Christophe ROGIER

Abstract:

Abstract: The preventive measures against malaria recommended by the WHO include anti-vector procedures such as indoor residual spraying, the use of long-lasting insecticide-treated bed-nets, and the destruction of larval breeding sites. The presence of insecticide-treated materials inside the mosquito habitat has consequences for the vector's population, reducing density, survival, contact with humans, and feeding frequency. However, the effectiveness of these tools is being challenged by the emergence of insecticide resistance. The evolution of resistance to insecticides in Anopheles threatens to thwart the goal of decreasing malaria transmission, in an arms race between malaria control programmes and the vector populations. Multiple mechanisms of resistance to insecticides have been observed in Anopheles populations, including target site mutation (knockdown resistance), increased metabolic detoxification, and remarkable behavioural adaptation. These disturbing observations all show the capacity of Anopheles to adapt to and circumvent strategies aimed at reducing malaria transmission. Thus, by using nets to protect ourselves, are we providing Anopheles with the entire arsenal needed to hit much harder?

Abstract: Although the World Health Organization recommends replacing quinine (QN) by artesunate due to its increased efficacy and the higher tolerance to the drug in both adults and children, QN remains a first-line treatment for severe malaria, especially in Africa. Investigations of microsatellite Pfnhe-1 ms4760 polymorphisms in culture-adapted isolates from around the world have revealed that an increase in the number of DNNND amino acid motifs was associated with decreased QN susceptibility, whereas an increase in the number of DDNHNDNHNND motifs was associated with increased QN susceptibility.

Abstract: Approximately 125 million travellers visit malaria-endemic countries annually and about 10,000 cases of malaria are reported after returning home. Due to the fact that malaria is insect vector transmitted, the environment is a key determinant of the spread of infection. Geo-climatic factors (such as temperature, moisture, water quality) determine the presence of Anopheles breeding sites, vector densities, adult mosquito survival rate, longevity and vector capacity. Several studies have shown the association between environmental factors and malaria incidence in autochthonous population. The association between the incidence of clinical malaria cases among non-immune travellers and environmental factors is yet to be evaluated. The objective of the present study was to identify, at a country scale (Ivory Coast), the environmental factors that are associated with clinical malaria among non-immune travellers, opening the way for a remote sensing-based counselling for malaria risk prevention among travellers.

Abstract: This study aimed to determine the epidemiological impact of rice cultivation in inland valleys on malaria in the forest region of western Cote d'Ivoire. The importance of malaria was compared in terms of prevalence and parasite density of infections and also in terms of clinical malaria incidence between three agro-ecosystems: (i) uncultivated inland valleys, (R0), (ii) inland valleys with one annual rice cultivation in the rainy season, (R1) and (iii) developed inland valleys with two annual rice cultivation cycles, (R2).

Abstract: Case management of imported malaria within the context of malaria pre-elimination is increasingly considered to be relevant because of the risk of resurgence. The assessment of malaria importation would provide key data i) to select countries with propitious conditions for pre-elimination phase and ii) to predict its feasibility. Recently, a sero-prevalence study in Djibouti indicated low malaria prevalence, which is propitious for the implementation of pre-elimination, but data on the extent of malaria importation remain unknown.

Abstract: Antibody responses against Anopheles salivary proteins can indicate individual exposure to bites of malaria vectors. The extent to which these salivary proteins are species-specific is not entirely resolved. Thus, a better knowledge of the diversity among salivary protein repertoires from various malaria vector species is necessary to select relevant genus-, subgenus- and/or species-specific salivary antigens. Such antigens could be used for quantitative (mosquito density) and qualitative (mosquito species) immunological evaluation of malaria vectors/host contact. In this study, salivary gland protein repertoires (sialomes) from several Anopheles species were compared using in silico analysis and proteomics. The antigenic diversity of salivary gland proteins among different Anopheles species was also examined.

Abstract: Malaria transmission occurs during the blood feeding of infected anopheline mosquitoes concomitant with a saliva injection into the vertebrate host. In sub-Saharan Africa, most malaria transmission is due to Anopheles funestus s.s and to Anopheles gambiae s.l. (mainly Anopheles gambiae s.s. and Anopheles arabiensis). Several studies have demonstrated that the immune response against salivary antigens could be used to evaluate individual exposure to mosquito bites. The aim of this study was to assess the use of secreted salivary proteins as specific biomarkers of exposure to An. gambiae and/or An. funestus bites.

Abstract: In tropical Africa, where malaria is highly endemic, low grade infections are asymptomatic and the diagnosis of clinical malaria is usually based on parasite density. Here we investigate how changes in malaria control and endemicity modify diagnostic criteria of Plasmodium falciparum attacks.

Abstract: High malaria transmission heterogeneity in an urban environment is basically due to the complex distribution of Anopheles larval habitats, sources of vectors. Understanding 1) the meteorological and ecological factors associated with differential larvae spatio-temporal distribution and 2) the vectors dynamic, both may lead to improving malaria control measures with remote sensing and high resolution data as key components. In this study a robust operational methodology for entomological malaria predictive risk maps in urban settings is developed.

Abstract:

Abstract: Screening for in vitro susceptibility to pyrimethamine and sequencing of the pfmdr2 and pfdhfr genes were performed in 140 Plasmodium falciparum isolates. The risk of in vitro resistance to pyrimethamine was analyzed with a logistic regression model. The mutation F423Y in pfmdr2 (odds ratio [OR] = 2.12 [confidence interval {CI}, 1.02 to 4.59]; P = 0.0489) and the mutation N51I, C59R, or S108N in pfdhfr (OR = 42.34 [CI, 5.52 to 324.61]; P = 0.0003) were independently associated with in vitro resistance to pyrimethamine.

Abstract: One of the major complications of Plasmodium falciparum infection is cerebral malaria (CM), which causes one million deaths worldwide each year, results in long-term neurological sequelae and the treatment for which is only partially effective. Statins are recognized to have an immunomodulatory action, attenuate sepsis and have a neuroprotective effect. Atorvastatin (AVA) has shown in vitro anti-malarial activity and has improved the activity of mefloquine (MQ) and quinine.

Abstract: The origin of Plasmodium falciparum in South America is controversial. Some studies suggest a recent introduction during the European colonizations and the transatlantic slave trade. Other evidence--archeological and genetic--suggests a much older origin. We collected and analyzed P. falciparum isolates from different regions of the world, encompassing the distribution range of the parasite, including populations from sub-Saharan Africa, the Middle East, Southeast Asia, and South America. Analyses of microsatellite and SNP polymorphisms show that the populations of P. falciparum in South America are subdivided in two main genetic clusters (northern and southern). Phylogenetic analyses, as well as Approximate Bayesian Computation methods suggest independent introductions of the two clusters from African sources. Our estimates of divergence time between the South American populations and their likely sources favor a likely introduction from Africa during the transatlantic slave trade.

Abstract: The aim of the present work was to assess i) ex vivo activity of pyronaridine (PND) and piperaquine (PPQ), as new components of artemisinin-based combination therapy (ACT), to define susceptibility baseline, ii) their activities compared to other partner drugs, namely monodesethylamodiaquine (MDAQ), lumefantrine (LMF), mefloquine (MQ), artesunate (AS) and dihydroartemisinin (DHA) against 181 Plasmodium falciparum isolates from African countries, India and Thailand, and iii) in vitro cross-resistance with other quinoline drugs, chloroquine (CQ) or quinine (QN).

Abstract: Malaria intermittent preventive treatment of malaria in infant with sulphadoxine-pyrimethamine (IPTi-SP) reduced the incidence of malaria and anaemia by 30% and 20% respectively. The strategy is now a recommended policy for malaria control. However, there was no published study on the impact of the strategy on mortality. The present study assessed the impact of the implementation of IPTi-SP in health services in Mali on all-cause mortality.

Abstract: As a result of the widespread resistance to chloroquine and sulphadoxine-pyrimethamine, artemisinin-based combination therapy (ACT) (including artemether-lumefantrine and artesunate-amodiaquine) has been recommended as a first-line anti-malarial regimen in Senegal since 2006. Intermittent preventive treatments with anti-malarial drugs based on sulphadoxine-pyrimethamine are also given to children or pregnant women once per month during the transmission season. Since 2006, there have been very few reports on the susceptibility of Plasmodium falciparum to anti-malarial drugs. To estimate the prevalence of resistance to several anti-malarial drugs since the introduction of the widespread use of ACT, the presence of molecular markers associated with resistance to chloroquine and sulphadoxine-pyrimethamine was assessed in local isolates at the military hospital of Dakar.

Abstract: Malaria control efforts and elimination in Africa are being challenged by the development of resistance of parasites to antimalarial drugs and vectors to insecticides. We investigated whether the combination of long-lasting insecticidal mosquito nets (LLINs) with indoor residual spraying (IRS) or carbamate-treated plastic sheeting (CTPS) conferred enhanced protection against malaria and better management of pyrethroid-resistance in vectors than did LLINs alone.

Abstract: Urban malaria can be a serious public health problem in Africa. Human-landing catches of mosquitoes, a standard entomological method to assess human exposure to malaria vector bites, can lack sensitivity in areas where exposure is low. A simple and highly sensitive tool could be a complementary indicator for evaluating malaria exposure in such epidemiological contexts. The human antibody response to the specific Anopheles gSG6-P1 salivary peptide have been described as an adequate tool biomarker for a reliable assessment of human exposure level to Anopheles bites. The aim of this study was to use this biomarker to evaluate the human exposure to Anopheles mosquito bites in urban settings of Dakar (Senegal), one of the largest cities in West Africa, where Anopheles biting rates and malaria transmission are supposed to be low.

Abstract: Aedes mosquitoes are important vectors of re-emerging diseases in developing countries, and increasing exposure to Aedes in the developed world is currently a source of concern. Given the limitations of current entomologic methods, there is a need for a new effective way for evaluating Aedes exposure. Our objective was to evaluate specific antibody responses to Aedes aegypti saliva as a biomarker for vector exposure in a dengue-endemic urban area. IgG responses to saliva were strong in young children and steadily waned with age. Specific IgG levels were significantly higher in persons living in sites with higher Ae. aegypti density, as measured by using entomologic parameters. Logistic regression showed a significant correlation between IgG to saliva and exposure level, independently of either age or sex. These results suggest that antibody responses to saliva could be used to monitor human exposure to Aedes bites.

Abstract: Formerly known as a hypoendemic malaria country, the Republic of Djibouti declared the goal of pre-eliminating malaria in 2006. The aim of the present study was to evaluate the prevalence of Plasmodium falciparum, Plasmodium vivax and mixed infections in the Djiboutian population by using serological tools and to identify potential determinants of the disease and hotspots of malaria transmission within the country.

Abstract: BACKGROUND: The effectiveness of anti-vectorial malaria protective measures in travellers and expatriates is hampered by incorrect compliance. The objective of the present study was to identify the determinants of compliance with anti-vectorial protective measures (AVPMs) in this population that is particularly at risk because of their lack of immunity. METHODS: Compliance with wearing long clothing, sleeping under insecticide-impregnated bed nets (IIBNs) and using insect repellent was estimated and analysed by questionnaires administered to 2,205 French military travellers from 20 groups before and after short-term missions (approximately four months) in six tropical African countries (Senegal, Ivory Coast, Chad, Central African Republic, Gabon and Djibouti). For each AVPM, the association of "correct compliance" with individual and collective variables was investigated using random-effect mixed logistic regression models to take into account the clustered design of the study. RESULTS: The correct compliance rates were 48.6%, 50.6% and 18.5% for wearing long clothing, sleeping under bed nets and using repellents, respectively. Depending on the AVPM, correct compliance was significantly associated with the following factors: country, older than 24 years of age, management responsibilities, the perception of a personal malaria risk greater than that of other travellers, the occurrence of life events, early bedtime (i.e., before midnight), the type of stay (field operation compared to training), the absence of medical history of malaria, the absence of previous travel in malaria-endemic areas and the absence of tobacco consumption.There was no competition between compliance with the different AVPMs or between compliance with any AVPM and malaria chemoprophylaxis. CONCLUSION: Interventions aimed at improving compliance with AVPMs should target young people without management responsibilities who are scheduled for non-operational activities in countries with high risk of clinical malaria. Weak associations between compliance and history of clinical malaria or variables that pertain to threat perception suggest that cognition-based interventions referencing a "bad experience" with clinical malaria could have only a slight impact on the improvement of compliance. Further studies should focus on the cognitive and behavioural predictors of compliance with AVPMs.

Abstract: The geometric mean 50% inhibitory concentration (IC50) for Proveblue, a methylene blue complying with the European Pharmacopoeia, was more active on 23 P. falciparum strains than chloroquine, quinine, mefloquine, monodesethylamodiaquine, and lumefantrine. We did not find significant associations between the Proveblue IC50 and polymorphisms in the pfcrt, pfmdr1, pfmdr2, pfmrp, and pfnhe-1 genes or the copy numbers of the pfmdr1 and pfmdr2 genes, all of which are involved in antimalarial resistance.

Abstract: BACKGROUND: The aim of this study was to evaluate the cultivation system in which the proper atmospheric conditions for growing Plasmodium falciparum parasites were maintained in a sealed bag. The Genbag(R) system associated with the atmospheric generators for capnophilic bacteria Genbag CO2(R) was used for in vitro susceptibility test of nine standard anti-malarial drugs and compared to standard incubator conditions. METHODS: The susceptibility of 36 pre-identified parasite strains from a wide panel of countries was assessed for nine standard anti-malarial drugs (chloroquine, quinine, mefloquine, monodesethylamodiaquine, lumefantrine, dihydroartemisinin, atovaquone and pyrimethamine) by the standard 42-hour 3H-hypoxanthine uptake inhibition method using the Genbag CO2(R) system and compared to controlled incubator conditions (5% CO2 and 10% O2). RESULTS: The counts per minute values in the control wells in incubator atmospheric conditions (5% CO2 and 10% O2) were significantly higher than those of Genbag(R) conditions (2738 cpm vs 2282 cpm, p < 0.0001). The geometric mean IC50 estimated under the incubator atmospheric conditions was significantly lower for atovaquone (1.2 vs 2.1 nM, p = 0.0011) and higher for the quinolines: chloroquine (127 vs 94 nM, p < 0.0001), quinine (580 vs 439 nM, p < 0.0001), monodesethylamodiaquine (41.4 vs 31.8 nM, p < 0.0001), mefloquine (57.5 vs 49.7 nM, p = 0.0011) and lumefantrine (23.8 vs 21.2 nM, p = 0.0044). There was no significant difference of IC50 between the 2 conditions for dihydroartemisinin, doxycycline and pyrimethamine.To reduce this difference in term of anti-malarial susceptibility, a specific cut-off was estimated for each drug under Genbag(R) conditions by regression. The cut-off was estimated at 77 nM for chloroquine (vs 100 nM in 10% O2), 611 nM for quinine (vs 800 nM), 30 nM for mefloquine (vs 30 nM), 61 nM for monodesethylamodiaquine (vs 80 nM) and 1729 nM for pyrimethamine (vs 2000 nM). CONCLUSIONS: The atmospheric generators for capnophilic bacteria Genbag CO2(R) is an appropriate technology that can be transferred to the field for epidemiological surveys of drug-resistant malaria. The present data suggest the importance of the gas mixture on in vitro microtest results for anti-malarial drugs and the importance of determining the microtest conditions before comparing and analysing the data from different laboratories and concluding on malaria resistance.

Abstract: Mapping and anticipating risk is a major issue in the fight against malaria, a disease causing an estimated one million deaths each year. Approximately half the world's population is at risk and it is of prime importance to evaluate the burden of malaria at the spatial as well as the temporal level. The role of the environment with regard to the determinants of transmission and burden of the disease are described followed by a discussion of special issues such as urban malaria, human population mapping and the detection of changes at the temporal scale. Risk maps at appropriate scales can provide valuable information for targeted control and the present review discusses the essentials of principles, methods, advantages and limitations of remote sensing along with a presentation of ecological, meteorological and climatologic data which rule the distribution of malaria. The panel of commonly used analytic methods is examined and the methodological limitations are highlighted. A review of the literature details the increasing interest in the use of remotely sensed data in the study of malaria, by mapping or modeling several malariometric indices such as prevalence, morbidity and mortality, which are discussed with reference to vector breeding, vector density and entomological inoculation rate, estimates of which constitute the foundation for understanding endemicity and epidemics.

Abstract: BACKGROUND: The evaluation of malaria transmission intensity is a crucial indicator for estimating the burden of malarial disease. In this respect, entomological and parasitological methods present limitations, especially in low transmission areas. The present study used a sensitive multiplex assay to assess the exposure to Plasmodium falciparum infection in children living in an area of low endemicity. In three Senegalese villages, specific antibody (IgG) responses to 13 pre-erythrocytic P. falciparum peptides derived from Lsa1, Lsa3, Glurp, Salsa, Trap, Starp, Csp and Pf11.1 proteins were simultaneously evaluated before (June), at the peak (September) and after (December) the period of malaria transmission, in children aged from 1 to 8 years. RESULTS: Compared to other antigens, a high percentage of seropositivity and specific antibody levels were detected with Glurp, Salsa1, Lsa3NR2, and Lsa1J antigens. The seropositivity increased with age for all tested antigens. Specific IgG levels to Glurp, Salsa1, Lsa3NR2, and Lsa1J were significantly higher in P. falciparum infected children compared to non-infected and this increase is significantly correlated with parasite density. CONCLUSION: The multiplex assay represents a useful technology for a serological assessment of rapid variations in malaria transmission intensity, especially in a context of low parasite rates. The use of such combined serological markers (i.e. Glurp, Lsa1, Lsa3, and Salsa) could offer the opportunity to examine these variations over time, and to evaluate the efficacy of integrated malaria control strategies.

Abstract: BACKGROUND: Substantial reductions in malaria have been reported in several African countries after distribution of insecticide-treated bednets and the use of artemisinin-based combination therapies (ACTs). Our aim was to assess the effect of these policies on malaria morbidity, mosquito populations, and asymptomatic infections in a west African rural population. METHODS: We did a longitudinal study of inhabitants of Dielmo village, Senegal, between January, 2007, and December, 2010. We monitored the inhabitants for fever during this period and we treated malaria attacks with artesunate plus amodiaquine. In July, 2008, we offered longlasting insecticide (deltamethrin)-treated nets (LLINs) to all villagers. We did monthly night collections of mosquitoes during the whole study period, and we assessed asymptomatic carriage from cross-sectional surveys. Our statistical analyses were by negative binomial regression, logistic regression, and binomial or Fisher exact test. FINDINGS: There were 464 clinical malaria attacks attributable to Plasmodium falciparum during 17,858 person-months of follow-up. The incidence density of malaria attacks averaged 5.45 (95% CI 4.90-6.05) per 100 person-months between January, 2007, and July, 2008, before the distribution of LLINs. Incidence density decreased to 0.41 (0.29-0.55) between August, 2008, and August, 2010, but increased back to 4.57 (3.54-5.82) between September and December, 2010--ie, 27-30 months after the distribution of LLINs. The rebound of malaria attacks were highest in adults and children aged 10 years or older: 45 (63%) of 71 malaria attacks recorded in 2010 compared with 126 (33%) of 384 in 2007 and 2008 (p<0.0001). 37% of Anopheles gambiae mosquitoes were resistant to deltamethrin in 2010, and the prevalence of the Leu1014Phe kdr resistance mutation increased from 8% in 2007 to 48% in 2010 (p=0.0009). INTERPRETATION: Increasing pyrethroid resistance of A gambiae and increasing susceptibility of older children and adults, probably due to decreasing immunity, caused the rebound and age shift of malaria morbidity. Strategies to address the problem of insecticide resistance and to mitigate its effects must be urgently defined and implemented. FUNDING: Institut de Recherche pour le Developpement and the Pasteur Institute of Dakar.

Abstract: BACKGROUND: Quinine is still recommended as an effective therapy for severe cases of Plasmodium falciparum malaria, but the parasite has developed resistance to the drug in some cases. Investigations into the genetic basis for quinine resistance (QNR) suggest that QNR is complex and involves several genes, with either an additive or a pairwise effect. The results obtained when assessing one of these genes, the plasmodial Na+/H+ exchanger, Pfnhe-1, were found to depend upon the geographic origin of the parasite strain. Most of the associations identified have been made in Asian strains; in contrast, in African strains, the influence of Pfnhe on QNR is not apparent. However, a recent study carried out in Kenya did show a significant association between a Pfnhe polymorphism and QNR. As genetic differences may exist across the African continent, more field data are needed to determine if this association exists in other African regions. In the present study, association between Pfnhe and QNR is investigated in a series of isolates from central Africa. METHODS: The sequence analysis of the polymorphisms at the Pfnhe-1 ms4760 microsatellite and the evaluation of in vitro quinine susceptibility (by isotopic assay) were conducted in 74 P. falciparum isolates from the Republic of Congo. RESULTS: Polymorphisms in the number of DNNND or NHNDNHNNDDD repeats in the Pfnhe-1 ms4760 microsatellite were not associated with quinine susceptibility. CONCLUSIONS: The polymorphism in the microsatellite ms4760 in Pfnhe-1 that cannot be used to monitor quinine response in the regions of the Republic of Congo, where the isolates came from. This finding suggests that there exists a genetic background associated with geographic area for the association that will prevent the use of Pfnhe as a molecular marker for QNR. The contribution of Pfnhe to the in vitro response to quinine remains to be assessed in other regions, including in countries with different levels of drug pressure.

