Open access peer-reviewed chapter

Molecular and Biotechnological Approaches in the Diagnosis of Leprosy

Written By

Mayara Ingrid Sousa Lima, Emilly Caroline dos Santos Moraes, Jaqueline Diniz Pinho, Gustavo Henrique Corrêa Soares and Ítalo Vinícius Cantanhêde Santos

Submitted: 29 March 2017 Reviewed: 15 February 2018 Published: 06 February 2019

DOI: 10.5772/intechopen.75506

From the Edited Volume

Hansen's Disease - The Forgotten and Neglected Disease

Edited by Wellman Ribòn

Chapter metrics overview

1,267 Chapter Downloads

View Full Metrics

Abstract

Leprosy is a worldwide health problem, which needs the development of new and innovative strategies to be controlled. Early diagnosis of leprosy is an important contribution to reducing the incidence of the disease; thus, the development of biotechnology platforms, which include the mapping of antigens with potential to be used in immunodiagnostic and molecular methods for the detection of Mycobacterium leprae, is an important tool to confirm the clinical diagnostic. Molecular biology and biotechnological methods have been used to assist in the diagnosis of this disease, each one with its advantages and drawbacks. Enzyme-linked immunosorbent assay (ELISA) is the used method for leprosy diagnosis, and it allows the detection of infection-related antigens. Alternatively, due to their versatility to perform the same functions as the protein and non-protein natural antigens, mimetic peptides are considered an important tool. On the other hand, lateral flow assay (LFA) and optical and electrochemical biosensors are rapid and portable methods, capable of performing diagnosis in the field without sample preparation. This chapter presents such techniques, their uses in the diagnosis and detection of M. leprae, as well as the potential for the development of new techniques and strategies that can help to control and understand mycobacteriosis.

Keywords

  • Mycobacterium leprae
  • immunoassays
  • molecular tests
  • biomarkers

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.

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.

Advertisement

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

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, 20, 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-treatment patients [17, 18, 19, 20, 21, 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].

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]

Advertisement

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, 30, 31, 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

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, 39, 40, 41, 42, 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].

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, 49, 50, 51, 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.

Advertisement

4. Complementary molecular 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, 68, 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]

Advertisement

5. New biotechnological tools in the diagnosis of leprosy

5.1. Mapping of new markers

Many studies have used post genomic procedures for the discovery of new antigens that can be used in the diagnosis of leprosy [71, 72, 73, 74, 75]. These studies have explored sequences of M. leprae for the identification of proteins or peptides that can be used in the serodiagnosis of the different clinical forms of leprosy [76].

The antigens ML0405, ML2331 and ML2055—the first two of previously unknown function and the latter a membrane protein - were used for serological tests in in multibacillary patients of the clinical forms boderline lepromatous (BL) and LL untreated [77]. The ML0308 and ML2498 proteins, a conserved hypothetical protein and an enoyl-CoA hydratase respectively, showed humoral and cellular immunogenicity and can be used in the diagnosis of tuberculoid and lepromatous forms [78]. These antigens were used in the production of fusion proteins, such as LID-1 (leprosy IDRI diagnostic-1) [42] and PADL [45, 79].

The tools of bioinformatics, genomic analysis and proteomics are also being used for mapping in silico of important antigenic targets of M. leprae [80]. This type of analysis was used to define a group of 50 potential antigens in mycobacteria, some being restricted to M. leprae [78].

Peptides derived from specific and immunogenic proteins of M. leprae have also been tested in patients with leprosy and controls [81]. Peptides obtained of proteins from M. leprae were promising as indicators of exposure [82].

The peptides are small in size and can be expressed on the surface of bacteriophage to select peptides that mimic different targets (pathogens, cellular receptors or antibodies) [83]. Mimetic peptides may have important applications in the diagnosis of leprosy, mimicking antigens such as PGL-1 [84] or other M. leprae antigens [85, 86]. Alternatively, due to their versatility to perform the same functions as the protein and non-protein natural antigens, mimetic peptides are considered an important tool in immunodiagnostic of infectious disease.

5.2. Biosensors as platforms for the diagnosis of leprosy.

The post-genomic, the identification and obtainment of hundreds of molecules with immunogenic potential have broadened the versatility of detection platforms and contributed to an optimal diagnostic test, especially for tropical diseases [87]. In recent decades, biosensors have been gaining more space in scientific research and diagnosis of various diseases [88].

