Evaluation of the application of national malaria treatment guidelines in private pharmacies in a rural area in the Democratic Republic of Congo.

In the Democratic Republic of the Congo, the first recourse in case of suspected malaria in the health system is the private pharmacy sector. This study was therefore designed to assess private provider adherence to national case management guidelines in Kimpese, a rural area of Central Kongo province. A descriptive cross-sectional survey of 103 pharmacies took place in March 2016. The study included 97 pharmacies. The artemether-lumefantrine combination recommended as the first-line treatment for uncomplicated P. falciparum malaria was available in 100% of pharmacies but only 3% stocked quality-assured medicines. The sulfadoxine-pyrimethamine recommended for intermittent preventive treatment of malaria in pregnant women and quinine, which is no longer part of national policy, were widely available (>97.0% of pharmacies). Among providers, fewer than 20% were aware of the national malaria treatment guidelines. The main reasons for non-adherence to national guidelines among private dispensers was the high cost (up to 10 times more expensive than sulfadoxine-pyrimethamine treatment) and adverse effects of artemisinin-based combination therapies. Governmental interventions to improve private sector engagement in implementation of the national guidelines and to prevent the spread of ineffective and non-quality assured antimalarial medicines must be intensified.

Access to artemisinin-based combination therapies and other anti-malarial drugs in Kinshasa.

OBJECTIVE: Artemisinin-based combination therapies have been available since 2005 in the Democratic Republic of the Congo to treat malaria and to overcome the challenge of anti-malarial drug resistance as well as to improve access to effective treatments. The private sector is the primary distribution source for anti-malarial drugs and thus, has a key position among the supply chain actors for a rational and proper use of anti-malarial drugs. We aimed to assess access to nationally recommended anti-malarial drugs in private sector pharmacies of the capital-city of Kinshasa. METHOD: We performed a cross-sectional survey of 404 pharmacies. RESULTS: Anti-malarial drugs were stocked in all surveyed pharmacies. Non-artemisinin-based anti-malarial therapies such as quinine or sulfadoxine-pyrimethamine, were the most frequently stocked drugs (93.8% of pharmacies). Artemisinin-based combination therapies were stocked in 88% of pharmacies. Artemether-lumefantrine combinations were the most frequently dispensed drugs (93% of pharmacies), but less than 3% were quality-assured products. Other non-officially recommended artemisinin-based therapies including oral monotherapies were widely available. CONCLUSION: Artemisinin-based combination therapies were widely available in the private pharmacies of Kinshasa. However, the private sector does not guarantee the use of nationally recommended anti-malarial drugs nor does it give priority to quality-assured anti-malarial drugs. These practices contribute to the risk of emergence and spread of resistance to anti-malarial drugs and to increasing treatment costs.

Microaerophilic respiratory metabolism of Plasmodium falciparum mitochondrion as a drug target.

The Plasmodium falciparum mitochondrion is an organelle that presents structural and physiological characteristics different from mitochondria in other eukaryotes. Moreover, there are substantial differences in the properties of asexual and sexual mitochondria. One of the reasons is the adaptation of the parasite to different environments, in particular the great differences in oxygen tension between the host and the mosquito. In this review, we present a synthesis of the recent data on the ultrastructure, the genome and the physiology of the mitochondrion. We try to clarify the mitochondrial role in the intraerythrocytic environment and particularly focus on mitochondrial metabolic pathways that relate to oxidative phosphorylation, including the tricarboxylic acid cycle, de novo pyrimidine biosynthesis via dihydroorotate dehydrogenase and the particularities of the electron transport chain. In addition, we provide details on certain characteristics like the lack of pyruvate dehydrogenase, the existence of a rotenone-insensitive NADH-dehydrogenase, the possible existence of an alternative oxidase, and uncoupled proteins. Such unique particularities of parasite mitochondria could be promising targets for development of a new therapy. The elucidation of the role of this organelle in microaerophilic respiratory metabolism and the association of antimalarial drugs with hyperbaric oxygen therapy might provide new treatments for infection by P. falciparum.

Pharmacokinetics and pharmacodynamics of a new ACT formulation: Artesunate/Amodiaquine (TRIMALACT) following oral administration in African malaria patients.

