Circulating immune complexes in leprosy sera: demonstration of antibodies against mycobacterial glycolipidic antigens in isolated immune complexes.

Circulating immune complexes (CIC) were assayed in sera of leprosy patients. Using an immunoassay for two mycobacterial antigens--phenolic glycolipid-I (PGL-I) and glycolipid IV (SL-IV)--sera from 65 patients with leprosy (38 lepromatous, 18 borderline, and 9 tuberculoid) were studied. The CIC were isolated by polyethylene glycol (PEG) precipitation, washed, treated with an acid buffer, neutralized, and tested using an enzyme-linked immunosorbent assay (ELISA). We demonstrated that CIC could contain IgG and IgM antibodies reacting against PGL-I and SL-IV. The high levels of antibodies in the precipitable CIC showed concordance with high levels in the original sera, although some patients presented high levels of precipitable CIC in the absence of high titers of antibodies in their sera. It was concluded that some of the CIC observed in patients with leprosy were composed of IgG and IgM immunoglobulins against specific mycobacterial antigens.

Comparison of bis-di-octadecylamide of trehalose dicarboxylic acid (BDA.TDA) with glycolipid SL-IV as ELISA antigens for the serodiagnosis of leprosy.

Two glycolipids--one synthetic and non-natural (BDA.TDA), the other natural and Mycobacterium tuberculosis species-specific (SL-IV)--were tested to determine their serological activity in sera obtained from leprosy patients, and to determine their discriminating ability in the detection of disease. The ELISA results obtained in the IgG antibody class show that both were useful substances capable of detecting multibacillary and paucibacillary disease in about 2 out of 3 leprosy patients. When these antigens were tested in parallel, the sensitivity of the ELISA test was increased by 10% without a decrease in specificity.

Relationships between titers of antibodies immunoreacting against glycolipid antigens from Mycobacterium leprae and M. tuberculosis, the Mitsuda and Mantoux reactions, and bacteriological loads: implications in the pathogenesis, epidemiology and serodiagnosis of leprosy and tuberculosis.

Analysis of cell-mediated immunity [(CMI) as judged from the Mantoux, Fernandez, and Mitsuda reactions and the presence of granulomas in biopsy material] against humoral immunity (measurements of anti-PGL-I, PGL-Tb1, and SL-IV IgG and IgM antibody titers by ELISA) were performed in selected human populations. The investigations yielded data indicating that humoral (B-cell) responses preceded protective CMI in both tuberculosis and leprosy. The B-cell responses were unrelated to (unfavorable) cell-mediated delayed-type hypersensitivity (DTH). Notwithstanding the difficulty in inferring sequential events from studies in humans, it was shown that in humoral responses there was an initial rise of specific IgM immunoglobulins that switched afterward to IgG production during subclinical tuberculosis and leprosy infections. In patent tuberculosis disease the IgM-to-IgG switch was observed in the majority of patients; in patent leprosy disease the switch was impaired in the majority of patients. The clinical, immunological, and laboratory data indicated that the B-cell responses were suppressed as protective CMI was re-established in the patients during the protracted subclinical infection. According to the data, the diagnosis of subclinical tuberculosis and leprosy may be accomplished using ELISA. The yearly risk of tuberculosis in apparently healthy persons but with significant antibody titers was estimated at 44%; the yearly risk for leprosy has not yet been established. The clinical, epidemiologic, and diagnostic implications of these findings are discussed.

DOT-ELISA for detection of phenolic glycolipid PGL-Tb1 and diacyl-trehalose antigens of Mycobacterium tuberculosis.

A DOT-ELISA method for detection of 2,3-diacyl-trehalose (DAT, previously referred to as SL-IV antigen) and triglycosyl phenol phthiocerol di-mycocerosate (PGL-Tb1) antigens from Mycobacterium tuberculosis is described. The method enabled the detection of both antigens in 14 clinical isolates of M. tuberculosis from different geographic origins; the presence of the glycolipids was confirmed by chemical analysis. It was therefore concluded that the synthesis of both of these compounds is characteristic of the species.

Mycobacterial flora of the skin in leprosy.

