The aim of this study was to perform a molecular characterization of highly polymorphic markers in
P. falciparum clinical isolates. In this first study, conducted after the introduction of artemisinin-based combination therapy (ACT), it was observed that
P. falciparum field isolates in Cameroon exhibited a high degree of genetic polymorphism in
P. falciparum msp-
1 and
msp-
2 markers. These genes encoding for individual functional proteins expressed on the surface of the merozoite appear to play an essential role in the invasion of the red blood cell [
39] and are of interest as potential vaccine candidates and as drug targets for inhibiting blood-stage replication [
40]. This study identified a high proportion of multiclonal isolates and MOI and a total of 16 different
Pfmsp-1 and 27 different
Pfmsp-
2 gene types. This figure is not exhaustive since it is obvious that with nested PCR genotyping some of the sub-populations present in mixed infections would not be fully typed by all amplification steps [
21]. Moreover, more amplified product was found for the
msp2 gene than
msp1, this could be explained by non-synonymous substitutions introduced in template DNA that could jeopardize the proper annealing of the primer at its binding site in
msp1 gene or could be explained by the fact that natural selection is more efficient when acting on
msp-
1 than
msp-
2 [
39,
41]. This suggests that MSP-1 as compared with MSP-2 proteins are under strong functional constraints in a complex interaction with the host leading to an increased in host’s immunological response. The two markers including
msp-
1 (16 genotypes) and
msp-
2 (27 genotypes) revealed considerably greater parasite diversity than
glurp (5 genotypes). Thus, the observed genetic polymorphism in the these two
P. falciparum major merozoite surface proteins,
Pfmsp1 and
Pfmsp2 could be explained by balancing selection occurring as a result of different mechanisms of interaction with the host [
42]. This distribution of families of
msp-
1 and
msp-
2 and their allelic variations were similar to that reported from other countries with meso- to high endemicity of malaria [
43]. Genotyping procedure recommended in anti-malarial drug trials stipulates consecutive analysis of the three markers starting with
msp2 or
glurp, and then
msp1 [
23]. Based on allelic profile of each gene obtained in this study, parasitological outcome assessment could be more accurate when both markers
msp-
1 and msp-
2 are included in the genotyping of recurrent parasitaemias in anti-malarial drug trials, consistent with other studies [
21]. The results of this study equally raise concern over the use of
glurp genotyping in anti-malarial drug trials since this marker showed limited allelic families as compared to
msp-
1 and
msp-
2. However, genotyping by SNPs and indels employing NGS could show more allelic families than identified by nPCR [
44,
45].
Genetic diversity for msp-1 and msp-2 allelic families
The present study reported higher numbers of alleles (43 alleles for both MSP-1 and MSP-2) than previously reported to be circulating in the study area and in the central region of Cameroon [
11,
24]. A peculiarity of this study is that, RO33 was found to be monomorphic and the most predominant allele type of msp1 compared to the polymorphic K1 and MAD20 allelic families in agreement with previous studies where RO33 was found to be the most predominant allelic family for
msp1 locus [
19,
35,
43]. However, the results of this study does not corroborate other studies in which RO33 was identified as the least predominant allelic variant type while MAD20 was the most predominant allelic family of the
msp1 locus [
16,
24]. This discrepancy could be attributed to the difference in the degree of transmission intensity. Another peculiarity of our study is that, the RO33 family of
msp-1 did not show any polymorphism, with only 1 variant (155 bp) detected. This result differs from that of Gabon and West Uganda, where the Ro33 family was polymorphic with three and four allelic variants, respectively [
8,
46], but was close to that in Senegal [
47], and Brazil [
48], showing the monomorphic nature of RO33 family of
msp-
1. The allelic variant K1 was the second highly distributed after RO33. This does not corroborate previous finding in the same region of Cameroon [
24], but is consistent with most findings in areas of holoendemic, mesoendemic, and hyperendemic malaria, in which the allelic variant K1 was predominant [
19]. The predominance of RO33 and K1 allelic family could be attributed to the balancing selection acting on these two allelic variants. The MAD20 allelic variant was the least predominant among the
Pfmsp1 allelic family in agreement with other studies conducted Africa including the Gambia, Nigeria, and Gabon [
35]. This result does not corroborate with other studies reporting the predominance of MAD20 allelic variant over K-1 and RO33 allelic variants [
13,
19]. The low distribution of MAD20 allelic variant could be partly explained by purifying selection acting on MAD20 allelic type as compared with RO33 and K1 allelic type. This could equally be attributed to a single nucleotide substitution in DNA template that hinders proper annealing with primers designed to amplify the MAD20 allelic type [
49]. An association between the distribution of K1 allelic families with severe malaria has been investigated [
43], while the RO33 allelic family has been frequently reported in asymptomatic malaria cases [
16,
50]. The predominance of RO33 seems to be less harmful for the host since the presence of this allele type is related to reduced risk of clinical malaria [
43,
51].