Abstract: BACKGROUND: Duffy blood group polymorphisms are important in areas where Plasmodium vivax is present because this surface antigen is thought to act as a key receptor for this parasite. In the present study, Duffy blood group genotyping was performed in febrile uninfected and P. vivax-infected patients living in the city of Nouakchott, Mauritania. METHODS: Plasmodium vivax was identified by real-time PCR. The Duffy blood group genotypes were determined by standard PCR followed by sequencing of the promoter region and exon 2 of the Duffy gene in 277 febrile individuals. Fisher's exact test was performed in order to assess the significance of variables. RESULTS: In the Moorish population, a high frequency of the FYBES/FYBES genotype was observed in uninfected individuals (27.8%), whereas no P. vivax-infected patient had this genotype. This was followed by a high level of FYA/FYB, FYB/FYB, FYB/FYBES and FYA/FYBES genotype frequencies, both in the P. vivax-infected and uninfected patients. In other ethnic groups (Poular, Soninke, Wolof), only the FYBES/FYBES genotype was found in uninfected patients, whereas the FYA/FYBES genotype was observed in two P. vivax-infected patients. In addition, one patient belonging to the Wolof ethnic group presented the FYBES/FYBES genotype and was infected by P. vivax. CONCLUSIONS: This study presents the Duffy blood group polymorphisms in Nouakchott City and demonstrates that in Mauritania, P. vivax is able to infect Duffy-negative patients. Further studies are necessary to identify the process that enables this Duffy-independent P. vivax invasion of human red blood cells.

Abstract: Plasmodium falciparum malaria is usually transmitted by mosquitoes. We report 2 cases in France transmitted by other modes: occupational blood exposure and blood transfusion. Even where malaria is not endemic, it should be considered as a cause of unexplained acute fever.

Abstract: Although malaria has become a serious public health problem in Mauritania since the late 1990s, few documented data on its epidemiology exist. The objective of this study was to assess the morbidity of clinical malaria among children in Nouakchott. Three hundred and one febrile children, consulting at three health facilities of Nouakchott, were screened for malaria in 2009 (n=216) and 2010 (n=85). Plasmodium species identification and parasite density were determined by microscopic examination of Giemsa-stained thin and thick films and confirmed by rapid diagnostic test and nested PCR. Of 301 febrile children, 105 (34.9%) were malaria-positive by nested PCR and 87 (28.9%) by microscopy. Plasmodium vivax represented 97.1% (102/105) and P. falciparum accounted for 2.9% (3/105) of positive cases. All positive children under five years old were infected with P. vivax. The highest numbers of malaria positives were found during or shortly after the rainy season and the lowest during the dry season. Fifty-four of 105 (51.4%) malaria cases, all with P. vivax, had never travelled outside Nouakchott. Individuals belonging to the Moors ethnic group represented 97.0% of P. vivax cases. Results of the present study indicate that malaria is endemic in Nouakchott and that P. vivax is the principal causative agent. Regular surveillance is required to monitor malaria prevalence and incidence, and further measures are needed to counter the possible spread of malaria in the country.

Abstract:

Abstract: Over the past decade, advances in proteomic and mass spectrometry techniques and the sequencing of the Plasmodium falciparum genome have led to an increasing number of studies regarding the parasite proteome. However, these studies have focused principally on parasite protein expression, neglecting parasite-induced variations in the host proteome. Here, we investigated P. falciparum-induced modifications of the infected red blood cell (iRBC) membrane proteome, taking into account both host and parasite proteome alterations. Furthermore, we also determined if some protein changes were associated with genotypically distinct P. falciparum strains. Comparison of host membrane proteomes between iRBCs and uninfected red blood cells using fluorescence-based proteomic approaches, such as 2D difference gel electrophoresis revealed that more than 100 protein spots were highly up-represented (fold change increase greater than five) following P. falciparum infection for both strains (i.e. RP8 and Institut Pasteur Pregnancy Associated Malaria). The majority of spots identified by mass spectrometry corresponded to Homo sapiens proteins. However, infection-induced changes in host proteins did not appear to affect molecules located at the outer surface of the plasma membrane. The under-representation of parasite proteins could not be attributed to deficient parasite protein expression. Thus, this study describes for the first time that considerable host protein modifications were detected following P. falciparum infection at the erythrocyte membrane level. Further analysis of infection-induced host protein modifications will improve our knowledge of malaria pathogenesis.

Abstract: BACKGROUND: As a result of widespread chloroquine and sulphadoxine-pyrimethamine resistance, artemisinin-based combination therapy (ACT) (which includes artemether-lumefantrine and artesunate-amodiaquine) has been recommended as a first-line anti-malarial regimen in Senegal since 2006. Since then, there have been very few reports on the ex vivo susceptibility of Plasmodium falciparum to anti-malarial drugs. To examine whether parasite susceptibility has been affected by the widespread use of ACT, the ex vivo susceptibility of local isolates was assessed at the military hospital of Dakar. METHODS: The ex vivo susceptibility of 93 P. falciparum isolates from Dakar was successfully determined using the Plasmodium lactate dehydrogenase (pLDH) ELISA for the following drugs: chloroquine (CQ), quinine (QN), mefloquine (MQ), monodesethylamodiaquine (MDAQ), lumefantrine (LMF), dihydroartemisinin (DHA) and doxycycline (DOX). RESULTS: After transformation of the isolate IC50 in ratio of IC50 according to the susceptibility of the 3D7 reference strain (isolate IC50/3D7 IC50), the prevalence of the in vitro resistant isolates with reduced susceptibility was 50% for MQ, 22% for CQ, 12% for DOX, 6% for both QN and MDAQ and 1% for the drugs LMF and DHA. The highest significant positive correlations were shown between responses to CQ and MDAQ (r = 0.569; P < 0.0001), LMF and QN (r = 0.511; P < 0.0001), LMF and DHA (r = 0.428; P = 0.0001), LMF and MQ (r = 0.413; P = 0.0002), QN and DHA (r = 0.402; P = 0.0003) and QN and MQ (r = 0.421; P = 0.0001). CONCLUSIONS: The introduction of ACT in 2002 has not induced a decrease in P. falciparum susceptibility to the drugs DHA, MDAQ and LMF, which are common ACT components. However, the prevalence of P. falciparum isolates with reduced susceptibility has increased for both MQ and DOX. Taken together, these data suggest that intensive surveillance of the P. falciparum in vitro susceptibility to anti-malarial drugs in Senegal is required.

Abstract: Inhibition of hemozoin biocrystallization is considered the main mechanism of action of 4-aminoquinoline antimalarials including chloroquine (CQ) but cannot fully explain the activity of ferroquine (FQ) which has been related to redox properties and intramolecular hydrogen bonding. Analogues of FQ, methylferroquine (Me-FQ), ruthenoquine (RQ), and methylruthenoquine (Me-RQ), were prepared. Combination of physicochemical and molecular modeling methods showed that FQ and RQ favor intramolecular hydrogen bonding between the 4-aminoquinoline NH group and the terminal amino group in the absence of water, suggesting that this structure may enhance its passage through the membrane. This was further supported by the use of Me-FQ and Me-RQ where the intramolecular hydrogen bond cannot be formed. Docking studies suggest that FQ can interact specifically with the {0,0,1} and {1,0,0} faces of hemozoin, blocking crystal growth. With respect to the structure-activity relationship, the antimalarial activity on 15 different P. falciparum strains showed that the activity of FQ and RQ were correlated with each other but not with CQ, confirming lack of cross resistance. Conversely, Me-FQ and Me-RQ showed significant cross-resistance with CQ. Mutations or copy number of pfcrt, pfmrp, pfmdr1, pfmdr2, or pfnhe-1 did not exhibit significant correlations with the IC(50) of FQ or RQ. We next showed that FQ and Me-FQ were able to generate hydroxyl radicals, whereas RQ and me-RQ did not. Ultrastructural studies revealed that FQ and Me-FQ but not RQ or Me-RQ break down the parasite digestive vacuole membrane, which could be related to the ability of the former to generate hydroxyl radicals.

Abstract: The saliva of haematophagous arthropods contains an array of anti-haemostatic, anti-inflammatory and immunomodulatory molecules that contribute to the success of the blood meal. The saliva of haematophagous arthropods is also involved in the transmission and the establishment of pathogens in the host and in allergic responses. This survey provides a comprehensive overview of the pharmacological activity and immunogenic properties of the main salivary proteins characterised in various haematophagous arthropod species. The potential biological and epidemiological applications of these immunogenic salivary molecules will be discussed with an emphasis on their use as biomarkers of exposure to haematophagous arthropod bites or vaccine candidates that are liable to improve host protection against vector-borne diseases.

Abstract: BACKGROUND: Even though the efficacy of Intermittent Preventive Treatment in infants (IPTi) with Sulfadoxine-Pyrimethamine (SP) against clinical disease and the absence of its interaction with routine vaccines of the Expanded Immunization Programme (EPI) have been established, there are still some concerns regarding the addition of IPTi, which may increase the work burden and disrupt the routine EPI services especially in Africa where the target immunization coverage remains to be met. However IPTi may also increase the adherence of the community to EPI services and improve EPI coverage, once the benefice of strategy is perceived. METHODS: To assess the impact of IPTi implementation on the coverage of EPI vaccines, 22 health areas of the district of Kolokani were randomized at a 1:1 ratio to either receive IPTi-SP or to serve as a control. The EPI vaccines coverage was assessed using cross-sectional surveys at baseline in November 2006 and after one year of IPTi pilot-implementation in December 2007. RESULTS: At baseline, the proportion of children of 9-23 months who were completely vaccinated (defined as children who received BGG, 3 doses of DTP/Polio, measles and yellow fever vaccines) was 36.7% (95% CI 25.3% -48.0%). After one year of implementation of IPTi-SP using routine health services, the proportion of children completely vaccinated rose to 53.8% in the non intervention zone and 69.5% in the IPTi intervention zone (P <0.001).The proportion of children in the target age groups who received IPTi with each of the 3 vaccinations DTP2, DTP3 and Measles, were 89.2% (95% CI 85.9%-92.0%), 91.0% (95% CI 87.6% -93.7%) and 77.4% (95% CI 70.7%-83.2%) respectively. The corresponding figures in non intervention zone were 2.3% (95% CI 0.9% -4.7%), 2.6% (95% CI 1.0% -5.6%) and 1.7% (95% CI 0.4% - 4.9%). CONCLUSION: This study shows that high coverage of the IPTi can be obtained when the strategy is implemented using routine health services and implementation results in a significant increase in coverage of EPI vaccines in the district of Kolokani, Mali.

Abstract: Mosquito salivary proteins are involved in several biological processes that facilitate their blood feeding and have also been reported to elicit an IgG response in vertebrates. A growing number of studies have focused on this immunological response for its potential use as a biological marker of exposure to arthropod bites. As mosquito saliva collection is extremely laborious and inefficient, most research groups prefer to work on mosquito salivary glands (SGs). Thus, SG protein integrity is a critical factor in obtaining meaningful data from immunological and biochemical analysis. Current methodologies rely on an immediate freezing of SGs after their collection. However, the maintenance of samples in a frozen environment can be hard to achieve in field conditions. In this study, SG proteins from two mosquito species (Aedes aegypti and Anopheles gambiae s.s.) stored in different media for 5 days at either +4 degrees C or room temperature (RT) were evaluated at the quantitative (i.e., ELISA) and qualitative (i.e., SDS-PAGE and immunoblotting) levels. Our results indicated that PBS medium supplemented with an anti-protease cocktail seems to be the best buffer to preserve SG antigens for 5 days at +4 degrees C for ELISA analysis. Conversely, cell-lysis buffer (Urea-Thiourea-CHAPS-Tris) was best at preventing protein degradation both at +4 degrees C and RT for further qualitative analysis. These convenient storage methods provide an alternative to freezing and are expected to be applicable to other biological samples collected in the field.

Abstract: Mosquito-borne diseases are major health problems worldwide. Serological responses to mosquito saliva proteins may be useful in estimating individual exposure to bites from mosquitoes transmitting these diseases. However, the relationships between the levels of these IgG responses and mosquito density as well as IgG response specificity at the genus and/or species level need to be clarified prior to develop new immunological markers to assess human/vector contact. To this end, a kinetic study of antibody levels against several mosquito salivary gland extracts from southeastern French individuals living in three areas with distinct ecological environments and, by implication, distinct Aedes caspius mosquito densities were compared using ELISA. A positive association was observed between the average levels of IgG responses against Ae. caspius salivary gland extracts and spatial Ae. caspius densities. Additionally, the average level of IgG responses increased significantly during the peak exposure to Ae. caspius at each site and returned to baseline four months later, suggesting short-lived IgG responses. The species-specificity of IgG antibody responses was determined by testing antibody responses to salivary gland extracts from Cx. pipiens, a mosquito that is present at these three sites at different density levels, and from two other Aedes species not present in the study area (Ae. aegypti and Ae. albopictus). The IgG responses observed against these mosquito salivary gland extracts contrasted with those observed against Ae. caspius salivary gland extracts, supporting the existence of species-specific serological responses. By considering different populations and densities of mosquitoes linked to environmental factors, this study shows, for the first time, that specific IgG antibody responses against Ae. caspius salivary gland extracts may be related to the seasonal and geographical variations in Ae. caspius density. Characterisation of such immunological-markers may allow the evaluation of the effectiveness of vector-control strategies or estimation of the risk of vector-borne disease transmission.

Abstract: BACKGROUND: Previous studies in Dakar have highlighted the spatial and temporal heterogeneity of Anopheles gambiae s.l. biting rates. In order to improve the knowledge of the determinants of malaria transmission in this city, the present study reports the results of an extensive entomological survey that was conducted in 45 areas in Dakar from 2007 to 2010. METHODS: Water collections were monitored for the presence of anopheline larvae. Adult mosquitoes were sampled by human landing collection. Plasmodium falciparum circumsporozoite (CSP) protein indexes were measured by ELISA (enzyme-linked immunosorbent assay), and the entomological inoculation rates were calculated. RESULTS: The presence of anopheline larvae were recorded in 1,015 out of 2,683 observations made from 325 water collections. A water pH of equal to or above 8.0, a water temperature that was equal to or above 30 degrees C, the absence of larvivorous fishes, the wet season, the presence of surface vegetation, the persistence of water and location in a slightly urbanised area were significantly associated with the presence of anopheline larvae and/or with a higher density of anopheline larvae. Most of the larval habitats were observed in public areas, i.e., freely accessible. A total of 496,310 adult mosquitoes were caught during 3096 person-nights, and 44967 of these specimens were identified as An.gambiae s.l. The mean An. gambiae s.l. human-biting rate ranged from 0.1 to 248.9 bites per person per night during the rainy season. Anopheles arabiensis (93.14%), Anopheles melas (6.83%) and An. gambiae s.s. M form (0.03%) were the three members of the An. gambiae complex. Fifty-two An. arabiensis and two An. melas specimens were CSP-positive, and the annual CSP index was 0.64% in 2007, 0.09% in 2008-2009 and 0.12% in 2009-2010. In the studied areas, the average EIR ranged from 0 to 17.6 infected bites per person during the entire transmission season. CONCLUSION: The spatial and temporal heterogeneity of An. gambiae s.l. larval density, adult human-biting rate (HBR) and malaria transmission in Dakar has been confirmed, and the environmental factors associated with this heterogeneity have been identified. These results pave the way for the creation of malaria risk maps and for a focused anti-vectorial control strategy.

Abstract: We have analyzed the profiles of 23 of Plasmodium falciparum strains for their in vitro chemosusceptibilities to piperaquine (PPQ), dihydroartemisinin (DHA), chloroquine, monodesethylamodiaquine, quinine, mefloquine, lumefantrine, atovaquone, pyrimethamine, and doxycycline (DOX) in association with polymorphisms in genes involved in quinoline resistance (Plasmodium falciparum crt [pfcrt], pfmdr1, pfmrp, and pfnhe). The 50% inhibitory concentrations (IC(50)s) for PPQ ranged from 29 to 98 nM (geometric mean = 57.8 nM, 95% confidence interval [CI] = 51 to 65) and from 0.4 to 5.8 nM for DHA (geometric mean = 1.8 nM, 95% CI = 1.4 to 2.3). We found a significant positive correlation between the responses to PPQ and DHA (r(2) = 0.17; P = 0.0495) and between the responses to PPQ and DOX (r(2) = 0.41; P = 0.001). We did not find a significant association between the PPQ IC(50) (0.0525 < P < 0.9247) or the DHA IC(50) (0.0138 < P < 0.9018) and polymorphisms in the pfcrt, pfmdr1, pfmrp, and pfnhe-1 genes. There was an absence of cross-resistance with quinolines, and the IC(50)s for PPQ and DHA were found to be unrelated to mutations in the pfcrt, pfmdr1, pfmrp, and pfnhe-1 transport protein genes, which are involved in quinoline antimalarial drug resistance. These results confirm the interest in and the efficacy of the combination of PPQ and DHA for areas in which parasites are resistant to chloroquine or other quinolines.

Abstract: BACKGROUND: The effectiveness of malaria chemoprophylaxis is limited by the lack of compliance whose determinants are not well known. METHODS: The compliance with malaria chemoprophylaxis has been estimated and analysed by validated questionnaires administered before and after the short-term missions (about four months) in five tropical African countries of 2,093 French soldiers from 19 military companies involved in a prospective cohort study. "Correct compliance" was defined as "no missed doses" of daily drug intake during the entire mission and was analysed using multiple mixed-effect logistic regression model. RESULTS: The averaged prevalence rate of correct compliance was 46.2%, ranging from 9.6%to 76.6% according to the companies. Incorrect compliance was significantly associated with eveningness (p = 0.028), a medical history of clinical malaria (p < 0.001) and a perceived mosquito attractiveness inferior or superior to the others (p < 0.007). Correct compliance was significantly associated with the systematic use of protective measures against mosquito bites (p < 0.001), the type of military operations (combat vs. training activities, p < 0.001) and other individual factors (p < 0.05). CONCLUSIONS: The identification of circumstances and profiles of persons at higher risk of lack of compliance would pave the way to specifically targeted strategies aimed to improve compliance with malaria chemoprophylaxis and, therefore, its effectiveness.

Abstract: BACKGROUND: Malaria microscopy and rapid diagnostic tests are insensitive for very low-density parasitaemia. This insensitivity may lead to missed asymptomatic sub-microscopic parasitaemia, a potential reservoir for infection. Similarly, mixed infections and interactions between Plasmodium species may be missed. The objectives were first to develop a rapid and sensitive PCR-based diagnostic method to detect low parasitaemia and mixed infections, and then to investigate the epidemiological importance of sub-microscopic and mixed infections in Rattanakiri Province, Cambodia. METHODS: A new malaria diagnostic method, using restriction fragment length polymorphism analysis of the cytochrome b genes of the four human Plasmodium species and denaturing high performance liquid chromatography, has been developed. The results of this RFLP-dHPLC method have been compared to 1) traditional nested PCR amplification of the 18S rRNA gene, 2) sequencing of the amplified fragments of the cytochrome b gene and 3) microscopy. Blood spots on filter paper and Giemsa-stained blood thick smears collected in 2001 from 1,356 inhabitants of eight villages of Rattanakiri Province have been analysed by the RFLP-dHPLC method and microscopy to assess the prevalence of sub-microscopic and mixed infections. RESULTS: The sensitivity and specificity of the new RFLP-dHPLC was similar to that of the other molecular methods. The RFLP-dHPLC method was more sensitive and specific than microscopy, particularly for detecting low-level parasitaemia and mixed infections. In Rattanakiri Province, the prevalences of Plasmodium falciparum and Plasmodium vivax were approximately two-fold and three-fold higher, respectively, by RFLP-dHPLC (59% and 15%, respectively) than by microscopy (28% and 5%, respectively). In addition, Plasmodium ovale and Plasmodium malariae were never detected by microscopy, while they were detected by RFLP-dHPLC, in 11.2% and 1.3% of the blood samples, respectively. Moreover, the proportion of mixed infections detected by RFLP-dHPLC was higher (23%) than with microscopy (8%). CONCLUSIONS: The rapid and sensitive molecular diagnosis method developed here could be considered for mass screening and ACT treatment of inhabitants of low-endemicity areas of Southeast Asia.

