RESUMEN
Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), a diverse family of multidomain proteins expressed on the surface of malaria-infected erythrocytes, is an important target of protective immunity against malaria. Our group recently studied transcription of the var genes encoding PfEMP1 in individuals from Papua, Indonesia, with severe or uncomplicated malaria. We cloned and expressed domains from 32 PfEMP1s, including 22 that were upregulated in severe malaria and 10 that were upregulated in uncomplicated malaria, using a wheat germ cell-free expression system. We used Luminex technology to measure IgG antibodies to these 32 domains and control proteins in 63 individuals (11 children). At presentation to hospital, levels of antibodies to PfEMP1 domains were either higher in uncomplicated malaria or were not significantly different between groups. Using principal component analysis, antibodies to 3 of 32 domains were highly discriminatory between groups. These included two domains upregulated in severe malaria, a DBLß13 domain and a CIDRα1.6 domain (which has been previously implicated in severe malaria pathogenesis), and a DBLδ domain that was upregulated in uncomplicated malaria. Antibody to control non-PfEMP1 antigens did not differ with disease severity. Antibodies to PfEMP1 domains differ with malaria severity. Lack of antibodies to locally expressed PfEMP1 types, including both domains previously associated with severe malaria and newly identified targets, may in part explain malaria severity in Papuan adults.
Asunto(s)
Malaria Falciparum , Malaria , Adulto , Anticuerpos Antiprotozoarios , Niño , Eritrocitos , Humanos , Indonesia , Proteínas de la Membrana/genética , Plasmodium falciparum/genética , Proteínas Protozoarias/genéticaRESUMEN
Zoonotic Plasmodium infections in humans in many Southeast Asian countries have been increasing, including in countries approaching elimination of human-only malaria transmission. Most simian malarias in humans are caused by Plasmodium knowlesi, but recent research shows that humans are at risk of many different simian Plasmodium species. In Southeast Asia, simian Plasmodium species are mainly transmitted by mosquitoes in the Anopheles leucosphyrus and Anopheles dirus complexes. Although there is some evidence of species outside the Leucosphyrus Group transmitting simian Plasmodium species, these await confirmation of transmission to humans. The vectors of monkey malarias are mostly found in forests and forest fringes, where they readily bite long-tailed and pig-tailed macaques (the natural reservoir hosts) and humans. How changing land-uses influence zoonotic malaria vectors is still poorly understood. Fragmentation of forests from logging, agriculture and other human activities is associated with increased zoonotic Plasmodium vector exposure. This is thought to occur through altered macaque and mosquito distributions and behaviours, and importantly, increased proximity of humans, macaques, and mosquito vectors. Underlying the increase in vector densities is the issue that the land-use change and human activities create more oviposition sites and, in correlation, increases availably of human blood hosts. The current understanding of zoonotic malaria vector species is largely based on a small number of studies in geographically restricted areas. What is known about the vectors is limited: the data is strongest for distribution and density with only weak evidence for a limited number of species in the Leucosphyrus Group for resting habits, insecticide resistance, blood feeding habits and larval habitats. More data are needed on vector diversity and bionomics in additional geographic areas to understand both the impacts on transmission of anthropogenic land-use change and how this significant disease in humans might be controlled.
Asunto(s)
Anopheles , Malaria , Plasmodium knowlesi , Animales , Asia Sudoriental , Macaca nemestrina , Mosquitos VectoresRESUMEN
Within the human host, the malaria parasite Plasmodium falciparum is exposed to multiple selection pressures. The host environment changes dramatically in severe malaria, but the extent to which the parasite responds to-or is selected by-this environment remains unclear. From previous studies, the parasites that cause severe malaria appear to increase expression of a restricted but poorly defined subset of the PfEMP1 variant, surface antigens. PfEMP1s are major targets of protective immunity. Here, we used RNA sequencing (RNAseq) to analyse gene expression in 44 parasite isolates that caused severe and uncomplicated malaria in Papuan patients. The transcriptomes of 19 parasite isolates associated with severe malaria indicated that these parasites had decreased glycolysis without activation of compensatory pathways; altered chromatin structure and probably transcriptional regulation through decreased histone methylation; reduced surface expression of PfEMP1; and down-regulated expression of multiple chaperone proteins. Our RNAseq also identified novel associations between disease severity and PfEMP1 transcripts, domains, and smaller sequence segments and also confirmed all previously reported associations between expressed PfEMP1 sequences and severe disease. These findings will inform efforts to identify vaccine targets for severe malaria and also indicate how parasites adapt to-or are selected by-the host environment in severe malaria.
