Variable numbers of TTC repeats in Mycobacterium leprae DNA from leprosy patients and use in strain differentiation

Strain differentiation of Mycobacterium leprae would be of great value for epidemiological investigation to identify the infectious sources of leprosy, to understand transmission patterns, and to distinguish between relapse and reinfection. From the M. leprae genome sequence database, TTC DNA repeats were identified. Primer sets designed to amplify the region flanking TTC repeats revealed PCR products of different sizes, indicating that the number of repeats at each locus may be variable among M. leprae strains. The TTC repeats were not found in Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium marinum, or human tissues, which indicated their specificity to M. leprae. Sequence analysis of the TTC repeat region in each of the M. leprae strains showed a variation of 10 to 37 repeats. In the M. leprae strains of 34 multibacillary patients at Cebu, Philippines, M. leprae with 24 and 25 TTC repeats was most common, and this was followed by strains with 14, 15, 20, 21, and 28 repeats. This study thus indicates that there are variable numbers of TTC repeats in a noncoding region of M. leprae strains and that the TTC region may be useful for strain differentiation for epidemiological investigations of leprosy.     

Rapid Variable-Number Tandem-Repeat Genotyping for Mycobacterium leprae Clinical Specimens

Mycobacterium leprae is the noncultivable pathogen of leprosy. Since the genome sequence of an isolate of M. leprae has become available, multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) has been explored as a tool for strain typing and identification of chains of transmission of leprosy. In order to discover VNTRs and develop methods transferable to clinical samples, MLVA was applied to a global collection of M. leprae isolates derived from leprosy patients and propagated in armadillo hosts. PCR amplification, agarose gel electrophoresis, and sequencing methods were applied to DNA extracts from these infected armadillo tissues (n = 21). We identified polymorphisms in 15 out of 25 short-tandem-repeat (STR) loci previously selected by in silico analyses of the M. leprae genome. We then developed multiplex PCR for amplification of these 15 loci in four separate PCRs suitable for fluorescent fragment length analysis and demonstrated STR profiles highly concordant with those from the sequencing methods. Subsequently, we extended this method to DNA extracts from human clinical specimens, such as skin biopsy specimens (n = 30). With these techniques, mapping of multiple loci and differentiation of genotypes have been possible using total DNA extracts from limited amounts of clinical samples at a reduced cost and with less time. These practical methods are therefore available and applicable to answer focused epidemiological questions and to allow monitoring of the transmission of M. leprae in different countries where leprosy is endemic.The causative pathogen of leprosy is Mycobacterium leprae. A continued incidence, defying global campaigns to eliminate leprosy even after years of rigorous case finding and the availability of multidrug therapy regimens (28, 29, 30, 31), is attributed to subclinical human and environmental reservoirs of the pathogen (1, 8, 13). In recent years, molecular strain-typing methodologies have complemented conventional infectious disease epidemiology. With the publication in 2001 of the complete genome sequence of an isolate from Tamil Nadu, India, called the TN strain (4), selection of potential polymorphic genomic markers for strain typing was feasible. The first genetic markers that showed polymorphism were short tandem repeats (STRs) in the M. leprae genome. One was a 6-bp intragenic sequence in the rpoT gene, and the second, a trinucleotide (TTC) repeat element upstream of a pseudogene (17, 23). These sequences exhibit variable numbers of tandem repeats (VNTRs) when sequenced in different isolates. Based on these observations, we short-listed 44 loci (including the rpoT and TTC loci) by in silico analyses of the M. leprae genome and accomplished the screening of 11 STR loci, of which 9 were polymorphic when tested in a small panel of four human isolates derived from passage through armadillos (6). Five were minisatellites (6- to 50-bp repeat units), and four were microsatellites (1- to 5-bp repeat units). Since then, others have also shown that VNTR loci exist in M. leprae isolates (25, 33, 34). Three single-nucleotide polymorphisms have also been discovered by comparing sequences of a limited number of strains (20).The goal of our work has been to discover and apply DNA variation among M. leprae isolates to identify sources and chains of transmission of leprosy in regions of endemicity. There are, however, physiological and practical issues relevant to strain typing of M. leprae in the clinical setting, such as the long incubation period and low transmissibility of leprosy and the requirement for clinical specimens such as slit skin smears and skin biopsy specimens from leprosy patients due to the inability of M. leprae to grow in culture. During the course of the last 4 years, field studies in which STR mapping was implemented have been reported. Matsuoka et al. (16) applied the microsatellite locus (TTC)21 to type M. leprae strains obtained from nasal swabs and slit skin smears from patients grouped by village, dwelling, or household in Indonesia, while Young et al. (33) combined (AT)15, (GTA)9, and (TTC)21 VNTR loci for the identification of short- and long-range M. leprae transmission chains in areas within and surrounding the city of Hyderabad, India. Monot et al. mapped five M. leprae STR loci in patients from Mali, Africa (19). The results from those studies demonstrated heterogeneity in prevalent haplotypes, indicating that genotype mapping with a small panel of one to five microsatellite VNTR loci was insufficient to discern strain relatedness. However, within an intrafamilial case, three markers were congruent (33). The authors of those studies concluded that in these areas of endemicity, multiple rather than single dominant isolates are found and that additional genomic markers are necessary for strain typing.For these reasons, assays for the amplification and differentiation of multiple genomic loci are needed. When these requirements have been met, it becomes possible to undertake systematic strain-typing studies that include suitable sampling strategies and conventional epidemiology methods for monitoring transmission and detecting clusters of cases. In light of these laboratory, field, and clinical issues, we further explored multiple-locus VNTR analysis (MLVA) techniques. In this paper, we report the development and testing of multiplex-PCR methods for MLVA for reference armadillo-derived M. leprae isolates and clinical materials and address allelic properties of individual loci and the reproducibility and feasibility of the techniques. In a future paper, we will apply and extend these methods to the data from population-based studies in Cebu, the Philippines (R. M. Sakamuri, M. Kimura, W. Li, K. Madanahally, H.-C. Kim, H. Lee, M. Balagon, R. Gelber, W. C. Black, S.-N. Cho, P. J. Brennan, and V. Vissa, submitted for publication).     

