**1. Introduction**

Leprosy is a chronic infectious disease, dermato neurological and incapacitating, which has *Mycobacterium leprae* as its causal agent [1]. Even with the worldwide effort to eliminate this disease as a public health problem [2], countries such as India and Brazil still present a higher number of cases than the World Health Organization (WHO) recommended [3]. In this sense, this organization defined as a global strategy the reduction in the incidence of new cases as a priority [4], highlighting the importance of early diagnosis, which aims to reduce the transmission of the disease in the community [5], which includes correctly diagnosing cases with suspicion of the disease and to identify subclinical infection.

**2.1. Dermato-neurological clinical exam, bacilloscopy, histopathology and** 

For the diagnosis of leprosy, it is essential to perform clinical-dermatological exams that search for lesions in the epidermis, areas with changes in sensitivity (may be thermal, painful and tactile) and motor impairment-searching for thickened nerve trunks-which are classic signs of the disease [7]. In addition to clinical examination, there is a class of diagnostic exams and tests that are widely standardized and used in reference networks and centers [7, 15].

Molecular and Biotechnological Approaches in the Diagnosis of Leprosy

http://dx.doi.org/10.5772/intechopen.75506

65

Bacilloscopy is the most commonly used exam in clinical practice and, together with the dermato-neurological clinical test, is the most useful methodology for diagnosis [16]. The test presents advantages such as reduced cost and aid in the confirmation of new cases and patients with relapse [15]. The methodology used to perform the exam is a dermal smear of sites, these being: ear lobes (LO), elbows (C) and active lesions, where it is possible to analyze

As a result, a Bacilloscopy Index (BI), proposed by Ridley and Joplin, is provided, where there is a logarithmic scale ranging from 0 to 6 [19–21]. Thus, after an average of the fields analyzed, the result may vary from BI = 0, associated with patients of the tuberculoid (TT) pole and BI = 3+ to 6+, associated with lepromatous patients (LL). It is still possible to analyze the morphological index (MI) of the bacilli arranged on the slide, describing aspects of their morphological integrity [20]. Thus, bacilli may present three aspects related to their structure, classified as integral, fragmented or granular [21, 22]. Integral bacilli are considered viable, that is, they are related to host susceptibility to the parasite. These bacilli exhibit cell structure with preserved ends and uniform staining and are seen in smears of patients who are either non-treated or have relapse. Both fragmented and granular bacilli present flaws in the cell wall structure, being considered unviable or dead and more observed in post-

Bacilloscopy is effective when associated with the results of clinical exams [7, 15]. However, it is an exam that presents low sensitivity, since 50% of the smears of the sick individuals are negative. In addition, the exam requires adequate laboratory infrastructure and trained professional apparatus, factors that are not always a reality in hyperendemic regions and where

Histopathology is commonly performed for the diagnosis of diseases caused by obligate intracellular parasites and has good indices of sensitivity and specificity in the detection of leprosy [23]. However, the method encounters issues related to cost, time of analysis and false-negative results, besides of being an invasive exam [24]. Exactly for these reasons, histopathology is only recommended for individuals where it is impossible to assess degrees of cutaneous sensitivity, such as in children, elderly and mentally handicapped people; when it is not possible to classify the dimorphic clinical form; or when there is uncertainty whether the diagnosis is leprosy or other diseases that cause local hypoesthesia [23]. The diagnosis using this exam also depends on the association with the clinical aspects and bacilloscopy. In addition, biopsies of peripheral nerve branches are not recommended and should only be

the presence of the bacillus using a specific staining and optic microscopy [17, 18].

**Mitsuda test**

treatment patients [17–22].

medical and financial resources are reduced [7].

performed in the last instance [23, 24].

The clinical and epidemiological diagnosis of leprosy represents the gold standard for confirmation of the disease [6]. Bacilloscopy and Mitsuda's reaction are important tests to identify the etiological agent [7, 8], but there is a need to use complementary tests that allow a more accurate diagnosis with high sensitivity and specificity. In this context, stand out the standardization of serological and molecular tests, important for the understanding of the epidemiological profile of the disease.

The use of *M. leprae* specific antigens in serological tests has been the subject of research. The use of phenol-glycolipid-1 (PGL-1) [9], lipoarabinomannan (LAM) [1] and heat shock proteins (GroES and GroEL) [10] as antigens for the enzyme-linked immunosorbent assay (ELISA) and in immunosensors can be validated as methods for detecting new cases of the disease and for early diagnosis [11]. In addition, molecular tests aid in the identification of specific *M. leprae* sequences in clinical samples, which can be amplified through the polymerase chain reaction (PCR) technique, allowing DNA detection of the infectious agent [12] and/or through the use of real-time PCR technology that allows the evaluation of bacterial load [13] and also the monitoring of drug resistance [14].

Thus, this chapter will present an overview of the laboratory diagnosis of leprosy in the world. Initially, we will present a review of the main tests traditionally used in clinical routine and regulated by the WHO, in addition to the complementary tests that have been focus of research as a future perspective for the early diagnosis of the disease. These diagnostic approaches may contribute to a reduction in the number of cases of the disease, since they allow the monitoring of populations and endemic and hyperendemic areas.

