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Malaria is one of the world׳s most widespread parasitic diseases. The parasitic protozoans of the genus Plasmodium have developed resistance to several antimalarial drugs. Some patients are therefore infected by two or more strains with different levels of antimalarial drug sensitivity. We previously developed a model to estimate the drug concentration (IC50) that inhibits 50% of the growth of the parasite isolated from a patient infected with one strain. We propose here a new Two-Slopes model for patients infected by two strains. This model involves four parameters: the proportion of each strain and their IC50, and the sigmoidicity parameter. To estimate the parameters of this model, we have developed a new algorithm called PGBO (Population Genetics-Based Optimizer). It is based on the Metropolis-Hasting algorithm and is implemented in the statistical software R. We performed a simulation study and defined three evaluation criteria to evaluate its properties and compare it with three other algorithms (Gauss-Newton, Levenberg-Marquardt, and a simulated annealing). We also evaluated it using in vitro data and three ex vivo datasets from the French Malaria Reference Center. Our evaluation criteria in the simulation show that PGBO gives good estimates of the parameters even if the concentration design is poor. Moreover, our algorithm is less sensitive than Gauss-Newton algorithms to initial values. Although parameter estimation is good, interpretation of the results can be difficult if the proportion of the second strain is close to 0 or 1. For these reasons, this approach cannot yet be implemented routinely., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Background: Malaria Rapid Diagnostic Tests (RDTs) are widely used to diagnose malaria. The present study evaluated a new RDT, the Clearview® Malaria pLDH test targeting the pan-Plasmodium antigen lactate dehydrogenase (pLDH)., Methods: The Clearview® Malaria pLDH test was evaluated on fresh samples obtained in returned international travellers using microscopy corrected by PCR as the reference method. Included samples were Plasmodium falciparum (139), Plasmodium vivax (22), Plasmodium ovale (20), Plasmodium malariae (7), and 102 negative., Results: Overall sensitivity for the detection of Plasmodium spp was 93.2%. For P. falciparum, the sensitivity was 98.6%; for P. vivax, P. ovale and P. malariae, overall sensitivities were 90.9%, 60.0% and 85.7% respectively. For P. falciparum and for P. vivax, the sensitivities increased to 100% at parasite densities above 100/μl. The specificity was 100%. The test was easily to perform and the result was stable for at least 1 hour., Conclusion: The Clearview® Malaria pLDH was efficient for the diagnosis of malaria. The test was very sensitive for P. falciparum and P. vivax detection. The sensitivities for P. ovale and P. malariae were better than other RDTs.

Artemisinin combination therapy (ACT) paves the way for new opportunities to eliminate malaria in the tropics. However, the huge increase of ACT consumption raises major concerns about their availability over the next few years. At the same time a decrease in their efficacy has already been reported. Alongside the deployment of multifocal control programs, the process ranging from artemisia crop production to accreditation of new ACT combinations urgently needs to be strengthened to supply sufficient quantities of high-quality drugs. New suppliers will have the opportunity to enter this market to develop new formulations, and bioequivalence studies are required to validate these new formulations. It is thus crucial for national malaria control teams to be able to better scrutinize the dossier of these new formulations., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Since the 1970's, the diversity of Plasmodium parasites in African great apes has been neglected. Surprisingly, P. reichenowi, a chimpanzee parasite, is the only such parasite to have been molecularly characterized. This parasite is closely phylogenetically related to P. falciparum, the principal cause of the greatest malaria burden in humans. Studies of malaria parasites from anthropoid primates may provide relevant phylogenetic information, improving our understanding of the origin and evolutionary history of human malaria species. In this study, we screened 130 DNA samples from chimpanzees (Pan troglodytes) and gorillas (Gorilla gorilla) from Cameroon for Plasmodium infection, using cytochrome b molecular tools. Two chimpanzees from the subspecies Pan t. troglodytes presented single infections with Plasmodium strains molecularly related to the human malaria parasite P. ovale. These chimpanzee parasites and 13 human strains of P. ovale originated from a various sites in Africa and Asia were characterized using cytochrome b and cytochrome c oxidase 1 mitochondrial partial genes and nuclear ldh partial gene. Consistent with previous findings, two genetically distinct types of P. ovale, classical and variant, were observed in the human population from a variety of geographical locations. One chimpanzee Plasmodium strain was genetically identical, on all three markers tested, to variant P. ovale type. The other chimpanzee Plasmodium strain was different from P. ovale strains isolated from humans. This study provides the first evidence of possibility of natural cross-species exchange of P. ovale between humans and chimpanzees of the subspecies Pan t. troglodytes.

