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1.
Anal Chem ; 93(4): 2097-2105, 2021 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-33464825

RESUMO

In many countries targeting malaria elimination, persistent malaria infections can have parasite loads significantly below the lower limit of detection (LLOD) of standard diagnostic techniques, making them difficult to identify and treat. The most sensitive diagnostic methods involve amplification and detection of Plasmodium DNA by polymerase chain reaction (PCR), which requires expensive thermal cycling equipment and is difficult to deploy in resource-limited settings. Isothermal DNA amplification assays have been developed, but they require complex primer design, resulting in high nonspecific amplification, and show a decrease in sensitivity than PCR methods. Here, we have used a computational approach to design a novel isothermal amplification assay with a simple primer design to amplify P. falciparum DNA with analytical sensitivity comparable to PCR. We have identified short DNA sequences repeated throughout the parasite genome to be used as primers for DNA amplification and demonstrated that these primers can be used, without modification, to isothermally amplify P. falciparum parasite DNA via strand displacement amplification. Our novel assay shows a LLOD of ∼1 parasite/µL within a 30 min amplification time. The assay was demonstrated with clinical samples using patient blood and saliva. We further characterized the assay using direct amplicon next-generation sequencing and modified the assay to work with a visual readout. The technique developed here achieves similar analytical sensitivity to current gold standard PCR assays requiring a fraction of time and resources for PCR. This highly sensitive isothermal assay can be more easily adapted to field settings, making it a potentially useful tool for malaria elimination.


Assuntos
DNA de Protozoário/genética , Malária Falciparum/diagnóstico , Técnicas de Amplificação de Ácido Nucleico/métodos , Plasmodium falciparum/genética , Sequências Repetitivas de Ácido Nucleico/genética , DNA de Protozoário/isolamento & purificação , Humanos , Limite de Detecção , Plasmodium falciparum/isolamento & purificação , Reprodutibilidade dos Testes
2.
Exp Parasitol ; 218: 107967, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32858044

RESUMO

Reported incidence rates of cryptosporidiosis in Ireland are consistently among the highest in Europe. Despite the national prevalence of this enteric parasite and the compulsory nature of incidence surveillance and reporting, in-depth analyses seeking to genotype clinical isolates of Cryptosporidium on an intra-species level are rarely undertaken in Ireland. This molecular epidemiology study of 163 clinical Cryptosporidium isolates was conducted in Southern Ireland, from 2015 to 2018, in order to ascertain population subtype heterogeneity. Analysis was conducted via real-time PCR amplification and gp60 gene sequencing, which successfully determined the subtype designation of 149 of the 163 (91.4%) tested isolates. Overall, 12 C. parvum and five C. hominis subtypes were identified, with the incidence of the regionally predominant C. parvum species found to primarily occur during springtime months, while C. hominis incidence was largely confined to late summer and autumnal months. Additionally, one C. parvum and four C. hominis subtypes were newly reported by this study, having not been previously identified in clinical or livestock infection in Ireland. Overall, these data give insight into the diversification of the Cryptosporidium population and emergent subtypes, while also allowing comparisons to be made with clinical epidemiological profiles reported previously in Ireland and elsewhere.


Assuntos
Criptosporidiose/parasitologia , Cryptosporidium/classificação , Zoonoses/parasitologia , Animais , Criptosporidiose/epidemiologia , Cryptosporidium/genética , Cryptosporidium/isolamento & purificação , Cryptosporidium parvum/classificação , Cryptosporidium parvum/genética , Cryptosporidium parvum/isolamento & purificação , DNA de Protozoário/isolamento & purificação , Fezes/parasitologia , Gastroenterite/parasitologia , Glicoproteínas/química , Glicoproteínas/genética , Humanos , Incidência , Irlanda/epidemiologia , Estudos Longitudinais , Prevalência , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , RNA Ribossômico 18S/genética , Reação em Cadeia da Polimerase em Tempo Real , Estudos Retrospectivos , Estações do Ano , Alinhamento de Sequência
3.
PLoS One ; 15(8): e0237118, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32764795

RESUMO

The objective of this study was to evaluate the effect of supplementation with 100ppm sodium monensin or 0.15% of a blend of functional oils (cashew nut oil + castor oil) on the intestinal microbiota of broilers challenged with three different Eimeria spp. The challenge was accomplished by inoculating broiler chicks with sporulated oocysts of Eimeria tenella, Eimeria acervulina, and Eimeria maxima via oral gavage. A total of 864, day-old male broiler chicks (Cobb) were randomly assigned to six treatments (eight pens/treatment; 18 broilers/pen) in a 3 × 2 factorial arrangement, composed of three additives (control, monensin or blend), with or without Eimeria challenge. Intestinal contents was collected at 28 days of age for microbiota analysis by sequencing 16s rRNA in V3 and V4 regions using the Illumina MiSeq platform. Taxonomy was assigned through the SILVA database version 132, using the QIIME 2 software version 2019.1. No treatment effects (p > 0.05) were observed in the microbial richness at the family level estimated by Chao1 and the biodiversity assessed by Simpson's index, except for Shannon's index (p < 0.05). The intestinal microbiota was dominated by members of the order Clostridiales and Lactobacillales, followed by the families Ruminococcaceae, Bacteroidaceae, and Lactobacillaceae, regardless of treatment. When the controls were compared, in the challenged control group there was an increase in Erysipelotrichaceae, Lactobacillaceae, Bacteroidaceae, Streptococcaceae, and Peptostreptococcaceae, and a decrease in Ruminococcaceae. Similar results were found for a challenged group that received monensin, while the blend partially mitigated this variation. Therefore, the blend alleviated the impact of coccidiosis challenge on the microbiome of broilers compared to monensin.


