Tuberculosis

Companies: TB Alliance

Introduction/Definitions:

The genus Mycobacterium consists of about 100 species, including pathogens and saprophytes, and may be divided into three groups on the basis of clinical significance. The first group includes obligate pathogens in humans and animals, i.e., the Mycobacterium tuberculosis complex (M. africanum, M. bovis, M. canettii, M. caprae, M. microti, M. pinnipedii, and M. tuberculosis), M. leprae, and M. lepraemurium, which are generally not found in the environment. The second group comprises mycobacteria that are potentially pathogenic to humans or animals. The majority of these species have been isolated from various terrestrial and aquatic environments and may cause disease under certain circumstances, e.g. skin lesions, pulmonary or immune dysfunctions and chronic diseases. Examples are M. avium and other members of the so-called ÔM. avium complexÕ (MAC). The third group consists of saprophytic species that are non-pathogenic or only exceptionally pathogenic.

Mummies from the Stone Age, ancient Egypt and Peru provide evidence that tuberculois (TB) is an ancient human disease. Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb) infection, was among the top 10 causes of death worldwide in 2017 with about 1.5 million registered deceases. One to five bacilli may suffice to transmit the infection by air.

Tuberculosis (TB), caused by members of the Mycobacterium tuberculosis complex, is one of the most common human infectious diseases, causing three million deaths a year world-wide. While the disease is associated with impoverished economic conditions, TB is on the rise in many industrialized nations. The spread in TB is due to immigration, the emergence of drug resistant strains, and the AIDS epidemic.

Exposure to M tuberculosis infrequently leads to symptomatic disease. Thus, although the statistic that one-third of the world’s population is infected with M tuberculosis sounds alarming, only about 12% of these immune-sensitised individuals actually develop disease. Disease development is a function of the host’s immunocompetence; individuals with HIV, for example, are at increased risk of progression to active disease. However, disease development is also a reflection of the evolutionary strategy of M tuberculosis as a pathogen, which during human existence has needed to ensure transmission to the next host. M tuberculosis has to undertake a delicate balancing act: cause enough disease to ensure transmission but not so much that patients rapidly die, taking the pathogen’s progeny with them. The solution to this equation in modern, mostly urban high-density human populations might be very different than in historic low-density hunter gatherer human populations, which might be reflected in newly emerging strain clades. See Dheda 

Primary Tuberculosis: 

The minimum infectious dose for lung infection is low, around 10 bacterial cells. Alveolar macrophages phagocytose these cells, but they are not killed and continue to multiply inside the macrophages. This period of hidden infection is asymptomatic or is accompanied by mild fever. Some bacteria escape from teh lungs into the blood and lymphatics. After 3-4 weeks, the immune system mounts a complex, cell mediated response. The large influx of mononuclear cells into the lungs plays a part in the formation of sepcific infection sites called tubercles.

Latent/Secondary (Reactivation) Tuberculosis:

Although the majority of adequately treated TB patietns recover more or less completely form the primary episode of infection, live bacteria can remain dormant and become reactivated weeks, months or years later, especially in people iwth weakened immunity.

LTBI is the presence of M. tuberculosis organisms (tubercle bacteriologic evidence of TB disease. Therefore, persons with LTBI do not experience clinical illness; they are asymptomatic, and their infection is not transmissible. The only evidence of infection might be a reaction to a TST or a positive IGRA test. In persons with LTBI, TB infection can persist for decades, and those with M. tuberculosis infection can remain at risk for progressing to TB disease, especially if the immune system becomes impaired. An estimated 13 million persons have LTBI in the United States.

Since the early 1980s, TB prevention and control in the United States has expanded with treatment of persons with LTBI to prevent TB disease. Approximately 80% of the active TB disease cases in the United States are believed to be caused by reactivation of LTBI (34,35). As TB disease rates in the United States decrease, finding and treating persons at high risk for LTBI has become a higher priority and a cornerstone strategy for TB elimination. LTBI treatment is important because it can substantially reduce the risk that persons infected with M. tuberculosis will progress to TB disease. However, LTBI treatment can be associated with adverse events; therefore, the goal of preventive therapy is to treat those for whom prophylaxis for LTBI carries substantially more benefit than potential harm. See MMWR

Agent of the disease:

Mycobacterium tuberculosis is the main TB causing micro-organisms in human. The mycobacteria include species in the genus,

The zoonotic disease bovin TB is caused by a closely related M. bovis. It can pose a significant threat to human health and can be responsible for up to 10% of human TB cases. Thus, both human and bovine TB should be targeted for efficient control strategy.  CDC regulates nonhuman primate importation and quarantine under the Public Health Service Act (42 US Code 264). All NHP entering the US must be imported by CDC registered facilites and are required to undergo quarantine and TB ttesting under 42 CFR 71.53.  Importers are required to submit samples from any NHP that dies or is euthranized druing quarantine and is suspected to have tB for confirmatory culture. nonhman primate (NHP) iomportation and quarantine under the Public Health Service Act (42 US Code 264). ALL NHP entering the US must be imported by CDC registered facilities and are requried to undergo quarantine and tuberculosis testing under 42 Code of Federal Regulations Seciton 71.53. Importted NHP must have at least three negative TB skin tests. (Swisher “Outbreak of Mycobacterium oregulates CDC rynomolgus Macagues imported form Southeast Asia -United States, February – May 023).

Structure: These small, non-motile, rod-shaped bacilli are obligate aerobes which grow most successfully in tissues with a high oxygen content, such as the lungs. They are distinguished by a complex, lipid rich cell envelope possessing large amounts of mycolic acid. The presence of mycolic acid makes the bacteria very difficult to stain, which is responsible for their characterization acid-fast bacilli. Recent drug penetration studies have suggested that the cell wall contributes to phenotypic drug resistance observed in nonreplicating M. tuberculosis

Prevention/Transmission:

Transmission: M. tuberculosis is transmitted usually by airborne droplets which must penetrate deep into the respiratory tree. Predisposing factors which can influence the onset of clinical disease include HIV infection , diabetes, smoking, alcoholism, malnutrition, and overcrowded living conditions. Clinical tuberuculosis invariably starts with the pulmonary form of the disease; however, the pathogen can subsequently disseminate via the circulatory or lymphatic systems and multiply in extrapulmonary host sites such as the skin, lymph nodes, central nervous system, genitourinary tract, and skeleton.

Diagnosis:

Commercially available confirmatory tests include various nucleic acid amplification tests (NAATs), some of which include testing for drug resistance. Sputum samples can be directly assessed with the WHO-endorsed Gene Xpert MTB/RIF60 and Hain MTBDRplus assays. The Xpert assay works well for patients with suspected pulmonary tuberculosis and for specific forms of extrapulmonary tuberculosis (meningitis in people with HIV63 and lymphadenitis, but not pleural, pericardial, or abdominal tuberculosis). Several next-generation NAAT technologies are in advanced stages of development, including Xpert cartridges with alternative technology able to detect second-line drug resistance. Genotypic drug- resistance tests, including to second-line drugs, might be undertaken with the Hain MTBDRs/ assay (with isolates or smear-positive sputum). See Dheda 

Mantoux tuberculosis skin test (TEST): This test involves injecting a small amoutn of TB protein derivative into a pateint’s forearm and then observing the injection site 48-72 hours after the injection. A positive TEST test indicates that a patient has been infected with TB. A sptum culture — collecting and culturing phelgm from the upper resperatory tract — is used to determine whether an infected patient actually has TB as distinguished from a latent infection. An ID injection of tuberculin, purified protein derivative (PPD) of the cell wall stimulates pre-primed CD4-helper cells at the injection site that secrete  and lead to a positive result. PPD skin conversion occurs about 4 weeks after exposure. A few people with active TB convert to negative (anergy) through an unknown mechanism. Immunization results in positivity.

