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1.
Mem Inst Oswaldo Cruz ; 115: e190284, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32049097

RESUMO

Despite some phlebotomines being well recognised as vectors of leishmaniasis agents, vector importance of those belonging to the genus Trichophoromyia has not been extensively studied. The present study provides evidence regarding the putative vector role played by some species of Trichophoromyia on leishmanine enzootics, based on literature reports and findings obtained from field experiments conducted in the ecotopes of Pará State, Brazil. The species Th. ubiquitalis, Th. velascoi, Th. auraensis, Th. ininii and Th. brachipyga possess minimal criteria to be included in the list of suspected leishmanine vectors. However, knowledge on man-biting behavior, substantiation of vector competence and determination of epidemiological implications are limited for all of the above mentioned species. Published studies together with present data draw attention to prioritize these phlebotomine species in entomological surveillance programs and studies on experimental susceptibility to Leishmania spp. infection.


Assuntos
Insetos Vetores/parasitologia , Leishmania/classificação , Psychodidae/parasitologia , Animais , DNA de Protozoário/genética , Leishmania/genética , Leishmaniose/transmissão , Reação em Cadeia da Polimerase
2.
Parasitol Res ; 119(2): 675-682, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31901995

RESUMO

Sarcocystis neurona is the main agent associated with equine protozoal myeloencephalitis (EPM). Apart from horses, S. neurona has been occasionally described causing neurologic disease in several other terrestrial animals as well as mortality in marine mammals. Herein, we describe the clinical, pathological, and molecular findings of a fatal case of S. neurona-associated meningoencephalitis in a domestic cat. The causing agent was analyzed by multilocus genotyping, confirming the presence of S. neurona DNA in the tissue samples of the affected animal. Significant molecular differences were found in relation to S. neurona isolates detected in other regions of the Americas. In addition, the parasite was identical to Sarcocystis sp. identified in opossum sporocysts in Brazil at molecular level, which suggests that transmission of. S. neurona in Brazil might involve variants of the parasite different from those found elsewhere in the Americas. Studies including more samples of S. neurona would be required to test this hypothesis, as well as to assess the impact of this diversity.


Assuntos
Doenças do Gato/parasitologia , Infecções Protozoárias do Sistema Nervoso Central/veterinária , Encefalomielite/parasitologia , Meningoencefalite/parasitologia , Sarcocystis/isolamento & purificação , Sarcocistose/veterinária , Animais , Brasil , Gatos , DNA de Protozoário/genética , Doenças dos Cavalos/parasitologia , Cavalos , Gambás/parasitologia , Sarcocystis/genética
3.
BMC Infect Dis ; 20(1): 16, 2020 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-31910816

RESUMO

BACKGROUND: Cryptosporidium is a genus of common intestinal protozoa, members of which cause diarrhea in a wide variety of hosts. Previous studies on Cryptosporidium in China have mainly focused on diarrhea sufferers, children, and immunodeficient individuals such as HIV/AIDS patients. However, the epidemiological characteristics of Cryptosporidium in the population in rural areas remain unclear. Herein, we investigated the prevalence of, and risk factors for, Cryptosporidium in rural areas of Binyang County, Guangxi Zhuang Autonomous Region, China, and genetically characterized the Cryptosporidium isolates we obtained. METHODS: From August to December 2016, two villages in Binyang County, Guangxi, were sampled using a random cluster sampling method. Fresh fecal samples were collected from all eligible residents (residence time > 6 months). Molecular characterization of Cryptosporidium was carried out based on its SSU rRNA, gp60, actin and hsp70 gene sequences. Fisher's exact test were conducted to assess the risk factors for Cryptosporidium infection. RESULTS: A total of 400 fecal samples were collected from 195 males (48.8%) and 205 females (51.2%). Two samples (0.5%) were positive for Cryptosporidium and were identified as C. viatorum and C. occultus respectively. Moreover, a new C. viatorum subtype XVaA3h was identified based on the sequence of the gp 60 gene. CONCLUSIONS: To our knowledge, this is the first report of C. viatorum and C. occultus infections in humans in China and of C. viatorum subtype XVaA3h. The findings provide important information on the prevalence of Cryptosporidium in the Chinese population, and expand the range of Cryptosporidium species known to infect people in China.


