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Objective/Background: Clinical diagnosis of indeterminate and tuberculoid leprosy is often difficult due to limited and confounding signs and symptoms. In the current study, we evaluated the utility of new multiplex polymerase chain reaction (PCR) using Mycobacterium leprae-specific DNA sequences in the pseudogene regions of ML1545, ML2180, and ML2179 for PCR-based diagnosis of indeterminate leprosy (IND) and leprosy cases across the immunological spectrum. The sensitivity was compared with that of RLEP PCR. Methods: DNA was extracted from paraffin-embedded skin biopsy specimens of 220 leprosy cases, which were divided into IND (41), tuberculoid form (3), borderline tuberculoid (42), midborderline (3), borderline lepromatous (n=59), and lepromatous leprosy (72) cases. PCR positivity of both multiplex and RLEP PCR were compared in all the samples. A decision tree was constructed using the classification and regression trees algorithm to predict the probability of PCR positivity with the new multiplex PCR scheme in various clinical groups of leprosy. Sensitivity of each pseudogene target was determined using real-time PCR assays, and specificity was confirmed by PCR amplification of DNA extracted from three other mycobacterial species and skin biopsies of 44 non-leprosy cases. Results: A multiplex PCR positivity of 75.61% was noted in IND cases when compared to that of 58.54% using RLEP PCR (P < 0.05). Enhanced multiplex PCR positivity was noted across various clinical groups in comparison to RLEP PCR. The decision tree classifier has predicted statistically significant probability for multiplex PCR positivity among RLEP-PCR negative group and clinical groups with a low bacillary load. Conclusion: This new multiplex PCR scheme can support the diagnosis of indeterminate and tuberculoid forms of leprosy with limited clinical manifestations and can be implemented in basic clinical/diagnostic setting that possess conventional PCR facilities.

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.

Objective/background: Clinical diagnosis of indeterminate and tuberculoid leprosy is often difficult due to limited and confounding signs and symptoms. In the current study, we evaluated the utility of new multiplex polymerase chain reaction (PCR) using Mycobacterium leprae-specific DNA sequences in the pseudogene regions of ML1545, ML2180, and ML2179 for PCR-based diagnosis of indeterminate leprosy (IND) and leprosy cases across the immunological spectrum. The sensitivity was compared with that of RLEP PCR., Methods: DNA was extracted from paraffin-embedded skin biopsy specimens of 220 leprosy cases, which were divided into IND (41), tuberculoid form (3), borderline tuberculoid (42), midborderline (3), borderline lepromatous (n=59), and lepromatous leprosy (72) cases. PCR positivity of both multiplex and RLEP PCR were compared in all the samples. A decision tree was constructed using the classification and regression trees algorithm to predict the probability of PCR positivity with the new multiplex PCR scheme in various clinical groups of leprosy. Sensitivity of each pseudogene target was determined using real-time PCR assays, and specificity was confirmed by PCR amplification of DNA extracted from three other mycobacterial species and skin biopsies of 44 non-leprosy cases., Results: A multiplex PCR positivity of 75.61% was noted in IND cases when compared to that of 58.54% using RLEP PCR (P < 0.05). Enhanced multiplex PCR positivity was noted across various clinical groups in comparison to RLEP PCR. The decision tree classifier has predicted statistically significant probability for multiplex PCR positivity among RLEP-PCR negative group and clinical groups with a low bacillary load., Conclusion: This new multiplex PCR scheme can support the diagnosis of indeterminate and tuberculoid forms of leprosy with limited clinical manifestations and can be implemented in basic clinical/diagnostic setting that possess conventional PCR facilities.

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., (Copyright © 2016 Asian-African Society for Mycobacteriology. Published by Elsevier Ltd. All rights reserved.)

The molecular basis for determination of resistance to anti-leprosy drugs is the presence of point mutations within the genes of Mycobacterium leprae (M. leprae) that encode active drug targets. The downstream structural and functional implications of these point mutations on drug targets were scarcely studied. In this study, we utilized computational tools to develop native and mutant protein models for 5 point mutations at codon positions 53 and 55 in 6-hydroxymethyl-7, 8-dihydropteroate synthase (DHPS) of M. leprae, an active target for dapsone encoded by folp1 gene, that confer resistance to dapsone. Molecular docking was performed to identify variations in dapsone interaction with mutant DHPS in terms of hydrogen bonding, hydrophobic interactions, and energy changes. Schrodinger Suite 2014-3 was used to build homology models and in performing molecular docking. An increase in volume of the binding cavities of mutant structures was noted when compared to native form indicating a weakening in interaction (60.7 Å(3) in native vs. 233.6 Å(3) in Thr53Ala, 659.9 Å(3) in Thr53Ile, 400 Å(3) for Thr53Val, 385 Å(3) for Pro55Arg, and 210 Å(3) for Pro55Leu). This was also reflected by changes in hydrogen bonds and decrease in hydrophobic interactions in the mutant models. The total binding energy (ΔG) decreased significantly in mutant forms when compared to the native form (-51.92 Kcal/mol for native vs. -35.64, -35.24, -46.47, -47.69, and -41.36 Kcal/mol for mutations Thr53Ala, Thr53Ile, Thr53Val, Pro55Arg, and Pro55Leu, respectively. In brief, this analysis provided structural and mechanistic insights to the degree of dapsone resistance contributed by each of these DHPS mutants in leprosy., (© 2015 Wiley Periodicals, Inc.)