Genotyping of Mycobacterium leprae on the basis of the polymorphism of TTC repeats for analysis of leprosy transmission

The polymorphism of TTC repeats in Mycobacterium leprae was examined using the bacilli obtained from residents in villages at North Maluku where M. leprae infections are highly endemic (as well as from patients at North Sulawesi of Indonesia) to elucidate the possible mode of leprosy transmission. TTC genotypes are stable for several generations of passages in nude mice footpads and, hence, are feasible for the genotyping of isolates and epidemiological analysis of leprosy transmission. It was found that bacilli with different TTC genotypes were distributed among residents at the same dwelling in villages in which leprosy is endemic and that some household contacts harbored bacilli with a different genotype from that harbored by the patient. Investigations of a father-and-son pair of patients indicated that infections of bacilli with 10 and 18 copies, respectively, had occurred. Genotypes of TTC repeats were found to differ between a son under treatment and two brothers. These results reveal the possibility that in addition to exposure via the presence of a leprosy patient with a multibacillary infection who was living with family members, there might have been some infectious sources to which the residents had been commonly exposed outside the dwellings. A limited discriminative capacity of the TTC polymorphism in the epidemiological analysis implies the need of searching other useful polymorphic loci for detailed subdivision of clinical isolates.     

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.     

Drug and multidrug resistance among Mycobacterium leprae isolates from Brazilian relapsed leprosy patients

Skin biopsy samples from 145 relapse leprosy cases and from five different regions in Brazil were submitted for sequence analysis of part of the genes associated with Mycobacterium leprae drug resistance. Single nucleotide polymorphisms (SNPs) in these genes were observed in M. leprae from 4 out of 92 cases with positive amplification (4.3%) and included a case with a mutation in rpoB only, another sample with SNPs in both folP1 and rpoB, and two cases showing mutations in folP1, rpoB, and gyrA, suggesting the existence of multidrug resistance (MDR). The nature of the mutations was as reported in earlier studies, being CCC to CGC in codon 55 in folP (Pro to Arg), while in the case of rpoB, all mutations occurred at codon 531, with two being a transition of TCG to ATG (Ser to Met), one TCG to TTC (Ser to Phe), and one TCG to TTG (Ser to Leu). The two cases with mutations in gyrA changed from GCA to GTA (Ala to Val) in codon 91. The median time from cure to relapse diagnosis was 9.45 years but was significantly shorter in patients with mutations (3.26 years; P = 0.0038). More than 70% of the relapses were multibacillary, including three of the mutation-carrying cases; one MDR relapse patient was paucibacillary.     

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.     

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.     

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.     

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.     

Lower numbers of natural killer T cells in HIV-1 and Mycobacterium leprae co-infected patients

Natural killer T (NKT) cells are a heterogeneous population of lymphocytes that recognize antigens presented by CD1d and have attracted attention because of their potential role linking innate and adaptive immune responses. Peripheral NKT cells display a memory-activated phenotype and can rapidly secrete large amounts of pro-inflammatory cytokines upon antigenic activation. In this study, we evaluated NKT cells in the context of patients co-infected with HIV-1 and Mycobacterium leprae. The volunteers were enrolled into four groups: 22 healthy controls, 23 HIV-1-infected patients, 20 patients with leprosy and 17 patients with leprosy and HIV-1-infection. Flow cytometry and ELISPOT assays were performed on peripheral blood mononuclear cells. We demonstrated that patients co-infected with HIV-1 and M. leprae have significantly lower NKT cell frequencies [median 0.022%, interquartile range (IQR): 0.007-0.051] in the peripheral blood when compared with healthy subjects (median 0.077%, IQR: 0.032-0.405, P < 0.01) or HIV-1 mono-infected patients (median 0.072%, IQR: 0.030-0.160, P < 0.05). Also, more NKT cells from co-infected patients secreted interferon-γ after stimulation with DimerX, when compared with leprosy mono-infected patients (P = 0.05). These results suggest that NKT cells are decreased in frequency in HIV-1 and M. leprae co-infected patients compared with HIV-1 mono-infected patients alone, but are at a more activated state. Innate immunity in human subjects is strongly influenced by their spectrum of chronic infections, and in HIV-1-infected subjects, a concurrent mycobacterial infection probably hyper-activates and lowers circulating NKT cell numbers.     