## **2. Leprosy diagnosis: traditional exams**

The diagnosis of leprosy includes clinical observation of the patient, dermato-neurological clinical exams and complementary laboratory tests. Therefore, identify lesions and damaged nerves and analyze the life history of the patient are essential tools in the identification and detection of disease. These practices combined with other tools and methodologies are able to assist in epidemiological and disease control strategies, helping to map index cases and individuals who may develop leprosy, such as household contacts.

## **2.1. Dermato-neurological clinical exam, bacilloscopy, histopathology and Mitsuda test**

**1. Introduction**

64 Hansen's Disease - The Forgotten and Neglected Disease

miological profile of the disease.

monitoring of drug resistance [14].

**2. Leprosy diagnosis: traditional exams**

Leprosy is a chronic infectious disease, dermato neurological and incapacitating, which has *Mycobacterium leprae* as its causal agent [1]. Even with the worldwide effort to eliminate this disease as a public health problem [2], countries such as India and Brazil still present a higher number of cases than the World Health Organization (WHO) recommended [3]. In this sense, this organization defined as a global strategy the reduction in the incidence of new cases as a priority [4], highlighting the importance of early diagnosis, which aims to reduce the transmission of the disease in the community [5], which includes correctly diagnosing cases with

The clinical and epidemiological diagnosis of leprosy represents the gold standard for confirmation of the disease [6]. Bacilloscopy and Mitsuda's reaction are important tests to identify the etiological agent [7, 8], but there is a need to use complementary tests that allow a more accurate diagnosis with high sensitivity and specificity. In this context, stand out the standardization of serological and molecular tests, important for the understanding of the epide-

The use of *M. leprae* specific antigens in serological tests has been the subject of research. The use of phenol-glycolipid-1 (PGL-1) [9], lipoarabinomannan (LAM) [1] and heat shock proteins (GroES and GroEL) [10] as antigens for the enzyme-linked immunosorbent assay (ELISA) and in immunosensors can be validated as methods for detecting new cases of the disease and for early diagnosis [11]. In addition, molecular tests aid in the identification of specific *M. leprae* sequences in clinical samples, which can be amplified through the polymerase chain reaction (PCR) technique, allowing DNA detection of the infectious agent [12] and/or through the use of real-time PCR technology that allows the evaluation of bacterial load [13] and also the

Thus, this chapter will present an overview of the laboratory diagnosis of leprosy in the world. Initially, we will present a review of the main tests traditionally used in clinical routine and regulated by the WHO, in addition to the complementary tests that have been focus of research as a future perspective for the early diagnosis of the disease. These diagnostic approaches may contribute to a reduction in the number of cases of the disease, since they

The diagnosis of leprosy includes clinical observation of the patient, dermato-neurological clinical exams and complementary laboratory tests. Therefore, identify lesions and damaged nerves and analyze the life history of the patient are essential tools in the identification and detection of disease. These practices combined with other tools and methodologies are able to assist in epidemiological and disease control strategies, helping to map index cases and

allow the monitoring of populations and endemic and hyperendemic areas.

individuals who may develop leprosy, such as household contacts.

suspicion of the disease and to identify subclinical infection.

For the diagnosis of leprosy, it is essential to perform clinical-dermatological exams that search for lesions in the epidermis, areas with changes in sensitivity (may be thermal, painful and tactile) and motor impairment-searching for thickened nerve trunks-which are classic signs of the disease [7]. In addition to clinical examination, there is a class of diagnostic exams and tests that are widely standardized and used in reference networks and centers [7, 15].

Bacilloscopy is the most commonly used exam in clinical practice and, together with the dermato-neurological clinical test, is the most useful methodology for diagnosis [16]. The test presents advantages such as reduced cost and aid in the confirmation of new cases and patients with relapse [15]. The methodology used to perform the exam is a dermal smear of sites, these being: ear lobes (LO), elbows (C) and active lesions, where it is possible to analyze the presence of the bacillus using a specific staining and optic microscopy [17, 18].

As a result, a Bacilloscopy Index (BI), proposed by Ridley and Joplin, is provided, where there is a logarithmic scale ranging from 0 to 6 [19–21]. Thus, after an average of the fields analyzed, the result may vary from BI = 0, associated with patients of the tuberculoid (TT) pole and BI = 3+ to 6+, associated with lepromatous patients (LL). It is still possible to analyze the morphological index (MI) of the bacilli arranged on the slide, describing aspects of their morphological integrity [20]. Thus, bacilli may present three aspects related to their structure, classified as integral, fragmented or granular [21, 22]. Integral bacilli are considered viable, that is, they are related to host susceptibility to the parasite. These bacilli exhibit cell structure with preserved ends and uniform staining and are seen in smears of patients who are either non-treated or have relapse. Both fragmented and granular bacilli present flaws in the cell wall structure, being considered unviable or dead and more observed in posttreatment patients [17–22].

