**3. Complementary immunological tests**

The discovery of the lipid apparatus present in the bacillus capsule and the characterization of a range of important lipidic and proteic components in the immunogenicity [30] allowed innovations in the leprosy serology [27].

Serological techniques are based on the detection of specific antibodies against the bacillus, since immunoglobulin production occurs in response to the antigenic signal of stimulation. These tests are important because they represent a class of complementary tests capable of detecting leprosy cases, besides the possibility of diagnosis recommendation, disease prevalence determination, infection evaluation in endemic and hyperendemic areas, and household contacts monitoring.

#### **3.1. ELISA with native PGL-1 and its synthetic molecules**

Many researches have used natural *M. leprae* antigens for the immunodiagnosis of leprosy [19, 28]. The elucidation of the structure of the PLG-1-the first *M. leprae* specific antigen to be isolated and the main antigenic glycolipid of the bacillus [29]-is a clear example of the widespread use of these molecules.

ELISA (enzyme-linked immunosorbent assay) has been widely used as a research tool for the detection of anti-PGL-1-native antibodies [29–32]. The technique consists of a quantitative test based on the IgM class antibodies response. In this scenario, important aspects in the biology and epidemiology of the disease, such as the determination and comparison of the positivity of patients and home contacts in several areas, besides the fluctuations in the reactivity profile in individuals from the hyperendemic area [33, 34] have been described from the studies with PGL-1.

Anti-PGL-1 antibodies are present in large numbers in untreated multibacillary patients, but paucibacillary patients naturally have low circulating antibody concentrations. For this reason, some of these patients present negative results against the diagnosis, even showing positive clinical signs [35, 36].

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

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

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 forms that can vary intensely from one area to another.

#### **3.2. ELISA with recombinant proteins**

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

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

The discovery of the lipid apparatus present in the bacillus capsule and the characterization of a range of important lipidic and proteic components in the immunogenicity [30] allowed

Serological techniques are based on the detection of specific antibodies against the bacillus, since immunoglobulin production occurs in response to the antigenic signal of stimulation. These tests are important because they represent a class of complementary tests capable of detecting leprosy cases, besides the possibility of diagnosis recommendation, disease prevalence determination, infection evaluation in endemic and hyperendemic areas, and house-

Many researches have used natural *M. leprae* antigens for the immunodiagnosis of leprosy [19, 28]. The elucidation of the structure of the PLG-1-the first *M. leprae* specific antigen to be isolated and the main antigenic glycolipid of the bacillus [29]-is a clear example of the wide-

ELISA (enzyme-linked immunosorbent assay) has been widely used as a research tool for the detection of anti-PGL-1-native antibodies [29–32]. The technique consists of a quantitative test based on the IgM class antibodies response. In this scenario, important aspects in the biology and epidemiology of the disease, such as the determination and comparison of the positivity of patients and home contacts in several areas, besides the fluctuations in the reactivity profile in individuals from the hyperendemic area [33, 34] have been described from the studies with PGL-1. Anti-PGL-1 antibodies are present in large numbers in untreated multibacillary patients, but paucibacillary patients naturally have low circulating antibody concentrations. For this reason, some of these patients present negative results against the diagnosis, even showing positive

to analyze the late cellular response of patients [8, 25].

66 Hansen's Disease - The Forgotten and Neglected Disease

**3. Complementary immunological tests**

**3.1. ELISA with native PGL-1 and its synthetic molecules**

innovations in the leprosy serology [27].

hold contacts monitoring.

spread use of these molecules.

clinical signs [35, 36].

negatives [8, 24, 26]

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

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 potential of early detection of the disease using this protein, besides the possibility of its immobilization in different platforms [40].

**4. Complementary molecular tests**

diagnosis [62].

tion of resistance to treatment [14].

DNA in contacts of leprosy patients [48, 67–69].

After the advent of the genome sequencing of the *M. leprae* bacterium [53], species-specific genetic sequences have been searched in order to standardize diagnostic tests based on DNA analysis. These sequences can be amplified through the PCR technique, which allows the

Molecular and Biotechnological Approaches in the Diagnosis of Leprosy

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

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The first works using the PCR technique were performed a little over 20 years, but the data were not satisfactory for the identification of *M. leprae* DNA in paucibacillary (PB) patients [54]. Thus, the methodology of molecular biology began to be used as an alternative method to traditional diagnostic methods. These data stimulated the search for new specific sequences