Assuntos
Sequência de Bases/genética , Criptosporidiose/epidemiologia , Cryptosporidium/classificação , Cryptosporidium/genética , DNA de Protozoário/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Criança , Pré-Escolar , China/epidemiologia , Estudos Transversais , Criptosporidiose/parasitologia , Cryptosporidium/isolamento & purificação , Diarreia/parasitologia , Fezes/parasitologia , Feminino , Genótipo , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Filogenia , Prevalência , Fatores de Risco , Inquéritos e Questionários , Adulto Jovem
4.
Eur J Protistol ; 72: 125659, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31825791

RESUMO

Three species of tintinnines, namely Tintinnopsis tentaculata Nie and Cheng, 1947, Tintinnopsis orientalis Kofoid and Campbell, 1929, and Eutintinnus lususundae (Entz, 1885) Kofoid and Campbell, 1939, were isolated from coastal waters of China. The morphology of each was investigated based on observations of live and protargol-stained specimens, and their SSU rDNA- and LSU rDNA-based phylogenetic relationships were analyzed. The ciliary patterns of these species are revealed for the first time. Based on the original descriptions and data from the present study, an improved diagnosis is given for each species. Unlike its congeners, the second dorsal kinety of Eutintinnus lususundae is displaced below the left ciliary field, which may suggest that the second dorsal kinety is evolving into a posterior kinety by a migration process. The ventral kinety in Eutintinnus is redefined. A neotype is fixed for T. tentaculata to stabilize the species name objectively, mainly because of the unavailability of type material.


Assuntos
Cilióforos/classificação , Água do Mar/parasitologia , China , Cilióforos/citologia , Cilióforos/genética , DNA de Protozoário/genética , Filogenia , RNA Ribossômico 18S/genética , Subunidades Ribossômicas Maiores de Eucariotos/genética , Especificidade da Espécie
5.
Parasitol Int ; 74: 101925, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31077806

RESUMO

Tick-borne diseases cause significant losses to livestock production in tropical and subtropical regions. However, information about the tick-borne infections in cattle in Xinjiang Uygur Autonomous Region (XUAR), northwestern China, is scarce. In this study, nested polymerase chain reaction (PCR) assays and gene sequencing were used to detect and analyze epidemiological features of Babesia bovis, B. bigemina, Coxiella burnetii and Anaplasma bovis infections in XUAR. Out of 195 samples tested, 24 (12.3%), 67 (34.4%), 40 (20.5%) and 10 (5.1%) were positive for B. bovis, B. bigemina, C. burnetii and A. bovis, respectively. Sequencing analysis indicated that B. bovis SBP-4, B. bigemina Rap1a, C. burnetii htpB and A. bovis 16S rRNA genes from XUAR showed 99%-100% identity with documented isolates from other countries. Phylogenetic analyses revealed that B. bovis SBP-4, B. bigemina Rap1a, C. burnetii htpB and A. bovis 16S rRNA gene sequences clustered in the same clade with isolates from other countries. To the best of our knowledge, this is the first report of C. burnetii infection of cattle in XUAR. Furthermore, this study provides important data for understanding the distribution of tick-borne pathogens, and is expected to improve the approach for prevention and control of tick-borne diseases in China.


Assuntos
Anaplasma/isolamento & purificação , Babesia/isolamento & purificação , Doenças dos Bovinos/epidemiologia , Coxiella burnetii/isolamento & purificação , Febre Q/veterinária , Doenças Transmitidas por Carrapatos/veterinária , Anaplasma/genética , Anaplasmose/epidemiologia , Animais , Babesia/genética , Babesiose/epidemiologia , Bovinos , Doenças dos Bovinos/microbiologia , Doenças dos Bovinos/parasitologia , China/epidemiologia , Coxiella burnetii/genética , DNA Bacteriano/genética , DNA de Protozoário/genética , Filogenia , Febre Q/epidemiologia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Doenças Transmitidas por Carrapatos/epidemiologia , Carrapatos/microbiologia
6.
Rev Soc Bras Med Trop ; 52: e20180541, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31800918

RESUMO

INTRODUCTION: Chagas disease is a major public health problem that is endemic in Brazil and Latin America. This study aimed to determine the socioeconomic, demographic, and clinical characteristics of 171 patients (mean age, 45 years; female, 65%) with Chagas disease at Hospital Universitário de Brasília, Federal District, Brazil. METHODS: We implemented this cross-sectional study using a clinical epidemiological questionnaire, electrocardiography, echocardiography, and quantitative detection of Trypanosoma cruzi DNA in blood using qRT-PCR. RESULTS: Among the patients, 26.3% had a full elementary education, and 13.2% were illiterate. Most (63.6%) were economically classified as class C, and 51.5% were born in Bahia state. A total of 62.0% participants reported previous contact with the triatomine bug. The clinical forms of the disease were indeterminate (69.51%), cardiac (15.24%), digestive (10.37%), and mixed (4.88%). The most common electrocardiographic abnormality was complete right bundle branch block in association with a divisional anterosuperior block. Only 14.6% of the patients complied with benznidazole medication for at least 60 days, and 164 of them were assessed by echocardiography. The parasite load was positive in 56% of the patients. CONCLUSIONS: Chagas disease affected mostly women, with the indeterminate chronic form of the disease.