Abstract:

Abstract: BACKGROUND: Doxycycline is used in combination with quinine for malaria treatment or alone for malaria chemoprophylaxis. However, the occurrence of malaria after doxycycline chemoprophylaxis has been reported. Identification of genetic determinants that contribute to the susceptibility of Plasmodium falciparum to doxycycline will be important for the detection and surveillance of doxycycline resistance. METHODS: Sequence analysis of 11 genes (pftufA, pfEF-TS, pfmdt, pftetQ, pfrps3, pfrps7, pfrps8, pfrps9, pfrps11, pfrps14, and pfrps17) and evaluation of pfmdt and pftetQ copy numbers by quantitative real-time polymerase chain reaction were conducted in 90 African P. falciparum isolates that were obtained from 14 countries and that belonged to phenotypic groups differing in their doxycycline median inhibitory concentrations. RESULTS: We found that pfmdt copy number of >1 (adjusted odds ratio [OR], 7.09 [95% confidence interval {CI}, 1.58-31.82]; P=.011), pftetQ copy number of >1 (adjusted OR, 5.23 [95% CI, 1.06-25.77]; P=.042), and KYNNNN amino acid motif repeats of <3 (adjusted OR, 3.00 [95% CI, 1.02-8.86]; P=.046) were independently associated with decreased susceptibility to doxycycline. CONCLUSIONS: Our findings suggest that pfmdt and pftetQ copy numbers and pftetQ sequence polymorphisms are potential molecular markers of decreased in vitro susceptibility to doxycycline in African P. falciparum isolates.

Abstract: BACKGROUND: Intermittent preventive treatment in infants (IPTi) with sulphadoxine-pyrimethamine (SP) given during routine vaccinations is efficacious in preventing malaria disease and shows no interaction with the vaccines. However, there is a fear that IPTi may result in a rapid increase of parasite resistance to SP. METHODS: To evaluate the impact of IPTi on SP-resistance point mutations, the 22 health sub-districts in the district of Kolokani, Mali, were randomized in a 1:1 ratio and starting in December 2006, IPTi with SP was implemented in 11 health sub-districts (intervention zone), while the other 11 health sub-districts served as the control (non-intervention zone). Blood smears and blood dots on filter paper were obtained from children aged 0-5 years, randomly selected in each of heath sub-districts during two cross-sectional surveys. The first survey was conducted in May 2007 before the start of the transmission season to collect baseline prevalence of the molecular markers of resistance to SP and the second in December 2007 after the end of the transmission season and one year after implementation of IPTi. A total of 427 and 923 randomly selected blood samples from the first and second surveys respectively were analysed by PCR for dhfr and dhps mutations. RESULTS: Each of the three dhfr mutations at codons 51, 59 and 108 was present in 35% and 57% of the samples during the two surveys with no significant differences between the two zones. Dhps mutations at codons 437 and 540 were present respectively in about 20% and 1% of the children during the two surveys in both zones at similar proportion. The prevalence of quadruple mutants (triple dhfr-mutants + dhps-437G) associated with in-vivo resistance to SP in Mali after one year implementation of IPTi was also similar between the two zones (11.6% versus 11.2%, p = 0.90) and to those obtained at baseline survey (10.3% versus 8.1%). CONCLUSION: This study shows no increase in the frequency of molecular markers of SP resistance in areas where IPTi with SP was implemented for one year.

Abstract: OBJECTIVES: The aim of the study was to assess the in vitro potentiating effects of atorvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, in combination with mefloquine, chloroquine or monodesethylamodiaquine against Plasmodium falciparum and to evaluate whether the effects of atorvastatin could be associated with mutations or gene copy number in multidrug resistance (MDR)-like protein genes. METHODS: The susceptibilities of 21 parasite strains to combinations of atorvastatin with mefloquine, chloroquine or monodesethylamodiaquine were assessed using the in vitro isotopic microtest. Genotypes and gene copy number were assessed for pfmdr1, pfmdr2 and pfmrp genes. RESULTS: Atorvastatin demonstrated synergistic effects in combination with mefloquine. The mefloquine IC(50) (50% inhibitory concentration) was reduced by 7%, 24% and 37% in the presence of atorvastatin at concentrations of 0.1, 0.5 and 1.0 microM, respectively. The synergistic effect of atorvastatin on the response to mefloquine was significantly associated with pfmdr1 copy number. The concentration of atorvastatin that could reduce the IC(50) of mefloquine by 50% was 2.4 +/- 1.3 microM for the 12 strains that contained one copy of pfmdr1 and 5.8 +/- 2.1 microM for the 9 strains that contained two copies or more. The synergistic effect of atorvastatin in combination with mefloquine was found to be significantly unrelated to mutations in pfmdr1, pfmdr2 or pfmrp genes. CONCLUSIONS: The synergy of the effect of mefloquine at concentrations relevant to its achievable plasma concentrations in patients taking 80 mg of atorvastatin daily suggests that atorvastatin will be a good candidate in combination with mefloquine for malaria treatment.

Abstract: BACKGROUND: The emergence of Plasmodium falciparum resistance to most anti-malarial compounds has highlighted the urgency to develop new drugs and to clarify the mechanisms of anti-malarial drugs currently used. Among them, doxycycline is used alone for malaria chemoprophylaxis or in combination with quinine or artemisinin derivatives for malaria treatment. The molecular mechanisms of doxycycline action in P. falciparum have not yet been clearly defined, particularly at the protein level. METHODS: A proteomic approach was used to analyse protein expression changes in the schizont stage of the malarial parasite P. falciparum following doxycycline treatment. A comparison of protein expression between treated and untreated protein samples was performed using two complementary proteomic approaches: two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and isobaric tagging reagents for relative and absolute quantification (iTRAQ). RESULTS: After doxycycline treatment, 32 and 40 P. falciparum proteins were found to have significantly deregulated expression levels by 2D-DIGE and iTRAQ methods, respectively. Although some of these proteins have been already described as being deregulated by other drug treatments, numerous changes in protein levels seem to be specific to doxycycline treatment, which could perturb apicoplast metabolism. Quantitative reverse transcription polymerase chain reaction (RT-PCR) was performed to confirm this hypothesis. CONCLUSIONS: In this study, a specific response to doxycycline treatment was distinguished and seems to involve mitochondrion and apicoplast organelles. These data provide a starting point for the elucidation of drug targets and the discovery of mechanisms of resistance to anti-malarial compounds.

Abstract: BACKGROUND: Plasmodium falciparum infections could lead to severe malaria, principally in non-immune individuals as children and travellers from countries exempted of malaria. Severe malaria is often associated with the sequestration of P. falciparum-infected erythrocytes in deep micro-vascular beds via interactions between host endothelial receptors and parasite ligands expressed on the surface of the infected erythrocyte. Although, serological responses from individuals living in endemic areas against proteins expressed at surface of the infected erythrocyte have been largely studied, seldom data are available about the specific targets of antibody response from travellers. METHODS: In order to characterize antigens recognized by traveller sera, a comparison of IgG immune response against membrane protein extracts from uninfected and P. falciparum-infected red blood cells (iRBC), using immunoblots, was performed between non exposed individuals (n = 31) and briefly exposed individuals (BEI) (n = 38) to malaria transmission. RESULTS: Immune profile analysis indicated that eight protein bands from iRBC were significantly detected more frequently in the BEI group. Some of these antigenic proteins were identified by an original immuno-proteomic approach. CONCLUSION: Collectively, these data may be useful to characterize the singular serological immune response against a primary malaria infection in individuals briefly exposed to transmission.

Abstract: The increasing usage of long-lasting insecticide-treated nets allows protection of millions of people from malaria infection. Monitoring studies should be planned during any wide-scale malaria control program integrating insecticide-treated materials, to evaluate their effects and effectiveness on epidemiologically relevant parameters. Such operational control interventions may be challenged by insecticide resistance spread within vector populations, as a result of wide insecticide pressure. A nationwide distribution of long-lasting insecticidal nets was implemented throughout Niger in 2005. We studied the population genetic structure of major malaria vectors across Nigerien Sahel, and investigated potential effects of this large malaria control intervention. Wild-caught Anopheles gambiae sensu lato females from seven villages and two wet seasons were genotyped at 12 microsatellite loci. The genetic diversity within both species appeared homogenous between villages and years. The estimated genetic differentiation among samples was very low within both species, indicating high gene flow across the area. An absence of differentiation was also found between 2005 and 2006 wet seasons, for all samples but one, showing that the net distribution did not impact significantly the genetic diversity and structure of vector populations in a single year. We provide valuable results participating to document effects of large malaria control programs, to maximize the efficiency of available tools in future interventions.

Abstract: BACKGROUND: In 2006, the Senegalese National Malaria Control Programme (NMCP) has recommended artemisinin-based combination therapy (ACT) as the first-line treatment for uncomplicated malaria and, in 2007, mandated testing for all suspected cases of malaria with a Plasmodium falciparum HRP-2-based rapid diagnostic test for malaria (RDT(Paracheck). Given the higher cost of ACT compared to earlier anti-malarials, the objectives of the present study were i) to study the accuracy of Paracheck compared to the thick blood smear (TBS) in two areas with different levels of malaria endemicity and ii) analyse the cost-effectiveness of the strategy of the parasitological confirmation of clinically suspected malaria cases management recommended by the NMCP. METHODS: A cross-sectional study was undertaken in the villages of Dielmo and Ndiop (Senegal) nested in a cohort study of about 800 inhabitants. For all the individuals consulting between October 2008 and January 2009 with a clinical diagnosis of malaria, a questionnaire was filled and finger-prick blood samples were taken both for microscopic examination and RDT. The estimated costs and cost-effectiveness analysis were made considering five scenarios, the recommendations of the NMCP being the reference scenario. In addition, a sensitivity analysis was performed assuming that all the RDT-positive patients and 50% of RDT-negative patients were treated with ACT. RESULTS: A total of 189 consultations for clinically suspected malaria occurred during the study period. The sensitivity, specificity, positive and negative predictive values were respectively 100%, 98.3%, 80.0% and 100%. The estimated cost of the reference scenario was close to 700 euros per 1000 episodes of illness, approximately twice as expensive as most of the other scenarios. Nevertheless, it appeared to us cost-effective while ensuring the diagnosis and the treatment of 100% of malaria attacks and an adequate management of 98.4% of episodes of illness. The present study also demonstrated that full compliance of health care providers with RDT results was required in order to avoid severe incremental costs. CONCLUSIONS: A rational use of ACT requires laboratory testing of all patients presenting with presumed malaria. Use of RDTs inevitably has incremental costs, but the strategy associating RDT use for all clinically suspected malaria and prescribing ACT only to patients tested positive is cost-effective in areas where microscopy is unavailable.

Abstract: Diseases caused by arthropod-borne viruses are a significant threat to the health of human and animal populations throughout the world. Better knowledge of the molecules synthesized in the salivary gland and saliva of hematophagous arthropods could be of use for improving the control of pathogen transmission. Recently, a sialome analysis of three Aedes aegypti mosquito colonies (PAEA, Rockefeller, and Formosus) carried out in our laboratory allowed us to identify 44 saliva proteins. Of these secreted proteins, none was exclusively expressed in one colony, suggesting that expression of salivary proteins is highly conserved across populations. In another study, we reported that some of these salivary proteins could be used as the genus-specific markers for travelers' exposure to mosquito vectors. Here, comparison of salivary gland protein profiles between these same three Ae. aegypti colonies was performed using the one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) difference gel electrophoresis method. As observed at the saliva level, no significant differences were detected between these three colonies. The salivary gland protein repertoire from the Ae. aegypti mosquito was analyzed using a proteomic approach. One hundred and twenty proteins were identified in these salivary glands representing the largest description of the Ae. aegypti salivary gland protein catalog. We succeeded in identifying 15 secreted proteins, some of which have already been reported as being involved in blood feeding. A comparison of the proteins identified between the salivary glands and the sialome is discussed.

Abstract: BACKGROUND: Buruli ulcer, the third mycobacterial disease after tuberculosis and leprosy, is caused by the environmental mycobacterium M. ulcerans. Various modes of transmission have been suspected for this disease, with no general consensus acceptance for any of them up to now. Since laboratory models demonstrated the ability of water bugs to transmit M. ulcerans, a particular attention is focused on the transmission of the bacilli by water bugs as hosts and vectors. However, it is only through detailed knowledge of the biodiversity and ecology of water bugs that the importance of this mode of transmission can be fully assessed. It is the objective of the work here to decipher the role of water bugs in M. ulcerans ecology and transmission, based on large-scale field studies. METHODOLOGY/PRINCIPAL FINDINGS: The distribution of M. ulcerans-hosting water bugs was monitored on previously unprecedented time and space scales: a total of 7,407 water bugs, belonging to large number of different families, were collected over one year, in Buruli ulcer endemic and non endemic areas in central Cameroon. This study demonstrated the presence of M. ulcerans in insect saliva. In addition, the field results provided a full picture of the ecology of transmission in terms of biodiversity and detailed specification of seasonal and regional dynamics, with large temporal heterogeneity in the insect tissue colonization rate and detection of M. ulcerans only in water bug tissues collected in Buruli ulcer endemic areas. CONCLUSION/SIGNIFICANCE: The large-scale detection of bacilli in saliva of biting water bugs gives enhanced weight to their role in M. ulcerans transmission. On practical grounds, beyond the ecological interest, the results concerning seasonal and regional dynamics can provide an efficient tool in the hands of sanitary authorities to monitor environmental risks associated with Buruli ulcer.

Abstract: ABSTRACT: BACKGROUND: The United Nations forecasts that by 2050, more than 60% of the African population will live in cities. Thus, urban malaria is considered an important emerging health problem in that continent. Remote sensing (RS) and geographic information systems (GIS) are useful tools for addressing the challenge of assessing, understanding and spatially focusing malaria control activities. The objectives of the present study were to use high spatial resolution SPOT (Satellite Pour l'Observation de la Terre) satellite images to identify some urban environmental factors in Dakar associated with Anopheles arabiensis densities, to assess the persistence of these associations and to describe spatial changes in at-risk environments using a decadal time scale. METHODS: Two SPOT images from the 1996 and 2007 rainy seasons in Dakar were processed to extract environmental factors, using supervised classification of land use and land cover, and a calculation of NDVI (Normalized Difference Vegetation Index) and distance to vegetation. Linear regressions were fitted to identify the ecological factors associated with An. arabiensis aggressiveness measured in 1994-97 in the South and centre districts of Dakar. Risk maps for populated areas were computed and compared for 1996 and 2007 using the results of the statistical models. RESULTS: Almost 60% of the variability in anopheline aggressiveness measured in 1994-97 was explained with only one variable: the built-up area in a 300-m radius buffer around the catching points. This association remained stable between 1996 and 2007. Risk maps were drawn by inverting the statistical association. The total increase of the built-up areas in Dakar was about 30% between 1996 and 2007. In proportion to the total population of the city, the population at high risk for malaria fell from 32% to 20%, whereas the low-risk population rose from 29 to 41%. CONCLUSIONS: Environmental data retrieved from high spatial resolution SPOT satellite images were associated with An. arabiensis densities in Dakar urban setting, which allowed to generate malaria transmission risk maps. The evolution of the risk was quantified, and the results indicated there are benefits of urbanization in Dakar, since the proportion of the low risk population increased while urbanization progressed.

Abstract: ABSTRACT: BACKGROUND: Assessment exposure and immunity to malaria is an important step in the fight against the disease. Increased malaria infection in non-immune travellers under anti-malarial chemoprophylaxis, as well as the implementation of malaria elimination programmes in endemic countries, raises new issues that pertain to these processes. Notably, monitoring malaria immunity has become more difficult in individuals showing low antibody (Ab) responses or taking medications against the Plasmodium falciparum blood stages. Commonly available techniques in malaria seroepidemiology have limited sensitivity, both against pre-erythrocytic, as against blood stages of the parasite. Thus, the aim of this study was to develop a sensitive tool to assess the exposure to malaria or to bites from the vector Anopheles gambiae, despite anti-malarial prophylactic treatment. METHODS: Ab responses to 13 pre-erythrocytic P. falciparum-specific peptides derived from the proteins Lsa1, Lsa3, Glurp, Salsa, Trap, Starp, CSP and Pf11.1, and to 2 peptides specific for the Anopheles gambiae saliva protein gSG6 were tested. In this study, 253 individuals from three Senegalese areas with different transmission intensities and 124 European travellers exposed to malaria during a short period of time were included. RESULTS: The multiplex assay was optimized for most but not all of the antigens. It was rapid, reproducible and required a small volume of serum. Proportions of Ab-positive individuals, Ab levels and the mean number of antigens (Ags) recognized by each individual increased significantly with increases in the level of malaria exposure. CONCLUSION: The multiplex assay developed here provides a useful tool to evaluate immune responses to multiple Ags in large populations, even when only small amounts of serum are available, or Ab titres are low, as in case of travellers. Finally, the relationship of Ab responses with malaria endemicity levels provides a way to monitor exposure in differentially exposed autochthonous individuals from various endemicity areas, as well as in travellers who are not immune, thus indirectly assessing the parasite transmission and malaria risk in the new eradication era.

Abstract: BACKGROUND: Studies on human genetic factors associated with malaria have hitherto concentrated on their role in susceptibility to and protection from disease. In contrast, virtually no attention has been paid to the role of human genetics in eliciting the production of parasite transmission stages, the gametocytes, and thus enhancing the spread of disease. METHODS AND FINDINGS: We analysed four longitudinal family-based cohort studies from Senegal and Thailand followed for 2-8 years and evaluated the relative impact of the human genetic and non-genetic factors on gametocyte production in infections of Plasmodium falciparum or P. vivax. Prevalence and density of gametocyte carriage were evaluated in asymptomatic and symptomatic infections by examination of Giemsa-stained blood smears and/or RT-PCR (for falciparum in one site). A significant human genetic contribution was found to be associated with gametocyte prevalence in asymptomatic P. falciparum infections. By contrast, there was no heritability associated with the production of gametocytes for P. falciparum or P. vivax symptomatic infections. Sickle cell mutation, HbS, was associated with increased gametocyte prevalence but its contribution was small. CONCLUSIONS: The existence of a significant human genetic contribution to gametocyte prevalence in asymptomatic infections suggests that candidate gene and genome wide association approaches may be usefully applied to explore the underlying human genetics. Prospective epidemiological studies will provide an opportunity to generate novel and perhaps more epidemiologically pertinent gametocyte data with which similar analyses can be performed and the role of human genetics in parasite transmission ascertained.

Abstract: ABSTRACT: BACKGROUND: Urban malaria is a major health priority for civilian and militaries populations. A preliminary entomologic study has been conducted in 2006-2007, in the French military camps of the two mains towns of Gabon: Libreville and Port-Gentil. The aim was to assess the malaria transmission risk for troops. METHODS: Mosquitoes sampled by human landing collection were identified morphologically and by molecular methods. The Plasmodium falciparum circumsporozoite (CSP) indexes were measured by ELISA, and the entomological inoculation rates (EIR) were calculated for both areas. Molecular assessments of pyrethroid knock down (kdr) resistance and of insensitive acetylcholinesterase resistance were conducted. RESULTS: In Libreville, Anopheles gambiae s.s. S form was the only specie of the An. gambiae complex present and was responsible of 9.4 bites per person per night. The circumsporozoite index was 0.15% and the entomological inoculation rate estimated to be 1.23 infective bites during the four months period. In Port-Gentil, Anopheles melas (75.5% of catches) and An. gambiae s.s. S form (24.5%) were responsible of 58.7 bites per person per night. The CSP indexes were of 1.67% for An. gambiae s.s and 0.28% for An. melas and the EIRs were respectively of 1.8 infective bites per week and of 0.8 infective bites per week. Both kdr-w and kdr-e mutations in An. gambiae S form were found in Libreville and in Port-Gentil. Insensitive acetylcholinesterase has been detected for the first time in Gabon in Libreville. CONCLUSION: Malaria transmission exists in both town, but with high difference in the level of risk. The co-occurrence of molecular resistances to the main families of insecticide has implications for the effectiveness of the current vector control programmes that are based on pyrethroid-impregnated bed nets.

Abstract: BACKGROUND: Quinine (QN) remains the first line anti-malarial drug for the treatment of complicated malaria in Europe and Africa. The emergence of QN resistance has been documented. QN resistance is not yet a significant problem, but there is an urgent need to discover partners for use in combination with QN. The aim of the study was to assess the in vitro potentiating effects of atorvastatin (AVA), a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, in combination with QN against Plasmodium falciparum and to evaluate whether the effects of AVA could be associated with gene copy number or mutations in genes involved in QN resistance, such as pfcrt, pfmdr1, pfmrp and pfnhe. METHODS: The susceptibilities to combination of AVA with QN were assessed against 21 parasite strains using the in vitro isotopic microtest. Genotypes and gene copy number were assessed for pfcrt, pfmdr1, pfmdr2, pfmrp genes. In addition, the number of DNNND, DDNHNDNHNN repeats in pfnhe-1 ms4760 and the ms4760 profile were determined for each strains of P. falciparum. RESULTS: AVA demonstrated synergistic effects in combination with QN against 21 P. falciparum strains. The QN IC50 was reduced by 5% (0% to 15%; 95%CI: 1%-8%), 10% (3% to 23%; 95%CI: 7%-14%) and 22% (14% to 40%; 95%CI: 19%-25%) in presence of AVA at concentrations of 0.1, 0.5 and 1.0 microM, respectively. These reductions were all significant (p < 0.009). The reduction in the QN IC50 in presence of AVA was not significantly correlated with the QN IC50 (r = 0.22, P = 0.3288) or the AVA IC50 (r = 0.03, P = 0.8946). The synergistic effect of AVA in combination with QN was not significantly associated with polymorphisms in the pfcrt, pfmdr1, pfmrp, and pfnhe-1 genes that could be involved in QN resistance. The synergistic effect of AVA on QN responses was not significantly associated with pfmdr1 copy number (P = 0.0428). CONCLUSION: The synergistic effect of AVA in combination with QN was found to be unrelated to mutations occurring in transport protein genes involved in QN drug resistance. The different mechanisms of drug uptake and/or mode of action for AVA compared to the other anti-malarial drugs, as well as the AVA-mediated synergy of the anti-malarial effect of QN, suggests that AVA will be a good candidate for combinatorial malaria treatment. All of these observations support calls for both an in vivo evaluation with pharmacokinetic component and clinical trials of AVA as an anti-malarial therapy.

