Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 3.346
Filtrar
1.
BMC Res Notes ; 13(1): 497, 2020 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-33109270

RESUMO

OBJECTIVE: Nigeria bears 25% of global malaria burden despite concerted efforts towards its control and elimination. The emergence of drug resistance to first line drugs, artemisinin combination therapies (ACTs), indicates an urgent need for continuous molecular surveillance of drug resistance especially in high burden countries where drug interventions are heavily relied on. This study describes mutations in Plasmodium falciparum genes associated with drug resistance in malaria; Pfk13, Pfmdr1, PfATPase6 and Pfcrt in isolates obtained from 83 symptomatic malaria patients collected in August 2014, aged 1-61 years old from South-west Nigeria. RESULTS: Two Pfmdr1, N86 and Y184 variants were present at a prevalence of 56% and 13.25% of isolates respectively. There was one synonymous (S679S) and two non-synonymous (M699V, S769M) mutations in the PATPase6 gene, while Pfcrt genotype (CVIET), had a prevalence of 45%. The Pfk13 C580Y mutant allele was suspected by allelic discrimination in two samples with mixed genotypes although this could not be validated with independent isolation or additional methods. Our findings call for robust molecular surveillance of antimalarial drug resistance markers in west Africa especially with increased use of antimalarial drugs as prophylaxis for Covid-19.


Assuntos
Combinação Arteméter e Lumefantrina/uso terapêutico , ATPases Transportadoras de Cálcio/genética , Malária Falciparum/tratamento farmacológico , Proteínas de Membrana Transportadoras/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Mutação , Plasmodium falciparum/efeitos dos fármacos , Proteínas de Protozoários/genética , Adolescente , Adulto , Antimaláricos/uso terapêutico , Artemisininas/uso terapêutico , Criança , Pré-Escolar , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/prevenção & controle , Resistência a Medicamentos/genética , Feminino , Expressão Gênica , Genótipo , Humanos , Lactente , Malária Falciparum/epidemiologia , Malária Falciparum/parasitologia , Pessoa de Meia-Idade , Epidemiologia Molecular , Nigéria/epidemiologia , Pandemias/prevenção & controle , Plasmodium falciparum/genética , Plasmodium falciparum/crescimento & desenvolvimento , Pneumonia Viral/epidemiologia , Pneumonia Viral/prevenção & controle
2.
Nature ; 585(7826): 579-583, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32939086

RESUMO

Malaria has had a major effect on the human genome, with many protective polymorphisms-such as the sickle-cell trait-having been selected to high frequencies in malaria-endemic regions1,2. The blood group variant Dantu provides 74% protection against all forms of severe malaria in homozygous individuals3-5, a similar degree of protection to that afforded by the sickle-cell trait and considerably greater than that offered by the best malaria vaccine. Until now, however, the protective mechanism has been unknown. Here we demonstrate the effect of Dantu on the ability of the merozoite form of the malaria parasite Plasmodium falciparum to invade red blood cells (RBCs). We find that Dantu is associated with extensive changes to the repertoire of proteins found on the RBC surface, but, unexpectedly, inhibition of invasion does not correlate with specific RBC-parasite receptor-ligand interactions. By following invasion using video microscopy, we find a strong link between RBC tension and merozoite invasion, and identify a tension threshold above which invasion rarely occurs, even in non-Dantu RBCs. Dantu RBCs have higher average tension than non-Dantu RBCs, meaning that a greater proportion resist invasion. These findings provide both an explanation for the protective effect of Dantu, and fresh insight into why the efficiency of P. falciparum invasion might vary across the heterogenous populations of RBCs found both within and between individuals.


Assuntos
Antígenos de Grupos Sanguíneos/genética , Eritrócitos/citologia , Eritrócitos/parasitologia , Malária Falciparum/patologia , Malária Falciparum/prevenção & controle , Plasmodium falciparum/metabolismo , Polimorfismo Genético , Antígenos de Grupos Sanguíneos/classificação , Antígenos de Grupos Sanguíneos/metabolismo , Criança , Eritrócitos/metabolismo , Eritrócitos/patologia , Feminino , Genótipo , Humanos , Quênia , Ligantes , Masculino , Merozoítos/metabolismo , Merozoítos/patogenicidade , Microscopia de Vídeo , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium falciparum/patogenicidade
3.
Nat Commun ; 11(1): 4015, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32782246

RESUMO

Intracellular pathogens mobilize host signaling pathways of their host cell to promote their own survival. Evidence is emerging that signal transduction elements are activated in a-nucleated erythrocytes in response to infection with malaria parasites, but the extent of this phenomenon remains unknown. Here, we fill this knowledge gap through a comprehensive and dynamic assessment of host erythrocyte signaling during infection with Plasmodium falciparum. We used arrays of 878 antibodies directed against human signaling proteins to interrogate the activation status of host erythrocyte phospho-signaling pathways at three blood stages of parasite asexual development. This analysis reveals a dynamic modulation of many host signalling proteins across parasite development. Here we focus on the hepatocyte growth factor receptor (c-MET) and the MAP kinase pathway component B-Raf, providing a proof of concept that human signaling kinases identified as activated by malaria infection represent attractive targets for antimalarial intervention.


