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Canine visceral leishmaniasis: a comparative study of real-time PCR, conventional PCR, and direct agglutination on sera for the detection of Leishmania infantum infection.
Vet Parasitol. 2013 Feb 18; 192(1-3):83-90.VP

Abstract

Canine visceral leishmaniasis (CVL) is endemic in northwestern Iran. This study aimed to compare real-time PCR, conventional PCR, and the direct agglutination test (DAT) for the diagnosis Leishmania infantum infection in 167 serum samples of domestic dog. Bone marrow was used for parasitological examination (smears and/or culture) in symptomatic visceral leishmaniasis, and serum was used for detection of L. infantum kinetoplast DNA (kDNA) by both conventional PCR and real-time PCR, while anti-L. infantum antibodies in sera were measured by DAT. The sera were collected from 37 symptomatic and 112 asymptomatic dogs during April to May 2011. Eighteen presumed negative samples were obtained from healthy dogs kept in non-endemic areas with no history of CVL and used as controls. All 18 samples were negative by DAT and Dipstick rK39. DAT confirmed previous exposure to L. infantum for all 149 serum samples collected from symptomatic and asymptomatic dogs in CVL endemic areas of Iran. Among the 37 symptomatic dogs, 20 (54%), 25 (67.6%), 36 (97.3%), and 37 (100%) showed L. infantum infection by parasitological methods, conventional PCR, real-time PCR, and DAT (≥ 1:80), respectively. Of 112 asymptomatic dogs, 79 (70.5%), 111 (99.1%), and 112 (100%) were shown to be positive by conventional PCR, and DAT (≥ 1:80), respectively. For ethical reasons, no asymptomatic or healthy control dogs were examined by parasitological methods. Three (16.7%) control dogs were positive by real-time PCR, but were negative by DAT, dipstick rK39, and conventional PCR methods. Parasitemia levels were measured by real-time PCR targeting kDNA using SYBR(®) green assay. This quantitative technique detected infection in 89.9% (150/167) of the domestic dogs that harbored L. infantum kDNA, ranging from 0.01 49 to 310.1 parasites/ml. The average was 16.60 parasites/ml. A good agreement (0.97) was found between real-time PCR and DAT at ≥ 1:80 titer, used as cut-off value by Kappa analysis. Thus, real-time PCR as a quantitative PCR assay on serum samples represents a valuable tool for initial diagnosis of CVL when whole blood is not available.

Authors+Show Affiliations

Department of Medical Parasitology & Mycology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Comparative Study
Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

23153824

Citation

Mohammadiha, A, et al. "Canine Visceral Leishmaniasis: a Comparative Study of Real-time PCR, Conventional PCR, and Direct Agglutination On Sera for the Detection of Leishmania Infantum Infection." Veterinary Parasitology, vol. 192, no. 1-3, 2013, pp. 83-90.
Mohammadiha A, Haghighi A, Mohebali M, et al. Canine visceral leishmaniasis: a comparative study of real-time PCR, conventional PCR, and direct agglutination on sera for the detection of Leishmania infantum infection. Vet Parasitol. 2013;192(1-3):83-90.
Mohammadiha, A., Haghighi, A., Mohebali, M., Mahdian, R., Abadi, A. R., Zarei, Z., Yeganeh, F., Kazemi, B., Taghipour, N., Akhoundi, B., Barati, M., & Mahmoudi, M. R. (2013). Canine visceral leishmaniasis: a comparative study of real-time PCR, conventional PCR, and direct agglutination on sera for the detection of Leishmania infantum infection. Veterinary Parasitology, 192(1-3), 83-90. https://doi.org/10.1016/j.vetpar.2012.10.013
Mohammadiha A, et al. Canine Visceral Leishmaniasis: a Comparative Study of Real-time PCR, Conventional PCR, and Direct Agglutination On Sera for the Detection of Leishmania Infantum Infection. Vet Parasitol. 2013 Feb 18;192(1-3):83-90. PubMed PMID: 23153824.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Canine visceral leishmaniasis: a comparative study of real-time PCR, conventional PCR, and direct agglutination on sera for the detection of Leishmania infantum infection. AU - Mohammadiha,A, AU - Haghighi,A, AU - Mohebali,M, AU - Mahdian,R, AU - Abadi,A R, AU - Zarei,Z, AU - Yeganeh,F, AU - Kazemi,B, AU - Taghipour,N, AU - Akhoundi,B, AU - Barati,M, AU - Mahmoudi,M R, Y1 - 2012/10/26/ PY - 2012/01/11/received PY - 2012/10/08/revised PY - 2012/10/11/accepted PY - 2012/11/17/entrez PY - 2012/11/17/pubmed PY - 2014/12/24/medline SP - 83 EP - 90 JF - Veterinary parasitology JO - Vet Parasitol VL - 192 IS - 1-3 N2 - Canine visceral leishmaniasis (CVL) is endemic in northwestern Iran. This study aimed to compare real-time PCR, conventional PCR, and the direct agglutination test (DAT) for the diagnosis Leishmania infantum infection in 167 serum samples of domestic dog. Bone marrow was used for parasitological examination (smears and/or culture) in symptomatic visceral leishmaniasis, and serum was used for detection of L. infantum kinetoplast DNA (kDNA) by both conventional PCR and real-time PCR, while anti-L. infantum antibodies in sera were measured by DAT. The sera were collected from 37 symptomatic and 112 asymptomatic dogs during April to May 2011. Eighteen presumed negative samples were obtained from healthy dogs kept in non-endemic areas with no history of CVL and used as controls. All 18 samples were negative by DAT and Dipstick rK39. DAT confirmed previous exposure to L. infantum for all 149 serum samples collected from symptomatic and asymptomatic dogs in CVL endemic areas of Iran. Among the 37 symptomatic dogs, 20 (54%), 25 (67.6%), 36 (97.3%), and 37 (100%) showed L. infantum infection by parasitological methods, conventional PCR, real-time PCR, and DAT (≥ 1:80), respectively. Of 112 asymptomatic dogs, 79 (70.5%), 111 (99.1%), and 112 (100%) were shown to be positive by conventional PCR, and DAT (≥ 1:80), respectively. For ethical reasons, no asymptomatic or healthy control dogs were examined by parasitological methods. Three (16.7%) control dogs were positive by real-time PCR, but were negative by DAT, dipstick rK39, and conventional PCR methods. Parasitemia levels were measured by real-time PCR targeting kDNA using SYBR(®) green assay. This quantitative technique detected infection in 89.9% (150/167) of the domestic dogs that harbored L. infantum kDNA, ranging from 0.01 49 to 310.1 parasites/ml. The average was 16.60 parasites/ml. A good agreement (0.97) was found between real-time PCR and DAT at ≥ 1:80 titer, used as cut-off value by Kappa analysis. Thus, real-time PCR as a quantitative PCR assay on serum samples represents a valuable tool for initial diagnosis of CVL when whole blood is not available. SN - 1873-2550 UR - https://www.unboundmedicine.com/medline/citation/23153824/Canine_visceral_leishmaniasis:_a_comparative_study_of_real_time_PCR_conventional_PCR_and_direct_agglutination_on_sera_for_the_detection_of_Leishmania_infantum_infection_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0304-4017(12)00560-2 DB - PRIME DP - Unbound Medicine ER -