Background
The last decade, tremendous concerted efforts have been made to control malaria [
1], and the success observed in some countries (Tanzania, Ethiopia) has raised hope in the global community. This decline in the disease burden with 655,000 deaths in 2011 could be attributed to control interventions like insecticide-treated nets, indoor insecticide spraying, deployment of artemisinin-combination therapy (ACT) and intermittent preventive treatment for pregnant women [
2,
3].
The challenges facing all those fighting malaria are the elaboration of efficient tools that could be used in any endemic country and reached all the affected population. In that perspective, malaria vaccine would be the best weapon if efficient and affordable by poor people [
4,
5].
In areas where malaria is endemic, immunity to
Plasmodium falciparum malaria develops slowly and is hardly ever complete. This phenomenon is explained by the fact that many parasite strains, differing in the sequences of key protective antigens circulate within any given malaria endemic area [
6]. The genetic and antigenic diversity of
P. falciparum strains has been reported as a major obstacle for the development of an effective malaria vaccine. Indeed, it has been shown geographical diversity of
P. falciparum strains in asymptomatic and symptomatic isolates [
7] and it is quite difficult to identify the best immune targets that will lead to the malaria vaccine, eventually. There is a need to understand why some children do not develop malaria episodes while others have repeatedly malaria attacks under the same malaria and socio-economic exposure. Human genetic background like sickle cell trait carriage, blood group and other mutations have been reported to influence susceptibility to clinical malaria [
8,
9] of some exposed individuals but the mechanisms remain poorly understood.
The merozoite surface protein−2 (MSP-2) of
P. falciparum is considered as a good candidate for inclusion into a malaria vaccine. Several studies have reported the relationship between protection and humoral immune responses to MSP-2 antigens [
10,
11]. Moreover, merozoite surface protein-2 gene (
msp2) is also commonly used as a single marker for the molecular characterization of field malaria parasites [
12], because it appears to be more or at least as equal reliable as the merozoite surface protein-1 (
msp1) marker [
6,
13]. These genes are represented as a single copy on
P. falciparum genome [
14] and high degree of polymorphism has been reported in the block 2 for
msp1 gene [
15] and in the central variable region for
msp2 gene [
14]. Typing of these different polymorphic
P. falciparum genome regions have permitted to determine malaria infection indicators e.g. diversity of
P. falciparum strains and multiplicity of infection (MOI), which may contribute to the description of malaria situation in a given location.
Against this background, many sub-Saharan African countries cannot, because of limited resources, report updated data on the malaria situation and specifically on the genetic diversity of malaria parasites circulating in their areas. These data would assist in identifying the most appropriate strategies for control and also to evaluate the impact of control interventions.
In the Republic of Congo, since 2009, the Central Africa Network on Tuberculosis, HIV/AIDS and Malaria (CANTAM) has initiated baseline epidemiological studies for collecting in vivo and in vitro data on sensitivity to anti-malarials and characterization of malaria parasites infecting children living in Brazzaville. The major objective of the present study carried out in the southern part of Brazzaville was to evaluate P. falciparum genetic diversity and multiplicity of infection in isolates from children who did not have any malaria episode (considered as “protected” against clinical malaria) and children who presented three or more uncomplicated malaria episodes (considered as “non-protected”) during one year follow-up. The secondary objective was to characterize the msp2 profiles of P. falciparum isolates collected from successive malaria episodes in ten children who had four or more clinical episodes during the follow up.
Discussion
In malaria endemic areas, people are exposed to diverse
P. falciparum strains and this contributes to the development of natural immunity including clinical and parasite-immunity. Determining
P. falciparum genetic diversity and MOI from field samples may be useful and helpful indicators to describe malaria infection in a place and to relate to the level of immunity at the time of infection [
21].
This study carried out in 2009 in Southern part of Brazzaville after the introduction of ACTs showed that
P. falciparum genetic diversity in isolates from Congolese children did not change and remains at about 20
msp2 alleles. To the best of our knowledge, this study is the first to describe
P. falciparum infections in clinical cases in Congolese children after the introduction of ACTs in the Republic of Congo. Based on the one year follow up of children, two clinical groups were considered according to the number of malaria episodes presented over the study period: “protected” and “unprotected” referring to the absence of malaria episodes or more than two malaria episodes respectively. Interestingly,
msp2 genetic diversity in the “protected” group was higher compared to the unprotected children. A possible explanation could be the better control of
Plasmodium falciparum strains by protected children under a threshold leading to fever [
22].
