**4. Complementary molecular tests**

potential of early detection of the disease using this protein, besides the possibility of its immo-

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

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.

contributing to the generation of accurate diagnoses and quality.

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,

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.

bilization in different platforms [40].

68 Hansen's Disease - The Forgotten and Neglected Disease

the diagnosis of the disease [45].

**3.3. Lateral flow tests**

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

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

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 diagnosis [62].

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 determination of resistance to treatment [14].

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

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]