Assuntos
Antimaláricos/farmacologia , Eritrócitos/metabolismo , Plasmodium falciparum/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais , Eritrócitos/parasitologia , Interações Hospedeiro-Parasita , Humanos , Concentração Inibidora 50 , Estágios do Ciclo de Vida/efeitos dos fármacos , Malária Falciparum/metabolismo , Malária Falciparum/parasitologia , Fosforilação/efeitos dos fármacos , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium falciparum/metabolismo , Plasmodium falciparum/fisiologia , Análise Serial de Proteínas , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-met/metabolismo , Transdução de Sinais/efeitos dos fármacos
4.
PLoS One ; 15(7): e0235798, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32673324

RESUMO

During the course of the asexual erythrocytic stage of development, Plasmodium spp. parasites undergo a series of morphological changes and induce alterations in the host cell. At the end of this stage, the parasites egress from the infected cell, after which the progeny invade a new host cell. These processes are rapid and occur in a time-dependent manner. Of particular importance, egress and invasion of erythrocytes by the parasite are difficult to capture in an unsynchronized culture, or even a culture that has been synchronized within a window of one to several hours. Therefore, precise synchronization of parasite cultures is of paramount importance for the investigation of these processes. Here we describe a method for synchronizing Plasmodium falciparum and Plasmodium knowlesi asexual blood stage parasites with ML10, a highly specific inhibitor of the cGMP-dependent protein kinase (PKG) that arrests parasite growth approximately 15 minutes prior to egress. This inhibitor allows parasite cultures to be synchronized so that all parasites are within a window of development of several minutes, with a simple wash step. Furthermore, we show that parasites remain viable for several hours after becoming arrested by the compound and that ML10 has advantages, owing to its high specificity and low EC50, over the previously used PKG inhibitor Compound 2. Here, we demonstrate that ML10 is an invaluable tool for the study of Plasmodium spp. asexual blood stage biology and for the routine synchronization of P. falciparum and P. knowlesi cultures.


Assuntos
Eritrócitos/parasitologia , Malária/parasitologia , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium knowlesi/crescimento & desenvolvimento , Técnicas de Cultura de Células/métodos , Humanos , Malária Falciparum/parasitologia , Plasmodium falciparum/efeitos dos fármacos , Plasmodium knowlesi/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Fatores de Tempo
5.
PLoS One ; 15(6): e0233840, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32555601

RESUMO

BACKGROUND: Immunization with radiation-attenuated sporozoites (RAS) by mosquito bite provides >90% sterile protection against Plasmodium falciparum (Pf) malaria in humans. RAS invade hepatocytes but do not replicate. CD8+ T cells recognizing parasite-derived peptides on the surface of infected hepatocytes are likely the primary protective mechanism. We conducted a randomized clinical trial of RAS immunization to assess safety, to achieve 50% vaccine efficacy (VE) against controlled human malaria infection (CHMI), and to generate reagents from protected and non-protected subjects for future identification of protective immune mechanisms and antigens. METHODS: Two cohorts (Cohort 1 and Cohort 2) of healthy, malaria-naïve, non-pregnant adults age 18-50 received five monthly immunizations with infected (true-immunized, n = 21) or non-infected (mock-immunized, n = 5) mosquito bites and underwent homologous CHMI at 3 weeks. Immunization parameters were selected for 50% protection based on prior clinical data. Leukapheresis was done to collect plasma and peripheral blood mononuclear cells. RESULTS: Adverse event rates were similar in true- and mock-immunized subjects. Two true- and two mock-immunized subjects developed large local reactions likely caused by mosquito salivary gland antigens. In Cohort 1, 11 subjects received 810-1235 infected bites; 6/11 (55%) were protected against CHMI vs. 0/3 mock-immunized and 0/6 infectivity controls (VE 55%). In Cohort 2, 10 subjects received 839-1131 infected bites with a higher first dose and a reduced fifth dose; 9/10 (90%) were protected vs. 0/2 mock-immunized and 0/6 controls (VE 90%). Three/3 (100%) protected subjects administered three booster immunizations were protected against repeat CHMI vs. 0/6 controls (VE 100%). Cohort 2 uniquely showed a significant rise in IFN-γ responses after the third and fifth immunizations and higher antibody responses to CSP. CONCLUSIONS: PfRAS were generally safe and well tolerated. Cohort 2 had a higher first dose, reduced final dose, higher antibody responses to CSP and significant rise of IFN-γ responses after the third and fifth immunizations. Whether any of these factors contributed to increased protection in Cohort 2 requires further investigation. A cryobank of sera and cells from protected and non-protected individuals was generated for future immunological studies and antigen discovery. TRIAL REGISTRATION: ClinicalTrials.gov NCT01994525.


