A Potential Diagnostic Methodology for Leprosy ... - ARCA â Fiocruz

Technical Note pubs.acs.org/ac

Skin Imprinting in Silica Plates: A Potential Diagnostic Methodology for Leprosy Using High-Resolution Mass Spectrometry Estela de Oliveira Lima,†,⊥ Cristiana Santos de Macedo,‡,§,⊥ Cibele Zanardi Esteves,† Diogo Noin de Oliveira,† Maria Cristina Vidal Pessolani,§ José Augusto da Costa Nery,∇ Euzenir Nunes Sarno,∥ and Rodrigo Ramos Catharino*,† †

INNOVARE Biomarkers Laboratory, School of Pharmaceutical Sciences, University of Campinas, Campinas, SP Brazil, 13083-877 Center for Technological Development in Health (CDTS), Oswaldo Cruz Foundation (FIOCRUZ), Rio de Janeiro, RJ Brazil, 21040-361 § Cellular Microbiology Laboratory, Oswaldo Cruz Institute, Oswaldo Cruz Foundation (FIOCRUZ), Rio de Janeiro, RJ Brazil, 21040-360 ∇ Souza Araújo Outpatient Clinic, Oswaldo Cruz Foundation (FIOCRUZ), Rio de Janeiro, RJ Brazil, 21040-360 ∥ Leprosy Laboratory, Oswaldo Cruz Institute, Oswaldo Cruz Foundation (FIOCRUZ), Rio de Janeiro, RJ Brazil, 21040-360 ‡

S Supporting Information *

ABSTRACT: Leprosy is a chronic infectious disease caused by Mycobacterium leprae, which primarily infects macrophages and Schwann cells, affecting skin and peripheral nerves. Clinically, the most common form of identification is through the observation of anesthetic lesions on skin; however, up to 30% of infected patients may not present this clinical manifestation. Currently, the gold standard diagnostic test for leprosy is based on skin lesion biopsy, which is invasive and presents low sensibility for suspect cases. Therefore, the development of a fast, sensible and noninvasive method that identifies infected patients would be helpful for assertive diagnosis. The aim of this work was to identify lipid markers in leprosy patients directly from skin imprints, using a mass spectrometric analytical strategy. For skin imprint samples, a 1 cm2 silica plate was gently pressed against the skin of patients or healthy volunteers. 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 different lipid markers, for example, mycobacterial mycolic acids, inflammatory and apoptotic molecules were identified as leprosy patients’ markers. Otherwise, phospholipids and gangliosides were pointed as healthy volunteers’ skin lipid markers, according to normal skin composition. Results indicate that silica plate skin imprinting associated with ESI-HRMS is a promising fast and sensible leprosy diagnostic method. With a prompt leprosy diagnosis, an early and effective treatment could be feasible and thus the chain of leprosy transmission could be abbreviated.

L

disabilities that worsen through time. Since ancient times, it has been seen as a terrifying disease, and this concept currently still persists in most countries.4−6 Patients present a wide range of clinical and histopathological manifestations, which may occur in two main forms: tuberculoid

eprosy, which is also known as Hansen’s disease, is a longlasting infection caused by Mycobacterium leprae that affects primarily epidermal macrophages and peripheral nerves. Despite the fact that the disease prevalence has fallen substantially in the past 50 years,1 it still remains endemic in the tropics, especially in underdeveloped or developing countries, where it is considered to be an important public health concern.2,3 Being a chronic disease, leprosy results in long-term physical and social effects, stigmatizing patients because of the severe skin conditions and © XXXX American Chemical Society

