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2.
Gene ; 716: 144016, 2019 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-31377318

RESUMO

Drug resistance of malaria parasites remains a problem affecting antimalarial treatment and control of the disease. We previously synthesized an antimalarial endoperoxide, N-89, having high antimalarial effects in vitro and in vivo. In this study we seek to understand the resistant mechanism against N-89 by establishing a highly N-89-resistant clone, named NRC10H, of the Plasmodium falciparum FCR-3 strain. We describe gene mutations in the parent FCR-3 strain and the NRC10H clone using whole-genome sequencing and subsequently by expression profiling using quantitative real-time PCR. Seven genes related to drug resistance, proteolysis, glycophosphatidylinositol anchor biosynthesis, and phosphatidylethanolamine biosynthesis exhibited a single amino acid substitution in the NRC10H clone. Among these seven genes, the multidrug resistance protein 2 (mdr2) variant A532S was found only in NRC10H. The genetic status of the P. falciparum endoplasmic reticulum-resident calcium binding protein (PfERC), a potential target of N-89, was similar between the NRC10H clone and the parent FCR-3 strain. These findings suggest that the genetic alterations of the identified seven genes, in particular mdr2, in NRC10H could give rise to resistance of the antimalarial endoperoxide N-89.


Assuntos
Antimaláricos/farmacologia , Compostos Heterocíclicos com 2 Anéis/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Compostos de Espiro/farmacologia , Resistência a Medicamentos/genética , Genômica , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , RNA Mensageiro/metabolismo , Sequenciamento Completo do Genoma
3.
Pan Afr Med J ; 32: 183, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31312296

RESUMO

Introduction: Molecular characterization of malaria parasites from different localities is important to improve understanding of acquisition of natural immunity to Plasmodium falciparum, to assist in identifying the most appropriate strategies for control and to evaluate the impact of control interventions. This study aimed to determine the genetic diversity and the multiplicity of infection in Plasmodium falciparum isolates from Pointe-Noire, Republic of Congo. Methods: Plasmodium falciparum isolates were collected from 71 children with uncomplicated malaria; enrolled into the study for evaluating the therapeutic efficacy of artemether-lumefantrine combination. Both msp-1 and msp-2 genes were genotyped. Results: From 296 distinct fragments detected, 13 msp-1 and 27 msp-2 different alleles were identified. For msp-1, RO33 family was poorly polymorphic. The K1 family has shown the trend of predominance (41%), followed by Mad20 (35%). Comparatively to msp-2, 49.6% and 48.8% fragments belonged to 3D7 and FC27 respectively. Taking together msp-1 and msp-2 genes, the overall multiplicity of infection has been increased to 2.64 and 86% harbored more than one parasite genotype. Parasite density was not influenced by age as well as the multiplicity of infection which was not influenced neither by age nor by parasite density. Conclusion: Genetic diversity of Plasmodium falciparum in isolates from patients with uncomplicated malaria in Pointe-Noire is high and consisted mainly of multiple clones. The overall multiplicity of infection has been largely increased when considering msp-1 and msp-2 genes together. With the changes in malaria epidemiology, the use of both msp-1 and msp-2 genes in the characterization of Plasmodium falciparum infection is recommended.


Assuntos
Antígenos de Protozoários/genética , Malária Falciparum/parasitologia , Proteína 1 de Superfície de Merozoito/genética , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Alelos , Antimaláricos/administração & dosagem , Combinação Arteméter e Lumefantrina/administração & dosagem , Criança , Pré-Escolar , Congo/epidemiologia , Feminino , Variação Genética , Genótipo , Humanos , Lactente , Malária Falciparum/tratamento farmacológico , Malária Falciparum/epidemiologia , Masculino , Plasmodium falciparum/isolamento & purificação
4.
Parasite ; 26: 40, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31298995

RESUMO

A better understanding of malaria transmission at a local scale is essential for developing and implementing effective control strategies. In the framework of a randomized controlled trial (RCT), we aimed to provide an updated description of malaria transmission in the Korhogo area, northern Côte d'Ivoire, and to obtain baseline data for the trial. We performed human landing collections (HLCs) in 26 villages in the Korhogo area during the rainy season (September-October 2016, April-May 2017) and the dry season (November-December 2016, February-March 2017). We used PCR techniques to ascertain the species of the Anopheles gambiae complex, Plasmodium falciparum sporozoite infection, and insecticide resistance mechanisms in a subset of Anopheles vectors. Anopheles gambiae s.l. was the predominant malaria vector in the Korhogo area. Overall, more vectors were collected outdoors than indoors (p < 0.001). Of the 774 An. gambiae s.l. tested in the laboratory, 89.65% were An. gambiae s.s. and 10.35% were An. coluzzii. The frequencies of the kdr allele were very high in An. gambiae s.s. but the ace-1 allele was found at moderate frequencies. An unprotected individual living in the Korhogo area received an average of 9.04, 0.63, 0.06 and 0.12 infected bites per night in September-October, November-December, February-March, and April-May, respectively. These results demonstrate that the intensity of malaria transmission is extremely high in the Korhogo area, especially during the rainy season. Malaria control in highly endemic areas such as Korhogo needs to be strengthened with complementary tools in order to reduce the burden of the disease.


