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Background: In the Republic of Congo, hot temperature and seasons distortions observed may impact the development of malaria parasites. We investigate the variation of malaria cases, parasite density and the multiplicity of Plasmodium falciparum infection throughout the year in Brazzaville., Methods: From May 2015 to May 2016, suspected patients with uncomplicated malaria were enrolled at the Hôpital de Mfilou, CSI « Maman Mboualé», and the Laboratoire National de Santé Publique. For each patient, thick blood was examined and parasite density was calculated. After DNA isolation, MSP1 and MSP2 genes were genotyped., Results: A total of 416, 259 and 131 patients with suspected malaria were enrolled at the CSI «Maman Mboualé», Hôpital de Mfilou and the Laboratoire National de Santé Publique respectively. Proportion of malaria cases and geometric mean parasite density were higher at the CSI «Maman Mboualé» compared to over sites (P-value <0.001). However the multiplicity of infection was higher at the Hôpital de Mfilou (P-value <0.001). At the Laboratoire National de Santé Publique, malaria cases and multiplicity of infection were not influenced by different seasons. However, variation of the mean parasite density was statistically significant (P-value <0.01). Higher proportions of malaria cases were found at the end of main rainy season either the beginning of the main dry season at the Hôpital de Mfilou and the CSI «Maman Mboualé»; while, lowest proportions were observed in September and January and in September and March respectively. Higher mean parasite densities were found at the end of rainy seasons with persistence at the beginning of dry seasons. The lowest mean parasite densities were found during dry seasons, with persistence at the beginning of rainy seasons. Fluctuation of the multiplicity of infection throughout the year was observed without significance between seasons., Conclusion: The current study suggests that malaria transmission is still variable between the north and south parts of Brazzaville. Seasonal fluctuations of malaria cases and mean parasite densities were observed with some extension to different seasons. Thus, both meteorological and entomological studies are needed to update the season's periods as well as malaria transmission intensity in Brazzaville.

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., Competing Interests: The authors declare no competing interests.

Background: In the Republic of Congo, artemisinin-based combinations have been recommended for the treatment of uncomplicated malaria since 2006. However, the emergence of resistant parasites again these combinations in Southeast Asia is a threat for the control of this disease, especially in sub-Saharan Africa where the weight of the disease is important. Indeed, polymorphisms in Plasmodium falciparum K13-propeller gene have been involved in variations of drug sensitivity of Plasmodium falciparum to artemisinin-based combinations. The aim of the current study is to determine the prevalence of mutations of this gene in isolates collected in three health centers in Brazzaville., Methods: From May 2015 to May 2016, a total of 131, 259 and 416 samples from patients with suspected malaria were collected at the Laboratoire National de Santé Publique, Hôpital de Mfilou, and the CSI «Maman Mboualé» respectively. After DNA isolation, genotyping and sequencing of Plasmodium falciparum K13-propeller were performed in positive Plasmodium falciparum isolates identified after msp-2 gene genotyping., Results: All 806 samples collected were msp-2 genotyped and Plasmodium falciparum infections were confirmed in 287 samples with 43, 85, 159 samples from Laboratoire National de Santé Publique, Hôpital de Mfilou, and the CSI «Maman Mboualé» respectively. Of these 287 msp-2 positives samples, K13-propeller nested PCR products were successfully obtained from 145 (50.52%) isolates and sequences were generated from 127(87.58%) nested products. None of mutations that were associated with ACTs resistance in Southeast Asia were detected on the samples from three different study sites from Brazzaville. However, one mutation type was observed at position 578, where alanine was substituted by serine (A578S) in two isolates (1.57%, 2/127), those from the Hôpital de Mfilou. No mutation was found in isolates from the two other sites., Conclusion: The current study shows a very limited polymorphism in the K13-propeller gene in isolates from the Republic of Congo and K13 polymorphisms associate with ACT resistance are not present in this country. However, permanent and large surveillance of resistant parasite population using K13-propeller gene is recommended.

