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Area of Vietnam
Masanori Kai1, Nhu Ha Nguyen Phuc2, Hoang An Nguyen2, Thi Hoang Bich Diu
Pham2, Khanh Hoa Nguyen2, Yuji Miyamoto1, Yumi Maeda1, Yasuo Fukutomi1,
Noboru Nakata1, Masanori Matsuoka1, Masahiko Makino1, and Thanh Tan Nguyen2
+ Author Affiliations
1Department of Mycobacteriology, Leprosy Research Center, National Institute of
Infectious Diseases, Higashimurayama, Tokyo, Japan
2Quyhoa National Leprosy and Dermato-Venereology Hospital, Quynhon City,
Binhdinh, Vietnam
Correspondence: Masanori Kai, MD, Department of Mycobacteriology, Leprosy
Research Center, National Institute of Infectious Diseases, 4-2-1 Aoba-cho,
Higashimurayama, Tokyo 189-0002, Japan (mkai@nih.go.jp).
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Abstract
(See brief report by Ramien and Wong, e133-e135.)
Background.Multidrug therapy has effectively reduced the number of leprosy
cases in the world. However, the rate of reduction has decelerated over the years,
giving early detection of Mycobacterium leprae and epidemiological study of relapse
renewed relevance in attempts to eliminate the disease.
Methods.A molecular epidemiological survey for drug-resistant M. leprae was
conducted in the central and highland regions of Vietnam. A total of 423 samples
taken from patients, including 83 patients with new cases, 321 patients receiving
treatment, and 19 patients with relapse, were studied for detection of M. leprae with
mutations relating to drug resistance by sequencing the drug resistance
determining region of the folP1, rpoB, and gyrA genes, which are responsible for
dapsone, rifampicin, and ofloxacin resistance, respectively.
Results.Nineteen mutations were found in the folP1 gene samples, and no
mutations relating to drug resistance were found in either the rpoB or gyrA genes.
Samples from patients with relapse showed folP1 mutation rates as high as 57%,
and the mutation rates in samples from new and recent cases were <10%. Patients
with relapse who had histories of treatment with dapsone monotherapy showed
high mutation rates (78%), compared with patients with relapse who had previously
only received multidrug therapy (33%).
Conclusions.Our study indicated high rates of dapsone resistance in patients with
relapse, compared with patients with new and recent cases of leprosy. Moreover, it
was observed that many of the patients with relapse who had dapsone-resistant
mutations had histories of treatment with dapsone monotherapy.
Leprosy is a chronic infectious disease caused by infection with Mycobacterium
leprae. The present strategy for leprosy control is based on the multidrug therapy
(MDT), recommended by the World Health Organization (WHO) [1], which has
successfully reduced the number of leprosy cases in the world. However, transition
in the number of registered cases and new cases amounting to 210,000 and
250,000, respectively, has almost come to a standstill [2]. Drug-resistant strains
were first found in 1964, 1976, and 1997 [35]. MDT was designed to prevent the
emergence and spread of drug-resistant strains. However, a strain showing
resistance to both dapsone and rifampicin was reported in 1993 [6], and at present,
there are further reports indicating the emergence of M. leprae strains resistant to
multiple drugs [5, 7]. At present, the rapid detection and control of such drugresistant strains is essential in countries approaching leprosy elimination levels,
such as Vietnam.
MDT has been quite successful in Vietnam, and elimination of leprosy (prevalence
rate, < 1/10,000 population) was achieved on the national level in 1995 [8]. The
prevalence rate per 10,000 population in 2006 was .07 [8, 9]. However, the majority
of patients with leprosy are found in the central and highland regions of Vietnam
[10], consisting of 11 provinces, including 4 provinces in the highland region and 7
provinces in the delta region. In 2005, the number of patients with leprosy was 236,
spread through 4 provinces of the highland region; the prevalence rate of newly
detected cases was 3.5 cases/10,000 population, although the overall prevalence
rate was .25 cases/100,000 population on the national level. The rate of newly
detected cases in the 7 delta region provinces was 1.38 cases/10,000 population [8,
9]. These cases not only present the danger of being possible infectious sources for
leprosy but also harbor the risk of developing into relapse cases. However, little is
known regarding the effects of drug-resistant M. leprae in patients with leprosy,
especially in cases of relapse.
Therefore, in the present study, molecular epidemiological studies on drug-resistant
strains were conducted in 11 provinces primarily in the central and highland regions
that represent the areas where leprosy is endemic in Vietnam.
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MATERIALS AND METHODS
Sensitivity of Polymerase Chain Reaction
The number of bacilli isolated from nude mice footpads was counted using the
method described by Shepard et al [11]. Serial 10-fold dilutions of the enumerated
M. leprae bacilli were used for polymerase chain reaction (PCR) in our study.
Clinical Specimens
Samples (from slit-skin smears or punch biopsies) were taken from patients with
leprosy after receipt of informed consent in primarily the central and highland
regions of Vietnam (including 11 provinces: Danang, Quangnam, Quangngai,
Binhdinh, Phuyen, Khanhhoa, Ninhthuan, Kontum, Gialai, Daknong, and Daklak), and
the samples were classified as new (before starting MDT), recent (receiving MDT),
and relapse cases. Relapse was defined as development of new skin lesions after
completion of MDT and increase in bacterial index by >2 log units in any lesion.
The total of 423 samples included those from 83 patients with new cases, 321
patients with recent cases (receiving treatment), and 19 patients with relapse
(collection period: March 2004August 2009). Among 16 patients with relapse who
had positive results of M. lepraespecific PCR, 9 cases were determined to be
relapse after dapsone monotherapy (720 years), 3 as relapse after complete MDT,
2 as second relapse (the first after dapsone monotherapy and the second after
MDT), and 2 as relapse after ofloxacin treatment. Samples were obtained from the
skin lesions of patients (smear on blade or biopsy soaked in 1 mL of 70% ethanol at
room temperature in the field, before being sent to Quyhoa National Leprosy &
Dermato-Venereology Hospital laboratory).
