Genetic diversity of Plasmodium falciparum and genetic profile after artemisinin-based combination therapy (ACTs) deployment in Cameroon for the management of uncomplicated malaria in Children CURRENT STATUS: POSTED

Background Plasmodium falciparum is the number one cause of malaria morbidity and mortality. Several methods of intervention have been deployed in Cameroon with an attempt to reduce malaria transmission. But evaluation methods mostly based on microscopy and immunology have proven to be cumbersome and expensive. This study aimed at analyzing the genetic diversity of P.falciparum and the impact of ACTs deployment on MOI Method 350 clinical isolates were collected between 2012 and 2013 and, three P. falciparum loci namely, msp-1(block2), msp-2 (block3), and glurp, (region II) characterized by nested PCR and DNA sequencing. Results From this study, a total of 16 different pfmsp1 were identified, including K1, MAD20 and RO33 allelic families. The K1 and MAD20 were the predominant polymorphic allelic types at the msp-1 gene, whereas alleles belonging to 3D7/IC were more frequent at the msp-2 gene. A peculiarity of this study is that RO33 revealed a monomorphic pattern among the msp-1 allelic type. Msp-1 and msp-2 revealed considerably greater parasite diversity than glurp. A total of 27 different msp-2 genotypes were recorded of which 15 belonged to the 3D7-type and 12 to the FC27 allelic families. Alignment of peptides encoded by pfmsp1 and Pfmsp2 reveals that K1 polymorphism had the highest similarity in the P.fmsp1 and Pfmsp2 clade followed by MAD20 with 93% to 100% homology. Indicating that P. falciparum isolates from Cameroon present high identity with allelic sequences from other areas in Africa, suggesting that vaccine developed with k1 and MAD20 of Pfmsp1 allelic variant could be protective for Africa children. The MOI ranged from 2.51 for msp1 to 3.82 for msp2. The overall heterozygosity ranged from 0.55 for msp-1 to 0.96 for msp-2 consistent with the genetic pattern observed in hyperendemic areas. Conclusion The present study reveals that isolates from South West Region of Cameroon are mainly polyclonal with high MOI and highly diverse in respect to both msp-1 and msp-2 despite ACTs deployment aiming at reducing malaria transmission. This study lays emphasis on the use of MOI and genotyping of both msp-1 and msp-2 in the evaluation of malaria control intervention in malaria endemics countries.

Background Plasmodium falciparum is the number one cause of malaria morbidity and mortality.
Several methods of intervention have been deployed in Cameroon with an attempt to reduce malaria transmission.But evaluation methods mostly based on microscopy and immunology have proven to be cumbersome and expensive.This study aimed at analyzing the genetic diversity of P.falciparum and the impact of ACTs deployment on MOI Method 350 clinical isolates were collected between 2012 and 2013 and, three P. falciparum loci namely, msp-1(block2), msp-2 (block3), and glurp, (region II) characterized by nested PCR and DNA sequencing.Results From this study, a total of 16 different pfmsp1 were identified, including K1, MAD20 and RO33 allelic families.The K1 and MAD20 were the predominant polymorphic allelic types at the msp-1 gene, whereas alleles belonging to 3D7/IC were more frequent at the msp-2 gene.A peculiarity of this study is that RO33 revealed a monomorphic pattern among the msp-1 allelic type.Msp-1 and msp-2 revealed considerably greater parasite diversity than glurp.A total of 27 different msp-2 genotypes were recorded of which 15 belonged to the 3D7-type and 12 to the FC27 allelic families.Alignment of peptides encoded by pfmsp1 and Pfmsp2 reveals that K1 polymorphism had the highest similarity in the P.fmsp1 and Pfmsp2 clade followed by MAD20 with 93% to 100% homology.Indicating that P. falciparum isolates from Cameroon present high identity with allelic sequences from other areas in Africa, suggesting that vaccine developed with k1 and MAD20 of Pfmsp1 allelic variant could be protective for Africa children.The MOI ranged from 2.51 for msp1 to 3.82 for msp2.The overall heterozygosity ranged from 0.55 for msp-1 to 0.96 for msp-2 consistent with the genetic pattern observed in hyperendemic areas.

Conclusion
The present study reveals that isolates from South West Region of Cameroon are mainly polyclonal with high MOI and highly diverse in respect to both msp-1 and msp-2 despite ACTs deployment aiming at reducing malaria transmission.This study lays emphasis on the use of MOI and genotyping of both msp-1 and msp-2 in the evaluation of malaria control intervention in malaria endemics countries.