Background: Regular monitoring of the levels of anti-malarial resistance of Plasmodium falciparum is an essential policy to adapt therapy and improve malaria control. This monitoring can be facilitated by using molecular tools, which are easier to implement than the classical determination of the resistance phenotype. In Cameroon, chloroquine (CQ), previously the first-line therapy for uncomplicated malaria was officially withdrawn in 2002 and replaced initially by amodiaquine (AQ) monotherapy. Then, artemisinin-based combination therapy (ACT), notably artesunate-amodiaquine (AS-AQ) or artemether-lumefantrine (AL), was gradually introduced in 2004. This situation raised the question of the evolution of P. falciparum resistance molecular markers in Yaoundé, a highly urbanized Cameroonian city., Methods: The genotype of pfcrt 72 and 76 and pfmdr1 86 alleles and pfmdr1 copy number were determined using real-time PCR in 447 P. falciparum samples collected between 2005 and 2009., Results: This study showed a high prevalence of parasites with mutant pfcrt 76 (83%) and pfmdr1 86 (93%) codons. On the contrary, no mutations in the pfcrt 72 codon and no samples with duplication of the pfmdr1 gene were observed., Conclusion: The high prevalence of mutant pfcrt 76T and pfmdr1 86Y alleles might be due to the choice of alternative drugs (AQ and AS-AQ) known to select such genotypes. Mutant pfcrt 72 codon was not detected despite the prolonged use of AQ either as monotherapy or combined with artesunate. The absence of pfmdr1 multicopies suggests that AL would still remain efficient. The limited use of mefloquine or the predominance of mutant pfmdr1 86Y codon could explain the lack of pfmdr1 amplification. Indeed, this mutant codon is rarely associated with duplication of pfmdr1 gene. In Cameroon, the changes of therapeutic strategies and the simultaneous use of several formulations of ACT or other anti-malarials that are not officially recommended result in a complex selective pressure, rendering the prediction of the evolution of P. falciparum resistance difficult. This public health problem should lead to increased vigilance and regular monitoring.

Background: In order to prepare the field site for future interventions, the prevalence of asymptomatic Plasmodium falciparum infection was evaluated in a cohort of children living in Brazzaville. Plasmodium falciparum merozoite surface protein 2 gene (msp2) was used to characterize the genetic diversity and the multiplicity of infection. The prevalence of mutant P. falciparum chloroquine resistance transporter (pfcrt) allele in isolates was also determined., Methods: Between April and June 2010, 313 children below 10 years of age enrolled in the cohort for malaria surveillance were screened for P. falciparum infection using microscopy and polymerase chain reaction (PCR). The children were selected on the basis of being asymptomatic. Plasmodium falciparum msp2 gene was genotyped by allele-specific nested PCR and the pfcrt K76T mutation was detected using nested PCR followed by restriction endonuclease digestion., Results: The prevalence of asymptomatic P. falciparum infections was 8.6% and 16% by microscopy and by PCR respectively. Allele typing of the msp2 gene detected 55% and 45% of 3D7 and FC27 allelic families respectively. The overall multiplicity of infections (MOI) was 1.3. A positive correlation between parasite density and multiplicity of infection was found. The prevalence of the mutant pfcrt allele (T76) in the isolates was 92%., Conclusion: This is the first molecular characterization of P. falciparum field isolates in Congolese children, four years after changing the malaria treatment policy from chloroquine (CQ) to artemisinin-based combination therapy (ACT). The low prevalence of asymptomatic infections and MOI is discussed in the light of similar studies conducted in Central Africa.