Biosensors are analytical devices that have specific reactions and/or specific interactions mediated by a diversity of components (antigens, antibodies, enzymes, DNA fragments, organelles, receptors and even mimetic peptides) that, in contact with a transducer, have the conversion of a biological signal‑a result of the interaction between specific components‑in a measurable signal proportional to the analyte concentration [89]. These platforms can be electrochemical, piezoelectric, thermal, optical and based on surface plasmon resonance, depending on the type of transducer used [90]. There are still specific classes of biosensors such as immunosensors [91], which evaluate interactions between antibodies and antigens, and genosensores, based on the hybridization of DNA-specific ribbons [87].

There is a wide range of studies showing the efficacy of biosensors for the detection of various diseases such as leishmaniasis, bacterial diseases, cystic fibrosis, dengue and leprosy itself [92, 93, 94, 95, 96]. In Brazil, a genosensor for M. leprae was constructed using the immobilization of a bacillus single‑stranded DNA (ssDNA) on functionalized graphite electrodes. The interaction between the immobilized sequence and M. leprae double‑stranded DNA (dsDNA) is measured electrochemically by reductions in the peak oxidation current and using ferrocenecarboxyaldehyde as the hybridization indicator. The result was very promising, showing efficient detection in only 3 minutes [87, 88, 96].

Currently, Brazilian research groups are betting on the use of electrochemical biosensors as an indispensable tool in the diagnosis and control of diseases. This innovation is mainly because these sensors are sensitive, reliable, fast response and operate in conditions that pre-treat the samples. In addition, these techniques are capable of providing exceptionally low detection limits.

In this scenario, mimetic peptides of proteins and glycolipids present in the bacillus capsule have been validated for the immunogenic potential and immobilized in these electrochemical detection platforms. Thus, the proposals consist of using different biological fluids such as blood, secretion and saliva, ensuring a less invasive and more comfortable test to the patient and the manipulator. Subsequently, these platforms will be tested in hyperendemic areas, in order to evaluate their detection potential and help in the epidemiological control of the disease.

Advertisement

6. Conclusion

The early diagnosis of leprosy is one of the goals of the WHO for the control and reduction of new cases of the disease. This strategy will be implemented with the development of new diagnostic tools more sensitive and can be applied in large-scale monitoring. Molecular techniques and new biotechnological approaches can be used as complementary tests.The qualitative PCR, RLEP and real time PCR have been used for the detection of M. leprae in samples of different tissues of patients or of household contacts.

Immunodiagnosis can be done using different native M. leprae antigens such as PGL-1, LAM or their synthetic derivatives. Post-genomic technologies can be used for the production of recombinant chimeric proteins, peptides obtained in silico or mimetic peptides. Immunodiagnosis can be performed by ELISA, lateral flow tests and biological sensors.

Biotechnology and molecular biology have contributed to the development of research and improve the diagnosis of leprosy. Significant advances in laboratory diagnosis contribute to improving clinical practice.

