Course:PATH4172019W2/Case 3

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Niamh Kelly
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Case 3: From India to Canada

53-year-old Robert immigrated from India about a year ago. Over the past month he has had fevers, chills, night sweats and a chronic productive cough. He goes to see his family doctor who confirms a fever of 38.5°C. Upon auscultation she also finds crackles in the right lung and decreased breath sounds in the right lower lung field.  She sends Robert for a chest X-ray and gives him three sterile containers with instructions to generate three deep sputum samples over three mornings.

The samples are examined in the Microbiology Laboratory and Robert is informed that he has TB.  The Public Health Unit is notified and Robert is sent to the local hospital for further assessment (and treatment).

Q1. The Body Systems Questions

(i) What are the signs (objective characteristics usually detected by a healthcare professional) and symptoms (characteristics experienced by the patient, which may be subjective). What is ‘deep sputum’ and, in this particular infectious scenario, where is it produced?

(ii) Which body system is affected? In what way has the normal physiological functioning of this area of the body been disturbed by the infection (without going into detail on the cause of this disturbance as this will be dealt with in questions 3 and/or 4). Representing this diagrammatically is always helpful.

(iii) What are treatments will be offered to Robert and how do these work?

(iv) Why was the Public Health unit notified in this instance?

Q2. The Microbiology Laboratory Questions

(i) Including the bacteria implicated, what are the most common bacterial pathogens associated with this type of infectious scenario.

(ii) What samples are taken for laboratory testing and how important is the Microbiology Laboratory in the diagnosis of this infectious disease?

(iii) Explain the tests that will be performed on the samples in order to detect any of the potential bacterial pathogens causing this disease.

(iv) What are the results expected from these tests that might allow the identification of the bacteria implicated in this case.

Q3. Bacterial Pathogenesis Questions

Using the following pathogenic steps outline the pathogenesis of the bacteria implicated as being responsible for this infection.

(i) Encounter: where does the organism normally reside, geographically and host wise, and what are the bacterial characteristics that leave it suited to these places of residence. How might our patient have come in contact with this bacteria

(ii) Entry: how does the bacteria enter into the human host and take up residence. What are the molecular, cellular and/or physiological factors at play in this site specificity and in the initial adherence step (referencing both the bacteria and the host)..

(iii) Multiplication and Spread: does the organism remain at the entry site and/or does it spread beyond the initial site. Are there, for instance, secondary sites of infection. Does the organism remain extracellular and/or does it enter into cells - and what are the molecular and cellular determinants of these events.

(iv) Bacterial Damage: do the bacteria cause any direct damage to the host (or is the damage fully attributable to the host response, as indicated below) and, if so, what is the nature of the bacterial damage. Can it be linked to any of the signs and symptoms in this case?

Q4. The Immune Response Questions

(i) Host response: what elements of the innate and adaptive (humoral and cellular) immune response are involved in this infection.

(ii) Host damage: what damage ensues to the host from the immune response?

(iii) Bacterial evasion: how do the bacteria attempt to evade these host response elements.

(iv) Outcome: is the bacteria completely removed, does the patient recover fully and is there immunity to future infections with this infectious agent?

Reports: From India to Canada

53-year-old Robert immigrated from India about a year ago. Over the past month he has had fevers, chills, night sweats and a chronic productive cough. He goes to see his family doctor who confirms a fever of 38.5°C. Upon auscultation she also finds crackles in the right lung and decreased breath sounds in the right lower lung field.  She sends Robert for a chest X-ray and gives him three sterile containers with instructions to generate three deep sputum samples over three mornings.

The samples are examined in the Microbiology Laboratory and Robert is informed that he has TB.  The Public Health Unit is notified and Robert is sent to the local hospital for further assessment (and treatment).

Q1. The Body System Questions

Section (i)

What are the signs (objective characteristics usually detected by a healthcare professional) and symptoms (characteristics experienced by the patient, which may be subjective). What is ‘deep sputum’ and, in this particular infectious scenario, where is it produced?
Table 1: Differences between latent TB infection and active TB disease (2)

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis in humans (1). It is important to note that TB can exist in both a latent and active form. Latent TB infection means that the bacteria is present in the body, but the immune system is able to keep it under control; this differs from active TB disease (2). People with latent TB usually do not have symptoms, feel sick, and are not infectious, therefore they cannot spread the infection to others (2). Many people who have latent TB do not develop active TB disease (2). In some people, the Mycobacterium tuberculosis can remain inactive throughout their lives without ever causing active disease. However, people with weaker immune systems can have latent TB infections progress to active disease at any time (2). Some people can also become sick years after initial infection when their immune system becomes compromised for any number of reasons (e.g. aging, co-infection with other disease, underlying health complications) (2). Once the immune system is no longer able to control the bacteria’s growth, they can become active and multiply to eventually cause active TB disease (2). People who have active TB disease experience symptoms of sickness and are infectious to others (2). TB disease progression depends on the specific strain of Mycobacterium tuberculosis and the response of each person to the disease can depend on prior exposure, vaccination, and immune status (2). Robert most likely has active TB disease that is infectious due to the signs and symptoms that he is exhibiting.

Signs are objective characteristics usually detected by a healthcare professional; the patient may or may not be aware of the signs of their illness. The TB signs that Robert exhibits are a chronic productive cough, 38.5°C fever, crackles in right lung upon auscultation, and decreased breath sounds in right lower lung field. Symptoms are characteristics experienced by the patient (e.g. felt or observed) that may be subjective. The TB symptoms that Robert exhibits are fevers, chills, night sweats, and a chronic productive cough. Other clinical signs and symptoms that TB patients may present with include easy fatiguability, weight loss, and extrapulmonary manifestations (e.g. lymphadenitis, kidney/bone/joint involvement, meningitis, disseminate miliary disease) (3).

Figure 1: Colonies of Mycobacterium tuberculosis on Lowenstein-Jensen medium (2)

Deep Sputum is mucus coughed up from deep inside lungs (lower airways: trachea and bronchi) that is usually thick, cloudy, and sticky (4). Irritation of the respiratory system causes both inflammation of air passages and a marked increase in mucus secretions (5). Inflammation of mucosa is often responsible for sputum production (6, 7). Expectorated sputum contains secretions from lower respiratory tract, nose, mouth, pharynx, as well as cellular debris and microorganisms (8). It is important to note that sputum is not saliva (spit); saliva comes from mouth and is thin, clear, and watery. Do not collect saliva for a TB test (4). Three sequential sputum samples are required for diagnosis to ensure accuracy of results. Furthermore, some patients shed mycobacteria irregularly and in small numbers, thus, increasing the number of specimens would increase the yield and therefore accuracy of results (9). It is recommended that deep sputum be collected in the early morning before the patient eats, drinks, smokes, brushes their teeth, or uses mouthwash (10). This also follows the assumption that M. tuberculosis would be present in maximum concentration in sputum after pooling overnight in the respiratory tract (9). Positive sputum smears and cultures for acid-fast bacilli (AFB) indicate the presence of active TB disease in lungs (2, 3). To detect Mycobacterium tuberculosis (MTB) in a sputum sample, an excess of 10,000 organisms per mL of sputum are required to visualize the bacilli with a 100x microscope objective (1000x magnification) (2). One acid-fast bacillus/slide is regarded as "suspicious" of an MTB infection (2).


1) Horne DJ, Narita M. Pulmonary tuberculosis [Internet]. Pulmonary tuberculosis - Symptoms, diagnosis and treatment | BMJ Best Practice. 2018 [cited 2020Mar14]. Available from:

2) Todar K. Mycobacterium tuberculosis and Tuberculosis. Wisconsin: University of Wisconsin Department of Bacteriology; 2012.

3) McMurray DB. Mycobacteria and Nocardia. In: Baron S, editor. Medical Microbiology. 4th ed. Galveston (TX): University of Texas Medical Branch at Galveston; 1996.

4) BC Centre for Disease Control. Sputum Collection for Tuberculosis (TB) Testing [Internet]. HealthLink BC. 2019 [cited 2020Mar6]. Available from:

5) Richardson M. The physiology of mucus and sputum production in the respiratory system. Nursing Times. 2003Jun10;99(23):63–4.

6) Jeffery P, Zhu J. Mucin-Producing Elements and Inflammatory Cells. Mucus Hypersecretion in Respiratory Disease Novartis Foundation Symposia. 2008;:51–75.

7) Maestrelli P, Saetta M, Mapp CE, Fabbri LM. Remodeling in Response to Infection and Injury. American Journal of Respiratory and Critical Care Medicine. 2001;164(supplement_2).

8) King M. Physiology of mucus clearance. Paediatric Respiratory Reviews. 2006;7.

9) Bates JH. Diagnosis of Tuberculosis. Chest. 1979;76(6):757–63.

10) BC Centre for Disease Control. Sputum Collection for Tuberculosis (TB) Testing [Internet]. HealthLink BC. HealthLink BC; 2017 [cited 2020Mar14]. Available from:

Section (ii)

Which body system is affected? In what way has the normal physiological functioning of this area of the body been disturbed by the infection (without going into detail on the cause of this disturbance as this will be dealt with in questions 3 and/or 4). Representing this diagrammatically is always helpful.
Figure 1. Mechanisms and radiographic features associated with airflow obstruction and restrictive ventilatory defects in patients with a history of tuberculosis
Figure 1.2a:  Mechanisms and radiographic features associated with airflow obstruction [1.2 1]
Figure 1.2b: Major Interaction Points (IPs) of the respiratory system

TB is a disease that has been around for at least 15000 years and is caused by Mycobacterium tuberculosis (MTB). This disease mainly affects the lungs, which is part of the respiratory system. TB disease can manifest in all compartments of the respiratory tract, from the nose and sinuses to the pharynx, larynx, trachea, then to the bronchi, bronchioles and the lungs. [1.2 2] Judging by Robert’s signs, symptoms, and risk factors, he likely has a pulmonary tuberculosis infection which primarily affects the respiratory system. This system can be divided up into the upper and lower respiratory tracts, which include the mucosal surfaces from the nose and pharynx, down to the alveoli in the lungs. The respiratory system is directly exposed to the air, and has a significantly large surface area to enable gas exchange. As it is constantly in contact with microbes from both outside and inside the body, it uses a unique and highly regulated local immune defense system which efficiently allows for microbial clearance while minimizing damaging inflammatory responses. As a prototypic host-adapted airborne pathogen, Mycobacterium tuberculosis traverses through the lung and uses several interaction points (IPs) to cause the infection [1.2 3]. The major IPs include: 1. trachea and main stem bronchi which bifurcate to enter each lung, division of bronchi into bronchioles, 2. terminal bronchioles, transitional bronchioles and respiratory bronchioles, 3. the division of respiratory bronchioles into alveolar ducts, then the alveoli, and 4. the interaction between MTB and granuloma in the lungs. [1.2 4] Transmission of TB often occurs from individuals infected with the pulmonary form of TB disease and infection results from the inhalation of aerosolized droplets containing the bacterium.

A notable feature of pulmonary TB infection is the heterogeneity in pathology (Fig. 1.2a), in terms of the magnitude of resultant pulmonary function, ranging from no impairment to severe dysfunction and the specific types of ventilatory defects. Four primary processes (Fig. 1.2b) underlie the lung remodelling during and after pulmonary TB infection, and include pulmonary cavitation, pulmonary fibrosis, bronchiectasis, and general pulmonary impairment (Table 1.2a).

Table 1.2a: Four primary processes that underlie the lung remodelling, during and after pulmonary TB infection
Pulmonary Cavitation Normal pulmonary tissue is destroyed, becoming spaces or cavities in the lung
Pulmonary Fibrosis Long-term lung tissue injury characterized by extreme extracellular matrix deposition in the lung: replace lung parenchyma with collagenous tissue can lead to thickening and hardening of the lung
Bronchiectasis Irreversible bronchial dilation and thickening of the bronchial wall- elasticity of the lung decrease
General Pulmonary Impairment Broad term that refers to lung dysfunction and includes airflow obstruction, restrictive ventilatory defects and impaired gas exchange
Figure 2. Normal Alveoli vs. Alveoli with Pulmonary cavitation
Figure 1.2c:  Normal Alveoli vs. Alveoli with Pulmonary cavitation [1.2 5]

Normally, the negative pressure created in the lung by the respiratory muscles causes air to flow in, where diffusion can occur in the alveoli between the blood and the air. Alveoli are air sacs in your lungs that are surrounded by tiny blood vessels called capillaries. The air sacs have thin walls that allow the exchange of gases. When blood flows through the capillaries around the air sacs, it picks up oxygen from the air and dumps off carbon dioxide from the blood. Pulmonary cavitation (Fig. 1.2c) is where such normal pulmonary tissue is obliterated, and instead becoming gas-filled or cavity spaces in the lung. This process starts with the necrosis of pneumonia lesions, producing caseous pneumonia. During caseation, alveolar cells and septa that make up this region are destroyed, in addition to the neighbouring vessels and bronchi. Cavities form when these regions of caseous pneumonia liquefy, fragment and come apart upon coughing [1.2 1]. The alveoli swell and fill with inflammatory cells and fluid, containing white blood cells, red blood cells, macrophages, fibrin, cell debris, and microorganisms, which causes the cough and makes it hard to breathe [1.2 5]. This inhibits the lungs ability to comfortably pull in air, as well as reducing the overall surface area for diffusion [1.2 1].

Figure 3. Normal Lungs vs. Lungs with Pulmonary Fibrosis
Figure 1.2d:  Normal Lungs vs. Lungs with Pulmonary Fibrosis [1.2 6]
Figure 1.2d:  Normal versus damaged bronchus with bronchiectasis
Figure 1.2e:  Normal versus damaged bronchus with bronchiectasis [1.2 7]

In order to accommodate the inhaled air, the lung generally expands in volume. The lung tissue has lower levels of collagen than other organs in order to have a lower stiffness in order to allow for this stretching. Pulmonary fibrosis (Fig. 1.2d) is a condition that may result from long-term lung tissue injury and it is characterised by excessive extracellular matrix deposition in the lung. By replacing the normal lung parenchyma with collagenous tissue, the lung goes through architectural changes such as thickening and stiffening of the lung walls. This makes it more difficult to breathe as the lung is more resistant to volume changes [1.2 6]. Similarly, bronchiectasis (Fig. 1.2e) is the irreversible bronchial dilatation and thickening of the bronchial wall. Normal elastic and muscular components of the bronchial wall are destroyed, where the cause may be due to multiple factors, including traction from surrounding tissue fibrosis, caseous necrosis invading the bronchi, and elevated luminal pressure due to coughing. The process of bronchiectasis can also predispose to recurrent exacerbations of purulent sputum production and possibly bacterial pneumonia in subsequent years [1.2 7].