Received: January 8, 2015 Accepted: March 18, 2015

A

DOI: 10.1021/acs.analchem.5b00097 Anal. Chem. XXXX, XXX, XXX−XXX

Technical Note

Analytical Chemistry (TT) or lepromatous leprosy (LL).5 However, the majority of patients fall into a broad borderline category between these two polar forms; this is subdivided into borderline lepromatous (BL), midborderline (BB), and borderline tuberculoid (BT). These unspecific clinical manifestations significantly hamper the correct diagnosis.6 For that reason, in 1988, the World Health Organization (WHO) established a simplified classification method, where leprosy patients were divided into paucibacillary (PB) (up to five lesions, and/or only one nerve trunk involved) and multibacillary (MB) (more than five skin lesions and/or more than one nerve trunk involved). Along with the clinical evaluation, PB patients comprise TT and BT leprosy forms, wherein MB patients comprise LL, LB, and BB leprosy forms.7,8 A patient presenting skin lesions or symptoms suggestive of nerve damage, whose cardinal signs are absent or doubtful, should be called as a suspect case in the absence of any immediately obvious alternate diagnosis.9 This uncertainty in diagnosis impedes the correct treatment and allows the development of disabling complications.10 The clinical differential diagnosis of leprosy is extremely complex, because of the variety of clinical manifestations and several skin diseases that present similar lesions. In order to enhance the accuracy in diagnosis, there are different laboratorial tests available; the routine laboratory diagnosis of leprosy is, however, essentially based on histopathological examination of skin biopsy sample and on skin smear microscopy (bacilloscopy). The first is based on Hematoxylin-eosin and Fite-Faraco tissue staining (or its variations) and evaluates histopathological characteristics according to the criteria established by Ridley and Jopling, as well as allowing the investigation of acid-fast bacilli (AFB) on skin samples.5,11 The bacilloscopy exam can be used for the semiquantitative enumeration of acid-fast bacilli in infected skin and it is also useful in the followup of patients during and after treatment.6,7 Although the smear is positive in the multibacillary group (MB), bacilloscopy sensitivity is low in the suspect cases and paucibacillary group (PB), in which smear is often negative.12 Despite the availability of these methods, laboratorial diagnosis is time-consuming, exhibits low sensitivity, and is dependent on uncomfortable and invasive sampling. Because of uncertainties or delays in diagnosis, the multidrug therapy (MDT) recommended by WHO13 is delayed, mainly on suspect cases. This allows infected people to develop worse conditions and negatively influences in the eradication of leprosy as a public health problem. Presently, priority has been given in leprosy research to identify specific markers for M. leprae and develop sensitive and noninvasive material collection for laboratory tests, enabling accurate diagnosis, especially on suspect cases or those with very few symptoms.11 In this context, lipidomics has recently emerged as a crucial field of research providing an integrated view on the role of lipids involved in a wide range of cellular functions, including cellular and subcellular partitioning, maintenance of electrochemical gradients, cell signaling, energy storage, etc. Lipids are traditionally analyzed by gas chromatography (GC); however, identification and quantification of lipid in complex samples remains difficult, because of extensive extraction procedures and molecular lability. Furthermore, hydrolysis and derivatization reactions used in GC analyses make this technique more timeconsuming and may subject lipids to the formation of byproducts, thus interfering in the final results. To cope with these issues, mass spectrometry has been increasingly standing out as being an important tool for lipid analysis; the development of soft ionization techniques, such as matrix-assisted laser

desorption/ionization (MALDI), atmospheric pressure chemical ionization (APCI), and electrospray ionization (ESI) made sensible and fast lipid evaluation in complex samples possible. For that reason, lipidomics research associated with mass spectrometry can be a powerful combination to elucidate phenotypic changes, especially in disease conditions.14 Previous studies on leprosy metabolomics showed an enhanced fatty acid metabolism on serum and skin from leprosy patients before MDT, especially of polyunsaturated fatty acids (omega-3 and omega-6), which are precursors of eicosanoids, substances involved in the regulation of inflammation and immunity.15,16 After MDT, eicosanoids proceeded to normal levels, pointing to the importance of these lipid mediators on leprosy pathophysiology. Recent study on lipidomics of skin from leprosy patients showed differences on phospholipid and sphingolipid signal intensities and distribution before and after MDT, evidencing the importance of lipids on leprosy pathogenesis. The objective of this study is to develop a new, noninvasive skin sample collection method, coupled with high-resolution mass spectrometry analysis (HRMS) to help electing lipids that can be used as potential diagnosis biomarkers.

■

PATIENTS AND METHODS Patients. Control subjects and leprosy patients were recruited at Souza Araújo Outpatient Clinic from Oswaldo Cruz Institute−Rio de Janeiro, and all human procedures were approved by the CONEP−Research Ethical National Comitee/ Brazil (No. 30621514.4.0000.5248). A total of eight healthy control subjects and eight leprosy patients prior to MDT, all male, 18−60 years of age, were evaluated (see Table S-1 in the Supporting Information). Control subjects presented neither skin disorders nor systemic diseases such as diabetes and kidney dysfunctions, which could have influenced epidermal function. Patients were clinically diagnosed as multibacillary, which was later confirmed by laboratory diagnosis (Table S-1 in the Supporting Information). Sample Obtainment. For sample obtainment (skin imprints), a 1 cm2 silica plate (TLC Silica Gel 60 plate, Merck, Darmstadt, Germany) was gently pressed against the subjects’ back for 1 min. For leprosy patients, samples from two distinct regions were collected: leprosy lesion (L) and unaffected skin (S) imprints. In addition, no cleaning procedures were performed on skin or lesion sites before sample collection, as that could affect the lipid composition of the sample. High-Resolution Electrospray Mass Spectrometry (ESIHRMS) Analysis. Lipids were extracted from each skinimprinted silica plate by extracting them with 1000 μL of methanol directly in a plastic tube. Formic acid was then added to a concentration of 0.2% on each sample. For accurate identification of chemical markers, all samples were analyzed by high-resolution ESI-HRMS. For spectra acquisition, samples were directly infused in an ESI-LTQ-XL Orbitrap Discovery instrument (Thermo Scientific, Bremen, Germany) with nominal resolution of 30 000 (fwhm). All analyses were performed in the positive ion mode under the following conditions: sheath gas at 5 arbitrary units, flow rate of 10 μL min−1, spray voltage of 5 kV, and capillary temperature of 280 °C. Spectra were acquired over 60 s at the m/z range of 600−1500 for all samples. Statistical Analysis and Chemical Markers Identification. PCA was performed using Unscrambler v.9.7 (CAMO Software, Trondheim, Norway). The software has clustered B