Assuntos
Mordeduras e Picadas de Insetos/imunologia , Malária/prevenção & controle , Esporozoítos/imunologia , Vacinação/métodos , Vacinas Atenuadas/efeitos adversos , Adulto , Animais , Anopheles/parasitologia , Anopheles/fisiologia , Feminino , Raios gama , Humanos , Malária/imunologia , Masculino , Pessoa de Meia-Idade , Mosquitos Vetores/parasitologia , Mosquitos Vetores/fisiologia , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium falciparum/imunologia , Plasmodium falciparum/patogenicidade , Proteínas de Protozoários/imunologia , Esporozoítos/patogenicidade , Esporozoítos/efeitos da radiação , Vacinação/efeitos adversos
6.
Nat Commun ; 11(1): 2763, 2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32488076

RESUMO

Malaria parasites complete their intra-erythrocytic developmental cycle (IDC) in multiples of 24 h suggesting a circadian basis, but the mechanism controlling this periodicity is unknown. Combining in vivo and in vitro approaches utilizing rodent and human malaria parasites, we reveal that: (i) 57% of Plasmodium chabaudi genes exhibit daily rhythms in transcription; (ii) 58% of these genes lose transcriptional rhythmicity when the IDC is out-of-synchrony with host rhythms; (iii) 6% of Plasmodium falciparum genes show 24 h rhythms in expression under free-running conditions; (iv) Serpentine receptor 10 (SR10) has a 24 h transcriptional rhythm and disrupting it in rodent malaria parasites shortens the IDC by 2-3 h; (v) Multiple processes including DNA replication, and the ubiquitin and proteasome pathways, are affected by loss of coordination with host rhythms and by disruption of SR10. Our results reveal malaria parasites are at least partly responsible for scheduling the IDC and coordinating their development with host daily rhythms.


Assuntos
Ritmo Circadiano/fisiologia , Eritropoese/fisiologia , Interações Hospedeiro-Parasita/fisiologia , Malária/metabolismo , Proteínas de Protozoários/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Alcaloides de Triptamina e Secologanina/metabolismo , Animais , Proteínas de Caenorhabditis elegans , Modelos Animais de Doenças , Feminino , Expressão Gênica , Interações Hospedeiro-Parasita/genética , Humanos , Malária/parasitologia , Camundongos , Camundongos Knockout , Plasmodium chabaudi/genética , Plasmodium chabaudi/crescimento & desenvolvimento , Plasmodium falciparum/genética , Plasmodium falciparum/crescimento & desenvolvimento , Proteínas de Protozoários/genética , Receptores Acoplados a Proteínas-G/genética , Roedores , Transcriptoma
7.
PLoS One ; 15(6): e0235119, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32574179

RESUMO

BACKGROUND: Colombia has officially adopted the parasite density levels of severe malaria established by the WHO (>50,000 parasites/µl). These values have been inferred from areas of high transmission in Africa and are not consistent with the dynamics of low and unstable transmission in Colombia. The objective of this study was therefore to determine the parasite density values observed in patients with severe malaria and their distribution in the different ecoepidemiological regions of Colombia. METHODS: A retrospective and descriptive study of confirmed cases of severe malaria was conducted in endemic areas of malaria in Colombia over the period 2014-2017. Data were collected from secondary sources of the Subnational Programs of Malaria Prevention and Control. Person, place, and time variables were selected. The official definition of severe malaria was adopted, and compliance with these criteria was determined. Univariate and bivariate analyses were conducted with absolute and relative frequency measures, and the relevant statistical tests were applied. RESULTS: The overall parasite density values in Colombia showed a geometric mean of 5,919 parasites/µl (95% CI: 5,608-6,248). By parasite species, the values were 6,151 (95% CI: 5,631-6,718) for Plasmodium falciparum and 5,815 (95% CI: 5,428-6,230) for Plasmodium vivax. The highest parasite density values were recorded in the Amazon ecoepidemiological region (8,177; 95% CI: 6,015-11,116), and the lowest values were recorded in the Andean region (5,026; 95% CI: 2,409-10,480). CONCLUSIONS: In endemic areas of low and unstable malaria transmission in the Colombian territory, the parasite density levels observed in populations with severe malaria are lower than the officially established values. The parasite density criterion is not really a relevant criterion for the definition of severe cases in Colombia and it certainly not be used to make a clinical decision about the severity of the disease.


Assuntos
Malária Falciparum/epidemiologia , Malária Vivax/epidemiologia , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium vivax/crescimento & desenvolvimento , Densidade Demográfica , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Colômbia/epidemiologia , Doenças Endêmicas/prevenção & controle , Feminino , Geografia , Humanos , Lactente , Malária Falciparum/parasitologia , Malária Falciparum/transmissão , Malária Vivax/parasitologia , Malária Vivax/transmissão , Masculino , Pessoa de Meia-Idade , Plasmodium falciparum/fisiologia , Plasmodium vivax/fisiologia , Prevalência , Estudos Retrospectivos , Índice de Gravidade de Doença , Adulto Jovem
8.
Parasitol Res ; 119(6): 1753-1765, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32363442

RESUMO

RbAp46/RBBP7 and RbAp48/RBBP4 are WD40-repeat histone chaperones and chromatin adaptors that reside in multiple complexes involved in maintenance of chromatin structure. RbAp48 is the essential subunit of the chromatin assembly factor-1 (CAF-1) complex, therefore also named as CAF-1C. A detailed in silico sequence and structure analysis of homologs of RbAp46/48 in Plasmodium falciparum (PF3D7_0110700 and PF3D7_1433300) exhibited conservation of characteristic features in both the protein-seven-bladed WD40 ß-propeller conformation and different binding interfaces. A comparative structural analysis highlighted species-specific features of the parasite, yeast, drosophila, and human RbAp46/48. In the present study, we report cloning, expression, and characterization of P. falciparum PF3D7_0110700, a putative RbAp46/48 (PfRbAp46/48). PfRbAp46/48 was cloned into pTEM11 vector in fusion with 6xHistidine tag and over-expressed in Escherichia coli B834 cells. The protein was purified by Ni-NTA followed by gel permeation chromatography. The protein expressed in all the three asexual blood stages and exhibited nuclear localization. We showed direct interaction of the purified rPfRbAp46/48 with the histone H4. These findings further our understanding of RbAp46/48 proteins and role of these proteins in the parasite biology.


