Tuberculosis: Pathogenesis, Protection, and Control

This chapter reviews the current epidemiology of tuberculosis (TB) in the world. Although the authors concentrate on the number of new cases of deaths from this disease, they also aim at presenting the exact numbers of new cases of TB and deaths from TB that occur each year. The ability of the tuberculin skin test to detect the presence of Mycobacterium tuberculosis infection can be used to measure the prevalence of infection. The annual risk of infection is the probability that any individual will be infected with M. tuberculosis in 1 year. It is estimated that about 1,700 million people are infected with M. tuberculosis. Primary resistance is defined as the presence of drug resistance to at least one anti-TB drug in a TB patient who has never received prior treatment. Without recognition of the TB crisis confronting the world and prompt, effective action, the TB epidemic can be expected to worsen for several reasons. First, demographic forces are at work. Children born in past decades in regions with high population growth rates are now reaching the ages at which morbidity and mortality for TB are high. Second, famine, war, and natural disasters that create large populations of displaced, malnourished people in crowded living conditions may cause increases in TB case rates. Third, age-specific TB incidence rates can be expected to rise in those areas of the world where immunity of the population is seriously challenged by HIV infection.

As centuries and millennia passed, human beings began to live in larger and larger communities, and with this shift came environmental changes that were associated with a change in the delicate balance between humans and the tubercle bacillus. Two alternative theories have been proposed to explain the epidemic spread and subsequent decline of tuberculosis that followed. In the 1700s and early 1800s, tuberculosis prevalence peaked in Western Europe and the United States and was undoubtedly the largest cause of death, and 100 to 200 years later, it had spread in full force to Eastern Europe, Asia, Africa, and South America. The epidemic grew over the next two centuries and spread through Western Europe. During this phase of the epidemic, almost all Western Europeans became infected with M. tuberculosis, and about one in four deaths were due to tuberculosis. Army medical officers from Great Britain noted that tuberculosis was unknown in those parts of Africa where European immigration had not occurred. By matching microbial drug susceptibility patterns, one patient with tuberculosis laryngitis was identified as particularly infectious. Modern parallels are presented by microepidemics in poorly ventilated areas, of which few are more dramatic than the one described by Catanzaro just 100 years after Koch's demonstration of the tubercle bacillus.

The clinical expression of infection with Mycobacterium tuberculosis is quite varied and depends on a number of identified factors. Immunization with bacillus of Calmette and Guérin (BCG) in persons with intact cell-mediated immunity minimizes the risk of early disseminated tuberculosis, especially in children. Systemic manifestations of the disease, including fever, malaise, and weight loss, are likely mediated by cytokines, especially tumor necrosis factor alpha (TNF-α). Weight loss, weakness, and malaise appear to be less common but are more difficult to quantify. Cough is the most common symptom of pulmonary tuberculosis. Tuberculosis that occurs relatively early in the course of HIV infection tends to have the typical radiographic findings described. Due to the frequency of extrapulmonary tuberculosis among HIV-infected patients, diagnostic specimens from any suspected site of disease should be examined for mycobacteria. Paravertebral or other para-articular abscesses may develop, with occasional formation of sinus tracts. Although weight-bearing joints are the most common sites for skeletal tuberculosis, any bone or joint may be involved. Meningitis is the most frequent form of central nervous system tuberculosis; solitary or multiple tuberculomas occur less commonly. The epidemiologic pattern of central nervous system tuberculosis is quite different from either pulmonary or other forms of extrapulmonary tuberculosis in that the peak incidence is in children in the zero-4-year age group, but an appreciable number of cases occur in adults. The chest pain may occasionally mimic angina but usually is described as being dull, aching, and often affected by position and by inspiration.

Study of the epidemiology of tuberculosis has been greatly assisted by the availability of a test for infection, the tuberculin test, that enables one to distinguish those who are infected but without disease from those who are uninfected. The principal risk for acquiring infection with Mycobacterium tuberculosis is breathing. The concept that microbes may exist in sufficient concentration in the air to cause airborne infection and disease was controversial until the middle of this century. Tuberculosis due to reactivation of latent bacilli is presumed to result from a failure in immune surveillance. Until relatively recently, rates of tuberculosis in developed countries had shown large and consistent declines for many decades. The epidemic of HIV infection has radically changed the epidemiology of tuberculosis. Recent outbreaks of multiple-drug-resistant (MDR) tuberculosis in AIDS units and other congregate settings suggest an increased risk of primary infection with M. tuberculosis. The demonstration of exogenous reinfection raises the issue of the effect of HIV on primary transmission of M. tuberculosis in developed countries. HIV has profoundly changed the epidemiology of tuberculosis, disrupting the balance between tuberculosis infection and control in both developing and developed countries.

