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.     

Recognition of Mycobacterium leprae recombinant 18-kDa proteins in leprosy.

Three different, purified, Escherichia coli-derived, recombinant preparations of the Mycobacterium leprae 18K protein were compared for their immunological recognition in leprosy. The preparations tested were 18K fusion proteins containing 70% (amino acids 38-148) of the full 18K protein fused to either a short leader sequence containing six asparagine residues or to beta-galactosidase, and the full length 18K protein. All three recombinant antigens were recognized by IgG antibodies which were restricted mostly to lepromatous leprosy patients. The 18K antigen with the asparagine leader sequence showed better reactivity with IgG antibodies compared with the other two 18K preparations. In lymphocyte proliferation assays, the truncated 18K and the full-length 18K showed equivalent responses in the same donors with strongest recognition in donors who were also strongly responsive to the M. leprae soluble sonicate. These results indicate that the major human B- and T-cell epitopes are located within the segment 38-148, although some individuals may recognize additional epitopes at the NH2-terminal end.     

Adenosine triphosphate content of Mycobacterium leprae from leprosy patients

Mycobacterium leprae obtained from randomly selected lepromatous leprosy patients were used to evaluate the ATP assay technique for detecting viability of these cells. The findings were further confirmed by the standard mouse foot pad technique. While the latter takes about 8-12 months to obtain any valid information on the status of M. leprae, the ATP data can be generated within hours and at much lower cost. It is hoped that the ATP data could also instantaneously identify viable bacilli from patients taking dapsone and thereby identify dapsone-resistant patients so that alternative treatment could be given. The advantages of this method over other currently available methods are discussed.     

Enzyme linked immunosorbent assay for identification of whole Mycobacterium bovis isolates with the monoclonal antibodies

Objective: The development of a test system for identification of Mycobacterium bovis with the use of monoclonal antibodies (Mabs).Design: BALB/c mice were immunized with whole gamma-radiation-exposed M. bovis strains. The splenocytes of the immunized mice were used for producing hybridomes which generated Mabs specific to M. bovis. The specificity of the Mabs was determined with the ELISA procedure for the whole cells of the 11 mycobacteria of various species fixed in the wells of a microtitre plate. The optimal conditions for performing identification were selected.Results: Three Mabs specific to M. bovis have been produced which identify various epitopes of the protein with a molecular weight of 31 kD. The ELISA tests with the 2A1 Mab have been conducted for 213 strains of 15 species of mycobacteria. The technique has been shown to be highly specific and reproducible.Conclusion: An ELISA test system with a Mab for identification of M. bovis has been developed.     

Analysis of circulating immune complexes from leprosy patients for Mycobacterium leprae antigens.

Circulating immune complexes (CICs) from 31 leprosy patients (16 tuberculoid, 15 lepromatous) and 12 healthy volunteers, precipitated by 3.5% polyethylene glycol, were individually subjected to SDS-PAGE and immunoblotting using a variety of monoclonal and polyclonal antibodies against Mycobacterium leprae. A common mycobacterial antigen of an apparent molecular size of 65 kDa was identified in CICs from about 40% of the patients. No correlation was observed between the positivity for this antigen and any of the following parameters: bacterial index, M. leprae-specific antibody titers, motor nerve involvement, duration of disease or treatment. Nevertheless, patients with a relatively recent and massive infection were more frequently positive for antigen than the others.     

Preliminary study of cellular immunity to Mycobacterium leprae protein in contacts and leprosy patients.

Because of the good results obtained in the mononuclear cell (T lymphocyte) proliferative response in tuberculoid leprosy patients and family contacts and healthy Mitsuda-positive volunteers using Mycobacterium leprae soluble extract, we prepared different protein fractions from the soluble extract. We used the T-cell Western blot technique with separation by electrophoresis in SDS-polyacrylamide gels and transfer onto nitrocellulose membranes. Each unstained blot was converted into 18 fractions of antigen-bearing particles and tested with peripheral blood mononuclear cells from 21 individuals including Mitsuda-positive contacts, vaccinated lepromatous leprosy (LL) patients, borderline tuberculoid (BT) patients, and unvaccinated lepromatous patients. The stimulation index (SI) of the contacts was higher to the different fractions in comparison with the leprosy patients. They showed four peaks of stimulation to fractions 66-55, 45-29, 22-18, and 14 kDa. The second highest responders were BT patients, followed by vaccinated LL patients. The unvaccinated patients did not respond significantly to any of the fractions (SI less than 1).     