Assuntos
Doença de Chagas/epidemiologia , Trypanosoma cruzi/genética , Adulto , Idoso , Brasil/epidemiologia , Doença de Chagas/parasitologia , Estudos Transversais , DNA de Protozoário/genética , Ecocardiografia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Carga Parasitária , Reação em Cadeia da Polimerase em Tempo Real , Fatores Socioeconômicos , Trypanosoma cruzi/isolamento & purificação , Adulto Jovem
7.
BMC Infect Dis ; 19(1): 1014, 2019 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-31783809

RESUMO

BACKGROUND: Clonorchiasis, caused by Clonorchis sinensis (C. sinensis) infection, is a serious food-borne zoonotic disease that is often asymptomatic or shows only mild symptoms, which leads to delayed treatment and chronic clonorchiasis and results in various complications, such as cholelithiasis, cholangitis, cholecystitis and cholangiocarcinoma. However, acute shock caused by C. sinensis infection has not been reported. Here, for the first time, we describe a fatal case of acute shock caused by C. sinensis infection. CASE PRESENTATION: A patient with a history of eating raw or undercooked freshwater fish was hospitalized with acute shock caused by severe abdominal pain. Physical examination suggested acute abdomen with severe abdominal pain and rigidity. Computed tomography (CT) detection indicated acute cholecystitis and cholelithiasis. After cholecystectomy, several liver flukes were found in the drainage tube. Furthermore, morphological analysis and polymerase chain reaction (PCR) identified the pathogen as C. sinensis. The liver gradually restored normal function after anthelmintic therapy with praziquantel. CONCLUSIONS: In this fatal case, C. sinensis infection was the cause of acute shock, which is rarely found in the clinic environment. This report aims to increase awareness of the hazards and complications related to clonorchiasis. The PCR diagnosis method used in this report might be helpful in reducing the misdiagnosis of clonorchiasis and unnecessary cholecystectomy.


Assuntos
Clonorquíase/diagnóstico , Clonorchis sinensis/isolamento & purificação , Choque/diagnóstico , Dor Abdominal/etiologia , Doença Aguda , Animais , Clonorquíase/complicações , Clonorquíase/parasitologia , Clonorchis sinensis/genética , DNA de Protozoário/genética , DNA de Protozoário/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Choque/etiologia , Tomografia Computadorizada por Raios X
8.
PLoS Negl Trop Dis ; 13(12): e0007900, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31830038

RESUMO

Whole genome sequencing (WGS) is increasingly used for molecular diagnosis and epidemiology of infectious diseases. Current Leishmania genomic studies rely on DNA extracted from cultured parasites, which might introduce sampling and biological biases into the subsequent analyses. Up to now, direct analysis of Leishmania genome in clinical samples is hampered by high levels of human DNA and large variation in parasite load in clinical samples. Here, we present a method, based on target enrichment of Leishmania donovani DNA with Agilent SureSelect technology, that allows the analysis of Leishmania genomes directly in clinical samples. We validated our protocol with a set of artificially mixed samples, followed by the analysis of 63 clinical samples (bone marrow or spleen aspirates) from visceral leishmaniasis patients in Nepal. We were able to identify genotypes using a set of diagnostic SNPs in almost all of these samples (97%) and access comprehensive genome-wide information in most (83%). This allowed us to perform phylogenomic analysis, assess chromosome copy number and identify large copy number variants (CNVs). Pairwise comparisons between the parasite genomes in clinical samples and derived in vitro cultured promastigotes showed a lower aneuploidy in amastigotes as well as genomic differences, suggesting polyclonal infections in patients. Altogether our results underline the need for sequencing parasite genomes directly in the host samples.


Assuntos
Genótipo , Leishmania/classificação , Leishmania/genética , Leishmaniose Visceral/parasitologia , Manejo de Espécimes/métodos , Sequenciamento Completo do Genoma/métodos , Adolescente , Criança , Pré-Escolar , DNA de Protozoário/química , DNA de Protozoário/genética , Humanos , Lactente , Leishmania/isolamento & purificação , Nepal
9.
Ann Agric Environ Med ; 26(4): 538-543, 2019 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-31885225

RESUMO

INTRODUCTION AND OBJECTIVE: It is easier and non-invasive to obtain faecal samples compared with blood samples. Molecular techniques may enable detection of parasites even in tiny amounts of blood-containing faeces. We aimed to compare the sensitivity of detection of three Babesia species and Hepatozoon canis in blood and faecal samples, including samples derived from naturally infected hosts. MATERIAL AND METHODS: Three groups were involved: 1) Nine BALB/c mice infected with Babesia microti sampled during acute (n=3), post-acute (n=3) and chronic phases of infection (n=3); 2) Eight dogs with symptoms of babesiosis; 3) Six red foxes infected with B. vulpes, one fox infected with B. canis, four foxes infected with H. canis. Genomic DNA was extracted from blood and faeces by use of commercial kits and amplified with genus-specific primers in one-step or nested PCR reactions. Selected PCR products were sequenced. RESULTS: No positive results for faecal samples were obtained from H. canis-positive foxes in contrast to Babesia spp. infections. Positive results from PCRs were obtained for all BALB/c mice (100%), five dogs (62.5%) and four of seven foxes (57.1%). Successful sequencing was obtained for six selected murine samples (B. microti), four canine samples (B. canis) and for one fox sample (B. vulpes). The success of B. microti detection in murine faecal samples from acute, post-acute and chronic phases was identical (100%). CONCLUSIONS: Detectability of Babesia spp. infections was lower in naturally infected dogs and foxes, compared to experimentally infected mice. Detection of DNA in faecal samples can be useful in the detection of Babesia infection in populations from which blood samples are hard to obtain, but due regard must be given to the possibility that prevalence of infection may be severely underestimated.