Background
In spite of enhanced control efforts, malaria continues to be a major public health problem in sub-Saharan Africa and Southeast Asia, and Plasmodium falciparum infection is prevalent in most of the endemic countries.The World Health Organization (WHO) estimated that, 219 million malaria cases occurred worldwide of which 78% was in Africa.About 407,000 fatal cases were registered, 92% in Africa and 61% of the global death in children under 5 years of age P. falciparum being the major cause of all deaths [1,2].Malaria in Cameroon is caused by three human malaria species: P. falciparum, P. ovale and P. malariae.The prevalence of P. falciparum is about 99% while P. malariae and P. ovale share the remaining 1%.The most deadly malaria is caused by P. falciparum.Malaria transmission in Cameroon depends on the population of Anopheles arabiensis, a member of the An.
gambiae Giles complex, a principal malaria vector in Cameroon [3,4].Malaria is a leading public health problem in Cameroon and is reported as the first cause of morbidity and mortality [5,6], accounting for 16% outpatient visits, 20% hospital admissions and 27% inpatient deaths [6][7][8].
Despite the current efforts to control malaria in Cameroon, the situation has not improved, mainly due to the increasing problems of vector resistance to insecticides [10] and resistance to almost all antimalarial drugs including ACTs [11][12][13][14].Merozoite surface protein (MSP-1) and merozoite surface protein-2 (MSP-2) are 2 proteins causing immune responses in humans [15,16] and are important candidates for development of blood stage malaria vaccines [17][18][19].The msp-1 gene is located on chromosome 9 and contains 17 blocks of sequences, of which 7 are variable, flanked by conserved regions [20].The block 2 msp-1 is particularly polymorphic and 3 distinct allelic families have been described as MAD20, K1 and Ro33 [21][22][23][24][25].The msp-2 gene is located on chromosome 2 composed of five blocks of which the most polymorphic is the central block 3 [26].The polymorphic central domain of the gene encoding MSP-2 belongs to 2 distinct families; 3D7 and Fc27 [27][28][29].Allelic forms of these antigen genes have been reported from different parts of the world [18,[23][24][25][26][27][28][29][30][31][32].Differences in allelic types as well as the number of repeat sequences in msp-1 and msp-2 can be detected by PCR, followed by dot-blot hybridization [33].Size polymorphism is also used not only for the msps but also for other markers including Glurp and Csp in which variations are also detected by sequencing.Genetic diversity of P. falciparum populations determines the intensity of malaria transmission [34][35][36][37], thus providing baseline data for any antimalarial drug efficacy trial and the possibility of implementing control strategies based on modern intervention or vaccines.Merozoite surface proteins 1 and 2 genotyping is widely used in malaria molecular epidemiology studies to assess the allelic diversity and multiplicity of infection as a proxy of transmission level in molecular monitoring of interventions [32,38].ThePfmsp1, Pfmsp2 and glurp are commonly selected because they are located on different chromosomes, and this reduces the likelihood of linkage disequilibrium [39,40].
Genotyping of these genes has been effectively used to trace individual clones over time in cohort studies and to measure duration of infection [41,42].Different clones of P. falciparum can be determined using regions of highly polymorphic genes coding for MSP-1 and MSP-2, and GLURP.
These markers are equally essential in distinguishing recrudescence from reinfection of the parasite in an anti-malarial treatment trial [29,[43][44][45][46], since the discriminating power of these markers is dependent on the extent of allelic diversity and on the frequency of each allele within a population [29,47].Genetic diversity of P. falciparum has been used to implement specific strategies for control and to evaluate the impact of interventions on changes in malaria epidemiology [10,111].However, in Cameroon, there are no or limited data on MOI and P. falciparum genetic diversity respectively, especially when msp-1, msp-2 and glurp need to be considered together as molecular markers in genotyping studies.Thus, the aim of this study was to characterize the highly polymorphic genetic markers of P. falciparum field isolates, including the merozoite surface protein 1 and 2 (msp-1), the merozoite surface protein 2 (msp-2) and glutamate rich protein (glurp) and to determine the allele distribution and factors influencing the MOI and heterozygosity as indicator of malaria parasite transmission dynamics Methods 1.