Background: Chloroquine resistance (CR) decreased after the removal of chloroquine from national treatment guidelines in Malawi, Kenia and Tanzania. In this investigation the prevalence of the chloroquine resistance (CQR) conferring mutant pfcrt allele and its associated chromosomal haplotype were determined before and after the change in Gabonese national treatment guidelines from chloroquine (CQ) to artesunate plus amodiaquine (AQ) in 2003., Methods: The prevalence of the wild type pfcrt allele was assessed in 144 isolates from the years 2005 - 07 by PCR fragment restriction digest and direct sequencing. For haplotype analysis of the chromosomal regions flanking the pfcrt locus, microsatellite analysis was done on a total of 145 isolates obtained in 1995/96 (43 isolates), 2002 (47 isolates) and 2005 - 07 (55 isolates)., Results: The prevalence of the mutant pfcrt allele decreased from 100% in the years 1995/96 and 2002 to 97% in 2005 - 07. Haplotype analysis showed that in 1995/96 79% of the isolates carried the same microsatellite alleles in a chromosomal fragment spanning 39 kb surrounding the pfcrt locus. In 2002 and 2005 - 07 the prevalence of this haplotype was 62% and 58%, respectively. Pfcrt haplotype analysis showed that all wild type alleles were CVMNK., Conclusion: Four years after the withdrawal of CQ from national treatment guidelines the prevalence of the mutant pfcrt allele remains at 97%. The data suggest that the combination of artesunate plus AQ may result in continued selection for the mutant pfcrt haplotype even after discontinuance of CQ usage.

Background: The characterization of malaria parasite populations circulating in an area is part of site characterization, as a basis for evaluating the impact of malaria interventions on genetic diversity, parasite species, and multiplicity of infection. The present study was aimed at analysing genetic diversity of Plasmodium falciparum merozoite surface proteins 1 and 2 (MSP-1 and MSP-2) and to determine the multiplicity of infection in clinical isolates collected from children living in the Southern district of Brazzaville in the Republic of Congo., Methods: A total of 125 isolates from patients with uncomplicated malaria attending Terinkyo and Madibou health centres were collected between January and June 2005 while evaluating the therapeutic efficacy of amodiaquine-artesunate combination. DNA was extracted and msp-1 and msp-2 genes were genotyped using allele-specific nested-PCR., Results: Out of 468 distinct fragments detected, 15 msp-1 and 20 msp-2 genotypes were identified. For the msp-1 gene, K1 family was the predominant allelic type carried alone or in association with RO33 and Mad20 types, whereas the 3D7 family was the most prevalent in the msp-2 gene. Overall, the mean multiplicity of infection was 2.2. Out of 125 samples, 104 (83%) harboured more than one parasite genotype. There was no statistical significant difference in the multiplicity of infection by either sex or age of patients. However, a statistically significant correlation was found between parasite densities and the number of genotypes., Conclusion: Polymorphism in P. falciparum clinical isolates from Brazzaville was high and mainly of multiple clones. The basis for the positive association between parasite densities and multiplicity of infection is discussed.

We evaluated the relationship between immune response markers and the multiplicity of Plasmodium falciparum infections in order to assess the validity of the latter as an indicator of the acquisition of anti-malarial immunity. Parasite populations present during malaria episodes of 64 Gabonese children who presented with at least 4 such attacks during active follow-up over a 7-year period were characterized using MSP-1 and MSP-2 PCR-based methods. Plasma samples taken at healthy and parasite-free phase were used to measure P. falciparum antigen-specific antibody and cytokine activity. We found evidence of intra- and inter-individual variation in the number of parasite genotypes present in different malaria episodes, although in 72% of isolates no more than 2 parasite genotypes were detectable. Samples with the highest multiplicity were from children with significantly lower (p < 0.03) antibody responses to specific asexual stage antigens. Additionally, the whole blood interferon-gamma production capacity was significantly higher (p < 0.02) in those with lower infection multiplicity. Malaria episodes with multiple clones indeed reflect a low level of acquired immunity and a consequently poor capacity to control the infection. These findings suggest that the multiplicity of falciparum infection may be a potentially useful parameter in the evaluation of malaria control interventions.

In Lambaréné (Gabon), where a high level of Plasmodium falciparum resistance to chloroquine has been reported, we assessed the relationship between polymorphisms in the P. falciparum chloroquine resistance transporter (Pfcrt) and multidrug resistance-1 (Pfmdr1) genes and the clinical severity of malaria. Ninety-one and 60 P. falciparum isolates from children with uncomplicated or severe malaria were collected in 1996 and 2002, respectively. Single nucleotide mutations at codon 76 in the Pfcrt gene and at codons 86, 184, 1034, 1042, and 1246 in the Pfmdr1 gene were assessed by PCR-RFLP. All P. falciparum isolates presented the Pfcrt K76T mutation, whatever the clinical status. A high prevalence (>80%) of the Pfmdr1 86Tyr and 184Phe mutations was detected at both time points and in both clinical groups. We did not identify any specific mutation in the Pfmdr1 gene associated with the severity of disease, and the multiplicity of P. falciparum infection was also similar in both groups. Our results showed no change in the polymorphism of Pfcrt and Pfmdr1 genes in P. falciparum isolates collected in 1996 and 2002, and the severity of the disease was not associated with specific mutations neither in the Pfcrt nor in the Pfmdr1 genes in the study site.