A significant correlation has been established between the genetic diversity of the
msp-1 gene of
P. falciparum and parasite density. Likewise an association has been observed between
msp-
1 allele diversity and age group on one hand, and between
msp-
1 allele diversity and gender among asymptomatic patients on the other hand [
52].
The
msp-
2 allelic families IC/3D7 and FC27 were almost of equal frequencies which is consistent with other findings [
36]. In contrast, previous reports showed a significant predominance of FC27 over the 3D7/IC allelic family [
16]. The frequencies of individual
msp2 genotypes were low with 88.9% occurring at a frequency ≤ 10%. However 2 (16.7%) genotypes, belonging to the FC27 and 1 (6.7%) belonging to the 3D7/IC allelic family were found at frequencies of above 10%. High genetic diversity and low allelic frequencies have been reported previously from other sites including Gabon [
8], Uganda [
22], Senegal [
53], and Burkina Faso [
54].
Diversity, expressed as expected heterozygote (
He), ranged from 0.55 for
msp-
1 to 0.96 for
msp-
2 suggesting that the parasite population in Cameroon, exhibits intermediate to higher heterozygosity reflecting intermediate to high transmission pattern [
14] consistent with the findings in Uganda, Congo and Zimbabwe, showing a high heterozygote ranges between 0.78 and 0.8 for
msp-
2 [
16,
55]. The correlation between the genetic variation of
P. falciparum and malaria endemicity has been described [
16,
46]. In areas with declining endemicity, it is reported that the number and diversity of alleles decrease with decreasing
P. falciparum transmission [
55]. The present study reveals that the
msp-2 gene is highly polymorphic as compared with
msp 1 gene. This observation is different in low transmission settings where high diversity has been recorded for
msp-1 as compared with the
msp-2 gene [
13]. Hence, the high allelic diversity together with the low frequency of individual circulating alleles observed in the present study increase the discriminatory power of
msp-1 and
msp-2 to differentiate between recrudescence and re-infection. Thus, this study reinforce the importance for the genotyping of
P. falciparum based on
msp-1 and
msp-2 in anti-malarial drug efficacy trials, to distinguish between re-infection as recrudescence and emphasize on the importance of implementing
msp-
1 and
msp-
2 genotyping in effective malaria management and malaria control strategies in Cameroon and in other endemic areas.
