Genotyping of Mycobacterium leprae strains from a region of high endemic leprosy prevalence in India

Infection, Genetics and Evolution 36 (2015) 256–261

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Genotyping of Mycobacterium leprae strains from a region of high endemic leprosy prevalence in India Mallika Lavania, Rupendra Jadhav ⁎,1, Ravindra P. Turankar, Itu Singh, Astha Nigam, U. Sengupta Stanley Browne Research Laboratory, The Leprosy Mission Community Hospital, NandNagri, Shahdara, New Delhi 110093, India

a r t i c l e

i n f o

Article history: Received 8 July 2015 Received in revised form 28 September 2015 Accepted 1 October 2015 Available online xxxx Keywords: STRs M. leprae SNP typing Allele Subtyping Phylogenetic

a b s t r a c t Leprosy is still a major health problem in India which has the highest number of cases. Multiple locus variable number of tandem repeat analysis (MLVA) and single nucleotide polymorphism (SNP) have been proposed as tools of strain typing for tracking the transmission of leprosy. However, empirical data for a defined population from scale and duration were lacking for studying the transmission chain of leprosy. Seventy slit skin scrapings were collected from Purulia (West Bengal), Miraj (Maharashtra), Shahdara (Delhi), and Naini (UP) hospitals of The Leprosy Mission (TLM). SNP subtyping and MLVA on 10 VNTR loci were applied for the strain typing of Mycobacterium leprae. Along with the strain typing conventional epidemiological investigation was also performed to trace the transmission chain. In addition, phylogenetic analysis was done on variable number of tandem repeat (VNTR) data sets using sequence type analysis and recombinational tests (START) software. START software performs analyses to aid in the investigation of bacterial population structure using multilocus sequence data. These analyses include data summary, lineage assignment, and tests for recombination and selection. Diversity was observed in the cross-sectional survey of isolates obtained from 70 patients. Similarity in fingerprinting profiles observed in specimens of cases from the same family or neighborhood locations indicated a possible common source of infection. The data suggest that these VNTRs including subtyping of SNPs can be used to study the sources and transmission chain in leprosy, which could be very important in monitoring of the disease dynamics in high endemic foci. The present study strongly indicates that multi-case families might constitute epidemic foci and the main source of M. leprae in villages, causing the predominant strain or cluster infection leading to the spread of leprosy in the community. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Leprosy (Hansen's disease) is a disease of great antiquity, having been recognized from Vedic times in India and from Biblical times in the Middle East. Armauer Hansen discovered the leprosy bacillus in 1873, in Bergen, Norway. With this discovery M. leprae became the first bacterial pathogen to be associated with any human disease. Leprosy may be defined as a chronic, infectious disease of low contagiousness caused by an acid-fast bacillus, Mycobacterium leprae that affects primarily the superficial parts of the body especially nerves and appendages of the skin like sweat and sebaceous glands in susceptible hosts after a varying incubation period. Implementation of WHO multidrug treatment (MDT) regimen in the treatment of leprosy globally for last two and half decades has resulted in a dramatic decline in the prevalence of leprosy although there is a

⁎ Corresponding author at: Department of Microbiology, Government Institute of Science, Mumbai 400032, India. E-mail address: [email protected] (R. Jadhav). 1 Formerly Head, Stanley Browne Laboratory, TLM Community Hospital, NandNagari, New Delhi 110093, India.

http://dx.doi.org/10.1016/j.meegid.2015.10.001 1567-1348/© 2015 Elsevier B.V. All rights reserved.

much slower decline in the detection of new cases. In 1981, when the presence of the disease was considered to be at its peak in India, the prevalence rate (PR) was 57 per ten thousand population. MDT was introduced in India 1982 onwards. In April 2014 it had fallen to 0.68/ 10,000 (NLEP, 2014). According to WHO during 2014, globally 180,618 leprosy patients were on record for treatment. The prevalence rate was estimated as 0.32 per 10,000 population and the new case detection rate globally was 3.81 per 100,000 population (WHO, 2014). Three decades after the introduction of multi-drug therapy (MDT) the prevalence of leprosy has come down from 4.2 to 0.68/10,000 from 2002 to 2014 in India (NLEP, 2014). The remaining pockets of endemicity are localized to the states of Bihar and Maharashtra, parts of Uttar Pradesh, some parts of West Bengal, Jharkhand and Orissa. This does indicate the continued transmission of the disease in these areas. The exact mode or mechanism of transmission is not known. It is believed that the main reservoir of M. leprae are the highly bacillated lepromatous patients, but occasionally, also the sparsely bacillated tuberculoid cases may be a source of infection. Inanimate objects or fomites like the articles used by infectious patients can theoretically spread infection, but sputum or nasal mucus and sneezed droplets