Mycobacterium leprae in the striated muscle of tuberculoid leprosy patients.

Striated muscle specimens from 24 untreated proved cases of tuberculoid leprosy and five healthy normal individuals were studied histopathologically for the evidence of leprous pathology. Atrophy or damage to the muscle fibre was not observed in any patient. Nineteen (79.16%) cases showed evidence of leprosy in striated muscles. Seventeen (70.83%) cases showed scanty histiocytic infiltrate between the muscle fibres. Thirteen (54.16%) cases had acid fast bacilli mostly inside the muscle. There was no correlation between the location of the bacilli and that of the histiocytes; in two cases, acid fast bacilli were seen without the histocyte. The bacilli were solidly staining and were lying singly in the undamaged muscle. There was no evidence of tuberculosis and, in the Control group, none showed any AFB or infiltrate. The presence of lepra bacilli did not depend upon the location of the muscle. Two of the muscle specimens not underneath the cutaneous lesions also had acid fast bacilli. 21.05% of these cases also showed simultaneous involvement of liver and lymph nodes. These are strong evidences of systemic nature of disease in tuberculoid leprosy as well.     

Recombinant fusion protein identified by lepromatous sera mimics native Mycobacterium leprae in T-cell responses across the leprosy spectrum.

Pooled polyvalent sera from lepromatous leprosy patients were used to screen a lambda gt11 recombinant DNA expression library of Mycobacterium leprae in order to identify the relevant antigens recognized by the human immune response. Of the 300,000 phages screened, 4 clones were identified that coded for fusion proteins of the same molecular mass. The fusion protein from clone LSR2 was tested for immunoreactivity in assays using peripheral blood cells and sera from 11 laboratory personnel and 105 patients across the leprosy spectrum. LSR2 protein appears to be predominantly a T-cell antigen. It evokes similar lymphoproliferative responses as the native bacillus both at the individual level and in the leprosy spectrum as a whole. Though only 50% of patient sera with anti-M. leprae antibodies reacted with the fusion protein, the pattern of reactivity in the antibody responses was also similar for the various clinical types. The coding regions of clones LSR1 and LSR2 are identical. They show no homology with sequences stored in data banks and encode a protein of 89 amino acids with a calculated molecular mass of approximately 10 kDa.     

Immunological evaluation of a 12-kilodalton protein of Mycobacterium tuberculosis by enzyme-linked immunosorbent assay

Objective: To purify and study the seroreactivity of native and recombinant 12-kilodalton protein of Mycobacterium tuberculosis H37Rv.Design: M. tuberculosis H37Rv cells and Escherichia coli XL-1 containing the plasmid PRL4 encoding the M. tuberculosis heat shock protein GroES homolog were used as sources for the purification of native and recombinant 12 kD of M. tuberculosis respectively. The seroreactivity of the 12 kDs was studied by ELISA using sera from 35 leprosy and 25 active pulmonary tuberculosis (TB) patients, and from 10 normal healthy controls.Results: The 12 kD protein was purified from H37Rv extract (s12 kD) and from recombinant E. coli (r12 kD) by ultrafiltration and MonoQ fast pressure liquid chromatography (FPLC). Analysis of s12 kD and r12 kD by SDS-PAGE revealed a single protein band in both cases with an approximate molecular weight of 12 000 which was recognized by monoclonal antibody SA-12 in immunoblotting. Both the proteins exhibited a pI of ~4.6 by isoelectric focusing. Both the 12 kD proteins exhibited 96% positivity with TB sera as compared to normal control sera (P < 0.01). Only one serum sample from the 35 leprosy sera tested exhibited binding to both the s12 kD and r12 kD proteins. Delayed type hypersensitivity reaction to the 12 kD proteins was elicited in guinea pigs that had been immunized with H37Rv sonicate.Conclusion: The 12 kD protein could be easily purified and could serve as a valuable serodiagnostic tool in the screening of TB cases from a large population in an endemic area.     