Asunto(s)
Antígenos de Protozoos/genética , Antígenos de Superficie/genética , Malaria/parasitología , Plasmodium falciparum/genética , Proteínas Protozoarias/genética , Transcriptoma , Regulación de la Expresión Génica , Humanos , Malaria/patología , Plasmodium falciparum/aislamiento & purificación , Plasmodium falciparum/metabolismo , Análisis de Secuencia de ARNRESUMEN
BACKGROUND: Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) variants are encoded by var genes and mediate pathogenic cytoadhesion and antigenic variation in malaria. PfEMP1s can be broadly divided into three principal groups (A, B and C) and they contain conserved arrangements of functional domains called domain cassettes. Despite their tremendous diversity there is compelling evidence that a restricted subset of PfEMP1s is expressed in severe disease. In this study antibodies from patients with severe and uncomplicated malaria were compared for differences in reactivity with a range of PfEMP1s to determine whether antibodies to particular PfEMP1 domains were associated with severe or uncomplicated malaria. METHODS: Parts of expressed var genes in a severe malaria patient were identified by RNAseq and several of these partial PfEMP1 domains were expressed together with others from laboratory isolates. Antibodies from Papuan patients to these parts of multiple PfEMP1 proteins were measured. RESULTS: Patients with uncomplicated malaria were more likely to have antibodies that recognized PfEMP1 of Group C type and recognized a broader repertoire of group A and B PfEMP1s than patients with severe malaria. CONCLUSION: These data suggest that exposure to a broad range of group A and B PfEMP1s is associated with protection from severe disease in Papua, Indonesia.
Asunto(s)
Anticuerpos Antiprotozoarios/sangre , Malaria Falciparum/inmunología , Proteínas Protozoarias/inmunología , Adolescente , Adulto , Preescolar , Femenino , Humanos , Indonesia , Masculino , Adulto JovenRESUMEN
The 4-aminoquinoline naphthoquine (NQ) and the thiazine dye methylene blue (MB) have potent in vitro efficacies against Plasmodium falciparum, but susceptibility data for P. vivax are limited. The species- and stage-specific ex vivo activities of NQ and MB were assessed using a modified schizont maturation assay on clinical field isolates from Papua, Indonesia, where multidrug-resistant P. falciparum and P. vivax are prevalent. Both compounds were highly active against P. falciparum (median [range] 50% inhibitory concentration [IC50]: NQ, 8.0 nM [2.6 to 71.8 nM]; and MB, 1.6 nM [0.2 to 7.0 nM]) and P. vivax (NQ, 7.8 nM [1.5 to 34.2 nM]; and MB, 1.2 nM [0.4 to 4.3 nM]). Stage-specific drug susceptibility assays revealed significantly greater IC50s in parasites exposed at the trophozoite stage than at the ring stage for NQ in P. falciparum (26.5 versus 5.1 nM, P = 0.021) and P. vivax (341.6 versus 6.5 nM, P = 0.021) and for MB in P. vivax (10.1 versus 1.6 nM, P = 0.010). The excellent ex vivo activities of NQ and MB against both P. falciparum and P. vivax highlight their potential utility for the treatment of multidrug-resistant malaria in areas where both species are endemic.
