Homologs of Mycobacterium leprae 18-kilodalton and Mycobacterium tuberculosis 19-kilodalton antigens in other mycobacteria.

Most of the antigens of Mycobacterium leprae and M. tuberculosis that have been identified are members of stress protein families, which are highly conserved throughout many diverse species. Of the M. leprae and M. tuberculosis antigens identified by monoclonal antibodies, all except the 18-kDa M. leprae antigen and the 19-kDa M. tuberculosis antigen are strongly cross-reaction between these two species and are coded within very similar genes. Studies of T cell reactivity against mycobacterial antigens have indicated that M. tuberculosis bears epitopes that are cross-reactive with the M. leprae 18-kDa antigen, but attempts to identify an 18-kDa antigen-like protein or protein coding sequence in M. tuberculosis have been unsuccessful. We have used a combination of low-stringency DNA hybridization and polymerase chain reaction techniques to identify, isolate, and sequence genes from M. avium and M. intracellulare that are very similar to the 18-kDa antigen gene of M. leprae and others that are homologs of the 19-kDa antigen gene of M. tuberculosis. Unlike M. leprae, which contains a single 18-kDa antigen gene, M. avium and M. intracellulare each have two 18-kDa antigen coding sequences. Although the M. leprae, M. avium, and M. intracellulare 18-kDa antigen genes are all very similar to one another, as are the M. tuberculosis, M. avium, and M. intracellulare 19-kDa antigen genes, we have been unable to detect any 18-kDa antigen-like coding sequences in DNA from M. tuberculosis.

DNA hybridization analysis of mycobacterial DNA using the 18-kDa protein gene of Mycobacterium leprae.

DNA hybridization studies using a 611-base pair (bp) probe, encoding the entire 18-kDa protein of Mycobacterium leprae, demonstrated that M. simiae, M. intracellulare, M. kansasii, M. terrae, ADM-2, M. avium, M. scrofulaceum, M. gordonae and M. chelonei appear to possess DNA sequences homologous to the 18-kDa protein gene of M. leprae. RFLP analysis revealed that the restriction sites in the M. leprae 18-kDa gene were not conserved in the putative gene homologs of M. simiae and M. intracellulare. The restriction patterns observed with the 611-bp probe were useful in differentiating M. intracellulare, M. simiae, and M. leprae from each other, as well as in distinguishing strains of M. simiae serovar 1. Finally, the presence of homologous sequences in various mycobacteria did not affect the specificity of a previously described PCR test for detection of M. leprae, based on the M. leprae 18-kDa protein gene.

Comparative evaluation of enzyme immunoassays based on synthetic glycoconjugates and phenolic glycolipid-I for immunodiagnosis of leprosy.

Enzyme immunoassays (EIAs) based on synthetic glycoconjugates containing the terminal monosaccharide (M-BGG) or disaccharide (ND-BSA) residue of the trisaccharide component of phenolic glycolipid-I (PGL-I), for immunodiagnosis of leprosy are described. The results of the assays were compared with that of the EIA using PGL-I. All the three assays were highly specific for leprosy. The per cent positivity of active lepromatous leprosy (LL) patients with M-BGG was 78.05 in comparison to 85.36 with ND-BSA and 82.11 with PGL-I. Similarly, the positivity of tuberculoid (TT) leprosy patients in M-BGG assay was lower than that in EIAs using ND-BSA or PGL-I. However, the difference in the positivity of individual category of leprosy patients in the three EIAs was not statistically significant. The correlation between absorbance values of leprosy sera in EIAs based on M-BGG and PGL-I, as well as that in assays using ND-BSA and PGL-I was statistically significant.

Antibody response to phenolic glycolipid I and Mycobacterium w antigens and its relation to bacterial load in M. leprae-infected mice and leprosy patients.

Twenty-six inbred BALB/cBy mice were infected with live Mycobacterium leprae by injecting 6 x 10(3) bacilli in the hind footpad. Bleeds were collected at monthly intervals. After 6 months, acid-fast bacilli (AFB) were harvested monthly from the footpad of mice. The sera were analysed in enzyme immunoassay for antibodies against phenolic glycolipid I (PGL-I) of M. leprae and antigens of Mycobacterium w (M. w); 21 out of 26 (80.7%) mice demonstrated the presence of antibodies against PGL-I and M. w . Anti-M. w antibodies appeared slightly earlier than did anti-PGL-I antibodies. The titre of anti-M. w antibodies was higher than that of anti-PGL-I antibodies. The mice giving a positive antibody response had more than 7 x 10(5) AFB/footpad. The coefficient of correlation (r) between the number of AFB and antibody titres at the time of harvest was 0.566 for PGL-I and 0.628 for M. w. The value of r for bacterial index and antibody titres in 188 leprosy patients was 0.510 for PGL-I and 0.418 for M. w; these values were statistically significant (P < 0.001). The decrease in bacterial index and antibody titres in treated lepromatous leprosy patients correlated with increase in the duration of chemotherapy. The measurement of anti-PGL-I antibodies of IgM class may serve as an adjunct to skin biopsy and skin-slit smear for serial monitoring of the bacterial load in the course of chemotherapy in leprosy control programmes.

Generation and characterization of a human monoclonal antibody against phenolic glycolipid-I of M. leprae.

The development of an Epstein-Barr virus transformed human B-cell line secreting a monoclonal antibody (MoAb), KR2/B5 is described. KR2/B5 is an IgM type of antibody and is highly specific for phenolic glycolipid-I (PGL-I) a component unique to M. leprae. The MoAb appears to be directed against the terminal sugar residue of the immunodominant trisaccharide component of PGL-I.

