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1.
Rev. Soc. Bras. Med. Trop ; 53: e20190535, 2020. tab
Article in English | LILACS, ColecionaSUS, SES-SP | ID: biblio-1136801

ABSTRACT

Abstract Since the early 20th century, the detection of intestinal parasites has improved with the development of several techniques for parasitic structures recovery and identification, which differ in sensitivity, specificity, practicality, cost, and infrastructure demand. This study aims to review, in chronological order, the stool examination techniques and discuss their advantages, limitations, and perspectives, and to provide professionals and specialists in this field with data that lays a foundation for critical analysis on the use of such procedures. The concentration procedures that constitute the main techniques applied in routine research and in parasitological kits are a) spontaneous sedimentation; b) centrifugation-sedimentation with formalin-ethyl acetate; and c) flotation with zinc sulfate solution. While selecting a technique, one should consider the purpose of its application and the technical-operational, biological, and physicochemical factors inherent in the procedures used in stool processing, which may restrict its use. These intrinsic limitations may have undergone procedural changes driven by scientific and technological development and by development of alternative methods, which now contribute to the improvement of diagnostic accuracy.


Subject(s)
Humans , Animals , History, 20th Century , History, 21st Century , Parasitology/history , Specimen Handling/history , Feces/parasitology , Intestinal Diseases, Parasitic/diagnosis , Parasitology/methods , Specimen Handling/methods , Sensitivity and Specificity
2.
Rev. bras. parasitol. vet ; 29(2): e000420, 2020. tab, graf
Article in English | LILACS | ID: biblio-1138065

ABSTRACT

Abstract Angiostrongylus vasorum is a pulmonary artery parasite of domestic and wild canid. On molluscs, intermediate host, first stage larvae (L1) are found after the first day of infection, in the 8th L2 and in the 30 th L3. It was evaluated L1, L2 and L3 recovered by Baermann technique from Achatina fulica infected with 1000 L1. Fifty larvae/stage were incubated with antibodies anti-β-tubulin, anti-α-tubulin, anti- α-actin, anti-β-actin and anti-collagen, and then with Alexa 633. Fifty larvae/stage were observed with picrosirius red and Oil Red O. It was also observed in the anterior region of L1 the beginning of the chitinous stems development, in the initial portion of the intestine and genital primordium. In L2 anterior region, the papillae, chitinous canes juxtaposed to the mouth and intestines bigger than L1. The L3 musculature is well defined, next to the chitinous stems, there are two round distally arranged from each other. It was observed the whole extension of the intestine genital primordium and intense cellularity in the L3 distal portion. With the picrosirius red the L1, L2 and L3 musculature could be observed, as the nerve ganglia on L3. Oil Red O revealed that L1, L2 and L3 store energy on lipid droplets.


Resumo Angiostrongylus vasorum é um parasito de artérias pulmonares dos canídeos domésticos e silvestres. Nos moluscos, hospedeiros intermediários, encontram-se no primeiro dia após a infecção, larvas de primeiro estágio (L1), ao 8° L2 e ao 30° L3. Avaliou-se L1, L2 e L3 recuperadas pela técnica de Baermann de Achatina fulica infectada com 1.000 L1. Incubou-se 50 larvas/estádio com anticorpos anti-β-tubulina, anti-α-tubulina, anti-β-actina e anti-colágeno e, em seguida, com anticorpo Alexa 633. Observaram-se também 50 larvas/estádio com picrosirius red e Oil Red O, na região anterior da L1, o início do desenvolvimento de hastes quitinosas, a porção inicial do intestino e o primórdio genital. Na região anterior de L2, papilas, bastões quitinosos justapostos à boca e ao intestino maior que em L1. A musculatura de L3 é bem desenvolvida, próximo às hastes quitinosas, há duas estruturas redondas dispostas distalmente uma da outra. Observaram-se também toda a extensão do intestino, o primórdio genital e a intensa celularidade na porção distal da L3. Com o picrosirius red observou-se a musculatura de L1, L2 e L3, assim como, gânglios nervosos na L3. Oil Red O revelou que L1, L2 e L3 armazenam energia em gotículas lipídicas.


Subject(s)
Animals , Parasitology/methods , Gastropoda/parasitology , Angiostrongylus/anatomy & histology , Larva/anatomy & histology
3.
Rev. medica electron ; 40(2): 454-462, mar.-abr. 2018. ilus
Article in Spanish | LILACS, CUMED | ID: biblio-902298

ABSTRACT

RESUMEN La larva migrans visceral es una enfermedad que se produce al ingerir huevos infectantes de nematodos parásitos de gatos y perros (Toxocaracanis y Toxocaracati); los cuales eclosionan en el intestino del hombre y las larvas se distribuyen en todo el organismo, principalmente hígado, pulmón, corazón y cerebro. Las larvas en su migración dejan trazos de hemorragias, necrosis y células inflamatorias; algunas son destruidas por la respuesta inmune del huésped y otras forman granulomas eosinofílicos. Los síntomas dependen del tejido u órgano afectado, de la intensidad de la infección y del grado de la respuesta inmunológica inducida. Se presenta un caso del sexo masculino de 72 años que ingresa en el Servicio de Medicina del Hospital Militar Docente “Dr. Mario Muñoz Monroy”, de Matanzas, por cuadro de fiebre, diarreas, tos seca, astenia, anorexia y pérdida de peso al que se le diagnosticó larva migrans visceral. Por lo atípico de la edad del paciente y la complejidad del diagnóstico decidimos presentar este caso (AU).


