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Avian haemosporidian parasites (order Haemosporida, phylum Apicomplexa) are blood and tissue parasites transmitted by blood-sucking dipteran insects. Three genera (Plasmodium, Haemoproteusand Leucocytozoon) have been most often found in birds, with over 270 species described and named in avian hosts based mainly on the morphological characters of blood stages. A broad diversity of Haemoproteusparasites remains to be identified and characterized morphologically and molecularly, especially those infecting birds of prey, an underrepresented bird group in haemosporidian parasite studies. The aim of this study was to investigate and identify Haemoproteusparasites from a large sample comprising accipitriform raptors of 16 species combining morphological and new molecular protocols targeting the cytbgenes of this parasite group. This study provides morphological descriptions and molecular characterizations of two Haemoproteusspecies, H. multivacuolatusn. sp. and H. nisiPeirce and Marquiss, 1983. Haemoproteusparasites of this group were so far found in accipitriform raptors only and might be classified into a separate subgenus or even genus. Cytbsequences of these parasites diverge by more than 15% from those of all others known avian haemosporidian genera and form a unique phylogenetic clade. This study underlines the importance of developing new diagnostic tools to detect molecularly highly divergent parasites that might be undetectable by commonly used conventional tools.

Background/AimsAcanthamoebakeratitis (AK) is a rare but sight-threatening infection. Molecular diagnosis of corneal scraping has improved the diagnosis of AK. Different molecular targets and conditions have been used in diagnosis thus far. In this study, we prospectively compared the performance of five PCR assays on corneal samples for the diagnosis of AK.Methods1217 corneal scraping samples were obtained from patients, for whom an AK was suspected. Sample processing involved both molecular diagnostics and culture. AcanthamoebaPCR assays detected different regions of the Acanthamoebanuclear small-subunit rRNA gene: three final point PCR assays using Nelson, ACARNA and JDP1–JDP2 pairs of primers, and two real-time PCR assays using Acant primer-probe. Human DNA and internal control were co-amplified in the real-time PCR assay to ensure scraping quality and the absence of inhibitors. In the absence of a gold standard, the performance of each test was evaluated using latent class analysis. Genotypes of Acanthamoebaisolates were also characterised.ResultsEstimated prevalence of AK was 1.32%. The sensitivity of Acanthamoebadiagnostic PCRs (73.3% to 86.7%) did not differ significantly from that of culture (66.7%), or according to the target sequence or the technology. Sensitivity could be increased to 93.8% or 100% by combining two or three assays, respectively. PCR specificity (99.3% to 100%) differed between the assays. T4 was the predominant Acanthamoebagenotype (84.6%).ConclusionsCulture and a single PCR assay could lead to misdiagnosing AK. A combination of different PCR assays and improved sample quality could increase diagnosis sensitivity.

Artemisinin resistance (delayed P. falciparumclearance following artemisinin-based combination therapy), is widespread across Southeast Asia but to date has not been reported in Africa1–4. Here we genotyped the P. falciparum K13(Pfkelch13) propeller domain, mutations in which can mediate artemisinin resistance5,6, in pretreatment samples collected from recent dihydroarteminisin-piperaquine and artemether-lumefantrine efficacy trials in Rwanda7. While cure rates were >95% in both treatment arms, the Pfkelch13R561H mutation was identified in 19 of 257 (7.4%) patients at Masaka. Phylogenetic analysis revealed the expansion of an indigenous R561H lineage. Gene editing confirmed that this mutation can drive artemisinin resistance in vitro. This study provides evidence for the de novo emergence of Pfkelch13-mediated artemisinin resistance in Rwanda, potentially compromising the continued success of antimalarial chemotherapy in Africa.