DNA Extraction, Nested PCR, and Sequencing
M. leprae templates from both dilutions of M. leprae bacilli and slit-skin smears were
prepared by treatment with lysis buffer at 60C overnight, as described elsewhere
[12]. Nested PCR amplification of the RLEP regions of M. leprae was performed
under conditions described elsewhere with minor modifications, using the primers
listed in Table 1 [13]. In brief, PCR amplification using special reagents (20 mM TrisHCl [pH, 7.5], 8 mM magnesium chloride, 7.5 mM DTT, 2.5 mg BSA, 150 M
deoxynucleotides, 1.5 mM magnesium sulphate, and 2.5 units KOD-plus-Ver.2 DNA
polymerase [Toyobo]) was performed using sample DNA as templates. Both first and
second PCR conditions were as follows; strand separation at 94C for 4 min,
denaturing at 94C for 40 s, annealing at 55C for 1 min, and extension at 72C for
20 s plus 1-s increment per cycle for 25 cycles. Products from the first PCR (0.5 uL)
were used as templates in the second PCR. The nested PCR for DRDR was
performed using the primer pairs listed in Table 1. Mutations were measured on the
folP1 gene for dapsone [14], the rpoB gene for rifampicin, and the gyrA gene for
ofloxacin [15, 16]. Nested PCR conditions for drug resistance were different from
that for RLEP-nested PCR. In brief, PCR amplification using standard reagents (10
mM Tris-HCl [pH, 8.3], 2 mM magnesium chloride, 250 M dNTPs, and 2.5 units
TaKaRa Ex Taq DNA polymerase [Takara shuzo]) was performed using sample
genomic DNA as templates. The primer pairs used to amplify the specific drugresistant genes are shown in Table 1. The reaction condition was 30 s at 94C, 30 s
at 60C, and 1 min at 72C for 35 cycles.
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Table 1.
Sequences of Primers Used in this Study
The amplicons were visualized by agarose gel electrophoresis, and DNA was
recovered from the gel using Mini-Elute gel extraction kits (Qiagen). The recovered
DNA molecules were sequenced using the ABI Prism BigDye Terminator Cycle
Sequencing kit (Perkin-Elmer Applied Biosystems) and run on an ABI Prism 3130
Genetic Analyzer (Applied Biosystems). The sequence data were analyzed by DNA
analysis program Genetyx-MAC, version 15 (GENETYX), and were compared with
those in the GenBank database.
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RESULTS
PCR Sensitivity
Serial dilutions of the bacilli of 1 1081 100 were prepared to determine PCR
sensitivities. Genomic DNAs were extracted from the diluents with use of methods
described under Materials and Methods [11]. The previously reported RLEP-nested
PCR (named RLEP-L) was capable of detecting 1 102 bacilli in samples (Figure 1a)
[13]. The newly designed RLEP-nested PCR, using K1 and K2 primers for the first
PCR and LP1 and LP2 primers for the second PCR (named RLEP-K), is capable of
detecting comparable counts of bacilli (Figure 1b), and RLEP-K products are
visualized more clearly with less smear bands. Therefore, the new RLEP-K system
was used for detection in further experimentation with use of clinical samples.
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Figure 1.
Sensitivity of nested polymerase chain reaction (PCR). The nested PCR products
were visualized on 2 % agarose gel. A, RLEP-nested PCR (RLEP-L) using primers,
LP1-LP4 (final products size, 99 bp). B, RLEP-nested PCR (RLEP-K) using primers, K1,
K2, LP1, and LP2 (final products size, 129bp). C, folP1-nested PCR using F1-F4. D,
rpoB-nested PCR using R1-R4. E, gyrA-nested PCR using G1-G4.
Using DNAs extracted from the serial dilutions of M. leprae, we determined the
sensitivity of the nested PCR for DRDRs. The limit of amplification by PCR was 1
1031 104 bacilli (Figure 1 ce).
RLEP-nested PCR for Clinical Samples
The PCR methods were applied on 423 clinical samples collected from areas of
endemicity in Vietnam. First, we tested RLEP-K for detection of M. leprae after
extraction of DNA from smear samples. Positive bands were obtained by gel
electrophoresis using RLEP-K on 290 samples. The positivity rate was 69%. The
patients supplying the 290 samples were divided into 3 categories: new, relapse,
and recent cases. Positive rates of RLEP-K by category were 75%, 84%, and 66%,
respectively (Table 2).
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Table 2.
Polymerase Chain Reaction Positivity in New, Relapse, and Recent Cases
Mutations in Clinical Samples
Samples positive by RLEP-nested PCR were applied for mutation experiments on the
DRDRs of folP1, rpoB, and the gyrA gene. Nineteen mutations were found in 187
folP1 samples, but no mutations related to drug resistance were noted in 163 rpoB
and 147 gyrA gene samples. The mutations detected on folP1 were as follows: 6
cases of ACC to ATC in codon 53(threonine to isoleucine), 9 cases of CCC to CGC in
codon 55 (proline to arginine), and 4 cases of CCC to CTC (proline to leucine). Two
new cases, 8 relapse cases, and 9 recent cases had mutations on folP1. Mutation
rates in the 3 categories were 6.1%, 57%, and 6.4%, respectively (Table 3).
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Table 3.
Number of Mutations on folP1
Some missense mutations, of which the association with drug resistance is
unknown, were detected in the rpoB gene from clinical samples. The mutations
were detected in 7 patients at codons 517, 532, and 556. One patient with relapse
showed a mutation from CAG (glutamine) to CAT (histidine) at codon 517. One new
patient showed 2 mutations at codon 517 from CAG (glutamine) to CAT (histidine)
and at codon 532 from GCG (alanine) to TCG (serine). Sequence electropherograms
indicated double peaks of a second nucleotide at codon 556 in 3 patients
categorized as having recent cases. One peak was G (identical to that of wild-type),
and the other peak was T, which changed the amino acid from glycine (GGC) to
valine (GTC; data not shown).
The Relation between Treatment and Drug-Resistant Mutations in Patients with
Relapse
Patients with relapse were categorized into 4 groups, by treatment history (Table 4).
