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The members of the Palurea Study Group were provided in such a way that they could not be indexed as collaborators on PubMed. The publisher apologizes for this error and is pleased to list the members of the group here.

In Africa, malaria kills one child each minute. It is also responsible for about one million deaths worldwide each year. Plasmodium falciparum, is the protozoan responsible for the most lethal form of the disease, with resistance developing against the available anti-malarial drugs. Among newly proposed anti-malaria targets, are the P. falciparum cyclin-dependent kinases (PfCDKs). There are involved in different stages of the protozoan growth and development but share high sequence homology with human cyclin-dependent kinases (CDKs). We previously reported the synthesis of CDKs inhibitors that are structurally-related to (R)-roscovitine, a 2,6,9-trisubstituted purine, and they showed activity against neuronal diseases and cancers. In this report, we describe the synthesis and the characterization of new CDK inhibitors, active in reducing the in vitro growth of P. falciparum (3D7 and 7G8 strains). Six compounds are more potent inhibitors than roscovitine, and three exhibited IC50 values close to 1 µM for both 3D7 and 7G8 strains. Although, such molecules do inhibit P. falciparum growth, they require further studies to improve their selectivity for PfCDKs.

Artemisinin-based combination therapies (ACTs) are the main option to treat malaria, and their efficacy and susceptibility must be closely monitored to avoid resistance. We assessed the association of Plasmodium falciparum polymorphisms and ex vivo drug susceptibility with clinical effectiveness. Patients enrolled in an effectiveness trial comparing artemether-lumefantrine ( n = 96), fixed-dose artesunate-amodiaquine ( n = 96), and sulfadoxine-pyrimethamine ( n = 48) for the treatment of uncomplicated malaria 2007 in Benin were assessed. pfcrt , pfmdr1 , pfmrp1 , pfdhfr , and pfdhps polymorphisms were analyzed pretreatment and in recurrent infections. Drug susceptibility was determined in fresh baseline isolates by Plasmodium lactate dehydrogenase enzyme-linked immunosorbent assay (ELISA). A majority had 50% inhibitory concentration (IC 50 ) estimates (the concentration required for 50% growth inhibition) lower than those of the 3D7 reference clone for desethylamodiaquine, lumefantrine, mefloquine, and quinine and was considered to be susceptible, while dihydroartemisinin and pyrimethamine IC 50 s were higher. No association was found between susceptibility to the ACT compounds and treatment outcome. Selection was observed for the pfmdr1 N86 allele in artemether-lumefantrine recrudescences (recurring infections) (4/7 [57.1%] versus 36/195 [18.5%]), and of the opposite allele, 86Y, in artesunate-amodiaquine reinfections (new infections) (20/22 [90.9%] versus 137/195 [70.3%]) compared to baseline infections. The importance of pfmdr1 N86 in lumefantrine tolerance was emphasized by its association with elevated lumefantrine IC 50 s. Genetic linkage between N86 and Y184 was observed, which together with the low frequency of 1246Y may explain regional differences in selection of pfmdr1 loci. Selection of opposite alleles in artemether-lumefantrine and artesunate-amodiaquine recurrent infections supports the strategy of multiple first-line treatment. Surveillance based on clinical, ex vivo , molecular, and pharmacological data is warranted.

The online version of this article (doi:10.1007/s00134-016-4356-x) contains supplementary material, which is available to authorized users.; International audience; PURPOSE: Prospective data on potential factors associated with severity of imported Plasmodium falciparum malaria are lacking. We evaluated whether several host- and parasite-related biomarkers may improve early severity evaluation.METHODS: Prospective multicenter observational study comparing uncomplicated and severe imported falciparum malaria in adults conducted in France in 52 units, from 2007 to 2010. Association of several host- and parasite-related biomarkers with severity of malaria was tested using univariate and multivariate analyses.RESULTS: Of 295 patients, 140 had uncomplicated malaria and 155 severe malaria (including very severe and less severe cases according to predefined criteria). Curative intravenous quinine treatment was used in 154/155 patients with severe malaria and atovaquone/proguanil in 74 % of patients with uncomplicated malaria. Hospital mortality was 5.2 % (8 patients), all in the severe malaria group. Among host-related biomarkers, CRP, procalcitonin, and sTREM-1 were significantly higher and albumin was significantly lower in severe versus uncomplicated malaria; only the last three biomarkers also differed significantly between the very and less severe malaria groups. Among parasite-related biomarkers, only plasma PfHRP2 was significantly higher in severe versus uncomplicated malaria and in very severe versus less severe malaria; parasitemia did not differ between very and less severe malaria. By multivariate analysis, only lower plasma albumin and higher sTREM-1 were associated with greater severity, with intermediate accuracies.CONCLUSIONS: During imported malaria, the most useful biomarkers associated with severity seem to be plasma albumin and sTREM-1; and among parasite-related parameters, PfHRP2 was more strongly associated with severity than parasitemia was.