This chapter provides scientists and technicians a basic orientation to health and safety practices appropriate for the control of hazards associated with the handling of Mycobacterium tuberculosis in the research laboratory. The most important message in this chapter is that M. tuberculosis should be handled only by those who have mastered basic safe practices. The laboratory in which this work is carried out has certain design and operational features that provide protection from exposure to M. tuberculosis for those other facility occupants who do not require access to the laboratory. The chapter discusses the importance of safety considerations and precautions that will help the user understand both the limitations and the benefits of this equipment. In the class I cabinet, the intake airflow passes through the work space of the cabinet. Class II cabinets can protect experimental materials from airborne contamination. Airborne contamination within the room air is prevented from reaching the work space by the downward airflow of high-efficiency particulate air (HEPA)-filtered air, which is supplied at the top of the cabinet's interior work space. Biological spills outside biological safety cabinets generate aerosols that can be dispersed in the air throughout the laboratory. A symptom check should be provided by a physician for all exposed individuals who were previously tuberculin positive. It is imperative that the tuberculin skin test always be placed and interpreted by an expert.

Individual investigators have different reasons for cultivating Mycobacterium tuberculosis in their laboratories; the medium to be used and the conditions of incubation should be tailored to the specific goals, taking into account the physiologic characteristics of this organism. A classic description of M. tuberculosis, which would be only partially accurate, might characterize this organism as a fastidious, slowly growing, strictly aerobic, lipid-rich, hydrophobic, acid-fast bacterial rod. Much of the early research on the tubercle bacillus was directed toward production of tuberculin or other crude chemical products of the bacilli, such as unique lipids or polysaccharides. The growth of tubercle bacilli in the synthetic media cited is difficult to follow in quantitative terms, as it cannot be measured optically and since simple plating is not accurate because of the severe clumping of the bacilli. Media based on a Tween 80-albumin formulation are satisfactory for cultivation of tubercle bacilli for studies of growth rates and kinetics and, in most cases, for preparation of bacilli for chemical extraction. Glycerol has commonly been used in media when large crops of bacilli are desired, since this carbohydrate markedly stimulates growth of M. tuberculosis (note that glycerol inhibits growth of fresh isolates of the closely related Mycobacterium bovis). M. tuberculosis is known to produce enzymes of anaerobic metabolism, and these are especially prominent when bacilli are tested after direct harvest from host tissues; there is a general shift away from O2-dependent pathways to anaerobic or facultative anaerobic pathways in host derived bacilli compared to cultured organisms.

This chapter presents a broad picture of activities in a diagnostic mycobacteriology laboratory. It addresses the methodology currently available in most laboratories as well as the techniques that require further standardization and therefore have been implemented in only a few laboratories, mostly in conjunction with research and development. It provides short descriptions of these new methods in order to review their possible application in clinical laboratories. It also gives a condensed description of biosafety practices. In order to present a comprehensive review of all activities that take place in a clinical mycobacteriology laboratory, the chapter focuses on biosafety in the mycobacteriology laboratory, and specimen collection, smear examination, and inoculation of primary culture media. It then describes methods for identification of Mycobacterium tuberculosis. Finally, the chapter talks about drug susceptibility testing for detection of initial and acquired drug resistance.

This chapter describes how the mouse model has evolved over the past century from the simple but beautiful experiments of Koch to present-day models based on sophisticated gene targeting. In the process, the authors describe the course of the infection in the mouse after inoculation by various routes and their growing picture of how the host immune response is mobilized against the infecting organism. They also describe various mouse models that involve immunodeficiency; these may prove useful not only in the further dissection of the cellular immune response but also in applied strategies of chemotherapy and immunotherapy. The growth of Mycobacterium tuberculosis in mice has been extremely well characterized, with the organism giving rise to highly characteristic distribution patterns in target organs after inoculation. A week or so after inoculation of mice with a sublethal intravenous dose of M. tuberculosis, a population of CD4 cells that are capable of adoptively transferring protective immunity emerges in the spleen. In the mouse model of tuberculosis, enriched populations of immune CD8 T cells transferred some degree of resistance, albeit rather weakly, and in vivo depletion of CD8 T cells by intravenous administration of monoclonal antibody was shown to diminish resistance to some extent. The cytokine response to M. tuberculosis probably begins almost immediately after infection of host macrophages, as these cells begin to transcribe message from a number of early response genes.

Recent studies of the comparative biology of the guinea pig have revealed a number of remarkable similarities between Mycobacterium tuberculosis and humans. Guinea pigs have been employed in the testing of both biological reagents and drugs for use in human beings with tuberculosis. Guinea pigs respond quite well to many of the antibiotics currently used to treat tuberculosis patients. The chapter presents guinea pig model of pulmonary tuberculosis, and discusses application of modern immunological techniques in the guinea pig. A model of endogenous reactivation disease would contribute to ones understanding of the factors associated with persistence of M. tuberculosis in the tissues as well as of the events that allow the dormant mycobacteria to reappear in large numbers. The advantages of the guinea pig model of tuberculosis are as follows: (i) animals can be infected reproducibly by the pulmonary route with very small numbers of virulent human tubercle bacilli; (ii) the course of disease following pulmonary infection, which includes bacillemia and hematogenous reseeding of the lung, is similar to that in humans; (iii) the similarities between the granulomatous and hypersensitivity responses of guinea pigs and humans to M. tuberculosis are remarkable; (iv) the degree of protection induced by vaccination with BCG is excellent, and one can discriminate between various degrees of protection; and (v) there is the potential for modeling other clinical forms of tuberculosis in the guinea pig.

