Dynamics of Plasmodium falciparum genetic diversity in symptomatic and asymptomatic children in three endemic areas of Gabon

Dynamics of Plasmodium falciparum genetic diversity in symptomatic and asymptomatic children in three endemic areas of Gabon | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Dynamics of Plasmodium falciparum genetic diversity in symptomatic and asymptomatic children in three endemic areas of Gabon Lady Charlène Kouna, Sandrine Lydie Oyegue-Liabagui, Dominique Fatima Voumbo-Matoumona, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7462994/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Malaria cases in Gabon vary, from asymptomatic infection to severe malaria. The present study explores the allelic diversity of the merozoïte surface protein genes Pfmsp1 and Pfmsp2 among Plasmodium falciparum isolates in three endemic areas, and their potential correlation with virulence. Methods This cross-sectional study involved elementary schools in Gabon's urban, semi-urban and rural areas. 102 samples were analysed using PCR to examine Pfsmp1 and Pfmsp2. Results Forty-seven allele types were identified in the Pfmsp1 block, K1 allele type was the most prevalent (72.2%), followed by the RO33 allele type (49.5%) and the Mad20 allele type (36.6%). Forty-four alleles were detected in Pfmsp2 ; the FC27-like allele was the most prevalent (74.6%), followed by the 3D7-like allele (50.7%). The Mad20 allelic family was more prevalent in symptomatic isolates than in asymptomatic ones. Some Mad20, K1 and RO33 genotypes were found only in symptomatic patients, while other genotypes were found only in asymptomatic patients. The number of Pfmsp-1 and Pfmsp-2 genotypes per isolate ranged from 1 to 5 and 1 to 4, respectively. The MOI ranged from 1.95 to 2.78. The diversity index ( He ) ranged from 0.6 to 0.9 for both genes and was higher for Pfmsp_2 than for Pfmsp-1. The estimated fixation index (Fst) per site was 0.08202, 0.10413 and 0.13634 between Franceville/Lastoursville, then between Lastoursville/Makokou and finally between Franceville/Makokou. Conclusion The study revealed genetic diversity in the area, with some genotypes being asymptomatic and others symptomatic. ​This data could help develop a vaccine to reduce the disease. Plasmodium falciparum Pfmsp1 Pfmsp2 virulence rural semi-rural urban Gabon Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 BACKGROUND Malaria remains the deadliest parasitic disease in the world, despite many efforts for its control this disease. In 2021, it was responsible for 247 million new cases, including 619,000 deaths of which occurred in sub-Saharan Africa [ 1 ]. In Gabon, malaria represents 45% and 71% of the reasons for consultation among children and pregnant women respectively [ 2 ]. In addition, malaria remains the leading cause of death and absenteeism at school and professional levels. It is also responsible for prenatal and infant-juvenile complications [ 2 , 3 ]. Malaria elimination involves key global strategies, including rapid and effective diagnosis and management of all cases, intermittent preventive treatment (IPT) of malaria in pregnancy, and integrated vector management (IVM). However, if symptomatic cases are addressed and treated, asymptomatic carriers remain the foundation that fuels parasite transmission. However, to successfully eliminate malaria, in addition to the actions that are being carried out, it will be necessary to know the variations of the parasite genome in different geographic areas and better understand the factors that determine gene flow between sites. Plasmodium falciparum merozoïte surface protein-1 ( Pfmsp-1 ) and Plasmodium falciparum merozoïte surface protein-2 ( Pfmsp-2 ), which are asexual blood stage antigens, are considered as prime candidates for malaria vaccine development and are also suitable markers for the identification of genetically distinct populations of Plasmodium falciparum parasites [ 3 ]. However, since the publication of the complete genome of the reference clone 3D7 of P. falciparum [ 4 ] and the sequencing of other plasmodial isolates, there has been some clarification of the genetic variations responsible for phenotypes such as chemoresistance and parasite virulence [ 5 ]. Although in these studies, both symptomatic and asymptomatic cases are taken into account. One of the difficulties of this elimination is that most strategies concern symptomatic carriers of Plasmodium. Thus, little work on genetic polymorphism and virulence markers of P. falciparum has been done on asymptomatic infections. Moreover, the results of studies carried out between symptomatic and asymptomatic carriers have been mixed, showing an association of save alleles with virulence. School-age children are often neglected in malaria control interventions, and thus constitute a reservoir of infections and contribute substantially to malaria transmission [ 4 – 9 ]. In high-transmission settings, approximately 70% of school-age children (5 to 14 years of age) have a higher prevalence of malaria than children under five [ 6 ]. Children under five years of age are often at risk of symptomatic malaria, including severe malaria and death, while school-age children mainly carry asymptomatic malaria infections due to acquired immunity as a result of repeated exposure to parasites [ 10 – 13 ]. Thus, studies generally target children under five years of age, while surveys focusing on school-age children are scarce [ 4 , 8 ]. A report from Senegal showed that the genetic characteristics of parasite populations were different between asymptomatic and symptomatic malaria carriers [ 14 ]. In Gabon, the link between Plasmodium falciparum virulence and the virulence markers studied is not clearly established. However, studies on the genetic polymorphism of P. falciparum have described the genetic diversity of this parasite in different regions of the country, both in patients with uncomplicated to severe malaria and in asymptomatic children with virulence. [ 15 – 17 ]. These studies based on the amplification of the Pfmsp-1 and Pfmsp-2 genes aimed to characterize the genetic profile of Plasmodium falciparum isolates with the ultimate goal of malaria elimination. The study of Pfmsp-1 shows a high allelic diversity of this gene, which would be compatible with the high level of malaria transmission in Libreville [ 16 ]. This genetic diversity of P. falciparum is very variable from one region to another, as in Franceville, a low diversity has been described in comparison with Libreville [ 16 , 17 ]. Moreover, a predominance of the K1 allele has been reported in Gabon [ 15 , 17 ]. Some studies show that RO33 was weakly polymorphic in Gabon, but a study by William et al describes10 different alleles of RO33 [ 15 , 16 , 18 ]. Some authors have suggested that Mad20 allelic diversity would increase with disease severity [ 16 ]. As for the allelic diversity of Pfmsp-2 , the predominant allelic family is 3D7 [ 15 ]. Several studies have also shown that multi-clonal infections can vary depending on the clinical status of the individual (symptomatic or asymptomatic) [ 16 , 19 ]. We hypothesize that the genetic diversity of Plasmodium falciparum could vary between asymptomatic and symptomatic subjects in children in Gabon. And the parasite populations would be genetically different depending on the locality. It would therefore be interesting to describe the genotype of these strains in relation to the profile found in symptomatic patients and asymptomatic subjects in different epidemiological areas. Thus, the study of the genetic diversity of P. falciparum populations in these different epidemiological areas is an important step both towards a thorough knowledge of the strains circulating in Gabon and towards the development and/or evaluation of malaria vaccines. The aim of this study was to explore the genetic diversity of P. falciparum in asymptomatic and symptomatic malaria infections in three epidemiological areas of Gabon during a specific period. METHODS Study sites and Period A cross-sectional study was conducted between 2019 and 2020 in three areas of Gabon: Franceville, the administrative capital of the Haut-Ogooué Province and an urban area in South-Eastern Gabon (1°37′15″S and 13°34′58″E); Makokou, the administrative capital of the Ogooué-Ivindo Province and a semi-urban area in North-Eastern Gabon (0°33′33″N and 12°50′48″E); and Lastoursville, the capital of the Mulundu department and a rural region of Central-Eastern Gabon (0° 49′ S, 12° 42′ E). Blood samples containing P. falciparum were obtained from symptomatics and asymptomatics children presenting at local health center and school. The children aged between 6 and 180 months (15 years) were enrolled in our study. The number of participants per school and per health centers was determined by their availability and willingness. In Franceville, the capital of the Haut-Ogooué province (urban area), the prevalence of plasmodial infection was estimated at 21.2% [ 20 ] targeting only symptomatic patients. In Lastoursville, a rural area, the prevalence of plasmodial infection was estimated at 77.3% among febrile children [ 21 ]. A retrospective study carried out in Makokou, a semi-urban area located in the Ogooué-Ivindo province, shows that the prevalence of malaria among febrile children consulting pediatricians was high, contrary to what is observed in urban areas [ 22 ]. However, this study is the first to evaluate the genetic diversity of Plasmodium falciparum in asymptomatic and symptomatic children in our study area.The asymptomatic population was recruited in primary schools of Ombélé in Franceville, Notre Dame de Victoire A, in Makokou, public schools of Matsatsa, Mana-Mana and Malende, in Mulundu department, and composed of children with a temperature < 37.5°C who did not present a fever or history of fever during the seven days before and after enrollment. Microscopy and Blood sample collection Thick blood smear microscopy slides were prepared according to the Lambaréné method as described in a previous study [ 1 ] and stained with a 20% Giemsa solution for 10 min. All blood smears were read by two different experienced technicians, and an internal quality control was performed by a third experienced reader for 10% of slides. The result is the arithmetic mean of the results found by the two technicians. An amount of 5 ml of venous blood was collected in tubes containing ethylene diamine tetra acetic acid (EDTA) for the diagnostic of malaria. Blood elements were separated by centrifugation. Plasma was aliquoted and stored at − 80°C until use and blood pellets were used for DNA extraction. DNA extraction DNA extraction was performed using the DNA Blood Omega Bio-tek E.Z.N.A® (Omega Bio-tek, Nor-cross, GA, USA) method according to the manufacturer’s protocol as previously described [ 23 ]. Briefly, 250 µl of blood, 25 µl of Omega Biotek (OB) protease (20 mg/ml), and 250 µl of lysis buffer were mixed and heated to 65°C for 30 min before adding 260 µl of isopropanol. The mixture was transferred to a column and centrifuged at 10,000 rpm for 1 min. The column was washed twice at 13,000 rpm for 2 min, and DNA was eluted with 90 µl of sterile water preheated to 65°C. DNA samples were kept at − 20°C until use. Plasmodium species identification and Pfmsp-1/Pfmsp-2 genotyping by PCR Nested PCRs targeting the 18 S rRNA gene were performed for genus and species identification, as described by Snounou and al [ 24 ]. The polymorphism region of the mérozoïtes surface protein gen Pfmsp-1 (Block-2), Pfmsp-2 (Block-3) were amplified by nested PCR. In the first round of PCR, oligonucleotide primers were used to target conserved genomic regions within Pfmsp-1 (Block-2) msp-2 (Block-3). In the second round of PCR, the polymorphic families of Pfmsp-1 (K1, MAD20 and RO33) and Pfmsp-2 (3D7 and FC27) alleles were amplified with specific primers. The primers used for the first and second round PCR were as described by different authors [ 25 – 27 ]. The primer sequences used as well as the amplification program can be found in Supplementary Table (Table.S1). The PCR products were revealed on a 2% agarose gel using a 100bp size marker, and a semi-logarithmic curve for determining the size of the bands. Multiplicity of infection The average MOI of all samples collected was determined as the ratio of the total number of distinct parasites clones (distinct fragments) obtained for a specific marker relative to the number of samples that tested positive for that marker. Isolated carrying more than one family of alleles were considered to correspond to polyclonal infections, whereas the presence of single allele family was considered to indicate a monoclonal infection. Data Analysis The data collected were entered in an Excel spreadsheet, and then processed and transformed into a c.s.v or .txt file before being imported into R software version R 64x 3.5.0 for analysis ( https://cran.r-project.org/bin/windows/base/old/3.5.0/R-3.5.0-win.exe ). Pearson’s χ 2 test for categorical variables was used to compare group means. The non-parametric Kruskal–Wallis and Fisher’s exact tests were used to compare multiple groups of data. The Mann–Whitney U-nonparametric test was used for pairwise comparisons. The statistical significance was set at p < 0.05 . Multiplicity of