A new fixed-dose combination of artesunate (AS) plus amodiaquine (AQ) (TRIMALACT) was recently developed for the treatment of uncomplicated falciparum malaria. The originality of this combination lies in its galenic formulation which consists of a three-layer tablet with two layers containing each of the active ingredients, i.e. AS and AQ, and these are separated by a middle layer containing an antioxidant compound. To evaluate the efficacy and tolerability of this combination, adults with uncomplicated malaria received three administrations of two tablets (100:300 mg AS/AQ) in a 24-h interval, in Democratic Republic of Congo. Parasitemia and fever were measured and the plasma levels of parent compounds and metabolites [dihydroartemisinin (DHA) and monodesethylamodiaquine (MdAQ)] were determined by high-performance liquid chromatography. In addition, we determined the prevalence of molecular markers of resistance to chloroquine (CQ) and sulfadoxine/pyrimethamine (SP). The AS/AQ combination TRIMALACT demonstrated a good efficacy resulting in an excellent clinical and parasitological response rate of 100% after correction for PCR results. Treatment regimen was well tolerated. The main disposition parameters to AS+AQ were: for DHA, AUC = 632 +/- 475 ng h/ml and Cmax = 432 +/- 325 ng/ml, and for MdAQ = 14268 +/- 4114 ng h/ml and Cmax = 336 +/- 225 ng/ml (mean +/- standard deviation). Parasite genotyping show high frequencies of molecular SP- and CQ-resistance markers with more 80% of the samples showing more than three mutations linked to SP resistance and 93.48% carrying parasite with the CQ-resistant haplotype. This study shows that the AS/AQ combination TRIMALACT is safe and effective in the treatment of highly drug-resistant falciparum malaria.

Chimio-sensibilité de plasmodium falciparum aux antipaludiques au Niger

Nous avons réalisé un test thérapeutique in vivo à la chloroquine, couplé à une étude in vitro de chimiosensibilité et à une étude des gènes de mutation associés à la résistance aux antipaludiques en Septembre et Octobre 2001 à Niamey au Niger. Au total, 244 enfants ont été inclus. Une réponse clinique adéquate du traitement par chloroquine a été observée chez 78,3% des enfants de 1-15 ans,78,9% des enfants de 1-5 ans, 77,9% des enfants de 6-10 ans et 78% des enfants de 11-15 ans. L'échec thérapeutique a été constaté dans 13,1% des cas dont 9,4% d'échec thérapeutique précoce et 3,7% d'échec thérapeutique tardif. Au vu de ces résultats, le Programme National de Lutte contre le Paludisme a décidé de maintenir la chloroquine comme traitement de première intention du paludisme simple à P. falciparum à Niamey. Concernant les tests in vitro, sur les 244 souches, 26 seulement ont pu être cultivées, par défaut du transporteur. Sur ces 26 souches, 15 étaient sensibles à la chloroquine par les tests in vitro isotopiques. Concernant la sensibilité aux autres antipaludiques, 4 étaient résistantes la pyriméthamine, 5 au cycloguanil, 3 à l'atovaquone. Les tests moléculaires ont été effectués sur les souches qui avaient pu être isolées en cultures. Nous présentons ainsi les résultats de la prévalence des mutations des gènes pfcrt, dhfr et dhps et discutons ces résultats par rapport à ceux des tests classiques

In vitro activity of iron-binding compounds against Senegalese isolates of Plasmodium falciparum.

OBJECTIVES: The in vitro activities of FR160, a synthetic catecholate siderophore, and two iron-binding agents, desferrioxamine and doxycycline, were evaluated against Plasmodium falciparum isolates. Correlations between these compounds and standard antimalarial drugs (chloroquine, quinine, amodiaquine, pyronaridine, artemether, artesunate, atovaquone, cycloguanil and pyrimethamine) were assessed to determine any degree of cross-resistance. METHODS: Between October 1997 and February 1998, and September and November 1998, 189 P. falciparum isolates were obtained in Dielmo and Ndiop (Dakar). Their susceptibilities were assessed using an isotopic, microwell format, drug susceptibility test. RESULTS: The 137 inhibitory concentrations (IC(50)) values of FR160 ranged from 0.1 to 10 microM and the geometric mean IC(50) was 1.48 microM (95% CI = 1.29-1.68 microM). The geometric mean IC(50) of doxycycline for 121 isolates was 18.9 microM (95% CI = 16.8-21.3 microM) and that of desferrioxamine for 73 isolates was 20.7 microM (95% CI = 17.3-24.8 microM). FR160 was significantly less active against the chloroquine-resistant isolates (P < 0.0001). The mean IC(50)s of doxycycline were significantly higher for the chloroquine-susceptible isolates than for the resistant parasites (P = 0.0447). There was a weak correlation between the responses to FR160, desferrioxamine or doxycycline and those to the other antimalarial compounds (r(2) < 0.22). CONCLUSIONS: The activities of FR160 and desferrioxamine, determined for P. falciparum clones, were confirmed against 137 isolates. The coefficients of determination between the responses to FR160, doxycycline or desferrioxamine and those to all the antimalarial drugs tested are too weak to suggest cross-resistance. FR160 could be a rationale partner to use in combination with doxycycline.