Technical Note

Analytical Chemistry

Figure 1. Representative fingerprinting of the (A) control and (B) leprosy groups. The imaging represents a sum of all ions within mass range m/z 600− 1500 on positive ion mode. (C) Principal component analysis of skin samples. Ion chemical markers of each group separated by principal component analysis. [CT = control group; L = leprosy lesion group.]

samples, according to the relationship between m/z and intensity, with the results expressed as groups of samples with

the same characteristics when considering these parameters. The mass range was established between m/z 600−1500, and a signalC


Technical Note


Figure 2. Representative fingerprinting of (A) control and (B) leprosy groups. The imaging represents a sum of all ions within mass range m/z 600− 1500 on positive ion mode. (C) Principal component analysis of skin samples. Ion chemical markers of each group separated by principal component analysis. [CT = control group; S = unaffected leprosy skin group.]

to-noise threshold ratio of 3:1 was established after extracting spectral data in tables of mass times intensity. No normalization

was required, since all the samples were acquired within the same parameters. The LIPID MAPS online database (University of D


Technical Note

Analytical Chemistry Table 1. Lipid Chemical Markers Identified via HR-FTMS of Skin Imprints (Positive Ion Mode)a moleculeb [PA(O-16:0/17:1) + Na]+ [PA(18:0/22:1(11Z)) + H]+ [Ganglioside GM3(d18:1/22:1(13Z)) + H]+ [M(IP)2C(t18:0/16:0(2OH)) + Na]+ [Ganglioside GM2(d18:0/12:0) + Na]+ [Phthioceranic acid (C40) + Na]+ [GlcCer(d14:2(4E,6E)/16:0) + Na]+ [PS(18:4/15:1) + Na]+ [α-Semegma mycolic acid + H]+ [(13E-Tetranor-16-oxo-16-CoA-LTE4) + Na]+ d

experimental mass (m/z)

theoretical mass (m/z)

Control Skin 669.4839 669.4830 759.0909 759.5898 1235.7965 1235.7987 1240.5958 1240.5979 1323.7412 1323.7395 Leprosy Lesion and Unaffected Leprosy Skin 615.6048 615.6050 664.4755 664.4759 762.4340 762.4345 1124.1640 1124.1658 1181.2124 1181.2123

error (ppm)

IDc

−1.34 −1.45 1.77 1.69 −1.28

LMGP10020009 LMGP10010318 HMDB11930 LMSP03030102 HMDB11894

0.32 0.60 0.66 1.60 0.76

LMFA01020302 LMSP0501AA52 LMGP03010430 LMFA01160003 HMDB12576

a

Identification is based on the comparison between the exact and theoretical masses of each compound and lipid maps and human metabolome databases. bAbbreviations: L, leprosy lesion skin group; S, leprosy unaffected skin group; GlcCer, glucosylceramides; M(IP), Myo-inositolphosphate; PA, phosphatidic acid; PS, phophatidylserine; LTE, leukotriene; ppm, parts per million. cLM = lipid maps ID; HMDB = human metabolome database ID. dExclusive for Leprosy lesion skin group (L).

In order to study skin lipidomic profile and identify leprosy chemical markers, we have developed a new, comfortable and noninvasive procedure for sample obtainment based on a thinlayer chromatography (TLC) plate. TLC consists of a silica-gel matrix, forming an adsorbent layer upon an aluminum surface, and it is widely used as an analytical tool for chromatography studies because of its simplicity, relative low cost, and inertness. Therefore, the goal of our study was using the TLC plate as a “patch” for skin lipid adsorption, without the interference of any other components. Once the samples were collected and analyzed by mass spectrometry, the comparative analysis by the two PCA (Table 1) elected some chemical markers present in regular epidermis, with an explained variance of >85%. The elected molecules showed Na+ or H+ adducts, coherent to ESI ionization mode in which several types of ions may be formed, depending on the compound, sample, solvent, and ESI parameters.18 When the skin imprint was the studied sample, the sodium and hydrogen adducts were consistent with the analyzed specimen. One of the reasons is that skin homeostasis is mainly maintained by sweat glands and skin appendages that produce our primary source of cooling and hydration: sweat.19 The most concentrated solute in sweat is NaCl,20 which justifies the presence of Na+ and the formation of sodium adducts. In addition, sodium is one of the most common cations forming adducts in ESI ionization; therefore, the NaCl in sweat can contribute to the sodium adducts found in our samples. With regard to the hydrogen adducts, the skin surface (stratum corneum (SC)) presents an inherent acidic nature,21 with a pH range of 4−6.22 SC acidification is a key factor in permeability barrier homeostasis, antimicrobial defense and stratum corneum integrity, wherein there are potential contributors to SC acidity: the presence of urocanic acid,23 the generation of free fatty acids (FFA) from phospholipids (see the work of Fluhr et al.24), and a non-energydependent sodium-proton exchange (NHE1).25 All these factors contribute to maintain the ideal pH for skin enzymes activity, such as lipid hydrolases, β-glucocerebrosidases, and sphingomyelinases, which makes the ideal microenvironment to the generation of molecules, just like FFA and ceramides, required for epidermal permeability barrier homeostasis and to inhibit colonization by pathogenic microorganisms.26 Therefore, skin compounds can be proton and sodium donors, coherent with the resulting adducts, as demonstrated in Table 1. As a common rule,