Single nucleotide polymorphisms typing of Mycobacterium leprae reveals focal transmission of leprosy in high endemic regions of India

Earlier studies indicate that genotyping of Mycobaterium leprae based on single-nucleotide polymorphisms (SNPs) is useful for analysis of the global spread of leprosy. In the present study, we investigated the diversity of M. leprae at eight SNP loci using 180 clinical isolates obtained from patients with leprosy residing mainly in Delhi and Purulia (West Bengal) regions. It was observed that the frequency of SNP type 1 and subtype D was most predominant in the Indian population. Further, the SNP type 2 subtype E was noted only from East Delhi region and SNP type 2 subtype G was noted only from the nearby areas of Hoogly district of West Bengal. These results indicate the occurrence of focal transmission of M. leprae infection and demonstrate that analysis by SNP typing has great potential to help researchers in understanding the transmission of M. leprae infection in the community.     

Diversity of potential short tandem repeats in Mycobacterium leprae and application for molecular typing

A recent advance in molecular typing for tracing the transmission of leprosy is the discovery of short tandem repeats (STRs) in Mycobacterium leprae. To substantiate polymorphic loci from STR as promising candidates for molecular typing tools in leprosy epidemiology, 44 STR loci including 33 microsatellites and 11 minisatellites were investigated among 27 laboratory strains by sequencing PCR products. Not all STRs were necessarily polymorphic. Thirty-two out of the 44 loci were polymorphic. Nine polymorphic loci were suitable for identifying genotypes according to the discriminatory capacity, stability, and reproducibility. All the strains were classified into independent genotypes by the selected nine loci. Three multi-case households were subjected to molecular typing. M. leprae obtained from household cases showed identical copy numbers by TTC triplet alone, but the isolates from one family contact case were divided into different genotypes by adding eight other polymorphic loci. The combination of information from multiple loci allows increasing levels of discrimination and it is likely that the generation and documentation of data will result in the choice of a potential molecular typing tool for leprosy epidemiology.     

