Genetic diversity of Plasmodium falciparum infection among children with uncomplicated malaria living in Pointe-Noire, Republic of Congo

Introduction Molecular characterization of malaria parasites from different localities is important to improve understanding of acquisition of natural immunity to Plasmodium falciparum, to assist in identifying the most appropriate strategies for control and to evaluate the impact of control interventions. This study aimed to determine the genetic diversity and the multiplicity of infection in Plasmodium falciparum isolates from Pointe-Noire, Republic of Congo. Methods Plasmodium falciparum isolates were collected from 71 children with uncomplicated malaria; enrolled into the study for evaluating the therapeutic efficacy of artemether-lumefantrine combination. Both msp-1 and msp-2 genes were genotyped. Results From 296 distinct fragments detected, 13 msp-1 and 27 msp-2 different alleles were identified. For msp-1, RO33 family was poorly polymorphic. The K1 family has shown the trend of predominance (41%), followed by Mad20 (35%). Comparatively to msp-2, 49.6% and 48.8% fragments belonged to 3D7 and FC27 respectively. Taking together msp-1 and msp-2 genes, the overall multiplicity of infection has been increased to 2.64 and 86% harbored more than one parasite genotype. Parasite density was not influenced by age as well as the multiplicity of infection which was not influenced neither by age nor by parasite density. Conclusion Genetic diversity of Plasmodium falciparum in isolates from patients with uncomplicated malaria in Pointe-Noire is high and consisted mainly of multiple clones. The overall multiplicity of infection has been largely increased when considering msp-1 and msp-2 genes together. With the changes in malaria epidemiology, the use of both msp-1 and msp-2 genes in the characterization of Plasmodium falciparum infection is recommended.


Introduction
Malaria, a disease mainly caused by Plasmodium falciparum, remains a public health concern. Although massive interventions deployed in sub-Saharan Africa have reduced the global malaria morbidity and mortality at 212 million and 429,000 deaths respectively in 2015, some sub-Saharan Africa region had provided partial data regarding the impact of certain interventions [1]. In the Republic of Congo, malaria is still the leading cause of attendance in health facilities. The latest estimations from the National Malaria Control Program indicate that clinical malaria account for 47.9% of all outpatient consultations in public hospitals, 64.8% of hospital admissions and 18.4% of deaths [2]. The high levels of resistance of Plasmodium falciparum to chloroquine as well as the inefficacy of sulphadoxine-pyrimethamine and amodiaquine either singly or in combination for the treatment of uncomplicated malaria have been well documented [3][4][5][6]. Thus, the Republic of Congo has changed its anti-malarial drug policy for treating uncomplicated malaria to artemisinin-combination therapies (ACTs) in 2006 [7]. Studies aiming to evaluate the efficacy of these combinations have been conducted mainly in Brazzaville [8][9][10]. Only one was done in Owando, located in the north part of the Republic of Congo [11].
Although the efficacy of artesunate-amodiaquine and artemetherlumefantrine is still high as reported in these previous studies, it is important to extend the efficacy assessment as well as to evaluate the impact of these combinations on the malaria parasite population dynamic and the multiplicity of Plasmodium falciparum infection (MOI) in other localities, nine years after the implementation of artesunate-amodiaquine and artemether-lumefantrine in Republic of Congo.
Genetic diversity of circulating Plasmodium falciparum strain, the occurrence of variant forms of the parasite in different geographic areas and variation of MOI after recombination between genetically distinct gametocytes, constitute the major obstacles to the design of a malaria vaccine [12,13]. Consequently, these two parameters allow characterization of malaria parasites in human populations and improve understanding of acquisition of natural immunity to Plasmodium falciparum and would assist in identifying the most appropriate strategies for control and also to evaluate the impact of control interventions [14,15]. The merozoite surface protein-1 (msp-1) and merozoite surface protein-2 (msp-2) are asexual blood stage antigens that are considered prime candidates for the development of malaria vaccine, and also suitable markers used extensively to identify genetically distinct Plasmodium falciparum parasite sub-populations in many malaria endemic countries as well as to distinguish recrudescence to re-infection in anti-malarial drug trials and efficacy [16][17][18]. It has been proposed that with the change of malaria epidemiology, both msp-1 and msp-2 allele frequency and genetic diversity should be monitored regularly to ensure the reliability of the PCR (polymerase chain reaction)-adjusted treatment outcome [18]. To our knowledge, in the Republic of Congo, after the introduction of artemisinin combination therapy (ACTs), the most epidemiological studies on Plasmodium falciparum genetic diversity have been conducted in Brazzaville [15,19]. However, only one has been done in one health facility in the city of Pointe-Noire, the second largest city in the country, using msp-2 gene as marker [20].

