Jean Bernard Lekana-Douki

Increased prevalence of the Plasmodium falciparum Pfmdr1 86N genotype among field isolates from Franceville, Gabon after replacement of chloroquine by artemether–lumefantrine and artesunate–mefloquine

Despite global antimalarial measures, Plasmodium falciparum malaria remains a major public health problem. WHO has recommended the use of arteminisin-based combination therapy to limit the emergence of antimalarial drug resistance. However, ACT treatment failures have been linked to the selection of the wild types 86N genotype of P. falciparum multidrug resistance 1 (Pfmdr1) and the 76K genotype of P. falciparum chloroquine resistance (Pfcrt) genes. The aim of this study was to investigate the molecular impact of widespread implementation of artemether-lumefantrine and artesunate-mefloquine on local parasite population in Franceville, Gabon. We analyzed 230 pediatric field isolates (96 from 2004 and 134 from 2009). Routine hematological parameters were collected. Pfmdr1 codons 86 and 1246 and Pfcrt codon 76 were genotyped using PCR-RFLP and the prevalence of the genotypes was compared. The childrens mean age did not differ between 2004 and 2009 (respectively 31.8 (6-84) months vs 38.6 (6-84) months, p=0.32), and neither did mean parasitemia [16,750 (1000-96,234) and 14,587 (1093-83,941) parasites/μL, respectively (p=0.21)]. The mean hemoglobin level was higher in 2009 than in 2004 (11.0 ± 2.4 vs 7.8 ± 2.0 g/dL, respectively; p=0.04). More interesting, the prevalence of Pfmdr1 wild type 86N increased from 15.6% (n=15/96) in 2004 to 31.3% (n=42/134) in 2009 (p=0.007). A significant increase combining pure and mixed genotypes (86N+86N/Y) was also found between 2004 and 2009 (p=0.02), while the prevalence of genotypes Pfmdr1 1246D, Pfcrt wild type 76T and all mixed genotypes (Pfmdr1 86N/Y and 1246D/Y, and 76K/T) remained stable. The complexity of isolates was high (around 2.9 and 2.4) and the FC27 allele of Pfmsp2 was more prevalent. These findings show a substantial benefice of artemether-lumefantrine and artesunate-mefloquine and of new control measures. The selection, in the general population, of wild type Pfmdr1 86N, which is associated with antiplasmodial resistance against some drugs, has been induced underlining the need for molecular surveillance of the impact of ACT on antimalarial resistance.

In vitro antiplasmodial activity and cytotoxicity of nine plants traditionally used in Gabon.

AIM OF THE STUDY As part of a project to identify new compounds active on malarial parasites, we tested the in vitro antiplasmodial activity of nine plants traditionally used to treat malaria symptoms in Haut-Ogooué Province, South-East Gabon. MATERIALS AND METHODS Dichloromethane and methanolic extracts of each plant were tested for their antiplasmodial activity on two chloroquine-resistant strains of Plasmodium falciparum (FCB and W2), based on lactate dehydrogenase activity. Cytotoxicity was assessed with the MTT test on MRC-5 human diploid embryonic lung cells. RESULTS The methanolic extract of Staudtia gabonensis and the dichloromethane extract of Adhatoda latibracteata showed high antiplasmodial activity (IC₅₀<1 μg/ml) and low cytotoxicity, with selectivity indexes of about 58.25 and 16.43, respectively. The methanolic extract of Monodora myristica and the dichloromethane extract of Afromomum giganteum also showed promising activity (1<IC₅₀<10 μg/ml) and low cytotoxicity, with selectivity indexes about 15.70 and 12.48, respectively. Dichloromethane extracts of Monodora myristica and Leonotis Africana showed moderate activity (10<IC₅₀<40 μg/ml), with selectivity indexes about 6.07 and 28.89, respectively. Both extracts of Culcasia lancifolia had IC₅₀ values of 10-40 μg/ml but high cytotoxicity (selectivity indexes <2.77). The methanolic extract of Dorstenia klaineana had moderate antiplasmodial activity (IC₅₀ around 17 μg/ml) but strong cytotoxicity (0.43 μg/ml), giving a selectivity index of about 0.03. CONCLUSIONS Most extracts of nine selected plants traditionally used to treat malaria in Gabon had interesting antiplasmodial activity in vitro. This supports continued investigations of traditional medicines in the search for new antimalarial agents. The compounds responsible for the observed antiplasmodial effects are under investigation.