Assuntos
Coccidiose/veterinária , Eimeria/isolamento & purificação , Microbioma Gastrointestinal/efeitos dos fármacos , Monensin/administração & dosagem , Óleos Vegetais/administração & dosagem , Doenças das Aves Domésticas/dietoterapia , Anacardium/química , Ração Animal , Animais , Galinhas/parasitologia , Coccidiose/dietoterapia , Coccidiose/imunologia , Coccidiose/parasitologia , DNA de Protozoário/isolamento & purificação , Eimeria/genética , Eimeria/imunologia , Eimeria/patogenicidade , Microbioma Gastrointestinal/imunologia , Masculino , Oocistos/patogenicidade , Doenças das Aves Domésticas/imunologia , Doenças das Aves Domésticas/parasitologia , RNA Ribossômico 16S/genética , Ricinus/química
4.
Exp Parasitol ; 216: 107941, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32622940

RESUMO

Distinct antigens have been evaluated with diagnostic purpose for canine and human visceral leishmaniasis (VL), and variable sensitivity and specificity values have been obtained in the assays. In the present study, a Leishmania infantum hypothetical protein called LiHyG, which was identified in an immunoproteomics study in Leishmania infantum amastigote extracts by antibodies in VL dogs sera; was cloned, expressed, purified and evaluated as a recombinant protein (rLiHyG) for the diagnosis of canine and human disease. The recombinant amastigote-specific A2 protein (rA2) and a soluble L. infantum protein extract (SLA) were used as controls. For canine VL, the sensitivity values were of 100%, 57.29% and 48.57%, when rLiHyG, rA2 and SLA were used, respectively, while the specificity values were of 100%, 81.43% and 88.57%, respectively. In addition, AUC values were of 1.00, 0.72 and 0.65, when rLiHyG, rA2 and SLA were used, respectively, while accuracy was of 100%, 72.38% and 75.24%, respectively. For human VL, the sensitivity values were of 100%, 84.00% and 88.00%, when rLiHyG, rA2 and SLA were used, respectively, while the specificity values were of 100%, 58.75% and 73.75%, respectively. In addition, AUC values were of 1.00, 0.76 and 0.83, when rLiHyG, rA2 and SLA were used, respectively, while accuracy was of 100%, 64.8% and 66.6%, respectively. The prognostic role of rLiHyG in the human VL was also evaluated, by means of post-therapeutic serological follow-up with sera samples collected before and six months after treatment. Results showed that treated patients presented significant reductions in the anti-rLiHyG IgG, IgG1, and IgG2 antibody levels, with results being similar to those found in healthy subjects. Testing the rA2 protein and SLA as antigens, lower IgG, IgG1, and IgG2 levels were also found, although they were higher after treatment than those obtained for rLiHyG. In conclusion, results suggested that rLiHyG could be considered for future studies as a diagnostic and/or prognostic marker for canine and human VL.


Assuntos
Antígenos de Protozoários/isolamento & purificação , Doenças do Cão/parasitologia , Leishmania infantum/imunologia , Leishmaniose Visceral/diagnóstico , Adulto , Idoso , Sequência de Aminoácidos , Animais , Antígenos de Protozoários/genética , Medula Óssea/parasitologia , Biologia Computacional , DNA de Protozoário/química , DNA de Protozoário/isolamento & purificação , Doenças do Cão/diagnóstico , Cães , Ensaio de Imunoadsorção Enzimática , Epitopos de Linfócito B/química , Feminino , Humanos , Imunoglobulina G/sangue , Leishmania infantum/genética , Leishmaniose Visceral/parasitologia , Leishmaniose Visceral/veterinária , Masculino , Pessoa de Meia-Idade , Prognóstico , Proteínas de Protozoários/química , Sensibilidade e Especificidade , Alinhamento de Sequência , Testes Sorológicos , Baço/parasitologia , Adulto Jovem
5.
BMC Infect Dis ; 20(1): 530, 2020 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-32698764

RESUMO

BACKGROUND: Plasmodium falciparum dihydrofolate reductase (Pfdhfr) and dihydropteroate synthetase (Pfdhps) mutations compromise the effectiveness of sulfadoxine-pyrimethamine (SP) for treatment of uncomplicated malaria, and are likely to impair the efficiency of intermittent preventive treatment during pregnancy (IPTp). This study was conducted to determine the level of Pfdhfr-Pfdhps mutations, a decade since SP was limited for IPTp use in pregnant women in Tanzania. METHODS: P. falciparum genomic DNA was extracted from dried blood spots prepared from a finger prick. Extracted DNA were sequenced using a single MiSeq lane by combining all PCR products. Genotyping of Pfdhfr and Pfdhps mutations were done using bcftools whereas custom scripts were used to filter and translate genotypes into SP resistance haplotypes. RESULTS: The Pfdhfr was analyzed from 445 samples, the wild type (WT) Pfdhfr haplotype NCSI was detected in 6 (1.3%) samples. Triple PfdhfrIRNI (mutations are bolded and underlined) haplotype was dominant, contributing to 84% (number [n] = 374) of haplotypes while 446 samples were studied for Pfdhps, WT for Pfdhps (SAKAA) was found in 6.7% (n = 30) in samples. Double Pfdhps haplotype (SGEAA) accounted for 83% of all mutations at Pfdhps gene. Of 447 Pfdhfr-Pfdhps combined genotypes, only 0.9% (n = 4) samples contained WT gene (SAKAA-NCSI). Quintuple (five) mutations, SGEAA-IRNI accounted for 71.4% (n = 319) whereas 0.2% (n = 1) had septuple (seven) mutations (AGKGS-IRNI). The overall prevalence of Pfdhfr K540E was 90.4% (n = 396) while Pfdhps A581G was 1.1% (n = 5). CONCLUSIONS: This study found high prevalence of Pfdhfr-Pfdhps quintuple and presence of septuple mutations. Mutations at Pfdhfr K540E and Pfdhps A581G, major predictors for IPTp-SP failure were within the recommended WHO range. Abandonment of IPTp-SP is recommended in settings where the Pfdhfr K540E prevalence is > 95% and Pfdhps A581G is > 10% as SP is likely to be not effective. Nonetheless, saturation in Pfdhfr and Pfdhps haplotypes is alarming, a search for alternative antimalarial drug for IPTp in the study area is recommended.