General pulmonary impairment is a broad term that refers to lung dysfunction and includes airflow obstruction, restrictive ventilatory defects and impaired gas exchange. Pulmonary impairment after TB is likely related to a variety of lung remodelling events including the ones mentioned above, where some may even manifest as symptoms and pulmonary disability over a period of time. For this case study, the crackles in the right lung and decreased breath sounds in the right lower lung field during auscultation are likely related to a combination of these rearrangements where the crackles are related to pneumonia like fluid build up, and the decreased breath sounds are related to thickening and stiffening of the tissue in the region.

Figure 1.2e:  Changes to mucociliary clearance processes caused by TB infection
Figure 1.2f:  Changes to mucociliary clearance processes caused by TB infection [1.2 3]

Tracheobronchial secretions are generally kept at unnoticeable levels by the mucociliary clearance mechanism (Fig. 1.2f). The mucosal lining of the tracheobronchial tree is comprised of pseudostratified ciliated columnar epithelium containing eight different cell types including basal, Kultschitzsky, intermediate, brush, serous, Goblet (mucous), ciliated and club (Clara) cells [1.2 8]. Basal cells are stem/progenitor cells of ciliated and mucosal cells, and thus are central to pulmonary host defense. Cigarette smoke affects the stem cell’s capacity to regenerate the epithelium and thus this mechanism is likely the reason for this risk factor [1.2 9]. Kultschitzsky, intermediate and brush cells have unknown functions in lung diseases. Serous and Goblet cells release watery and mucin-rich viscoelastic mucous to act as a lubricant and protect the respiratory system lining [1.2 3]. This mucus is moved by beating ciliated cells contributing to mucociliary clearance, which may trap and remove TB droplets, thus limiting their access to the alveoli. Club cells act as both protectors, by secreting mucus-like surfactant rich in glycosaminoglycans, and regenerators, by acting as stem cells to restore damaged bronchial and alveolar epithelia. Generally we lack understanding of how each specific cell may contribute to TB pathogenesis. However, we understand that the airway inflammation can disrupt lung protection and regeneration by inducing swelling of mucous membranes lining the bronchi, which increases the bronchial mucous production, and decreasing movement of the thick mucus by ciliated cells, thus limiting microbial clearance [1.2 3]. Wet cough can result in this situation when the mucociliary function is ineffective or insufficient as a proper balance between the formation and clearance is required to effectively trap and remove the impurities of the inspired air while preventing the excessive accumulation of secretions . This highlights chronic productive cough, the other symptom in this case study.

Figure 1.2g: Progression of natural course of events in an immunocompetent individual following exposure to droplet nuclei containing MTB [1.2 10]

In addition to the respiratory system, if TB can overcome the host immune system (Fig. 1.2g), it can also affect other organ systems of the body, including the gastrointestinal system, the lymphoreticular system, the skin, the central nervous system (CNS), the musculoskeletal system, the reproductive system and the liver.


  1. 1.0 1.1 1.2 Ravimohan, Shruthi; Kornfeld, Hardy; Weissman, Drew; Bisson, Gregory P. (2018-03-31). "Tuberculosis and lung damage: from epidemiology to pathophysiology". European respiratory review : an official journal of the European Respiratory Society. 27 (147). doi:10.1183/16000617.0077-2017. ISSN 0905-9180. PMC 6019552. PMID 29491034. Retrieved 2020-03-07.CS1 maint: PMC format (link)
  2. Adigun, Rotimi; Singh, Rahulkumar (2020). "Tuberculosis". StatPearls. Treasure Island (FL): StatPearls Publishing. PMID 28722945. Retrieved 2020-03-14.
  3. 3.0 3.1 3.2 3.3 Torrelles, Jordi B.; Schlesinger, Larry S. (2017-08). "Integrating Lung Physiology, Immunology and Tuberculosis". Trends in microbiology. 25 (8): 688–697. doi:10.1016/j.tim.2017.03.007. ISSN 0966-842X. PMC 5522344. PMID 28366292. Retrieved 2020-03-07. Check date values in: |date= (help)CS1 maint: PMC format (link)
  4. Torrelles, Jordi B.; Schlesinger, Larry S. (2017-08-01). "Integrating Lung Physiology, Immunology, and Tuberculosis". Trends in Microbiology. 25 (8): 688–697. doi:10.1016/j.tim.2017.03.007. ISSN 1878-4380 0966-842X, 1878-4380 Check |issn= value (help). PMID 28366292. Retrieved 2020-03-14.
  5. 5.0 5.1 "Pneumonia anatomy PI - UpToDate". Retrieved 2020-03-07.
  6. 6.0 6.1 "Idiopathic Pulmonary Fibrosis | National Heart, Lung, and Blood Institute (NHLBI)". Retrieved 2020-03-07.
  7. 7.0 7.1 "Bronchiectasis". Cleveland Clinic. Retrieved 2020-03-07.
  8. Mercer, R R; Russell, M L; Roggli, V L; Crapo, J D (1994-06-01). "Cell number and distribution in human and rat airways". American Journal of Respiratory Cell and Molecular Biology. 10 (6): 613–624. doi:10.1165/ajrcmb.10.6.8003339. ISSN 1044-1549. Retrieved 2020-03-07.
  9. Mercer, R R; Russell, M L; Roggli, V L; Crapo, J D (1994-06-01). "Cell number and distribution in human and rat airways". American Journal of Respiratory Cell and Molecular Biology. 10 (6): 613–624. doi:10.1165/ajrcmb.10.6.8003339. ISSN 1044-1549. Retrieved 2020-03-07.
  10. "Pathogenesis, Immunology, and Diagnosis of Latent Mycobacterium tuberculosis Infection". ResearchGate. Retrieved 2020-03-14.

Section (iii)

What treatments will be offered to Robert and how do these work?

The high lipid content of MTB’s cell wall compared to that of other bacteria is thought to contribute to the resistance of MTB to several antibiotics, the difficulty with Gram staining and several other stains, and the ability to survive under extreme conditions (e.g. acidity or alkalinity), in anoxic environments and intracellularly within macrophages (2).


As a preventative measure, the Bacille Calmette-Guérin (BCG) vaccination is available for the TB disease but is not widely used (9). It consists of a live-attenuated strain that is derived from M. bovis; this strain has remained avirulent for over sixty years (1). The BCG vaccine does not protect people from being infected 100%, and studies suggest only a 60-80% effective rate in children (1). In Canada, the BCG vaccine is not recommended for routine use in any population and is only considered for a select group of people who meet specific criteria and are in consultation with a TB expert (10). This includes infants, children and some healthcare workers (9). Those who are immunocompromised and/or pregnant should not receive the BCG vaccine since it is a live vaccine (10).

The BCG vaccine is not widely administered for several reasons. The vaccine cannot prevent disease reactivation in previously exposed individuals (9). The vaccine does not prevent infection, only disease (9). Therefore, in order for the vaccine to be considered efficacious, the entire population would have to be vaccinated (9). The vaccine may complicate the way the tuberculin skin test is read since vaccination may cause a positive result from the TB skin test (9). In places that do not vaccinate, the skin test may be used to monitor the effectiveness of antibiotic therapy (9).

Active Infection

The treatment goal for active TB is to eradicate infection in affected patient and to prevent spread of disease to contacts (30). TB can be treated and cured by taking several drugs for 6-12 months (5). It is of utmost importance that people with the TB disease finish and take the medications exactly as prescribed (5). If the medications are not taken correctly, resistant strains of the MTB may arise (5). This is not ideal since resistant strains of TB are harder and more expensive to treat and often have non-ideal treatment outcomes (5). In some situations, staff of the local health department meet regularly with patients who have TB to monitor their medication usage (5). This is called directly observed therapy (DOT), and it helps patients to complete treatment regimens in the least amount of time (5). Patient compliance (i.e. completing course of medications) is probably the single most important variable affecting treatment outcome (32). Most patients are treated in outpatient settings; patients should wear a simple surgical mask when there are other people in the room. Patients should be in respiratory isolation (airborne pathogen precautions, negative pressure room) until 3 consecutive negative sputum acid-fast bacilli smears on specimens obtained at least 8 hours apart (one of which is obtained as an early-morning specimen) have been demonstrated (34).

Treatment of confirmed TB disease always requires combination therapy (2). Monotherapy should never be used for TB since the administration of a single drug often leads to the development of bacterial resistance (1). Effective regimens for the treatment of TB must contain multiple drugs to which the organisms are susceptible (1). When two or more drugs are used simultaneously, each helps prevent the emergence of resistance to the others (1). TB diseases are most often treated by taking four different antimicrobial agents for 6-9 months (1). Studies show that patients who are treated with a four-drug regimen are more likely to be cured and not relapse when compared to patients who are treated for the same length of time but with a three-drug regimen (1). The most common and core four-drug regimen includes isoniazid, rifampicin, ethambutol and pyrazinamide; vitamin B6 is always given with isoniazid to prevent neural damages or neuropathies (2). There is an intensive phase for 2 months, followed by a continuation phase of either 4 or 7 months; this makes up a total of 6 to 9 months for treatment (2).

Figure 1. Structure of isoniazid (1)

First-line Medication, Group 1

1.     Isoniazid (NIH) – competitive inhibitor (2)

This drug is used to prevent outgrowth of resistant strains because if either isoniazid or ethambutol is used alone, MTB can quickly develop resistance (1). It functions by inhibiting mycolic acid synthesis in mycobacteria (1). It also disrupts DNA, lipid, carbohydrate, and nicotinamide adenine dinucleotide (NAD) synthesis and/or metabolism (32). Since it is an analog of pyridoxine (vitamin B6), it may inhibit pyridoxine-catalyzed reactions as well (1). Isoniazid is activated by a mycobacterial peroxidase enzyme and destroys several targets in the cell (1). All patients taking isoniazid are required to take a liver function test (2).

2.     Ethambutol (EMB) – competitive inhibitor (2)

This drug is used to prevent outgrowth of resistant strains for the same reasons stated above. It functions by inhibiting incorporation of mycolic acids into the mycobacterial cell wall, therefore disrupting cell structure and function (1). All patients taking ethambutol must be monitored for retinopathies (2).

Figure 2. Structure of rifampicin (1)

3.     Rifampicin (RIF) – effects on nucleic acid (2)

This drug is a semi-synthetic derivative of rifamycin that is active against MTB, Gram-positive and some Gram-negative bacteria (1). It acts specifically on the bacterial RNA polymerase by binding to the beta subunit of the polymerase and blocks the entry of the first nucleotide (1). The first nucleotide is necessary for the activation of the RNA polymerase, therefore, rifampicin blocks mRNA synthesis (1). Studies have also found that rifampicin has greater bactericidal effect against MTB than other anti-TB drugs (1). It has largely replaced isoniazid as one of the front-line drugs used to treat TB disease, especially when the patient is resistant to isoniazid (1). Rifampicin can also be used to treat bacterial meningitis since it is effective orally and can penetrate the cerebrospinal fluid (CSF) (1).

4.     Pyrazinamide (PZA) (2)

This prodrug is a synthetic pyrazinoic acid amide derivative with bactericidal properties (11). Its mechanism of action is unknown due to its unusual properties; however, it is particularly active against slow-multiplying intracellular bacilli (11). PZA and its analog, 5-chloro-PZA may inhibit the fatty acid synthetase I (FASI) enzyme of M. tuberculosis (33). Pyrazinamidase removes the amide group to produce active pyrazinoic acid and is required for the bactericidal action of pyrazinamide (11). When PZA is used as part of combination therapy, it appears to accelerate the sterilizing effect of isoniazid and rifampicin (33).

5.     Rifabutin (2)

This drug is a semi-synthetic ansamycin antibiotic with potent antimycobacterial properties (12). It functions by inhibiting bacterial DNA-dependent RNA polymerase, thereby suppressing the initiation of RNA formation (12). This also leads to the inhibition of RNA synthesis and transcription (12).

6.     Rifapentine (2)

This drug is a rifamycin antibiotic that is similar in structure and activity to rifampin and rifabutin (13). It is often used in combination with other agents as therapy of TB; however, it can cause acute liver injuries (13).

Second-line Medication, Group 2

1.    Injectable aminoglycosides – protein synthesis inhibitors (1)

These drugs function by binding to bacterial ribosomes and preventing the initiation of protein synthesis (1)

A.   Amikacin

This drug is a broad-spectrum semi-synthetic aminoglycoside antibiotic (14). It is derived from kanamycin with antimicrobial properties (14).

B.    Kanamycin

This drug functions by binding to the ribosomal 30S subunit to prevent it from joining to the 50S subunit during protein synthesis (1). This leads to cytoplasmic accumulation of dissociated 30S subunits, which is lethal to the cells (1).

Figure 3. Structure of streptomycin (1)

C.    Streptomycin

This drug functions by binding to the 30S bacterial ribosomal subunit, specifically to the S12 protein, which is involved in the initiation of protein synthesis (1). It also prevents the normal dissociation of ribosomes into their subunits, leaving them in the 70S form and preventing the formation of polysomes (1). Overall, the drug functions by distorting ribosomal functions.

2.     Injectable polypeptides – block bacterial ribosomal translocation (2)

A.   Capreomycin

B.    Viomycin

Second-line Anti-TB Drugs, Group 3, Oral and Injectable

Fluoroquinolones – quinolones are nucleic acid synthesis inhibitors (1)

This is a class of broad-spectrum antimicrobial agents that is active against both Gram-positive and Gram-negative bacteria (1). They function via the inhibition of DNA gyrase, a type II topoisomerase, which is needed to separate replicated DNA (1). This inhibits cell division (1).

1.     Levofloxacin

2.     Moxifloxacin

3.     Ofloxacin

4.     Gatifloxacin

Second-line Anti-TB Drugs, Group 4

1.     Para-aminosalicylic acid (PAS) – competitive inhibitor (2)

This drug is an anti-folate and functions similarly to the sulfonamides (1). This was once a primary anti-TB drug, but is now a secondary agent, after having been largely replaced by ethambutol (1).

2.     Cycloserine (2)

This drug has broad-spectrum antibiotic and glycinergic activities (15). It functions by interfering with bacterial cell wall synthesis (15).

3.     Ethionamide (2)

This drug is a nicotinamide derivate with antibacterial activity (16). The exact mechanism of action is unknown, and it may either be bacteriostatic or bactericidal, depending on the concentration of drug at site of infection and susceptibility of the organism involved (16). It perhaps inhibits bacterial cell wall synthesis by inhibiting the synthesis of mycolic acid (16). This eventually leads to bacterial cell wall disruption and cell lysis (16).

4.     Prothionamide (2)

This drug is a thioamide derivative with anti-TB activity (17). It functions by competitively inhibiting an enzyme that is essential for mycolic acid synthesis (17). This results in cell wall permeability and decreased resistance against cell injury, leading to cell lysis and death (17).

5.     Linezolid (2)

This drug is a synthetic oxazolidinone derivative (18). It selectively inhibits an early step in bacterial protein synthesis (18).

