Dermatoscopy in leprosy and its correlation with clinical spectrum and histopathology: a prospective observational study.

BACKGROUND: Leprosy, a chronic granulomatous infection has varied clinical presentations spanning across different spectrums. The scope of dermatoscopy is vast and has been studied for other granulomatous disorders like sarcoidosis. OBJECTIVES: The objective of this study was to describe the dermatoscopic features of the entire spectrum of leprosy and to correlate with clinical and histopathological findings. METHODS: This was a prospective observational study of treatment naïve leprosy patients over a period of 1 year. The study patients were categorized as per Ridley-Jopling classification based on clinical, slit skin smear and histopathological findings. Most representative lesions were photographed, evaluated by dermatoscopy and were biopsied. RESULTS: A total of 30 patients (21 males and 9 females) were recruited; 2 cases of tuberculoid leprosy, 12 cases of borderline tuberculoid (3 with type 1 reaction), 8 cases of borderline lepromatous, 6 cases of lepromatous leprosy (3 with type 2 reaction) and 2 cases of Histoid leprosy. The dermatoscopic featues consistently seen were yellowish orange areas and vascular structures like linear branching vessels and crown vessels correlating with the presence of dermal granulomas and dilated vessels. Broken pigment network, white chrysalis like areas were seen in addition. Tuberculoid spectrum also had absence of or diminished hair follicles and eccrine duct openings correlating with presence of peri-appendageal granuloma and appendageal destruction. Scaling and follicular plugs were other features in lesions of type 1 reaction. CONCLUSION: Yellowish-orange areas and vascular structures are the common dermatoscopic features of leprosy. Broken pigment network and paucity of appendageal structures are additional specific features.

Effect of unique Mycobacterium leprae phenolic glycolipid-I (PGL-I) on tumour necrosis factor production by human mononuclear cells.

Mycobacterium leprae cell wall-associated components are found in large amounts in the tissues of leprosy patients, particularly those at the lepromatous pole. Among these molecules, the phenolic glycolipid-I (PGL-I), unique to M. leprae, has been involved in the selective anergy observed in the lepromatous patients. Armadillo-derived M. leprae retains only a small proportion of the total PGL-I found in infected tissues. Therefore, the addition of PGL-I to M. leprae in vitro is important for a better understanding of M. leprae effects in vivo. We have studied the influence of PGL-I on TNF production by normal human peripheral blood mononuclear cells (PBMC) and by a human monocytic leukaemia cell line (THP-1) following stimulation with killed M. leprae. PGL-I alone did not induce TNF secretion by PBMC, but when associated with a sub-optimal dose of armadillo-derived M. leprae increased the release of this cytokine. In agreement with these results, M. leprae-exposed THP-1 cells did not secrete detectable levels of TNF unless PGL-I was simultaneously added to the culture. This increase in TNF production suggests that PGL-I plays a role in the induction of TNF during the natural infection. In addition, the modulatory effect of PGL-I on TNF release by THP-1 cells reinforces that monocytes are one of the possible targets of this molecule.

Mycolactone: a polyketide toxin from Mycobacterium ulcerans required for virulence.

Mycobacterium ulcerans is the causative agent of Buruli ulcer, a severe human skin disease that occurs primarily in Africa and Australia. Infection with M. ulcerans results in persistent severe necrosis without an acute inflammatory response. The presence of histopathological changes distant from the site of infection suggested that pathogenesis might be toxin mediated. A polyketide-derived macrolide designated mycolactone was isolated that causes cytopathicity and cell cycle arrest in cultured L929 murine fibroblasts. Intradermal inoculation of purified toxin into guinea pigs produced a lesion similar to that of Buruli ulcer in humans. This toxin may represent one of a family of virulence factors associated with pathology in mycobacterial diseases such as leprosy and tuberculosis.

Molecular interaction of CD1b with lipoglycan antigens.

The ability of human CD1b molecules to present nonpeptide antigens is suggested by the T cell recognition of microbial lipids and lipoglycans in the presence of CD1b-expressing antigen-presenting cells. We demonstrate the high-affinity interaction of CD1b molecules with the acyl side chains of known T cell antigens, lipoarabinomannan, phosphatidylinositol mannoside, and glucose monomycolate. Furthermore, CD1b-antigen binding was optimal at acidic pH, consistent with the known requirement for endosomal acidification in CD1b-restricted antigen presentation. The mechanism for CD1b-ligand interaction involves the partial unfolding of the alpha helices of CD1b at acidic pH, revealing a hydrophobic binding site that could accommodate lipid. These data provide direct evidence that the CD1b molecule has evolved unique biochemical properties that enable the binding of lipid-containing antigens from intracellular pathogens.