M. Lavania et al. / Infection, Genetics and Evolution 36 (2015) 256–261

(Weddell and Palmere, 1963; Barton, 1974), particularly the infected dust particles or air-borne droplets discharged from a highly bacillated patient are more likely to propagate and spread the infection. The relationship between environment and transmission is also reported to be important in continued incidence of the disease (Lavania et al., 2006; Turankar et al., 2012). Molecular typing using genetic markers are expected to be important in establishing strain specific polymorphisms in M. leprae which should be helpful for improving our understanding of the epidemiology of leprosy. The current approach of molecular typing largely involves locus-to-locus comparisons, such as microsatellite analysis (Groathouse et al., 2004). Unlike locus-directed methods of genotyping, current advances in technology do allow insertion, deletion, and singlenucleotide polymorphism detection on a genome-wide basis. For bacterial organisms that are not cultivable, it is difficult to obtain sufficient amounts of genomic DNA, particularly from clinical material. Since M. leprae cannot be cultured amplification-based techniques are potentially more applicable and preferred for M. leprae as compared to other organisms. Molecular typing is useful to study the global and geographical distribution of different strains of M. leprae and the potential application in studying the transmission dynamics. This would also provide some insight into the historical and phylogenetic evolution of the bacillus (Monot et al., 2005, 2009). While the potential of different genotyping methods has been speculated, actual usefulness of these markers to identify genotypic differences has not been investigated adequately. Very little information about the genetic diversity among M. leprae is known from India which still has a large pool of infection. Using these targets, new molecular system/schemes need to be developed. Our study focuses on the identification of M. leprae strains from different parts of India by using 10 polymorphic loci short tandem repeats (STRs) and SNP subtyping. 2. Material and methods 2.1. Ethical approval Informed consents were obtained from all the patients and the study was approved by the Organisation Ethical Committee of The Leprosy Mission, India. 2.2. Collection of specimens Overall, 70 slit scrapings were obtained from BI positive leprosy patients who attended the Out Patient Department of TLM hospitals at Shahdara (Delhi), Naini (UP), Purulia (West Bengal) and Miraj (Maharashtra) during 2007–2010 (Fig. 1). Among these 70 samples 41, 14, 12 and 3 were from Shahdara, Miraj, Purulia and Naini respectively. All cases were diagnosed and classified as multibacillary (MB) type following standard clinical criteria (NLEP guidelines — http://nlep.nic.in/). 2.3. Isolation of DNA DNA was isolated from slit scrapings by following a procedure described earlier (Jadhav et al., 2001). Smears collected in 1 ml of 70% ethanol were centrifuged at 10,000 rpm (8000 ×g) for 10 min. Supernatants were discarded and pellets were air dried for the removal of ethanol. After ethanol removal samples were kept overnight in lysis buffer (100 mM Tris buffer pH 8.5 with 1 mg/ml proteinase K and 0.05% Tween 20) at 60 °C. The reaction was terminated at 97 °C for 15 min. This lysate preparation was further used for PCR. PCR targeting the RLEP gene region was performed to confirm the diagnosis of leprosy. Reference M. leprae NHDP63 and Thai 53 DNA (obtained from Colorado State University, USA) were used as positive controls and distilled water

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used as negative controls included in each set of experiment (data not shown).

2.4. Detection of VNTRs using PCR and fragment length analysis The sequences of primer pairs as described by Gillis et al. (2009) for the 10 loci (AC8b, GTA9, GGT5, 21-3, AC9, AC8a, 27-5, 6-7, 12-5 and 23-3) were listed in Table 1a. PCR was performed using the Hotstart-PCR kit (Qiagen). Each reaction mixture (20-μl final volume) was comprised of 10 μl of 2 × Qiagen PCR master mix, 2 μl Q solution, 2 μl (each) of the forward- and reverse-primer (described by the IDEAL group2) working stocks, and 2 μl of template DNA; the volume was adjusted with distilled water. The final concentration of each primer was thus 0.2 μM. Following an initial denaturation step at 95 °C for 15 min, 40 cycles of PCR were run as follows: denaturation at 94 °C for 30 s, primer annealing at 60 °C for 90 s, and primer extension at 72 °C for 90 s. The PCR was terminated with a final extension at 72 °C for 10 min. 5 μl of PCR products were electrophoresed in a 2% agarose gel (Sigma, India) using Trisborate EDTA buffer (1×) at 50 V constant current for approximately 2 h. After electrophoresis DNAs were further sent for commercial fragment length analysis (FLA) to Xplorigen Technologies Ltd., Delhi, India.