The susceptible rabbit families developed hematogenously spread tuberculosis resembling that found in infants and immunocompromised individuals. The resistance to tuberculosis of some of the American rabbit breeds may be more uniform than that of the New Zealand White rabbits on the market today, and among such breeds, new resistant or susceptible strains may be discovered. Two factors influence resistance to the establishment of tuberculosis: (i) the trapping of tubercle bacilli in the lung and (ii) the initial inactivation of these bacilli. The trapping is partly dependent on the ability of the alveolar macrophages to phagocytize the bacilli. The severity of tuberculosis was determined by both the susceptibility of the host and the virulence of the infecting bacilli. Human strains of tubercle bacilli are less virulent for rabbits than the bovine strains. Recovery from infection with them is the rule, even in genetically susceptible rabbits. The use of these strains of bacilli has made possible the development of one of the most precise tests available for native and acquired resistance to tuberculosis. BCG is usually given intradermally, so the response of Lurie’s resistant and susceptible rabbits to this route of infection is described in this chapter. The acquired immunity from BCG vaccination was higher in resistant than susceptible animals. Lurie obtained a hybrid strain of rabbits (F1) by crossing one of his highly resistant strains with one of his highly susceptible strains. The degree of resistance to tuberculosis of this F1 generation was intermediate between that of the two parent strains.

Different animal species vary in their susceptibilities to infection by the different types of virulent tubercle bacilli: Mycobacterium tuberculosis, M. africanum, M. bovis, and M. avium. Liquefication promotes extracellular multiplication of tubercle bacilli to tremendous numbers, and cavity formation allows these bacilli to spread through the air passages to other parts of the lung and to other people. When pasteurization was adopted, there was usually a concomitant decrease in tuberculosis in children. Tubercle bacilli that are inhaled usually lodge in alveolar spaces, where they are ingested by alveolar macrophages. When the organism multiplies within the phagocyte, the host cell may die, resulting in the development of a microscopic tubercle. Wild mammals found to have tuberculous lesions at necropsy after natural death are usually without prior suspicion of tuberculosis. Tuberculous lesions from camelines, cervines, and wild bovines closely resemble those of domestic bovines. In nonhuman primates, M. bovis, M. africanum, and M. tuberculosis can produce extensive disease involving the parenchyma of the lung as well as extrapulmonary tissues. Recently, there has been increased interest in the isolation of M. avium complex serovars 1, 4, and 8 from patients with AIDS and from nonimmunocompromised patients. Some of the same serovars have been isolated from domestic and wild animals. Although M. avium has been isolated from environmental specimens (i.e., soil and water), no definitive information on a common source(s) of these bacteria for animals and humans is available.

This chapter describes the ways that a systematic use of phages led to the development of novel cloning vectors and ultimately transformation of mycobacteria. In addition, it describes how the detailed characterization of one phage, L5, has provided an abundance of novel insights into and genetic tools for mycobacterial molecular genetics. Lastly, it also describes how the combination of a reporter gene with a mycobacteriophage has breathed new life into rapid assessment of drug susceptibilities in clinical samples of Mycobacterium tuberculosis and is also providing a useful tool for screening novel antituberculosis compounds. Mycobacteriophage L5 is the best characterized of the mycobacteriophages. L5 lysogens of M. smegmatis contain a copy of the L5 prophage integrated site specifically into the bacterial chromosome. The introduction of the BACTEC system for clinical analysis of M. tuberculosis has considerably shortened the time needed for drug susceptibility determination. Luciferase reporter mycobacteriophages have the potential of greatly reducing the time needed for M. tuberculosis analysis in a simple and relatively inexpensive assay. The attractions in using luciferase reporter phages for drug screening are many. First, they can provide a readout of results in as little as 2 h. Second, they can be easily adapted to an automated system that uses microtiter plates. The finding of even a single new antituberculosis drug could have an important impact on the control of tuberculosis, and the authors believe that the search for such a drug warrants efforts to develop luciferase reporter antibiotic screening systems for M. tuberculosis.

This chapter reviews what is known concerning plasmids of the genus Mycobacterium. It includes methods for plasmid isolation, analysis of plasmid distribution, and identification of plasmid-encoded genes. It also discusses the background for use of mycobacterial plasmids as vectors. There are several obstacles to isolation and characterization of mycobacterial plasmids. The first is the slow growth rate of most mycobacteria. Second, all mycobacterial cells are difficult to lyse, making efficient isolation of intact circular plasmid DNA difficult. Third, mycobacterial cell walls contain complex lipids and polysaccharides that can contaminate DNA preparations, rendering them unsuitable for molecular biologic techniques. The demonstration of related plasmids in different Mycobacterium avium, M. intracellular, and M. scrofulaceum strains suggests that mycobacterial plasmids are capable of horizontal transfer. The permeability barrier of mycobacteria that results in resistance to antibiotics, intracellular killing by macrophages, and environmental stresses is likely to limit the ability of DNA to traverse the cell wall and membrane. Introduction of kanamycin resistance or other antibiotic resistance genes into various mycobacterial plasmids and the use of electroporation to introduce the plasmids into other strains may allow analysis of plasmid functions.

