Evaluation of PCR for Diagnosis of American Cutaneous Leishmaniasis in an Area of Endemicity in Northeastern Brazil

Abstract

PCR-based approaches targeting kinetoplast DNA were evaluated for the diagnosis of American cutaneous leishmaniasis (ACL) in regions of endemicity in northeastern Brazil. A total of 119 cutaneous biopsy specimens from patients with ACL and nonleishmaniasis cutaneous lesions were studied. Two PCR-based systems were used; one was specific for the subgenus Viannia, and the other was specific for the genus Leishmania. The PCR specific for the subgenus Viannia had a sensitivity of 95.4%, whereas the genus-specific PCR detected the target DNA in 88.2% of the samples tested. The specificities of the assays, determined with samples from a group with nonleishmaniasis cutaneous lesions, was 100%. The results of the conventional tests indicate that the sensitivities of the PCR-based methods were significantly higher than those of smear examination, histological staining, and isolation by culture (P < 0.05). Antibodies specific for Leishmania braziliensis were detected by indirect immunofluorescence in 82.9% of the patients tested. Parasites were isolated from 40 of 86 patients (46.5%). Sixty-seven percent of dermal scrapings and 66.2% of stained tissue sections were positive by microscopy. Amplified products from the subgenus-specific PCR hybridized with the Leishmania panamensis minicircle, confirming infection consistent with L. braziliensis. The evidence available at present incriminates L. braziliensis as the only causative agent of ACL in the state of Pernambuco in Brazil.

Detection, Differentiation, and Quantitation of Pathogenic Leishmania Organisms by a Fluorescence Resonance Energy Transfer-Based Real-Time PCR Assay

“…Nevertheless, the fact that all six tissue biopsy specimens available tested positive in our assay, while only two tested positive by culture and three by histology, suggests that the sensitivity of PCR may be superior. This is consistent with other findings concerning PCR-based diagnosis of CL (31). The comparably low sensitivity of culture (23,31) is attributable to the fact that several samples were sent to our laboratory over long distances, probably leaving no parasites alive.…”

“…This is consistent with other findings concerning PCR-based diagnosis of CL (31). The comparably low sensitivity of culture (23,31) is attributable to the fact that several samples were sent to our laboratory over long distances, probably leaving no parasites alive. Consequently, it can be considered an additional benefit of PCR that DNA can be detected even from dead parasites or old samples.…”

Clinical, epidemiological and laboratory aspects of patients with American cutaneous leishmaniasis in the State of Pernambuco

“…The amplification consisted of 35 cycles: 94ºC (1 min), 65ºC (1 min) and 72ºC (1 min), preceded by an initial denaturation of 5 minutes at 94ºC. PCR products (10µl) were analyzed by means of electrophoresis on 1% agar gel with TAE buffer (Tris-acetate 40mM, EDTA 1mM), which was bleached with ethidium bromide in order to view the 750bp target (corresponding to the Viannia species) in a UV transilluminator 19 .…”

Clinical epidemiological profile of American tegumentary leishmaniasis at the Pinto Sugar Mill in Moreno Municipality, Greater Metropolitan Recife, Pernambuco State, Brazil

“…Laboratory diagnosis is based on demonstration of the parasite and immune assays. Such methods include direct identification of amastigote forms by light microscopy, in vitro isolation of the parasite, polymerase chain reaction (PCR), indirect immunofluorescence (IIF), ELISA, and the Montenegro skin test (MST) 5,6 .…”