The presence of mycobacteria on the skin of healthy people and in leprosy lesions has been documented previously. The present study observed the mycobacterial flora on the hands (by the hand-washing method) and fingers (by the inoculated culture medium using scraped material obtained during the preparation of slit-skin smears) in 89 untreated leprosy patients. We also evaluated the slit-skin smears from fingers for the diagnosis of leprosy. In 16 patients (17.9%) mycobacteria were cultured from scrapings and hand washings. The frequency of isolates from lepromatous (LL) leprosy cases (52.9%) was significantly higher than from tuberculoid (TT) leprosy cases (5.2%). It was observed that Mycobacterium avium and M. scrofulaceum were the only opportunistic mycobacteria isolated from multibacillary patients, and two hypotheses are discussed to explain these findings. The slit-skin smears from fingers were as satisfactory as smears from other sites for the diagnosis of leprosy, but they were less satisfactory for estimating the morphological index.

Use of the SL-IV and the PGL-Tb1 glycolipid antigens in ELISA for the diagnosis of tuberculosis in AIDS patients.

At a predetermined specificity of 100.0%, the sensitivity of ELISA using the PGL-Tb1 and SL-IV antigens and IgG assays was 35.0% for the diagnosis of tuberculosis in AIDS patients (44.1% when tuberculosis was diagnosed before AIDS, 21.7% when AIDS was diagnosed before tuberculosis). Serial assays in sera collected from 11 AIDS patients before tuberculosis was diagnosed indicated that significant antibody titres were detected 10 months before the onset of clinical tuberculosis. Consequently, it was proposed that serodiagnosis using the glycolipid specific antigens should help in deciding on preventive antituberculosis treatment in these patients.

Evaluation of a novel 2,3-diacyl-trehalose-2'-sulphate (SL-IV) antigen for case finding and diagnosis of leprosy and tuberculosis.

Serum IgG and IgM antibodies against a 2,3-diacyl-trehalose-2'-sulphate (SL-IV) antigen using ELISA were determined in controls (n = 288) and in leprosy (n = 210) and tuberculosis (n = 99) patients. In all assays, the amount of antigen per well was 100.0 ng and sera were diluted 1/250. In the case of leprosy, anti-SL-IV IgG and IgM antibody titres increased from the tuberculoid towards the lepromatous pole of the spectrum. In the tested population, the sensitivity of the assay was 93.2% in multibacillary leprosy and 33.3% in paucibacillary leprosy (specificity of 88.7%). Multibacillary patients with erythema nodosum leprosum (ENL) had lower titres than non-ENL. ELISA results were similar to those obtained using the Mycobacterium leprae phenolic glycolipid-I (PGL-I) antigen. In the case of tuberculosis (pulmonary and extrapulmonary), significant titres of anti SL-IV IgG and IgM antibodies were detected in about 75% of the patients using a cutoff point of 0.150, and in 51.6% using a cutoff of 0.300 (specificities were, respectively, 88% and 100%). We concluded that the determination of IgG and IgM antibodies against SL-IV was useful in leprosy and tuberculosis case finding program using a cutoff point of 0.150, and for serodiagnosis using a cutoff of 0.300.

IgG and IgM antibodies immunoreacting with a 2,3-diacyl trehalose-2'-sulphate in sera from leprosy patients.

The distribution of IgG and IgM antibodies immunoreacting with the sulpholipid I (SLI) and sulpholipid IV (SLIV) of Mycobacterium tuberculosis was examined in sera from leprosy patients. It was found that the immunological reactions correlated with the clinical spectrum of leprosy; and in multibacillary patients, antibody titres declined in response to successful treatment. The serological patterns were similar to the PGL I patterns, however, the IgG responses towards the sulpholipids were predominant over the IgM responses in the case of the sulpholipid antigens.

Antigenicity and specificity of selected glycolipid fractions from Mycobacterium tuberculosis.

Antigenicity of Mycobacterium tuberculosis glycolipids polyphthienoyl trehalose (PPTR), phenolic glycolipid (PGL-Tb1), tetraacyl trehalose-2'-sulphate (SL-I) and diacyl trehalose-2'-sulphate (SL-IV) was examined in rabbits. PPTR did not induce production of IgG antibodies in rabbits, while PGL-Tb1, SL-I and SL-IV glycolipids were efficient in this respect. Immune sera raised in rabbits immunoreacted exclusively with the corresponding antigens, which indicated that they were remarkably specific. Specificity of the immune sera was further examined using crude extracts of representative strains of 39 mycobacterial species, and the data showed that these immune sera reacted only with extracts of M. tuberculosis and M. africanum. An antiserum raised against whole cells of M. leprae immunoreacted with the purified SL-IV antigen from tubercle bacilli.

Isoenzymes as tools to discriminate various subdivisions in the Mycobacterium fortuitum complex.

