Comparison of apoptosis in human primary pulmonary endothelial cells and a brain microvascular endothelial cell line co-cultured with Plasmodium falciparum field isolates.

BACKGROUND: Plasmodium falciparum infection can progress unpredictably to severe forms including respiratory distress and cerebral malaria. The mechanisms underlying the variable natural course of malaria remain elusive. METHODS: The cerebral microvascular endothelial cells-D3 and lung endothelial cells both from human were cultured separately and challenged with P. falciparum field isolates taken directly from malaria patients or 3D7 strain (in vitro maintained culture). The capacity of these P. falciparum isolates to induce endothelial cell apoptosis via cytoadherence or not was then assessed. RESULTS: Overall, 27 P. falciparum isolates were collected from patients with uncomplicated malaria (n = 25) or severe malaria (n = 2). About half the isolates (n = 17) were able to bind brain endothelial cells (12 isolates, 44%) or lung endothelial cells (17 isolates, 63%) or both (12 isolates, 44%). Sixteen (59%) of the 27 isolates were apoptogenic for brain and/or lung endothelial cells. The apoptosis stimulus could be cytoadherence, direct cell-cell contact without cytoadherence, or diffusible soluble factors. While some of the apoptogenic isolates used two stimuli (direct contact with or without cytoadherence, plus soluble factors) to induce apoptosis, others used only one. Among the 16 apoptogenic isolates, eight specifically targeted brain endothelial cells, one lung endothelial cells, and seven both. CONCLUSION: These results indicate that the brain microvascular cell line was more susceptible to apoptosis triggered by P. falciparum than the primary pulmonary endothelial cells and may have relevance to host-parasite interaction.

Non-malarial infectious diseases of antenatal care in pregnant women in Franceville, Gabon.

BACKGROUND: In sub-tropical countries, infectious diseases remain one of the main causes of mortality. Because of their lack of active immunity, pregnant women and their unborn children represent the most susceptible people. In Gabon, data on infectious diseases of pregnant women such as syphilis and rubella are either scarce or very old. Few studies have assessed T. gondii infection during pregnancy in the country. Here, we evaluate seroprevalence of HIV, HTVL-1, syphilis and T. gondii and rubella infection during antenatal care among women living in Franceville, Gabon. METHODS: A retrospective study was conducted on data collected from May 2007 to July 2010. After signing an informed written consent form, all pregnant women consulting in two hospitals of Franceville (Gabon) and in offices of maternity and childbirth health centers were included. Demographic and clinical data were collected. Serum samples were collected and analysed using immunological assays relevant for HIV (Genscreen HIV-1 version 2, Bio-Rad®, Marne la Roquette, France).HTLV-1 (Vironostika HTLV-1, Biomérieux®, Marcy l'Etoile, France), T. pallidum (TPHA/VDRL), BIOLABO®SA), rubella virus (Vidas Biomerieux®, Marcy l'Etoile, France) and T. gondii (Vidas Biomerieux®, Marcy l'Etoile, France) diagnoses were performed. Data analysis was done using the Stat view 5.0 software. RESULTS: A total of 973 pregnant women were assessed. The mean age was 25.84 ± 6.9 years, with a minimum age of 14.0 years and a maximum of 45.0 years. Women from 26 to 45 years old and unemployed women were the most prevalent: 41.93% and 77.18%, respectively. The prevalence of studied infectious diseases were 2.50% for syphilis, 2.88% for HTLV-1, 4.00% for HIV with no significant difference between them (p = 0.1). Seropositivity against rubella was higher (87.56%, n = 852) than seropositivity against T. gondii (57.35%, n = 557), (p < 0.0001). Only 5 (0.51%) co-infection cases were found: 2 co-infected with HIVand T. pallidum, 2 co-infected with HIV and HTLV-1, and one co-infected with T. pallidum and HTLV-1. Sixty-two pregnant women were seronegative against toxoplasmosis and rubella (6.37%). CONCLUSION: High levels of seropositivity against T. gondii and the rubella virus were observed. The prevalence of T. pallidum and HTLV-1 were lowest but HIV prevalence in young women was worrying.

Malaria in urban, semi-urban and rural areas of southern of Gabon: comparison of the Pfmdr 1 and Pfcrt genotypes from symptomatic children.