ABSTRACT Visceral larva migrans is a disease produced after the ingestion of infectant eggs of cat´s and dog´s nematode parasites (Toxocara canis and Toxocara cati). These parasites harch in the men´s intestines and the larvas are distributed around the organism, mainly in the following organs: liver, lungs, hearth and brain. In their migration, the larvas leave traces of hemorrhage, necrosis and inflammatory cells; several of them are destroyed by the host´s immune answer and others form eosinophilic granulomas. The symptoms depend on the affected tissue or organ, on the infection intensity and on the level of induced immunologic answer. The case of a male patient, aged 72 years-old is presented. He entered the Medicine Service of the Teaching Military Hospital “Dr. Mario Muñoz Monroy”, of Matanzas with fever, diarrhea, dry cought, asthenia, anorexia and weight loss.Visceral larva migrans was diagnosed. The presentation of the case was decided because of the atypical patient´s age and the complexity of the diagnosis (AU).


Subject(s)
Humans , Male , Aged , Parasitic Diseases/prevention & control , Toxocara , Larva Migrans, Visceral/complications , Larva Migrans, Visceral/diagnosis , Larva Migrans, Visceral/etiology , Larva Migrans, Visceral/drug therapy , Larva Migrans, Visceral/diagnostic imaging , Toxocara canis , Parasitology/methods , Communicable Disease Control , Risk Factors , Clinical Laboratory Techniques , Diagnostic Tests, Routine , Latin America/epidemiology
4.
Rev. bras. parasitol. vet ; 27(1): 86-89, Jan.-Mar. 2018. tab
Article in English | LILACS | ID: biblio-1042462

ABSTRACT

Abstract Duddingtonia flagrans has been tested as an alternative parasite control, but data from in vitro experiments based on in vivo calculations describing nematophagous fungi predation in nematodes are restricted. The objective of this work was to determine the efficacy of D. flagrans against sheep nematode larvae in vitro using in vivo calculations. Fecal samples were introduced to fungi in different concentrations: 0.0/control; 0.05; 0.1; 0.2; 0.4; 0.8; 1.6; 3.2; and 6.4 g corresponding, respectively, to 583.000; 1.166.000; 2.332.000; 4.664.000; 9.328.000; 18.656.000; 37.312.000 and 74.624.000 chlamydospores/kg of body weight. The material was incubated for 14 days, before the larvae recovery (Assay 1). Assay 2 was carried out with the doses of 0.00625; 0.0125; and 0.025 g. The results showed a negative correlation between fungal concentrations and larval numbers for both assays. The fungus demonstrated an efficacy above 89% in both assays. Thus, we consider that the data from in vitro studies based on in vivo calculations may optimize the fungi quantities for field experiments.


Resumo Duddingtonia flagrans tem sido testado como uma alternativa no controle de parasitos, entretanto, trabalhos in vitro da predação de nematoides por fungos nematófagos correlacionados com cálculos baseados para testes in vivo são restritos. O objetivo deste trabalho foi determinar a eficácia in vitro de D. flagrans contra larvas de nematoides de ovinos tendo como base cálculos in vivo. Amostras fecais receberam a adição do fungo em diferentes concentrações: 0.0/controle; 0,05; 0,1; 0,2; 0,4; 0,8; 1,6; 3,2 e 6,4 gramas correspondendo, respectivamente, às seguintes dosagens: 583.000; 1.166.000; 2.332.000; 4.664.000; 9.328.000; 18.656.000; 37.312.000 e 74.624.000 clamidósporos/Kg de peso vivo animal. O material foi incubado por 14 dias, para recuperação das larvas (Ensaio 1). O Ensaio 2 foi realizado com concentrações de 0,00625; 0,0125 e 0,025 g. Foi observada correlação negativa entre a concentração fúngica e o número de larvas, nos dois ensaios. O fungo demonstrou eficácia acima de 89% em ambos os ensaios. A partir destes dados, acreditamos que ensaios in vitro baseados em cálculos in vivo podem aprimorar as dosagens para a realização de experimentos a campo.


Subject(s)
Animals , Female , Sheep Diseases/parasitology , Sheep Diseases/therapy , Sheep/parasitology , Duddingtonia , Biological Control Agents/therapeutic use , Parasitology/methods , Treatment Outcome , Larva/microbiology , Nematoda/microbiology
5.
Rev. bras. parasitol. vet ; 27(1): 90-93, Jan.-Mar. 2018. tab, graf
Article in English | LILACS | ID: biblio-1042461

ABSTRACT

Abstract Cyathostomins are the most prevalent nematodes of horses, and multidrug resistance has been reported worldwide. There is a need to implement alternative drug monitoring analytical tests. The objective of this study was to determine the consistency (5 repetitions) of the larval migration on agar test (LMAT) using ivermectin, moxidectin, pyrantel or albendazole against cyathostomin infective-stage larvae in eight different concentrations. LMAT showed a strong coefficient of determination (R2 > 0.91), between the test repetitions (n=5). The average 50% effective concentration (EC50) for ivermectin, moxidectin, pyrantel and albendazole were 0.0404, 0.0558, 0.0864 and 0.0988 nMol, respectively. The results of the EC50 for albendazole showed the greatest range of concentration. Ivermectin and moxidectin had the lowest in between-test variation. In the future, internationally certified susceptible isolates could be used for screening new drug candidates, or to follow up the pattern of drug efficacy from field populations.