Polyclonal infection expressed as MOI values
In this study, most patients with
P. falciparum infections were infected with multiple genetically distinct parasite variants with high level of polyclonal infections observed among
msp-2 (88.6%) and
msp-1 (33.8%) positive isolates. Such a pattern of parasite structure is typical among
P. falciparum populations in areas of high transmission, where more than 10 variants can be routinely detected in an individual [
41], and selection among these variants in the host is likely to play an important role in parasite diversity. In contrast, in areas of low transmission, such as in Asia or in Latin America, patients may have infections with as few as a single variant [
17]. No data on MOI was available in Cameroon before the introduction of ACT. This renders it difficult to draw a conclusive statement on the impact of ACT on MOI. Nevertheless, in this study, the polyclonal infection expressed as the MOI values were heterogeneous across the different loci, and the mean MOI was highest for
msp-2 than
msp-
1 in accordance with previous studies in neighbouring countries with high intensity of malaria transmission including Congo and Gabon [
56]. Higher MOI in Cameroon can be the results of multiple infectious mosquito bites or transmission of genetically diverse sporozoite inoculum from a single mosquito bite. Genetically distinct malaria parasites in natural populations have an extremely high rate of genetic recombination during the sexual stages resulting in multiple strains being transmitted simultaneously [
57]. Effective recombination of parasites and mutation occurring in several rounds of DNA replication cycles will likely continue to maintain this genetic diversity. During genetic recombination, novel combinations of alleles can be generated offering beneficial features to the parasite, as driven by positive selection enabling spread alleles through the population. Since the evolutionary selection of malaria occurs both within individual hosts and within populations, determining the number of strain in an infection might be an important indicator of transmission intensity [
58]. Effective malaria control measures (ACT, distribution of ITNs) have successfully reduced malaria transmission in many hyperendemic regions of sub-Saharan Africa. After an intervention, the malaria parasite population structure and transmission rate in these regions is expected to become similar to the low transmission rates of the regions of Southeast Asia and South America. The declining malaria transmission, as a result of scaling up interventions, has been shown to affect the genetic diversity pattern and population structure of
P. falciparum [
18]. Therefore, the high MOI observed in this study reflects a high intensity of malaria transmission in Cameroon, despite several control strategies deployed at the health facilities and in the community. This is in agreement with previous findings that observed an increase of MOI with an increase in malaria endemicity and a low MOI for
msp-1 and
msp -
2 correlated with a low intensity of malaria transmission [
17].
Therefore, regular molecular epidemiological surveys need to be performed in order to monitor the genetic diversity of
P. falciparum populations in different regions of Cameroon, and then correlate parasite genotypes to the disease phenotypes [
52,
59]. Previous studies reported a reduced risk of clinical malaria associated with polyclonal infections and high rate of severe malaria in individual harbouring mono-infections and very common genotypes [
60]. This study showed an increase in the mean MOI according to parasite density, but not according to the gender of patients which is consistent with previous studies showing significantly high MOI in patients with moderate to high transmission [
57]. In the present study, most of the positive samples were from children aged 0–5 years, and this limited range of age constraint examining the correlation between the MOI and age. However, previous studies showed a pattern of greater MOI in older individuals than younger individuals reflecting more previous exposure to infection [
61] while conflicting findings, indicated decreased MOI with age [
62]. Thus, determining the MOI in endemic areas is very important since it can be used to predict clinical outcome and target population with higher attention.