Study site
This study was performed in the (2,000-10,000 mm) [48].The mean annual rainfall is 2,625 mm, relative humidity is constantly high (75%-80%), and the temperature varies from 18°C in August to 27°C in March [3].In general, malaria transmission is intense and perennial in the South West region, with peak periods corresponding to the rainy season [48].Three anopheles vectors including Anopheles gambiae, A. funestus and A. nili have been identified in malaria transmission in South West Region.A. gambiae the most dominant in terms of aggressiveness and activity [3,4,22] accounts for up to 72.7% of transmission, Infection rates of 87 infective bites/person/year [4] and overall entomologic inoculation rate ( EIR) estimated recently at 3.93 infective bites/person/night [22].In addition, houses are grouped into camps which are structured buildings provided by the company to accommodate the workers and their families.

2.
Sampling and Parasite collection P.falciparum isolates were collected from microscopically diagnosed P. falciparum positive patients aged six months to 6 years, in clinics and hospitals of CDC Cameroon.Consent to participate in this study was obtained from parents or guardians and blood sample was taken from each patient using finger prick for thick and thin blood films.2-3 drops of blood were collected on 3 MM Whatman filter paper.A total of 315 blood samples were collected from 23 clinics and 2 hospitals of the CDC Cameroon belonging to 2 different health districts.

Microscopy
Blood films were stained with diluted Giemsa stain and then examined microscopically for the presence of malaria parasites; 200 fields under 1000× magnification were examined from the thick film before the slide was considered negative.For positive slides, parasitaemia (parasite density) was determined by counting only the asexual stages against 200 white blood cells (WBC) and then multiplied by 25, assuming the average of total WBC count of individuals equal to 8000 cells/μl of blood.The level of parasitaemia was graded as low (< 1000 parasites/μl of blood), moderate (1000 -9999 parasites/μl of blood) and severe (≥ 10,000 parasites/μl of blood).

Molecular identification and genotyping
Genomic DNA was extracted from blood spots collected on filter papers.Briefly, a disc of the filter paper was punched out from the blood spot using a paper puncher and placed in 1.5 ml centrifuge tubes using clean forceps.Genomic DNA was extracted using Qiagen blood and tissue kit (QIAGEN, Germany) according to the manufacturer's instruction.

Nested PCR amplification and allele detection
DNA was eluted using 50 μl AE (10 mM Tris-Cl; 0.5 mM EDTA; pH 9.0) elution buffer (QIAGEN, DNeasy® Blood & Tissue Kit, Cat.no.69506, Germany) and kept at -20°C until used for PCR.Genomic DNA was amplified by Nested PCR using allelic specific Primers (Table 1) and conditions for PCR amplification were followed as previously described [15,39,49] for family specific allele analysis of msp-1 (block 2), msp-2 (block 3) and Glurp (region II).In the primary PCR, a 25 μl PCR mixture was used containing 1 μl of DNA template, 0.2 µM of each primer, 1x TBE buffer, 250 μM of dNTPs, nuclease free water, 1 U of Taq polymerase enzyme, 2 mM MgCl2, 10 mM KCl, and 10mM Tris-HCl, at pH 8.3.All reagents were from TIAGEN (Biotechnology, Inc., Beijing).Cycling conditions for the primary PCR were as follows; starting with three single steps of denaturation at 94°C for 5 minutes, annealing at 58°C for 2 minutes and extension at 72°C for 2 minutes.This was followed by 35 cycles of denaturation at 94°C for 1 minute, annealing at 58°C for 1 minute and extension at 72°C for 1 minute, then a single annealing step at 58°C for 2 minutes and final extension at 72°C for 10 minutes.
P. falciparum genotypes were further analyzed by amplification of the two highly polymorphic regions of msp-1 (Block 2) and msp-2 (Block 3) using nested-PCR as previously described [49] with slight modifications for the cycling conditions of the secondary PCR.Briefly, oligonucleotide primers sets (Suppl.1, Table1), were used for detecting the different families (K1, MAD20 and RO33 in MSP-1; FC27 and IC in MSP-2).Three microliters of primary PCR products were used as the DNA templates in the secondary PCR which had similar concentrations to the primary PCR.The cycling conditions for the secondary PCR were as follows: starting with a single step of denaturation at 95°C for 10 minutes followed by 35 cycles of denaturation at 94°C for 30 seconds, annealing at 58°C for 30 seconds and extension at 72°C for 1 minute, and a final extension at 72°C for 10 minutes.PCR reaction mixtures were incubated in a thermal cycler.
The secondary PCR products were separated by electrophoresis at 100 volts on 1.5 % molecular grade agarose gel (Caisson, Utah, USA), stained with ethidium bromide, submerged in 0.5 X TBE (Trisborate EDTA) buffer and visualized by UV transilluminator (BioDoc-It UVP, Cambridge, UK) at 302 nm on gel documentation system.The number and size of DNA fragments were estimated based on their mobility related to a 100bp DNA ladder (Vivantis, Selangor Darul Ehsan, Malaysia).DNA fragment sizes were binned into different classes of 20 and 50 bp ranges with each bin assigned as an allele.
Alleles in paired samples were considered a match for msp2 and msp1 if within 20 base pairs and for glurp if within 50 base pairs [27,30] 5.

