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International audience; Background: Since several malaria parasite species are usually present in a particular area, co-infections with more than one species of Plasmodium are more likely to occur in humans infected in these areas. In many mixed infections, parasite densities of the cryptic species may be low and often not recognized in clinical practice.Case presentation: Two children (3 and 6 years old) adopted recently from Central African Republic were admitted to hospital because of intermittent fever. Thin blood smears stained with Giemsa showed Plasmodium falciparum and Plasmodium malariae co-infection for both children at admission. They were both treated with atovaquone-proguanil combination for 3 days. At day 7, both thin blood smears examination remained negative but at day 28, thin blood smear was positive for P. malariae trophozoites and for Plasmodium ovale for the girl and her brother, respectively. Samples collected at day 1 and day 28 were submitted to real-time PCR showing the presence of the three parasite species (P. falciparum, P malariae and P. ovale) in admission blood samples from the two children and only P. ovale at day 28.Conclusions: Twenty-eight days follow-up after treatment led to detection of a third parasite species in the blood of these two patients suggesting covert co-infection and a delayed appearance of one cryptic species following treatment. Concurrently infecting malaria species could be mutually suppressive, with P. falciparum tending to dominate other species. These observations provide more evidence that recommendations for treatment of imported malaria should take into account the risk of concurrent or cryptic infection with Plasmodium species. Clinicians and biologists should be aware of the underestimated frequency of mixed infections with cryptic species and of the importance of patient follow-up at day 28. Future guidelines should shed more light on the treatment of mixed infection and on the interest of using artemisinin-based combinations for falciparum and non-falciparum species.

Background Plasmodium vivax is the second most important human malaria parasite, widely spread across the world. This parasite is associated with important issues in the process toward malaria elimination, including potential for relapse and increased resistance to chloroquine. Plasmodium vivax multi-drug resistant (pvmdr1) is suspected to be a marker of resistance although definitive evidence is lacking. Progress has been made in knowledge of biological factors affecting parasite growth, including mechanisms of regulated cell death and the suspected role of metacaspase. Plasmodium vivax metacaspase1 (PvMCA1-cd) has been described with a catalytic domain composed of histidine (H372) and cysteine (C428) residues. The aim of this study was to test for a link between the conserved histidine and cysteine residues in PvMCA1-cd, and the polymorphism of the P. vivax multi-drug resistant gene (pvmdr1). Results Thirty P. vivax isolates were collected from Mauritania, Sudan, and Oman. Among the 28 P. vivax isolates successfully sequenced, only 4 samples showed the conserved His (372)–Cys (428) residues in PvMCA1-cd. Single nucleotide polymorphisms observed were H372T (46.4%), H372D (39.3%), and C428R (85.7%). A new polymorphic catalytic domain was observed at His (282)–Cys (305) residues. Sequences alignment analysis of pvmdr1 showed SNP in the three codons 958, 976 and 1076. A single SNP was identified at the codon M958Y (60%), 2 SNPs were found at the position 976: Y976F (13%) and Y976V (57%), and 3 SNPs were identified at the position 1076: F1076L (40%), F1076T (53%) and F1076I (3%). Only one isolate was wildtype in all three codons (MYF), 27% were single MYL mutants, and 10% were double MFL mutants. Three new haplotypes were also identified: the triple mutant YVT was most prevalent (53.3%) distributed in the three countries, while triple YFL and YVI mutants (3%), were only found in samples from Sudan and Mauritania. Conclusions Triple or quadruple mutants for metacaspase genes and double or triple mutants for Pvmdr1 were observed in 24/28 and 19/28 samples. There was no difference in the frequency of mutations between PvMCA1-cd and Pvmdr1 (P > 0.2). Histidine and cysteine residues in PvMCA1-cd are highly polymorphic and linkage disequilibrium with SNPs of Pvmdr1 gene may be expected from these three areas with different patterns of P. vivax transmission.

Background Improving management of patients suffering from cerebral malaria is needed to reduce the devastating mortality and morbidity of the disease in endemic areas. Intravenous artesunate is currently the first-line treatment, but the lack of material and skills in the field make it difficult to implement in endemic areas. Intranasal route provides a very easy and direct gateway to blood and brain to deliver medications, by-passing the brain blood barrier. Therefore, it could be helpful and suitable to administer artesunate in the context of cerebral malaria, especially in young children. In this study, intranasal administration of artesunate to rescue from cerebral malaria using a murine model was tested. Methods CBA/J mice infected with Plasmodium berghei ANKA strain received artesunate (20 mg/kg) or a placebo solution intranasally, either on day 5, 6 or 7 post-infection, during a controlled, blinded, randomized trial. Primary endpoint was mortality on day 12 post-infection. Secondary endpoints were parasitaemia and clinical stage. Pharmacokinetics data following administration were collected in blood and brains of treated mice. Local toxicity was evaluated by histopathologic examination of brain and nasal sections in blinded manner. Results Intranasal administration of artesunate dramatically reduced the mortality rate (p