Methods for the characterization of catalase, peroxidase, beta-glucosidase, esterase, and beta-lactamase mycobacterial isoenzymes were described. These methods were applied to examine strains of the M. fortuitum complex. M. fortuitum, M. peregrinum, M. chelonae- M. abscessus and an unnamed species had distinct isoenzyme profiles. M. chelonae and M. abscessus could not be satisfactorily differentiated using the described methods.

Isoenzymes profiles in slowly-growing and difficult-to-grow mycobacteria.

The cell-free extracts of 13 slowly-growing mycobacteria were run on polyacrilamide gels (Page) and the various protein bands obtained were tested for peroxidase and catalase enzyme activities. The results obtained were compared to those obtained with M. fortuitum and M. chelonae. Based only on a limited numbers of strains employed, it is suggested that these isoenzyme patterns may permit a better separation of "Wood-pigeon" mycobacteria from both M. avium and M. paratuberculosis and also give distinct profiles for other species used. These results further suggest the potential of isoenzymes as taxonomic markers.

Non-cultivable mycobacteria in ulcers of the skin.

In biopsies of 54 patients suffering from chronic dermatological lesions (mostly ulcers of the skin) acid-fast bacilli were found in 14. In these 14 cases in 4 were lesions caused by M. tuberculosis, in 1 the lesion was caused by M. avium-intracellulare, in 1 the lesion was caused by M. fortuitum and in 2 the lesions were caused by non-cultivable mycobacteria (Feldmann-Hershfield ulcer?). In 2 cases the cultures were heavily contaminated, and the diagnosis remained uncertain. In the remaining 4 cases the mycobacteria were considered occasional isolates without clinical significance.

Immunological response to homologous and heterologous phenolic glycolipid antigens in tuberculosis and leprosy.

The occurrence of IgM antibodies immunoreacting in an ELISA test with five phenolic-glycolipids (GPL) antigens (PGL-Tb 1, from M. tuberculosis; PGL-I, from M. leprae; PGK-K-I, from M. kansasii; Mycoside G, from M. marinum; and Mycoside B, from M. bovis), was examined in the sera of 46 tuberculous patients, 48 multibacillary leprosy patients, 40 paucibacillary leprosy patients and in 134 healthy controls. The sensitivity (97.9) and the specificity (91.8) observed in tuberculous patients with the homologous antigen PGL-Tb 1 underlined the interest of this antigen for case finding in tuberculosis epidemiology. The sensitivity and the specificity observed in multibacillary leprosy patients, respectively 91.7 and 91.8, and in paucibacillary leprosy patients, respectively, 35.0 and 91.7, confirmed the limited value of homologous antigen PGL-I for the serological case finding of leprosy patients in endemic areas with a strong incidence of paucibacillary leprosy forms. The data obtained with the heterologous PGL antigens in tuberculosis and multibacillary leprosy serology were higher than those observed in healthy controls and in paucibacillary leprosy patients. However the ELISA using the heterologous antigens was not useful in diagnosis of active tuberculosis or multibacillary leprosy forms. Healthy controls showed low immunoreactivity against PGL antigens, with the exception of mycoside B.

Comparative intracellular growth of difficult-to-grow and other mycobacteria in a macrophage cell line.

We have recently developed a murine macrophage cell line (J-774) model which permits the growth of various mycobacteria (8). The purpose of the present investigation was to compare the intracellular growth of various difficult-to-grow mycobacteria (Mycobacterium paratuberculosis, M. ulcerans), and other pathogenic (M. tuberculosis H37Rv, M. kansasii, M. bovis) and nonpathogenic or avirulent (M. tuberculosis H37Ra, M. bovis BCG, M. gastri) mycobacteria. Electron microscopic studies were also performed to elucidate whether the formation of an electron-transparent zone (ETZ) around phagocytized bacilli was linked to their intramacrophagic survival. Furthermore, the comparison of intracellular growth of a pathogenic (M. kansasii) and nonpathogenic (M. gastri) mycobacteria sharing the same phenolic glycolipid antigen at their surface (Mycoside-A, 5), suggested that these antigens did not play a primary role in intracellular survival and multiplication of these bacteria. Also, we were unable to propagate M. ulcerans inside J-774 macrophages, which were massively lyzed after infection (due to a characteristic toxin secreted inside the macrophages?). These results are discussed in terms of the validity of the J-774 model for studying intracellular growth of mycobacteria.