Many studies have been carried out involving different sequences and target genes, with the aim of increasing sensitivity and specificity in the identification of bacillus, especially in patients with low bacillary load. The literature reports the use of sequences that amplify gene regions encoding the 36 kDa [56], 18-kDa [57] and 65-kDa antigens [58], complex 85 [59], 16S rDNA [60] as well as for repetitive sequences of *M. leprae* (RLEP) [61]. By comparing these sequences, RLEP has been shown to be more sensitive and more specific than the bacilloscopic index. This could be explained by the number of copies, estimated to be at least 28 units, of the RLEP sequence in the *M. leprae* genome. In addition, this sequence generates a 130pb amplicon, which is considerably small compared to the sequences mentioned above, that is an important factor in the best efficiency in conventional PCR. Having a specific sequence is of great importance, since the PCR technique may be useful in the differential dermatological

A significant advance in increasing bacillus identification occurred with the use of real-time PCR technology. This methodology has been used in the follow-up of leprosy patients undergoing treatment [63] evaluation of bacterial load [13] viable bacterial load [60] and determina-

In clinical practice, detection of *M. leprae* by PCR in patients with negative bacilloscopy or inconclusive histopathology is of great value to define the correct diagnosis and treatment scheme [64]. In the same way, the methodology can be useful, for those patients with the pure neural form (PNL), who usually do not have cutaneous lesions and because of this they have deficient treatment scheme [65, 66]. Further, the PCR technique may be useful in early identification, since a considerable number of studies have addressed the positivity of *M. leprae*

In a study carried out in a hyperendemic area in cases of leprosy, it was possible to identify DNA from the bacterium in buccal and nasal swab samples in individuals with subclinical infection with multibacillary or paucibacillary index cases [48]. The identification of DNA from the Hansen bacillus in buccal and nasal swab raises considerations about the participation of this risk group in the transmission chain, besides the route of infection of the bacillus [70]. Although PCR can be a useful tool for identification, few studies associate the presence of *M. leprae* DNA to the development of the disease [71, 72], highlighting the importance of the use of serological tools and the follow-up of patients with subclinical infection [73, 69]

for the identification of the bacillus, as well as the use of several clinical samples [55].

detection of bacillus DNA from small amounts of *M. leprae* cells [12].

Also in this scenario, in 2009 a chimeric protein with multiple epitopes (PADL), from the fusion of epitopes of recombinant proteins (ML0405, ML0049, ML0050, ML0091, ML0411, ML2055 and ML2311) was designed. The chimeric molecule was tested in serum from pauci and multibacillary patients living in Brazil and the endemic controls from the Philippines. The results demonstrated that all the portions that formed the protein have specific binding capacity to antibodies and the same showed great effectiveness in the diagnosis of MB patients and no specific response to the serum of the endemic controls, showing promise in the diagnosis of the disease [45].

#### **3.3. Lateral flow tests**

In addition to the ELISA immunoassays, leprosy serology may also be performed with the lateral flow test, known as ML-flow. The development of this immunochromatographic semiquantitative assay was due, in particular, to the possibility of field use. The test was developed by Burker-Sékula et al. [46], aiming the detection of IgM antibodies against several antigenic molecules such as PGL-1 and its synthetic derivatives, recombinant proteins and peptides [30].

The ML-flow test is not a diagnostic method, but assists in the classification of patients and presents low cost and easy execution, making its use possible in health services routine, especially in regions where laboratory resources are not available [30, 35, 46, 47]. Therefore, ML-flow is a methodology widely used in hyperendemic areas, especially in Brazil, where populations of Maranhão, São Paulo, Pará and Minas Gerais states were tested and reaffirm the importance of detection and control of disease cases through simple but reliable methodologies [32, 48–52].

In the search for increasingly fast and accurate tests, in 2012 the Brazilian Institute of Infectious Research together with the company OrangeLife developed a test capable of offering a diagnosis in only 10 minutes using only one drop of blood of the patient. The tool has received approval from the Brazilian National Sanitary Surveillance Agency (ANVISA) and has been field tested to evaluate the potential of the new platform. The assay is based on immunochromatography aspects, where recombinant proteins like LID-1, used as antigen, are immobilized on nitrocellulose membranes. Detection can be performed by the presence of IgM and IgG antibodies from various samples such as whole blood, plasma and serum.

Among the advantages of the test, it is mentioned the low cost of the tool, the possibility of early detection, agile and minimally invasive. In addition, it is associated with software stored on smartphones, further facilitating the interpretation of the exam and ensuring the availability of information in databases. Parallel analyzes of the rapid detection tool show the ability to diagnose, in most cases, the presence of the infection before clinical symptoms appear, contributing to the generation of accurate diagnoses and quality.

The interdisciplinary researches related to leprosy provided an amount of laboratory tools used as alternative methodologies for the more accurate and efficient diagnosis of the disease. Although there are still difficulties linked to the detection of paucibacillary forms, subclinical infections and contact monitoring, all research reinforces the importance of the search and use of efficient platforms and able to ally reduced cost and good indexes of sensitivity and specificity for the disease.