Assuntos
Anopheles/parasitologia , Ecologia , Resistência a Inseticidas , Inseticidas , Malária/transmissão , Animais , Anopheles/genética , Mordeduras e Picadas/epidemiologia , Costa do Marfim , Feminino , Humanos , Malária/prevenção & controle , Controle de Mosquitos , Mosquitos Vetores/genética , Mosquitos Vetores/parasitologia , Plasmodium falciparum/genética , Ensaios Clínicos Controlados Aleatórios como Assunto , Estações do Ano
5.
BMC Infect Dis ; 19(1): 600, 2019 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-31288760

RESUMO

BACKGROUND: Oxidative stress plays a vital role in the pathogenesis of both Sickle Cell Disease (SCD) and Plasmodium falciparum malaria. However, there are limited studies on the effect of P. falciparum malaria infection on oxidative stress in SCD patients. METHODS: A cross-sectional study was undertaken to compare levels of biomarkers of oxidative stress in isolates from SCD patients with uncomplicated P.falciparum malaria. The biomarkers namely: malondialdehyde (MDA), reduced glutathione (GSH), catalase (CAT) and glutathione peroxidase (GPx) were determined in plasma samples from SCD malaria positive, malaria positive, SCD malaria negative and healthy control participants. The genetic diversity of P.falciparum was determined by nested polymerase chain reaction of merozoite surface protein-2 (MSP-2) gene. RESULTS: Out of 207 participants, 54 (26%) were SCD malaria positive, 51 (24%) malaria positive, 51 (24%) SCD controls and 51 (24%) healthy control individuals. The mean concentration of MDA was significantly higher in SCD malaria positive than SCD controls (P < 0.0001). In contrast, the mean concentration of GSH (P < 0.0001) and GPx (P < 0.0001) were significantly lower in SCD malaria than SCD controls. Although not significantly different, the mean concentration of MDA was higher (P = 0.0478), but the geometric mean parasite density (P = 0.2430) and multiplicity of infection (P = 0.3478) were lower in SCD malaria samples than in malaria samples. The most prevalent MSP2 allelic family was IC3D7 in SCD malaria (72%) and Malaria (76%) samples. The biomarkers of oxidative stress were not significantly different between IC3D7 and FC27 allelic families of MSP2. CONCLUSION: We identified severe oxidative stress in Sickle cell disease patients with uncomplicated P.falciparum malaria.


Assuntos
Anemia Falciforme/diagnóstico , Malária Falciparum/diagnóstico , Estresse Oxidativo , Adulto , Anemia Falciforme/complicações , Anemia Falciforme/patologia , Antígenos de Protozoários/genética , Biomarcadores/metabolismo , Catalase/metabolismo , Estudos Transversais , Teste em Amostras de Sangue Seco , Feminino , Variação Genética , Genótipo , Glutationa/metabolismo , Humanos , Malária Falciparum/parasitologia , Masculino , Malondialdeído/metabolismo , Estresse Oxidativo/genética , Plasmodium falciparum/genética , Plasmodium falciparum/isolamento & purificação , Proteínas de Protozoários/genética , Índice de Gravidade de Doença , Uganda
6.
Parasit Vectors ; 12(1): 355, 2019 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-31319880

RESUMO

BACKGROUND: Malaria is the most important vector-borne disease in the world. Epidemiological and ecological studies of malaria traditionally utilize detection of Plasmodium sporozoites in whole mosquitoes or salivary glands by microscopy or serological or molecular assays. However, these methods are labor-intensive, and can over- or underestimate mosquito transmission potential. To overcome these limitations, alternative sample types have been evaluated for the study of malaria. It was recently shown that Plasmodium could be detected in saliva expectorated on honey-soaked cards by Anopheles stephensi, providing a better estimate of transmission risk. We evaluated whether excretion of Plasmodium falciparum nucleic acid by An. stephensi correlates with expectoration of parasites in saliva, thus providing an additional sample type for estimating transmission potential. Mosquitoes were exposed to infectious blood meals containing cultured gametocytes, and excreta collected at different time points post-exposure. Saliva was collected on honey-soaked filter paper cards, and salivary glands were dissected and examined microscopically for sporozoites. Excreta and saliva samples were tested by real time polymerase chain reaction (RT-rtPCR). RESULTS: Plasmodium falciparum RNA was detected in mosquito excreta as early as four days after ingesting a bloodmeal containing gametocytes. Once sporogony (the development of sporozoites) occurred, P. falciparum RNA was detected concurrently in both excreta and saliva samples. In the majority of cases, no difference was observed between the Ct values obtained from matched excreta and saliva samples, suggesting that both samples provide equally sensitive results. A positive association was observed between the molecular detection of the parasites in both samples and the proportion of mosquitoes with sporozoites in their salivary glands from each container. No distinguishable parasites were observed when excreta samples were stained and microscopically analyzed. CONCLUSIONS: Mosquito saliva and excreta are easily collected and are promising for surveillance of malaria-causing parasites, especially in low transmission settings or in places where arboviruses co-circulate.


