International Journal of Mycobacteriology 5 ( 2 0 1 6 ) 1 3 5 1 4 1

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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

V. Sundeep Chaitanya a,*, Madhusmita Das a, Tiffany L. Eisenbach b, Angela Amoako b,

Lakshmi Rajan c, Ilse Horo d, Mannam Ebenezer a
a
Molecular Biology and Immunology Division, Schieffelin Institute of Health Research and Leprosy Center, Karigiri, Vellore, Tamil Nadu, India
b
Biology Department, Regents Hall of Natural Science, St. Olaf College, Northfield, MN, USA
c
Department of Laboratories, Schieffelin Institute of Health Research and Leprosy Center, Karigiri, Vellore, Tamil Nadu, India
d
Department of Dermatology, Schieffelin Institute of Health Research and Leprosy Center, Karigiri, Vellore, Tamil Nadu, India

A R T I C L E I N F O A B S T R A C T

Article history: With the absence of an effective diagnostic tool for leprosy, cases with negative bacteriolog-
Received 9 January 2016 ical index and limited clinical manifestations often pose diagnostic challenges. In this
Accepted 23 January 2016 study, we investigated the utility of a novel Mycobacterium leprae specific 112-bp DNA
Available online 16 February 2016 sequence in the promoter region of probable 4-alpha-glucanotransferase (pseudogene,
ML1545) for polymerase chain reaction (PCR) based diagnosis of leprosy in comparison to
Keywords: that of the RLEP gene. DNA was extracted from slit skin scrapings of 180 newly diagnosed
Bacteriological index untreated leprosy cases that were classified as per Ridley Jopling classifications and bacte-
Mycobacterium leprae riological index (BI). Primers were designed using Primer Blast 3.0 and PCR was performed
PCR-based early diagnosis with annealing temperatures of 61 C for ML1545 and 58 C for the RLEP gene using conven-
Probable 4-alpha-glucanotransferase tional gradient PCR. The results indicated a significant increase in PCR positivity of ML1545
Pseudogene 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.
2016 Asian-African Society for Mycobacteriology. Production and hosting by Elsevier Ltd.
All rights reserved.

