Mycobacterium leprae typing by genomic diversity and global distribution of genotypes

The genetic diversity and related global distribution of 51 Mycobacterium leprae isolates were studied. Isolates were obtained from leprosy patients from 12 geographically distinct regions of the world and two were obtained from nonhuman sources. Polymerase chain reaction (PCR) followed by DNA sequencing was performed targeting the rpoT gene of M. leprae. Isolates were classified into two groups based on the number of tandem repeats composed of 6 base pairs in the rpoT gene. Isolates from Japan (except Okinawa) and Korea belonged to one group, while those from Southeast Asian countries, Brazil, Haiti and Okinawa in Japan belonged to a second genotype. M. leprae obtained from two nonhuman sources (an armadillo and a mangabey monkey) revealed the latter genotype. These results demonstrate the genetic diversity of M. leprae and the related genotype-specific distribution in the world.     

SNP genotypes of Mycobacterium leprae isolates in Thailand and their combination with rpoT and TTC genotyping for analysis of leprosy distribution and transmission

Based on the discovery of three single nucleotide polymorphisms (SNPs) in Mycobacterium leprae, it has been previously reported that there are four major SNP types associated with different geographic regions around the world. Another typing system for global differentiation of M. leprae is the analysis of the variable number of short tandem repeats within the rpoT gene. To expand the analysis of geographic distribution of M. leprae, classified by SNP and rpoT gene polymorphisms, we studied 85 clinical isolates from Thai patients and compared the findings with those reported from Asian isolates. SNP genotyping by PCR amplification and sequencing revealed that all strains like those in Myanmar were SNP type 1 and 3, with the former being predominant, while in Japan, Korea, and Indonesia, the SNP type 3 was found to be more frequent. The pattern of M. leprae distribution in Thailand and Myanmar is quite similar, except that SNP type 2 was not found in Thailand. In addition, the 3-copy hexamer genotype in the rpoT gene is shared among the isolates from these two neighboring countries. On the basis of these two markers, we postulate that M. leprae in leprosy patients from Myanmar and Thailand has a common historical origin. Further differentiation among Thai isolates was possible by assessing copy numbers of the TTC sequence, a more polymorphic microsatellite locus.     

Mutations in genes related to drug resistance in Mycobacterium leprae isolates from leprosy patients in Korea

OBJECTIVE: Identification of the presence and drug resistance of Mycobacterium leprae is key to the diagnosis and treatment of leprosy in non-endemic country like Korea. The aim of this study was to screen the drug target DNA such as folP, rpoB, gyr, and 23S rRNA of drug resistance strain of M. leprae. PATIENTS AND METHODS: Sequences of those genes were analyzed for the 104 bacterial index positive cases out of 171 leprosy patients in Korea using touchdown PCR, single stranded conformational polymorphism. RESULTS: Twenty (19.2%) cases have shown the mutations in folP gene of dapsone-resistant M. leprae in which three (2.89%) cases were mutations in two genes, folP and rpoB, of multidrugs resistant strains to dapsone and rifampin, and two (1.92%) cases in folP and gyr genes of resistance to dapsone and oflaxacin, respectively. Besides double mutation for folP gene was one case (0.96%) and for rpoB gene one case, respectively. There was no mutant isolates in 23S rRNA gene against clarithromycin. CONCLUSIONS: This result should leads to a better understanding of the status of multidrug resistant leprosy in Korea and may assist in the rapid diagnosis of drug resistant M. leprae and the choice of the appropriate treatment regimens.     

鼻分泌物中麻风分枝杆菌的聚合酶链反应检测及应用

目的 了解鼻分泌物中麻风分枝杆菌对麻风病传播的影响.方法 对流行区患者与密切接触者在治疗前、后采用扩增重复序列片段套式PCR法,并对患者皮肤损伤组织与鼻分泌物麻风分枝杆菌可变串联重复序列(VNTR)基因型进行比对.采用X2检验.结果 常规PCR检测38例麻风病患者阳性率为47.4%,31例接触者和29例流行村一般人群阳性率为8.3%,套式PCR检测阳性率分别为71.0%和40.0%.其中15份PCR阳性标本经DNA测序,确认鼻分泌物中有麻风分枝杆菌,而非流行区健康人鼻分泌物中无麻风分枝杆菌.未经治疗的多菌型与少菌型患者鼻分泌物中麻风分枝杆菌阳性率比较,差异有统汁学意义(P=0.039);分组及队列比较显示,治疗前和治疗6个月时,多菌型患者分泌物中麻风分枝杆菌阳性率无明显下降,但街切接触者治疗前后阳性率差异有统计学意义.患者皮肤损伤部位和鼻分泌物中的麻风分枝杆菌基因型一致.结论 多菌型患者鼻分泌物排菌是导致高流行区接触者、一般人群易感原因.短期治疗不能阻断鼻排菌.提示早期发现和治疗对阻断麻风病的传播尤为重要.     