Asunto(s)
Antimaláricos/farmacología , Azul de Metileno/farmacología , Plasmodium falciparum/efectos de los fármacos , Plasmodium vivax/efectos de los fármacosRESUMEN
Chloroquine (CQ) has been the mainstay of malaria treatment for more than 60 years. However, the emergence and spread of CQ resistance now restrict its use to only a few areas where malaria is endemic. The aim of the present study was to investigate whether a novel combination of a CQ-like moiety and an imipramine-like pharmacophore can reverse CQ resistance ex vivo. Between March to October 2011 and January to September 2013, two "reversed chloroquine" (RCQ) compounds (PL69 and PL106) were tested against multidrug-resistant field isolates of Plasmodium falciparum (n = 41) and Plasmodium vivax (n = 45) in Papua, Indonesia, using a modified ex vivo schizont maturation assay. The RCQ compounds showed high efficacy against both CQ-resistant P. falciparum and P. vivax field isolates. For P. falciparum, the median 50% inhibitory concentrations (IC50s) were 23.2 nM for PL69 and 26.6 nM for PL106, compared to 79.4 nM for unmodified CQ (P < 0.001 and P = 0.036, respectively). The corresponding values for P. vivax were 19.0, 60.0, and 60.9 nM (P < 0.001 and P = 0.018, respectively). There was a significant correlation between IC50s of CQ and PL69 (Spearman's rank correlation coefficient [r s] = 0.727, P < 0.001) and PL106 (rs = 0.830, P < 0.001) in P. vivax but not in P. falciparum. Both RCQs were equally active against the ring and trophozoite stages of P. falciparum, but in P. vivax, PL69 and PL106 showed less potent activity against trophozoite stages (median IC50s, 130.2 and 172.5 nM) compared to ring stages (median IC50s, 17.6 and 91.3 nM). RCQ compounds have enhanced ex vivo activity against CQ-resistant clinical isolates of P. falciparum and P. vivax, suggesting the potential use of reversal agents in antimalarial drug development. Interspecies differences in RCQ compound activity may indicate differences in CQ pharmacokinetics between the two Plasmodium species.
Asunto(s)
Antimaláricos/uso terapéutico , Cloroquina/uso terapéutico , Plasmodium falciparum/efectos de los fármacos , Plasmodium vivax/efectos de los fármacos , Antimaláricos/farmacología , Cloroquina/farmacología , Humanos , Concentración 50 Inhibidora , Malaria/tratamiento farmacológico , Pruebas de Sensibilidad MicrobianaRESUMEN
BACKGROUND: The World Health Organization recommends malaria be diagnosed by standard microscopy or rapid diagnostic test (RDT) before treatment. RDTs have been used with greater frequency in the absence of matching blood slide confirmation in the majority of RDT reported cases in Mimika Regency, Papua Province, Indonesia. Given the importance of RDT in current health system as point-of-care tool, careful validation of RDT product performance for providing accurate malaria diagnosis is critical. METHODS: Plasmotec Malaria-3 (XW-P07) performance was evaluated by comparing it with paired blood film microscopy and quantitative real-time PCR (qPCR). Consecutive whole blood samples were derived from one clinic in Mimika as part of routine passive malaria case detection. RDT results were read by two trained technicians and interpreted by consensus. Expert microscopic examination of blood slides was cross-checked by observer-blinded second reader and a third examiner if discordant between examinations. qPCR was used as the 'gold standard', followed by microscopy for the outcome/disease variable. Comparison analysis included sensitivity (Sn), specificity (Sp), positive and negative predictive values (PPV & NPV), and other diagnostic screening performance measures for detecting Plasmodium falciparum and Plasmodium vivax infections. RESULTS: Overall malaria positive samples from qPCR was 42.2% (175/415 samples); and from matching blood slides 40.5% (168/415) of which those infections with relatively low parasite densities ≤100/µl blood was 5.7% of P. falciparum and 16.5% of P. vivax samples examined. Overall RDT performance when compared with microscopy for detecting P. falciparum was Sn:92%, Sp:96.6%, PPV:88%, NPV:97.8%, Kappa:0.87; and for P. vivax Sn:72.9%, Sp:99.1%, PPV:95.4%, NPV:93.4%, Kappa:0.79. Overall RDT performance when compared with qPCR for detecting P. falciparum was Sn:92%, Sp:96.6%, PPV:88%, NPV:97.8%, Kappa:0.87; and for P. vivax Sn:66%, Sp:99.1%, PPV:95.4%, NPV:90.9%, Kappa:0.73. CONCLUSIONS: Plasmotec Malaria-3 test showed good overall performance scores in precision for detecting P. falciparum, but lower values regarding sensitivity and negative likelihood ratio for detecting P. vivax, a finding partly associated with greater frequency of lower density P. vivax infections compared to P. falciparum in this study. In particular, the negative likelihood ratio (>0.1) for P. vivax detection indicates RDT lacked sufficient discriminating exclusion power falling below general acceptance criteria.