Resumo Ciatostomíneos são os nematodas mais prevalentes em equinos e a resistência múltipla foi relatada em todo o mundo. Existe a necessidade de implementar o monitoramento dos produtos com testes analíticos alternativos. O objetivo deste estudo foi determinar a consistência (5 repetições) do teste de migração larval em ágar (TMLA) usando ivermectina, moxidectina, pirantel e albendazole contra larvas infectantes de ciatostomíneos em oito concentrações diferentes. O TMLA demonstrou um coeficiente de determinação (R2) acima de 0,91 entre as repetições do teste. A concentração efetiva para 50% (CE50) para ivermectina, moxidectina, pirantel e albendazole foi de 0,0404; 0,0558; 0,0864 e 0,0988 nMol, respectivamente. A CE50 do albendazole demonstrou a maior amplitude entre os testes. A ivermectina e a moxidectina tiveram as menores variações das doses entre as repetições. No futuro, isolados certificados susceptíveis poderão ser testados com o TMLA para indicação de novos produtos e mesmo para acompanhar o perfil de eficácia de populações do campo.


Subject(s)
Animals , Horses/parasitology , Nematoda/drug effects , Antiparasitic Agents/pharmacology , Parasitology/methods , Pyrantel/pharmacology , Ivermectin/pharmacology , Albendazole/pharmacology , Macrolides/pharmacology , Larva/drug effects
6.
Rev. bras. parasitol. vet ; 27(1): 19-25, Jan.-Mar. 2018. tab, graf
Article in English | LILACS | ID: biblio-899313

ABSTRACT

Abstract The aim of the present study was to evaluate the growth rate of Balantidium coli in three xenic media cultures. Between 2013 and 2015, 10 B. coli isolates obtained from feces of Cynomolgus macaques, and 30 isolates from feces of pigs were studied. An inoculum of 500 trophozoites was transferred to tubes containing LES, TYSGM-9 and Pavlova media. These cultures were evaluated at incubation times of 24, 48, 72 and 96 hours. In most of strains analyzed wasn't showed significant difference in the growth rate comparing TYSGM-9 and Pavlova media (Wilcoxon p>0.016). In Pavlova medium, the trophozoites showed a maximum growth at 72 hours with significant difference when compared with the times of 24 h and 96 h (Wilcoxon <0.008). In LES, viable trophozoites were observed until 24 hours, with a significant difference (Friedman p<0.05, Wilcoxon p<0.016) in the number of parasite cells compared with Pavlova and TYSGM-9 media cultures. Thus, LES medium seemed to be less adequate than the other media for maintenance of B. coli. Despite the satisfactory results in TYSGM-9, Pavlova medium was considered ideal for the maintenance of this protozoan strain, guaranteeing the viability of the parasite with subculture every three days, presenting lower costs.


Resumo O objetivo do presente estudo foi avaliar a taxa de crescimento de Balantidium coli em três meios de cultura xênicos. Entre 2013 e 2015 foram estudados 10 isolados de B. coli obtidos de Cynomolgus macaques e 30 isolados de suínos. Um inóculo contendo 500 trofozoítos foi transferido para tubos contendo os meios LES, TYSGM-9 e Pavlova. Os cultivos foram avaliados com tempos de incubação de 24, 48, 72 e 96 horas. Na maioria das cepas analisadas não foi observado diferença significativa na taxa de crescimento comparando TYSGM-9 e Pavlova (Wilcoxon p>0,016). Em Pavlova, os trofozoítos apresentaram máximo de crescimento a 72 h com diferença significativa quando se comparou com os tempos de 24 h e 96 h (Wilcoxon <0,008). Em LES observou-se trofozoítos viáveis até 24 horas com diferença significativa (Friedman p<0,05 e Wilcoxon p<0,016), na quantidade de células parasitárias, quando comparado com Pavlova e TYSGM-9. Dessa forma, o meio LES mostrou-se ser menos adequado do que os outros, para a manutenção de B. coli. Apesar do resultado satisfatório em TYSGM-9, Pavlova foi considerado ideal para manutenção do protozoário, por garantir a viabilidade do parasito com subcultivos a cada três dias, além de apresentar menor custo.


Subject(s)
Animals , Balantidium/growth & development , Culture Media , Parasitology/methods , Swine/parasitology , Balantidium/isolation & purification , Macaca/parasitology
7.
Rev. bras. parasitol. vet ; 25(3): 286-292, July-Sept. 2016. tab, graf
Article in English | LILACS | ID: lil-795078

ABSTRACT

Abstract Balantidium coli is a protozoon that can cause dysentery in humans, pigs and nonhuman primates, with zoonotic potential. In the literature, there is still little information on the effectiveness of different laboratory techniques for diagnosing this disease. This study compared and evaluated the performance of the Lutz, modified Ritchie, Faust, modified Sheather and direct examination techniques for detecting cysts of this protozoon. Between 2012 and 2014, 1905 fecal samples were collected from captive animals in the state of Rio de Janeiro. Of these, 790 were obtained from the rectum of pigs and 1115 from enclosures occupied by nonhuman primates. B. coli cysts were most evident through direct examination (22.4% of the samples) and the Lutz technique (21%). Fair agreement (Kappa = 0.41; p < 0.05) was observed only between direct examination and Lutz. The flotation techniques (Faust and modified Sheather) did not show good recovery of cysts. A statistically significant difference (p < 0.05) in the frequency of cysts between pigs and nonhuman primates could only be observed through direct examination and the Lutz technique. The most efficient method for diagnosing this parasitosis was seen to an association between direct examination and the spontaneous sedimentation technique.