International audience; BACKGROUND: Theoretical and experimental data support the geographic differentiation strategy as a valuable tool for detecting loci under selection. In the context of Plasmodium falciparum malaria, few populations have been studied, with limited genomic coverage. METHODS: We examined geographic differentiation in P. falciparum populations on the basis of 12 single-nucleotide polymorphisms (SNPs) in 4 genes encoding drug resistance determinants, 5 SNPs in 2 genes encoding antigens, and a set of 17 putatively neutral SNPs dispersed on 13 chromosomes. We sampled 326 parasite isolates representing 7 P. falciparum populations from regions with varied levels of malaria transmission (Gabon, Kenya, Madagascar, Mali, Mayotte, Haiti, and the Philippines). RESULTS: Frequencies of drug resistance alleles varied considerably among populations (mean F(ST), 0.52). In contrast, allele frequencies varied significantly less for antigenic and neutral SNPs (mean F(ST), 0.16 and 0.24, respectively). This contrasting pattern was more pronounced when only the African populations were considered. Signature of selection was detected for most of the resistant SNPs but not for the antigenic SNPs. CONCLUSION: These data further validate the utility of geographic differentiation for identifying loci under strong positive selection, such as drug resistance loci. This study also provides frequencies of molecular makers of resistance in some overlooked populations.

International audience; We sought to test the association of polymorphisms in Plasmodium falciparum nhe-1 (Pfnhe-1, gene PF13_0019) with in vitro susceptibility to quinine, which was previously reported in a limited number of reference strains or culture-adapted isolates. Determination of in vitro susceptibility to quinine, genotyping of Pfnhe-1 ms4760 microsatellite and polymorphism in codon 76 of Pfcrt were performed for 83 isolates obtained from symptomatic malaria-infected travelers returning from various African countries to France or from subjects living in Madagascar. Nineteen different ms4760 microsatellite profiles of Pfnhe-1 were found including 14 not previously described. Multivariate analysis showed no significant association between the in vitro susceptibility to quinine with particular ms4760 profiles. Contrary to previous reports, we only observed that the number of NHNDNHNNDDD repeats was positively associated with the increased IC50 of QN (P = 0.01). We concluded that the studied polymorphisms in Pfnhe-1 did not appear as valid molecular markers of in vitro susceptibility to quinine in P. falciparum isolates from Africa. Because we did not include any isolate of Asian origin in our series, these results did not exclude the possibility of regional associations, for example in South-East Asia.

Objectives: To study the relative impact of HIV-1 infection and associated immunodepression on the severity of Plasmodium falciparum malaria in adults returning from areas of endemic malaria. Methods: We conducted a cross-sectional study, based on data from 104 HIV-infected patients from the French Hospital Database on HIV cohort (FHDH-ANRS CO4) and 161 HIV-negative patients from Bichat hospital, with a diagnosis of imported P. falciparum malaria between 2000 and 2003. The severity of falciparum malaria episode was graded with World Health Organization (WHO) criteria 2000 or on 2007 French recommendations. Result: Depending on criteria used, 40% (WHO) and 28% (2007 French recommendations) of episodes of imported P. falciparum malaria in HIV-infected patients were classified as severe, compared with 21 % (WHO) and 11 % (2007 French recommendations) of episodes among HIV-negative patients. Among HIV-infected patients, the episodes were severe in between 22 (CD4 cell counts ≥350/μl) and 51 % (CD4 cell counts

Summary objective To investigate whether the severity of Plasmodium falciparum attack in endemic areas was associated with the multiplicity of infection (MOI) and ⁄ or with a particular genotype(s). method In two areas of different malaria transmission pattern in Madagascar (Sainte-Marie – mesoendemic and Tsiroanomandidy – hypoendemic) the number and the proportions of msp-2 genotypes within isolates were determined for each patient using a capillary electrophoresis genotyping method. DNA sequencing was performed to identify the msp-2 allelic family of dominant clones. results Eighty six uncomplicated and 33 severe cases were included in Sainte-Marie and 48 uncomplicated and 69 severe cases were included in Tsiroanomandidy. We found no association between the MOI and severity of malaria as the same mean number of msp-2 genotypes was found in isolates from uncomplicated and from severe malaria cases (3.72 and 3.73, respectively, P>0.05). The study of the association of dominant clones with clinical status showed no particular genotype or allelic family associated with malaria severity. conclusions Severity of malaria was not associated with higher MOI in our study. Severity did not appear restricted to some particular genotypes either. On the contrary, severe malaria appeared to be caused by very common genotypes in the studied areas. More comprehensive explorations including immunity and genetic factors of the host are needed to acquire new information about this complex condition.