Mycobacterial lipoarabinomannan: an extraordinary lipoheteroglycan with profound physiological effects.

Detailed structural and functional studies over the last decade have led to current recognition of the mycobacterial lipoarabinomannan (LAM) as a phosphatidylinositol anchored lipoglycan with diverse biological activities. Fatty acylation has been demonstrated to be essential for LAM to maintain its functional integrity although the focus has largely been on the arabinan motifs and the terminal capping function. It has recently been shown that the mannose caps may be involved not only in attenuating host immune response, but also in mediating the binding of mycobacteria to and subsequent entry into macrophages. This may further be linked to an intracellular trafficking pathway through which LAM is thought to be presented by CD1 to subsets of T-cells. The implication of LAM as major histocompatibility complex (MHC)-independent T-cell epitope and the ensuing immune response is an area of intensive studies. Another recent focus of research is the biosynthesis of arabinan which has been shown to be inhibitable by the anti-tuberculosis drug, ethambutol. The phenomenon of truncated LAM as synthesized by ethambutol resistant strains provides an invaluable handle for dissecting the array of arabinosyltransferases involved, as well as generating much needed structural variants for further structural and functional studies. It is hoped that with more systematic investigations based on clinical isolates and human cell lines, the true significance of LAM in the immunopathogenesis of tuberculosis and leprosy can eventually be explained.

Novel O-methylated terminal glucuronic acid characterizes the polar glycopeptidolipids of Mycobacterium habana strain TMC 5135.

Mycobacterium "habana" strain TMC 5135, which has been proposed as a vaccine against both leprosy and tuberculosis, is considered to be a strain of serotype I of the recognized species Mycobacterium simiae. We have now shown that each of these strains possesses characteristic polar glycopeptidolipids (GPL) which are sufficiently different to allow unequivocal strain identification. Thin layer chromatographic analysis demonstrated that M. habana synthesizes a family of apolar GPLs and three distinct polar GPLs (pGPL-I to -III) which exhibited migration patterns different from those of M. simiae serotype I (pGPL-Sim). Using a combination of chemical, mass spectrometric, and proton-NMR analyses, the GPLs from M. habana were determined to be based on the same generic structure as those from the M. avium complex, namely N-fatty acyl-D-Phe-(O-saccharide)-D-allo-Thr-D-Ala-L-alaninyl-O-m onosaccharide. The de-O-acetylated apolar GPLs contain a 3-O-Me-6-deoxy-Tal attached to the allo-Thr and either a 3-O-Me-Rha or a 3,4-di-O-Me-Rha attached to the alaninol. In the pGPLs, oligosaccharides were found to be attached to the allo-Thr. The oligoglycosyl alditol reductively released from the least polar pGPL-I was fully characterized as L-Fucp alpha 1 in --7 with 3-(6-O-Me)-D-Glcp beta 1 in --7 with 3-(4-O-Me)-L-Rhap alpha 1 in --7 with 3-L-Rhap alpha 1 in --7 with 2-(3-O-Me)-6-deoxy-Tal. In pGPl-II and -III, the terminal Fuc residue is further 3-O-methylated and 4-O-substituted with an additional 2,4-di-O-Me-D-GlcA and 4-O-Me-D-GlcA, respectively. The corresponding oligosaccharide from pGPL-Sim was shown to be of identical molecular weight to pGPL-II but terminating with a 3,4-di-O-Me-GlcA. Enzyme-linked immunosorbent assay-based serological studies using anti-M. habana and anti-M. simiae sera against whole cells and purified pGPLs firmly established the polar GPLs as important antigens and indicated that the terminal epitopes L-Fuc-, 2,4-di-O-Me-D-GlcA, and 4-O-Me-D-GlcA uniquely present in pGPL-I, -II, and -III, respectively, confer sufficient specificity for the identification of M. habana as a distinct serotype of M. simiae.

CD1-restricted T cell recognition of microbial lipoglycan antigens.