Assuntos
Anopheles/parasitologia , Fezes/parasitologia , Malária/transmissão , Mosquitos Vetores/parasitologia , Plasmodium/isolamento & purificação , Saliva/parasitologia , Animais , DNA de Protozoário/genética , Feminino , Malária Falciparum/transmissão , Masculino , Plasmodium/genética , Plasmodium falciparum/genética , Plasmodium falciparum/isolamento & purificação , Plasmodium vivax/genética , Plasmodium vivax/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real , Esporozoítos/genética , Esporozoítos/isolamento & purificação
7.
BMC Infect Dis ; 19(1): 659, 2019 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-31340774

RESUMO

BACKGROUND: It is estimated that over a third of the world population is infected by malaria and helminthiases mainly among communities with high poverty indices. The distribution of these parasitic infections overlaps in many epidemiological settings and have varying outcomes in the host. In this paper we report the prevalence of malaria and intestinal helminthiases coinfections among malaria suspected patients and the association of helminthiases with the occurrence of malaria and its outcomes in Wondo Genet, southern Ethiopia. METHODS: In a cross-sectional study conducted from December 2009 to July 2010 in Kella, Aruma and Busa Health Centers in Wondo Genet, a total of 427 consenting febrile patients were screened for malaria and intestinal helminths infections. Malaria parasite detection and quantification were done using Giemsa stained thick and thin blood films. Helminth infections were screened and quantified by Kato-Katz thick smear method. Haemoglobin level was assessed using haemocue machine (HemoCue HB 201+). Difference in proportions and means were tested by Student's t test and ANOVA while logistic regression analysis was used to determine the association between variables. RESULTS: Of the total examined, 196 (45.90%) were positive for at least one helminth infection while 276 (64.64%) were positive for malaria. The prevalence of Plasmodium falciparum and P. vivax infections were 47.31 and 16.62%, respectively. The most common helminth parasites detected were Ascaris lumbricoides (33.96%), Trichuris trichiura (21.55%), Schistosoma mansoni (13.35%), and hookworms (6.79%). The overall malaria-helminthiases coinfection was 33.96%. The prevalence of anaemia was 43.12%. Helminthiases coinfection showed a positive correlation with the occurrence of malaria (AOR = 2.17, 95% CI: 1.44-3.28; P < 0.001). Schistosoma mansoni coinfection was associated with the increased risk of developing malaria associated anaemia (OR = 14.4, 95% CI: 1.37-150.80; P = 0.026). CONCLUSION: Malaria and helminth coinfections are important causes of morbidities among the population in Wondo Genet necessitating integrated control measures. Nevertheless, further detailed studies on the consequences and pathogenesis of these coinfections are needed to institute sound control and intervention measures.


Assuntos
Coinfecção/epidemiologia , Helmintíase/epidemiologia , Enteropatias Parasitárias/epidemiologia , Malária/epidemiologia , Adolescente , Adulto , Idoso , Animais , Criança , Coinfecção/parasitologia , Coinfecção/prevenção & controle , Comorbidade , Estudos Transversais , Etiópia/epidemiologia , Feminino , Febre/epidemiologia , Febre/parasitologia , Helmintíase/parasitologia , Helmintíase/prevenção & controle , Helmintos/genética , Helmintos/isolamento & purificação , Humanos , Enteropatias Parasitárias/parasitologia , Enteropatias Parasitárias/prevenção & controle , Malária/parasitologia , Malária/prevenção & controle , Masculino , Pessoa de Meia-Idade , Pacientes Ambulatoriais , Plasmodium falciparum/genética , Plasmodium falciparum/isolamento & purificação , Prevalência , Viverridae , Adulto Jovem
8.
Malar J ; 18(1): 192, 2019 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-31185976

RESUMO

BACKGROUND: Mutational analysis of the Plasmodium falciparum kelch 13 (k13) gene is routinely performed to track the emergence and spread of artemisinin resistance. Surveillance of resistance markers has been impeded by the difficulty of extracting sufficient DNA from low parasite density infections common in low-transmission settings, such as Southeast Asia. This problem can be overcome by collecting large volumes of venous blood. Efficient methods for extracting and amplifying k13 from dried blood spots (DBS) would facilitate resistance surveillance. METHODS: Methods for k13 amplification from standard Whatman 3MM DBS (stored for 14 days at 28 °C with 80% relative humidity) were optimized by systematically testing different extraction conditions. Conditions that improved parasite DNA recovery as assessed by quantitative polymerase chain reaction (PCR) of 18S rDNA were then tested for their impact on k13 PCR amplification. RESULTS: The optimized protocol for amplification of k13 from DBS is markedly more sensitive than standard methods using commercial kits. Using this method, k13 was successfully amplified from laboratory-created DBS samples with parasite densities as low as 500 parasites/mL. Importantly, the method recovers both DNA and RNA, making it compatible with RNA-based ultrasensitive techniques currently in use. CONCLUSIONS: The optimized DBS protocol should facilitate drug resistance surveillance, especially in low-transmission settings where clinical malaria infections with high parasite densities are rare.


Assuntos
Artemisininas/farmacologia , Sangue/parasitologia , DNA de Protozoário/isolamento & purificação , Resistência a Medicamentos , Malária Falciparum/parasitologia , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Antimaláricos/farmacologia , Ásia Sudeste , DNA de Protozoário/química , DNA de Protozoário/genética , Dessecação/métodos , Humanos , Plasmodium falciparum/efeitos dos fármacos , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Manejo de Espécimes/métodos
9.
Parasitol Int ; 72: 101938, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31201923

RESUMO

Plasmodium falciparum is a blood protozoan parasite, transmitted by Anopheles mosquitoes vectors, that can cause morbidity and even leads to mortality in tropical countries. Strategies are directed to combat malaria including development of diagnostic tools, serological markers and vaccinations. A target under intensive studies is Merozoite Surface Protein (MSP)-3. The aim of this study is to express and purify recombinant MSP3 of P. falciparum (rPfMSP3) using silkworm expression system as a host for its large-scale production and to investigate its potential effectiveness for sero-diagnosis. The rPfMSP3 formed oligomers in a blue-native PAGE and its N-glycosylation was confirmed by periodic acid-Schiff staining and PNGase F treatment. The amyloid-like morphology of the rPfMSP3 oligomers was observed. Enzyme-linked immunosorbent assay showed that 60-70% of human samples from subjects living in malaria endemic areas in Indonesia detected the rPfMSP3. Western blot results showed that the rPfMSP3 was recognized by a malaria infected human serum but not by an uninfected human serum. The rPfMSP3 was successfully expressed in silkworm as a soluble protein and has the potential to be used in serological measurement for detecting PfMSP3-specific antibodies in sera from individuals living in endemic areas.