Assuntos
Babesia/isolamento & purificação , Babesiose/parasitologia , Sangue/parasitologia , Coccídios/isolamento & purificação , Coccidiose/parasitologia , DNA de Protozoário/genética , Fezes/parasitologia , Animais , Babesia/genética , Coccídios/genética , DNA de Protozoário/sangue , Cães/parasitologia , Raposas/parasitologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Reação em Cadeia da Polimerase
10.
Ann Agric Environ Med ; 26(4): 656-660, 2019 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-31885241

RESUMO

INTRODUCTION AND OBJECTIVE: Free-living animals can play an important role as a reservoir of Toxoplasma gondi;, however, data concerning this issue in Poland are still limited.The aim of study was to assess the occurrence of T. gondii infection by using molecular methods in free-living animals in selected regions of Poland. MATERIAL AND METHODS: Tissues samples of 396 animals (foxes, muskrats, birds, martens, badgers, polecats, raccoons, minks, raccoon dogs, otters, small rodents and insectivores, and grass snakes were collected from various regions of Poland. After samples digestion, DNA was isolated using QIAmp DNA Mini Kit (Qiagen). DNA extraction from small rodents and insectivores samples was performed without digestion. Next, nested PCR (B1 gene) and, for a part of nested PCR positive amplicons, RFLP PCR, were performed according to the method by Grigg and Boothroyd (2001). The other part of nested PCR positive DNA isolates were genotyped using 5 genetic markers: SAG1, SAG2 (5'- and 3'), SAG3, BTUB and GRA6, based on the method by Dubey et al. (2006). These PCR products were sequenced and compared with the NCBI database using Blast. RESULTS: In total, in 50 of the 396 examined animals DNA of T. gondii was detected (12.6%). The highest percentages of positive results in PCR was obtained in martens (40.9%) and badgers (38.5%), lower in birds (27.3%) and the lowest in foxes (7.4%). The RFLP and multilocus PCR analysis showed the dominance of T. gondii clonal type II (or II/III). CONCLUSIONS: The results of this study indicate the frequent T. gondii infection among free-living animals in Poland, especially martens and badgers, which may indirectly indicate that these animals contribute to the spread of the parasite in the sylvatic environment in Poland. The genotyping analysis showed the dominance of T. gondii clonal type II (or II/III).


Assuntos
Animais Selvagens/parasitologia , Toxoplasma/isolamento & purificação , Toxoplasmose Animal/parasitologia , Animais , DNA de Protozoário/genética , Raposas/parasitologia , Genótipo , Vison/parasitologia , Mustelidae/parasitologia , Polônia/epidemiologia , Cães Guaxinins/parasitologia , Roedores/parasitologia , Toxoplasma/classificação , Toxoplasma/genética , Toxoplasmose Animal/epidemiologia
11.
PLoS One ; 14(12): e0225822, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31851687

RESUMO

The order Piroplasmida encompasses two main families: Babesiidae and Theileriidae, containing tick-borne pathogens of veterinary and medical importance worldwide. While only three genera (Babesia, Cytauxzoon and Theileria) comprising piroplasm parasites are currently recognised, phylogenetic studies at the 18S rRNA (18S) gene suggest that these organisms represent at least ten lineages, one of which comprises the relatively unique and highly diverse Theileria spp. from Australian marsupials and ticks. As an alternative to analysing 18S sequences alone, sequencing of mitochondrial genes has proven to be useful for the elucidation of evolutionary relationships amongst some groups of piroplasms. This research aimed to characterise piroplasms from Australian native mammals and ticks using multiple genetic markers (18S, cytochrome c, oxidase subunit III (cox3) and cytochrome B (cytB)) and microscopy. For this, nearly complete piroplasm-18S sequences were obtained from 32 animals belonging to six marsupial species: eastern bettong (Bettongia gaimardi), eastern quoll (Dasyurus viverrinus), eastern grey kangaroo (Macropus giganteus), swamp wallaby (Wallabia bicolor), quokka (Setonix brachyurus) and Gilbert's potoroo (Potorous gilbertii). The organisms detected represented eight novel Theileria genotypes, which formed five sub-clades within the main marsupial clade containing previously reported Australian marsupial and tick-derived Theileria spp. A selection of both novel and previously described Australian piroplasms at the 18S were also successfully characterised, for the first time, at the cox3 and cytB loci, and corroborated the position of Australian native theilerias in a separate, well-supported clade. Analyses of the cox3 and cytB genes also aided in the taxonomic resolution within the clade of Australian Piroplasmida. Importantly, microscopy and molecular analysis at multiple loci led to the discovery of a unique piroplasm species that clustered with the Australian marsupial theilerias, for which we propose the name Theileria lupei n. sp.