Background: To extend our previous findings showing an imbalanced distribution of immunoglobulin G2 (IgG2) antibodies to Plasmodium falciparum merozoite surface protein 2 (MSP2) and a higher frequency of infection with multiple P. falciparum strains in Gabonese children with sickle cell trait (hemoglobin AS), human Fc gamma receptor (Fc gamma R) IIa (CD32) polymorphism and the rate of in vitro invasion of red blood cells (RBCs) from subjects with either hemoglobin AA or AS by multiple P. falciparum strains were investigated., Methods: Fc gamma RIIa mutation at amino acid position 131 (arginine or histidine) was detected by polymerase chain reaction, and in vitro cultures for parasites were used to assess the invasion rate., Results: Fc gamma RIIa polymorphism is normally distributed in this population, with no preferential carriage by children with hemoglobin AS. Lower levels of IgG2 subclass antibodies to MSP2 peptides were independently associated with the Fc gamma RIIa-R131 allele and with carriage of hemoglobin AS. Our data suggest that IgG3 antibody responses to MSP2 epitopes could be exacerbated by lower IgG2 levels in children with hemoglobin AS., Conclusions: The higher rate of invasion of RBCs in the presence of multiple strains may indicate that several invasion pathways are solicited simultaneously, and the longer persistence of ring forms in RBCs from the subjects with hemoglobin AS might reflect a slower multiplication phase, leading to a longer circulation and enhanced phagocytosis of these nonpathogenic parasite forms. This may contribute to the protection against P. falciparum malaria observed in children with hemoglobin AS.

Chloroquine (CQ) resistance in Plasmodium falciparum has been particularly associated with mutations in the pfcrt gene. The present study was carried out in the malaria hyperendemic town of Brazzaville (Republic of Congo, Central Africa) where CQ is still recommended and used as a first-line drug for P. falciparum malaria. We assessed the efficacy of CQ in vivo, and the association between pfcrt mutation at codon 76 and treatment outcome in 50 children with uncomplicated malaria. The failure rate on day 28 was 95.7% and the pfcrt K76T mutation was present in 100% of isolates. No variation in the multiplicity of infection was observed in pre- and post-treatment isolates. In further 87 isolates from uncomplicated patients not treated with CQ, the mutation was detected in 98.5% of isolates. This study confirms the high level of in vivo resistance to CQ and shows the high prevalence of pfcrt K76T mutation in the Republic of Congo.

Summary In malaria-endemic regions, pregnant women are more susceptible to malarial infections than non-pregnant women. The main objective of this study, which was conducted in the malaria hyperendemic town of Lambaréné (Gabon, Central Africa), was to characterize Plasmodium falciparum infections in peripheral, placental and cord blood from women of different gravidities with submicroscopic infections. Using the P. falciparum merozoite surface protein 2 (MSP 2)* gene as a polymorphic marker in polymerase chain reactions, we analysed genetic diversity and multiplicity of infection in isolates from all three kinds of samples of 184 pregnant women at delivery. We detected infection in 44% of the women who were originally negative by microscopy. Equally important was the finding that the placenta had the highest prevalence of infection (P < 0.001). There was no correlation with gravidity status or age of the patients. The multiplicities of infection in the peripheral and placental blood samples did not differ and single infection was observed in cord blood, independently of the gravidity. The FC27/MSP 2 was the predominant allelic family. The major FC27 alleles detected in the peripheral, placental and cord blood were sequenced and found to be closely related to the published K1 form sequence. Below microscopy level, the placenta remains the most infected organ and this submicroscopic carriage of parasites may contribute to the development and maintenance of immunity to malaria during pregnancy.