Mycobacterium leprae DNA in peripheral blood may indicate a bacilli migration route and high-risk for leprosy onset

Leprosy epidemiological studies have been restricted to Mycobacterium leprae DNA detection in nasal and oral mucosa samples with scarce literature on peripheral blood. We present the largest study applying quantitative real-time PCR (qPCR) for the detection of M. leprae DNA in peripheral blood samples of 200 untreated leprosy patients and 826 household contacts, with results associated with clinical and laboratory parameters. To detect M. leprae DNA a TaqMan qPCR assay targeting the M. leprae ML0024 genomic region was performed. The ML0024 qPCR in blood samples detected the presence of bacillus DNA in 22.0% (44/200) of the leprosy patients: 23.2% (16/69) in paucibacillary (PB), and 21.4% (28/131) in multibacillary (MB) patients. Overall positivity among contacts was 1.2% (10/826), with similar percentages regardless of whether the index case was PB or MB. After a follow-up period of 7 years, 26 contacts have developed leprosy. Comparing the results of healthy contacts with those that become ill, ML0024 qPCR positivity at the time of diagnosis of their index case represented an impressive 14.78-fold greater risk for leprosy onset (95% CI 3.6–60.8; p <0.0001). In brief, contacts with positive PCR in blood at diagnosis of index cases are at higher risk of later leprosy onset and this marker might be combined with other prognostic markers for management of contacts, which requires further studies.     

Evaluation of real-time and conventional PCR targeting complex 85 genes for detection of Mycobacterium leprae DNA in skin biopsy samples from patients diagnosed with leprosy

In spite of the decrease in the number of registered leprosy patients, the number of new cases diagnosed each year (400,000) has remained essentially unchanged. Leprosy diagnosis is difficult due to the low sensitivity of current methodologies to identify new cases. In this study, conventional and TaqMan real-time PCR assays for detection of Mycobacterium leprae DNA were compared to current classification based on clinical, bacteriological, and histological evaluation. M. leprae DNA was extracted from frozen skin biopsy specimens from 69 leprosy patients enrolled in the study and was amplified using specific primers for either the antigen 85B-coding gene or the 85A-C intergenic region by using conventional and real-time PCR. The detection rate was 100% among multibacillary (MB) patients and ranged from 62.5% to 79.2% among paucibacillary (PB) patients according to the assay used. The TaqMan system for 85B gene amplification showed the highest sensitivity, although conventional PCR using the 85A-C gene as a target was also efficient. The cycle threshold (C(T)) values obtained using the TaqMan system were able to statistically (P < 0.0001) differentiate MB (mean C(T), 28.06; standard deviation [SD], 4.51) from PB (mean C(T), 33.06; SD, 2.24) patients. Also, there was a correlation between C(T) values and the bacteriological index for MB patients (Pearson's r, -0.444; P = 0.008). Within the PB patients' group, we tested normal skin from six patients exhibiting the pure neuritic form of leprosy (PNL). Five out of six PNL patients were positive for the presence of M. leprae DNA, even in the absence of skin lesions. In conclusion, the TaqMan real-time PCR developed here seems to be a useful tool for rapidly detecting and quantifying M. leprae DNA in clinical specimens in which bacilli were undetectable by conventional histological staining.     

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.     

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.     

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).     

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.     

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.     

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.     

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.     

Identification and characterization of antigenic determinants of Mycobacterium leprae that react with antibodies in sera of leprosy patients.

Antigenic determinants of Mycobacterium leprae were identified by screening a lambda gt11::M. leprae genomic library with two separate pools of sera from leprosy patients. A total of 45 recombinant clones were detected with pooled sera from 21 lepromatous (LL) leprosy patients and 5 additional clones specified polypeptides that reacted with antibodies in pooled sera from 30 borderline tuberculoid or tuberculoid leprosy patients. The recombinant clones that specified antigenic determinants that reacted with sera from LL patients were condensed into eight groups on the basis of DNA hybridization experiments among the M. leprae DNA insert fragments. In addition, 11 of the 45 recombinant clones did not hybridize to members of the eight groups nor to one another; these represent unique recombinant clones. None of the recombinant clones identified by screening with sera from tuberculoid leprosy patients hybridized to each other or to any of the 45 LL recombinant clones. The polypeptides specified by the recombinant clones were usually fusion proteins with beta-galactosidase, ranging in size from 117 to 175 kilodaltons (kDa). Members of hybridization group III specified nonfusion proteins of 45 kDa. Only members of hybridization group I reacted with any of 30 monoclonal antibodies prepared against M. leprae proteins; recombinant proteins from these clones reacted with a single monoclonal antibody directed against the M. leprae 65-kDa protein. Thus, at least 22 new antigenic determinants of M. leprae have been identified on the basis of their reactivity to antibodies in sera from LL patients or sera from tuberculoid leprosy patients or both.