Complementary exams in the diagnosis of American tegumentary leishmaniasis

Abstract

The diagnosis of American Tegumentary Leishmaniasis is a difficult but essential task when considering the high toxicity profile of the drugs available. Since the discovery of its etiologic agent, numerous diagnostic tests have been developed. None of the tests available today can be considered as the gold standard, since they do not add enough accuracy for the disease detection. Good epidemiological and clinical knowledge of the disease are fundamental precepts of the dermatology practice and precede the rational use of existing diagnostic tests. In this article we aim, through extensive literature review, to recall fundamental concepts of any diagnostic test. Subsequently, based on this information, we will weave important comments about the characteristics of existing diagnostic tests, including immunological tests such as Montenegro's skin test, serology and detection of parasites by direct examination, culture or histopathology. Finally we will discuss the new technologies and options for the diagnosis of Cutaneous Leishmaniasis. The molecular biology technique is considered a promising tool, promoting the rapid identification of the species involved. We also aim to educate dermatologists about a disease with high morbidity and assist in its difficult recognition.

FIGURE 1

Initial clinical lesion, with one - month evolution, in a case of localized cutaneous leishmaniasis. Patient evolved with ulceration and development of a typical ATL cutaneous lesion

FIGURE 2

ATL ulcer with elevated borders: evolution of the case after five months.

FIGURE 3

Case of cutaneous leishmaniasis with 10 years of evolution. An intense destruction of the nasal septum can be observed. This case represents a severe unusual clinical lesion, however it illustrates the potential severity of mucosal leishmaniasis.

FIGURE 4

Montenegro skin test: intradermal application of a solution containing antigenic preparation of promastigote forms of Leishmania. The result should be evaluated within 48 hours with a ballpoint pen, being positive if the papule formed is equal or greater than 5mm

FIGURE 5

Indirect search for antibodies. Positive result with fluorescence of the promastigote forms of Leishmania

FIGURE 6

Histopatholo gical exam showing several amastigote forms in a case of cutaneous leishmaniasis (Giemsa 400x)

FIGURE 7

Histopatholo gical exam showing intense pseudoepitheliomatous hyperplasia (H&E 10x)

FIGURE 8

Solid culture medium for Leishmania - Neal, Novy, Nicolle (NNN). The sample material aspirated from the border of the cutaneous ulcer can be inoculated in this medium

FIGURE 9

In vivo culture: C57BL/6 strain mouse, Mus musculus species, adult female infected subcutaneously on the right paw with 3.57 x 106 promastigotes of Leishmania (L.) amazonensis, in the metacyclic phase.

FIGURE 10

Collection technique of an imprint in paper filter of material from incisional biopsy. The collected material can be easily stored, sent for DNA extraction and polymerase chain reaction

FIGURE 11

Agarose gel print of PCR amplified material showing positivity for Leishmania spp. Sample collected from a lesion, in a case of mucosal leishmaniasis, by paper filter imprinting (6) and nasal swab (7). Demonstration of positivity on incisional biopsy sample, in a case of cutaneous leishmaniasis (9). Positive control (10). Negative control (11). 100 bp molecular marker (12). Exam performed at the Dermatology Service Laboratory, HC-FMRP-USP, Ribeirão Preto, SP, Brazil

FIGURE 12

10% acrylamide gel electrophoresis after enzymatic digestion of PCR amplified material - PCR-RFLP. Digestion by the enzyme HaeIII in species L. donovani (4); L. (V.) braziliensis (5); L (L.) amazonensis (6) kept in culture. Digestion by the enzyme BsrI in species L. donovani (12); L. (V.) braziliensis (13); L (L.) amazonensis (14) kept in culture. Depicts 50 bp and 100bp molecular markers respectively (7, 8). Exam performed at the Dermatology Service Laboratory, HC-FMRP-USP, Ribeirão Preto, SP, Brazil