In vitro antiplasmodial activity of crude extracts of Tetrapleura tetraptera and Copaifera religiosa

BackgroundMalaria remains a major public health problem, especially in tropical and subtropical regions because of the emergence and widespread of antimalarial drug resistance. Traditional medicine represents one potential source of new treatments. Here, we investigated the in vitro antiplasmodial activity of bark extracts from two Fabaceae species (Tetrapleura tertaptera and Copaifera religiosa) traditionally used to treat malaria symptoms in Haut-Ogooué province, Gabon.FindingsThe antiplasmodial activity of dichloromethane and methanolic extracts was tested on P. falciparum strains FCB (chloroquine-resistant) and 3D7 (chloroquine-sensitive) and on fresh clinical isolates, using the DELI method. Host cell toxicity was analyzed on MRC-5 human diploid embryonic lung cells using the MTT test.The dichloromethane extracts of the two plants had interesting activity (IC50 between 8.5 ± 4.7 and 13.4 ± 3.6 μg/ml). The methanolic extract of Tetrapleura tetraptera was less active (IC50 around 30 μg/ml) and the methanolic extract of Copaifera religiosa was inactive. The selectivity index (toxicity/antiplasmodial activity) of the dichloromethane extract of Tetrapleura tetraptera was high (around 7), while the dichloromethane extract of Copaifera religiosa had the lowest selectivity (0.6). The mean IC50 values for field isolates were less than 1.5 μg/ml for dichloromethane extracts of both plants, while methanolic extracts of Tetrapleura tetraptera showed interesting activity (IC50 = 13.1 μg/ml). The methanolic extract of Copaifera religiosa was also inactive on field isolates.ConclusionsDichloromethane extracts of Tetrapleura tetraptera and Copaifera religiosa, two plants used to treat malaria in Gabon, had interesting antiplasmodial activity in vitro. These data provide a scientific rationale for the traditional use of these plants against malaria symptoms. Bioactivity-guided phytochemical analyses are underway to identify the active compounds.

Inhibition of Plasmodium falciparum Field Isolates-Mediated Endothelial Cell Apoptosis by Fasudil: Therapeutic Implications for Severe Malaria

Plasmodium falciparum infection can abruptly progress to severe malaria, a life-threatening complication resulting from sequestration of parasitized red blood cells (PRBC) in the microvasculature of various organs such as the brain and lungs. PRBC adhesion can induce endothelial cell (EC) activation and apoptosis, thereby disrupting the blood-brain barrier. Moreover, hemozoin, the malarial pigment, induces the erythroid precursor apoptosis. Despite the current efficiency of antimalarial drugs in killing parasites, severe malaria still causes up to one million deaths every year. A new strategy targeting both parasite elimination and EC protection is urgently needed in the field. Recently, a rho-kinase inhibitior Fasudil, a drug already in clinical use in humans for cardio- and neuro-vascular diseases, was successfully tested on laboratory strains of P. falciparum to protect and to reverse damages of the endothelium. We therefore assessed herein whether Fasudil would have a similar efficiency on P. falciparum taken directly from malaria patients using contact and non-contact experiments. Seven (23.3%) of 30 PRBC preparations from different patients were apoptogenic, four (13.3%) acting by cytoadherence and three (10%) via soluble factors. None of the apoptogenic PRBC preparations used both mechanisms indicating a possible mutual exclusion of signal transduction ligand. Three PRBC preparations (42.9%) induced EC apoptosis by cytoadherence after 4 h of coculture (“rapid transducers”), and four (57.1%) after a minimum of 24 h (“slow transducers”). The intensity of apoptosis increased with time. Interestingly, Fasudil inhibited EC apoptosis mediated both by cell-cell contact and by soluble factors but did not affect PRBC cytoadherence. Fasudil was found to be able to prevent endothelium apoptosis from all the P. falciparum isolates tested. Our data provide evidence of the strong anti-apoptogenic effect of Fasudil and show that endothelial cell-P. falciparum interactions are more complicated than previously thought. These findings may warrant clinical trials of Fasudil in severe malaria management.

Possible role of Rickettsia fells in acute febrile illness among children in Gabon

Infection is widespread but most prevalent among young, rural residents with fever.

