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 Table of Contents  
ORIGINAL ARTICLE
Year : 2017  |  Volume : 1  |  Issue : 1  |  Page : 42-48

Microsatellite typing of Mycobacterium leprae strains in newly diagnosed multibacillary leprosy patients to trace out the transmission pattern


1 Department of Microbiology and Molecular Biology, National JALMA Institute for Leprosy and Other Mycobacterial Diseases, Agra, Uttar Pradesh, India
2 Department of Clinical Medicine, National JALMA Institute for Leprosy and Other Mycobacterial Diseases, Agra, Uttar Pradesh, India
3 Department of Animal Experiments, National JALMA Institute for Leprosy and Other Mycobacterial Diseases, Agra, Uttar Pradesh, India

Date of Web Publication24-Jul-2017

Correspondence Address:
Umesh Datta Gupta
National JALMA Institute for Leprosy and Other Mycobacterial Diseases, Agra, Uttar Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/bbrj.bbrj_11_17

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  Abstract 

Background: Leprosy was eliminated from India in December 2005 imprinting a prevalence rate of <1/10,000 population. Still some endemic pockets exist in India where the new cases of leprosy continue to occur at a constant rate. It means the active transmission of leprosy is still continuing. In the present study, we aimed to elucidate the transmission pattern of Mycobacterium leprae using three microsatellite loci. Methods: Slit-skin samples from 36 newly diagnosed multi-bacillary leprosy cases from Ghatampur, Kanpur Nagar, Uttar Pradesh, India, were collected in a sterile centrifuge tube containing Tris-EDTA (TE) buffer. DNA was isolated, and the microsatellite loci, namely, (GT)6, (TA)18, and (TA)8CA3 were amplified using the in-house designed primers. The amplified products were recovered through polymerase chain reaction cleanup kit and sequenced by Sangers method in a 16 capillary genetic analyzer. Results: Sequences were searched by Basic Local Alignment Search Tool, and phylogenetic analysis was done to trace out the transmission pattern of M. leprae. Out of the three microsatellite loci, (TA)18 was unable to define the transmission pattern while (GT)6 and (TA)8CA3 were endowed with distinct signature for transmission of M. leprae in the study segment of the population. Conclusions: We found that nine types of M. leprae strains were circulating in the Ghatampur area and the same type of strains were found in the same villages or in neighboring villages. To conclude the study, we accentuated that (TA)8CA3 is a better microsatellite locus for strain typing of M. leprae.

Keywords: Leprosy, microsatellite, Mycobacterium leprae, short tandem repeat


How to cite this article:
Naaz F, Mohanty PS, Bansal AK, Kumar D, Gupta UD. Microsatellite typing of Mycobacterium leprae strains in newly diagnosed multibacillary leprosy patients to trace out the transmission pattern. Biomed Biotechnol Res J 2017;1:42-8

How to cite this URL:
Naaz F, Mohanty PS, Bansal AK, Kumar D, Gupta UD. Microsatellite typing of Mycobacterium leprae strains in newly diagnosed multibacillary leprosy patients to trace out the transmission pattern. Biomed Biotechnol Res J [serial online] 2017 [cited 2019 Aug 20];1:42-8. Available from: http://www.bmbtrj.org/text.asp?2017/1/1/42/211405


  Introduction Top


Leprosy is a slow and chronic infection caused by Mycobacterium leprae, affecting both sexes and all age groups, in many parts of the world. India achieved “elimination” (prevalence <1/10,000) in December 2005, but new cases of leprosy continue to be detected in some endemic pockets, confirming active dissemination of the disease.

The most likely route of transmission of leprosy is through aerosols,[1] with contacts closest to a patient with leprosy, in particular within household contacts, having the highest risk of acquiring the infection.[2],[3] Detection of leprosy is based on clinical signs and classified into paucibacillary (≤5 skin lesions) and multibacillary (>5 skin lesions) leprosy. Currently, the main strategies to control leprosy are early detection of cases and treatment with multidrug therapy. Chemoprophylaxis and immune-prophylaxis are both potential interventions but are not yet routinely available. Numerous studies suggest that leprosy is transmitted from person to person by close contact of a healthy individual with an infectious patient.[4],[5],[6],[7] Till date, the exact mechanisms of leprosy transmission are not clearly understood. Even the widely advocated methods of spread including person to person contact or contact with respiratory secretions from infected individuals have not been conclusively established so far.[8] To understand the transmission of M. leprae in the general population, we specifically amplified and sequenced a novel microsatellite locus along with two other microsatellite loci and compared them for tracing out possible transmission pattern of leprosy.