Assuntos
Marsupiais/parasitologia , Mitocôndrias/genética , RNA Ribossômico 18S/genética , Theileria , Theileriose/parasitologia , Carrapatos/parasitologia , Animais , Austrália , DNA de Protozoário/genética , Loci Gênicos , Filogenia , RNA de Protozoário/genética , Theileria/classificação , Theileria/genética , Theileria/isolamento & purificação
12.
Mikrobiyol Bul ; 53(4): 408-418, 2019 Oct.
Artigo em Turco | MEDLINE | ID: mdl-31709938

RESUMO

Leishmaniasis is a parasitic disease that is transmitted to humans by the bites of infected female phlebotomine sandflies. In the diagnosis of cutaneous leishmaniasis (CL), in the smear samples, the demonstration of the parasite by microscope remains a gold standard method. However, it becomes difficult to diagnose the parasite since the number of amastigotes in chronic cases with a lesion of one year or longer is very low. Due to many factor such as patients primarily do not to take any notice these lesions in their bodies, do not apply to health institutions or late applied, receive wrong treatment; the diagnosis and treatment are delayed. In addition, it is been worse prognosis by add secondary infection to lesions and wounds become chronic. For this reason, molecular methods are used in addition to microscopic examination in chronic suspected CL cases. It was aimed to reveal of the molecular diagnostic value in chronic suspected CL cases by polymerase chain reaction (PCR) in the smear belonging to Turkish patients that reported to be evaluated clinically because it can not be seen Leishmania amastigotes in microscopic examination. Smear of 50 Turkish patients who were clinically reported of the evaluation of chronic CL were selected. These samples were smears belonging to suspected CL patients that applied Hatay Mustafa Kemal University, Faculty of Medicine, Parasitology laboratory from different polyclinics and were decided to be evaluated clinically as a result of microscopic examination because they came from endemic regions (such as Hassa, Altinözü, Yayladagi). DNA was isolated from selected samples and PCR was performed using 13A, 13B primers targeting the kinetoplastid DNA (kDNA) region. The samples found positive by PCR were typed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis using LITSR and L5.8S primers targeting internal transcribed spacer (ITS-1) region. Of the 50 smear samples, 17 (34%) were determined positive with 13A, 13B primers targeting the kinetoplastid DNA (kDNA) region. Positive samples were also found to be positive with LITSR and L5.8S primers targeting ITS-1 region. The PCR products obtained from PCR with ITS-1 gene region were digested with the restriction endonucleases BsuRI (HaeIII). As a result of PCR-RFLP analysis, it was determined that 11 of Leishmania tropica, one of Leishmania major and five of Leishmania infantum/donovani out of 17 samples. Chronic CL can be confused with skin diseases such as sarcoidosis, tuberculosis, malignant tumors. In particular, chronic CL cases can be escaped the attention for many reasons such as failure to diagnose correctly, insufficient microscope experience, fail to see due to low number of parasites. For this reason, it was concluded that PCR, which is a molecular method, should be used in chronic suspected CL samples which are negative for the parasite by microscopic examination.


Assuntos
DNA de Protozoário , Leishmania , Leishmaniose Cutânea , Reação em Cadeia da Polimerase , DNA de Protozoário/genética , Feminino , Humanos , Leishmania/genética , Leishmaniose Cutânea/diagnóstico , Microscopia , Polimorfismo de Fragmento de Restrição , Reprodutibilidade dos Testes , Turquia
13.
PLoS Negl Trop Dis ; 13(11): e0007698, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31697673

RESUMO

Leishmaniasis, caused by protozoan parasites of the Leishmania genus, represents an important health problem in many regions of the world. Lack of effective point-of-care (POC) diagnostic tests applicable in resources-limited endemic areas is a critical barrier to effective treatment and control of leishmaniasis. The development of the loop-mediated isothermal amplification (LAMP) assay has provided a new tool towards the development of a POC diagnostic test based on the amplification of pathogen DNA. LAMP does not require a thermocycler, is relatively inexpensive, and is simple to perform with high amplification sensitivity and specificity. In this review, we discuss the current technical developments, applications, diagnostic performance, challenges, and future of LAMP for molecular diagnosis and surveillance of Leishmania parasites. Studies employing the LAMP assay to diagnose human leishmaniasis have reported sensitivities of 80% to 100% and specificities of 94% to 100%. These observations suggest that LAMP offers a good molecular POC technique for the diagnosis of leishmaniasis and is also readily applicable to screening at-risk populations and vector sand flies for Leishmania infection in endemic areas.


