Detection of phenolic glycolipid I of Mycobacterium leprae in sera from leprosy patients before and after start of multidrug therapy

A total of 100 untreated new leprosy patients were recruited prospectively and examined for the presence of phenolic glycolipid I (PGL-I) antigen in their serum specimens by dot enzyme-linked immunosorbent assay (ELISA) using rabbit anti-PGL-I antiserum. The presence of circulating PGL-I antigen was closely related to the bacterial indices (BI) of the patients. The PGL-I antigen was detectable in 27 (93.1%) of 29 patients with a BI of 4.0 or above and in 15 (68.2%) of 22 patients with a BI of 3.0 to 3.9. However, none of the 37 patients with a BI of less than 1.9 had detectable PGL-I antigen by the methods used in this study. The level of PGL-I in serum declined rapidly by about 90% 1 month after the start of multidrug therapy. This study showed clearly that anti-PGL-I IgM antibodies and circulating PGL-I antigen levels reflect the bacterial loads in untreated leprosy patients. The serological parameters based on the PGL-I antigen may therefore be useful in the assessment of leprosy patients at the time of diagnosis and possibly in monitoring patients following chemotherapy.     

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.     

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 activity of a characteristic phenolic glycolipid from Mycobacterium leprae in sera from patients with leprosy and tuberculosis

Serological activity against a purified phenolic glycolipid from Mycobacterium leprae, which may be obtained in large amounts from M. leprae infected armadillo liver, was investigated using immunodiffusion and an enzyme linked immunosorbent assay (ELISA). Generally a good correlation was obtained between these techniques, but the ELISA was more sensitive and convenient. Relatively high IgG and IgM anti-glycolipid antibody levels were found in lepromatous leprosy patients. The antibody titres to the glycolipid were, however, low when compared with antibody titres to crude sonicates. Since the glycolipid is present in large quantities, this suggests that it is not very immunogenic. Antibody against the glycolipid especially of the IgM class, was demonstrable in some tuberculoid leprosy patients, although at much lower titres than in the lepromatous leprosy sera. In lepromatous leprosy patients that were skin smear negative after more than 5 years of treatment the IgG anti-M. leprae derived glycolipid activity had decreased markedly. The anti-IgG and IgM glycolipid antibody levels in tuberculosis patients did not differ significantly from the levels in appropriate normal healthy subjects. The glycolipid antibody levels in patients infected with M. kansasii, M. avium or M. intracellulare also fell within the range of normal healthy individuals.     

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.     

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.     

Pyrazinamide resistance among South African multidrug-resistant Mycobacterium tuberculosis isolates

Pyrazinamide is important in tuberculosis treatment, as it is bactericidal to semidormant mycobacteria not killed by other antituberculosis drugs. Pyrazinamide is also one of the cornerstone drugs retained in the treatment of multidrug-resistant tuberculosis (MDR-TB). However, due to technical difficulties, routine drug susceptibility testing of Mycobacterium tuberculosis for pyrazinamide is, in many laboratories, not performed. The objective of our study was to generate information on pyrazinamide susceptibility among South African MDR and susceptible M. tuberculosis isolates from pulmonary tuberculosis patients. Seventy-one MDR and 59 fully susceptible M. tuberculosis isolates collected during the national surveillance study (2001 to 2002, by the Medical Research Council, South Africa) were examined for pyrazinamide susceptibility by the radiometric Bactec 460 TB system, pyrazinamidase activity (by Wayne's assay), and sequencing of the pncA gene. The frequency of pyrazinamide resistance (by the Bactec system) among the MDR M. tuberculosis isolates was 37 of 71 (52.1%) and 6 of 59 (10.2%) among fully sensitive isolates. A total of 25 unique mutations in the pncA gene were detected. The majority of these were point mutations that resulted in amino acid substitutions. Twenty-eight isolates had identical mutations in the pncA gene, but could be differentiated from each other by a combination of the spoligotype patterns and 12 mycobacterial interspersed repetitive-unit loci. A high proportion of South African MDR M. tuberculosis isolates were resistant to pyrazinamide, suggesting an evaluation of its role in patients treated previously for tuberculosis as well as its role in the treatment of MDR-TB.     

