PCR-Based Techniques for Leprosy Diagnosis: From the Laboratory to the Clinic

Abstract

In leprosy, classic diagnostic tools based on bacillary counts and histopathology have been facing hurdles, especially in distinguishing latent infection from active disease and diagnosing paucibacillary clinical forms. Serological tests and IFN-gamma releasing assays (IGRA) that employ humoral and cellular immune parameters, respectively, are also being used, but recent results indicate that quantitative PCR (qPCR) is a key technique due to its higher sensitivity and specificity. In fact, advances concerning the structure and function of the Mycobacterium leprae genome led to the development of specific PCR-based gene amplification assays for leprosy diagnosis and monitoring of household contacts. Also, based on the validation of point-of-care technologies for M. tuberculosis DNA detection, it is clear that the same advantages of rapid DNA detection could be observed in respect to leprosy. So far, PCR has proven useful in the determination of transmission routes, M. leprae viability, and drug resistance in leprosy. However, PCR has been ascertained to be especially valuable in diagnosing difficult cases like pure neural leprosy (PNL), paucibacillary (PB), and patients with atypical clinical presentation and histopathological features compatible with leprosy. Also, the detection of M. leprae DNA in different samples of the household contacts of leprosy patients is very promising. Although a positive PCR result is not sufficient to establish a causal relationship with disease outcome, quantitation provided by qPCR is clearly capable of indicating increased risk of developing the disease and could alert clinicians to follow these contacts more closely or even define rules for chemoprophylaxis.

Presence of Mycobacterium leprae DNA and PGL-1 antigen in household contacts of leprosy patients from a hyperendemic area in Brazil

“…The detection percentages of M. leprae DNA can vary from 1 to 10%, likely because of the use of different primers, amplification techniques, and clinical form of the index case Martinez et al, 2014). In our study, no significant association was observed between the bacillary load of the index case and the detection of bacterial DNA.…”

Characterization of MicroRNA Expression Profiles and Identification of Potential Biomarkers in Leprosy

“…Currently, the gold standard for leprosy diagnosis is based on clinical examination and skin biopsy. Techniques based on PCR technology and serological analysis have been developed but were not able to diagnose leprosy with acceptable sensitivity and specificity taking into consideration the different clinical forms and/or bacterial burden (11,(14)(15)(16)(18)(19)(20)(21). Accordingly, identification of biomarkers that allow the diagnosis of leprosy with a greater sensitivity and specificity is still needed.…”

“…Molecular techniques using PCR technology and serological tests were developed; however, specificity and sensitivity were limited (11,(14)(15)(16)(17)(18)21). In this context, the identification of biomarkers that allow early diagnosis of leprosy and differentiation between forms of the disease with an adequate sensitivity and specificity is still required.…”

Critical analysis: use of polymerase chain reaction to diagnose leprosy

“…Clinical diagnosis is possible only when the patient is symptomatic, exhibiting skin lesions or presenting the clinical criteria of cardinal signs of leprosy (Martinez et al, 2006). Polymerase chain reaction may be an important tool for helping diagnose leprosy in cases in which clinical findings and SSS are inconclusive, such as with pure neural leprosy, paucibacillary cases, and atypical clinical presentation (Martinez et al, 2014).…”

“…Different PCR primers have been used to amplify the DNA of M. leprae from clinical samples (Kang, Kim, Lee, 2003). The PCR targets that are used to detect M. leprae include genes that encode 36 kDa (known as proline-rich antigen [pra] (Parkash et al, 2004;Torres et al, 2003;Hartskeerl, De Wit, Klatser, 1989), 18 kDa (Chae et al, 2002), 85 kDa (Martinez et al, 2014), and 65 kDa (Plikaytis, Gelber, Shinnick, 1990) protein antigens, 16S rRNA (Phetsuksiri, et al, 2006), and repetitive sequences (Donoghue, Holton, Spigelman, 2001). Cox, Kempsell, Fairclough (1991) and Arnoldi et al (1992) detected 8.3 bacilli in skin biopsy samples using primers that amplified a specific sequence that was 172 bp from 16S rRNA.…”