Assuntos
Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/imunologia , Malária Falciparum/diagnóstico , Proteínas de Protozoários/imunologia , Animais , Antígenos de Protozoários/genética , Bombyx/genética , Ensaio de Imunoadsorção Enzimática , Humanos , Imunoglobulina G/sangue , Malária Falciparum/imunologia , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Merozoítos/imunologia , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Testes Sorológicos
10.
BMC Infect Dis ; 19(1): 559, 2019 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-31242863

RESUMO

BACKGROUND: Blood smear microscopy remains the gold-standard method to diagnose and quantify malaria parasite density. In addition, parasite genotyping of select loci is the most utilized method for distinguishing recrudescent and new infections and to determine the number of strains per sample. In research settings, blood may be obtained from capillary or venous compartments, and results from these matrices have been used interchangeably. Our aim was to compare quantitative results for parasite density and strain complexity from both compartments. METHODS: In a prospective observational study, children and adults presenting with uncomplicated Plasmodium falciparum malaria, simultaneous capillary and venous blood smears and dried blood spots were collected over 42-days following treatment with artemether-lumefantrine. Blood smears were read by two microscopists, any discrepancies resolved by a third reader. Parasite DNA fingerprinting was conducted using six microsatellites. Bland Altman analysis and paired t-test/McNemar's test were used to assess the difference in density readings and measurements. RESULTS: Two hundred twenty-three participants were included in the analysis (177 children (35 HIV-infected/142 HIV-uninfected), 21 HIV-uninfected pregnant women, and 25 HIV-uninfected non-pregnant adults). Parasite density measurements did not statistically differ between capillary and venous blood smears at the time of presentation, nor over the course of 42-day follow-up. Characterization of merozoite surface protein-2 (MSP-2) genetic polymorphism demonstrated a higher level of strain diversity at the time of presentation in venous samples, as compared with capillary specimens (p = 0.02). There was a high degree of variability in genotype-corrected outcomes when pairs of samples from each compartment were compared using MSP-2 alone, although the variability was reduced with the use of multiple markers. CONCLUSIONS: Parasite density measurements do not statistically differ between capillary and venous compartments in all studied demographic groups at the time of presentation with malaria, or over the course of follow-up. More strains were detected by MSP-2 genotyping in venous samples than in capillary samples at the time of malaria diagnosis. The use of multiple polymorphic markers reduces the impact of variability in strain detection on genotype-corrected outcomes. This study confirms that both capillary and venous compartments can be used for sampling with confidence in the clinical research setting. TRIAL REGISTRATION: The trial was registered at ClinicalTrials.gov under registration no. NCT01717885 .


Assuntos
Capilares/parasitologia , Malária Falciparum/parasitologia , Carga Parasitária/métodos , Plasmodium falciparum/genética , Veias/parasitologia , Infecções Oportunistas Relacionadas com a AIDS/tratamento farmacológico , Infecções Oportunistas Relacionadas com a AIDS/parasitologia , Adolescente , Adulto , Idoso , Animais , Antimaláricos/farmacocinética , Antimaláricos/uso terapêutico , Combinação Arteméter e Lumefantrina/farmacocinética , Combinação Arteméter e Lumefantrina/uso terapêutico , Criança , Pré-Escolar , Monitoramento de Medicamentos/métodos , Feminino , Genótipo , Técnicas de Genotipagem/métodos , HIV , Infecções por HIV/complicações , Infecções por HIV/parasitologia , Humanos , Lactente , Malária Falciparum/sangue , Malária Falciparum/diagnóstico , Malária Falciparum/tratamento farmacológico , Masculino , Pessoa de Meia-Idade , Parasitemia/sangue , Parasitemia/complicações , Parasitemia/diagnóstico , Parasitemia/tratamento farmacológico , Plasmodium falciparum/isolamento & purificação , Uganda , Adulto Jovem
11.
Nat Commun ; 10(1): 2665, 2019 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-31209259

RESUMO

Estimates of Plasmodium falciparum migration may inform strategies for malaria elimination. Here we elucidate fine-scale parasite population structure and infer recent migration across Southeast Asia using identity-by-descent (IBD) approaches based on genome-wide single nucleotide polymorphisms called in 1722 samples from 54 districts. IBD estimates are consistent with isolation-by-distance. We observe greater sharing of larger IBD segments between artemisinin-resistant parasites versus sensitive parasites, which is consistent with the recent spread of drug resistance. Our IBD analyses reveal actionable patterns, including isolated parasite populations, which may be prioritized for malaria elimination, as well as asymmetrical migration identifying potential sources and sinks of migrating parasites.