California, San Diego, CA, www.lipidmaps.org) and HMDB version 3.6 (Human Metabolome database, www.hmdb.ca) were consulted to help guide the choice for potential lipid markers. Structural propositions were performed using high resolution as the main parameter. Mass accuracy was calculated and expressed in terms of ppm shifts.17

■

RESULTS Metabolic Fingerprinting. The leprosy lesion (L), unaffected skin from leprosy patient (S), and control skin (CT) groups were subjected to HR-FTMS analysis, as described in the “Patients and Methods” section. The obtained metabolic fingerprints presented clear distinction when comparing L with CT (Figure 1) and S with CT (Figure 2). In order to evaluate these differences statistically, PCA was performed, confirming this observation. Statistical Analysis. The multivariate data analysis PCA was performed, comparing the m/z and intensity of the L and CT or S and CT precursor ion groups. As shown in Figures 1C and 2C, all groups were clearly discriminated with accuracies of >85%, and chemical markers of each group were selected for the structural elucidation. Structural Elucidation. Experimental m/z ratios obtained by high-resolution ESI-MS were compared to their theoretical equivalent, taken from LIPID MAPS and HMDB. The maximum error was set at 2 ppm. Table 1 organizes this dataset as the identified lipid species in each sample group and the mass error for each signal selected from the PCA. It is possible to note that CT markers (phosphatidic acids, gangliosides, and myo-inositolphosphate) were the same for the two PCAs performed, whereas the selected markers for S and L were interestingly the same (phthioceranic acid, glucosylceramide, phosphatidylserine, αsmegma mycolic acid), except for Leukotriene E4, which appears only as an L biomarker.

■

DISCUSSION Traditional diagnostic methods for leprosy are based in clinical evaluation and invasive sample obtainment, but alone, none of these tests is considered enough to diagnose the disease, mainly in suspect cases.11 Lipidomics and HMRS were the tools of choice to determine chemical markers on the skin that distinguish healthy individuals from leprosy patients, aiming to make them useful for leprosy diagnosis. E


Technical Note


disease within a copious environment of exotic lipids of its own making.41 The other mycobacterial molecule identified, αsmegma mycolic acid, is the major mycolic acid in most mycobacterial species and was first isolated and described in Mycobacterium smegmatis in 1966.42 However, as shown in our findings, 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 characteristic constituents of the mycobacterial cell wall. Changes in the structure or composition of mycolic acids have been associated with modification of cell wall permeability and attenuation of pathogenic mycobacterial strains,43 which turns this molecule into a potential new target in the M. leprae discovery field. The finding of mycobacterial lipids outside leprosy lesions can be explained by the presence of M. leprae on normal-appearing skin of leprosy patients.44 For example, AFB, cellular infiltrate (histiocytes and lymphocytes), and degenerative changes were observed in erector pili muscle in all manifestations of leprosy, being more significative in multibacillary patients.44,45 Hence, the presence of AFB on normalappearing skin corroborates our findings and would have implications on leprosy persistence and dissemination.45 In addition to mycobacterial molecules, lipids related to inflammation and apoptosis were elected by PCA for S and L groups’ analysis, such as phosphatidylserine and glucosylceramide. Leukotriene E4, which is another chemical marker elected by PCA, was the only one elected for the L group. Leukotrienes (LTs) are lipid inflammatory mediators derived from the 5lipoxygenase pathway of arachidonic acid metabolism and, of the three cysteinyl leukotrienes (cys-LTs) (LTC4, LTD4, LTE4), LTE4 is the most stable and might be active for a prolonged time after its synthesis.46 Leukotriene E4 is generated in vivo by mast cells, eosinophils, basophils, and macrophages,47 and it is capable of inducing persistent eosinophilia and is potent enough to generate vasoactive cutaneous responses, mainly Th2 inflammatory responses, such as bronchial asthma.46,48 Leprosy patients evaluated in our study were clinically diagnosed with multibacillary leprosy forms (LL and BL), indicating that the predominant immune response is based on the synthesis of Th2 cytokines.49 Thus, the identification of LTE4 as a M. leprae lesion chemical marker is coherent with the inflammatory response developed at the injured site, and this can be considered to be a significant lipid marker for leprosy lesions. Probably, the PCA result for leprosy skin samples (S) did not show LTE4 as a chemical marker because this sample was collected from nonaffected leprosy skin, where there is no visible inflammatory response, such as edema, erythema, and inflammatory cell infiltration.50 It is possible that LTE4 concentration on uninjured skin sites was lower than lesion skin sites, what corroborates with PCA chemical markers’ election. Previous studies on metabolomics of skin from leprosy patients showed an increased eicosanoid metabolism before multidrug treatment (MDT), which proceeded to normal levels after MDT,16 supporting the observation of LTE4 on leprosy lesions. Another identified lipid chemical marker was phosphatidylserine, which is one of the major phospholipids present in the cell membrane. Normally, it is confined to the inner leaflet of the membrane bilayer but, during apoptosis, it is externalized to the outer surface of the cell membrane. Increasing numbers of bacterial pathogens have been identified as mediators of apoptosis in vitro,51 including mycobacteria.52 According to Santucci et al.,53 under mycobacterial infection, apoptotic cells present outer membrane-exposed phosphatidylserine. This