References

  1. 1. Goulart B, Penna O, Cunha G. Imunopatologia da hanseníase: a complexidade dos mecanismos da resposta imune do hospedeiro ao Mycobacterium leprae. Revista da Sociedade Brasileira de Medicina Tropical. 2002;35:363-375. DOI: 10.1590/S0037-86822002000400014
  2. 2. Alberts J, Smith C, Meima A, Wang L, Richardus H. Potential effect of the World Health Organization’s 2011-2015 global leprosy strategy on the prevalence of grade 2 disability: A trend analysis. Bulletin of the World Health Organization. 2011;89:487-495. DOI: 10.2471/BLT.10.085662
  3. 3. World Health Organization, Department of Control of Neglected Tropical Diseases. Global leprosy update, 2013; reducing disease burden. Weekly Epidemiological Record. 2014;89:389-400
  4. 4. World Health Organization SEARO, Department of Control of Neglected Tropical Diseases. Enhanced global strategy for further reducing the disease burden due to leprosy: 2011-2015. Leprosy Review. 2009;80:353-354
  5. 5. World Health Organization. Department of Control of neglected tropical diseases. Global leprosy: Update on the 2012 situation. Weekly Epidemiological Record. 2013;88:365-379
  6. 6. Penna G, Pereira G, Moreira M. Guia Para o Controle da Hanseníase-Cadernos da Atenção Básica n° 10. 1st ed. Brasília: Ministério da Saúde; 2002. p. 90
  7. 7. International Leprosy Association. The diagnosis and classification of leprosy. International Journal of Leprosy. 2002;23-31(2002):70
  8. 8. Mitsuda K. On the value of a skin reaction to a suspension of leprous nodules. International Journal of Leprosy. 1953;21(3):347-358
  9. 9. Cabral E, Junior C, Alexandre C, Alexandre A, Thially B, Tereza C. Anti-PGL1 salivary IgA/IgM, serum IgG/IgM, and nasal Mycobacterium leprae DNA in individuals with household contact with leprosy. International Journal of Infectious Diseases. 2013;17(11):1005-1010. DOI: 10.1016/j.ijid.2013.05.011
  10. 10. Hussain R, Shahid F, Zafar S, Dojki M, Dockrell M. Immune profiling of leprosy and tuberculosis patients to 15-mer peptides of Mycobacterium leprae and M. tuberculosis GroES in a BCG vaccinated area: Implications for development of vaccine and diagnostic reagents. Immunology. 2004;111(4):462-471. DOI: 10.1111/j.0019-2805.2004.01839.x
  11. 11. Lobato J, Costa P, Reis E, Gonçalves A, Spencer J, Brennan J. Comparison of three immunological tests for leprosy diagnosis and detection of subclinical infection. Leprosy Review. 2011;82(4):389-401
  12. 12. Hartskeerl A, De Wit Y, Klatser R. Polymerase chain reaction for the detection of Mycobacterium leprae. Journal of General Micorbiology. 1989;135(9):2357-2364. DOI: 10.1099/00221287-135-9-2357
  13. 13. Azevedo C, Ramuno M, Fachin R, Tassa M, Rosa S, Belone F. qPCR detection of Mycobacterium leprae in biopsies and slit skin smear of different leprosy clinical forms. The Brazilian Journal of Infectious Diseases. 2017;21(1):71-78. DOI: 10.1016/j.bjid.2016.09.017
  14. 14. Araujo S, Goulart R, Truman W, Goulart B, Vissa V, Li W. qPCR-high resolution melt analysis for drug susceptibility testing of Mycobacterium leprae directly from clinical specimens of leprosy patients. PLoS Neglected Tropical Diseases. 2017;11(6):e0005506. DOI: 10.1371/journal.pntd.0005506
  15. 15. Nath I, Saini C, Valluri L. Immunology of leprosy and diagnostic challenge. Clinical Dermatology. 2015;33(1):90-98. DOI: 10.1016/j.clindermatol.2014.07.005
  16. 16. Martelli T, Stefani A, Penna O, Andrade S. Endemias e epidemias brasileiras, desafios e perspectivas de investigação científica: hanseníase. Revista Brasileira de Epidemiologia. 2002;5(3):220-225. DOI: 10.1590/S1415-790X2002000300006
  17. 17. BRAZIL. Ministry of Health. Guide to Technical Procedures Bacilloscopy in Leprosy. Brasília; 2010
  18. 18. Stretch R. Presentation and treatment of Hansen’s disease. Nursing Times. 1999;95(29):46-47
  19. 19. ARAUJO G. Hanseníase no Brasil. Revista da Sociedade Brasileira de Medicina Tropical. 2003;36(3):373-382
