Antimycobacterial antibody levels in pleural fluid as reflection of passive diffusion from serum.

The objective of this study was the prospective evaluation of the relationship between serum and pleural fluid antibody levels to mycobacterial antigens and their role in the diagnosis of tuberculous pleuritis. The setting was a tertiary care medical center. Thirteen patients with tuberculous pleuritis and 53 control subjects with pleural effusion (22 with carcinoma, 17 with cardiac failure, and 14 with empyema or parapneumonic effusion) were studied. The level of IgG was measured by ELISA. The median titers of antibody to both Mycobacterium tuberculosis and M avium were significantly higher in the serum and pleural fluid of the patients with tuberculosis than in the control patients. There was a very close relationship between the levels of M tuberculosis (r = 0.95) and M avium (r = 0.94) antibodies in the serum and pleural fluid. We concluded that the levels of antimycobacterial IgG in pleural fluid, adjusted to constant protein concentration, are very closely related to the serum levels. Therefore, these antibodies in the pleural fluid probably result from passive diffusion from serum and not local production. Measurement of pleural fluid antibody levels will not add diagnostic sensitivity or specificity to that achieved with serodiagnosis.

In Vitro Model of Hypoxically Induced Nonreplicating Persistence of Mycobacterium tuberculosis.

Great progress has been made in the latter half of the twentieth century in the understanding of the immunology of tuberculosis and of strategies for chemotherapeutic management of this disease. Indeed, given the evidence that the dominant, and perhaps sole, ecologic niche of Mycobacterium tuberculosis is the infected human host, it seemed reasonable to hope that the disease could not only be controlled, but eradicated by the end of this century (1). These hopes are dashed by the periodic resurgence of tuberculosis in various populations. Undoubtedly socioeconomic factors have played a major role in the failure to eradicate this disease, but another, neglected, factor is the apparent ability of the tubercle bacillus to remain in a relatively quiescent state in the host, neither replicating and causing progression of disease, nor dying off and disappearing from the host's tissues, in spite of apparently adequate immune responses and aggressive chemotherapy.

The impact of new technology on the laboratory's contribution to the diagnosis and management of mycobacterial disease.

From the time of the discovery of the tubercle bacillus in the late nineteenth century until the introduction of chemotherapy in the mid-twentieth, the role of the laboratory in the management of tuberculosis was limited because the treatment of the disease was nonspecific. The advent of specific chemotherapy and the recognition of human diseases caused by a number of mycobacterial species other than M. tuberculosis increased the scope and importance of the clinical laboratory in guiding the diagnosis and management of mycobacterial disease. This included the isolation of mycobacteria, the identification of the isolates, the determination of their susceptibilities to chemotherapeutic agents and their subtyping for epidemiologic purposes. In spite of the enhanced role it has played in the past forty years, the laboratory's contribution has been impeded by the slow growth of mycobacteria, which causes delays of weeks or months between submission of a specimen and the availability of a definitive report. In the meantime both the urgency and the complexity of diagnosis and management of mycobacterial disease have increased with the emergence of epidemics of these diseases associated with the acquired immunodeficiency syndrome (AIDS). This development has also increased the need for recognition of tubercle bacilli in such specimens as blood and stools, which were only infrequently studied in past years. Recent developments in microchemical and immunologic technology, and especially molecular biology, are greatly reducing the time needed to get information that contributes to diagnosis and management of mycobacterial disease.(ABSTRACT TRUNCATED AT 250 WORDS)

Selection of characters for an Adansonian analysis of mycobacterial taxonomy.

Tests which are most frequently employed to provide characters for numerical analysis of bacterial taxonomy provide little differentiation among the slow-growing members of the genus Mycobacterium. A special scheme was proposed to deal with this problem. Characters were based, to some extent, on quantitative reactions to tests, and were defined, for each test, in terms of natural clustering behavior of a large collection of assorted strains. Inter- and intrataxon analyses were based on comparisons of reaction patterns with tentative hypothetical median strain patterns. This permitted the analysis of larger numbers of cultures than can be practically studied in a conventional n x n table.

Synchronized replication of Mycobacterium tuberculosis.

When Mycobacterium tuberculosis was grown in Tween-albumin broth without any agitation, the bacilli replicated in the upper, oxygen-rich portion of the medium at a rate that was just balanced by the rate at which the bacilli settled toward the bottom of the tube. When the organisms that accumulated in the sediment were suspended and diluted into fresh medium, they exhibited synchronous replication. The bacilli initiated ribonucleic acid synthesis immediately upon suspension, but marked deoxyribonucleic acid synthesis was not apparent until after the first cellular division was completed, about 14 h after suspension.

Numerical taxonomy and cooperative studies: roles and limits.