Assuntos
Antimaláricos/uso terapêutico , Di-Hidropteroato Sintase/genética , Malária Falciparum/epidemiologia , Malária Falciparum/prevenção & controle , Mutação , Plasmodium falciparum/genética , Complicações Parasitárias na Gravidez/epidemiologia , Complicações Parasitárias na Gravidez/prevenção & controle , Proteínas de Protozoários/genética , Pirimetamina/uso terapêutico , Sulfadoxina/uso terapêutico , Tetra-Hidrofolato Desidrogenase/genética , DNA de Protozoário/genética , DNA de Protozoário/isolamento & purificação , Combinação de Medicamentos , Resistência Microbiana a Medicamentos/genética , Feminino , Haplótipos , Humanos , Malária Falciparum/parasitologia , Plasmodium falciparum/enzimologia , Reação em Cadeia da Polimerase , Gravidez , Prevalência , Tanzânia/epidemiologia , Resultado do Tratamento
6.
Exp Appl Acarol ; 81(4): 599-607, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32676999

RESUMO

Babesia bovis and Babesia bigemina are tick-transmitted piroplasms that cause severe damage to the livestock industry in tropical regions of the world. Recent studies demonstrated differences in infection levels of these haemoparasites among bovine breeds and variation between individual cows regarding resistance to these diseases. This study aimed to estimate the repeatability and correlations between B. bovis and B. bigemina using two cattle breeding systems, an individual system (IS) and a collective paddock system (CPS). All animals were Holstein breed, and the levels of B. bovis and B. bigemina in blood samples were estimated by quantitative polymerase chain reaction (qPCR). The estimated correlations for the B. bigemina and B. bovis DNA copy number for IS and CPS were moderate and high, respectively, whereas repeatability estimates for both systems and both Babesia species were moderate. Although we cannot infer that the type of rearing system directly influenced the correlation and repeatability coefficients, it appears that the bovine parasitemia burden may be dependent on (or determine) the parasitemia burden on ticks because the bovines remained in the same place for a longer time in both systems. Thus, the babesiosis infection levels of the ticks may have been uniform, a phenomenon that also ensures greater uniformity in cattle infection. This factor may have favored the occurrence of infected ticks leading to higher repeatability estimates and correlations. Our study confirms high variability in resistance/susceptibility between breeds, and the high correlations found may be linked to this characteristic and the most intensive breeding type of dairy cattle. Besides, under the present study conditions, the estimated correlations suggest that measuring an infection level of one Babesia species can predict the level of infection of the other.


Assuntos
Babesia bovis , Babesia , Babesiose/epidemiologia , Doenças dos Bovinos , Bovinos/parasitologia , Animais , Babesia/isolamento & purificação , Babesia bovis/isolamento & purificação , Cruzamento , Doenças dos Bovinos/epidemiologia , Doenças dos Bovinos/parasitologia , DNA de Protozoário/isolamento & purificação , Indústria de Laticínios , Parasitemia
7.
Exp Parasitol ; 217: 107956, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32659234

RESUMO

The species name Cryptosporidium bollandi n. sp. is proposed for Cryptosporidium piscine genotype 2 based on morphological, biological and molecular characterisation. Phylogenetic analyses of 18S rRNA (18S) sequences revealed that C. bollandi n. sp. was most closely related to piscine genotype 4 (5.1% genetic distance) and exhibited genetic distances of 10.0%, 12.2% and 25.2% from Cryptosporidium molnari, Cryptosporidium huwi and Cryptosporidium scophthtalmi, respectively. At the actin locus, C. bollandi n. sp. was again most closely related to piscine genotype 4 (6.8% genetic distance) and exhibited 15.5% (C. molnari), 18.4% (C. huwi), 22.9% (C. scophthalmi) and up to 27.5% genetic distance from other Cryptosporidium spp. (Cryptosporidium felis). Phylogenetic analysis of concatenated 18S and actin sequences showed that C. bollandi n. sp. exhibited 12.9% (C. molnari) to 21.1% (C. canis) genetic distance from all other Cryptosporidium spp. Genetic data as well as previous histological analysis clearly supports the validity of C. bollandi n. sp. as a separate species.