Assuntos
Chaperonas de Histonas/química , Chaperonas de Histonas/metabolismo , Plasmodium falciparum/química , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Núcleo Celular/metabolismo , Cromatina/metabolismo , Expressão Gênica , Chaperonas de Histonas/genética , Histonas/metabolismo , Estágios do Ciclo de Vida/genética , Plasmodium falciparum/genética , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium falciparum/metabolismo , Ligação Proteica , Conformação Proteica , Proteínas de Protozoários/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo
9.
Science ; 368(6492): 754-759, 2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32409472

RESUMO

The blood stage of the infection of the malaria parasite Plasmodium falciparum exhibits a 48-hour developmental cycle that culminates in the synchronous release of parasites from red blood cells, which triggers 48-hour fever cycles in the host. This cycle could be driven extrinsically by host circadian processes or by a parasite-intrinsic oscillator. To distinguish between these hypotheses, we examine the P. falciparum cycle in an in vitro culture system and show that the parasite has molecular signatures associated with circadian and cell cycle oscillators. Each of the four strains examined has a different period, which indicates strain-intrinsic period control. Finally, we demonstrate that parasites have low cell-to-cell variance in cycle period, on par with a circadian oscillator. We conclude that an intrinsic oscillator maintains Plasmodium's rhythmic life cycle.


Assuntos
Relógios Circadianos/fisiologia , Eritrócitos/parasitologia , Interações Hospedeiro-Parasita/fisiologia , Estágios do Ciclo de Vida , Malária Falciparum/sangue , Malária Falciparum/parasitologia , Plasmodium falciparum/crescimento & desenvolvimento , Animais , Relógios Circadianos/genética , Expressão Gênica , Genes de Protozoários/fisiologia , Interações Hospedeiro-Parasita/genética , Camundongos , Plasmodium falciparum/genética , Transcriptoma
10.
Nature ; 582(7810): 104-108, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32427965

RESUMO

Malaria caused by Plasmodium falciparum remains the leading single-agent cause of mortality in children1, yet the promise of an effective vaccine has not been fulfilled. Here, using our previously described differential screening method to analyse the proteome of blood-stage P. falciparum parasites2, we identify P. falciparum glutamic-acid-rich protein (PfGARP) as a parasite antigen that is recognized by antibodies in the plasma of children who are relatively resistant-but not those who are susceptible-to malaria caused by P. falciparum. PfGARP is a parasite antigen of 80 kDa that is expressed on the exofacial surface of erythrocytes infected by early-to-late-trophozoite-stage parasites. We demonstrate that antibodies against PfGARP kill trophozoite-infected erythrocytes in culture by inducing programmed cell death in the parasites, and that vaccinating non-human primates with PfGARP partially protects against a challenge with P. falciparum. Furthermore, our longitudinal cohort studies showed that, compared to individuals who had naturally occurring anti-PfGARP antibodies, Tanzanian children without anti-PfGARP antibodies had a 2.5-fold-higher risk of severe malaria and Kenyan adolescents and adults without these antibodies had a twofold-higher parasite density. By killing trophozoite-infected erythrocytes, PfGARP could synergize with other vaccines that target parasite invasion of hepatocytes or the invasion of and egress from erythrocytes.


Assuntos
Apoptose/imunologia , Peptídeos e Proteínas de Sinalização Intercelular/imunologia , Malária Falciparum/imunologia , Malária Falciparum/prevenção & controle , Parasitos/imunologia , Plasmodium falciparum/citologia , Plasmodium falciparum/imunologia , Proteínas de Protozoários/imunologia , Adolescente , Adulto , Animais , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/química , Antígenos de Protozoários/imunologia , Aotidae/imunologia , Aotidae/parasitologia , Caspases/metabolismo , Criança , Estudos de Coortes , DNA de Protozoário/química , DNA de Protozoário/metabolismo , Ativação Enzimática , Eritrócitos/parasitologia , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/química , Quênia , Vacinas Antimaláricas/imunologia , Malária Falciparum/parasitologia , Masculino , Camundongos , Parasitos/citologia , Parasitos/crescimento & desenvolvimento , Plasmodium falciparum/crescimento & desenvolvimento , Proteínas de Protozoários/química , Tanzânia , Trofozoítos/citologia , Trofozoítos/crescimento & desenvolvimento , Trofozoítos/imunologia , Vacúolos/imunologia
11.
Lancet Infect Dis ; 20(8): 964-975, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32275867