Assuntos
Resistência a Medicamentos/genética , Monitoramento Epidemiológico , Genoma de Protozoário/genética , Malária Falciparum/microbiologia , Plasmodium falciparum/genética , Antimaláricos/farmacologia , Antimaláricos/uso terapêutico , Artemisininas/farmacologia , Artemisininas/uso terapêutico , Ásia Sudeste , Biodiversidade , Genótipo , Geografia Médica , Malária Falciparum/tratamento farmacológico , Malária Falciparum/prevenção & controle , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/isolamento & purificação , Polimorfismo de Nucleotídeo Único
12.
Malar J ; 18(1): 214, 2019 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-31234871

RESUMO

BACKGROUND: Undesirable consequences of donor Plasmodium falciparum parasitaemia on stored donor blood have been reported. Therefore, it is imperative that all prospective blood donors are screened for P. falciparum infections using sensitive techniques. In this study, the sensitivities of microscopy, rapid diagnostic test (RDT), loop-mediated isothermal amplification (LAMP) assay and selective whole genome amplification (sWGA) technique in detecting P. falciparum infections in blood donors was assessed. METHODS: Randomly selected blood donors from 5 districts in Greater Accra Region of Ghana were screened for asymptomatic P. falciparum infections. Each donor sample was screened with SD Bioline RDT kit for P. falciparum histidine rich protein 2 and Plasmodium lactate dehydrogenase antigens, sWGA and 18s-rRNA LAMP. Crude DNA LAMP (crDNA-LAMP) was compared to purified DNA LAMP (pDNA-LAMP). RESULTS: A total of 771 blood donors were screened. The respective overall prevalence of P. falciparum in Ghana by microscopy, RDT, crDNA-LAMP, pDNA-LAMP and sWGA was 7.4%, 11.8%, 16.9%, 17.5% and 18.0%. Using sWGA as the reference test, the sensitivities of microscopy, RDT, crDNA-LAMP and pDNA-LAMP were 41.0% (95% CI 32.7-49.7), 65.5% (95% CI 56.9-73.3), 82.6% (95% CI 75.8-88.3) and 95.7% (95% CI 90.1-98.4), respectively. There was near perfect agreement between LAMP and sWGA (sWGA vs. crDNA-LAMP, κ = 0.87; sWGA vs. pDNA-LAMP, κ = 0.96), while crDNA-LAMP and pDNA-LAMP agreed perfectly (κ = 0.91). Goodness of fit test indicated non-significant difference between the performance of LAMP and sWGA (crDNA-LAMP vs. sWGA: x2 = 0.71, p = 0.399 and pDNA-LAMP vs. sWGA: x2 = 0.14, p = 0.707). Finally, compared to sWGA, the performance of LAMP did not differ in detecting sub-microscopic parasitaemia (sWGA vs. crDNA-LAMP: x2 = 1.12, p = 0.290 and sWGA vs. pDNA-LAMP: x2 = 0.22, p = 0.638). CONCLUSIONS: LAMP assay agreed near perfectly with sWGA with non-significant differences in their ability to detect asymptomatic P. falciparum parasitaemia in blood donors. Therefore, it is recommended that LAMP based assays are employed to detect P. falciparum infections in blood donors due to its high sensitivity, simplicity, cost-effectiveness and user-friendliness.


Assuntos
Testes Diagnósticos de Rotina/normas , Malária Falciparum/diagnóstico , Técnicas de Amplificação de Ácido Nucleico/normas , Plasmodium falciparum/genética , Adulto , Infecções Assintomáticas , Testes Diagnósticos de Rotina/métodos , Feminino , Gana , Humanos , Masculino , Técnicas de Amplificação de Ácido Nucleico/economia , RNA Ribossômico 18S/genética , Adulto Jovem
13.
Mikrobiyol Bul ; 53(2): 239-244, 2019 Apr.
Artigo em Turco | MEDLINE | ID: mdl-31130128

RESUMO

Plasmodium falciparum malaria causes about 450.000 deaths every year, mostly in children around the world. The infection is seen in cases coming from abroad and may lead to deaths in Turkey. Many native P.falciparum malaria cases and deaths due to this infection were observed in Turkey during mid 1900's when malaria was epidemic. But only two native cases were reported in the last 50 years, both from Manisa. First case was a one-year old baby who has come to Manisa from Urfa with his family and has never been abroad. He has diagnosed with Plasmodium vivax malaria and treated with chloroquine and primaquine. A previously obtained thin blood film was examined and characteristic P.falciparum rings in red blood cells were observed and the case was published together with photographs as probable P.falciparum and P.vivax mixed infection. After this case, microscopists working in Malaria Control Unit of Manisa were informed about the differentiation of malaria species in thin blood samples. Soon afterward, another case who have never been abroad before were also diagnosed with P.falciparum and P.vivax mixed infection and this case was also published with photographs taken from thin blood samples. As molecular diagnostic methods were not improved and widespread in those years, it could not be applied in both cases. A Giemsa stained thin blood sample of the baby case was incidentally found 22 years afterwards and with the aim of molecular diagnosis, the blood sample on the slide previously processed for DNA isolation, then analysed with "FTD Malaria Differentiation (Fast Track Diagnostics, Luxembourg)" multiplex kit with real-time polymerase chain reaction by using probes special for P.falciparum, P.ovale, P.malariae, P.vivax species. DNA's belonging to P.falciparum and P.vivax were found to be positive, the case is molecularly proved to have P.falciparum and P.vivax mixed infection. This case indicated that Turkey is convenient for the expansion of P.falciparum malaria in terms of the climate and vectors and suggested that the potential danger may increase with the effects of global warming, wars and migrations and may jump to Europe over Turkey. The case which molecularly proved the existence of native P.falciparum malaria in the near future in Turkey, was presented to draw attention to the danger of this infection for Turkey and Europe.