BACKGROUND: Artesunate-amodiaquine (AS-AQ) and artemether-lumefantrine (AL) are first- and second-line treatments for uncomplicated Plasmodium falciparum malaria in Gabon. AL remains highly efficacious, but its widespread use has led to molecular selection of the NFD haplotype on Pfmdr1 and K76 in Pfcrt. In this study, plasmodial infection characteristics and the distribution of the Pfmdr1 and Pfcrt genotypes involved in reduced efficacy of artemisinin-based combination therapy (ACT) were investigated in four Gabonese localities. METHODS: A cross-sectional study was conducted in the paediatric units of rural (Lastourville and Fougamou), semi-urban (Koula-Moutou) and urban (Franceville) areas. Malaria was diagnosed with the rapid diagnostic test Optimal-IT(®) and confirmed by blood smear. Pfmdr1 codons 86, 184 and 1246 and Pfcrt codon 76 were genotyped by PCR-RFLP and sequencing. RESULTS: Among 1129 included children, the prevalence of plasmodial infection was 79.5 % at Lastourville, 53.6 % at Fougamou, 36.1 % at Koula-Moutou, and 21.2 % at Franceville. The prevalence was significantly higher among children over 60 months of age in both semi-urban (p = 0.01) and urban (p = 0.004) areas. The prevalence of Pfmdr1 wild-type N86 differed significantly between Lastourville (57.8 %) and Koula-Moutou (45.4 %) (p = 0.039). No difference in 184F-carrying parasites was found between Lastourville (73.8 %), Fougamou (81.6 %), Koula-Moutou (83.2 %), and Franceville (80.6 %) (p = 0.240). The prevalence of wild-type D1246 was significantly different between Lastourville (94.1 %), Koula-Moutou (85.6 %) and Franceville (87.3 %) (p = 0.01). The frequency of wild-type K76 was not significantly different across the four sites: Lastourville (16.5 %), Fougamou (27.8 %), Koula-Moutou (17.4 %), and Franceville (29.4 %) (p = 0.09). The mixed genotypes were only found in Lastourville and Franceville. The NFD, YFD and NYD haplotypes were mainly Lastourville (46.6, 25.8, 14.0 %), Fougamou (45.5, 9.1, 42.4 %), Koula-Moutou (35, 6.7, 40.4 %), and Franceville (40.0, 16.0, 32.0 %). CONCLUSION: This study shows an increase in the prevalence of childhood plasmodial infection in Gabon according to the low socio-economic level, and a high frequency of markers associated with AL treatment failure. Close monitoring of ACT use is needed.

[Malaria in Gabon: results of a clinical and laboratory study at the Chinese-Gabonese Friendship Hospital of Franceville]. / Paludisme au gabon: résultats d'une étude bioclinique à l'hôpital de l'amitié sino-gabonaise de Franceville.

Despite progress in the control of malaria, it remains a serious public health problem. Substantial declines in malaria transmission, morbidity and mortality have nonetheless been reported in several countries where new malaria control strategies have been implemented. We conducted this molecular and epidemiological analysis of malaria in the pediatric department of the Chinese-Gabon Friendship Hospital (HCGC) in Franceville in 2010. Franceville is the third largest town in Gabon, and malaria transmission is high year-round. We included 945 children, 756 of them febrile. Malaria was diagnosed based on the detection of P. falciparum in thick blood films, with Lambarene's method. Malaria prevalence among the febrile children included in this study was 17.9% (n=135). The burden of malaria is thus lower than in the past; it is now the second leading cause of pediatric hospital visits, rather than the leading cause as it was in 2004. The children's mean age was 48.5 ± 3.9 months, older than in 2004 (p<0.05). We also analysed the molecular drug resistance marker, Pfmdr1. The prevalence of the wild-type genotype N86 of Pfmdr1 was 47.4% (n=64), higher than in 2004 (p<0.001). The increased prevalence of codon 1246 was not significant. Socio-economic factors and known malaria risk factors were analysed. We found that the use of Insecticide-treated mosquito nets and the provision of information (education or communication) to parents and guardians about malaria were protective factors against the disease. In conclusion, a larger study of the entire region over a longer period is necessary to characterise malaria in Franceville today. Transmission factors must also be studied.

Prevalence of malaria, prevention measures, and main clinical features in febrile children admitted to the Franceville Regional Hospital, Gabon.

Recently, major progress has been made in controlling malaria in Africa. However, in Gabon, little information is available on the role of malaria in childhood febrile syndromes, the use and efficacy of preventive measures, and Plasmodium species distribution. Here, we characterized malaria in febrile children in Franceville, Gabon through a cross-sectional study at the pediatric unit of the Franceville Regional Hospital. We registered 940 febrile children. Their general condition was markedly altered in 11.7% of cases (n = 89/760); among them 19 (21.4%) had a severely altered condition. Malaria was the second most frequent etiology (22.0%; n = 162/738), after respiratory tract infections (37.3%; n = 275/738). Children with malaria (63 ± 39 months) were older than children without malaria (40 ± 37 months) (p = 0.0013). Hemoglobin, red blood cell, white blood cell, and platelet values were lower in children with malaria than in those without malaria (p < 0.0001). Anemia was the most common feature of severe malaria (70.6%; n = 12/17), followed by neurological involvement (23.5%; n = 4/17). The prevalence of malaria was significantly higher in children older than 60 months than in younger children (40% vs. 15.5%; p < 0.0001). Plasmodium falciparum accounted for 97.5% of cases (158/162), followed by Plasmodium malariae (2.5%; n = 4/162). Bed net use was high (74.4%; n = 697/936) and contributed to malaria prevention (p = 0.001). Good basic knowledge of malaria also had a preventive effect (p < 0.0001). The prevalence of malaria in children in Franceville did not decrease significantly from 2009 to 2012, remaining at about 20%, highlighting that preventive measures should be reinforced.

Marked Rise in the Prevalence of Asymptomatic Plasmodium falciparum Infection in Rural Gabon.

