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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 5 ( 2 0 1 6 ) 1 3 5 1 4 1

1. Introduction specific and sensitive M. leprae gene targets that aid in diagno-
sis of leprosy cases with negative BI or very minimal bacillary
Leprosy, a debilitating disease of the skin and peripheral load using simple conventional PCR.
nerves, continues to remain as a constant peril with approx- In this study, we examined M. leprae specific and unique
imately 135,752 cases from India alone in 2013 [1]. Mycobac- DNA sequences in pseudogenes and identified a 112-bp
terium leprae, the causative organism for this disease, has a sequence in the promoter region of ML1545 (probable
long incubation period of 37 years during which the bacteria 4-alpha-glucanotransferase [pseudogene]) which possess sub-
remain dormant or multiply in the Schwann cells of the neu- stantial specificity. Further, we investigated the utility of this
ronal myelin and manifest a spectrum of clinical outcomes gene sequence as a target for PCR-based detection of M. leprae
[2]. These outcomes can range from a self-limiting, cell- DNA in the clinical isolates of leprosy patients. The PCR
mediated immunity predominant tuberculoid pole (TT) with positivity was compared to that of RLEP to determine the
minimal bacillary load in the skin and nerves to a more sys- sensitivity in detection of M. leprae DNA in leprosy cases
temic, humoral-immunity predominant lepromatous pole across the spectrum of the disease.
with high bacillary load. These polar forms are interspersed
by three intermediary unstable borderline forms (borderline 2. Materials and methods
tuberculoid [BT], midborderline, and borderline lepromatous)
in which the diagnosis of the disease is often based on clinical 2.1. Study patients
manifestations and histopathological features [3]. These
clinical signs are very minimal and often confounding in A total of 180 newly diagnosed untreated leprosy cases from
the TT and BT forms and hence pose diagnostic challenges. the Dermatology Outpatient Department of Schieffelin Insti-
World Health Organization (WHO) has introduced a more tute of Health-Research and Leprosy Center, Karigiri in Vel-
convenient field based classification called the multibacillary lore, Tamil Nadu, India, were enrolled in the study following
(MB) leprosy with greater than five skin lesions and pauci- the institutional ethical guidelines. An informed and written
bacillary (PB) leprosy with less than five skin lesions [4]. consent for participation was obtained from all the patients
During the incubation phase between the infection and before enrolling in the study, following the ethical guidelines
manifestation of clinical symptoms, the disease may remain laid down by the Indian Council of Medical Research. All pro-
subclinical and chronically progress to a systemic phase cedures conducted in the study were in accordance with the
which predisposes nerve damage and subsequent deformities guidelines of the Institutional Ethics Committee of Schieffelin
[5]. Therefore, it is important to diagnose the disease early in Institute of Health-Research and Leprosy Center and with the
a subclinical state in order to treat it rapidly and prevent ethical standards as laid down in the 1964 Declaration of
deformities. In this context, an early diagnostic test has long Helsinki and its later amendments or comparable ethical
been desired for leprosy [6]. To date, there is no specific standards. All the cases were examined by a clinician/leprolo-
screening test and/or assay that aids in early diagnosis of gist and clinical, demographic, and histopathological details
leprosy with substantial sensitivity and specificity [7]. While were recorded at diagnosis (Table 1). In addition, six healthy
the bacteriological index (BI) determination in the slit skin individuals who reside in a low endemic area and without
smears and histopathological examination of the skin biopsy any direct contacts with active leprosy cases, were enrolled
sections remained as clinical and pathological methods for as controls. DNA extracted from Mycobacterium tuberculosis,
determination of bacillary load, the sensitivity of these tests Mycobacterium smegmatis, and Mycobacterium phlei was used
is limited to the few microscopic sections being examined. as a technical control to ascertain the specificity of ML1545.
Detection of M. leprae gene targets (RLEP, 16SrRNA, SodA,
Ag85A, PRA, etc.) in the DNA extracted from the skin biopsies 2.2. Sample types
and peripheral blood samples of leprosy patients compara-
tively presented higher sensitivity especially in the indetermi- Part of the 5 mm  5 mm excisional skin biopsy that was
nate, TT, and BT forms where the BI remains negative due to collected for routine histopathological examination was used
low bacterial counts [8,9]. However, some of these gene tar- for the experiments in the current study. Ridley Jopling classifi-
gets have questionable specificity. cation and BI were recorded at diagnosis for all the participants.
To date, the known gene target that remains specific to The clinical characteristics of the participants were represented
M. leprae genome is the RLEP gene [10], which is a repetitive in Table 1.
element in the genome of M. leprae [11]. Real-time polymerase
chain reaction (PCR) based detection using RLEP PCR assays 2.3. DNA extraction
could detect 70% of PB leprosy in which the BI is negative
[12]. However, simple conventional nested PCR assay with a DNA was extracted from skin biopsy specimens following the
129-bp fragment could detect only 55.5% of the BI negative lysis protocol which was reported earlier, with minor modifi-
PB cases [13]. While it was reported that RLEP real-time PCR cations [15]. Briefly, 25 mg of tissue was thoroughly homoge-
assay can detect M. leprae DNA in as little as 8 fg (indicating nized using a manual homogenizer and homogenate was
3 bacterial cells per sample, taking a 3.2-mbp genome into added to 1 mL of 1X phosphate buffer saline and centrifuged
calculation), the 129-bp fragment missed on identifying at 8000 rpm for 10 min at 4 C. The pellet was dried for 1 h at
3050% of the BI negative PB cases when used in a conven- 50 C on a dry block incubator and was later used in the lysis
tional PCR [14]. Hence there is a need for identification of 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% TATTGCGTC-30 . The designed primers were obtained com-
Tween 20) was added to the homogenate in a 1.5-mL micro- mercially from Eurofins Scientific Inc., Germany. The PCR
centrifuge tube, vortexed thoroughly, and incubated at 60 C amplifications with these primers were compared with those
for 16 h in a water bath (Cole-Parmer Polystat Inc., Chicago, of the RLEP whose primers were taken from the earlier reports
IL, USA). Post incubation, proteinase K was deactivated at [16].
95 C for 15 min. Later the lysate was allowed to cool to room
temperature and DNA was eluted using phenol chloroform 2.5. PCR conditions and cycling parameters
extraction. DNeasy Kit (Cat No: 69504; Qiagen Inc., Valencia,
CA, USA) was used in some samples where the tissue content The PCR amplifications were performed in PTC-150 MiniCycler
was apparently low. DNA extraction from cultures of M. tuber- (MJ Research, MA, USA). A 20-lL reaction mix was prepared
culosis, M. Smegmatis, and M. phlei was also performed using using 1.25 units of AmpliTaq Gold DNA polymerase with
the same protocol. Buffer I (N8080240: Life Technologies Inc., Carlsbad, CA, USA),
0.35 mM of each kind of deoxynucleotides, 0.25 mM MgCl2,
2.4. Selection of the gene target (ML1545) and primer 0.25 lM each of forward and reverse primers, and 2 ng of
design 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
A sequential search of M. leprae specific DNA sequences in 2 min, and 72 C for 2 min. This was followed by 40 cycles of
pseudogenes was performed using National Center for 94 C for 30 s, 61 C for 30 s, and 72 C for 45 s. The reaction
Biotechnology Information (NCBI) Basic Local Alignment was terminated with a final extension of 72 C for 10 min and
Search Tool (BLAST) and PatricBRC Portal (https://www.pa- the tubes were cooled to 25 C for 10 min. The amplicons were
tricbrc.org). A set of eight genes that include ML1127, electrophoresed on a 2% agarose gel and gel images were
ML2110, ML2211, ML0203, ML0307, ML1545, ML2177, and captured using gel documentation system coupled with a UV
ML0466 were identified to harbor sequences that remain camera. PCR-based amplification of the RLEP gene target
specific for M. leprae genome. However, upon further analysis was performed as described earlier [16]. DNA extracted from
of these individual sequences using NCBI BLAST with various M. leprae strain Br4923 (Courtesy: BEI Resources, catalog num-
database search parameters like NCBI Genomes (chromo- ber: NR-19351) was used as a positive control in the
somes) and Reference genomic sequences within Mycobac- experiments.
teria and Mycobacteriaceae, a DNA sequence that traverses
the promoter region of probable 4-alpha-glucanotransferase 2.6. PCR conditions for other Mycobacteria
(ML1545) gene was chosen as it remained highly specific to
the genome. Primers were designed while maintaining the DNA was extracted from pure cultures of M. tuberculosis (pro-
specificity using Primer BLAST version 3 from NCBI. The vided by Department of Microbiology, CMC Vellore, Tamil
sequence for the forward primer is 50 -GTCCTCCGTCTTGCTG Nadu, India), M. Smegmatis, and M. phlei. ML1545 and RLEP
ACTG-30 and for the reverse primer is 50 -CATACCGGCCA genes were amplified using the above mentioned PCR
138 International Journal of Mycobacteriology 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