Detection of Mycobacterium leprae nasal carriers in populations for which leprosy is endemic.

In order to better understand the role of Mycobacterium leprae nasal carriage in the maintenance of infection reservoirs and transmission of leprosy, we applied a polymerase chain reaction (PCR) that detected a 531-bp fragment of the pra gene of M. leprae on nasal swab specimens collected through a total population survey from individuals living in an area in which leprosy is endemic. Among the total tested population of 1,228 people, 7.8% were found to be PCR positive. PCR positivity was shown to be randomly distributed among the population for which leprosy is endemic. No association was observed between PCR positivity, age, or sex. The observed distribution of PCR positivity among households of different sizes confirmed the expected values, with the exception of two households, each with three people with PCR-positive nasal swab specimens. Although nasal carriage does not necessarily imply infection or excretion of bacilli, the finding of nasal carriage supports the theory of a disseminated occurrence of M. leprae in populations for which leprosy is endemic.     

Cellular immune responses of leprosy contacts to fractionated Mycobacterium leprae antigens.

Antigens of armadillo-derived Mycobacterium leprae sonic extract were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted onto a nitrocellulose membrane, and the unstained blot was converted into 20 fractions of antigen-bearing particles. These were tested in cellular proliferation assays, and reproducible results were obtained between batches of fractions. Peripheral blood mononuclear cells from healthy contacts of leprosy patients (presumed to have protective immunity) were tested with the fractions to investigate which antigens they recognized. A small group of tuberculoid leprosy patients were also tested. Both groups showed a wide range of responses. Almost every fraction stimulated proliferation with at least one donor, yet none was clearly immunodominant or inhibitory in either group. Thus, protective immunity did not appear to be associated with proliferation caused by any single fraction.     

How to prevent immunological reactions in leprosy patients and interrupt transmission of Mycobacterium leprae to healthy subjects: two hypotheses

The basis of World Health Organization strategy for leprosy elimination is that the only source and reservoir for infection are patients with the disease. It was assumed that multi drug therapy (MDT) would reduce transmission of Mycobacterium leprae, but there is no convincing evidence for this. Furthermore, even if MDT has been proved to be extremely effective against the infectious disease, a noticeable proportion of leprosy patients can suffer from immunologic hypersensitivity reactions which are now the most significant issue in the managements of the disease. In endemic areas it was found that: M. leprae survives outside human body; healthy individuals harbor M. leprae bacilli in nasal cavity and shed micro-organisms in environment; there is widespread subclinical transmission of M. leprae with transient infection of the nose resulting in the development of a mucosal immune response. This disparate clinical, epidemiologic, and microbiologic evidence leads to the first hypothesis: that antigenic load in local tissues, sufficient to trigger the immune response, comes from external supply of M. leprae organisms. The hypothiocyanite anion (OSCN(-)) is generated in vivo by the reaction of thiocyanate with hydrogen peroxide catalyzed by lactoperoxidase. OSCN(-) is an antimicrobial oxidizing agent that prevents growth of bacteria, fungi, and viruses. OSNC(-) exists in lower airway secretions and lung has never been reported to be affected by leprosy. There is a sufficient concentration of OSCN(-) in the saliva, and accordingly mouth is rarely affected by leprosy. By contrast, the concentration of this compound is low or nil in nasal and lacrimal secretions and leprosy very often affects nose and eyes. The second hypothesis is that OSCN(-) may also protect from leprosy. Recently a method of OSCN(-) production, not involving enzymatic steps or use of toxic heavy-metal salts, has been patented. Studies on the susceptibility of M. leprae to hypothiocyanite could be carried out and, in case of positive results, the substance might be used in order to sterilize the nasal cavity of healthy carriers and prevent transmission of M. leprae to healthy subjects and to leprosy patients in whom it may trigger an immune response.     