RESUMO

BACKGROUND: (+)-SJ000557733 (SJ733) is a novel, orally bioavailable inhibitor of Plasmodium falciparum ATP4. In this first-in-human and induced blood-stage malaria phase 1a/b trial, we investigated the safety, tolerability, pharmacokinetics, and antimalarial activity of SJ733 in humans. METHODS: The phase 1a was a single-centre, dose-escalation, first-in-human study of SJ733 allowing modifications to dose increments and dose-cohort size on the basis of safety and pharmacokinetic results. The phase 1a took place at St Jude Children's Research Hospital and at the University of Tennessee Clinical Research Center (Memphis, TN, USA). Enrolment in more than one non-consecutive dose cohort was allowed with at least 14 days required between doses. Participants were fasted in seven dose cohorts and fed in one 600 mg dose cohort. Single ascending doses of SJ733 (75, 150, 300, 600, 900, or 1200 mg) were administered to participants, who were followed up for 14 days after SJ733 dosing. Phase 1a primary endpoints were safety, tolerability, and pharmacokinetics of SJ733, and identification of an SJ733 dose to test in the induced blood-stage malaria model. The phase 1b was a single-centre, open-label, volunteer infection study using the induced blood-stage malaria model in which fasted participants were intravenously infected with blood-stage P falciparum and subsequently treated with a single dose of SJ733. Phase 1b took place at Q-Pharm (Herston, QLD, Australia) and was initiated only after phase 1a showed that exposure exceeding the threshold minimum exposure could be safely achieved in humans. Participants were inoculated on day 0 with P falciparum-infected human erythrocytes (around 2800 parasites in the 150 mg dose cohort and around 2300 parasites in the 600 mg dose cohort), and parasitaemia was monitored before malaria inoculation, after inoculation, immediately before SJ733 dosing, and then post-dose. Participants were treated with SJ733 within 24 h of reaching 5000 parasites per mL or at a clinical score higher than 6. Phase 1b primary endpoints were calculation of a parasite reduction ratio (PRR48) and parasite clearance half-life, and safety and tolerability of SJ733 (incidence, severity, and drug-relatedness of adverse events). In both phases of the trial, SJ733 hydrochloride salt was formulated as a powder blend in capsules containing 75 mg or 300 mg for oral administration. Healthy men and women (of non-childbearing potential) aged 18-55 years were eligible for both studies. Both studies are registered with ClinicalTrials.gov (NCT02661373 for the phase 1a and NCT02867059 for the phase 1b). FINDINGS: In the phase 1a, 23 healthy participants were enrolled and received one to three non-consecutive doses of SJ733 between March 14 and Dec 7, 2016. SJ733 was safe and well tolerated at all doses and in fasted and fed conditions. 119 adverse events were recorded: 54 (45%) were unrelated, 63 (53%) unlikely to be related, and two (2%) possibly related to SJ733. In the phase 1b, 17 malaria-naive, healthy participants were enrolled. Seven participants in the 150 mg dose cohort were inoculated and dosed with SJ733. Eight participants in the 600 mg dose cohort were inoculated, but two participants could not be dosed with SJ733. Two additional participants were subsequently inoculated and dosed with SJ733. SJ733 exposure increased proportional to the dose through to the 600 mg dose, then was saturable at higher doses. Fasted participants receiving 600 mg exceeded the target area under the concentration curve extrapolated to infinity (AUC0-∞) of 13 000 µg × h/L (median AUC0-∞ 24 283 [IQR 16 135-31 311] µg × h/L, median terminal half-life 17·4 h [IQR 16·1-24·0], and median timepoint at which peak plasma concentration is reached 1·0 h [0·6-1·3]), and this dose was tested in the phase 1b. All 15 participants dosed with SJ733 had at least one adverse event. Of the 172 adverse events recorded, 128 (74%) were mild. The only adverse event attributed to SJ733 was mild bilateral foot paraesthesia that lasted 3·75 h and resolved spontaneously. The most common adverse events were related to malaria. Based on parasite clearance half-life, the derived log10PRR48 and corresponding parasite clearance half-lives were 2·2 (95% CI 2·0-2·5) and 6·47 h (95% CI 5·88-7·18) for 150 mg, and 4·1 (3·7-4·4) and 3·56 h (3·29-3·88) for 600 mg. INTERPRETATION: The favourable pharmacokinetic, tolerability, and safety profile of SJ733, and rapid antiparasitic effect support its development as a fast-acting component of combination antimalarial therapy. FUNDING: Global Health Innovative Technology Fund, Medicines for Malaria Venture, and the American Lebanese Syrian Associated Charities.


Assuntos
Antimaláricos/uso terapêutico , Compostos Heterocíclicos de 4 ou mais Anéis/uso terapêutico , Isoquinolinas/uso terapêutico , Malária Falciparum/tratamento farmacológico , Malária Falciparum/parasitologia , Plasmodium falciparum/efeitos dos fármacos , Inibidores da Bomba de Prótons/uso terapêutico , Adulto , Antimaláricos/administração & dosagem , Antimaláricos/efeitos adversos , Antimaláricos/farmacocinética , Estudos de Casos e Controles , Eritrócitos/efeitos dos fármacos , Eritrócitos/parasitologia , Feminino , ATPase Trocadora de Hidrogênio-Potássio/metabolismo , Compostos Heterocíclicos de 4 ou mais Anéis/administração & dosagem , Compostos Heterocíclicos de 4 ou mais Anéis/efeitos adversos , Compostos Heterocíclicos de 4 ou mais Anéis/farmacocinética , Humanos , Isoquinolinas/administração & dosagem , Isoquinolinas/efeitos adversos , Isoquinolinas/farmacocinética , Estágios do Ciclo de Vida/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium falciparum/metabolismo , Inibidores da Bomba de Prótons/administração & dosagem , Inibidores da Bomba de Prótons/efeitos adversos , Inibidores da Bomba de Prótons/farmacocinética , Resultado do Tratamento , Adulto Jovem
12.
Transfusion ; 60(5): 955-964, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32282944