Background Malaria rapid diagnostic tests (RDTs) are a useful tool in endemic malaria countries, where light microscopy is not feasible. In non-endemic countries they can be used as complementary tests to provide timely results in case of microscopy inexperience. This study aims to compare the new VIKIA Malaria Ag Pf/Pan™ RDT with PCR-corrected microscopy results and the commonly used CareStart™ RDT to diagnose falciparum and non-falciparum malaria in the endemic setting of Bamako, Mali and the non-endemic setting of Lyon, France. Methods Blood samples were collected during a 12-months and six-months period in 2011 from patients suspected to have malaria in Lyon and Bamako respectively. The samples were examined by light microscopy, the VIKIA Malaria Ag Pf/Pan™ test and in Bamako additionally with the CareStart™ RDT. Discordant results were corrected by real-time PCR. Sensitivity, specificity, positive predictive value and negative predictive value were used to evaluate test performance. Results Samples of 877 patients from both sites were included. The VIKIA Malaria Ag Pf/Pan™ had a sensitivity of 98% and 96% for Plasmodium falciparum in Lyon and Bamako, respectively, performing similar to PCR-corrected microscopy. Conclusions The VIKIA Malaria Ag Pf/Pan™ performs similar to PCR-corrected microscopy for the detection of P. falciparum, making it a valuable tool in malaria endemic and non-endemic regions.

Background Cerebral malaria carries an unacceptable case fatality rate in children despite timely and adequate chemotherapy. To improve the survival rate, adjunctive therapies previously tested mainly focused on the modulation of the inflammatory response, without definitive effect in humans. In this context, a new adjunctive strategy using a neuroprotective drug: erythropoietin (epoietin-beta, Epo) was proposed. Methods An open-labelled study including cerebral malaria children (Blantyre coma score below 3) was conducted in Mali. The objective was to assess the short-term safety (seven days) of erythropoietin at high doses (1,500 U/kg/day during three days) combined to quinine. Results 35 patients with unrousable coma were included in the study. None of expected side effects of erythropoietin were observed during the seven days follow-up. No significant increase in the case fatality rate (7/35 patients) was observed compared to other studies with mortality rates ranging from 16 to 22% in similar endemic areas. Conclusion These data provide the first evidence of the short-term safety of erythropoietin at high doses combined to quinine. A multicentre study is needed to assess the potential of Epo as an adjunctive therapy to increase the survival during cerebral malaria. Clinical registration number ClinicalTrials.gov ID: NCT00697164

Background In Guyana, chloroquine + primaquine is used for the treatment of vivax malaria. A worldwide increase of chloroquine resistance in Plasmodium vivax led to questioning of the current malaria treatment guidelines. A therapeutic efficacy study was conducted using artemether-lumefantrine + primaquine against P. vivax to evaluate a treatment alternative for chloroquine. Methods From 2009 to 2010, a non-controlled study in two hospitals in Guyana was conducted. A total 61 patients with P. vivax infection were treated with artemether-lumefantrine as a six-dose regimen twice a day for three days with additional 0.25 mg/kg/d primaquine at day 0 for 14 days. Clinical and parasitological parameters were followed on days 0,1,2,3,7,14 and 28 in agreement with WHO guidelines. Plasmodium vivax DNA from eight patients was analysed for pvmdr1, molecular marker of resistance. Results Artemether-lumefantrine cleared 100% of parasites on day 1, but two patients (3%) had recurrence of parasites on day 28, suggesting relapse. No pvmdr1 Y976F polymorphism was detected. The treatment regimen was well tolerated. Conclusions In Guyana, artemether-lumefantrine represents an adequate treatment option against P. vivax when combined with primaquine. Availability of this alternative will be of great importance in case of emerging chloroquine resistance against P. vivax.

Background Four of five Plasmodium species infecting humans are present in Madagascar. Plasmodium vivax remains the second most prevalent species, but is understudied. No data is available on its susceptibility to sulphadoxine-pyrimethamine, the drug recommended for intermittent preventive treatment during pregnancy. In this study, the prevalence of P. vivax infection and the polymorphisms in the pvdhfr and pvdhps genes were investigated. The correlation between these polymorphisms and clinical and parasitological responses was also investigated in P. vivax-infected patients. Methods Plasmodium vivax clinical isolates were collected in eight sentinel sites from the four major epidemiological areas for malaria across Madagascar in 2006/2007. Pvdhfr and pvdhps genes were sequenced for polymorphism analysis. The therapeutic efficacy of SP in P. vivax infections was assessed in Tsiroanomandidy, in the foothill of the central highlands. An intention-to-treat analysis of treatment outcome was carried out. Results A total of 159 P. vivax samples were sequenced in the pvdhfr/pvdhps genes. Mutant-types in pvdhfr gene were found in 71% of samples, and in pvdhps gene in 16% of samples. Six non-synonymous mutations were identified in pvdhfr, including two novel mutations at codons 21 and 130. For pvdhps, beside the known mutation at codon 383, a new one was found at codon 422. For the two genes, different combinations were ranged from wild-type to quadruple mutant-type. Among the 16 patients enrolled in the sulphadoxine-pyrimethamine clinical trial (28 days of follow-up) and after adjustment by genotyping, 3 (19%, 95% CI: 5%–43%) of them were classified as treatment failure and were pvdhfr 58R/117N double mutant carriers with or without the pvdhps 383G mutation. Conclusion This study highlights (i) that genotyping in the pvdhfr and pvdhps genes remains a useful tool to monitor the emergence and the spread of P. vivax sulphadoxine-pyrimethamine resistant in order to improve the national antimalarial drug policy, (ii) the issue of using sulphadoxine-pyrimethamine as a monotherapy for intermittent preventive treatment of pregnant women or children.