Detection of Mycobacterium leprae DNA for 36kDa protein in urine from leprosy patients: a preliminary report

We have searched for Mycobacterium leprae DNA for 36kDa protein in urine using a M. leprae specific PCR technique. A limited number of 16 patients (of which 11 belonged to lepromatous leprosy and five to tuberculoid leprosy) and eight healthy individuals were included for the present study. The number of urine samples positive by PCR were 36.4% (4/11) in lepromatous patients and 40% (2/5) in tuberculoid patients. None of the samples from healthy individuals was positive. To our knowledge, the results indicate, for the first time, the presence of M. leprae DNA in urine from leprosy patients. Another important finding obtained out of the study is that amongst treated patients 66.6% (4/6) were positive whereas amongst untreated only 20% (2/10) were positive. From the present indicative data it appears that treatment improves the PCR results with urine as a sample. Thus, the approach could prove to be useful for monitoring the treatment response of individual patients and needs to be further evaluated with a large number of patients.     

T-cell responses of leprosy patients and healthy contacts toward separated protein antigens of Mycobacterium leprae.

Sonicated extracts of Mycobacterium leprae were separated by two-dimensional gel electrophoresis and electroeluted into 400 distinct soluble fractions. These fractions were probed with T lymphocytes from leprosy patients of different disease types, healthy contacts, and unexposed healthy individuals. Proliferative responses were visualized using three-dimensional stimulation profiles. T cells from many patients and contacts responded to a multitude of antigen fractions of different molecular masses and isoelectric points. T cells from unexposed individuals gave significant responses to lysates or whole organisms of M. leprae, but no or only marginal responses to separated antigen fractions. T cells of polar tuberculoid (TT) and the majority of polar lepromatous (LL) leprosy patients responded only to separated antigen fractions but not to lysates or whole organisms of M. leprae. The remaining LL patients were totally unresponsive and even failed to respond to separated M. leprae fractions. Thus, in some leprosy patients unresponsiveness to M. leprae seems to be caused by distinct components and can be broken by using separated antigen fractions; whereas in others, anergy remains. T cells of borderline tuberculoid (BT) patients, who were under chemotherapy, responded to separated antigen fractions as well as to lysates of M. leprae organisms. In contrast, BT patients who were untreated failed to react with any of the M. leprae preparations. Similarly, T cells of the majority of LL patients responding to separated fractions were under chemotherapy; whereas T cells from untreated LL patients gave no or only marginal responses to any of the M. leprae antigen preparations. These findings suggest some linkage between the degree of T-cell responsiveness and antileprosy drug treatment.     

Enzyme linked immunosorbent assay for the diagnosis of human neurocysticercosis: a preliminary report

The diagnostic efficacy of an indirect enzyme linked immunosorbent assay (ELISA) was evaluated in cases of neurocystecercosis. Subjects studied included 22 cases and 30 controls (comprising 12 cases of surgically confirmed hydatid disease, 5 cases each of tuberculoma and P.U.O. and 8 cases of pyogenic meningitis). A standard ELISA was performed using porcine cysticerci as antigen. Sensitivity and specificity of the test were calculated for serum and cerebrospinal fluid separately. The test was found to give a sensitivity of 68 and 33 per cent for serum and C.S.F. respectively. The specificity for confirmed case was found to be cent per cent for both with the criteria of reporting used. The sensitivity of the test may be increased by using purified specific antigen and/or sandwich ELISA instead of indirect ELISA technique.     

Susceptibility of strains of Mycobacterium leprae isolated prior to 1977 from patients with previously untreated lepromatous leprosy

Because of the recent spate of reports of primary resistance to dapsone among patients with lepromatous leprosy, largely to small concentrations of the drug, a survey was made of the results of dapsone-susceptibility testing of strains of Mycobacterium leprae isolated before 1977 among six laboratories which employed the mouse foot pad technique for this work prior to that time. Data have been found for strains that had been isolated from 73 patients, representing 19 countries and dependencies, with previously untreated lepromatous leprosy; all 73 strains were inhibited from multiplication by dapsone administered to mice in a concentration of 0.0001 g per 100 g mouse diet. These data suggest that the properties of M. leprae isolated from previously untreated patients with respect to susceptibility to dapsone have changed since the years preceding 1977.     

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.     

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.     

Immunosuppression and cellular immunity reactions in leprosy patients treated with a mixture of Mycobacterium leprae and BCG

Suppressor reactivity was studied in a group of leprosy patients before and after immunotherapy with a mixture of Mycobacterium leprae and BCG. The treatment increases the responses in lymphocyte transformation tests to levels which are comparable to those observed in BT-TT patients and reduces suppressor activity. The soluble extract of M. leprae appears to be more sensitive than purified intact bacilli in the lymphocyte transformation tests, but this preparation did not induce suppressor reactivity with the regularity observed when using a Dharmendra preparation.