6.     Thioacetazone

7.     Terizidone

Third-line Anti-TB drugs, Group 5

These are medications with variable but unproven efficacy against TB (2). They are used as a last resort for total drug-resistant TB (2).

1.     Clofazimine (2)

This drug has both anti-mycobacterial and anti-inflammatory activities (19). However, the exact effect is unknown. We do know that it binds preferentially to mycobacterial DNA, thereby inhibiting DNA replication and cell growth (19).

2.     Imipenem/Cilastatin (2)

This drug binds to and inactivates penicillin-binding proteins (PBPs) on the inner membrane of bacterial cell walls (20). PBPs facilitate the last stages of assembling and reshaping of the bacterial cell wall during growth and division (20). The usage of this drug results in weakening of bacterial cell wall and eventually cell lysis (20).

3.     Clarithromycin (2)

This drug binds to the 50S bacterial ribosomal subunit and inhibits RNA-dependent protein synthesis in susceptible organisms (21).

4.     Amoxicillin and clavulanic acid

The combination of these two drugs either make clavamox or augmentin (1). This is a combination of a beta-lactam antibiotic and a beta lactamase enzyme inhibitor (1).

5.     Linezolid

Function stated above.

Latent TB

The treatment goal for latent TB is to prevent development of active TB; however, the decision to treat latent infections requires balancing risk of toxicity against likelihood of developing active infection (30). Isoniazid is usually prescribed with vitamin B6, pyridoxine, for patients with latent TB (2). This regimen should be continued for 6 or 9 months (2). The WHO recommends either a 6-months or 9-months regimen of isoniazid daily, a 3-months regimen of rifapentine and isoniazid weekly, a 3-months or 4-months regimen of isoniazid and rifampicin daily, or a 3-months or 4-months regimen of rifampicin alone daily (2).

Multidrug Resistant TB (MDR-TB)

MDR-TB is becoming increasingly common, especially when anti-TB drugs are misused or mismanaged (2). MDR-TB is resistant to more than one type of anti-TB drugs and at least one of isoniazid or rifampin (2). This is problematic since isoniazid and rifampin are considered first-line drugs that are used to treat all persons with the TB disease (2). For patients with MDR-TB, the combination of first- and second-line medications are used at high doses (2). If further drugs are required because of resistance or intolerance, then drugs such as bedaquiline (especially if the patient is quinolone-resistant) or delamanid are added; combination of these two drugs are not recommended (2).

Extensively Drug-resistant TB (XDR-TB)

XDR-TB is a rare type of MDR-TB that is resistant to both isoniazid and rifampin, plus any fluoroquinolone and at least one of three injectable second-line drugs (i.e. amikacin, kanamycin or capreomycin) (2). Treatment is usually 18-24 months with 4 to 6 second- and third- line drugs. Since XDR-TB is resistant to both first- and second-line drugs, patients are often left with less effective and desirable treatment options (1). This often leads to worse treatment outcomes (1).

Co-Management of HIV and Active TB Disease

Patients who are HIV-positive and have pulmonary TB disease are at a higher risk of acquiring rifampicin resistance (2). They are also at a higher risk for MDR-TB and XDR-TB infection since they are more immunosuppressed than the general public (2). Once infection occurs, this population is more likely to develop the TB disease and have a higher risk of death from the disease (1). TB patients with positive HIV status and/or living in HIV-prevalent settings should receive daily TB treatment at least during the intensive phase (2). It is recommended that their treatment lasts for longer periods (9-12 months) (31).

Co-trimoxazole preventative therapy should be initiated as soon as possible and given throughout the TB treatment (2). This therapy can greatly reduce mortality in HIV-positive TB patients (2). Antiretroviral therapy (ART) has also been shown to improve survival in HIV-positive patients who have TB (2). This treatment should be initiated for all who live with HIV and active TB disease (2). TB treatment should begin first, followed by ART as soon as possible and within the first 8 weeks of starting the TB treatment (2).

Extrapulmonary TB

Patients with extrapulmonary TB are usually treated with isoniazid, rifampicin, ethambutol and pyrazinamide for 2 months, followed by 4-9 months of isoniazid plus rifampicin (if confirmed susceptible) (30). Longer treatment may be required for TB meningitis (9-12months) (30). With TB meningitis and some cases of pericarditis, an addition of corticosteroids is recommended for the first 1-2 months (30). Patients with healed, quiescent fibrotic disease should be treated with a regiment for latent infection (30).

Toxicity and Side Effect Management

There are often side effects associated with the most commonly used anti-TB drugs (2). It is important for healthcare professionals to closely monitor for these side effects. Most can be managed by adjusting dose but in some cases, the medication may need to be discontinued (2). Second-line therapy should be considered in these cases if alternatives are not available (2).

Isoniazid can cause asymptomatic elevation of aminotransferases, hepatitis, or peripheral neurotoxicity and hypersensitivity (2). Rifampin can cause pruritis, nausea and vomiting, flulike symptoms, hepatotoxicity and orange discolouration of bodily fluids (2). Rifabutin can cause neutropenia, uveitis, polyarthralgia and hepatotoxicity (2). Rifapentine can cause side effects similar to that of rifampin (2). Pyrazinamide can cause hepatotoxicity, nausea and vomiting, polyarthralgia, acute gouty arthritis, rash and photosensitive dermatitis (2). Ethambutol can cause retrobulbar neuritis (2).


With the assumption that Robert has active pulmonary TB, he should be treated with a combination of the four first-line drugs – isoniazid, rifampicin, pyrazinamide and ethambutol.


1.     Todar KG. 2012, posting date. Mycobacterium tuberculosis and Tuberculosis. In Todar K (ed), Todar’s Online Textbook of Bacteriology. University of Wisconsin-Madison Dept. of Bacteriology, Madison, WI.

2.     Rotimi A, Rahulkumar S. 2019. Tuberculosis. In StatPearls (ed), Tuberculosis [Internet]. StatPearls Publishing, Treasure Island, FL.

3.     CDC. 2016. How TB Spreads. Division of Tuberculosis Elimination, U.S. Department of Health & Human Services.

4.     HealthLinkBC. 2017. Sputum Collection for Tuberculosis (TB) Testing. BC Centre for Disease Control, BC.

5.     CDC. 2016. Latent TB Infection and TB Disease. Division of Tuberculosis Elimination, U.S. Department of Health & Human Services.

6.     Farzan S. 1990. Chapter 38, Cough and Sputum Production. In Walker HK, Hall WD, Hurst JW (ed), Clinical Methods: The History, Physical, and Laboratory Examinations, 3rd ed. Butterworth Publishing, Boston, MA.

7.     Torrelles JB, Schlesinger LS. 2017. Integrating Lung Physiology, Immunology, and Tuberculosis. Trends Microbiol.

8.     Stek C, Allwood B, Walker NF, Wilkinson RJ, Lynen L, Meintjes G. 2018. The Immune Mechanisms of Lung Parenchymal Damage in Tuberculosis and the Role of Host-Directed Therapy. Front Microbiol.

9.     CDC. 2016. Vaccines. Division of Tuberculosis Elimination, U.S. Department of Health & Human Services.

10.  Government of Canada. 2020. Bacille Calmette-Guérin (BCG) vaccine: Canadian Immunization Guide. Government of Canada.

11.  National Center for Biotechnology Information. PubChem Database. Pyrazinamide, CID=1046, (accessed on Mar. 7, 2020)

12.  National Center for Biotechnology Information. PubChem Database. Rifabutin, CID=135398743, (accessed on Mar. 7, 2020)

13.  National Center for Biotechnology Information. PubChem Database. Priftin, CID=135403821, (accessed on Mar. 7, 2020)

14.  National Center for Biotechnology Information. PubChem Database. Amikacin, CID=37768, (accessed on Mar. 7, 2020)

15.  National Center for Biotechnology Information. PubChem Database. Cycloserine, CID=6234, (accessed on Mar. 7, 2020)

16.  National Center for Biotechnology Information. PubChem Database. Ethionamide, CID=2761171, (accessed on Mar. 7, 2020)

17.  National Center for Biotechnology Information. PubChem Database. Protionamide, CID=666418, (accessed on Mar. 7, 2020)

18.  National Center for Biotechnology Information. PubChem Database. Linezolid, CID=441401, (accessed on Mar. 7, 2020)

19.  National Center for Biotechnology Information. PubChem Database. Clofazimine, CID=2794, (accessed on Mar. 7, 2020)

20.  National Center for Biotechnology Information. PubChem Database. Imipenem, CID=104838, (accessed on Mar. 7, 2020)

21.  National Center for Biotechnology Information. PubChem Database. Clarithromycin, CID=84029, (accessed on Mar. 7, 2020)

22.  Edemekong PF, HuangB. 2019. Epidemiology of Prevention of Communicable Diseases. In StatPearls (ed), Epidemiology of Prevention of Communicable Diseases [Internet]. StatPearls Publishing, Treasure Island, FL.

23.  NHS. 2019. Causes Tuberculosis (TB). National Health Service, UK.

24.  WHO. 2019. Tuberculosis. World Health Organization.

25.  Government of Canada. 2019. Tuberculosis: For health professionals. Government of Canada.

26.  BCCDC. 2019. Communicable Disease Control Manual Chapter 4: Tuberculosis, Tuberculosis. BC Centre for Disease Control, BC.

27.  BCCDC. 2019. Communicable Disease Control Manual Chapter 4: Tuberculosis, Appendix B: Infection Prevention and Control. BC Centre for Disease Control, BC.

28.  Medline Plus. 2020. Reportable diseases. U.S. National Library of Medicine. Bethesda, MD.

29.  Ahmad S. 2011.Pathogenesis, Immunology, and Diagnosis of Latent Mycobacterium tuberculosis Infection. J Immunol Res.

30.   Elsevier Point of Care. Clinical Overview Tuberculosis. ClinicalKey. 2019Apr25.

31.  McMurray DB. Mycobacteria and Nocardia. In: Baron S, editor. Medical Microbiology. 4th ed. Galveston (TX): University of Texas Medical Branch at Galveston; 1996.

32.  Berning SE, Peloquin CA. Antimycobacterial agents: Isoniazid. In: Yu V, Merigan T, Barriere S, editors. Antimicrobial Therapy and Vaccines. Williams and Wilkins, Baltimore 1998.

33.  Zimhony O, Cox JS, Welch JT, et al. Pyrazinamide inhibits the eukaryotic-like fatty acid synthetase I (FASI) of Mycobacterium tuberculosis. Nat Med 2000; 6:1043.

34.  Centers for Disease Control and Prevention. TB Infection Control in Health Care Settings [Internet]. Centers for Disease Control and Prevention. Centers for Disease Control and Prevention; 2019 [cited 2020Mar7]. Available from:

Section (iv)

Why was the Public Health unit notified in this instance?

Tuberculosis is a legally reportable disease in every Canadian province and territory. This means that cases must be reported to the corresponding provincial and territorial department of health within one business day (1). Reportable diseases are diseases that are considered to be of great public health importance (2). In the U.S. and Canada, it is required that these diseases be reported when they are diagnosed by doctors or laboratories (2). The reports allow statistical measurements that show how often the disease occurs (2). There are reasons for this outlined below.

The global burden of TB has been immense. A total of 1.5 million people died from TB in 2018 (including 251 000 people with HIV) (3). Worldwide, TB is one of the top 10 causes of death and the leading cause from a single infectious agent (above HIV/AIDS) (3). TB is a communicable disease, meaning that it can be spread between people through various means (4). TB is spread from person to person through the air. When people with lung TB cough, sneeze or spit, they propel the TB germs into the air (3). A person needs to inhale only a few of these germs to become infected (3). Since it is so easy to spread in a population it is important to contain it and keep track of who is infected in places (like Canada) where it has not yet been an epidemic and it is contained.

Additionally, this information is also shared with CDC for the purpose of research to understand the lineages of each TB case. This is important in understanding where each case emerged from and if there are any drug-resistant strains circulating, and too which drugs. Bioinformaticians use the data collected by Whole Genome Sequencing (WGS) of MTB, and through bioinformatics analysis of the reported cases determine the lineage of each case and determine what lineages have the most susceptibility to certain anti-TB drugs. This information is very useful for additional research on drugs, TB, and its progression around the world.

Figure 1: Multidrug- and Extensively Drug-Resistant Mycobacterium tuberculosis (MTB) by lineage as provided by CDC.


  1. Public Health Agency of Canada. Tuberculosis: For health professionals. Published November 22, 2019. Accessed March 7, 2020.
  2. Medline Plus. 2020. Reportable diseases. U.S. National Library of Medicine. Bethesda, MD.
  3. Tuberculosis (TB). World Health Organization. Accessed March 7, 2020.
  4. Edemekong PF, HuangB. 2019. Epidemiology of Prevention of Communicable Diseases. In StatPearls (ed), Epidemiology of Prevention of Communicable Diseases [Internet]. StatPearls Publishing, Treasure Island, FL.
  5. Figure 1 - Multidrug- and Extensively Drug-Resistant Mycobacterium tuberculosis Beijing Clades, Ukraine, 2015 - Volume 26, Number 3-March 2020 - Emerging Infectious Diseases journal - CDC. Centers for Disease Control and Prevention. Accessed March 14, 2020.

Q2. The Microbiology Laboratory

Question (i)

(i) Including the bacteria implicated, what are the most common bacterial pathogens associated with this type of infectious scenario.

Mycobacterium tuberculosis (MTB) is the etiological agent of Tuberculosis (TB) in humans. For this fairly large, nonmotile, rod-shaped bacterium (2-4μm in length and 0.2-0.5μm in width), humans are the only reservoir. Because MTB are obligate aerobes, they are always found in the well-aerated upper lobes of the lungs. They are also facultative intracellular parasites that can replicate in macrophages; this physiological characteristic contributes to its virulence (1). Once the bacteria are carried to the lymph nodes through the macrophages, the bacterium can spread hematogenously to other sites. Most patients are asymptomatic during this time and usually develop no evidence of disease. However, once cell-mediated immunity is developed, tests of TB (tuberculin skin test and IFN-y release assays) become positive. At this point, the bacterium’s pathogenesis may cease and the person remains asymptomatic, conferring a latent TB infection (2). MTB are characterized by a complex cell wall rich in mycolic acids, a complex polysaccharide molecule that surrounds the cell membrane, along with peptidoglycan and arabinogalactan (3). When MTB are tested through gram-staining, the bacterium shows neither chemical properties of positive or gram negative bacteria, although the bacteria do contain peptidoglycan in their cell wall (1). Instead, MTB are considered acid fast bacteria where they show positive results for the Ziehl-Neelsen stain, which will be described in the later questions (1).

Many non-pathogenic mycobacteria are components of our the normal flora found most often in dry and oily locales (1). Of the 175 mycobacteria species recognized, slow growing mycobacteria are the most suspected pathogens for humans (4).