Assuntos
Leishmaniose/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Sistemas Automatizados de Assistência Junto ao Leito , Animais , DNA de Protozoário/genética , Bases de Dados Factuais , Doenças do Cão/diagnóstico , Doenças do Cão/parasitologia , Cães , Humanos , Leishmania/genética , Programas de Rastreamento/métodos , Técnicas de Amplificação de Ácido Nucleico/economia , Psychodidae/parasitologia , Sensibilidade e Especificidade , Fatores de Tempo
14.
Parasitol Res ; 118(12): 3469-3478, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31691003

RESUMO

Blastocystis is a prevalent parasite that has a wide distribution. In order to design HRM real-time PCR, primers were selected from SSU rRNA gene to amplify specific fragment with different melting temperatures for each subtype of Blastocystis. Subsequently, HRM real-time PCR was performed and melting curve analysis was done by Rotor-Gene Q software. The results of HRM real-time PCR was then compared with sequence results of "barcoding region" of SSU rRNA gene of Blastocystis. To evaluate sensitivity of test, 10-fold serial dilutions of the parasite were prepared from ~ 106 to 1 parasite per mL of stool sample and were investigated by HRM real-time PCR. In order to determine specificity of method, HRM real-time PCR was done for some microorganisms and Blastocystis-negative stool samples. In silico analysis showed that all seventeen subtypes of Blastocystis were distinguish. In vitro analysis revealed that the test discriminated subtypes with specific melting temperatures.


Assuntos
Infecções por Blastocystis/parasitologia , Blastocystis/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Blastocystis/classificação , Blastocystis/genética , Infecções por Blastocystis/diagnóstico , Primers do DNA/química , Primers do DNA/genética , DNA de Protozoário/química , DNA de Protozoário/genética , Testes Diagnósticos de Rotina , Fezes/parasitologia , Humanos , Sensibilidade e Especificidade
15.
Parasitol Res ; 118(12): 3555-3559, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31722067

RESUMO

The aim of this study was to survey the Cryptosporidium species in peafowls (Pavo cristatus) in Henan Province, China. A total of 143 fecal specimens collected from a breeding farm were tested for Cryptosporidium by nested PCR targeting the small subunit rRNA (SSU rRNA), 70-kDa heat shock protein (HSP70), and actin genes of Cryptosporidium followed by sequence analysis. Only one isolate from an asymptomatic host was obtained, and the isolate differed from a new C. xiaoi-like genotype by one nucleotide and from C. xiaoi or C. bovis at the SSU rRNA locus by six nucleotides. Likewise, the actin gene shared 99% identity with the C. xiaoi-like genotype, accompanied by four nucleotide mutations. A complete sequence of the HSP70 gene was obtained, and exhibited 96% similarity with that from C. xiaoi and differed by one nucleotide from that with the C. xiaoi-like genotype. Phylogenetic analysis of the current isolate revealed genetic relatedness to the C. xiaoi-like genotype and distinction from C. xiaoi and C. bovis. Therefore, our results provided the first documentation of avian infection with a C. xiaoi-like genotype in China and further insight into the diversity of Cryptosporidium spp. in avians.


Assuntos
Doenças das Aves/parasitologia , Criptosporidiose/parasitologia , Cryptosporidium/isolamento & purificação , Animais , Doenças das Aves/epidemiologia , China/epidemiologia , Criptosporidiose/epidemiologia , Cryptosporidium/classificação , Cryptosporidium/genética , DNA de Protozoário/genética , Fezes/parasitologia , Galliformes/parasitologia , Genótipo , Filogenia , Reação em Cadeia da Polimerase/veterinária , RNA Ribossômico/genética
16.
Parasitol Res ; 118(12): 3491-3496, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31728723

RESUMO

In this study, 36.8% (28/76) of tissue samples collected from domestic pigs (Sus scrofa) contained sarcocysts, as determined by light microscopy. The organisms were identified as Sarcocystis miescheriana and Sarcocystis suihominis based on their morphological and molecular characteristics. Four genetic markers, i.e., 18S rDNA, 28S rDNA, ITS-1 region (ITS-1), and the mitochondrial COX1 gene (COX1), of the two parasites were sequenced and analyzed, and the 28S rDNA and ITS-1 of S. suihominis obtained from pigs constituted the first records of these markers in GenBank. The sequences of the four loci (18S rDNA, 28S rDNA, ITS-1, and COX1) of S. miescheriana shared high identities with those of S. miescheriana obtained from domestic and/or wild pigs in GenBank, with similarities of 99.6%, 99.6%, 95.9%, and 95.4%, respectively. The 18S rDNA sequences of S. suihominis exhibited 99.4% identity with those of S. suihominis from domestic and wild pigs. The comparison of the newly obtained sequences of the four genetic markers between the two parasites revealed that the interspecific similarities of 18S rDNA, 28S rDNA, ITS-1, and COX1 were 97.7%, 96.6%, 80.3%, and 81.2%, respectively. Therefore, the two species could be better discriminated with ITS-1 and mitochondrial COX1 compared with 18S rDNA or 28S rDNA. The phylogenetic analysis using 28S rDNA indicated that the two Sarcocystis species in domestic pigs had a close relationship.


