title: Mycobacterium leprae specific genomic target in the promoter region of probable 4-alpha-glucanotransferase (ML1545) gene with potential sensitivity for polymerase chain reaction based diagnosis of leprosy.
abstract: With the absence of an effective diagnostic tool for leprosy, cases with negative bacteriological index and limited clinical manifestations often pose diagnostic challenges. In this study, we investigated the utility of a novel Mycobacterium leprae specific 112-bp DNA sequence in the promoter region of probable 4-alpha-glucanotransferase (pseudogene, ML1545) for polymerase chain reaction (PCR) based diagnosis of leprosy in comparison to that of the RLEP gene. DNA was extracted from slit skin scrapings of 180 newly diagnosed untreated leprosy cases that were classified as per Ridley Jopling classifications and bacteriological index (BI). Primers were designed using Primer Blast 3.0 and PCR was performed with annealing temperatures of 61°C for ML1545 and 58°C for the RLEP gene using conventional gradient PCR. The results indicated a significant increase in PCR positivity of ML1545 when compared to RLEP across the study groups (164/180 [91.11%] were positive for ML1545 whereas 114/180 (63.33%) were positive for RLEP [p<.0001, z=6.3]). Among 58 leprosy cases with negative BI, 28 (48.28%) were positive for RLEP and 48 (82.76%) were positive for ML1545 (p=.0001, z=3.8). Of the 42 borderline tuberculoid leprosy cases, 23 (54.76%) were positive for RLEP whereas 37 (88.09%) were positive for ML1545 (p<.0001, z=3.9). Increase in PCR positivity for ML1545 was also noted in lepromatous leprosy and BI-positive groups. ML1545 can be a potential gene target for PCR-based diagnosis of leprosy especially in cases where clinical manifestations were minimal.
authors.affiliation: Molecular Biology and Immunology Division, Schieffelin Institute of Health Research and Leprosy Center, Karigiri, Vellore, Tamil Nadu, India. Electronic address: sundeepchaitanya@gmail.com.
authors.forename: V
authors.initials: V
authors.lastname: Sundeep Chaitanya
authors.affiliation: Molecular Biology and Immunology Division, Schieffelin Institute of Health Research and Leprosy Center, Karigiri, Vellore, Tamil Nadu, India.
authors.forename: Madhusmita
authors.initials: M
authors.lastname: Das
authors.affiliation: Biology Department, Regents Hall of Natural Science, St. Olaf College, Northfield, MN, USA.
authors.forename: Tiffany L
authors.initials: TL
authors.lastname: Eisenbach
authors.affiliation: Biology Department, Regents Hall of Natural Science, St. Olaf College, Northfield, MN, USA.
authors.forename: Angela
authors.initials: A
authors.lastname: Amoako
authors.affiliation: Department of Laboratories, Schieffelin Institute of Health Research and Leprosy Center, Karigiri, Vellore, Tamil Nadu, India.
authors.forename: Lakshmi
authors.initials: L
authors.lastname: Rajan
authors.affiliation: Department of Dermatology, Schieffelin Institute of Health Research and Leprosy Center, Karigiri, Vellore, Tamil Nadu, India.
authors.forename: Ilse
authors.initials: I
authors.lastname: Horo
authors.affiliation: Molecular Biology and Immunology Division, Schieffelin Institute of Health Research and Leprosy Center, Karigiri, Vellore, Tamil Nadu, India.
authors.forename: Mannam
authors.initials: M
authors.lastname: Ebenezer
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D016680:Genome, Bacterial; D006004:Glycogen Debranching Enzyme System / Q000235:genetics; D006801:Humans; D007918:Leprosy / Q000175:diagnosis* / Q000382:microbiology; D009166:Mycobacterium leprae / Q000201:enzymology / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011401:Promoter Regions, Genetic; D012680:Sensitivity and Specificity
chemical_list: D001426:Bacterial Proteins; D017931:DNA Primers; D006004:Glycogen Debranching Enzyme System; C020569:4 alpha-glucanotransferase
keywords: Bacteriological index; Mycobacterium leprae; PCR-based early diagnosis; Probable 4-alpha-glucanotransferase; Pseudogene