Estela de Oliveira Lima(1), Cristiana S. de Macedo(2), Cibele Z. Esteves(1), Diogo N. de Oliveira(1), Maria C. V. Pessolani(2), José A.C. Nery(2), Euzenir N. Sarno(2), and Rodrigo R. Catharino(1)
(1) University of Campinas, (2) Oswaldo Cruz Foundation (FIOCRUZ)
Leprosy is an infectious disease caused by Mycobacterium leprae, primarily present at skin macrophages and Schwann cells. Presently, the available laboratorial diagnostic methods for Leprosy are invasive, expensive, and present low sensibility for the asymptomatic cases. Therefore, this work intended to develop a noninvasive, fast and sensible method for leprosy diagnosis, associated to high-resolution mass spectrometry. Our data analysis has elected two mycobacterial biomarkers at leprosy skin patients, absent at control samples. These results indicate that our new methodology can be candidate as a fast and sensible leprosy diagnostic method, even for patients without clinical skin manifestations.
Introduction: Leprosy is an infectious disease caused by the obligate intracellular microorganism Mycobacterium leprae, which primarily infects skin macrophages and peripheral Schwann cells. It is one of the most frequently reported diseases in history and its reports date back to 3000 years ago. Leprosy is a highly contagious disease in which patients, if left untreated, develop serious skin lesions, even mutilations that can make the leper incapable of the simplest tasks. All these factors contribute with social effects, stigmatizing patients due to the severe skin conditions and disabilities that worsen through time. The leprosy patients present a wide range of clinical and histopathological manifestations, which may occur in two main forms: paucibacillary (up to five lesions, and/or only one nerve trunk involved) and multibacillary (more than five skin lesions and/or more than one nerve trunk involved). However, clinically, up to 30% of infected patients may not present specific manifestations, wherein the differential diagnosis is extremely complex. Currently, the gold standard diagnostic test for leprosy is based on skin lesion biopsy and bacilloscopy, which are invasive and present low sensibility for suspect cases. Therefore, the development of a fast, sensible and noninvasive method that identifies infected patients, independently of a characteristic skin lesion, would prove helpful for assertive diagnosis.
Objective: The aim of this work was to develop a new noninvasive procedure for skin sample obtainment in leprosy patients and to identify lipid leprosy chemical markers, using a mass spectrometric analytical strategy.
Patients and methods: A total of eight healthy control subjects and eight leprosy patients prior to MDT, all male, 18−60 years of age, were evaluated in this study. For skin imprint samples obtainment, a 1 cm2 silica plate was gently pressed against patients’ or healthy volunteers’ skin. Multibacilary leprosy patients were divided into two different groups, according to the local of skin imprinting: leprosy lesion group (L) and unaffected skin group (S). Imprinted silica lipids were extracted and submitted to direct-infusion electrospray ionization high-resolution mass spectrometry (ESI-HRMS). All samples were differentiated using a lipidomics-based data workup employing multivariate data analysis, which helped electing markers from distinct lipid classes.
Results: For leprosy patients’ samples (L and S groups) we could identify two mycobacterial mycolic acids, phthioceranic acid and α-smegma mycolic acid. The first one is part of the specific M. leprae antigen phenolic glycolipid (PGL-I), what provides specificity for the new methodology developed here. The second one is the major mycolic acid in most mycobacterial species, but this is the first report of α-smegma mycolic acid in M. leprae. Further studies are needed to understand the role of this molecule in leprosy infection, since mycolic acids are important constituents of the mycobacterial cell wall. Furthermore, other molecules were elected as biomarkers for leprosy patients’ samples, like leukotriene E4 – LTE4 (inflammatory molecule), phosphatidylserine and glucosylceramide (apoptotic molecules), coherent with the disease characteristics. However, LTE4 was the only one elected exclusively for the L group, what is coherent with the inflammatory response developed at the injured site, and can be considered a significant lipid marker for leprosy lesions. Otherwise, phospholipids (myo-inositol-phosphate and phosphatidic acids) and gangliosides (GM2 and GM3) were pointed as healthy volunteers’ skin lipid markers, according to normal skin composition, where the outermost skin layer - the epidermis - has been extensively studied by many researchers, and its lipid composition is divided into phospholipids, neutral lipids, ceramides, cholesterol, gangliosides, and other glycosphingolipids.
Conclusions: The results obtained above indicate that silica plate skin imprinting associated with ESI-HRMS is a promising fast and sensible leprosy diagnostic method, because our method was capable of differentiate healthy and leprosy skin and identify a specific M. leprae chemical marker at multibacillary leprosy patients. Furthermore, the sample obtainment was not harmful for the patients, contrasting with current laboratory methods for leprosy diagnosis. One of the most interesting results in this research was the fact that leprosy lesion markers were similar to the leprosy uninjured skin markers. This allows us to suggest that this new diagnosis methodology can potentially be used for identifying suspected cases of leprosy (subclinical infection), where there is no characteristic skin lesion or well-defined clinical signals. With an early leprosy diagnosis, an early and effective treatment can be feasible and thus the chain of leprosy transmission can be abbreviated.