Background: Malaria diagnosis is vital to efficient control programmes and the recent advent of malaria rapid diagnostic tests (RDTs) provides a reliable and simple diagnostic method. However a characterization of the efficiency of these tests and the proteins they detect is needed to maximize RDT sensitivity., Methods: Plasmodial lactate dehydrogenase (pLDH) gene of wild isolates of the four human species of Plasmodium from a variety of malaria endemic settings were sequenced and analysed., Results: No variation in nucleotide was found within Plasmodium falciparum, synonymous mutations were found for Plasmodium malariae and Plasmodium. vivax; and three different types of amino acid sequence were found for Plasmodium ovale. Conserved and variable regions were identified within each species., Conclusion: The results indicate that antigen variability is unlikely to explain variability in performance of RDTs detecting pLDH from cases of P. falciparum, P. vivax or P. malariae malaria, but may contribute to poor detection of P. ovale.

Intrinsic resistance of Plasmodium falciparum is clearly a major determinant of the clinical failure of antimalarial drugs. However, complex interactions between the host, the parasite and the drug obscure the ability to define parasite drug resistance in vivo. The in vitro antimalarial drug susceptibility assay determines ex-vivo growth of parasite in the presence of serial drug concentrations and, thus, eliminates host effects, such as drug metabolism and immunity. Although the sensitivity of the parasite to various antimalarials provided by such a test provides an important indicator of intrinsic parasite susceptibility, there are fundamental methodological issues that undermine comparison of in vitro susceptibility both between laboratories and within a single laboratory over time. A network of laboratories is proposed that will agree on the basic parameters of the in vitro test and associated measures of quality control. The aim of the network would be to establish baseline values of sensitivity to commonly used antimalarial agents from key regions of the world, and create a global database, linked to clinical, molecular and pharmacology databases, to support active surveillance to monitor temporal trends in parasite susceptibility. Such a network would facilitate the rapid detection of strains with novel antimalarial resistance profiles and investigate suitable alternative treatments with retained efficacy.

Congenital malaria (CM) has been considered to be rare, even in malaria-endemic areas but the disease can result in significant neonatal morbidity. Because of its rarity, the disease may go undiagnosed for a prolonged period in a seriously ill infant. We report the first case of Plasmodium malariae CM from a HIV mother. HIV could have facilitated the transfer of erythrocytic persistent P. malariae through the placenta to the fetus.

Background: The study of urban malaria is an area undergoing rapid expansion, after many years of neglect. The problem of over-diagnosis of malaria, especially in low transmission settings including urban areas, is also receiving deserved attention. The primary objective of the present study was to assess the frequency of malaria among febrile outpatients seen in private and public primary care facilities of Antananarivo. The second aim was to determine, among the diagnosed malaria cases, the contribution of autochthonous urban malaria., Methods: Two cross-sectional surveys in 43 health centres in Antananarivo in February 2003 (rainy season) and in July 2003 (dry season) were conducted. Consenting clinically suspected malaria patients with fever or history of fever in the past 48 hours were included. Malaria rapid diagnostic tests and microscopy were used to diagnose malaria. Basic information was collected from patients to try to identify the origin of the infection: autochthonous or introduced., Results: In February, among 771 patients, 15 (1.9%) positive cases were detected. Three malaria parasites were implicated: Plasmodium. falciparum (n = 12), Plasmodium vivax (n = 2) and Plasmodium. ovale (n = 1). Only two cases, both P. falciparum, were likely to have been autochthonous (0.26%). In July, among 739 blood smears examined, 11 (1.5%) were positive: P. falciparum (n = 9) and P. vivax (n = 2). Three cases of P. falciparum malaria were considered to be of local origin (0.4%)., Conclusion: This study demonstrates that malaria cases among febrile episodes are low in Antananarivo and autochthonous malaria cases exist but are rare.