Recombination by transposition occurs without homology between the sequences of the transposon and the target sequence. A large number of different mobile genetic elements have been discovered in prokaryotes. The most basic is known as insertion sequences (ISs). In the cases of Tn5 and Tn10, only one of the ISs can mediate transposition. The chapter focuses on mycobacterial transposable elements. Work on constructing transposons from ISs for use as genetic tools in the mycobacteria has also been initiated. The analysis of cointegrates isolated from different Mycobacterium smegmatis strains each having one copy of Tn670 inserted has demonstrated that transposition occurs randomly with no specific target sites. Transposable elements from mycobacteria are potentially useful sources of genetic tools for the manipulation of mycobacteria in general and for the investigation of virulence mechanisms in pathogenic strains. Useful insertion elements for the development of mutagenesis systems for mycobacteria should (i) have a high frequency of transposition, (ii) not be present in the bacterial strains in which they will be used for mutagenesis, and (iii) exhibit no site or regional specificity. In mycobacteria, nonreplicative vectors have been used to demonstrate the transposition of IS6100, IS900, and IS6110 in M. smegmatis. Transposons can be used for purposes other than mutagenesis. Genes of interest can be cloned in them and introduced as a stable single copy into the chromosome. Most ISs isolated from mycobacteria are species specific. Primers have been derived from these sequences and used for identification in polymerase chain reaction tests.

This chapter discusses the mechanisms involved in homologous recombination and DNA repair, what is known about these systems in mycobacteria, and recent information on the unusual structure of the recA gene in the pathogenic mycobacteria. The RecA protein of Escherichia coli induces the expression of several genes in response to DNA damage, resulting in an increase in cell survival. The RecA protein is essential for homologous recombination in E. coli. Homologous recombination provides a tool for creating and characterizing specific mutations. "Gene knockout" techniques, for example, involve the replacement of a wild-type gene with a disrupted, nonfunctional mutated gene. Homologous recombination has been reported in the fast-growing mycobacterium Mycobacterium smegmatis. When DNA encoding the intein of Mycobacterium tuberculosis was used to probe genomic DNA of a range of mycobacteria, no hybridization was obtained. The presence of independently acquired inteins in the recA genes of the two major mycobacterial pathogens raised the possibility that they are a common feature of mycobacterial recA genes. Recent studies of the recA genes of mycobacteria have indicated that the major human pathogens M. tuberculosis and M. leprae have an unusual recA structure, suggesting that these organisms may have evolved novel mechanisms for dealing with DNA damage and effecting genetic recombination.

The aims of this chapter are to present the current status of the Mycobacterium tuberculosis genome project, to summarize the main findings of that project, and to discuss its potential impact on future tuberculosis research. The chromosomal inserts carried by the latter clones can be excised by digestion with PacI and are destined for systematic DNA sequence analysis. The rapid mapping of genes is achieved by hybridization of specific probes to cosmid grids, and since the distribution of cosmid clones within a contig is relatively uniform, genes can usually be positioned with a precision of 10 to 20 kb. Sequences are considered finished when both strands have been completely sequenced at an indel error rate of about 1/5,000 and when the sequence has been analyzed for genes and open reading frames (ORFs). Finished sequences are submitted electronically to GenBank and a mycobacterial mapping and sequence database, MycDB, based on the ACEDB software. Analysis of the TBC2 sequence revealed 23 putative genes, including 14 encoding polypeptides with homologies to other known proteins and 9 ORFs. The M. tuberculosis enzyme is highly homologous to Bacillus subtilis PUR3. A putative transport protein, encoded by ant, shows weak homology to the E. coli ArsB proton pump, P-glycoprotein, and other transport proteins.

This chapter reviews the development of genetic systems for the expression of foreign genes in mycobacteria. Mycobacteriophage have been used for many years to type mycobacterial isolates and have more recently been modified as vectors for efficient delivery of foreign DNA into mycobacteria. Plasmid-based expression systems extend the capabilities of phage-based systems by providing increased cloning capacity, increased copy number in the mycobacterial host, and ease of manipulation. Various derivatives of the pAL5000 plasmid replicon have been combined with Escherichia coli plasmid replication origins, such as ColEl and pl5A, by several groups to fashion E. coli-mycobacterium shuttle vectors. Expression of foreign proteins in recombinant organisms is influenced by many factors. Much of the increased interest in mycobacteria is a result of the emergence of multidrug-resistant Mycobacterium tuberculosis. Complex surface macromolecules, such as oligosaccharides and glycolipids, require the products of many genes and the correct cell envelope structure for proper assembly. BCG, the current vaccine against tuberculosis, has provided variable protective efficacy against tuberculosis in different vaccine trials. Immunization of chickens with recombinant Mycobacterium smegmatis expressing an Eimeria acervulina surface antigen elicits partial protection against coccidiosis.