ESAT-6 protines: Oxford Immunotec Ltd has developed a TB test using a unique protein called ESAT-6 that is produced by M. tuberculosis. The test uses specified concentrations of eight peptides that are components of ESAT0-6 which are contacted with a population of T cells form the hoest in vitro and detecting an IFN-gamma secretion from the T cells.

rpoB gene:Scientists from Roche adn the Mayo Foundation for Medical Education and Research sequenced the rpoB gene from MTB and discovered that the gene contains eleven position specific signature nucleotidesthat are only present in MTB but not in other bacteria. These signature nucleotides can thus be used to identify MTB. See US Patent No: 5,643,723

QFT-Plus is a major scientific advance over the 100-year-old TB skin test (sometimes called Mantoux, tuberculin skin test, TST or PPD). QFT-Plus uses four unique blood collection tubes that enable immediate exposure of viable blood lymphocytes (immune cells) to highly specific TB antigens and test controls coated on the inner surface of the tubes. Exposure to these TB antigens causes lymphocytes (specifically CD4 and CD8 T cells) to produce a quantifiable small molecule called Interferon-γ (IFN-γ). Interferon-gamma production is correlated to the presence or absence of TB infection, and this IFN-γ response is measured in a laboratory to aid in the diagnosis of TB infection. see Qiagen

Interferon-Gamma Release Assays (IGRAs): Quest sells an IGRA whole-blood test that detects the immuen system’s response to M. tuberculosis. M. tuberculosis or TB bacterail usually attack the lungs. However, TB bacteria can attack any par to fthe boyd such as the spine, kidneys and brain. In fact, no everyone that becomes infected with TB will become sick. As a result, two TB related conditions exist: latent TB infection (LTBI) and TB disease. This test does not differential between the two. LTBI is a carrier state of TB that can last for weeks or years before developing into TB disease. Active TB is when TB overwhilm a person’s immune syste and symptoms start to appear. This test can be a first step in determing if one has latent TB or TB disease. Further tesitng may be required if the blood test is positive.

Pathology:

Inhibition of phogolysosome fusion: Following engulfment by alveolar macrophages, M. tuberculosis replicate freely in the cell because they evade phagocytic destruction by inhibiting phagolysosome fusion. In a healthy adult exposed to low numbers of bacteria, the TH1 response and collections of activated macrophages (called granulomas) appear early enough to stop infection. But viable bacteria may remain with potential for future reactivation.

To ensure survival in the macrophage, M. tuberculosis blocks fusion between the early phagosome and lysosome of the host macrophage. The underlying mechanism of action is complex, and at least three essential mycobacterial protein kinases—PknG, SapM, and PtpA—are involved that directly interact with host proteins. See Kaufmann

Granuloma formation: is the hallmark of TB infection. Granulomas are formed by activated macrophages and other host components that surround infected lung tissue, isolating the infected cells in an organized structure and creating an environment that suppresses MTB replication. Granulomas are thought to limit bacterial growth in a variety of ways including oxygen and nutrient deprivation, acidic pH, and production of host factors such as nitric oxide. Of these, hypoxia is the best-studied, with much work focused on in vitro models of hypoxia-induced dormancy. Tuberculosis bacilli exposed to hypoxia in vitro cease replicating but can remain viable and virulent for years. However, even in this non-replicating hypoxic condition, M. tuberculosis metabolizes exogenous and endogenous energy sources for maintenance functions.

A nonreplicating cell state is induced in response to human hypoxic stress, such as controlled, low oxygen tension in rich medium culture. Low oxygen tension is sensed by the two-component system DosR/DosS triggering transcriptional synthesis of the DosR regulon genes and synthesis of proteins such as HspX.

Metabolism:

Importance of oxidases: The bioenergetic mechanisms by which Mycobacterium tuberculosis survives hypoxia are poorly understood. Current models assume that the bacterium shifts to an alternate electron acceptor or fermentation to maintain membrane potential and ATP synthesis. Counterintuitively, one study finds that oxygen itself is the principal terminal electron acceptor during hypoxic dormancy. M. tuberculosis can metabolize oxygen efficiently at least two orders of magnitude below the concentration predicted to occur in hypoxic lung granulomas. Despite a difference in apparent affinity for oxygen, both the cytochrome bcc:aa3 and cytochrome bd oxidase (tranwfers H to oxygen) respiratory branches are required for hypoxic respiration. Simultaneous inhibition of both oxidases blocks oxygen consumption, reduces ATP levels, and kills M. tuberculosis under hypoxia. The capacity of mycobacteria to scavenge trace levels of oxygen, coupled with the absence of complex regulatory mechanisms to achieve hierarchal control of the terminal oxidases, may be a key determinant of long-term M. tuberculosis survival in hypoxic lung granulomas. See Pethe