The number of travellers in malaria striken areas increases each year (2). The risk of infection is high in Sub-Saharan Africa, but appropriate chemoprophylaxis can reduce the morbidity and mortality rate of malaria. Half of the samples of malaria cases received by the National reference centre of malaria chemosensibility (CNRCP) for chemosensibility analysis came from two hospitals in the north of Paris: Bichat Claude Bernard in Paris and Delafontaine in Saint-Denis. In 2000, quite all the malaria cases (n = 387) observed at the Bichat and Delafontaine Hospitals came from Africa (99%). Plasmodium falciparum remains the most represented (87.6%) species, with an average parasitic density of 0.3%. Patients with P falciparum came for medical advice on the tenth day after return (median, extremes 0-174 days). More than half of the patients (58%) did not take any medication for chemoprophylaxis and even if they took some, it was irregular or inappropriate. The most used drug chemoprophylaxis is the association of chloroquine and proguanil or Savarine. In 15% of the cases, the travellers took chloroquine as a prophylaxis and 4% other medicine not recommended by the French authorities. An average of 43.7% of these travellers took inappropriate chemoprophylaxis. In total, 27 chemoprophylaxis failures are reported. Some patients (22%) have already taken self treatment which was readjusted during admission at hospital. The first treatment of malaria in 2000 was monotherapy with quinine (P. falciparum) and chloroquine (P. ovale, malariae, vivax). The treatment associations in case of suspicious resistance were quinine + doxycycline and atovaquone + proguanil. Treatment failure was infrequent and resulted above all from a bad observance. More information should be given to travellers as well as doctors about recommendations and treatments.

Background: The differentiation of malaria from other causes of fever is difficult. The development of tools for rapid and specific clinical diagnosis is of paramount importance for the identification of individuals infected with malaria., Method: A 4-year prospective study to identify the clinical and biological variables associated with malaria included all patients suspected of having malaria who presented in the emergency department (ED) of a French hospital., Results: Of 783 patients admitted to the ED with suspected malaria, 145 had positive findings of a thick smear for Plasmodium species, mainly Plasmodium falciparum (90.3%). In univariate analysis, the following 12 variables were significantly associated with diagnosis of malaria: older than 30 years, male sex, immigration to France from an area where malaria is endemic, a visit to sub-Saharan Africa, insufficient antimalaria prophylaxis, fever, chills, absence of diarrhea, a leukocyte count within the reference range, thrombocytopenia, and increased lactate dehydrogenase and bilirubin levels. In multivariate analysis, the factors predictive of malaria included a visit to sub-Saharan Africa (odds ratio [OR], 7.7; 95% confidence interval [CI], 2.8-21.3), a temperature of at least 38.5 degrees C (OR, 6.2; 95% CI, 2.8-13.3), chills (OR, 3.0; 95% CI, 1.4-6.6), thrombocytopenia (OR, 16.5; 95% CI, 7.1-38.3), and abnormally high total bilirubin levels (OR, 21.5; 95% CI, 6.4-72.5). However, alone or combined, these features had insufficient sensitivity (95.0%) and low specificity (55.0%) for the diagnosis of malaria., Conclusions: Malaria should be suspected in all patients presenting with complaints after travel to an area where malaria is endemic, and these patients should undergo blood microscopy.

The quantitative buffy coat system (QBC Test) was designed for rapid diagnosis of malaria by identifying the presence of hemoparasites. The main drawback of the technique is failure to identify the Plasmodium species. The purpose of this study was to attempt to remedy this problem by studying the distribution of the parasites at the bottom of the test tube. Indeed since the QBC Test is based on gradient centrifugation of blood components, the distribution of the parasites in the test tube depends on density. Blind QBC Tests were performed on specimens obtained from two different batches, i.e., one from France and the other from Burkina Faso. Distribution curves were obtained by counting the number of parasites in all microscopic fields in the five-millimeter test tube. Our findings showed differences in distribution curve depending on species. For Plasmodium falciparum, the number of parasites was nearly the same in all fields suggesting that the arrangement of the parasites in the QBC Test tube was linear. For Plasmodium vivax and Plasmodium ovale, the number of parasites was markedly lower near the cap of the tube suggesting that a non-linear arrangement with a decreasing number of parasites toward the top of the tube. In 97 p. 100 of cases, we were able to propose a differential diagnosis of Plasmodium falciparum versus Plasmodium vivax or Plasmodium ovale. However it was not possible to distinguish between Plasmodium vivax and Plasmodium ovale. In case of mixed infection it would be difficult to distinguish Plasmodium falciparum from the other species. The ability to identify Plasmodium species would add to the advantages of the rapid and sensitive QBC Test.