Asunto(s)
Pruebas Diagnósticas de Rutina/métodos , Malaria Falciparum/diagnóstico , Malaria Vivax/diagnóstico , Microscopía/normas , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , Adolescente , Adulto , Anciano , Niño , Preescolar , Humanos , Indonesia , Lactante , Malaria Falciparum/parasitología , Malaria Vivax/parasitología , Persona de Mediana Edad , Plasmodium falciparum/aislamiento & purificación , Plasmodium vivax/aislamiento & purificación , Adulto JovenRESUMEN
BACKGROUND: The emergence and spread of multidrug-resistant Plasmodium falciparum and Plasmodium vivax highlights the need for objective measures of ex vivo drug susceptibility. Flow cytometry (FC) has potential to provide a robust and rapid quantification of ex vivo parasite growth. METHODS: Field isolates from Papua, Indonesia, underwent ex vivo drug susceptibility testing against chloroquine, amodiaquine, piperaquine, mefloquine, and artesunate. A single nucleic acid stain (i.e., hydroethidine (HE) for P. falciparum and SYBR Green I (SG) for P. vivax) was used to quantify infected red blood cells by FC-based signal detection. Data derived by FC were compared to standard quantification by light microscopy (LM). A subset of isolates was used to compare single and double staining techniques. RESULTS: In total, 57 P. falciparum and 23 P. vivax field isolates were collected for ex vivo drug susceptibility testing. Reliable paired data between LM and FC was obtained for 88 % (295/334) of these assays. The median difference of derived IC50 values varied from -5.4 to 6.1 nM, associated with 0.83-1.23 fold change in IC50 values between LM and FC. In 15 assays (5.1 %), the derived difference of IC50 estimates was beyond the 95 % limits of agreement; in eleven assays (3.7 %), this was attributable to low parasite growth (final schizont count < 40 %), and in four assays (1.4 %) due to low initial parasitaemia at the start of assay (<2000 µl(-1)). In a subset of seven samples, LM, single and double staining FC techniques generated similar IC50 values. CONCLUSIONS: A single staining FC-based assay using a portable cytometer provides a simple, fast and versatile platform for field surveillance of ex vivo drug susceptibility in clinical P. falciparum and P. vivax isolates.
Asunto(s)
Antimaláricos/farmacología , Citometría de Flujo/métodos , Pruebas de Sensibilidad Parasitaria/métodos , Plasmodium falciparum/efectos de los fármacos , Plasmodium vivax/efectos de los fármacos , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Eritrocitos/parasitología , Femenino , Humanos , Indonesia , Masculino , Persona de Mediana Edad , Plasmodium falciparum/crecimiento & desarrollo , Plasmodium vivax/crecimiento & desarrollo , Coloración y Etiquetado/métodos , Adulto JovenRESUMEN
Renewed global efforts toward malaria eradication have highlighted the need for novel antimalarial agents with activity against multiple stages of the parasite life cycle. We have previously reported the discovery of a novel class of antimalarial compounds in the imidazolopiperazine series that have activity in the prevention and treatment of blood stage infection in a mouse model of malaria. Consistent with the previously reported activity profile of this series, the clinical candidate KAF156 shows blood schizonticidal activity with 50% inhibitory concentrations of 6 to 17.4 nM against P. falciparum drug-sensitive and drug-resistant strains, as well as potent therapeutic activity in a mouse models of malaria with 50, 90, and 99% effective doses of 0.6, 0.9, and 1.4 mg/kg, respectively. When administered prophylactically in a sporozoite challenge mouse model, KAF156 is completely protective as a single oral dose of 10 mg/kg. Finally, KAF156 displays potent Plasmodium transmission blocking activities both in vitro and in vivo. Collectively, our data suggest that KAF156, currently under evaluation in clinical trials, has the potential to treat, prevent, and block the transmission of malaria.