Lsr2 protein: M. tuberculosis causes nearly two million deaths per year and infects nearly one-third of the world population. The success of this aerobic pathogen is due in part to its ability to successfully adapt to constantly changing oxygen availability throughout the infectious cycle, from the high oxygen tension during aerosol transmission to anaerobiosis within necrotic lesions. An understanding of how M. tuberculosis copes with these changes in oxygen tension is critical for its eventual eradication. Using a mutation in lsr2, it has been demonstrated demonstrated that the Lsr2 protein present in all mycobacteria is a global transcriptional regulator in control of genes required for adaptation to changes in oxygen levels. M. tuberculosis lacking lsr2 was unable to adapt to both high and very low levels of oxygen and was defective in long-term anaerobic survival. Lsr2 was also required for disease pathology and for chronic infection in a mouse model of TB. The Lsr2 protein in mycobacterial species is similar to H-NS from Escherichia coli. H-NS works mainly to repress gene transcription by binding to AT-rich sequences in a sequenceindependent fashion and has been implicated in virulence in Shigella flexneri and Vibrio cholerae. H-NS is able to activate gene expression as well, but examples of activation are less common. Lsr2 from M. tuberculosis and from Mycobacterium smegmatis has been shown in vitro to bind directly to DNA and protect it against H2O2. An M. smegmatis lsr2 deletion mutant was also transiently more susceptible to H2O2 stress. A separate study found that M. tuberculosis Lsr2 protein is not important for protection against DNA damage. The Lsr2 protein is, however, a global transcriptional regulator important for allowing M. tuberculosis to respond to changes in oxygen levels that would be experienced during both transmission and infection. See Voskull

Immune Reaction:

Innate Immunity: Initially innate immunity may abort the infection through the activities of alveolar macrophages and other cells which are recruited, such as neutrophils and natural killer (NK) cells. When innate immunity fails, the bacteria multiply intracellularly and adaptive immunity determines the formation of the tuberculous granuloma. The arrival of macrophages and lymphocytes controls the bacterial proliferation, although some bacilli will survive in a latent form. Early in the primary infection of a ‘naive’ host, bacteria are transported to regional lymph nodes, causing an intense reaction. The granulomatous reaction and necrosis in the lymph nodes are known as the Ranke complex, characteristic of tuberculosis in childhood.

When inhaled, Mtb encounters a first line of defense consisting of airway epithelial cells (AECs) and “professional” phagocytes (neutrophils, monocytes and dendritic cells). If this first line succeeds in eliminating the Mtb rapidly, the infection aborts. Otherwise, phagocytes are infected and the Mtb reproduces inside the cells, initially causing few, if any, clinical manifestations. The establishment of the infection, the development of active TB (ATB) rather than latent TB infection (LTBI) and the eventual evolution of LTBI to ATB depends on the complex relation between bacterial and host factors. AECs are the first cells to come in contact with Mtb. Beyond their major role as physical barriers, they display several immunological functions albeit being traditionally considered as “non-professional” immune cells. Through pattern recognition receptors (PRRs), AECs can perceive the presence of Mtb and consequently modulate the composition of the airways surface liquid improving its antimicrobial capacity.

Recent advances in multicolor flow cytometry have revealed that pulmonary myeloid effector cells are vastly heterogeneous, with lung resident pulmonary DCs and macrophage subsets in addition to a plethora of effector populations, such as neutrophils, IMs, inflammatory monocyte-derived dendritic cells (mDCs), plasmacytoid DCs and cDCs being recruited to the site of infection.

–Alveolar macrophages: Infection with Mtb occurs via the aerosol route, and consequently, lung resident myeloid cells are the primary cells initiating “first contact” with the bacilli. Alveolar macrophages (AMs) are long-lived, specialized innate immune cells that reside in pulmonary alveoli and ingest the inhaled bacteria, and therefore, AMs are critical in setting the stage for the subsequent immune response against Mtb. Phagocytosis of Mtb is facilitated by binding to complement receptors, mannose receptor (MR), surfactant molecules, and DC-SIGN (dendritic cell-specific intracellular adhesion molecule-3–grabbing nonintegrin). In addition, AMs express a large array of pattern recognition receptors (PRR), including Toll-like receptors (TLRs), C-type lectin receptors (CLRs), and Nod-like receptors (NLRs), all of which have been shown to participate in Mtb recognition. Among the TLRs, TLR-2, -4, and -9 are of particular importance in sensing Mtb, with NOD2, Mincle, Dectin-1, MR, and DC-SIGN contributing to PRR-driven macrophage activation. See Barber