In vitro antimalarial susceptibility and molecular markers of drug resistance in Franceville, Gabon

BackgroundMalaria remains a major public health problem, due largely to emergence and widespread P. falciparum drug resistance. WHO recommends artemisinine combination based therapy (ACT) to overcome P. falciparum drug resistance, but reports of declining ACT efficacy have been published. A thorough understanding of the molecular bases of P. falciparum resistance to existing drugs is therefore needed. The aims of this study were to analyze the in vitro sensitivity of P. falciparum field isolates from Franceville, Gabon, to chloroquine (CQ), mefloquine (MF), dihydroartemisinine (DHA) and monodesethylamodiaquine (MDAQ), and to investigate polymorphisms associated with drug resistance.MethodsWe conducted a cross-sectional study of 53 field isolates. Field isolates sensitivity to CQ, MF, DHA and MDAQ was assessed using the colorimetric DELI test. The Pfmdr1 codons 86 and 1246, Pfcrt (haplotype codon 72 to 76) and the PfATPAse6 codons 110 and 2694 were analysed by PCR-RFLP. Associations between drug sensitivity and parasite gene polymorphisms were evaluated with the Chi square test, and routine hematological parameters were analyzed with Fisher’s exact test implemented with Epinfo software. In all statistical tests, significance was assumed at p<0.05.ResultsA total of 46 P. falciparum isolates were successfully cultured in vitro and their sensitivity was tested. The proportions of isolates resistant to CQ, MF and MDAQ were 43.5%, 23.4% and 56.5%, respectively. Some isolates (23.9%) had DHA IC50 values higher than 10 nM. The median IC50 values were 71.67 (interquartile range (IQR, 1–438.2), 6.59 (IQR, 0.08-96), 64.79 (IQR, 0.09-448) and 6.45 nM (IQR, 0.09-23) for CQ, MF, MDAQ and DHA, respectively. The strongest correlation between diminished DHA sensitivity and MF resistance was observed (r2=0.73), followed by correlation between diminished DHA sensitivity and CQ resistance. Cross-resistance between CQ and MF was also observed. The prevalence of the 86Y and 1246Y mutations in Pfmdr1, 76T in Pfcrt, and 110A and 2694T in PfATPase6 was respectively 42% and 17.1%, 97.8%, and 0% and 22.2%.ConclusionThese high levels of antimalarial drug resistance in Franceville, Gabon, call for reinforced surveillance of drug efficacy.

Molecular Detection of Fastidious and Common Bacteria as Well as Plasmodium spp. in Febrile and Afebrile Children in Franceville, Gabon

Malaria was considered as the main cause of fever in Africa. However, with the roll back malaria initiative, the causes of fever in Africa may change. This study aimed to evaluate the prevalence of bacteria and Plasmodium spp. in febrile and afebrile (controls) children from Franceville, Gabon. About 793 blood samples from febrile children and 100 from controls were analyzed using polymerase chain reaction (PCR) coupled with sequencing. Plasmodium spp. was the microorganism most detected in febrile (74.5%, 591/793) and controls (13%, 13/100), P < 0.0001. Its coinfection with bacteria was found only in febrile children (P = 0.0001). Staphylococcus aureus was the most prevalent bacterium in febrile children (2.8%, 22/793) and controls (3%, 3/100). Eight cases of Salmonella spp. (including two Salmonella enterica serovar Paratyphi) and two of Streptococcus pneumoniae were found only among febrile children. Borrelia spp. was found in 2 controls while Rickettsia felis was detected in 10 children (in 8 febriles and 2 afebriles). No DNA of other targeted microorganisms was detected. Plasmodium spp. remains prevalent while Salmonella spp., Staphylococcus aureus, and Streptococcus pneumoniae were common bacteria in Gabon. Two fastidious bacteria, Rickettsia felis and Borrelia spp., were found. Inclusion of controls should improve the understanding of the causes of fever in sub-Saharan Africa.

Co-circulation of Plasmodium and Bacterial DNAs in Blood of Febrile and Afebrile Children from Urban and Rural Areas in Gabon.