Assuntos
Ciclídeos/parasitologia , Criptosporidiose/parasitologia , Cryptosporidium/fisiologia , Doenças dos Peixes/parasitologia , Actinas/química , Actinas/genética , Animais , Sequência de Bases , Evolução Biológica , Criptosporidiose/epidemiologia , Cryptosporidium/classificação , Cryptosporidium/genética , Cryptosporidium/ultraestrutura , DNA de Protozoário/química , DNA de Protozoário/isolamento & purificação , Doenças dos Peixes/epidemiologia , Pesqueiros , Genótipo , Funções Verossimilhança , Microscopia Eletrônica de Transmissão/veterinária , Filogenia , Reação em Cadeia da Polimerase/veterinária , Prevalência , RNA Ribossômico 18S/química , Washington/epidemiologia , Austrália Ocidental/epidemiologia
8.
Parasitol Res ; 119(8): 2679-2686, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32588173

RESUMO

Rodents and other micromammals constitute important reservoirs of infectious diseases; their role in the life cycle of apicomplexan parasites such as Toxoplasma gondii, Neospora caninum, and Sarcocystis spp. still needs clarification. In the present study, we analyzed by PCR and Sanger sequencing methods the presence of specific parasite DNA within brain and heart tissues of 313 individuals of five synanthropic small mammal species (Apodemus sylvaticus, Mus spretus, M. musculus, Rattus rattus, and Crocidura russula) collected in Barcelona metropolitan area (NE Spain). In addition, PCR-RFLP and microsatellites were also used as tools for genotypic characterization of T. gondii and N. caninum, respectively. Specific DNA of T. gondii, N. caninum, and Sarcocystis spp. was detected in 0.3% (n = 1), 1.3% (n = 4), and 3.8% (n = 12) of the animals, respectively. No mixed infections were observed. Crocidura russula stood out as the main host for Sarcocystis spp. Toxoplasma gondii-specific DNA detected in a house rat was genetically characterized by PCR-RFLP, presenting type II and III alleles (SAG1 [II], SAG3 [II], GRA6 [II], c22-8 [III], Apico [III]). Also, unsuccessful DNA sequencing and microsatellite typing were attempted in N. caninum-positive samples, which suggested a lack of PCR specificity and open avenues to speculate the host competence of rodents for N. caninum. Likewise, Sarcocystis spp. identity was studied by alignment and phylogenetic analyses of cox1 and 28S rRNA sequences from the 14 positive samples. It resulted in at least three unknown organisms closely similar (95.7-100% cox1-sequence homology) to Sarcocystis pantherophisi from the Eastern rat snake (Pantherophis alleghaniensis) (KU891603), suggesting together with 28S rRNA sequences analyses, three Sarcocystis sp. with a life cycle conformed by rodents as intermediate host (IH) and snakes as definitive hosts (DH) infecting the periurban micromammals surveyed. Prevalence figures found in this first survey carried out in Spain agree with other international studies focused on periurban areas. Further surveys should be conducted in farms and their surroundings in order to unravel the role of wild micromammals in the epidemiology of such protozoan parasites affecting our livestock, and therefore human population.


Assuntos
Coccidiose/veterinária , Mamíferos/parasitologia , Infecções Protozoárias em Animais/parasitologia , Sarcocystidae/genética , Animais , Coccidiose/epidemiologia , Coccidiose/parasitologia , DNA de Protozoário/genética , DNA de Protozoário/isolamento & purificação , Genótipo , Mamíferos/classificação , Encistamento de Parasitas , Filogenia , Infecções Protozoárias em Animais/epidemiologia , Sarcocystidae/classificação , Sarcocystidae/isolamento & purificação , Espanha/epidemiologia
9.
PLoS One ; 15(6): e0234445, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32579586

RESUMO

This study aimed to describe the sand fly fauna and detect trypanosomatids in these insects from Casa Branca, state of Minas Gerais, Brazil, an endemic area of both visceral (VL) and tegumentary leishmaniasis (TL). Sand flies were collected bimonthly from May 2013 to July 2014, using automatic light traps exposed for three consecutive nights in peridomiciliary areas of nine houses with previous reports of VL and TL. ITS1-PCR and DNA sequencing were performed for trypanosomatids identification. A total of 16,771 sand flies were collected belonging to 23 species. The most abundant species was Nyssomyia whitmani (Antunes & Coutinho, 1939) (70.9%), followed by Lutzomyia longipalpis (Lutz & Neiva, 1912) (15.2%) and Migonemyia migonei (França, 1920) (9.1%). Leishmania amazonensis DNA was detected in Ny. whitmani (four pools) and Le. braziliensis DNA was detected in Psychodopygus lloydi (one pool). In seven pools of Ny. whitmani and in one pool of Lu. longipalpis positive for Leishmania DNA, the parasite species was not determined due to the low quality of the sequences. Moreover, DNA of Herpetomonas spp. was detected in Ny. whitmani (two pools) and Cortelezzii complex (one pool). DNA of Crithidia spp. was detected in Ny. whitmani and Ps. lloydi (both one pool). Our results suggest that Ny. whitmani may be involved in the transmission of Le. amazonensis in the study area. The molecular detection of Le. amazonensis suggests the presence of this species in a sylvatic cycle between vertebrate and invertebrate hosts in the region of Casa Branca. Our data also reveal the occurrence of other non-Leishmania trypanosomatids in sand flies in Casa Branca District.


Assuntos
Biodiversidade , Insetos Vetores/parasitologia , Leishmania/isolamento & purificação , Phlebotomus/parasitologia , Psychodidae/parasitologia , Animais , Brasil/epidemiologia , DNA de Protozoário/isolamento & purificação , Doenças Endêmicas/prevenção & controle , Feminino , Humanos , Leishmania/genética , Leishmaniose/epidemiologia , Leishmaniose/parasitologia , Leishmaniose/prevenção & controle , Leishmaniose/transmissão , Análise de Sequência de DNA
10.
J Parasitol ; 106(3): 395-399, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32556163

RESUMO

The objective of the present study was to determine the characterization of Toxoplasma gondii in cats, rats, and chickens in the border areas of Yunnan Province. A total of 259 samples was collected from 10 border areas in Yunnan Province including 94 cats, 58 rats, and 107 chickens. Samples were screened by a nested polymerase chain reaction (PCR) assay and the positive products were analyzed by multilocus PCR-restriction fragment length polymorphism (RFLP) to determine the genotypes. Toxoplasma gondii deoxyribonucleic acid (DNA) was detected from 15.96% of 94 cats, 15.52% of 58 rats, and 6.54% of 107 chickens, respectively, and the average infection rate is 11.97%. Using the multilocus PCR-RFLP, we found that the genotype of T. gondii in cats and rats was ToxoDB#9. Because of low DNA concentration, no genotype was determined from chickens. These results fill the gaps of knowledge in the prevalence and genotype of T. gondii in the border areas of Yunnan Province and have implications for the better control of T. gondii infection in humans and animals.