2.5. SNP typing SNP loci 1, 2, and 3 of M. leprae (nucleotide positions 14676, 1642875, and 2935685, respectively, on the sequenced TN strain) were amplified using previously reported primer sequences (Monot et al., 2005). Details of primers used for genotyping are given in Table 1b. 20 μl of reactions contained M. leprae DNA from different samples, 10 mM Tris–HCl (pH 9.0), 50 mM KCl, 1.5 mM MgCl2, 1.5 U Taq DNA polymerase (Qiagen, India) and 200 nM of forward and reverse primers. PCR conditions were similar as per Monot et al. (2005). After PCR amplification restriction digestion assays were done according to the protocol described by Sakamuri et al (2009) using SmlI, CviKI-1, and BstUI (New England Biolabs, MA) for SNP loci 1, 2, and 3, respectively. The PCR products (5 μl) were digested with 1 unit of the enzymes. The SmlI, BstUI, and CviKI-1 reactions were performed at 55 °C, 60 °C, and 37 °C, respectively, for 1 h. The SmlI- and BstUI-uncut and -cut DNAs were subjected to electrophoresis on 3% agarose gels. The gels were stained with ethidium bromide and visualized by UV transillumination using a gel documentation system.

2.6. PCR for SNP sub typing Subtyping of SNP loci 1 (nucleotide positions 8453, 313361, 61425 and 1642879 respectively) were amplified using previously reported primer sequences as mentioned in Table 1b (Monot et al., 2009). The reaction mix (25 μl) consisted of 10 mM Tris–HCl (pH 9.0), 50 mM KCl, 1.5 mM MgCl2, 1.5 U Taq DNA polymerase (Qiagen, India), 200 nM of each primers and 2 μl DNA sample. PCR was carried out using initial denaturation at 95 °C for 5 min followed by 40 cycles consisting of denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min and extension at 72 °C for 2 min, with a final extension at 72 °C for 10 min in a thermal cycler. PCR products were submitted for commercial sequencing (The Centre of Genomic Application, Delhi, India). Sequence data were analyzed by using CodonCode Aligner.

2 IDEAL (Initiative for Diagnostic and Epidemiological Assays for Leprosy) group — we are one of the member of this consortium. The IDEAL consortium consists of thirty leprosy research groups, half of which is established in countries where leprosy still occurs.

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M. Lavania et al. / Infection, Genetics and Evolution 36 (2015) 256–261

Fig. 1. Sampling sites of various Indian populations used under this study.

2.7. Analysis of genotypic data with respect to patients/geographical distribution In order to delineate relatedness among these Indian strains, the variable number tandem repeat (VNTR) and single nucleotide polymorphism (SNP) subtyping data sets were subjected to phylogenetic analysis using the sequence type analysis and recombinational tests (START) software. In the START software an unweighted pair group method with arithmetic mean (UPGMA) programme was used for analysis. The nearest neighbor (N-N) analysis was applied to all the strains. A minimum spanning tree (MST) was also constructed with the 10 typed loci and compared with the results of N-N analysis (http:// pubmlst.org/analysis/). Discriminatory index (D) values were calculated with the help of LIAN 3.5 (http://pubmlst.org/analysis/). LIAN tests the null hypothesis of linkage equilibrium for multilocus data. The program was written by Bernhard Haubold and Richard Hudson.

Table 1a List of primers used (Gillis et al, 2009) in the study for molecular typing. VNTR Number of repeats locus (TN strain) AC8a

2

AC8b

2

AC9

2

GGT5

3

GTA9

3

6-7

6

12-5

12

21-3

21

23-3

23

27-5

27

F R F R F R F R F R F R F R F R F R F R

Primer sequence

Product size (bp)