infection was assessed by calculating the MOI. Genetic diversity was assessed by calculating Nei’s unbiased expected heterozygosity (He) from haploid data as following lows: H e = [ n /( n − 1)] [(1 − pi)] (n = the number of isolates sampled, pi = the frequency of the itch allele [ 28 ]. Population genetic differentiation was assessed with Wright’s F statistic [ 29 ]. Population genetic parameters were computed with FSTAT software, v2.9.4 [ 29 ]. RESULTS Study profile A total of 102 participants infected with Plasmodium falciparum were recruited from three Gabonese localities. Among these participants, 51 were symptomatic and 51 were asymptomatic. Genotyping of Pfmsp-1 and Pfmsp-2 genes Among the one hundred and two (102) samples genotyped, 91.2% (93/102) of the amplifications were successful for the Pfmsp-1 gene and 65.7% (67/102) for the Pfmsp-2 gene. The overall frequency of the K1, Mad20 and RO33 allelic families was respectively 72.02% (67/93), 36.6% (34/93) and 49.5% (46/93) for the Pfmsp-1 gene. The overall frequency of the K1 allelic family was significantly higher than that of Mad20 and RO33 ( P<0.05 ). For this gene ( Pfmsp-1 ) in general, a significant difference in the RO33 allelic family was observed between the three sites (Makokou = 52.2% (24/46), Lastoursville =32.6% (15/46), Franceville =15.2% (7/46), P=0.008 ). In addition, Mad20 and K1 allelic families were more frequent in Franceville compared to RO33, P=0.02. However, the frequency of the Mad20 allelic family was higher in symptomatic subjects (61.8%; 21/34) than in asymptomatic subjects (38.2%; 13/34), P=0.05 . Concerning the Pfmsp-2 gene, the overall frequency of the FC27 allelic family (74.6% (50/67) was significantly higher than that of 3D7 (50.7% (34/67), P=0.004 . For this gene ( Pfmsp-2) , the frequency of the 3D7 allelic family was significantly related to geographical area, P=0.01 . (Table 1). Table1 : Frequency of allelic families by locality and by clinic status Gene Allelic families Localities Clinical status msp-1 Franceville % (n/T) Lastoursville % (n/T) Makokou % (n/T) p Symptomatic % (n/T) Asymptomatic % (n/T) p* K1 31.1(21/67) 35.8 (22/67) 32.8(24/67) 0.8 43.3(29/67) 56.7 (38/67) 0.1 Mad20 35.8(12/34) 23.5 (8/34) 41.7 (14/34) 0.4 61.8(21/34) 38.2 (13/34) 0.05 Ro33 52.2(24/46) 32.6 (15/46) 15.2 (7/46) 0.008 45.7 (21/46) 54.3 (25/46) 0.4 msp-2 3D7 20.6(7/34) 26.5 (9/34) 52.8(18/34) 0.01 58.8 (20/34) 41.2 (14/34) P >0.05 F27 32(16/50) 44 (22/50) 24 (12/50) 0.1 46 (23/50) 54 (27/50) P >0.05 p= p.value between three localities; p*= p. value between two clinical status Polyclonal infections of the Pfmsp-1 and Pfmsp-2 genes according to clinical status and locality Combinations of the K1, Mad20 and RO33 allelic families were detected in 49.5% (46/93) of samples. Overall, the K1_RO33 combination was the most frequent with a frequency of 50% (23/46), followed by K1_MAD20 with a frequency of 21.7% (10/46), the other two allelic family combinations (RO33-Mad20 and K1_Mad20_Ro33) had frequencies of less than 20%. The proportion of polyclonal infections was higher in Makokou (semi-urban, 45.7% (21/46)) and Lastoursville (32.6% (10/46) rural) than in Franceville (urban, 21.7% (10/46), P=0.04 . Overall, the K1_Mad20 combination was the most frequent with a frequency of 40% (4/10), followed by K1_RO33 with a frequency of 30% (3/10) the other two allelic family combinations (RO33-Mad20 and K1_Mad20_Ro33) had frequencies of less than 20% in Franceville (Fig1.a). Overall, in Lastoursville, the K1_Ro33 combination was the most frequent with a frequency of 67% (10/15), the other three allelic family combination had frequencies of less than 20% (Fig1.c).In Makokou, the K1_Ro33 combination was overall the most frequent with a frequency of 47.6% (10/21) the other three allelic family combination had frequencies of less than 20%. (Fig.1b). The K1_Mad20 combination was significantly higher in symptomatic isolates (70%; 7/10) compared to asymptomatic isolates (30%), P=0.03 (Fig1.d). For the Pfmsp-2 gene, a combination of 3D7/FC27 allelic families was detected in 25.7% (17/67) of the samples. The 3D7_FC27 association was significantly higher in symptomatic isolates (70.6%; 12/17) compared to asymptomatic isolates (29.4%), P=0.02 . (Fig.2) Allelic diversity and fragment size length polymorphism The distribution of Pfmsp-1 and Pfmsp-2 alleles varied widely among sites. The identified alleles were classified according to fragment size" better. A total of 91 individual alleles of the two genes were identified in all three sites and all subjects. Twenty-three alleles for K1, Mad20 and one for RO33 were found, with band sizes ranging from 195 to 400 base pairs. In addition, 44 different Pfmsp-2 alleles with fragment sizes ranging from 394 to 600 base pairs were found, of which 19 belonging to the 3D7 allelic family (fragment range 400-600 bp) and 25 belonging to the FC27 allelic family (fragment range 394-556 bp). The number of alleles detected for Pfmsp-2 was not significantly different from pfmsp-1 , P=0.7 . The RO33 allelic family was found to be monomorphic (with an amplified fragment size of 270 bp) and accounted for 2.1% (1/47) of all Pfmsp-1 genotypes. K1 and Mad20 alleles predominated with frequencies of 48.9% (23/47) each, P<0.05 . Among the 23 K1 and Mad20 alleles of the Pfmsp1 gene, K1_327 (14%), K1_273 (12%) K1_343 (10%), and Mad20_360 (14%), Mad20_267 (13%) were the most frequent Fig3.a and Fig3.b). In Pfmsp-2 allelic families, FC27_429 (11.8%), 3D7_480 (16%), and 3D7_560 (14%) were the most frequently encountered, with the frequency of other alleles being less than 10%. (Fig3.c and Fig3.d). The different alleles were classified by sites, 27 and 16 different alleles were identified in Franceville (urban areas) for Pfmsp-2 and Pfmsp-1 genes (11 FC27 type and 5 3D7 type) and (12 Mad20 type, 14 K1 type and 1 RO33 type) respectively. 3D7_438 (25%), 3D7_462 (37.5%), Mad20_360, alleles were the most frequent. Twenty-two (22) and, 20 different alleles of Pfmsp1 (9 of Mad20 type and 12 of K1 type and 1 of RO33 type) and Pfmsp-2 (13 of FC27 type and 7 of 3D7 type) genes, were respectively identified in Lastoursville (rural areas), alleles FC27_429 (35.7%) were the most frequent. In Makokou (semi-urban areas) 18 different Pfmsp-2 alleles (7 FC27 type and 11 3D7 type) and 17 Pfmsp-1 alleles (7 Mad20 type, 10 K1 type and 1 RO33 type) were identified and the most frequent alleles were, Mad20_260 (42.9%) and K1_327 (35.7%). All other alleles from the different allelic families of the two genes had frequencies below 20% in all three sites. Furthermore, certain types of alleles were observed in semi-urban areas as well as in urban and rural areas. For the Pfmsp-2 gene, 6 FC27 alleles and 2 3D7 alleles were identified in both Franceville (urban) and Lastoursville (rural areas). Three 3D7 alleles were identified in both Lastoursville (rural) and Makokou (semi-urban areas). For the Pfmsp-1 gene, five allelic profiles (Mad20_240, K1_273, K_327, K1_328, and K1_355) common to the three sites were identified. In addition, the Franceville and Lastoursville had 5 common allelic profiles (3 Mad20 and 2 K1), but the Makokou and Lastoursville had 2 common allelic profiles, all K1. Apart from the common allelic profiles in the three localities, no common profile in Franceville and Makokou was found. (Fig.4) The different allelic profiles varied according to the clinical symptomatology . For the Pfmsp-2 gene, 31 different alleles were identified in asymptomatic children (13 of type 3D7 and 18 of type FC27) and 28 alleles were identified in symptomatic isolates (17 of type FC27 and 11 of type 3D7). Eight (8) alleles of type 3D7 and eight (8) alleles of type FC27 were only observed in isolates from asymptomatic children. Similarly, 7 alleles of type FC27 and 5 alleles of type 3D7 were only identified in symptomatic isolates. These different alleles had frequencies below 20%. However, for alleles identified in both symptomatic and asymptomatic isolates no significant difference in frequencies was observed P>0.05 . For the Pfmsp-1 gene, 29 different alleles were found in asymptomatic isolates (10 Mad20 and 19 K1) and 36 different alleles were observed in symptomatic isolates (K1=17, Mad20=19). Three (3) alleles of type Mad20 and four (4) alleles of type K1 were only observed in asymptomatic isolates. Similarly, four (4) alleles of type K1 and thirteen (13) alleles of type Mad20 were only identified in symptomatic isolates with frequencies less than 20%. Some alleles were common to both groups with no significant difference in frequency, P>0.05 . (Fig.5) MOI, Diversity Index (HE) and Genetic differentiation (FST) The estimated MOI at the three sites and by clinical status is summarized in Table 2. The overall number of Pfmsp-1 and Pfmsp-2 genotypes per isolate ranged from 1 to 5 and 1 to 4 respectively. In Lastoursville the number of Pfmsp-1 genotypes per isolate varied from 1 to 4, on the other hand for Franceville and Makokou as well as for the two clinical statuses, the number of genotypes per isolate varied from 1 to 5. The number of Pfmsp-2 genotypes varied from 1 to 4 in each site and depending on the clinical status. The overall MOI was 2.06±1.07, 2.13±1.07 and 2.57±0.99 for Pfmsp-1 , Pfmsp-2 and Pfmsp-1 + Pfmsp-2 respectively. The mean MOI for Pfmsp-2 and Pfmsp-2, or Pfmsp-1+ Pfmsp-2 genotypes was not significantly different between the three sites. Similarly, no significant difference in MOI was detected between symptomatic and asymptomatic isolates (Table 2). Table 2 : Number of clones (MOI) detected with msp1, msp2 and msp1+2 according to sites and clinical status. Variable No. of isolates No. of genotype MOI (SD) Pfmsp1 Pfmsp2 Pfmsp1 Pfmsp2 Pfmsp1 P Pfmsp2 P Pfmsp1 +2 P Overall 93 67 1-5 1-4 2.06(1.07) - 2.13 (1.07) - 2.57(0.99) - By site Franceville 29 20 1-5 1-4 2(1.16) NS 2.25 (1.06) NS 2.55(1.24) NS Lastoursville 31 25 1-4 1-4 1.96(0.98) 2(0.95) 2.54(0.85) Makokou 33 22 1-5 1-4 2.21(1.08) 2.18(1.22) 2.62(0.92) By clinical status Asymptomatic 46 31 1-5 1-4 1.91(1.05) NS 2.03(0.91) 2.32(0.81) NS Symptomatic 47 36 1-5 1-4 2.21(1.08) 2.22(1.19) 2.78(1.08) No.= Number, Ns=Non significant; SD= Standard deviation Genetic diversity ( He ) was estimated based on study sites and clinical status. For the Pfmsp-1 gene the genetic diversity was 0.59893±0.5183; 0.596±0.5174 and 0.5036 ±0.4371 in Franceville, Lastoursville and Makokou respectively. For Pfmsp-2 the genetic diversity was 0.9005±0.0277; 0.9115±0.0354 and 0.8584 ±0.0653 in Franceville, Lastoursville and Makokou respectively. The genetic diversity of the Pfmsp-1 and Pfmsp-2 genes was almost the same in the three study sites. On the other hand, the genetic diversity was more important for the Pfmsp-2 marker compared to the Pfmsp-1 marker. Furthermore, the genetic diversity of Pfmsp-1 , Pfmsp-2 and Pfmsp-1 + Pfmsp-2 was almost identical between symptomatic ( Pfmsp-1 =0.7265±0.3381, Pfmsp-2 =0.9267±0.0117 and Pfmsp-1 + Pfmsp-2 =0.802±0.2612 and asymptomatic ( Pfmsp-1 = 0.6746±0.3706, Pfmsp-2 = 0.9232±0.0545 and Pfmsp-1 + Pfmsp-2 =0.7764±0.2968) (Table 3) Table 3: Genetic diversity (He) according to sites and clinical status Site He (SD) Pfmsp-1 Pfmsp-2 Pfmsp-1+2 Urban 0.5983 (0.5183) 0.9005 (0.0277) 0.8137 (0.1322) Rural 0.596 (0.5174) 0.9115 (0.0354) 0.7652 (0.2736) Semi-urban 0.5036 (0.4371) 0.8584 (0.0653) 0.6850 (0.3089) Clinical status He (SD) Pfmsp-1 Pfmsp-2 Pfmsp-1+2 Asymptomatic Symptomatic 0.6746 (0.3706) 0.9232 (0.0545) 0.7764 (0.2968) 0.7265 (0.3381) 0.9267 (0.0117) 0.802 (0.2612) SD= Standard deviation The genetic diversity of the two combined markers was gradual from South to North: diversity was first greater in Franceville, then in Lastoursville and finally in Makokou. The estimated fixation index (Fst) measuring the differentiation of the parasite population due to the genetic structure at each site was 0.08202, 0.10413 and 0.13634 between Franceville and Lastoursville, then between Lastoursville and Makokou, finally between Franceville and Makokou respectively. Overall the Fst between the populations of the different localities was moderate. A greater proximity of populations between Franceville and Lastoursville then between Lastoursville and Makokou was observed. But a greater difference in genetic differentiation was observed between Franceville and Makokou (Figure 6). This result is consistent with the proportion of genotypes common to the various sites. The highest proportion of allele sharing was observed between Franceville and Lastoursville, then between Lastoursville and Makokou (Fig.6). The study of the flow of parasite populations between symptomatic and asymptomatic has made it possible to observe a close proximity of populations between symptomatic and asymptomatic (Fst= 0.03134) DISCUSSION Understanding the genetic diversity and transmission dynamics of Plasmodium falciparum provides information on the intensity of malaria transmission, necessary for evaluating malaria control interventions. The analysis of the genetic profile of Plasmodium falciparum. , obtained after PCR, according to the type of infection, can provide useful information on the characteristics of certain specific parasitic clones in order to design intervention strategies targeting virulence factors [ 30 ]. It has been shown that most alleles fluctuate considerably