Resumo Balantidium coli é um protozoário que pode determinar disenteria em humanos, suínos e primatas não humanos apresentando potencial zoonótico. Na literatura ainda são escassas as informações sobre a eficiência das diferentes técnicas laboratoriais para o diagnóstico dessa parasitose. Este estudo comparou e avaliou o desempenho das técnicas de Lutz, Ritchie modificada, Faust, Sheather modificada e do exame direto para a detecção de cistos desse protozoário. Entre 2012 e 2014, foram coletadas 1905 amostras fecais de animais cativos no Estado do Rio de Janeiro. Dessas, 790 foram obtidas da ampola retal de suínos e 1115 dos recintos de primatas não humanos. Cistos de B. coli foram 22,4 % mais evidenciados pelo exame direto; e pela técnica de Lutz, 21% das amostras. Concordância regular (Kappa = 0,41; p < 0,05) foi observada somente entre exame direto e Lutz. As técnicas de flutuação, Faust et al. e Sheather modificada não apresentaram boa recuperação dos cistos. Diferença estatística significativa (p < 0,05) na frequência de cistos entre suínos e primatas não humanos pode ser observada somente no exame direto e na técnica de Lutz. A metodologia mais eficiente para diagnóstico dessa parasitose foi observada pela associação do exame direto e da técnica de sedimentação espontânea.


Subject(s)
Animals , Primates/parasitology , Balantidiasis/veterinary , Balantidium/isolation & purification , Feces/parasitology , Parasitology/methods , Swine/parasitology , Balantidiasis/diagnosis , Clinical Laboratory Techniques/veterinary , Cysts/parasitology , Cysts/veterinary
8.
Pesqui. vet. bras ; 35(5): 466-469, May 2015. ilus
Article in English | LILACS | ID: lil-759380

ABSTRACT

Trematodes belonging to the family Eucotylidae, including Tanaisia (Paratanaisia)bragai Santos, 1934are parasites of the kidney and ureter that affect several species of domestic and wild birds. Tanaisia bragai is considered a low pathogenic parasite, but high worm burdens may determine clinical complications, including signs of apathy, weight loss, diarrhea and death...


Os trematódeos da família Eucotylidae, incluindo Tanaisia (Paratanaisia)bragai Santos, 1934, são parasitos de rins e ureteres de várias espécies de aves domésticas e silvestres. Tanaisia bragai é considerada uma espécie pouco patogênica, mas que pode determinar complicações clínicas como apatia, perda de peso, diarreia e morte, quando em cargas parasitárias elevadas...


Subject(s)
Animals , Poultry Diseases/diagnosis , Poultry Diseases/parasitology , Trematoda/parasitology , Parasitology/methods , Parasite Load/veterinary , Parasite Egg Count/methods , Parasite Egg Count/veterinary , Parasites/parasitology
9.
Article in English | WPRIM | ID: wpr-130562

ABSTRACT

Wuchereria bancrofti, Dirofilaria immitis, and Dirofilaria repens are filarial nematodes transmitted by mosquitoes belonging to Culex, Aedes, and Anopheles genera. Screening by vector dissection is a tiresome technique. We aimed to screen filarial parasites in their vectors by single and multiplex PCR and evaluate the usefulness of multiplex PCR as a rapid xenomonitoring and simultaneous differentiation tool, in area where 3 filarial parasites are coexisting. Female mosquitoes were collected from 7 localities in Assiut Governorate, were microscopically identified and divided into pools according to their species and collection site. Detection of W. bancrofti, D. immitis, and D. repens using single PCR was reached followed by multiplex PCR. Usefulness of multiplex PCR was evaluated by testing mosquito pools to know which genera and species are used by filarial parasites as a vector. An overall estimated rate of infection (ERI) in mosquitoes was 0.6%; the highest was Culex spp. (0.47%). W. bancrofti, D. immitis, and D. repens could be simultaneously and differentially detected in infected vectors by using multiplex PCR. Out of 100 mosquito pools, 8 were positive for W. bancrofti (ERI of 0.33%) and 3 pools each were positive for D. immitis and D. repens (ERI 0.12%). The technique showed 100% sensitivity and 98% specificity. El-Nikhila, El-Matiaa villages, and Sahel Seleem district in Assiut Governorate, Egypt are still endemic foci for filarial parasites. Multiplex PCR offers a reliable procedure for molecular xenomonitoring of filariasis within their respective vectors in endemic areas. Therefore, it is recommended for evaluation of mosquito infection after lymphatic filariasis eradication programs.


