Aetiological agents and methods for detection

by Patrícia D. Deps,

Department of Social Medicine, Post-Graduate Program in Infectious Diseases, Federal University of Espírito Santo, Vitória, Espírito Santo, Brazil.

João Marcelo Antunes

Universidade Federal Rural do Semi-Árido, Jerônimo Dix-Huit Rosado Maia Veterinary Hospital , Mossoró, Rio Grande do Norte, Brazil.

and Simon M. Collin.

Public Health England, London, United Kingdom.

Aetiological agents

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

Bacilloscopy

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).

Histopathology

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

Figure 1. Morphological aspects of M. leprae using the Ziehl-Neelsen method in a patient with virchowian Hansen’s disease. Magnification x100.

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).

Figure 2: Immunoexpression of BCG antigen in virchowian Hansen’s disease. Magnification x40.

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.

Sorological methods

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.^23-25Table 2 shows the antigens used in serological techniques.

Table 2 - antigens used in serological techniques for diagnosing Hansen’s disease

Genomic methods

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

PCR

Polymerase chain reaction (PCR) and its derivations have assisted in the diagnosis of Hansen’s disease.^31,32Different 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.^33,34Similarly, 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

Real Time-PCR (qPCR); Sensitivity (Sn); Specificity (Sp); MB (Multibacillary); PB (Paucibacillary)

Academic Collaborators

Génèse Faïrana Godeline Essali,

Júlia Salarini Carneiro e

Lavínia Damacena Perin

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