Effective vaccination of mice against leprosy bacilli with subunits of Mycobacterium leprae.

Model vaccines against leprosy bacilli have consisted of nonvirulent, live, attenuated Mycobacterium bovis BCG and irradiated, heat-killed, or autoclaved intact M. leprae. We report that immunization with various cell wall fractions of M. leprae, progressively depleted of lipids, carbohydrates, and soluble proteins, as well as a partially purified protein(s) derived from a pellet fraction of sonicated M. leprae, conferred significant protection against subsequent infection with live leprosy bacilli. Moreover, lymphocytes from regional lymph nodes and spleens of mice immunized with these M. leprae-derived subunits responded by proliferation when stimulated with M. leprae in vitro. Our results provide the first evidence that vaccination with M. leprae-derived fractions protects mice against leprosy bacilli.     

In situ demonstration of Mycobacterium leprae antigens in leprosy lesions using monoclonal antibodies

Cryostat sections of skin and nerve lesions of leprosy were stained with monoclonal antibodies recognising Mycobacterium leprae antigens and indirect immunofluorescence. In both the tuberculoid and lepromatous lesions, PGL1, 55-65-kDa, 17-kDa protein antigens and cross-reactive non-protein antigens were present. 65-kDa antigens were seen mainly in the skin lesions of lepromatous leprosy. The infiltrates in both the skin and nerve granulomas of tuberculoid and lepromatous leprosy showed membranous staining with monoclonal antibodies recognising PGL1 and 55-65-kDa antigens. Bacilli in the lesions and the cells in the lymph node granulomas of patients with tuberculosis or the infiltrates in the lesions of tinea corporis or sections of normal skin did not show any staining with these monoclonal antibodies. These results confirm that M. leprae antigens are present and are expressed on the infiltrating cells of leprosy lesions.     

Presence of viable Mycobacterium leprae in environmental specimens around houses of leprosy patients

Purpose: Leprosy is a chronic systemic infectious disease caused by Mycobacterium leprae, one of the first organisms to be established as the cause for disease in humans. Because of high prevalence pockets of leprosy in the endemic regions, it is necessary to identify the possible sources of M. leprae in the environment and its mode of transmission. Materials and Methods: Slit skin smears (SSSs) from lesions were collected in 70% ethanol from 50 leprosy cases staying in the leprosy resettlement village and hospital from a high endemic area. One hundred and sixty soil samples were collected from different areas around the leprosy hospital and from the resettlement village of cured leprosy patients where active cases also resided at the time of sample collection. M. leprae specific gene region (RLEP 129 bp) and 16S rRNA targets were used for polymerase chain reaction (PCR) based detection for the presence and viability of M. leprae. An rpoT region was also amplified to determine presence of numbers of 6 bp tandem repeats. Results: All the SSS samples collected from patients showed three copies of rpoT region (6 bp tandem repeat, an ancient Indian type). Fifty-two soil samples showed presence of M. leprae DNA whereas M. leprae specific 16S rRNA gene was amplified in sixteen of these samples. PCR amplification and fragment length analysis showed 91 bp, i.e., three copies of the rpoT 6 bp tandem repeats from soil samples and similar three copies observed in patient samples. Conclusion: Presence of viable M. leprae in the soil having same rpoT genotype of M. leprae noted in patients suggests that it could be the same strain of M. leprae. M. leprae found in the soil could be the one that is excreted out by the patient. Significance of its viability in the environment and its pathogenicity with respect to transmission needs to be further explored. Findings of this study might provide possible insights for further exploration into understanding transmission patterns in leprosy and also will throw light on identifying potential for existence of extra human source or reservoirs of M. leprae, if any.     