Control strategies implemented a decade ago led to a marked reduction in the prevalence of malaria in many countries. In Dienga, southeastern Gabon, the prevalence of microscopic P. falciparum infection was 7% in 2003, close to the pre-elimination threshold of 5%. The aim of this work was to determine the prevalence of P. falciparum infection in the same community a decade later. A cohort of 370 individuals aged from 3 to 85 years living in Dienga was investigated for P. falciparum infection; during six passages (P) in 15-month period. Demographic data were collected, along with behaviors and attitudes towards malaria. Plasmodium infection was diagnosed by microscopy (ME), followed by PCR to detect submicroscopic infection. The prevalence of P. falciparum infection in P1, P2, P3, P4, P5 and P6 was respectively 43.5% (25.1% ME+, 18.4% PCR+); 40.9% (27.0% ME+, 13.9% PCR+), 52.7% (26.1% ME+, 26.6% PCR+); 34.1% (14.1% ME+, 20% PCR+), 57.7% (25.4.% ME+, 32.3% PCR+); and 46.2% (21.4% ME+, 24.8% PCR+) with an overall average of 45.9% (95%CI [37.0-54.7], 23.2% ME+ and 22.7% PCR+). P4 and P5 prevalences were statically different throughout the six passages. Microscopic prevalence was significantly higher than that observed ten years ago (23% [n = 370] vs 7% [n = 323], p < 0.001). Asymptomatic infections were the most frequent (96%). Gametocytes were detected in levels ranging from 5.9% to 13.9%. Insecticide-treated nets, indoor residual insecticides, and self-medication were used by respectively 33.2% (95%CI [29.0-37.4]), 17.7% (95%CI [15.5-19.9]) and 12.1% (95%CI [10.6-13.6]) of the study population. A near-threefold increase in P. falciparum infection has been observed in a rural area of southeastern Gabon during a 10-year period. Most infections were asymptomatic, but these subjects likely represent a parasite reservoir. These findings call for urgent reinforcement of preventive measures.

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.

Synthesis and in vitro antiplasmodial activity of ferrocenyl aminoquinoline derivatives.

The aim of this study was to synthesize a series of ferrocenyl 4-aminoquinolines and to evaluate their activities against Plasmodium falciparum F32 (chloroquine-sensitive) and FCB1 and K1 (chloroquino-resistant). Some of the ferrocenyl compounds exhibited in vitro antiplasmodial activity in the nM range. In particular, (1R,4R)-N1-(7-chloroquinolin-4-yl)-N4-(ferrocenylmethyl)-N4-methylcyclohexane-1,4-diamine 17 presented the lowest IC50 value (26 nM) against CQ-resistant strains.

Complete genome sequence of mumps virus genotype g from a vaccinated child in franceville, southeastern Gabon, in 2013.

The genome of mumps virus (MuV), a member of the family Paramyxoviridae of the genus Rubulavirus, consists of a single-stranded, negative-sense, nonsegmented RNA. Here, we report the first whole-genome sequence of 15,263 nucleotides of a mumps virus strain from a 6-year-old vaccinated boy in Franceville, southeastern Gabon.

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

BACKGROUND: Malaria 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. FINDINGS: The 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. CONCLUSIONS: Dichloromethane 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.

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 children's 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

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 inhibitor 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.

Relationship between in vivo synchronicity of Plasmodium falciparum and allelic diversity.

Plasmodium falciparum cells tend to grow in synchronicity during their cyclic intraerythrocytic development in vivo. Both host and parasite factors appear to be involved in this synchronization. We examined the link between mixed-allelic-family P. falciparum infection and synchronicity in parasitized red blood cells (PRBC) from symptomatic children. The distribution of rings and trophozoites in each PRBC sample was determined by standard microscopy. P. falciparum was genotyped by using a polymerase chain reaction (PCR) targeting three loci (merozoite surface proteins (MSP) 1 and 2, and 175-kD erythrocyte binding antigen (EBA), allowing us to distinguish parasite clones belonging to a single-allelic family (SAF) and those belonging to a mixed-allelic family (MAF). Parasite development was considered synchronous when peripheral blood contained at least 95% of rings or 95% of trophozoites. Parasite development was synchronous in 22 (21.2%) of the 104 children studied. Twenty (90.9%) of these infections were SAF and two (9.1%) were MAF. Rings and trophozoites predominated in respectively 12 (60%) and 8 (40%) SAF infections. Respectively 17.1% and 82.9% of the 82 asynchronous cases corresponded to SAF and MAF infection. Parasite synchronicity was therefore significantly related to single-allelic-family infection (p<2x10(-10)). Twenty different MSP-1 alleles and thirteen different MSP-2 alleles were identified. Only three isolates from patients with SAF infection comprised a single allele or genotype, the other isolates harboring at least two alleles. The mean number of alleles or clones was respectively 3.0 and 10.0 in SAF and MAF infection. These results reflect the allelic diversity of the MSP loci and show that SAF infection can correspond to multiple parasite clones (or genotypes) but, in general, fewer than in MAF infection (p