Fig. 1 Two percent agarose gel electrophoresis of the Comparative assessment of PCR positivity among the leprosy
ML1545 gene amplicons from a representative set of two patients revealed that 164/180 (91.11%) were positive for
leprosy cases (S1 and S2). Note. NC = negative control ML1545 when compared with 114/180 (63.33%) for the RLEP
(nuclease free water); PC = positive control (DNA extracted (p < .0001, z = 6.3). Of the 180 samples, 108 (60.00%) samples
from Mycobacterium leprae Br4923). 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
conditions. The presence of DNA in the samples was confirmed (3.33%) samples were negative for ML1545 and positive for
through the amplification of the rpoB gene region in all three the RLEP (p < .0001, z = 7.1; Table 2). Of the ML1545 positive
species using specific primers and PCR conditions [17]. and RLEP negative samples (n = 56), 24 (42.85%) samples had
a negative BI, 6 (10.71%) were cases of PB leprosy, 17
2.7. Statistical analysis (30.35%) were cases of BT and indeterminate leprosy, and 9
(16.07%) were cases with other clinical forms of leprosy.
Statistical analysis of the data was performed using GraphPad
Prism 6 software (GraphPad Software, Inc. CA, USA). Z test of 3.3. Comparison of PCR positivity of ML1545 and RLEP
proportions was used to compare the PCR positivity between across various clinical forms in leprosy
the two gene targets. An association with p < 0.05 is consid-
ered statistically significant. Attribution of PCR positivity of ML1545 and RLEP to various
clinical forms in leprosy as classified according to WHO regi-
3. Results men of MDT, RJ classification, and BI revealed that 103
(63.58%) of 162 MB leprosy cases were positive for RLEP and
3.1. PCR and sequence determination of 112-bp fragment 150/162 (92.59%) were positive for ML1545 (p = .0001, z = 6.0).
flanking the promoter region of ML1545 Of the 42 BT leprosy cases, 23 (54.76%) were positive for RLEP
whereas 37 (88.09%) were positive for ML1545 (p = .0006,
The PCR amplification of ML1545 revealed a 112-bp fragment z = 3.4). In the lepromatous pole group, 52 (72.22%) out of
(Fig. 1) whose sequence was confirmed through Sanger DNA the 72 samples were positive for RLEP whereas 69 (95.83%)
sequencing. BLAST alignment with the known genomic were positive for ML1545 (p = .0002, z = 3.7). Importantly,
repository of NCBI (Gen Bank Accession: FM211192) for among the 58 leprosy cases with negative BI, 28 (48.28%) were