Abstract: We describe clinical and parasitologic features of in vivo and in vitro Plasmodium falciparum resistance to quinine in a nonimmune traveler who returned to France from Senegal in 2007 with severe imported malaria. Clinical quinine failure was associated with a 50% inhibitory concentration of 829 nmol/L. Increased vigilance is required during treatment follow-up.

Abstract: ABSTRACT: BACKGROUND: The aim of the present work was to assess the in vitro cross-resistance of pyronaridine with other quinoline drugs, artesunate and several other commonly used anti-malarials and to evaluate whether decreased susceptibility to pyronaridine could be associated with genetic polymorphisms in genes involved in reduced quinoline susceptibility, such as pfcrt, pfmdr1, pfmrp and pfnhe. METHODS: The in vitro chemosusceptibility profiles of 23 strains of Plasmodium falciparum were analysed by the standard 42-hour 3H-hypoxanthine uptake inhibition method for pyronaridine, artesunate, chloroquine, monodesethylamodiaquine, quinine, mefloquine, lumefantrine, atovaquone, pyrimethamine and doxycycline. Genotypes were assessed for pfcrt, pfmdr1, pfnhe-1 and pfmrp genes. RESULTS: The IC50 values for pyronaridine ranged from 15 to 49 nM (geometric mean = 23.1 nM). A significant positive correlation was found between responses to pyronaridine and responses to artesunate (r2 = 0.20; P = 0.0317) but too low to suggest cross-resistance. No significant correlation was found between pyronaridine IC50 and responses to other anti-malarials. Significant associations were not found between pyronaridine IC50 and polymorphisms in pfcrt, pfmdr1, pfmrp or pfnhe-1. CONCLUSION: There was an absence of cross-resistance between pyronaridine and quinolines, and the IC50 values for pyronaridine were found to be unrelated to mutations in the transport protein genes pfcrt, pfmdr1, pfmrp or pfnhe-1, known to be involved in quinoline resistance. These results confirm the interest and the efficacy of the use of a combination of pyronaridine and artesunate in areas in which parasites are resistant to quinolines.

Abstract: ABSTRACT: BACKGROUND: Mixing repellent and organophosphate (OP) insecticides to better control pyrethroid resistant mosquito vectors is a promising strategy developed for bed net impregnation. Here, we investigated the opportunity to adapt this strategy to personal protection in the form of impregnated clothes. METHODS: We compared standard permethrin impregnated uniforms with uniforms manually impregnated with the repellent KBR3023 alone and in combination with an organophosphate, Pirimiphos-Methyl (PM). Tests were carried out with Aedes aegypti, the dengue fever vector, at dusk in experimental huts. RESULTS: Results showed that the personal protection provided by repellent KBR3023-impregnated uniforms is equal to permethrin treated uniforms and that KBR3023/PM-impregnated uniforms are more protective. CONCLUSION: The use of repellents alone or combined with OP on clothes could be promising for personal protection of military troops and travellers if residual activity of the repellents is extended and safety is verified.

Abstract: The efficacy of malaria control and elimination on islands may depend on the intensity of new parasite inflow. On the Comoros archipelago, where falciparum malaria remains a major public health problem because of spread of drug resistance and insufficient malaria control, recent interventions for malaria elimination were planned on Moheli, 1 of 4 islands in the Comoros archipelago. To assess the relevance of such a local strategy, we performed a population genetics analysis by using multilocus microsatellite and resistance genotyping of Plasmodium falciparum sampled from each island of the archipelago. We found a contrasted population genetic structure explained by geographic isolation, human migration, malaria transmission, and drug selective pressure. Our findings suggest that malaria elimination interventions should be implemented simultaneously on the entire archipelago rather than restricted to 1 island and demonstrate the necessity for specific chemoresistance surveillance on each of the 4 Comorian islands.

Abstract: BACKGROUND: Variant surface antigens (VSA) expressed on the surface of Plasmodium falciparum-infected red blood cells constitute a key for parasite sequestration and immune evasion. In distinct malaria pathologies, such as placental malaria, specific antibody response against VSA provides protection. This study investigated the antibody response specifically directed against VSA expressed by parasites isolated from individuals presenting a given type of clinical presentation. METHODS: Plasma and isolates were obtained from four groups of Beninese subjects: healthy adults, patients presenting uncomplicated malaria (UM), cerebral malaria (CM), or pregnancy-associated malaria (PAM). The reactivity of plasma samples from each clinical group was measured by flow cytometry against parasites isolated from individuals from each clinical group. RESULTS: Antibody responses against VSAUM were predominant in CM, UM and HA plasmas. When analysed according to age in all plasma groups, anti-VSACM and -VSAUM antibody levels were similar until six years of age. In older groups (6-18 and >19 years of age), VSAUM antibody levels were higher than VSACM antibody levels (P = .01, P = .0008, respectively). Mean MFI values, measured in all plasmas groups except the PAM plasmas, remained low for anti-VSAPAM antibodies and did not vary with age. One month after infection the level of anti-VSA antibodies able to recognize heterologous VSACM variants was increased in CM patients. In UM patients, antibody levels directed against heterologous VSAUM were similar, both during the infection and one month later. CONCLUSIONS: In conclusion, this study suggests the existence of serologically distinct VSACM and VSAUM. CM isolates were shown to share common epitopes. Specific antibody response to VSAUM was predominant, suggesting a relative low diversity of VSAUM in the study area.

Abstract: Atorvastatin (AVA) is a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor. AVA exposure resulted in the reduced in vitro growth of 22 Plasmodium falciparum strains, with the 50% inhibitory concentrations (IC(50)s) ranging from 2.5 microM to 10.8 microM. A significant positive correlation was found between the strains' responses to AVA and mefloquine (r = 0.553; P = 0.008). We found no correlation between the responses to AVA and to chloroquine, quinine, monodesethylamodiaquine, lumefantrine, dihydroartemisinin, atovaquone, or doxycycline. These data could suggest that the mechanism of AVA uptake and/or the mode of action of AVA is different from those for other antimalarial drugs. The IC(50)s for AVA were unrelated to the occurrence of mutations in the transport protein genes involved in quinoline antimalarial drug resistance, such as the P. falciparum crt, mdr1, mrp, and nhe-1 genes. Therefore, AVA can be ruled out as a substrate for the transport proteins (CRT, Pgh1, and MRP) and is not subject to the pH modification induced by the P. falciparum NHE-1 protein. The absence of in vitro cross-resistance between AVA and chloroquine, quinine, mefloquine, monodesethylamodiaquine, lumefantrine, dihydroartemisinin, atovaquone, and doxycycline argues that these antimalarial drugs could potentially be paired with AVA as a treatment for malaria. In conclusion, the present observations suggest that AVA is a good candidate for further studies on the use of statins in association with drugs known to have activities against the malaria parasite.

Abstract: Flaviviruses are positive-stranded RNA viruses that are a public health problem because of their widespread distribution and their ability to cause a variety of diseases in humans. West Nile virus is a mosquito-borne member of this genus and is the etiologic agent of West Nile encephalitis. Clinical manifestations of West Nile virus infection are diverse, and their pathogenic mechanisms depend on complex virus-cell interactions. In the present work, we used proteomics technology to analyze early Vero cell response to West Nile infection. The differential proteomes were resolved 24 h postinfection using two-dimensional DIGE followed by mass spectrometry identification. Quantitative analysis (at least 2-fold quantitative alteration, p < 0.05) revealed 127 differentially expressed proteins with 68 up-regulated proteins and 59 down-regulated proteins of which 93 were successfully identified. The implication for mammalian cellular responses to this neurotropic flavivirus infection was analyzed and made possible more comprehensive characterization of the virus-host interactions involved in pathogenesis. The present study thus provides large scale protein-related information that should be useful for understanding how the host metabolism is modified by West Nile infection and for identifying new potential targets for antiviral therapy.

Abstract: Atorvastatin (AVA) is a synthetic inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA). ...

Abstract: A series of acridine derivatives were synthesised and their in vitro antimalarial activity was evaluated against one chloroquine-susceptible strain (3D7) and three chloroquine-resistant strains (W2, Bre1 and FCR3) of Plasmodium falciparum. Structure-activity relationship showed that two positives charges as well as 6-chloro and 2-methoxy substituents on the acridine ring were required to exert a good antimalarial activity. The best compounds possessing these features inhibited the growth of the chloroquine-susceptible strain with an IC(50)0.07 microM, close to that of chloroquine itself, and that of the three chloroquine-resistant strains better than chloroquine with IC(50)0.3 microM. These acridine derivatives inhibited the formation of beta-hematin, suggesting that, like CQ, they act on the haem crystallization process. Finally, in vitro cytotoxicity was also evaluated upon human KB cells, which showed that one of them 9-(6-ammonioethylamino)-6-chloro-2-methoxyacridinium dichloride 1 displayed a promising antimalarial activity in vitro with a quite good selectivity index versus mammalian cell on the CQ-susceptible strain and promising selectivity on other strains.

Abstract: BACKGROUND: Genetic evidence for diversifying selection identified the Merozoite Surface Protein1 block2 (PfMSP1 block2) as a putative target of protective immunity against Plasmodium falciparum. The locus displays three family types and one recombinant type, each with multiple allelic forms differing by single nucleotide polymorphism as well as sequence, copy number and arrangement variation of three amino acid repeats. The family-specific antibody responses observed in endemic settings support immune selection operating at the family level. However, the factors contributing to the large intra-family allelic diversity remain unclear. To address this question, population allelic polymorphism and sequence variant-specific antibody responses were studied in a single Senegalese rural community where malaria transmission is intense and perennial. RESULTS: Family distribution showed no significant temporal fluctuation over the 10 y period surveyed. Sequencing of 358 PCR fragments identified 126 distinct alleles, including numerous novel alleles in each family and multiple novel alleles of recombinant types. The parasite population consisted in a large number of low frequency alleles, alongside one high-frequency and three intermediate frequency alleles. Population diversity tests supported positive selection at the family level, but showed no significant departure from neutrality when considering intra-family allelic sequence diversity and all families combined. Seroprevalence, analysed using biotinylated peptides displaying numerous sequence variants, was moderate and increased with age. Reactivity profiles were individual-specific, mapped to the family-specific flanking regions and to repeat sequences shared by numerous allelic forms within a family type. Seroreactivity to K1-, Mad20- and R033 families correlated with the relative family genotype distribution within the village. Antibody specificity remained unchanged with cumulated exposure to an increasingly large number of alleles. CONCLUSION: The Pfmsp1 block2 locus presents a very large population sequence diversity. The lack of stable acquisition of novel antibody specificities despite exposure to novel allelic forms is reminiscent of clonal imprinting. The locus appears under antibody-mediated diversifying selection in a variable environment that maintains a balance between the various family types without selecting for sequence variant allelic forms. There is no evidence of positive selection for intra-family sequence diversity, consistent with the observed characteristics of the antibody response.

Abstract: Abstract Diseases caused by arthropod-borne viruses are a significant threat to the health of human and animal populations throughout the world. Better knowledge of the molecules synthesized in the salivary gland and saliva of hematophagous arthropods could be of use for improving the control of pathogen transmission. Recently, a sialome analysis of three Aedes aegypti mosquito colonies (PAEA, Rockefeller, and Formosus) carried out in our laboratory allowed us to identify 44 saliva proteins. Of these secreted proteins, none was exclusively expressed in one colony, suggesting that expression of salivary proteins is highly conserved across populations. In another study, we reported that some of these salivary proteins could be used as the genus-specific markers for travelers' exposure to mosquito vectors. Here, comparison of salivary gland protein profiles between these same three Ae. aegypti colonies was performed using the one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) difference gel electrophoresis method. As observed at the saliva level, no significant differences were detected between these three colonies. The salivary gland protein repertoire from the Ae. aegypti mosquito was analyzed using a proteomic approach. One hundred and twenty proteins were identified in these salivary glands representing the largest description of the Ae. aegypti salivary gland protein catalog. We succeeded in identifying 15 secreted proteins, some of which have already been reported as being involved in blood feeding. A comparison of the proteins identified between the salivary glands and the sialome is discussed.

Abstract: BACKGROUND: Several strategies are currently deployed in many countries in the tropics to strengthen malaria control toward malaria elimination. To measure the impact of any intervention, there is a need to detect malaria properly. Mostly, decisions still rely on microscopy diagnosis. But sensitive diagnosis tools enabling to deal with a large number of samples are needed. The molecular detection approach offers a much higher sensitivity, and the flexibility to be automated and upgraded. METHODS: Two new molecular methods were developed: dot18S, a Plasmodium-specific nested PCR based on the 18S rRNA gene followed by dot-blot detection of species by using species-specific probes and CYTB, a Plasmodium-specific nested PCR based on cytochrome b gene followed by species detection using SNP analysis. The results were compared to those obtained with microscopic examination and the "standard" 18S rRNA gene based nested PCR using species specific primers. 337 samples were diagnosed. RESULTS: Compared to the microscopy the three molecular methods were more sensitive, greatly increasing the estimated prevalence of Plasmodium infection, including P. malariae and P. ovale. A high rate of mixed infections was uncovered with about one third of the villagers infected with more than one malaria parasite species. Dot18S and CYTB sensitivity outranged the "standard" nested PCR method, CYTB being the most sensitive. As a consequence, compared to the "standard" nested PCR method for the detection of Plasmodium spp., the sensitivity of dot18S and CYTB was respectively 95.3% and 97.3%. Consistent detection of Plasmodium spp. by the three molecular methods was obtained for 83% of tested isolates. Contradictory results were mostly related to detection of Plasmodium malariae and Plasmodium ovale in mixed infections, due to an "all-or-none" detection effect at low-level parasitaemia. CONCLUSION: A large reservoir of asymptomatic infections was uncovered using the molecular methods. Dot18S and CYTB, the new methods reported herein are highly sensitive, allow parasite DNA extraction as well as genus- and species-specific diagnosis of several hundreds of samples, and are amenable to high-throughput scaling up for larger sample sizes. Such methods provide novel information on malaria prevalence and epidemiology and are suited for active malaria detection. The usefulness of such sensitive malaria diagnosis tools, especially in low endemic areas where eradication plans are now on-going, is discussed in this paper.

Abstract:

Abstract: For decades malarial control has been implemented to control the impact of the disease on the health of populations living in endemic zones. The use of artemisinine combination therapy, intermittent preventive treatment for children and pregnant women, vector-control methods such as long-lasting insecticide-impregnated mosquito nets and indoor remanent insecticide spraying has proven to be effective. These practices have lead to such an extensive reduction of the malaria burden in some endemic areas that the objective of eradication that was unimaginable a few years ago is now back to the forefront. Regardless of the method chosen, careful evaluation and surveillance of its effectiveness in man is necessary. Achieving epidemiologic impact is the main goal of malaria control methods. The main measures for evaluation involve parasitological and clinical aspects of human malaria. The purpose of this article is to review methods used for epidemiologic evaluation of malaria burden.

Abstract: Most new vector control methods against malaria involve the use of pesticides. Prior to release of these products for general use, their efficacy, persistence, and cross-resistance must be tested on mosquito colonies raised in the laboratory (phase I) then on wild mosquitoes in the field (small-scale), individual dwellings, or experimental huts (phase II). The goal of phase III studies is to evaluate the efficacy and effectiveness of the vector-control product or method against malaria in a population at regular risk for transmission. The main objective of phase III tests is to measure the epidemiologic impact, e.g. on the incidence or prevalence of malaria in humans. This article presents guidelines for carrying out phase III tests of vector-control methods against malaria (e.g. home insecticide spraying or insecticide-impregnated bednet use). It was written by participants in a workgroup formed to define recommendations for the WHOPES (WHO Pesticide Evaluation Scheme).

Abstract: BACKGROUND: Urbanization has a great impact on the composition of the vector system and malaria transmission dynamics. In Dakar, some malaria cases are autochthonous but parasite rates and incidences of clinical malaria attacks have been recorded at low levels. Ecological heterogeneity of malaria transmission was investigated in Dakar, in order to characterize the Anopheles breeding sites in the city and to study the dynamics of larval density and adult aggressiveness in ten characteristically different urban areas. METHODS: Ten study areas were sampled in Dakar and Pikine. Mosquitoes were collected by human landing collection during four nights in each area (120 person-nights). The Plasmodium falciparum circumsporozoite (CSP) index was measured by ELISA and the entomological inoculation rates (EIR) were calculated. Open water collections in the study areas were monitored weekly for physico-chemical characterization and the presence of anopheline larvae. Adult mosquitoes and hatched larvae were identified morphologically and by molecular methods. RESULTS: In September-October 2007, 19,451 adult mosquitoes were caught among which, 1,101 were Anopheles gambiae s.l. The Human Biting Rate ranged from 0.1 bites per person per night in Yoff Village to 43.7 in Almadies. Seven out of 1,101 An. gambiae s.l. were found to be positive for P. falciparum (CSP index = 0.64%). EIR ranged from 0 infected bites per person per year in Yoff Village to 16.8 in Almadies. The An. gambiae complex population was composed of Anopheles arabiensis (94.8%) and Anopheles melas (5.2%). None of the An. melas were infected with P. falciparum. Of the 54 water collection sites monitored, 33 (61.1%) served as anopheline breeding sites on at least one observation. No An. melas was identified among the larval samples. Some physico-chemical characteristics of water bodies were associated with the presence/absence of anopheline larvae and with larval density. A very close parallel between larval and adult densities was found in six of the ten study areas. CONCLUSION: The results provide evidence of malaria transmission in downtown Dakar and its surrounding suburbs. Spatial heterogeneity of human biting rates was very marked and malaria transmission was highly focal. In Dakar, mean figures for transmission would not provide a comprehensive picture of the entomological situation; risk evaluation should therefore be undertaken on a small scale.

Abstract: Despite national and international efforts, malaria remains a major public health problem and the fight to control the disease is confronted by numerous hurdles. Study of space and time dynamics of malaria is necessary as a basis for making appropriate decision and prioritizing intervention including in areas where field data are rare and sanitary information systems are inadequate. Evaluation of malarial risk should also help anticipate the risk of epidemics as a basis for early warning systems. Since 1960-70 civilian satellites launched for earth observation have been providing information for the measuring or evaluating geo-climatic and anthropogenic factors related to malaria transmission and burden. Remotely sensed data gathered for several civilian or military studies have allowed setup of entomological, parasitological, and epidemiological risk models and maps for rural and urban areas. Mapping of human populations at risk has also benefited from remotely sensing. The results of the published studies show that remote sensing is a suitable tool for optimizing planning, efficacy and efficiency of malaria control.

Abstract: Identification of the anopheline mosquito species involved in local transmission as well as knowledge of its biology and behavior is necessary for malaria vector control. To allow such study, two methods are usually used to capture adult mosquitoes, i.e., night catches on human volunteers and light-trap collections with human bait. The purpose of this article is to describe these two methods including their advantages and disadvantages as well as a method of surveying breeding sites as implemented by French Army personnel.

Abstract: BACKGROUND: A number of molecular tools have been developed to monitor the emergence and spread of anti-malarial drug resistance to Plasmodium falciparum. One of the major obstacles to the wider implementation of these tools is the absence of practical methods enabling high throughput analysis. Here a new Zip-code array is described, called FlexiChip, linked to a dedicated software program, which largely overcomes this problem. METHODS: Previously published microarray probes detecting single-nucleotide polymorphisms (SNP) associated with parasite resistance to anti-malarial drugs (ResMalChip) were adapted for a universal microarray FlexiChip format. To evaluate the overall sensitivity of the FlexiChip package (microarray + software), the results of FlexiChip were compared to ResMalChip microarray, using the same extension probes and with the same PCR products. In both cases, sequence results were used as gold standard to calculate sensitivity and specificity. FlexiChip results obtained with a set of field isolates were then compared to those assessed in an independent reference laboratory. RESULTS: The FlexiChip package gave results identical to the ResMalChip results in 92.7% of samples (kappa coefficient 0.8491, with a standard error 0.021) and had a sensitivity of 95.88% and a specificity of 97.68% compared to the sequencing as the reference method. Moreover the method performed well compared to the results obtained in the reference laboratories, with 99.7% of identical results (kappa coefficient 0.9923, S.E. 0.0523). CONCLUSION: Microarrays could be employed to monitor P. falciparum drug resistance markers with greater cost effectiveness and the possibility for high throughput analysis. The FlexiChip package is a promising tool for use in poor resource settings of malaria endemic countries.

