Use of Parasitological Culture to Detect <i>Leishmania (Leishmania) chagasi</i> in Naturally Infected Dogs

Almeida, Arleana do Bom Parto Ferreira de; Sousa, Valéria Régia Franco; Sorte, Eveline da Cruz Boa; Figueiredo, Fabiano Borges; Paula, Daphine Ariadne Jesus de; Pimentel, Maria Fernanda Aranega; Dutra, Valéria; Madeira, Maria de Fátima

doi:10.1089/vbz.2011.0723

Cited by 26 publications

(21 citation statements)

References 26 publications

(26 reference statements)

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“…This lack of consensus is related to the clinical diversity of CVL and the animal's disease stage. [13][14][15][16][17][18] For example, bone marrow (BM) is a widely used specimen in many different diagnostic techniques and provides acceptable sensitivity values for both symptomatic and asymptomatic dogs. 14,17 Given the importance of direct examination, this study aimed to assess different concentration techniques, such as thick smear and cytocentrifugation, using BM for parasitological confirmation of CVL.…”

Section: Introductionmentioning

confidence: 99%

Thick Smear is a Good Substitute for the Thin Smear in Parasitological Confirmation of Canine Visceral Leishmaniasis

Mello

Figueiredo

Júnior

et al. 2016

The American Journal of Tropical Medicine and Hygiene

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Abstract. Although direct examination methods are important for diagnosing leishmaniasis, such methods are often neglected because of their low sensitivity relative to other techniques. Our study aimed to evaluate the performance of bone marrow (BM) thick smears and cytocentrifugation tests as alternatives to direct examination for diagnosing canine visceral leishmaniasis (CVL). Ninety-two dogs exhibiting leishmaniasis seroreactivity were evaluated. The animals were euthanized; and healthy skin, spleen, popliteal lymph node, and BM puncture samples were cultured. BM cultures were used as the reference standard. Of the 92 dogs studied, 85.9% exhibited positive cultures, and Leishmania infantum (synonym Leishmania chagasi) was confirmed in all positive culture cases. The sensitivity rates for cytocentrifugation as well as thin and thick smears were 47.1%, 52.8%, and 77%, respectively. However, no association between the dogs' clinical status and culture or direct examination results was found. To our knowledge, this was the first study to use thick smears and cytocentrifugation for diagnosing CVL. Our results indicate that BM thick smears have a good sensitivity and their use reduces the time required to read slides. Therefore, thick smears can provide a rapid and safe alternative to parasitological confirmation of seroreactive dogs.

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Section: Introductionmentioning

confidence: 99%

Thick Smear is a Good Substitute for the Thin Smear in Parasitological Confirmation of Canine Visceral Leishmaniasis

Mello

Figueiredo

Júnior

et al. 2016

The American Journal of Tropical Medicine and Hygiene

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“…Nonetheless, parasitological culture, PCR, and serological assays present disadvantages that prevent their use alone for the routine diagnosis of L. infantum infection in dogs. The parasitological culture is time consuming, taking from 5 to 30 days (on average, 15 days) to be completed, and there are only a small number of reference centers worldwide currently using MLEE (1,22). In addition, this method is susceptible to microbiologic contamination, which in many cases prevents its use in samples collected in the field, where proper storage and sterile conditions may be difficult to attain (1,10).…”

Section: Discussionmentioning

confidence: 99%

“…Each specimen obtained was divided into two samples. One of them was immersed in sterile saline with antimicrobials (22) and submitted for parasitological culture. The other fragment was fixed in 10% neutral buffered formalin and processed for routine paraffin embedding (23).…”

Section: Samplesmentioning

confidence: 99%

Sensitivity and Specificity of In Situ Hybridization for Diagnosis of Cutaneous Infection by Leishmania infantum in Dogs

et al. 2013

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An accurate diagnosis of infection by Leishmania infantum in dogs is fundamental for the control of zoonotic visceral leishmaniasis (VL). Histopathology (HP) and immunohistochemistry (IHC) are frequently used for the histological diagnosis of L. infantum in dogs but have shown limited accuracy. To improve the sensitivity and specificity of the histological diagnosis of VL, we evaluated automated in situ hybridization (ISH) using a generic probe for Leishmania and a specific probe for L. infantum in surgical skin biopsy specimens of dogs. The ISH results were compared with those of HP and IHC, using parasitological culture as the reference standard. Skin samples from 51 dogs with cutaneous L. infantum infection and 51 noninfected dogs were randomly selected from samples of dogs from various cities in Brazil where canine VL is endemic. These samples were processed for parasitological culture, HP, IHC, and ISH using both probes. The sensitivities of ISH using the specific probe, ISH using the generic probe, IHC, and HP were, respectively, 74.5%, 70.6%, 69.5%, and 57.6%. The specificity of both ISH probes tested was 100%, and there was no cross-hybridization of the generic and specific probes with selected pathogenic fungi and protozoa. The specific probe discriminated L. infantum from the other species of Leishmania that infect dogs in the New World. ISH is highly sensitive and specific for the diagnosis of L. infantum in histologic samples of skin from infected dogs and can be used on routine biopsy material to make a diagnosis of leishmaniasis.

