Aetiological agents and methods for detection
by Patrícia D. Deps,
João Marcelo Antunes
and Simon M. Collin.
M. leprae is a slightly-curved immobile acid-fast obligate intracellular bacillus, not cultivable in artificial environments, and with an affinity for skin cells (macrophages) and peripheral nerves (Schwann cells). Its cell wall is composed of a covalent complex linked to peptidoglicanarabinogalactane-mycolic acid, similar to that found in the cell walls of other mycobacteria.1 Glycolipid-phenol-1 (PGL-1), predominant in the cell wall, is a glycolipid that confers the most striking immunogenic capacity of M. leprae and may be involved in the interaction with the Schwann cell laminine.
Although M. leprae does not grow in culture medium, the bacillus multiplies on the mouse foot pad and also in armadillos, where it reproduces a similar clinical picture to Hansen’s disease in humans.2
In 2008 a new species called Mycobacterium lepromatosis was identified in Mexico as a cause of diffuse virchowiana Hansen’s disease.3 It was later detected in Africa, Asia, and Brazil.4 Comparative genomic analysis has shown that M. lepromatosis appears to date further back on the evolutionary scale than M. leprae.5
Methods used in the routine diagnosis of Hansen’s disease
A skin smear is a laboratory test that provides information about the presence of Hansen's bacillus in the body of a patient with suspected Hansen’s disease. By microscopic examination, an attempt is made to detect the bacillus in intradermal scrapes of dermatological lesions or desensitized areas, if any, of the earlobes and elbows.6,7
The bacillary index proposed by Ridley is based on a logarithmic scale for quantitative evaluation of the number of bacilli found in lymph smears taken from four skin sites. The bacillary index is standardized and a positive result is presented as shown in Table 1. The bacillary index was adopted by the Ministry of Health (Brazil) in 1989.
Table 1 – Bacillary index: classification criteria adopted by the Ministry of Health (Brazil).
The different immune responses of the host determine the clinical and histopathological forms of the Hansen’s disease spectrum. The stains used are hematoxylin and eosin, and the specific stains for acid-fast bacilli, Ziehl-Neelsen, Wade or Fite-Faraco.6 The slides are observed using an optical microscope.
By the Ziehl-Neelsen (Figure 1), Wade and Fite-Faraco methods, the bacilli are stained evenly in red and, when feasible, in the form of rods, isolated or grouped, forming globules that are characteristic of M. leprae.8 These stainings are widely used for the identification of bacilli in tissues, mainly in skin fragments. The immunohistochemistry technique with BCG marker (Figure 2) can also be used to identify M. leprae, the bacillus being visualised in golden brown colour. False positive results can occur with these methods by identification of Gram-positive bacteria, mast cells and melanophages by the same dyes.9,10
The Ridley-Jopling classification was developed for scientific research but is used in clinical practice in many parts of the world.11,12 The Ridley-Jopling system classifies Hansen’s disease as an immune-mediated disease with the tuberculoid (TT) form at one end of the spectrum and virchowian Hansen’s disease (VV) at the other. The TT form correlates immunologically with strong cell-mediated immunity (CMI), while the VV form correlates with weak CMI. Between these two ends is the clinically unstable spectrum, subdivided into borderline-tuberculoid (BT), borderline-borderline (BB), and borderline-virchowian (BV).
Tuberculoid Hansen’s disease (TT) has granulomas with or without Langhans giant cells, nerves damaged and infiltrated by the inflammatory process, and epithelial cells arranged side by side. Bacilli are rare, demonstrating complete phagocytosis, and when they occur are almost exclusively on nerve branches.
Borderline-tuberculoid Hansen’s disease (BT) is similar to TT, but with occasional bacilli, usually on nerves, and a spare inflammatory zone may occur in the subepidermal region.
Borderline-borderline Hansen’s disease (BB) presents epithelial cells, histiocytes, focal lymphocytes, increased cellularity in the nerves, presence of localized bacilli in the nerves and spare subepidermal zone.
