Comparison between Colorimetric In Situ Hybridization, Histopathology, and Immunohistochemistry for the Diagnosis of New World Cutaneous Leishmaniasis in Human Skin Samples

Abstract

New world cutaneous leishmaniasis (NWCL) is an anthropozoonosis caused by different species of the protozoan Leishmania. Colorimetric in situ hybridization (CISH) was shown to satisfactorily detect amastigote forms of Leishmania spp. in animal tissues, yet it was not tested for the diagnosis of human NWCL. The aim of this study was to compare CISH, histopathology (HP), and immunohistochemistry (IHC) techniques to diagnose NWCL in human cutaneous lesions. The sample comprised fifty formalin-fixed, paraffin-embedded skin biopsy specimens from patients with NWCL caused by L. (V.) braziliensis. These specimens were analyzed by CISH, using a generic probe for Leishmania, IHC, and HP to assess the sensitivity of these methods by using a parasitological culture as a standard reference. Additional specimens from three patients diagnosed with cutaneous mycoses were also included to evaluate cross-reactions between CISH and IHC. The sensitivities of IHC, CISH, and HP for detecting amastigotes was 66%, 54%, and 50%, respectively. IHC, unlike CISH, cross-reacted with different species of fungi. Together, these results demonstrate that CISH may be a complementary assay for the detection of amastigote in the laboratorial diagnosis routine of human NWCL caused by L. (V.) braziliensis.

Keywords: Leishmania braziliensis; diagnosis; immunoperoxidase; in situ hybridization; tegumentary leishmaniasis.

Figure 1

Positive staining for amastigotes in samples fixed in 10% formalin and paraffin-embedded of cutaneous lesions from patients with new world cutaneous leishmaniasis caused by L. (V.) braziliensis. (A) Dark-blue-stained amastigote forms (arrows) inside macrophages. Colorimetric in situ hybridization, 100× objective. (B) Brown-stained amastigote forms (arrows) inside macrophages. Immunohistochemistry, 100× objective. (C) Amastigote forms inside the parasitophorous vacuoles of macrophages (arrows and inset) in the dermis amidst an inflammatory reaction consisting mainly of macrophages, with fewer lymphocytes and plasma cells and rare neutrophils. Histopathology, 100× objective.

Figure 2

Cross-reaction evaluation by immunohistochemistry (IHC) using polyclonal rabbit anti-Leishmania sp. serum and cross-hybridization by colorimetric in situ hybridization (CISH) using a generic probe for Leishmania spp. in samples fixed in 10% formalin and paraffin-embedded of cutaneous lesions from fungal-infected patients. (A) Infected skin by Candida sp. Brown stained hyphae are observed (arrows). IHC, 100× objective. (B) Infected skin by Candida sp. There was no staining of hyphae using the generic probe for Leishmania spp. (arrows). CISH, 100× objective. (C) Infected skin by Sporothrix sp. Brown-stained yeasts are observed (arrows). IHC, 100× objective. (D) Infected skin by Sporothrix sp. There was no staining of yeasts using the generic probe for Leishmania spp. (arrows). CISH, 100× objective. (E) Infected skin by Histoplasma sp. showing brown-stained yeasts. (arrow). IHC, 100× objective. (F) Infected skin by Histoplasma sp. There was no cross-staining using the generic probe for Leishmania spp. (arrows). CISH, 100× objective.