Group 1 comprised those treated with dapsone monotherapy. Group 2 was treated
with MDT for 24 months. Group 3 included patients who had received a diagnosis of
second relapseonce after treatment with dapsone monotherapy and,
subsequently, after MDT for 24 months. Group 4 was treated with ofloxacine
monotherapy. Eight of the 14 patients with folP1-amplified relapse cases (57%) had
mutations on the folP1 gene. Seven (78%) of 9 patients with relapse who were
categorized in groups 1 and 3 also had folP1 mutations. However, 2 patients in
group 4 had no mutations on any of the 3 genes.
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Table 4.
Mutations Noted in RLEP-Positive Relapse Cases, by Treatment Group
Monitoring of Mutations in Patients
One hundred seven slit-skin smear samples from 43 patients were taken with
consents at different times from each patient for monitoring mutations under
treatment. Table 5 shows the difference in mutation results between 5 such
patients. The other 38 patients showed no mutation during monitoring. Patients A,
B, and C, who had new cases, showed a similar pattern, with no mutation at first
testing and mutation in codon 53 on the folP1 gene during MDT. However, double
peaks of T and C in the second base were observed on folP1 in the 3 patients.
Patients D and E, who had relapse cases and finished dapsone monotherapy 20
years earlier, had a mutation on folP1 in 2005 and no mutation after MDT.
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Table 5.
Monitoring of 5 Patients with Multibacillary Leprosy for folP1 Mutation
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DISCUSSION
The most popular PCR method for M. leprae detection with high sensitivity and
specificity is probably the RLEP-nested PCR method, because the RLEP regions are
specific for M. leprae, with >28 copies dispersed through the M. leprae genome
[17]. New primers were designed for the RLEP-nested PCR in our study. This system
using the new primers was termed RLEP-K. RLEP-K products appear to be a
somewhat sharper and stronger band on agarose gel electrophoresis, compared
with that that of previous RLEP-nested PCR (ie, RLEP-L). The RLEP-K detected M.
leprae in 69% of the Vietnam samples. The remaining 31% of the samples were
deduced as being cases either cleared of M. leprae by chemotherapy or those
having <100 bacilli, which was below the detection limit of RLEP-K. We also
designed new primers for amplification and sequencing of DRDR in the drugresistance related genes folP1, rpoB, and gyrA, which were applied in examining the
Vietnam samples. The mutation rates of folP1 in new and recent cases were 6.1%
and 6.4%, respectively. In contrast, the mutation rate in relapse cases was quite
high, at 57%. The result indicated a strong correlation between mutation rate and
relapse. Two possible reasons were conceived regarding the high positive rate of
dapsone resistance in patients with relapse: (1) reinfection by the primary drugresistant strain (7 of 8 samples indicating relapse were collected in the province in
central Vietnam, which had the highest prevalence of leprosy and high rate of
relapse (data not shown) and (2) reactivation of dapsone-resistant strains capable of
persisting after chemotherapy, discussed below. Although it is still unclear whether
the relapses are caused by reinfection by M. leprae or by reactivation of persistent
M. leprae, close correlation between drug resistance and relapse have been
recognized in several studies [18, 19].
The proportion of samples showing mutation on the folP1 gene related to dapsone
resistance was 10.2% (19 of 187) in samples from the central and highland regions
of Vietnam (Table 3). Comparison with previous reports from South Korea (19.2%)
indicates lower rates of relapse in these regions of Vietnam [20].
No mutation was found in the DRDR regions of rpoB in all samples. Mutation
frequencies of the rpoB gene are also very low in other reports. Regarding other
areas in Southeast Asia, no cases of rifampicin resistance have been detected in the
Philippines, 1 (1.9%) of 54 cases in Myanmar, and 4 (3.3%) of 121 cases in
Indonesia. However, in Japan, where the prevalence of leprosy is very low, the
reported rate of rifampicin resistance is very high, at 29.5% (26 of 88 cases) [21].
The long-term use of drugs outside the standard MDT regimen in Japanese leprosy
cases might have been instrumental in promoting this rifampicin resistance.
As such, no mutations have been found in the DRDR of the M. leprae rpoB gene
derived from patients with leprosy, including relapse cases in Vietnam. A possible
explanation for this could be the success of leprosy control in Vietnam and efficacy
of properly administered MDT in which rifampicinwith its bactericidal properties
was effective in suppressing the occurrence of drug-resistant bacilli. In contrast,
dapsone (not bactericidal in itself, although capable of suppressing growth), which
had previously been used as monotherapy, may have enabled bacteria surviving in
the patient receiving treatment to develop mutations, giving them resistance
against the drug. Although occurrence of drug-resistant M. leprae was kept very low
after application of MDT, 7 of 9 samples with drug-resistant mutations had
previously been treated by dapsone monotherapy (Table 4). Jing et al [22] reported
that patients with multibacillary leprosy who were retreated with MDT after dapsone
monotherapy may have lower risk of early relapse while continuing to carry the risk
of late relapse. Our observations suggest the possibility that efficacy of MDT may be
hampered in some patients by the presence of surviving dapsone-resistant M.
leprae in their bodies, which could develop into late relapse. Similar observations
have been reported, suspecting involvement of the effects of dapsone monotherapy
in patients with relapse [23].
There was no mutation in the major sites for drug resistance on the rpoB gene.
However, we observed mutations at 3 positions, codons 517, 532, and 556, which
have not been associated with rifampicin resistance. These mutations in the rpoB
gene are a finding calling for further clarification.
To reveal the possible relation between treatment and gene mutation, some
patients with leprosy were monitored for gene mutations in light of drug treatments.
The results showed incidence of dapsone-resistant M. leprae in patients receiving
MDT, suggesting that some of the patients with relapse who were previously treated
with dapsone monotherapy might have persistent infections with dapsone-resistant
M. leprae. Furthermore, samples derived from different sites of lesions in the same
patient sometimes showed different results (Table 5). The results suggest that we
need to know the relation between the situation of patients with leprosy and drug
resistance.
Overall, our study indicated a high ratio of dapsone resistance in patients with
relapse, compared with the other patients with leprosy. In contrast, an unexpected
outcome of our study was that we were unable to find mutations on the rpoB gene
in patients with relapse. Moreover, it was shown that many of the patients with
relapse who had dapsone-resistant mutations had histories of treatment with
dapsone monotherapy. To clarify the relationship between relapse, drug resistance,
and dapsone monotherapy, it might be necessary to investigate persistence of
drug-resistant M. leprae through large-scale surveillance.