  20. 20. BRAZIL. Ministry of Health. Secretariat of Health Policies. Department of Basic Attention. Technical Area of Sanitary Dermatology. Legislation on the Control of Leprosy in Brazil. Brasília: Ministry of Health; 2002
  21. 21. Leiker DL, McDougall AC. Guia técnico baciloscopia da hanseníase. 2nd ed. Wurzburg: DAHW; 1987
  22. 22. Baptista D, Sartori S, Trino M. Guia de conduta para realização do exame baciloscópico. Hansenologia Internationalis. 2006;31(2):39-41
  23. 23. Ura S, Barreto A. Papel da biópsia cutânea no diagnóstico de hanseníase. Hansenologia Internationalis. 2004;9(2):141-144
  24. 24. Alves ED, Ferreira TL, Fereira IN, editors. Hanseníase, avanços e desafios. 1st Editors. Brasília: NESPOM; 2014. 492p. ISBN 978-85-64593-22-0
  25. 25. Scollard D, Adams L, Gillis T, Krahenbuhl J, Truman R, Williams D. The continuing challenges of leprosy. Clinical Microbiology Reviews. 2006;19(2):338-381. DOI: 10.1128/CMR.19.2.338-381.2006
  26. 26. Opromolla D. Noções de Hansenologia. 1st ed. Bauru: Centro de Estudos Dr Reynaldo Quagliato; 2000. pp. 1-123
  27. 27. Bührer-Sékula S. Sorologia PGL-1 na hanseníase. Revista da Sociedade Brasileira de Medicina Tropical. 2008;41(2):3-5. DOI: 10.1590/S0037-86822008000700002
  28. 28. Geluk A, Spencer J, Bobosha K, Pessolani M, Pereira G, Banu S, Honoré N, Reece S, MacDonald M, Sapkota B, Ranjit C, Franken K, Zewdie M, Aseffa A, Hussain R, Stefani M, Cho S, Oskam L, Brennan P, Dockrell H. From genome-based in silico predictions to ex vivo verification of leprosy diagnosis. Clinical and Vaccine Immunology. 2009;16(3):352-359. DOI: 10.1128/CVI.00414-08
  29. 29. Hunter S, Brennan PA. A novel phenolic glycolipid from Mycobacterium leprae possibly involved in immunogenicity and pathogenicity. Journal of Bacteriology. 1981;147(3):728-735
  30. 30. Baumgart K, Britton W, Basten A, Bagshawe A. Use of phenolic glycolipid I for serodiagnosis of leprosy in a high prevalence village in Papua New Guinea. Transactions of the Royal Society of Tropical Medicine and Hygiene. 1987;81(6):1030-1032. DOI: 10.1016/0035-9203(87)90388-9
  31. 31. Brasil M, Oliveira L, Rímoli N, Cavallari S, Gonçalves O, Lessa Z, Rotta O. Anti PGL-1 serology and the risk of leprosy in a highly endemic area in the state of São Paulo, Brazil: Four-year follow-up. Revista Brasileira de Epidemiologia. 2003;6(3):262-271. DOI: 10.1590/S1415-790X2003000300010
  32. 32. Cunha M, Silvestre M, Queiroz M, Xavier M. Profile of anti-PGL-1 antibodies in healthy people from endemic areas of leprosy in Pará state, BraziL. Revista Pan-Amazônica de Saúde. 2012;3(3):41-47. DOI: 10.5123/S2176-62232012000300005
  33. 33. Fine P et al. Seroepidemio-logical studies of leprosy in northern Malawii, based on na enzyme-linked immunosorbent assay using synthetic glycoconjugate antigen. International Journal of Leprosy. 1988;6:243-225
  34. 34. Krishnamurthy P et al. Seroepidemiological study of leprosy in a highly endemic population of South India based on Elisa using synthetic PGL-1. International Journal of Leprosy. 1991;59:426-431
  35. 35. Moura R, Calado K, Oliveira M, Bührer-Sékula S. Leprosy serology using PGL-I: A systematic review. Revista da Sociedade Brasileira de Medicina Tropical. 2008;41(2):11-18. DOI: 10.1590/S0037-86822008000700004
  36. 36. Oskam L, Slim E, Bührer-Sékula S. Serology: Recent developments, strengths, limitations and prospects: A state of the art overview. Leprosy Review. 2003;74(3):196-205
  37. 37. Stefani M. Challenges in the post genomic era for the development of tests for leprosy diagnosis. Revista da Sociedade Brasileira de Medicina Tropical. 2008;41(2):89-94. DOI: 10.1590/S0037-86822008000700018
  38. 38. Araóz R, Honoré N, Banu S, Demangel C, Cissoko Y, Arama C, Uddin M, Hadi S, Monot C, Cho SN, Ji B, Brennan P, Sow S, Cole S. Towards an immunodiagnostic test for leprosy. Microbes and Infection. 2006;6(6):2270-2276. DOI: 10.1016/j.micinf.2006.04.002
