Background
Despite significant reduction in malaria-related morbidity and mortality in the recent past, malaria remains endemic in the tropics and sub-tropics including sub-Saharan Africa. About 225 million clinical cases and 781,000 deaths were reported worldwide in 2009, whereby almost 90% occurred in sub-Saharan Africa [
1]. Current malaria control strategies include the use of insecticide-treated bed nets (ITNs), indoor residual spraying of insecticide, intermittent preventive treatment to young children and pregnant women, and early parasitological diagnosis and treatment of clinical cases using artemisinin-based combination therapy (ACT) [
2‐
4]. Deployment of these strategies has had significant impact on malaria in many endemic areas.
Although the impact of current malaria control strategies is encouraging, much more remains to be done to attain elimination. In the meantime, malaria vaccine is considered as an additional necessary arsenal. However, genetic diversity in
Plasmodium falciparum is a major limitation for the successful development of an effective malaria vaccine, as it influences the level and efficacy of acquired protective immunity to malaria. Therefore, screening of the genetic diversity of malaria parasite populations in different endemic settings is an important step towards the development and/or the evaluation of malaria vaccines [
5].
Genotyping of merozoite surface protein-2 (
msp2) is the commonly used method to study the genetic diversity of
P. falciparum. This gene presents polymorphisms in the number of repeats and sequence type [
6]. The analysis of
msp2 alleles involves two major allelic families, FC27 and 3D7. The
msp2 gene is also the basis of determining the multiplicity of infections (MOI) in infected individuals, which is defined as the number of distinguishable
P. falciparum clones
per infected individual. MOI is a good indicator of acquired immunity or premunition of the populations living in endemic areas and is also correlated to the transmission intensity [
7,
8]. It has been demonstrated that MOI could be influenced by parasite density and age [
9], and haemoglobin (Hb) type carriage [
10].
Makélékélé is an administrative division in the south-eastern part of Brazzaville (Republic of Congo), and has been well characterized as a highly endemic area with perennial malaria transmission [
11,
12]. However, there is a paucity of information on the genetic diversity of
P. falciparum populations. Following the replacement of chloroquine (CQ) with ACT (artesunate-amodiaquine and artemether-lumefantrine as first and second line treatment respectively) in 2006 for the treatment of uncomplicated malaria in the Republic of Congo [
13], CQ drug pressure has since been reduced. In Malawi, absence of CQ pressure was followed by the re-emergence of CQ-susceptible malaria, based on genotyping of CQ resistance biomarker,
P. falciparum chloroquine resistance transporter gene (
pfcrt) [
14‐
16]. In Congo Brazzaville, molecular analysis of the parasite population, including the CQ resistance
pfct K76T mutation has not been investigated since the change of anti-malarial treatment policy in 2006. Therefore, the present study was conducted in the semi-urban area of Makélékélé to determine (i) the prevalence and the multiplicity of
P. falciparum asymptomatic infection; (ii) the genetic diversity of the
msp2 gene; and (iii) the prevalence of
pfcrt K76T mutation in
P. falciparum isolates.
Methods
Study site
The study conducted under the auspices of the Central Africa Network on Tuberculosis, HIV/AIDS and Malaria (CANTAM) project, was carried out in three districts of Makélékélé health division: Ngoko, Kinsana and Mbouono. These semi-urban divisions with about 6,000 inhabitants are located in the southern part of Brazzaville along the Congo River where malaria is transmitted throughout the year [
12].
Plasmodium falciparum is the main plasmodial species and
Anopheles gambiae the main mosquito vector.
Study population
Children aged one to nine years and permanent residents of the study area were enrolled in a cohort for malaria surveillance study (Ndounga et al, unpublished). From October 2009 to June 2010, a demographic survey was conducted and 313 children under 10 years were enrolled. Malaria incidence is 0.9 malaria episode/year/child. Inclusion criteria were absence of clinical malaria in the last two weeks and at least one week after enrolment, and with an axillary temperature of < 37.5°C. Recruitment was done from April to June 2010, and informed consent was obtained from parents or guardians. Thick and thin blood smears were made from each child. About 4 ml of whole blood were also collected in EDTA tube for haemoglobin concentration measurement and DNA extraction. This study was approved by the Institutional Ethics Committee for Research on Heath Sciences of the Republic of Congo.
Microscopic examination
Thick and thin blood films were stained with 10% Giemsa for 15 min and read by two independent competent microscopists to determine malaria species and the parasite density. Asexual parasites were counted against 200 leucocytes and expressed as the number of asexual parasites/μl of blood, assuming the leucocyte count of 8,000/μl of blood.
Genomic DNA was extracted from 200 μl of whole blood sample using the QIAmp DNA Blood Mini kit (QIAGEN Gmbh, Hilden, Germany) following the manufacturer's instruction. DNA was recovered in 200 μl of elution buffer provided in the kit and stored at -20°C until use.
msp 2 genotyping of plasmodium falciparum isolates
To characterize parasite infection,
P. falciparum genotyping was performed using a nested PCR for
msp2 central region, which comprises repeats of varying lengths. The FC27 and 3D7 allele families were analysed. The specific oligonucleotide primers and PCR conditions were as previously described by Ntoumi
et al[
17].
Pfcrt K76T PCR-typing
The nested PCR followed by restriction enzyme digestion PCR reactions were used to genotype the Lysine to Threonine mutation in codon 76 of
pfcrt as described by Mayengue
et al[
18], Djimde
et al[
19] and Mayor
et al[
20]. All isolates successfully amplified using
msp2 PCR were assayed with the
pfcrt PCR.