Borderline-virchowian Hansen’s disease (BV) presents histiocytes, few epithelial cells, foamy or Virchow's cells (macrophages or histiocytes containing large number of bacilli), presence of bacilli in the nerves, and spare subepidermal zone.
Virchowian Hansen’s disease consists of a granuloma of the histio-monocyte type. Presence of Virchow's cells. The picture is still composed of few lymphocytes, numerous bacilli in the nerves, minimal intraneural cell infiltration, and spare subepidermal zone.
Indeterminate Hansen’s disease has a non-specific inflammatory infiltrate, consisting of undifferentiated lymphocytes and histiocytes around the nerves and skin appendages. Rare bacilli.
Serological techniques such as ELISA and lateral flow (ML Flow) detect the presence of IgM anti-PGL-1 antibodies.13 The PGL-1 molecule is composed of trisaccharide 3,6-di-O-methyl-beta-D-glucopyranosyl-(1-4)-2,3-di-O-methyl-alpha-L-ramnopyranosyl-(1-2)-3-O-methyl-alpha-L-ramnopyranosyl.14 Removal of terminal sugar results in loss of binding of most antibodies, while removal of the long fatty acid chain from the PGL-1 molecule has no effect on antibody binding.
This suggests that chemical synthesis of the last part of the disaccharide produces an antigen that stimulates the production of monoclonal antibodies used to detect the presence of PGL-1.15 Sugar synthesis (natural di- or trisaccharides, ND or NT, respectively), followed by identification of the terminal sugar as the primary determinant antigen of PGL-1 and its binding to bovine serum albumin (BSA) with an octyl (O) ring, or a phenolic (P) ring, can produce semi-synthetic antigens.16 The ND-O-BSA antigen has been considered equal to or better than other PGL-1 antigen derivatives, both natural and synthetic.17 The potent immune response to PGL-1, with IgM production, is proportional to the bacillary load,18 and is species-specific.19
LID-1 is a fusion of two M. leprae proteins (ML0405 and ML2331) to detect IgG antibodies,20 while NDO-LID combines LID-1 with a synthetic PGL-1 IgM mimtope.21 rMLP15 is a purified recombinant polypeptide of six proteins (ML1358, ML2055, ML0885, ML1811, ML1812, and ML1214).22
Systematic reviews have been unable to identify conclusively a serological technique that performs better in terms of sensitivity and specificity due to heterogeneity between studies, but all serological techniques perform poorly in the detection of paucibacillary Hansen’s disease.23-25 Table 2 shows the antigens used in serological techniques.
Table 2 - antigens used in serological techniques for diagnosing Hansen’s disease
An important advance in understanding the biology of M. leprae has been the sequencing of its genome, which has a lower content of Guanine and Cytosine compared to that found in M. tuberculosis.26 It has approximately 4,000 genes that encode proteins, 27% are from degenerated genes (pseudogenes), and 2% are composed of repetitive sequences.
After sequencing the M. leprae genome, researchers began to find repetitive sequences in the genome, variable number tandem repeats (VNTRs) and Short Tandem Repeats (STRs),27 and single nucelotide polymorphisms (SNPs) in order to differentiate strains of this bacterium. These techniques have been used to understand the genetic diversity of this pathogen, as well as to elucidate aspects of Hansen’s disease dissemination and epidemiological gaps.28
Initially, these analyses revealed only four SNPs (SNP types 1, 2, 3 and 4).29 However, more recent approaches, involving 78 informational SNPs, have made it possible to classify M. leprae into 16 subtypes, for which a correlation between the geographical origin of the Hansen’s disease and the SNP profile has also been made.30
Polymerase chain reaction (PCR) and its derivations have assisted in the diagnosis of Hansen’s disease.31,32 Different sequences were used as targets for PCR. The genes encoding the 36-kDa antigen, the 18-kDa antigen, the 65-kDa antigen, 85 complex, 16S rDNA, hsp65 (heat shock protein 65) and the RLEP repetitive sequence were studied. Real-Time PCR (qPCR) techniques have improved the sensitivity and specificity of M. leprae detection.33
Although PCR is potentially useful in the diagnosis of difficult cases such as pure neural Hansen’s disease, paucibacillary Hansen’s disease and patients with atypical clinical presentation, it is not used in routine clinical practice.33,34 Similarly, genomic techniques such as next-generation sequencing (NGS) are useful in scientific research but are unlikely to be of clinical use in Hansen’s disease-endemic settings.35 Table 3 demonstrates the markers and some of their characteristics used in PCR techniques.