There is no longer malaria transmission in Europe and North America, while the transmission decreases in sub-tropical areas and increases in tropical countries. Most of malarias are now due to Plasmodium falciparum and happen in Africa. In the regions where the transmission is high, malaria is stable, baby mortality is high, and protective immunity is achieved in early childhood. Falciparum resistant malaria originates from mutations on drug target decreasing affinity to antifols, or mutations preventing accumulation of chloroquine in parasitized red blood cells. Resistance is a rapid event following large use of antifols, even associated, while falciparum chloroquine resistance is now widespread. Resistance to quinine, mefloquine and halofantrine is still at low levels out of Thailand, as their use remains through medical hands. Non resistance was observed yet with artemisinin derivatives.

The plasma concentrations of the enantiomers of halofantrine and its N-desbutyl metabolite in six patients with malaria were measured after oral administration of 3 x 750 mg doses of micronised, racemic halofantrine hydrochloride given at 6-hour intervals. Significant differences were observed between the plasma concentrations of the enantiomers both of halofantrine and its N-monodesbutyl metabolite. AUC(0)84h values were higher for (+)halofantrine (9917 micrograms.ml-1.h) than for (-)-halofantrine (6127 micrograms.ml-1.h). The clinical significance of these observations is not known. The isomers have equipotent activity in vitro but their relative toxicity has not yet been assessed.

The efficacy of a loading dose of 20 mg of chloroquine per kg of body weight per os given at intervals during the first day was evaluated in 27 patients in Madagascar with Plasmodium falciparum malaria. The conventional regimen of 25 mg/kg over 3 days (schedule 1) was thus compared with a regimen of 30 mg/kg over 2 days (schedule 2; one dose of 10 mg/kg followed by two doses of 5 mg/kg at 6-h intervals on the first day and two doses of 5 mg/kg at 12-h intervals on the second day) in terms of their clinical and parasitological efficacies, tolerance, and drug concentration-time curves. At 24 h schedule 2 gave higher chloroquine levels in blood, which induced a more rapid decrease in parasitemia. The time required for a 50% decrease in the initial parasitemia was shorter in patients on schedule 2 (14.3 +/- 1.6 h) than it was in patients on schedule 1 (35.5 +/- 5.4 h; P less than 0.01). Moreover, negative blood smears were obtained more rapidly with schedule 2 (50.8 +/- 3.7 h) than with schedule 1 (72 +/- 8.7 h). As predicted by the drug concentration-time curve, no high, potentially toxic peak drug concentration appeared and no adverse effects were observed with the loading dose regimen (schedule 2). These findings support the idea that a loading dose of 20 mg/kg given at intervals during the first 12 h is well tolerated and can be used to obtain a more rapid decrease in parasitemia and to shorten the treatment time of uncomplicated chloroquine-susceptible falciparum malaria in the field.

Mefloquine and halofantrine are quinine-related aminoalcohols recently introduced for the oral treatment (both drugs) and oral chemoprophylaxis (mefloquine) of malaria. Soon after these new drugs were launched the volume of sales testified to the need for alternative treatments in view of the resistance of Plasmodium falciparum to a variety of drugs, including chloroquine, amodiaquine and sulfadoxine-pyrimethamine. Quinine remains effective but it is difficult to handle. Both mefloquine and halofantrine are remarkably effective against the intraerythrocytic pathogenic forms of malaria; they also have a rapid and prolonged action and a low toxicity. Halofantrine is sometimes incompletely absorbed. Neurological side-effects, usually moderate, have been reported with mefloquinone. Partial cross-resistance has been demonstrated within each class of antimalarials: aminoalcohols, amino-4-quinoleins (chloroquine, amodiaquine) and antifolics-antifolinics (sulfamides, pyrimethamine, proguanil), but not between the classes. Further studies are necessary concerning the absorption and tolerability of mefloquine and halofantrine, as well as the regional level of P. falciparum sensitivity to these new drugs.