It has long been the paradigm that T cells recognize peptide antigens presented by major histocompatibility complex (MHC) molecules. However, nonpeptide antigens can be presented to T cells by human CD1b molecules, which are not encoded by the MHC. A major class of microbial antigens associated with pathogenicity are lipoglycans. It is shown here that human CD1b presents the defined mycobacterial lipoglycan lipoarabinomannan (LAM) to alpha beta T cell receptor-bearing lymphocytes. Presentation of these lipoglycan antigens required internalization and endosomal acidification. The T cell recognition required mannosides with alpha(1-->2) linkages and a phosphotidylinositol unit. T cells activated by LAM produced interferon gamma and were cytolytic. Thus, an important class of microbial molecules, the lipoglycans, is a part of the universe of foreign antigens recognized by human T cells.

Inositol phosphate capping of the nonreducing termini of lipoarabinomannan from rapidly growing strains of Mycobacterium.

Previous studies have demonstrated that the nonreducing termini of the lipoarabinomannan (LAM) from Mycobacterium tuberculosis are extensively capped with mannose residues, whereas those from a fast growing Mycobacterium sp., once thought to be an attenuated strain of M. tuberculosis, are not. The noncapped LAM, termed AraLAM, is known to be more potent than the mannose-capped LAM (ManLAM) in inducing functions associated with macrophage activation. Using a combination of chemical and enzymatic approaches coupled with fast atom bombardment-mass spectrometry analysis, we demonstrated that LAMs from all M. tuberculosis strains examined (Erdman, H37Ra, and H37Rv), as well as the attenuated Mycobacterium bovis BCG strain, are mannose-capped with the extent of capping varying between 40 and 70%. The nonreducing termini of LAM from Mycobacterium leprae were also found to be capped with mannoses but at a significantly lower level. A novel inositol phosphate capping motif was identified on a minor portion of the otherwise uncapped arabinan termini of LAMs from the fast growing Mycobacterium sp. and Mycobacterium smegmatis ATCC 14468 and mc(2)155. In addition, an inositol phosphate tetra-arabinoside was isolated from among endoarabinase digestion products of AraLAM and was shown to induce tumor necrosis factor-alpha production. Accordingly, we concluded that AraLAM is characteristic of some rapidly growing Mycobacterium spp. It is distinct from ManLAMs of M. tuberculosis, M. bovis BCG, and Mycobacterium leprae not only in the absence of mannose-capping but also in containing some terminal inositol phosphate substituents which may account for its particular potency in inducing macrophage activation.

Structural definition of acylated phosphatidylinositol mannosides from Mycobacterium tuberculosis: definition of a common anchor for lipomannan and lipoarabinomannan.

Based on chemical analysis, we have previously concluded that the biologically important lipoarabinomannan (LAM) and lipomannan (LM) from Mycobacterium are multiglycosylated forms of the phosphatidylinositol mannosides (PIMs), the characteristic cell envelope mannophosphoinositides of mycobacteria. Using definitive analytical techniques, we have now re-examined the reported multiacylated nature of PIMs in order to gain a better insight into their possible roles as biosynthetic precursors of LM and LAM. High-sensitivity fast atom bombardment-mass spectrometry analyses of the perdeuteroacetyl and permethyl derivatives of PIMs from Mycobacterium tuberculosis and Mycobacterium leprae enabled us to define the exact fatty acyl compositions of the multiacylated, heterogeneous PIM families, notably the dimannoside (PIM2) and the hexamannoside (PIM6). Specifically, in conjunction with other chemical and gas chromatography-mass spectrometry (GC-MS) analyses, the additional C16 fatty acyl substituent on PIM2 and its lyso form were defined as attached to the C6 position of mannose. We also present evidence for triacylated mannophosphoinositide as a common lipid anchor for both LM and LAM, and further postulate that acylation of PIM2 may constitute a key regulatory step in their biosynthesis.

Macrophage activation: lipoarabinomannan from avirulent and virulent strains of Mycobacterium tuberculosis differentially induces the early genes c-fos, KC, JE, and tumor necrosis factor-alpha.