GTG TTA CGC GGA ACC AGG CA CCA TCT GTT GGT ACT ACT GA GAT GCG ACT ATC ACT CGC ACG CAG TT GCT GGT TTC CTT CTA GTC CC GCC TGG TGC CCG GAC AAT GC ACA TCA CAC TGA TCT CGC CGG CGC T TCA CCA TCG ACG CTC CGG GT TCG GCC TGG TTG TCT GCC TT CGC AGA TGCAACGAT CAC AAT ATG CAT GCC GGT GGT CTA CTT GCG CGC CAC CGC CA GCC GTC GCC AGG TTT TGC AG CTG GTC CAC TTG CGG TAC GAC GGA GAA GGA GGC CGA ATA CA TGT TGA AAT TTG GCG GCC AT TGC AAG GAG TGC TCA GCT AT CAG TCG CCC GGA TAC TGT TA TAA ATC CGC TCC CAA ATC TT GTG CTG TGC CTG CAG CCG TT TCC CCA AAG CCG CCG AAT CC

124

3. Results 3.1. VNTR typing based on genomic variation of M. leprae in patients in India Slit skin smears were taken from leprosy patients at the time of registering procedures at our hospitals. VNTR analysis was performed for the total of 70 specimens using PCR and FLA analysis to determine the number of repeats. VNTR data for 10 loci from 70 specimens from Northern India were analyzed for allelic diversity. Phylogenetic analysis showed evidence of clustering associated with the geographical origin of the strain detected (Fig. 2).

3.2. Allelic diversity of M. leprae in India Index diversity among these loci calculated from 70 Indian patients and compared for each of the 10 loci among different hospitals is summarized in Table 2. The allelic index for each locus was found to range from 0.1 to 0.7 (Table 2). The allelic diversity of the other 5 more loci (AT-17, rpoT, TTC 21, 18-8 and TA 10 (not included in this study)) was 0–0.9. These loci are either showing high polymorphism (TTC 21, TA 10, AT 17) or less polymorphism (rpoT and 18-8).

140 146 161 122 191 289 180 190 270

Table 1b Primers for SNP typing and subtyping (Monot et al, 2005, 2009). Primers for SNP typing SNP 14676

AATGGAATGCTGGTGAGAGC

SNP 1642875 SNP 2935685

CTCGTCACAAATCCGAGTTTGA AT ATCTGGTCCGGGTAGGAATC

CAATGCATGCTAGCCTTAAT GA GTAGTAGTCTTCCAAGTTGT GGTG ACCGGTGAGCGCACTAAG

194 114

CAATAGCGCTCAGACACGAC CTCGGAGACCAAACTTCTCG CCAGAACACCGAGGGAATAA TTGAAGGACGGACAACTATGC

211 200 243 203

180

Primers for subtyping SNP 8453 SNP 313361 SNP 61425 SNP 832152

GGTCTGCGGACAAGTTGGTA CACCGGAGACAAAGCTGAT TCGTCAAGCCGAAAGAGTTT CAGCTCGGTGTTGATGTGTG

M. Lavania et al. / Infection, Genetics and Evolution 36 (2015) 256–261

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Fig. 2. A minimum spanning tree was constructed using VNTR data to represent clusters. Samples from Delhi have been clustered. Each sample is represented as having a distinct profile which may be attributed to polymorphic VNTR loci.

The observed allele numbers for each of the 10 polymorphic loci are presented in Table 2. 3.3. SNP subtyping of M. leprae in patients of India All the samples belonged to SNP type 1 which is predominant in India and other South East Asian countries (Monot et al 2005). Further we categorized into subtypes (according to the protocol described by Monot et al., 2009). We found that 55 of these belonged to SNP subtype 1D 12 with 1C, one sample with 1A and 2 with 2E only (Supplementary Table 1). In our study we genotyped 70 strains of M. leprae from 4 different states of India and found two groups of SNP type 1 and type 2. Samples collected from Delhi and nearby states were in cluster. 3.4. MLVA clusters by minimum-spanning tree (MST) MST was created based on 66 different MLVA types of 70 M. leprae strains from four different sources as described in Fig. 2. From the analysis of strains from villages of Purulia, West Bengal, intrafamilial transmission appeared to occur in some cases (Table 3). Matching alleles were detected at all ten loci, with the same SNP subtype indicating a common infectious source and recent transmission within the household. By the use of combination of ten STR's clustering of strains from several defined geographical areas and villages was observed. It was observed that patients belonging to the same families (Table 3, Fig. 2) had similar strains as well as subtypes whereas others had different profiles. However, allelic differences across several loci seen within patient pairs of families suggest that the family members harbored different strains also indicating circulation of many strains in the community. Strains isolated from East Delhi were having a similar subtype but they differed in one of the VNTR loci (Table 3).