Between 1978 and 1989, some Plasmodium falciparum chloroquino-resistant (C.Q.R.) strains have been imported in all intertropical African countries. Evidence of sudden occurrence of C.Q.R. foci was obtained by some prophylactic or therapeutic failures, and culture of strains from non-immune travellers back in Europe or U.S.A., then by carrying out some field surveys. C.Q.R. heterogeneity is the rule in Africa, efficiency of other antimalarial drugs diminishes rapidly in the foci where C.Q.R. remains at high level, probably in relation with drug pressure. Non typical clinical manifestations raising of child death-rate death not avoidable by chloroquino-prophylaxis, confused therapeutic attitudes were all of them the main facts accompanying the occurrence of C.Q.R. in Africa. It is not possible nowadays to foresee the evolution of C.Q.R. situation and of its social consequences in low-immune African populations.

The good results achieved in the treatment of malaria with a 7 and 5 days one of quinine (orally dose 8 mg/kg/8 h), incited the authors to try a 3 days long treatment at the same dose. They experimented this protocol on hospitalised children. All patients were free of parasites at day 7 with an average residual concentration quinine of 2.7 +/- 0.8 mg/l. Among the 7 patients with malaria at day 14, 4 didn't require another treatment and 2 presented new infestation. 50% of plasmodial isolated strains were chloroquine resistant and 40% amodiaquine resistant. The efficiency of this protocol could be in favour of its larger use.

One thousand and twenty six P. falciparum strains isolated from cases imported in France and field surveys in four regions of Africa and Madagascar were studied in vitro against chloroquine, monodesethylamodiaquine, quinine and mefloquine, 917 in vivo tests were performed during field studies with chloroquine (10 and 25 mg/kg) and amodiaquine (10, 25, 35 mg/kg). In Madagascar, the chemoresistance remained low and stable during the study period, concerning mostly chloroquine (11% in vitro and in vivo) without obvious geographical variation. 25 mg/kg chloroquine or amodiaquine were satisfactory as respectively first and second line therapeutic regimen. In Central Africa, chemoresistance emerged with an epidemic profile and increased dramatically in disseminated urban focus. High level and prevalence of chloroquine resistance and multiresistance were observed few months after the index cases in these foci. In South West Cameroon, amodiaquine remained efficient as curative treatment but only at a dose of 35 mg/kg/5 days. Decrease of in vitro sensitivity and in vivo efficacy of quinine is a matter of concern. Given the heterogeneous and evolutive situation of drug resistance, the need for epidemiological surveillance and monitoring of P. falciparum drug sensitivity in Africa is obvious to adjust therapeutic regimen.

The synchronized cultures of Plasmodium falciparum were used to stimulate in vitro the chemiluminescence of human polymorphonuclear leukocytes in the presence of immune serum. The schizonts were concentrated by Percoll gradient centrifugation method (density 1.085 and osmolarity 285 mOsmol), and placed in culture, treated 6 hours later by sorbitol. Under incubation at constant temperature and pressure, the rate of synchronization reached 85% for schizonts during 5 replicative cycles. Every asexual stages of Plasmodium falciparum were used separately to stimulate polymorphonuclear leukocytes: merozoites were the most effective, followed by schizonts, trophozoites, and lastly supernatants of cultures containing degradation products of parasites.

We present two cases of Plasmodium falciparum malaria contracted in Douala despite adequate prophylaxis by Fansidar for one and by chloroquine for the other. Failure of curative treatment by Fansidar for the first case (in vitro chloroquine-resistant strain) and by amodiaquine plus erythromycin for the second. After these therapeutic failures, both patients presented without fever, but with splenomegaly and anaemia. The successful therapeutic was mefloquine.

The authors report a case of malaria from Burundi, due to chloroquine resistant Plasmodium falciparum. It is the second case of chloroquine resistance, confirmed by in vitro tests, in this country. Decreased sensitivity to quinine is demonstrated by in vivo study, and quinine assay. The isolate was sensitive to amodiaquine and mefloquine in vitro.