Lipoarabinomannan (LAM) is a major cell-wall associated glycolipid produced by Mycobacterium tuberculosis and Mycobacterium leprae. Previous work demonstrated that LAM from avirulent (H37Ra) and virulent (Erdman) strains of M. tuberculosis differ in structure at their non-reducing termini. In this study the effects of the H37Ra and Erdman LAM on the activation of murine bone marrow-derived macrophages has been investigated. Their abilities to elicit immediate early gene responses at mRNA (c-fos, JE, KC) and protein (TNF-alpha secretion) levels, and nitrite production, was examined. H37Ra LAM, but not Erdman LAM, elicited TNF-alpha secretion at 1000 ng/ml. Neither stimulated production of reactive nitrogen intermediates (RNI). Addition of 25 U/ml IFN-gamma enhanced TNF-alpha secretion in response to H37Ra LAM, reducing the threshold level of LAM required to 10 to 100 ng/ml. In contrast, Erdman LAM at concentrations up to 1000 ng/ml could not induce macrophage TNF-alpha secretion even in the presence of 25 U/ml IFN-gamma. H37Ra LAM also synergized with IFN-gamma to stimulate enhanced production of RNI, whereas IFN-gamma and Erdman LAM did not elicit RNI production. Examination of events before TNF-alpha and RNI production revealed that H37Ra LAM, like LPS, was able to induce increased levels of mRNA expression for c-fos, KC, and JE, with similar kinetics but reduced potency compared with LPS. Erdman LAM in concentrations up to 2500 ng/ml was unable to stimulate c-fos, KC, or JE expression. IFN-gamma at 25 U/ml was itself a potent stimulus of JE expression, and synergized with 1000 ng/ml H37Ra, and to a lesser extent, Erdman LAM for the induction of JE. In contrast, IFN-gamma inhibited H37Ra LAM stimulation of KC expression. The phenomenon of avoiding the stimulation of macrophage immediate early gene expression may be an important determinant of mycobacterial virulence.

Tumor necrosis factor production in patients with leprosy.

The spectrum of host responses to Mycobacterium leprae provides a model for investigating the role of cytokines in the pathogenesis of mycobacterial disease. Of particular interest is tumor necrosis factor (TNF), a cytokine which may have both antimycobacterial and immunopathologic effects. To evaluate the potential role of TNF in leprosy, we measured TNF production in response to M. leprae and its defined constituents by peripheral blood mononuclear cells from patients across the spectrum of disease. The levels of TNF induced through the stimulation of cells with M. leprae or its dominant "lipopolysaccharide," lipoarabinomannan, were higher in patients with the tuberculoid form of the disease than in those with the lepromatous form. In patients with erythema nodosum leprosum (ENL), a reactional state of lepromatous leprosy, the levels of TNF release by peripheral blood mononuclear cells were higher than in any other form of the disease. Treatment of ENL patients with thalidomide reduced TNF secretion by more than 90%. The mycolylarabinogalactan-peptidoglycan complex of Mycobacterium species, the protein-peptidoglycan complex, and muramyl dipeptide all elicited significant TNF release. Therefore, TNF release appears to be triggered by at least two major cell wall structural constituents of M. leprae, lipoarabinomannan and segments of the cell wall skeleton. The prominent TNF release in patients with the paucibacillary tuberculoid form of the disease compared with that in patients with the multibacillary lepromatous form suggests that this cytokine contributes to a resistant immune response to mycobacterial infection. However, the marked TNF release in patients with ENL indicates that TNF may also mediate immunopathologic effects, such as fever and tissue damage.

Lipoarabinomannan. Multiglycosylated form of the mycobacterial mannosylphosphatidylinositols.

The lipopolysaccharides of mycobacteria, lipoarabinomannan (LAM) and lipomannan (LM), of key importance in host-pathogen interaction, were recently shown to contain a phosphatidylinositol "anchoring domain." We now have established that LAM and LM are based on the phosphatidylinositol mannosides, the characteristic glycophospholipids of mycobacteria. Digestion of the arabinose-free LM with an endo-alpha 1----6-mannosidase yielded evidence for the presence of the 1-(sn-glycerol-3-phospho)-D-myo-inositol-2,6-bis-alpha-D-mannopyranoside unit, indistinguishable from that derived from phosphatidylinositol dimannoside. This same inositol substitution pattern was shown to be present in LAM by methylation analysis before and after dephosphorylation. Positions C-2 and C-6 of the inositol unit of LAM are occupied by mannosyl residues and C-1 by a phosphoryl group. Partial acid hydrolysis of per-O-methylated LAM and comparison by gas chromatography-mass spectrometry of the resulting derivatized oligosaccharides with like products from phosphatidylinositol hexamannoside demonstrated that the C-6 of inositol is the point of attachment of the mannan core of LAM, which consists of an alpha 1----6-linked backbone with considerable alpha-1----2 side chains. Thus, a structural and presumably biosynthetic relationship is established between some of the membranous mannosylphosphatidylinositols described some 25 years ago and the newly emerging, biologically active lipopolysaccharides of mycobacteria.

