beta 2-microglobulin (beta 2m) was found to interact with many group A streptococcal strains. The interaction appeared to require multipoint attachment, since monomeric beta 2m in solution showed no binding, whereas both beta 2m monomers bound to liposomes, and beta 2m in aggregates showed affinity for the bacteria. Aggregated HLA antigens (-A, -B and -C) and aggregated beta 2m exhibited the same binding patterns when tested in binding experiments with various group A streptococcal strains. Furthermore, beta 2m aggregates in excess completely blocked the binding of aggregated HLA antigens, thereby demonstrating that beta 2m is able to interact with streptococcal surface structures also when it is part of the HLA antigen complex. M protein-positive group A streptococcal strains bound significantly more beta 2m than M protein-negative variants of these strains. Purified M 12 protein partly inhibited the binding of radiolabelled beta 2m aggregates to whole streptococci, and in gel filtration and affinity chromatography experiments, the M 12 protein interacted with beta 2m. These various data suggest that the interaction between beta 2m and group A streptococci could be mediated by M protein. Lipoteichoic acid (LTA) is a constituent of the streptococcal cell wall that has been reported to form complexes with M protein at the bacterial cell surface. However, LTA did not influence the interaction between beta 2m and streptococci, suggesting that the binding of beta 2m to streptococcal M protein represents a pure protein-protein interaction. In vivo such an interaction could be established between infecting streptococci and host cells. Among 45 strains of different M types large differences in beta 2m binding were recorded, whereas among 60 strains of the classical nephritogenic M types 12 and 49, all were highly beta 2m-reactive, which points towards a role for beta 2m in streptococcal pathogenicity.

A polymerase chain reaction for the specific detection of mycobacteria belonging to the Mycobacterium tuberculosis complex was developed. Using a single primer pair derived from the nucleotide sequence of protein antigen b of M. tuberculosis, we achieved specific amplification of a 419-base-pair DNA fragment in M. tuberculosis and M. bovis. After DNA was extracted from mycobacteria by using a simple, safe lysis procedure, we detected the 419-base-pair sequence in samples containing few mycobacteria. Preliminary data suggested that this technique could be applied to clinical specimens for early and specific diagnosis of tuberculosis.

The diagnosis of pulmonary tuberculosis (TB) relies on the bacteriological examination of sputum. However, microscopy of smears made directly from sputum has a low sensitivity and there is an urgent need for improved methods. We have compared microscopy of smears made directly from sputum with microscopy after liquefaction of sputum with household bleach (NaOCl) and concentration of bacteria by centrifugation. In 3 studies performed in Ethiopia and India, the use of the NaOCl method increased the number of samples positive for acid-fast bacilli by more than 100%. The technique is appropriate for developing countries and its application would increase the efficiency of TB control programmes. As a potent disinfectant, NaOCl also has the advantage of lowering the risk of laboratory infection.

The colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was standardized for direct detection of rifampin-resistant Mycobacterium tuberculosis in sputum samples. The sensitivity and specificity of the direct MTT assay matched those of the standard indirect susceptibility assay on 7H10 medium, and interpretable results were obtained for 98.5% of the samples within 2 weeks. Traditional methods of in vitro drug susceptibility testing are time consuming and laborious. Susceptibility tests on clinical isolates require 6 to 9 weeks, and tests conducted directly on smear-positive samples take about 3 weeks (International Union Against Tuberculosis and Lung Disease, The public health service national tuberculosis reference laboratory and the national laboratory network. Minimum requirements, role and operation in a low-income country, Paris, France, 1998, and P. T. Kent and G. P. Kubica, Public health mycobacteriology. A guide for the level III laboratory, Centers for Disease Control and Prevention, Atlanta, Ga., 1985). More-rapid methods are available but are very expensive for routine use under program conditions in countries with high levels of tuberculosis endemicity.A colorimetric assay based on 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) was previously standardized and evaluated using a BACTEC radiometric method as a "gold standard" for indirect detection of rifampin resistance (1,8). MTT is a yellow tetrazolium salt that is converted into blue formazan by dehydrogenases of live cells (7). The amount of blue or purple formazan formation is proportional to the number of live mycobacteria in a sample (8). The results of the MTT assay matched fully with the results obtained using the BACTEC method (1).This study was conducted with the objectives of standardizing the MTT assay for the detection of rifampin resistance directly on smear-positive sputum samples and evaluating the assay against the traditional method as the gold standard.Standardization and evaluation. Sputum samples from 16 new smear-positive cases of pulmonary tuberculosis were used to standardize the assay. Sputum samples from 74 smear-positive retreatment cases were pelleted after digestion and decontamination with 4% NaOH (5). The pellets were neutralized and resuspended in 3 ml of sterile 7H9 broth each. An aliquot of 500 l was added to tubes containing 3 ml of 7H9 Middlebrook broth supplemented with 10% oleic-acid-albumin-dextrose-catalase, glycerol (0.05%), and PANTA (Becton Dickinson, Paramus, N.J.) (75 l). Rifampin (Sigma) at a final concentration of 2 g/ml was added to some of the tubes. The experimental approach is summarized in Fig. 1. All tubes were incubated at 37°C until the day of the MTT assay.MTT assay. The MTT assay was done each week for 3 weeks. Contamination was checked by growing subcultures on nutrient agar medium. Each week, an MTT assay was done using one bacterial control tube with no rifampin and another tube with bacteria and rifampin. The assay was done as described ...

Tuberculosis remains a major global cause of morbidity and mortality. There is an urgent need for improved bacteriologic diagnosis of Mycobacterium tuberculosis infection. Three methods for rapid identification of M. tuberculosis in sputum samples (direct microscopy, gas chromatography-mass spectrometry [GC-MS], and polymerase chain reaction [PCR]), were compared with culture on Lowenstein-Jensen medium. Growth of M. tuberculosis was observed in 38 of 145 sputum samples. Detection of acid-fast bacilli by direct microscopy gave a sensitivity of 66% and a specificity of 100%. Detection of tuberculostearic acid by GC-MS gave a sensitivity of 55% and a specificity of 87%. Amplification by PCR of a fragment of the insertion sequence IS6110 gave a sensitivity of 95% and a specificity of 93% compared with culture and a corrected specificity of 99% compared with both culture and clinical data. This study indicates that PCR can be adapted for clinical use and is the method of choice for rapid diagnosis of pulmonary tuberculosis.