over the years and can vary from one endemic region to another [ 31 ]. The present study is the first to evaluate the virulence factors of Plasmodium falciparum . In Franceville (urban area), Lastoursville (rural) and Makokou (semi-urban) settings in asymptomatic and symptomatic children in the South-East and North-East of Gabon. Overall, the results of our study showed that the three allelic families K1, Mad20 and RO33 of the Pfmsp-1 gene and the two allelic families 3D7 and FC27 of the Pfmsp-2 gene were present in the three study sites. The distribution of these allelic families was different according to the site, the RO33 allelic families were more frequent in urban areas than in semi-urban and rural areas. And the frequency of 3D7 was higher than FC27 in semi-rural settings. This supports the hypothesis that the frequency of Pfmsp-1 and Pfmsp-2 allelic families may vary between localities and even within the same locality [ 15 , 26 , 32 ]. The K1 and FC27 allelic families are the most frequently encountered. Studies in Gabon, Burkina Faso and Côte d'Ivoire described a high frequency of the K1 and 3D7 allelic families, but in this study the frequency of the FC27 allelic family was lower than that of 3D7 [ 15 , 32 ]. The Mad20 allelic family was most frequent in isolates from symptomatic versus asymptomatic children in all samples in both urban and rural settings. This allelic family could play an important role in symptomatic malaria, at least in Gabon. This is different from the data described in the literature because the K1 and 3D7 allelic families have often been associated with symptomatic infection [ 32 ] than FC27. The frequency of FC27 and 3D7 allelic families was not different between symptomatic and asymptomatic, which proves that there would not exist specific Pfmsp-2 variants that could be associated with clinical status. These results are supported by studies that have shown that there is no association between the FC27 or 3D7 allelic families and the type of infection [156]. Conversely, another study reported a frequency of the FC27 allelic family twice as high in symptomatic isolates compared to asymptomatic [ 33 ]. However, further studies are needed to clarify the interpretation of these results. Overall, our study reported high genetic diversity with forty-seven genotypes for the Pfmsp-1 gene and 44 genotypes for the Pfmsp-2 gene in parasites found in the Southeast and Northeast from Gabon. These different genotypes were distributed as follows, 23 K1 alleles and Mad20 1 Ro33 allele (fragment sizes 195-600bp) for the msp1 gene. In the Pfmsp2 gene, 19 alleles for 3D7 (fragment range of 400–600 bp) and 27 different alleles for FC27 (fragment range of 394-600bp) have been demonstrated. This observation is consistent with that of some studies conducted in different malaria-endemic countries in Africa [ 27 , 32 , 34 ]. The high genetic diversity of the parasites may be related to the high prevalence of Plasmodium infection in the study area where the prevalence is ranging from 20% to about 70% have been found [ 21 ]. This great diversity observed could also be associated with the high risk of being infected by several parasitic clones of different genotypes. Similarly, the implementation of control methods and the use of antimalarials could also lead to high genetic diversity [ 26 ]. Thirty-one (31) and twenty-eight (28) different alleles of the msp-2 gene were identified in isolates from asymptomatic and symptomatic children respectively. The allele frequency of 3D7 and FC27 was not significantly different between isolates from symptomatic and asymptomatic children. This suggests that the allelic diversity of FC27 and 3D7 in both groups was the same and that there is no relationship between the clinical status and the polymorphism of these alleles. For the msp1 gene, 29 different genotypes and 36 different genotypes were identified in isolates from asymptomatic and symptomatic children respectively. K1 and Mad20 allelic variants had similar frequencies between isolates from asymptomatic and symptomatic children. Suggesting a high polymorphism of this allele in asymptomatic and symptomatic isolates. This result is different from that of other studies that have shown a link between asymptomatic infection and the polymorphism of the K1 allele [ 34 , 35 ]. The different genotypes of Pfmsp-1 and Pfmsp-2 gene alleles identified in both symptomatic and asymptomatic isolates did not have significantly different frequencies. This suggests that the clinical status of the children included in the study was not related to genetic polymorphism. asymptomatic but were not different between the two groups [ 15 , 36 ]. Indeed, genetic polymorphism is strongly affected by the level of endemicity. With high endemicity, multiclonal infections and crosses are predominant [ 27 ]. In addition, 12 Mad20 genotypes, 8 FC27 genotypes, 4 K1 genotypes and 8 3D7 genotypes were only found in symptomatic isolates. And 4 K1 genotypes, 3 Mad20 genotypes, 7 FC27 genotypes and 5 different 3D7 genotypes were only found in the asymptomatic isolates. These results support the idea that certain parasitic clones could be responsible for the clinical state of an individual. However, the link between the clinical condition and any P. falciparum genotype remains a controversial subject. Because many studies have shown that the presence of subsequent malaria episodes could be due to new infectious parasites [ 37 ]. A high genetic polymorphism within the allelic families of the msp1 and 2 genes was observed in isolates from urban areas (27 alleles for msp-1 and 17 alleles for msp-2), from rural areas (22 alleles) for Pfmsp-1 and 20 alleles for Pfmsp-2 ) and semi-urban areas (Franceville, 18 msp-2 alleles and 17 msp-1 alleles). Our results are lower than those described in Libreville (30 alleles), Dielmo in Senegal (33 alleles) in 1995 and Burkina Faso in 2009 (41 alleles) and identical to those of Bakoumba near Franceville (25 alleles) in 1999 [ 16 , 18 , 27 , 35 ]. This allelic diversity could be explained by the fact that the genetic diversity of P. falciparum varies from one region to another and within the same population [ 16 ]. Some genotypes were common between rural and urban areas, then between semi-urban and rural areas, and finally between the three localities. This observation could lead to the conclusion that certain genotypes could easily circulate between urban and rural areas due to the proximity of these two localities. Also the demographic facies of the rural environment and that of the semi-urban environment being almost identical, it is therefore not surprising that these two localities share a greater number of parasitic genotypes compared to the urban and semi-urban environment. This difference in allele frequencies in the three localities could also be due to the difference in prevalence observed here. In fact, in semi-urban and rural areas, the prevalence of plasmodial infection was higher than in urban areas. The number of Pfmsp-1 and Pfmsp-2 genotypes per isolate ranged from 1 to 5 and 1 to 4 respectively. The MOI is an indicator of the level of malaria transmission because it was expected to be higher in high transmission areas and lower in low transmission areas [ 38 ]. Our study is in line with this hypothesis because high MOIs were found in all localities with values ​​ranging from 1.96 to 2.78. MOI could also be a useful indicator of immune status. MOI has been suggested to be associated with symptoms of Plasmodium infections [ 19 , 39 ]. However, although the MOI was high in all localities, no significant difference in MOI between symptomatic and asymptomatic in P. falciparum was demonstrated in our study. This high MOI value could be explained by the fact that malaria transmission could be high in the areas studied due to the high prevalence of P. falciparum observed in this study. The high genetic diversity observed in our study is confirmed by the value of the diversity index (He) ranging from 0.6 to 0.9 for the two genes. The H e value was higher for Pfmsp-2 compared to Pfmsp-1 . Moreover, the diversity index was not significantly different between the sites, and between the two clinical statuses for the two genes. This lack of variability between the levels of genetic diversity observed within the country is not in accordance with the local epidemiological parameters of the country. Because the level of diversity is expected to be high in areas with high transmission. In addition, the genetic diversity of the two markers combined more or less follows a gradient from South to North: the diversity was first greater in urban areas, then in rural areas and finally in semi-urban areas. In this study, the He does not reflect the level of prevalence, because here the prevalence of plasmodial infection was higher in rural and semi-urban areas compared to urban areas. Contrary results were found in Senegal and Mali where He values ​​increase with the transmission gradient from north to south [ 40 ]. The high heterozygosity observed in these localities could indicate a reduction in clonal expansion and an increase in genetic diversity in these localities. It would also mean that the genetic diversity of P. falciparum would increase in areas with high transmission and decrease when transmission declines [ 41 ]. Concerning the Fst, we found moderate genetic differentiations of the parasite populations of P. falciparum between the three sites. The average values ​​of Fst found between the populations of P. falciparum show that there would be a greater proximity of the populations between the urban and rural areas on the one hand, then between the rural and semi-urban areas on the other hand. But a greater difference in genetic differentiation was observed between urban and semi-urban areas. This result is consistent with the proportion of genotypes common to the various sites. These data also reveal gene flow between urban and rural parasite populations facilitated by extensive human migrations between these two localities and subsequently causing vector displacement. In addition, the greatest proportion of allele sharing was observed between urban and rural areas, then between rural and semi-urban areas. Although our results are superior to those from Senegal, they confirm that there could be gene flow between parasite populations facilitated by human migration events between endemic regions and facilitating vector movement [ 38 , 42 , 43 ]. Genetic differentiation studies have not yet been carried out in our different localities, but studies carried out in other countries have reported a lack of genetic differentiation [ 38 , 44 ]. This is the case of our study because the study of the flow of parasitic populations between symptomatic and asymptomatic has made it possible to observe a close proximity of the populations between the two clinical groups (Fst = 0.03134). However, other analyzes such as sequencing by next-generation techniques are necessary in order to confirm the existence or not of parasitic clones responsible for the clinical state of an individual. One of the limitations of this study is the small number. Another limitation is the low rate of amplification of the Pmfp2 gene CONCLUSION Our study revealed a high genetic diversity of the pfmsp1 and pfmsp2 genes. high to high MOI was also observed. The Mad20 allelic family of msp1 was the most prevalent among the asymptomatic group, this allele may play an important role in symptomatic malaria. Additionally, some alleles of the msp1 and msp2 allelic families were only observed in symptomatic individuals. These alleles could be responsible for virulence in symptomatic patients, and could be of key interest for vaccine research. Declarations Institutional Review Board Statement This study has been approved by Gabon’s National Ethics Committee (CNER) under number: PROT 0023/2013/ SG/CNER. Informed Consent Statement Written informed consent was obtained from the parents or guardians before each child’s participation in the study. No non-consenting participants were included in the present study. Funding statment This study was supported by CIRMF, the Gabonese Government, CANTAM organisation and Total Gabon. Author Contribution L.C.K., S.L.O.-L. and J.B.L.-D. designed the study. LCK, participated in the field survey and sample collection. FDVM. and LCK carried out the microscopic and moleccular analysis in the laboratory. L.C.K. carried out data analyses, and wrote the manuscript. S.L.O.-L. and J.B.L.-D. reviewed the document. All authors have read and agreed to the published version of the manuscript. Acknowledgement The authors thank the children and their parents for agreeing to participate in this study. We also wish to thank the staff of the pediatric wards of the Lastoursville health centre and Amissa Bongo regional hospitals in Makokou and Franceville, respectively. We are also grateful to the staff of the Unit of Evolution, Epidemiology and Parasitic Resistances (UNEEREP) of CIRMF. References WHO (2021) https : //wwwwhoint/teams/global-malaria-programme/reports/world-malaria-report-2021 (accessed Jan 03, 2022) 2021 Santé) (2013) D-EDGdlSdG-EDed: Enquête Démographique et de Santé au Gabon-Phase II (EDSG II), 501p Mdl santé-SNIS (2016) Mission de collecte des donnés en vue d’une cartographie pour établir un état des lieux, des équipements, des usages et perception des TIC des établissements concernés par le Système National d’Information Sanitaire (SNIS). Rapport final de mission, Libreville, p 139 . 