BACKGROUND Recent gains in reducing the global burden of malaria are threatened by the emergence of Plasmodium falciparum resistance to artemisinins. The discovery that mutations in portions of a P. falciparum gene encoding kelch (K13)–propeller domains are the major determinant of resistance has provided opportunities for monitoring such resistance on a global scale. METHODS We analyzed the K13-propeller sequence polymorphism in 14,037 samples collected in 59 countries in which malaria is endemic. Most of the samples (84.5%) were obtained from patients who were treated at sentinel sites used for nationwide surveillance of antimalarial resistance. We evaluated the emergence and dissemination of mutations by haplotyping neighboring loci. RESULTS We identified 108 nonsynonymous K13 mutations, which showed marked geographic disparity in their frequency and distribution. In Asia, 36.5% of the K13 mutations were distributed within two areas — one in Cambodia, Vietnam, and Laos and the other in western Thailand, Myanmar, and China — with no overlap. In Africa, we observed a broad array of rare nonsynonymous mutations that were not associated with delayed parasite clearance. The gene-edited Dd2 transgenic line with the A578S mutation, which expresses the most frequently observed African allele, was found to be susceptible to artemisinin in vitro on a ring-stage survival assay. CONCLUSIONS No evidence of artemisinin resistance was found outside Southeast Asia and China, where resistance-associated K13 mutations were confined. The common African A578S allele was not associated with clinical or in vitro resistance to artemisinin, and many African mutations appear to be neutral.

Adequate clinical and parasitologic cure by artemisinin combination therapies relies on the artemisinin component and the partner drug. Polymorphisms in the Plasmodium falciparum chloroquine resistance transporter (pfcrt) and P. falciparum multidrug resistance 1 (pfmdr1) genes are associated with decreased sensitivity to amodiaquine and lumefantrine, but effects of these polymorphisms on therapeutic responses to artesunate-amodiaquine (ASAQ) and artemether-lumefantrine (AL) have not been clearly defined. Individual patient data from 31 clinical trials were harmonized and pooled by using standardized methods from the WorldWide Antimalarial Resistance Network. Data for more than 7,000 patients were analyzed to assess relationships between parasite polymorphisms in pfcrt and pfmdr1 and clinically relevant outcomes after treatment with AL or ASAQ. Presence of the pfmdr1 gene N86 (adjusted hazards ratio = 4.74, 95% confidence interval = 2.29 – 9.78, P < 0.001) and increased pfmdr1 copy number (adjusted hazards ratio = 6.52, 95% confidence interval = 2.36–17.97, P < 0.001) were significant independent risk factors for recrudescence in patients treated with AL. AL and ASAQ exerted opposing selective effects on single-nucleotide polymorphisms in pfcrt and pfmdr1. Monitoring selection and responding to emerging signs of drug resistance are critical tools for preserving efficacy of artemisinin combination therapies; determination of the prevalence of at least pfcrt K76T and pfmdr1 N86Y should now be routine.

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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.

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. 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.Resistance to currently available drugs is still a cause of therapeutic failures and of the persistence of a high level of morbidity due to malaria. Combined therapy with artemisinin derivatives plus amodiaquine or lumefantrine is now implemented in Africa. Epidemiological assessment of Plasmodium falciparum drug susceptibility is thus necessary to sustain health recommendations for malaria treatment and prophylaxis in countries where malaria is endemic. Even though molecular techniques are improving, few genes responsible for clinical resistance have been identified. The in vitro assay, which allows the phenotypic determination of P. falciparum susceptibility to antimalarials, is thus an essential tool. The multiclonal structure of clinical Plasmodium falciparum isolates is the rule, and a mixture of susceptible and resistant parasites gives a biphasic concentration response (10, 22). As malaria parasites divide every 2 days, cloning is not a workable routine before testing for drug susceptibility. Our previous experience has shown that the in vitro response of a clinical isolate to chloroquine was monotonic at a frequency of Ͼ95%. This suggested that the majority of parasites which are present in the patient had homogeneous susceptibilities. The concentration-response relationship is a classical pharmacological sigmoid response, and the correct modeling of this response may give an accurate assessment of the drug susceptibilities of malaria parasites. Two methods are in common use for assessment of the in vitro parasite growth inhibited by drugs: schizont counting on thick films, known as the WHO Schizont Maturation assay (21), and incorporation of a radiolabeled nucleic acid precursor, [8-3 H...

