A Genotyping Study in Benin Comparing the Carriage of Plasmodium falciparum Infections Before Pregnancy and in Early Pregnancy: Story of a Persistent Infection.

BACKGROUND: Malaria infections in the first trimester of pregnancy are frequent and deleterious for both mother and child health. To investigate if these early infections are newly acquired or already present in the host, we assessed whether parasites detected before pregnancy and those detected in early pregnancy are the same infection. METHODS: We used data from the preconceptional "RECIPAL" study (Benin, 2014-2017). Sixty-three pregnant women of 411 included who had a malaria infection detected by quantitative polymerase chain reaction both before pregnancy and at the first antenatal care (ANC) visit were selected for this study. Two highly polymorphic markers, msp-2 and glurp, and a fragment-analysis method were used to enumerate the Plasmodium falciparum genotypes and to quantify their proportions within isolates. An infection was considered as persistent when identical msp-2 and glurp genotypes were found in the corresponding prepregnancy and early-pregnancy samples. RESULTS: The median time between the 2 malaria screenings was 3 months. The median gestational age at the first ANC visit was 6.4 weeks. Most infections before pregnancy were submicroscopic infections. Based on both msp-2 and glurp genotyping, the infection was similar before and in early pregnancy in 46% (29/63) of cases. CONCLUSIONS: Almost half of P. falciparum infections detected in the first trimester originate before pregnancy. Protecting young women from malaria infection before pregnancy might reduce the prevalence of malaria in early pregnancy and its related poor maternal and birth outcomes.

From genomic to LC-MS/MS evidence: Analysis of PfEMP1 in Benin malaria cases.

BACKGROUND: PfEMP1 is the major protein from parasitic origin involved in the pathophysiology of severe malaria, and PfEMP1 domain subtypes are associated with the infection outcome. In addition, PfEMP1 variability is endless and current publicly available protein repositories do not reflect the high diversity of the sequences of PfEMP1 proteins. The identification of PfEMP1 protein sequences expressed with samples remains challenging. The aim of our study is to identify the different PfEMP1 proteins variants expressed within patient samples, and therefore identify PfEMP1 proteins domains expressed by patients presenting uncomplicated malaria or severe malaria in malaria endemic setting in Cotonou, Benin. METHODS: We performed a multi-omic approach to decipher PfEMP1 expression at the patient's level in different clinical settings. Using a combination of whole genome sequencing approach and RNA sequencing, we were able to identify new PfEMP1 sequences and created a new custom protein database. This database was used for protein identification in mass spectrometry analysis. RESULTS: The differential expression analysis of RNAsequencing data shows an increased expression of the var domains transcripts DBLα1.7, DBLα1.1, DBLα2 and DBLß12 in samples from patients suffering from Cerebral Malaria compared to Uncomplicated Malaria. Our approach allowed us to attribute PfEMP1 sequences to each sample and identify new peptides associated to PfEMP1 proteins in mass spectrometry. CONCLUSION: We highlighted the diversity of the PfEMP1 sequences from field sample compared to reference sequences repositories and confirmed the validity of our approach. These findings should contribute to further vaccine development strategies based on PfEMP1 proteins.

Dynamics of Plasmodium falciparum gametocyte carriage in pregnant women under intermittent preventive treatment with sulfadoxine-pyrimethamine in Benin.

BACKGROUND: In sub-Saharan Africa, malaria is a major cause of morbidity and mortality, in particular in children and pregnant women. During pregnancy, Plasmodium falciparum infected red blood cells expressing VAR2CSA are selected from circulation by selective cytoadherence to chondroitin sulfate proteoglycan receptors expressed in the placenta, leading to an increased susceptibility to malaria, long-lasting infections and poor pregnancy outcome. Partly because of these long-lasting infections, women were reported to have a higher density of gametocytes in their peripheral blood, and are considered as a potential reservoir for malaria transmission. To improve pregnancy outcome in areas of high malaria transmission, The WHO recommends intermittent preventive treatment with sulfadoxine/pyrimethamine (IPTp-SP) during antenatal care visits. The effect of IPTp-SP on gametocyte carriage in infected pregnant women was studied. METHODS: The levels of transcription of three gametocytes stage-specific genes Pfs16 (expressed by sexually-committed ring stage parasites and fully matured gametocytes), Pfs25 (expressed by female mature gametocytes) and Pfs230 (expressed by male mature gametocytes) were assessed by real-time PCR in 50 P. falciparum infected women at early pregnancy (before implementation of IPTp-SP), and in 50 infected women at delivery. Sex ratios of male and female gametocytes were determined in these women to assess the effect of IPTp-SP on the gametocyte populations. RESULTS: The data show that the three transcript types specific to Pfs16, Pfs25 and Pfs230 were detected in all samples, both at inclusion and delivery. Levels of Pfs25 and Pfs230 transcripts were higher at delivery than at inclusion (p = 0.042 and p = 0.003), while the opposite was observed for Pfs16 (p = 0.048). The ratio of male/female gametocyte transcript levels was higher at delivery than at inclusion (p = 0.018). Since a mixed gender late stage gametocyte culture was used as a positive control, male and female gametocytes could not be quantified in an absolute way in the samples. However, the amplification reliability of the Pfs25 and Pfs230 markers in the samples could be checked. A relative quantity of each type of Pfs transcript was, therefore, used to calculate the sex ratio proxy. CONCLUSION: This study demonstrates that IPTp-SP treatment contributes to modify the parasite populations' structure during pregnancy. In line with previous studies, we suggest that the continued use of SP in pregnant women as IPTp, despite having a beneficial effect on the pregnancy outcome, could be a risk factor for increased transmission. This reinforces the need for an alternative to the SP drug for malaria prevention during pregnancy.