The genetic diversity of the
msp2 is limited in both clinical groups at asymptomatic phase. However, it is observed that the 3D7 allelic family, which is the most prevalent in the general population [
19] is also the most prevalent in protected children, whereas the FC27 allelic family predominated in the unprotected group, pointing out that FC27 allelic types are the less successful controlled strains in unprotected children and probably acquisition of natural semi-immunity in this population has to include the control of these specific strains. In many countries (Tanzania, Burkina Faso, Malawi and Uganda), Mwingira
et al.[
23] showed that the 3D7 family was the most predominant in clinical isolates. Contrary, in Gabon [
24] and Cameroon [
25], the FC27 family was found to be the most predominant parasite allelic types. This difference can be explained by the fact that genetic diversity of
P. falciparum differs according to geographic areas, and the level of transmission [
26].
With regard to the parasite density, during the asymptomatic phase, a significantly higher parasite density was observed in unprotected children. These findings are in agreement with another study suggesting the association of asymptomatic parasitaemia of higher parasite density with a higher risk of symptomatic malaria in children [
25]. Considering the molecular epidemiological studies carried out in this population before the introduction of ACT [
27,
28], the level of parasitaemia in clinical cases is reduced in the present study. This suggests that intermittent treatment of asymptomatic infections with a high parasitaemia would be an efficient tool of preventing clinical episodes of malaria in childhood. To implement such an intervention, the definition of a parasitaemia threshold for Congolese patients [
22], which has not been defined, would be imperative.
The MOI is considered to be a key indicator of malaria infection in humans and may reflect to some degree malaria transmission and immunity [
6]. The mean MOI was slightly higher in unprotected children and this result correlates with the higher parasite density in unprotected children with asymptomatic infection. This finding could be interpreted as a reduce acquired immunity in unprotected children confirming a higher risk of occurrence of clinical malaria [
6,
21,
29]. However, the mean MOI in clinical isolates is lower than expected from previous studies conducted in the same area and the same age group. It is important to note the low MOI of about 1.3 and the high number of children harbouring one malaria genotype in the study group with regard to the level of malaria transmission [
19] This is different to what was reported from other endemic areas in Central Africa [
23,
30]. This could be explained by the massive distribution of impregnated mosquito nets by the government and supporting agencies and the deployment of artemisinin-combination therapy in the country. As a result, there has been a decrease of the burden of malaria parasites, reflected in lower parasite densities, but this did not influence the diversity of parasites in circulation.
In a second step of this study,
P. falciparum infections during successive clinical attacks in unprotected children have been characterized by analysing each isolate using three molecular markers:
msp1,
msp2 and
glurp when necessary [
23]. The discrimination between recrudescence and new infections has been carefully analysed. It appears that the successive clinical episodes experienced by each child were caused by genetically distinct parasite populations. This gives a confirmation that each episode was a true one instead of being a recrudescence and only one child for one episode was identified as recrudescence. Therefore, we can claim that malaria episodes in these children were caused by new inoculated parasites. These findings are in line with previous reports from Soudan, Senegal, and Gabon [
31‐
33]. It is worth noting that the FC27 fragment of 400bp, which was the most prevalent in clinical episodes of malaria, was also observed with a high frequency in other studies among symptomatic malaria children [
34,
35]. This may indicates an association between this FC27 allelic type and clinical episodes, hence predicts a possible candidate antigene that can be considered in designing malaria vaccine.
As a conclusion, this study shows that the introduction of ACT in the Republic of Congo has reduced the multiplicity of infection but not the genetic diversity of P. falciparum isolates from children living in Southern districts of Brazzaville. It also points out that children exposed to the same malaria transmission and socio-economic conditions might have different susceptibility to malaria infections. The two groups described here are important for designing additional studies to investigate human and parasite genetic factors that may be involved in the susceptibility/resistance to malaria in this area.
Competing interests
The authors declare that they have no competing interest.
Authors’ contributions
RIO participated in genomic DNA extraction, molecular genetic study and writing of the manuscript. FKK contributed in data analysis and writing of the manuscript. CJV participated in data analysis. VM participated in molecular genetic study. MN and NPC designed and supervised the field work. AS contributed in writing of the manuscript. JRI participated in correction of the manuscript. FN supervised the different steps of the work and participated in writing of the manuscript. All authors contributed to the final manuscript.