Evaluation of enzyme immunoassays using purified protein derivative (PPD) and its pooled fractions 3 and 4 for diagnosis of pulmonary tuberculosis.

A critical evaluation of two enzyme immunoassays (EIAs) for diagnosis of pulmonary tuberculosis is reported. Purified protein derivative (PPD) or its pooled fractions 3 and 4 were used as antigens for detection of antibodies in sera from 53 patients with active pulmonary tuberculosis and 10 normal healthy individuals. The cut-off point for each EIA was based on the absorbance (mean + 3 SD) of normal sera with the respective antigens. All the normal sera were negative in both the assays. The positivity of tuberculosis patients in either assay was 86.8 per cent. Thus, for serodiagnosis of tuberculosis fractions 3 and 4 of PPD could serve as a good substitute for whole PPD. Sera from 45 leprosy patients were also analysed to assess the specificity of the EIAs. The mean reactivity of tuberculoid leprosy sera was comparable to that of normal sera. The ratio of the mean absorbance of lepromatous leprosy (LL) sera and normal sera was 16.73 with PPD, in comparison to 21.95 for pooled fractions 3 and 4. Out of 10 LL patients 9 (90%) were positive with fractions 3 and 4, in comparison to 10 (100%) with PPD. 71.1 per cent of leprosy patients belonging to different categories were positive in assay based on PPD in comparison to 64.4% in EIA using fractions 3 and 4. The high false positivity of leprosy sera in an assay designed for detection of pulmonary tuberculosis has immense implications in interpretation of results of the assay for diagnostic and epidemiological purposes.

Serological pattern of hepatitis B virus markers (HBsAg, anti-HBs, IgM anti-HBc and HBV specific DNA polymerase) in leprosy patients.

Sera of 134 lepromatous (LL/BL) and 57 tuberculoid (TT/BT) leprosy patients were analysed for four HBV markers. HBsAg was detected in 6.71% of lepromatous and 3.5% of tuberculoid sera. The per cent positivity of lepromatous and tuberculoid sera for anti-HBs antibodies was 30.59% and 35.08%, respectively. The positivity of normal sera for HBsAg and anti-HBs was 3.60% and 21.69%, respectively. The difference in the positivity of three groups of sera (lepromatous, tuberculoid and normal) for HBsAg or anti-HBs was not statistically significant. Anti-HBc (IgM) antibodies were detected in 6% of lepromatous sera. HBV-specific DNA-polymerase activity was found in 22.22% of HBsAg positive (but anti-HBc negative) sera, and 66.66% of anti-HBc positive (but HBsAg negative) sera. The pattern of acute HBV infection in leprosy patients followed the typical pattern prevalent in the normal population.

Detection of lepromatous leprosy patients shedding M. leprae in nasal droppings by enzyme immunoassay.

Enzyme immunoassays (EIAs) for detection of lepromatous leprosy (LL) patients harbouring M. leprae in nasal mucosa are described. One EIA measures IgM antibodies against the synthetic disaccharide (ND-BSA) residue of phenolic glycolipid I of M. leprae, whereas the other titrates primarily IgG antibodies against sonicate supernatant antigens of Mycobacterium w. (M.w.). Fifty coded leprosy sera were analysed by EIAs under a double blind code. Amongst the 20 LL patients with positive nasal smear, 18 (90%) were positive in EIA based on ND-BSA, in comparison to 19 (95%) in EIA using M.w. antigens. The assays can be performed on fresh serum samples or on blood samples collected on filter paper discs. These assays can be useful for leprosy control programmes.

Evaluation of an enzyme immunoassay based on sonicate supernatant antigens of Mycobacterium w for immunodiagnosis of leprosy.

An enzyme immunoassay (EIA) based on sonicate supernatant antigens of a cultivable, atypical bacterium, Mycobacterium w (M. w), for immunodiagnosis of leprosy is described. M. w was selected after screening of sonicate supernatant antigens of seven cultivable mycobacteria in EIA. The results of the assay were compared with that of EIA using phenolic glycolipid-I (PGL-I). The M. w assay was more sensitive than PGL-I based EIA, for detection of leprosy patients of all categories, including long term treated patients with low bacterial load. The M. w assay was highly sensitive (93.49%) for detection of active LL patients, and the difference in the positivity of the two assays for LL patients was statistically significant (p 0.05). The combined positivity of the assays with M. w and PGL-I for LL was higher than that with either antigen alone. M. w assay, in addition, was also highly sensitive for detection of patients with active pulmonary tuberculosis.

A dot enzyme immunoassay for detection of IgM antibodies against phenolic glycolipid-I in sera from leprosy patients.

A visual dipstick dot enzyme immunoassay (EIA) for diagnosis of leprosy is described. The assay is based on detection of IgM antibodies against phenolic glycolipid (PGL-I) in sera from leprosy patients. The antigen (PGL-I or synthetic disaccharide of PGL-I) was dotted on a nitrocellulose pad stuck on a plastic strip (dipstick). Sera were used at a dilution of 1:200. Peroxidase coupled mouse anti-human IgM monoclonal antibodies were used as the conjugate. A positive test gave a blue dot against a white background. The test was highly specific for leprosy, and was quite sensitive for detection of bacilliferous (BL/LL) leprosy. The antigen dotted and preblocked dipsticks stored at room temperature upto 4 months of observation period, were unable in the assay.