Evaluation of the performance of selected in-house and commercially available PCR and real-time PCR assays for the detection of Leishmania DNA in canine clinical samples.
Protozoa of the genus Leishmania are the causative agents of leishmaniosis. Although the polymerase chain reaction (PCR) has proved very effective in the detection of Leishmania DNA, a standardized method does not exist. In this study we attempt a comparative evaluation between one real time PCR (Method D), two in-house (Methods A and C), and a commercially available PCR assay (Method B) for the detection of Leishmania DNA, in order to support reliable diagnostic investigation of leishmaniosis. This evaluation was performed in regard to relative specificity and sensitivity, minimum detection limit (MDL), repeatability and reproducibility using cultured isolates and clinical samples. All the methods under study produced the expected result with the positive and negative controls. However with regard to clinical samples, Method C showed a statistically significant higher level of positivity. Relative sensitivity and specificity of Methods A, B and D in comparison to C was calculated respectively at 50.7%, 43%, 40%, and 90.8%, 93.4% and 89.5%. The MDL for Methods A-D was defined respectively at 30.7, 5, 3.7, and 5 promastigotes/ml. Repeatability and reproducibility were excellent in all cases with only the exception of Method A regarding reproducibility with a different brand of PCR reagents. The results that were recorded indicate that evaluation of PCR assays before their application for research and clinical diagnosis can provide useful evidence for their reliable application. Within this context the use of internal amplification controls and the confirmation of the specificity of the amplification product is recommended.
Agricultural University of Athens, Laboratory of Anatomy and Physiology, 11855 Athens, Greece.
Margarita
M
Andreadou
Emmanouil
E
Liandris
Ioannis N
IN
Kasampalidis
Styliani
S
Taka
Maria
M
Antoniou
Pantelis
P
Ntais
Anna
A
Vaiopoulou
Georgios
G
Theodoropoulos
Maria
M
Gazouli
John
J
Ikonomopoulos
D000818:Animals; D017931:DNA Primers / Q000592:standards; D016054:DNA, Protozoan / Q000302:isolation & purification*; D004283:Dog Diseases / Q000175:diagnosis / Q000469:parasitology*; D004285:Dogs; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D007891:Leishmania / Q000235:genetics* / Q000302:isolation & purification; D007896:Leishmaniasis / Q000175:diagnosis / Q000469:parasitology / Q000662:veterinary*; D057230:Limit of Detection; D016133:Polymerase Chain Reaction / Q000379:methods / Q000592:standards*; D060888:Real-Time Polymerase Chain Reaction / Q000592:standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D017931:DNA Primers; D016054:DNA, Protozoan