Asunto(s)
Antimaláricos/farmacología , Imidazoles/farmacología , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/transmisión , Piperazinas/farmacología , Animales , Concentración 50 Inhibidora , Ratones , Ratones Endogámicos ICR , Plasmodium falciparum/efectos de los fármacos , Esporozoítos/efectos de los fármacosRESUMEN
The declining efficacy of artemisinin derivatives against Plasmodium falciparum highlights the urgent need to identify alternative highly potent compounds for the treatment of malaria. In Papua Indonesia, where multidrug resistance has been documented against both P. falciparum and P. vivax malaria, comparative ex vivo antimalarial activity against Plasmodium isolates was assessed for the artemisinin derivatives artesunate (AS) and dihydroartemisinin (DHA), the synthetic peroxides OZ277 and OZ439, the semisynthetic 10-alkylaminoartemisinin derivatives artemisone and artemiside, and the conventional antimalarial drugs chloroquine (CQ), amodiaquine (AQ), and piperaquine (PIP). Ex vivo drug susceptibility was assessed in 46 field isolates (25 P. falciparum and 21 P. vivax). The novel endoperoxide compounds exhibited potent ex vivo activity against both species, but significant differences in intrinsic activity were observed. Compared to AS and its active metabolite DHA, all the novel compounds showed lower or equal 50% inhibitory concentrations (IC(50)s) in both species (median IC(50)s between 1.9 and 3.6 nM in P. falciparum and 0.7 and 4.6 nM in P. vivax). The antiplasmodial activity of novel endoperoxides showed different cross-susceptibility patterns in the two Plasmodium species: whereas their ex vivo activity correlated positively with CQ, PIP, AS, and DHA in P. falciparum, the same was not apparent in P. vivax. The current study demonstrates for the first time potent activity of novel endoperoxides against drug-resistant P. vivax. The high activity against drug-resistant strains of both Plasmodium species confirms these compounds to be promising candidates for future artemisinin-based combination therapy (ACT) regimens in regions of coendemicity.
Asunto(s)
Adamantano/análogos & derivados , Antimaláricos/farmacología , Artemisininas/farmacología , Compuestos Heterocíclicos con 1 Anillo/farmacología , Peróxidos/farmacología , Plasmodium falciparum/efectos de los fármacos , Plasmodium vivax/efectos de los fármacos , Compuestos de Espiro/farmacología , Adamantano/farmacología , Amodiaquina/farmacología , Artesunato , Cloroquina/farmacología , Resistencia a Múltiples Medicamentos/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Quinolinas/farmacologíaRESUMEN
BACKGROUND: The surveillance and control of mosquito-borne diseases is dependent upon understanding the bionomics and distribution of the vectors. Most studies of mosquito assemblages describe species abundance, richness and composition close to the ground defined often by only one sampling method. In this study, we assessed Australian mosquito species near the ground and in the sub-canopy using two traps baited with a variety of lures. METHODS: Mosquitoes were sampled using a 4 × 4 Latin square design at the Cattana Wetlands, Australia from February to April 2020, using passive box traps with octenol and carbon dioxide and three variations of a sticky net trap (unbaited, and baited with octenol or octenol and carbon dioxide). The traps were deployed at two different heights: ground level (≤ 1 m above the ground) and sub-canopy level (6 m above the ground). RESULTS: In total, 27 mosquito species were identified across the ground and sub-canopy levels from the different traps. The abundance of mosquitoes at the ground level was twofold greater than at the sub-canopy level. While the species richness at ground and sub-canopy levels was not significantly different, species abundance varied by the collection height. CONCLUSIONS: The composition of mosquito population assemblages was correlated with the trap types and heights at which they were deployed. Coquillettidia species, which prefer feeding on birds, were mainly found in the sub-canopy whereas Anopheles farauti, Aedes vigilax and Mansonia uniformis, which have a preference for feeding on large mammals, were predominantly found near the ground. In addition to trap height, environmental factors and mosquito bionomic characteristics (e.g. larval habitat, resting behaviour and host blood preferences) may explain the vertical distribution of mosquitoes. This information is useful to better understand how vectors may acquire and transmit pathogens to hosts living at different heights.