Assuntos
Malária Falciparum , Malária Vivax , Plasmodium falciparum , Plasmodium vivax , Criança , Coinfecção/parasitologia , Europa (Continente) , Humanos , Lactente , Malária Falciparum/diagnóstico , Malária Falciparum/parasitologia , Malária Vivax/diagnóstico , Malária Vivax/parasitologia , Masculino , Patologia Molecular , Plasmodium falciparum/genética , Plasmodium vivax/genética , Turquia
14.
Malar J ; 18(1): 154, 2019 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-31039781

RESUMO

BACKGROUND: The unexpected high proportion of submicroscopic malaria infections in areas with low transmission intensity challenges the control and elimination of malaria in the Americas. The current PCR-based assays present limitations as most protocols still rely on amplification of few-copies target gene. Here, the hypothesis was that amplification of different plasmodial targets-ribosomal (18S rRNA) and non-ribosomal multi-copy sequences (Pvr47 for Plasmodium vivax and Pfr364 for Plasmodium falciparum)-could increase the chances of detecting submicroscopic malaria infection. METHODS: A non-ribosomal real-time PCR assay targeting Pvr47/Pfr364 (NR-qPCR) was established and compared with three additional PCR protocols, two of them based on 18S rRNA gene amplification (Nested-PCR and R-qPCR) and one based on Pvr47/Pfr364 targets (NR-cPCR). The limit of detection of each PCR protocol, at single and artificial mixed P. vivax/P. falciparum infections, was determined by end-point titration curves. Field samples from clinical (n = 110) and subclinical (n = 324) malaria infections were used to evaluate the impact of using multiple molecular targets to detect malaria infections. RESULTS: The results demonstrated that an association of ribosomal and non-ribosomal targets did not increase sensitivity to detect submicroscopic malaria infections. Despite of that, artificial mixed-malaria infections demonstrated that the NR-qPCR was the most sensitive protocol to detect low-levels of P. vivax/P. falciparum co-infections. Field studies confirmed that submicroscopic malaria represented a large proportion (up to 77%) of infections among asymptomatic Amazonian residents, with a high proportion of infections (~ 20%) identified only by the NR-qPCR. CONCLUSIONS: This study presents a new species-specific non-ribosomal PCR assay with potential to identify low-density P. vivax and P. falciparum infections. As the majority of subclinical infections was caused by P. vivax, the commonest form of malaria in the Amazon area, future studies should investigate the potential of Pvr47/Pfr364 to detect mixed-malaria infections in the field.


Assuntos
Coinfecção/diagnóstico , Malária/diagnóstico , RNA Ribossômico 18S/genética , Reação em Cadeia da Polimerase em Tempo Real , Adulto , Infecções Assintomáticas , Brasil , Coinfecção/parasitologia , Feminino , Humanos , Limite de Detecção , Malária/sangue , Malária Falciparum/sangue , Malária Falciparum/diagnóstico , Malária Vivax/sangue , Malária Vivax/diagnóstico , Masculino , Pessoa de Meia-Idade , Técnicas de Diagnóstico Molecular , Plasmodium falciparum/genética , Plasmodium falciparum/isolamento & purificação , Plasmodium vivax/genética , Plasmodium vivax/isolamento & purificação , Adulto Jovem
15.
Malar J ; 18(1): 156, 2019 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-31046769

RESUMO

BACKGROUND: Malaysia has declared its aim to eliminate malaria with a goal of achieving zero local transmission by the year 2020. However, targeting the human reservoir of infection, including those with asymptomatic infection is required to achieve malaria elimination. Diagnosing asymptomatic malaria is not as straightforward due to the obvious lack of clinical manifestations and often subpatent level of parasites. Accurate diagnosis of malaria is important for providing realistic estimates of malaria burden and preventing misinformed interventions. Low levels of parasitaemia acts as silent reservoir of transmission thus remains infectious to susceptible mosquito vectors. Hence, the aim of this study is to investigate the prevalence of asymptomatic submicroscopic malaria (SMM) in the District of Belaga, Sarawak. METHODS: In 2013, a total of 1744 dried blood spots (DBS) were obtained from residents of 8 longhouses who appeared healthy. Subsequently, 251 venous blood samples were collected from residents of 2 localities in 2014 based on the highest number of submicroscopic cases from prior findings. Thin and thick blood films were prepared from blood obtained from all participants in this study. Microscopic examination were carried out on all samples and a nested and nested multiplex PCR were performed on samples collected in 2013 and 2014 respectively. RESULTS: No malaria parasites were detected in all the Giemsa-stained blood films. However, of the 1744 samples, 29 (1.7%) were positive for Plasmodium vivax by PCR. Additionally, of the 251 samples, the most prevalent mono-infection detected by PCR was Plasmodium falciparum 50 (20%), followed by P. vivax 39 (16%), P. knowlesi 9 (4%), and mixed infections 20 (8%). CONCLUSIONS: This research findings conclude evidence of Plasmodium by PCR, among samples previously undetectable by routine blood film microscopic examination, in local ethnic minority who are clinically healthy. SMM in Belaga district is attributed not only to P. vivax, but also to P. falciparum and P. knowlesi. In complementing efforts of programme managers, there is a need to increase surveillance for SMM nationwide to estimate the degree of SMM that warrant measures to block new transmission of malaria.