Multiplicity of infection (MOI) and heterozygosity (He)
Multiclonal infections were defined as those having more than one allele in at least one locus out of the loci genotyped.The MOI was determined by calculating the number of different alleles at any one locus detected in the sample [19]; single infections were those with only one allele per locus at all of the genotyped loci.Isolates with more than one genotype were considered as polyclonal infection while the presence of a single allele was considered as monoclonal infection.The mean multiplicity of infection (MOI) was determined as the quotient of the total number of P. falciparum genotypes detected in MSP-1 or MSP-2 by the number of samples positive for either msp-1 or msp-2 [50].
As a measure for genetic diversity, the expected heterozygosity (HE) which represents the probability of being infected by two parasites with different alleles at a given locus and ranging between 0 and 1 [29] was calculated by using the following formula: where n is the number of isolates sampled and pi is the allele frequency at a given locus.

Statistical analysis
Data was analyzed using the SPSS for windows software version 17.For descriptive analysis, proportion was used to present the distribution of different allelic families while the mean was used to present the multiplicity of infection (MOI).Independent chi square test was used to compare the mean MOI according to gender, and parasitaemia.A p-value ≤ 0.05 was considered indicative of a statistically significant difference.

msp-1 and msp-2 sequence analysis
Purified PCR products of isolates representing different alleles of msp-1 and msp-2 were sequenced in both directions with the primers of the secondary PCR using the ABI PRISM® BigDyeTM terminator Ready Reaction Cycle Sequencing Kit (Biometra Thermocycler, England) according to the manufacturer's instruction.The sequences were then analyzed using the DNASTAR software package (DNASTAR, Madison, WI).The sequences were used to correct the estimated molecular weight and to confirm the nature and size of the amplified product.To understand the identity of Cameroonian isolates with respect to isolates of other regions, sequence data available in public domains were downloaded for allelic families of msp-1 and msp-2 and aligned using ClustalW method (EMBL-EBI, Hixton, and Cambridge, UK), then analysed by MEGA version 5.1 (http://mega.software.informer.com/5.1b/).We sought to identify sequence differences using BLASTN sequence homology searches.Each allelic family msp-1 (including K1, MAD20 and R033) and msp-2 (including FC27 and 3D7/IC) were analyzed separately in order to estimate the average number of nucleotide substitutions for each msp-2 allele, and to examine the mode of evolution of FC27 type repeats.We assembled and aligned individual repeat units with DNAstar and BLAST searched in GenBank to analyze the distribution of the pairwise proportion of nucleotide differences among individual repeat units using isolates derived from GeneBank, namely the isolate from Gabon accession number AY372506 for FC27, the isolate HM568631 from India for 3D7/IC allelic family.As for msp-1 alleles, the isolate from Brazil (accession:JX416338) for the alignment of K1-type allele, while the isolates EU032224 from central Sub Sahara Africa and AY138508 from Iran were used as reference isolate for the sequence analysis of R033 and MAD20, respectively.

Study profile
Out of 315 DNA samples at baseline, 137 samples for msp-1 and 307 samples for msp-2 were successfully amplified by PCR, among which 107 for msp-1 and 243 for msp-2 were successfully sequenced.