* Corresponding author at: Molecular Biology and Immunology Division, Schieffelin Institute of Health Research and Leprosy Center,
Karigiri, Vellore, Tamil Nadu 632106, India.
E-mail address: sundeepchaitanya@gmail.com (V. Sundeep Chaitanya).
Peer review under responsibility of Asian African Society for Mycobacteriology.
http://dx.doi.org/10.1016/j.ijmyco.2016.01.002
2212-5531/ 2016 Asian-African Society for Mycobacteriology. Production and hosting by Elsevier Ltd. All rights reserved.
136 International Journal of Mycobacteriology
1. Introduction
Leprosy, a debilitating disease of the skin and peripheral
nerves, continues to remain as a constant peril with approx-
imately 135,752 cases from India alone in 2013 [1]. Mycobac-
terium leprae, the causative organism for this disease, has a
long incubation period of 37 years during which the bacteria
remain dormant or multiply in the Schwann cells of the neu-
ronal myelin and manifest a spectrum of clinical outcomes
[2]. These outcomes can range from a self-limiting, cell-
mediated immunity predominant tuberculoid pole (TT) with
minimal bacillary load in the skin and nerves to a more sys-
temic, humoral-immunity predominant lepromatous pole
with high bacillary load. These polar forms are interspersed
by three intermediary unstable borderline forms (borderline
tuberculoid [BT], midborderline, and borderline lepromatous)
in which the diagnosis of the disease is often based on clinical
manifestations and histopathological features [3]. These
clinical signs are very minimal and often confounding in
the TT and BT forms and hence pose diagnostic challenges.
World Health Organization (WHO) has introduced a more
convenient field based classification called the multibacillary
(MB) leprosy with greater than five skin lesions and pauci-
bacillary (PB) leprosy with less than five skin lesions [4].
During the incubation phase between the infection and
manifestation of clinical symptoms, the disease may remain
subclinical and chronically progress to a systemic phase
which predisposes nerve damage and subsequent deformities
[5]. Therefore, it is important to diagnose the disease early in
a subclinical state in order to treat it rapidly and prevent
deformities. In this context, an early diagnostic test has long
been desired for leprosy [6]. To date, there is no specific
screening test and/or assay that aids in early diagnosis of
leprosy with substantial sensitivity and specificity [7]. While
the bacteriological index (BI) determination in the slit skin
smears and histopathological examination of the skin biopsy
sections remained as clinical and pathological methods for
determination of bacillary load, the sensitivity of these tests
is limited to the few microscopic sections being examined.
Detection of M. leprae gene targets (RLEP, 16SrRNA, SodA,
Ag85A, PRA, etc.) in the DNA extracted from the skin biopsies
and peripheral blood samples of leprosy patients compara-
tively presented higher sensitivity especially in the indetermi-
nate, TT, and BT forms where the BI remains negative due to
low bacterial counts [8,9]. However, some of these gene tar-
gets have questionable specificity.
To date, the known gene target that remains specific to
M. leprae genome is the RLEP gene [10], which is a repetitive
element in the genome of M. leprae [11]. Real-time polymerase
chain reaction (PCR) based detection using RLEP PCR assays
could detect 70% of PB leprosy in which the BI is negative
[12]. However, simple conventional nested PCR assay with a
129-bp fragment could detect only 55.5% of the BI negative
PB cases [13]. While it was reported that RLEP real-time PCR
assay can detect M. leprae DNA in as little as 8 fg (indicating
3 bacterial cells per sample, taking a 3.2-mbp genome into
calculation), the 129-bp fragment missed on identifying
3050% of the BI negative PB cases when used in a conven-
tional PCR [14]. Hence there is a need for identification of
5 ( 2 0 1 6 ) 1 3 5 1 4 1
specific and sensitive M. leprae gene targets that aid in diagno-
sis of leprosy cases with negative BI or very minimal bacillary
load using simple conventional PCR.
In this study, we examined M. leprae specific and unique
DNA sequences in pseudogenes and identified a 112-bp
sequence in the promoter region of ML1545 (probable
4-alpha-glucanotransferase [pseudogene]) which possess sub-
stantial specificity. Further, we investigated the utility of this
gene sequence as a target for PCR-based detection of M. leprae
DNA in the clinical isolates of leprosy patients. The PCR
positivity was compared to that of RLEP to determine the
sensitivity in detection of M. leprae DNA in leprosy cases
across the spectrum of the disease.
2. Materials and methods
2.1. Study patients
A total of 180 newly diagnosed untreated leprosy cases from
the Dermatology Outpatient Department of Schieffelin Insti-
tute of Health-Research and Leprosy Center, Karigiri in Vel-
lore, Tamil Nadu, India, were enrolled in the study following
the institutional ethical guidelines. An informed and written
consent for participation was obtained from all the patients
before enrolling in the study, following the ethical guidelines
laid down by the Indian Council of Medical Research. All pro-
cedures conducted in the study were in accordance with the
guidelines of the Institutional Ethics Committee of Schieffelin
Institute of Health-Research and Leprosy Center and with the
ethical standards as laid down in the 1964 Declaration of
Helsinki and its later amendments or comparable ethical
standards. All the cases were examined by a clinician/leprolo-
gist and clinical, demographic, and histopathological details
were recorded at diagnosis (Table 1). In addition, six healthy
individuals who reside in a low endemic area and without
any direct contacts with active leprosy cases, were enrolled
as controls. DNA extracted from Mycobacterium tuberculosis,
Mycobacterium smegmatis, and Mycobacterium phlei was used
as a technical control to ascertain the specificity of ML1545.
2.2. Sample types
Part of the 5 mm
5 mm excisional skin biopsy that was
collected for routine histopathological examination was used
for the experiments in the current study. Ridley Jopling classifi-
cation and BI were recorded at diagnosis for all the participants.
The clinical characteristics of the participants were represented
in Table 1.
2.3. DNA extraction
DNA was extracted from skin biopsy specimens following the
lysis protocol which was reported earlier, with minor modifi-
cations [15]. Briefly, 25 mg of tissue was thoroughly homoge-
nized using a manual homogenizer and homogenate was
added to 1 mL of 1X phosphate buffer saline and centrifuged
at 8000 rpm for 10 min at 4 C. The pellet was dried for 1 h at
50 C on a dry block incubator and was later used in the lysis
protocol. Two hundred microliters of lysis buffer (containing
 International Journal of Mycobacteriology 5 ( 2 0 1 6 ) 1 3 5 1 4 1 137