Mycobacterium leprae is identified in the oral mucosa from paucibacillary and multibacillary leprosy patients

In leprosy, the nasal mucosa is considered as the principal route of transmission for the bacillus Mycobacterium leprae. The objective of this study was to identify M. leprae in the oral mucosa of 50 untreated leprosy patients, including 21 paucibacillary (PB) and 29 multibacillary (MB) patients, using immunohistochemistry (IHC), with antibodies against bacillus Calmette-Guérin (BCG) and phenolic glycolipid antigen-1 (PGL-1), and polymerase chain reaction (PCR), with MntH-specific primers for M. leprae, and to compare the results. The material was represented by 163 paraffin blocks containing biopsy samples obtained from clinically normal sites (including the tongue, buccal mucosa and soft palate) and visible lesions anywhere in the oral mucosa. All patients and 158 available samples were included for IHC study. Among the 161 available samples for PCR, 110 had viable DNA. There was viable DNA in at least one area of the oral mucosa for 47 patients. M. leprae was detected in 70% and 78% of patients using IHC and PCR, respectively, and in 94% of the patients by at least one of the two diagnostic methods. There were no differences in detection of M. leprae between MB and PB patients. Similar results were obtained using anti-BCG and anti-PGL-1 antibodies, and immunoreactivity occurred predominantly on free-living bacteria on the epithelial surface, with a predilection for the tongue. Conversely, there was no area of predilection according to the PCR results. M. leprae is present in the oral mucosa at a high frequency, implicating this site as a potential means of leprosy transmission.     

ML0405 and ML2331 are antigens of Mycobacterium leprae with potential for diagnosis of leprosy

Despite the success of multidrug therapy in reducing the number of registered leprosy cases worldwide, evidence suggests that Mycobacterium leprae continues to be transmitted. A serological diagnostic test capable of identifying and allowing treatment of early-stage disease could reduce transmission and prevent the onset of the disability, a common complication of the disease in later stages. Serological diagnosis based on antibody recognition of phenolic glycolipid I (PGL-I) cannot reliably identify individuals with lower bacterial indices (BI). One strategy that might improve this situation is the provision of highly specific serological antigens that may be combined with PGL-I to improve the sensitivity of diagnosis. Using serological expression cloning with a serum pool of untreated lepromatous leprosy (LL) patients, we identified 14 strongly reactive M. leprae proteins, 5 of which were previously unstudied. We present results suggesting that two of these proteins, ML0405 and ML2331, demonstrate the ability to specifically identify LL/borderline lepromatous (BL) patients on the basis of immunoglobulin G (IgG) reactivity. In a household contact study, LL index cases were identified on the basis of this reactivity, while household contacts of these patients demonstrated undetectable reactivity. At a serum dilution of 1:800, suitable to reduce background PGL-I IgM reactivity, two BL patients with a BI of <4 showed anti-human polyvalent immunoglobulin G, A, and M reactivity measured with a combination of ML0405, ML2331, and natural disaccharide O-linked human serum albumin (NDOHSA) (synthetic PGL-I) that was markedly higher than IgM reactivity to NDOHSA alone. We suggest that ML0405 and ML2331 may have utility in serological leprosy diagnosis.     

Detection of leprosy in ancient human skeletal remains by molecular identification of Mycobacterium leprae

We isolated ancient DNA from skeletal remains obtained from a South German ossuary (approximately 1400-1800 AD) and from a 10th century Hungarian cemetery partially indicating macromorphologic evidence of leprosy. In samples taken of 2 skulls from Germany and of 1 hard palate from Hungary, Mycobacterium leprae-specific fragments of RLEP1 and RLEP3 were amplified using polymerase chain reaction (PCR), thereby confirming their specificity by sequencing. In another case, PCR with primers targeting IS6110 of Mycobacterium tuberculosis gave positive results only for a mandibular specimen. No signal for any mycobacterial DNA was observed in samples from 2 Hungarian foot bones. In ancient material, osseous involvement of M leprae may be detected and distinguished from other mycobacterial infections by specific PCR. In the small bones of leprous hands and feet, not enough M leprae DNA seems to be present for detection. This supports the view that rhinomaxillary leprous alterations result from direct bacterial involvement, while osseous mutilations of hands and feet result from a nervous involvement and/or secondary infections due to small lacerations of the overlying soft tissues.     