Because of the wide array of specialized tests needed for classification of mycobacteria and the complexity of some of these tests, the International Working Group on Mycobacterial Taxonomy was organized in 1967 to conduct cooperative studies and provide a broad data base for numerical taxonomic analysis of the genus. Certain parameters, such as immunologic properties and lipid composition, were withheld from the numerical analyses to provide independent corroboration of the observed clustering. Overall, these studies have yielded confirmation of the validity of many species, as well as detailed description of these species. The numerical analyses do not resolve all taxonomic questions in the genus Mycobacterium, but they do provide phylogenetic hypotheses based on phenetic clustering. These hypotheses presently are being tested by techniques for measuring DNA relatedness and protein sequence conservation. Concurrently, cooperative studies are continuing in order to provide adequate description of some of the more obscure mycobacterial species.

The "atypical" mycobacteria: recognition and disease association.

Although techniques based on immunologic or chromatographic analyses have been described for identifying mycobacteria in clinical laboratories, most microbiologists continue to rely on a series of specialized physiological and biochemical tests for this purpose. The recognition of additional significant species over the past decade has required the addition of more tests to the battery used for mycobacterial identification. This paper will review briefly the taxonomic status of species likely to be encountered in clinical specimens and the most useful tests for characterizing them. Strategies will be presented for using these tests in the most efficient way to provide optimal resolution of taxa without use of an unreasonably large battery of tests. A brief survey of techniques that may become more practical in the future will also be included.

Dormancy of Mycobacterium tuberculosis and latency of disease.

There is ample circumstantial evidence from observation of the natural history of tuberculosis in humans and experimental animals that Mycobacterium tuberculosis is capable of adapting to prolonged periods of dormancy in tissues, and that these dormant bacilli are responsible for latency of the disease itself. Furthermore, the dormant bacilli are resistant to killing by antimycobacterial agents. A systematic evaluation of the mechanism of dormancy, and of attempts to abrogate latency will require a better understanding of the physiologic events that attend the shiftdown into dormancy. There are probably two or more stages in the shiftdown of Mycobacterium tuberculosis from active replication to dormancy as bacilli in unagitated cultures settle through a self-generated O2 gradient into a sediment where O2 is severely limited. One step involves a shift from rapid to slow replication. The other involves complete shutdown of replication, but not death. Presumably this last step includes completion of a round of DNA synthesis. The shiftup on resumption of aeration includes at least three discrete sequential steps, the production of RNA, the ensuing synchronized cell division and, finally, the initiation of a new round of synthesis of DNA. Three markers of the process of shiftdown of Mycobacterium tuberculosis to dormancy have been described, namely the changes in tolerance to anaerobiosis, the production of a unique antigen and the ten-fold increase in glycine dehydrogenase production. Additional markers represented in the shiftup and shiftdown process may yet be discovered, and determination of their specific functions should provide insights into the mechanisms of dormancy and latency in tuberculosis, and into strategies for preventing reactivation of the bacilli and development of disease.

Microbiology of tubercle bacilli.

Based on conventional taxonomic analyses, as well as molecular level studies on DNA and catalases, the tuberculosis complex (M. tuberculosis, M. microti, M. bovis, and M. africanum) appears to consist of a single species. This species does not occur normally free in nature, but depends on host-to-host transmission for its continued existence. Members of the complex are sufficiently distinct genetically from known free-living mycobacteria so that it is unlikely that they would evolve from the free-living species at a significant frequency in nature, although that is presumably how they arose in the first place. Therefore, eradication of tuberculosis seems to be a realistic hope. The technical means now exist for control of the disease; some improvements in diagnostic technology can be expected to accelerate the control efforts. At a basic science level, more information is needed about the ability of M. tuberculosis to survive in a latent state in a host, only to revive years later, if the control of tuberculosis is to progress to eradication.

Dynamics of submerged growth of Mycobacterium tuberculosis under aerobic and microaerophilic conditions.

When Mycobacterium tuberculosis is grown in detergent-containing medium under continous agitation, multiplication is known to follow a logarithmic mode. When the cultures are not continuously shaken, but only agitated a few times a week to resuspend the bacilli and permit turbidity to be measured, the net increase suggests an arithmetic growth mode. It is shown here that a single pulse of aeration of an unshaken submerged culture of M. tuberculosis causes an almost instantaneous acceleration of growth, followed rapidly by a cessation of growth. Whether or not the bacilli will subsequently resume growth depends on the bacillary population density of the cuture at the time of application of the pulse of aeration. If the bacilli are permitted to grow in the depths of Dubos Tween Albumin broth without any agitation, they exhibit net arithmetic growth and attain a maximal population density greater than is seen in cultures exposed to occasional pulses of aeration. By the use of isotopically labeled cells, it has been shown that replication occurs ar a logarithmic rate amoung the small proportion of the bacilli that remain suspended in nonagitated cultures. This replication is balanced by settling of cells, resulting in a net appearance of arithmetic multiplication. The cells that have settled into the sediment replicate at a very slow rate, if at all, but do retain their viability for 4 weeks or longer. This suggests a possible analogy to quiescent tubercle bacilli in vivo.

Phenol-Soluble Antigens from Mycobacterium kansasii, Mycobacterium gastri, and Mycobacterium marinum.