Table 1 Clinical and demographic characteristics of the leprosy cases and control individuals.
Si. No. Characteristics Types Leprosy group (n = 180) Nonleprosy group (control) (n = 6)
No % No %

1 Sex Male 133 73.89 5 83.33

Female 47 26.11 1 16.67
2 Age 115 14 7.78 1 16.67
1630 29 16.11 1 16.67
3150 67 37.22 2 33.33
>50 70 38.89 2 33.33
3 WHO classification PB 18 10.00
MB 162 90.00
4 RJ classification Indeterminate 1 0.56
TT 3 1.67
BT 42 23.33
BB 3 1.67
BL 59 32.78
LL 72 40.00
5 BI Status Negative 58 32.22
12 25 13.89
2.254 67 37.22
4.256 30 16.67
Note: BI = Bacteriological index; BL = Borderline Lepromatous; BT = Borderline Tuberculoid; LL = Lepromatous leprosy; BB = Mid-borderline;
RJ = Ridley Jopling; TT = Tuberculoid pole; WHO = World Health Organization, PB = Paucibacillary, MB = Multibacillary.

100 mM Tris buffer at pH 8.5, 1 mg/mL proteinase K, and 0.05%
Tween 20) was added to the homogenate in a 1.5-mL micro-
centrifuge tube, vortexed thoroughly, and incubated at 60 C
for 16 h in a water bath (Cole-Parmer Polystat Inc., Chicago,
IL, USA). Post incubation, proteinase K was deactivated at
95 C for 15 min. Later the lysate was allowed to cool to room
temperature and DNA was eluted using phenol chloroform
extraction. DNeasy Kit (Cat No: 69504; Qiagen Inc., Valencia,
CA, USA) was used in some samples where the tissue content
was apparently low. DNA extraction from cultures of M. tuber-
culosis, M. Smegmatis, and M. phlei was also performed using
the same protocol.
2.4. Selection of the gene target (ML1545) and primer
design
A sequential search of M. leprae specific DNA sequences in
pseudogenes was performed using National Center for
Biotechnology Information (NCBI) Basic Local Alignment
Search Tool (BLAST) and PatricBRC Portal (https://www.pa-
tricbrc.org). A set of eight genes that include ML1127,
ML2110, ML2211, ML0203, ML0307, ML1545, ML2177, and
ML0466 were identified to harbor sequences that remain
specific for M. leprae genome. However, upon further analysis
of these individual sequences using NCBI BLAST with various
database search parameters like NCBI Genomes (chromo-
somes) and Reference genomic sequences within Mycobac-
teria and Mycobacteriaceae, a DNA sequence that traverses
the promoter region of probable 4-alpha-glucanotransferase
(ML1545) gene was chosen as it remained highly specific to
the genome. Primers were designed while maintaining the
specificity using Primer BLAST version 3 from NCBI. The
sequence for the forward primer is 50 -GTCCTCCGTCTTGCTG
ACTG-30 and for the reverse primer is 50 -CATACCGGCCA
TATTGCGTC-30 . The designed primers were obtained com-
mercially from Eurofins Scientific Inc., Germany. The PCR
amplifications with these primers were compared with those
of the RLEP whose primers were taken from the earlier reports
[16].
2.5. PCR conditions and cycling parameters
The PCR amplifications were performed in PTC-150 MiniCycler
(MJ Research, MA, USA). A 20-lL reaction mix was prepared
using 1.25 units of AmpliTaq Gold DNA polymerase with
Buffer I (N8080240: Life Technologies Inc., Carlsbad, CA, USA),
0.35 mM of each kind of deoxynucleotides, 0.25 mM MgCl2,
0.25 lM each of forward and reverse primers, and 2 ng of
DNA. The reaction conditions included an initial denaturation
of 95 C for 7 min, one cycle of 94 C for 2 min, 61 C for
2 min, and 72 C for 2 min. This was followed by 40 cycles of
94 C for 30 s, 61 C for 30 s, and 72 C for 45 s. The reaction
was terminated with a final extension of 72 C for 10 min and
the tubes were cooled to 25 C for 10 min. The amplicons were
electrophoresed on a 2% agarose gel and gel images were
captured using gel documentation system coupled with a UV
camera. PCR-based amplification of the RLEP gene target