  39. 39. Corstjens P, de Dood C, van der Ploeg-van Schip J, Wiesmeijer K, Riuttamäki T, van Meijgaarden K, Spencer JS, Tanke H, Ottenhoff T, Geluk A. Lateral flow assay for simultaneous detection of cellular- and humoral immune responses. Clinical Biochemistry. 2011;44(14-15):1241-1246. DOI: 10.1016/j.clinbiochem.2011.06.983
  40. 40. Duthie M, Truman R, Goto W, O’Donnell J, Hay M, Spencer J, Carter D, Reed S. Insight toward early diagnosis of leprosy through analysis of the developing antibody responses of Mycobacterium leprae-infected armadillos. Clinical and Vaccine Immunology. 2011;18(2):254-259. DOI: 10.1128/CVI.00420-10
  41. 41. Duthie M, Goto W, Ireton G, Reece S, Cardoso L, Martelli C, Stefani M, Nakatani M, Jesus R, Netto E, Balagon M, Tan E, Gelber R, Maeda Y, Makino M, Hoft D, Reed S. Use of protein antigens for early serological diagnosis of leprosy. Clinical and Vaccine Immunology. 2007;14(11):1400-1408. DOI: 10.1128/CVI.00299-07
  42. 42. Reece S, Ireton G, Mohamath R, Guderian J, Goto W, Gelber R, Groathouse N, Spencer J, Brennan P, Reed S. ML0405 and ML2331 are antigens of Mycobacterium leprae with potential for diagnosis of leprosy. Clinical and Vaccine Immunology. 2006;13(3):333-340. DOI: 10.1128/CVI.13.3.333-340.2006
  43. 43. Spencer J, Brennan P. The role of Mycobacterium leprae phenolic glycolipid I(PGL-I) in Serodiagnosis and in the pathogenesis of leprosy. Leprosy Review. 2011;82:344-357
  44. 44. Duthie M, Ireton G, Kanaujia G, Goto W, Liang H, Bhatia A, Busceti J, MacDonald M, Neupane K, Ranjit C, Sapkota B, Balagon M, Esfandiari J, Carter D, Reed S. Selection of antigens and development of prototype tests for point-of-care leprosy diagnosis. Immunology. 2008;15(10):1590-1597. DOI: 10.1128/CVI.00168-08
  45. 45. Duthie M, Hay M, Morales C, Carter L, Mohamath R, Ito L, Oyafuso L, Manini M, Balagon M, Tan E, Saunderson P, Reed S, Carter D. Rational design and evaluation of a multiepitope chimeric fusion protein with the potential for leprosy diagnosis. Clinical and Vaccine Immunology. 2010;17(2):298-303. DOI: 10.1128/CVI.00400-09
  46. 46. Bührer-Sékula S, Smits H, Gussenhoven G, van Leeuwen J, Amador S, Fujiwara T, Klatser P, Oskam L. Simple and fast lateral flow test for classification of leprosy patients and identification of contacts with high risk of developing leprosy. Journal of Clinical Microbiology. 2003;41(5):1991-1995. DOI: 10.1128/JCM.41.5.1991-1995.2003
  47. 47. Martinez T, Figueira M, Costa A, Gonçalves M, Goulart L, Goulart I. Oral mucosa as a source of Mycobacterium leprae infection and transmission, and implications of bacterial DNA detection and the immunological status. Clinical Microbiology and Infection. 2010;17:1653-1658. DOI: 10.1111/j.1469-0691.2010.03453.x
  48. 48. Pinho J, Rivas P, Bonfim M, Soares R, Costa G, Nascimento F, Paiva M, Aquino D, Figueireido I, Santos A, Pereira S. Presence of Mycobacterium leprae DNA and PGL-1 antigen in household contacts of leprosy patients from a hyperendemic area in Brazil. Genetics and Molecular Research. 2015;14(4):14479-14487. DOI: 0.4238/2015.November.18.10
  49. 49. Contin L, Alves C, Fogagnolo L, Nassif P, Barreto J, Lauris J, Nogueira M. Uso do teste ML-Flow como auxiliar na classificação e tratamento da hanseníase. Anais Brasileiros de Dermatologia. 2011;86(1):91-95
  50. 50. Gonçalves MC, Queiroz MFA, Martins LC, Moura AA, Franco ACA, Xavier MB. Assessing serology tests for leprosy complementary diagnosis. Rev Pan-Amaz Saude. 2014;5(4):23-28. DOI: 10.5123/S2176-62232014000400003
  51. 51. Ferreira IN, Ferreira IL, Evangelista MS, Alvarez RR. The use of ML flow test in school children diagnosed with leprosy in the district of Paracatu, Minas Gerais. Revista da Sociedade Brasileira de Medicina Tropical. 2008;41(2):77-80. DOI: 10.1590/S0037-86822008000700016
  52. 52. Andrade AR, Grossi MA, Bührer-Sékula S, Antunes CM. Seroprevalence of ML flow test in leprosy contacts from state of Minas Gerais, Brazil. Revista da Sociedade Brasileira de Medicina Tropical. 2008;41(2):56-59. DOI: 10.1590/S0037-86822008000700012