Subject(s)
Aedes/parasitology , Animals , Anopheles/parasitology , Culex/parasitology , Dirofilaria immitis/genetics , Dirofilaria repens/genetics , Egypt , Entomology/methods , Female , Multiplex Polymerase Chain Reaction/methods , Parasitology/methods , Sensitivity and Specificity , Wuchereria bancrofti/genetics
10.
Article in English | WPRIM | ID: wpr-130555

ABSTRACT

Wuchereria bancrofti, Dirofilaria immitis, and Dirofilaria repens are filarial nematodes transmitted by mosquitoes belonging to Culex, Aedes, and Anopheles genera. Screening by vector dissection is a tiresome technique. We aimed to screen filarial parasites in their vectors by single and multiplex PCR and evaluate the usefulness of multiplex PCR as a rapid xenomonitoring and simultaneous differentiation tool, in area where 3 filarial parasites are coexisting. Female mosquitoes were collected from 7 localities in Assiut Governorate, were microscopically identified and divided into pools according to their species and collection site. Detection of W. bancrofti, D. immitis, and D. repens using single PCR was reached followed by multiplex PCR. Usefulness of multiplex PCR was evaluated by testing mosquito pools to know which genera and species are used by filarial parasites as a vector. An overall estimated rate of infection (ERI) in mosquitoes was 0.6%; the highest was Culex spp. (0.47%). W. bancrofti, D. immitis, and D. repens could be simultaneously and differentially detected in infected vectors by using multiplex PCR. Out of 100 mosquito pools, 8 were positive for W. bancrofti (ERI of 0.33%) and 3 pools each were positive for D. immitis and D. repens (ERI 0.12%). The technique showed 100% sensitivity and 98% specificity. El-Nikhila, El-Matiaa villages, and Sahel Seleem district in Assiut Governorate, Egypt are still endemic foci for filarial parasites. Multiplex PCR offers a reliable procedure for molecular xenomonitoring of filariasis within their respective vectors in endemic areas. Therefore, it is recommended for evaluation of mosquito infection after lymphatic filariasis eradication programs.


Subject(s)
Aedes/parasitology , Animals , Anopheles/parasitology , Culex/parasitology , Dirofilaria immitis/genetics , Dirofilaria repens/genetics , Egypt , Entomology/methods , Female , Multiplex Polymerase Chain Reaction/methods , Parasitology/methods , Sensitivity and Specificity , Wuchereria bancrofti/genetics
11.
Rev. cuba. hig. epidemiol ; 52(2): 196-209, Mayo.-ago. 2014.
Article in Spanish | LILACS | ID: lil-743997

ABSTRACT

El agua contaminada puede transmitir infinidad de patógenos con comportamientos y resistencias diversas. Dentro de los patógenos a determinar los parásitos son de especial relevancia pues se destacan por su alta resistencia a los diversos factores ambientales además se encuentran relacionados con altos índices de morbilidad y mortalidad en los países en desarrollo, especialmente en la población infantil. El objetivo de este trabajo es destacar la importancia de la vigilancia sanitaria de los parásitos en la calidad del agua según su uso y en su relación con el ambiente. Los huevos de helmintos son el principal riesgo a la salud debido al uso seguro del agua residual o lodos en la agricultura. Los quistes de los protozoarios como Giardia y Cryptosporidium, son difíciles de eliminar del agua de consumo sin tratar, debido a su pequeño tamaño y resistencia a oxidantes usados comúnmente como el cloro. Aunque no se recomienda su monitoreo de rutina en el agua, sí es necesario realizar investigaciones para detectar su presencia y establecer normativas propias adecuadas a nuestras condiciones(AU)


Polluted water can transmit lots of pathogens with various behaviors and resistances. Among the pathogens, the parasites are particularly important since they stand out for their high resistance to various environmental factors in addition to being associated to high morbidity and mortality rates in the developing countries, particularly children. The objective of this paper was to highlight the importance of health surveillance of parasites in water quality according to use and its relationship with the environment. The helminth eggs are the main health risk due to the safe use of wastewater or sludge in agriculture. Protozoan cysts like Cryptosporidium and Giardia, are difficult to remove from untreated drinking water due to its small size and resistance to commonly used oxidants such as chlorine. Although the routine monitoring of these cysts in water is not recommended, it is necessary to conduct research to detect its presence and to establish suitable guidelines according to our conditions(AU)


Subject(s)
Humans , Water Quality/standards , Health Surveillance/standards , Water Consumption (Environmental Health) , Parasitology/methods , Water Purification , Intestinal Diseases, Parasitic/prevention & control , Water Microbiology , Parasitology/prevention & control , Environmental Health Surveillance
12.
Rev. peru. med. exp. salud publica ; 31(2): 222-227, abr.-jun. 2014. ilus, tab
Article in Spanish | LILACS, LIPECS | ID: lil-719497

ABSTRACT

Objetivos. Comparar dos protocolos de extracción de ADN de Trypanosoma cruzi para su uso en la amplificación de ADN de minicírculos de kinetoplasto (ADNk) mediante la técnica de Reacción en Cadena de Polimerasa (PCR). Materiales y métodos. Se cultivaron epimastigotas de T. cruzi en condiciones exénicas obteniéndose masas entre 1,5 hasta 100 x 10(6) parásitos. A partir de estas se procedió a la extracción de ADN mediante dos protocolos: extracción con solventes orgánicos (fenol/cloroformo), y empleo de resina (Chelex®100), a partir de los diferentes sedimentos parasitarios. La concentración y pureza del ADN se determinó por espectrofotometría y la integridad se evaluó mediante electroforesis en geles de agarosa. Se realizó el análisis de varianza y comparaciones de medias mediante la prueba de Tukey, utilizando el software Statistix 8.0. Resultados. Se realizaron diez extracciones de ADN de cada una de las diferentes cantidades de parásitos sedimentados. En la extracción de ADN con la resina Chelex®100 se obtuvo mayor rendimiento, pero menor pureza e integridad respecto a la extracción con solventes orgánicos. Sin embargo, permitió la amplificación del producto de 330 pb de ADNk de T. cruzi. Conclusiones. Aun cuando la técnica de Chelex®100 proporcionó menor pureza e integridad del ADN, permitió la amplificación con éxito de ADNk por PCR, evitando el uso de técnicas laboriosas y solventes orgánicos tóxicos.