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“…Fueron seleccionados animales procedentes del Departamento Central, destinados a eutanasia humanitaria a cargo del Programa Nacional ) fueron sometidas a lavado con buffer salino fosfato (PBS 1X) esterilizado, pH 7,2, con el fin de aislar promastigotes en medio de cultivo bifásico NNN suplementado con medio Schneider (Sigma, EEUU), gentamicina, estreptomicina 200 µg/mL y suero fetal bovino al 10% inactivado por calor (16). Los cultivos fueron mantenidos en incubadoras a 26 ºC.…”

Section: Materiales Y Métodosunclassified

Estandarización de la técnica de PCR-RFLP de la región ITS1, para la caracterización molecular de Leishmania (L) infantum, en muestras caninas

Chaves¹,

Portillo²,

Thomaz-Soccol

et al. 2016

Mem. Inst. Investig. Cienc. Salud

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Cómo referenciar este artículo/ How to reference this article:Céspedes Chaves E, Portillo N, Thomaz-Soccol V, Gonçalves AL, González Brítez N. Estandarización de la técnica de PCR-RFLP de la región ITS1, para la caracterización molecular de Leishmania (L.) infantum, en muestras caninas. Mem. Inst. Investig. Cienc. Salud. 2016;14(3):24-33 R E S U M E NLa leishmaniasis es una enfermedad producida por protozoarios parásitos del género Leishmania. Estos parásitos infectan a hospedadores mamíferos, entre los cuales los perros han sido implicados como reservorios del parásito. Este trabajo planteó estandarizar la técnica de la PCR-RFLP luego de la amplificación de la región ITS1 de Leishmania spp, como herramienta útil para la detección y caracterización molecular. Se utilizaron promastigotes de cultivo y muestras de biopsias procedentes de perros con leishmaniasis visceral previamente diagnosticados en el Centro Antirrábico Nacional. La región ITS1 del ADN genómico nuclear de Leishmania spp. fue amplificada utilizando los cebadores LITSR y L5,8S. La técnica ITS1 PCR-RFLP aplicada, permitió la detección de Leishmania (Leishmania) infantum en 10/10 aislados de parásitos mantenidos en medio NNN, en 10/18 muestras de bazo y 10/18 muestras de ganglio linfático poplíteo. Las condiciones óptimas de reacción fueron 0,2 mM de dNTPs, 0,1 pmol de cada cebador y 1U de Taq polimerasa. La sensibilidad de la PCR fue de 3 ng/µL de ADN en aislados de cultivo NNN y 60 ng/µL de ADN en muestras de biopsias, mientras que la especificidad fue de 100% para la detección de Leishmania sp. La enzima de restricción Hae III, determinó fragmentos de 184, 72 y 55 pb., que resultaron específicos para la especie Leishmania (L.) infantum. El marcador utilizado resultó confiable para la detección y caracterización de Leishmania sp. en perros procedentes de zonas endémicas, lo cual podría ser útil para verificar las especies de parásitos circulantes entre los perros.Palabras claves: Leishmania infantum, perros, ITS -1 (Espaciador transcripto interno 1). Standardization of PCR-RFLP technique of the ITS-1 region for Leishmania (L.) infantum molecular characterization in canine samples A B S T R A C TLeishmaniasis is a disease caused by protozoan parasites of the genus Leishmania. These parasites infect to mammalian hosts, including canines that have been implicated as reservoirs of the parasite. The aim of this research was to standardize the technique of PCR-RFLP after amplification of the ITS1 region of Leishmania (Leishmania) infantum, as a useful tool for detection and molecular characterization. Promastigotes from culture and biopsies from dogs with visceral Leishmaniasis previously diagnosed by the Centro Antirrábico Nacional. The ITS1 region of the genomic DNA of Leishmania sp. was amplified using LITSR and L5,8S primers. The technique ITS1 PCR-RFLP applied, allowed the detection of

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Use of Parasitological Culture to Detect Leishmania (Leishmania) chagasi in Naturally Infected Dogs

Cited by 26 publications

References 26 publications

Thick Smear is a Good Substitute for the Thin Smear in Parasitological Confirmation of Canine Visceral Leishmaniasis

Thick Smear is a Good Substitute for the Thin Smear in Parasitological Confirmation of Canine Visceral Leishmaniasis

Sensitivity and Specificity of In Situ Hybridization for Diagnosis of Cutaneous Infection by Leishmania infantum in Dogs

Estandarización de la técnica de PCR-RFLP de la región ITS1, para la caracterización molecular de Leishmania (L) infantum, en muestras caninas

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