Bacilloscopy is effective when associated with the results of clinical exams [7, 15]. However, it is an exam that presents low sensitivity, since 50% of the smears of the sick individuals are negative. In addition, the exam requires adequate laboratory infrastructure and trained professional apparatus, factors that are not always a reality in hyperendemic regions and where medical and financial resources are reduced [7].

Histopathology is commonly performed for the diagnosis of diseases caused by obligate intracellular parasites and has good indices of sensitivity and specificity in the detection of leprosy [23]. However, the method encounters issues related to cost, time of analysis and false-negative results, besides of being an invasive exam [24]. Exactly for these reasons, histopathology is only recommended for individuals where it is impossible to assess degrees of cutaneous sensitivity, such as in children, elderly and mentally handicapped people; when it is not possible to classify the dimorphic clinical form; or when there is uncertainty whether the diagnosis is leprosy or other diseases that cause local hypoesthesia [23]. The diagnosis using this exam also depends on the association with the clinical aspects and bacilloscopy. In addition, biopsies of peripheral nerve branches are not recommended and should only be performed in the last instance [23, 24].

The Mitsuda test or Mitsuda reaction does not present diagnostic value, but it is an alternative prognostic tool that assists in assessing susceptibility to lepromatous forms [27]. It is an exam based on the use of heat-killed bacilli (lepromin), derived from extract of the inactivated "leprosy bacillus" under the skin of LL patients. The test consists of inoculating the Mitsuda antigen intradermally and reading them between days 21 and 28 after the challenge in order to analyze the late cellular response of patients [8, 25].

In addition, there's still a great limitation in obtaining the native molecule, restricted to the growth of *M. leprae* in armadillos. As an alternative, several synthetic analogous molecules associated with the tri or disaccharides of PGL-1 have been produced from the conjugation of these elements with BSA (Bovine serum albumin) and Phenol (P) or Octyl (O). The literature shows several semisynthetic analogues, among which the most well known are: monosaccharide-octyl-BSA (MO-BSA), disaccharide-BSA (D-BSA), natural disaccharide-octyl-BSA, natural octyl-HSA (ND-O -BSA and ND-O-HSA), natural trisaccharide-phenol-BSA (NT-P-BSA)

Molecular and Biotechnological Approaches in the Diagnosis of Leprosy

http://dx.doi.org/10.5772/intechopen.75506

67

A study carried out in the hyperendemic region of the Brazilian Amazon points to the potential of synthetic molecules to identify new cases of leprosy and, similar to glycolipid, they have good detection rates in multibacillary patients. In addition, research shows that the molecules exhibit behavior related to the spectral immunology of the disease, where the LL pole has a higher antibody titer that decays at the boderline and tuberculoid poles [35]. When comparing two molecules derived from PGL-1 in the region, it was possible to observe that NT-P-BSA was very effective in the monitoring of home contacts and MB patients, whereas ND-O-BSA

ELISA requires skilled labor and specific equipment which is not always available. Therefore, the use of serological tests of both the native molecule and synthetic derivatives is important to validate increasingly adequate methods for the seroepidemiology of both endemic and hyperendemic regions, besides helping to standardize the positivity indices of the clinical

If the characterization of PGL-1 was important for the synthesis of several derived molecules with similar immunological aspects, the decoding of the *M. leprae* genome was essential for the identification of proteins and peptides with applicability in the laboratory detection of the disease [38]. Several advantages are associated with the use of these molecules, especially for reducing the cost of the assays and reflecting the spectral character of leprosy immunology. Thus, assays using recombinant proteins indicate high levels of antibodies in LL patients that decay in patients of the boderline and tuberculoid poles [38–43].Currently, there are a large number of *M. leprae* recombinant proteins, normally identified as ML, and several studies have evaluated the potential of these proteins [44]. In 2007, a survey conducted in Brazil, the Philippines and Japan evaluated the cellular and humoral response to 33 recombinant proteins across a broad population spectrum and identified three proteins (ML0405, ML2055 and ML2331) capable of inducing the humoral response in multibacillary (MB), production of IgG, as well as the cellular response in PB. Comparison between sites identified differential response patterns between populations, however, in all locations ML0405 and ML2331

Due to the potential of these two proteins, the Leprosy IDRI Diagnostic protein (LID-1) was generated, resulting from the fusion between the two MLs. LID-1 was produced in order to maintain the reactivity profile of both proteins and was subsequently evaluated in several populations of Japan, Brazil, Venezuela, the Philippines and Nepal. The results pointed to the

obtained better sensitivity and specificity indices in paucibacillary individuals [37].

which are used as antigens in immunodiagnosis [30, 36].

forms that can vary intensely from one area to another.

showed similar results to PGL-1 serology in MB33 patients [40].

**3.2. ELISA with recombinant proteins**

Mitsuda's reaction has good agreement when related to bacilloscopy. Typically, individuals with a diameter reaction greater than 10 mm are considered resistant; they do not get sick or develop the TT shape, being Mitsuda positive [24]. While reactions with a diameter between 3 and 5 mm are indicative of dimorphic leprosy and, below this value, the test indicates anergy of the host's immune system to the bacillus, associated with patients in the LL pole, being Mitsuda negatives [8, 24, 26]