Objectives. To compare two extraction protocols of Trypanosoma cruzi DNA for use in DNA amplification of kinetoplast minicircles (kDNA) through the technique of Polymerase Chain Reaction (PCR). Materials and methods. Epimastigotes of T. cruzi were cultured in axenic conditions and masses from 1.5 to 100 x 106 parasites were obtained. DNA extraction was performed using two protocols: extraction with organic solvents (phenol/chloroform), and with resin (Chelex®100), from different parasitic sediments. Concentration and purity of DNA was determined by spectrophotometry, and integrity was assessed by agarose gel electrophoresis. Analysis of variance and comparisons of means were performed through Tukey’s test, using the Statistix 8.0 software. Results. Ten DNA extractions were done of each one of the different amounts of parasitic sediments. In the DNA extraction with Chelex®100 resin, a higher performance was obtained but a lower purity and integrity compared to the extraction with organic solvents. However, it allowed a product amplification of 330 bp of T. cruzi kDNA. Conclusions. Although the technique of Chelex®100 provided less purity and integrity of DNA, it allowed a successful amplification of kDNA by PCR, avoiding the use of laborious techniques and toxic organic solvents.


Subject(s)
Axenic Culture , DNA, Kinetoplast/isolation & purification , Polymerase Chain Reaction/methods , Trypanosoma cruzi/genetics , Parasitology/methods
13.
Rev. bras. parasitol. vet ; 23(2): 179-186, 06/2014. tab, graf
Article in English | LILACS | ID: lil-714794

ABSTRACT

The aim of the present study was to evaluate the serological methods using ELISA with recombinant-rK39 (ELISA-rK-39) and soluble extract-SE (ELISA-SE) antigens, the indirect fluorescence antibody test (IFAT) in comparison to an immunochromatography rapid diagnostic test (RDT-rK39) and with a direct parasitological exam (PA) for Canine Visceral Leishmaniasis (CVL) diagnosis. The results showed that 89% (60/67) of the dogs were positive for at least one serological diagnostic test. ELISA-SE was the test that detected anti-Leishmania antibodies in the serum of the highest number of dogs (71.6%) followed by ELISA-rK39 (65.7%), IFAT (65.7%) and RDT-rK39 (55.2%). PA detected the lowest numbers (40.3%) of positive dogs. In relation to the total of examined dogs, the Kappa indexes (p ≤ 0.05) showed a good agreement between ELISA-SE and IFAT (88.1%; k = 0.7237), and it was also observed in the comparison of RDT-rK39 with ELISA-SE (83.6%, k= 0.6561), IFAT (83.5%, k= 0.6605) and PA (85.0%, k= 0.7074). A bad agreement was detected in any association of ELISA-rk39 with the other tests in either symptomatic or asymptomatic animals. ELISA as well as RDT using recombinant antigenic protein (rK39) were the methods that detected the lowest prevalence rates (33.3%) of CVL in asymptomatic dogs. In conclusion, only one test does not adequately identify dogs with CVL and it is necessary the association of two or more diagnostic tests. Because of the good agreement indexes of RDT-rK39 when evaluated with ELISA-SE, IFAT and PA it was suggested as a complementary method to be used in association with either ELISA-SE or IFAT, particularly in the symptomatic dogs. Furthermore, new studies are recommended in order to improve the sensitivity of tests mainly for asymptomatic dogs.


O objetivo do presente estudo foi avaliar os métodos sorológicos usando ELISA (Ensaio Imunoenzimático Indireto) com o antígeno recombinante rK39 (ELISA-rK39) e o antígeno extrato solúvel bruto (ELISA-ES) e a RIFI (Reação de Imunofluorescência Indireta) em comparação com o método imunocromatográfico rápido (RDT-rK39) e o parasitológico direto (PA), para o diagnóstico da Leishmaniose Visceral Canina (LVC) em cães de Ilha Solteira, São Paulo, Brasil. Os resultados mostraram que 89% (60/67) dos cães foram positivos por pelo menos um teste diagnóstico sorológico (RIFI, ELISA-ES, ELISA-rk39 ou RDT-rK39) e somente 40,3% (27/67) foram positivos pelo PA. O ELISA-ES foi o teste que detectou anticorpos anti-Leishmania em maior número de cães (71,6%) seguido por ELISA-rK39, RIFI (65,7%) e por RDT-rK39 (55,2%). No total de cães analisados (assintomáticos e sintomáticos), o índice Kappa de concordância (p ≤ 0,05) foi considerado de boa concordância entre ELISA-ES e IFAT (88,1%; k= 0,7237) e entre RDT-rK39 com ELISA-ES (83,6%, k= 0,6561), RIFI (83,5%, k= 0,6605) e PA (85,0%, k= 0,7074). O índice de concordância ruim foi observado em qualquer associação de ELISA-rk39 com todos os outros testes nos animais sintomáticos e nos assintomáticos. Tanto o ELISA como o RDT com proteínas recombinantes (rK39) detectaram a menor porcentagem de cães assintomáticos (33,3%) em relação aos outros testes sorológicos. Em conclusão, somente um método diagnóstico não foi suficiente para identificar todos os cães positivos com LVC, principalmente os assintomáticos e por isso foi necessário a associação de dois ou mais métodos. Em função da boa concordância do teste RDT-rK39 com ELISA-ES, RIFI e PA, o mesmo foi sugerido como um teste complementar ao ELISA-ES ou RIFI para o diagnóstico da LVC, principalmente dos cães sintomáticos. No entanto, novos estudos são recomendados para melhorar a sensibilidade dos testes principalmente para cães assintomáticos.