Abstract: Vector-control measures are a component of integrated malaria control strategies. After evaluation in phase III pilot studies, these measures are currently being deployed in many endemic malaria zones. Their effectiveness must be evaluated under actual conditions of use but it is not ethically acceptable to use unexposed individuals for control groups. In a attempt to overcome this problem, a case-control study was undertaken to evaluate the effectiveness of long-lasting insecticide treated mosquito nets (LLITN) against clinical malaria attacks due to Plasmodium falciparum in an endemic area of southern Benin. During a 4-month period (July to October 2008), 35 clinically documented cases of uncomplicated malaria (fever + parasite density > 3000/microL) were diagnosed in children less than 5 years old from 6 villages in the Tori Bossito medical district. The parents of these children were interviewed at the same time as the parents of 181 children randomly selected from the same 6 villages. A total of 115 of the randomly selected children who had not been feverish during study period were used as controls. The proportion of children having consistently slept under LLITN throughout the study period was 46% in the case group and 78% in the control group (OR=0.32, 95%CI: 0.15-0.71). These data show that the LLITN provided a significant level of protection, i.e., 68% (IC95%: 29%-85%). This case-control study shows that vector control measures can be effectively evaluated after deployment in population. The limitations of this methodology are discussed.

Abstract: Aedes aegypti is responsible for the transmission of arboviruses. The Yellow Fever, Dengue and Chikungunya viruses are transmitted to the vertebrate host by injection of infected saliva during the blood meal of its vectors. Saliva contains different components with various biochemical activities; anti-hemostatic, angiogenic, inflammatory, and immunomodulatory. This work compares the sialomes of three Ae. aegypti colonies (Rockefeller, PAEA, and Formosus), where the repertoire of salivary proteins from these colonies was analyzed by a proteomic approach. This study indicated that major proteins were detectable in the three colonies. However, differences in the abundance of some saliva proteins have been observed between the three Ae. aegypti colonies.

Abstract: BACKGROUND: Despite low endemicity, malaria remains a major health problem in urban areas where a high proportion of fevers are presumptively treated using anti-malarial drugs. Low acquired malaria immunity, behaviour of city-dwellers, access to health care and preventive interventions, and heterogenic suitability of urban ecosystems for malaria transmission contribute to the complexity of the malaria epidemiology in urban areas. METHODS: The study was designed to identify the determinants of malaria transmission estimated by the prevalence of anti-circumsporozoite (CSP) antibodies, the prevalence and density of Plasmodium falciparum infection, and the prevalence of malarial disease in areas of Ouagadougou, Burkina-Faso. Thick blood smears, dried blood spots and clinical status have been collected from 3,354 randomly chosen children aged 6 months to 12 years using two cross-sectional surveys (during the dry and rainy seasons) in eight areas from four ecological strata defined according to building density and land tenure (regular versus irregular). Demographic characteristics, socio-economic information, and sanitary and environmental data concerning the children or their households were simultaneously collected. Dependent variables were analysed using mixed multivariable models with random effects, taking into account the clustering of participants within compounds and areas. RESULTS: Overall prevalences of CSP-antibodies and P. falciparum infections were 7.7% and 16.6% during the dry season, and 12.4% and 26.1% during the rainy season, respectively, with significant differences according to ecological strata. Malaria risk was significantly higher among children who i) lived in households with lower economic or education levels, iii) near the hydrographic network, iv) in sparsely built-up areas, v) in irregularly built areas, vi) who did not use a bed net, vii) were sampled during the rainy season or ii) had traveled outside of Ouagadougou. CONCLUSION: Malaria control should be focused in areas which are irregularly or sparsely built-up or near the hydrographic network. Furthermore, urban children would benefit from preventive interventions (e.g. anti-vectorial devices or chemoprophylaxis) aimed at reducing malaria risk during and after travel in rural areas.

Abstract: The distribution and range of 50% inhibitory concentrations (IC(50)s) of doxycycline were determined for 747 isolates obtained between 1997 and 2006 from patients living in Senegal, Republic of the Congo, and Gabon and patients hospitalized in France for imported malaria. The statistical analysis was designed to answer the specific question of whether Plasmodium falciparum has different phenotypes of susceptibility to doxycycline. A triple normal distribution was fitted to the data using a Bayesian mixture modeling approach. The IC(50) geometric mean ranged from 6.2 microM to 11.1 microM according to the geographical origin, with a mean of 9.3 microM for all 747 parasites. The values for all 747 isolates were classified into three components: component A, with an IC(50) mean of 4.9 microM (+/-2.1 microM [standard deviation]); component B, with an IC(50) mean of 7.7 microM (+/-1.2 microM); and component C, with an IC(50) mean of 17.9 microM (+/-1.4 microM). According to the origin of the P. falciparum isolates, the triple normal distribution was found in each subgroup. However, the proportion of isolates predicted to belong to component B was most important in isolates from Gabon and Congo and in isolates imported from Africa (from 46 to 56%). In Senegal, 55% of the P. falciparum isolates were predicted to be classified as component C. The cutoff of reduced susceptibility to doxycycline in vitro was estimated to be 35 microM.

Abstract: We evaluated the relationship between immune response markers and the multiplicity of Plasmodium falciparum infections in order to assess the validity of the latter as an indicator of the acquisition of anti-malarial immunity. Parasite populations present during malaria episodes of 64 Gabonese children who presented with at least 4 such attacks during active follow-up over a 7-year period were characterized using MSP-1 and MSP-2 PCR-based methods. Plasma samples taken at healthy and parasite-free phase were used to measure P. falciparum antigen-specific antibody and cytokine activity. We found evidence of intra- and inter-individual variation in the number of parasite genotypes present in different malaria episodes, although in 72% of isolates no more than 2 parasite genotypes were detectable. Samples with the highest multiplicity were from children with significantly lower (p < 0.03) antibody responses to specific asexual stage antigens. Additionally, the whole blood interferon-gamma production capacity was significantly higher (p < 0.02) in those with lower infection multiplicity. Malaria episodes with multiple clones indeed reflect a low level of acquired immunity and a consequently poor capacity to control the infection. These findings suggest that the multiplicity of falciparum infection may be a potentially useful parameter in the evaluation of malaria control interventions.

Abstract: Endemic malaria has been eradicated from France, but some falciparum malaria cases have been described in patients who have never travelled outside the country. Ms. V. 21 year-old and Mr. M. 23 year-old living together in Paris were on holiday in Saint Raphaël (French Riviera). They presented with fever, vertigo and nausea. A blood smear made to control thrombocytopaenia revealed intra-erythrocytic forms of Plasmodium falciparum. The parasitaemia level was 0.15% for Ms. V and 3.2% for Mr. M. This couple had no history of blood transfusion or intravenous drug use. They had never travelled outside metropolitan France, but had recently travelled around France: to Saint Mard (close to Paris Charles de Gaulle (CdG) airport), to Barneville plage (in Normandy) and finally to Saint Raphaël. The most probable hypothesis is an infection transmitted in Saint Mard by an imported anopheline mosquito at CdG airport. The DNA analysis of parasites from Ms. V.'s and Mr. M.'s blood revealed identical genotypes. Because it is unlikely that two different anopheline mosquitoes would be infected by exactly the same clones, the two infections must have been caused by the infective bites of the same infected mosquito.

Abstract: BACKGROUND: Chikungunya virus (CHIKV), an arbovirus, is responsible for a two-stage disabling disease, consisting of an acute febrile polyarthritis for the first 10 days, frequently followed by chronic rheumatisms, sometimes lasting for years. Up to now, the pathophysiology of the chronic stage has been elusive. Considering the existence of occasional peripheral vascular disorders and some unexpected seronegativity during the chronic stage of the disease, we hypothesized the role of cryoglobulins. METHODS: From April 2005 to May 2007, all travelers with suspected CHIKV infection were prospectively recorded in our hospital department. Demographic, clinical and laboratory findings (anti-CHIKV IgM and IgG, cryoglobulin) were registered at the first consultation or hospitalization and during follow-up. RESULTS: Among the 66 travelers with clinical suspicion of CHIKV infection, 51 presented anti-CHIKV IgM. There were 45 positive with the serological assay tested at room temperature, and six more, which first tested negative when sera were kept at 4 degrees C until analysis, became positive after a 2-hour incubation of the sera at 37 degrees C. Forty-eight of the 51 CHIKV-seropositive patients were screened for cryoglobulinemia; 94% were positive at least once during their follow-up. Over 90% of the CHIKV-infected patients had concomitant arthralgias and cryoglobulinemia. Cryoglobulin prevalence and level drop with time as patients recover, spontaneously or after short-term corticotherapy. In some patients cryoglobulins remained positive after 1 year. CONCLUSION: Prevalence of mixed cryoglobulinemia was high in CHIKV-infected travelers with long-lasting symptoms. No significant association between cryoglobulinemia and clinical manifestations could be evidenced. The exact prognostic value of cryoglobulin levels has yet to be determined. Responsibility of cryoglobulinemia was suspected in unexpected false negativity of serological assays at room temperature, leading us to recommend performing serology on pre-warmed sera.

Abstract: Liver-stage antigen 3 (LSA-3) is a new vaccine candidate that can induce protection against Plasmodium falciparum sporozoite challenge. Using a series of long synthetic peptides (LSP) encompassing most of the 210-kDa LSA-3 protein, a study of the antigenicity of this protein was carried out in 203 inhabitants from the villages of Dielmo (n = 143) and Ndiop (n = 60) in Senegal (the level of malaria transmission differs in these two villages). Lymphocyte responses to each individual LSA-3 peptide were recorded, some at high prevalences (up to 43%). Antibodies were also detected to each of the 20 peptides, many at high prevalence (up to 84% of responders), and were directed to both nonrepeat and repeat regions. Immune responses to LSA-3 were detectable even in individuals of less than 5 years of age and increased with age and hence exposure to malaria, although they were not directly related to the level of malaria transmission. Thus, several valuable T- and B-cell epitopes were characterized all along the LSA-3 protein, supporting the antigenicity of this P. falciparum vaccine candidate. Finally, antibodies specific for peptide LSP10 located in a nonrepeat region of LSA-3 were found significantly associated with a lower risk of malaria attack over 1 year of daily clinical follow-up in children between the ages of 7 and 15 years, but not in older individuals.

Abstract: Polymorphisms in the Plasmodium falciparum crt (Pfcrt), Pfmdr1, and Pfmrp genes were not significantly associated with quinine (QN) 50% inhibitory concentrations (IC(50)s) in 23 strains of Plasmodium falciparum. An increased number of DNNND repeats in Pfnhe-1 microsatellite ms4760 was associated with an increased IC(50) of QN (P = 0.0007). Strains with only one DNNND repeat were more susceptible to QN (mean IC(50) of 154 nM). Strains with two DNNND repeats had intermediate susceptibility to QN (mean IC(50) of 548 nM). Strains with three DNNND repeats had reduced susceptibility to QN (mean IC(50) of 764 nM). Increased numbers of NHNDNHNNDDD repeats were associated with a decreased IC(50) of QN (P = 0.0020). Strains with profile 7 for Pfnhe-1 ms4760 (ms4760-7) were significantly associated with reduced QN susceptibility (mean IC(50) of 764 nM). The determination of DNNND and NHNDNHNNDDD repeats in Pfnhe-1 ms4760 could be a good marker of QN resistance and provide an attractive surveillance method to monitor temporal trends in P. falciparum susceptibility to QN. The validity of the markers should be further supported by analyzing more isolates.

Abstract: BACKGROUND: Malaria remains a major threat, to both travellers and military personnel deployed to endemic areas. The recommendations for travellers given by the World Health Organization is based on the incidence of malaria in an area and do not take the degree of exposure into account. The aim of this article is to evaluate the exposure of travellers by entomologic methods, which are the commonly used measures of the intensity of malaria transmission. METHODS: From February 2004 to June 2004, five groups of 30 military personnel were stationed in up to 10 sites in western Côte d'Ivoire, from one week to several months. Adult mosquitoes were collected by human landing catches at each site during the five months and the level of exposure to malaria transmission of each group was estimated. RESULTS: The level of transmission varied from one site to another one from less than one to approximately more than 100 infective bites per month. In the majority of sites, at least two anopheline species were involved in transmission. The cumulative EIR over the study period varied according to the groups from 29 infected bites per person/per mission to 324. CONCLUSION: The level of malaria transmission and malaria risk varies widely (varying by a factor of eleven) between groups of travellers travelling in the same region and at the same time. Physicians involved in travel medicine or supporting expatriated populations or refugees should consider this heterogeneity and emphasize the importance of combining appropriate measures, such as chemoprophylaxis and protective measures against mosquitoes.

Abstract: BACKGROUND: The time necessary for malaria parasite to re-appear in the blood following treatment (re-infection time) is an indirect method for evaluating the immune defences operating against pre-erythrocytic and early erythrocytic malaria stages. Few longitudinal data are available in populations in whom malaria transmission level had also been measured. METHODS: One hundred and ten individuals from the village of Ndiop (Senegal), aged between one and 72 years, were cured of malaria by quinine (25 mg/day oral Quinimax in three equal daily doses, for seven days). Thereafter, thick blood films were examined to detect the reappearance of Plasmodium falciparum every week, for 11 weeks after treatment. Malaria transmission was simultaneously measured weekly by night collection of biting mosquitoes. RESULTS: Malaria transmission was on average 15.3 infective bites per person during the 77 days follow up. The median reappearance time for the whole study population was 46.8 days, whereas individuals would have received an average one infective bite every 5 days. At the end of the follow-up, after 77 days, 103 of the 110 individuals (93.6%; CI 95% [89.0-98.2]) had been re-infected with P. falciparum. The median reappearance time ('re-positivation') was longer in subjects with patent parasitaemia at enrolment than in parasitologically-negative individuals (58 days vs. 45.9; p = 0.03) and in adults > 30 years than in younger subjects (58.6 days vs. 42.7; p = 0.0002). In a multivariate Cox PH model controlling for the sickle cell trait, G6PD deficiency and the type of habitat, the presence of parasitaemia at enrolment and age >/= 30 years were independently predictive of a reduced risk of re-infection (PH = 0.5 [95% CI: 0.3-0.9] and 0.4; [95% CI: 0.2-0.6] respectively). CONCLUSION: Results indicate the existence of a substantial resistance to sporozoites inoculations, but which was ultimately overcome in almost every individual after 2 1/2 months of natural challenges. Such a study design and the results obtained suggest that, despite a small sample size, this approach can contribute to assess the impact of intervention methods, such as the efficacy vector-control measures or of malaria pre-erythrocytic stages vaccines.

Abstract: Plasmodium vivax isolates from French Guiana were studied for the presence of mutations associated with sulfadoxine/pyrimethamine (SP) drug resistance. Ninety-six blood samples were collected from 2000 to 2005 from symptomatic malaria patients. SP drug resistance was predicted by determining point mutations in the dihydrofolate reductase (pvdhfr) and dihydropteroate synthase (pvdhps) genes. All samples showed mutant genotypes in both genes with a prevalence > 90% for the 58R, 117N, 382C, and 383G. A new mutation (116G) in pvdhfr was found at a frequency of 3.3%. Six different pvdhfr/dhps multilocus genotypes were observed with the predominance of the quintuple mutant-type 58R/117N/173L-382C/383G (59.3%). No significant differences were observed between the prevalence of haplotypes and the year of collection. Our results indicate that, in this area, the fixation of SP drug-resistant parasites in the P. vivax population is stable.

Abstract: Airport malaria is a particular form of autochthonous malaria: it happens when the Plasmodium infected Anopheles genus mosquito travels from an endemic area to a malaria free airport. Since 1969, 30 cases of airport malaria have been reported in France, 2 during summer 2008. The severity of airport malaria is explained by the frequency of Plasmodium falciparum infecting non immune individuals and an often important diagnosis delay. It is a compulsory notification disease in France. The International Health Regulations (IHR) require states to check that airplanes coming from malaria or arboviral endemic area are systematically disinsected. Vector control measures have to be implemented within a distance of at least 400 meters around the perimeter of airports in malaria or arboviral endemic areas. In France, this measure applies to all airports of French overseas territories, except for the island of Saint-Pierre and Miquelon.

Abstract: BACKGROUND: Homeless people commonly present with ectoparasite-based pruritus. We evaluated the efficacy of a single dose of ivermectin to reduce the pruritus prevalence in a homeless population. METHODS: We conducted a randomized, double-blind, placebo-controlled trial from January 2006 to April 2006 in two homeless shelters in the city of Marseille, France. Homeless people complaining of pruritus were randomized to receive either ivermectin (24 mg) or placebo. Follow-up visits were planned at day 14 and day 28 after the inclusion to assess the outcome of pruritus. RESULTS: Forty-two subjects with pruritus were randomized to the ivermectin group and 40 to the placebo group. On day 14, pruritus was reported by significantly more subjects in the placebo group than those in the ivermectin group for both the per-protocol (PP) population (91.42% versus 68.57%, P = 0.014) and the intention-to-treat (ITT) population (92.5% versus 73.80%, P = 0.038). No significant effect was observed at day 28. Ivermectin was the only independent factor associated with the absence of pruritus at day 14 in both PP population [OR: 4.60 (95% CI:1.13; 18.73), P = 0.033] and ITT population [OR: 4.38 (95% CI: 1.07; 17.77), P = 0.039]. CONCLUSIONS: A single dose of oral ivermectin has a transient beneficial effect on the reduction of the prevalence of pruritus in the homeless population. More studies are required to assess the efficacy of multiple repeated treatments with ivermectin to reduce scabies and body lice endemic among homeless people with pruritus and the impact of such treatment on this population.

Abstract: Plasmodium falciparum malaria episodes may vary considerably in their severity and clinical manifestations. There is good evidence that host genetic factors contribute to this variability. To date, most genetic studies aiming at the identification of these genes have used a case/control study design for severe malaria, exploring specific candidate genes. Here, we performed a family-based genetic study of falciparum malaria related phenotypes in two independent longitudinal survey cohorts, as a first step towards the identification of genes and mechanisms involved in the outcome of infection. We studied two Senegalese villages, Dielmo and Ndiop that differ in ethnicity, malaria transmission and endemicity. We performed genome-scan linkage analysis of several malaria-related phenotypes both during clinical attacks and asymptomatic infection. We show evidence for a strong genetic contribution to both the number of clinical falciparum malaria attacks and the asymptomatic parasite density. The asymptomatic parasite density showed linkage to chromosome 5q31 (LOD = 2.26, empirical p = 0.0014, Dielmo), confirming previous findings in other studies. Suggestive linkage values were also obtained at three additional chromosome regions: the number of clinical malaria attacks on chromosome 5p15 (LOD = 2.57, empirical p = 0.001, Dielmo) and 13q13 (LOD = 2.37, empirical p = 0.0014 Dielmo), and the maximum parasite density during asymptomatic infection on chromosome 12q21 (LOD = 3.1, empirical p<10(-4), Ndiop). While regions of linkage show little overlap with genes known to be involved in severe malaria, the four regions appear to overlap with regions linked to asthma or atopy related traits, suggesting that common immune related pathways may be involved.

Abstract:

Abstract: BACKGROUND: Nonimmune travelers in malaria-endemic areas are exposed to transmission and may experience clinical malaria attacks during or after their travel despite using antivectorial devices or chemoprophylaxis. Environment plays an essential role in the epidemiology of this disease. Remote-sensed environmental information had not yet been tested as an indicator of malaria risk among nonimmune travelers. METHODS: A total of 1,189 personnel from 10 French military companies traveling for a short-duration mission (about 4 mo) in sub-Saharan Africa from February 2004 to February 2006 were enrolled in a prospective longitudinal cohort study. Incidence rate of clinical malaria attacks occurring during or after the mission was analyzed according to individual characteristics, compliance with antimalaria prophylactic measures, and environmental information obtained from earth observation satellites for all the locations visited during the missions. RESULTS: Age, the lack of compliance with the chemoprophylaxis, and staying in areas with an average Normalized Difference Vegetation Index higher than 0.35 were risk factors for clinical malaria. CONCLUSIONS: Remotely sensed environmental data can provide important planning information on the likely level of malaria risk among nonimmune travelers who could be briefly exposed to malaria transmission and could be used to standardize for the risk of malaria transmission when evaluating the efficacy of antimalaria prophylactic measures.

Abstract: Plasmodium falciparum is one of the most lethal parasite responsible for human malaria. Until now, the only one solution to counter malaria is the use of antimalarial drugs. Unfortunately, the extensively use of drugs, such as quinolines (i.e. chloroquine, quinine or mefloquine), have led to the emergence of drug resistance. Chloroquine and probably other quinolines act in interfering in the detoxification of hematin in the digestive vacuole. Quinolines are accumulated in P. falciparum digestive vacuole and the accumulation varies from a susceptible strain to a resistant one. Nevertheless, the mechanisms of quinoline resistance are still investigating. Genetic polymorphisms in some strains have been linked to drug resistance. The modifications observed are mutations on genes that encode transport proteins localized in the membrane of digestive vacuole. Three transporters were involved in quinoline resistance: PfCRT (Plasmodium falciparum chloroquine resistance transporter), Pgh1 (P-glycoprotein homologue 1) and PfMRP (Plasmodium falciparum multidrug resistance protein). They could be involved in accumulation or efflux mechanisms of drugs. In order to understand their role in resistance, localization, encoding gene structure, protein structure and endogenous function of these three transporters are reported. Some molecules that have no intrinsic antimalarial effect have been shown to reverse drug resistance when they are combined to chloroquine, quinine or mefloquine. These molecules are a solution to counter resistance but also they are precious tools to elucidate the resistance mechanisms. The molecules that have already shown a capacity to reverse chloroquine, quinine or mefloquine resistances were reported. Some of them could act on one of the three transporters involved in drug resistance, by confirming their role in quinoline resistance. Here we summarize the main elements of quinoline resistance and reversion of quinoline resistance related to malaria.