  53. 53. Cole ST, Eiglmeier K, Parkhill J, James KD, Thomson NR, Wheeler PR, Honoré N, Garnier T, Churcher C, Harris D, Mungall K, Basham D, Brown D, Chillingworth T, Connor R, Davies RM, Devlin K, Duthoy S, Feltwell T, Fraser A, Hamlin N, Holroyd S, Hornsby T, Jagels K, Lacroix C, Maclean J, Moule S, Murphy L, Oliver K, Quail MA, Rajandream MA, Rutherford KM, Rutter S, Seeger K, Simon S, Simmonds M, Skelton J, Squares R, Squares S, Stevens K, Taylor K, Whitehead S, Woodward JR, Barrell BG. Massive gene decay in the leprosy bacillus. Nature. 2001;409(6823):1007-1011. DOI: 10.1038/35059006
  54. 54. Williams DL, Gillis TP, Booth RJ, Looker D, Watson JD. The use of a specific DNA probe and polymerase chain reaction for the detection of Mycobacterium leprae. The Journal of Infectious Diseases. 1990;162:193-200
  55. 55. Santos AR, De Miranda AB, Sarno EN, Suffys PN, Degrave WM. Use of PCR-mediated amplification of Mycobacterium leprae DNA in different types of clinical samples for the diagnosis of leprosy. Journal of Medical Microbiology. 1993;39:298-304. DOI: 10.1099/00222615-39-4-298
  56. 56. Kampirapap K, Singtham N, Klatser PR, Wiriyawipart S. DNA amplification for detection of leprosy and assessment of efficacy of leprosy chemotherapy. International Journal of Leprosy and Other Mycobacterial Diseases. 1998;66:16-21
  57. 57. Scollard DM, Gillis TP, Williams DL. Polymerase chain reaction assay for the detection and identification of Mycobacterium leprae in patients in the United States. American Journal of Clinical Pathology. 1998;109:642-646
  58. 58. Plikaytis BB, Gelber RH, Shinnick TM. Rapid and sensitive detection of Mycobacterium leprae using a nested-primer gene amplification assay. Journal of Clinical Microbiology. 1990;28:1913-1917
  59. 59. Martinez AN, Britto CF, Nery JA, Sampaio EP, Jardim MR, et al. Evaluation of real-time and conventional PCR targeting complex 85 genes for detection of Mycobacterium leprae DNA in skin biopsy samples from patients diagnosed with leprosy. Journal of Clinical Microbiology. 2006, 2006;44(9):3154-944: 3154-3159). DOI: 10.1128/JCM.02250-05
  60. 60. Martinez AN, Lahiri R, Pittman TL, Scollard D, Truman R, Moraes MO, Williams DL. Molecular determination of Mycobacterium leprae viability by use of real-time PCR. Journal of Clinical Microbiology. 2009;47:2124-2130. DOI: 10.1128/JCM.00512-09
  61. 61. Adams LB, Pena MT, Sharma R, Hagge DA, Schurr E, Truman RW. Insights from animal models on the immunogenetics of leprosy: A review. Memórias do Instituto Oswaldo Cruz. 2012;107(1):197-208. DOI: 10.1590/S0074-02762012000900028
  62. 62. Martinez NA, Talhari C, Moraes MO, Talhari S. PCR-based techniques for leprosy diagnosis: From the laboratory to the clinic. PLoS Neglected Tropical Diseases. 2014;8:e2655. DOI: 10.1371/journal.pntd.0002655
  63. 63. Martinez AN, Ribeiro-Alves M, Sarno EN, Moraes MO. Evaluation of qPCR-based assays for leprosy diagnosis directly in clinical specimens. PLoS Neglected Tropical Diseases. 2011;5:e1354. DOI: 10.1371/journal.pntd.0001354
  64. 64. Chaitanya VS, Cuello L, Das M, Sudharsan A, Ganesan P, Kanmani K, Rajan L, Ebenezer M. Analysis of a novel multiplex polymerase chain reaction assay as a sensitive tool for the diagnosis of indeterminate and tuberculoid forms of leprosy. International Journal of Mycobacteriology. 2017;6(1):1-8. DOI: 10.4103/2212-5531.201885
  65. 65. Jardim MR, Antunes SL, Santos AR, Nascimento OJ, Nery JA, Sales AM, Illarramendi X, Duppre N, Chimelli L, Sampaio EP, Sarno EP. Criteria for diagnosis of pure neural leprosy. Journal of Neurology. 2003;250:806-809. DOI: 10.1007/s00415-003-1081-5
  66. 66. Bezerra Da Cunha FM, Werneck MC, Scola RH, Werneck LC. Pure neural leprosy: Diagnostic value of the polymerase chain reaction. Muscle & Nerve. 2006;33:409-414. DOI: 10.1002/mus.20465