Subject(s)
Animals , Dogs , Dog Diseases/diagnosis , Dog Diseases/parasitology , Leishmaniasis, Visceral/veterinary , Enzyme-Linked Immunosorbent Assay , Fluorescent Antibody Technique, Indirect , Leishmaniasis, Visceral/diagnosis , Parasitology/methods
14.
Article in English | WPRIM | ID: wpr-190460

ABSTRACT

The oocyst wall is severed by means of mechanical injury or chemical agents. This study reports the percentage of in vitro sporocyst release following mechanical shaking in the presence of varying sizes of glass beads. Glass beads measured 0.5, 1, and 3 mm in diameter and were shaken with the oocysts for different times ranging from 5 sec to 5 min. Approximately 80% of sporocysts were released with 5 min of shaking in the presence of 3 mm glass beads, as well as 30 sec with 0.5 mm beads and 1 mm glass beads. The release of sporocysts of E. tenella was most efficient using 1 mm glass beads and treatment times of 30 sec to 1 min. Therefore, the use of 1 mm glass beads with 30 sec to 1 min of agitation is recommended in order to maximize sporocyst release and recovery and to improve the yield of viable sporozoites for use in biochemical, tissue culture, and immunological applications of coccidia.


Subject(s)
Eimeria tenella/physiology , Glass , Mechanical Phenomena , Microspheres , Oocysts/physiology , Parasitology/methods , Stress, Physiological , Time Factors
15.
Article in English | WPRIM | ID: wpr-210968

ABSTRACT

The mature domain of a cysteine protease of Spirometra erinacei plerocercoid larva (i.e., sparganum) was expressed in Escherichia coli, and its value as an antigen for the serodiagnosis of sparganosis was investigated. The recombinant protein (rSepCp-1) has the molecular weight of 23.4 kDa, and strongly reacted with the sparganum positive human or mice sera but not with negative sera by immunoblotting. ELISA with rSepCp-1 protein or sparganum crude antigen (SeC) was evaluated for the serodiagnosis of sparganosis using patient's sera. The sensitivity and specificity of ELISA using rSepCp-1 protein were 95.0% (19/20) and 99.1% (111/112), respectively. In contrast, the sensitivity and specificity of ELISA with SeC were 100% (20/20) and 96.4% (108/112), respectively. Moreover, in experimentally infected mice, the sensitivity and specificity of both ELISA assays were 100% for the detection of anti-sparganum IgG. It is suggested that the rSepCp-1 protein-based ELISA could provide a highly sensitive and specific assay for the diagnosis of sparganosis.


Subject(s)
Animals , Antigens, Helminth/chemistry , Cloning, Molecular , Cysteine Proteases/chemistry , Enzyme-Linked Immunosorbent Assay , Escherichia coli/genetics , Gene Expression , Humans , Mice , Molecular Weight , Parasitology/methods , Recombinant Proteins/chemistry , Sensitivity and Specificity , Serologic Tests/methods , Sparganosis/diagnosis , Spirometra/enzymology
16.
Rev. cuba. hig. epidemiol ; 51(3): 278-288, sep.-dic. 2013.
Article in Spanish | LILACS | ID: lil-699699

ABSTRACT

Introducción: el control de la calidad del diagnóstico de las parasitosis intestinales es un proceder de gran importancia en la práctica de la salud pública; sin embargo, no está tan difundido como en otras ramas del diagnóstico del laboratorio clínico y solo ha sido incorporado en los últimos años. Objetivos: evaluar la calidad del diagnóstico parasitológico en cuatro municipios de La Habana. Métodos: el estudio se efectuó en 15 policlínicos de los municipios La Lisa, Arroyo Naranjo, La Habana del Este y Cerro, de la provincia de La Habana, en el período comprendido entre marzo de 2011 a mayo de 2012. El universo de trabajo estuvo constituido por 747 muestras de heces analizadas en los laboratorios de dichos policlínicos. Para determinar la concordancia entre observadores se calculó el coeficiente Kappa para dos observadores y dos categorías. Resultados: solo en un policlínico hubo grado de acuerdo casi perfecto en el diagnóstico parasitario (coeficiente de concordancia kappa de 0.90, p < 0,05). En una cuarta parte de los policlínicos evaluados fue posible establecer la concordancia en el diagnóstico parasitario y de ellos solo en uno hubo calidad satisfactoria. Conclusiones: los principales errores en el diagnóstico son para Ascaris lumbricoides y Blastocystis spp. Estos resultados sugieren perfeccionar constantemente la capacitación del personal que realiza este tipo de exámenes


Introduction: quality control in the diagnosis of intestinal parasitoses is a very important public health procedure. However, it is not as well known as it is in other branches of clinical laboratory diagnosis, and has only been incorporated in recent years. Objectives: Evaluate the quality of parasitological diagnosis in four Havana municipalities. Methods: the study was conducted at 15 polyclinics from the municipalities of La Lisa, Arroyo Naranjo, Habana del Este and Cerro, in the province of Havana, from March 2011 to May 2012. The study universe was composed of 747 stool samples analyzed in laboratories of the aforementioned polyclinics. The kappa coefficient was estimated for two observers and two categories, in order to determine the degree of agreement between observers. Results: only in one polyclinic was there an almost perfect degree of agreement in parasitological diagnosis (a kappa concordance coefficient of 0.90, p < 0,05). Conclusions: it was possible to establish the concordance in parasitological diagnosis. Only in one case was quality satisfactory. The main diagnostic errors corresponded to Ascaris lumbricoides and Blastocystis spp. Based on these results, permanent improvement is recommended in the training of the personnel performing these tests