Abstract: Plasmodium falciparum infection can lead to a life threatening disease and the pathogenetic mechanisms of severe manifestations are not fully understood. Here, we investigated the capacity of P. falciparum-parasitized red blood cells (PRBC) from 45 children with clinical malaria to induce endothelial cell (EC) apoptosis. In all subjects, PRBC that cytoadhered to ECs could be found albeit to a variable degree. By contrast, PRBC that induce EC apoptosis were found only in nine (20%) subjects. Interestingly, children with neurological manifestations were significantly more likely to harbour apoptogenic strains. There was no quantitative relationship between the capacity of these isolates to cytoadhere and apoptosis induction. We hypothesize that P. falciparum-encoded molecules could be responsible for apoptosis induction and therefore suggest new insights in the pathogenesis of P. falciparum malaria. Further investigations are currently in progress to determine whether these results can be confirmed and to identify putative parasite apoptogenic factors.

Abstract: Previous studies have shown that antibodies from humans exposed continuously to malaria recognize the Plasmodium falciparum asexual blood-stage antigen Pf332. Here we analysed the antibody responses to a C-terminal fragment of Pf332, designated C231, in individuals from Senegal, by measuring the serum levels of immunoglobulin M (IgM), IgG class and subclass and IgE antibodies. IgG antibody reactivity with crude P. falciparum antigen was detected in all the donors, while many of the children lacked or had low levels of such antibodies against C231. The antibody levels increased significantly with age for both crude P. falciparum antigen and C231, and in the older age groups most of the donors displayed antibodies to C231. This was also true for IgM, IgE and IgG subclass reactivity against C231. Moreover, the ratio of IgG1/IgG2 was considerably lower for C231 than for crude P. falciparum antigen, and in age groups 10-14 and 15-19 years the levels of IgG2 against C231 even exceeded that of IgG1. The IgG2/IgG3 ratios suggest that C231 gives similar levels of IgG2 and IgG3, except for children aged 4-9 years, where IgG3 was higher. Raw IgM, IgG class and subclass and IgE antibody levels to C231 tended to be higher in those who did not experience a malaria attack, but following linear multivariate analysis the trends were not significant.

Abstract: The capacity of ten molecules for reversing resistance in Plasmodium falciparum in vitro to quinoline antimalarial drugs, such as chloroquine (CQ), quinine (QN), mefloquine (MQ) and monodesethylamodiaquine (MDAQ), was assessed against 27 Plasmodium falciparum isolates. Four of these compounds were 9,10-dihydroethanoanthracene derivatives (DEAs). These DEAs reversed 75 to 92% of the CQ resistant strains. These synthetic compounds were more effective in combination with CQ than verapamil, ketotifen, chlorpromazine, reserpine or nicardipine, which reversed less than 50% of the CQ resistant strains. DEAs significantly reversed 67 to 100% of MDAQ resistant parasites. These compounds were more effective in combination with MDAQ than ketotifen (60% of reversal), chlorpromazine (45%), verapamil (33%), reserpine (30%) or nicardipine (9%). The reversal activity of MQ resistance was less pronounced, regardless of the molecule tested, and was homogeneous with a rate ranging from 42% for ketotifen to 58% for reserpine, nicardipine, verapamil and cyproheptadine. The four DEAs significantly reversed 50 to 55% of the parasites resistant to MQ. Fifty-six to 78 % of the QN resistant parasites were reversed by the synthetic DEAs. There were few differences in the rate of reversal activity on QN resistant strains between the ten compounds, with rates ranging between 56 to 78% for the ten chemosensitizers. The use of DEAs in combination with quinoline seems to be thus a promising strategy for limiting the development of drug resistant strains and for treating patients in drug resistant areas.

Abstract: ABSTRACT: BACKGROUND: The burden of Plasmodium falciparum malaria has worsened because of the emergence of chloroquine resistance. Antimalarial drug use and drug pressure are critical factors contributing to the selection and spread of resistance. The present study explores the geographical, socio-economic and behavioural factors associated with the use of antimalarial drugs in Africa. METHODS: The presence of chloroquine (CQ), pyrimethamine (PYR) and other antimalarial drugs has been evaluated by immuno-capture and high-performance liquid chromatography in the urine samples of 3,052 children (2-9y), randomly drawn in 2003 from the general populations at 30 sites in Senegal (10), Burkina-Faso (10) and Cameroon (10). Questionnaires have been administered to the parents of sampled children and to a random sample of households in each site. The presence of CQ in urine was analysed as dependent variable according to individual and site characteristics using a random - effect logistic regression model to take into account the interdependency of observations made within the same site. RESULTS: According to the sites, the prevalence rates of CQ and PYR ranged from 9% to 91% and from 0% to 21%, respectively. In multivariate analysis, the presence of CQ in urine was significantly associated with a history of fever during the three days preceding urine sampling (OR=1.22, p= 0.043), socio-economic level of the population of the sites (OR=2.74, p = 0.029), age (2-5y = reference level; 6-9y OR = 0.76, p =0.002), prevalence of anti-circumsporozoite protein (CSP) antibodies (low prevalence : reference level; intermediate level OR = 2.47, p = 0.023), proportion of inhabitants who lived in another site one year before (OR= 2.53, p= 0.003), and duration to reach the nearest tarmacked road (duration less than one hour = reference level, duration equal to or more than one hour OR= 0.49, p = 0.019). CONCLUSIONS: Antimalarial drug pressure varied considerably from one site to another. It was significantly higher in areas with intermediate malaria transmission level and in the most accessible sites. Thus, P. falciparum strains arriving in cross-road sites or in areas with intermediate malaria transmission are exposed to higher drug pressure, which could favour the selection and the spread of drug resistance.

Abstract: The in vitro activity of ferroquine (FQ) (SR97193), a 4-aminoquinoline antimalarial compound that contains a ferrocenic nucleus, against 15 Plasmodium falciparum strains was assessed and compared with those of chloroquine (CQ), quinine (QN), monodesethylamodiaquine (MDAQ), and mefloquine (MQ). These 15 strains were genotyped for polymorphisms in quinoline resistance-associated genes such as Pfcrt, Pfmdr1, Pfmrp, and Pfnhe-1. FQ was highly active against CQ-resistant parasites or in parasites with reduced susceptibility to QN, MDAQ, or MQ. Encouragingly, we did not find a correlation between responses to FQ and those to other quinoline drugs. These results suggest that no cross-resistance exits between FQ and CQ or quinoline antimalarial drugs. Mutations in codons 74, 75, 76, 220, 271, 326, 356, and 371 of the Pfcrt gene; codons 86, 184, 1034, 1042, and 1246 of the Pfmdr1 gene; and codons 191 and 437 of the Pfmrp gene were not significantly associated with P. falciparum susceptibility to FQ. Neither the number of ms4760 DNNND or DDNHNDNHNN repeats in Pfnhe-1 nor the profile of ms4760 was significantly associated with the FQ in vitro response. These data suggest the FQ may not interact with transport proteins in quinoline-resistant parasites. The present results justify further clinical trials of FQ in multidrug resistance areas.

Abstract: Aedes aegypti is responsible for the transmission of arboviruses. The Yellow Fever, Dengue and Chikungunya viruses are transmitted to the vertebrate host by injection of infected saliva during the blood meal of its vectors. Saliva contains different components with various biochemical activities; anti-hemostatic, angiogenic, inflammatory, and immunomodulatory. This work compares the sialomes of three Ae. aegypti colonies (Rockefeller, PAEA, and Formosus), where the repertoire of salivary proteins from these colonies was analyzed by a proteomic approach. This study indicated that major proteins were detectable in the three colonies. However, differences in the abundance of some saliva proteins have been observed between the three Ae. aegypti colonies.

Abstract: ABSTRACT: BACKGROUND: According to entomological studies conducted over the past 30 years, there was low malaria transmission in suburb of Dakar but little evidence of it in the downtown area. However; there was some evidence of local transmission based on reports of malaria among permanent residents. An entomological evaluation of malaria transmission was conducted from May 2005 to October 2006 in two areas of Dakar. METHODS: Mosquitoes were sampled by human landing collection during 34 nights in seven places in Bel-air area (238 person-nights) and during 24 nights in five places in Ouakam area (120 person-nights). Mosquitoes were identified morphologically and by molecular methods. The Plasmodium falciparum circumsporozoite indexes were measured by ELISA, and the entomological inoculation rates (EIR) were calculated for both areas. Molecular assessments of pyrethroid knock down resistance (Kdr) and of insensitive acetylcholinesterase resistance were conducted. RESULTS: From May 2005 to October 2006, 4,117 and 797 Anopheles gambiae s.l. respectively were caught in Bel-air and Ouakam. Three members of the complex were present: Anopheles arabiensis (>98%), Anopheles melas (<1%) and An. gambiae s.s. molecular form M (<1%). Infected mosquitoes were caught only during the wintering period between September and November in both places. In 2005 and 2006, annual EIRs were 9,5 and 4, respectively, in Bel-air and 3 and 3, respectively, in Ouakam.. The proportion of host-seeking An. gambiae s.l. captured indoors were 17% and 51% in Bel air and Ouakam, respectively. Ace 1 mutations were not identified in both members of the An. gambiae complex. Kdr mutation frequency in An. arabiensis was 12% in Bel-air and 9% in Ouakam. CONCLUSION: Malaria is transmitted in Dakar downtown area. Infected mosquitoes were caught in two subsequent years during the wintering period in two distant quarters of Dakar. These data agree with clinical data from a Senegalese military Hospital of Dakar (Hospital Principal) where most malaria cases occurred between October and December. It was the first detection of An. melas in Dakar.

Abstract:

Abstract: The emergence and rapid spread of Plasmodium falciparum resistance to various antimalarials compounds is gradually reducing the clinician's options for treating malaria and for adapting prophylaxis to each traveler and destination. In this context doxycycline is an increasingly useful alternative except in individuals with contraindications, mainly children under the age of eight and pregnant women. Already used successfully in association with quinine for treatment of malaria in areas with multiresistance, doxycline has also proven to be effective and well tolerated for prophylaxis of malaria. No resistance to doxycycline has been observed to date. Most reported prophylactic failures have been related to poor compliance during the month following return from the endemic zone. The mechanisms of action of doxycycline on the parasite are still unclear. Identification of the molecular targets of doxycycline would open the way for the design of more active structural analogues with longer half-life.

Abstract: BACKGROUND: Little is known about children undergoing critical care for malaria. The purpose of this survey was to evaluate the outcome in African children requiring endotracheal intubation for life-threatening malaria. METHODS: All children with a primary diagnosis of severe malaria (2000 WHO definition) requiring endotracheal intubation, hospitalised over a five-year period, within a tertiary-care hospital in Dakar, Senegal, were enrolled in a retrospective cohort study. RESULTS: 83 consecutive patients were included (median PRISM h24 score: 14; IQR: 10-19, multiple organ dysfunctions: 91.5%). The median duration of ventilation was 36 hrs (IQR: 4-72). Indications for intubation were deep coma (Glasgow score </=7, n = 16), overt cortical or diencephalic injury, i.e, status epilepticus/decorticate posturing (n = 20), severe brainstem involvement, i.e., decerebrate posturing/opisthotonus (n = 15), shock (n = 15), cardiac arrest (n = 13) or acute lung injury (ALI) (PaO2/FiO2 <300 Torr, n = 4). Death occurred in 50 cases (case fatality rate (CFR), 60%) and was associated with multiple organ dysfunctions (median PELODh24 scores: 12.5 among non-survivors versus 11 among survivors, p = 0.02). Median PRISMh24 score was significantly lower when testing deep coma against other indications (10 vs 15, p < 0.001), ditto for PELODh24 score (2.5 vs 13, p = 0.02). Multivariate analysis identified deep coma as having a better outcome than other indications (CFR, 12.5% vs 40.0 to 93.3%, p < 0.0001). Decerebrate posturing/opisthotonus (CFR 73.3%, adjusted relative risk (aRR) 10.7, 95% CI 2.3-49.5) were associated with a far worse prognosis than status epilepticus/decorticate posturing (CFR 40.0%, aRR 5.7, 95% CI 1.2-27.1). Thrombocytopaenia (platelet counts <100,000/mm3) was associated with death (aRR 2.6, 95% CI 1.2-5.8) and second-line anticonvulsant use (clonazepam or thiopental) with survival (aRR 0.4, 95% CI 0.2-0.9). Complications, mostly nosocomial infections (n = 20), ALI/ARDS (n = 9) or sub-glottic stenosis (n = 3), had no significant prognostic value. CONCLUSION: In this study, the outcome of children requiring intubation for malaria depends more on clinical presentation and progression towards organ failures than on critical care complications per se. In sub-Saharan Africa, mechanical ventilation for life-threatening childhood malaria is feasible, but seems unlikely to dramatically improve the prognosis.

Abstract: BACKGROUND: Inadequate treatment practices with antimalarials are considered major contributors to Plasmodium falciparum resistance to chloroquine, pyrimethamine and sulfadoxine. The longitudinal survey conducted in Dielmo, a rural Senegalese community, offers a unique frame to explore the impact of strictly controlled and quantified antimalarial use for diagnosed malaria on drug resistance. METHODOLOGY/PRINCIPAL FINDINGS: We conducted on a yearly basis a retrospective survey over a ten-year period that included two successive treatment policies, namely quinine during 1990-1994, and chloroquine (CQ) and sulfadoxine/pyrimethamine (SP) as first and second line treatments, respectively, during 1995-1999. Molecular beacon-based genotyping, gene sequencing and microsatellite analysis showed a low prevalence of Pfcrt and Pfdhfr-ts resistance alleles of Southeast Asian origin by the end of 1994 and their effective dissemination within one year of CQ and SP implementation. The Pfcrt resistant allele rose from 9% to 46% prevalence during the first year of CQ reintroduction, i.e., after a mean of 1.66 CQ treatment courses/person/year. The Pfdhfr-ts triple mutant rose from 0% to 20% by end 1996, after a mean of 0.35 SP treatment courses/person in a 16-month period. Both resistance alleles were observed at a younger age than all other alleles. Their spreading was associated with enhanced in vitro resistance and rapidly translated in an increased incidence of clinical malaria episodes during the early post-treatment period. CONCLUSION/SIGNIFICANCE: In such a highly endemic setting, selection of drug-resistant parasites took a single year after drug implementation, resulting in a rapid progression of the incidence of clinical malaria during the early post-treatment period. Controlled antimalarial use at the community level did not prevent dissemination of resistance haplotypes. This data pleads against reintroduction of CQ in places where resistant allele frequency has dropped to a very low level after CQ use has been discontinued, unless drastic measures are put in place to prevent selection and spreading of mutants during the post-treatment period.

Abstract: In more than one way, the development of antiparasitic vaccines is challenging, but major efforts have been made. A large number of clinical trials have been carried out and a few antigens have been tested in the endemic zone, especially against malaria. So far, no vaccine candidate has shown a sufficient and long-lasting effectiveness that would be useful for public health. However, the trials have shown without ambiguity that a certain level of clinical immunity against paludism, schistosomiasis or leishmaniasis could be induced by vaccination, in the experimental setting or in the field.

Abstract: OBJECTIVE: The main objective of this study was to assess the influence of gas mixtures on in vitro Plasmodium falciparum growth and 50% inhibitory concentration (IC50) for chloroquine. METHODS: The study was performed between February 2004 and December 2005. 136 Plasmodium falciparum isolates were used to evaluate gas mixtures effect on IC50 for chloroquine by isotopic microtest. The oxygen effect on asexual blood cycle of 3D7 and W2 clones was determined by thin blood smears examination and tritiated hypoxanthine uptake. RESULTS: From 5% O2 to 21% O2 conditions, no parasiticide effect of O2 concentration was observed in vitro on the clones 3D7 and W2. A parasitostatic effect was observed during the exposure of mature trophozoites and schizonts at 21% O2 with an increase in the length of schizogony. The chloroquine IC50 at 10% O2 were significantly higher than those at 21% O2, means of 173.5 nM and 121.5 nM respectively (p < 0.0001). In particular of interest, among the 63 isolates that were in vitro resistant to chloroquine (IC50 > 100 nM) at 10% O2, 17 were sensitive to chloroquine (IC50 < 100 nM) at 21% O2. CONCLUSION: Based on these results, laboratories should use the same gas mixture to realize isotopic microtest. Further studies on comparison of isotopic and non-isotopic assays are needed to establish a standardized in vitro assay protocol to survey malaria drug resistance.

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Abstract: O'Farrel described a method allowing two-dimensional (2D) protein separation more than 30 years ago. Since then the original technique has made enormous progress. This progress has been accompanied by advances in mass spectrometry technology as well as various genome-sequencing programs. Today 2D electrophoresis has become the workhorse of proteomics, allowing resolution of complex structures containing thousands of proteins and providing a global view of the state of a proteome. This article presents the different steps and limitations of proteomic analysis: preparation of biological material, 2D electrophoresis, protein detection systems, and available tools for protein identification. Alternative proteomic approaches to 2D electrophoresis are also presented. A few applications are described as examples to illustrate the utility of proteomic analysis for studying the mechanisms underlying virulence, resistance to antimalarial therapies and immune response against pathologic agents.

Abstract: BACKGROUND: Surrogate markers of protective immunity to malaria in humans are needed to rationalize malaria vaccine discovery and development. In an effort to identify such markers, and thereby provide a clue to the complex equation malaria vaccine development is facing, we investigated the relationship between protection acquired through exposure in the field with naturally occurring immune responses (i.e., induced by the parasite) to molecules that are considered as valuable vaccine candidates. METHODS AND FINDINGS: We analyzed, under comparative conditions, the antibody responses of each of six isotypes to five leading malaria vaccine candidates in relation to protection acquired by exposure to natural challenges in 217 of the 247 inhabitants of the African village of Dielmo, Senegal (96 children and 121 older adolescents and adults). The status of susceptibility or resistance to malaria was determined by active case detection performed daily by medical doctors over 6 y from a unique follow-up study of this village. Of the 30 immune responses measured, only one, antibodies of the IgG3 isotype directed to merozoite surface protein 3 (MSP3), was strongly associated with clinical protection against malaria in all age groups, i.e., independently of age. This immunological parameter had a higher statistical significance than the sickle cell trait, the strongest factor of protection known against Plasmodium falciparum. A single determination of antibody was significantly associated with the clinical outcome over six consecutive years in children submitted to massive natural parasite challenges by mosquitoes (over three parasite inoculations per week). Finally, the target epitopes of these antibodies were found to be fully conserved. CONCLUSIONS: Since anti-MSP3 IgG3 antibodies can naturally develop along with protection against P. falciparum infection in young children, our results provide the encouraging indication that these antibodies should be possible to elicit by vaccination early in life. Since these antibodies have been found to achieve parasite killing under in vitro and in vivo conditions, and since they can be readily elicited by immunisation in naive volunteers, our immunoepidemiological findings support the further development of MSP3-based vaccine formulations.

Abstract: T cells are thought to play a critical role in cerebral malaria pathogenesis. However, available evidences are restricted to rodent models in which V beta specific T cell expansion has been associated with neurological syndrome suggesting involvement of superantigens or dominant antigens. Using flow cytometry, we studied the peripheral V beta T cell repertoire of Ghanaian children with cerebral malaria, uncomplicated malaria and asymptomatic control children, to look for either expansion or deletion of specific V beta associated with cerebral malaria. At admission, the general pattern of the repertoire of the patients was very similar, with no major distortion compared to the control group a part a significant increase of the frequency of the V beta 21.3 subset correlating with disease severity and attributed to the CD4 subset. During convalescence very limited fluctuations were observed including a significant decrease of the V beta 21.3 subset and increase of the V beta 20 subset, a subset not detected at admission. The remarkable stability of the V beta repertoire observed in acute malaria either cerebral or uncomplicated argues against the idea that cerebral malaria would result from a T cell-mediated inflammatory shock syndrome driven by some dominant super-antigenic activity(ies). The significance of the reproducible increase of the CD4+V beta 21.3T cell subset deserves further investigations.

Abstract: We report 2 cases of Plasmodium falciparum malaria in southern France in a French woman and an American man of Togolese origin who reported no recent travel to malaria-endemic countries. Both infections occurred after a stay near Marseille, which raises the possibility of autochthonous transmission. Entomologic and genotypic investigations are described.