Few immigrant travelers have Plasmodium falciparum infections greater than 2 months after leaving malaria-endemic areas. We conducted a case-control study to identify factors associated with prolonged P. falciparum infection in immigrant travelers. Results suggest that P. falciparum infection should be systematically suspected even months after travel especially in pregnant women and first-arrival immigrants. (authors)

Background Controversy exists about the management of patients with imported Plasmodium falciparum malaria. We postulated that rapid parasite clearance supports ambulatory care, or, conversely, that factors associated with longer parasite clearance time (PCT) could be used as hospitalization criteria. Methods Hospitalized patients with imported falciparum malaria recruited through one single travel clinic between 1993 and 2000. We used a linear regression to identify factors independently associated with PCT defined as the time in hours from antimalarial drug administration until the first negative malaria smear. Results Among 400 patients hospitalized with falciparum malaria, mean (range) PCT was 58 (1–189) hours. In multivariate analysis, severe malaria, gastrointestinal signs, initial temperature greater than or equal to 40°C, parasitemia greater than or equal to 1%, and platelet counts less than 50,000/μL were associated with longer PCT. Offering ambulatory care to patients aged 15 to 64 years with none of the factors associated with longer PCT in the study would have resulted in 147 (37%) patients receiving outpatient care. Conclusion Factors identified in this model may help physicians determine which P falciparum malaria patients can be treated on an ambulatory basis, and contribute to revisions of national guidelines for imported malaria management.

Background. Usefulness of sulfadoxine-pyrimethamine as first-line therapy for uncomplicated Plasmodium falciparum malaria and intermittent preventive treatment in pregnancy throughout sub-Saharan Africa is compromised by the spread of dhfr alleles associated with pyrimethamine resistance. A predominant haplotype associated with the N51I+C59R+S108N triple-mutant dhfr allele has been reported recently in 4 African countries. A more comprehensive picture of the evolution of this mutant allele in Africa is lacking. Methods. Seventy-five P. falciparum isolates carrying the wild-type dhfr allele and 204 carrying the triple-mutant dhfr allele from 11 African countries were selected. The genetic diversity of the chromosomes bearing these alleles was analyzed with 4 microsatellite markers closely linked to the dhfr gene. Results. Seventy-three different 4-locus haplotypes carrying the wild-type dhfr allele were found. By contrast, 175 (85%) of 204 isolates carrying the triple-mutant dhfr allele shared a unique haplotype, identical to the one identified in Thailand. For the remaining triple-mutant isolates and one isolate with the quadruple-mutant dhfr allele (N51I+C59R+S108N+I164L), haplotypes were closely related to the predominant haplotype by mutation or recombination. Conclusions. Migration of parasites carrying an ancestral triple-mutant dhfr allele drives the spread of dhfr alleles associated with pyrimethamine resistance throughout West and Central Africa.

Plasmodium falciparum malaria is a serious health hazard for travelers to malaria-endemic areas and is often diagnosed on return to the country of residence. We conducted a retrospective study of imported falciparum malaria among travelers returning to France from malaria-endemic areas from 1996 through 2003. Epidemiologic, clinical, and parasitologic data were collected by a network of 120 laboratories. Factors associated with fatal malaria were identified by logistic regression analysis. During the study period, 21,888 falciparum malaria cases were reported. There were 96 deaths, for a case-fatality rate of 4.4 per 1,000 cases of falciparum malaria. In multivariate analysis, risk factors independently associated with death from imported malaria were older age, European origin, travel to East Africa, and absence of chemoprophylaxis. Fatal imported malaria remains rare and preventable. Pretravel advice and malaria management should take into account these risk factors, particularly for senior travelers.

A method for simultaneous analysis of chloroquine, proguanil and their metabolites from a whole blood sample (80 microL) dried on a filter paper was developed. Sample preparation included a liquid extraction from the filter paper, followed by a solid-phase extraction (C18 Bond Elut cartridge). Separation was obtained by reverse-phase liquid chromatography (HPLC) using a gradient elution on an X-Terra column; UV detection was made at 254 nm. This assay was linear between 150 and 2500 ng mL(-1) for chloroquine (and metabolite) and 300 and 2500 ng mL(-1) for proguanil and cycloguanil. The lower limit of quantification was close to 50 ng mL(-1) for chloroquine (and its metabolite) and 100 ng mL(-1) for proguanil (and its metabolite). No chromatographic interference from endogenous compounds or other tested anti-malarial drugs was evidenced. Chromatographic separation takes about 40 min with a coefficient of variation below 10.3% for within- and between-batch precision. The paper sampling method was validated in 10 healthy subjects treated by Savarine. The stability of compounds and metabolites on the filter paper was evaluated at four temperatures (-20, +4, 20 and 50 degrees C) and for 1, 5 and 20 days. Cycloguanil concentrations were not influenced by storage conditions, whereas, high temperatures and prolonged storage decreased chloroquine and proguanil levels. The proposed HPLC assay is accurate, precise and cost-effective; it can be used for pharmacokinetic and epidemiological studies on anti-malarial treatments.