  Methods Top


Ethical clearance

Ethical clearance for the study was obtained from the Institutional Ethical Committee before the initiation of the study.

Collection of samples

Slit-skin samples of 36 leprosy patients were collected from Ghatampur area. All the samples were put in sterilized 2 ml centrifuge tube containing TE buffer, labeled for patient identification number, and transported to National JALMA Institute for Leprosy and Other Mycobacterial Diseases, Agra, India, for molecular experimentation. Patient consent was clearly explained to the patients, and the consent was taken before taking the sample.

Extraction of DNA

DNA extraction was done by the method described by Zolan and Pukkila.[9] In brief, TE buffer containing slit-skin samples was centrifuged at 21,000 rpm for 10 min, TE buffer was discarded, and 700 μl of extraction buffer was added to the pellet and mixed by vortexing. The mixture was incubated at 65°C for 1 h, and an equal volume of chloroform-isoamyl alcohol (24:1) was added, vortexed, and centrifuged for 10 min at 10,000 g. The aqueous phase was precipitated with cold isopropanol and centrifuged at 10,000 g for 10 min. The pellet was washed with 70% ethanol, air-dried, and resuspended in 15 μl of TE buffer. The DNA samples were quantified in a BioPhotometer (Eppendorf) for the absorbance at 260 nm (for DNA), 280 nm (for protein), and 230 nm (for RNA). Samples were diluted to 20 ng/μl for conducting short tandem repeat (STR) amplification.

Short tandem repeat amplification and sequencing

To depict the better variability among strains, a total of three STRs, namely, (GT)6, (TA)18, and (TA)8CA3 were amplified and sequenced. STRs were amplified by primers as shown in [Table 1]. For each polymerase chain reaction (PCR), 50 μl of mixture was prepared. Three STRs for all 52 isolates were amplified using pfu polymerase (Stratagene) which had proofreading activity to avoid single nucleotide mismatch amplification. The amplification was done using standard PCR protocol with different annealing temperatures [Table 1]. The amplified products were electrophorized in 1.5% agarose gel and visualized in gel document system. PCR products of the STRs were purified using PCR cleanup kit. All the polymerase chain reaction products were sequenced directly in the ABI 3031XL BigDye Terminator sequencer according to the manufacturer's instructions.
Table 1: Primers used in this study showing annealing temperature and band size

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Basic Local Alignment Search Tool search and sequence alignment

Initially, the sequences were subjected to the BLAST search at the NCBI to determine their molecular taxonomic identity. For BLAST search, sequences were converted into FASTA format and entered into the NCBI web page (http://blast.ncbi.nlm.nih.gov/blast), selecting the reference data domain as nucleotide collection (nt/nr) for highly similar MegaBLAST search. The taxonomic identities of the strains were determined after comparing the search results. Five sequences from BLAST search results were aligned with our sequences. The sequence alignment was done using MEGA version 4.0 software (http://www.megasoftware.net/).

Phylogenetic analysis

Phylogenetic analysis was performed by maximum parsimony (MP) method. MP is a nonparametric, statistical method commonly used in computational phylogenetics for estimating phylogenies. Under parsimony, the preferred phylogenetic tree is the tree that requires the least evolutionary change to explain some observed data. In this study, a most likely parsimony tree was constructed to check the evolutionary history of M. leprae strains. The evolutionary history was inferred using the MP method.[10] Distances between isolates were measured by maximum likelihood method. The MP tree was obtained using the close-neighbor-interchange algorithm[11] with search level 3 in which the initial trees were obtained with the random addition of sequences (10 replicates). Branch lengths were calculated using the average pathway method in the units of the number of changes over the whole sequence. Phylogenetic analysis was conducted using MEGA4 software.[12]


  Results Top


Short tandem repeat amplification and sequencing

PCR amplification for three STR regions, namely, (TA)8CA3, (GT)6, and (TA)18 was done. Sequencing was done for 36 strains of M. leprae which were Repetitive sequences of leprosy (RLEP)-PCR positive. Sequencing result of the (TA)8CA3 showed that the dinucleotide repeat of TA varies from 8 copy to 13 copy and CA remains in three copies in all the strains. In case of (GT)6, the copy number varies from 6 to 8 copy number while we were unable to detect any change in copy number for the dinucleotide repeat (TA)18.

