Malaria is the most important parasitic disease in the world, affecting 300-500 million people and killing one million people every year. Imported malaria is a growing problem in non-endemic areas throughout the world. Annually, up to 4,000 cases of malaria inFranceand 30,000 cases in all industrialized countries are imported with an average case fatality rate ranging from 1% to 4%. Every year, 500 to 800 French soldiers have malaria.
Parasitological component of malaria control is based on rapid and accurate diagnosis and appropriate treatment of acute malarial infections.
Over the past 20 years, many strains of Plasmodium falciparum have become resistant to chloroquine and other antimalarial drugs. This development has prompted the search for an effective alternative antimalarial drug with minimal side effects. Since 2001, more than 60 countries have officially adopted artemisinin-based combination therapies (ACTs) for the treatment of falciparum malaria, and the official first-line antimalarial treatment inAfrica is now ACTs. However, the first clinical failures have been described inCambodia.
Each year, Plasmodium vivax is responsible for 100 millions of malaria cases. Classically, clinical cases of P. vivax malaria are simple. But more and more severe manifestations are reported (neurological, haematological forms and acute respiratory distress syndrom) as those observed with P. falciparum. Moreover, the therapeutic capacities are now limited by the emergence of P. vivax resistance to the different antimalarial compounds.
In this context, there is a need to provide an active surveillance to monitor temporal trends in parasite susceptibility of P. falciparum and P. vivax, to identify molecular markers that predict resistance and to develop new antimalarial drugs.
We need also to assess travelers exposure to vector bites and to evaluate the protective efficacy of antivectorial devices and the risk of pathogen transmission.
The current gold standard for malaria diagnosis is microscopic examination of blood films. Therefore, there is a clear need to improve the current laboratory procedures to establish malaria diagnosis. Three alternative laboratory tools can be developed to achieve this goal: rapid diagnostic test (RDT), PCR, and mass spectrometry.
The use of RDT in low- and high-transmission areas inAfricarequires further evaluation for possible introduction and full integration of RDT in the health care system. Preliminary studies conducted inYaounde,Cameroon(coll with Health personnel of the Catholic missionary dispensary,Yaounde,Cameroon), suggested that RDT can be a major user-friendly tool that can radically improve the current malaria control efforts. A positive result indicates a rapid need to treat for malaria, whereas a negative result suggests that other infectious (or non-infectious) entities may be the cause of fever and that ACTs should not be prescribed. RDT may be the key element to avoid presumptive antimalarial treatment based on the history of recent fever.
In a first methodological approach, a series of randomized studies will be conducted to compare cost-efficacy of the presumptive treatment arm (malaria diagnosis based on clinical evaluation only and antimalarial treatment for all febrile patients) versus RDT arm (treatment prescribed after RDT result). The accuracy of RDT will be validated by a retrospective microscopic examination of blood films.
In an other health center inYaounde, where malaria diagnosis is routinely established only by microscopic examination of blood films, microscopy will be entirely replaced by RDT. Different trademarks of RDT will be evaluated, and social acceptability of this novel diagnostic method will be also evaluated, both among the prescriptors and patients. A retrospective microscopic examination of blood films will be performed to ensure the high sensitivity and specificity of different RDTs.
Several protocols for the detection and distinction of different Plasmodium species by real-time PCR have been published since 2001. The most appropriate protocol should at least distinguish between P. falciparum and non-P. falciparum infection with high sensitivity and specificity. As the results of this study are intended to be applicable and used both at the Emergency Department of a hospital inFrance and outposts inAfrica with very limited resources, a simplified and rapid protocol is required. The existing, published protocols will be evaluated, and the technology transferred to the points of care (POC). The isolates will be collected in hospitals from Marseille for diagnosis by real-time PCR and mass spectrometry (coll with URMITE, B. La Scola & Ph. Parola)
Preliminary studies on P. falciparum reference clones in continuous culture by MALDI did not yield consistent results. The current protocol for parasite preparation is too long to obtain a rapid diagnosis. Further modifications of the protocol are required to eliminate human leukocytes and uninfected erythrocytes, which may be the origin of artefacts in spectrometric measures. When consistent results with MALDI are obtained with P. falciparum reference clones and fresh isolates from imported malaria, the study will be extended to cover 3 other Plasmodium species (and possibly P. knowlesi, claimed by some to be the fifth human malaria parasites).