Once engulfed by the macrophage, Mtb potently inhibits macrophage activation and becomes highly resistant to clearance. Virulent Mtb manipulates the response of infected cells to avoid detection and elimination through a variety of immune evasion strategies, including inhibition of phago-lysosome fusion and detoxification of nitrogen and oxygen radicals and dormancy.  See Barber

Adaptive Immune Response:

The initiation of the CD4 T-cell response to Mtb is notoriously slow, as CD4 T cells first arrive in the lungs of infected mice several weeks after exposure. This lag time between the establishment of infection and the arrival of T cells at the site of infection likely contributes to the inability of the host to clear the organism by allowing the bacteria to increase significantly in number before adaptive immune cells can mediate their protective effects.

One likely contributor to delayed T-cell responses during M. tuberculosis infection is the early induction of pathogen-specific, Foxp3⁺ regulatory T (Treg) cells. Treg cells proliferate and accumulate at sites of infection in both murine and human Tb. Inflammation associated with M. tuberculosis infection, and IL-2 produced by effector T cells, are both insufficient to drive the non-specific expansion of Treg cells, but Treg cells recognizing M. tuberculosis antigens expand preferentially. See Ernst

Symptoms:

Symptoms of the disease include fever, coughing, bloody sputum.

Prevention/Vaccination: 

BCG Vaccine: There is only one licensed vaccine M. bovis Bacillus Calmette-Guerin (BCG) with variable efficacy (WO 2005/023867). It has been distributed since the 1920 and more than 3 billion people have received the vaccine. BCG vaccination, however, remains a matter of debate due to safety aspects, loss of sensitivity to tuberculin as a diagnostic reagent, and varying efficacy (form 0 to 85%) in different BCG vaccine trials.

The only TB vaccine currently in use is the live attenuated M. bovis strain, BCG. It protects against severe forms of childhood TB but not against pulmonary TB at all stages of life. Thus, BCG has outlived its usefulness, and new vaccines are needed for efficient TB control

TB subunit vaccines have been developed, mainly based on M. tuberculosis secreted components such as early secretory antigenic target, ESAT-6 and the antigen 85B (Ag85B). Both antigens have an impressive track record of studies. ESAT-6 and Ag85B are common to both M. tuberculosis and M. bovis.

(Floss, J. Biomedicine and Biotechnology, 2010, article ID 27434) discloses a fusion between elastin-like peptide (ELP) and Ag85B and ESAT-6 which are produced in plants. Mice and piglets immunized with the TBAg-ELP fusion exhibited exhibited a mycobacterial specific immune response with no side effects. The ain reactivity of the TBAg-ELP induced antibodies against Ag85B.

(Olsen, Infect Immun, 69(5), 2773-8, 2001) discloses a TB subunit vaccine based on fusion proteins of ESAT-6 and antigen 85. The fusion proteins were adminsitered to mice in the adjuvant combination dimethyl dioctadecylammonium bromide-monophosphoryl lipid A such that a strong dose dependent immune response was induced to both single components as well as to the fusion proteins.

Treatment:

Treatment requires at least 2 agents like streptomycin and and is more than 90% successful even in AIDS patients. Almost 10% of new M. tuberculosis patients in the US show resistance to at least one of the first line antituberculosis drugs (isoniazid (INH), pyrazinamide (PZA), rifampin (RIF), ethambutol (EMB) and streptomycin (STR) with about 2-3% of cases resistant to both INH and RIF. The term multi-drug-resistant tuberculosis (MDR-TB) is sometimes used to refer to tuberculosis which is resistant to at least isoniazid and rifampicin, the two most common anti-TB drugs.

The evidence base for the recommended regimen for drug-sensitive tuberculosis (isoniazid and rifampicin for 6 months, together with pyrazinamide and ethambutol for the first 2 months) was established four decades ago, but the regimen is highly effective.