Subject(s)
Parasitology/methods , Parasitology/prevention & control , Clinical Diagnosis/education , Diagnostic Errors/prevention & control , Intestinal Diseases, Parasitic/diagnosis , Health Services Research , Quality Control
18.
Mem. Inst. Oswaldo Cruz ; 108(2): 155-159, abr. 2013. tab, graf
Article in English | LILACS | ID: lil-670407

ABSTRACT

Parasite findings in sambaquis (shell mounds) are scarce. Although the 121 shell mound samples were previously analysed in our laboratory, we only recently obtained the first positive results. In the sambaqui of Guapi, Rio de Janeiro, Brazil, paleoparasitological analysis was performed on sediment samples collected from various archaeological layers, including the superficial layer as a control. Eggs of Acanthocephala, Ascaridoidea and Heterakoidea were found in the archaeological layers. We applied various techniques and concluded that Lutz's spontaneous sedimentation technique is effective for concentrating parasite eggs in sambaqui soil for microscopic analysis.


Subject(s)
Animals , Archaeology , Geologic Sediments/parasitology , Helminths/isolation & purification , Paleopathology , Acanthocephala/isolation & purification , Ascaridoidea/isolation & purification , Brazil , Parasite Egg Count , Parasitology/methods
19.
Article in English | WPRIM | ID: wpr-197171

ABSTRACT

Opisthorchis viverrini and Clonorchis sinensis are parasites known to be carcinogenic and causative agents of cholangiocarcinoma in Asia. The standard method for diagnosis for those parasite infections is stool examination to detect parasite eggs. However, the method has low sensitivity, and eggs of O. viverrini and C. sinensis are difficult to distinguish from each other and from those of some other trematodes. Here, we report a multiplex real-time PCR coupled with high resolution melting (HRM) analysis for the differentiation of O. viverrini and C. sinensis eggs in fecal samples. Using 2 pairs of species-specific primers, DNA sequences from a portion of the mitochondrial NADH dehydrogenase subunit 2 (nad 2) gene, were amplified to generate 209 and 165 bp products for O. viverrini and C. sinensis, respectively. The distinct characteristics of HRM patterns were analyzed, and the melting temperatures peaked at 82.4+/-0.09degrees C and 85.9+/-0.08degrees C for O. viverrini and C. sinensis, respectively. This technique was able to detect as few as 1 egg of O. viverrini and 2 eggs of C. sinensis in a 150 mg fecal sample, which is equivalent to 7 and 14 eggs per gram of feces, respectively. The method is species-specific, rapid, simple, and does not require fluorescent probes or post-PCR processing for discrimination of eggs of the 2 species. It offers a new tool for differentiation and detection of Asian liver fluke infections in stool specimens.


Subject(s)
Animals , Asia , Clonorchis sinensis/classification , Feces/parasitology , Humans , Multiplex Polymerase Chain Reaction/methods , NADH Dehydrogenase/genetics , Opisthorchis/classification , Parasitology/methods , Real-Time Polymerase Chain Reaction/methods , Sensitivity and Specificity , Transition Temperature , Zygote
20.
Article in English | WPRIM | ID: wpr-197169

ABSTRACT

To increase public health awareness for prevention of opisthorchiasis caused by eating raw freshwater fish, the distribution and abundance of Opisthorchis viverrini metacercariae (OV MC) was investigated in freshwater fish obtained from 20 provinces in northeastern Thailand between April 2011 and February 2012. A cross-sectional survey was conducted on 12,890 fish consisting of 13 species randomly caught from 26 rivers, 10 dams, and 38 ponds/lakes. Fish, were collected in each of the rainy and winter seasons from each province. Fish were identified, counted, weighed, and digested using pepsin-HCl. Samples were examined for OV MC by a sedimentation method, and metacercariae were identified under a stereomicroscope. OV MC were found in 6 species of fish; i.e., Cyclocheilichthys armatus, Puntius orphoides, Hampala dispar, Henicorhynchus siamensis, Osteochilus hasselti, and Puntioplites proctozysron from localities in 13 provinces. Among the sites where OV MC-infected fish were found, 70.0% were dams, 23.7% were ponds/lakes, and 7.7% were rivers. The mean intensity of OV MC ranged from 0.01 to 6.5 cysts per fish (or 1.3-287.5 cysts per kg of fish). A high mean intensity of OV MC per fish (>3 cysts) was found in 5 provinces: Amnat Charoen (6.5 cysts), Nakhon Phanom (4.3), Mukdahan (4.1), Khon Kaen, (3.5) and Si Sa Ket (3.4). In conclusion, OV MC are prevalent in natural cyprinid fish, with the infection rate varying according to fish species and habitats.


Subject(s)
Animals , Cross-Sectional Studies , Cyprinidae/parasitology , Fish Diseases/epidemiology , Metacercariae/isolation & purification , Microscopy , Opisthorchiasis/epidemiology , Opisthorchis/isolation & purification , Parasitology/methods , Prevalence , Thailand
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