Table 3 – Markers used in the detection of M. leprae DNA by PCR
Génèse Faïrana Godeline Essali,
Júlia Salarini Carneiro e
Lavínia Damacena Perin
Draper, P., Kandler, O. & Darbre, A. Peptidoglycan and arabinogalactan of Mycobacterium leprae. J. Gen. Microbiol. 133, 1187–1194 (1987).
Storrs, E. E. The nine-banded armadillo: a model for leprosy and other biomedical research. Int J Lepr Mycobact Dis 39, 703–714 (1971).
Han, X. Y. et al. A new Mycobacterium species causing diffuse lepromatous leprosy. Am. J. Clin. Pathol. 130, 856–864 (2008).
Han, X. Y., Aung, F. M., Choon, S. E. & Werner, B. Analysis of the leprosy agents Mycobacterium leprae and Mycobacterium lepromatosis in four countries. Am. J. Clin. Pathol. 142, 524–532 (2014).
Singh, P. et al. Insight into the evolution and origin of leprosy bacilli from the genome sequence of Mycobacterium lepromatosis. Proc. Natl. Acad. Sci. U. S. A. 112, 4459–4464 (2015).
Ministério da Saúde. Guia para o controle da Hanseníase. (2002).
Ministério da Saúde. Normas Técnicas para a Eliminação da Hanseníase no Brasil. (2001).
Hastings, R. C., Gillis, T. P., Krahenbuhl, J. L. & Franzblau, S. G. Leprosy. Clin. Microbiol. Rev. 1, 330–348 (1988).
Schettini, A. P. et al. Enhancement in the histological diagnosis of leprosy in patients with only sensory loss by demonstration of mycobacterial antigens using anti-BCG polyclonal antibodies. Int J Lepr Mycobact Dis 69, 335–340 (2001).
Deps, P. D., Michalany, N. S. & Tomimori-Yamashita, J. False positive reaction of the immunohistochemistry technique using anti-BCG polyclonal antibodies to identify Mycobacterium leprae in wild nine-banded armadillos. Int J Lepr Mycobact Dis 72, 327–330 (2004).
Meyer, M. Leprosy. in Tropical infectious diseases: principles, pathogens and practice (eds. Guerrant, R. L., Walker, D. H. & Weller, P. F.) (Saunders/Elsevier, 2011).
Opromolla, P. A. & Laurenti, R. Controle da hanseníase no Estado de São Paulo: análise histórica. Rev. Saúde Pública 45, 195–203 (2011).
Bührer, S. S., Smits, H. L., Gussenhoven, G. C., van Ingen, C. W. & Klatser, P. R. A simple dipstick assay for the detection of antibodies to phenolic glycolipid-I of Mycobacterium leprae. Am. J. Trop. Med. Hyg. 58, 133–136 (1998).
Hunter, S. W., Fujiwara, T. & Brennan, P. J. Structure and antigenicity of the major specific glycolipid antigen of Mycobacterium leprae. J. Biol. Chem. 257, 15072–15078 (1982).
Young, D. B., Khanolkar, S. R., Barg, L. L. & Buchanan, T. M. Generation and characterization of monoclonal antibodies to the phenolic glycolipid of Mycobacterium leprae. Infect. Immun. 43, 183–188 (1984).
Fujiwara, T., Hunter, S. W., Cho, S. N., Aspinall, G. O. & Brennan, P. J. Chemical synthesis and serology of disaccharides and trisaccharides of phenolic glycolipid antigens from the leprosy bacillus and preparation of a disaccharide protein conjugate for serodiagnosis of leprosy. Infect. Immun. 43, 245–252 (1984).
