American Tegumentary Leishmaniasis: Effectiveness of an Immunohistochemical Protocol for the Detection of Leishmania in Skin

Abstract

BackgroundAmerican tegumentary leishmaniasis (ATL) is endemic in Latin America, where Brazil has over 27 thousand cases per year. The aim of the present study was to develop an immunohistochemical method (IHC) for ATL diagnosis. For this purpose, we used serum from a dog naturally infected with Leishmania (L) infantum (canine hyperimmune serum) as the primary antibody, followed by a detection system with a secondary biotinylated antibody.MethodologySkin samples were obtained from 73 patients in an endemic area of Caratinga, Minas Gerais (MG) State, Brazil all testing positive for ATL with the Montenegro skin test, microscopy, and PCR. Canine hyperimmune serum of a dog naturally infected with Leishmania (L.) infantum was employed as a primary antibody in an immunohistochemical diagnostic method using streptavidin-biotin peroxidase. To assess the specificity of this reaction, IHC assays employing two monoclonal antibodies were carried out. As the polymer-based technology is less time-consuming and labor intensive than the IHC labeled streptavidin-biotin peroxidase method, we compared the two methods for all samples.ResultsThe IHC method detected ATL in 67 of the 73 cases (91.8%). Immunolabeled parasites were primarily detected inside macrophages either in the superficial or the deep dermis. Detection was facilitated by the high contrast staining of amastigotes (dark brown) against the light blue background. A lower detection rate (71.2%) was observed with the both of the monoclonal Leishmania antibodies compared to the canine hyperimmune serum. This may have been due to a non-specific background staining observed in all histological samples rendering positive detection more difficult. The higher efficacy of the canine hyperimmune serum in the IHC method was confirmed by the method using streptavidin-biotin peroxidase as well as that with the polymer-based technology (biotin-avidin-free system).ConclusionsThe data are encouraging with regard to validating IHC as a standard alternative method for ATL diagnosis.

Press Imprint Smear: A Rapid, Simple, and Cheap Method for the Diagnosis of Cutaneous Leishmaniasis Caused by Leishmania (Viannia) braziliensis

“…[8][9][10] These results are in agreement with our results (44%). It is important to emphasize that all patients in our study are from areas where the only parasite ever identified in cutaneous leishmaniasis has been L. (V.) braziliensis, 11,12 which makes the sensitivity of our method very good for this species of leishmania.…”

Pustular dermatitis in dogs affected by leishmaniosis: 22 cases

“…The reaction highlighted the parasitic organisms and allowed detection of small numbers of amastigotes. Cross‐reactivity between human anti‐ Leishmania hyperimmune serum and fungal forms with immunohistochemistry has been reported in humans and may occur in dogs. However, special stains such as PAS or Grocott stain may help to differentiate these diseases, and all cases in this series were negative for fungal elements with PAS stain.…”

Proteins Selected in Leishmania (Viannia) braziliensis by an Immunoproteomic Approach with Potential Serodiagnosis Applications for Tegumentary Leishmaniasis

“…Parasitological diagnosis is definitive and consists of the microscopic examination of Giemsa-stained biopsy specimen smears, of histopathological examination or immunohistochemical approaches in lesion fragment triturates, and by parasite in vitro cultures, as well as PCR. However, these tests' sensitivity varies according to when the lesions occurred, and a low number of parasites can be present in the late lesions, hampering the sensitivity of the tests (8)(9)(10)(11)(12)(13). Additionally, these tests require invasive procedures of sample collection, which limit their use.…”