This chapter is concerned with how knowledge of other bacterial pathogens may provide insight into the pathogenesis of tuberculosis. In 1882, Robert Koch’s landmark paper demonstrated that tuberculosis was caused by Mycobacterium tuberculosis. The necessity for defining virulence determinants stringently can readily be illustrated from the authors' studies of M. tuberculosis. They have analyzed the M. tuberculosis H37Rv inserts from clones isolated from different experiments that had the ability to preferentially localize in spleen and found several clones with overlapping DNA fragments. The introduction of linear DNA fragments into both M. tuberculosis and BCG results in their incorporation primarily in nonhomologous sites around the M. tuberculosis chromosome. It has long been appreciated that there are significant homologs of many virulence genes of gram-negative pathogens, even in different genera. Clearly, the demonstration that such homologs are involved in mycobacterial virulence will depend on fulfillment of Koch's molecular postulates, namely, mutation and transfer to avirulent strains.

Mycobacterium tuberculosis was an early object of study by electron microscopy, consonant with its importance as a human pathogen. This chapter describes what is understood of the ultrastructure of M. tuberculosis and points out problems of interpretation and areas where further investigation, probably involving the development of new techniques, is needed. Most bacteria do not possess the elaborate system of internal compartments found in eukaryotic cells, and the information obtained about mycobacteria by electron microscopy is primarily information about the envelope layers, a discussion of which forms the major part of the chapter. It also describes the interaction between bacterium and host cells and some aspects of the ultrastructure of this interaction. Membranes of mycobacteria, including M. tuberculosis, appear in ultrathin sections as classic bilayers, with two electrondense layers separated by a transparent layer. If the hypothesis is that the walls of M. tuberculosis and other mycobacteria form permeability barriers somewhat analogous to the outer membranes of gram-negative bacteria, then the space between the outer leaflet of the membrane and the wall forms a compartment analogous to the periplasmic space of gram-negative bacteria. The commonest type of host cell is the macrophage; inside this cell, the bacteria occur within vacuoles, apparently the phagosomes formed as the bacteria are engulfed by the cells.

This chapter presents a broad structural definition of complex mycobacterial lipids such as mycolyl-arabinogalactan-peptidoglycan complex (mAGP), lipoarabinomannan (LAM), lipomannan (LM), phosphatidyl-myo-inositol mannoside (PIM), sulfatide (SL), trehalose 6,6'-dimycolate (cord factor), other acylated trehaloses, phenolic glycolipid, lipooligosaccharides, and attenuation indicator lipid. It then discusses the possible roles of the lipids found within Mycobacterium tuberculosis. A lipid apparently not present in other actinomycetes was isolated from M. tuberculosis by Noll and was degraded by lithium aluminum hydride to yield two products. The phospholipids found within mycobacterial species are almost invariably phosphodiacylglycerols based on phosphatide acid. Researchers observed that M. tuberculosis grows in the form of serpentine cords and also that avirulent and saprophytic species could be distinguished by an ability to absorb the cationic phenazine dye natural red. The development of thin-layer chromatographic systems has proved invaluable for the assignment of superficial mycolate patterns. Mycolates could be separated by their polar functions, giving rise to multispot thin-layer chromatograms. The most characteristic feature of the mycobacterial cell wall is the chemotype IV peptidoglycan, which is composed of substantial quantities of meso-diaminopimelic acid. A more comprehensive study of the L forms of mycobacteria may provide additional information concerning both the physical characterization and the biological functions of the complex carbohydrates and lipids of M. tuberculosis.

The proteins of the causative agent of tuberculosis, Mycobacterium tuberculosis, have been a major research topic almost since the days of the discovery of the organism by Robert Koch in 1882. The goals of subsequent efforts have been to identify antigens that may be important in conferring protection against tuberculosis. The search for improved diagnostic reagents such as improved skin test reagents and/or serological markers is another aspect of such studies. Obviously, each protein of M. tuberculosis, like proteins of any other organism, serves different functions. As discussed in this chapter, many bacterial proteins are highly conserved, not only within the genus Mycobacterium but also in a broad range of other bacterial species. One example is the group of stress or heat shock proteins that are produced in abundant quantities by M. tuberculosis and that exhibit at least 50% homology at the amino acid level with stress proteins from other bacterial species. A new group of researchers were thereby recruited to the field from other disciplines, and the microorganism was soon approached at both the DNA level and the protein level by the development of monoclonal antibodies (MAbs) raised against M. tuberculosis. Five recombinant antigens have been produced in large enough quantities to be distributed to interested researchers through a WHO-organized "antigen bank". In gram-negative bacteria, the cell wall consists of three layers: the cytoplasmic membrane, the peptidoglycan layer, and an outer lipopolysaccharide-containing cell membrane.

As in other mycobacterial species, cells of Mycobacterium tuberculosis are covered by a lipid-rich cell wall. What is most striking in the cell wall is the presence of a large amount of mycolic acids, most of which are covalently linked to the underlying arabinogalactan, which in turn is covalently linked to the peptidoglycan. There are significant differences in drug susceptibility among mycobacteria, and some mycobacterial species other than M. tuberculosis are more resistant to some of the traditional antimycobacterial agents. By using the Zimmermann-Rosselet procedure, the permeability of M. chelonae cell wall to small nutrient molecules was estimated. It was assumed that the nutrients cross the cell wall by a simple diffusion process and then are taken up by the active transport systems located in the cytoplasmic membrane. The mechanisms of inducer exclusion and inducer expulsion are found in organisms that use phosphotransferase system (PTS) transport and demonstrate an ordered hierarchy of carbon source utilization, usually headed by glucose. It is possible to measure the relative contributions of the proton and electrochemical gradients to the activated membrane state.