The chapter talks about the mechanisms of resistance to antiparasitic agents. Plasmodium falciparum, the most virulent species, which adapts more easily to environmental constraints in tropical areas, has become the dominant species, and its resistance to chloroquine, to sulfadoxine-pyrimethamine, and, further, to all known drugs has developed to various degrees. True resistance is more likely in anthroponotic forms of leishmaniasis, such as those caused by Leishmania donovani and Leishmania tropica, because the zoonotic species that infect primarily animals, with humans as an occasional host, rarely encounter drugs. Laboratory or field isolates with resistance to pentavalent antimonials are also killed effectively by paromomycin and miltefosine. Several factors contribute to the emergence of drug- resistant parasites. Public health interventions to correct these factors have not always been successful and would benefit from a better understanding of the drug resistance mechanisms used by parasites. These mechanisms are very diverse and have been difficult to study, but recent technological advances now provide long-awaited tools that will facilitate the task. The genome sequences for P. falciparum, T. vaginalis, L. major, T. brucei, and S. mansoni have been compiled. When these data are used, for example, in combination with microarray technology, where the DNA of drug-resistant parasite strains is compared to that of drugsusceptible strains, identification of the genes that confer resistance should proceed even more rapidly than in the last few years.

Drug susceptibility tests fall into four broad categories: in vivo tests, in vitro tests, tests with experimental animals, and molecular tests. These different categories of tests provide complementary information. At one end of the spectrum, molecular tests analyze parasites at their most basic biologic level, without any outside interference. Most drug resistance tests in malaria concern Plasmodium falciparum, the most prevalent and virulent species and the most prone to the development of resistance. Incorporating pharmacokinetic parameters is now considered essential for true resistance identification within partners of artemisinins, which are frequently poorly absorbed or slowly eliminated drugs. While treatment failures for Trichomonas vaginalis infections have often been disregarded as being due to poor patient compliance or rapid reinfection, there is an increasing recognition that true clinical resistance exists and may be increasing, thus necessitating accurate drug susceptibility testing of patient isolates. Treatment failure in leishmaniasis may result from either true drug resistance or patient immunodeficiencies that preclude effective chemotherapeutic action. Differentiation of these two possibilities is important for both individual patient care and general public health. A standardized in vivo method for detection of drug resistance that was designed for testing trypanosomes that infect cattle can also be applied to those that infect humans. Investigation of drug resistance mechanisms of schistosomes is more difficult and time-consuming than those associated with protozoan infections.

The extension of drug resistance among malaria-causing Plasmodium falciparum parasites in Africa necessitates implementation of new combined therapeutic strategies. Drug susceptibility phenotyping requires precise measurements. Until recently, schizont maturation and isotopic in vitro assays were the only methods available, but their use was limited by technical constraints. This explains the revived interest in the development of replacement methods, such as the Plasmodium lactate dehydrogenase (pLDH) immunodetection assay. We evaluated a commercially controlled pLDH enzyme-linked immunosorbent assay (ELISA; the ELISA-Malaria antigen test; DiaMed AG, Cressier s/Morat, Switzerland) to assess drug susceptibility in a standard in vitro assay using fairly basic laboratory equipment to study the in vitro resistance of malaria parasites to major antimalarials. Five Plasmodium falciparum clones and 121 clinical African isolates collected during 2003 and 2004 were studied by the pLDH ELISA and the [8- 3 H]hypoxanthine isotopic assay as a reference with four antimalarials. Nonlinear regression with a maximum effect model was used to estimate the 50% inhibitory concentration (IC 50 ) and its confidence intervals. The two methods were observed to have similar reproducibilities, but the pLDH ELISA demonstrated a higher sensitivity. The high correlation ( r = 0.98) and the high phenotypic agreement (κ = 0.88) between the two methods allowed comparison by determination of the IC 50 s. Recently collected Plasmodium falciparum African isolates were tested by pLDH ELISA and showed drug resistance or decreased susceptibilities of 62% to chloroquine and 11.5% to the active metabolite of amodiaquine. No decreased susceptibility to lumefantrine or the active metabolite of artemisinin was detected. The availability of this simple and highly sensitive pLDH immunodetection assay will provide an easier method for drug susceptibility testing of malaria parasites.

In Congo, urgent efforts are needed to help with the revision of the national antimalarial drug policy. Despite its high resistance level, chloroquine (CQ) is still extensively used as the first-line treatment for uncomplicated Plasmodium falciparum malaria. The study was conducted in children under 5 years with uncomplicated malaria in Pointe-Noire and Brazzaville, the two largest cities that contain approximately 60% of the population of Congo. We investigated by polymerized chain reaction and sequencing methods the frequency distribution of molecular markers for antimalarial drug resistance, including mutations in P. falciparum chloroquine resistance transporter (pfcrt) gene associated with CQ resistance and mutations in dihydrofolate reductase (dhfr) and dihydropteroate synthetase (dhps) genes conferring resistance to sulphadoxine/pyrimethamine (SP) among pre-treatment P. falciparum isolates, as well as assessing antimalarial drug use in the community. pfcrt (K76T) mutation was present in most isolates (96.4%, n = 138) and high frequency (69.2%, n = 133) of triple-mutant dhfr-S108N, N51I, C59R was observed. The quintuple mutant (dhfr-S108N, N51I, C59R and dhps-A437G or S436A, K540E) considered as molecular marker for SP treatment failure was not found because dhps-K540E mutation was absent in isolates tested; this is a clear evidence for the excellent efficacy of SP that we previously described in the same population. The complete absence of the dhps-K540E mutation is a deterrent component for using this molecular marker as an early warning tool for SP resistance testing in that population. Poor compliance issues related to the antimalarial drug use including inappropriate manufacturing practices reported in this study require intensive attention and should be taken into account when implementing drug policy change. If Congo changes its treatment policy from CQ to SP monotherapy, this will not last long. The strategy of combining SP with other affordable and effective antimalarial drugs such as the artemisinin derivatives to improve efficacy and to delay the development of parasite resistance is essential.