Mycobacterium bovis is the etiological agent of TB mainly in cows. While both humans and cows are reservoirs for Mycobacterium bovis, humans can only be infected by the consumption of unpasteurized milk. Infection can lead to extrapulmonary TB, and as seen in history, can lead to bone infections to causes hunched back (1).

Mycobacterium avium causes a TB-like disease, particularly in patients with AIDs due to their weakened immune system (1). It is believed M. avium enters intracellularly through the gastrointestinal tract (3).

Furthermore, with the emergence of many immunodeficiency diseases, including HIV and cancer, opportunistic micro-organisms like Nontuberculosis Mycobacterium (NTM) are becoming prevalent pathogens in these patients (4).

The most common types of Nontuberculous mycobacteria include Mycobacterium avium and Mycobacterium intracellulare which are categorized into a group known as Mycobacterium avium complex (MAC) based on the similarities in clinical conditions that they cause (2). A difference between TM and nontuberculous mycobacteria is the latter cannot be transmitted from patient to patient. These pathogens are ubiquitous in the environment and can cause colonization, infection and pseudo outbreaks in health care settings. NTM’s are able to cause pneumonia, lung abscess, pleural infection, meningitis, lymphadenitis and many other infections of the skin and soft tissue. These bacteria are all acid fast bacteria and difficult to differentiate from MTB using primary staining and microscopy laboratory procedures (4).

Another bacterium that produces similar symptoms as TB includes Mycoplasma pneumonia. It is a bacterium which most commonly causes mild infections of the respiratory system and tracheobronchitis. Some symptoms of tracheobronchitis include productive coughs, fatigue, fever and chills which are similar to some symptoms of TB in this scenario (3).

Another important pathogen in the Mycobacterium genus is Mycobacterium leprae, the causative agent of leprosy (1). M. leprae are obligate intracellular parasites that preferentially enter Schwann cells and macrophages in the endoneural space (3).

Although not a bacterial pathogen, the World Health Organization acknowledges infection with HIV poses a 16-27% increased risk of developing TB than those living within HIV. In 2015, 11% of the 10.4 million global TB cases were HIV patients. In the same year, almost 60% of tuberculosis cases among the HIV patients were not diagnosed or treated, resulting in 390,000 tuberculosis related deaths among people living with HIV (5).

S. pneumoniae is a gram-positive, non-sporulating, nonmotile cocci bacteria that is often arranged in pairs (diplococcus) or short chains. S. pneumoniae is between 0.5 and 1.25 micrometers in diameter. There are around 90 serotypes and its surface capsule is the major virulence factor. S. pneumoniae is transmitted from person to person via direct contact with respiratory sections such as saliva or mucus [7]. The type of pneumonia that Robert may have is called community-acquired pneumonia (CAP) that is acquired outside hospitals or healthcare setting (Mercola). S.pneumoniae colonizes the mucosal surfaces of the upper respiratory airway and nasopharynx with infection appearing as bacteria migrate into sterile parts of the airway. S. pneumoniae is alpha-hemolytic, in that it can break down red blood cells through producing hydrogen peroxide (H2O2). The production of H2O2 by bacterial infection can also cause damage to DNA, killing cells within the lungs causing fluid build up. Symptoms vary and can include high fever, difficulty breathing and coughs producing pink to rusty coloured sputum. In addition, S.pneumoniae can lead to chronic fever, pleuritic pain, arthritis, sinusitis and can enter the bloodstream, causing septicaemia, if not treated. This bacteria affects primarily people younger than two years old and older than sixty-five. Other increased risk factors include alcoholism, influenza, diabetes mellitus and absence of normal spleen function.

Robert K. symptoms are present for both MTB and Streptococcus pneumoniae bacteria and could be caused by either. However, Mycobacterium tuberculosis is more likely due to Robert’s immigration from India, a country with the highest prevalence of MTB in the world [1]. In addition, Robert’s symptoms have persisted for a month, with chronic coughing being a key indicator of tuberculosis. It could have been that Robert got infected with MTB while in India and the bacteria could have been in its latent state, getting activated a month ago leading to his chronic coughs, night sweats and fever.


1.         Tuberculosis [Internet]. [cited 2020 Mar 3]. Available from:

2.         Dunn JJ, Starke JR, Revell PA. Laboratory Diagnosis of Mycobacterium tuberculosis Infection and Disease in Children. J Clin Microbiol. 2016 Jun 1;54(6):1434–41.

3.         Mycobacterium Tuberculosis - an overview | ScienceDirect Topics [Internet]. [cited 2020 Mar 6]. Available from:

4.         Azadi D, Motallebirad T, Ghaffari K, Shojaei H. Mycobacteriosis and Tuberculosis: Laboratory Diagnosis. Open Microbiol J. 2018 Mar 30;12:41–58.

5.         WHO | Tuberculosis and HIV [Internet]. WHO. [cited 2020 Mar 6]. Available from:

6.         LABORATORY INVESTIGATION OF MYCOBACTERIA TUBERCULOSIS AND OTHER MYCOBACTERIA SPECIES [Internet]. Oxford University Hospitals. [cited 2020 Mar 6]. Available from:

7.         Diagnosing Latent TB infection & Disease | Testing & Diagnosis | TB | CDC [Internet]. 2018 [cited 2020 Mar 4]. Available from:

8.         TB Culture Test - TB diagnosis, resistance testing [Internet]. TBFacts. [cited 2020 Mar 6]. Available from:

9.         Lewinsohn DM, Leonard MK, LoBue PA, Cohn DL, Daley CL, Desmond E, et al. Official American Thoracic Society/Infectious Diseases Society of America/Centers for Disease Control and Prevention Clinical Practice Guidelines: Diagnosis of Tuberculosis in Adults and Children. Clin Infect Dis. 2017 Jan 15;64(2):111–5.

10.       Fact Sheets | Testing & Diagnosis | Fact Sheet - Recommendations for Human Immunodeficiency... Clinics | TB | CDC [Internet]. 2019 [cited 2020 Mar 6]. Available from:

Question (ii)

(ii) What samples are taken for laboratory testing and how important is the Microbiology Laboratory in the diagnosis of this infectious disease?

A person with symptoms of tuberculosis, should have their clinical specimens examined in the Microbiology Laboratory. All samples should be collected aseptically. According to the Center for Disease Control and Prevention, mucus that comes from the nose and throat are not good specimens for laboratory testing. All individuals (even asymptomatic ones) who are suspected of having TB at any site in the body must have sputum specimens collected for an AFB smear and culture. A minimum of 3 consecutive sputum specimens are necessary, and each must be collected in intervals of 8- 24 hour intervals with at least one specimen being an early morning specimen. All specimens should be collected in an airborne infection room or another well-ventilated area if possible

There are 4 specimen collection methods for pulmonary TB disease: coughing, induced sputum, bronchoscopy, and gastric aspiration. Coughing is the most common method used for sputum collection, (4) and involves the patient coughing up sputum into a sterile container. Sputum induction involves a patient inhaling a saline mist which causes the patient to cough deeply. A bronchoscopy is when a bronchoscope is passed through either the mouth or nose of the patient and goes directly into the diseased portion of the lung. This results in removal of either sputum or lung tissue. Gastric aspiration is a process in which a tube is inserted through the patient’s mouth or nose and is passed into the stomach to remove a small sample of gastric secretions that contain sputum that has been coughed up into the throat and then swallowed by the patient.

If the diagnosis is suspected to be tuberculous meningitis, congenital TB or infants with disseminated disease, sterile body fluids, such as cerebral spinal fluid (CSF) and pleural fluid, can be collected for laboratory testing. If a small volume of CSF is collected from the initial lumbar puncture and results from cell counts and protein analysis suggest TB meningitis, a second procedure should be done to collect a larger volume and improve chances of achieving a positive culture. Urine samples may also be requested and should be collected in the early mornings on 3 consecutive days in a universal container.

The previous examples show two types of specimen that may be collected: specimen that are

normally contaminated with resident flora or specimen from sterile sites. For only specimen

collected from areas containing resident bacteria, a decontamination step before culture is

required to reduce the likelihood of overgrowth by organism other than Mycobacteria.

Examples of clinical specimens needed for TB culture at the London Health Sciences Center Microbiology Laboratory include:

Abscess or Aspirate Fluid - Collect as much material/ fluid as possible in a sterile container

Blood - Collect into a 6 mL Sodium Heparin top Vacutainer tube and do not spin the blood. Samples must be retrieved in SPS or a heparin tube. Blood samples must be transported at room temperature and it is important not to freeze or refrigerate them (5).

Body Fluids - Collect as much as possible (10-15 mL minimum) in a sterile container.

Bone - Collect in a sterile container.

Bone Marrow - Collect into 6 mL Green top Vacutainer tube or sterile container.

CSF - Collect a minimum of 2mL in a sterile container.

Faeces - Collect a minimum of 2 grams in a sterile container.

Gastric Lavage - Obtain Gastric Lavage Transport media from the Microbiology Laboratory. Collect fasting, early morning sample to recover sputum swallowed during sleep. Following collection, the sample must be neutralized immediately by transferring it to the transport container which contains sodium carbonate.


·       Bronchial Alveolar Lavages - Collect a minimum of 5 mL in a sterile container. Submit a post-bronchoscopy sputum as well.

·       Sputum - Collect 5-10 mL of expectorated or induced sputum. Patient can expectorate several times per collection to obtain minimum volume.

o   Ensure patient does not rinse mouth before expectoration or expectorate saliva or postnasal discharge.

    • For specimens such as sputum that are contaminated with normal bacterial flora, a selective medium containing antimicrobial agents should be used (2).
    • As seen in Robert’s case 3 separate sputum samples over 3 mornings was taken, this is to increase the sensitivity of laboratory tests(6). Specimens collected early morning are recommended in order to obtain sputum swallowed during deep sleep (6) .
    • When transporting samples, they should be sent to the lab as soon as possible. If transport is delayed, the specimen should be stored in a refrigerator until transported to the lab.

·       Tracheal Aspiration - Collect as much as possible in a sterile container.

Tissue - Collect 1 gram of tissue in a sterile container. Select caseous portion if available. A small amount (<1 mL) of sterile saline may be added to keep specimen from drying. Tissue submitted in anaerobic transport is not acceptable for culture.

Urine - Send minimum 40 mL volume of first morning urine in sterile container. Over three consecutive days, one specimen must be collected each day. If the samples can’t be transported within one hour or greater, refrigeration is possible. However, it is important that samples aren’t frozen(5).

The Importance of Laboratory Testing

The culturing and identification of mycobacteria in specimens by the Microbiology Laboratory is necessary for diagnosis of TB. Some TB infections could be unknowingly transmitted to others if laboratory testing was not conducted to confirm the pathogen causing the symptoms. Furthermore, testing in the laboratory is essential to monitor levels of drug resistant strains as well as to implement a proper treatment for the TB depending on the strain. laboratory testing is extremely important to determining that the patient does in fact have TB, and that it has not been misdiagnosed as pneumonia or lung cancer. Microbiology Laboratory testing is required to determine if the infection is indeed TB, as radiological findings can not confer this diagnosis. Without proper treatment, TB disease can lead to the formation of cavities in the lungs, which can further result in bleeding or increase the risks of developing bacterial infections characterized by abscesses. In addition, it is possible for holes between the airways to develop and for airway blockage to occur. Most importantly, without proper treatment, Robert’s illness can be fatal.


1.         Tuberculosis [Internet]. [cited 2020 Mar 6]. Available from:

2.         McMurray DN. Mycobacteria and Nocardia. In: Baron S, editor. Medical Microbiology [Internet]. 4th ed. Galveston (TX): University of Texas Medical Branch at Galveston; 1996 [cited 2020 Mar 6]. Available from:

3.         Pneumonia | Mycoplasma pneumoniae | Signs and Symptoms | CDC [Internet]. 2019 [cited 2020 Mar 6]. Available from:

4.         TB Culture Test - TB diagnosis, resistance testing [Internet]. TBFacts. [cited 2020 Mar 13]. Available from:

5.         Tans-Kersten J. Specimen collection. :68.

6.         Dunn JJ, Starke JR, Revell PA. Laboratory Diagnosis of Mycobacterium tuberculosis Infection and Disease in Children. J Clin Microbiol. 2016;54(6):1434–41.

7.         File:Acid Fast Stain.pdf. In: Wikipedia [Internet]. [cited 2020 Mar 6]. Available from:

8.         Microbiology and Molecular Diagnosis in Pathology | ScienceDirect [Internet]. [cited 2020 Mar 6]. Available from:

9.         Laboratory Test Information Guide [Internet]. [cited 2020 Mar 6]. Available from:

10.       Muchwa C, Akol J, Etwom A, Morgan K, Orikiriza P, Mumbowa F, et al. Evaluation of Capilia TB assay for rapid identification of Mycobacterium tuberculosis complex in BACTEC MGIT 960 and BACTEC 9120 blood cultures. BMC Res Notes. 2012 Jan 19;5(1):44.

11.       Azadi D, Motallebirad T, Ghaffari K, Shojaei H. Mycobacteriosis and Tuberculosis: Laboratory Diagnosis. Open Microbiol J. 2018 Mar 30;12:41–58.

12.       Figure 1: M. tuberculosis stained by fluorescence auramine–rhodamine. [Internet]. ResearchGate. [cited 2020 Mar 6]. Available from:

13.       Tuberculosis Skin Test (PPD): Reading, Results, Side Effects & Risks [Internet]. MedicineNet. [cited 2020 Mar 6]. Available from:

14.       Tuberculosis radiology. In: Wikipedia [Internet]. 2020 [cited 2020 Mar 6]. Available from:

15.       Bhalla GS, Sarao MS, Kalra D, Bandyopadhyay K, John AR. Methods of phenotypic identification of non-tuberculous mycobacteria. Pract Lab Med. 2018 Nov 1;12:e00107.

Question (iii)

(iii) Explain the tests that will be performed on the samples in order to detect any of the potential bacterial pathogens causing this disease.