ABSTRACTThe pharmacogenetics of antimalarial agents are poorly known, although the application of pharmacogenetics might be critical in optimizing treatment. This population pharmacokinetic-pharmacogenetic study aimed at assessing the effects of single nucleotide polymorphisms (SNPs) in cytochrome P450 isoenzyme genes (CYP, namely, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4, and CYP3A5) and the N-acetyltransferase 2 gene (NAT2) on the pharmacokinetics of artemisinin-based combination therapies in 150 Tanzanian patients treated with artemether-lumefantrine, 64 Cambodian patients treated with artesunate-mefloquine, and 61 Cambodian patients treated with dihydroartemisinin-piperaquine. The frequency of SNPs varied with the enzyme and the population. Higher frequencies of mutant alleles were found in Cambodians than Tanzanians for CYP2C9*3, CYP2D6*10(100C→T), CYP3A5*3, NAT2*6, and NAT2*7. In contrast, higher frequencies of mutant alleles were found in Tanzanians for CYP2D6*17(1023C→T and 2850C→T), CYP3A4*1B, NAT2*5, and NAT2*14. For 8 SNPs, no significant differences in frequencies were observed. In the genetic-based population pharmacokinetic analyses, none of the SNPs improved model fit. This suggests that pharmacogenetic data need not be included in appropriate first-line treatments with the current artemisinin derivatives and quinolines for uncomplicated malaria in specific populations. However, it cannot be ruled out that our results represent isolated findings, and therefore more studies in different populations, ideally with the same artemisinin-based combination therapies, are needed to evaluate the influence of pharmacogenetic factors on the clearance of antimalarials.

ABSTRACTThe declining efficacy of artemisinin derivatives against Plasmodium falciparumin western Cambodia is a major concern. The knowledge gap in the understanding of the mechanisms involved hampers designing monitoring tools. Here, we culture-adapted 20 isolates from Pailin and Ratanakiri (areas of artemisinin resistance and susceptibility in western and eastern Cambodia, respectively) and studied their in vitroresponse to dihydroartemisinin. No significant difference between the two sets of isolates was observed in the classical isotopic test. However, a 6-h pulse exposure to 700 nM dihydroartemisinin (ring-stage survival assay -RSA]) revealed a clear-cut geographic dichotomy. The survival rate of exposed ring-stage parasites (ring stages) was 17-fold higher in isolates from Pailin (median, 13.5%) than in those from Ratanakiri (median, 0.8%), while exposed mature stages were equally and highly susceptible (0.6% and 0.7%, respectively). Ring stages survived drug exposure by cell cycle arrest and resumed growth upon drug withdrawal. The reduced susceptibility to artemisinin in Pailin appears to be associated with an altered in vitrophenotype of ring stages from Pailin in the RSA.

ABSTRACTMolecular investigations performed following the emergence of sulfadoxine-pyrimethamine (SP) resistance in Plasmodium falciparumhave allowed the identification of the dihydrofolate reductase (DHFR) enzyme as the target of pyrimethamine. Although clinical cases of Plasmodium malariaeare not usually treated with antifolate therapy, incorrect diagnosis and the high frequency of undetected mixed infections has probably exposed non-P. falciparumparasites to antifolate therapy in many areas. In this context, we aimed to assess the worldwide genetic diversity of the P. malariaedhfrgene in 123 samples collected in Africa and Asia, areas with different histories of SP use. Among the 10 polymorphic sites found, we have observed 7 new mutations (K55E, S58R, S59A, F168S, N194S, D207G, and T221A), which led us to describe 6 new DHFR proteins. All isolates from African countries were classified as wild type, while new mutations and haplotypes were recognized as exclusive to Madagascar (except for the double mutations at nucleotides 341 and 342 [S114N] found in one Cambodian isolate). Among these nonsynonymous mutations, two were likely related to pyrimethamine resistance: S58R (corresponding to C59R in P. falciparumand S58R in Plasmodium vivax; observed in one Malagasy sample) and S114N (corresponding to S108N in P. falciparumand S117N in P. vivax; observed in three Cambodian samples).