Fig. 2 Basic Local Alignment Search Tool report of the 112-bp genomic target in the promoter region of ML1545.
Note. Sbjct = subject.
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 4. Discussion
(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%) Diagnosis of leprosy has always remained a challenging task
were positive for ML1545 (p < .0001, z = 5.1). These results indi- for clinicians and dermatologists because the disease follows
cate that ML1545 has statistically significant higher PCR posi- an immunological spectrum wherein TT and BT forms are
tivity in various clinical forms of leprosy when compared with often misdiagnosed due to the lack of clear and evident clin-
RLEP using conventional gradient PCR. Comparisons within ical manifestations, especially in a setting where histopatho-
other groups were not detailed due to low sample numbers logical examination is not available. PCR has proved to be a
in each group; however, they were represented in Table 3. great support in diagnosis of TT and PB leprosy where the BI
These observations provide a preliminary lead to the utility is negative. In the past 20 years, PCR has been definitively
of ML1545 as a PCR target for diagnosis of leprosy, especially used in detecting M. leprae DNA in clinical specimens of vari-
among leprosy cases with low bacillary load. ous types, including slit skin scrapings, nerves, nasal swabs,
oral swabs, ocular lesions, urine, and blood [1822]. Various
3.4. PCR assessments in control groups and other M. leprae gene targets have been chosen and analyzed for
mycobacterial species their sensitivity and specificity in a diagnostic setting. A
530-bp gene encoding a proline-rich antigen [23] was used
Our results indicated that six nonendemic healthy controls in the detection process and later genes like Ag85B [9], sodA,
were PCR negative for RLEP or ML1545. Specificity assess- and 16SrRNA [12] have also been analyzed for their utility in
ments with DNA extracted from M. tuberculosis, M. smegmatis, diagnosis. Identification of RLEP, a repetitive sequence of M.
and M. phlei revealed that all three species were negative for leprae which demonstrated higher sensitivity and specificity
PCR with RLEP as well as ML1545. Presence of bacterial DNA than earlier reported gene targets, enabled detection of low
in the samples was determined using PCR for rpoB gene region levels of M. leprae DNA [24]. Donoghue et al. [10] first reported
of all three species which indicated positive PCR results. the amplification of the 129-bp fragment of the RLEP gene
These control experiments indicate that RLEP as well as using nested PCR in DNA extracted from skin biopsies of
ML1545 are specific for M. leprae and do not show any cross leprosy cases. It has been noted that conventional RLEP PCR
amplification with other mycobacterial species. 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 5 ( 2 0 1 6 ) 1 3 5 1 4 1

BI and around 80% in cases with acid fast bacilli in the smears Dr. Joy S. Michael for the provision of DNA of M. tuberculosis
[25]. The quantitative PCR with RLEP revealed 87.1% sensitiv- (H37Rv) from Department of Microbiology, Christian Medical
ity [26] and the application of RLEP real-time PCR assays has College and Hospital, Vellore, Tamil Nadu, India for the con-
enabled the detection of 75% of PB leprosy cases [14]. While trol experiments.
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-
R E F E R E N C E S
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
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Conflicts of interest
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and Leprosy Center who were involved in sample collection leprae from multicase families of leprosy patients and their
and analysis of data. We give special thanks to Dr. Richard surroundings to understand the transmission of leprosy,
who helped in the statistical analysis and to Dr. Balaji and Clin. Microbiol. Infect. 20 (2014) 142149.
International Journal of Mycobacteriology 5 ( 2 0 1 6 ) 1 3 5 1 4 1 141

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