  1. Middlebrook’s medium (agar based medium) and Lowenstein-Jensen medium (egg based medium): M. tuberculosis colonies are small and off-white colored when grown on either agar based or egg based medium. Both types of media contain inhibitors to keep contaminants from out-growing M. tuberculosis. It will take 4-6 weeks to see visual colonies on either medium. (4) The advantage of culture on solid media vs. both media is that the solid media allows for visualization of colony morphology and pigmentation. This is extremely useful in distinguishing colonies of M. tuberculosis from non tuberculosis mycobacteria. (7)
  2. Acid-fast staining method (The Ziehl-Neelsen strain):  Mycobacteria are classified as acid-fast bacteria due to their impermeability by certain dyes and stains. Upon being stained, acid-fast bacteria retain dyes when they are heated and treated with acidified organic compounds. (4) In this test, the M. tuberculosis smear is fixed and stained with a pink dye called “carbol-fuchsin” and is decolorized with acid-alcohol. This smear is then counterstained with methylene-blue (or other dyes that contrast). Acid-fast bacilli will appear as pink or red rods. They are often beaded, banded, coccoid, or filamentous, and range from 1-10 μm long or 0.2-0.6 μm wide. A positive acid-fast bacteria smear will provide the first indication of mycobacterial infection and potential disease with tuberculosis. This method can evaluate a variety of specimens, including sputum, tissue, and bodily fluids. Unfortunately, acid-fast bacteria microscopy is unable to distinguish between viable and dead bacteria, and is unable to distinguish NTM from MTB.
  3. Fluorescent Acid-fast staining method (Auramine Rhodamine Stain): Rhodamine Auramine is commonly used as the stain in the direct detection of mycobacteria from clinical specimens. The dye used in this method will bind with mycolic acids and becomes fluorescent underneath ultraviolet light. Acid-fast bacteria such as Mycobacteria will appear yellow or orange under UV light. This method is used more for screening for mycobacteria, as the fluorescent stain is only sensitive for the detection of mycobacteria but is not specific. However, the presence of beaded rods is critical in determining that the stain is positive. (8)
  4. Skin Testing (Tuberculin Test/Mantoux Test): Purified protein derivative (PPD) is the test antigen during the Mantoux test. In this test, 0.1 mL volume containing 5 tuberculin units of PPD is injected into the patient’s forearm intradermally. (4) 48-72 hours later, the transverse diameter of induration is measured. The interpretation of this test will vary depending on the lesion (is there any redness? Swelling? Is it raised and hard?) 90% of patients who have a lesion with a diameter of 10 mm or greater are infected with MTB, or have previously been exposed to the pathogen, while 100% of patients with a lesion of 15 mm of greater are currently infected with MTB or were previously exposed to MTB. In the Mantoux Test, false positives may occur if the patient has had prior exposure to vaccines or other mycobacteria.
  5. Rapid Broth Systems (BACTEC): The BACTEC system uses media that contains radio-labeled palmitate as its sole carbon source. (4) As MTB multiplies, it will break down the palmitate and free the radio-labeled CO2. The BACTEC method allows for MTB growth to be detected within 9-16 days.
  6. Nucleic Acid Amplification Test (NAAT): This is a molecular test for TB. NAAT is able to detect the genetic material of mycobacteria. This type of test is used when the AFB smear returns as positive, or when infection with TB is highly suspected. (6) The results from the NAAT test are available anywhere between 1-3 days. Molecular methods of testing for TB are approved for use with respiratory samples, however it is pertinent that they are confirmed with an AFB culture as well.
  7. Chest X-ray: During a chest x-ray, patients  who have infection with TB will have an x-ray that appears normal. However, patients who have TB disease will have an x-ray that shows lesions.
  8. Capilia TB Rapid Diagnostic Test: This is a strip-test that detects the MPB64 antigen that is specifically secreted by the Mycobacterium tuberculosis complex from either positive liquid or solid cultures. This test can confirm the presence of TB bacteria in culture samples. (9)
  9. Niacin Reduction Test: All mycobacteria produce the vitamin niacin. However, certain mycobacteria, such as M. simaie and M. tuberculosis lack the enzyme that is necessary for conversion of niacin. (1) Therefore, accumulation of niacin in the culture medium will indicate the presence of M. simaie and M. tuberculosis. For all other bacteria, this test will be negative.
  10. Nitrate reduction test: Certain mycobacteria are able to reduce nitrate to nitrite. (1) M. tuberculosis is one of these mycobacteria. This nitrate reduction test will show positive results for the following mycobacteria:  M. tuberculosis, M. kanasii, M. szulgai, and M. fortuitum.
  11. Catalase test: Catalase is an enzyme that breaks hydrogen peroxide into water and oxygen. (1) In a catalase test, the presence of oxygen bubbles in the mixture is indicative of a positive test result for mycobacteria, as mycobacteria possess catalase enzymes.
  12. Culture: The purpose of culturing is to determine the presence of MTB. (5) This test is recommended by the World Health Organization as it is the gold standard for diagnosis of TB disease. (2) There are two types of media that are most often used to grow MTB cultures: Middlebrook’s Medium, which is agar based, and Lowenstein-Jensen, which is an egg based medium. (4) These mediums allow for MTB to grow easier. While creating a culture using the patient’s sputum as a sample, the sputum is first treated with NaOH in order to kill all the remaining bacteria, thus leaving only NaOH. This is a result of MTB’s physiological resistance to alkaline compounds by virtue of their lipid layer. (4) The culture may take 4-6 weeks before visual colonies become present on either type of media. This is a long time for a patient to have to wait for test results, and thus the BACTEC system may be used as well as it has much faster results. In this test, chains of cells in smears that are made from in-vitro grown colonies will often form distinctive serpentine cords. (4)
  13. IFN-Gamma release assays (IGRAs): The IGRA test is able to measure either the IFN-gamma that is secreted by the patient’s T cells or the amount of IFN-gamma secreting lymphocytes upon in vivo simulation with MTB specific antigens. These antigens can not be found in most NTM species, or in BCG vaccine strains. (2) These antigens are mixed with fresh blood samples. Either the IFN-gamma concentration or the amount of IFN-gamma producing cells is measured. (3) Two TB blood tests have been approved by the U.S. FDA that are currently available: the QFT-GIT and the T-Spot test. One of the major advantages of using the IGRA is that previous vaccination with BCG does not result in a false-positive IGRA test result. In addition, in pediatric studies it has been demonstrated that IGRAs possess higher specificities that that of TST for tuberculosis infection. (2)
  14. Gram-staining: In regards to infection with MT, the classic laboratory technique of gram-staining is ineffective. This is because MTB has neither gram-positive or gram-negative chemical properties. (4) As a result, the stain will appear very weak, or show no stain at all.


1. Azadi D, Motallebirad T, Ghaffari K, Shojaei H. Mycobacteriosis and Tuberculosis: Laboratory Diagnosis. Open Microbiol J. 2018 Mar 30;12:41–58.

2. Dunn JJ, Starke JR, Revell PA. Laboratory Diagnosis of Mycobacterium tuberculosis

Infection and Disease in Children. J Clin Microbiol. 2016 Jun 1;54(6):1434–41.

3. Fact Sheets | Testing &amp; Diagnosis | Fact Sheet - Recommendations for Human

Immunodeficiency... Clinics | TB | CDC [Internet]. 2019 [cited 2020 Mar 6]. Available


4. Tuberculosis [Internet]. [cited 2020 Mar 3]. Available from:

5. TB Culture Test - TB diagnosis, resistance testing [Internet]. TBFacts. [cited 2020 Mar 6].

Available from:

6. Laboratory Test Information Guide [Internet]. [cited 2020 Mar 6]. Available from:

7. McMurray DN. Mycobacteria and Nocardia. In: Baron S, editor. Medical Microbiology [Internet]. 4th ed. Galveston (TX): University of Texas Medical Branch at Galveston; 1996 [cited 2020 Mar 6]. Available from:

8. Microbiology and Molecular Diagnosis in Pathology | ScienceDirect [Internet]. [cited 2020 Mar 6]. Available from:

9. Muchwa C, Akol J, Etwom A, Morgan K, Orikiriza P, Mumbowa F, et al. Evaluation of Capilia TB assay for rapid identification of Mycobacterium tuberculosis complex in BACTEC MGIT 960 and BACTEC 9120 blood cultures. BMC Res Notes. 2012 Jan 19;5(1):44.

Question (iv)

(iv) What are the results expected from these tests that might allow the identification of the bacteria implicated in this case.

M. tuberculosis  Test Result
Interferon-Gamma Release Assays (IGRAs) (IGRAs) are blood tests which help to measure a person’s immune response to bacteria causing TB. This is a more accurate measure than the skin test, as it can detect the presence of biomarkers like specific cytokines, known as interferon gamma, which are present during infection. Interferon-gamma will be released in the presence of Mycobacterium tuberculosis.
Acid-fast staining method

The Ziehl-Neelsen stain

Robert’s test will show Acid-fast bacilli appear pink/red red rods (1–10 μm long and 0.2–0.6 μm wide) as beaded, banded, coccoid or filamentous (1)
Nitrate Reduction Test Positive test is indicated by a change of color from amber to pink or red. If no change of color, it is negative (3).
Chest- X ray For people who have TB infection the X-ray would appear normal, people who have TB disease would reveal lesions.
Niacin Test While B group vitamins are produced by all Mycobacteria, only 2 lack the enzyme for conversion of niacin, a B vitamin, to ribonucleotides: M. simiae and M. tuberculosis. This leads to the accumulation of niacin which is important for the identification of M.tuberculosis (4).

Positive test = pink or red colour

Negative test = yellow colour

Nucleic Acid Amplification Test (NAAT) Robert’s NAAT test will test positive for genetic material of mycobacteria.
Pleural fluid testing Exudate fluid is an indication of infection. If the patient is infected with Mycobacterium tuberculosis, the plural fluid will be a serous exudate. If the patient is infected with Streptococcus pneumoniae, the fluid will appear clear (5).
Capilia TB Rapid Diagnostic Test Robert’s strip test will show 2 pink lines to indicate positive detection of MPB64 antigen specifically secreted by the Mycobacterium tuberculosis complex (MTC) sample.
Skin Testing

Tuberculin Test/ Mantoux Test

Robert’s skin test will show a lesion of 15 mm or greater. False positives can occur if there was prior exposure to vaccines or other mycobacteria.
Fluorescent Acid-fast staining method – Auramine Rhodamine Stain Robert’s results will  have mycobacteria appear yellow or orange beaded rods under ultraviolet light (2).
Rapid Broth Systems– Bactec Using the BACTEC system,  Robert’s results will show radio-labelled CO2.  
Catalase Test Catalase is a soluble intracellular enzyme that breaks down hydrogen peroxide into water and oxygen. The presence of oxygen bubbles in mixture indicates a positive test (4). Results for MTB should be negative for both the 68 degree catalase test or SQ catalase test
Middlebrook's medium – agar based medium

Lowenstein-Jensen medium – an egg based medium.

Robert’s test will show small and off-white coloured MTB colonies grown on either medium, which will indicate a positive MTB result (1).


  1. Tuberculosis [Internet]. [cited 2020 Mar 6]. Available from:
  2. Microbiology and Molecular Diagnosis in Pathology | ScienceDirect [Internet]. [cited 2020 Mar 6]. Available from:
  3. Bhalla GS, Sarao MS, Kalra D, Bandyopadhyay K, John AR. Methods of phenotypic identification of non-tuberculous mycobacteria. Pract Lab Med. 2018 Nov 1;12:e00107.
  4. Azadi D, Motallebirad T, Ghaffari K, Shojaei H. Mycobacteriosis and Tuberculosis: Laboratory Diagnosis. Open Microbiol J. 2018 Mar 30;12:41–58.
  5. Karkhanis, V., & Joshi, J. (2012). Pleural effusion: diagnosis, treatment, and management. Open Access Emergency Medicine, 31. doi: 10.2147/oaem.s29942

Q3. Bacterial Pathogenesis Questions

Using the following pathogenic steps outline the pathogenesis of the bacteria implicated as being responsible for this infection.

Question (i)

(i) Encounter: where does the organism normally reside, geographically and host wise, and what are the bacterial characteristics that leave it suited to these places of residence. How might our patient have come in contact with this bacteria?

Tuberculosis (TB) is an airborne disease caused by the bacteria Mycobacterium tuberculosis (M. tuberculosis), and it is the leading cause of death in the world from a bacterial disease [1]. M. tuberculosis is an obligate aerobe bacteria, so it is able to survive in the lungs due to the constant supply of oxygen [1]. Specifically, TB can be found in the upper lobes or upper areas of lower lobes which are the most oxygenated areas. Humans are the only reservoir for this bacterium.  Within the body, it mainly impacts the lungs and larynx, but can also cause infection in the brain, spinal cord, kidneys, or lymph nodes in severe cases [2]. Robert exhibits symptoms of infection, particularly those for TB of the lungs, so he definitely has TB disease. Within one year, patients with this form of active TB disease are capable of infecting up to 5-15 others via close contact [3].

Normal Geographical Residence and Relevant Characteristics

TB occurs in every part of the world and affects about 1.8 billion people per year [1]. TB is most commonly found in the Western Pacific/South-East Asian area and Africa [4]. The World Health Organization has a TB a “high burden country” list for the 2016-2020 period, which accounts for 85-89% of the global burden. The TB HBC list includes Angola, Bangladesh, Brazil, Cambodia, China, Congo, Central African Republic, DPR Korea, DR Congo, Ethiopia, India, Indonesia, Kenya, Lesotho, Liberia, Mozambique, Myanmar, Namibia, Nigeria, Pakistan, Papua New Guinea, Philippines, Russian Federation, Sierra Leone, South Africa, Thailand, the United Republic of Tanzania, Vietnam, Zambia and Zimbabwe [5].

In 2018, eight of the 30 HDCs accounted for two thirds of total TB. India led the statistics, followed by China, Indonesia, the Philippines, Pakistan, Nigeria, Bangladesh and South Africa (World Health Organization, n.d.). This is of relevance to our case because Robert immigrated from India. Worldwide, TB is one of the top 10 causes of death (leading cause from a single infectious agent), and in 2018, a total of 1.5 million people died from this disease [6]. People infected with the TB bacteria have a 5-15% lifetime risk of falling ill with TB.

There are predisposing factors to TB, including close contact with large populations of people (or overcrowding), poor nutrition, IV drug use, alcoholism, poor healthcare and HIV infection (the largest predisposing factor) may contribute to the increased risk of TB in India and many other developing countries [1]. Factors such as compromised immune systems, malnutrition, infection with HIV/AIDS, or smoking can increase this risk. However, it is still important to note that even in low TB endemic countries, such as the Netherlands, clinicians still face patients who have a suspected mycobacterial infection. Even though there are high burden countries, TB can occur worldwide (Figure 1) [7] .

Various TB strains exist, all with different geographical spread. TB bacteria can be divided into “generalists,” with worldwide distribution, and “specialists,” that have a localized ecological niche [8]. “Generalists” show a slightly increased diversity of their antigens compared to specialists, allowing them to spread more globally [8]. The global distribution and genetic diversity and Mycobacterium tuberculosis lineages are shown in Figure 2 and 3 [9]. For example, Lineage 2 strains were more likely to cause transmission chains and are associated with drug resistance, possibly reflecting how they can spread quickly through populations [10][11]. Examples of “specialists” are the West African lineages 5 and 6 strains, which are geographically restricted to West Africa. The reasons for this are largely unknown, however, clinical characteristics of the patients (such as being HIV-positive and malnourished) are thought to play a role [9][12][13]. Another example of a “specialist” is the M. tuberculosis sub-lineage LAM9C3, which showed phylogeographical specificity to the Old World (considered Africa, Asia, and Europe). This could be due to migration histories and adaptations to human hosts of specific M. tuberculosis clones [14]. The host-pathogen relationship in TB is sympatric, meaning that the host and pathogen usually share a common geographical origin [15], which can help explain the prevalence and distribution of TB.