Assuntos
Sarcocystis/crescimento & desenvolvimento , Sarcocystis/genética , Sarcocistose/veterinária , Doenças dos Suínos/parasitologia , Animais , China , DNA de Protozoário/genética , DNA Ribossômico/genética , Genes Mitocondriais , Filogenia , Reação em Cadeia da Polimerase , RNA Ribossômico 18S/genética , Sarcocystis/classificação , Sarcocystis/isolamento & purificação , Sarcocistose/parasitologia , Sus scrofa/parasitologia , Suínos
17.
PLoS Negl Trop Dis ; 13(11): e0007748, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31730650

RESUMO

BACKGROUND: We identified the species of Leishmania isolated from traveling and migrant patients attended in a reference center from 2000 to 2015, we performed the georeferencing of these species in Rio de Janeiro (RJ) state and we had knowledge about the human flows between the likely location of infection (LLI) and place of residence (PR) in RJ state, Brazil. METHODOLOGY/PRINCIPAL FINDINGS: This is a retrospective cross-sectional study including 171 patients diagnosed with ATL. Google Maps, OpenStreetMap, and Bing Maps were tools used to georeference LLI and PR. For etiological identification, we used isoenzyme electrophoresis, polymerase chain reaction-restriction fragment length polymorphism (molecular target hsp70C with restriction enzymes HaeIII and BstUI), and sequencing of the internal transcribed spacer of ribosomal DNA. ARCGIS software was used to create maps of the geographic distribution of Leishmania species in the state and municipality of RJ, together with flows between the LLI and PR. Isolates from 104 patients were identified as: L. (Viannia) braziliensis (80.8%), L. (V.) naiffi (7.7%), L. (V.) guyanensis (6.7%), L. (Leishmania) amazonensis (1%), and genetic variants of L. (V.) braziliensis (3.8%). The flow maps showed that the LLI included 4 countries, 19 Brazilian states, and 18 municipalities of RJ state. The Brazilian states with the highest density of cases were Amazonas (n = 32), Bahia (n = 18), and Ceará (n = 15). CONCLUSIONS/SIGNIFICANCE: This work is the first contribution to the knowledge of the routes of Leishmania species introduced in RJ state by migrants and travelers patients. L. (V.) braziliensis, L. (V.) guyanensis, L. (V.) naiffi, L. (L.) amazonensis, and genetic variants of L. (V.) braziliensis were identified in RJ state. To determine whether the autochthonous transmission of these imported species is possible it is necessary the adaptation of these species to environmental conditions as well as the presence of reservoirs and phlebotomine vectors in this region.


Assuntos
Leishmania/classificação , Leishmania/isolamento & purificação , Leishmaniose/epidemiologia , Leishmaniose/parasitologia , Migrantes , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Brasil/epidemiologia , Criança , Pré-Escolar , Estudos Transversais , DNA de Protozoário/genética , DNA Ribossômico/genética , Feminino , Humanos , Leishmania/genética , Leishmaniose/diagnóstico , Masculino , Pessoa de Meia-Idade , Filogenia , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Estudos Retrospectivos , Adulto Jovem
18.
Rev Bras Parasitol Vet ; 29(1): e009819, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31691734

RESUMO

The aim of this study was to confirm the emergence of canine visceral leishmaniasis among dogs in Foz do Iguaçu. The disease was diagnosed through the isolation and molecular identification of Leishmania infantum. In the first sample collection stage (2012), three lymph node aspirates and 46 buffy coat samples were obtained mostly from the dogs that were seroreagents for leishmaniasis. In the second sample collection stage (2013), the buffy coat samples were collected from 376 dogs located close to Paraguay, Paraná river, center and peripheral parts of the city. The DNA from the six isolates, four from the first sampling stage (4/49) and two from the second sampling stage (2/376), was subjected to polymerase chain reaction using the K26F/R primers. The isolate was confirmed as L. infantum by sequencing. As none of the dogs had ever left the city, the isolates were confirmed as autochthonous. Further, the study confirmed the emergence of canine visceral leishmaniasis in Paraná through the identification of L. infantum among dogs in Foz do Iguaçu city. Hence, collaborative control measures should be designed and implemented by the public agencies and research institutions of Brazil, Argentina, and Paraguay to control the spread of visceral leishmaniasis.