was performed as described earlier [16]. DNA extracted from
M. leprae strain Br4923 (Courtesy: BEI Resources, catalog num-
ber: NR-19351) was used as a positive control in the
experiments.
2.6. PCR conditions for other Mycobacteria
DNA was extracted from pure cultures of M. tuberculosis (pro-
vided by Department of Microbiology, CMC Vellore, Tamil
Nadu, India), M. Smegmatis, and M. phlei. ML1545 and RLEP
genes were amplified using the above mentioned PCR
138 International Journal of Mycobacteriology
Fig. 1 Two percent agarose gel electrophoresis of the
ML1545 gene amplicons from a representative set of two
leprosy cases (S1 and S2). Note. NC = negative control
(nuclease free water); PC = positive control (DNA extracted
from Mycobacterium leprae Br4923).
conditions. The presence of DNA in the samples was confirmed
through the amplification of the rpoB gene region in all three
species using specific primers and PCR conditions [17].
2.7. Statistical analysis
Statistical analysis of the data was performed using GraphPad
Prism 6 software (GraphPad Software, Inc. CA, USA). Z test of
proportions was used to compare the PCR positivity between
the two gene targets. An association with p < 0.05 is consid-
ered statistically significant.
3. Results
3.1. PCR and sequence determination of 112-bp fragment
flanking the promoter region of ML1545
The PCR amplification of ML1545 revealed a 112-bp fragment
(Fig. 1) whose sequence was confirmed through Sanger DNA
sequencing. BLAST alignment with the known genomic
repository of NCBI (Gen Bank Accession: FM211192) for
Fig. 2 Basic Local Alignment Search Tool report of the 112-bp genomic target in the promoter region of ML1545.
Note. Sbjct = subject.
5 ( 2 0 1 6 ) 1 3 5 1 4 1
M. leprae BR4923 and M. leprae TN strains revealed that the
sequence spans nucleotide positions 18681371868248 in the
BR4923 strain and 187412187523 in the TN strain (Fig. 2). This
sequence was chosen owing to its specificity to M. leprae and
the absence of this sequence in other mycobacteria.
3.2. Comparison of PCR positivity between ML1545 and
RLEP
Comparative assessment of PCR positivity among the leprosy
patients revealed that 164/180 (91.11%) were positive for
ML1545 when compared with 114/180 (63.33%) for the RLEP
(p < .0001, z = 6.3). Of the 180 samples, 108 (60.00%) samples
were positive for both of the gene targets, 10 (5.56%) samples
were negative for both ML1545 and RLEP, 56 (31.11%) samples
were positive for ML1545 and negative for RLEP, and six
(3.33%) samples were negative for ML1545 and positive for
the RLEP (p < .0001, z = 7.1; Table 2). Of the ML1545 positive
and RLEP negative samples (n = 56), 24 (42.85%) samples had
a negative BI, 6 (10.71%) were cases of PB leprosy, 17
(30.35%) were cases of BT and indeterminate leprosy, and 9
(16.07%) were cases with other clinical forms of leprosy.
3.3. Comparison of PCR positivity of ML1545 and RLEP
across various clinical forms in leprosy
Attribution of PCR positivity of ML1545 and RLEP to various
clinical forms in leprosy as classified according to WHO regi-
men of MDT, RJ classification, and BI revealed that 103
(63.58%) of 162 MB leprosy cases were positive for RLEP and
150/162 (92.59%) were positive for ML1545 (p = .0001, z = 6.0).
Of the 42 BT leprosy cases, 23 (54.76%) were positive for RLEP
whereas 37 (88.09%) were positive for ML1545 (p = .0006,
z = 3.4). In the lepromatous pole group, 52 (72.22%) out of
the 72 samples were positive for RLEP whereas 69 (95.83%)
were positive for ML1545 (p = .0002, z = 3.7). Importantly,
among the 58 leprosy cases with negative BI, 28 (48.28%) were
 International Journal of Mycobacteriology 5 ( 2 0 1 6 ) 1 3 5 1 4 1 139