Researchers in mycobacterial biochemistry have almost exclusively concentrated their efforts on those aspects of metabolism that appear to be unique to members of the genus Mycobacterium and have, in the absence of information to the contrary, assumed that other aspects of metabolism will be more or less the same as those of other, more amenable bacteria. In this chapter, the authors have chosen to follow the same elective pathway, concentrating on those aspects of metabolism that appear to be at least in some way unique to the mycobacteria and are, moreover, of relevance to the growth of Mycobacterium tuberculosis as a pathogen within the tissues and fluids of its host. The peptidoglycan in mycobacteria is of a type common in many bacteria but with two slight differences. First, there are interpeptide linkages between two diaminopimelate residues as well as the usual D-alanyl-diaminopimelate linkages. Second, the usual N-acetylmuramic acid is replaced with N-glycolyl-muramic acid in M. bovis and in other mycobacteria. An approach that might be appropriate would be to make probes for M. tuberculosis DNA based on appropriate genes identified in muramic acid metabolism from other microbes, as these genes would be expected to have some sequence similarity in all bacteria. There are many interesting and important implications for the new field of mycobacterial genetics: the complex organization of these microbes demands multiple genes for fatty acid biosynthesis and seemingly innumerable glycosyltransferase genes.

This chapter focuses on the immune mechanisms involved in protective immunity against tuberculosis, with the awareness that in most cases the immune response activated during infection with M. tuberculosis may be remarkably powerful yet insufficient. M. Lurie and E. Suter independently found that macrophages from immune animals expressed tuberculostatic activities, whereas those from normal animals permitted unrestricted bacillary multiplication. Although these studies suggested involvement of specific immune mechanisms, the investigators did not contest alternative strategies when they realized that immune serum did not influence tuberculostasis by mononuclear phagocytes (MP). The use of oxygen radical scavengers to probe the significance of reactive oxygen intermediates (ROI) in the antimycobacterial function of macrophages can potentially generate misleading information because of nonspecific effects of peroxynitrite anion, NO, superoxide anion chemicals. More importantly, the role that RNIs play in defense against pathogens has not been established in humans. Mycobactins, a group of iron-chelating growth factors of mycobacteria, have been considered a possible virulence factor of M. tuberculosis. In tuberculosis, the port of entry as well as the major organ of disease is the lung. Due the relationship between M. tuberculosis and host immunity underlying infection is a labile one, any diminution of protective immunity will cause progression into clinical disease.

The immune response to tuberculosis is a double-edged sword that may contribute to both clearance of infection and tissue damage. In addition, recent evidence suggests that tuberculosis promotes progression of disease due to human immunodeficiency virus (HIV). Most persons who become infected with Mycobacterium tuberculosis mount a protective immune response and remain clinically well, the only evidence of infection being development of a positive tuberculin skin test. Disseminated tuberculosis reflects an ineffective immune response, manifested by a high frequency of negative tuberculin skin tests and failure of T lymphocytes to proliferate in vitro in response to M. tuberculosis antigens. Tumor necrosis factor (TNF) synergizes with gamma interferon to increase production of nitric oxide metabolites and mycobacterial killing and is essential for granuloma formation to contain mycobacterial infection. Published data on cytokine production by human T cells in response to M. tuberculosis are conflicting. In patients with tuberculous pleuritis, expression of mRNA for the Th1 cytokines gamma interferon and IL-2 is greater in pleural fluid mononuclear cells than in blood mononuclear cells, and concentrations of gamma interferon are 15-fold higher than those in serum. More comprehensive studies are needed to assess the role of CD8+ cells in human antituberculosis defenses. One strategy for developing antituberculosis vaccines is to characterize the T cells and cytokines that mediate clearance of bacilli during the primary immune response as well as the memory T-cell subpopulations and cytokines that protect against tuberculosis from exogenous reinfection and endogenous reactivation.

This chapter talks about the progress in the study of immune reactions mainly to antigens of Mycobacterium tuberculosis that have been firmly identified and the largely characterized protein structure. An important breakthrough in the characterization of antigens was achieved initially by the production of monoclonal antibodies to mycobacterial antigens and particularly by the development of recombinant DNA systems for efficient expression of mycobacterial genes in Escherichia coli. Heat shock proteins (hsp) have highly conserved amino acid sequences. Their functions as molecular chaperones in the assembly and disassembly of proteins into oligomers during transport through the cell or as proteases that degrade misfolded or foreign proteins are vital for each cell. The significance of lipoproteins for the immune response to mycobacteria has been in focus in view of reports that acylation of proteins enhances their ability to induce delayed-type hypersensitivity (DTH) and that synthetic lipopeptides have the ability to prime cytotoxic T cells. Proteins secreted from viable bacteria could be available for immune recognition at an early stage of infection and may therefore play a special role in protective immune mechanisms. Antibody levels following immunization with soluble extracts of tubercle bacilli in adjuvant demonstrated the codominant control by the H-2 IA locus of the response to individual epitopes. Analysis of the specificity of T-cell responses to the mycobacterial 65-kDa heat shock protein has so far attracted more attention in relation to its potential role in autoimmunity than in respect to tuberculosis.