Basic Local Alignment Search Tool search and alignment of the short tandem repeats

GT6

The STR regions of the GT6 were amplified and sequenced for 36 strain of M. leprae. The STR region is present as flanking region between conserved protein (ML1825) and cobinamide kinase pseudogene (ML1826). From the alignment of 36 sequences of GT6, it was found that the copy number of the STR region varied from 6 to 8 copy number [Figure 1]. Variation in the copy number was also reported in reference strain of M. leprae (TN, AL583917) [Figure 2]. The result confirms that three types of strains were circulating in the Ghatampur area on the basis of GT6 copy number analysis. We also found one single nucleotide polymorphism (SNP) at the nucleotide position 161.
Figure 1: GT6 short tandem repeat sequences' alignment of the strains of Mycobacterium leprae showing variability in the short tandem repeat region

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Figure 2: Comparison between field strain of Mycobacterium leprae with reference strain TN showing variability in the copy number in the sequenced short tandem repeat region and also showing the single nucleotide polymorphism position

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TA18

The STR regions of the TA18 were amplified and sequenced for 36 strains of M. leprae. The STR region is present in the precursor of probable membrane protein pseudogene (ML0830). From the alignment of 36 sequences of TA18, we were unable to detect any variation in copy number [Figure 3] and [Figure 4]. The result confirms that there was a single type of strain which was circulating in the Ghatampur area on the basis of TA18 copy number analysis. We also found one SNP at the nucleotide position which was obtained in the sequenced region of the gene.
Figure 3: TA18 short tandem repeat sequence alignment of the strains of Mycobacterium leprae showing no variability in the short tandem repeat region

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Figure 4: Comparison between field strain of Mycobacterium leprae with reference strain TN showing no variability in the copy number in the sequenced short tandem repeat region

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(TA)8CA3

The STR regions of the (TA)8CA3 were amplified and sequenced for 36 strains of M. leprae. The STR region is present at the flanking region of hypothetical protein (ML0009) and hypothetical protein pseudogene (ML0010). From the alignment of 36 sequences of TA13, we found that the copy number of dinucleotide repeat TA varied from 8 to 13 [Figure 5]. Variation in the copy number was also reported in reference strain of M. leprae (TN, AL583917) [Figure 6]. The result confirms that six types of strains were circulating in the Ghatampur area on the basis of (TA)8CA3 copy number analysis. We also detected one SNP at the nucleotide position 263 in the sequenced region.
Figure 5: (TA)8CA3 short tandem repeat sequence alignment of the strains of Mycobacterium leprae showing variability in the short tandem repeat region

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Figure 6: Comparison between field strain of Mycobacterium leprae with reference strain TN showing variability in the copy number in the sequenced short tandem repeat region and also showing the single nucleotide polymorphism position

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Phylogenetic analysis

To elucidate the relationship among strains, a combined MP tree was constructed using three STR regions sequenced in this study. A phylogenetic tree was constructed from the combined dataset of TA8CA3, GT6, and TA18 regions of 36 sequences aligned. A total of 702 characters were included in the study. The consensus tree inferred from six most parsimonious trees is shown in [Figure 7]. The consistency index was 0.822175, retention index was 0.866389, and composite index was 0.986461 (0.914451) for all sites and parsimony-informative sites (in parentheses). The MP tree was differentiated into five main clusters from A to E. In cluster A, the strains of M. leprae collected from Ghuchupur and Ghugua were grouped together, showing the similarity among them. In cluster B, the strains collected from Maraaha, Sargaon, Bihupur, Rahasitalpur, and Paras were found. The cluster B was not a perfect cluster and it was paraphyletic group of clusters C, D, and E. The strains found in cluster B were unique in their SNP positions. In cluster C, the strains from Aswarmau, Koron, Sukhapur, NibiaKhera, Katari, Durgaganj, Girsi, and Madhepur were grouped together. In cluster D, strains collected from Chawar, Paras, Jasara, NibiaKhera, Kotra, Rahasitalpur, Raipura, and Ramsari were clustered together. In cluster E, strains collected from Bandh, Sahpur, Benda, Aliyapur, Bhadras, Srinagar, Bari Maithan, and Tilsida were clustered. From the cluster analysis, it was found that nine types of strains were circulating in the Ghatampur area.
Figure 7: Phylogenetic tree obtained by maximum parsimony analysis of the TA8CA3, GT6, and TA18 regions showing phylogenetic relationships among strains of Mycobacterium leprae circulating in Ghatampur area