Assuntos
Doenças do Gato/epidemiologia , Galinhas/parasitologia , Doenças das Aves Domésticas/epidemiologia , Ratos/parasitologia , Doenças dos Roedores/epidemiologia , Toxoplasmose Animal/epidemiologia , Animais , Encéfalo/parasitologia , Doenças do Gato/parasitologia , Gatos , China/epidemiologia , DNA de Protozoário/química , DNA de Protozoário/isolamento & purificação , Marcadores Genéticos , Técnicas de Genotipagem/veterinária , Tipagem de Sequências Multilocus/veterinária , Reação em Cadeia da Polimerase/veterinária , Polimorfismo de Fragmento de Restrição , Doenças das Aves Domésticas/parasitologia , Prevalência , Doenças dos Roedores/parasitologia , Toxoplasma/classificação , Toxoplasma/genética , Toxoplasmose Animal/parasitologia
11.
Nat Immunol ; 21(7): 790-801, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32424361

RESUMO

Plasmodium parasite-specific antibodies are critical for protection against malaria, yet the development of long-lived and effective humoral immunity against Plasmodium takes many years and multiple rounds of infection and cure. Here, we report that the rapid development of short-lived plasmablasts during experimental malaria unexpectedly hindered parasite control by impeding germinal center responses. Metabolic hyperactivity of plasmablasts resulted in nutrient deprivation of the germinal center reaction, limiting the generation of memory B cell and long-lived plasma cell responses. Therapeutic administration of a single amino acid to experimentally infected mice was sufficient to overcome the metabolic constraints imposed by plasmablasts and enhanced parasite clearance and the formation of protective humoral immune memory responses. Thus, our studies not only challenge the current model describing the role and function of blood-stage Plasmodium-induced plasmablasts but they also reveal new targets and strategies to improve anti-Plasmodium humoral immunity.


Assuntos
Imunidade Humoral , Malária/imunologia , Plasmócitos/metabolismo , Plasmodium falciparum/imunologia , Adolescente , Adulto , Aminoácidos/administração & dosagem , Aminoácidos/metabolismo , Animais , Anticorpos Antiprotozoários/sangue , Anticorpos Antiprotozoários/imunologia , Anticorpos Antiprotozoários/metabolismo , Antimaláricos/administração & dosagem , DNA de Protozoário/isolamento & purificação , Modelos Animais de Doenças , Centro Germinativo/citologia , Centro Germinativo/imunologia , Centro Germinativo/metabolismo , Interações Hospedeiro-Parasita/imunologia , Humanos , Malária/sangue , Malária/tratamento farmacológico , Malária/parasitologia , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Nutrientes/metabolismo , Plasmócitos/imunologia , Plasmócitos/parasitologia , Plasmodium falciparum/genética , Plasmodium falciparum/isolamento & purificação , Estudo de Prova de Conceito , Adulto Jovem
12.
Exp Parasitol ; 215: 107931, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32464222

RESUMO

Chagas disease is a public health problem in America. Its parasite, Trypanosoma cruzi, presents different discrete typing units (DTUs), colonizes organs of mammalian hosts in chronic infections, and presents tropism for particular organs in experimental infections. We evaluated T. cruzi tropism towards organs on the naturally infected rodent Octodon degus, identifying the parasites' DTUs, by means of conventional PCR and hybridization. Almost all the analyzed organs presented T. cruzi. More than 42% of the tested oesophagus, skin, skeletal muscle, brain and intestine showed T. cruzi DNA. Other nine types of organs were infected in over 15%. These results suggest that there is some tropism by T. cruzi in chronically infected O. degus. DTU TcV was present in 92.5% of infected organs with identified DTUs; this DTU is frequently reported in human infections in the Southern Cone of South America. Few organs showed mixed DTU infections. This is one of the few reports on the outcome of chronic natural T. cruzi-infection in wild mammal hosts exposed to naturally infected vectors.


Assuntos
Doença de Chagas/veterinária , Octodon/parasitologia , Doenças dos Roedores/patologia , Doenças dos Roedores/parasitologia , Animais , Animais Selvagens , Doença de Chagas/parasitologia , Doença de Chagas/patologia , DNA de Protozoário/isolamento & purificação , Feminino , Masculino , Trypanosoma cruzi/classificação , Trypanosoma cruzi/genética
13.
J Parasitol ; 106(2): 312-315, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32330280

RESUMO

The purpose of this study was to describe the prevalence and incidence of Neospora caninum infection in dogs that are in close contact with dairy cattle and to identify possible risk factors associated with the infection in this population. Twenty-four dogs located in 8 different dairy farms of Aguascalientes, Mexico, were evaluated for a 6-mo period. Once a month a sample of serum and a sample of peripheral blood was collected. The serum was used to detect antibodies against N. caninum by means of the indirect immunofluorescence technique, and the blood was used to detect parasite's DNA. The association between seroprevalence and possible risk factors was estimated using logistic regression. The prevalence of anti-N. caninum antibodies was 54% in the first month, 62% in the last month, and the incidence was 8.69%. One farm had no positive cases. Antibody titers ranged from 1:50 to 1:800. Parasite DNA was not detected in any of the samples. Only the age (>6 yr) of the dogs was identified as a risk factor for infection by N. caninum (P ≤ 0.05).