4, Nankabirwa JBS, Clarke SE, Fernando D, Gitonga CW, Schellenberg D et al (2014) : Malaria in school-age children in Africa: an increasingly important challenge. 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Franceville","correspondingAuthor":false,"prefix":"","firstName":"Sandrine","middleName":"Lydie","lastName":"Oyegue-Liabagui","suffix":""},{"id":505832328,"identity":"3cfb4876-8091-4d83-95e5-1888eeb08238","order_by":2,"name":"Dominique Fatima Voumbo-Matoumona","email":"","orcid":"","institution":"Université Marien Ngouabi","correspondingAuthor":false,"prefix":"","firstName":"Dominique","middleName":"Fatima","lastName":"Voumbo-Matoumona","suffix":""},{"id":505832329,"identity":"96419f7e-7381-4411-9b11-d8f389e57c78","order_by":3,"name":"Jean Bernard Lekana-Douki","email":"","orcid":"","institution":"Ecole Doctorale Régionale d’Afrique Centrale en Infectiologie Tropicale","correspondingAuthor":false,"prefix":"","firstName":"Jean","middleName":"Bernard","lastName":"Lekana-Douki","suffix":""}],"badges":[],"createdAt":"2025-08-26 12:38:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7462994/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7462994/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":90317592,"identity":"5cb34f80-4600-4e45-801b-0229c95160dd","added_by":"auto","created_at":"2025-09-01 10:27:22","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":129061,"visible":true,"origin":"","legend":"\u003cp\u003eDistribution of polyclonal infections of the \u003cem\u003emsp-1\u003c/em\u003e gene by locality and by clinical status\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7462994/v1/8d26379c88b31b8dc3cf19ac.png"},{"id":90320182,"identity":"662f84a8-66eb-4d92-a52d-84589295c7c2","added_by":"auto","created_at":"2025-09-01 10:43:22","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":83745,"visible":true,"origin":"","legend":"\u003cp\u003eDistribution of polyclonal infection of the \u003cem\u003ePfmsp-2\u003c/em\u003egene by sites and by clinical status (\u003cstrong\u003eMKK=Makokou, LTV= Lastoursville, FCV= Franceville)\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7462994/v1/bcb15dce2435e177dfc9211e.png"},{"id":90320180,"identity":"c40f01ba-5f1d-4e5c-a91f-fd225c4af1d6","added_by":"auto","created_at":"2025-09-01 10:43:22","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":312729,"visible":true,"origin":"","legend":"\u003cp\u003eAllelic diversity of different \u003cem\u003ePfmsp-1 and 2\u003c/em\u003e gene alleles in the whole population\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7462994/v1/59c5eebfb731e577da8530f6.png"},{"id":90320869,"identity":"914c226a-4d7a-49a9-8a35-54930c67c4ed","added_by":"auto","created_at":"2025-09-01 10:51:22","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":371691,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of allele frequencies of \u003cem\u003ePfmsp1 and 2\u003c/em\u003e genes by locality.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7462994/v1/ba509fd41716c9e835b29c7f.png"},{"id":90318666,"identity":"7045d9af-43e8-4c05-8bf6-162a4e6438e8","added_by":"auto","created_at":"2025-09-01 10:35:22","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":182034,"visible":true,"origin":"","legend":"\u003cp\u003eAllele frequencies of the \u003cem\u003ePfmsp-1 and 2\u003c/em\u003e gene in symptomatic and asymptomatic isolates in the general population.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7462994/v1/22211c9a899b38fed72a2960.png"},{"id":90317609,"identity":"a3dd407f-4c15-41e9-b95d-6bdd3892a332","added_by":"auto","created_at":"2025-09-01 10:27:22","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":322665,"visible":true,"origin":"","legend":"\u003cp\u003eGeographic distribution and genetic differentiation of parasite populations (source: \u003ca href=\"https://www.cosmovisions.com/cartes/gb-map.gifm\"\u003ehttps://www.cosmovisions.com/cartes/gb-map.gifm\u003c/a\u003e. Amended_modify)\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7462994/v1/4fe4cc4c2852a0dcb8665ec6.png"},{"id":91148129,"identity":"86d8d8f0-c802-40d9-9fa0-3b96a6664815","added_by":"auto","created_at":"2025-09-12 06:42:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2437371,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7462994/v1/f6037654-036c-4302-bc64-8e7e5aa948c4.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Dynamics of Plasmodium falciparum genetic diversity in symptomatic and asymptomatic children in three endemic areas of Gabon","fulltext":[{"header":"BACKGROUND","content":"\u003cp\u003eMalaria remains the deadliest parasitic disease in the world, despite many efforts for its control this disease. In 2021, it was responsible for 247\u0026nbsp;million new cases, including 619,000 deaths of which occurred in sub-Saharan Africa [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In Gabon, malaria represents 45% and 71% of the reasons for consultation among children and pregnant women respectively [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In addition, malaria remains the leading cause of death and absenteeism at school and professional levels. It is also responsible for prenatal and infant-juvenile complications [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eMalaria elimination involves key global strategies, including rapid and effective diagnosis and management of all cases, intermittent preventive treatment (IPT) of malaria in pregnancy, and integrated vector management (IVM). However, if symptomatic cases are addressed and treated, asymptomatic carriers remain the foundation that fuels parasite transmission. However, to successfully eliminate malaria, in addition to the actions that are being carried out, it will be necessary to know the variations of the parasite genome in different geographic areas and better understand the factors that determine gene flow between sites. \u003cem\u003ePlasmodium falciparum merozo\u0026iuml;te surface protein-1\u003c/em\u003e (\u003cem\u003ePfmsp-1\u003c/em\u003e) and \u003cem\u003ePlasmodium falciparum merozo\u0026iuml;te surface protein-2\u003c/em\u003e (\u003cem\u003ePfmsp-2\u003c/em\u003e), which are asexual blood stage antigens, are considered as prime candidates for malaria vaccine development and are also suitable markers for the identification of genetically distinct populations of \u003cem\u003ePlasmodium falciparum\u003c/em\u003e parasites [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. However, since the publication of the complete genome of the reference clone 3D7 of \u003cem\u003eP. falciparum\u003c/em\u003e [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] and the sequencing of other plasmodial isolates, there has been some clarification of the genetic variations responsible for phenotypes such as chemoresistance and parasite virulence [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Although in these studies, both symptomatic and asymptomatic cases are taken into account. One of the difficulties of this elimination is that most strategies concern symptomatic carriers of Plasmodium. Thus, little work on genetic polymorphism and virulence markers of \u003cem\u003eP. falciparum\u003c/em\u003e has been done on asymptomatic infections. Moreover, the results of studies carried out between symptomatic and asymptomatic carriers have been mixed, showing an association of save alleles with virulence.\u003c/p\u003e\u003cp\u003eSchool-age children are often neglected in malaria control interventions, and thus constitute a reservoir of infections and contribute substantially to malaria transmission [\u003cspan additionalcitationids=\"CR5 CR6 CR7 CR8\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In high-transmission settings, approximately 70% of school-age children (5 to 14 years of age) have a higher prevalence of malaria than children under five [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Children under five years of age are often at risk of symptomatic malaria, including severe malaria and death, while school-age children mainly carry asymptomatic malaria infections due to acquired immunity as a result of repeated exposure to parasites [\u003cspan additionalcitationids=\"CR11 CR12\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Thus, studies generally target children under five years of age, while surveys focusing on school-age children are scarce [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. A report from Senegal showed that the genetic characteristics of parasite populations were different between asymptomatic and symptomatic malaria carriers [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn Gabon, the link between \u003cem\u003ePlasmodium falciparum\u003c/em\u003e virulence and the virulence markers studied is not clearly established. However, studies on the genetic polymorphism of \u003cem\u003eP. falciparum\u003c/em\u003e have described the genetic diversity of this parasite in different regions of the country, both in patients with uncomplicated to severe malaria and in asymptomatic children with virulence. [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. These studies based on the amplification of the \u003cem\u003ePfmsp-1\u003c/em\u003e and \u003cem\u003ePfmsp-2\u003c/em\u003e genes aimed to characterize the genetic profile of \u003cem\u003ePlasmodium falciparum\u003c/em\u003e isolates with the ultimate goal of malaria elimination. The study of \u003cem\u003ePfmsp-1\u003c/em\u003e shows a high allelic diversity of this gene, which would be compatible with the high level of malaria transmission in Libreville [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. This genetic diversity of \u003cem\u003eP. falciparum\u003c/em\u003e is very variable from one region to another, as in Franceville, a low diversity has been described in comparison with Libreville [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Moreover, a predominance of the K1 allele has been reported in Gabon [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Some studies show that RO33 was weakly polymorphic in Gabon, but a study by William et al describes10 different alleles of RO33 [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Some authors have suggested that Mad20 allelic diversity would increase with disease severity [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. As for the allelic diversity of \u003cem\u003ePfmsp-2\u003c/em\u003e, the predominant allelic family is 3D7 [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Several studies have also shown that multi-clonal infections can vary depending on the clinical status of the individual (symptomatic or asymptomatic) [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eWe hypothesize that the genetic diversity of Plasmodium falciparum could vary between asymptomatic and symptomatic subjects in children in Gabon. And the parasite populations would be genetically different depending on the locality.\u003c/p\u003e\u003cp\u003eIt would therefore be interesting to describe the genotype of these strains in relation to the profile found in symptomatic patients and asymptomatic subjects in different epidemiological areas. Thus, the study of the genetic diversity of \u003cem\u003eP. falciparum\u003c/em\u003e populations in these different epidemiological areas is an important step both towards a thorough knowledge of the strains circulating in Gabon and towards the development and/or evaluation of malaria vaccines. The aim of this study was to explore the genetic diversity of \u003cem\u003eP. falciparum\u003c/em\u003e in asymptomatic and symptomatic malaria infections in three epidemiological areas of Gabon during a specific period.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eStudy sites and Period\u003c/h2\u003e\n \u003cp\u003eA cross-sectional study was conducted between 2019 and 2020 in three areas of Gabon: Franceville, the administrative capital of the Haut-Ogoou\u0026eacute; Province and an urban area in South-Eastern Gabon (1\u0026deg;37\u0026prime;15\u0026Prime;S and 13\u0026deg;34\u0026prime;58\u0026Prime;E); Makokou, the administrative capital of the Ogoou\u0026eacute;-Ivindo Province and a semi-urban area in North-Eastern Gabon (0\u0026deg;33\u0026prime;33\u0026Prime;N and 12\u0026deg;50\u0026prime;48\u0026Prime;E); and Lastoursville, the capital of the Mulundu department and a rural region of Central-Eastern Gabon (0\u0026deg; 49\u0026prime; S, 12\u0026deg; 42\u0026prime; E). Blood samples containing \u003cem\u003eP. falciparum\u003c/em\u003e were obtained from symptomatics and asymptomatics children presenting at local health center and school. The children aged between 6 and 180 months (15 years) were enrolled in our study. The number of participants per school and per health centers was determined by their availability and willingness. In Franceville, the capital of the Haut-Ogoou\u0026eacute; province (urban area), the prevalence of plasmodial infection was estimated at 21.2% [\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e] targeting only symptomatic patients. In Lastoursville, a rural area, the prevalence of plasmodial infection was estimated at 77.3% among febrile children [\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e]. A retrospective study carried out in Makokou, a semi-urban area located in the Ogoou\u0026eacute;-Ivindo province, shows that the prevalence of malaria among febrile children consulting pediatricians was high, contrary to what is observed in urban areas [\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e]. However, this study is the first to evaluate the genetic diversity of \u003cem\u003ePlasmodium falciparum\u003c/em\u003e in asymptomatic and symptomatic children in our study area.The asymptomatic population was recruited in primary schools of Omb\u0026eacute;l\u0026eacute; in Franceville, Notre Dame de Victoire A, in Makokou, public schools of Matsatsa, Mana-Mana and Malende, in Mulundu department, and composed of children with a temperature\u0026thinsp;\u0026lt;\u0026thinsp;37.5\u0026deg;C who did not present a fever or history of fever during the seven days before and after enrollment.