RESUMO

BACKGROUND: Malaria remains a leading transfusion associated infectious risk in endemic areas. However, the prevalence of malaria parasitemia has not been well characterized in blood donor populations. This study sought to determine the prevalence of Plasmodium in red blood cell (RBC) and whole blood (WB) units after the rainy season in Uganda. METHODS AND MATERIALS: Between May and July 2018, blood was collected from the sample diversion pouch of 1000 WB donors in Kampala and Jinja, Uganda. The RBC pellet from ethylenediamine tetraacetic acid (EDTA) anticoagulated blood was stored at -80°C until testing. DNA was extracted and nested PCR was used to screen samples at the genus level for Plasmodium, with positive samples further tested for species identification. RESULTS: Malaria parasitemia among asymptomatic, eligible blood donors in two regions of Uganda was 15.4%; 87.7% (135/154) of infections were with P. falciparum, while P. malariae and P. ovale were also detected. There were 4.3% of blood donors who had mixed infection with multiple species. Older donors (>30 years vs. 17-19 years; aPR = 0.31 [95% CI = 0.17-0.58]), females (aPR = 0.60 [95% CI = 0.42-0.87]), repeat donors (aPR = 0.44 [95% CI = 0.27-0.72]) and those donating near the capital city of Kampala versus rural Jinja region (aPR = 0.49 [95% CI = 0.34-0.69]) had a lower prevalence of malaria parasitemia. CONCLUSIONS: A high proportion of asymptomatic blood donors residing in a malaria endemic region demonstrate evidence of parasitemia at time of donation. Further research is needed to quantify the risk and associated burden of transfusion-transmitted malaria (TTM) in order to inform strategies to prevent TTM.


Assuntos
Doadores de Sangue/estatística & dados numéricos , Malária/epidemiologia , Parasitemia/epidemiologia , Adolescente , Adulto , Infecções Assintomáticas/epidemiologia , Transfusão de Sangue/estatística & dados numéricos , Estudos Transversais , Feminino , Humanos , Malária/sangue , Malária Falciparum/sangue , Malária Falciparum/epidemiologia , Masculino , Pessoa de Meia-Idade , Parasitemia/sangue , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium falciparum/isolamento & purificação , Plasmodium malariae/crescimento & desenvolvimento , Plasmodium malariae/isolamento & purificação , Plasmodium ovale/crescimento & desenvolvimento , Plasmodium ovale/isolamento & purificação , Prevalência , Uganda/epidemiologia , Adulto Jovem
13.
Malar J ; 19(1): 147, 2020 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-32268910

RESUMO

BACKGROUND: A previous study reported that the malaria parasite Plasmodium falciparum enters an altered growth state upon extracellular withdrawal of the essential amino acid isoleucine. Parasites slowed transit through the cell cycle when deprived of isoleucine prior to the onset of S-phase. METHODS: This project was undertaken to study at higher resolution, how isoleucine withdrawal affects parasite growth. Parasites were followed at regular intervals across an extended isoleucine deprivation time course across the cell cycle using flow cytometry. RESULTS: These experiments revealed that isoleucine-deprived parasites never exit the cell cycle, but instead continuously grow at a markedly reduced pace. Moreover, slow growth occurs only if isoleucine is removed prior to the onset of schizogony. After S-phase commenced, the parasite is insensitive to isoleucine depletion and transits through the cell cycle at the normal pace. CONCLUSIONS: The markedly different response of the parasite to isoleucine withdrawal before or after the onset of DNA replication is reminiscent of the nutrient-dependent G1 cell cycle checkpoints described in other organisms.


Assuntos
Ciclo Celular/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , DNA de Protozoário/fisiologia , Eritrócitos/parasitologia , Isoleucina/deficiência , Plasmodium falciparum/crescimento & desenvolvimento , Replicação do DNA/fisiologia , Plasmodium falciparum/citologia , Plasmodium falciparum/efeitos dos fármacos
14.
Nat Commun ; 11(1): 1411, 2020 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-32179747

RESUMO

The disease-causing blood-stage of the Plasmodium falciparum lifecycle begins with invasion of human erythrocytes by merozoites. Many vaccine candidates with key roles in binding to the erythrocyte surface and entry are secreted from the large bulb-like rhoptry organelles at the apical tip of the merozoite. Here we identify an essential role for the conserved protein P. falciparum Cytosolically Exposed Rhoptry Leaflet Interacting protein 1 (PfCERLI1) in rhoptry function. We show that PfCERLI1 localises to the cytosolic face of the rhoptry bulb membrane and knockdown of PfCERLI1 inhibits merozoite invasion. While schizogony and merozoite organelle biogenesis appear normal, biochemical techniques and semi-quantitative super-resolution microscopy show that PfCERLI1 knockdown prevents secretion of key rhoptry antigens that coordinate merozoite invasion. PfCERLI1 is a rhoptry associated protein identified to have a direct role in function of this essential merozoite invasion organelle, which has broader implications for understanding apicomplexan invasion biology.