A species-specific repetitive sequence in Mycobacterium leprae DNA

A 2.2-kilobase Mycobacterium leprae DNA insert fragment from a recombinant genomic library (pYA1065) was found to hybridize to at least 19 fragments of chromosomal M. leprae DNA by Southern hybridizations. The probe hybridized to identical fragments of chromosomal DNA from four M. leprae isolates (two from patients with leprosy, one from a naturally infected armadillo, and one from a naturally infected Mangabey monkey) whether the chromosomal DNA was digested with BamHI, BstEII, PstI, or SacI. The pYA1065 probe is specific for M. leprae; it did not hybridize to chromosomal DNA from 14 cultivable slow- and fast-growing mycobacterial species. Dot-blot hybridizations between pYA1065 and purified M. leprae chromosomal DNA indicate that the probe can detect DNA equivalent to 4 x 10(3) M. leprae cells in a spot. The probe can also hybridize to DNA in M. leprae cells spotted on a filter from homogenized skin biopsy specimens from patients with lepromatous leprosy.     

Multiple mutations in the rpoB gene of Mycobacterium leprae strains from leprosy patients in Thailand

A new finding is reported of multiple mutations in the rpoB gene of 9 Mycobacterium leprae strains from leprosy patients in Thailand, who did not respond to therapy even when rifampicin, the main drug in multi-drug therapy was used. By means of sequence analysis of 9 Thai M. leprae strains, various mutations in 289 bps of the rpoB gene revealed forms of mutation never before described, such as multiple mutations (ie, mutation at two, three, six, seven, eight and nine positions in the rpoB gene), most of which were point-mutation substitutions (a few of which were silent), and some insertions. This investigation demonstrates that mutation in the rpoB gene of M. leprae strains from Thailand involves more variety than previously reported for rpoB mutation patterns in rifampicin resistance M. leprae strains.     

Active surveillance of leprosy contacts in country with low prevalence rate

For advanced control of leprosy in Pakistan where the World Health Organization leprosy elimination goal was achieved in 1996, we conducted surveillance of Mycobacterium leprae-seropositive patients and their contacts and drug resistant strains of M. leprae.We measured anti-PGL-I antibody level in sera from leprosy patients and their contacts for early detection of M. leprae infection. Out of 34 leprosy patients undergoing treatment, 4 lepromatous leprosy patients were antibody positive, and 6.8 to 23.7 percent of occupational or household contacts were seropositive. Furthermore, three cases (1.2%) had a high antibody titer. For surveillance of drug resistant strains of M. leprae, dapsone and rifampin were targeted. Four out of 18 polymerase chain reaction (PCR) positive samples had mutation in folP gene, and among 10 PCR positive samples, one had a mutation in the rpoB gene.These results indicate that serological analysis of patient contacts might be useful to find out high risk individuals, and there are M. leprae strains resistant to chemotherapeutic agents in Pakistan.     

A second case of multidrug-resistant Mycobacterium leprae isolated from a Japanese patient with relapsed lepromatous leprosy

Emergence of drug resistant strains of Mycobacterium leprae was reported soon after the introduction of dapsone (diamino-diphenyl sulphone, DDS) for leprosy treatment (6, 10, 11). Three cases of multidrug-resistant strains of M. leprae have been reported recently (2, 8, 9, 13). In order to prevent multiple drug resistant strains of M. leprae from developing, current leprosy control strategies are based on early detection of cases and treatment with multidrug therapy (MDT) as recommended by the World Health Organization (WHO). We report here the identification of a multidrug-resistant strain of M. leprae from a patient who received inadequate therapy for leprosy. The drug resistant profile of the isolated strain was confirmed by the mouse footpad method and the identification of mutations in genes previously shown to be associated with resistance to each drug was made.     