Assuntos
Infecções Assintomáticas/epidemiologia , Portador Sadio/epidemiologia , Reservatórios de Doenças/parasitologia , Malária/epidemiologia , Parasitemia/diagnóstico , Plasmodium/genética , Adolescente , Adulto , Portador Sadio/parasitologia , Criança , Pré-Escolar , Coinfecção/epidemiologia , Coinfecção/parasitologia , Feminino , Humanos , Lactente , Recém-Nascido , Malária/transmissão , Malária Falciparum/epidemiologia , Malária Vivax/epidemiologia , Malásia/epidemiologia , Masculino , Microscopia , Parasitemia/epidemiologia , Plasmodium/isolamento & purificação , Plasmodium falciparum/genética , Plasmodium falciparum/isolamento & purificação , Plasmodium knowlesi/genética , Plasmodium knowlesi/isolamento & purificação , Plasmodium vivax/genética , Plasmodium vivax/isolamento & purificação , Reação em Cadeia da Polimerase , Adulto Jovem
16.
MBio ; 10(2)2019 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-31040236

RESUMO

The clinical presentation of severe Plasmodium falciparum malaria differs between children and adults, but the mechanistic basis for this remains unclear. Contributing factors to disease severity include total parasite biomass and the diverse cytoadhesive properties mediated by the polymorphic var gene parasite ligand family displayed on infected erythrocytes. To explore these factors, we performed a multicohort analysis of the contribution of var expression and parasite biomass to severe malaria in two previously published pediatric cohorts in Tanzania and Malawi and an adult cohort in India. Machine learning analysis revealed independent and complementary roles for var adhesion types and parasite biomass in adult and pediatric severe malaria and showed that similar var profiles, including upregulation of group A and DC8 var, predict severe malaria in adults and children. Among adults, patients with multiorgan complications presented infections with significantly higher parasite biomass without significant differences in var adhesion types. Conversely, pediatric patients with specific complications showed distinct var signatures. Cerebral malaria patients showed broadly increased expression of var genes, in particular group A and DC8 var, while children with severe malaria anemia were classified based on high transcription of DC8 var only. This study represents the first large multisite meta-analysis of var expression, and it demonstrates the presence of common var profiles in severe malaria patients of different ages across distant geographical sites, as well as syndrome-specific disease signatures. The complex associations between parasite biomass, var adhesion type, and clinical presentation revealed here represent the most comprehensive picture so far of the relationship between cytoadhesion, parasite load, and clinical syndrome.IMPORTANCE P. falciparum malaria can cause multiple disease complications that differ by patient age. Previous studies have attempted to address the roles of parasite adhesion and biomass in disease severity; however, these studies have been limited to single geographical sites, and there is limited understanding of how parasite adhesion and biomass interact to influence disease manifestations. In this meta-analysis, we compared parasite disease determinants in African children and Indian adults. This study demonstrates that parasite biomass and specific subsets of var genes are independently associated with detrimental outcomes in both childhood and adult malaria. We also explored how parasite var adhesion types and biomass play different roles in the development of specific severe malaria pathologies, including childhood cerebral malaria and multiorgan complications in adults. This work represents the largest study to date of the role of both var adhesion types and biomass in severe malaria.


Assuntos
Variação Genética , Genótipo , Malária Falciparum/patologia , Malária Falciparum/parasitologia , Plasmodium falciparum/classificação , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Adulto , Criança , Pré-Escolar , Estudos de Coortes , Feminino , Humanos , Índia , Lactente , Aprendizado de Máquina , Malaui , Masculino , Carga Parasitária , Tanzânia
17.
MBio ; 10(2)2019 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-31040246

RESUMO

The global spread of Plasmodium falciparum chloroquine resistance transporter (PfCRT) variant haplotypes earlier caused the widespread loss of chloroquine (CQ) efficacy. In Asia, novel PfCRT mutations that emerged on the Dd2 allelic background have recently been implicated in high-level resistance to piperaquine, and N326S and I356T have been associated with genetic backgrounds in which resistance emerged to artemisinin derivatives. By analyzing large-scale genome sequencing data, we report that the predominant Asian CQ-resistant Dd2 haplotype is undetectable in Africa. Instead, the GB4 and previously unexplored Cam783 haplotypes predominate, along with wild-type, drug-sensitive PfCRT that has reemerged as the major haplotype. To interrogate how these alleles impact drug susceptibility, we generated pfcrt-modified isogenic parasite lines spanning the mutational interval between GB4 and Dd2, which includes Cam783 and involves amino acid substitutions at residues 326 and 356. Relative to Dd2, the GB4 and Cam783 alleles were observed to mediate lower degrees of resistance to CQ and the first-line drug amodiaquine, while resulting in higher growth rates. These findings suggest that differences in growth rates, a surrogate of parasite fitness, influence selection in the context of African infections that are frequently characterized by high transmission rates, mixed infections, increased immunity, and less recourse to treatment. We also observe that the Asian Dd2 allele affords partial protection against piperaquine yet does not directly impact artemisinin efficacy. Our results can help inform the regional recommendations of antimalarials, whose activity is influenced by and, in certain cases, enhanced against select PfCRT variant haplotypes.IMPORTANCE Our study defines the allelic distribution of pfcrt, an important mediator of multidrug resistance in Plasmodium falciparum, in Africa and Asia. We leveraged whole-genome sequence analysis and gene editing to demonstrate how current drug combinations can select different allelic variants of this gene and shape region-specific parasite population structures. We document the ability of PfCRT mutations to modulate parasite susceptibility to current antimalarials in dissimilar, pfcrt allele-specific ways. This study underscores the importance of actively monitoring pfcrt genotypes to identify emerging patterns of multidrug resistance and help guide region-specific treatment options.