Assuntos
Eritrócitos/parasitologia , Malária Falciparum/parasitologia , Merozoítos/metabolismo , Plasmodium falciparum/crescimento & desenvolvimento , Proteínas de Protozoários/metabolismo , Humanos , Merozoítos/genética , Merozoítos/crescimento & desenvolvimento , Organelas/parasitologia , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/genética
15.
Transfusion ; 60(4): 799-805, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32129497

RESUMO

BACKGROUND: Risk of transfusion-transmitted (TT) malaria is mainly associated with whole blood (WB) or red blood cell (RBC) transfusion. Risk mitigation relies mostly on donor deferral while a limited number of countries perform blood testing, both negatively impacting blood availability. This study investigated the efficacy of the pathogen reduction system using amustaline and glutathione (GSH) to inactivate Plasmodium falciparum in WB. STUDY DESIGN AND METHODS: WB units were spiked with ring stage P. falciparum infected RBCs. Parasite loads were measured in samples at time of infection, after 24 hours at room temperature (RT), and after a 24-hour incubation at RT post-treatment with 0.2 mM amustaline and 2 mM GSH. Serial 10-fold dilutions of the samples were inoculated to RBC cultures and maintained up to 4 weeks. Parasitemia was quantified by cytometry. RESULTS: The P. falciparum viability assay has a limit of detection of a single live parasite per sample. Input parasite titer was >5.7 log10 TCID50 per mL. A 24-hour incubation at RT paused parasite development in controls, but they retained viability and infectivity when tested in culture. In contrast, no infectious parasites were detected in the amustaline/GSH-treated sample after 4 weeks of culture. CONCLUSION: A robust level of P. falciparum inactivation was achieved in WB using amustaline/GSH treatment. Parasite log reduction was >5.7 log10 TCID50 per mL. Development of such a pathogen reduction system may provide an opportunity to reduce the risk of TT malaria and improve blood availability.


Assuntos
Acridinas/farmacologia , Glutationa/farmacologia , Malária Falciparum/prevenção & controle , Viabilidade Microbiana/efeitos dos fármacos , Compostos de Mostarda Nitrogenada/farmacologia , Segurança do Sangue/métodos , Eritrócitos/microbiologia , Eritrócitos/parasitologia , Humanos , Malária Falciparum/sangue , Malária Falciparum/transmissão , Carga Parasitária , Parasitemia/tratamento farmacológico , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/crescimento & desenvolvimento
16.
Int J Parasitol ; 50(3): 235-252, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32135179

RESUMO

With emerging resistance to frontline treatments, it is vital that new drugs are identified to target Plasmodium falciparum. One of the most critical processes during parasites asexual lifecycle is the invasion and subsequent egress of red blood cells (RBCs). Many unique parasite ligands, receptors and enzymes are employed during egress and invasion that are essential for parasite proliferation and survival, therefore making these processes druggable targets. To identify potential inhibitors of egress and invasion, we screened the Medicines for Malaria Venture Pathogen Box, a 400 compound library against neglected tropical diseases, including 125 with antimalarial activity. For this screen, we utilised transgenic parasites expressing a bioluminescent reporter, nanoluciferase (Nluc), to measure inhibition of parasite egress and invasion in the presence of the Pathogen Box compounds. At a concentration of 2 µM, we found 15 compounds that inhibited parasite egress by >40% and 24 invasion-specific compounds that inhibited invasion by >90%. We further characterised 11 of these inhibitors through cell-based assays and live cell microscopy, and found two compounds that inhibited merozoite maturation in schizonts, one compound that inhibited merozoite egress, one compound that directly inhibited parasite invasion and one compound that slowed down invasion and arrested ring formation. The remaining compounds were general growth inhibitors that acted during the egress and invasion phase of the cell cycle. We found the sulfonylpiperazine, MMV020291, to be the most invasion-specific inhibitor, blocking successful merozoite internalisation within human RBCs and having no substantial effect on other stages of the cell cycle. This has significant implications for the possible development of an invasion-specific inhibitor as an antimalarial in a combination based therapy, in addition to being a useful tool for studying the biology of the invading parasite.


Assuntos
Antimaláricos/farmacologia , Avaliação Pré-Clínica de Medicamentos , Plasmodium falciparum/efeitos dos fármacos , Animais , Eritrócitos/parasitologia , Humanos , Malária Falciparum/tratamento farmacológico , Malária Falciparum/parasitologia , Merozoítos/efeitos dos fármacos , Piperazina , Piperazinas/farmacologia , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium falciparum/metabolismo , Esquizontes/efeitos dos fármacos
17.
Nat Commun ; 11(1): 1503, 2020 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-32198457

RESUMO

In the malaria parasite Plasmodium falciparum, the switch from asexual multiplication to sexual differentiation into gametocytes is essential for transmission to mosquitos. The transcription factor PfAP2-G is a key determinant of sexual commitment that orchestrates this crucial cell fate decision. Here we identify the direct targets of PfAP2-G and demonstrate that it dynamically binds hundreds of sites across the genome. We find that PfAP2-G is a transcriptional activator of early gametocyte genes, and identify differences in PfAP2-G occupancy between gametocytes derived via next-cycle and same-cycle conversion. Our data implicate PfAP2-G not only as a transcriptional activator of gametocyte genes, but also as a potential regulator of genes important for red blood cell invasion. We also find that regulation by PfAP2-G requires interaction with a second transcription factor, PfAP2-I. These results clarify the functional role of PfAP2-G during sexual commitment and early gametocytogenesis.