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Acknowledgments
We thank all medical officers working in the local health centers of the 11 provinces
in the central and highland regions of Vietnam for their help in collecting the clinical
samples.
Financial support.Thos work was supported by Ministry of Health of Vietnam;
Quyhoa National Leprosy & Dermato-Venereology Hospital, Vietnam; Japan Health
Sciences Foundation; and a Health Science Research GrantResearch on Emerging
and Re-emerging Infectious Diseases, Ministry of Health, Labour and Welfare, Japan.
Potential conflicts of interest.All authors: no conflicts.
Received August 28, 2010.
Accepted December 7, 2010.
The Author 2011. Published by Oxford University Press on behalf of the Infectious
Diseases Society of America. All rights reserved. For Permissions, please email:journals.permissions@oup.com.
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Journal of Pathogens
This paper highlights the importance of the LDs as one of the most unique
determinant for M. leprae persistence and virulence. The formation of LDs in M.
leprae infected cells has been compared to LDs formation in macrophages infected
with M. tuberculosis. Thereafter, we will focus on the interplay between the LDs of
the host and the pathogen.
[29]. BODIPY stains infected SCs, indicating that LDs contain neutral lipids, such as
triacylglycerols (TAG) [5].
2.3. Regulation of Host-Signaling by M. leprae Induced-Lipid Droplets
In M. leprae infected-cells, LDs show high ability to fuse further to form giant LDs. In
most mammalian cells, dynein and extracellular signal-regulated kinase-2 (ERK2)
facilitate LDs move along the cytoskeletal network [55]. Extracellular signalregulated kinase-1/2 is one of the most upregulated signals after M. leprae inducedSCs demyelination and nerve injury [56]. Using NIH 3T3 cells, Andersson et al.
demonstrated that ERK2 is an essential signal in LDs formation [57] and also seem
to be crucial for the phospholipase D1- (PLD1-) mediated increases of LDs. The ERK2
mechanism is mediated by phosphorylation of dynein, which finally upregulates its
association in ADRP-containing LDs [57].
Lipid droplets induction in all type of cells associates with lipid droplets protein
adipophilin (ADFP) [58]. The classic model of LDs postulates that LDs formation take
place in the outer leaflet of the endoplasmic reticulum (ER) membrane (the place of
TAG synthesis). In this formation, the function of AFDP has been proposed in fatty
acid absorption and transport. Cruz et al. showed that ADFP is overexpressed in Llep lesions [29]. Furthermore, the authors also pinpointed similarity in overall
observations to one of the important metabolic disease atherosclerosis. Similar
observation in atherosclerosis showed that ADFP is the most expressed LDassociated protein found in macrophages and that the accumulation of LDs in
lesional macrophages is associated with ADFP aggregation [58].
Several reports have supported the inflammatory role of macrophages and SCs,
where LDs induction by these cells associated with modulation in production of
inflammatory mediators [5963]. Moreover, increased LDs formation accompanied
by PGE2 and transforming growth factor- (TGF-1) synthesis in BCG infected
macrophage while strong PGE2 was seen in foamy macrophages during M.
tuberculosis infection [64, 65]. Cytokines, chemokines, and growth factors seem to
be correlated with LDs formation by M. leprae. For instance, Persson et al. found
that IL-1 and TNF- inflate foamy macrophages formation [66]. M. leprae infection
significantly increased NF-B, TNF-, and IL-1 expression in NOD1- and NOD2transfected macrophages cells [67]. Another example, IL-10 and IL-12 secretion
correlated with bacilli-LD induction in L-lep lesions [32, 68]. Schwann cells are able
to produce several type of cytokines, including IL-1, IL-6, IL-8, IL-10, IL-12, PGE2,
TGF-, and TNF- [37, 69, 70]. PGE2 and IL-10 overexpression is associated with
clog of IL-12 and NO production in M. leprae-infected SCs [32, 68]. In late infection
state, M. leprae impede the expression of TNF signaling and induce expression of 9O-acetyl GD3 as smart strategy to avoid the cellular apoptosis and facilitate its
persistence inside the favored cell niche [37, 67, 71, 72]. Obviously, lipid
their M. leprae homologs enzymes involved in lipid metabolism in lipid bodies are
summarized in Table 1.
Table 1: Comparison between the enzymes involved in TAG metabolism in M.
tuberculosis and M. bovis BCG and their homologous in M. leprae. ND, not
determined.
M. tuberculosis can grow on fatty acids as sole carbon source and it has been
demonstrated that fatty acid oxidation is important for survival of the pathogen in
the lungs of mice [83, 84]. Fatty acids are oxidized via the -oxidation cycle and the
glyoxylate shunt, to replenish TCA cycle intermediated during growth [88]. The oxidation cycle consists of five biochemical reactions, where one molecule acetylCoA of the fatty acid is split off per cycle. The genome of M. tuberculosis encodes
around 100 genes, designated as fad genes (fatty acid degradation) with putative
roles in the -oxidation of fatty acids. While E. coli has only one enzyme for each
step of the -oxidation cycle, M. tuberculosis seems to have several backup
enzymes for each reaction [89]. M. leprae has approximately one-third as many
potential fad enzymes. However, there are three times more FadD acyl-CoA
synthases than there are FadE acyl-CoA dehydrogenases, whereas these are
predicted in equal amounts in M. tuberculosis [74]. The initial step of -oxidation is
the formation of acyl-CoA from free fatty acids and coenzyme A and is catalyzed by
acyl-CoA synthase. The Acyl-CoA synthase Rv1683 is suspected to be essential for
TAG hydrolysis and growth [80, 90].
Even though M. leprae genome contains less necessary -oxidation cycle genes
than M. tuberculosis, transcripts analysis revealed active expression of acyl-CoA
metabolic enzymes including echA1 (ML0120, putative enoyl-CoA hydratase),
echA12 (ML1241, possible enoyl-CoA hydratase), fadA2 (ML2564, acetyl-CoAacetyltransferase), fadB2 (ML2461, 3-hydroxyacyl-CoA dehydrogenase), fadD19
(ML0352, acyl-CoA synthase), fadD26 (ML2358, fatty acid-CoA-ligase), fadD29
(ML0132, probable fatty-acid-CoA synthetase), fadD28 (ML0138, possible fatty-acidCoA synthase), fadE25 (ML0737, probable acyl-CoA dehydrogenase), and fadE5
(ML2563, acyl-CoA dehydrogenase) [91, 92]. Combing observations from leprosy
lesions, this gives strong evidence that host lipids provide the main carbon and
energy sources for M. leprae during infection.