any lipid species can be found as either sodium or hydrogen adducts, among others. The observed presence of either [M + H]+ or [M + Na]+ species may be due to the characteristic of PCA to elect potential markers that explain the grouping of samples; for this reason, intrinsic to the discriminant analysis’ algorithm (with attributed variables as mass × relative intensity), the distribution of cationic species may be variable in the final result. Previous studies have shown that skin constitutes a large complex barrier between the body and the environment.27 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.28−31 Among these molecules, phospholipids like myo-inositol-phosphate and phosphatidic acids were noted as control group markers in our comparative analysis by PCA (Table 1). These results are in accordance with the work of Natarajan et al.,29 which reviewed healthy skin architecture and its lipids. The phospholipids identified in our research can be part of the keratinocyte differentiation process that will constitute the epidermal stratum corneum (SC) layer. For that, during this process, keratinocytes become full of membrane-coating granules, referred to as lamellar bodies (LB), which are lipid storages rich in glucosylceramides, sphingomyelin, and phospholipids. These lipids will be secreted in the extracellular space, followed by modification and cross-linking to produce the SC’s lipid matrix, the skin protective mantle.32 Thus, the skin’s barrier function is dependent on LB components, which can include the phospholipids found in our results. PCA analysis also elected as control group chemical markers, the gangliosides GM2 and GM3 (Table 1). Gangliosides are sialylated membrane glycosphingolipids ubiquitously found in tissues and body fluids,33 localized specifically in the outer leaflets of plasma membranes. These glycosphingolipids comprise 0.1% of the total epidermal lipids28 and are involved in cell−cell recognition, adhesion and signal transduction within specific cell surface microdomains, namely caveolae,34 lipid rafts,35 or glycosphingolipid-enriched microdomains.36 GM2 and GM3 are gangliosides present in keratinocytes and skin melanocytes, but previous studies have shown that there is a higher GM3 content compared to GM2.30,37 Several investigations have proposed that ganglioside GM3 is a regulator of EGFR function. In this way, membrane expression of GM3 is able to impact not only cell proliferation, but also other ligand-independent EGFR functions that require crosstalk, such as cell migration and invasiveness.38 On the other hand, GM2 function is not entirely clear in normal skin cells; therefore, investigations to understand the molecular mechanisms that lead to membrane-based events of ganglioside modulation will be critical for comprehending their function. As shown in several studies, infectious diseases may change the skin lipid profile, modifying the barrier function and skin homeostasis maintenance.29,39 The statistical analysis based on PCA elected specific mycobacterial molecules, such as the phthioceranic acid and α-smegma mycolic acid, present in both L and S groups, also with an explained variance of >85%. Phthioceranic acid or phthiocerol is part of the specific M. leprae antigen phenolic glycolipid (PGL-I), first reported in 1980.40 During the PGL-1 structure research, Hunter and Brennan defined the phthiocerol dimycocerosate of M. leprae as consisting of a mixture of phthiocerols homologues, and this discovery resulted in understanding the establishment of the F