Detection of Mycobacterium leprae DNA from soil samples by PCR targeting RLEP sequences

Despite near elimination of leprosy as a public health problem, several problems in leprosy still remain. These include early detection, determining efficacy of the treatment and differentiating relapses from re-infection. These aspects have important impact on the patients undergoing treatment and also have a bearing on understanding transmission dynamics in the community. While early diagnosis and management do not need major technological inputs, various reports have suggested that M. leprae is found in the environment and may have a role in continued transmission of disease. In earlier studies from other parts of world the presence of M. leprae DNA in the environment has been investigated both by microbiological and molecular studies. In the present study, an attempt was made to extract M. leprae DNA from soil samples, which were collected from eighteen different locations including 3 from our Institute area and 15 from different villages of Ghatampur area. We optimized a protocol for the extraction of DNA and amplified a fragment of M. leprae using specific primers targeting RLEP sequences. It was found that 33.3% of these soil samples collected from areas inhabited by leprosy cases gave positive result for M. leprae specific DNA. The utility of this method needs to be explored on a larger scale to establish the presence of M.leprae in the environment, and its role in the spread of the disease.     

Antigenic protein from Mycobacterium leprae released in macrophages in vitro as indicator of viability of bacteria

Peritoneal macrophages from randombred, Swiss white mice, when cultured and infected with Mycobacterium leprae for 24 hours, are able to show the presence of antigen(s) with binding affinity to antibodies present in the sera of bacteriologically positive, lepromatous leprosy patients. Such antibodies are not seen in sera from normal and healthy persons, tuberculoid leprosy patients, or long-term-treated, bacteriologically negative, lepromatous leprosy patients. The production of the antigen(s) is blocked by the anti-M leprae drug rifampin. Other mycobacteria when incubated with macrophages from mice show very little antigens in the lysate but the antigens have an equal affinity for antibodies in sera from both normal individuals and lepromatous patients. Only the lysates from macrophages exposed to live M. leprae could discriminate and could exhibit differential binding to sera from leprosy patients compared to sera from normal individuals. This antigen(s) does not have any binding ability to the monoclonal antibodies available to the antigens of M. leprae identified at present and shown to be specific to M. leprae. This indicates a separate identity of this product which has potential for further exploitation in exploring host-pathogen interactions related specifically to the leprosy infection and the tolerance of M. leprae inside cells.     

Identification of a Mycobacterium leprae specific protein antigen(s) and its possible application for the serodiagnosis of leprosy.

Acetone-killed Mycobacterium leprae separated from infected armadillo liver tissue without the use of proteases were treated with 0.2 M lithium acetate, 20 mM EDTA, pH 8.8 solution, and the concentrated antigen extract was analyzed by Ouchterlony immunodiffusion. The antigen extract gave a single immunoprecipitate when reacted with pooled lepromatous leprosy (LL) patients sera made highly specific for M. leprae by adsorption. Apparently identical precipitates were produced by reacting the antigen extract with sera of each of 15 treated LL patients, 5 of 7 patients with tuberculoid leprosy, and 3 of 4 M. leprae infected armadillos. Serum from 1 of 16 persons immunized with BCG and from none of 15 patients with chlamydial urethritis or brucellosis reacted with the antigen. Identically prepared extracts of M. smegmatis, M. phlei, M. vaccae, M. duvali and M. diernhoferi gave no immunoprecipitates with sera from LL patients or infected armadillos. Preliminary characterization indicates the antigen is protein since antigenicity was destroyed by pronase and/or heat treatment. The relative specificity of the protein antigen for M. leprae and the presence of antibody to this antigen in patients with leprosy suggest a possible role for this antigen in the serodiagnosis of leprosy.     

Electron microscopic study of Mycobacterium leprae membrane

We report the results of the study by transmission electron microscopy of normal Mycobacterium leprae in the tissues of experimentally infected armadillos. Several fixation procedures were used and compared to those previously employed in the study of M. leprae in lepromatous leprosy patients. The results show that the ultrastructure of M. leprae is identical in both hosts. The demonstration of a symmetric membrane profile in M. leprae in armadillos confirms our previous results. This characteristic of the M. leprae membrane is peculiar in that it is not shared by any of the easily cultivable species of mycobacteria we have studied so far.     