Once the first small caseous tuberculous lesion is established, all subsequent battles occur in a host capable of both tissue damaging and macrophage-activating immune responses. Both tissue-damaging and macrophage-activating immune responses can stop the growth (i.e., multiplication) of tubercle bacilli, because these bacilli do not multiply appreciably in nonliquefied caseous necrotic tissue. Various inflammatory mediators, e.g., clotting factors, eicosanoids, cytokines, hydrolytic enzymes, and reactive oxygen and nitrogen intermediates, are frequently involved in one or both processes. However, the principles described in this chapter are most easily understood if the reader accepts our simplified definitions, namely, that the tissue-damaging hypersensitivity process kills nonactivated macrophages that have allowed tubercle bacilli to multiply within them and that the cell-mediated immunity (CMI) process makes the microbicidal power of macrophages strong enough to kill or inhibit mycobacteria. The macrophage-activating immune response could not be responsible, because the susceptible host develops only weak cell-mediated immunity, and at this stage of the disease, the CMI of the resistant host is not yet fully developed. The majority of pulmonary tuberculous infections of human beings are arrested before they cause clinical disease. Activated macrophages can kill the tubercle bacilli they ingest. Under normal conditions, i.e., before infection begins, most of the alveolar macrophages are nonspecifically activated. Therapeutic agents to prevent liquefaction or to prevent its continuation are greatly needed to treat tuberculosis and limit the spread of this disease to other people.

In the 1890s, Robert Koch observed that a primary infection of guinea pigs with Mycobacterium tuberculosis in the skin produced a nonhealing lesion and that reinoculation of the animals after several weeks produced only a firm, red nodule that necrosed and finally healed. These observations first suggested the existence of immunity to tuberculosis infection. While T-cell-derived lymphokines and the activated macrophage represent a necessary condition for protection, it is certainly not the whole story even in the mouse. A large proportion of human peripheral blood γδ T cells, even from PPD-negative donors, will proliferate in response to mycobacteria. Recent experiments involving M. tuberculosis infection of transgenic mice are providing new and interesting information on the role of some lymphokines and cytokines. In many chronic infections or inflammatory diseases, there is a permanent or transient switch from a Th1 pattern of response to Th2. Such a switch appears to occur, for example, in schistosomiasis and syphilis patients. The tissue-damaging responses are evoked by tuberculin, which is a very crude culture supernatant from old autolysing bacterial cultures precipitated by trichloroacetic acid or ammonium sulfate. Such supernatants contain fragments of essentially all the antigens of M. tuberculosis. The known capacity of mycobacterium containing adjuvants (Freund's complete adjuvant) to facilitate experimental induction of autoimmunity and evidence that mycobacterial disease can be accompanied by a sterile arthropathy have reawakened speculation that some autoimmune syndromes may be cryptic infections or may be triggered by past encounters with mycobacterium-like organisms.

Tuberculosis among human immunodeficiency virus (HIV)-infected people has become an epidemic within an epidemic. Autopsy of HIV-positive tuberculosis patients frequently reveals old fibrous or calcified lesions of tuberculosis in the thorax that are adjacent to recent active lesions with bacilli. In 87% of cadavers, tuberculosis was disseminated to more than one organ, nearly always involving the lungs, liver, spleen, multiple internal lymph nodes, and bone marrow. The histologic patterns of tuberculosis reflect the integrity of the cellular immune response of the patient. Recent studies of HIV-infected individuals show that Th1 cells are progressively lost, shifting the ratio to a Th2-dominant population. Understanding the timing of Th1 function loss is key to understanding the altered host response in tuberculosis and to developing strategies for prophylaxis. Due to the high risk of developing tuberculosis by reactivation or reinfection, trials in Africa and elsewhere are under way to assess the value of antituberculosis prophylaxis in preventing or deferring the development of tuberculosis. The majority were diagnosed as having primary pulmonary or extrapulmonary tuberculosis. The radiographic appearances of advanced pulmonary tuberculosis with or without cavitation are well recognized. In our experience, HIV-associated miliary pulmonary tuberculosis is readily missed as a clinical diagnosis because the lesions are often too small to be visualized and sputum negativity is common.

This chapter reviews the progress in developing new tools and procedures for the rapid and reliable diagnosis of tuberculosis. A definitive diagnosis of tuberculosis requires the identification of Mycobacterium tuberculosis bacilli in patient specimens. A definitive diagnosis of tuberculosis requires the identification of M. tuberculosis bacilli in patient specimens. In general, the commercially available assays display sensitivities and specificities approaching 100% for the detection and identification of these species and can usually be completed in a few hours. One easily detected component of M. tuberculosis is tuberculostearic acid, which can be detected in femtomole quantities by gas-liquid chromatography. Several diagnositic procedures have been described for use with M. tuberculosis and include strand displacement amplification (SDA), polymerase chain reaction (PCR) amplification, transcription-mediated amplification (TMA), reporter phage systems, oligonucleotide ligation amplification, and Q-beta replicase amplification. If one uses a physician’s diagnosis of tuberculosis as the gold standard, then the MTD test displayed 95% sensitivity and 96% specificity for correctly identifying persons as having tuberculosis. Given the importance of drug-resistant tuberculosis, it would be advantageous to determine drug susceptibilities directly from clinical specimens and avoid the time required for culture. The luciferase reporter technology has been used to develop simple and rapid screening procedures to identify new antituberculosis agents.