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eMicroscopy and Blood sample collection\u003c/h3\u003e\n\u003cp\u003eThick blood smear microscopy slides were prepared according to the Lambar\u0026eacute;n\u0026eacute; method as described in a previous study [\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e] and stained with a 20% Giemsa solution for 10 min. All blood smears were read by two different experienced technicians, and an internal quality control was performed by a third experienced reader for 10% of slides. The result is the arithmetic mean of the results found by the two technicians. An amount of 5 ml of venous blood was collected in tubes containing ethylene diamine tetra acetic acid (EDTA) for the diagnostic of malaria. Blood elements were separated by centrifugation. Plasma was aliquoted and stored at \u0026minus;\u0026thinsp;80\u0026deg;C until use and blood pellets were used for DNA extraction.\u003c/p\u003e\n\u003ch3\u003eDNA extraction\u003c/h3\u003e\n\u003cp\u003eDNA extraction was performed using the DNA Blood Omega Bio-tek E.Z.N.A\u0026reg; (Omega Bio-tek, Nor-cross, GA, USA) method according to the manufacturer\u0026rsquo;s protocol as previously described [\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e]. Briefly, 250 \u0026micro;l of blood, 25 \u0026micro;l of Omega Biotek (OB) protease (20 mg/ml), and 250 \u0026micro;l of lysis buffer were mixed and heated to 65\u0026deg;C for 30 min before adding 260 \u0026micro;l of isopropanol. The mixture was transferred to a column and centrifuged at 10,000 rpm for 1 min. The column was washed twice at 13,000 rpm for 2 min, and DNA was eluted with 90 \u0026micro;l of sterile water preheated to 65\u0026deg;C. DNA samples were kept at \u0026minus;\u0026thinsp;20\u0026deg;C until use.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlasmodium species identification and\u003c/strong\u003e \u003cstrong\u003ePfmsp-1/Pfmsp-2\u003c/strong\u003e \u003cstrong\u003egenotyping by PCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNested PCRs targeting the \u003cem\u003e18 S rRNA\u003c/em\u003e gene were performed for genus and species identification, as described by Snounou and al [\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e]. The polymorphism region of the m\u0026eacute;rozo\u0026iuml;tes surface protein gen \u003cem\u003ePfmsp-1\u003c/em\u003e (Block-2), \u003cem\u003ePfmsp-2\u003c/em\u003e (Block-3) were amplified by nested PCR. In the first round of PCR, oligonucleotide primers were used to target conserved genomic regions within \u003cem\u003ePfmsp-1\u003c/em\u003e (Block-2) msp-2 (Block-3). In the second round of PCR, the polymorphic families of \u003cem\u003ePfmsp-1\u003c/em\u003e (K1, MAD20 and RO33) and \u003cem\u003ePfmsp-2\u003c/em\u003e (3D7 and FC27) alleles were amplified with specific primers. The primers used for the first and second round PCR were as described by different authors [\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e]. The primer sequences used as well as the amplification program can be found in Supplementary Table (Table.S1). The PCR products were revealed on a 2% agarose gel using a 100bp size marker, and a semi-logarithmic curve for determining the size of the bands.\u003c/p\u003e\n\u003ch3\u003eMultiplicity of infection\u003c/h3\u003e\n\u003cp\u003eThe average MOI of all samples collected was determined as the ratio of the total number of distinct parasites clones (distinct fragments) obtained for a specific marker relative to the number of samples that tested positive for that marker. Isolated carrying more than one family of alleles were considered to correspond to polyclonal infections, whereas the presence of single allele family was considered to indicate a monoclonal infection.\u003c/p\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003eData Analysis\u003c/h2\u003e\n \u003cp\u003eThe data collected were entered in an Excel spreadsheet, and then processed and transformed into a c.s.v or .txt file before being imported into R software version R 64x 3.5.0 for analysis (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cran.r-project.org/bin/windows/base/old/3.5.0/R-3.5.0-win.exe\u003c/span\u003e\u003c/span\u003e). Pearson\u0026rsquo;s \u0026chi;\u003csup\u003e2\u003c/sup\u003e test for categorical variables was used to compare group means. The non-parametric Kruskal\u0026ndash;Wallis and Fisher\u0026rsquo;s exact tests were used to compare multiple groups of data. The Mann\u0026ndash;Whitney U-nonparametric test was used for pairwise comparisons. The statistical significance was set at \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e. Multiplicity of infection was assessed by calculating the MOI. Genetic diversity was assessed by calculating Nei\u0026rsquo;s unbiased expected heterozygosity (He) from haploid data as following lows: \u003cem\u003eH\u003c/em\u003e\u003csub\u003e\u003cem\u003ee\u003c/em\u003e\u003c/sub\u003e = [\u003cem\u003en\u003c/em\u003e/(\u003cem\u003en\u003c/em\u003e\u0026thinsp;\u0026minus;\u0026thinsp;1)] [(1\u0026thinsp;\u0026minus;\u0026thinsp;pi)] (n\u0026thinsp;=\u0026thinsp;the number of isolates sampled, pi\u0026thinsp;=\u0026thinsp;the frequency of the itch allele [\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e]. Population genetic differentiation was assessed with Wright\u0026rsquo;s F statistic [\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e]. Population genetic parameters were computed with FSTAT software, v2.9.4 [\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e"},{"header":"RESULTS","content":"\u003ch3\u003e\u003cstrong\u003eStudy profile\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eA total of 102 participants infected with \u003cem\u003ePlasmodium falciparum\u003c/em\u003e were recruited from three Gabonese localities. Among these participants, 51 were symptomatic and 51 were asymptomatic. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenotyping of \u003cem\u003ePfmsp-1\u003c/em\u003e and \u003cem\u003ePfmsp-2\u003c/em\u003e genes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAmong the one hundred and two (102) samples genotyped, 91.2% (93/102) of the amplifications were successful for the \u003cem\u003ePfmsp-1\u003c/em\u003e gene and 65.7% (67/102) for the \u003cem\u003ePfmsp-2\u003c/em\u003e gene.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eThe overall frequency of the K1, Mad20 and RO33 allelic families was respectively 72.02% (67/93), 36.6% (34/93) and 49.5% (46/93) for the \u003cem\u003ePfmsp-1\u003c/em\u003e gene. The overall frequency of the K1 allelic family was significantly higher than that of Mad20 and RO33 (\u003cem\u003eP\u0026lt;0.05\u003c/em\u003e). For this gene (\u003cem\u003ePfmsp-1\u003c/em\u003e) in general, a significant difference in the RO33 allelic family was observed between the three sites (Makokou = 52.2% (24/46), Lastoursville =32.6% (15/46), Franceville =15.2% (7/46), \u003cem\u003eP=0.008\u003c/em\u003e). In addition, Mad20 and K1 allelic families were more frequent in Franceville compared to RO33, \u003cem\u003eP=0.02.\u003c/em\u003e However, the frequency of the Mad20 allelic family was higher in symptomatic subjects (61.8%; 21/34) than in asymptomatic subjects (38.2%; 13/34), \u003cem\u003eP=0.05\u003c/em\u003e. Concerning the \u003cem\u003ePfmsp-2\u003c/em\u003e gene, the overall frequency of the FC27 allelic family (74.6% (50/67) was significantly higher than that of 3D7 (50.7% (34/67), \u003cem\u003eP=0.004\u003c/em\u003e. For this gene (\u003cem\u003ePfmsp-2)\u003c/em\u003e, the frequency of the 3D7 allelic family was significantly related to geographical area, \u003cem\u003eP=0.01\u003c/em\u003e. (Table 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable1\u003c/strong\u003e: Frequency of allelic families by locality and by clinic status\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGene\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 50px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAllelic families\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" valign=\"top\" style=\"width: 282px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLocalities\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eClinical status\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"4\" valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003emsp-1\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 50px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFranceville\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e% (n/T)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLastoursville\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e% (n/T)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMakokou\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e% (n/T)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 30px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003ep\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSymptomatic\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e% (n/T)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAsymptomatic\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e% (n/T)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 41px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003ep*\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 50px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eK1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e31.1(21/67)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e35.8 (22/67)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e32.8(24/67)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 30px;\"\u003e\n \u003cp\u003e0.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e43.3(29/67)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e56.7 (38/67)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 41px;\"\u003e\n \u003cp\u003e0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 50px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMad20\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e35.8(12/34)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e23.5 (8/34)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e41.7 (14/34)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 30px;\"\u003e\n \u003cp\u003e0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e61.8(21/34)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e38.2 (13/34)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 41px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003e0.05\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 50px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRo33\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e52.2(24/46)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e32.6 (15/46)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e15.2 (7/46)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 30px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003e0.008\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e45.7 (21/46)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e54.3 (25/46)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 41px;\"\u003e\n \u003cp\u003e\u003cem\u003e0.4\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003emsp-2\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 50px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e3D7\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e20.6(7/34)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e26.5 (9/34)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e52.8(18/34)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 30px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003e0.01\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e58.8 (20/34)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e41.2 (14/34)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 41px;\"\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u0026gt;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 50px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eF27\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e32(16/50)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e44 (22/50)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e24 (12/50)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 30px;\"\u003e\n \u003cp\u003e0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e46 (23/50)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e54 (27/50)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 41px;\"\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u0026gt;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ep= p.value between three localities; p*= p. value between two clinical status\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePolyclonal infections of the \u003cem\u003ePfmsp-1 and Pfmsp-2\u003c/em\u003e genes according to clinical status and locality\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCombinations of the K1, Mad20 and RO33 allelic families were detected in 49.5% (46/93) of samples. Overall, the K1_RO33 combination was the most frequent with a frequency of 50% (23/46), followed by K1_MAD20 with a frequency of 21.7% (10/46), the other two allelic family combinations (RO33-Mad20 and K1_Mad20_Ro33) had frequencies of less than 20%.