Develop and Field Evolution of Single Tube Nested PCR, SYBRGreen PCR Methods, for the Diagnosis of Leprosy in Paraffin-embedded Formalin Fixed Tissues in Yunnan Province, a Hyper endemic Area of Leprosy in China

Abstract

BackgroundDetection and pathology analysis of Mycobacterium leprae using skin biopsy tissues are essential for leprosy diagnosis and monitoring response to treatment. Although formalin fixation of patient tissues may not be ideal for molecular studies, biopsy samples are the most accessible material from suspected cases. Therefore, clinical molecular laboratories must be able to utilize formalin-fixed, paraffin-embedded (FFPE) material.ObjectiveTo determine the best molecular method for diagnosing and monitoring leprosy in FFPE specimens, we developed a single-tube nested PCR (STNPCR) (131 bp) and SYBRGreen PCR (101 bp) assay using primers for the M. leprae-specific repetitive element (RLEP) gene and evaluated the results compared to those using previously established RLEP primers (372 bp).MethodsFFPE biopsy samples obtained from 145 leprosy patients (during or after multidrug therapy (MDT)) and patients with 29 other confounding dermatoses were examined by the bacteria index (BI) and by simple PCR, STNPCR, and SYBRGreen PCR using primers amplifying a 372-bp, 131-bp or 101-bp fragment of RLEP, respectively.ResultsIn leprosy patients receiving MDT, STNPCR showed a highest specificity of 100% and a positive predictive value (PPV) of 100%. For multibacillary (MB), paucibacillary (PB) and all leprosy patients, the highest sensitivities were 91.42%, 39.13%, and 67.92%, negative predictive values (NPVs) were 8.57%, 60.36%, and 32.07%, and the highest accuracies were 93.93%, 62.67%, and 74.81%, respectively, higher than the results of SYBRGreen PCR and simple PCR. For post-MDT leprosy patients, SYBRGreen PCR showed the highest sensitivity of 50.0%, highest specificity of 100%, a PPV of 100%, an NPV of 100% and the highest accuracy of 83.72% for MB patients, which were higher than those of STNPCR and simple PCR. STNPCR showed the highest sensitivity of 26.66% and 34.48%, highest specificity of 100% and 100%, a PPV of 100% and 100%, NPV of 72.50% and 60.21%, and highest accuracy of 75.00% and 67.24% for PB and leprosy patients, respectively, higher than those of SYBRGreen PCR and simple PCR.ConclusionsThese findings suggest that STNPCR or SYBRGreen PCR (131-bp and 101-bp fragment amplification, respectively) for RLEP using FFPE specimens performs better as a diagnostic test and for monitoring response to MDT than does simple PCR based on 372-bp fragment amplification. Additionally, STNPCR showed increased sensitivity for PB diagnosis using FFPE specimens, which can be transferred remotely or retrieved from previous leprosy patients.

Fluorescent Hybridization of Mycobacterium leprae in Skin Samples Collected in Burkina Faso

“…DNA amplification inhibitors from human surgical tissue also may explain the false-negative PCR result (15). Another potential explanation is the low sensitivity of the PCR techniques targeting a single copy of a gene in the genome (16)(17)(18) in a paucibacillary specimen (19).…”

Challenges and advances in serological and molecular tests to aid leprosy diagnosis

“…The qPCR, 85 nested PCR 84 and variations such as multiplex, 87,91 duplex, 92 and droplet digital (ddPCR) 93 have been reported for different M. leprae target sequences, with RLEP and 16S rRNA representing the most used ones. [94][95][96] A study showed that multiplex PCR helps detect early leprosy cases and household contact surveillance for leprosy. 97 Nested PCR is useful for amplifying genes from samples with low bacillary load in PB patients, as this technique amplifies sequences within the amplicon generated in the first amplification round.…”

“…98 Success has been reported in the use of formalin-fixed, paraffin-embedded skin biopsy specimens for genomic DNA extraction and subsequent amplification of the M. leprae RLEP-based sequences. 96 Peripheral whole blood tested by M. leprae ML0024 qPCR in untreated leprosy patients and household contacts followed up for 7 years showed that contacts with positive PCR in blood at diagnosis of index cases are at higher risk of later leprosy onset. 99 However, other studies show that blood may not be a suitable specimen to detect M. leprae DNA in MB and PB patients 90,99,100 in contrast with studies that succeeded detecting M. leprae in whole blood.…”

“…The qPCR, 85 nested PCR 84 and variations such as multiplex, 87,91 duplex, 92 and droplet digital (ddPCR) 93 have been reported for different M. leprae target sequences, with RLEP and 16S rRNA representing the most used ones. 94–96 A study showed that multiplex PCR helps detect early leprosy cases and household contact surveillance for leprosy. 97…”

Prevention and Treatment of Leprosy — China, 2009−2019

“…Furthermore, detection of M. leprae -specific DNA could be more sensitive. New primer sets for the M. leprae -specific repetitive element ( RLEP ) gene were designed by BTMRI and single tube nested PCR (STNPCR) and SYBR Green PCR assays were performed to test M. leprae for PB patients ( 19 ). SPIDV developed the droplet digital polymerase chain reaction (ddPCR) assay using primers for M. leprae –specific RLEP and groEL genes to detect M. leprae in PB patients with a high sensitivity of 79.5% ( 20 ).…”