Transmission of leprosy: a study of skin and nasal secretions of household contacts of leprosy patients using PCR

It is generally held that dissemination of Mycobacterium leprae is from nasal mucosa and not through the skin of infected patients. In this study, we evaluated M. leprae in the unbroken skin and nasal secretions of multibacillary (MB) leprosy patients and their contacts. Specimens were examined by direct microscopy and polymerase chain reaction (PCR) for M. leprae DNA. Results showed that 60% of untreated MB leprosy patients examined histologically had acid-fast bacilli in the keratin layer. By PCR studies it was found that 80% of the patients had M. leprae DNA in skin washings and 60% had M. leprae DNA on swabs obtained from the nasal mucosa. Ninety-three contacts of the untreated MB cases were also tested for exposure to M. leprae by analyzing skin washings and nasal secretions by PCR. PCR analysis showed significant skin (17% positive) and nasal muscosal (4%) exposure in contacts before instituting treatment of the index cases. After 2 months of treating the index cases, all contacts tested were negative for M. leprae DNA. These data suggested that both skin and nasal epithelia of untreated MB leprosy patients contribute to the shedding of M. leprae into the environment and contacts of untreated MB cases are at risk for contact with M. leprae through both the nasal mucosa and exposed surfaces of their skin.     

In vitro activation of neutrophils by suspensions of Mycobacterium leprae.

Activation, defined as an increase in the proportion of cells that reduce nitroblue-tetrazolium in vitro, is present in neutrophils from patients with reactional lepromatous leprosy but not in neutrophils from patients with non-reactional lepromatous leprosy. Neutrophils from patients with all forms of leprosy are equally well activated by endotoxin in vitro. We have now shown that in vitro activation induced by Mycobacterium leprae suspensions is of comparable magnitude in neutrophils from patients with all forms of leprosy (including lepromatous and reactional lepromatous leprosy). There is no intrinsic neutrophil anergy in patients with lepromatous leprosy vis-à-vis M. leprae as pertains to activation. Spontaneous activation in reactional lepromatous leprosy is likely due to an indirect mechanism, probably of immunologic nature, and not simply to the presence of circulating Mycobacterium leprae in the blood.     

Antibody responses to the 18-kDa protein of Mycobacterium leprae in leprosy and tuberculosis patients.

The 18-kDa protein of Mycobacterium leprae, as recognized by the monoclonal antibody L5, has a restricted species distribution, being confined to M. leprae and M. habana. We have developed a solid-phase ELISA using purified, recombinant M. leprae 18-kDa protein and compared the serological responses of Nepali leprosy and tuberculosis patients and endemic control subjects to the protein and the M. leprae phenolic glycolipid-I (PGL-I). Few control subjects had anti-18-kDa antibodies. A small proportion of paucibacillary (PB) leprosy and 42% of multibacillary (MB) leprosy patients had IgG anti-M. leprae antibodies. A similar proportion (47%) of Nepali tuberculosis (TB) patients were seropositive, and IgG anti-18-kDa antibody levels were significantly higher in MB and TB patients than in control subjects. By comparison, IgM anti-PGL-I antibodies were detected in 88% of MB leprosy patients and only 7% of TB patients. The possible reasons for the 18-kDa protein seroreactivity in TB patients are discussed, and the anti-18-kDa assay is compared with other antibody assays for protein and nonprotein antigens of M. leprae. It is concluded that the sensitivity and specificity of the anti-M. leprae 18-kDa ELISA are insufficient for the assay to be of clinical utility in leprosy patients.     

Cross-reactivity of antigens from the cytoplasm and cell walls of some corynebacteria and mycobacteria

Leprosy-derived corynebacteria (LDC) are non-acid-fast organisms isolated from leprosy lesions in humans. In this study 20 antigens of native LDC cytoplasm were identified by immunoelectrophoresis, and autoclaving yielded the M1 component, which strongly cross-reacted with antigen 60 of Mycobacterium bovis BCG (bacille Calmette-Guérin) and antigen 7 of Mycobacterium leprae. The polysaccharide moiety of M1 was immunologically related to the LDC cell wall polysaccharide previously characterized as arabinogalactomannan. The latter polysaccharide competitively inhibited the formation of immune complexes by labeled M1 and antisera to the LDC cell wall; cytoplasm and wall polysaccharides from other bacteria produced lower-level inhibition. In a radioimmunoassay with 125I-labeled antigen 7 of M. leprae, sera from patients with leprosy and antisera to the LDC cell wall yielded overlapping curves. Sera from patients with tuberculoid leprosy and those from patients with lepromatous leprosy afforded different levels of inhibition in this radioimmunoassay; this result indicated a difference in antibody specificity in the two forms of leprosy. In conclusion, the cell wall polysaccharide of LDC corresponds to the main thermostable cytoplasmic antigen M1, which strongly crossreacts with sera from patients with leprosy and, more specifically, with antigen 7 of M. leprae.     