Together with malate synthase, isocitrate lyase (ICL) is the key enzyme of the
glyoxylate cycle that catalyzes the cleavage of isocitrate to glyoxylate and
succinate [88, 93]. The M. leprae genome contains a gene, coding for an isocitrate
lyase, aceA. AceA is upregulated in both M. leprae-infected nude mouse and human
lesions. [91]. The amino acid sequence of aceA (ML1985c) shows 80% identity with
its homologue from M. tuberculosis ICL2 (Rv1915/1916). A second icl gene is not
present in M. leprae, as observed in M. tuberculosis. This finding is of particular
interest, because two M. tuberculosis isocitrate lyases, icl and icl2, are jointly
required for in vivo growth and virulence [83, 84]. Deletion of icl1 or icl2 has little
effect on bacterial growth in macrophages [83]. So far the evidence indicates that
M. leprae aceA might play a slightly different role in both isocitrate lyases in M.
tuberculosis.
4. M. leprae Has a Reduced Number of Triacylglycerol Synthase Genes
For persistent M. tuberculosis, TAGs seem to be the major carbon and energy
source. Biosynthesis of TAG consists of the sequential esterification of the glycerol
moiety with fatty acyl-residues by various acyltransferases. The fatty acids are
supplied by de novo biosynthesis or -oxidation. Esterification occurs via sequential
acylation of the sn-1,2 and 3 positions of glycerol-3-phosphate, and removal of the
phosphate group before the last acylation step. The terminal reaction is the
esterification of diacylglycerol (DAG) with acyl-CoA by an diacylglycerol
acyltransferase (Figure 2) [94]. Bacteria do not contain DGATs but only bifunctional
wax ester synthase/acyl-CoA: diacylglycerol acyltransferases (WS/DGAT). WS/DGATs
mediate next to TAG formation the synthesis of waxes by esterification of acyl-CoA
with alcohol [95]. The genome of M. tuberculosis codes for 15 genes which contain
the highly conserved putative active site motif of WS/DGATs (HHxxxDG). These
genes were designated as tgs, triacylglycerol synthases, but have only a weak
sequence similarity to other WS/DGAT sequences. All 15 expressed mycobacterial
tgs proteins show diacylglycerol acyltransferase activity and tgs1 has the highest
activity of all enzymes [26]. Tgs1 appears to be a major contributor to TAG
synthesis in M. tuberculosis so far [26, 78]. And moreover, two homologue proteins
to tgs1 and tgs2 (BCG3153c and BCG3794c) and another poorly characterized
acyltransferase (BCG1489c) were found to be exclusively associated to lipid bodies.
Disruption of the tgs genes BCG3153c, BCG3794c, and BCG1489c reduces TAG
accumulation during hypoxia-induced nonreplicating state, revealing that the
enzymes are involved in TAG synthesis during latency and pathogenicity [80].
Figure 2: Schematic representation of putative genes involved in TAG biosynthesis in
M. leprae. As a precursor, glycerol-3-phosphate further processed by sequential
acylation steps, until last the transesterification of diacylglycerol (DAG) to
triacylglycerol (TAG). Reaction 1 mediated by glycerol-3-phosphate acyltransferase,
1-acylglycerol-3-phosphate acyltransferase mediates reaction 2, phosphatidc acid
phosphatase processes reaction 3, and the last reaction 4 is mediated by acyl-CoA:
diacylglycerol acyltransferase enzyme.
Ten of the 15 tgs genes in M. tuberculosis are located adjacent or proximal to 11 lip
genes that are annotated as probable phospholipases or lipases-esterases
carboxylesterases. Some tgs genes may be cotranscribed with neighboring lip
genes and may synthesize triacylglycerols from the released fatty acids from the
host [26]. Lip gene products may be important for utilization of TAGs during
dormancy and upon reactivation after dormancy. The tgs gene Rv0221 is located
near LipC (Rv0220), lipW (Rv0217c), acyl-CoA synthetase (Rv0214), acyl-CoA
dehydrogenase (Rv0215c), and an integral membrane acyltransferase (Rv0228).
This clustering of genes of the fatty acid metabolism suggests that these genes may
be cotranscribed and may release fatty acid from host TAG, carry out the transport
of fatty acids and finally catalyze the resynthesis of TAGs in the pathogen. Rv0221
and LipC have to be shown to be catalytical active [26, 76].
The M. leprae genome shows only one predicted gene product which has a
significant degree of identity to any tgs enzymes from M. tuberculosis [26]. The tgs
gene product ML1244 shows 72% identity to Rv2484c from M. tuberculosis.
Rv2484c is located next to a carboxylesterase lipQ, (Rv2485c), a probable glycerol3-phosphate acyltransferase, (Rv2482c), a lysophosphatidic acid acyltransferaselike protein (Rv2483c), and a probable enoyl-CoA hydratase (Rv2486). The gene
cluster of lipid metabolism genes suggests a possible involvement of the gene
products in the synthesis of TAG [26]. A few tgs genes (Rv3234c, Rv3233c, Rv2285,
and Rv1425) are located proximally to lipoproteins, which may serve as donors or
acceptors of fatty acids [26].
Ag85A, a mycoly transferase that is known to catalyze the formation of the cord
factor was recently found to have additional DGAT activity [79]. The homologue
gene of M. tuberculosis Ag85A is expressed in M. leprae. Although, mycoly
transferase 85 complex genes (A, B, and C) transcripts were one of the major
secreted group during mycobacterium infection of the mouse, Ag85A is one of the
most genes shown to be relatively highly expressed either in infected nude mouse
or human skin lesions [91].
5. Cholesterol Accumulation in the Host Cell and Utilization by M. leprae
Macrophages, infected with M. leprae, alter the lipid metabolism. Especially an
increased accumulation of cholesterol and cholesterol esters has been found [42].