[Imported malaria at the Marseilles Hôpital-Nord, France: a prospective study on 352 cases between 2001 and 2003]. / Le paludisme d'importation à l'Hôpital-Nord de Marseille en 2001-2003: étude prospective de 352 cas.

OBJECTIVE: The authors had for aim to study epidemiological, clinical, and parasitological characteristics, as well as regimen received, of imported malaria cases hospitalised at the North University Hospital, in Marseilles, France. DESIGN: The patients presenting with imported malaria included in this study were hospitalised in the infectious and tropical diseases unit and in the pediatrics unit at the North University Hospital, from January 1, 2001 to December 31, 2003. Variables were prospectively collected and recorded. RESULTS: 352 patients including 240 adults and 112 children were included. Most of them (67% of the adults and 92% of the children) were contaminated during a trip to the Comoros Islands. Plasmodium falciparum was the most common species identified. 97.5% of adult and 98% of child patients back from Comoros did not take any chemoprophylaxis against malaria or took inadequate regimens. Halofantrin was the most commonly used drug for children to treat uncomplicated P. falciparum malaria. In adults, atovaquone-proguanil was used as a first line drug in the absence of vomiting, and a 3-day intravenous regimen of quinine-clindamycin in case of vomiting. CONCLUSION: The specificity of imported malaria in Marseilles is the high proportion of Comorian patients who go back home periodically to visit friends and relatives. A better education of the Comorian population in Marseilles, regarding malaria risks and prophylaxis, needs to be implemented.

[Chemosusceptibility analysis of Plasmodium falciparum imported from Comoros to Marseilles, France in 2001-2003]. / Chimiosensibilité de Plasmodium falciparum importé des Comores à Marseille en 2002-2003.

OBJECTIVES: The aim of this work was to study the chemosensitivity of Plasmodium falciparum strains isolated from patients presenting with malaria after having returned from Comoros Islands in 2002-2003, and hospitalized at the North University Hospital, in Marseilles, France. MATERIALS AND METHODS: In vitro drug susceptibility (for strains maintained in culture) and mutation-specific polymerase chain reaction (PCR) assays (for all strains) were performed. RESULTS: Out of 23 strains kept in culture, 50% were shown to be resistant in vitro to chloroquine, 50% were resistant to pyrimethamine, 40% to cycloguanil, 25% to atovaquone, and 7% to mefloquine. However all these strains were susceptible to quinine, halofantrine, and artemether. Moreover, 48 strains were tested by molecular methods. As a result, 69% were shown to have the Asp108 mutation in the dihydrofolate reductase gene (Pfdhfr), the basic mutation associated with antifolate resistance, and 54% had additional mutations Ile51 plus Arg59, associated with a high level of resistance. Furthermore, 90% of the 20 strains tested in 2003 were shown to have the point mutation Pfcrt76 in the P. falciparum chloroquine resistance transporter (Pfcrt) gene recently proposed as a molecular marker of chloroquine-resistance. CONCLUSION: Obtaining plasmodium strains from Comoros to be tested in Marseilles, where all laboratory facilities are available, is a unique opportunity to establish a surveillance of falciparum drug resistance in the Comoros islands.

Experimental infection of human erythrocytes from alcoholic patients with Bartonella quintana.

Bartonella spp. are found in the erythrocytes of their specific natural hosts and B. quintana bacteremia is associated epidemiologically with lice, alcoholism, and homelessness. The aim of our study was to compare the growth and the number of bacteria per erythrocyte in vitro in laboratory-infected red blood cells from alcoholic patients versus normal blood donor erythrocytes. Enumeration of bacteria was performed either with plate counting or with a real-time PCR quantitative assay. Number of bacteria per cell was determined using immunofluorescence assay and laser confocal microscopy. Although the number of bacteria after 4 days of incubation was similar in the two groups of erythrocytes, we found that the distribution of bacteria per erythrocyte in the two groups was different. Erythrocytes from alcoholics contain significantly more bacteria per cell than erythrocytes from blood donors. Our results suggest that there is a link between alcoholism and infections of B. quintana that may be due to the macrocytosis of erythrocytes.