Genetic diversity of VAR2CSA ID1-DBL2Xb in worldwide Plasmodium falciparum populations: impact on vaccine design for placental malaria.

In placental malaria (PM), sequestration of infected erythrocytes in the placenta is mediated by an interaction between VAR2CSA, a Plasmodium falciparum protein expressed on erythrocytes, and chondroitin sulfate A (CSA) on syncytiotrophoblasts. Recent works have identified ID1-DBL2Xb as the minimal CSA-binding region within VAR2CSA able to induce strong protective immunity, making it the leading candidate for the development of a vaccine against PM. Assessing the existence of population differences in the distribution of ID1-DBL2Xb polymorphisms is of paramount importance to determine whether geographic diversity must be considered when designing a candidate vaccine based on this fragment. In this study, we examined patterns of sequence variation of ID1-DBL2Xb in a large collection of P. falciparum field isolates (n=247) from different malaria-endemic areas, including Africa (Benin, Senegal, Cameroon and Madagascar), Asia (Cambodia), Oceania (Papua New Guinea), and Latin America (Peru). Detection of variants and estimation of their allele frequencies were performed using next-generation sequencing of DNA pools. A considerable amount of variation was detected along the whole gene segment, suggesting that several allelic variants may need to be included in a candidate vaccine to achieve broad population coverage. However, most sequence variants were common and extensively shared among worldwide parasite populations, demonstrating long term persistence of those polymorphisms, probably maintained through balancing selection. Therefore, a vaccine mixture including such stable antigen variants will be putatively applicable and efficacious in all world regions where malaria occurs. Despite similarity in ID1-DBL2Xb allele repertoire across geographic areas, several peaks of strong population differentiation were observed at specific polymorphic loci, pointing out putative targets of humoral immunity subject to positive immune selection.

Differential protein expression profiles between Plasmodium falciparum parasites isolated from subjects presenting with pregnancy-associated malaria and uncomplicated malaria in Benin.

BACKGROUND: Plasmodium falciparum is responsible for severe malaria, including pregnancy-associated malaria (PAM). During intra-erythrocytic maturation, the infected erythrocyte (iE) membrane is modified by insertion of parasite-derived proteins, primarily consisting of variant surface antigens such as P. falciparum erythrocyte membrane protein-1. METHODS: To identify new PAM-specific parasite membrane proteins, we conducted a mass spectrometry-based proteomic study and compared the protein expression profiles of 10 PAM and 10 uncomplicated malaria (UM) samples. RESULTS: We focused on the 454/1139 membrane-associated and hypothetical proteins for comparative analysis. Using filter-based feature-selection methods combined with supervised data analysis, we identified a subset of 53 proteins that distinguished PAM and UM samples. Up to 19/20 samples were correctly assigned to their respective clinical group. A hierarchical clustering analysis of these 53 proteins based on the similarity of their expression profiles revealed 2 main clusters of 40 and 13 proteins that were under- or over-expressed, respectively, in PAM. CONCLUSIONS: VAR2CSA is identified and associated with PAM, validating our experimental approach. Other PAM-predictive proteins included PFI1785w, PF14_0018, PFB0115w, PFF0325c, and PFA_0410w. These proteomics data demonstrate the involvement of selected proteins in the pathophysiology of PAM, providing new insights for the definition of potential new targets for a vaccine against PAM.

Plasmodium falciparum genotype population dynamics in asymptomatic children from Senegal.