\u003c/p\u003e\n\u003cp\u003eThe proportion of polyclonal infections was higher in Makokou (semi-urban, 45.7% (21/46)) and Lastoursville (32.6% (10/46) rural) than in Franceville (urban, 21.7% (10/46), \u003cem\u003eP=0.04\u003c/em\u003e. Overall, the K1_Mad20 combination was the most frequent with a frequency of 40% (4/10), followed by K1_RO33 with a frequency of 30% (3/10) the other two allelic family combinations (RO33-Mad20 and K1_Mad20_Ro33) had frequencies of less than 20% in Franceville (Fig1.a). Overall, in Lastoursville, the K1_Ro33 combination was the most frequent with a frequency of 67% (10/15), the other three allelic family combination had frequencies of less than 20% (Fig1.c).In Makokou, the K1_Ro33 combination was overall the most frequent with a frequency of 47.6% (10/21) the other three allelic family combination had frequencies of less than 20%. (Fig.1b). The K1_Mad20 combination was significantly higher in symptomatic isolates (70%; 7/10) compared to asymptomatic isolates (30%), \u003cem\u003eP=0.03\u003c/em\u003e (Fig1.d).\u003c/p\u003e\n\u003cp\u003eFor the \u003cem\u003ePfmsp-2\u003c/em\u003e gene, a combination of 3D7/FC27 allelic families was detected in 25.7% (17/67) of the samples. The 3D7_FC27 association was significantly higher in symptomatic isolates (70.6%; 12/17) compared to asymptomatic isolates (29.4%), \u003cem\u003eP=0.02\u003c/em\u003e. (Fig.2)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAllelic diversity and fragment size length polymorphism\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe distribution of \u003cem\u003ePfmsp-1\u003c/em\u003e and \u003cem\u003ePfmsp-2\u003c/em\u003e alleles varied widely among sites. The identified alleles were classified according to fragment size\u0026quot; better. A total of 91 individual alleles of the two genes were identified in all three sites and all subjects. Twenty-three alleles for K1, Mad20 and one for RO33 were found, with band sizes ranging from 195 to 400 base pairs. In addition, 44 different \u003cem\u003ePfmsp-2\u003c/em\u003e alleles with fragment sizes ranging from 394 to 600 base pairs were found, of which 19 belonging to the 3D7 allelic family (fragment range 400-600 bp) and 25 belonging to the FC27 allelic family (fragment range 394-556 bp). \u0026nbsp;The number of alleles detected for \u003cem\u003ePfmsp-2\u003c/em\u003e was not significantly different from \u003cem\u003epfmsp-1\u003c/em\u003e, \u003cem\u003eP=0.7\u003c/em\u003e. The RO33 allelic family was found to be monomorphic (with an amplified fragment size of 270 bp) and accounted for 2.1% (1/47) of all \u003cem\u003ePfmsp-1\u003c/em\u003e genotypes. K1 and Mad20 alleles predominated with frequencies of 48.9% (23/47) each, \u003cem\u003eP\u0026lt;0.05\u003c/em\u003e. Among the 23 K1 and Mad20 alleles of the \u003cem\u003ePfmsp1\u003c/em\u003e gene, K1_327 (14%), K1_273 (12%) K1_343 (10%), and Mad20_360 (14%), Mad20_267 (13%) were the most frequent Fig3.a and Fig3.b). In \u003cem\u003ePfmsp-2\u003c/em\u003e allelic families, FC27_429 (11.8%), 3D7_480 (16%), and 3D7_560 (14%) were the most frequently encountered, with the frequency of other alleles being less than 10%. (Fig3.c and Fig3.d).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe different alleles were classified by sites, 27 and 16 different alleles were identified in Franceville (urban areas) for \u003cem\u003ePfmsp-2\u003c/em\u003e and \u003cem\u003ePfmsp-1\u003c/em\u003egenes (11 FC27 type and 5 3D7 type) and (12 Mad20 type, 14 K1 type and 1 RO33 type) respectively. 3D7_438 (25%), 3D7_462 (37.5%), Mad20_360, alleles were the most frequent. Twenty-two (22) and, 20 different alleles of \u003cem\u003ePfmsp1\u003c/em\u003e (9 of Mad20 type and 12 of K1 type and 1 of RO33 type) and \u003cem\u003ePfmsp-2\u003c/em\u003e (13 of FC27 type and 7 of 3D7 type) genes, were respectively identified in Lastoursville (rural areas), alleles FC27_429 (35.7%) were the most frequent. In Makokou (semi-urban areas) 18 different \u003cem\u003ePfmsp-2\u003c/em\u003e alleles (7 FC27 type and 11 3D7 type) and 17 \u003cem\u003ePfmsp-1\u003c/em\u003e alleles (7 Mad20 type, 10 K1 type and 1 RO33 type) were identified and the most frequent alleles were, Mad20_260 (42.9%) and K1_327 (35.7%). All other alleles from the different allelic families of the two genes had frequencies below 20% in all three sites. Furthermore, certain types of alleles were observed in semi-urban areas as well as in urban and rural areas. For the \u003cem\u003ePfmsp-2\u003c/em\u003e gene, 6 FC27 alleles and 2 3D7 alleles were identified in both Franceville (urban) and Lastoursville (rural areas). Three 3D7 alleles were identified in both Lastoursville (rural) and Makokou (semi-urban areas). \u0026nbsp;For the \u003cem\u003ePfmsp-1\u003c/em\u003e gene, five allelic profiles (Mad20_240, K1_273, K_327, K1_328, and K1_355) common to the three sites were identified. In addition, the Franceville and Lastoursville had 5 common allelic profiles (3 Mad20 and 2 K1), but the Makokou and Lastoursville had 2 common allelic profiles, all K1. Apart from the common allelic profiles in the three localities, no common profile in Franceville and Makokou was found. (Fig.4)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe different allelic profiles varied according to the clinical symptomatology\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;For the \u003cem\u003ePfmsp-2\u003c/em\u003e gene, 31 different alleles were identified in asymptomatic children (13 of type 3D7 and 18 of type FC27) and 28 alleles were identified in symptomatic isolates (17 of type FC27 and 11 of type 3D7). Eight (8) alleles of type 3D7 and eight (8) alleles of type FC27 were only observed in isolates from asymptomatic children. Similarly, 7 alleles of type FC27 and 5 alleles of type 3D7 were only identified in symptomatic isolates. These different alleles had frequencies below 20%. However, for alleles identified in both symptomatic and asymptomatic isolates no significant difference in frequencies was observed \u003cem\u003eP\u0026gt;0.05\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eFor the \u003cem\u003ePfmsp-1\u003c/em\u003e gene, 29 different alleles were found in asymptomatic isolates (10 Mad20 and 19 K1) and 36 different alleles were observed in symptomatic isolates (K1=17, Mad20=19). Three (3) alleles of type Mad20 and four (4) alleles of type K1 were only observed in asymptomatic isolates. Similarly, four (4) alleles of type K1 and thirteen (13) alleles of type Mad20 were only identified in symptomatic isolates with frequencies less than 20%. Some alleles were common to both groups with no significant difference in frequency, \u003cem\u003eP\u0026gt;0.05\u003c/em\u003e. (Fig.5)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMOI, Diversity Index (HE) and Genetic differentiation (FST)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe estimated MOI at the three sites and by clinical status is summarized in Table 2. The overall number of \u003cem\u003ePfmsp-1 and Pfmsp-2\u003c/em\u003e genotypes per isolate ranged from 1 to 5 and 1 to 4 respectively. In Lastoursville the number of \u003cem\u003ePfmsp-1\u003c/em\u003egenotypes per isolate varied from 1 to 4, on the other hand for Franceville and Makokou as well as for the two clinical statuses, the number of genotypes per isolate varied from 1 to 5. The number of \u003cem\u003ePfmsp-2\u003c/em\u003e genotypes varied from 1 to 4 in each site and depending on the clinical status. The overall MOI was 2.06\u0026plusmn;1.07, 2.13\u0026plusmn;1.07 and 2.57\u0026plusmn;0.99 for \u003cem\u003ePfmsp-1\u003c/em\u003e, \u003cem\u003ePfmsp-2\u003c/em\u003e and \u003cem\u003ePfmsp-1\u003c/em\u003e +\u003cem\u003e\u0026nbsp;Pfmsp-2\u003c/em\u003e respectively. The mean MOI for \u003cem\u003ePfmsp-2\u003c/em\u003e \u003cem\u003eand Pfmsp-2, or Pfmsp-1+ Pfmsp-2\u003c/em\u003e genotypes was not significantly different between the three sites. Similarly, no significant difference in MOI was detected between symptomatic and asymptomatic isolates (Table 2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2\u003c/strong\u003e: Number of clones (MOI) detected with \u003cem\u003emsp1, msp2 and msp1+2\u003c/em\u003e according to sites and clinical status.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"633\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" rowspan=\"2\" valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eVariable\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNo. of isolates\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNo. of genotype\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMOI (SD)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 38px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003ePfmsp1\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003ePfmsp2\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003ePfmsp1\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003ePfmsp2\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003ePfmsp1\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 28px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eP\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003ePfmsp2\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 28px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eP\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003ePfmsp1\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e+2\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 38px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eP\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eOverall\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e1-5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e1-4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e2.06(1.07)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 28px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e2.13 (1.07)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 28px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e2.57(0.99)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 38px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eBy site\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFranceville\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e1-5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e1-4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e2(1.16)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 28px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e2.25 (1.06)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 28px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e2.55(1.24)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 38px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLastoursville\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e1-4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e1-4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e1.96(0.98)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e2(0.95)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e2.54(0.85)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMakokou\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e1-5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e1-4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e2.21(1.08)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e2.18(1.22)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e2.62(0.92)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBy clinical status\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAsymptomatic\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e1-5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e1-4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e1.91(1.05)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 28px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e2.03(0.91)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 28px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e2.32(0.81)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 38px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSymptomatic\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e1-5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e1-4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e2.21(1.08)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e2.22(1.19)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 28px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e2.78(1.08)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eNo.