Table 2 Comparative assessment of polymerase chain reaction positivity of RLEP and ML1545 in leprosy cases.
Si. No. Comparison No. %

1 Positive for both RLEP and ML1545 108 60.00

2 Negative for both RLEP and ML1545 10 5.56
3 Positive for RLEP and negative for ML1545 6 3.33
4 Positive for ML1545 and negative for RLEP 56 31.11

positive for RLEP and 48 (82.76%) were positive for ML1545
(p = .0001, z = 3.8). In the BI positive groups, among 122 leprosy
cases, 86 (70.49%) were positive for RLEP whereas 116 (95.08%)
were positive for ML1545 (p < .0001, z = 5.1). These results indi-
cate that ML1545 has statistically significant higher PCR posi-
tivity in various clinical forms of leprosy when compared with
RLEP using conventional gradient PCR. Comparisons within
other groups were not detailed due to low sample numbers
in each group; however, they were represented in Table 3.
These observations provide a preliminary lead to the utility
of ML1545 as a PCR target for diagnosis of leprosy, especially
among leprosy cases with low bacillary load.
3.4. PCR assessments in control groups and other
mycobacterial species
Our results indicated that six nonendemic healthy controls
were PCR negative for RLEP or ML1545. Specificity assess-
ments with DNA extracted from M. tuberculosis, M. smegmatis,
and M. phlei revealed that all three species were negative for
PCR with RLEP as well as ML1545. Presence of bacterial DNA
in the samples was determined using PCR for rpoB gene region
of all three species which indicated positive PCR results.
These control experiments indicate that RLEP as well as
ML1545 are specific for M. leprae and do not show any cross
amplification with other mycobacterial species.
4. Discussion
Diagnosis of leprosy has always remained a challenging task
for clinicians and dermatologists because the disease follows
an immunological spectrum wherein TT and BT forms are
often misdiagnosed due to the lack of clear and evident clin-
ical manifestations, especially in a setting where histopatho-
logical examination is not available. PCR has proved to be a
great support in diagnosis of TT and PB leprosy where the BI
is negative. In the past 20 years, PCR has been definitively
used in detecting M. leprae DNA in clinical specimens of vari-
ous types, including slit skin scrapings, nerves, nasal swabs,
oral swabs, ocular lesions, urine, and blood [1822]. Various
M. leprae gene targets have been chosen and analyzed for
their sensitivity and specificity in a diagnostic setting. A
530-bp gene encoding a proline-rich antigen [23] was used
in the detection process and later genes like Ag85B [9], sodA,
and 16SrRNA [12] have also been analyzed for their utility in
diagnosis. Identification of RLEP, a repetitive sequence of M.
leprae which demonstrated higher sensitivity and specificity
than earlier reported gene targets, enabled detection of low
levels of M. leprae DNA [24]. Donoghue et al. [10] first reported
the amplification of the 129-bp fragment of the RLEP gene
using nested PCR in DNA extracted from skin biopsies of
leprosy cases. It has been noted that conventional RLEP PCR
indicated a 3050% positivity in leprosy cases with negative

Table 3 Comparative analysis of polymerase chain reaction positivity of RLEP and ML1545 across various study groups and
clinical characteristics in leprosy.