L’etude de la diversite de Plasmodium falciparum passe par l’analyse des marqueurs de polymorphisme comme les genes msp-1 et msp-2. Ces genes, presents en copie unique dans le genome de Plasmodium, sont composes de differents blocs. Le bloc 2 de msp-1, compose de motifs repetes de facon variable, est le plus polymorphe. Nous avons mis au point une technique permettant d’etudier le polymorphisme de taille de ce bloc par analyse de la taille des fragments generes par amplification du bloc 2 avec une amorce fluorescente. Elle a ete appliquee a l’analyse de melanges de deux souches clonees : HB3-Honduras et FCM29-Cameroun et a l’etude des isolats de 19 patients en suivi post therapeutique rapproche. Le nombre, la proportion de clones presents, ainsi que leur variation au cours du temps ont ete compares aux variations du phenotype de resistance a la chloroquine. Les resultats ont montre une sensibilite superieure de la methode d’analyse de fragments msp-1 par rapport aux methodes classiques (PCR, PCR-RFLP). Cette methode est un bon outil permettant d’analyser precisement le polymorphisme de Plasmodium falciparum dans differents isolats.

Objectives: To assess the relationship between the presence of DHFR and DHPS mutations in Plasmodium falciparum, parasite in vitro resistance, and in vivo efficacy of sulfadoxine-pyrimethamine (SP) treatment. Patients and methods: Measurement of SP treatment efficacy in malaria-infected children in Gabon was combined with in vitro tests of susceptibility to pyrimethamine and cycloguanil, and molecular genotyping at several DHFR and DHPS loci of parasites isolated before treatment. DHFR was studied at codons 108, 51, and 59, whereas DHPS gene was typed at positions 436, 437, 540 and 581. Results: SP treatment was effective in 86% of children by day 28. Seventy-five percent of isolates were in vitro resistant to pyrimethamine and 65.5% to cycloguanil. No mutation was detected at codons 540 and 581 of the DHPS gene. Most isolates (71.8%) presented with the triple mutant DHFR genotype, whereas 64.3% combined at least three DHFR and one DHPS mutations. The increase in the number of DHFR mutations was associated with an increase in in vitro resistance to pyrimethamine and cycloguanil; three DHFR mutations conferred pyrimethamine and to a lesser extent cycloguanil resistance. Treatment failures only occurred with isolates presenting at least two DHFR mutations (S108N and C59R) and one DHPS mutation (S436A or A437G), but SP treatment of infections with such parasites gave treatment success in 82.0% of children. Conclusions: DHFR mutations that lead to high-level in vitro resistance to pyrimethamine plus 1-2 DHPS mutations are not sufficient to induce in vivo failure of SP treatment in young children from Gabon.

Drug-resistant malaria is primarily caused by Plasmodium falciparum, a species highly prevalent in tropical Africa, the Amazon region and South-east Asia. It causes severe fever or anaemia that leads to more than a million deaths each year. The emergence of chloroquine resistance has been associated with a dramatic increase in malaria mortality among inhabitants of some endemic regions. The rationale for chemoprophylaxis is weakening as multiple-drug resistance develops against well-tolerated drugs. Plasmodium falciparum drug-resistant malaria originates from chromosome mutations. Analysis by molecular, genetic and biochemical approaches has shown that (i). impaired chloroquine uptake by the parasite vacuole is a common characteristic of resistant strains, and this phenotype is correlated with mutations of the Pfmdr1, Pfcg2 and Pfcrt genes; (ii). one to four point mutations of dihydrofolate reductase (DHFR), the enzyme target of antifolates (pyrimethamine and proguanil) produce a moderate to high level of resistance to these drugs; (iii). the mechanism of resistance to sulfonamides and sulfones involves mutations of dihydropteroate synthase (DHPS), their enzyme target; (iv). treatment with sulphadoxine-pyrimethamine selects for DHFR variants Ile(51), Arg(59), and Asn(108) and for DHPS variants Ser(436), Gly(437), and Glu(540); (v) clones that were resistant to some traditional antimalarial agents acquire resistance to new ones at a high frequency (accelerated resistance to multiple drugs, ARMD). The mechanisms of resistance for amino-alcohols (quinine, mefloquine and halofantrine) are still unclear. Epidemiological studies have established that the frequency of chloroquine resistant mutants varies among isolated parasite populations, while resistance to antifolates is highly prevalent in most malarial endemic countries. Established and strong drug pressure combined with low antiparasitic immunity probably explains the multidrug-resistance encountered in the forests of South-east Asia and South America. In Africa, frequent genetic recombinations in Plasmodium originate from a high level of malaria transmission, and falciparum chloroquine-resistant prevalence seems to stabilize at the same level as chloroquine-sensitive malaria. Nevertheless, resistance levels may differ according to place and time. In vivo and in vitro tests do not provide an adequate accurate map of resistance. Biochemical tools at a low cost are urgently needed for prospective monitoring of resistance.