The increased cholesterol level was also associated with a lower level of esterase
activity in infected macrophages [42].
Mattos et al. recently showed by high-performance thin-layer chromatography that
the amount of cholesterol increases in SCs upon infection with M. leprae [5].
Cholesterol utilization was also identified to be required for mycobacterial
persistence [96]. In 2008, Pandey and Sassetti found that M. tuberculosis can grow
using cholesterol as a primary carbon source and that the mce4 transporter is
required for cholesterol uptake.
While the M. tuberculosis genome contains four homologous mce operons, mce1mce4, which are thought to encode lipid transporters [96, 97], M. leprae genome
encodes only one operon (mce1). All M. leprae five mce genes were overexpressed
during intracellular growth in mouse and human biopsies [91, 92]. This observation
suggests that the intracellular bacilli population induces cholesterol uptake of the
infected cell and subsequently uses the stored cholesterol as carbon and energy
source.
Cholesterol is also essential for survival of mycobacteria in macrophages.
Cholesterol accumulates at the site of mycobacterial entry in macrophages and
promotes mycobacterial uptake. For M. tuberculosis and M. leprae it has been
shown that cholesterol mediates the recruitment of TACO from the plasma
membrane to the phagosome [34]. TACO, also termed as coronin-1A (CORO1A), is a
coat protein that prevents phagosome-lysosome fusion and thus degradation of
mycobacteria in lysosomes [34, 35]. The entering of mycobacteria at cholesterolrich domains of the plasma membrane and their subsequent uptake in TACO-coated
phagosomes promotes intracellular survival [35, 36].
6. Conclusions
Studying M. leprae metabolisms during infection is as complex as the choice of
invasion mechanisms traffic and as their multifarious factors for entry are. In this
scenario, most of publications emphasize that leprosy maintains metabolic
remodeling between both host and pathogen. A hallmark of intracellular infection is
the formation of foamy host cells. Several results support the view that M. leprae
actively catabolizes fatty acids for energy and produces a wide array of secretory
proteins. Huge gaps remain in our knowledge of the structure and function of M.
leprae lipid bodies, as the host metabolism required alterations to switch the
organism to the utilization of lipids as alternate carbon sources. Investigating the
metabolic aspects of M. leprae especially LDs formation is an important topic to
learn more about metabolic pathways that could potentially be exploited for
developing novel drugs against leprosy and tuberculosis.
Acknowledgments
This work was supported by NOPERSIST (EC Grant Agreement no. 232188) grant
from European Commission, Seventh Framework Programme (FP7) to M. Singh.
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Go to:
Introduction
What was known?
Since its introduction, the multidrug therapy (MDT) regimens for the treatment of
leprosy have undergone several changes especially with regard to the duration of
treatment. The advantages of shortened duration of treatment need to be balanced
against the risk of relapse and a lot of controversies exist pertaining to this aspect.
The fixed-duration (FD) therapy is not popular among academicians and private
practitioners who prefer precise diagnosis and treatment with superior MDT
regimens and for a longer duration.
leprosy, a disease which had been a scourge of mankind for many decades was
considered a curable disease after the implementation of multidrug therapy (MDT)
by the World Health Organization (WHO) in 1982. MDT has proved its effectiveness
in bringing about a significant change in the leprosy scenario, both at the global and
national level, by bringing down the case loads to the extent that leprosy no longer
continues to be a disease of public health-care importance. In addition to being
effective, it has also been proved to be safe as well as acceptable to the patients as
well as program managers. In spite of these advantages, there are some
controversies regarding the current MDT regimens especially among academicians
and private practitioners who prefer precise diagnosis and treatment with superior
MDT regimens and for longer duration, but these are not cost effective, thereby not
warranting their implementation in the leprosy elimination programs.
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Global and Indian Scenario
The global registered prevalence of leprosy in the beginning of 2011 as per the
WHO official reports received during 2011, from 130 countries and territories, was
192,246 cases, whereas the number of new cases detected during 2010 was
228,474 and the figure for India was 1,26,800, which accounts for an alarming
55.5%.[1,2] Pockets of high endemicity still remain in some areas of India[1] like
Bihar and Chattisgarh which are yet to achieve elimination (with a prevalence rate
of 1.12 and 1.94, respectively).[3]
Go to:
History of MDT
The WHO Executive Board reviewed and endorsed the reports of the study Group
on Chemotherapy of Leprosy for Control Programmes on 17 May, 1982 which is the
official date of birth of MDT.[4] MDT was recommended for the following reasons:
[5]
To address resistance to dapsone and to discourage resistance to other drugs to be
used.
To promote compliance and to move away from long-term monotherapy such as
dapsone.
To retain rifampicin in all therapeutic regimens because of its powerful bactericidal
action and its effectiveness even when taken once a month.
To promote compliance and cost effectiveness.
There were four successive phases in the implementation of MDT which were as
follows:[4]
19821985: Introduction of MDT on a global basis.
19861990: Expansion of MDT (into the less difficult areas).
19911999: Elimination strategy.
2000 onward: A fourth period, planned to last six years, designated for the
Intensive elimination strategy or the Final push.
The changes in the classification of paucibacillary (PB) and multibacillary (PB)
leprosy and MDT regimens are presented in Table 1.[4,69]
Table 1
Classification of paucibacillary and multibacillary leprosy and MDT regimens:
Changes over time
Go to:
Differences in Classification of Leprosy Cases and MDT Implementation in India
Until 1995, leprosy cases with more than 10 lesions (counting number of skin and
nerve lesions involved) were classified as MB. In addition, all cases where skin
smear testing gave positive results were classified as MB, irrespective of the
number of skin and nerve lesions. Since 1996, the WHO criterion of six or more skin
lesions has been used for MB classification.[10] The criteria of the National Leprosy
Eradication Programme (NLEP) to classify PB and MB cases includes the number of
nerves involved and the slit-skin smear results apart from the number of skin
lesions to increase the sensitivity as well as to account for the pure neuritic cases.
[11] Thus since 2009, the classification of PB and MB cases in India as per the
recommendations of the NLEP is as follows:
PB: 15 skin lesions, no nerve or only one nerve with or without 15 skin lesions,
slit-skin smears negative at all sites.