Genotypic and phenotypic heterogeneity among Mycobacterium tuberculosis isolates from pulmonary tuberculosis patients

Although the heterogeneity of Mycobacterium tuberculosis populations and the existence of mixed infections are now generally accepted, systematic studies on their relative importance are rare. In the present study, 10 individual colonies of each M. tuberculosis isolate (primary isolate) from 97 tuberculosis patients in a primarily human immunodeficiency virus-negative population were screened for heterogeneity and detectable mixed infections by spoligotyping, IS6110-based restriction fragment length polymorphism analysis, and mycobacterial interspersed repetitive unit-variable number of tandem repeat typing. The MICs of antituberculosis drugs for colonies with divergent fingerprints were determined. Infections with different bacterial subpopulations were detected in the samples from eight patients (8.2%), and the frequency of detectable mixed infections in the study population was estimated to be 2.1%. Genotypic variations were found to be independent of the drug susceptibilities, and the various molecular markers evolved independently in most cases. The predominant strains and the primary isolates always had concordant drug susceptibility and MIC testing results. These findings have implications on the interpretation of molecular epidemiology results for patient follow-up and in transmission studies.     

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.     

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.     

Failure to induce delayed-type hypersensitivity to Mycobacterium leprae in long-term treated lepromatous leprosy patients.

Lepromatous leprosy (LL) patients whose bacillary load has decreased to almost undetectable levels by long-term chemotherapy failed to develop delayed-type hypersensitivity (DTH) to Mycobacterium leprae antigen following immunization with killed armadillo-derived M. leprae. When these LL patients were immunization with killed armadillo-derived M. leprae. When these LL patients were immunized with killed M. leprae in a mixture with live BCG, only DTH to purified protein derivative (PPD) was induced. These results are further evidence that immunological unresponsiveness to the leprosy antigen of patients with lepromatous leprosy is antigen-specific and non-reversible.     

Reduced suppressor cell response to Mycobacterium leprae in lepromatous leprosy.

We have previously shown that concanavalin A (ConA) induction of suppressor cell activity is impaired in patients with lepromatous leprosy (LL). In this study, we demonstrated that the proportion of cells bearing the Leu8 antigen (associated with suppressor-inducer cells) is low in LL patients and tends to normalize during the erythema nodosum leprosum (ENL) episode. Antigen-induced suppressor cell function was evaluated by a two-stage assay. In the first stage, peripheral blood mononuclear cells (PBMC) were cultured for 5 days either in the presence of gamma-irradiated Mycobacterium leprae or in tissue culture medium as a control. In the second stage, mitomycin C-treated suppressor or control cells were added to phytohemagglutinin (PHA)- or ConA-stimulated autologous PBMC. The results indicate that the ability of M. leprae to induce suppressor activity was lower in LL patients than in patients with tuberculoid (TT) and intermediate clinical (BB, BL, BT) forms and Mycobacterium bovis BCG-immunized normal controls. In ENL patients, the percent suppression was between that of TT and normal individuals. M. leprae-induced suppression was more effective on ConA- than on PHA-triggered T-cell proliferation in all groups. In contrast, normal PBMC cultured for 5 days in RPMI 1640 medium (N-C) and cells from patients with leprosy (TT-C and LL-C) had effects of their own on PHA- or ConA-induced proliferation. LL-C depressed the response to ConA and enhanced PHA-induced proliferation of autologous cells. Conversely, TT-C reduced PHA-induced proliferation and increased the ConA response. Suppression of proliferation could not be overcome with exogenous interleukin-2 and was not related to the induction of the Tac antigen. The abilities of LL, TT, ENL, and normal cells to proliferate upon PHA or ConA stimulus were similar, indicating that the defect in the generation of in vitro suppression by M. leprae in LL patients occurred during the induction period (step 1 of assay).     

Lymphocyte response of leprosy patients to human-derived and purified armadillo-derived Mycobacterium leprae, BCG and PPD.

The lymphocyte transformation test was applied to compare in vitro lymphocyte responses of tuberculoid (high resistant) and lepromatous (low resistant) leprosy patients to purified Mycobacterium leprae derived from experimentally infected armadillos and crude M. leprae derived from man, as well as to bacille Calmette-Guérin (BCG) and purified protein derivative (PPD). It was found that the purification procedure using enzymic digestion did not affect the immunogenicity of armadillo-derived M. leprae as compared with the crude human-derived preparation, although 2.5-5-fold higher doses of the purified organisms were required to elicitate equivalent lymphocyte responses. The result indicated the suitability of purified armadillo-derived M. leprae as the standard antigen for lymphocytes transformation tests in leprosy. The cross-reactivity studies show a close relationship between PPD and BCG, but not between M. leprae and PPD or BCG.     