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.

Little is known about severe imported malaria in nonendemic indus- in endemic areas. The defining criteria of severe and complitrialized countries. The purpose of this retrospective study was to cated falciparum malaria established by the World Health describe the clinical spectrum of severe imported malaria in adults Organization (WHO) in 1990 (5) and revised in 2000 (6, 7) and to determine factors that were present at admission and were were developed mainly on the basis of studies performed in associated with in–intensive care unit mortality. This retrospective tropical areas. In addition, few studies have focused on severe study evaluated the 188 patients who were admitted to our inten- imported falciparum malaria in patients admitted to the insive care unit in 1988–1999 with severe and/or complicated imported tensive care unit (ICU), and little is known about the relemalaria. Among them, 93 had strictly defined severe malaria, and vance of the WHO criteria in populations living in nonen95 had less severe malaria. The mean age was 38 years, 51% of demic industrialized areas. patients were nonimmune whites, 94% acquired Plasmodium falci- The objective of this retrospective study was to assist cliniparum in sub-Saharan Africa, and 96% had taken inadequate anti- cians in diagnosing and managing imported falciparum mamalarial chemoprophylaxis. Mortality was 11% (10 patients) in the laria by (1) describing the clinical spectrum of severe imported severe malaria group, whereas no patients died in the less severe malaria in adults, (2 ) identifying predictors of mortality in malaria group (p 0.002). In the bivariable analysis, the main fac- patients admitted to the ICU with major WHO criteria, and tors associated with death in the severe malaria group were the (3 ) assessing the relevance of WHO definitions in this populaSimplified Acute Physiology Score, shock, acidosis, coma, pulmotion. nary edema (p 0.001 for each), and coagulation disorders (p 0.002). Bacterial coinfection is not infrequent and may contribute METHODS

Many African countries currently use a sulfadoxine-pyrimethamine combination (SP) or amodiaquine (AQ) to treat uncomplicated Plasmodium falciparum malaria. Both drugs represent the last inexpensive alternatives to chloroquine. However, resistant P. falciparum populations are largely reported in Africa, and it is compulsory to know the present situation of resistance. The in vivo World Health Organization standard 28-day test was used to assess the efficacy of AQ and SP to treat uncomplicated falciparum malaria in Gabonese children under 10 years of age. To document treatment failures, molecular genotyping to distinguish therapeutic failures from reinfections and drug dosages were undertaken. A total of 118 and 114 children were given AQ or SP, respectively, and were monitored. SP was more effective than AQ, with 14.0 and 34.7% of therapeutic failures, respectively. Three days after initiation of treatment, the mean level of monodesethylamodiaquine (MdAQ) in plasma was 149 ng/ml in children treated with amodiaquine. In those treated with SP, mean levels of sulfadoxine and pyrimethamine in plasma were 100 μg/ml and 212 ng/ml, respectively. Levels of the three drugs were higher in patients successfully treated with AQ (MdAQ plasma levels) or SP (sulfadoxine and pyrimethamine plasma levels). Blood concentration higher than breakpoints of 135 ng/ml for MdAQ, 100 μg/ml for sulfadoxine, and 175 ng/ml for pyrimethamine were associated with treatment success (odds ratio: 4.5, 9.8, and 11.8, respectively; all P values were