Abstract: BACKGROUND: The antimalarial drug atovaquone specifically targets Plasmodium falciparum cytochrome b (Pfcytb), a mitochondrial gene with uniparental inheritance. Cases of resistance to atovaquone associated with mutant Pfcytb have been reported, justifying efforts to better document the natural polymorphism of this gene. To this end, a large molecular survey was conducted in several malaria endemic areas where atovaquone was not yet in regular use. METHODS: The polymorphism of the Pfcytb was analysed by direct sequencing of PCR products corresponding to the full length coding region. Sequence was generated for 671 isolates originating from three continents: Africa (Senegal, Ivory Coast, Central African Republic and Madagascar), Asia (Cambodia) and South America (French Guiana). RESULTS: Overall, 11 polymorphic sites were observed, of which eight were novel mutations. There was a large disparity in the geographic distribution of the mutants. All isolates from Senegal, Central African Republic and Madagascar displayed a Camp/3D7 wild type Pfcytb sequence, as did most samples originating from Cambodia and Ivory Coast. One synonymous (t759a at codon V253V) and two non-synonymous (t553g and a581g at codons F185V and H194R, respectively) singletons were detected in Ivory Coast. Likewise, two synonymous (a126t and c793t at codons -T42T and L265L, respectively) singletons were observed in Cambodia. In contrast, seven mutated sites, affecting seven codons and defining four mutant haplotypes were observed in French Guiana. The wild type allele was observed in only 14% of the French Guiana isolates. The synonymous c688t mutation at position L230L was highly prevalent; the most frequent allele was the c688t single mutant, observed in 84% of the isolates. The other alleles were singletons (a126t/a165c, a4g/a20t/a1024c and a20t/t341c/c688t corresponding to T42T/S55S, N2D/N71I/I342L, N71I/L114S/L230L, respectively" please replace with ' corresponding to T42T/S55S, N2D/N71I/I342L and N71I/L114S/L230L, respectively). The codon 268 polymorphisms associated with atovaquone resistance were not observed in the panel the isolates studied. Overall, the wild type PfCYTb protein isoform was highly predominant in all study areas, including French Guiana, suggesting stringent functional constraints. CONCLUSION: These data along with previously identified Pfcytb field polymorphisms indicate a clustering of molecular signatures, suggesting different ancestral types in South America and other continents. The absence of mutations associated with most atovaquone-proguanil clinical failures indicates that the atovaquone-proguanil association is an interesting treatment option in the study areas.

Abstract: The need for diagnosis of imported malaria is frequent in France. Diagnosis biological tools are different, according methods, sensitivity, interpretation and costs. Strategies for their use could be stratified according locally available methods, and experience of the practician.

Abstract: Exposure to vectors of infectious diseases has been associated with antibody responses against salivary antigens of arthropods among people living in endemic areas. This immune response has been proposed as a surrogate marker of exposure to vectors appropriate for evaluating the protective efficacy of antivectorial devices. The existence and potential use of such antibody responses in travellers transiently exposed to Plasmodium or arbovirus vectors in tropical areas has never been investigated. The IgM and IgG antibody responses of 88 French soldiers against the saliva of Anopheles gambiae and Aedes aegypti were evaluated before and after a 5-month journey in tropical Africa. Antibody responses against Anopheles and Aedes saliva increased significantly in 41% and 15% of the individuals, respectively, and appeared to be specific to the mosquito genus. A proteomic and immunoproteomic analysis of anopheles and Aedes saliva allowed for the identification of some antigens that were recognized by most of the exposed individuals. These results suggest that antibody responses to the saliva of mosquitoes could be considered as specific surrogate markers of exposure of travellers to mosquito vectors that transmit arthropod borne infections.

Abstract: Much effort has been devoted to developing anti-parasite vaccines. The main obstacles to overcome involve problems in cultivating parasites, variability in antigens of cultivated parasites, insufficient immune responses that do not provide full protection, lack of animal models and difficulty in evaluating immune protection acquired naturally or after vaccination in populations living in endemic areas. Numerous clinical trials have been conducted and several parasite antigens, in particular against malaria, have been tested in endemic areas. Up to now no candidate vaccine has shown sufficient, long-term efficacy to justify its inclusion in public health program. However trials using anti-parasite vaccination under both experimental and field conditions clearly demonstrate that a certain level of clinical immunity against malaria, bilharziasis, and leishmaniasis.

Abstract: The chemosusceptibility and genetic polymorphism of Plasmodium falciparum populations from 48 patients hospitalized for malaria at the Hospital Principal in Dakar, Senegal were investigated during the 2002 malaria transmission season. Sixty-two percent of the isolates collected were from patients with severe malaria and 38% were from patients with mild malaria. In vitro activities of chloroquine, quinine, cycloguanil, atovaquone, mefloquine, halofantrine, and artesunate were evaluated. The prevalence of mutations in the Plasmodium falciparum dihydrofolate reductase (dhfr) and dihyropteroate synthetase (dhps) genes and the P. falciparum chloroquine resistance transporter (Pfcrt) gene associated with cycloguanil, pyrimethamine, sulfadoxine, and chloroquine resistance were estimated. The genetic polymorphism of the parasite populations was evaluated by analysis of the highly polymorphic regions of merozoite surface protein 1 (msp1) block 2 and msp2. Seventy percent of the isolates were assessed by an in vitro assay. Fifty-two percent of the isolates were chloroquine resistant, 45% were cycloguanil resistant, and 24% were atovaquone resistant. Four percent had low susceptibility to quinine. The Pfcrt and dhfr mutations were associated with in vitro chloroquine- and antimetabolic drug-resistant isolates, respectively. Approximately 70% of the isolates contained two or more clones. Genetic diversity of P. falciparum was high. The prevalence of allelic family K1 of msp1 was 68%. Isolates of P. falciparum were highly resistant to chloroquine, cycloguanil and atovaquone. The transmission rate of malaria in Dakar is low but a high degree of genetic polymorphism can increase severe malaria, as shown by persons coming to Dakar from areas highly endemic for malaria. Areas with urban malaria should use vector control measures and efficient chemoprophylaxis for non-immune populations.

Abstract: The development and spread of resistance to antimalarial drugs poses a severe and increasing public health threat. Failures of prophylaxis or treatment with quinolines, hydroxynaphthoquinones, sesquiterpene lactones, antifolate drugs and sulfamides are involved in a return malaria-related morbidity and mortality. Resistance is associated with a decrease in accumulation of drugs into the vacuole, which results from a reduced uptake of the drug, an increased efflux or a combination of both. A number of candidate genes in P. falciparum have been proposed to be involved in antimalarial resistance, each concerned in membrane transport. Weaker or stronger associations are seen in P. falciparum between the resistance to quinolines or artemisinin derivatives and codon changes in Pfmdr1, a gene which encodes Pgh-1, an ortholog of one of the P-glycoproteins expressed in multi-drug resistant human cancer cells (ABC transporter). Further analysis has revealed a new gene, Pfcrt, encoding a PfCRT protein, which resembles an anion channel. Codon changes found in the Pfcrt sequence in drug resistant isolates could facilitate the drug efflux through a putative channel. It has been proposed that the reversal of quinoline resistance by verapamil is due to hydrophobic binding to the mutated PfCRT protein. Several compounds have demonstrated in the past decade a promising capability to reverse the antimalarial drug resistance in vitro in parasite isolates, in animal models and in human malaria. These drugs belong to different pharmacological classes such as calcium channel blockers, tricyclic antidepressants, antipsychotic calmodulin antagonists, histamine H1-receptor antagonists, analgesic and antipyretic drugs, non-steroidal anti-inflammatory drugs, and to different chemical classes such as synthetic surfactants, alkaloids from plants used in traditional medicine, pyrrolidinoaminoalkanes and anthracenic derivatives. Here we summarize the progress made in biochemical and genetic basis of antimalarial resistance, emphasizing the recent developments on drugs, which interfere with trans membrane proteins involved in drug efflux or uptake.

Abstract: We propose a stochastic model for simulating malaria tolerance. The model relates the probability of a clinical attack of malaria to the peripheral parasite densities via a pyrogenic threshold that itself responds dynamically to the parasite load. The parameters of the model have been estimated by fitting it to the relationship between incidence of clinical episodes and the entomologic inoculation rate, using age-specific incidence data from two villages in Senegal and one village in Tanzania. The model reproduces the shifts in age distribution of clinical episodes associated with variation in transmission intensity, and in keeping with the data, predicts a slightly higher lifetime number of episodes in the mesoendemic village of Ndiop than in the holoendemic village of Dielmo. This model provides a parsimonious explanation of counter-intuitive relationships between the overall incidence of clinical malaria and transmission intensity. In contrast to the theory of endemic stability, recently proposed to apply to P. falciparum, it does not assume any intrinsic age dependence in the outcome of infection. This model can be used to explore the consequences for predictions of the effects of different anti-malarial interventions on the incidence of clinical malaria.

Abstract: The development of a malaria vaccine has been accelerating in the last ten years. The number of clinical trials has increased and some malaria antigens have been tested in endemic areas. No potential vaccine has yet shown sufficient and lasting efficacy to justify its inclusion in a public health program. However, trials have unambiguously shown that some level of anti-malaria clinical immunity can be achieved by vaccination, both in experimental and in field conditions. Advances in malaria vaccine development are presented.

Abstract: An important vector control program is ongoing to lower the risk of malaria transmission in the French military camp of Port-Bouet, Abidjan (Cote d'Ivoire). However, some autochthonous malaria cases are regularly suspected. An entomological survey was conducted in June 2004 in the camp to assess malaria transmission and evaluate the pyrethroid and organophosphate resistance of the malaria vectors. The average mosquito biting rate was 178.0 bites per person per night. Mosquitoes belonging to the Anopheles gambiae (Diptera: Culicidae) complex and the Anopheles funestus group were collected. An. gambiae s.s. molecular form M was the only species of the An. gambiae complex present. The average number of An. gambiae bites was approximately 44.3 per person per night. The circumsporozoite index was 0.38% and the entomological inoculation rate estimated to be 1.2 infective bites per week for the study period. The kdr and ace1 gene frequencies in the An. gambiae population were 0.70 and 0.15, respectively. Personnel living in the French barracks of Port-Bouet are thus at high risk of being bitten by parasite-infected mosquitoes. Such an entomological inoculation rate, usually found in African peri-urban environments, was unexpected considering the extensive effort deployed to control mosquitoes in the camp. Insecticide resistance could explain the inefficacy of the vector control program but the spraying strategy is also questionable.

Abstract:

Abstract: We determined the level of in vitro resistance of Plasmodium falciparum parasites to standard antimalarial drugs, such as chloroquine, quinine, amodiaquine, halofantrine, mefloquine, cycloguanil, and pyrimethamine, and to new compounds, such as dihydroartemisinin, doxycycline, atovaquone, and lumefantrine. The in vitro resistance to chloroquine reached 75.5%. Twenty-eight percent of the isolates were intermediate or had reduced susceptibility to quinine. Seventy-six percent and 96% of the tested isolates showed in vitro resistance or intermediate susceptibilities to cycloguanil and pyrimethamine, respectively. Only 2% of the parasites demonstrated in vitro resistance to monodesethylamodiaquine. No resistance was shown with halofantrine, lumefantrine, dihydroartemisinin, or atovaquone. Halofantrine, mefloquine, and lumefantrine demonstrated high correlation. No cross-resistance was identified between responses to monodesethyl-amodiaquine, dihydroartemisinin, atovaquone, and cycloguanil. Since the level of chloroquine resistance in vitro exceed an unacceptable upper limit, high rates of in vitro resistance to pyrimethamine and cycloguanil and diminution of the susceptibility to quinine, antimalarial drugs used in combination, such as amodiaquine, artemisinin derivatives, mefloquine, lumefantrine, or atovaquone, seem to be appropriate alternatives for the first line of treatment of acute, uncomplicated P. falciparum malaria.

Abstract: The genetic variability and population structure of Plasmodium falciparum are key factors in malaria control strategies. Studies have suggested no P. falciparum population structure although linkage disequilibrium was observed in some African areas. We have assessed length polymorphism at 6-22 microsatellites in four urban and rural sites (Djibouti, Dakar, Niamey, and Zouan-Hounien, n = 240 blood samples). Results have shown a P. falciparum population structure in Africa (Fst = 0.17-0.24), lower genetic diversity in Djibouti (He = 0.53) than in the other sites (He = 0.73-0.76), and 3) significant linkage disequilibrium in Djibouti. These results could be related to geographic isolation and low flow of parasites between sites. They also suggest a potential effect of rural suburbs to generate genetic diversity in towns. This could affect the dispersal of selected drug resistance and should be considered when adapting urban malaria control strategies.

Abstract: OBJECTIVE: Little is known about the use of generic severity scores in severe childhood infectious diseases. The purpose of this prospective study was to evaluate the performance of the Pediatric Risk of Mortality (PRISM) scoring system in predicting the outcome of falciparum malaria in African children. DESIGN, SETTING, PATIENTS: All children admitted to a 120-bed pediatric ward in a tertiary care hospital in Dakar, Senegal, with a primary diagnosis of acute malaria were assigned a PRISM score after 24 hrs or at time of death. INTERVENTIONS: None. RESULTS: PRISM discrimination, evaluated by areas under receiver operating characteristic curves (AUC), was good both for all acute malaria cases (n = 311; lethality, 9%; AUC, 0.89; 95% confidence interval [CI], 0.85-0.92) and for severe malaria cases (n = 233; lethality, 12%; AUC, 0.86; 95% CI, 0.81-0.90). However, the number of children who died was greater than the number of deaths predicted by PRISM (standardized mortality ratio, 2.16; 95% CI, 1.46-2.87). CONCLUSION: This discrepancy observed in five classes of expected mortality (Hosmer-Lemeshow chi-square test, p < .001) may have been due to chance (sample size too small for a valid test), to a lower standard of care in Dakar than in the American hospitals where PRISM was designed, or to a failure of PRISM to classify risk in severe malaria.

Abstract: Protective devices against vectors are used by travelers in malaria-endemic areas but their efficacy for protection against mosquitoes has rarely been evaluated. The level of exposure to malaria transmission of 205 soldiers deployed in Africa and the efficacy of their anti-vector prophylaxis was evaluated by comparison of their IgM and IgG responses against five pre-erythrocytic Plasmodium falciparum antigens (circumsporozoite protein, sporozoite threonine- and asparagine-rich protein, sporozoite- and liver-stage antigen, liver stage antigen 1, and SR11.1) before and at the end of their deployment, and three months after returning to France for 106 of these soldiers. The immune responses increased significantly during the mission in 35% (95% confidence interval = 28-42%) of the individuals. The permanent use of insecticide-treated bed nets and long-sleeve battle dress at night were associated with protective efficacy. The analysis of these antibody responses was sensitive enough to evaluate exposure to malaria transmission and the efficacy of anti-vector devices in travelers using antimalarial chemoprophylaxis.

Abstract: 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.

Abstract: BACKGROUND: The ongoing drought in sub-Saharan countries has led to the colonisation of west African Savanna by Ornithodoros sonrai; this tick acts as a vector for Borrelia crocidurae, which causes tick-borne relapsing fever (TBRF). Our aim was to ascertain the incidence of TBRF in west Africa. METHODS: From 1990 to 2003, we monitored the incidence of TBRF in Dielmo, Senegal, by daily clinical surveillance and by blood testing of individuals with a fever. From 2002 to 2005, we investigated the presence of O sonrai in 30 villages in Senegal, Mauritania, and Mali, and measured by PCR the prevalence of B crocidurae. FINDINGS: The average incidence of TBRF over 14 years was 11 per 100 person-years (range from 4 in 1990 to 25 in 1997). All age-groups presented a high incidence of the disease. In addition to relapses, repeated infections in the same individuals were common, with some affected by up to six distinct infections during the study period. Epidemiological studies indicated that 26 of the 30 studied villages (87%) were colonised by the vector tick O sonrai and that the average B crocidurae infection rate of the vector was 31%. INTERPRETATION: The incidence of TBRF at the community level is the highest described in Africa for any bacterial disease. The presence of the vector tick in most villages investigated and its high infection rate suggest that TBRF is a common cause of fever in most rural areas of Senegal, Mauritania, and Mali.

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Abstract: Four parasite species, among which one lethal, at least 20 efficient vectors, spread on the most crowded and underdeveloped continents, where first antimalarials and insecticides were so extensively used for decades that they are now inefficient after resistances. It is today the sad medium on which malaria raged, spring of 1 million deaths and 300 millions cases a year, of which 90% in sub-Saharan Africa. In Africa, epidemiological patterns are widely different some to others, which explain unique control strategy failure and vanity. Waiting vaccines and new drugs, control must be centred on vectors control and multidrug therapy. France is the industrialised country with the highest number of imported cases (approximately equal to 7000 cases yearly), linked mainly to P. falciparum coming from sub-Saharan Africa and recurring in nearly 3/4 of cases in immigrants.

Abstract: The aim of this study in Curaca, Bahia, Northeast Brazil was to explore possible factors associated with the infestation of human dwellings by Triatoma brasiliensis or Triatoma pseudomaculata, two sylvatic triatomine species. We use multiple logistic regression analysis to show how structural features of the peridomiciliary area combine with the number of animals and sociological factors to allow infestation of some dwellings. It is suggested that T. brasiliensis is associated with human activities, while T. pseudomaculata is associated with vegetation and animals. Peridomiciliary farm animals are a strong risk factor for triatomine infestation.

Abstract: Analysis of Plasmodium falciparum isolates collected before, during, and after a 1999 malaria epidemic in Djibouti shows that, despite a high prevalence of resistance to chloroquine, the epidemic cannot be attributed to a sudden increase in drug resistance of local parasite populations.

Abstract: Rapid development of significant resistance to antimalarial drugs has been a major force driving research to identify and develop new compounds. The use of synthetic organometallic complexes seems to be promising for treatment of malaria infections. Recent progress in identification and development of new drugs promises to lead to a much greater range of antimalarial agents. Organometallic complexes and metalloporphyrins have shown in vitro activity against Plasmodium falciparum. Ferroquine (ferrocenyl chloroquine) is more active than chloroquine against strains and isolates of P. falciparum and shows efficacy against murine parasites.

Abstract: Many genes have been implicated in the risk of severe malaria, generally based on candidate gene studies in case/control populations. Among these genes, there has been conflicting reports for the implication of a variant of the intercellular adhesion molecule 1 (ICAM1), ICAM1(Kilifi), in the risk of severe malaria, while in vitro studies provided independent support for a functional role of this variant. In order to explore the possible implication of ICAM1 in the susceptibility/resistance to malaria and to try to understand its clinical relevance in the disease process, we have conducted linkage and association studies of ICAM1 in two Senegalese villages located in regions of endemic malaria. We explored the full genetic variability of ICAM1, and tested it on several clinical malarial traits which are under genetic control, focusing principally on variables related to the parasite density and the number of malarial attacks. Our study provides no evidence for a role of ICAM1 variability on the malarial phenotypes studied.

Abstract: During a national survey in 2002 in Djibouti, serum samples were collected using a valid sampling scheme from 2423 Djiboutians representing the general population of urban and rural districts. The HIV-1 seroprevalence was 2%. The HIV-1 polymerase gene from 53 untreated patients was amplified. Phylogenetic analysis of 34 isolates revealed a majority of subtype C (73%) as well as other subtypes, including CRF02_AG recombinants (18%), subtype D (6%), and subtype A (3%).

Abstract:

Abstract: There are no specific clinical signs or symptoms of malaria. Fever attacks, anemia, or signs of severity like coma or respiratory distress cannot easily be attributed to malaria in people who are infected most of the time. Ascribing clinical manifestations to malaria is problematic in populations that are regularly exposed to the transmission of human plasmodia. The more transmission is intense and regular, the higher the prevalence of asymptomatic infections. In areas of intense and perennial malaria transmission, more than 90% of the population may be infected and the simple detection of a plasmodial infection is not enough to attribute clinical manifestations to malaria. Naturally acquired anti-malaria immunity permitting asymptomatic infections is incomplete and temporary. It is an obstacle to the estimation of the malaria burden in endemic areas. The positive association between parasite density and fever allows the attribution of clinical attacks to malaria. The relationship between parasitaemia and the risk of fever is not continuous. An age- and endemicity-dependent threshold effect of parasite density has been demonstrated and can be used to distinguish clinical attacks due to malaria from others. Clinical diagnosis and evaluation of malaria are problematic in three situations: in public health to estimate the malaria burden for health services, in clinical research to evaluate treatments or prophylactic measures (drug, vaccine, anti-vectorial devices), and in basic research on pathophysiology, immunology or genetic susceptibility to clinical malaria. No one diagnostic definition nor procedure for detecting cases is adequate for all three purposes. Case detection may be passive (in health structures for example) or active (in population). The choice of methods for diagnosis and recruitment depends on the objectives and whether a "pragmatic" or "explicative" approach is used. The radical differences between these approaches are often unsuspected or ignored.