Chemical synthesis and seroreactivity of a neoantigen containing the oligosaccharide hapten of the Mycobacterium tuberculosis-specific phenolic glycolipid.

The report of a major triglycosyl phenol phthiocerol (phenolic) glycolipid in some strains of Mycobacterium tuberculosis that resembles the phenolic glycolipid I of Mycobacterium leprae raised the prospects of a specific serodiagnostic tool for human tuberculosis. The terminal diglycosyl unit of the M. tuberculosis product was synthesized and converted to a corresponding neoglycoprotein, the O-(2,3,4-tri-O-methyl-alpha-L-fucopyranosyl)-(1----3)-O-alpha-L- rhamnopyranosyl)-(1----9)-oxynonanoyl-bovine serum albumin, and applied, in ELISA, to sera from individuals with tuberculosis. Although the correlation coefficient between the synthetic product and the native glycolipid was excellent, the seroreactivity rate of tuberculous sera was disappointing; only 24 of 119 sera from tuberculosis patients showed evidence of anti-glycolipid antibodies. In isolates of M. tuberculosis from tuberculosis patients the glycolipid was present in only 1 of 11. A partially deglycosylated version was present in two other isolates; however, most isolates lacked the glycolipid. Accordingly, while the results, unlike those of others, do not portend a future for this form of serodiagnosis in the management of tuberculosis, they offer intriguing hints as to the basis of the variable immunogenicity and pathogenicity of strains of M. tuberculosis.

Structure and function of mycobacterial glycolipids and glycopeptidolipids.

Earlier work from this and other laboratories has revealed the presence within Mycobacterium spp. of three classes of glycolipid antigens which we have called the glycopeptidolipids, the lipooligosaccharides and the phenolic glycolipids. Representative structures of each from different species and sub-species have been proposed. More recently, new variants of these antigens and older structures have been analyzed by Fourier transform infrared, NMR, particularly at high temperatures, and, most notably, by fast atom bombardment and Californium desorption mass spectrometry. Extraordinary novelty and diversity were revealed, particularly at the distal non-reducing end of the oligosaccharide chains, marked by the presence of new branched-chain sugars, amino sugars and sugar acids. These epitopes and monoclonal antibodies to them have been used for the critical identification of mycobacteria. In addition, the pure antigens are the basis of specific serological tests for various mycobacterioses. The resurgence of interest in "atypical" mycobacteria stems from their occurrence as opportunistic pathogens in many patients with acquired immunodeficiency syndrome, although they have long been associated with pulmonary and other organ infections. Foremost among these mycobacteria are serovars of the Mycobacterium avium-Mycobacterium intracellulare complex (the M. avium complex). The surface antigens which differentiate these serovars are glycopeptidolipids, related to "mycoside C" and, accordingly, composed of a glycosylated lipopeptide "core", fatty acyl-D-Phe-D-alloThr-D-Ala-L-acanyl-O- (3,4-di-O-methyl-alpha-L-rhamnopyranoside), to which a haptenic oligosaccharide is linked at the threonine substituent; this oligoglycosyl unit is the source of type specificity.(ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis and immunoreactivity of neoglycoproteins containing the trisaccharide unit of phenolic glycolipid I of Mycobacterium leprae.