Geographic survival (and host!) of the bacteria primarily comes from its cell wall envelope [16].  It contains a-alkyl B-hydroxy mycolic acids which makes the permeability of these bacterial cells very low. As a result, they are able to live longer in various environments because the envelope prevents many extraneous substances from getting in. For example, the envelope provides resistance to a few disinfectants (ex. Chlorhexidine gluconate). Furthemore, the high concentration of lipids in the cell wall has been associated with impermeability to stains and dyes and resistance to antibiotics [1]. The bacterium can survive for months on dry, inanimate surfaces, for 74 days in sunlight-free conditions, and 8 weeks in feces [17].

Figure 1. Annual tuberculosis incidence (per 100,000 population), by region worldwide in 2018 [7]
Figure 2. Global distribution and genetic diversity of Mycobacterium tuberculosis complex phylogenetic lineages [9].
Figure 3. Distribution of Mycobacterium tuberculosis complex phylogenetic lineages by regions [9].

Normal Host Residence and Relevant Characteristics

M. tuberculosis is not part of the normal flora of humans [1]. As a result, humans are the only known reservoir for M. tuberculosis. However, many non-pathogenic mycobacteria can be part of the normal human flora in dry and oily regions. All age groups are at risk of contracting this disease, but it mainly affects adults in their most productive years [4]. Those who are infected with the bacterium have a 5-15% risk of progressing to active TB. Individuals with HIV are 19x more likely to develop active TB and immunocompromised patients are also at higher risk. For example, malnourished people are 3x more likely to progress into active TB. Furthermore, alcohol use increases risk of TB disease by a factor of 3.3, whereas tobacco smoking increases it by 1.6. M. tuberculosis needs a way to by-pass the upper airway, an area fraught with toll-like receptor (TLR) stimulating commensal bacteria [18]. To do this, the small aerosol infection droplets that the bacteria are contained in deliver M. tuberculosis directly into the alveolar spaces in the lower lung, which contains essentially no commensal bacteria [19].

Being an obligate aerobe, the bacteria normally resides within the lungs of its host [1]. TB mycobacteria can be found in the mouth and upper airway upon transmission, but usually resides in the lungs (specifically the alveoli in the lower lungs) and regional lymph nodes, which can cause lymphadenitis (Figures 4 and 5) [20][18]. Other extrapulmonary manifestations can be in the central nervous system, where it can cause meningitis (an inflammation of the protective membranes covering the brain and spinal cord), the urogenital tract, the digestive system, and cutaneously, known as lupus vulgaris [21]. M. tuberculosis can be present in granulomas at any site in the body if the infection has spread systemically [20].

M. tuberculosis is also a facultative intracellular parasite, and has mechanisms to survive within host cells, such as macrophages. Upon entry into the alveoli, the bacterium are thought to first encounter resident macrophages. To survive within the host, the bacteria possess a number of traits that help them achieve this.

Cell wall :

Over 60% of the mycobacterial cell wall is made out of lipids [1]. The bacteria have a complex cell wall  three main components: mycolic acids, cord factor, and wax-D. Mycolic acids are alpha-branched lipids that are located in the cell walls of the bacterium, and they are very hydrophobic and contribute to the formation of a lipid shell around the organism [20] [1]) (Figure 7). This peculiar cell wall structure provides a strong, impermeable barrier to noxious compounds and drugs. The lipid content consists of It is thought that they may confer resistance to attacks from cationic proteins, lysozyme, and oxygen radicals from the phagocytic granule. Furthermore, they protect extracellular mycobacteria from complement deposition in serum. This allows the bacteria to resist stress, letting M. tuberculosis maintain residence in the host [22][23].

Cord factor is a glycolipid on the cell walls of mycobacteria, which not only causes the bacteria to grow in serpentine cords (chains of cells), but is also toxic to mammalian cells and inhibits polymorphonuclear leukocyte migration [1]. Wax-D is a lipid that is a major component of Freund’s complete adjuvant. The high concentration of lipids in the cell wall have been associated with resistance to osmotic lysis from complement deposition, resistance to lethal oxidations and survival inside macrophages, and resistance to killing by acidic and alkaline compounds. Another glycolipid of the cell wall is LAM, a complex glycolipid that is an immunomodulator. It functions to decrease IFN-gamma production from macrophages, as well as scavenge oxygen radicals and inhibit the host protein kinase C.

Outer membrane proteins (OMPs) are incorporated in the cell wall and they function to facilitate the uptake of small, hydrophilic molecules as well as efflux mechanisms. They are also involved in attachment and invasion of host cells. Another cell wall surface protein is exported receptive protein (Erp), which is involved in multiplication of the bacteria in macrophages.

Resistance to Acidity:

Once in the alveoli, macrophages engulf the bacteria. However, M. tuberculosis can resist intracellular destruction, multiply, and kill the macrophage [20]. In this way, the bacteria can continue residing in the lung tissue. The mechanisms for this are still unknown, however, some explanations may include prevention of lysosome-phagosome fusion, prevention of acidification of the phagolysosome, or direct cytotoxicity of the bacterium to the macrophage [20]. For example, the bacteria can exclude a proton ATPase from the phagosome of non-activated macrophages [23]. Furthermore, when M. tuberculosis upregulates many genes when in the phagosome of macrophages, providing another mechanism for combating acidity [24].

OmpATb also particularly has the ability to function in extremely acidic environments (such as host phagosomes), which facilitates survival of the bacterium. The efflux properties of OMPs may also contribute to drug resistance by pumping out antibiotics. KefB is a protein that uptakes protons from the host phagosome and exports K+ from the bacterial cytoplasm in order to increase the luminal pH. This inhibits phagosomal acidification by macrophages, enabling the bacteria to evade these host defense mechanisms.

Secretion Proteins:

The bacterium possesses five type seven secretion systems (T7SS). These systems secrete certain immune modulators during the course of the macrophage infection cycle. One of the systems secretes proteins that are essential for moving the bacteria to the host cytosol, and another system induces apoptosis of host cells after this movement has taken place. The protein secretion system ESX1 allows bacteria to translocate from the phagosome into the safer cytosol by interfering with the integrity of the phagosomal membrane, leading to rupture [25]. ESX5 is thought to secrete proteins with immunomodulatory properties [22]. ESX3 is involved in zinc and iron uptake and homeostasis to facilitate growth (Figure 6) [26].

Host Cell Entry/Adherence:

Fibronectin binding protein (Fbp) is a bacterial complex consisting of 3 proteins that bind to fibronectin, which allows the bacteria to adhere to mucosal host surfaces and begin entry. It also helps make cell wall lipids. Mammalian cell entry (Mce) proteins allow mycobacteria to enter host cells and survive in macrophages (and cause latent TB), and these proteins are also involved in the transport of important molecules (ex. cholesterol). M. tuberculosis also has an abundance of lipoproteins which are involved in transport, cell wall metabolism, signalling, protein degradation, and cell adhesion.

Combating Oxidative Stress:

Upon phagocytosis of a bacterium, host macrophages will produce reactive oxygen species (ROS) and reactive nitrogen species (RNS) which can harm/kill the bacteria by damaging bacterial proteins, lipids, and other targets. The first defense the bacteria has against this, is it’s fairly impermeable cell wall. However, there are a number of proteins that also assist in evading these host defenses. Acr proteins are made in response to hypoxia (or nitric oxide) and may help promote long-term survival and viability of bacteria in latent TB. Rv2136c and PonA2 increase the tolerance of the bacteria to low pH conditions (such as in the phagosome), as well as to ROS and RNS. Alkyl hydroperoxide reductase C (AhpC) and catalase (some of which are upregulated during early infection) decreases organic peroxides enabling bacteria to hide from the damaging effects of the respiratory oxidative burst that occurs within macrophages [27][1]. Superoxide dismutase (Sod) functions to decrease the amount of ROS by facilitating the conversion of O2- to oxygen and hydrogen peroxide. M. tuberculosis also inhibits phagosome arresting by preventing phagosome-lysosome fusion. For example, PtpA is a tyrosine phosphate that inhibits a host protein that is needed for the membrane fusion of the phagosome and lysosome, thus preventing phagosome maturation. The Ndk protein (nucleoside diphosphate kinase) prevents the recruitment of effectors needed for phagosome maturation.

M. tuberculosis also contains mycothiol, a unique substance with cysteine residues, allowing the bacteria to combat oxidative stress [23]. UvrB, a subunit in the UvrABC enzyme complex in bacteria, repairs DNA damage and also protects against oxidative stress [28][29].

Evading the Host Defenses/Immune System:

Normally, the host will induce apoptosis of infected cells to limit the spread of the infection, however, M. tuberculosis has a few proteins that have enabled it to evade this defense mechanism. For example, NuoG is involved in the inhibition of the host TNF-a dependent apoptosis pathway, as well as the suppression of apoptosis in neutrophils. Because of M. tuberculosis’s slow generation time, the immune system may not readily recognize the bacteria or be triggered sufficiently to eliminate them [1]. Since the bacteria grow intracellularly, they cannot be targeted by host antibody immune responses [1].

Furthermore, M. tuberculosis can commonly form latent infections, where the bacteria can be re-activated to cause disease. Little is known about the establishment of latent infections; however, they may be determined by the interaction with adaptive immune responses. In this scenario, the host is able to control the infection but not completely eradicate the bacteria. In this way, the bacteria can continue to survive in the host, especially during stressful scenarios, and possibly reactive to continue spreading [30].

Another survival and virulence characteristic of M. tuberculosis is its use of various extracellular proteases to evade host defenses and induce damage to the host [31]. Metalloproteases use metals in their catalytic activities. M. tuberculosis has some zinc-dependent metalloproteases that contribute to bacterial survival in the host. Zmp1 cleaves host proteins that are part of a protein complex that contributes to the activation of inflammation. The removal of these proteins prevents the host from generating IL-1B, and this means a weaker host response to the TB infection. Rip1 is another zinc-dependent metalloprotease that degrades anti-sigma factors so that sigma factors are freed and capable of directing RNA polymerases to aid in bacterial cell envelope formation. Serine proteases are secreted by the bacteria and they function to cleave random, non-specific host proteins so that the bacteria have an abundance of importable peptides.

Other characteristics:

M. tuberculosis may also be capable of surviving the host by using the small amounts of carbon monoxide produced by immune cells as an energy source [32]. Nitrate reductase allows the bacteria to grow in anaerobic conditions, using NO3 as the final electron acceptor [1].

M. tuberculosis possesses almost 190 transcriptional regulators that are encoded in its genome [33]. Some of these have been shown to respond to environmental conditions that may impose a lot of stress onto the bacteria, such as extreme temperatures, nutrient starvation, and oxidative stress. To prolong their survival in these conditions, the bacterium has evolved to adapt to the environment by turning on and off transcription of specific regulators at certain times.

Lastly, M. tuberculosis may not always be able to take up residence. For example, some of the pathogens can be expelled by ciliated mucosal cells if they become trapped in the upper airways [34].

Figure 4. Presence of tuberculosis causing Mycobacteria in the human body [35]
Figure 5. Wider distribution of Mycobacteria tuberculosis in the human body [20].
Figure 7. Complex cell wall structure of Mycobacteria tuberculosis [20]

Our Patient and TB

Almost all M. tuberculosis infections occur by airborne transmission of droplet nuclei containing viable, virulent organisms from the infected individual [20]. This usually occurs when the patient comes in contact with air from a patient with active pulmonary TB [22]. When patients with TB in the lungs cough, sneeze, or spit, they transmit the TB bacteria into the surrounding air and environment (Figure 8).

Figure 8: Spread of droplet nuclei [1]
Figure 6. Mycobacteria tuberculosis protein excretion systems that aid in bacterial virulence [22].

Only a few inhaled bacteria are sufficient to cause one to be infected. This creates airborne droplets that contain the bacteria, and since these droplets are so small (no more than 3 bacilli), particles can remain suspended in the air for hours due to the small size of the droplet nuclei [1]. Coughing generates about 3000 droplet nuclei (talking for five minutes will do the same), but sneezing generates even more and can spread to people up to 10 feet away.

The body’s ability to effectively limit or eliminate the infection is determined by the individual’s immune status, genetic factors, and whether this is their primary or secondary exposure to the pathogen [36]. Furthermore, when people progress from latent to active TB, their symptoms may be mild for a long period of time [4]. As a result, they may be very delayed in seeking care and are capable of transmitting the disease to other people through close contact the whole time. TB begins once the droplets reach the alveoli; larger droplets may get lodged in the upper respiratory tract where infection is unlikely or slow to occur, but smaller droplet nuclei can reach the small spaces of the alveoli and initiate infection.

Once the bacteria are in the lungs, there can be a few possible outcomes. First, the host response and defense may sufficiently eliminate the bacteria, thus preventing the onset of TB. Second, the bacteria may start to invade and multiply immediately, initiating active TB. Third, the host response may control the bacteria, but not completely eliminate them, leading to latent TB (bacteria are dormant). Four, the latent bacteria may reactivate sometime in the future and progress into active TB (occurs in 5-15% of people with latent TB). However, TB is NOT spread by hand-to-hand contact, sharing food/drinks, touching bed linens or toilet seats, kissing, or sharing toothbrushes [37].

In Robert’s case, he likely contracted the disease by inhaling droplet nuclei from someone with TB. He may have also come into contact with the bacteria in compact areas, such as an airplane. The droplets may have been spread to Robert through an infected individual, who had laryngeal or pulmonary TB who sneezed, coughed, laughed or shouted. Robert may have become sick during his time in India. India has the largest number of TB cases in the world, with 2.79 million people becoming ill in 2016 in India [38]. Since TB can manifest as a latent infection, it is very likely that Robert came into contact with M. tuberculosis in India and had a reactivation of the disease while in the United States. This would explain why he is just starting to feel symptoms recently, even though he likely contracted the infection long ago.


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  25. van der Wel, N., Hava, D., Houben, D., Fluitsma, D., van Zon, M., Pierson, J., Brenner, M. and Peters, P. (2007). M. tuberculosis and M. leprae Translocate from the Phagolysosome to the Cytosol in Myeloid Cells. Cell, 129(7), pp.1287-1298.
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Question (ii)

(ii) Entry: how does the bacteria enter into the human host and take up residence. What are the molecular, cellular and/or physiological factors at play in this site specificity and in the initial adherence step (referencing both the bacteria and the host)..

Entry into host:

When an infected individual coughs, laughs, shouts or sneezes, the droplets expelled evaporate into droplet nuclei that are 1-micron-sized, having characteristics that enable them to remain suspended in the air, and be inhaled by another person, deep enough into their lungs (1). Following deposition of the bacteria in the alveoli of the lungs, the bacteria are engulfed by alveolar macrophages (2).