Asunto(s)
Distribución Animal , Culicidae/fisiología , Ecosistema , Mosquitos Vectores/fisiología , Aedes/fisiología , Animales , Anopheles/fisiología , Australia , Culex/fisiología , Culicidae/clasificación , Femenino , Masculino , Control de Mosquitos/métodos , Bosque Lluvioso , Árboles/parasitología , HumedalesRESUMEN
Drug resistant Plasmodium parasites are a major threat to malaria control and elimination. After reports of high levels of multidrug resistant P. falciparum and P. vivax in Indonesia, in 2005, the national first-line treatment policy for uncomplicated malaria was changed in March 2006, to dihydroartemisinin-piperaquine against all species. This study assessed the temporal trends in ex vivo drug susceptibility to chloroquine (CQ) and piperaquine (PIP) for both P. falciparum and P. vivax clinical isolates collected between 2004 and 2018, by using schizont maturation assays, and genotyped a subset of isolates for known and putative molecular markers of CQ and PIP resistance by using Sanger and next generation whole genome sequencing. The median CQ IC50 values varied significantly between years in both Plasmodium species, but there was no significant trend over time. In contrast, there was a significant trend for increasing PIP IC50s in both Plasmodium species from 2010 onwards. Whereas the South American CQ resistant 7G8 pfcrt SVMNT isoform has been fixed since 2005 in the study area, the pfmdr1 86Y allele frequencies decreased and became fixed at the wild-type allele in 2015. In P. vivax isolates, putative markers of CQ resistance (no pvcrt-o AAG (K10) insertion and pvmdr1 Y967F and F1076L) were fixed at the mutant alleles since 2005. None of the putative PIP resistance markers were detected in P. falciparum. The ex vivo drug susceptibility and molecular analysis of CQ and PIP efficacy for P. falciparum and P. vivax after 12 years of intense drug pressure with DHP suggests that whilst the degree of CQ resistance appears to have been sustained, there has been a slight decline in PIP susceptibility, although this does not appear to have reached clinically significant levels. The observed decreasing trend in ex vivo PIP susceptibility highlights the importance of ongoing surveillance.
Asunto(s)
Antimaláricos , Artemisininas , Malaria Falciparum , Antimaláricos/farmacología , Antimaláricos/uso terapéutico , Artemisininas/farmacología , Artemisininas/uso terapéutico , Cloroquina/farmacología , Cloroquina/uso terapéutico , Resistencia a Medicamentos/genética , Humanos , Indonesia/epidemiología , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/epidemiología , Plasmodium falciparum/genética , Plasmodium vivax/genética , Proteínas Protozoarias/genética , QuinolinasRESUMEN
The quest for new antimalarial drugs, especially those with novel modes of action, is essential in the face of emerging drug-resistant parasites. Here we describe a new chemical class of molecules, pyrazoleamides, with potent activity against human malaria parasites and showing remarkably rapid parasite clearance in an in vivo model. Investigations involving pyrazoleamide-resistant parasites, whole-genome sequencing and gene transfers reveal that mutations in two proteins, a calcium-dependent protein kinase (PfCDPK5) and a P-type cation-ATPase (PfATP4), are necessary to impart full resistance to these compounds. A pyrazoleamide compound causes a rapid disruption of Na(+) regulation in blood-stage Plasmodium falciparum parasites. Similar effect on Na(+) homeostasis was recently reported for spiroindolones, which are antimalarials of a chemical class quite distinct from pyrazoleamides. Our results reveal that disruption of Na(+) homeostasis in malaria parasites is a promising mode of antimalarial action mediated by at least two distinct chemical classes.