Malaria is considered to be the most common etiology of fever in sub-Saharan Africa while bacteremias exist but are under assessed. This study aimed to assess bacteremias and malaria in children from urban and rural areas in Gabon. DNA extracts from blood samples of 410 febrile and 60 afebrile children were analyzed using quantitative polymerase chain reaction. Plasmodium spp. was the microorganism most frequently detected in febrile (78.8%, 323/410) and afebrile (13.3%, 8/60) children, (P < 0.001). DNA from one or several bacteria were detected in 15 febrile patients (3.7%) but not in the controls (P = 0.1). This DNA was more frequently detected as co-infections among febrile children tested positive for Plasmodium (4.6%, 15/323) than in those tested negative for Plasmodium (0%, 0/87; P = 0.04). The bacteria detected were Streptococcus pneumoniae 2.4% (10/410), Staphylococcus aureus 1.7% (7/410), Salmonella spp. 0.7% (3/410), Streptococcus pyogenes 0.2% (1/410) and Tropheryma whipplei 0.2% (1/410) only in febrile children. Coxiella burnetii, Borrelia spp., Bartonella spp., Leptospira spp., and Mycobacterium tuberculosis were not observed. This paper reports the first detection of bacteremia related to T. whipplei in Gabon and shows that malaria decreases in urban areas but not in rural areas. Co-infections in febrile patients are common, highlighting the need to improve fever management strategies in Gabon.

High prevalence of Plasmodium falciparum antimalarial drug resistance markers in isolates from asymptomatic patients from the Republic of the Congo between 2010 and 2015

OBJECTIVES This study investigated the prevalence of haplotypes of the Pfdhps, Pfdhfr, Pfcrt, Pfmdr1 and PfK13 resistance markers in isolates from asymptomatic patients from the Republic of the Congo following implementation of artemisinin based-combination therapy (ACT). METHODS Peripheral blood was collected from asymptomatic children in 2010 and 2015 from Brazzaville in the south and in 2013 in the north of the Congo. Genotypes of Pfmdr1, Pfcrt, Pfdhps, Pfdhfr and PfK13 were assessed by PCR. RESULTS Children from 2010 were younger than those from 2015 (mean age 5.38 years vs. 8.67 years; P=0.003). The main Pfcrt haplotype was the wild-type CVMNK (84.85%) in 2010, whereas the mutant CVIET (61.64%) predominated in 2015 (P<0.001). In the north, 45.00% of samples were CVMNK and 10.00% were CVIET. Other samples harboured new haplotypes in the country or mixed alleles. No significant difference in Pfmdr1 haplotypes was observed in 2010 and 2015 and the main haplotypes were NYD and NFD (30.56% vs. 28.57% and 61.11% vs. 42.86% for 2010 and 2015, respectively). In the south, the Pfdhps haplotypes observed were AAKAA, AGKAA, SGKAA and SGEGA (87.50% vs. 0%, 12.50% vs. 33.33%, 0% vs. 33.33% and 0% vs. 33.33% for 2010 and 2015, respectively). For Pfdhfr, the IRNI haplotype was most prevalent (85.71% for 2010, 87.50% for 2013 and 100% for 2015). No PfK13 mutations were found. CONCLUSIONS Monitoring the efficacy of ACT and intermittent preventive treatment with sulfadoxine-pyrimethamine is necessary to ensure an epidemiological survey of asymptomatic malaria.

Pfmdr1 gene polymorphism of Plasmodium falciparum isolates from asymptomatic individuals of Dienga, southeastern Gabon

Background: In Gabon, studies on molecular epidemiology of malaria are mainly confined to urban and hospital settings; contrasting with rural areas where data are scarce, especially on asymptomatic P. falciparum infection. In this study, the polymorphism of P. falciparum multidrug resistance -1 gene (Pfmdr1) was investigated for the first time in P. falciparum specimens from asymptomatic individuals living in Dienga, a rural area in southeastern Gabon. Methods: P. falciparum specimens collected in 2013 and 2014 and diagnosed by standard microscopy (microscopic infection=MI) or by PCR (submicroscopic infection=SMI) were used in this study. The codons 86, 184 and 1246 of Pfmdr1 of P. falciparum specimens were genotyped by PCR and RFLP. Results: Among the three codons 86, 184 and 1246 of Pfmdr1, the wild-type alleles were the most prevalent in 2014. The frequency of mixed genotype N86Y was 19.23% in 2013 and 25.28% in 2014. The frequency of D1246Y mixed genotype was 10.40% in 2013 and 17.24% in 2014. On the other hand, no mixed genotype was observed for the codon 184. The most frequent haplotypes identified were NFY (33%) in 2013 and YYD (30.0%) in 2014. The frequencies of other haplotypes (YYY, YFD, NYD, YFY, NFD and NYY) did not show significant difference between 2013 and 2014. Conclusion: Data from Dienga showed moderate variations in the distribution of Pfmdr1 genotypes and a high haplotype diversity suggesting the low ACT drug pressure on parasites in this locality.