Sandrine Houzé

A Worldwide Map of Plasmodium falciparum K13-Propeller Polymorphisms.

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. (Funded by Institut Pasteur Paris and others.).

Assessment of the Drug Susceptibility of Plasmodium falciparum Clinical Isolates from Africa by Using a Plasmodium Lactate Dehydrogenase Immunodetection Assay and an Inhibitory Maximum Effect Model for Precise Measurement of the 50-Percent Inhibitory Concentration

ABSTRACT 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 [8-3H]hypoxanthine isotopic assay as a reference with four antimalarials. Nonlinear regression with a maximum effect model was used to estimate the 50% inhibitory concentration (IC50) 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 IC50s. 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.

Polymorphisms in Plasmodium falciparum chloroquine resistance transporter and multidrug resistance 1 genes: parasite risk factors that affect treatment outcomes for P. falciparum malaria after artemether-lumefantrine and artesunate-amodiaquine.

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.

Chimpanzee malaria parasites related to Plasmodium ovale in Africa.

Since the 1970s, 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.

Evaluation of the intra- and inter-specific genetic variability of Plasmodium lactate dehydrogenase.

BackgroundMalaria 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.MethodsPlasmodial 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.ResultsNo 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.ConclusionThe 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.

Leishmania Resistance to Miltefosine Associated with Genetic Marker

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.

Combined Deletions of pfhrp2 and pfhrp3 Genes Result in Plasmodium falciparum Malaria False-Negative Rapid Diagnostic Test

ABSTRACT 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.

Severe imported Plasmodium falciparum malaria, France, 1996-2003.

Little is known about severe imported Plasmodium falciparum malaria in industrialized countries where the disease is not endemic because most studies have been case reports or have included <200 patients. To identify factors independently associated with the severity of P. falciparum, we conducted a retrospective study using surveillance data obtained from 21,888 P. falciparum patients in France during 1996-2003; 832 were classified as having severe malaria. The global case-fatality rate was 0.4% and the rate of severe malaria was ≈3.8%. Factors independently associated with severe imported P. falciparum malaria were older age, European origin, travel to eastern Africa, absence of chemoprophylaxis, initial visit to a general practitioner, time to diagnosis of 4 to 12 days, and diagnosis during the fall-winter season. Pretravel advice should take into account these factors and promote the use of antimalarial chemoprophylaxis for every traveler, with a particular focus on nonimmune travelers and elderly persons.

Prolonged Plasmodium falciparum Infection in Immigrants, Paris

Few immigrant travelers have Plasmodium falciparum infections >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.

PfHRP2 and PfLDH antigen detection for monitoring the efficacy of artemisinin-based combination therapy (ACT) in the treatment of uncomplicated falciparum malaria

BackgroundAn assessment of the accuracy of two malaria rapid diagnostic tests (RDT) for the detection of Plasmodium falciparum histidine-rich protein 2 (Pf HRP2) or Pf lactate dehydrogenase (Pf LDH) was undertaken in children aged between six and 59 months included in an anti-malarial efficacy study in Benin.MethodsIn Allada (Benin), 205 children aged 6-59 months with falciparum malaria received either artesunate-amodiaquine (ASAQ), artemether-lumefantrine (AL), or sulphadoxine-pyrimethamine (SP). Children included in the study were simultaneously followed by both RDT and high-quality microscopy for up to 42 days.ResultsAt the time of inclusion, Pf HRP2-based tests were positive in 203 children (99%) and Pf LDH-based tests were positive in 204 (99.5%). During follow-up, independent of the treatment received, only 17.3% (28/162) of children effectively cured were negative with the Pf HRP2 RDT at day 3, with a gradual increase in specificity until day 42. The specificity of antigen detection with the Pf LDH test was 87% (141/162) on day 3, and between 92% and 100% on days 7 to 42. A statistical difference was observed between the persistence of Pf HRP2 and Pf LDH antigenaemia during follow-up in children treated with artemisinin-based combination therapy (ACT) but not with SP.ConclusionAlthough both RDTs are as sensitive as microscopy in detecting true malaria cases, the Pf HRP2 RDT had very low specificity during follow-up until day 28. On the other hand, the Pf LDH test could be used to detect failures and, therefore, to assess anti-malarial efficacy.