In vitro testing of drug resistance (B. Pradines, B. Pouvelle, L. Basco)
We will survey malaria resistance among imported cases (coll INVS, French hospitals, Fund INVS) and in endemic areas such asSenegal(coll Senegalese Health Military Service, Dakar Pasteur Institute & URMITE, JF. Trape. Fund "Schéma directeur paludisme Etat Major des Armées") andCameroon(coll with Health personnel of the Catholic missionary dispensary,Yaounde,Cameroon).
We are associated to the FrenchNationalReferenceCenterfor malaria for 10 years and to the WorldWide Antimalarial Resistance Network for 3 years. We investigate in vitro P. falciparum isolates susceptibility to all of the drugs used in malaria prophylaxis and therapeutic, by in vitro culture test and genetic polymorphisms implicated in resistance.
In vitro evaluation of resistance is and will be assessed every 2-3 years in Senegaland at least once in Cameroon. A clinical evaluation of the efficacy of quinine will be realized in military hospital in Dakarand compared to in vitro results. In Cameroon, therapeutic efficacy and tolerance of atovaquone-proguanil and artesunate-atovaquone-proguanil will be compared to those of artesunate-amodiaquine in randomized studies in children aged less than 5 years old. Fingerprick capillary blood samples will be collected prior to treatment and at the time of treatment failure. Parasites will be characterized by molecular biology. P. falciparum cytochrome b will be sequenced. Pre-treatment and post-treatment isolates will be compared according to the recommended WHO protocol (msp-1, msp-2, glurp markers) to distinguish between recrudescence and reinfection. For some, but not all, children (more than 2 or 3 years old), venous blood will be collected to perform in vitro drug sensitivity assay and determine atovaquone IC50.
The current gold standard for in vitro drug sensitivity assay is hypoxanthine-based radioisotope assay. Isotopic assay is not an appropriate technology for most endemic countries. Alternative, non-isotopic assays will therefore be developed. ELISA-based assays have recently been evaluated and developed by several other research groups. SYBR Green I-based fluorescence assay and FACS-based assay will be developed in our institute. These alternative assay systems will be conducted in parallel with ELISA-based and radioisotope assays to evaluate their reproducibility.
Molecular approaches (H. Bogreau, C. Rogier, S. Briolant, B. Pradines)
High-throughput drug resistance monitoring requires identification of molecular markers associated with resistance. Then, simple molecular methods are used to detect polymorphism associated with the resistance within plasmodial populations. So far, parasites have developed resistance against almost all antimalarial drugs, notably the new drug combinations (ACT) and Quinine. But associations between these molecules and genetic molecular markers still are elusive and unclear.
Resistant strains according to test in vivo results will be collected from Senegal, RCA, Djibouti and Cambodia (Projet GENANTIPAL, coll Military health department, Republic of Djibouti; Pasteur Institute of Bangui RCA; Senegalese Health Military Service; Fund DGA). Confirmation of drug resistance in vitro will be assessed. Resistant strains will be genotyped with microarray high density technology (Affymetrix) resistant strain and identification of polymorphisms associated with resistance (SNP, Insertion/Deletion, gene amplification) or genetic linkage disequilibrium will be assessed. Then, we will develop molecular test to high-throughput screening.
In addition, we identified some transport proteins that could be implicated in drug resistance (quinine, doxycycline, amodiaquine or artemisinin derivatives) and which can be validated now. Mutagenesis studies will be realized to validate the implication of these markers (coll French hospitals, Pasteur Institute of French Guyana, Senegalese Health Military Service). These molecular markers that predict resistance can provide an active surveillance method to monitor temporal trends in parasite susceptibility.