Cellular immune response to the cell walls of Mycobacterium leprae in leprosy patients and healthy subjects exposed to leprosy.

Cell walls of M. leprae consist of complex arrangements of carbohydrate, lipid, peptidoglycan and protein molecules. Recently, extractable proteins of a wide range of molecular weights were identified as components of the cell wall. We have examined the cellular immune responses of Nepali leprosy patients to a cell wall preparation of M. leprae enriched for these proteins. Strong lymphocyte proliferative responses to the antigens were present in half of the paucibacillary leprosy patients and in the majority of healthy control subjects with occupational exposure to leprosy. Patients with multibacillary disease responded poorly and patients with tuberculosis had intermediate responses. Proliferative responses to the cell wall protein fraction were strongly correlated to the proliferative responses to sonicates of the whole leprosy bacillus. Immunization of mice with cell wall proteins resulted in inhibition of growth of M. leprae following foot-pad inoculation with viable organisms. Therefore cell-mediated immune responses to the extractable proteins of the cell wall may play a role in protective immunity against M. leprae infection.     

Human T-cell clones with reactivity to Mycobacterium leprae as tools for the characterization of potential vaccines against leprosy.

T-cell clones with the T4 phenotype were established from patients with tuberculoid leprosy. The antigen reactivity of these clones ranged from stringent specificity for Mycobacterium leprae to broad cross-reactivity with other mycobacteria. Killed M. leprae had a weak stimulatory capacity which could be enhanced by ultrasonication. Among the three candidate antileprosy vaccines, M. leprae, M. bovis BCG, and the ICRC (Indian Cancer Research Center) strain, the last was superior in stimulating cross-reactive T4 clones. This finding argues for a differential masking of similar or identical membrane antigens in various mycobacterial species. T-cell clones with defined reactivity patterns for mycobacterial antigens could be helpful tools for the characterization of an antileprosy vaccine.     

Antigens of Mycobacterium leprae identified by immunoprecipitation with sera from leprosy and tuberculosis patients.

Mycobacterial antigens which react with human B lymphocytes were investigated by immunoprecipitation of radiolabelled sonicates of Mycobacterium leprae and M. bovis (BCG) with sera from patients with leprosy and tuberculosis in the presence of Staphylococcus aureus. SDS-PAGE analysis of the immunoprecipitates demonstrated that dense bands of Mr 12,000 (12K), 15K, 27K, 32-33K, 36K and 48K were the major antigens of M. leprae recognized by antibodies in lepromatous leprosy sera. Of these, only the 15-16K band reacted significantly with sera from patients with tuberculoid leprosy and tuberculosis. Other antigens including the T cell immunogens of Mr 18K and 70K reacted with some of the BL/LL sera tested. There were differences in the pattern of antigens precipitated from BCG sonicate by leprosy sera with the 65K antigen and a high molecular weight band (greater than 94K) being readily detected. These results differ in part to these obtained by probing immunoblots of M. leprae sonicate with leprosy sera. Factors contributing to these differences are discussed.     

Serological specificity of phenolic glycolipid I from Mycobacterium leprae and use in serodiagnosis of leprosy

The serological activities of the specific phenolic glycolipid I from Mycobacterium leprae, its dissected parts, and related glycolipids from other mycobacteria were examined by enzyme-linked immunosorbent assay against hyperimmune anti-M. leprae rabbit antiserum and sera from patients with leprosy and other mycobacterial diseases. High anti-phenolic glycolipid I immunoglobulin M antibodies were found in 23 of 24 (96%) of lepromatous leprosy patients on short term chemotherapy and in 8 of 13 tuberculoid leprosy patients (62%). Sera from patients with tuberculosis or atypical mycobacterial infections were devoid of anti-phenolic glycolipid I activity. The structurally related phenolic glycolipids from Mycobacterium kansasii and Mycobacterium bovis and the aglycone segments of the M. leprae product showed no significant activity. Thus, the trisaccharide determinant of phenolic glycolipid I is specific in its structure, serological activity, and, to a lesser extent, the antibody class it evokes.