Dot blot assay for detection of antidiacyltrehalose antibodies in tuberculous patients.

A simple dot blot test with diacyltrehalose (DAT) as the antigen was developed to detect anti-DAT antibodies in tuberculous patients. To enhance antigen-antibody reaction detection, rabbit serum raised against human immunoglobulins was used prior to incubation with a protein A-colloidal gold complex. With the dot blot system, it was possible to obtain a sensitivity similar to that of enzyme-linked immunosorbent assay (ELISA) and a specificity of 97.14%, versus a specificity of 94.29% by the ELISA. We conclude that this simple and fast assay could be used in places where ELISA equipment is not easy available and that it might also be applicable with other Mycobacterium tuberculosis immunogenic antigens.

Development of an enzyme-linked immunosorbent assay (ELISA) combined with a streptavidin-biotin and enzyme amplification method to detect anti-2,3-di-o-acyltrehalose (DAT) antibodies in patients with tuberculosis.

IgG antibodies against the 2,3-di-o-acyltrehalose glycolipid of Mycobacterium tuberculosis were determined in a set of 49 sera from patients with pulmonary tuberculosis and 65 negative control subjects. We compared a conventional ELISA method using a beta-galactosidase anti-human IgG conjugate developed with ONPG, with an amplification ELISA system constituted of an anti-human IgG biotinylated conjugate, a streptavidin-alkaline phosphatase complex, and NADP as a substrate. The resulting NAD was measured by using a redox enzymatic recycling system of alcohol dehydrogenase, diaphorase and iodonitrotetrazolium as chromogen. With specificity set at 92.31% in both methods, we obtained a sensitivity of 42.86% in the conventional method and a sensitivity of 61.22% in the amplified method. We conclude that by using a more sensitive method we can detect cases that otherwise could be identified as false negatives.

Screening of synthetic trehalose 6,6'-diesters and trehalose 6-monoesters as potential immunoreactants for the serodiagnosis of tuberculosis.

The absence of serological cross-reactivity between trehalose 2,3-diester (DAT, formerly SL-IV) and synthetic trehalose 6,6'-diesters and trehalose 6-monoesters was established by ELISA testing using polyclonal immune sera raised in rabbits sensitized with "DAT". From the screening of fifteen synthetic trehalose 6,6'- and 6-esters, "mirror" pseudo cord factor no. 1, "mirror" amides no. 5 and 6, cord factor analogues 7 and 8 and trehalose 6-monoesters 10 and 11 were selected for future, more extensive serological analysis. Paired comparisons of analogues among these fifteen substances showed that serodiagnostic discrimination power was more a function of the carbon chain length of their substituent groups--as well as of their position--than of the "mirror" constitution of the molecules. More exhaustive testing of these seven compounds is needed to select the synthetic product most efficient in the ELISA serodiagnosis of tuberculosis.

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.

Evaluation of synthetic pseudo cord-factor-like glycolipids for the serodiagnosis of tuberculosis.

A number of glycolipids were evaluated in an ELISA test for their serodiagnostic usefulness in tuberculosis. One hundred and twelve (112) sera belonging to bacteriologically confirmed TB patients, patients with pathologies other than tuberculosis and healthy individuals were examined against several synthetic "mirror" pseudo cord factors (analogues of trehalose-6,6'-dimycolate or TDM) using natural cord factor and another recently described natural glycolipid (SL-IV) of Mycobacterium tuberculosis as control antigens. Analysis of the results shows that all synthetic "mirror" pseudo cord factors, except one with a short 8-carbon chain, were better recognized by the sera of tuberculosis patients than natural cord factor, with sensitivity and specificity values in the ELISA test similar to those reported for M. tuberculosis species-specific SL-IV. Of all antigens tested in this study, BDA. TDA, a bis(N,N-dioctadecylamide) of "trehalose dicarboxylic acid", [(alpha-D-glucopyranosyluronic acid) (alpha-D-glucopyranosiduranic acid)], showed the highest serodiagnostic discriminating power (93% sensitivity and specificity). We postulate that either these artificial molecules are cross-reactants of similarly structured native glycolipids of M. tuberculosis or that they bear closer resemblance to actual phagosome-lysosome-modified antigens than to native mycobacterial ones.

Radiometric diagnosis of mycobacteria.

The aim of this study was to determine the impact of recently developed rapid radiometric techniques on the clinical diagnostic operations of a reference laboratory for mycobacteria. Selective inhibition by rho-nitro-alpha-acetylamino-beta-hydroxypropiophenone was used to rapidly screen submitted cultures for identification of mycobacterial species other than Mycobacterium tuberculosis. The radiometric drug susceptibility test was applied only to those cultures presumptively identified as belonging to the Mycobacterium tuberculosis complex. All referred cultures were tested without additional subculture. The results showed that non-pigmented mycobacteria other than Mycobacterium tuberculosis can be screened with about 99% reliability, most of them within 24 hours. Unnecessary drug susceptibility testing of mycobacteria other than tubercle bacilli can be avoided at an early stage, thus shortening the average reporting time of the Mycobacterium tuberculosis complex to nine days following the receipt of the cultures. Ways of limiting erroneous reporting are discussed.

Conventional and radiometric drug susceptibility testing of Mycobacterium tuberculosis complex.

A recently developed method of drug susceptibility testing of Mycobacterium tuberculosis which measures the evolution of labeled CO2 from [1-14C]palmitic acid (BACTEC 460 system) was compared to three conventional methods. The proportion method of drug susceptibility testing was the standard against which all test results were compared. Indirect drug susceptibility to isoniazid, streptomycin, rifampin, and ethambutol of 245 isolates belonging to the M. tuberculosis complex was determined. In 95% of the cases, results obtained by the radiometric method were available within 1 week, as opposed to 3 to 6 weeks needed in conventional methodology. Overall agreement was 96.4%. Specificity values ranged from 0.98 to 1.0; sensitivity values of 1.0 for rifampin, 0.96 for streptomycin, 0.91 for isoniazid, and 0.18 for ethambutol were obtained. The specificity of the absolute concentration and resistance ratio drug susceptibility testing methods were 0.99 and 1.0, respectively. The sensitivity of the former was higher than that of the radiometric method (0.99 verus 0.92), whereas that of the latter was lower (0.88 verus 0.96). Further testing indicated that the low sensitivity determined for ethambutol may be due to the choice of the critical concentration used, rather than to a shortcoming of the procedure. The radiometric method thus does not significantly differ in reliability from conventional methods of drug susceptibility testing of M. tuberculosis.

Simple amidase test for identification of mycobacteria.

A modified amidase test for differentiation of mycobacteria is described. A total of 224 atypical mycobacteria, 154 Mycobacterium tuberculosis, and 26 M. bovis strains were classified by this procedure. Of the 404 strains of various species studied, 400 exhibited an amidase spectrum identical to the established pattern. The simplicity of this method may promote its application in routine examinations.