Molecular and Histopathology Diagnosis of Human Tegumentary Leishmaniasis Using Glycol Methacrylate-Embedded Samples: A New Approach for Referral Centers

Abstract

Tegumentary leishmaniasis (TL) is a neglected tropical disease that affects approximately one million new patients living in endemic areas with a lack of health service infrastructure. In many countries, including Brazil, public referral centers provide diagnostic support using high-sensitivity and high-specificity tests. In this context, PCR has been increasingly used for diagnosis and other downstream applications, employing different clinical specimens and types of storage. However, new protocols must be developed to enable the use of PCR in specific tissue processing methods, such as those involving glycol methacrylate (GMA). This study aimed to evaluate the applicability of GMA-embedded biopsies as clinical specimens for diagnosis of TL by PCR. Thirty-five 3-µm sections were incubated at 55°C for 12 h in Tris-EDTA-NaCl buffer containing NP-40 detergent and proteinase K for DNA extraction. Twenty-five patients with clinical suspicion of TL were included. PCR detected Leishmania the kinetoplast DNA (kDNA) in 19 out of 23 (82.6%) patients with cutaneous lesions. Additionally, 13 out of 15 (81.2%) patients with cutaneous lesions displaying a histopathological pattern compatible with TL were also kDNA PCR positive. Only two patients with mucosal lesions were evaluated, and both tested positive by PCR. Our results demonstrate that GMA-embedded samples are suitable for diagnosis of TL by Leishmania kDNA PCR, highlighting their potential for clinical applications.