sequencing is inferior by the inability to detect polymor phisms at minor frequencies especially in high transmission areas where super-infection is common.However, this can be resolved by the next-generation sequencing (NGS) technique that detects novel mutations and a minority allele variant in mixed infections, permitting allele frequency quantification in heterozygous genotypes.Nevertheless, this study provides relevant data intersecting the human-to-mosquito phenotypic attributes of drug resistance transmission, revealing that Anopheles mosquitoes contribute to maintaining the circulation and differential transmission of mutant variants in nature on a background of optimal parasite drug selection pressure in the human host.In conclusion, this study reveals a high prevalence of asymptomatic P. falciparum and P. malariae parasites harboring multiple drug resistance-associated mutations.Despite this observation, the low frequency of parasites with the quintuple dhps540E allele suggests the continuous efficacy of SP as a chemoprophylaxis regimen in Cameroon.Therapeutic efficacy studies accompanied by molecular analysis must be routinely implemented to monitor the clinical efficacy of SP and the parasitological impact on the emergence of resistant parasites for guiding effective anti-malaria policy

Dry blood spot collection from the human population and malaria parasite determination
Demographic data were collected from each participant using a questionnaire with capillary blood collected from each participant aged between 2 years and 50 + years in Mibellon (September 2020) (36) and Elende (August 2021).In Gounougou (March 2021), only the 3 to 15 years age group was recruited for finger-prick blood sampling as previously published (37).A community-based cross-sectional design was employed to actively recruit participants.Inclusion criteria comprised of individuals willing to participate and signed an informed consent form including approval from parents/ guardian for children ≤ 18 years and residents in the village for >1 year.Exclusion criteria included non-consenting individuals, signs of chronic illness, and severe anemia (Hb level < 5 g/dL).Enrollment consists of assigning an identification code to each partici pant, and data collected were entered in a database.Temperature, weight, and height were collected for each participant.The CareStart RDT and Giemsa-based microscopic examination of blood films were used to determine the parasitological prevalence of malaria following standard protocols (38).Briefly, air-dried thin blood film was fixed in 75% methanol, and both thick and thin blood films were stained using 10% Giemsa solution for 20 min.The slides were then microscopically examined for the presence of malaria parasites.Malaria parasites were enumerated against 200 white blood cells (WBC) (or 500 WBCs in the case of very low parasitemia) in thick blood films.Parasite density was expressed as the number of parasites per microliter (μL) of blood, assuming a WBC count of 8,000 leucocytes per μL of blood.Parasitemia was categorized as low (<1,000 parasites/μL blood), moderate (10,00-4,999 parasites/μL blood), high (5,000-99,999 parasites/μL blood), and hyperparasitemia (≥100,000 µL) (39).

DNA extraction and Plasmodium molecular species identification
The abdomen (Abd) and head/thorax (H/T) of a subset of female Anopheles mosqui toes were partitioned to discriminate between midgut (denoting the non-committed blood bolus asexual, zygote, and/or oocyst stages) and salivary gland (indicating sporozoite stage) infection.Extraction of genomic DNA from the H/T and Abd of individual dissected female mosquitoes from all the localities (except Obout for which whole mosquito samples were used) was done using the LIVAK method (40).Molecu lar identification was performed to discriminate against An.funestus siblings and the An.gambiae s.l species complex (41).Furthermore, the TaqMan assay employing the real-time PCR MX 3005 (Agilent, Santa Clara, CA, USA) system was used to detect Plasmodium infection in DNA extracts of wild Anopheles mosquito populations as previously published (11).In contrast, Whatman paper dry blood spots (DBS) were obtained from each human participant for genomic DNA extraction and molecular analysis of Plasmodium species composition.Genomic DNA was extracted from two dried blood spots on Whatman paper using chelex-100 (42) .

P. falciparum and P. malariae dhfr and dhps PCR gene amplification
The samples positive for P. falciparum and P. malariae from infected mosquitoes and human samples were each subjected to separate dhfr (28,45) and dhps (29) nested PCR gene amplification.A 20-µL primary PCR volume was prepared for each gene amplification constituting 5 µL of the genomic DNA extract, 0.51 µL each outer forward and reverse primers, 0.12 µL each kappa Taq enzyme (Kappa Biosystems, Wilmington, MA, USA), 1.875 µL BSA and dNTP mix, 0.75 µL MgCl 2 , 1.5 µL kappa Taq buffer, and 11.62 µL distilled water.Similarly, the nested PCR followed the same master mix composition as the primary except that inner forward and reverse primers were used.After amplification, the nested PCR products of both genes were loaded on 2% agarose gel to reveal appropriate band sizes.Exo-SAP purification was done on randomly selected amplicons and sequenced commercially (Microsynth Seqlab GmbH, Germany) using the Sanger method.