  67. 67. Araújo S, Lobato J, Reis Ede M, Souza DO, Gonçalves MA, Costa AV, Goulart LR, Goulart IM. Desvelando portadores saudáveis e infecções subclínicas entre os contatos familiares de pacientes com hanseníase que desempenham papéis potenciais na cadeia de transmissão da doença. Memórias do Instituto Oswaldo Cruz. 2012;107(Suppl. 1):55-59
  68. 68. Arunagiry K, Sangeetha G, Sugashini PK, Balaraman S, Showkath MK. Nasal PCR assay for the detection of Mycobacterium leprae pra gene to study subclinical infection in a community. Microbial Pathogenesis. 2017;104:336-339. DOI: 10.1016/j.micpath.2017.01.046
  69. 69. Romero-Montoya M, Beltran-Alzate JC, Cardona-Castro N. Evaluation and monitoring of Mycobacterium leprae transmission in household contacts of patients with Hansen’s disease in Colombia. PLoS Neglected Tropical Diseases. 2017;11(1):e0005325. DOI: 10.1371/journal.pntd.0005325
  70. 70. Araujo S, Freitas LO, Goulart LR, Goulart IM. Molecular evidence for the aerial route of infection of Mycobacterium leprae and the role of asymptomatic carriers in the persistence of leprosy. Clinical Infectious Diseases. 2016;63(11):1412-1420. DOI: 10.1093/cid/ciw570
  71. 71. Reis EM, Araujo S, Lobato J, Neves AF, Costa AV, Gonçalves MA, Goulart LR, Goulart IM. Mycobacterium leprae DNA in peripheral blood may indicate a bacilli migration route and high-risk for leprosy onset. Clinical Microbiology and Infection. 2014;20:447-452. DOI: 10.1111/1469-0691.12349
  72. 72. Goulart IM, Araujo S, Filho AB, de Paiva PH, Goulart LR. Asymptomatic leprosy infection among blood donors may predict disease development and suggests a potential mode of transmission. Journal of Clinical Microbiology. 2015;53:3345-3348. DOI: 10.1128/JCM.01305-15
  73. 73. Goulart IM, Bernardes Souza DO, Marques CR, Pimenta VL, Goncalves MA, Goulart LR. Risk and protective factors for leprosy development determined by epidemiological surveillance of household contacts. Clinical and Vaccine Immunology. 2008;15:101-105. DOI: 10.1128/CVI.00372-07
  74. 74. Bobosha K, Jolien S, Danuza E, Guimarães MM, Martins MV, Bekele Y, Fantahun Y, Aseffa A, Kees LMCF, Gismondi RC, Pessolani MCV, Ottenhoff THM, Pereira GMB, Geluk A. Peptides derived from Mycobacterium leprae ML1601c discriminate between leprosy patients and healthy endemic controls. Journal of Tropical Medicine. 2012:132049. DOI: 10.1155/2012/132049
  75. 75. Geluk A, Bobosha K, van der Ploeg-van Schip JJ, Spencer JS, Banu S, Martins MV, Cho SN, Franken KL, Kim HJ, Bekele Y, Uddin MK, Hadi SA, Aseffa A, Pessolani MC, Pereira GM, Dockrell HM, Ottenhoff TH. New biomarkers with relevance to leprosy diagnosis applicable in areas hyperendemic for leprosy. Journal of Immunology. 2012;188(10):4782-4791. DOI: 10.4049/jimmunol.1103452
  76. 76. Geluk A, Duthie MS, Spencer JS. Postgenomic Mycobacterium leprae antigens for cellular and serological diagnosis of M. leprae exposure, infection and leprosy disease. Leprosy Review. 2011;82(4):402-421
  77. 77. Duthie MS, Goto W, Ireton GC, Reece ST, Sampaio LH, Grassi AB, Sousa AL, Martelli CM, Stefani MM, Reed SG. Antigen-specific T-cell responses of leprosy patients. Clinical and Vaccine Immunology. 2008:1659-1665. DOI: 10.1128/CVI.00234-08
  78. 78. Aráoz R, Honoré N, Cho S, Kim JP, Cho SN, Monot M, Demangel C, Brennan PJ, Cole ST. Antigen discovery: A postgenomic approach to leprosy diagnosis. Infection and Immunity. 2006;74(1):175-182. DOI: 10.1128/IAI.74.1.175-182.2006
  79. 79. de Souza MM, Netto EM, Nakatani M, Duthie MS. Utility of recombinant proteins LID-1 and PADL in screening for Mycobacterium leprae infection and leprosy. Transactions of the Royal Society of Tropical Medicine and Hygiene. 2014;108(8):495-501. DOI: 10.1093/trstmh/tru093
  80. 80. Wiker HG, Tomazella GG, de Souza GA. A quantitative view on Mycobacterium leprae antigens by proteomics. Journal of Proteomics. 2011;74(9):1711-1719. DOI: 10.1016/j.jprot.2011.01.004
  81. 81. GELUK A, van der Ploeg J, Teles RO, Franken KL, Prins C, Drijfhout JW, Sarno EN, Sampaio EP, Ottenhoff TH. Rational combination of peptides derived from different Mycobacterium leprae proteins improves sensitivity for immunodiagnosis of M. leprae infection. Clinical and Vaccine Immunology. 2008;15(3):522-533. DOI: 10.1128/CVI.00432-07