Assuntos
Coccidiose/veterinária , Doenças do Cão/parasitologia , Neospora , Fatores Etários , Animais , Anticorpos Antiprotozoários/sangue , Bovinos , Coccidiose/epidemiologia , Coccidiose/parasitologia , DNA de Protozoário/sangue , DNA de Protozoário/isolamento & purificação , Indústria de Laticínios , Doenças do Cão/epidemiologia , Cães , Feminino , Incidência , Masculino , México/epidemiologia , Neospora/genética , Neospora/imunologia , Prevalência , Fatores de Risco
14.
Malar J ; 19(1): 123, 2020 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-32228599

RESUMO

BACKGROUND: Malaria surveillance requires powerful tools and strategies to achieve malaria elimination. Rapid diagnostic tests for malaria (RDTs) are easily deployed on a large scale and are helpful sources of parasite DNA. The application of sensitive molecular techniques to these RDTs is a modern tool for improving malaria case detection and drug resistance surveillance. Several studies have made it possible to extract the DNA of Plasmodium falciparum from RDTs. The knowledge of gametocyte carriage in the population is important to better assess the level of parasite transmission in elimination settings. The aim of this study was to detect P. falciparum gametocytes from used RDTs by quantitative PCR for molecular monitoring of malaria transmission. METHODS: DNA was extracted from 303 RDT devices (SD Bioline Malaria Pf) using the Chelex-100 protocol. qPCR was performed in a 20 µL reaction to detect and quantify transcripts of the pfs25 gene. The cycle threshold (Ct) was determined by the emission fluorescence corresponding to the initial amount of amplified DNA. RESULTS: The study found an overall prevalence of 53.47% with an average Ct of 32.12 ± 4.28 cycles. In 2018, the prevalence of gametocytes was higher in the Ranérou district (76.24%) than in the Saint-Louis district (67.33%) where an increase in the number of gametocyte carriers in 2018 was noted, in comparison with 2017. CONCLUSIONS: RDTs are a good source of DNA for molecular monitoring of gametocyte carriage. This method is a simple and effective tool to better understand the level of malaria transmission with a view to elimination.


Assuntos
DNA de Protozoário/isolamento & purificação , Testes Diagnósticos de Rotina , Malária Falciparum/diagnóstico , Plasmodium falciparum/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real , Monitoramento Epidemiológico , Senegal
15.
J Parasitol ; 106(2): 295-307, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32316032

RESUMO

Cyclospora cayetanensis is a coccidian parasite of humans of known and growing importance. However, we are surprisingly naïve as to our understanding of how to diagnose it and how it develops inside the human body. Here we provide details of the developmental stages of C. cayetanensis in the gallbladder of a 33-yr-old male with human immunodeficiency virus. The gallbladder was removed surgically in 2001 because of severe abdominal pain. For the present study, the archived paraffin block of gallbladder was processed for light microscopy and transmission electron microscopy (TEM). Histological sections were examined after staining with hematoxylin and eosin (HE) or using the periodic acid Schiff (PAS) reaction. Immature and mature asexual stages, gamonts, and oocysts were seen in epithelial cells, both in the superficial epithelium and in glands. The merozoites were present singly, in pairs, and 3 or more in a single parasitophorous vacuole in the host cytoplasm. Up to 6 nuclei were seen in immature schizonts without evidence of merozoite formation. Mature schizonts were 7.6 × 5.1 µm and contained up to 10, 3-4 µm long merozoites. Merozoites were 0.6 to 2.0 µm wide, and their shape varied from pear-shaped to slender. Merozoites were generally PAS-positive; however, some were intensely positive, some had only minute granules, while others were PAS-negative. The microgamonts (male) were 6.6 × 5.2 µm and contained fewer than 20 microgametes around a residual body. The microgametes were up to 2 µm long and were flagellated. Macrogamonts (female) contained distinctive eosinophilic wall-forming bodies that varied in size and were less than 1 µm in HE-stained sections. Macrogamonts were 5.8-6.5 × 5.3-6.5 µm. Oocysts in sections were unsporulated and had a diameter of 5.7-7.5 µm. The TEM examination confirmed the histologic findings. The DNA extracted from paraffin sections was confirmed as C. cayetanensis with real-time PCR. The detailed description of the life cycle stages of C. cayetanensis reported here in an immunosuppressed patient could facilitate histopathologic diagnosis of this parasite. We have shown that the parasite's development more closely resembles that of Cystoisospora than Eimeria and that the parasite has multiple nuclei per immature meront indicating schizogony, and we have undermined evidence for a Type II meront.


Assuntos
Cyclospora/crescimento & desenvolvimento , Ciclosporíase/parasitologia , Vesícula Biliar/parasitologia , Infecções por HIV/complicações , Adulto , Cyclospora/genética , Cyclospora/ultraestrutura , Ciclosporíase/imunologia , DNA de Protozoário/genética , DNA de Protozoário/isolamento & purificação , Feminino , Vesícula Biliar/patologia , Vesícula Biliar/ultraestrutura , Humanos , Hospedeiro Imunocomprometido , Estágios do Ciclo de Vida , Masculino , Microscopia Eletrônica de Transmissão , Reação em Cadeia da Polimerase em Tempo Real
16.
PLoS Negl Trop Dis ; 14(4): e0008148, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32282820