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  Discussion Top


Leprosy is still a public health problem in several countries including India. The disease is said to be transmitted from person to person by respiratory secretions from infected individuals, and this theory has not been established so far. Several endemic pockets exist in India where leprosy is still continued to occur, giving an impression of active dissemination of the disease. Several studies put insight into the nonhuman sources of M. leprae.[8],[13],[14]

The methods such as SNP typing,[15] restriction length polymorphisms,[16] and variable number of tandem repeats at the mycobacterial-interspersed repetitive units loci have not produced any significant result in associating M. leprae strains.

Microsatellite markers are used for the study of genetic variability, population size differentiation, geographical differentiation, and for the study of transmission pattern of a particular gene of reference. The heterozygosity and mutable nature of the marker laid it for genetic characterization of species/strains. They are highly mutable markers often with 15 or more alleles in any given population. This means that allelic identity-by-descent can be readily established (unlike with bi-allelic SNPs) and linkage can be determined. On the other hand, SNPs are great genetic markers, but because of their low heterozygosity (the likelihood that a marker in any individual will appear heterozygous), we need to type lots of them.

In this study, STR analysis showed that the same types of strains were circulating in the same or nearby village. The microsatellite locus (TA)18 would not be able to provide any information regarding the transmission of M. leprae strains in the population as the copy number of the locus (TA)18 was found to be fixed in all the 36 strains. It was also evident from the BLAST search result that the locus (GT)6 provided the type of M. leprae strains circulating in the population up to some extent. The copy number of the locus varied from 6 to 8, and the locus differentiated all the 36 strains into three groups. The genetic differentiation was found to be greater for the locus (TA)8CA3. The locus (TA)8CA3 was able to differentiate all the 36 M. leprae strains into six groups implying six types of strains circulating in the population. The variability of the loci (GT)6 and (TA)8CA3 was also evident from the identities of BLAST search result. To elucidate a better picture of transmission, we made a combined analysis of all the three microsatellite loci by inferring a MP tree. From the MP tree, we found that nine types of M. leprae strains were circulating in the population of Ghatampur and also it was confirmed that the single-type strain was circulating in a particular village or in the neighboring village. Our results also corroborate with the results provided by Shinde et al.[17] In this study, one imperfect microsatellite (TA)8CA3 was used for strain typing and showed a high degree of variability among all the 36 strains and could be used for further strain typing of M. leprae. The study also found that some of the strains circulating in the population of Ghatampur (cluster B) were acquired by the patients from somewhere else as the strains had one SNP in the loci (GT)6 and (TA)8CA3.


  Conclusions Top


In this study, we used three microsatellite loci to elucidate the type of strains circulating in the population of Ghatampur, a high endemic zone for leprosy, and found that nine types of strains were circulating in the population. We also conclude that the imperfect repeat TA8CA3 could be useful for the further study of strain typing.

Acknowledgment

The authors are highly grateful to Shailendra Chauhan, Momd Wasim, Dinesh Singh, Atul Saraswat, Pradip Mishra, Ashok Tiwari, Jitendra Chaurasia, Narendra Singh, Mahendra Singh, Shivkaran, Ram Singh, Nause, Pawan, and Raju for their constant support in the field at the time of sample collection and transportation.

Financial support and sponsorship

This study was financially supported by the Indian Council of Medical Research.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
 
 
    Tables

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PLOS ONE. 2019; 14(4): e0214051
[Pubmed] | [DOI]



 

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