Drug resistance and genotypic analysis of Mycobacterium tuberculosis strains from Thai tuberculosis patients

The aim of this study was the molecular characterization of primary drug‐resistant Mycobacterium tuberculosis strains in Thailand. We examined a group of M. tuberculosis isolates from newly registered tuberculosis (TB) cases, collected at the largest university hospital, the Siriraj Hospital, in Thailand. Of 76 selected drug‐resistant M. tuberculosis strains recovered from previously untreated pulmonary TB patients whose sputum samples were sent to this hospital, 29 (38%) were single‐drug resistant, 26 (34%) multidrug resistant and two (2.6%) extensively drug resistant. Fifty (66%) strains belonged to Beijing genotype. The study demonstrate a severe problem of drug resistance among recently detected TB patients, and two large clusters of genetically similar strains indicated ongoing transmission of drug‐resistant TB.     

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.     

Major proteins of mycobacterial strain ICRC and Mycobacterium leprae, identified by antibodies in sera from leprosy patients and their contacts.

Sera from leprosy patients across the clinical spectrum, healthy contacts, tuberculosis patients, and healthy donors were tested for their reactivity with antigens of mycobacterial strain ICRC (a cultivable mycobacterium) and Mycobacterium leprae by immunoprecipitation technique. Using M. leprae antigens, it was not possible to distinguish between reactivities of sera from lepromatous, borderline lepromatous, borderline tuberculoid, and tuberculoid leprosy patients. All these sera identified M. antigens with molecular masses of 47, 36, 21, and 14 kDa. When the same sera were tested for their reactivities with antigens of mycobacterial strain ICRC, several differences were observed. The 21-kDa antigen of mycobacterial strain ICRC was exclusively precipitated by sera from all lepromatous leprosy patients and from those undergoing erythema nodosum leprosum reaction. Sera from all the other donors tested failed to identify the 21-kDa antigen of mycobacterial strain ICRC. The 14-kDa protein of mycobacterial strain ICRC was identified by sera from a few lepromatous leprosy patients (5 of 26) and all their contacts. Our studies indicate that antigens present on cultivable mycobacteria rather than species-specific antigens may prove to be useful in the serodiagnosis of leprosy.     

Detection of multidrug resistance in Mycobacterium tuberculosis

We developed a DNA sequencing-based method to detect mutations in the genome of drug-resistant Mycobacterium tuberculosis. Drug resistance in M. tuberculosis is caused by mutations in restricted regions of the genome. Eight genome regions associated with drug resistance, including rpoB for rifampin (RIF), katG and the mabA (fabG1)-inhA promoter for isoniazid (INH), embB for ethambutol (EMB), pncA for pyrazinamide (PZA), rpsL and rrs for streptomycin (STR), and gyrA for levofloxacin, were amplified simultaneously by PCR, and the DNA sequences were determined. It took 6.5 h to complete all procedures. Among the 138 clinical isolates tested, 55 were resistant to at least one drug. Thirty-four of 38 INH-resistant isolates (89.5%), 28 of 28 RIF-resistant isolates (100%), 15 of 18 EMB-resistant isolates (83.3%), 18 of 30 STR-resistant isolates (60%), and 17 of 17 PZA-resistant isolates (100%) had mutations related to specific drug resistance. Eighteen of these mutations had not been reported previously. These novel mutations include one in rpoB, eight in katG, one in the mabA-inhA regulatory region, two in embB, five in pncA, and one in rrs. Escherichia coli isolates expressing individually five of the eight katG mutations showed loss of catalase and INH oxidation activities, and isolates carrying any of the five pncA mutations showed no pyrazinamidase activity, indicating that these mutations are associated with INH and PZA resistance, respectively. Our sequencing-based method was also useful for testing sputa from tuberculosis patients and for screening of mutations in Mycobacterium bovis. In conclusion, our new method is useful for rapid detection of multiple-drug-resistant M. tuberculosis and for identifying novel mutations in drug-resistant M. tuberculosis.     

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.     

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.