Figure 1. M. tuberculosis entry into host

Residence in cells:

The first contact of M. tuberculosis is usually with alveolar resident macrophages, and this interaction is mediated by bacterial contact with macrophage mannose and/or complement receptors, resulting in receptor-mediated phagocytosis of bacteria (3). M. tuberculosis binds directly to mannose receptors on macrophages through LAM (the cell wall-associated mannosylated glycolipid), or indirectly via Fc receptors or complement receptors (4).

Mannose receptor activity can be upregulated by surfactant protein A, a glycoprotein on alveolar surfaces, which in turn enhances the binding and uptake of M. tuberculosis (3). Surfactant protein D blocks mannosyl oligosaccharide residues on bacterial cell surfaces, which interact with mannose receptors on macrophages, to inhibit phagocytosis of M. tuberculosis (5). TLR2 (human toll-like receptor 2) is also involved in M. tuberculosis uptake (3). Upon entering a host macrophage, M. tuberculosis initially resides in the phagosome (an endocytic vacuole) (3).

Specifically, M. tuberculosis enters macrophages by using the complement receptors CR1, CR3, and CR4 present on macrophages (6). Complement receptor CR3 is the main integrin of phagocytic cells (polymorphonucleocytes and mononuclear phagocytes), which functions in homotypic aggregation and adhesion of leukocytes to endothelial cells, phagocytosis and subsequent passage into inflamed organs (6). These processes are mediated by the interaction of CR3 with ligands such as fibrinogen, C3bi, and ICAM-1 (CD54) (6). CR4 performs the same functions as CR3 with the same ligands and is an important phagocytic receptor for this bacterium on alveolar macrophages (6). CR1 (a monovalent transmembrane protein) phagocytoses M. tuberculosis that is opsonized by C3b, one of the degradation products of the complement component C3 (7). LFA-1 (β2 integrin lymphocyte function-associated antigen 1) also plays a role in adhesion, specifically in leukocyte adhesion to endothelial cells (6). LFA-1 does so by interacting with one of its three counter receptors ICAM-1, ICAM-2 or ICAM-3, which are members of the immunoglobulin superfamily (6).

Figure 2. Mannose and complement receptors of a macrophage

During phagocytosis, macrophages produce oxidative bursts of reactive oxygen and nitrogen intermediates (8). To escape these oxidative bursts, M. tuberculosis uses its thick cell wall and phospholipase D to block the action of ROS (reactive oxygen species) (8). Furthermore, through the production of catalase peroxidase and using its peroxidase activity, M. tuberculosis can resist reactive nitrogen and oxygen intermediates (8). The genes of the TrxB2 and KatG enzymes of M. tuberculosis are increased by NO and H2O2, which in turn provide resistance to an oxidative environment (8).

Cellular and Molecular Factors:


HBHA (a surface exposed fibronectin binding protein), through its C-terminal lysine rich domain, interacts with heparan sulphate proteoglycans on the cell surface to facilitate adherence of mycobacteria to epithelial cells (9). HBHA binds M. tuberculosis to epithelial cells but not phagocytic cells, possibly playing a role in extra-pulmonary spread (4).


M. tuberculosis also produce pili, which could be involved in colonization initially (4). These pili, called MTP (Mycobacterium tuberculosis pili), are made of thin coiled, aggregated fibers, and act as adhesins (4). MTP bind to laminin (an extracellular matrix protein), functioning as an adherence factor (10). The pili are also involved in biofilm formation and bacterial aggregation (10). MTP function as an invasin and adhesin of M. tuberculosis, allowing this bacterium to adhere to and invade host macrophages (10).


ManLAM (Mannose-capped lipoarabinomannan) is an amphipathic lipoglycan that mediates M. tuberculosis entry into phagocytes in the alveolar space (11). It does so by mediating M. tuberculosis binding to PRRs such as MR (mannose receptor), DC-SIGN (on dendritic cells), TLRs (on macrophages), and others (11).


Another protein involved in adherence is PE-PGRS, a polymorphic acidic, glycine-rich protein, which is also involved in fibronectin binding (4). M. tuberculosis PE-PGRS facilitates entry into macrophages through interaction with TLR2 (12). Activation of TLR2 by PE-PGRS activates the pro-adhesive pathway in macrophages by enhancing avidity of CR3 for mycobacteria (12).

Figure 3. Cell wall structure of M. tuberculosis


1. Talbot EA, Raffa BJ. Molecular Medical Microbiology [Internet]. 2nd Hanover, NH, USA: Elsevier; 2015. Chapter 92, Mycobacterium tuberculosis. [cited 2020 Mar 6]. Available from: (Links to an external site.)

2. McMurray DN. Mycobacteria and Nocardia. In: Baron S, editor. Medical Microbiology. 4th ed. Galveston (TX): University of Texas Medical Branch at Galveston; 1996. Chapter 33. Available from: (Links to an external site.)

3. Smith I. Mycobacterium tuberculosis pathogenesis and molecular determinants of virulence. Clin Microbiol Rev [Internet]. 2003 Jul [cited 2020 Mar 6];16(3):463–496. Available from: (Links to an external site.). DOI:10.1128/cmr.16.3.463-496.2003

4. Todar K. Todar's Online Textbook of Bacteriology [Internet]. Madison, Wisconsin: University of Wisconsin Department of Bacteriology; 2008-2012. Available from: (Links to an external site.)

5. Ferguson JS, Voelker DR, McCormack FX, Schlesinger LS. Surfactant Protein D Binds to Mycobacterium tuberculosis Bacilli and Lipoarabinomannan via Carbohydrate-Lectin Interactions Resulting in Reduced Phagocytosis of the Bacteria by Macrophages. J Immunol [Internet]. 1999 Jul 1 [cited 2020 Mar 13];163(1):312-321. Available from: (Links to an external site.).

6. DesJardin LE, Kaufman TM, Potts B, et al. Mycobacterium tuberculosis-infected human macrophages exhibit enhanced cellular adhesion with increased expression of LFA-1 and ICAM-1 and reduced expression and/or function of complement receptors, FcγRII and the mannose receptor. Access Microbiol [Internet]. 2002 Oct 1 [cited 2020 Mar 6];148(10):3161-3171. Available from: (Links to an external site.). DOI: (Links to an external site.)

7. Hu C, Mayadas-Norton T, Tanaka K, et al. Mycobacterium tuberculosis Infection in Complement Receptor 3-Deficient Mice. J Immunol [Internet]. 2000 Sep 1 [cited 2020 Mar 6];165(5):2596-2602. Available from: (Links to an external site.). DOI: 10.4049/jimmunol.165.5.2596

8. Zhai W, Wu F, Zhang Y, Fu Y, Liu Z. The Immune Escape Mechanisms of Mycobacterium Tuberculosis. Int J Mol Sci. [Internet]. 2019 Jan 15 [cited 2020 Mar 10];20(2):340. Available from: (Links to an external site.). DOI:10.3390/ijms20020340

9. Esposito C, Marasco D, Delogu G, et al. Heparin-binding hemagglutinin HBHA from Mycobacterium tuberculosis affects actin polymerization. Biochem Biophys Res Commun [Internet]. 2011 Jul 1 [cited 2020 Mar 6];410(2):339-344. Available from: (Links to an external site.). DOI: (Links to an external site.)

10. Ramsugit S, Pillay M. Mycobacterium tuberculosis Pili Promote Adhesion to and Invasion of THP-1 Macrophages. Jpn J Infect Dis [Internet]. 2014 [cited 2020 Mar 6];67(6):476-478. Available from: (Links to an external site.). DOI: (Links to an external site.)

11. Turner J, Torrelles JB. Mannose-capped lipoarabinomannan in Mycobacterium tuberculosis pathogenesis. Pathog Dis [Internet]. 2018 Jun [cited 2020 Mar 6];76(4):1-15. Available from: (Links to an external site.). DOI:10.1093/femspd/fty026

12. Palucci I, Camassa S, Cascioferro A, et al. PE_PGRS33 Contributes to Mycobacterium tuberculosis Entry in Macrophages through Interaction with TLR2. PLoS One [Internet]. 2016 Mar 15 [cited 2020 Mar 6];11(3):1-15. Available from: (Links to an external site.). DOI:10.1371/journal.pone.0150800

Question (iii)

(iii) Multiplication and Spread: does the organism remain at the entry site and/or does it spread beyond the initial site. Are there, for instance, secondary sites of infection. Does the organism remain extracellular and/or does it enter into cells - and what are the molecular and cellular determinants of these events.

Molecular and cellular determinants of bacterial spread

Does the bacteria remain at the entry site?

In short, M. tuberculosis does not necessarily remain at the entry site and can spread to secondary sites of infection. In order to escape its initial site of infection, M. tuberculosis can use adhesins to adhere to and invade the barrier cells and use toxins to lyse these cells in order to increase barrier permeability, ultimately crossing the barriers (Bermudez et al., 2002). These adhesins include HBHA (heparin-binding haemagglutinin) and the secreted effector ESAT-6 (Bermudez et al., 2002). HBHA fosters M. tuberculosis adherence to AECs (alveolar epithelial cells), resulting in extrapulmonary dissemination (Bermudez et al., 2002). ESAT-6 is a laminin-binding adhesin that binds AECs and is also a pore-forming toxin that lyses AECs and macrophages (Bermudez et al., 2002).

When bacterial numbers reach a certain threshold within a macrophage, the host cell undergoes apoptosis and the bacteria can return to the extracellular space. Then, new, uninfected macrophages will be recruited and  the pathogen will be phagocytosed again, allowing the bacteria to spread. This coordinated macrophage death and re-phagocytosis is organised by the bacterial protein secretion system ESX-1, most likely through ESAT 6, which has been shown to induce apoptosis of infected cells and recruit macrophages (Davis and Ramakrishnan, 2009). Overaction of host MMP9, a class of enzymes that are involved in the degradation of the extracellular matrix, can also contribute to increased granuloma formation (Volkman et al., 2009).

How does the granuloma form?

Figure 1. Multiplication and spread of Mycobacteria tuberculosis in a human host (Cambier, Falkow and Ramakrishnan, 2014)

As M. tuberculosis invades the tissue, chemokines produced by macrophages and pneumocytes will attract immune cells, such as NK cells, T cells, and neutrophils, which will promote inflammation and tissue remodelling (Feng et al., 2006). M. tuberculosis will replicate and eventually stimulate an inflammatory focus that matures into a granulomatous lesion (or tubercle), a mononuclear cell infiltrate surrounding a core of degenerating epithelioid and multinucleated giant (Langhans) cells. TNF-α and IFN-γ accelerate immune cell infiltration; however, they are not essential for granuloma initiation (Flynn et al., 1995). Nonetheless, the T cells that are recruited to the site of infection will initiate a cell-mediated response that will contribute to the granuloma. Specifically, a Th1-type immune response as well as pro-inflammatory cytokines, such as IFNγ, IP-10, RANTES, TNF‐α, and IL-12, are involved in forming and maintaining the integrity of the granuloma. Paradoxically, however, this response will also lead to cell death at the center of the lesion, resulting in the formation of a caseous lesion.

How does the granuloma contribute to bacterial spread?

Eventually, liquefication of the caseous material occurs and erosion of the tubercle into an adjacent airway can result in cavitation (the forming of a cavity) and the release of large numbers of bacteria into the sputum (McMurray, 1996). This is called a transitive granuloma, and it is characterized by immunopathology (immunosuppression), neutrophil dominance, bacterial growth, Th1/Th2 disbalance, and superinfection (Ehlers and Schaible, 2013). Moreover, host TNF deficiency can allow bacteria to grow inside macrophages that subsequently die and release the pathogen, while excess host TNF causes macrophages to undergo necrosis (through RIP1 and RIP3 kinase that induce reactive oxygen species in the mitochondria) (Roca and Ramakrishnan, 2013).

Secondary sites of infection

How does the bacteria spread to secondary sites?

As the tubercle grows, several options can occur. Firstly, the tubercle can invade a bronchus and spread to other regions of the lung (Todar, 2012). Secondly, the tubercle may begin to erode into a pulmonary vessel and enter into the blood circulation (McMurray, 1996). Once this occurs, secondary lesions can appear in the genitourinary system, bones, joints, and lymph nodes in a progression called military tuberculosis (Todar, 2012).

Furthermore, if the mycobacteria spread to regional lymph nodes, they can enter the blood circulation via the thoracic duct, infect other organs and eventually infect other regions of the lungs to form secondary lung lesions (McMurray, 1996). These secondary lung lesions may persist many years later leading to reactivation of clinical disease.

What are the secondary infection sites?

Without a doubt, the most common site of infection with Mtb is the lungs, representing about 51% of UK cases in a 2014 research. Nonetheless, M. tuberculosis can also spread beyond the lungs, usually due to reactivation of latent infection, in a process called extrapulmonary tuberculosis (EPTB). Common sites of EPTB are lymph nodes (19%), pleura (7%), gastrointestinal tract (4%), bone (6%), CNS (3%) and genitourinary system (1%) (Houston and Macallan, 2014). This infection tends to occur more frequently in patients that are immunocompromised, thus they are at increased risk. As a result, patients with the following factors tend to have increased risk of developing EPTB:

  • HIV/AIDs
  • Chemotherapy
  • Diabetes
  • Underweight
  • Organ Transplants
  • Other lung diseases (ex. silicosis)
  • Previous infection with TB
  • Chronic kidney failure

Other common secondary infection sites:

Figure 2. Pathogenesis of tuberculosis in lungs and other sites of the host (McMurray, 1996)

Peripheral lymph nodes are most commonly infected, often affecting young adults and children. The most common symptom is lymphadenopathy, and less commonly fever. Later, cervical lymph nodes are affected, followed by axillary and inguinal lymph nodes (Sharma and Mohan, 2019).

Abdominal TB includes TB of the peritoneum, gastrointestinal tract, omentum, mesentery and its nodes, pancreas, liver, spleen, and biliary tract (Sharma and Mohan, 2019).

Neurological TB can occur, particularly TB meningitis, characterized by vomiting, fever, headache, raised intracranial tension, and focal neurological deficits (Sharma and Mohan, 2019).

Pericardial TB occurs in the heart, as a result of direct spread of infection through a lymphohematogenous route or from adjacent mediastinal lymph nodes (Sharma and Mohan, 2019).

Skeletal TB affects nearly all bones, including the spine, hip joint, foot bones, hand bones, knee, elbow, and shoulder joint (Sharma and Mohan, 2019).

Genitourinary TB results from hematogenous spread from an active TB site which causes further spread of infection, scarring, and inflammation (Sharma and Mohan, 2019).

Ocular TB, affecting all parts of the eye, but most often the choroid (Sharma and Mohan, 2019).


Bermudez, L. E., Sangari, F. J., Kolonoski, P., Petrofsky, M., & Goodman, J. (2002). The efficiency of the translocation of Mycobacterium tuberculosis across a bilayer of epithelial and endothelial cells as a model of the alveolar wall is a consequence of transport within mononuclear phagocytes and invasion of alveolar epithelial cells. Infection and immunity, 70(1), 140-146.