= Number, Ns=Non significant; SD= Standard deviation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGenetic diversity (\u003cem\u003eHe\u003c/em\u003e) was estimated based on study sites and clinical status. For the \u003cem\u003ePfmsp-1\u003c/em\u003e gene the genetic diversity was 0.59893\u0026plusmn;0.5183; 0.596\u0026plusmn;0.5174 and 0.5036 \u0026plusmn;0.4371 in Franceville, Lastoursville and Makokou respectively. For \u003cem\u003ePfmsp-2\u003c/em\u003e the genetic diversity was 0.9005\u0026plusmn;0.0277; 0.9115\u0026plusmn;0.0354 and 0.8584 \u0026plusmn;0.0653 in Franceville, Lastoursville and Makokou respectively. The genetic diversity of the \u003cem\u003ePfmsp-1\u003c/em\u003eand \u003cem\u003ePfmsp-2\u003c/em\u003e genes was almost the same in the three study sites. On the other hand, the genetic diversity was more important for the \u003cem\u003ePfmsp-2\u003c/em\u003e marker compared to the \u003cem\u003ePfmsp-1\u0026nbsp;\u003c/em\u003emarker. Furthermore, the genetic diversity of \u003cem\u003ePfmsp-1\u003c/em\u003e, \u003cem\u003ePfmsp-2\u003c/em\u003e and \u003cem\u003ePfmsp-1\u003c/em\u003e+\u003cem\u003e\u0026nbsp;Pfmsp-2\u003c/em\u003e was almost identical between symptomatic (\u003cem\u003ePfmsp-1\u003c/em\u003e =0.7265\u0026plusmn;0.3381, \u003cem\u003ePfmsp-2\u003c/em\u003e =0.9267\u0026plusmn;0.0117 and \u003cem\u003ePfmsp-1\u003c/em\u003e+\u003cem\u003e\u0026nbsp;Pfmsp-2\u003c/em\u003e =0.802\u0026plusmn;0.2612 and asymptomatic (\u003cem\u003ePfmsp-1 =\u003c/em\u003e 0.6746\u0026plusmn;0.3706, \u003cem\u003ePfmsp-2\u003c/em\u003e = 0.9232\u0026plusmn;0.0545 and \u003cem\u003ePfmsp-1\u003c/em\u003e+\u003cem\u003e\u0026nbsp;Pfmsp-2\u003c/em\u003e =0.7764\u0026plusmn;0.2968) (Table 3)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3:\u003c/strong\u003e Genetic diversity (He) according to sites and clinical status\u003c/p\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"577\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 180px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSite\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 397px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eHe (SD)\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 122px;\"\u003e\n \u003cp\u003e\u003cem\u003ePfmsp-1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e\u003cem\u003ePfmsp-2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 143px;\"\u003e\n \u003cp\u003e\u003cem\u003ePfmsp-1+2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 180px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eUrban\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 122px;\"\u003e\n \u003cp\u003e0.5983 (0.5183)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e0.9005 (0.0277)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 143px;\"\u003e\n \u003cp\u003e0.8137 (0.1322)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 180px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRural\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 122px;\"\u003e\n \u003cp\u003e0.596 (0.5174)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e0.9115 (0.0354)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 143px;\"\u003e\n \u003cp\u003e0.7652 (0.2736)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 180px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSemi-urban\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 122px;\"\u003e\n \u003cp\u003e0.5036 (0.4371)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e0.8584 (0.0653)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 143px;\"\u003e\n \u003cp\u003e0.6850 (0.3089)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 180px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eClinical status\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 397px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eHe (SD)\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 122px;\"\u003e\n \u003cp\u003e\u003cem\u003ePfmsp-1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e\u003cem\u003ePfmsp-2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 143px;\"\u003e\n \u003cp\u003e\u003cem\u003ePfmsp-1+2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 180px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAsymptomatic\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eSymptomatic\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 122px;\"\u003e\n \u003cp\u003e0.6746 (0.3706)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e0.9232 (0.0545)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 143px;\"\u003e\n \u003cp\u003e0.7764 (0.2968)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 122px;\"\u003e\n \u003cp\u003e0.7265 (0.3381)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e0.9267 (0.0117)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 143px;\"\u003e\n \u003cp\u003e0.802 (0.2612)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eSD= Standard deviation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe genetic diversity of the two combined markers was gradual from South to North: diversity was first greater in Franceville, then in Lastoursville and finally in Makokou.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;The estimated fixation index (Fst) measuring the differentiation of the parasite population due to the genetic structure at each site was 0.08202, 0.10413 and 0.13634 between Franceville and Lastoursville, then between Lastoursville and Makokou, finally between Franceville and Makokou respectively. Overall the Fst between the populations of the different localities was moderate. A greater proximity of populations between Franceville and Lastoursville then between Lastoursville and Makokou was observed. But a greater difference in genetic differentiation was observed between Franceville and Makokou (Figure 6). This result is consistent with the proportion of genotypes common to the various sites. The highest proportion of allele sharing was observed between Franceville and Lastoursville, then between Lastoursville and Makokou (Fig.6). \u0026nbsp;The study of the flow of parasite populations between symptomatic and asymptomatic has made it possible to observe a close proximity of populations between symptomatic and asymptomatic (Fst= 0.03134)\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eUnderstanding the genetic diversity and transmission dynamics of Plasmodium falciparum provides information on the intensity of malaria transmission, necessary for evaluating malaria control interventions. The analysis of the genetic profile of \u003cem\u003ePlasmodium falciparum.\u003c/em\u003e, obtained after PCR, according to the type of infection, can provide useful information on the characteristics of certain specific parasitic clones in order to design intervention strategies targeting virulence factors [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. It has been shown that most alleles fluctuate considerably over the years and can vary from one endemic region to another [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The present study is the first to evaluate the virulence factors of \u003cem\u003ePlasmodium falciparum\u003c/em\u003e. In Franceville (urban area), Lastoursville (rural) and Makokou (semi-urban) settings in asymptomatic and symptomatic children in the South-East and North-East of Gabon.\u003c/p\u003e\u003cp\u003eOverall, the results of our study showed that the three allelic families K1, Mad20 and RO33 of the \u003cem\u003ePfmsp-1\u003c/em\u003e gene and the two allelic families 3D7 and FC27 of the \u003cem\u003ePfmsp-2\u003c/em\u003e gene were present in the three study sites. The distribution of these allelic families was different according to the site, the RO33 allelic families were more frequent in urban areas than in semi-urban and rural areas. And the frequency of 3D7 was higher than FC27 in semi-rural settings. This supports the hypothesis that the frequency of \u003cem\u003ePfmsp-1\u003c/em\u003e and \u003cem\u003ePfmsp-2\u003c/em\u003e allelic families may vary between localities and even within the same locality [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The K1 and FC27 allelic families are the most frequently encountered. Studies in Gabon, Burkina Faso and C\u0026ocirc;te d'Ivoire described a high frequency of the K1 and 3D7 allelic families, but in this study the frequency of the FC27 allelic family was lower than that of 3D7 [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The Mad20 allelic family was most frequent in isolates from symptomatic versus asymptomatic children in all samples in both urban and rural settings. This allelic family could play an important role in symptomatic malaria, at least in Gabon. This is different from the data described in the literature because the K1 and 3D7 allelic families have often been associated with symptomatic infection [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] than FC27. The frequency of FC27 and 3D7 allelic families was not different between symptomatic and asymptomatic, which proves that there would not exist specific \u003cem\u003ePfmsp-2\u003c/em\u003e variants that could be associated with clinical status. These results are supported by studies that have shown that there is no association between the FC27 or 3D7 allelic families and the type of infection [156]. Conversely, another study reported a frequency of the FC27 allelic family twice as high in symptomatic isolates compared to asymptomatic [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. However, further studies are needed to clarify the interpretation of these results.\u003c/p\u003e\u003cp\u003eOverall, our study reported high genetic diversity with forty-seven genotypes for the \u003cem\u003ePfmsp-1\u003c/em\u003e gene and 44 genotypes for the \u003cem\u003ePfmsp-2\u003c/em\u003e gene in parasites found in the Southeast and Northeast from Gabon. These different genotypes were distributed as follows, 23 K1 alleles and Mad20 1 Ro33 allele (fragment sizes 195-600bp) for the msp1 gene. In the \u003cem\u003ePfmsp2\u003c/em\u003e gene, 19 alleles for 3D7 (fragment range of 400\u0026ndash;600 bp) and 27 different alleles for FC27 (fragment range of 394-600bp) have been demonstrated. This observation is consistent with that of some studies conducted in different malaria-endemic countries in Africa [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The high genetic diversity of the parasites may be related to the high prevalence of Plasmodium infection in the study area where the prevalence is ranging from 20% to about 70% have been found [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. This great diversity observed could also be associated with the high risk of being infected by several parasitic clones of different genotypes. Similarly, the implementation of control methods and the use of antimalarials could also lead to high genetic diversity [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Thirty-one (31) and twenty-eight (28) different alleles of the msp-2 gene were identified in isolates from asymptomatic and symptomatic children respectively. The allele frequency of 3D7 and FC27 was not significantly different between isolates from symptomatic and asymptomatic children. This suggests that the allelic diversity of FC27 and 3D7 in both groups was the same and that there is no relationship between the clinical status and the polymorphism of these alleles. For the msp1 gene, 29 different genotypes and 36 different genotypes were identified in isolates from asymptomatic and symptomatic children respectively. K1 and Mad20 allelic variants had similar frequencies between isolates from asymptomatic and symptomatic children. Suggesting a high polymorphism of this allele in asymptomatic and symptomatic isolates. This result is different from that of other studies that have shown a link between asymptomatic infection and the polymorphism of the K1 allele [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. The different genotypes of \u003cem\u003ePfmsp-1\u003c/em\u003e and \u003cem\u003ePfmsp-2\u003c/em\u003e gene alleles identified in both symptomatic and asymptomatic isolates did not have significantly different frequencies. This suggests that the clinical status of the children included in the study was not related to genetic polymorphism. asymptomatic but were not different between the two groups [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Indeed, genetic polymorphism is strongly affected by the level of endemicity. With high endemicity, multiclonal infections and crosses are predominant [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In addition, 12 Mad20 genotypes, 8 FC27 genotypes, 4 K1 genotypes and 8 3D7 genotypes were only found in symptomatic isolates. And 4 K1 genotypes, 3 Mad20 genotypes, 7 FC27 genotypes and 5 different 3D7 genotypes were only found in the asymptomatic isolates. These results support the idea that certain parasitic clones could be responsible for the clinical state of an individual. However, the link between the clinical condition and any \u003cem\u003eP. falciparum\u003c/em\u003e genotype remains a controversial subject. Because many studies have shown that the presence of subsequent malaria episodes could be due to new infectious parasites [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eA high genetic polymorphism within the allelic families of the msp1 and 2 genes was observed in isolates from urban areas (27 alleles for msp-1 and 17 alleles for msp-2), from rural areas (22 alleles) for \u003cem\u003ePfmsp-1\u003c/em\u003e and 20 alleles for \u003cem\u003ePfmsp-2\u003c/em\u003e) and semi-urban areas (Franceville, 18 msp-2 alleles and 17 msp-1 alleles). Our results are lower than those described in Libreville (30 alleles), Dielmo in Senegal (33 alleles) in 1995 and Burkina Faso in 2009 (41 alleles) and identical to those of Bakoumba near Franceville (25 alleles) in 1999 [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. This allelic diversity could be explained by the fact that the genetic diversity of \u003cem\u003eP. falciparum\u003c/em\u003e varies from one region to another and within the same population [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Some genotypes were common between rural and urban areas, then between semi-urban and rural areas, and finally between the three localities. This observation could lead to the conclusion that certain genotypes could easily circulate between urban and rural areas due to the proximity of these two localities. Also the demographic facies of the rural environment and that of the semi-urban environment being almost identical, it is therefore not surprising that these two localities share a greater number of parasitic genotypes compared to the urban and semi-urban environment. This difference in allele frequencies in the three localities could also be due to the difference in prevalence observed here. In fact, in semi-urban and rural areas, the prevalence of plasmodial infection was higher than in urban areas.