A comprehensive analysis of drug resistance molecular markers and Plasmodium falciparum genetic diversity in two malaria endemic sites in Mali.

BACKGROUND: Drug resistance is one of the greatest challenges of malaria control programme in Mali. Recent advances in next-generation sequencing (NGS) technologies provide new and effective ways of tracking drug-resistant malaria parasites in Africa. The diversity and the prevalence of Plasmodium falciparum drug-resistance molecular markers were assessed in Dangassa and Nioro-du-Sahel in Mali, two sites with distinct malaria transmission patterns. Dangassa has an intense seasonal malaria transmission, whereas Nioro-du-Sahel has an unstable and short seasonal malaria transmission. METHODS: Up to 270 dried blood spot samples (214 in Dangassa and 56 in Nioro-du-Sahel) were collected from P. falciparum positive patients in 2016. Samples were analysed on the Agena MassARRAY® iPLEX platform. Specific codons were targeted in Pfcrt, Pfmdr1, Pfdhfr, and Pfdhps, Pfarps10, Pfferredoxin, Pfexonuclease and Pfmdr2 genes. The Sanger's 101-SNPs-barcode method was used to assess the genetic diversity of P. falciparum and to determine the parasite species. RESULTS: The Pfcrt_76T chloroquine-resistance genotype was found at a rate of 64.4% in Dangassa and 45.2% in Nioro-du-Sahel (p = 0.025). The Pfdhfr_51I-59R-108N pyrimethamine-resistance genotype was 14.1% and 19.6%, respectively in Dangassa and Nioro-du-Sahel. Mutations in the Pfdhps_S436-A437-K540-A581-613A sulfadoxine-resistance gene was significantly more prevalent in Dangassa as compared to Nioro-du-Sahel (p = 0.035). Up to 17.8% of the isolates from Dangassa vs 7% from Nioro-du-Sahel harboured at least two codon substitutions in this haplotype. The amodiaquine-resistance Pfmdr1_N86Y mutation was identified in only three samples (two in Dangassa and one in Nioro-du-Sahel). The lumefantrine-reduced susceptibility Pfmdr1_Y184F mutation was found in 39.9% and 48.2% of samples in Dangassa and Nioro-du-Sahel, respectively. One piperaquine-resistance Exo_E415G mutation was found in Dangassa, while no artemisinin resistance genetic-background were identified. A high P. falciparum diversity was observed, but no clear genetic aggregation was found at either study sites. Higher multiplicity of infection was observed in Dangassa with both COIL (p = 0.04) and Real McCOIL (p = 0.02) methods relative to Nioro-du-Sahel. CONCLUSIONS: This study reveals high prevalence of chloroquine and pyrimethamine-resistance markers as well as high codon substitution rate in the sulfadoxine-resistance gene. High genetic diversity of P. falciparum was observed. These observations suggest that the use of artemisinins is relevant in both Dangassa and Nioro-du-Sahel

Hepatitis C Virus Infection May Lead to Slower Emergence of P. falciparum in Blood

International audienceBACKGROUND: Areas endemic for Plasmodium falciparum, hepatitis B virus (HBV) and hepatitis C virus (HCV) overlap in many parts of sub-Saharan Africa. HBV and HCV infections develop in the liver, where takes place the first development stage of P. falciparum before its further spread in blood. The complex mechanisms involved in the development of hepatitis may potentially influence the development of the liver stage of malaria parasites. Understanding the molecular mechanisms of these interactions could provide new pathophysiological insights for treatment strategies in Malaria. METHODOLOGY: We studied a cohort of 319 individuals living in a village where the three infections are prevalent. The patients were initially given a curative antimalarial treatment and were then monitored for the emergence of asexual P. falciparum forms in blood, fortnightly for one year, by microscopy and polymerase chain reaction. PRINCIPAL FINDINGS: At inclusion, 65 (20.4%) subjects had detectable malaria parasites in blood, 36 (11.3%) were HBV chronic carriers, and 61 (18.9%) were HCV chronic carriers. During follow-up, asexual P. falciparum forms were detected in the blood of 203 patients. The median time to P. falciparum emergence in blood was respectively 140 and 120 days in HBV- and HBV+ individuals, and 135 and 224 days in HCV- and HCV+ individuals. HCV carriage was associated with delayed emergence of asexual P. falciparum forms in blood relative to patients without HCV infection. CONCLUSIONS: This pilot study represents first tentative evidence of a potential epidemiological interaction between HBV, HCV and P. falciparum infections. Age is an important confounding factor in this setting however multivariate analysis points to an interaction between P. falciparum and HCV at the hepatic level with a slower emergence of P. falciparum in HCV chronic carriers. More in depth analysis are necessary to unravel the basis of hepatic interactions between these two pathogens, which could help in identifying new therapeutic approaches against malaria

Indian Anti-Malaria OMARIA Is Effective against African Drug Resistant P. falciparum Field Isolates and Laboratory Strains; without Toxicity

ABSTRACT OMARIA which is used to treat malaria in Odisa province, India, was investigated in Africa. The in-vitro anti-malarial activity of OMARIA was tested on P. falciparum strains FCB (chloroquine-resistant) and 3D7 (chloroquine-sensitive) and on fresh clinical isolates from Gabon, using the DELI method. Host cell toxicity was analysed with the MTT test. Interesting activity was observed. Inhibition concentrations (IC 50 ) were 20.6 ± 5.2 µg/ml and 14.1 ± 4.3µg/ml respecttively on FCB and 3D7 strains. On clinical isolates, the mean of IC 50 was 10.65 ± 4.8µg/ml. OMARIA is highly potent against all field isolates tested by us (Gabon includes Pfmdr1 N86). Lethal dose on Vero cells being 165 ± 10.7 µg/ml indicate a selective index of 13 for FCB, i.e., non-toxic. Data substantiates scientific rationale for use of OMARIA. This information and such understanding can be used in searching African phyto parables (for use in Africa with similar results as in India) and in new drug design. With Indian assistance, Punica granatum can also be cultivated in Central Africa, and OMARIA can be made, with an aim to Fight Malaria at Home

<it>In vitro </it>antiplasmodial activity of crude extracts of <it>Tetrapleura tetraptera </it>and <it>Copaifera religiosa</it>

Abstract 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.</p

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

BACKGROUND: Malaria 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. METHODS: We 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. RESULTS: A 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 IC(50) values higher than 10 nM. The median IC(50) 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 (r(2)=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%. CONCLUSION: These high levels of antimalarial drug resistance in Franceville, Gabon, call for reinforced surveillance of drug efficacy

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

Abstract 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