Assuntos
Resistência a Múltiplos Medicamentos , Aptidão Genética , Genótipo , Malária Falciparum/parasitologia , Proteínas de Membrana Transportadoras/genética , Plasmodium falciparum/crescimento & desenvolvimento , Proteínas de Protozoários/genética , África/epidemiologia , Ásia/epidemiologia , Frequência do Gene , Genética Populacional , Malária Falciparum/epidemiologia , Proteínas Mutantes/genética , Plasmodium falciparum/classificação , Plasmodium falciparum/genética , Plasmodium falciparum/isolamento & purificação
19.
Malar J ; 18(1): 180, 2019 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-31126288

RESUMO

BACKGROUND: HIV-infected individuals on antiretroviral therapy (ART) require treatment with artemisinin-based combination therapy (ACT) when infected with malaria. Artemether-lumefantrine (AL) is the most commonly used ACT for treatment of falciparum malaria in Africa but there is limited evidence on the safety and efficacy of AL in HIV-infected individuals on ART, among whom drug-drug interactions are expected. Day-42 adequate clinical and parasitological response (ACPR) and incidence of adverse events was assessed in HIV-infected individuals on efavirenz-based ART with uncomplicated falciparum malaria treated with AL. METHODS: A prospective, open label, non-randomized, interventional clinical trial was conducted at St Paul's Hospital in northern Zambia, involving 152 patients aged 15-65 years with uncomplicated falciparum malaria, who were on efavirenz-based ART. They received a 3-day directly observed standard treatment of AL and were followed up until day 63. Day-42 polymerase chain reaction (PCR)-corrected ACPRs (95% confidence interval [CI]) were calculated for the intention-to-treat population. RESULTS: Enrolled patients had a baseline geometric mean (95% CI) parasite density of 1108 (841-1463) parasites/µL; 16.4% (25/152) of the participants had a recurrent malaria episode by day 42. However, PCR data was available for 17 out of the 25 patients who had malaria recurrence. Among all the 17 patients, PCR findings demonstrated malaria re-infection, making the PCR-adjusted day-42 ACPR 100% in the 144 patients who could be evaluated. Even when eight patients with missing PCR data were considered very conservatively as failures, the day-42 ACPR was over 94%. None of the participants, disease or treatment characteristics, including day-7 lumefantrine concentrations, predicted the risk of malaria recurrence by day 42. AL was well tolerated following administration. There were only two cases of grade 3 neutropaenia and one serious adverse event of lobar pneumonia, none of which was judged as probably related to intake of AL. CONCLUSIONS: AL was well tolerated and efficacious in treating uncomplicated falciparum malaria in HIV co-infected adults on efavirenz-based ART. However, a higher than anticipated proportion of participants experienced malaria re-infection, which highlights the need for additional malaria prevention measures in this sub-population after treatment with AL. Trial registration Pan African Clinical Trials Registry (PACTR): PACTR201311000659400. Registered on 4 October 2013. https://pactr.samrc.ac.za/Search.aspx.


Assuntos
Antimaláricos/uso terapêutico , Combinação Arteméter e Lumefantrina/uso terapêutico , Benzoxazinas/uso terapêutico , Malária Falciparum/tratamento farmacológico , Inibidores da Transcriptase Reversa/uso terapêutico , Adolescente , Adulto , Idoso , Antimaláricos/efeitos adversos , Combinação Arteméter e Lumefantrina/efeitos adversos , Feminino , Infecções por HIV/tratamento farmacológico , Humanos , Masculino , Pessoa de Meia-Idade , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/genética , Reação em Cadeia da Polimerase , Estudos Prospectivos , Adulto Jovem , Zâmbia
20.
Parasitol Int ; 71: 186-193, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31028841

RESUMO

Plasmodium falciparum, an obligate intracellular protozoan parasite which causes the severe form of human malaria, exports numerous proteins to the infected red blood cell that are important for its survival and of severe pathological effect to its host. These proteins and their export mechanisms are candidates for drug and vaccine development, and among them is the Plasmodium SURFIN family of proteins. Previously we showed that the N-terminal region along with the sequence surrounding the transmembrane domain of SURFIN4.1 is essential for its export to Maurer's clefts in the red blood cell cytoplasm. We proposed that this region is recognized by a machinery responsible for protein translocation across the parasitophorous vacuole membrane surrounding the parasite. To understand the export mechanism further, we utilized a fluorescent protein-tagged mini-SURFIN4.1 consisting of the minimum essential components for export. Alanine scanning of all charged amino acids within the N-terminal region revealed that replacement of 3 glutamic acid and 2 lysine residues significantly impairs the export efficiency of this protein across the parasitophorous vacuole membrane. In addition, N-terminally Myc-tagged mini-SURFIN4.1 and mini-SURFIN4.2 with similar architectures were detected with anti-Myc antibody at Maurer's clefts, indicating that elements required for export to Maurer's clefts are conserved between SURFIN4.1 and SURFIN4.2, and that N-terminal sequences of these SURFIN members are not cleaved during export. Our results implicate a conserved nature of SURFIN export to the red blood cell, particularly an important role of multiple glutamic acid and lysine residues in the SURFIN N-terminal region.


Assuntos
Aminoácidos/química , Eritrócitos/parasitologia , Interações Hospedeiro-Parasita , Proteínas de Membrana/química , Plasmodium falciparum/genética , Proteínas de Protozoários/química , Ácido Glutâmico/química , Humanos , Lisina/química , Transporte Proteico
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