Study area
The present study was conducted at the "Centre de Santé Intégré

Plasmodium falciparum msp-1 and msp-2 genotyping
Samples genotyping of Plasmodium falciparum was performed using the nested polymerase chain reactions (PCRs) technique. The merozoite surface protein-1 (msp-1) and merozoite surface protein-2 (msp-2) genes in their highly polymorphic loci, namely msp-1 block 2 and msp-2 central region were used as markers for this genotyping as described previously [16,17,22,23]. PCR amplification was performed following a 2-step amplification procedure, in which the initial amplifications were followed by individual nested PCR reactions using specific primers for K1, Mad20 and RO33 allelic families for msp-1, and FC27 and 3D7 allelic families for msp-2 (Table 1) (Table 1), assuming that one band represented one amplified PCR fragment derived from a single copy of Plasmodium falciparum msp-1 and msp-2 genes. Alleles in each family were considered the same fragment size was within 20 bp interval [17].

Data and statistical analysis
The frequency of msp-1 and msp-2 allele was calculated as the proportion of allele found for the allelic family out of the alleles detected in isolates. The mean expected heterozygosity (He) value for each allelic family of msp-1 and msp-2 genes was calculated as described by Mohd Abd Razak et al. [24]. The detection of one msp-

Characteristics of patients and parasite density
A total of 71 patients with malaria and microscopically confirmed Plasmodium falciparum only were enrolled in the study, with 34 (47.9%) and 37 (52.1%) being males and females respectively. Twenty-six and 45 were children aged between 1 to 4 years old and 5 to 12 years old respectively. The geometric mean parasite density was 36,699.6 parasites/µl of blood with the range of 1,435-349,700 parasites/µl (Table 2).

Plasmodium falciparum genotyping of msp-1 and msp-2
All 71 samples were analyzed for polymorphisms on msp-   (Table 3), as well as the MOI was not associated with parasite density (Table 4). Moreover, the age was not associated significantly with the parasite density (p-value = 0.381). Taking together, no statistical significant difference was observed in the parasite densities and the MOI according to the age (Figure 3).

Discussion
The genetic diversity may be an important element for implementing malaria control strategies in the country, as elimination may influence genetic diversity [25]. In the Republic of Congo, most of the studies on the Plasmodium falciparum genetic diversity using merozoite surface protein genes which are considered like robust and suitable markers [22,25,26] have been conducted in Brazzaville, the political capital [5,15,17,19,27].
With the expanding access to ACT and current changes in malaria epidemiology, it has been encouraged to monitor regularly the Plasmodium falciparum msp-1 and msp-2 allele frequencygenetic diversity [18], despite some debate around their usefulness as markers of population structure [14,28]. Allelic specific PCR typing of msp-1 and msp-2 genes showed a high genetic diversity in the Plasmodium falciparum population in the analyzed isolates. The degree of polymorphism found in the present study is also consistent with the previous findings in the same area using microsatellites markers [29]. The msp-2 gene has showed a high diversity as previously reported in Pointe-Noire [20].
Although msp-1 gene was amplified in all isolates, genetic diversity still low compared to msp-2 gene as previously described in the country [17]. With regard to msp-1, the presence of Mad20, K1 and R033 allelic families in the 71 isolates was noticed. RO33 family was poorly polymorphic with only one allele as reported in previous studies in Brazzaville and other countries such as Senegal, Iran, Nigeria and Brazil [13,17,[30][31][32] in contrast to findings in Gabon and Côte d'Ivoire [33]. Although the K1 family has shown the trend of predominance (41%), followed by Mad20 (35%), the difference in prevalence between these free families was not statistically significant. Comparatively to msp-2, no predominance to any allelic family was found. Taking  2 genes and this was higher than values obtained from Brazzaville, even before or after the introduction of ACTs [5,17,19,20].
Despite the lack of entomological data from Pointe-Noire and Brazzaville, the number of clones coinfecting a single host can be used as an indicator of the level of malaria transmission or the level of host acquired immunity [12,28,33,37]. Therefore, the discrepancies on the MOI may suggest the different level of malaria transmission between these two cities, with Brazzaville being more Brazzaville, Tanzania and Sudan [17,26,41,42], but concordant with those in Ethiopia and Mali [25,39]. Therefore, regardless of the parasite densities, and the fact that the samples collection has been done during the peak malaria transmission, the prevalence of multi clonal infections affected all the two age groups.