Assay of fipronil efficacy to prevent canine monocytic ehrlichiosis in endemic areas.

Our objective was to evaluate the efficacy of fipronil for the prevention of Ehrlichia canis transmission to dogs by Rhipicephalus sanguineus in two endemic areas situated in Africa (Dakar and Djibouti). We carried out controlled trials in kennels for 1 year on 248 dogs, mainly police dogs and military working dogs. Eight groups were studied in a multi-centre study. Fifty five fipronil treated dogs were located in two separated kennels (G3, 37 dogs in Djibouti and G8, 18 dogs in Dakar). G1 (66 dogs) and G2 (60 dogs) were untreated control groups located in Djibouti, whereas G4 (32 dogs), G5 (13 dogs), G6 (18 dogs) and G7 (4 dogs) were the control groups located in Dakar. The epidemiological status of each group is known. G1 and G2 dogs were not kept in kennels, whereas G3, G4, G5, G6, G7, G8 dogs were housed in equivalent kennels. Tick infestation, clinical status and Ehrlichia seroprevalence were assessed during 1 year (duration of the study). Dog treated with fipronil showed neither canine monocytic ehrlichiosis (CME) nor tick infestations. In all groups of untreated control animals, R. sanguineus tick infestations were frequent, particularly in kennels (G5, G6 and G7) as well as morbidity and mortality due to CME. E. canis infection rates were low for fipronil treated animals: 2.7% (1/37) for G3 and 5.5% (1/18) for G8 group. Among control animals, seroprevalence was maximum (100%) in dogs kept in kennels (G5, G6 and G7 groups) and high among native dogs in Djibouti (G1 group): 69.7% (46/66) and in Dakar (G4 group): 50% (16/32). Dogs belonging to expatriate citizens (G2 group) were less likely to be infected: 21.7% (13/60). The comparison of serological results among French army dogs and French citizen dogs that were introduced in Djibouti for an average of 10 months shows a statistically significant (P<0.001) difference. Among fipronil treated animals (G3 group), 2 dogs out of 55 seroconverted (3.6%) compared to 13 out of 60 dogs (21.7%) in the control G2 group. The results of our study indicate the preventative efficacy of a fipronil monthly treatment to avoid CME in endemic areas. Epidemiological data concerning animals that live in the same endemic areas are an example of the serious consequences (in terms of mortality and morbidity) that are related to the absence of efficient methods for tick-control. In order to protect dogs that are in transit in endemic areas against tick-transmitted diseases, the use of an adapted acaricide product is recommended.

Iron chelators as antimalarial agents: in vitro activity of dicatecholate against Plasmodium falciparum.

The present study was undertaken to explore the antimalarial effect of a series of dicatecholate iron chelators. They may be made more or less lipophilic by increasing or reducing the length of the R substituent on the nitrogen. In vitro activity against the W2 and 3D7 clones of Plasmodium falciparum, toxicity on Vero cells and toxicity on uninfected erythrocytes by measure of the released haemoglobin were assessed for each compound. These findings were compared with the ability of iron(III), iron(II) and ferritin to reverse the inhibitory effect of catecholates. This study shows that increased lipid solubility of catecholate iron chelators does not lead to improved antimalarial activity. However, their activity is well correlated with their interaction with iron and with their toxicity against Vero cells. This study demonstrates a potent antimalarial effect of FR160 (R = C9H19) on five different strains of P. falciparum in vitro. FR160 inhibited parasite growth with an IC50 between 0.8 and 1.5 micro M. The effects of FR160 on mammalian cells were minimal compared with those obtained with malaria parasites. FR160 acted on parasites at considerably higher rates than desferrioxamine, and at all stages of parasite growth. The drug was more effective at the late trophozoite and young schizont stages, although FR160 affected rings and schizonts as well. Ascorbic acid, a free radical scavenger, reduced the activities of FR160 and artesunate. FR160 might induce formation of free radicals, which could explain why FR160 antagonized the effects of artesunate and dihydroartemisinin.