Assuntos
DNA de Protozoário/genética , Doenças do Cão/parasitologia , Leishmania infantum/genética , Leishmaniose Visceral/veterinária , Animais , Brasil/epidemiologia , Doenças do Cão/diagnóstico , Doenças do Cão/epidemiologia , Cães , Leishmania infantum/isolamento & purificação , Leishmaniose Visceral/diagnóstico , Leishmaniose Visceral/epidemiologia , Reação em Cadeia da Polimerase em Tempo Real/veterinária
19.
BMC Infect Dis ; 19(1): 895, 2019 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-31660874

RESUMO

BACKGROUND: Leishmania infantum, the etiological agent of visceral leishmaniasis, is a neglected zoonosis that requires validation and standardization of satisfactory diagnostic methodologies. Thus, the aim of the present study was to evaluate the effectiveness of cathepsin L-like protease as a target for making molecular diagnoses and as a phylogenetic marker enabling to understand the intraspecies variations and evolutionary history of L. infantum in Brazil. METHODS: We used 44 isolates of L. infantum. The cathepsin L-like gene fragments were amplified, sequenced, manually aligned and analyzed using inference methods. The sequences generated were used to search and design oligonucleotide primers to be used in reactions specific to the target parasite. RESULTS: The cathepsin L-like gene did not show any intraspecies variability among the isolates analyzed. The pair of primers proposed amplified the target deoxyribonucleic acid (DNA) of L. infantum isolates and were effective for DNA amplification at concentrations of as low as 10- 11 ng/µl. The proposed marker did not present cross-reactions with other hemoparasites. When used for making the diagnosis in a panel of clinical samples from dogs, a positivity rate of 49.03% (102/208) was obtained, versus 14.42% (30/208) for a ribosomal internal transcribed spacer (ITS) marker. In samples from sandflies, the rate was 6.25% and from humans, 14.28%. CONCLUSIONS: The results described in this work allow us to infer that CatLeish-PCR is a sensitive and specific marker for use in diagnostic trials of L. infantum and in clinical and epidemiological surveys.


Assuntos
Catepsinas/genética , Leishmania infantum/enzimologia , Leishmaniose Visceral/diagnóstico , Filogenia , Animais , Sequência de Bases , Biomarcadores , Brasil , Ensaios Enzimáticos Clínicos/normas , Reações Cruzadas/imunologia , Primers do DNA/genética , DNA de Protozoário/genética , Doenças do Cão/parasitologia , Cães , Humanos , Leishmania infantum/classificação , Doenças Negligenciadas , Reação em Cadeia da Polimerase , Psychodidae/parasitologia , Padrões de Referência , Zoonoses/parasitologia
20.
Parasit Vectors ; 12(1): 502, 2019 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-31661007

RESUMO

BACKGROUND: Cryptosporidium viatorum is a minor Cryptosporidium pathogen in humans. Currently, there is limited information regarding the prevalence and genotypes of C. viatorum in animals in China. METHODS: In this study, 228 faecal samples were collected from two wild rat species (Leopoldamys edwardsi and Berylmys bowersi) in Chongqing Municipality and Guangdong Province, China. These specimens were analyzed for C. viatorum and then subtyped it using PCR and sequence analysis of the small subunit ribosomal RNA (SSU rRNA) and 60-kilodalton glycoprotein (gp60) genes, respectively. RESULTS: A total of 25 (11.0%) faecal samples were tested positive for C. viatorum by SSU rRNA assay. Of these samples, 4 (3.6%) came from L. edwardsi and 21 (18.0%) from B. bowersi. Of the 25 C. viatorum-positive samples, 17 were successfully amplified at the gp60 gene locus, which represented four subtypes belonging to two subtype families, including XVa (XVaA6, XVaA3g, XVaA3h) and XVc (XVcA2G1). Phylogenetic analysis based on the gp60 amino acid sequences indicated that all of the C. viatorum isolates grouped together, supporting the conclusion that C. viatorum from the wild rats represent two subtype families. CONCLUSIONS: These results indicate an occurrence of C. viatorum XVa subtype family from rats which is genetically identical to those found in humans. Our findings suggest that wild rats may be a potential source of human cryptosporidiosis.


Assuntos
Criptosporidiose/parasitologia , Cryptosporidium/isolamento & purificação , Murinae/parasitologia , Doenças dos Roedores/parasitologia , Sequência de Aminoácidos , Animais , China/epidemiologia , Criptosporidiose/epidemiologia , Criptosporidiose/transmissão , Cryptosporidium/classificação , Cryptosporidium/genética , DNA de Protozoário/genética , DNA de Protozoário/isolamento & purificação , Fezes/parasitologia , Humanos , Filogenia , Reação em Cadeia da Polimerase/veterinária , Prevalência , RNA Ribossômico/química , Doenças dos Roedores/epidemiologia , Doenças dos Roedores/transmissão , Sialoglicoproteínas/química , Sialoglicoproteínas/genética
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