Genotyping by gel electrophoresis and direct sequencing
In this study, the polymorphic surface antigens
msp2,
glurp, and
msp1 genes for 304 isolates were successfully amplified by primary and nested PCR and genotyped using agarose gel electrophoresis of which 127
msp-
1 and 297
msp-
2 gene fragments with single band were selected for further characterization by direct sequencing for identity and MSA purposes, but not to determine the entire parasite population. In this study, the lengths of the repeat units, whose number varies between the different allelic variants, was taken into account to set bin width of 20 bp for
msp-
1 and
msp2 then 50 bp for
glurp [
14]. However, variations in bin width [
22] and the different fragment sizing methods need to be standardized to facilitate the comparison of data for a particular marker between studies. Moreover, genotyping using agarose gel electrophoresis only groups alleles of similar size, but cannot distinguish the presence/absence of SNPs across the gene markers. Length polymorphic markers could introduce bias during the amplification process as this method is known to preferentially amplify shorter fragments [
37,
63]. Although, genotyping method using gel electrophoresis may face some confounding factors including the variability in the electrophoretic migration of a given DNA fragment, this genotyping method could be very important where alternative method are not available and is widely used for
P. falciparum genotyping [
27,
28,
36,
64,
65]. Capillary electrophoresis-PCR (CE-PCR) was not used in this study, which could increase the allele resolution in an agarose gel by determining differences between 2 and 3 bp sizes among bands [
14]. Beside, capillary electrophoresis is recognized to have higher resolution power than gel electrophoresis in the ability to distinguish between allelic variants of amplified fragments [
20]. However, PCR artefacts are a challenge for this method and MOI determination is often underestimated not only with gel electrophoresis but also with capillary electrophoresis analysis especially when genotyping strategy do not consider separate nPCRs for each allelic family [
21]. In contrast to previous studies, a separate nPCR followed by direct sequencing was performed in the present study enabling accurate determination of the fragments sizes for
msp1 and
msp2 allelic families as well as sequence motifs and nucleotide differences. The development of single-nucleotide polymorphism-based (SNP) genotyping techniques and next-generation sequencing (NGS), might provide highly diverse haplotype markers with sufficient resolution to detect minority population in a mixed infection [
44,
45]. Thus, next-generation sequencing technologies and genome-wide characterization is an alternative strategy to accurately analyse polyclonal infections although complete haplotype characterization of multiclonal infections remains a challenge due to PCR artefacts and sequencing errors [
66]. Therefore, in order to accurately study the competition and selection between variants in mixed malaria infection, new tools, more sensitive to detect minority populations and quantitative for relative parasite population sizes have being developed including DADA2, PASEC, HaplotypR, SeekDeep among others [
45].
Another strategy could be the combination of sequencing method and efficient computational tool for an effective characterization of allelic variants. A good number of software packages are being developed to analyse genome-wide SNP data of field isolates for the estimation of the presence of multiple genotypes, especially minor allele in multiclonal infections [
67]. However, DNA electrophoresis followed by direct sequencing should be used where alternative method are not yet available while waiting for the implementation of newly developed methods for allele genotyping, especially in the surveillance of malaria transmission. This would provide a guideline to policy makers to redefine the malaria control strategies.
Sequence analysis of genetic polymorphism of msp-1 and msp-2
This study showed highly diverse nature of
P. falciparum isolates of Cameroon in respect to length and sequence motifs. Sequencing and gene alignment confirmed the identity
Pfmsp1 and
Pfmsp2 polymorphisms. Thus, when performing the gene alignment, high similarity was observed between the peptides of
Pfmsp1 and
Pfmsp2 in Cameroon and those of other regions in Africa. However, from all peptides analysed, the region of the alignment corresponding K1 polymorphism had the highest similarity among all the species in the
Pfmsp clade included in this study ranging from 93% to 99% homology with previously described polymorphism in isolate from Kenya and Tanzania. The MAD20 peptide sequence polymorphism was the second most conserved with 83% to 100% homology between
P. falciparum isolates in Cameroon and those of other regions of Africa as well as with those of other regions of the world. Thus, the development of a vaccine based on K1 and MAD20 allelic variant could likely be effective in providing immune protection against malaria in those regions in Africa, although it is not yet known to what extent the high allelic diversity within the K1-like and MAD20-like allelic types is of immunological significance [
42].
However, previous analysis indicated more serological variation among the allelic sequences of the K1-like compared to the MAD20-like type [
31], and more effort has been made to incorporate the repeat sequence variation of the K1-like alleles in recombinant antigens towards design of a multivalent vaccine [
41,
49] and more than 500 different
msp1 block 2 allelic sequences has been described, providing a reference for molecular epidemiological studies and potentially for design of a multi-allelic vaccine [
42]. Sequencing and immunological characterization of other allelic variants such as MAD20 for
Pfmsp1, alongside with 3D7/IC for
Pfmsp 2 should be conducted to obtain more useful information.