Genetic diversity and haplotype frequency
The number of genotypes observed at each marker is shown in Table 2. Sixteen ( 16) different msp-1 genotypes were observed, representing K1 (9 genotypes), MAD20 (6 genotypes) and RO33 (1 genotype) allelic families.The msp-1 fragment sizes ranged from 153 bp -335 bp, while the msp-2 fragments ranged from 140 to 568 bp both for the FC27 and IC allelic families (Table 2).
The majority (87.5%) of these genotypes especially for the k1 (100%) and MAD20 (97.5 %) belonging to the msp1 allelic family occurred at a frequency below 10% (Table 2).However, one genotype the MAD20 family (167-187bp) occurred above 10%.The R033 family was found to be monomorphic with an amplified fragment size of 155 bp and occurred at a frequency of 78.1% (107/137) (Table 3) of the overall msp1 genotypes.
The glurp diversity and genotype frequencies are shown in Figure 4.A total of 5 different glurp genotypes (size range from 597 to 817 bp) were recorded with the majority of the allelic families occurring at above 10 %.However, only two alleles occurred below 10% of which, 4.67% and 9.34% belonged to the 550-600 bp and 652-702 bp allelic group.
In msp-1, more than halve of the msp-1 positive samples harbored all the three types of alleles of the msp-1 gene.Thus the combination of RO33, MAD20 and K1 allelic families was identified with an overall frequency of 57.50% (Figure 4a).The RO33 allelic family was predominant as it was identified in 78.10% (107/137) of the samples.One third (33%) of the blood samples positive for msp-1 were identified as monoclonal infections while two-third (66 %) exhibited a polyclonal pattern of K1, MAD20 and R033 combination in a set of two or three alleles (Figure 4a).Among the polyclonal infections, K1/RO33, K1/MAD20 and Ro33/MAD20 constituted 6.66%, 1.6% and 0.83% of the msp-1 positive isolates respectively.The distribution of the identified msp-2 allelic families is illustrated in Figure 4b.
Overall, 11.42 % of msp-2 positive isolates were identified as monoclonal infections either for 3D7/IC or FC27 allelic families against 88.56% msp-2 positive isolates (figure 4b) displaying a polyclonal pattern of infections.The frequency of the 3D7/IC and FC27 haplotypes combinations was found to be higher than the frequency of samples with only 3D7 or FC27 allelic families (figure 4b).

Multiplicity of infection and heterozygosity
P. falciparum isolates in this study had higher rates of multiple genotypes infection with an overall mean multiplicity of infection of 3.16.The mean MOI for msp-2 loci was the highest (3.82), as compared with msp-1 (2.51) Table 3).In the children, there was no difference in mean MOI between male and female (P=0.1)(Table 4).However, the influence of parasite density on genetic diversity showed that children with parasite density ranging from 2000 to 5000 had the lowest MOI (Table 5).
Meanwhile those with parasite ranging from 20000 and above exhibited the highest MOI difference (P=0.001), as compared with various range of parasite densities.
The mean MOI was found to be higher in male patients whose parasitaemia ranged from moderate to high level, than that of male patients with low parasitaemia.The expected heterozygosity (HE) was calculated which used to estimate the fraction of all parasites that would be heterozygous for any of the two loci.Table 3 shows generally high HE values in each allelic family ranging from 0.5 to 0.65 for msp-1 and from 0.94 t0 0.96 for msp-2.
The influence of parasite density on genetic diversity showed that children with parasite density ranging from 2000 to 5000 had the lowest MOI (table 5) meanwhile those with parasite ranging from 20000 and above exhibited the highest MOI as compared with various ranges of parasite densities.

Sequence analysis of genetic polymorphism of msp-1 block 2 and msp-2 block3
The msp-1 block 2 and msp-2 block 3 DNA fragments from PCR products with either similar or different molecular sizes in agarose gels were sequenced with the aim of further estimating the genetic diversity of the parasite population.Partial gene sequences were obtained from each end of the PCR product using the same primers for the nested PCR reactions, then DNA sequences assembled and BLAST searched in GenBank to identify identical or similar sequences.Analysis of msp-1 and msp-2 sequence data revealed above 80% identity of study isolates among themselves in general and above 83% with isolates from other countries with a few exceptions (Suppl.2; Table 6).
Allelic families of msp-2, FC27 and 3D7/IC showed 97 to 100% identity with isolates of Gambia [57],Brazil (accession no.DQ115969) Tanzania (accession no.AY378316), and Gabon (accession no.AY372518).No significant identity was found with either the Nigerian or the Brazilian isolates with respect to the 3D7/IC allelic family of this study.However 89 to 97 % identity were recorded with isolates from Gambia and Thailand [57] with the highest identity recorded with the isolates from India (97%) [58].