\u003c/p\u003e\u003cp\u003eThe number of \u003cem\u003ePfmsp-1\u003c/em\u003e and \u003cem\u003ePfmsp-2\u003c/em\u003e genotypes per isolate ranged from 1 to 5 and 1 to 4 respectively. The MOI is an indicator of the level of malaria transmission because it was expected to be higher in high transmission areas and lower in low transmission areas [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Our study is in line with this hypothesis because high MOIs were found in all localities with values ​​ranging from 1.96 to 2.78. MOI could also be a useful indicator of immune status. MOI has been suggested to be associated with symptoms of Plasmodium infections [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. However, although the MOI was high in all localities, no significant difference in MOI between symptomatic and asymptomatic in \u003cem\u003eP. falciparum\u003c/em\u003e was demonstrated in our study. This high MOI value could be explained by the fact that malaria transmission could be high in the areas studied due to the high prevalence of \u003cem\u003eP. falciparum\u003c/em\u003e observed in this study.\u003c/p\u003e\u003cp\u003eThe high genetic diversity observed in our study is confirmed by the value of the diversity index (He) ranging from 0.6 to 0.9 for the two genes. The \u003cem\u003eH\u003c/em\u003ee value was higher for \u003cem\u003ePfmsp-2\u003c/em\u003e compared to \u003cem\u003ePfmsp-1\u003c/em\u003e. Moreover, the diversity index was not significantly different between the sites, and between the two clinical statuses for the two genes. This lack of variability between the levels of genetic diversity observed within the country is not in accordance with the local epidemiological parameters of the country. Because the level of diversity is expected to be high in areas with high transmission. In addition, the genetic diversity of the two markers combined more or less follows a gradient from South to North: the diversity was first greater in urban areas, then in rural areas and finally in semi-urban areas. In this study, the \u003cem\u003eHe\u003c/em\u003e does not reflect the level of prevalence, because here the prevalence of plasmodial infection was higher in rural and semi-urban areas compared to urban areas. Contrary results were found in Senegal and Mali where He values ​​increase with the transmission gradient from north to south [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. The high heterozygosity observed in these localities could indicate a reduction in clonal expansion and an increase in genetic diversity in these localities. It would also mean that the genetic diversity of \u003cem\u003eP. falciparum\u003c/em\u003e would increase in areas with high transmission and decrease when transmission declines [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eConcerning the Fst, we found moderate genetic differentiations of the parasite populations of \u003cem\u003eP. falciparum\u003c/em\u003e between the three sites. The average values ​​of Fst found between the populations of \u003cem\u003eP. falciparum\u003c/em\u003e show that there would be a greater proximity of the populations between the urban and rural areas on the one hand, then between the rural and semi-urban areas on the other hand. But a greater difference in genetic differentiation was observed between urban and semi-urban areas. This result is consistent with the proportion of genotypes common to the various sites. These data also reveal gene flow between urban and rural parasite populations facilitated by extensive human migrations between these two localities and subsequently causing vector displacement. In addition, the greatest proportion of allele sharing was observed between urban and rural areas, then between rural and semi-urban areas. Although our results are superior to those from Senegal, they confirm that there could be gene flow between parasite populations facilitated by human migration events between endemic regions and facilitating vector movement [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Genetic differentiation studies have not yet been carried out in our different localities, but studies carried out in other countries have reported a lack of genetic differentiation [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. This is the case of our study because the study of the flow of parasitic populations between symptomatic and asymptomatic has made it possible to observe a close proximity of the populations between the two clinical groups (Fst\u0026thinsp;=\u0026thinsp;0.03134). However, other analyzes such as sequencing by next-generation techniques are necessary in order to confirm the existence or not of parasitic clones responsible for the clinical state of an individual. One of the limitations of this study is the small number. Another limitation is the low rate of amplification of the \u003cem\u003ePmfp2\u003c/em\u003e gene\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eOur study revealed a high genetic diversity of the pfmsp1 and pfmsp2 genes. high to high MOI was also observed. The Mad20 allelic family of msp1 was the most prevalent among the asymptomatic group, this allele may play an important role in symptomatic malaria. Additionally, some alleles of the msp1 and msp2 allelic families were only observed in symptomatic individuals. These alleles could be responsible for virulence in symptomatic patients, and could be of key interest for vaccine research.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eInstitutional Review Board Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study has been approved by Gabon’s National Ethics Committee (CNER) under number: PROT 0023/2013/ SG/CNER.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed Consent Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWritten informed consent was obtained from the parents or guardians before each child’s participation in the study. No non-consenting participants were included in the present study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding statment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by CIRMF, the Gabonese Government, CANTAM organisation and Total Gabon.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eL.C.K., S.L.O.-L. and J.B.L.-D. designed the study. LCK, participated in the field survey and sample collection. FDVM. and LCK carried out the microscopic and moleccular analysis in the laboratory. L.C.K. carried out data analyses, and wrote the manuscript. S.L.O.-L. and J.B.L.-D. reviewed the document. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors thank the children and their parents for agreeing to participate in this study. We also wish to thank the staff of the pediatric wards of the Lastoursville health centre and Amissa Bongo regional hospitals in Makokou and Franceville, respectively. We are also grateful to the staff of the Unit of Evolution, Epidemiology and Parasitic Resistances (UNEEREP) of CIRMF.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWHO (2021) \u003cem\u003ehttps\u003c/em\u003e:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e//wwwwhoint/teams/global-malaria-programme/reports/world-malaria-report-2021\u003c/span\u003e\u003cspan address=\"http:////wwwwhoint/teams/global-malaria-programme/reports/world-malaria-report-2021\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e \u003cem\u003e(accessed Jan 03, 2022)\u003c/em\u003e 2021\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSant\u0026eacute;) (2013) D-EDGdlSdG-EDed: Enqu\u0026ecirc;te D\u0026eacute;mographique et de Sant\u0026eacute; au Gabon-Phase II (EDSG II), 501p\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMdl sant\u0026eacute;-SNIS (2016) Mission de collecte des donn\u0026eacute;s en vue d\u0026rsquo;une cartographie pour \u0026eacute;tablir un \u0026eacute;tat des lieux, des \u0026eacute;quipements, des usages et perception des TIC des \u0026eacute;tablissements concern\u0026eacute;s par le Syst\u0026egrave;me National d\u0026rsquo;Information Sanitaire (SNIS). 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Am J Trop Med Hyg 54:632\u0026ndash;643 [PubMed] [CrossRef] [Google Scholar]\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Supplementary Table","content":"\u003cp\u003eSupplementary Table is not available with this version.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Plasmodium falciparum, Pfmsp1, Pfmsp2, virulence, rural, semi-rural, urban, Gabon","lastPublishedDoi":"10.21203/rs.3.rs-7462994/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7462994/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003eBackground\u003c/b\u003e\u003c/p\u003e\u003cp\u003eMalaria cases in Gabon vary, from asymptomatic infection to severe malaria. The present study explores the allelic diversity of the merozo\u0026iuml;te surface protein genes Pfmsp1 and Pfmsp2 among Plasmodium falciparum isolates in three endemic areas, and their potential correlation with virulence.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMethods\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThis cross-sectional study involved elementary schools in Gabon's urban, semi-urban and rural areas. 102 samples were analysed using PCR to examine Pfsmp1 and Pfmsp2.\u003c/p\u003e\u003cp\u003e\u003cb\u003eResults\u003c/b\u003e\u003c/p\u003e\u003cp\u003eForty-seven allele types were identified in the \u003cem\u003ePfmsp1\u003c/em\u003e block, K1 allele type was the most prevalent (72.2%), followed by the RO33 allele type (49.5%) and the Mad20 allele type (36.6%). Forty-four alleles were detected in \u003cem\u003ePfmsp2\u003c/em\u003e; the FC27-like allele was the most prevalent (74.6%), followed by the 3D7-like allele (50.7%). The Mad20 allelic family was more prevalent in symptomatic isolates than in asymptomatic ones. Some Mad20, K1 and RO33 genotypes were found only in symptomatic patients, while other genotypes were found only in asymptomatic patients. The number of \u003cem\u003ePfmsp-1 and Pfmsp-2\u003c/em\u003e genotypes per isolate ranged from 1 to 5 and 1 to 4, respectively. The MOI ranged from 1.95 to 2.78. The diversity index (\u003cem\u003eHe\u003c/em\u003e) ranged from 0.6 to 0.9 for both genes and was higher for \u003cem\u003ePfmsp_2\u003c/em\u003e than for \u003cem\u003ePfmsp-1.\u003c/em\u003e The estimated fixation index (Fst) per site was 0.08202, 0.10413 and 0.13634 between Franceville/Lastoursville, then between Lastoursville/Makokou and finally between Franceville/Makokou.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusion\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe study revealed genetic diversity in the area, with some genotypes being asymptomatic and others symptomatic. ​This data could help develop a vaccine to reduce the disease.\u003c/p\u003e","manuscriptTitle":"Dynamics of Plasmodium falciparum genetic diversity in symptomatic and asymptomatic children in three endemic areas of Gabon","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-01 10:27:17","doi":"10.21203/rs.3.rs-7462994/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"41cc4b0d-0c2b-4255-a154-aec0599eb1ef","owner":[],"postedDate":"September 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-09-01T10:27:20+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-01 10:27:17","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7462994","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7462994","identity":"rs-7462994","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}