Many of the demonstrable antigens derived from mycobacteria are common to members of different species. Agglutination tests have yielded the most specific characterizations. An antigen which may be associated with the specific agglutination is soluble in phenol and can be extracted and separated from other antigens by use of this solvent. Phenol-soluble antigens have been extracted from representative cultures of Mycobacterium kansasii, M. gastri, and M. marinum. Most representatives of each of these species yielded an antigen which was characteristic of the species but was distinct from antigen derived from the other two species.

Intrinsic catalase dot blot immunoassay for identification of Mycobacterium tuberculosis, Mycobacterium avium, and Mycobacterium intracellulare.

The heat-labile T class of mycobacterial catalase exhibits peroxidase activity with some substrates. Most species of mycobacteria produce T-catalase, which is serologically characterized by a combination of shared epitopes and unique, species-specific epitopes. Antibodies to T-catalases from Mycobacterium tuberculosis, Mycobacterium avium, and Mycobacterium intracellulare have been cross absorbed with T-catalases from heterologous species and applied as dots to nitrocellulose membranes. When these membranes were incubated with crude sonic extracts of 93 strains of mycobacteria that produce sufficient T-catalase, and were then exposed to 3,3'-diaminobenzidine peroxidase substrate, only those extracts made from one of the three species represented yielded a discrete brown dot at the site of the corresponding globulin. The sensitivity of the test was at least 96.5%, and the specificity was in excess of 99.5%.

Detection of a novel catalase in extracts of Mycobacterium avium and Mycobacterium intracellulare.

A novel class of catalase, which differs from the previously described M- and T-catalases of mycobacteria, was detected in strains of Mycobacterium avium and M. intracellulare. Designated A-catalase, this enzyme resisted inactivation at 68 degrees C, was inactivated by 3-amino-1,2,4-triazole (aminotriazole), and exhibited no peroxidase activity. All of these properties distinguished the enzyme from T-catalase. The A-catalase exhibited a Km of 70 mM H2O2, which is between the upper and lower extremes of the ranges reported for T- and M-catalases, respectively. The A-catalase appeared to be more hydrophobic than M-catalase and did not react with antiserum to a representative sample of this class. The banding patterns of T- and M-catalases seen by polyacrylamide gel electrophoresis (PAGE) were essentially unaffected by the incorporation of sodium dodecyl sulfate (SDS) into the PAGE system, whereas the single band of A-catalase seen by PAGE without SDS resolved into as many as five bands in the presence of SDS; these bands were all of slower mobility than the original band. The banding pattern seen with SDS appeared to be related more to counterion charge effects than to molecular size increases that could be attributed to SDS complexed to the protein. It remains to be determined whether the multiple A-catalase bands reflect different proteins or different SDS micellar complexes of a single protein.

Base composition of deoxyribonucleic acid isolated from mycobacteria.

Guanine plus cytosine values of deoxyribonucleic acid derived from 30 cultures representing 14 mycobacterial species or varieties are presented. These data provide impressive reasons for maintaining the separation between the genera Corynebacterium and Mycobacterium; no conclusions can be arrived at from these data with respect to the Nocardia-Mycobacterium relationship. A bimodal clustering, in terms of guanine plus cytosine composition, is apparent within the genus Mycobacterium. In general, all members of any single phenetic species appear to fit into one or another of these clusters. The phenetic separation of species is, in some cases, confirmed by separation in terms of guanine plus cytosine values. The bimodal separation of guanine plus cytosine values within the genus Mycobacterium does not correspond to a division of the species into slow and rapid growers; it thus provides no justification for splitting Mycobacterium into two genera, composed of slow and rapid growers. This is not to say that such a split would not be useful, only that these data do not contribute to such a decision. Any further attempts to correlate phenetic classification with properties of mycobacterial deoxyribonucleic acid will require more specific techniques, such as molecular hybridization.

Diagnostic key to mycobacteria encountered in clinical laboratories.

A diagnostic key has been developed which will permit identification of most mycobacteria encountered in clinical laboratories. The key is based on performance of a few simple tests. The efficiency and accuracy of the key was evaluated in terms of correlation between identifications based on the few tests and those arrived at through application of the techniques of numerical taxonomy, which involves a large battery of tests. Of 679 cultures of mycobacteria other than Mycobacterium tuberculosis, 86.5% were correctly identified by use of the key, and only 1.8% of the cultures were erroneously identified. The remaining cultures required further examination.

Comparison of methods for tuberculosis bacteriology.

To improve efficiency of isolation of tubercle bacilli from clinical specimens, the following recommendations are presented. (i) Employ multiple specimens consisting of a combination of morning sputums for the early detection of positives, along with 24-hr sputum pools for the greatest total yield of positives. (ii) When timing is rigorously controlled, Zephiran-trisodium phosphate and sodium hydroxide-acetylcysteine are comparable, but if timing cannot rigidly be controlled, employ the Zephiran-trisodium phosphate digestion procedure to allow the greatest freedom in exposure time with the lowest kill rate to tubercle bacilli. (iii) Employ both an agar medium incubated in 5% CO(2), for the early detection of positives as well as positives in the presence of contaminants, and an egg medium, preferably with CO(2), to increase the yield of positives.