Sickle cell trait
β-globin genotypes HbAA, HbAS and HbSS were determined using allele specific PCR method as described by Wu
et al[
21].
Data analysis
The prevalence of FC27 and 3D7 alleles was determined as the presence of PCR products for each type in the total number of amplified bands for the corresponding locus. The MOI was determined as the number of different
msp2 genotypes per isolate, and the mean MOI was calculated as the total number of detected
Plasmodium falciparum msp2 genotypes/total number of infected children [
18]. The
msp2 allelic frequency represents the ratio of one specific allele out of the total number of alleles identified in the isolates. The polyclonality (percentage of isolates with more than one amplified PCR fragment) was estimated for each group. Similarly, proportions of wild and mutant
pfcrt alleles were calculated as the ratio of each allele type out of the total of
P. falciparum positive samples. Statistical analysis was done using XLSTAT software (Version 2011.2.08). The chi-square test was used to compare quantitative variables such as parasite prevalence and MOI between different groups. The Spearman's rank correlation coefficient was calculated to assess association between MOI and parasite density. Differences were considered statistically significant at
P values < 0.05.
Discussion
In the Republic of Congo, less attention had been put on the investigation of the genetic diversity of P. falciparum among asymptomatic parasite carriers. Besides, this analysis is necessary to develop strategies for malaria control including the design of malaria vaccines interventions. The current study is the first of its kind to evaluate genetic diversity of P. falciparum isolates circulating among children younger than 10 years old, and to present the molecular analysis of pfcrt gene since the replacement of chloroquine with ACT as the first line anti-malarial drug in Congo Brazzaville.
The prevalence of
P. falciparum carriage of 8.6% (microscopically) and 16% (by PCR) is low compared to those reported previously from other malaria endemic areas [
9,
10,
22]. However, these findings are in line with the results of a recent study conducted in Gabon that showed a relatively low prevalence of asymptomatic
P. falciparum infection of 5% by microscopy among young children [
23]. In the recent past, a large number of ITNs were available to children under five years of age and to pregnant women in the Republic of Congo. The observed low prevalence could be attributed to the wide use of the ITNs. Further investigations involving more children and adults would be of great importance to confirm this finding.
Allele-specific genotyping of
msp2 has shown that
P. falcuparum population in this part of the Republic of Congo have a high genetic diversity. Dispite the fact that a high proportion of isolates (74%) carried one genotype in comparison to those having two and three genotypes, different types of both 3D7 and FC27 alleles were found. The proportion of 3D7 alleles (55%) was slightly high compared with that of FC27 (45%). This is similar to findings in Papua New Guinea [
22].
The MOI (number of concurrent
msp2 genotypes in individual infections) ranged from 1 to 3. The mean MOI of 1.3 is relatively low compared to those reported in Gabon [
24], Senegal [
9,
25], and Ghana [
26] in asymptomatic cases. This might be explained by the fact that diversity of
P. falciparum population differs according to the geographic location, and the level of transmission [
27,
28]. In the same study area [
29], the MOI in isolates from symptomatic children was 2.2. The interpretation of this increase in the mean number of parasite genotype/infected child could be the presence of the uncontrolled strain causing the symptoms.
Age is one of the suggested important factors involved in the acquisition of immunity against
P. falciparum and influences MOI [
9]. On the contrary, these results show that age has no effect on MOI. The absence of influence of age on MOI has also been previously reported in the same area among
P. falciparum symptomatic children by Pembe
et al[
29].
Previous studies on association between MOI and parasite densities showed that high parasite densities increase the probability of detecting concurent clones in an individual [
30,
31]. Expectedly, a positive correlation between MOI and parasite density was found in the present study.
The impact of sickle cell trait on MOI in this study is different from findings in Gabon by Ntoumi
et al[
10], in which, a correlation between sickle cell trait and MOI was found. However, this observation corroborates reports from Senegale by Konate
et al[
32] and Vafa
et al[
9] whereby no correlation was found. These discrepancies can be explained by differences in age ranges of the study population and the number of individuals with sickle cell trait enrolled.
The prevalence of
pfcrt K76T was also assessed, the mutation associated with CQ resistance. It was anticipated that the sensitivity of the parasite to CQ may have been restored several years after its withdrawal as was the case in Malawi [
16]. The prevalence of
Pfcrt K76T, an important molecular marker surveillance tool that can contribute to the evaluation of local parasite strains sensitivity to CQ in addition to
in vitro studies [
33], was 98% in Congo before withdrawal of CQ [
18]. Almost five years later, this study shows that the prevalence of this mutation remains high, 92%. This observation is similar to a recent report by Efunshile
et al in Nigeria [
34]. Therefore, CQ cannot be re-introduced in Congo now despite its many advantages over other anti-malarials.
Acknowledgements
We thank Aymar Ndounga, Prisca Casimiro, Brunelle Matondo for their participation in the field study. We are grateful to the children who participated in the study. FKK, VM, CJV, and PIM received support of CANTAM network. We are grateful to Professor Theonest Mutabingwa for the critical reading and editing of the manuscript.
This work has been supported through the Central Africa Network on Tuberculosis, HIV/AIDs and Malaria (CANTAM), which is a network of excellence supported by EDCTP.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
FKK performed DNA extraction, molecular genetic studies, data analysis, and drafted the manuscript. VM and PIM participated in molecular genetic studies, CJV participated in the data analysis. MN designed and supervised the field study. FN supervised the different steps of the work. All authors contributed to the final manuscript.