Crossreactivity between Mycobacterium leprae and various actinomycetes and related organisms

Serological crossreactivity was analyzed between M. leprae and strains of various species of Corynebacterium, Mycobacterium, Nocardia, Rhodococcus, Streptomyces, and related organisms. M. leprae shares antigens with most of these organisms, and sera from patients with lepromatous leprosy contain antibodies against them. The results demonstrate that M. leprae shares more antigens with the mycobacteria than with strains of the other tested genera, thus supporting the view that the leprosy organism belongs to the genus Mycobacterium. One precipitinogen (designated p beta) was found to be common to M. leprae and the streptomycetes, and sera from patients with lepromatous leprosy contain antibodies against this antigen.     

New data on the ultrastructure of the membrane of Mycobacterium leprae

In previous reports on the ultrastructure of Mycobacterium leprae, we described the occurrence of symmetric membranes in normal-looking bacilli from fresh or frozen samples primarily fixed with aldehydes. In those reports we admitted that such a symmetric profile, which is not found in the other normal mycobacteria, would not represent the structure of the normal membrane of the leprosy bacillus. We, therefore, re-analyzed the ultrastructure of the membrane of M. leprae. In the present work the micromorphology of the M. leprae membrane was studied by transmission electronmicroscopy after the fixation of fresh samples by OsO4 plus calcium followed by glutaraldehyde plus formaldehyde and calcium followed by uranyl acetate. The study of samples from two patients with lepromatous (LL) leprosy, three armadillos with natural leprosy, and one nude mouse with experimental leprosy showed that normal-looking bacilli present in lead-stained sections had asymmetric membranes with a thickness of 6.49 +/- 0.36 nm. These membranes showed periodic acid-Schiff (PAS)-positive components exclusively located in the outer half of the bilayer. We demonstrated that the symmetric profile of the M. leprae membrane described in our previous reports corresponds, as admitted in those reports, to an abnormal membrane structure. Such an abnormality was now found to result from the use of primary fixation with aldehydes or of samples stored frozen before fixation. These results indicate that, although ultrastructurally similar to that of the other mycobacteria, the membrane of M. leprae has a peculiar sensitivity to fixation by aldehydes. Such a characteristic, which was not found in M. lepraemurium, M. aurum, M. avium, and M. tuberculosis H37Ra, must reflect a unique membrane molecular structure, which is presently unknown.     

Efficient mapping of protein antigenic determinants.

A recombinant DNA expression strategy has been used to deduce the amino acid sequences of six different antigenic determinants in a single protein of Mycobacterium leprae, the etiologic agent of leprosy. The gene encoding the M. leprae 65-kDa antigen was sequenced and a lambda gt11 gene sublibrary was constructed with fragments of the gene. Recombinant DNA clones producing specific antigenic determinants were isolated by screening with monoclonal antibodies, and the sequences of their insert DNAs were determined with a rapid primer-extension method. The amino acid sequence of each determinant was deduced from the minimum overlap of insert DNAs from multiple antibody-positive DNA clones. Amino acid sequences for six different epitopes were elucidated. A peptide containing sequences for one of these epitopes was synthesized and shown to bind the appropriate monoclonal antibody; this antigenic determinant is unique to M. leprae. The approach described here can be used to rapidly elucidate protein epitopes that are recognized by antibodies or T cells.     

Study of 39 documented relapses of multibacillary leprosy after treatment with rifampin

Among 39 strains of Mycobacterium leprae isolated from patients with multibacillary leprosy who relapsed after treatment with rifampin (RMP), 22 strains were resistant to RMP and 17 were susceptible. All of the RMP-resistant strains were recovered from patients who had been treated with more than 50 doses of RMP, usually given as monotherapy. RMP-susceptible strains were recovered from only six patients who had received more than 50 doses of RMP, and from 11 patients who had received no more than seven doses. The median time to relapse after the beginning of RMP therapy was 9 years (range 1-12 years) among the patients harboring RMP-resistant strains of M. leprae, and the median time to relapse after discontinuation of RMP treatment was 7 years (range 1-11 years) among the patients harboring RMP-susceptible strains. These data suggest that monotherapy with more than a few doses of RMP can be responsible for the emergence of RMP-resistant strains of M. leprae, thus emphasizing the need to employ RMP only in combination with other effective drugs in the chemotherapy of multibacillary leprosy.