Discussion
The aim of this study was to perform a molecular characterisation of highly polymorphic markers in P.
falciparum clinical isolates.In this first study conducted after the introduction of artemisinincombination therapies, 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 [59,60] and have captivated a lot of interest as potential vaccine candidates and as drug targets for inhibiting blood-stage replication [61][62][63].Our findings revealed 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 [42,63].Moreover, more amplified product was found for the msp2 gene than msp1 amplified products, 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 [59,64]suggesting that msp-1 as compared with msp2 proteins are under strong functional constraints in a complex interaction with the host leading to an increased in host's immunological response [65].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 [66].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 [67,68].Genotyping procedure recommended in antimalarial drug trials stipulates consecutive analysis of the three markers starting with msp2 or glurp, and then msp1 [111].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 parasitemias in antimalarial drug trials, consistent with other studies [42].
Our findings equally raise concern over the use of glurp genotyping in antimalarial drug trials since this marker revealed limited allelic families as compare to msp-1 and msp-2.

Genetic diversity for msp-1 and msp-2 Allelic famillies
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 [22,27] .
A peculiarity of in this study is thatRO33 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 [39,54,[67][68][69][70].However, our finding 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 [22,34].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 [15,71], but was close to that in Senegal [72] and Brazil [73], 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 [22].but is consistent with most findings in areas of holoendemic, mesoendemic, and hyperendemic malaria, in which the allelic variant K1 was predominant [39].The predominance of Ro33 and K1 allelic family could be attributed to the balancing selection acting on these two allelic variants [74].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 [ 54,70].This result is does not coroborate other studies reporting the predominance of MAD20 allelic variant over K-1 and RO33 allelic variants [23, 39, 74-76.This low distribution of MAD20 allelic variant could be partly explained by a 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 [77]..An association between the distribution of k1 allelic families with severe malaria has been investigated [67,78] while theRO33 allelic family has been frequently reported in asymptomatic malaria cases [21,34].The predominance of Ro33 seems to be beneficial for the host since the presence of this allele type is related to reduced risk of clinical malaria [67,78].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 [79].
In msp-2 the allelic famillies IC/3D7 and FC27 were almost of equal frequencies consistent with other findings [55], but in contrast with previous reports showing a significant predominance of FC27 over the 3D7/IC allelic family [34].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 [15], Uganda [44], Senegal [81], Burkina Faso [82]and Honduras [36].
Diversity, expressed as expected heterozygosity (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 [30] consistent with the findings in Uganda, congo and Zimbabwe showing a high heterozygosity ranges between 0.78 and0.8 for msp-2 [34,83].
The correlation between the genetic variation of P. falciparum and malaria endemicity has been described [34][35][36]71].In areas with declining endemicity, it is reported that the number and diversity of alleles (heterozygosity) decrease with decreasing P. falciparum transmission [32,83].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 [23,32].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 antimalarial 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 endemic areas.

Polyclonal infection expressed as the 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, such as in sub-Saharan Africa where more than 10 variants can be routinely detected in an individual [32,[62][63][64] 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 Latin America, patients may have infections with as few as a single variant [37,84].No data on MOI was available in Cameroon before the introduction of ACT renders it difficult to draw a conclusive statement on the impact of ACTs introduction 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 [85].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 [86].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 [87].Effective malaria control measures (ACTs deployment, 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 [38,112].Therefore the high MOI observed in this study is a reflect of high intensity of malaria transmission in Cameroon, despite several control strategies deployed at the health facilities and in the community such as distribution of free ACTs treatment and mosquito bed nets.This is in agreement with previous findings that observed an increase of MOI with an increase in malaria endemicity [35,36] and a low MOI for msp-1 and msp -2 correlated with a low intensity of malaria transmission [37].
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 the world, and then correlate parasite genotypes to the disease phenotypes [79,[88][89][90].Previous studies reported a reduced risk of clinical malaria associated with polyclonal infections [91] and high rate of severe malaria in individual harbouring mono-infections and very common genotypes [92,93].Our findings revealed 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 [55,94] .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 [95] while conflicting findings, indicated decreased MOI with age [96,97].Thus, determining the MOI in endemic area including Cameroon 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 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 characterisation by direct sequencing.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 50 bp and msp2 then glurp [30].However, variations in bin width [44,98] and the different fragment sizing methods need to be standardized to facilitate the comparison of data for a particular marker between studies.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 [88,89] beside capillary electrophoresis [40,69,99,100]recognise to have higher resolution power than gel electrophoresis in the ability to distinguish between allelic variants of amplified fragments.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 considered separate nPCRs for each allelic family [42].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 genotyping techniques and next-generation sequencing, might provide highly diverse haplotype markers with sufficient resolution to detect minority population in a mixed infection [101].Thus, Next generation sequencing technologies and genomewide characterization could be an alternative strategy to accurately analyse polyclonal infections [103] although complete haplotype characterization of multiclonal infections remains a challenge due to PCR artefacts and sequencing errors [103,104].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 are required [105].
Another strategy could be the combination of sequencing method and efficient computational tool for an effective characterisation 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 [106,107] 4.