Rifampicin is a rifamycin derivative introduced in 1972 as an antituberculosis agent. It is one of the most effective anti-TB antibiotics and together with isoniazid constitutes the basis of the multidrug treatment regimen for TB. Rifampicin is active against growing and non-growing (slow metabolizing) bacilli. The mode of action of rifampicin in M. tuberculosis is by binding to the β-subunit of the RNA polymerase, inhibiting the elongation of messenger RNA. The majority of rifampicin-resistant clinical isolates of M. tuberculosis harbor mutations in the rpoB gene that codes for the β-subunit of the RNA polymerase. As a result of this, conformational changes occur that decrease the affinity for the drug and results in the development of resistance.  See martin

Fluoroquinolones are currently in use as second-line drugs in the treatment of MDR-TB. Both ciprofloxacin and ofloxacin are synthetic derivatives of the parent compound nalidixic acid, discovered as a by-product of the antimalarial chloroquine. The mode of action of fluoroquinolones is by inhibiting the topoisomerase II (DNA gyrase) and topoisomerase IV, two critical enzymes for bacterial viability. These proteins are encoded by the genes gyrA, gyrB, parC and parE, respectively.

The available drugs against TB all target metabolically active and replicating M. tuberculosis organisms. Hence, dormant M. tuberculosis with a highly reduced metabolic and replicative activity is phenotypically resistant against these drugs. It is generally accepted that novel drugs should target not only active M. tuberculosis, but also dormant M. tuberculosis. See Kaufmann

Isoniazid: was introduced in 1952 as an anti-TB agent and it remains, together with rifampicin, as the basis for the treatment of the disease. Unlike rifampicin, isoniazid is only active against metabolically-active replicating bacilli. Also known as isonicotinic acid hydrazide, isoniazid is a pro-drug that requires activation by the catalase/peroxidase enzyme KatG, encoded by the katG gene, to exert its effect. Isoniazid acts by inhibiting the synthesis of mycolic acids through the NADH-dependent enoyl-acyl carrier protein (ACP)-reductase, encoded by inhA

Ethambutol was first introduced in the treatment of TB in 1966 and is part of the current first-line regimen to treat the disease. Ethambutol is bacteriostatic against multiplying bacilli interfering with the biosynthesis of arabinogalactan in the cell wall.

Pyrazinamide was introduced into TB treatment in the early 1950s and constitutes now part of the standard first-line regimen to treat the disease. Pyrazinamide is an analog of nicotinamide and its introduction allowed reducing the length of treatment to six months. It has the characteristic of inhibiting semi-dormant bacilli residing in acidic environments such as found in the TB lesions. Pyrazinamide is also a pro-drug that needs to be converted to its active form, pyrazinoic acid, by the enzyme pyrazinamidase/nicotinamidase coded by the pncA gene. The proposed mechanism of action of pyrazinamide involves conversion of pyrazinamide to pyrazinoic acid, which disrupts the bacterial membrane energetics inhibiting membrane transport. See Martin

Streptomycin: Originally isolated from the soil microorganism Streptomyces griseus, streptomycin was the first antibiotic to be successfully used against TB. Unfortunately, as soon as it was prescribed, resistance to it emerged, a result of being administered as monotherapy. Streptomycin is an aminocyclitol glycoside active against actively growing bacilli and its mode of action is by inhibiting the initiation of the translation in the protein synthesis. More specifically, streptomycin acts at the level of the 30S subunit of the ribosome at the ribosomal protein S12 and the 16S rRNA coded by the genes rpsL and rrs, respectively.

IVIG: Jolles (WO2005/023867) discloses treating subjects with IVIG following infection with M. tuberculosis have significantly lower colony counts in the lungs and spleen.

Research Models:

The two key tasks for an antimycobacterial regimen are the prevention of the emergence of drug-resistant strains (early bactericidal activity) and the eradication of one or more, slowly metabolizing populations of bacilli that cause relapse (sterilizating activity). Only rifampin appears active in both these respect. Traditional in vitro measures of antimicobial activity predict the efficacy of drugs for the prevention of the emergence of drug resistance, not the sterilizing activity of antituberculous drugs. Although widely used, in vitro tests or the activity of drug comibnations do not appear to predict the results of multidrug treatment regimens. Thus although more expensive and labrious than in vitro assays, animal models have been the best prelicnical predictor of the efficacy of single drugs and multidrug regimens for tuberculosis (Burman, Am J Med Sci 1997, 313(6), 355-363).