In areas where malaria is endemic, infected individuals generally harbor a mixture of genetically distinct Plasmodium falciparum parasite populations. For the first time, we studied temporal variations of blood parasite densities and circulating genotypes in asymptomatic Senegalese children, at time intervals as short as 4-12 h. Twenty-one Senegalese children, presenting with an asymptomatic P. falciparum infection, were sampled eight times within three days. Parasite density was assessed by thick blood smears, and all infecting genotypes were quantified by the fragment-analysis method. Parasite densities showed dramatic fluctuations up to a 1 to 1,000 ratio, with at least one peak of parasite density. Polyclonal infections were detected in all children, with a multiplicity of infection of 5.2-6.8 genotypes per child. A single sample never reflected the full complexity of the parasite populations hosted by a given individual. Genotypes with different behaviors were detected in all children, some genotypes undergoing major fluctuations, while others were highly stable during the follow-up. A single peripheral blood sampling does not reflect the total parasite load. Repeated sampling is needed to have a more detailed scheme of parasite population dynamics and a better knowledge of the true complexity of an infection.

Epidemiology of drug-resistant malaria in Republic of Congo: using molecular evidence for monitoring antimalarial drug resistance combined with assessment of antimalarial drug use.

In Congo, urgent efforts are needed to help with the revision of the national antimalarial drug policy. Despite its high resistance level, chloroquine (CQ) is still extensively used as the first-line treatment for uncomplicated Plasmodium falciparum malaria. The study was conducted in children under 5 years with uncomplicated malaria in Pointe-Noire and Brazzaville, the two largest cities that contain approximately 60% of the population of Congo. We investigated by polymerized chain reaction and sequencing methods the frequency distribution of molecular markers for antimalarial drug resistance, including mutations in P. falciparum chloroquine resistance transporter (pfcrt) gene associated with CQ resistance and mutations in dihydrofolate reductase (dhfr) and dihydropteroate synthetase (dhps) genes conferring resistance to sulphadoxine/pyrimethamine (SP) among pre-treatment P. falciparum isolates, as well as assessing antimalarial drug use in the community. pfcrt (K76T) mutation was present in most isolates (96.4%, n = 138) and high frequency (69.2%, n = 133) of triple-mutant dhfr-S108N, N51I, C59R was observed. The quintuple mutant (dhfr-S108N, N51I, C59R and dhps-A437G or S436A, K540E) considered as molecular marker for SP treatment failure was not found because dhps-K540E mutation was absent in isolates tested; this is a clear evidence for the excellent efficacy of SP that we previously described in the same population. The complete absence of the dhps-K540E mutation is a deterrent component for using this molecular marker as an early warning tool for SP resistance testing in that population. Poor compliance issues related to the antimalarial drug use including inappropriate manufacturing practices reported in this study require intensive attention and should be taken into account when implementing drug policy change. If Congo changes its treatment policy from CQ to SP monotherapy, this will not last long. The strategy of combining SP with other affordable and effective antimalarial drugs such as the artemisinin derivatives to improve efficacy and to delay the development of parasite resistance is essential.

Demonstration of a high level of parasite population homology by quantification of Plasmodium falciparum alleles in matched peripheral, placental, and umbilical cord blood samples.

Plasmodium falciparum msp-1 and msp-2 genes were quantified by fragment analysis in matched placental, peripheral, and cord blood samples. In the three compartments, the multiplicity of infection values were similar, and parasite populations only partially overlapped, as reported. However, identical alleles represented 80 to 95% of the overall parasite populations of each compartment, demonstrating much more homogenous parasite populations than previously thought.

High prevalence of Plasmodium falciparum pfcrt K76T mutation in pregnant women taking chloroquine prophylaxis in Senegal.

OBJECTIVES: The risk of malaria infection is increased during pregnancy, and many countries recommend chloroquine prophylaxis in pregnant women, despite Plasmodium falciparum chloroquine resistance. Chloroquine resistance is associated with the pfcrt gene K76T mutation. The aim of this study was to compare the prevalence rate of pfcrt T76 mutation in P. falciparum isolates from infected pregnant and non-pregnant individuals from Senegal. METHODS: The study was conducted in the rural maternity hospital of Thiadiaye, Senegal, where malaria is seasonal. Sixty-nine P. falciparum isolates from infected women were collected at delivery. These women were part of a cohort study; they were followed from their first antenatal visit and advised to take chloroquine prophylaxis. For each woman, the earliest P. falciparum-infected blood sample was also used. A control group of 49 non-pregnant individuals with asymptomatic P. falciparum infection was enrolled. RESULTS: During pregnancy, prevalence of T76 mutant parasites was higher than in the 49 non-pregnant controls (P<0.001). Among pregnant women, this rate was highest at delivery (P=0.06), and tended to be higher in women who had taken chloroquine prophylaxis, as assessed in urine samples (P=0.08). CONCLUSIONS: Chloroquine prophylaxis is responsible for increased drug consumption and increased drug pressure that may lead to the selection of drug-resistant parasites. This is the first report showing that P. falciparum-infected pregnant women harbour pfcrt T76 mutant parasites more often than non-pregnant individuals, and that the prevalence of this mutation is higher at term than earlier during pregnancy.