Critical analysis: use of polymerase chain reaction to diagnose leprosy

Abstract

Leprosy is a neglected tropical disease and an important public health problem, especially in developing countries. It is a chronic infectious disease that is caused by Mycobacterium leprae, which has a predilection for the skin and peripheral nerves. Although it has low sensitivity, slit-skin smear (SSS) remains the conventional auxiliary laboratory technique for the clinical diagnosis of leprosy. Polymerase chain reaction (PCR) is a molecular biology technique that holds promise as a simple and sensitive diagnostic tool. In the present study, the performance of two PCR methods, using different targets, PCR-LP and PCR-P, were compared with SSS with regard to leprosy diagnosis in a reference laboratory. M. leprae DNA was extracted from 106 lymph samples of 40 patients who had clinical suspicion of leprosy. The samples were subjected to both PCR techniques and SSS. Amplification of the human b-globin gene was used as PCR inhibitor control. The specificity of both PCR techniques was 100%, and sensitivity was 0.007 and 0.015 µg/ml for PCR-LP and PCR-P, respectively. No significant difference was found between either the PCR-LP or PCR-P results and SSS results (p > 0.05). Although PCR is not yet a replacement for SSS in the diagnosis of leprosy, this technique may be used as an efficient auxiliary tool for early detection of the disease, especially in endemic regions. This strategy may also be useful in cases in which SSS results are negative (e.g., in paucibacillary patients) and cases in which skin biopsy cannot be performed. A hanseníase é uma doença tropical negligenciada e ainda um importante problema de saúde pública, especialmente nos países em desenvolvimento. É uma doença infecciosa crônica causada pelo Mycobacterium leprae, que tem predileção pela pele e nervos periféricos. Embora com baixa sensibilidade, o esfregaço de linfa (SSS) continua sendo o método laboratorial convencional auxiliar no diagnóstico clínico da hanseníase. A biologia molecular representada pela Reação em Cadeia da Polimerase (PCR) trouxe a expectativa de ser uma ferramenta diagnóstica simples e sensível. No presente estudo, o desempenho de dois métodos de PCR usando alvos diferentes, PCR-P e PCR-LP, foi comparado com SSS no diagnóstico da hanseníase em um laboratório de referência. DNA de M. leprae foi extraído de 106 amostras de linfa de 40 pacientes que apresentavam suspeita clínica de hanseníase. As amostras foram submetidas tanto a PCR como SSS. A amplificação do gene humano β-globina foi usada como controle de inibição da PCR. A especificidade de ambas as técnicas de PCR foi de 100% e a sensibilidade foi de 0,007 μg/mL e 0,015 μg/mL para a PCR-P e PCR-LP, respectivamente. Não se observou diferença estatística entre os resultados da PCR-LP e PCR-P, quando comparado com SSS (p > 0,05). Apesar de a PCR ainda não substituir o SSS no diagnóstico da hanseníase, esta técnica pode ser usada como ferramenta auxiliar eficiente para a detecção precoce da doença, especialmente em regiões endêmicas. Esta estratégia pode também ser útil nos cas...

Polymerase Chain Reaction (PCR) as a Potential Point of Care Laboratory Test for Leprosy Diagnosis—A Systematic Review

“…Martinez et al (2011) reported highest RLEP sensitivity (81%) as compared to other three PCR markers [34]. Maltempe et al (2016) reported RLEP PCR on SSS to be equally sensitive as AFB smear microscopy (24%) [28], which is the lowest (data not shown). Yan et al (2014) reported at least 72% of RLEP PCR positivity as compared to 35% on smear microscopy of paraffin-embedded biopsies among PB leprosy cases [36].…”

“…De Wit et al (1993) reported on the utility of PCR for detection of M. leprae in nasal swab specimens amplifying the 531-bp pra gene, demonstrating 79.6% positivity of PCR [22]. Kyeong-Han Yoon reported PCR on slit skin samples, which was subsequently reported by many others [23,24,25,26,27,28]. PCR on biopsies has been reported by Wichitwechkarn et al (1995), with a mean PCR positivity of 66% and subsequently by many others.…”

Development of a novel loop-mediated isothermal amplification assay for rapid detection of Mycobacterium leprae in clinical samples

“…In fact, the loop-mediated isothermal amplification assay performed better than the reported polymerase chain reactionbased molecular tests that exhibited a sensitivity of approximately 76%. 14,17 Furthermore, 8 out of 11 (72.7%) slit aspirates from paucibacillary patients tested positive for M. leprae as compared to two out of 11 (18.2%) by routine microscopy. Thus, based on the results observed in paucibacillary patients, our loop-mediated isothermal amplification assay can indeed be advantageous for detecting M. leprae in the early stage of the disease which could be crucial in controlling disease transmission.…”

“…The highly conserved repetitive sequence RLEP has been the preferred target due to its high copy number (n = 37), 10,14 although other targets such as rpoT, 16S rRNA and Sod A have also been exploited for detection of M. leprae by polymerase chain reaction/ quantitative polymerase chain reaction. 16,17 Several cost-effective isothermal amplification-based techniques have emerged to substitute expensive molecular methods. 18 Amongst these, loop-mediated isothermal amplification is simple, rapid, specific and sensitive and only a heating block or water bath capable to maintain a constant temperature (60 C to 65 ºC) is required.…”

Recent Trends in System-Scale Integrative Approaches for Discovering Protective Antigens Against Mycobacterial Pathogens

“…In fact, the advances in M. leprae structural and functional genomics has allowed the development of highly specific PCR-based gene amplification assays for early rapid M. leprae DNA detection with high sensitivity. PCR has also proved useful in the M. leprae viability determination, identification of routes of transmission and leprosy drug resistance ( Geluk et al, 2012 ; Martinez et al, 2014 ; Soto and Muñoz, 2015 ; Maltempe et al, 2016 ).…”