■

phospholipid can be recognized by a phosphatidylserine phagocyte receptor, which will initiate the uptake of apoptotic cell signals. Supporting this data, phosphatidylserine was observed by MALDI imaging MS of skin biopsies from leprosy patients, before and after MDT, presenting weak or almost absent signals on control skins. Taken together, it can be speculated that phosphatidylserine has an important function on leprosy pathophysiology, which remains to be studied. The last leprosy chemical marker elected by PCA was a glucosylceramide (GlcCer), which is a well-established class and core structure for over 300 glycolipids.54 GlcCer biosynthesis is modulated by multiple endogenous and exogenous compounds, and this can be synthesized using different pathways: from palmitoylCoA and serine via sphinganine to ceramide, from the hydrolysis of glycolipids, or from the hydrolysis of sphingomyelin to ceramide. Glucosylceramide can be used as the precursor of more highly glycosylated glycosphingolipids, such as lactosylceramide (galactosylglucosylceramide), which have both mitogenic55 and apoptotic roles.56 These cerebrosides are also converted into gangliosides and can be metabolized back to ceramide by hydrolysis.57 All these characteristics elucidate many questions about why has GlcCer been selected as a marker for leprosy patients. This glucosylceramide is probably derived from a ceramide synthesized via sphingomyelin hydrolysis, typical in leprosy-affected peripheral nerves.58 Another hypothesis is that GlcCer can be involved in the lipid accumulation inside infected foamy macrophages,59 just like the lipid storage disorder invovled with Gaucher’s disease.60 There are many hypotheses to explain this selected chemical marker, but the exact role of glucosylceramide in leprosy infection remains to be elucidated. Based on the results presented above, our method was capable of differentiate healthy and leprosy skin and identify a specific M. leprae chemical marker at lepromatous 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 (Figures 1 and 2). This allows us to infer that this new diagnosis methodology can potentially be used to identify suspected cases of leprosy (subclinical infection), where there is no characteristic skin lesion or well-defined clinical signals. Since leprosy is considered to be a systemic disease and presents skin tropism,61 even subclinical infections may be detected through the skin imprinting method on silica plate. Thus, we believe that this new diagnostic tool exhibits great potential to become a method to detect specific mycobacterial markers, such as phthiocerol and/or mycolic acid, even in the uninjured skin of leprosy suspect patients, independent of the presence of lesions. The development of a noninvasive and very sensitive diagnostic method is extremely important for the patient and the clinician, who can prescribe treatment early and accurately, which is a key element to break the chain of leprosy transmission and the individual stigmatization in society. Therefore, we believe that an additional step should be taken, which is the validation of this new method, establishing specific leprosy markers, considering other skin diseases. Nonetheless, the results found in this contribution are evidence that there is hope to devise a better and faster leprosy diagnosis.

Technical Note

ASSOCIATED CONTENT

S Supporting Information *

A table with detailed clinical data of the patients included in this study is available as Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org/.

■

AUTHOR INFORMATION

Corresponding Author

*Tel.: +55 19 35219138. E-mail: [email protected]. Author Contributions ⊥

These authors contributed equally.

Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS INNOVARE Biomarkers Laboratory would like to thank National Council for scientific and technological development (CNPq; Process No. 140456/2011-2) and São Paulo Research Foundation (FAPESP, Process Nos. 11/50400-0, 14/00302-0, and 14/23010-4). C.S.M. and M.C.V.P. would like to thank National Institute of Science and Technology for Innovation on Neglected Diseases (INCT-IDN).

■

REFERENCES

(1) Merle, C. S.; Cunha, S. S.; Rodrigues, L. C. Expert Rev. Vaccines 2010, 9, 209−222. (2) Pannikar, V. Lepr Rev. 2009, 80, 353−354. (3) Weekly Epidemiol. Rec. 2013, 88, 365−379. (4) Heijnders, M. Int. J. Lepr. Other Mycobact. Dis. 2004, 72, 437−447. (5) Ridley, D. S.; Jopling, W. H. Int. J. Lepr. Other Mycobact. Dis. 1966, 34, 255−273. (6) Scollard, D.; Adams, L.; Gillis, T.; Krahenbuhl, J.; Truman, R.; Williams, D. Clin. Microbiol. Rev. 2006, 19, 338−381. (7) Jacobson, R. R.; Krahenbuhl, J. L. Lancet 1999, 353, 655−660. (8) World Health Organisation (WHO), Expert Committee on Leprosy: Seventh Report, 1998. (9) World Health Organisation (WHO). Available via the Internet at: http://www.who.int/lep/diagnosis/en/, 2014. (10) (a) Smith, W. Lepr. Rev. 1997, 68, 195−200. (b) Noordeen, S. Lepr. Rev. 1992, 63, 1. (c) Deepak, S. Indian J. Lepr. 2003, 75, 127−146. (11) Lastória, J. C.; Abreu, M. A. M. M. D. An. Bras. Dermatol. 2014, 89, 389−401. (12) (a) Brasil, M. d. S., Saúde, S. d. V. e., Ed.; Guia de Procedimentos Técnicos: Baciloscopia em Hansenı ́ase (A Technical Guide for Bacilloscopy in Leprosy). D.O.U. (Official Government Publication), Brasil, Ministério da Saúde e Secretaria de Vigilância em Saúde (Ministry of Health and Health Surveillance Agency), 2010; pp 54. (b) Shepard, C. C.; McRae, D. Int. J. Lepr. Other Mycobact. Dis. 1967, 36, 78−82. (13) World Health Organisation (WHO), Chemotherapy of Leprosy for Control Programs; Report of a WHO Study Group, October 12−16, 1981, Geneva, 1982. (14) Carrasco-Pancorbo, A.; Navas-Iglesias, N.; Cuadros-Rodriguez, L. TrAC Trends Anal. Chem. 2009, 28, 263−278. (15) Al-Mubarak, R.; Vander Heiden, J.; Broeckling, C. D.; Balagon, M.; Brennan, P. J.; Vissa, V. D. PLoS Neglected Trop. Dis. 2011, 5, e1303. (16) Amaral, J. J.; Antunes, L. C. M.; de Macedo, C. S.; Mattos, K. A.; Han, J.; Pan, J.; Candéa, A. L.; Maria das Graças, M.; Ribeiro-Alves, M.; Borchers, C. H. PLoS Neglected Trop. Dis. 2013, 7, e2381. (17) Erve, J. C.; DeMaio, W.; Talaat, R. E. Rapid Commun. Mass Spectrom. 2008, 22, 3015−3026. (18) Cech, N. B.; Enke, C. G. Mass Spectrom. Rev. 2001, 20, 362−387. (19) Lu, C.; Fuchs, E. Cold Spring Harbor Perspect. Med. 2014, 4. (20) Robinson, S.; Robinson, A. H. Physiol. Rev. 1954, 34, 202−220. Slegers, J. F. Dermatologica 1966, 132, 152−174. (21) Schade, H.; Marchionini, A. J. Mol. Med. 1928, 7, 12−14. G


