Application of RLEP Real-Time PCR for Detection of M. leprae DNA in Paraffin-Embedded Skin Biopsy Specimens for Diagnosis of Paucibacillary Leprosy

Abstract

Abstract. The TaqMan real-time polymerase chain reaction (PCR) assay was evaluated systematically with respect to the standard curve, linear range, and used for detecting Mycobacterium leprae DNA in paraffin-embedded skin biopsy specimens from 60 confirmed leprosy patients and three healthy individuals and 29 other dermatoses and bacterial DNA from 21 different species. The test was further evaluated with 51 paucibacillary (PB) patients. The results showed that the test had good sensitivity (8 fg) and good specificity with no cross-reactivity with 21 other bacterial species and the control specimens, except one with Xanthomatosis. The real-time PCR detection rate for the 51 PB specimens was 74.5% (38 of 51). We conclude that the real-time PCR test is a useful adjunct test for diagnosing early stage or PB leprosy cases.

Detection of <em>Mycobacterium leprae</em> using real-time PCR in paucibacillary leprosy patients with negative acid-fast bacilli smears

“…According to theory and previous studies, PCR is a sensitive assay that can detect microorganisms even in low numbers. 3,7,9 The threshold of DNA detection for RT-PCR is as low as 8 femtograms or equal to 240 bacterial cells,3 which is more sensitive than conventional PCR. 6 The clinical sensitivity of RT-PCR in PB leprosy is also better than conventional PCR (79.2% versus 62.5%), with similar specificity.10 It is believed that PCR able to overcome the low sensitivity problems of prior diagnostic methods.…”

“…The threshold of DNA detection for RT-PCR is more sensitive than conventional PCR. 3,6 The clinical sensitivity of RT-PCR in PB leprosy is also better than conventional PCR with a similar specificity.10…”

“…Other advantages include more rapid examination time and the use of a closed system to minimize the risk of contamination. 3,11 Therefore, this molecular-based assay can be used to confirm diagnosis. 4,12 Based on the data, this study was aimed to identify the proportion of positive results from TaqMan® probe-based RT-PCR assay compared to histopathological examinations in PB leprosy patients with negative AFB smears.…”

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

“…Several authors have based their methods on the amplification of specific repetitive sequences of M. leprae (RLEP region, 32 repeats per genome) [26,[41][42][43], this technique has undergone modifications and using nPCR can amplify one-tenth of a single bacterial genome [27,[44][45][46]. Others have combined PCR and southern hybridization [47][48][49], mPCR [50] or qPCR [19,38,[51][52][53].…”

“…This type of sample increases the possibility of finding bacilli [48], and leprosy molecular diagnosis can be done in: fresh skin biopsies [61], frozen skin biopsies [19,30,33,38,40,44,45,50], in formalin (although is not the appropriate preservative for amplification) [30], paraffin embedded [33,37,53,97], in ethanol 70% [24], nerve biopsies [98], in post-biopsy swab [33] or filter paper biopsy imprints [40]. An internal control and DNA serial dilutions must be added [66], because this sample is more likely to contain inhibitors of polymerase as: crosslinks between proteins and DNA by formylation of nucleic acids, length of time of fixation in formalin, lengths time of fixation in formalin and impurities form DNA purification can inhibit the polymerase reaction [22,30,37].…”