Falciparum malaria may be asymptomatic, mild or severe. Human and parasite factors compose a system and each may impact this severity. So far, Human factors have been well documented but pathogen factors have been less studied. Nevertheless, small number of studies suggested genetic clusters between plasmodial populations from severe and mild malaria. These data suggest genetic specificity of strain responsible of severe malaria.
Isolates from severe and mild malaria were collected inMali,Cameroon,BeninandGabon(Project AGENPAL, coll Dr. C Eboumbou Moukoko, Univ of Douala, Cameroon; MRTC,Mali; MRC Lambéréné; IRD UMR216; Fund DGA). Molecular markers as antigenic protein gene (MSP1, MSP2 & GLURP) will be genotyped. Moreover, strains from uncomplicated and severe cases will be genotyped with microarray high density technology (Affymetrix) and identification of polymorphisms associated with severity will be assessed
Many strains of Plasmodium falciparum have become resistant to antimalarial drugs. There is an urgent need of development of new antimalarial drugs.
Previous studies showed that dihydroantracenes, atorvastatin and methylene blue potentiate the activities of antimalarial drugs such as quinine, mefloquine, lumefantrine, amodiaquine, artemisinin derivatives (Project OPTIPALASSOS, UMR MD1, CIML Luminy, Institut Jean Roche Marseille, INSERM U599, Provepharm. Fund DGA). In vivo evaluation will be assessed in rodent models. Atorvastatin and methylene blue will be quickly evaluated in combination with antimalarial drugs in human.
In addition, bioorganometallics may overcome resistance to established drugs via new and possibly metal-specific modes of action. a ferrocene analogue of CQ named ferroquine (FQ, SSR97193), was highly active against all P. falciparum clones tested, whatever their level of resistance to CQ, and moreover, did not show any significant cross resistance with other currently used antimalarials (UMR CNRS 8181, Lille; Inserm U547, Lille; UMR CNRS 8576, Lille; Département de Chimie Cape Town South Africa; Center of Biochemistry, Heidelberg, Germany; Instituto de Química, Chile. Fund Sanofi-Aventis). More recently, FQ was shown to have a potent ex vivo effect on P. vivax schizont maturation. Thus, it was suggested that FQ could be a suitable replacement for CQ in the treatment of drug resistant vivax malaria. FQ is currently being developed by Sanofi-Aventis and entered phase II clinical trials in association with artesunate in P. falciparum malaria. Additional series of derivatives have been synthesized for further evaluation, both in vitro and in vivo (rodent malaria).
Indolone N-oxide derivatives have been extensively developed as potential antimalarial drugs within the sixth framework programme of the European Union (University P Sabatier, Toulouse & Ideal Pharma, Lyon). Several promising leads have been identified in vitro and tested in vivo using the rodent malaria models. Additional series of derivatives have been synthesized for further evaluation, both in vitro and in vivo (rodent malaria).
Several derivatives of quinoa extracts have been purified and characterized (Universidad de La Serena,Chile& UMSA,Bolivia). Some of the crude extracts are part of the traditional medicine to treat fever in the Andean mountains. The purified molecules will be evaluated in vitro.
New 3-aminosterol compounds and derivatives analogous to squalamine showed efficacy against P. falciparum strains. These compounds as well as the underlying design rationale may find usefulness in the discovery and development of new antimalarial drugs. Current studies are under investigation and will be reported in due course (URMITE JM. Rolain & JM. Brunel).
The mechanisms of action of antimalarial drugs used alone or in combination will be investigated by cellular and ultrastructural approaches.