RESUMO

BACKGROUND: Echinococcosis is a life-threatening parasitic disease caused by Echinococcus spp. tapeworms with over one million people affected globally at any time. The Echinococcus spp. tapeworms in the human body release DNA to the circulatory system, which can be a biomarker for echinococcosis. Cell-free DNA (cfDNA) is widely used in medical research and has been applied in various clinical settings. As for echinococcosis, several PCR-based tests had been trialed to detect cell-free Echinococcus spp. DNA in plasma or serum, but the sensitivity was about 20% to 25%. Low sensitivity of PCR-based methods might be related to our limited understanding of the features of cell-free Echinococcus spp. DNA in plasma, including its concentration, fragment pattern and release source. In this study, we applied ultra-high-throughput sequencing to comprehensively investigate the characteristics of cell-free Echinococcus spp. DNA in plasma of echinococcosis patients. METHODOLOGY/PRINCIPAL FINDINGS: We collected plasma samples from 23 echinococcosis patients. Total plasma cfDNA was extracted and sequenced with a high-throughput sequencing platform. An average of 282 million read pairs were obtained for each plasma sample. Sequencing data were analyzed with bioinformatics workflow combined with Echinococcus spp. sequence database. After identification of cell-free Echinococcus spp. reads, we found that the cell-free Echinococcus spp. reads accounted for 1.8e-5 to 4.0e-9 of the total clean reads. Comparing fragment length distribution of cfDNA between Echinococcus spp. and humans showed that cell-free Echinococcus spp. DNA of cystic echinococcosis (CE) had a broad length range, while that of alveolar echinococcosis (AE) had an obvious peak at about 135 bp. We found that most of the cell-free Echinococcus spp. DNA reads were from the nuclear genome with an even distribution, which might indicate a random release pattern of cell-free Echinococcus spp. DNA. CONCLUSIONS/SIGNIFICANCE: With ultra-high-throughput sequencing technology, we analyzed the concentration, fragment length, release source, and other characteristics of cell-free Echinococcus spp. DNA in the plasma of echinococcosis patients. A better understanding of the characteristics of cell-free Echinococcus spp. DNA in plasma may facilitate their future application as a biomarker for diagnosis.


Assuntos
DNA de Protozoário/sangue , Equinococose/diagnóstico , Equinococose/parasitologia , Echinococcus/genética , Echinococcus/isolamento & purificação , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Adolescente , Adulto , Animais , Sequência de Bases , Biomarcadores , Criança , DNA de Protozoário/isolamento & purificação , Feminino , Genoma de Protozoário , Humanos , Masculino , Pessoa de Meia-Idade , Plasma , Reação em Cadeia da Polimerase/métodos , Sensibilidade e Especificidade , Análise de Sequência de DNA , Adulto Jovem
17.
PLoS Negl Trop Dis ; 14(4): e0008175, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32267840

RESUMO

We recently developed a superhydrophobic cone-based method for the collection of mosquito excreta/feces (E/F) for the molecular xenomonitoring of vector-borne parasites showing higher throughput compared to the traditional approach. To test its field applicability, we used this platform to detect the presence of filarial and malaria parasites in two villages of Ghana and compared results to those for detection in mosquito carcasses and human blood. We compared the molecular detection of three parasites (Wuchereria bancrofti, Plasmodium falciparum and Mansonella perstans) in mosquito E/F, mosquito carcasses and human blood collected from the same households in two villages in the Savannah Region of the country. We successfully detected the parasite DNA in mosquito E/F from indoor resting mosquitoes, including W. bancrofti which had a very low community prevalence (2.5-3.8%). Detection in the E/F samples was concordant with detection in insect whole carcasses and human blood, and a parasite not vectored by mosquitoes was detected as well.Our approach to collect and test mosquito E/F successfully detected a variety of parasites at varying prevalence in the human population under field conditions, including a pathogen (M. perstans) which is not transmitted by mosquitoes. The method shows promise for further development and applicability for the early detection and surveillance of a variety of pathogens carried in human blood.


Assuntos
Culicidae/parasitologia , DNA de Protozoário/isolamento & purificação , Fezes/parasitologia , Malária/parasitologia , Mosquitos Vetores/parasitologia , Animais , DNA de Helmintos/genética , DNA de Protozoário/genética , Características da Família , Gana/epidemiologia , Humanos , Malária/epidemiologia , Técnicas de Diagnóstico Molecular/métodos , Plasmodium falciparum/genética , Prevalência , Wuchereria bancrofti/genética
18.
Parasit Vectors ; 13(1): 108, 2020 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-32111234

RESUMO

BACKGROUND: Currently available short read genome assemblies of the tetraploid protozoan parasite Giardia intestinalis are highly fragmented, highlighting the need for improved genome assemblies at a reasonable cost. Long nanopore reads are well suited to resolve repetitive genomic regions resulting in better quality assemblies of eukaryotic genomes. Subsequent addition of highly accurate short reads to long-read assemblies further improves assembly quality. Using this hybrid approach, we assembled genomes for three Giardia isolates, two with published assemblies and one novel, to evaluate the improvement in genome quality gained from long reads. We then used the long reads to predict structural variants to examine this previously unexplored source of genetic variation in Giardia. METHODS: With MinION reads for each isolate, we assembled genomes using several assemblers specializing in long reads. Assembly metrics, gene finding, and whole genome alignments to the reference genomes enabled direct comparison to evaluate the performance of the nanopore reads. Further improvements from adding Illumina reads to the long-read assemblies were evaluated using gene finding. Structural variants were predicted from alignments of the long reads to the best hybrid genome for each isolate and enrichment of key genes was analyzed using random genome sampling and calculation of percentiles to find thresholds of significance. RESULTS: Our hybrid assembly method generated reference quality genomes for each isolate. Consistent with previous findings based on SNPs, examination of heterozygosity using the structural variants found that Giardia BGS was considerably more heterozygous than the other isolates that are from Assemblage A. Further, each isolate was shown to contain structural variant regions enriched for variant-specific surface proteins, a key class of virulence factor in Giardia. CONCLUSIONS: The ability to generate reference quality genomes from a single MinION run and a multiplexed MiSeq run enables future large-scale comparative genomic studies within the genus Giardia. Further, prediction of structural variants from long reads allows for more in-depth analyses of major sources of genetic variation within and between Giardia isolates that could have effects on both pathogenicity and host range.