Asunto(s)
Amidas/farmacología , Antimaláricos/farmacología , Bencimidazoles/farmacología , Eritrocitos/parasitología , Malaria/parasitología , Plasmodium falciparum/efectos de los fármacos , Pirazoles/farmacología , Sodio/metabolismo , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Femenino , Homeostasis/efectos de los fármacos , Humanos , Masculino , Plasmodium berghei/efectos de los fármacos , Plasmodium berghei/genética , Plasmodium berghei/metabolismo , Plasmodium falciparum/enzimología , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Proteínas ProtozoariasRESUMEN
Whole genome sequencing (WGS) of Plasmodium vivax is problematic due to the reliance on clinical isolates which are generally low in parasitaemia and sample volume. Furthermore, clinical isolates contain a significant contaminating background of host DNA which confounds efforts to map short read sequence of the target P. vivax DNA. Here, we discuss a methodology to significantly improve the success of P. vivax WGS on natural (non-adapted) patient isolates. Using 37 patient isolates from Indonesia, Thailand, and travellers, we assessed the application of CF11-based white blood cell filtration alone and in combination with short term ex vivo schizont maturation. Although CF11 filtration reduced human DNA contamination in 8 Indonesian isolates tested, additional short-term culture increased the P. vivax DNA yield from a median of 0.15 to 6.2 ng µl(-1) packed red blood cells (pRBCs) (p = 0.001) and reduced the human DNA percentage from a median of 33.9% to 6.22% (p = 0.008). Furthermore, post-CF11 and culture samples from Thailand gave a median P. vivax DNA yield of 2.34 ng µl(-1) pRBCs, and 2.65% human DNA. In 22 P. vivax patient isolates prepared with the 2-step method, we demonstrate high depth (median 654X coverage) and breadth (≥89%) of coverage on the Illumina GAII and HiSeq platforms. In contrast to the A+T-rich P. falciparum genome, negligible bias was observed in coverage depth between coding and non-coding regions of the P. vivax genome. This uniform coverage will greatly facilitate the detection of SNPs and copy number variants across the genome, enabling unbiased exploration of the natural diversity in P. vivax populations.
Asunto(s)
ADN Protozoario/genética , ADN Protozoario/aislamiento & purificación , Genoma de Protozoos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Malaria Vivax/parasitología , Plasmodium vivax/genética , Humanos , Malaria Vivax/diagnóstico , Plasmodium vivax/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
The goal for developing new antimalarial drugs is to find a molecule that can target multiple stages of the parasite's life cycle, thus impacting prevention, treatment, and transmission of the disease. The 4(1H)-quinolone-3-diarylethers are selective potent inhibitors of the parasite's mitochondrial cytochrome bc1 complex. These compounds are highly active against the human malaria parasites Plasmodium falciparum and Plasmodium vivax. They target both the liver and blood stages of the parasite as well as the forms that are crucial for disease transmission, that is, the gametocytes, the zygote, the ookinete, and the oocyst. Selected as a preclinical candidate, ELQ-300 has good oral bioavailability at efficacious doses in mice, is metabolically stable, and is highly active in blocking transmission in rodent models of malaria. Given its predicted low dose in patients and its predicted long half-life, ELQ-300 has potential as a new drug for the treatment, prevention, and, ultimately, eradication of human malaria.