Wu, Q. X., Ye, G. Y. & Li, X. Y. Serological activity of natural disaccharide octyl bovine serum albumin (ND-O-BSA) in sera from patients with leprosy, tuberculosis, and normal controls. Int J Lepr Mycobact Dis 56, 50–55 (1988).
Ng, V. et al. Role of the cell wall phenolic glycolipid-1 in the peripheral nerve predilection of Mycobacterium leprae. Cell 103, 511–524 (2000).
Brennan, P. J. & Barrow, W. W. Evidence for species-specific lipid antigens in Mycobacterium leprae. Int J Lepr Mycobact Dis 48, 382–387 (1980).
Duthie, M. S. et al. Specific IgG antibody responses may be used to monitor leprosy treatment efficacy and as recurrence prognostic markers. Eur. J. Clin. Microbiol. Infect. Dis. 30, 1257–1265 (2011).
Paula Vaz Cardoso, L. et al. Development of a quantitative rapid diagnostic test for multibacillary leprosy using smart phone technology. BMC Infect. Dis. 13, (2013).
Barbosa, M. dos S., de Sousa, I. B. A., Simionatto, S., Borsuk, S. & Marchioro, S. B. Recombinant polypeptide of Mycobacterium leprae as a potential tool for serological detection of leprosy. AMB Express 9, (2019).
de Oliveira, A. L. G. et al. Diagnostic accuracy of tests using recombinant protein antigens of Mycobacterium leprae for leprosy: A systematic review. J. Infect. Public Health (2020). doi:10.1016/j.jiph.2019.12.011
Espinosa, O. A., Benevides Ferreira, S. M., Longhi Palacio, F. G., Cortela, D. da C. B. & Ignotti, E. Accuracy of Enzyme-Linked Immunosorbent Assays (ELISAs) in Detecting Antibodies against Mycobacterium leprae in Leprosy Patients: A Systematic Review and Meta-Analysis. Can. J. Infect. Dis. Med. Microbiol. 2018, 1–11 (2018).
Gurung, P., Gomes, C. M., Vernal, S. & Leeflang, M. M. G. Diagnostic accuracy of tests for leprosy: a systematic review and meta-analysis. Clin. Microbiol. Infect. 25, 1315–1327 (2019).
Cole, S. T. et al. Massive gene decay in the leprosy bacillus. Nature 409, 1007–1011 (2001).
Gillis, T. et al. Characterisation of short tandem repeats for genotyping Mycobacterium leprae. Lepr. Rev. 80, 250–260 (2009).
Young, D. Prospects for molecular epidemiology of leprosy. Lepr. Rev. 74, 11–17 (2003).
Monot, M. et al. On the origin of leprosy. Science 308, 1040–1042 (2005).
Monot, M. et al. Comparative genomic and phylogeographic analysis of Mycobacterium leprae. Nat. Genet. 41, 1282–1289 (2009).
Hartskeerl, R. A., de Wit, M. Y. & Klatser, P. R. Polymerase chain reaction for the detection of Mycobacterium leprae. J. Gen. Microbiol. 135, 2357–2364 (1989).
Hackel, C. et al. Specific identification of Mycobacterium leprae by the polymerase chain reaction. Mol. Cell. Probes 4, 205–210 (1990).
Martinez, A. N., Talhari, C., Moraes, M. O. & Talhari, S. PCR-based techniques for leprosy diagnosis: from the laboratory to the clinic. PLoS Negl. Trop. Dis. 8, e2655 (2014).
Tatipally, S., Srikantam, A. & Kasetty, S. Polymerase Chain Reaction (PCR) as a Potential Point of Care Laboratory Test for Leprosy Diagnosis—A Systematic Review. Trop. Med. Infect. Dis. 3, 107 (2018).
Quan, M. et al. Leprosy in a low-incidence setting: Case report relevant to metagenomic next generation sequencing applications. Wien. Klin. Wochenschr. (2020). doi:10.1007/s00508-020-01644-7
Vera-Cabrera, L. et al. Mycobacterium lepromatosis Infections in Nuevo León, Mexico. J. Clin. Microbiol. 53, 1945–1946 (2015).