  82. 82. Bobosha K, Tang ST, van der Ploeg-van Schip JJ, Bekele Y, Martins MV, Lund O, Franken KL, Khadge S, Pontes MA, Gonçalves Hde S, Hussien J, Thapa P, Kunwar CB, Hagge DA, Aseffa A, Pessolani MC, Pereira GM, Ottenhoff TH, Geluk A. Mycobacterium leprae virulence-associated peptides are indicators of exposure to M. leprae in Brazil, Ethiopia and Nepal. Memórias do Instituto Oswaldo Cruz. 2012;1:112-123. DOI: 10.1590/S0074-02762012000900018
  83. 83. Smith GP. Filamentous fusion phage: Novel expression vectors that display cloned antigens on the virion surface. Science. 1985;228(4705):1315-1317. DOI: 10.1126/science.4001944
  84. 84. Youn JH, Myung HJ, Liav A, Chatterjee D, Brennan PJ, Choi IH, Cho SN, Shin JS. Production and characterization of peptide mimotopes of phenolic glycolipid-I of Mycobacterium leprae. FEMS Immunology and Medical Microbiology. 2004;41(1, 1):51-57. DOI: 10.1016/j.femsim.2004.01.001
  85. 85. Alban S, Moura JF, Minozzo JC, Mira MT, Soccol VT. Identification of mimotopes of Mycobacterium leprae as potential diagnostic reagents. BMC Infectious Diseases. 2013;13:42. DOI: 10.1186/1471-2334-13-42
  86. 86. Alban SM, Moura JF, Thomaz-Soccol V. Phage display and synthetic peptides as promising biotechnological tools for the serological diagnosis of leprosy. PLoS One. 2014. DOI: 10.1371/journal.pone.0106222
  87. 87. Teles FSR, Tavira LAP, Fonseca LP. Biosensors as rapid tests for tropical diseases. Critical Reviews in Clinical Laboratory Sciences. 2010;47(3):139-169. DOI: 10.3109/10408363.2010.518405
  88. 88. Goulart LR, Vieira CU, Freschi AP, Capparelli FE, Fujimura PT, Almeida JF, Ferreira LF, Goulart IM, Brito-Madurro AG, Madurro JM. Biomarkers for sérum diagnosis of infectious diseases and their potential application in novel sensor platforms. Critical Reviews in Immunology. 2010;30(2):201-222. DOI: 10.3109/10408363.2010.518405
  89. 89. Gronow M. Biosensors. Trends in Biochemical Sciences. 1984;9(8):336-340
  90. 90. D’orazio P. Biosensors in clinical chemistry. Clinica Chimica Acta. 2003;334:41-69. DOI: 10.1016/S0009-8981(03)00241-9
  91. 91. Periasamy AP, Umasankar Y, Chen SM. Nanomaterials - Acetylcholinesterase Enzyme Matrices for Organophosphorus Pesticides Electrochemical Sensors: A review. Sensors. 2009;9:4034-4055. DOI: 10.3390/s90604034
  92. 92. Perinoto ÂC, Maki RM, Colhone MC, Santos FR, Migliaccio V, Daghastanli KR, Stabeli RG, Ciancaglini P, Paulovich FV, de Oliveira MC, Oliveira ON Jr, Zucolotto V. Biosensors for efficient diagnosis of Leishmaniasis: Innovations in bioanalytics for a neglected disease. Analytical Chemistry. 2010;82(23):9763-9768. DOI: 10.1021/ac101920t
  93. 93. Nikkhoo N, Cumby N, Gulak PG, Maxwell KL. Rapid bacterial detection via an all-electronic CMOS biosensor. PLoS One. 2016;12:11(9). DOI: 10.1371/journal.pone.0162438
  94. 94. Feriotto G, Corradini R, Sforza S, Bianchi N, Mischiati C, Marchelli R, Gambari R. }Peptide nucleic acids and biosensor Technology for Real-Time Detection of the cystic fibrosis W1282X mutation by surface Plasmon resonance. Laboratory Investigation. 2001;81(10):1415-1412
  95. 95. Figueiredo A, Nirton CS, Vieira JFS, Janegitz BC, Aoki SM, Junior PP, Lovato RL, Nogueira ML, Zucolotto V, Guimarães FEG. Electrical detection of dengue biomarker using egg yolk immunoglobulin as the biological recognition element. Scientific Reports. 2015. DOI: 10.1038/srep07865
  96. 96. Afonso AS, Goulart LR, Moura M, Goulart IMB, Madurro JM. Detection of Mycobacterium leprae DNA onto Graphite for Leprosy Diagnostics. San Francisco: Fourth International Congress of Nanotechnology; 2007. pp. 1-2

Written By

Mayara Ingrid Sousa Lima, Emilly Caroline dos Santos Moraes, Jaqueline Diniz Pinho, Gustavo Henrique Corrêa Soares and Ítalo Vinícius Cantanhêde Santos

Submitted: 29 March 2017 Reviewed: 15 February 2018 Published: 06 February 2019