Serodiagnosis of Hansen's disease/leprosy by enzyme-linked immunosorbent assay using cord factor (trehalose 6,6'-dimycolate) as an antigen.

IgG and/or IgM antibodies against mycobacterial cord factor (trehalose 6,6'-dimycolate, TDM) in sera of 65 patients of Hansen's disease (21 cases with smear-positive and 44 cases with smear-negative) and 60 healthy individuals were tested by enzyme-linked immunosorbent assay (ELISA) with TDM purified from Mycobacterium tuberculosis H37Rv as an antigen. Of 65 patients with Hansen's disease, 58 cases (89.2%) had positive results (21 samples from 21 patients, 100% with acid-fast bacilli positive in the lesion, and 37 samples from 44 patients, 84.0% with acid-fast bacilli negative Hansen's disease diagnosed clinically). The sensitivity and specificity of anti-cord factor ELISA were higher than those of anti-phenolic glycolipid-I (PGL-I) agglutination test. Among the total, 34 patients were classified clinically into three types of the disease, lepromatous leprosy (LL), borderline lepromatous (BL) and borderline tuberculoid (BT). The antibody titer showed LL > BL > BT, indicating that the elevation of anti-cord factor antibody titers appeared to be parallel with the degree of humoral immune response against M. leprae. By using semisynthetic cord factor consisting of a single subclass of mycolic acid from M. tuberculosis, it was revealed that sera from patients with Hansen's disease were highly reactive against alpha-mycoloyl cord factor (alpha-TDM) and less reactive against methoxy mycoloyl TDM (methoxy TDM), differed from sera of tuberculosis patients, which were highly reactive against both methoxy and alpha-mycoloyl cord factor (alpha-TDM). Most of sera from patients with Hansen's disease were more reactive against TMM than TDM, differed from sera of tuberculosis patients which were highly reactive against TDM. ELISA using TDM as an antigen is simple, reproducible and useful for the rapid serodiagnosis of Hansen's disease, especially for smear-negative cases.

[Biochemistry and bioactivities of mycobacterial components].

The most characteristic pathological change in mycobacterial infection is caseous necrosis followed by tuberculous cavity formation due to the cellular immunity induced by antigenic proteins and adjuvant active cell wall components. Mycobacterial cell well contains unique hydrophobic compounds possessing mycolic acids (a long branched-chain high molecular weight fatty acid) and shows distinctive properties such as acid-fastness and wax-like hydrophobicity. Mycobacteria do not produce exotoxin and the virulence cannot be determined by a single toxic substance, but the cell wall components to contact with the host cells are the most important surface molecule at the early stage of infection. Cord factor (trehalose 6,6'-dimycolate) is the most classical virulence factor which is lethally toxic for mice. However, cord factor exists among various species of mycobacteria and even in Nocardia and Rhodococcus. Furthermore, cord factor can produce granulomas without protein antigen in mice and it shows antitumor or non-specific prevention promotion of infection and induction promotion of various cytokines. Sulfolipid (tetracyl trehalose sulfate) also plays a role as a virulence factor by phagocytic process inhibition. Glycopeptidolipid (GPL) from M. avium and phenolglycolipid (PGL) from M. leprae also appeared to be immunomodulatory molecules which inhibit the developing of cellular immunity. Lipoarabinomannan (LAM) is also unique amphipatic molecule, like gram-negative endotoxin. Here, we discuss the involvement of various surface molecutes to contribute to pathogenesis in mycobacterial infection and immunity.

Relationships between structure and biological activity of the mycolic acid-containing glycolipids from Nocardia asteroides "sensu stricto" and related species.

To reveal the taxonomical situation of Nocardia asteroides "sensu stricto", we compared the mycolic acid and mycolic acid-containing glycolipid composition and their granulomagenic activities in mice. The major glycolipids were glucose mono- and dimycolate, trehalose mono- and dimycolate and several unknown glycolipids, commonly, although the relative amount differed from strain to strains. On the other hand, molecular species composition of mycolic acids differed distinctively among the three closely related species: N. asteroides "sensu strico", N. farcinica and N. nova. GC/MS analysis showed the most abundant species of mycolic acids were C50(52) in N. asteroides, C54(52) in N. farcinica and C58(56) in N. nova, respectively with a different alpha-alkyl branch. Glucose mycolate and trehalose dimycolate possessing C50 mycolic acid showed a strong activity for granuloma formation in mice.

Rapid and precise diagnosis of atypical mycobacterial infection by chemotaxonomical and immunological methods.

The species of 205 strains of acid fast bacteria isolated from swine and human mycobacteriosis were identified chemotaxonomically and numericaltaxonomically. The species of the isolates which were identified numericaltaxonomically as Mycobacterium avium intracellulare (MAI) complex were further classified by using both thin-layer chromatography of the antigenic glycopeptidolipids (GPL) from the bacteria and seroagglutination test devised by Schaefer. These MAI complex from swine fell into serotype 8 (45 strains), serotype 4 (32 strains), serotype 9 (9 strains) and untypable (9 strains), respectively. In contrast to swine, human isolates covered more wide ranges of serotypes such as serovar 7, 12, 16 besides serovar 4, 8 and 9. Furthermore, enzyme-linked immunosorbent assay (ELISA) which is based on the type specific glycolipid antigen and infected swine/human sera was applied to distinguish serological variants of the MAI complex. Of the fourteen cases in swine and five in human that had been typed by both the seroagglutination reaction and the thin layer chromatography (TLC) the thirteen in swine and two in human cases showed clear coincidence with the results of ELISA. The results demonstrated that enzyme-linked immunosorbent assay using infected sera was especially useful, and it was recommended from the sensitivity and rapidity as an adjunct to seroagglutination test and thin layer chromatography for the identification of serotypes of MAI complex.