Assuntos
Malária/parasitologia , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Sistemas CRISPR-Cas , Eritrócitos/parasitologia , Regulação da Expressão Gênica , Genes de Protozoários/genética , Malária/transmissão , Malária Falciparum/parasitologia , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/sangue , Fatores de Transcrição/metabolismo
18.
Microb Genom ; 6(2)2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32017676

RESUMO

Proteins interacting with DNA are fundamental for mediating processes such as gene expression, DNA replication and maintenance of genome integrity. Accumulating evidence suggests that the chromatin of apicomplexan parasites, such as Plasmodium falciparum, is highly organized, and this structure provides an epigenetic mechanism for transcriptional regulation. To investigate how parasite chromatin structure is being regulated, we undertook comparative genomics analysis using 12 distinct eukaryotic genomes. We identified conserved and parasite-specific chromatin-associated domains (CADs) and proteins (CAPs). We then used the chromatin enrichment for proteomics (ChEP) approach to experimentally capture CAPs in P. falciparum. A topological scoring analysis of the proteomics dataset revealed stage-specific enrichments of CADs and CAPs. Finally, we characterized, two candidate CAPs: a conserved homologue of the structural maintenance of chromosome 3 protein and a homologue of the crowded-like nuclei protein, a plant-like protein functionally analogous to animal nuclear lamina proteins. Collectively, our results provide a comprehensive overview of CAPs in apicomplexans, and contribute to our understanding of the complex molecular components regulating chromatin structure and genome architecture in these deadly parasites.


Assuntos
Cromatina/metabolismo , Malária Falciparum/parasitologia , Plasmodium falciparum/metabolismo , Proteoma/metabolismo , Proteínas de Protozoários/metabolismo , Cromatina/genética , Regulação da Expressão Gênica , Genoma de Protozoário , Humanos , Plasmodium falciparum/genética , Plasmodium falciparum/crescimento & desenvolvimento , Ligação Proteica , Proteoma/genética , Proteínas de Protozoários/genética
19.
Malar J ; 19(1): 74, 2020 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-32070344

RESUMO

BACKGROUND: Gold standard microscopic examination of Plasmodium falciparum intraerythrocytic stage remains an important process for staging and enumerating parasitized erythrocytes in culture; however, microscopy is laborious and its accuracy is dependent upon the skill of the examiner. METHODS: In this study, ViSafe Green (VSG), which is a nucleic acid-binding fluorescent dye, was used for assessing in vitro development of P. falciparum using flow cytometry. RESULTS: Fluorescence intensity of VSG was found to depend on the developmental stage of parasites. Specifically, multiple-nuclei-containing schizonts were observed in the VSGhigh population, and growing trophozoites and ring-shaped forms were observed in the VSGintermediate and VSGlow populations. The efficacy of VSG-based assay was found to be comparable to the microscopic examination method, and it demonstrated an ability to detect as low as 0.001% of the parasitaemia estimated by Giemsa staining. Moreover, when applying VSG for anti-malarial drug test, it was able to observe the growth inhibitory effect of dihydroartemisinin, the front-line drug for malaria therapy. CONCLUSIONS: Taken together, the results of this study suggest the VSG-based flow cytometric assay to be a simple and reliable assay for assessing P. falciparum malaria development in vitro.


Assuntos
Eritrócitos/parasitologia , Citometria de Fluxo/métodos , Corantes Fluorescentes/química , Plasmodium falciparum/crescimento & desenvolvimento , Coloração e Rotulagem/instrumentação
20.
J Med Chem ; 63(4): 1750-1762, 2020 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-32011136

RESUMO

Ivermectin is a powerful endectocide, which reduces the incidence of vector-borne diseases. Besides its strong insecticidal effect on mosquito vectors of the disease, ivermectin inhibits Plasmodium falciparum sporogonic and blood stage development and impairs Plasmodium berghei development inside hepatocytes, both in vitro and in vivo. Herein, we present the first report on structural modification of ivermectin to produce dual-action molecular hybrids with good structure-dependent in vitro activity against both the hepatic and erythrocytic stages of P. berghei and P. falciparum infection, suggesting inclusion of ivermectin antimalarial hybrids in malaria control strategies. The most active hybrid displayed over threefold and 10-fold higher in vitro activity than ivermectin against hepatic and blood stage infections, respectively. Although an overwhelming insecticidal effect against Anopheles stephensi mosquitoes in laboratory conditions was not noticed, in silico docking analysis supports allosteric binding to glutamate-gated chloride channels similar to ivermectin.


Assuntos
Antimaláricos/farmacologia , Ivermectina/análogos & derivados , Ivermectina/farmacologia , Plasmodium berghei/efeitos dos fármacos , Plasmodium falciparum/efeitos dos fármacos , Animais , Anopheles/efeitos dos fármacos , Antimaláricos/síntese química , Sítios de Ligação , Canais de Cloreto/química , Canais de Cloreto/metabolismo , Desenho de Fármacos , Inseticidas/síntese química , Inseticidas/farmacologia , Ivermectina/metabolismo , Simulação de Acoplamento Molecular , Estrutura Molecular , Testes de Sensibilidade Parasitária , Plasmodium berghei/crescimento & desenvolvimento , Plasmodium falciparum/crescimento & desenvolvimento , Ligação Proteica , Relação Estrutura-Atividade
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA