Analysis of a novel multiplex polymerase chain reaction assay as a sensitive tool for the diagnosis of indeterminate and tuberculoid forms of leprosy.
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.



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.



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.



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.
Division of Molecular Biology and Immunology, Schieffelin Institute of Health Research and Leprosy Centre, Karigiri, Vellore, Tamil Nadu, India.
V Sundeep
VS
Chaitanya
Department of Biology, Regents Hall of Natural Science, Northfield, MN 55057, USA.
Luz
L
Cuello
Division of Molecular Biology and Immunology, Schieffelin Institute of Health Research and Leprosy Centre, Karigiri, Vellore, Tamil Nadu, India.
Madhusmita
M
Das
Division of Molecular Biology and Immunology, Schieffelin Institute of Health Research and Leprosy Centre, Karigiri, Vellore, Tamil Nadu, India.
A
A
Sudharsan
Division of Molecular Biology and Immunology, Schieffelin Institute of Health Research and Leprosy Centre, Karigiri, Vellore, Tamil Nadu, India.
Pavithrra
P
Ganesan
Division of Molecular Biology and Immunology, Schieffelin Institute of Health Research and Leprosy Centre, Karigiri, Vellore, Tamil Nadu, India.
K
K
Kanmani
Division of Molecular Biology and Immunology, Schieffelin Institute of Health Research and Leprosy Centre, Karigiri, Vellore, Tamil Nadu, India.
Lakshmi
L
Rajan
Division of Molecular Biology and Immunology, Schieffelin Institute of Health Research and Leprosy Centre, Karigiri, Vellore, Tamil Nadu, India.
Mannam
M
Ebenezer
D000293:Adolescent; D000328:Adult; D001706:Biopsy; D002648:Child; D002675:Child, Preschool; D004269:DNA, Bacterial; D003663:Decision Trees; D005260:Female; D006801:Humans; D007223:Infant; D015440:Leprosy, Lepromatous / Q000175:diagnosis* / Q000382:microbiology; D056005:Leprosy, Paucibacillary / Q000175:diagnosis* / Q000382:microbiology; D015441:Leprosy, Tuberculoid / Q000175:diagnosis*; D008297:Male; D008875:Middle Aged; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D009166:Mycobacterium leprae / Q000235:genetics* / Q000302:isolation & purification; D011544:Pseudogenes / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D012867:Skin / Q000382:microbiology / Q000473:pathology; D055815:Young Adult
D004269:DNA, Bacterial