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, we found high similarity between the peptides in Pfmsp1 and pfmsp2 of Cameroon and in the other regions in Africa.However, from all peptides analyzed, 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 of Cameroon and other region of Africa and other region 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 66].However, previous analysis indicated more serological variation among the allelic sequences of the K1-like compared to the MAD20-like type [108,109], 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 [64,77,110]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 [66].Sequencing and immunologic characterization of other allelic variants such as MAD20 for Pfmsp 1, alongside with 3D7/IC for pfmsp 2 should be conducted to obtain more useful information.

Conclusions
In this first study conducted five years after the introduction of artemisinin-based combination therapies, we investigated the genetic diversity of P. falciparum isolates from children aged 6 months to 6 years.The present study shows that field isolates of South West Region of Cameroon were found to be mainly polyclonal with high MOI and highly diverse in respect to both msp-1 (block 2) and msp-2 central repeat region, block 3).These markers appear to be highly polymorphic with low allelic frequencies as compared to glurp.This observation reinforced the value of msp-1 and msp-2 markers of P. falciparum for PCR correction of treatment outcomes in classifying recurrent post-treatment P. falciparum episodes as recrudescence or new-infections in drug clinical trials.This study lays emphasis on the use of both msp-1 and msp-2 genes in monitoring the trend of malaria epidemiology, and the use of MOI as an important indicator in the evaluation of malaria control interventions.The High MOI observed in this study is an indication that malaria transmission remains high in Cameroon despises a large distribution of ACTs and calls for intensifying control intervention.Besides, our findings reveal that population structure of P. falciparum isolates is identical in Cameroon as revealed by presence of common allelic composition and the high level of identity among allelic sequences from Cameroonian isolates and that of other areas in Africa and in the world and that P.falciparum population is a mixture of different strains.Therefore, vaccine developed with k1 and MAD20 of Pfmsp1 allelic variant could be protective for Africa children but this finding will require further genetic and immunological characterisation.It is anticipated that, clonal selection could not be the uniform across the country considering the varied climate range observed in Cameroon moving from the northern region (with sahel desert) to the south (with equatorial dense forest) corresponding to different of malaria epidemiology.Therefore, it will be relevant to carry out studies on the genetic diversity of P. falciparum isolates from other regions of Cameroon with varied malaria epidemiology as well as longitudinal studies involving other malaria transmission related markers such as CSP to further understand the clonal fluctuations associated with transmission intensity.AGA TGT TCA CAC TGA AC-3' CHG-OR: 5'-GTG GAATTGCTT TTTCTTCAACAC TAA-3'

Figures Figure 1
Figures Ocean at 0 meter and the highest point is Fako (peak of Mount Cameroon) at 4,095 meters above the sea level.The CDC is a para-stater company hosting rural communities in the South West region of the country which is about 30 km south of Buea town and 45 km from Douala (figure1).The CDC company has headquarters in Limbe town headed by a Director, with a population of about 45,000 Cameroon Development Corporation (CDC) which is an agroindustrial company based in the South West region of Cameroon specialised in the production of rubber, palm oil etc. Cameroon is distinguished by an overall climate which varies with terrain, from tropical rain forest with mean annual temperature of 24.5°C in the south coastal regions to 26.5 o C in the semiarid Northern regions.Concerning the Elevation extremes, the lowest point is the Atlantic

Table 2 .
Base pair range and number of detected genotypes of the respective msp-1 and msp-2 gene families in P.