To the Editor: During 2000–2010, serial Leishmania isolates obtained from an HIV-infected patient who was not responding to treatment showed a gradual decrease in in vitro miltefosine susceptibility. We performed L. donovani miltefosine transporter (Ldmt) gene analysis to identify an association between miltefosine resistance of reference L. donovani lines and variability in miltefosine response of L. infantum isolates. A new single-nucleotide polymorphism (SNP), L832F, was identified, which might be a marker of miltefosine resistance in leishmaniasis. The patient, a 46-year-old woman, had lived in France since 1994 but regularly returned to Algeria, her country of birth. HIV-1 infection was diagnosed in 1991. Antiretroviral therapy was initiated in 1993, leading to undetectable viral load and a CD4+ T-cell count of 185 cells/mm3 (reference >450/mm3). Concurrent conditions were thoracic herpes zoster in 1996, hairy leukoplakia of tongue, oropharyngeal candidiasis, and chronic renal failure of unknown cause since 2000. Visceral leishmaniasis was diagnosed in 1998 by culture of a bone marrow smear, which showed intracellular amastigotes. Use of meglumine antimonate (Glucantime; Sanofi, Paris, France), a drug of choice for the treatment of leishmaniasis, was contraindicated because of pancreatitis in the patient and in vitro isolate susceptibility variation; therefore, induction therapy consisted of liposomal amphotericin B (AmpB [AmBisome; Astellas Pharma US, Deerfield, IL, USA]) at a dose of 3 mg/kg/d for 5 consecutive days, then 1× week for 5 weeks (total dose 30 mg/kg) during 1998–2000 (Table). The same medication was administered for relapses at 4 mg/kg/d for 5 days, then 4 mg/kg 1× week for 5 weeks (total dose 40 mg/kg) during 2001–2010. Given the adverse effects of AmpB and the availability of oral miltefosine (Impavido; AEterna Zentaris Inc., Quebec City, Quebec, Canada), the latter drug was used for maintenance treatment during 2001–2007 at 50 mg 2×/d. Leishmaniasis was monitored by leukocytoconcentration and culture of blood samples on Novy-Nicolle-McNeal medium. Table Comparions of IC50 for AmpB and miltefosine against promastigotes and axenic amastigotes and distribution of LdMT SNPs in Leishmania infantum isolates and reference strains* When signs of biological and clinical relapse appeared, bone marrow was aspirated for parasite detection. After culture of the aspirate and isoenzyme determination, the strain was identified as L. infantum, zymodeme MON-24. Eleven relapses were documented; all were confirmed by positive direct examination of bone marrow or blood, but cultures of only 7 samples yielded positive results (Table). The susceptibility of 4 cryopreserved isolates (S1, S3, S4, and S6; Table) to AmpB and to miltefosine was studied in the in vitro promastigote and axenic amastigote form by determining the concentrations inhibiting parasite growth by 50% (1,2). The 50% inhibitory concentration (IC50) was determined in parallel for the following reference L. donovani lines: a wild-type L. donovani LV9 (MHOM/ET/67/HU3) line (LV9 WT), a wild-type L. donovani DD8 (MHOM/IN/80/DD8) line (DD8 WT), a laboratory miltefosine-resistant line obtained from LV9 WT (LV9 miltefosine-R, resistant to 90 μmol/L miltefosine), and the laboratory AmB-resistant line obtained from DD8 WT (DD8 AmB-R, resistant to 1.4 μmol/L AmB) on promastigote and axenic amastigote forms (3,4). The AmB susceptibility of the isolates did not change notably over time; IC50 values ranged from 0.09 µmol/L to 0.24 µmol/L, regardless of parasite form, similar to those of wild-type reference strains (Table). In contrast, the IC50 values of miltefosine increased greatly over time, from 5.00 µmol/L to 50.10 μmol/L. During the 6 years of follow-up with miltefosine maintenance therapy, the susceptibility of the isolate (S6) obtained 6 months after miltefosine treatment withdrawal in 2008 was 6-fold higher than that of the first isolate (S1) obtained in 2000. The L. donovani miltefosine transporter protein (LdMT) promotes miltefosine translocation (5), and LdMT inactivation in L. donovani promastigotes leads to miltefosine resistance at the promastigote and amastigote stages (6). In 2003 and 2006 studies, several mutations were linked to the inability of parasites to take up miltefosine and to miltefosine resistance (5,7). In a 2009 study, the weak expression of LdMT and its β subunit LdROS3 in L. braziliensis isolates was linked to diminished sensitivity (8). We sequenced the entire Ldmt gene (3,294 bp) in the reference strains and the clinical isolates for SNP analysis (5,7). Only 1 new SNP, L832F, was found in the miltefosine-resistant reference strain (LV9 miltefosine-R) and in clinical isolate S6. The L832 wild-type allele was found in isolate S1 and in the miltefosine-sensitive reference lines (LV9, DD8, and DD8 AmpB-R), whereas both alleles were found in isolates S3 and S4, with a decrease in the wild-type allele (Table). The last isolate, which was obtained 3 years after miltefosine withdrawal and could not be subcultured, had reverted to the wild-type allele (L832). These results point to a relation between the 832F allele and diminished susceptibility to miltefosine. Analysis of this case of miltefosine resistance in a patient co-infected with Leishmania sp. and HIV strongly suggests that an SNP (L832F) in the Ldmt gene could represent a molecular marker of miltefosine resistance in L. infantum and L. donovani.

We report a case of misdiagnosis of Plasmodium falciparum malaria from Brazil with negative PfHRP2-based rapid diagnostic tests (RDTs), leading to inappropriate case management. Genetic tests showed the deletion of both pfhrp2 and pfhrp3 genes. The detection of two distinct P. falciparum target antigens is then advisable.