Use of BCG (bacille Calmette-Guérin) vaccines increased in Europe after World War II and in developing countries starting in the 1950s to the extent that the vaccines have now been given to over 3 billion people. In a major evaluation of BCG's safety, researchers attempted to catalog all adverse events reported as attributed to BCG vaccination. Two conclusions are notable. First, the reports of severe neurologic or fatal sequelae indicate that the risk of such events is extremely rare, far less than that reported for the smallpox vaccine. Second, BCG vaccines are associated with a variety of more common minor adverse effects in addition to induration and ulceration of the vaccination site, which occur in almost all vaccines. The authors believe that such studies may identify useful surrogate markers for the evaluation of different preparations of BCG or new vaccines against tuberculosis. Although BCG is effective in developing type I CD4 T-cell responses, it is still unclear how effective it is in generating MHC class I-restricted CTL responses. It may ultimately be necessary to identify the molecules, either enzymes or lysins, that enable M. tuberculosis to escape from phagolysosomes into the cytoplasm in order to develop recombinant BCG vaccines that are effective in inducing both CD8- and CD4-type protective responses. Just as the BCG trials have provided our most detailed studies of the epidemiology of tuberculosis, so basic research directed at tuberculosis vaccines is leading the way in understanding pathogenesis of the disease.

This chapter focuses on the prospects for using a fundamental molecular approach to identification of novel lead compounds for new drug development. A section reviews existing and potential drug targets in Mycobacterium tuberculosis. The chapter then discusses distinctive features of mycobacteria relevant to drug design, and considers experimental approaches applicable to rational drug discovery programs. Most antibacterial agents inhibit biosynthetic pathways involved in the production of macromolecules. Streptomycin, the first antibiotic available for widespread use in treatment of tuberculosis, is a member of the aminoglycoside family that disrupts bacterial protein synthesis. An important strategy for enhancing the activity of sulfonamides against some bacteria has been their use in combination with trimethoprim, a drug that inhibits a subsequent step in the tetrahydrofolate pathway catalyzed by the enzyme dihydrofolate reductase. Rifampin is a key drug in mycobacterial therapy that has a broad antibacterial spectrum and a well-defined target. Ethambutol has a polyamine-like structure and was originally thought to interfere with RNA synthesis. M. tuberculosis isolates with defects in the katG gene encoding a catalase-peroxidase enzyme develop resistance to isoniazid (INH), indicating a possible role for the enzyme in intracellular activation of the drug. The recent development of molecular genetic systems for mycobacteria opens a range of novel opportunities for drug discovery.

Molecular epidemiology is the integration of molecular techniques to track specific strains of pathogens with conventional epidemiologic approaches to understanding the distribution of disease in populations. This chapter describes the genetic elements of Mycobacterium tuberculosis that may be exploited as strain-specific markers, the strain-typing methods that are based on these elements, and some of the DNA fingerprinting results obtained to date and speculates on future directions in this field. M. tuberculosis complex bacteria constitute a remarkably homogeneous group, as revealed by the inability of multilocus enzyme electrophoresis to differentiate individual strains and the minimal DNA polymorphism in restriction fragments of randomly chosen chromosomal DNA fragments. Most investigators use the technique of Southern blotting to exploit the presence of the above-described genetic elements to reveal restriction fragment length polymorphisms (RFLP) among M. tuberculosis strains. The application of molecular techniques in this setting suggests that tuberculosis control efforts should be focused on this population to prevent the emergence and spread of multidrug-resistant (MDR) tuberculosis in Europe. Differences in the genetic stabilities of such genetic elements can be used to exploit these elements as molecular clocks in the evolution of divergent strains. A major future challenge of DNA fingerprinting of M. tuberculosis will be to determine the paces of change of the various elements and to match these to study specific epidemiologic questions.

This chapter is organized into four sections: the burden of tuberculosis, BCG immunization, chemoprophylaxis, and case identification and treatment. In each section, the emphasis is on briefly reviewing relevant past work, identifying key constraints for effective tuberculosis control, and suggesting ways that operational research may provide answers. Of the four topics, the most important is case identification and cure. A reasonable estimate of prevalence can be derived by assuming that all purified protein derivative (PPD) responses with an induration size greater than the mode indicate true Mycobacterium tuberculosis infections. To demonstrate the burden of tuberculosis in terms comparable to those used for other health problems, it was essential to estimate the number of cases and deaths due to tuberculosis in various regions of the world. The success of BCG vaccination programs is largely attributable to the organizational, financial, and technical success of the Expanded Program of Immunization. Treatment of individuals who are known to be infected with M. tuberculosis on the basis of 12 months of skin tests with isoniazid is effective in preventing subsequent breakdown to clinical tuberculosis. One of the fundamental tenets of tuberculosis control is the primary importance of diagnosing and treating pulmonary sputum smear-positive tuberculosis.