Data analysis on malaria prevalence and associated indicators
Data collected were entered in MS Excel, imported into, and analyzed using GraphPad Prism V8 (GraphPad Software, La Jolla, California USA).Participants were categorized into <5 years, 5-15 years, 16-30 years, and >30 years age groups.Continuous variables were summarized into means and standard deviations (SD), and categorical variables reported as frequencies and percentages were used to evaluate the descriptive statistics.The differences in proportions were evaluated using Pearson's χ 2 .The level of agreement between parasite prevalence estimates determined by RDT and microscopy was inferred using Cohen's kappa test.Significant levels were measured at a 95% confidence interval with statistically acceptable differences set at P < 0.05.

Sequence analysis and genetic phylogeny
Sequence data FASTA files from the dhfr and dhps genes of P. falciparum and P. malariae were base called using BioEdit software and then submitted to the standard nucleo tide basic local alignment search tool (BLAST) database search program to confirm the sequence identities.The P. falciparum sequences were then aligned against the 3D7 wild-type Pfdhfr (PF3D7_0417200) and Pfdhps (PF3D7_0810800) sequences for reference obtained from PlasmoDB (www.Plasmodb.org).The P. malariae dhfr sequen ces were aligned with the Thailand isolates (Accession numbers: AY846633, AY846634, EF188271, EF188273, EF188274, and KX672034) as reference (46).Similarly, the Pmdhps isolates were matched with Thailand GenBank samples (Accession numbers: KJ400020, KJ400021, KJ400022, KJ400023, KJ400024, KJ400025, KX672040, KX672041, KX672042, and KX672043) (46).Particularly, the Pfdhfr and Pfdhps gene sequences were scanned for previously identified point mutations in resistance-associated codons.Identified SNPs related to drug resistance molecular markers were classified as either wild, mutant, or mixed.However, mixed genotypes were considered as mutant when constructing haplotypes.The haplotype diversity (the number of two random strains within the population having different haplotypes) of Pfdhfr/Pfdhps and Pmdhfr/Pmdhps in each sequenced field isolate was determined by exploring the variants in the coding region of the gene.DnaSP software (version 6.10.01) was used to interrogate the genetic diversity metrics of the parasite population per the host system and locality.Genealogi cal relationship between individual parasites and haplotypes was generated by MEGA X (47), Templeton, Crandall, and Sing (TCS) haplotype network and PopArt software (48).editing | Charles S. Wondji, Conceptualization, Data curation, Funding acquisition, Project administration, Resources, Supervision, Validation, Writing -review and editing

FIG 1
FIG 1 (a) Age stratification of malaria prevalence and (b) Plasmodium speciation in Elende.
Full-Length Text absence of mutations at codons 581 and 613 in the Pfdhps gene (Tables

FIG 3
FIG 3 Phylogenetic map of the sequenced Pfdhfr isolates revealing homogenous parasite cluster.

FIG 4
FIG4 Haplotype network of the sequenced Pfdhfr isolates identifying a dominant mutant population.

FIG 5
FIG 5 Phylogenetic plot of the sequenced Pfdhps isolates showing distinct population clusters.

FIG 6
FIG6 Haplotype network of the sequenced Pfdhps isolates identifying unique parasite assemblages.

FIG 7
FIG7 Phylogeny map of the sequenced Pmdhfr isolates revealing a high evolutionary relatedness of a major mutant clade.

FIG 8
FIG 8 Haplotype network of the sequenced Pmdhfr isolates identifying two dominant mutant parasite populations.

FIG 9
FIG9 Dendrogram of the sequenced Pmdhps isolates revealing cluster relatedness of the mutant parasite population backbone.

FIG 10
FIG10 Haplotype structure of the sequenced Pmdhps isolates identifying multiple mutant parasite lineages.

TABLE 1
Malaria prevalence and multispecies Plasmodium infection frequency in Elende