prevalence are sometimes associated/bordering areas with little or no disease. In some studies, it has been suggested that in endemic countries N50% of leprosy patients may have a history of intimate contact with an infected person (often a household member), and leprosy patients in non-endemic locales can identify such contact in only 10% of the time (Gelber, 2005). However, far less percentage was observed in studies from India (Bhatia et al., 1990; Job et al., 2008). Molecular typing of M. leprae presents several unique challenges. The organism cannot be cultivated in in-vitro conditions and must be maintained with passage in animals. Even among highly susceptible animal hosts, multiplication of M. leprae is quite slow and requires several months to years to manifest a suitable bacterial mass. Furthermore, samples from leprosy patients can have many limitations. These are often of poor quality, having low percent viability, are frequently contaminated with other cultivable agents, and the donors may have an unknown treatment history or infection with other diseases. Tandem repeats are usually classified among satellites, minisatellites and microsatellites. Both microsatellite and minisatellite loci including SNP subtyping have been investigated in this study to identify polymorphic loci as potential markers used as the tools for molecular typing of M. leprae. SNPs are capable of revealing a global pattern of lineages (Monot et al., 2009) and MLVA typing would be useful in tracking Table 2 Distribution of alleles of ten different VNTRs in the 70 Indian strains.

Locus

1

2

3

4

6

7

8

9

1

64

2

8

2

3

28

(GTA)9

21–3

65 5

12

13

14

18

10

6

1

2

0.1614

27–5

5

6–7

0.1328

12–5

62 3

15

17

20

10

3

2

4

51

5

7

39

25

1

2

65 3

52

0.7561

0.1328 5

2

11

5

(AC)8a

23–3

10

65

(AC)9

Leprosy is almost exclusively a disease of the developing world, affecting areas of Asia, Africa, Latin America, and the Pacific. Both Africa and Asia have the maximum disease burden. More than 80% of the world's cases occur in a few countries, namely, India, China, Myanmar, Indonesia, Brazil, Nigeria, Madagascar, and Nepal. Within endemic locales, the distribution of leprosy is not uniform, and areas of high

5

(AC)8b

(GGT)5

4. Discussion

Allelic diversity

Allele frequencies

8

0.7952 1

0.4511 0.1328 0.5694 0.2028 0.3968

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M. Lavania et al. / Infection, Genetics and Evolution 36 (2015) 256–261

Table 3 Similar allele present at VNTR loci in M. leprae from patients in the same area.

Area

(AC)8b

(GTA)9

(GGT)5

21–3

(AC)9

(AC)8a

27–5

6–7

12–5

23–3

SNP subtype

Relationship/ Remark

Miraj

7

9

4

2

9

8

5

6

4

2

D

Son

Miraj

7

9

4

2

9

8

5

6

4

2

D

Mother

Amravati

7

9

4

2

9

8

5

6

4

2

D

Different district in same state

Delhi

7

10

4

2

6

8

5

7

4

2

C

Delhi

7

10

4

2

6

8

5

7

4

2

D

East Delhi

7

9

4

1

8

8

5

6

4

2

D

East Delhi

7

9

4

2

8

8

5

6

4

2

D

East Delhi

7

12

4

2

6

8

5

6

4

2

D

Patients from old leprosy colony just behind The Leprosy

East Delhi

7

12

4

2

7

8

5

6

4

2

D

Mission Shahdara

East Delhi

7

9

4

2

8

7

5

6

4

3

D

East Delhi

7

9

4

2

9

8

5

6

4

3

D

East Delhi

7

12

4

2

8

8

5

6

4

3

D

East Delhi

7

12

4

2

8

8

5

6

4

3

D

Delhi Hospital

Shaded rows belong to the same family members with an identical profile.

transmission within communities across short distances and separating strains within and between countries (Cardona-Castro et al., 2009; Fontes et al., 2009, 2012; Sakamuri et al., 2009; Shinde et al., 2009, Kuruwa et al., 2012). Our study has been carried out with the aim of analysing various known genomic markers to elicit epidemiologically relevant information about the transmission of the disease and to compare strain types within the studied population with VNTRs. In our previous studies, we found a diversity of M. leprae at eight SNP loci from clinical isolates obtained from leprosy patients or from multicase families residing mainly in Delhi and Purulia (West Bengal) regions. It was observed that SNP subtype 1D was the most predominant in the Indian population, SNP subtype 2E was noted only from the East Delhi region and SNP type 2 subtype G was noted only from the nearby areas of the Hoogly district of West Bengal (Lavania et al., 2013, Turankar et al., 2014). In this present study we got similar observations for SNP subtypes. We found twelve SNP 1C in Purulia, Delhi and Miraj but we also isolated one strain with SNP 1A from Delhi. Delhi is a place where people are coming from different states for work so we are getting a mixture of all the subtypes in the Delhi population. East Delhi strains were similar in subtypes but dissimilar in VNTR profiles and some of the strains are from different areas but having similar profiles.