The therapeutic capacities are now limited by the emergence of P. vivax resistance to the different antimalarial compounds and the partial efficacy of primaquine used in the radical cure of hypnozoïtic forms of P. vivax parasite. The development of new molecules such as tafenoquine is required but their clinical evaluation will be difficult because of the absence of diagnosis markers for the detection of latent infection (hypnozoïtes forms) and to distinguish novel infections from relapses. The absence of standardized methods to evaluate the in vitro susceptibility of P. vivax isolates to antimalarial drugs and the difficulties to cultivate P. vivax limit the possibility to survey P. vivax chemosusceptibility. However, the complete sequence of P. vivax genome is now available and offers the possibility to identify molecular markers of resistance. The analysis of both humoral and cellular immune response to P. vivax infection could be explored by using peptides or recombinant proteins corresponding to preerythrocytic stages antigens and interferon gamma release assays.
To develop new chemosusceptibility in vitro tests, to identify molecular polymorphism of P. vivax isolates and biomarkers of latent infections should allow us 1) to enhance the therapeutic management of P. vivax malaria cases 2) to possess adapted tools to evaluate clinical efficacy of new antimalarial drugs and 3) to improve the knowledge of molecular epidemiology of P. vivax resistance.
Isolates of P. vivax were collected inNouakchott,Mauritania (Université Nouakchott,Mauritania & URMITE JF. Trape) and will be collected in Thailand and Guyana (Project OPAL, Shoklo Malaria Research Unit, Thailand; Mahidol University, Thailand; CNRCP, Guyana; Centre Hospitalier Général de Cayenne-EA3593. Fund DGA).
A focalized “epidemic” has been detected within the city of Nouakchott. Epidemiology of this malaria outbreak is currently under investigation. Mosquitoes have been collected weekly during the transmission period in 2007-2009, and more than 430 blood samples have been collected from febrile children and adults consulting three hospitals in the city during the same period. In 2010, captured mosquitoes were identified in Dakar, Senegal(IRD-Senegal), and Plasmodium species was confirmed by both classical and real-time PCR. Samples that were confirmed to be P. vivax by PCR will be further characterized by molecular biology techniques. Because the presence of P. vivax inMauritania has not been well documented in the past, additional field surveys, including mosquito and blood collection, and malaria monitoring will be conducted inNouakchott in 2010-2015.
Molecular markers that have been identified or suggested to be associated with drug resistance will be analyzed: pvdhfr for pyrimethamine, pvdhps for sulfonamides, and pvcrt and pvmdr1 for chloroquine. P. vivax isolates will be further characterized by microsatellites to evaluate the degree of diversity among them and deduce their origin and degree of relationship with other P. vivax isolates collected from other endemic regions inAfrica and elsewhere. In addition, the presence or absence of Duffy receptor will be analyzed.
Salivary proteins could be candidate markers to assess travelers exposure to vector bites and therefore to evaluate the protective efficacy of antivectorial devices and the risk of pathogen transmission.
The aims of the project are to identify of mosquito salivary proteins species specific for evaluation of individual human exposure to mosquito bites.
Recently, we showed that salivary proteins could induce a short-lived species-specific antibody response in human which was associated to the level of mosquito exposure. The development of a serological method based on specific antibody response against salivary proteins from malaria or arbovirose vectors could be a valuable tool to assess human exposure to mosquito bites. However, the composition of saliva protein cocktail of major mosquito species is not well characterized limiting the determination of species specific antigens.
In order to better identify salivary components from different mosquito genus and species (Anopheles gambiae, An. arabiensis, An. funestus, An. albimanus, An. stephensi; Aedes aegypti, Ae. albopictus, Ae. caspius; Culex quinquefasciatus, Culex pipiens) and to define species specific proteins as potential bite exposure candidates, several strategies are envisaged.
In silico comparisons of protein sequence database between the different selected mosquitoes. Large-scale proteomic approaches will be realized to define salivary glands proteome of the selected mosquitoes species (1D SDS-PAGE coupled to mass spectrometry, shot-gun proteomic and iTRAQ® technology). Based on these two first strategies, some proteins genus and/or species specifics will be selected to evaluate their antigenicity. Thus, recombinant proteins will be produce and further tested by ELISA and/or Luminex using sera bank available in the institute or in our collaborative partners.