In vitro activities of ferrochloroquine against 55 Senegalese isolates of Plasmodium falciparum in comparison with those of standard antimalarial drugs.

The in vitro activities of ferrochloroquine, chloroquine, quinine, mefloquine, halofantrine, amodiaquine, artesunate, atovaquone, cycloguanil and pyrimethamine were evaluated against Plasmodium falciparum isolates from Senegal (Dielmo, Ndiop), using an isotopic micro-drug susceptibility test. The IC50 values for ferrochloroquine ranged from 0.55 to 28.2 nM and the geometric mean IC50 for the 55 isolates was 7.9 nM (95% CI, 6.5-9.7 nM). Ferrochloroquine was 35 times more active than chloroquine (35-fold greater against chloroquine-resistant isolates), quinine, mefloquine, amodiaquine, cycloguanil and pyrimethamine. Weak positive correlations were observed between the responses to ferrochloroquine and that to chloroquine, quinine, and amodiaquine, but not compulsorily predictive of cross-resistance. There was no significant correlation between the response to ferrochloroquine and that to mefloquine, halofantrine, artesunate, atovaquone, cycloguanil and pyrimethamine. Ferrochloroquine may be an important alternative drug for the treatment of chloroquine-resistant malaria.

Description of three new polymorphisms in the intronic and 3'UTR regions of the human interferon gamma gene.

Interferon-gamma (IFN-gamma) is a key regulator of the development and functions of the immune system. In particular, this cytokine plays a major role in immune defense against infections by various human pathogens and polymorphisms in the IFN-gamma gene, including the transcription regulatory region, and might affect host resistance to infectious agents such as schistosomes. In this study on the genetics of human schistosomiasis we uncovered three new single nucleotide polymorphisms in the IFN-gamma genes. Two polymorphisms are located in the third intron and the third is in the 3'UTR region of this gene: an A to G transition at position +2109 from the transcription start and two G to A transitions at positions +3810 and +5134. In a SUDANESE population living in an endemic area of malaria and schistosomiasis, the allelic frequenciesare: 0.85 (+2109A), 0.15 (+2109G), 0.92 (+3810G), 0.08 (+3810A), (+5134G) and 0.04 (+5134A).

Two new polymorphisms in the human interferon gamma (IFN-gamma) promoter.

Two new single-nucleotide polymorphisms are described within the human interferon gamma (IFN-gamma) promoter in a Sudanese population. One is a G to T transition at position -183 from the transcription start. The other is a A to G transition at position -155. Allelic frequency analysis indicated frequencies of 0.927 (G) and 0.073 (T) at position -183 and 0.977 (A) and 0.023 (G) at position -155. These two polymorphisms have not been detected in the Centre d'Etude du Polymorphisme Humain (CEPH) reference population. The polymorphism -183(G-->T) may alter the AP-1 binding domain and the regulation of transcription. The polymorphism -155(A-->G) is located close to the nuclear factor-activated T-cell site (NFAT site) (-168 TAAAGGAAA-160) and may affect the stability of this region.

[Re-assessment of culture inhibition assays and reinvasion of P. falciparum for the appraisal of immunity of individuals living in an endemic area]. / Réévaluation des tests d'inhibition de culture et de réinvasion parasitaire de P. falciparum pour mesurer un degré de prémunition chez des individus vivant en zone d'endémie.

We conducted an analysis to reevaluate the in vitroculture inhibition assays as a reliable criteria of functional activity of anti-P. falcipanrm antibodies in premunized individuals. Several strains of P. falcipanrm adapted to in vitro culture were compared, and various technical conditions of parasite growth factors such as culture medium or incubation conditions were explored. A subsequent degree of variation was evidenced related to the parasite strain used and the culture conditions. The culture inhibition and the merozoite reinvasion inhibition assays were performed using a collection of plasma from premunized individuals living in two different endemic area of transmission in Senegal. High levels of inhibition were evidenced with a limited degree of variation according to the two different locations of individual's samples. A Significant relationship between anti-merozoite Ab levels and the culture inhibition assays was found contrary to the merozoite re-invasion inhibition assays. Taken together, our results show that such inhibition assays can be managed in facilities of southern laboratories near endemic areas. However, strictly defined culture conditions, homogeneous withdrawals of patients and standardized protocols are necessary for the assessment of such functional assaysas a potential markerof protection-associated mechanisms in longitudinal studies.