The trisaccharide segment, O-(3,6-di-O-methyl-beta-D-glucopyranosyl)-(1----4)-O-(2,3-di-O-methyl- alpha-L-rhamnopyranosyl)-(1----2)-3-O-methyl-L-rhamnopyranose, of the Mycobacterium leprae-specific phenolic glycolipid I has been synthesized as its 8-(methoxycarbonyl)octyl glycoside and coupled to a carrier protein, to produce a leprosy-specific neoglycoprotein, the so-called natural trisaccharide-octyl-bovine serum albumin (NT-O-BSA). Special features of the synthetic strategy were the use of silver trifluoromethanesulfonate (triflate) to promote glycosylation, resulting in the rhamnobiose in high yield and absolute stereospecificity. The terminal 3,6-di-O-methyl-D-glucopyranosyl group was introduced after O-deallylation of the rhamnobiose. Removal of protecting groups yielded the trisaccharide hapten suitable for coupling to carrier protein. Poly(acrylamide)-gel electrophoresis of the neoglycoprotein demonstrated its purity, and subsequent immunoblotting with a monoclonal antibody directed to the terminal 3,6-di-O-methyl-beta-D-glucopyranosyl epitope of the native glycolipid demonstrated its antigenicity. Comparative serological testing in enzyme-linked immunosorbent assays of NT-O-BSA, the corresponding disaccharide-containing products, and another trisaccharide-containing neoglycoprotein, O-(3,6-di-O-methyl-beta-D-glucopyranosyl)-(1----4)-O-(2,3-di-O- methyl-alpha-L-rhamnopyranosyl)-(1----2)-(3-O-methyl-alpha-L-rhamnopy ran osyl)- (1----4')-oxy-(3-phenylpropanoyl)-BSA (NT-P-BSA) [Fujiwara et al., Agric. Biol. Chem., 51 (1987) 2539-2547] against sera from leprosy patients and control populations showed concordance; the presence of the innermost sugar did not contribute significantly to sensitivity or specificity. The di- and tri-saccharide-containing neoantigens, on account of ready availability and solubility, provide greater flexibility than the native glycolipid for the serodiagnosis of leprosy.

Chemical synthesis and seroreactivity of O-(3,6-di-O-methyl-beta-D-glucopyranosyl)-(1----4)-O-(2,3-di-O-methyl- alpha-L-rhamnopyranosyl)-(1----9)-oxynonanoyl-bovine serum albumin--the leprosy-specific, natural disaccharide-octyl-neoglycoprotein.

The outer disaccharide segment, namely, O-(3,6-di-O-methyl-beta-D-glucopyranosyl)-(1----4)-2,3-di-O-methyl-alpha -L-rhamnopyranose, of the trisaccharide-containing, leprosy-specific, phenolic glycolipid I has been synthesized as the 8-(methoxycarbonyl)octyl glycoside in high yield and absolute stereospecificity by a series of modified Koenigs-Knorr and Helferich reactions. A particular feature of the synthetic pathway involves methylation of the 2-hydroxyl group of the rhamnose moiety under neutral conditions, after first preparing the 8-(methoxycarbonyl)octyl glycoside as the alpha anomer via the 1,2-orthoacetate, and thus precluding the possible formation of an anomeric mixture. The 8-(methoxycarbonyl)octyl O-(3,6-di-O-methyl-beta-D-glucopyranosyl)-(1----4)-2,3-di-O-methyl-alpha -L-rhamnopyranoside was converted into the crystalline hydrazide, and this was coupled to bovine serum albumin (BSA), via intermediate acyl-azide formation, to produce the corresponding neoglycoprotein, O-(3,6-di-O-methyl-beta-D-glucopyranosyl)- (1----4)-O-(2,3-di-O-methyl-alpha-L-rhamnopyranosyl)- (1----9)-oxynonanoyl-BSA, the so-called natural disaccharide-octyl-BSA. Extensive serological testing of this product against sera from leprosy patients and control populations, and comparison with the native glycolipid and previously synthesized neoglycoproteins, have shown that it is unparalleled in terms of sensitivity and specificity, and highly suited to replace the native glycolipid for the serodiagnosis of worldwide lepromatous leprosy.

A simplified serological test for leprosy based on a 3,6-di-O-methylglucose-containing synthetic antigen.

The recent advent of synthetic antigens containing the Mycobacterium leprae-specific epitope, 3,6-di-O-methyl-beta-D-glucopyranoside, has allowed the development of simple specific serological tests for leprosy. The incorporation of one such product, 8-carbonyloctyl O-[4-O-(3,6-di-O-methyl-beta-D-glucopyranosyl)-alpha-L- rhamnopyranoside]-BSA, into a simple "spot" test, diffusion-in-gel enzyme-linked immunosorbent assay (ELISA), allowed an over 90% detection rate of untreated lepromatous leprosy, and the results showed good concordance with conventional ELISA based on the native phenolic glycolipid I.