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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.Malaria diagnosis relies on blood smear examination as the reference. Recently, rapid diagnostic tests (RDTs) have been developed to improve the availability of malaria diagnosis (10). RDTs detect malarial antigens such as the Plasmodium falciparum-specific histidine-rich protein 2 (PfHRP2) and lactate dehydrogenase (PfLDH) and the panplasmodial aldolase (pAldolase) and lactate dehydrogenase (pLDH) (8). We report here a case of P. falciparum malaria in which the PfHRP2 test was negative, leading to inappropriate case management. We then provide the molecular basis underlying this falsenegative test.A 38-year-old French man traveled to the Brazilian Amazon region. Three days after returning to Paris, he started experiencing fever and headache. A blood film examination performed in a nonexpert laboratory showed Plasmodium (144,000 parasites/l; corresponding to 3.2% of infected erythrocytes). In the same laboratory, the Now ICT malaria test (Binax; Inverness Medical, France) was positive for pAldolase but negative for PfHRP2, suggesting non-P. falciparum malaria. The recent travel to Brazil, where P. falciparum chloroquine-resistant malaria is widespread, combined with the RDT results suggested Plasmodium vivax malaria, and the patient was treated with chloroquine. There was no clinical improvement 2 days later, when a second blood film examination in the same laboratory showed a parasitemia of 1,890,000 parasites/l (corresponding to 42% of infected erythrocytes). The patient was then transferred to our expert malaria center, where a new blood film examination was performed. Old trophozoites with hemozoin and Maurer's clefts were predominant compared to ring-stage parasites. Although unusual, this microscopic-stage pattern of parasites in peripheral blood is specific to P. falciparum. Earlier blood samples were sent to us for verification. We observed parasitized erythrocytes with hemozoin, old trophozoites, and schizonts, specific to P. falciparum. We then performed the Now ICT malaria test, which again failed to detect PfHRP2. The Palutop 4ϩ test (Alldiag, Strasbourg, France) was negative for PfHRP2 and positive for pLDH, and the OptiMal IT test (Diamed, Cressier, Switzerland) was positive for both pLDH and PfLDH. Quinine was given for 7 days, and the patient achieved full recovery.During the 7-day quinine course, eight blood samples were collected and analyzed. Parasitemias ranged from 10 to 1,890,000 parasites/l. RDTs were negative for PfHRP2 at any parasitemias but positive for pLDH and PfLDH until parasitemias dropped below the assay detection limit. The high parasitemias achieved during the infection rule out a falsenegative RDT for PfHRP2 because of sensitivity issues. DNA was extracted for...

Toxoplasmosis manifests no clinical signs in 80% of cases in immunocompetent patient, causing immunization characterized by the persistence of cysts, particularly in brain, muscles, and retina. Assessing the serological status, based on testing for serum toxoplasma IgG and IgM antibodies, is essential in cases that are increasingly at risk for the more severe disease forms, such as congenital or ocular toxoplasmosis. This disease also exposes immunosuppressed patients to reactivation, which can lead to more widespread forms and increased mortality. By interpreting the serological results, we can estimate the risk of contamination or reactivation and define appropriate prophylactic and preventive measures, such as hygienic and dietetic, therapeutic, biological, and clinical follow-up, according to the clinical context. We hereby propose practical approaches based on serological data, resulting from a consensus of a group of experts from the French National Reference Center Network for Toxoplasmosis, according to both routine and specific clinical situations.