Technical Note

Analytical Chemistry (22) (a) Dikstein, S.; Zlotogorski, A. Acta Derm.−Venereol. Suppl. 1993, 185, 18−20. (b) Rippke, F.; Schreiner, V.; Schwanitz, H.-J. Am. J. Clin. Dermatol. 2002, 3, 261−272. (c) Zlotogorski, A. Arch. Dermatol. Res. 1987, 279, 398−401. (23) Krien, P. M.; Kermici, M. J. Invest. Dermatol. 2000, 115, 414−420. (24) Fluhr, J. W.; Kao, J.; Jain, M.; Ahn, S. K.; Feingold, K. R.; Elias, P. M. J. Invest. Dermatol. 2001, 117, 44−51. (25) Behne, M. J.; Meyer, J. W.; Hanson, K. M.; Barry, N. P.; Murata, S.; Crumrine, D.; Clegg, R. W.; Gratton, E.; Holleran, W. M.; Elias, P. M. J. Biol. Chem. 2002, 277, 47399−47406. (26) (a) Elias, P. M.; Menon, G. K. Adv. Lipid Res. 1991, 24, 1−26. (b) Holleran, W. M.; Takagi, Y.; Menon, G. K.; Legler, G.; Feingold, K. R.; Elias, P. M. J. Clin. Invest. 1993, 91, 1656. (c) Schmuth, M.; Man, M.Q.; Weber, F.; Gao, W.; Feingold, K. R.; Fritsch, P.; Elias, P. M.; Holleran, W. M. J. Invest. Dermatol. 2000, 115, 459−466. (27) Elias, P. M.; Friend, D. S. J. Cell Biol. 1975, 65, 180−191. (28) Pappas, A. Derm.−Endocrinol. 2009, 1, 72−76. (29) Natarajan, V. T.; Ganju, P.; Ramkumar, A.; Grover, R.; Gokhale, R. S. Nat. Chem. Biol. 2014, 10, 542−551. (30) Paller, A. S.; Arnsmeier, S. L.; Robinson, J. K.; Bremer, E. G. J. Invest. Dermatol. 1992, 98, 226−232. (31) (a) Boddé, H. E.; Holman, B.; Spies, F.; Weerheim, A.; Kempenaar, J.; Mommaas, M.; Ponec, M. J. Invest. Dermatol. 1990, 95, 108−116. (b) Yardley, H. J.; Summerly, R. Pharmacol. Therapeut. 1981, 13, 357−383. (32) (a) Van Smeden, J.; Janssens, M.; Gooris, G.; Bouwstra, J. Biochim. Biophys. Acta, Mol. Cell Biol. Lipids 2014, 1841, 295−313. (b) Feingold, K. R.; Elias, P. M. Biochim. Biophys. Acta 2014, 3, 280−294. (33) Robert, K. Y.; Nakatani, Y.; Yanagisawa, M. J. Lipid Res. 2009, 50, S440−S445. (34) Anderson, R. G. Ann. Rev. Biochem. 1998, 67, 199−225. (35) Simons, K.; Toomre, D. Nat. Rev. Mol. Cell Biol. 2000, 1, 31−39. (36) Hakomori, S.-i.; Handa, K.; Iwabuchi, K.; Yamamura, S.; Prinetti, A. Glycobiology 1998, 8, xi−xviii. (37) Herlyn, M.; Thurin, J.; Balaban, G.; Bennicelli, J. L.; Herlyn, D.; Elder, D. E.; Bondi, E.; Guerry, D.; Nowell, P.; Clark, W. H.; et al. Cancer Res. 1985, 45, 5670−5676. (38) Bremer, E.; Schlessinger, J.; Hakomori, S.-I. J. Biol. Chem. 1986, 261, 2434−2440. Wang, X.-q.; Paller, A. S. Open Dermatol. J. 2009, 3, 159−162. (39) Pillai, S.; Oresajo, C.; Hayward, J. Int. J. Cosmetic Sci. 2005, 27, 17−34. (40) Brennan, P. J.; Barrow, W. W. Int. J. Lepr. Other Mycobact. Dis. 1980, 48, 382−387. (41) Spencer, J. S.; Brennan, P. J. Lepr. Rev. 2011, 82, 344. (42) Krembel, J.; Etemadi, A.-H. Tetrahedron 1966, 22, 1113−1119. Asselineau, J.; Lanéelle, G. Front. Biosci., Landmark Ed. 1998, 3, e164− e174. (43) Choi, K. H.; Kremer, L.; Besra, G. S.; Rock, C. O. J. Biol. Chem. 2000, 275, 28201−28207. (44) Job, C. K.; Jayakumar, J.; Aschhoff, M. Int. J. Lepr. Other Mycobact. Dis. 1999, 67 (2), 164−167. (45) Budhiraja, V.; Rastogi, R.; Khare, S.; Khare, A.; Krishna, A. Int. J. Infect. Dis. 2010, 14, e70−e72. (46) Lee, T. H.; Woszczek, G.; Farooque, S. P. J. Allergy Clin. Immunol. 2009, 124, 417−421. (47) (a) Ford-Hutchinson, A.; Rackman, A. Br. J. Dermatol. 1983, 109, 26−29. (b) Paruchuri, S.; Tashimo, H.; Feng, C.; Maekawa, A.; Xing, W.; Jiang, Y.; Kanaoka, Y.; Conley, P.; Boyce, J. A. J. Exp. Med. 2009, 206, 2543−2555. (c) Kanaoka, Y.; Boyce, J. A. J. Immunol. 2004, 173, 1503− 1510. (48) Laitinen, A.; Lindqvist, A.; Halme, M.; Altraja, A.; Laitinen, L. A. J. Allergy Clin. Immunol. 2005, 115, 259−265. (49) (a) World Health Organisation (WHO). Hospital practice (Office ed.), 1993; Vol. 28, pp 71−74, 77−80, 83, 84 (URL: www.lipidmaps. org; DOI: 10.1021/acs.analchem.5b00097; ISSN No. 0003-2700). (b) Mendonça, V. A.; Costa, R. D.; Melo, G. E. B. A. d.; Antunes, C. M.; Teixeira, A. L. An. Bras. Dermatol. 2008, 83, 343−350.