Characteristics RLEP ML1545 p for comparison

Positive % Negative % Positive % Negative % of PCR positive groups*

Leprosy group 114 63.33 66 36.67 164 91.11 16 8.89 <.0001

WHO PB (n = 18) 11 61.11 7 38.89 14 77.78 4 22.22
MB (n = 162) 103 63.58 59 36.42 150 92.59 12 7.41 .0001
RJ IND (n = 1) 0 0.00 1 100.00 1 100.00 0 0.00
TT (n = 3) 2 66.67 1 33.33 1 33.33 2 66.67
BT (n = 42) 23 54.76 19 45.23 37 88.09 5 11.90 .0006
BB (n = 3) 3 100.00 0 0.00 2 66.67 1 33.33
BL (n = 59) 34 57.62 25 42.37 54 91.52 5 8.47 <.0001
LL (n = 72) 52 72.22 20 27.77 69 95.83 3 4.16 .0002
BI Neg (n = 58) 28 48.28 30 51.72 48 82.76 10 17.24 .0001
12 (n = 25) 16 64.00 9 36.00 23 92.00 2 8.00 <.0001
2.254 (n = 67) 51 76.12 16 23.88 64 95.52 3 4.48
4.256 (n = 30) 19 63.33 11 36.67 29 96.67 1 3.33
Control group (n = 6) 0 0.00 6 100.00 0 0.00 6 100.00
Other Mycobacteria (n = 3) 0 0.00 3 100.00 0 0.00 3 100.00
Note: BI = Bacteriological index; BL = Borderline Lepromatous; BT = Borderline Tuberculoid; IND = Indeterminate; LL = Lepromatous leprosy;
BB = midborderline; Neg = Negative; PB = Paucibacillary leprosy; PCR = Polymerase Chain Reaction; RJ = Ridley Jopling; WHO = World Health
Organization.
* Comparison where p < .05 were only represented.
140 International Journal of Mycobacteriology
BI and around 80% in cases with acid fast bacilli in the smears
[25]. The quantitative PCR with RLEP revealed 87.1% sensitiv-
ity [26] and the application of RLEP real-time PCR assays has
enabled the detection of 75% of PB leprosy cases [14]. While
the target remained highly specific for M. leprae, the sensitiv-
ity values in patient groups especially in TT and BT forms of
the disease remained moderate. Although real-time quantita-
tive PCR using RLEP assays proved useful, their application is
limited due to costs associated with the assays. Hence, in
endemic countries and in resource limited field and patient
settings, a gene target that can demonstrate at least 8090%
sensitivity for M. leprae DNA using conventional gradient
PCR is important for confirmatory diagnosis especially in
cases with low bacillary load and where diagnosis is difficult.
Additionally, the specificity of RLEP gene amplification
using conventional PCR has not been adequately established.
RLEP gene also has conserved sequences which raises a pos-
sibility of the presence of homologous sequences in other
mycobacterial genomes [7,14].
We reported for the first time about the utility of ML1545
for PCR-based diagnosis of leprosy with conventional PCR.
This novel target was able to determine 82% of leprosy cases
with negative BI, demonstrating a higher sensitivity in cases
with low bacillary load. The target remained highly specific
for M. leprae as noted from the BLAST results with the ampli-
con sequence and also from the PCR amplifications in three
other related mycobacterial species. The results indicate only
a preliminary assessment of this M. leprae specific gene target
in clinical samples using conventional PCR; however, there is
scope for further assessments that include determination of
mRNA expression levels of the target across various study
groups. An assay which is similar to that of the RLEP real-
time PCR assay can be developed for ML1545 to determine
the sensitivity limit of this gene target in detecting M. leprae
DNA extracted from various tissue sources of leprosy
patients. Also, the utility of this specific target has not been
investigated in contacts of leprosy cases. Further studies in
this area may provide insights into the utility of ML1545 in
determining subclinical infection in leprosy, owing to its
higher sensitivity.
In conclusion, ML1545 can be a potential gene target for
PCR-based diagnosis of leprosy especially in leprosy cases
with low bacillary load and negative BI and in cases that are
in the TT and BT forms. The target may prove useful in a basic
diagnostic laboratory where conventional PCR machines are
available.
Conflicts of interest
The authors declare that they do not have any conflict of
interests.
Acknowledgments
The authors would like to thank all of the administration and
laboratory staff at Schieffelin Institute of Health-Research
and Leprosy Center who were involved in sample collection
and analysis of data. We give special thanks to Dr. Richard
who helped in the statistical analysis and to Dr. Balaji and
5 ( 2 0 1 6 ) 1 3 5 1 4 1
Dr. Joy S. Michael for the provision of DNA of M. tuberculosis
(H37Rv) from Department of Microbiology, Christian Medical
College and Hospital, Vellore, Tamil Nadu, India for the con-
trol experiments.
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