Assuntos
Benchmarking/métodos , Genoma de Protozoário , Giardia/genética , DNA de Protozoário/isolamento & purificação , Estudo de Associação Genômica Ampla , Genômica , Giardia lamblia/genética , Polimorfismo de Nucleotídeo Único , Sequências Repetitivas de Ácido Nucleico , Análise de Sequência de DNA
19.
PLoS One ; 15(3): e0230643, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32191777

RESUMO

In the Amazon basin, indigenous forest-dwelling communities typically suffer from a high burden of infectious diseases, including malaria. Difficulties in accessing these isolated ethnic groups, such as the semi-nomadic Yanomami, make official malaria data largely underestimated. In the current study, we longitudinally surveyed microscopic and submicroscopic malaria infection in four Yanomami villages of the Marari community in the northern-most region of the Brazilian Amazon. Malaria parasite species-specific PCR-based detection of ribosomal and non-ribosomal targets showed that approximately 75% to 80% of all malaria infections were submicroscopic, with the ratio of submicroscopic to microscopic infection remaining stable over the 4-month follow-up period. Although the prevalence of malaria infection fluctuated over time, microscopically-detectable parasitemia was only found in children and adolescents, presumably reflecting their higher susceptibility to malaria infection. As well as temporal variation, the prevalence of malaria infection differed significantly between villages (from 1% to 19%), demonstrating a marked heterogeneity at micro-scales. Over the study period, Plasmodium vivax was the most commonly detected malaria parasite species, followed by P. malariae, and much less frequently P. falciparum. Consecutive blood samples from 859 out of the 981 studied Yanomami showed that malaria parasites were detected in only 8% of the previously malaria-positive individuals, with most of them young children (median age 3 yrs). Overall, our results show that molecular tools are more sensitive for the identification of malaria infection among the Yanomami, which is characterized by heterogeneous transmission, a predominance of low-density infections, circulation of multiple malaria parasite species, and a higher susceptibility in young children. Our findings are important for the design and implementation of the new strategic interventions that will be required for the elimination of malaria from isolated indigenous populations in Latin America.


Assuntos
Malária/diagnóstico , Adolescente , Adulto , Idoso , Brasil/epidemiologia , Criança , Pré-Escolar , Estudos Transversais , DNA de Protozoário/isolamento & purificação , DNA de Protozoário/metabolismo , Feminino , Humanos , Lactente , Malária/epidemiologia , Malária/parasitologia , Malária/transmissão , Masculino , Pessoa de Meia-Idade , Plasmodium falciparum/genética , Plasmodium falciparum/isolamento & purificação , Plasmodium vivax/genética , Plasmodium vivax/isolamento & purificação , Prevalência , Estudos Prospectivos , RNA Ribossômico 18S/genética , RNA Ribossômico 18S/metabolismo , Adulto Jovem
20.
Exp Parasitol ; 212: 107872, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32165145

RESUMO

Theileriosis is a widespread and economically important disease of small ruminants in Pakistan. Ruminants are the intermediate hosts in the lifecycle of Theileria spp., with ticks of the family Ixodidae being the definitive hosts. To better understand the distribution and prevalence of theileriosis in Pakistan, a molecular survey was performed in small ruminants from the Lower Dir district of the Khyber Pakhtunkhwa province. A total of 200 healthy sheep and goats were screened from Maidan, Samar Bagh and Munda districts of district Dir Lower, Pakistan during December (2017) to April (2018). DNA samples were screened through nested PCR using universal primers. The amplified 492-498 bp amplicon was subjected to RLB analysis which was based on the hypervariable of the 18S rRNA gene to test for the presence of genotypes of Theileria in blood samples. A phylogeny was constructed to determine the species of Theileria genotypes. Nested PCR results indicated 53.5% prevalence of one or more Theileria genotypes in the blood of the host animal. From RLB assay, 27 animals (13.5%) showed infection with only a single species of Theileria while 80 animals (40%) showed coinfection by multiple Theileria spp. Based on the 18S rRNA phylogeny, the unknown genotype is of the species Theileria luwenshuni and is closely related to Chinese isolates. The present finding is the first report on molecular diagnosis of Theileria luwenshuni in small ruminants in Pakistan.


Assuntos
Doenças das Cabras/parasitologia , Doenças dos Ovinos/parasitologia , Theileria/isolamento & purificação , Theileriose/parasitologia , Distribuição por Idade , Análise de Variância , Animais , Coinfecção/epidemiologia , Coinfecção/parasitologia , DNA de Protozoário/química , DNA de Protozoário/genética , DNA de Protozoário/isolamento & purificação , DNA Ribossômico/química , DNA Ribossômico/genética , DNA Ribossômico/isolamento & purificação , Feminino , Genótipo , Doenças das Cabras/epidemiologia , Cabras , Hibridização Genética , Funções Verossimilhança , Modelos Lineares , Masculino , Análise Multivariada , Sondas de Oligonucleotídeos , Paquistão/epidemiologia , Filogenia , Reação em Cadeia da Polimerase , Prevalência , RNA Ribossômico 18S/genética , Fatores de Risco , Análise de Sequência de DNA , Distribuição por Sexo , Ovinos , Doenças dos Ovinos/epidemiologia , Theileria/classificação , Theileria/genética , Theileriose/epidemiologia
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