Anaemia during malaria is not completely understood. Fourteen cases of antierythrocyte autoimmunisation with anti-I specificity were investigated in a Paris hospital during a period of 2 years. The patients lived in Gabon and had not been taking antimalarial chemoprophylaxis. All had chronic malaria (strongly positive antimalarial immunofluorescent antibody tests). All the recognised caused for anti-erythrocyte autoimmunity were excluded. One possible explanation for these observations is some sort of interaction between I antigen and Plasmodium, the result of which facilitated penetration of the erythrocyte by the malarial parasite.

The authors report two additional cases of malaria due to chloroquine-resistant Plasmodium falciparum, one from Tanzania, the other from Kenya. In addition to the in vivo observations, these strains were thoroughly investigated by chloroquine assay and evaluation of ED50 using an original method (in vitro semi-microtest).

34 case-reports of type RI resistance of P. falciparum to chloroquine published between 1975 and 1982 are reviewed. The patients were non-immune travellers infected in East Africa and in the neighbouring islands, especially Tanzania, Kenya and Madagascar. The increase in number and level of resistance might be a problem in Africa. A monitoring system of the patient and in the field is proposed and the possibilities of prevention and control are discussed.

24 cases of Plasmodium falciparum malaria in a group of 73 non-immune subjects coming mostly from central Africa have been tested in vitro for chloroquine sensitivity: 5 are sensitive, 19 are resistant among whom one is also quinine-resistant. The comparison of the major clinical and biological features in the two groups by methods of statistical calculation shows that the parasitological diagnosis is retarded and that hemolysis is more frequent in the strains of Plasmodium falciparum resistant to chloroquine. Thrombopenia is more frequent with chemosensitives strains. In any case, the delay between the first symptoms and the parasitological diagnosis is increased with an average of 17 days in comparison to what was observed in the 1970's. Under the influence of several complex factors including human ones, the clinical aspects are changed in comparison to forms usually observed. In this context of an extension of the emergence of the chemoresistance and in the aim of efficiency, only the quinine I.V., with an average dose of 16.5 mg/kg per day of quinine-base, has been chosen as a curative treatment during 5 days allowing a rapid recovery without after-effects for all the cases.

The disposition of amopyroquin was investigated in 10 healthy volunteers after a single 2-mg/kg (body weight) intramuscular dose of amopyroquin base. The major form of the drug in plasma and in whole blood was nonmetabolized amopyroquin, and only very low levels of its primary amine derivative were detected. After a rapid absorption phase (15 min), levels in plasma declined, following a tri-exponential model with a terminal elimination half-life of 129.6 +/- 92.5 h. The apparent volume of distribution (V/F) and the systemic clearance (CL/F) were 238 +/- 75 liters/kg and 2,063 +/- 1,159 ml/min, respectively. The renal clearance, calculated by using urine excreted during the first 48 h, was 119 +/- 99 ml/min and represented about 6% of the systemic clearance. About 1.2 and 0.2% of the amopyroquin dose was excreted in the urine during the first 48 h as nonmetabolized amopyroquin and its primary amine metabolite, respectively. Twenty-two Plasmodium falciparum malaria patients were studied after treatment with one of the following regimens of intramuscularly injected amopyroquin base: 3 mg/kg (body weight), 6 mg/kg, or 6 mg/kg followed by 3 mg/kg 24 h later. Parasitemia was cleared at day 7 in one of six, four of seven, and seven of nine patients, respectively. On the basis of this study, a regimen of 12 mg/kg (body weight) administered in two or three injections is suggested.

In a patients with P. falciparum malaria contracted in Thailand, the course of the disease under treatment suggested resistance to both chloroquine and pyrimethamine-sulfadoxine, as well as reduced sensitivity to quinine. This was confirmed by in vitro tests on continuous culture of the strain. Therapeutic success was obtained with a quinine-cycline combination. The problems raised by the emergence of P. Falciparum strains resistant to polychemotherapy are emphasized.

Between April and June 1984, eight chloroquine-resistant P. falciparum malaria cases were observed in non-immune patients after their emergency sanitary return from NE Mozambique. In vivo resistance to amodiaquine and pyrimethamine were demonstrated from two isolates. Radical cure was obtained with mefloquine in all cases. These patients came from an expatriate community of 35 adult men among which six other chloroquine-resistant malarial cases were probable. The epidemic pattern of this chloroquine-resistant focus is underlined.