Analysis of allele frequencies of these VNTRs from published data from North India (Lavania et al., 2011), South India (Shinde et al., 2009) and Maharashtra (Kuruwa et al., 2012), suggested a number of demographic associations among strains of M. leprae. Strains from Maharashtra and South India have an identical predominant allele except (AC)8a and 6-7 with the 8 and 6 allele being predominant respectively in our strains. Along with this the strains from different countries like China (Xing et al., 2009), Brazil (Fontes et al., 2009), Thailand (Srisungnam et al., 2009), and the Philippines (Sakamuri et al., 2009) the VNTR loci (12-5, 23-3, 27-5, 6-7, (GTA)9) are showing similar dominant alleles across these countries except loci (AC)8b, (GGT)5 and 21-3 which have a different allele in Philippine specimens. On the basis of allelic diversity indices profiles of our strains were similar to those of South India and Maharashtra. (AC)9, (GTA)9 and 6-7 locus had high discriminatory index in our present study. The data generated suggests that molecular typing has a potential use as an effective tool to monitor drift and evolutionary changes occurring within M. leprae strains. The diversity by using these VNTRs is similar within these countries but different in other countries. There is a further need to carry out more in depth and prospective clinical as well as epidemiological studies using these loci in Indian strains. Its ultimate application will require a better

M. Lavania et al. / Infection, Genetics and Evolution 36 (2015) 256–261

understanding of the biological diversity of M. leprae in different environments and cohort studies to determine linkages with actual transmission of disease. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.meegid.2015.10.001. Authors' contributions M.L and R.J. conceived and designed the experiments. M.L., R.T., I.S., and A.N. performed the research work. M.L. and R.J. analyzed the data. R.J. and U.S. supervised the experiment. M.L., R.J. and U.S. wrote the paper. All authors read and approved the final manuscript. Acknowledgements We are likewise grateful to Mr. K.C. Lata and Atul Roy for assisting us in sample collection in the field. We also thank the Superintendent and staff of TLM, Purulia, TLM Shahdara, TLM Naini for their help and assistance during field work. We also acknowledge Dr. Sunil Anand, Director, TLM India, for his encouragement. This work was supported by a DBT grant (BT/Bio-CARe/08/000494/2010-11). References Barton, R.P., 1974. A clinical study of the nose in lepromatous leprosy. Lepr. Rev. 45, 135–144. Bhatia, V.N., Vanaja, G., Rao, S., Elango, T.V., 1990. Some observations on skin smear examination. Indian J. Lepr. 62, 338–345. Cardona-Castro, N., Beltrán-Alzate, J.C., Romero-Montoya, I.M., Meléndez, E., Torres, F., et al., 2009. Identification and comparison of Mycobacterium leprae genotypes in two geographical regions of Colombia. Lepr. Rev. 80, 316–321. Fontes, A.N., Sakamuri, R.M., Baptista, I.M., Ura, S., Moraes, M.O., et al., 2009. Genetic diversity of Mycobacterium leprae isolates from Brazilian leprosy patients. Lepr. Rev. 80, 302–315. Fontes, A.N., Gomes, H.M., Araujo, M.I., Albuquerque, E.C., Baptista, I.M., et al., 2012. Genotyping of Mycobacterium leprae present on Ziehl–Neelsen-stained microscopic slides and in skin biopsy samples from leprosy patients in different geographic regions of Brazil. Mem. Inst. Oswaldo Cruz 107, 143–149. Gelber, R.H., 2005. Leprosy (Hansen's disease). Harrison's Principals of Internal Medicine. The McGraw-Hill Companies (Chapter 159). Gillis, T., Vissa, V., Matsuoka, M., Young, S., Richardus, J.H., et al., 2009. Characterisation of short tandem repeats for genotyping Mycobacterium leprae. Lepr. Rev. 80, 250–260. Groathouse, N.A., Rivoire, B., Kim, H., Lee, H., Cho, S.N., et al., 2004. Multiple polymorphic loci for molecular typing of strains of Mycobacterium leprae. J. Clin. Microbiol. 42, 1666–1672.

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