Plasmodium falciparum chloroquine resistance was first detected in Cambodia in the early sixties. Treatment with chloroquine was abandoned 20 years ago. In vitro chloroquine sensitivity monitoring indicates that all eastern Cambodian isolates were sensitive to chloroquine, whereas most isolates collected from western provinces displayed reduced susceptibility to chloroquine. This indicates that the rate of chloroquine resistance remains high and stable in this region in the absence of chloroquine pressure. Characterization of codons 72 to 78 and 218 to 220 of pfcrt revealed six distinct haplotypes, four of which had never been described. The frequency of each haplotype depended on the geographical origin of the samples. The CVIETIF//ISS haplotype was detected in 92% of western Cambodian isolates and in 11% of isolates collected from the eastern province, where CVMNKIF//ISA and CVIDTIF//ISS predominate. The detection of an intermediate haplotype from a susceptible area with 76T/220A, suggests that acquisition of chloroquine resistance might be a stepwise process, during which accumulation of point mutations modulates the response to chloroquine. The association of the K76T mutation with chloroquine resistance was not clear. The mutation was detected in resistant and susceptible samples, suggesting that additional factors are involved in chloroquine resistance. By contrast, the pfcrt D/N75E mutation was strongly associated with the in vitro chloroquine resistance in Cambodian isolates. The N86 allelic form of pfmdr1 was detected in all isolates, consistent with a poor association with resistance to chloroquine. This indicates that in vitro resistance to chloroquine was associated with accumulation of point mutations in pfcrt.

ABSTRACTPlasmodium falciparumchloroquine resistance was first detected in Cambodia in the early sixties. Treatment with chloroquine was abandoned 20 years ago. In vitro chloroquine sensitivity monitoring indicates that all eastern Cambodian isolates were sensitive to chloroquine, whereas most isolates collected from western provinces displayed reduced susceptibility to chloroquine. This indicates that the rate of chloroquine resistance remains high and stable in this region in the absence of chloroquine pressure. Characterization of codons 72 to 78 and 218 to 220 of pfcrtrevealed six distinct haplotypes, four of which had never been described. The frequency of each haplotype depended on the geographical origin of the samples. The CVIETIF//ISS haplotype was detected in 92% of western Cambodian isolates and in 11% of isolates collected from the eastern province, where CVMNKIF//ISA and CVIDTIF//ISS predominate. The detection of an intermediate haplotype from a susceptible area with 76T/220A, suggests that acquisition of chloroquine resistance might be a stepwise process, during which accumulation of point mutations modulates the response to chloroquine. The association of the K76T mutation with chloroquine resistance was not clear. The mutation was detected in resistant and susceptible samples, suggesting that additional factors are involved in chloroquine resistance. By contrast, the pfcrtD/N75E mutation was strongly associated with the in vitro chloroquine resistance in Cambodian isolates. The N86 allelic form of pfmdr1was detected in all isolates, consistent with a poor association with resistance to chloroquine. This indicates that in vitro resistance to chloroquine was associated with accumulation of point mutations in pfcrt.

The increasing usage of long-lasting insecticide-treated nets allows protection of millions of people from malaria infection. Monitoring studies should be planned during any wide-scale malaria control program integrating insecticide-treated materials, to evaluate their effects and effectiveness on epidemiologically relevant parameters. Such operational control interventions may be challenged by insecticide resistance spread within vector populations, as a result of wide insecticide pressure. A nationwide distribution of long-lasting insecticidal nets was implemented throughout Niger in 2005. We studied the population genetic structure of major malaria vectors across Nigerien Sahel, and investigated potential effects of this large malaria control intervention. Wild-caught Anopheles gambiae sensu lato females from seven villages and two wet seasons were genotyped at 12 microsatellite loci. The genetic diversity within both species appeared homogenous between villages and years. The estimated genetic differentiation among samples was very low within both species, indicating high gene flow across the area. An absence of differentiation was also found between 2005 and 2006 wet seasons, for all samples but one, showing that the net distribution did not impact significantly the genetic diversity and structure of vector populations in a single year. We provide valuable results participating to document effects of large malaria control programs, to maximize the efficiency of available tools in future interventions.