Abstract: OBJECTIVE: To survey the frequency of genotypic antiretroviral resistance in drug-naive HIV-1-infected Djiboutians. METHODS: A national study was conducted in the general population of Djibouti in March 2002 to determine HIV-1 seroprevalence. Blood samples were collected anonymously and plasma samples scoring positive for HIV-1 antibodies were tested for viral load. Genotypic studies were performed with viral RNA from plasma using the consensus technique of the Agence Nationale de Recherche sur le SIDA (www.hivfrenchresistance.org). Mutations were identified using the International AIDS Society-USA resistance panel and resistant virus was defined according to the ANRS algorithm. RESULTS: A panel of 2423 individuals representing the general population of Djibouti was included. Antibodies were detected in 53 of 2423 samples tested. The HIV-1 seroprevalence in the general population was 2.2%. Genotype C was the most prevalent, and the other isolates were CRF_02 AG, or subtype A or D. Forty-seven of the 53 samples were tested for genotypic resistance, and mutations concerning all three classes of antiretrovirals were found. The most frequent were secondary mutations associated with protease inhibitors (PIs): M36I, R41K and K20I/R. A few strains displayed primary mutations (the non-nucleoside reverse transcriptase inhibitor [NNRTI]-associated mutations K101E, K103T, L100I and G190V; the PI-associated mutation N88D; and the NRTI-associated mutation K65R). The presence of these mutations may be due to the transmission of strains from treated patients. CONCLUSION: Substantial polymorphism and a few primary mutations are found in HIV-1 non-B subtype isolates from Djiboutian antiretroviral-drug-naive individuals. This needs to be taken into account to adapt antiretroviral regimens and prophylactic schedules locally.

Abstract: The efficacy of nets treated with lambda-cyhalothrin, a pyrethroid insecticide, on malaria infection and disease was assessed for the first time at the community level in Anopheles gambiae pyrethroid resistance areas. The study was carried out in northern Cote d'Ivoire, which is an area of kdr resistance. Four pairs of villages were selected and matched according to demographic, sociological, and ecological criteria. Among each pair, a village was randomly allocated to receive mosquito nets. More than 80% of beds were covered with nets treated with lambda-cyhalothrin and retreated after 6 months. In each village, 54 children aged 0-59 months were randomly selected and clinically monitored for 8 periods of 7 days throughout the year. Results showed that the efficacy of treated nets was maintained with a reduction of the prevalence of asymptomatic malaria infection by 12% and an estimated protective efficacy against malaria disease of 56%.

Abstract: The purpose of this article is to describe various models used for the study of malaria. The type of models depends on the focus of study. Until now the most models have been designed to study malaria transmission. In addition to giving a basic description of a few classic models, we show how simulation can be useful despite the inherent simplicity of models. In our opinion it is urgent to develop models for use in public health based on recent advances in computer, automation, and mathematical techniques.

Abstract: Seven repeated cross-sectional parasitological surveys, collecting a total of 13,912 blood samples, were carried out from September 1995 to February 1998 in three irrigated rice growing villages and three villages without irrigated agriculture in the area surrounding Niono, Mali. Parasite prevalence varied according to season and agricultural zone, but showed similar patterns for villages within the same zone. Overall, malaria prevalence was 47% in the villages without irrigated agriculture and 34% in the irrigated rice growing villages. In a village in the irrigated zone, and a village in the non-irrigated zone, 1067 and 608 children up to the age of 14 years, respectively, were followed in a passive malariological study for the period of 13 months. Fevers were attributed to malaria using a statistical method, taking into account the parasitaemia in afebrile controls from the cross-sectional surveys. The incidence of malaria fevers differed markedly between the two zones and over time. In the village in the irrigated zone, the incidence of malaria fevers was fairly constant over the year at 0.7 per 1000 children per day. In the village without irrigated agriculture, incidence was low during the dry season (at 0.6 per 1000 children per day), whereas it was high during the rainy season (at 3.2 per 1000 children per day). These results correspond well to the malaria transmission observed in a concurrent entomological survey. Rice cultivation in the semi-arid sub-Saharan environment altered the transmission pattern from seasonal to perennial, but reduced annual incidence more than two-fold.

Abstract: The ability of four 9,10-dihydroethanoanthracene derivatives (BG920, BG932, BG958, and BG996), as well as verapamil and promethazine, to reverse chloroquine resistance was assessed against 24 chloroquine-resistant and 10 chloroquine-susceptible strains of Plasmodium falciparum from different countries. The 9,10-dihydroethanoanthracene derivatives clearly increase chloroquine susceptibility only in chloroquine-resistant isolates.

Abstract:

Abstract: BG958 reverses resistance in chloroquine-resistant isolates from different countries. Five mutations in the Plasmodium falciparum crt (pfcrt) gene resulting in the amino acid changes K76T, M74I, N75E, A220S, and R371I are systematically identified in resistance-reversed Asian, African, and Brazilian parasites which possess the pfcrt (CIET) haplotype. In combination with BG958, the activity of chloroquine is increased in parasites with the N86Y mutation in pfmdr1.

Abstract:

Abstract: Our study demonstrates the capacity of FR160, a catechol iron chelator, to reach and accumulate into infected Plasmodium falciparum erythrocytes and parasites (cellular accumulation ratio between 12 and 43). Steady-state FR160 accumulation is obtained after 2 hr of exposure. After 2 hr exposure, it reaches intracellular levels that are 4- to 10-fold higher in infected red blood cells than those attained in normal erythrocytes. There is quite a good correlation between the accumulation of chloroquine and FR160 in the different strains (r=0.939) and in the IC(50) values (r=0.719). In contrast, the accumulation of FR160 and its activity is poorly correlated (r=0.500), suggesting that activity of FR160 may be independent of its penetration into infected erythrocytes. The mechanism of accumulation is yet unknown but based on inhibitor studies, the uptake of FR160 seems to be not associated with the calcium pump or channel, the potassium channel or the Na(+)/H(+) exchanger. Combinations of FR160 with verapamil, diltiazem, clotrimazole, amiloride, diazoxide, 4-aminopyridine, and picrotoxin should be avoided (antagonistic effects). The potent in vitro activity of FR160 on chloroquine-resistant strains or isolates, its lower toxicity against Vero cells, its mechanisms of action, its capacity to reach rapidly and accumulate into infected erythrocytes suggest that FR160 holds much promise as a new structural lead and effective antimalarial agent or at least a promising adjuvant in treatment of malaria.

Abstract: An epidemiological survey was conducted during a 4-month period of intense malaria transmission in Dielmo, a holoendemic Senegalese village. Two thick blood smears per inhabitant were collected weekly. The sex ratio of Plasmodium falciparum gametocytes (gamete precursors) was studied in 50 gametocyte carriers. All age classes were represented (mean 19.7 years; range: 2 months-75 years); 42 (84%) of them did not receive antimalarial treatment. Overall 668 thick smears were examined until 100 gametocytes had been counted or for 40 min. A total of 11204 gametocytes were observed with a mean sex ratio of 0.346 (95% CI 0.317-0.374), i.e. 2.89 females per 1 male. Among the 284 thick smears in which at least 10 gametocytes were observed, the mean percentage of male gametocytes was 27.8%, with a range of 0-82%. Great variability was observed between gametocyte carriers and also between thick smears from the same gametocyte carrier. A multivariate analysis was performed which highlighted the fact that only 2 variables had a significant effect on the sex ratio. Anaemia was associated with an increased percentage of males (Prevalence Rate Ratio [PPR] of male gametocytes was multiplied by 1.65 if haematocrit rate < 32%) and a wave of gametocytes was associated with an increased percentage of female gametocytes (PRR was multiplied by 0.48 during the peak of gametocytaemia and for the 2 weeks following this peak). The variables without significant effect on sex ratio were: age, sex, clinical status and sickle cell trait status of the gametocyte carrier, density of asexual parasites, quinine treatment, and gametocyte density (when taking account of its waves). These results are discussed in regard of possible differential production, mortality or sequestration of one gametocyte sex and selective advantages for the transmission of parasites.

Abstract: In sub-Saharan Africa, lowlands developed for rice cultivation favour the development of Anopheles gambiae s. l. populations. However, the epidemiological impact is not clearly determined. The importance of malaria was compared in terms of prevalence and parasite density of infections as well as in terms of disease incidence between three agroecosystems: (i) uncultivated lowlands, 'R0', (ii) lowlands with one annual rice cultivation in the rainy season, 'R1' and (iii) developed lowlands with two annual rice cultivation cycles, 'R2'. We clinically monitored 2000 people of all age groups, selected randomly in each agroecosystem, for 40 days (in eight periods of five consecutive days scheduled every 6 weeks for 1 year). During each survey, a systematic blood sample was taken from every sick and asymptomatic person. The three agroecosystems presented a high endemic situation with a malaria transmission rate of 139-158 infective bites per person per year. The age-standardized annual malaria incidence reached 0.9 malaria episodes per person in R0, 0.6 in R1 and 0.8 in R2. Children from 0 to 9-year-old in R0 and R2 had two malarial attacks annually, but this was less in R1 (1.4 malaria episodes per child per year). Malaria incidence varied with season and agroecosystem. In parallel with transmission, a high malaria risk occurs temporarily at the beginning of the dry season in R2, but not in R0 and R1. Development of areas for rice cultivation does not modify the annual incidence of malarial attacks despite their seasonal influence on malaria risk. However, the lower malaria morbidity rate in R1 could be explained by socio-economic and cultural factors.

Abstract: The epidemiological hallmark of the new millennium has been the emergence or recrudescence of transmissible diseases with high epidemic potential. Disease tracking is becoming an increasingly global task requiring implementation of more and more sophisticated control strategies and facilities for sustainable development. A promising initiative involves the use of satellite technology to monitor and forecast the spread of disease. The Health Early Warning System (HEWS) was designed based on successful application of satellite data in food programs as well as in other areas (e.g. weather, farming and fishing). The HEWS integrates data from communications, remote-sensing and positioning satellites. The purpose of this review is to present the main studies containing satellite data on public health in tropical areas. Satellite data has allowed development of more reactive epidemiological tracking networks better suited to increasing population mobility, correlation of environmental factors (vegetation index, rainfall and ocean surface color) with human, animal and insect factors in epidemiological studies and assessment of the role of such factors in the development or reappearance of disease. Satellite technology holds great promise for more efficient management of public health problems in tropical areas.

Abstract: A site-based characterization of Plasmodium falciparum infections in children living in two malaria hyperendemic urban areas from West and Central Africa was undertaken. A total of 58 and 46 children with either asymptomatic infections or uncomplicated (symptomatic) malaria were recruited in Gabon and Benin, respectively. Parasite density, hematological factors, the genetic diversity of P. falciparum merozoite surface protein 2 (msp2) and the complexity of infections (mean number of P. falciparum genotypes per infected child) were used for this characterization. Gabonese children with uncomplicated malaria presented a higher mean axillary temperature (39.2 vs 38.6, P=0.004) and a higher geometric mean parasite density (30,538 vs 18,921, P<0.001) associated with a significantly lower hemoglobin level ( P<0.01). A higher degree of msp2 polymorphism and the complexity of P. falciparum infections were also observed in children from Gabon ( P<0.05). With a similar level of malaria transmission in both urban sites, these results suggest an impact of malaria control interventions on the dynamics of concurrent P. falciparum infections.

Abstract: OBJECTIVE: To compare the efficacy of seven pyrethroid insecticides for impregnation of mosquito nets, six currently recommended by WHO and one candidate (bifenthrin), under laboratory conditions. METHODS: Tests were conducted using pyrethroid-susceptible and pyrethroid-resistant strains of Anopheles gambiae and Culex quinquefasciatus. Knock-down effect, irritancy and mortality were measured using standard WHO cone tests. Mortality and blood-feeding inhibition were also measured using a baited tunnel device. FINDINGS: For susceptible A. gambiae, alpha-cypermethrin had the fastest knock-down effect. For resistant A. gambiae, the knock- down effect was slightly slower with alpha-cypermethrin and much reduced following exposure to the other insecticides, particularly bifenthrin and permethrin. For susceptible C. quinquefasciatus, the knock-down effect was significantly slower than in A. gambiae, particularly with bifenthrin, and no knock-down effect was observed with any of the pyrethroids against the resistant strain. Bifenthrin was significantly less irritant than the other pyrethroids to susceptible and resistant A. gambiae but there was no clear ranking of pyrethroid irritancy against C. quinquefasciatus. In tunnels, all insecticides were less toxic against C. quinquefasciatus than against A. gambiae for susceptible strains. For resistant strains, mortality was significant with all the pyrethroids with A. gambiae but not with C. quinquefasciatus. Inhibition of blood-feeding was also high for susceptible strains of both species and for resistant A. gambiae but lower for resistant C. quinquefasciatus; bifenthrin had the greatest impact. CONCLUSIONS: Efficacy for impregnation of mosquito nets against A. gambiae was greatest with alpha-cypermethrin. Bifenthrin is likely to have a significant comparative advantage over other pyrethroids in areas with pyrethroid resistance because of its much stronger impact on the nuisance mosquito, C. quinquefasciatus, despite its slower knock-down effect and irritancy. Selection of pyrethroids for mosquito vector control and personal protection should take into account the different effects of these insecticides, the status of pyrethroid resistance in the target area, and the importance of nuisance mosquitoes, such as C. quinquefasciatus.

Abstract: French troops were sent to the Ivory Coast on September 22, 2002 within the framework of Operation Unicorn in response to the political unrest. From September 22 to October 20, a total of 37 cases of malaria were reported, i.e., 35.7 cases per 1000 man-months. As of October 11, the central headquarters of the Armed Services Health Corps decided to use doxycycline as the exclusive agent for drug prophylaxis in military personnel on duty in the Ivory Coast and to enhance vector control measures. The incidence of malaria decreased to 2 cases per 1000 man-months at the sixth month. A recrudescence of malaria to 15 cases per 1000 man-months was observed with the rainy season in April. During this period one person presenting severe malaria with coma required emergency evacuation to France. In May 2003, several studies were undertaken to determine the factors that caused this recrudescence. These studies included surveys to evaluate awareness concerning malaria and monitor compliance with drug prophylaxis and tolerance of doxycycline, a case-control study to identify factors related to malarious episodes and an entomological study. Awareness of malaria was high with 75% of the 477 respondents stating that malaria could be transmitted by single mosquito bite. The case-control study showed a correlation between occurrence of malarious bouts and non-compliance with drug prophylaxis (p < 10(-5)). The odds-ratio was 3.05 (95% confidence interval, 1.52-6.14) for subjects claiming zero to one incident of non-compliance per week and 7.51 (IC95%, 3.24-17.40) for those claiming more than one incident of non-compliance per week. Tolerance of doxycyline was good since 72% of respondents reported no adverse effects. The main vector was Anopheles gambiae. The number of bites per man per night ranged from 25 to 2 and the number of infected bites ranged from 2 to 3 per week. Treatment was initiated promptly using quinine at a total dose of 25 mg/kg in 3 daily doses for 7 days by the intravenous then oral route. This experience shows that malaria remains a major concern for military forces, that standardization of preventive measures in emergency situations is needed, and that enhanced vector control, verification of compliance with drug prophylaxis and prompt treatment based on the presence of a physician in each emergency outpost is crucial. These recommendation must be applied to all French military personnel in the Ivory Coast.

Abstract: Malaria is a major cause of childhood mortality and morbidity in endemic areas. The incidence and severity of malaria depends on various entomological, parasitological, environmental, and human factors. Clinical presentation and epidemiologic features vary according to genetic factors, personal behavior, and immune status. Populations exposed to frequent infection may develop partial labile protective malarial immunity at the price of high morbidity and mortality due to ineffectiveness of antimalarials. Malarial immunity affects not only mortality and severity of malaria but also uncomplicated malarial attacks and plasmodium infection. The number of determining factors involved in relatively limited but, like a game of chess, their possible combinations are numerous. A better understanding of malaria is necessary to plan and implement effective control strategies. That is the aim of this review.

Abstract: The relationship between Plasmodii transmission, infection, morbidity, genetic susceptibility and acquisition of natural immunity is studied among two cohorts in the Senegalese villages of Dielmo (300 inhabitants) and Ndiop (350 inhabitants) where malaria is holoendemic (about 200 P. falciparum infective bites/person/year) and mesoendemic (about 20 P. falciparum infective bites/person/year), respectively. The populations are under a daily active clinical survey. Blood samples are collected at least once per month. Plasma and red blood cells are stored in bio-libraries that allow longitudinal studies of the immune responses against plasmodial antigens and the investigation of the natural history of P. falciparum infections by molecular genotyping methods.

Abstract: Blackwater fever (BWF), one of the commonest causes of death of Europeans living in Africa at the beginning of the twentieth century, but rarely diagnosed since the 1950s, is related to Plasmodium falciparum malaria but there is considerable debate and controversy about its aetiology. From 1990 to 2000, the whole population of Dielmo, a village in Senegal, was involved in a prospective study of malaria. Three cases of BWF occurred in 3 children aged 4, 7 and 10 years, belonging to a subgroup of children who suffered malaria attacks every 4 to 6 weeks over many years, who had received repeated quinine treatment. The spread of chloroquine resistance, by increasing the use of more toxic alternative drugs, may expose endemic populations to a high incidence of severe side effects of antimalarials.

Abstract: The relevance of World Health Organization (WHO) criteria for severe malaria has not been assessed in non-immune children. The objectives of this study were (i) to evaluate the significance of 1990 WHO definition reconsidered in 2000 on distribution and lethality of severe cases in children admitted with falciparum malaria, and (ii) to contribute to the study of relevance of the WHO severe criteria in Dakar, an hypoendemic area in Senegal. PATIENTS AND METHODS: The 1990 WHO criteria, respiratory distress and platelet counts were prospectively collected in 1997-99 from children admitted to Hopital Principal de Dakar, Senegal, with falciparum malaria diagnosed on a thick blood film. This method allowed also the definition of severe cases according to 2000 WHO criteria. RESULTS: Among 311 patients (median age: 8 years old), according to the 2000 WHO criteria, the frequency of severe malaria cases was increased by 23% (75% versus 52%) and case-fatality rates thereof were decreased by 5% (17% versus 12%) compared with 1990 WHO definition. One death occurred among cases defined as severe on admission only according to criteria modified by WHO in 2000. A multivariate logistic regression model identified several independent prognostic factors: cerebral malaria, hypoglycaemia, respiratory distress, renal failure, collapse, abnormal bleedings, pupillary abnormalities and thrombocytopaenia defined as a platelet count below 100,000/mm3. A significant association (p < 0.001) was observed between platelet count increase and consciousness level improvement, evaluated on day of first platelet count control (time from admission: 1-7 d). Among survivors, a lesser improvement in coma score was associated with a decrease in platelet counts (p < 0.04). CONCLUSIONS: The 1990 WHO criteria, which predicted death among malaria cases in children living under stable falciparum transmission, are relevant in this series of non-immune children living in a low and seasonal transmission. Nevertheless new WHO criteria showed poor prognostic significance. However, the 2000 WHO definition was highly sensitive to detect severe malaria cases. These findings should be considered for managing severe malaria in migrant children.

Abstract: The effects of a series of dihydroethano- and ethenoanthracene derivatives on chloroquine (CQ) accumulation in CQ-susceptible strain 3D7 and CQ-resistant clone W2 were assessed. The levels of CQ accumulation increased little or none in CQ-susceptible strain 3D7 and generally increased markedly in CQ-resistant strain W2. At 10 microM, 28 compounds yielded cellular accumulation ratios (CARs) greater than that observed with CQ alone in W2. At 10 microM, in strain W2, 21 of 31 compounds had CQ CARs two or more times higher than that of CQ alone, 15 of 31 compounds had CQ CARs three or more times higher than that of CQ alone, 13 of 31 compounds had CQ CARs four or more times higher than that of CQ alone, and 9 of 31 compounds had CQ CARs five or more times higher than that of CQ alone. At 1 microM, 17 of 31 compounds had CQ CARs two or more times higher than that of CQ alone, 12 of 31 compounds had CQ CARs three or more times higher than that of CQ alone, 6 of 31 compounds had CQ CARs four or more times higher than that of CQ alone, and 3 of 31 compounds had CQ CARs five or more times higher than that of CQ alone. At 1 microM, 17 of 31 compounds were more potent inducers of CQ accumulation than verapamil and 12 of 31 compounds were more potent inducers of CQ accumulation than promethazine. The nature of the basic group seems to be associated with increases in the levels of CQ accumulation. At 1 and 10 microM, 10 of 14 and 13 of 14 compounds with amino group (amines and diamines), respectively, had CARs >or=3, while at 1 and 10 microM, only 1 of the 13 derivatives with amido groups had CARs >or=3. Among 12 of the 31 compounds which were more active inducers of CQ accumulation than promethazine at 1 microM, 10 had amino groups and 1 had an amido group.

Abstract: The spread of antimalarial drug resistance has major consequences for malaria control in tropical Africa. Here, the impact of chloroquine resistance on the burden of malaria is analyzed and its implications for the Roll Back Malaria initiative are examined. Malaria mortality has increased at least twofold during the past two decades. Combination therapy should be available for home treatment of young children. The potential toxicity of most antimalarials will require special surveillance programs. The main contribution to malaria control using methods to reduce the entomological inoculation rate is expected in areas with low or unstable transmission. Classic vector-control methods could potentially eliminate malaria in most urban areas and such programs deserve high priority.

Abstract: 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.

Abstract: 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.

Abstract: FR160, a catechol iron chelator, and tetracyclines or norfloxacin exert in vitro additive or synergistic activity against a chloroquine-resistant Plasmodium falciparum clone. FR160 shows antagonistic effects in association with macrolides, ofloxacin, and rifampin.