MB: 6 skin lesions, more than one nerve irrespective of the number of skin lesions,
slit-skin smears positive at any site irrespective of the number of skin lesions or
nerves.
The duration of treatment and MDT regimens for MB cases differed from those
recommended by the WHO during the initial phases. At the time of the introduction
of MDT in a district, the Indian Association of Leprologists recommended an
intensive phase of 21 days of daily rifampicin 600 mg followed by the monthly
pulsed dose recommended by the WHO for all prevalent MB cases. This was
modified by the NLEP by reducing the intensive phase to 14 days. Since 1986,
based on the reports of the Bombay Leprosy Project Study indicating the equal
efficacy of the WHO regimen and intensive phase regimen, the WHO regimen was
followed. However, the duration was not restricted to 24 months, but was continued
until negativity of smear.[11,12] NLEP introduced 24-month FD MDT in 1994 and 12month FD MDT since 1998. There has been no change with regard to the treatment
regimen or duration of PB cases from the introduction of MDT in 1982.[10]
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FD MDT Regimens
Currently, two FD MDT regimens are in vogue, the WHO regimen followed almost
worldwide and the Public Health Service regimen followed in the United States.
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Advantages of FD MDT
The effectiveness of FD MDT has been proved by its role in the elimination of
leprosy and the acceptability by the patients and public health-care administrators
worldwide. The validity of the efficacy of FD therapy in MB cases can be confirmed
based on the scientific basis by which the duration of the 24-month MDT was further
shortened to 12 months, which is as follows:
The modifications in the definitions of PB/MB cases over time have resulted in the
misclassification of many cases. There is an increased proportion of MB cases
among newly detected cases due to the classification of cases that would otherwise
be PB leprosy. Thus, the bacterial load of the majority of currently diagnosed MB
patients is significantly lesser than those in the past as also estimated by the WHO
wherein the newly detected cases with high bacillary indices (BI 3) are less than
15%.[15] The net result would be a decrease in the overall requirements of
chemotherapy for MB leprosy.
The rates of relapse among MB cases after 12 months of MDT, as estimated by the
routine control programs as well as the research projects were very low, about 0.2%
annually, similar to those observed after 24 months of MDT.[16] Nevertheless,
higher rates of relapse have been reported in some studies[17,18] involving MB
patients with a high initial bacillary index (BI) (average BI 4+). But, such patients
are only a minority in the current scenario of the widespread use of MDT, and hence
the relapses contributed by them will be small.
The dapsone-clofazimine combination is highly bactericidal as observed from nude
mouse experiments and clinical trials wherein daily treatment with this combination
killed more than 99.999% of viable Mycobacterium leprae in 36 months. Thus, this
combination ensures the effective elimination of the spontaneously occurring
rifampicin-resistant mutants which are usually not more than 10[4] organisms in an
untreated lepromatous leprosy (LL) case.[16]
According to some studies [Steering Committee on Chemotherapy of Mycobacterial
Diseases (THEMYC) and clinical trial in Malawi], the response of MB cases receiving
fewer than the standard 24 doses of MDT was as promising as those receiving 24
doses or more.[16,19,20]
A retrospective study done on the clinical and bacteriological progress of defaulted
MB cases revealed that treatment with less than 12 months of MDT still had
favorable therapeutic effects among the majority of MB patients.[21]
Four WHO-sponsored studies to evaluate the efficacy of a variety of drug
combinations and treatment periods have found that even though the bacillary load
was reduced, a positive BI was still present at the end of 24 or 12 months. But these
they look after the individual cases), are skeptical of FD MDT and find that the
clinical cure does not occur after FD MDT in all the cases of leprosy. Clinically cured
patients at the end of 12 months of MDT can still be bacteriologically active and
bacteriologically negative patients can still be clinically active. Thus an
overwhelming number of dermatologists give importance to clinical activity and do
not follow the FDT schedules as suggested by the WHO.
The limitations of the FD MDT pertaining to MB cases are based on the following
observations
It has been proved in a few studies that despite two years of regular therapy, 10%
of the patients continue to harbor viable persisters. In addition, incomplete killing of
M. leprae in patients treated for two years has been confirmed by the
demonstration of ATP in 19% of the bacterial suspensions from skin biopsies and the
presence of viable bacilli in the nerves of one-third of the patients as tested by the
mouse footpad. These observations signify the persistence of viable drug-sensitive
bacilli, probably the dormant bacilli that have escaped the bactericidal effect of
drugs including rifampicin in a proportion of patients. The fate of these organisms is
of prime importance because there is no evidence that MDT knocks down persisters
and there is no drug for leprosy that is known to act on dormant organisms.
