Leprosy Diagnosis: An Update on the Use of Molecular Tools Lucrecia

Leprosy is a chronic infectious disease caused by an obligatory intracellular mycobacteria Mycobacterium leprae, which presents tropism for Schwann cells and skin macrophages. Leprosy is a public health problem and early diagnosis is essential to avoid incapacities. The disease ́s clinical presentation varies from few to widespread lesions and its diagnosis continues to be a challenge due to the low sensibility of the conventional methods, based on bacillary counts of skin smears and histopathology. Molecular techniques, especially the methods to identify M. leprae DNA based on polymerase chain reaction (PCR) have emerged as a support of the conventional methods for the analysis of clinical samples in difficult to diagnose cases, such as pure neural leprosy, indeterminate and paucibacillary leprosy. The technique has also proved useful in the study of leprosy transmission and monitoring résistance to the WHO recommended Multidrug treatment. Different biological samples can be analysed and there is no consensus in the molecular diagnostic techniques respect of the most efficient nucleic acid extraction method, most appropriate methodology and genetic target for PCR. These methods provide a very valuable option for confirmation of difficult clinical cases with scarce bacilli but requires a well-equipped laboratory and the high cost makes it inaccessible to be used as a routine diagnostic tool in most endemic countries. *Corresponding author: Lucrecia Acosta Soto, Head of Molecular Biology and Investigation Unit, Sanatorium Fontilles. Ctra. Orba-Vall de Laguar, Km 4, 03791, Vall de Laguar, Alicante, Spain, Tel: +34965583350, E-mail: [email protected] Received September 22, 2015; Accepted October 24, 2015; Published October 31, 2015 Citation: Soto A, Muñoz PT (2015) Leprosy Diagnosis: An Update on the Use of Molecular Tools Lucrecia. Mol Biol 4: 139. doi:10.4172/2168-9547.1000139 Copyright: © 2015 Soto A, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Leprosy diagnosis Leprosy is a chronic infectious disease caused by an obligatory intracellular mycobacteria Mycobacterium leprae [1], which presents tropism for Schwann cells and skin macrophages [2,3]. At present, leprosy remains a public health problem. The main strategy to control leprosy is early detection and treatment with multidrug therapy (MDT) [4]. Despite being an ancient disease, known since Biblical times [5], its diagnosis continues to be a challenge due to the low sensibility of the conventional methods and impossibility to culture the bacillus “in vitro”. Leprosy develops after an estimated incubation period of 2 to 10 years and presents a complex spectrum of clinical forms [6]. In clinical practice, the diagnosis is mainly based on the observation of clinical symptoms and supported by bacteriological analysis (Zielh-Neelsen smear (ZNS) and histopathology). A negative ZNS only indicates that the concentration of bacilli is below 10,000 bacilli/mL [7], and this does not necessarily mean that the person is not infected [8]. This issue is especially problematic for individuals with pure neural leprosy (PNL), indeterminate (IL) and paucibacillary (PB) disease, which harbour a low burden of bacteria. On the other hand, with microscopic visualization all mycobacteria are phenotypically indistinguishable. Moreover, the serological techniques commercially available are inconclusive [9-12]. Many of the methods used in the diagnosis of other mycobacterial infections are not available in leprosy [13]. Research for the development of new diagnostic tools is particularly complicated since the only sources of bacteria are leprosy patients and a natural reservoir, the nine-banded armadillo (Dasypus novemcinctus) [14,15]. Thus, studying defined infections in mouse and armadillo models can provide insights into the host-pathogen interactions involved in this complex disease [16,17]. In leprosy, early diagnosis is essential and molecular techniques have emerged as a support of the conventional methods for the analysis of clinical samples. They offer culture-independent methods more sensitive and specific for the identification, confirmation and treatment of the infection, interrupting the chain of transmission and preventing the onset of disabilities [18,19]. M. leprae detection in clinical samples Definitive identification of M. leprae in clinical specimens using PCR will depend largely on the standardization and other related factors such as the number of copies of the target, the product size and the PCR conditions. Several non-commercial protocols using techniques based on the amplification of different sequences and targets have been developed. The most frequently used for diagnosis are: Amplifying the gene encoding an 18 kDa antigenic protein by conventional PCR (cPCR) [20-26], with a sensitivity limit of 100 bacilli/sample [22] or of approximately 30 bacilli/sample by nested PCR (nPCR) [27]. Another method targets a gene that encodes an antigenic 36 kDa protein known as proline-rich antigen (pra gen) by cPCR [28-33], and can detect up to a single bacteria in the sample [28]. The pra gen has also been amplified by multiplex PCR (mPCR) [34]. Plikaytis and coworkers, developed a nPCR that amplifies a heat shock protein 65 kDa called groEL, which can detect 3 fg of M. leprae-DNA (single bacteria) [35]. An 85-antigen complex has also been used as a target, which encodes an 85 kDa antigen of three structurally related components [36]; 85B intergenic region by cPCR [37] and for the 85A-C gene by cPCR [37] or quantitative PCR (qPCR) [37-39]. Amplification of specific regions of microsatellites, as well as an internal sequence of the high-affinity manganese transporter (Ml MntH; ML2098) gene of the bacillus can also be useful for detecting M. leprae [40]. Several authors have based their methods on the amplification of specific repetitive sequences of M. leprae (RLEP region, 32 repeats per genome) [26,41-43], this technique has undergone modifications and using nPCR can amplify one-tenth of a single bacterial genome [27,4446]. Others have combined PCR and southern hybridization [47-49], mPCR [50] or qPCR [19,38,51-53]. M. leprae can be identified combining PCR of the 16S rDNA internal transcribed spacer (ITS) region with restriction digestion of