(50) Soter, N. A.; Lewis, R. A.; Corey, E.; Austen, K. F. J. Invest. Dermatol. 1983, 80, 115−119. (51) Gallucci, S.; Matzinger, P. Curr. Opin. Immunol. 2001, 13, 114− 119. (52) Matzinger, P. In Seminars in Immunology; Academic Press: London, 1998; pp 399−415. (53) Santucci, M.; Amicosante, M.; Cicconi, R.; Montesano, C.; Casarini, M.; Giosue, S.; Bisetti, A.; Colizzi, V.; Fraziano, M. J. Infect. Dis. 2000, 181, 1506−1509. (54) Basu, S.; Kaufman, B.; Roseman, S. J. Biol. Chem. 1968, 243, 5802−5804. (55) Ogura, K.; Sweeley, C. C. Exp. Cell Res. 1992, 199, 169−173. (56) Marks, D. L.; Wu, K.; Paul, P.; Kamisaka, Y.; Watanabe, R.; Pagano, R. E. J. Biol. Chem. 1999, 274, 451−456. (57) Radin, N. S. Eur. J. Biochem. 2001, 268, 193−204. (58) Scollard, D. M. Lepr. Rev. 2008, 79, 242−253. (59) Rubin, R.; Strayer, D. S.; Rubin, E. Rubin’s Pathology: Clinicopathologic Foundations of Medicine; Lippincott Williams & Wilkins: Philadelphia, 2008. (60) Nilsson, O.; Svennerholm, L. J. Neurochem. 1982, 39, 709−718. Miller, S. P.; Zirzow, G. C.; Doppelt, S. H.; Brady, R. O.; Barton, N. W. J. Lab. Clin. Med. 1996, 127, 353−358. (61) Ridley, D. S. Pathogenesis of Leprosy and Related Diseases; Butterworth Scientific, Ltd.: Cambridge University Press, 1988.

H


A Potential Diagnostic Methodology for Leprosy ... - ARCA â Fiocruz

Recommend Documents

A Potential Diagnostic Methodology for Leprosy ... - ARCA â Fiocruz