Moreover, lepromatous patients with high initial BI have naturally a poor cellmediated immunity (CMI) and poor macrophage functioning to deal with these
remaining organisms. Hence, treatment cannot be stopped after one or two years,
as persisters are likely to grow, resulting in relapse. But, it may take a much longer
time than the incubation period of disease, because clofazimine remains in the
tissues for a long duration and maintains a check on the growth of M. leprae.[2527]
Higher rates of relapse of almost 39% have been reported in a subgroup of patients
with a large bacterial load (BI 4+) treated with 24 months of standard MDT,
during later years of follow-up in studies where long follow-up has been done.[28] It
has been observed that higher the BI or shorter the duration of therapy, the higher
the risk of relapse.[28] In a study by Girdhar, et al.,[29] the rate of relapse in
patients treated with MDT till they were smear negative was 1.1 per 100 personyears, whereas patients on FD MDT showed a relapse rate of 2.04 per 100 personyears. Similarly, the Marchoux Chemotherapy Study Group observed a relapse rate
of 3.4 per 100 person-years with FD MDT.[30]
It has been observed that the bacteriological relapses occurred earlier than clinical
worsening and this could not be picked up in many studies with a short follow-up
period as slit-skin smear is not routinely being used in the fields.[25]
There is also a difference noted in the rates of relapse based on whether they were
field studies or institutional studies. This could be attributed to the following
differences: Inclusion of all MB cases in the field studies in contrast to mainly LL/BL
(BL: Borderline lepromatous) cases in the institutional studies and more regular as
well as long-duration follow-up with periodic clinical and skin smear examination in
institutional studies.[25]
The endpoint of treatment of pure neuritic leprosy is difficult to establish due to the
highly controversial assessment and cure criteria. It has been shown that several
patients show advanced disease and are positive for AFB on histology both in the
nerves and in skin, even when one or few nerves are clinically affected. This
suggests that it would be better to include these patients in the MB group and give
a three-drug combination. The national program recommends that when upto one
nerve trunk is thickened, the patients could be treated as PB, whereas those with a
large number of affected nerves should receive MB therapy. These
recommendations seem justified from the point of view of control, as in these
patients bacilli are deep in the skin or nerves and are not discharged for
transmission. However, what happens in the long run is not clear as all the
organisms may not get killed with the limited treatment and may result in the
deterioration of the nerve function. Further, despite adequate treatment, in some
cases the nerve function can worsen on account of fibrosis, causing concern.[25]
The cost of treating either all or a targeted subpopulation of MB cases with MDT for
a prolonged period or carefully following the same sort of patients for a decade
might seem high initially, but might not be excessive. Nevertheless, this needs to be
weighed against the lower costs of retreatment of the relapses with a second course
of MDT, assuming that there is neither the development of drug-resistant disease
nor the development of additional disability in relapse cases.[28]
The limitations associated with FD PB MDT are as follows
The cure or endpoint of treatment of PB cases (smear-negative patients) has been
more difficult to define unlike in MB cases wherein the slit-skin smears are indicators
of disease activity.[31]
Continued visibility of a clinically active patch in a proportion of patients at the end
of six months of therapy has been observed in almost all studies varying from 10 to
67%. Studies conducted in the institutions have shown a larger proportion of
patients who are still active as compared to field situations.[25]
When the length of duration of treatment is not long enough for the body to clear
out the bacteria, continued inflammatory response results in persistent clinical
activity for up to 12-18 months. There is a delayed resolution in a significant
number of patients which sometimes may turn out to be failures of treatment. It has
also been observed in a few studies that if diamino diphenyl sulphone (DDS) is
continued for a further period of six months or MDT is administered for 12 months
instead of the recommended six months, the proportion of patients staying active
could be significantly reduced.[25]
In several reports, active granuloma has been found in over 50% of the patients on
completion of therapy and even six months later, suggesting persisting disease
activity.[25]
Studies at the Central JALMA Institute of Leprosy (CJLI), Agra, India have shown that
a good number of patients diagnosed as PB leprosy clinically were acid-fast (AFB)
bacilli positive in the skin smear, indicating their chances of getting inadequate
treatment and these patients would be prone for relapse.[31]
Go to:
Solutions to Overcome These Problems
The limitations of FD MDT can be overcome by adopting the following measures:
MB cases with an initial average BI 4+ should receive at least four years of
standard WHO MDT as proposed by the Marchoux chemotherapy study group.[30]
Though most of the patients with high BI continue to improve after the completion
of the 12-month regimen, an additional 12 months of MDT for MB leprosy may be
required for patients showing evidence of deterioration.[32]
The addition of immunotherapy by the Bacillus CalmetteGurin (BCG) or
Mycobacterium welchii vaccine can help to reduce the duration of treatment by
50%.[33]
A regimen of one year with the addition of ofloxacin and minocycline at a monthly
dose to the existing MDT can be carried out in an attempt to reduce the duration of
therapy as per the trials conducted by the NLEP and CJLI.[34]
Rigorous annual follow-up for at least five years and even more for PB cases and 10
years and even more for MB cases after being released from FD MDT is essential.
[24]
Ofloxacin and minocycline with potent bactericidal activity against M. leprae can
provide an effective single-dose therapy for PB leprosy when combined with
rifampicin as ROM (R: Rimfanpicin, O: Ofloxacin, M: Minocycline) regimen. For MB
leprosy, 24 monthly doses (pulsed) of supervised ROM can be advocated for
patients refusing or unable to take MDT. ROM is found to be as safe and effective as
MDT during dosing, causing no skin pigmentation, and conferring similar clinical,
bacteriologic, and histologic improvements, without increased rates of lepra
reactions. However, ROM costs four times more than MDT for similar duration
regimens, prohibiting mass administration, but patients treated with ROM may
remain anonymous to the community and even their families.[35]
The significance of abundant AFB- and MB-type histopathology in patients grouped
as PB leprosy should be resolved so that these patients could be given the drug
therapy and the duration of therapy they warrant. The techniques that directly
evaluate the type and severity of leprosy should be made relevant to the treatment,
especially in centers where they are available.[36]
As the workload and financial burden on leprosy organizations has significantly
decreased over the years with substantial decrease in the number of leprosy
patients, slit-skin smears may again be made an integral part of the leprosy
program.
Go to:
Newer MDT Regimens
Newer regimens that are more effective and operationally less demanding are the
need of the hour because of certain drawbacks with the existing MDT regimens,
such as:[3739]
The longer duration of therapy for MB leprosy is a disadvantage from the
operational point of view.
Daspone and clofazimine are only weakly bactericidal against M. leprae, and as
these weaker drugs determine the minimal effective duration of the current
regimen, further shortening of the duration of treatment might result in higher rates
of relapse.
Daily administration of dapsone and clofazimine are not directly supervised.
Side effects of the drugs in the current regimen like clofazimine-induced disoloration
of skin, hypersensitivity to dapsone, rifampicin-induced hepatitis, and so on.
Emergence of resistance to dapsone and rifampicin.
The various newer MDT regimens include the following:
MB cases
Rifampicin-susceptible MB patient:[40]
Fully supervisable, monthly administered regimen rifapentine 900 mg (or rifampicin
600 mg) plus moxifloxacin 400 mg plus clarithromycin 1000 mg (or minocycline 200
mg) administered once monthly under supervision for 12 months.
Rifampicin-resistant MB patient: Fully supervised regimen in two phases:[41]
An initial six-month intensive phase followed by an 18-month continuation phase.
The intensive phase: Moxifloxacin 400 mgclofazimine 50 mgclarithromycin 500
mgminocycline 100 mg all taken daily.
Go to:
Footnotes
Source of support: Nil
Conflict of Interest: Nil.
Go to:
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