The usefulness of atovaquone-proguanil (AP) as an antimalarial treatment is compromised by the emergence of atovaquone resistance during therapy. However, the origin of the parasite mitochondrial DNA (mtDNA) mutation conferring atovaquone resistance remains elusive. Here, we report a patient-based stochastic model that tracks the intrahost emergence of mutations in the multicopy mtDNA during the first erythrocytic parasite cycles leading to the malaria febrile episode. The effect of mtDNA copy number, mutation rate, mutation cost, and total parasite load on the mutant parasite load per patient was evaluated. Computer simulations showed that almost any infected patient carried, after four to seven erythrocytic cycles, de novo mutant parasites at low frequency, with varied frequencies of parasites carrying varied numbers of mutant mtDNA copies. A large interpatient variability in the size of this mutant reservoir was found; this variability was due to the different parameters tested but also to the relaxed replication and partitioning of mtDNA copies during mitosis. We also report seven clinical cases in which AP-resistant infections were treated by AP. These provided evidence that parasiticidal drug concentrations against AP-resistant parasites were transiently obtained within days after treatment initiation. Altogether, these results suggest that each patient carries new mtDNA mutant parasites that emerge before treatment but are killed by high starting drug concentrations. However, because the size of this mutant reservoir is highly variable from patient to patient, we propose that some patients fail to eliminate all of the mutant parasites, repeatedly producing de novo AP treatment failures.

Background The objective of this study was to evaluate the distribution of a series of independent doxycycline inhibitory concentration 50% (IC50) values to validate the trimodal distribution previously described and to validate the use of the pftetQ and pfmdt genes as molecular markers of decreased in vitro doxycycline susceptibility in Plasmodium falciparum malaria. Methods Doxycycline IC50 values, from 484 isolates obtained at the French National Reference Centre for Imported Malaria (Paris) between January 2006 and December 2010, were analysed for the first time by a Bayesian mixture modelling approach to distinguish the different in vitro phenotypic groups by their IC50 values. Quantitative real-time polymerase chain reaction was used to evaluate the pftetQ and pfmdt copy numbers of 89 African P. falciparum isolates that were randomly chosen from the phenotypic groups. Results The existence of at least three doxycycline phenotypes was demonstrated. The mean doxycycline IC50 was significantly higher in the group with a pftetQ copy number >1 compared to the group with a pftetQ copy number = 1 (33.17 μM versus 17.23 μM) and the group with a pfmdt copy number >1 (28.28 μM versus 16.11 μM). There was a significant difference between the combined low and medium doxycycline IC50 group and the high IC50 group in terms of the per cent of isolates with one or more copy numbers of the pftetQ gene (0% versus 20.69%) or pfmdt gene (8.33% versus 37.93%). In the logistic regression model, the pfmdt and pftetQ copy numbers >1 (odds ratio = 4.65 and 11.47) were independently associated with the high IC50 group. Conclusions Copy numbers of pftetQ and pfmdt are potential predictive molecular markers of decreased susceptibility to doxycycline.

The correlation between the structure of two short sequences from the Plasmodium falciparum cg2 gene and parasite chloroquine susceptibility was evaluated in unselected clinical isolates obtained from travellers returning mainly from Africa to France in 1995 and 1996. As determined by an isotopic semi-microtest, 74 isolates were susceptible to chloroquine (50% inhibitory concentration80 nM), 13 were intermediate (80 nM50% inhibitory concentration110 nM) and 53 were resistant (50% inhibitory concentration110 nM). Two polymerase chain reaction assays were developed, one for the kappa and one for the omega repeat domains of cg2 gene. The kappa and the omega repeat domains of 99 isolates were sequenced. A variation in the unit number of kappa and omega repeats was observed. Variations in repetitive sequences, which were not previously described, were found: three for the kappa repeat region: kappa9; kappa10 and kappa11 and three for the omega repeat region: omega8; omega9 and omega22. A polymorphism was observed inside the repeat units of kappa and omega regions. There were six possible kappa repeat units and seven possible omega repeat units. The presence of a particular pattern, containing kappa14 and omega16 repeat units, was associated with a lack of chloroquine susceptibility in 44 out of 46 cases. However, not all resistant isolates had this 'resistant' genotype. Among 43 resistant isolates, 36 (84%) had the kappa14 repeats sequence and 36 had the omega16 repeats sequence. These results lend further support to linkage between cg2 polymorphisms and chloroquine resistance without excluding the existence of other resistance component(s).

Fewer than 25 cases of falciparum malaria that failed to respond to atovaquone-proguanil (A-P) have been noted in published articles since the 1996 registration of Malarone (GlaxoSmithKline, Marly-le-Roi, France) (1,2). Well-documented cases that were not attributed to suboptimal dosage or impaired bioavailability essentially due to vomiting, diarrhea, or both, showed atovaquone-resistant parasites in the recrudescent isolate, with Y268S or Y268N cytochrome b mutations (2–10). We report the case of treatment failure that was not due to resistant parasites or impaired drug bioavailability.

The shelf lives of preserved antimalarial agent-predosed plates according to the type of wrapping and the temperature of storage were studied by measuring the 50% inhibitory concentrations of drug for Plasmodium falciparum 3D7. The shelf life of mefloquine was 8 weeks at 25°C; and those of artesunate, artemisinin, and dihydroartemisinin were a minimum of 24, 12, and 8 weeks, respectively, at 4°C.