Cambier, C. J., Falkow, S., & Ramakrishnan, L. (2014). Host evasion and exploitation schemes of Mycobacterium tuberculosis. Cell, 159(7), 1497-1509.

Davis, J. and Ramakrishnan, L. (2009). The Role of the Granuloma in Expansion and Dissemination of Early Tuberculous Infection. Cell, 136(1), pp.37-49.

Ehlers, S. and Schaible, U. (2013). The Granuloma in Tuberculosis: Dynamics of a Host–Pathogen Collusion. Frontiers in Immunology, 3.

Feng, C., Kaviratne, M., Rothfuchs, A., Cheever, A., Hieny, S., Young, H., Wynn, T. and Sher, A. (2006). NK Cell- Derived IFN-γ Differentially Regulates Innate Resistance and Neutrophil Response in T Cell-Deficient Hosts Infected with Mycobacterium tuberculosis. The Journal of Immunology, 177(10), pp.7086-7093.

Flynn, J., Goldstein, M., Chan, J., Triebold, K., Pfeffer, K., Lowenstein, C., Schrelber, R., Mak, T. and Bloom, B. (1995). Tumor necrosis factor-α is required in the protective immune response against mycobacterium tuberculosis in mice. Immunity, 2(6), pp.561-572.

Houston, A., & Macallan, D. C. (2014). Extrapulmonary tuberculosis. Medicine, 42(1), 18-22.

McMurray, D. (1996). Mycobacteria and Nocardia In: Baron S, ed. Medical Microbiology. 4th ed. Galveston: University of Texas Medical Branch at Galveston.

Roca, F. and Ramakrishnan, L. (2013). TNF Dually Mediates Resistance and Susceptibility to Mycobacteria via Mitochondrial Reactive Oxygen Species. Cell, 153(3), pp.521-534.

Sharma, S. K., & Mohan, A. (2019). Extrapulmonary Tuberculosis. In Mycobacterium Tuberculosis: Molecular Infection Biology, Pathogenesis, Diagnostics and New Interventions (pp. 37-53). Springer, Singapore.

Todar, K. (2012). Mycobacterium tuberculosis and Tuberculosis In: Todar's Online Textbook of Bacteriology. [ebook] Madison. Available at: [Accessed 2 Mar. 2020].

Volkman, H., Pozos, T., Zheng, J., Davis, J., Rawls, J. and Ramakrishnan, L. (2009). Tuberculous Granuloma Induction via Interaction of a Bacterial Secreted Protein with Host Epithelium. Science, 327(5964), pp.466-469.

Question (iv)

(iv) Bacterial Damage: do the bacteria cause any direct damage to the host (or is the damage fully attributable to the host response, as indicated below) and, if so, what is the nature of the bacterial damage. Can it be linked to any of the signs and symptoms in this case?

Some damages that can occur include tubercles and necrosis.

  1. Latent TB

Since the bacteria have not begun infection, no damage nor signs/symptoms to the host occurs from the bacteria.

  1. Active TB
    1. Direct damage: Although most of the damage is caused the host’s own immune system, M. tuberculosis can cause direct damage to some extent
      1. Granuloma-related: During active TB, lung damage may occur (Todar, K., 2012). As mentioned previously, it is known that M. tuberculosis has the ability to proliferate inside macrophages and induce cell death. In Stage 5, the bacteria may break out of the granuloma if the host system cannot contain the infection. This process causes the walls of nearby bronchi to become necrotic and then rupture, causing significant lung tissue damage. This tissue damage has been attributed to the action of bacterial proteases that are released when the bacilli are freed from the granuloma (Ollinger et al., n.d.). Figure 1 illustrates granuloma morphology which may favor host damage and bacterial persistence vs. a healing response.
        Figure 1. Granuloma formation favoring healing response vs. host damage and bacterial persistence (Kiran, D. et al., 2016)
      2. Caseous tubercle related: In up to 10% of infected individuals, the caseous tubercle softens, thus progressing the infection into tuberculosis disease. This softening results in the formation of a new lung cavity, providing the bacteria with an oxygen-rich environment, thus leading to increased stimulation of extracellular bacterial growth (Grosset, 2003). After the caseous tubercle has softened, it forms a tuberculous lung cavity that does not usually heal spontaneously, thus resulting in permanent tissue damage. Interestingly, the softening of the caseous tubercle is what allows M. tuberculosis to spread through coughing since the tuberculous lung cavity allows the bacteria to be aerosolized. Thus, the sign/symptom of coughing can be directly linked to the tuberculous lung cavity and spread of the bacterial infection to other people.
      3. Necrotizing Toxin: Tuberculosis does not really produce many toxins, however the tuberculosis Necrotizing Toxin (TNT) can induce necrotic death of macrophages. This is what allows TB to escape degradation by phagocytes, allowing activation and escape from the granuloma. In this process, bacterial proteases are released which destroy lung tissue (Ollinger at al., n.d.).
      4. Cord Factor: As mentioned in iii), M. tuberculosis can grow as corded bacteria. Cord factor, a glycolipid molecule found in the cell wall of these bacteria, is responsible for this cording. Cord factor is toxic to mammalian cells and inhibits polymorphonuclear leukocyte migration, thereby causing damage to the host (Todar, 2012). Although dead macrophages release lytic and lysosomal enzymes, released bacterial products may also result in host tissue damage, causing liquefaction (Saviola, 2013; Paustian, n.d.).
    2. Host Response Damage: Majority of the damage to the host is attributable to the host immune response in the cases of active  TB infections.
      1. Lung Tissue damage/local apoptotic cell deaths: As TB is most commonly a pulmonary disease, lung damage as a result of the disease is common and often inevitable. Patients who was diagnosed with TB face a risk for long-term respiratory impairment like chronic obstructive pulmonary disease (COPD) even after successful recovery (Ravimohan, S. et al., 2018). Delayed type hypersensitivity (DTH) is a cell-mediated immune response that causes tissue damage in response to TB. This process is mediated by T helper (Th) cells. First, IFN-gamma is released by Th1 cells and this activates macrophages. Activated macrophages can now release various interleukins IL1, IL2, IL6, TNFa, and hydrolytic enzymes. TNFa recruits more macrophages and causes local apoptotic cell deaths, which contributes to further host damage. These post-TB lung dysfunctions can go unnoticed despite its high prevalence and association with reduced quality of life (Ravimohan S, 2018). There is a large variation in the magnitude of pulmonary function of post-TB patients, ranging from no impairment to severe dysfunctions. Those with pulmonary dysfunctions can also develop a wide variety of diseases. Patients may present with “cavitation, fibrosis or nodular infiltrates, or a mix of these pulmonary pathologies” (Ravimohan S, 2018). Once the bacterial growth cannot be restrained by the host immune response, macrophage necrotic death is induced and granulomatous lesions increase, as a result of the recruitment of inflammatory cells (Amaral EP et al., 2016). The exacerbation of necrotic cell death can lead to extensive caseous lesions and rupture of granulomas into airways. Matrix metalloproteases (MMPs) are a family or proteases that can degrade the extracellular matrix, which can be upregulated during TB infection and are likely fundamental to TB-associated lung injury (Ravimohan et al., 2018; Subbian et al., 2015).
      2. Granulomatous inflammation & fibrosis: Activated macrophages eliminate the bacteria and contribute to the formation of the granuloma and can cause granulomatous inflammation. A granuloma is a mass of immune cells which prevent spread of infection. After elimination of the bacteria, inflammatory sites is induced by T cells and macrophages causing fibrosis (Wallis, R.S. et al., 2004).
      3. Airflow obstruction: Observable TB-associated lung pathology potentially linked to lung dysfunction were listed and discussed in Ravimohan’s study published in 2018. There is an increasing number of population-based studies that demonstrates that a history of TB increases risk for airflow obstruction and COPD. Airflow obstruction is “associated with decreased capacity to completely expel air out of the lungs”, which may be due to inflammation-induced narrowing of airways (Ravimohan S., 2018). Symptoms associated with airway obstruction include dyspnoea, reduced exercise capacity and chronic bronchitis (Ravimohan S., 2018). Since it is evident that pulmonary impairment is common among patients with a history of TB, future direction of new drug regimens for TB should consider possible impairments and targeting specific immunopathological mechanisms (Ravimohan S., 2018). It is important that host immune response is taken in consideration when developing anti-TB treatment regimen as well.
    3. Robert’s signs and symptoms
      1. Fevers: The fever can also be the result of our bodies normal response to get rid of the pathogen (D. Q., & L. X., 2016). Molecules such as cytokines (especially TNF-alpha) and interleukins are released by infected tissues and cause the hypothalamus to generate more heat. TNFa causes fever which were released by macrophages to increase body temperature to eliminate the bacteria, which caused sweating as well to lower the body temperature. The high body temperature of fevers may inhibit certain bacterial processes, such as replication, and therefore help eliminate the pathogen (D. Q., & L. X., 2016).
      2. Chills: Chills are associated with fevers as they function to cause core body temperature to rise (D. Q., & L. X., 2016).
      3. Night sweats: Since fevers are more dominant at night, sweating also occurs mainly at night. Sweating is the body’s natural mechanism to decrease increasing body temperature, such as those associated with fevers (D. Q., & L. X., 2016).
      4. Chronic productive cough: Coughing is one of the symptoms of active TB and it is usually the result of the inflammation in the lungs and airways  and openings in the alveoli, bronchi or bronchioles resulting in obstruction of air flow (CDC, 2019). This inflammation is caused by immune cells trying to eliminate the infection. His tissue damage from the immune response leads to the dry, persistent cough that worsens at night for TB patients. When the lung tissue damage worses due to the bacterial damage conferred from granuloma breakdown (Ollinger et al., n.d.), the contents of the granuloma may spill into airways and result in the cough becoming more productive and chronic. As the disease progresses, sputum of patients with active TB may present with blood stains as a result of tissue destruction and inflammation (CDC, 2019). Coughing is a symptom experienced by our patient Robert, and the causes listed above are likely to be the underlying reason for it. It is possible that M. tuberculosis directly affects host airway mucous characteristics to promote transmission, which may be causing Robert’s cough. A combination of the direct presence of bacteria and substances they produce, along with products of the host immune response, can decrease the coughing threshold and provoke cough directly (Turner and Bothamley, 2014). Moreover, TB can cause impaired gas exchange in the lungs due to a reduction in surface area (Ravimohan et al., 2018).
      5. Crackles in the right lung and decreased breath sounds in the right lower lung field: Decreases in breath sounds in the right lower lung could be due to the accumulation of fluid from liquefaction, the spilling of content from the cavity into the airway. Increased mucin secretion from the irritation and spillage of granuloma into the airway can also contribute to the crackles from his lungs (Wallis et al., 2004)


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Q4. The Immune Response Questions

Question (i)

(i) Host response: what elements of the innate and adaptive (humoral and cellular) immune response are involved in this infection.

Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis, which grows best in areas of the body that are well oxygenated and have ample blood flow [Q4-1 1]. For this reason, tuberculosis is most often found in the lungs, but can spread to other organs such as the kidneys, brain or spine through the blood [Q4-1 2]. Pulmonary tuberculosis is contagious and is transmitted through air droplets when an infected person coughs or speaks [Q4-1 2]. Other people may breathe in the M. tuberculosis suspended in the air and the pathogen can settle in the throat or lungs and begin to grow [Q4-1 2]. The host has a variety of protective mechanisms to tackle microbial diseases all with different onsets, durations, and mechanisms of action. These lines of defense can be separated into 1) Anatomical and Physical Barriers, 2) Innate Immunity, and 3) Adaptive Immunity.

Anatomical and Physical Barriers
Anatomical and Physical Barriers

Anatomical and Physical Barriers exist in all healthy human hosts and are the first lines of defense that a pathogen comes in contact with. These barriers are non-specific, are continually ready to respond, and prevent pathogens from entering the body. Anatomical and physical barriers can be split up into physiological barriers and chemical barriers. The first of the physiological barriers includes the skin where the tight packing of cells in the epidermis prevents entry of pathogens and the commensal bacteria out-compete pathogens for colonization [Q4-1 3]. Hair is an important physiological barrier that can trap, filter, and get rid of some large particles (i.e. respiratory system, sneeze reflex) [Q4-1 3]. Furthermore, mucous membranes of the conjunctivae, alimentary, respiratory, urogenital, and gastrointestinal systems secrete mucous that traps microorganisms [Q4-1 3]. The nasal passages and segmentation of the airways deposit particles ³ 10 mm in diameter onto mucus-coated surfaces and propel them towards the pharynx by the mucociliary system. Mucus has an important role, therefore, in entrapping microorganisms such as M. tuberculosis and preventing them from entering the lower respiratory tract. Mucus helps expose bacteria to lysozymes (degrading the glycosidic bonds of peptidoglycan), lactoferrin (sequestering iron from the bacteria), IgA and IgG, and defensins [Q4-1 4]. Since M. tuberculosis is 2-4 micrometers in length, it is often deposited into the upper respiratory tract and/or conducting airways, settling in the mucociliary surface [Q4-1 5]. The mucociliary system is capable of pushing bacteria upwards until they are caught in the oral secretions and swallowed [Q4-1 3]. In the stomach the low pH levels are capable of killing the bacteria. Body temperature is another physical factor that inhibits the growth of many pathogens. Fever responses which can elevate body temperature potentiate this response [Q4-1 3]. Chemical barriers including the low pH of stomach acid, kills most pathogens that are swallowed. Mucous, saliva, and tears contain enzymes which can damage the bacterial capsule. The dryness of certain body surfaces also creates uninhabitable environments for certain bacteria. In modern times TB infections are started by the respiratory route of exposure, so anatomical and physical barriers involving the respiratory system such as the mucociliary transport system, and mucous prevent the spread of infection to the lower respiratory system [Q4-1 6].   

Innate Immune Response
Innate Immunity Diagram Lungs

The second line of defense which is fast, non-specific, has no memory, and is associated with redness, pain, swelling, and heat is the innate immune response. This line of defense takes over once anatomical and physical barriers have been breached. The main functions of the innate immune response are: Phagocytosis, opsonization, activation of complement, chemotaxis (of phagocytic cells) and activation of the inflammatory response.

Airway epithelium

Since M. tuberculosis is inhaled through the nose and mouth, there are innate defences present in the airway, such as the respiratory mucosa [Q4-1 7]. Components of the respiratory mucosa include the: epithelium, lamina propria and a coating on the luminal surface [Q4-1 7]. The epithelium is a layer of airway epithelial cells (AECs) that form a physical barrier to prevent invasion [Q4-1 7]. The lamina propria is a layer of connective tissue and immune cells such as macrophages and lymphocytes [Q4-1 7].  On the luminal surface, is a coating of airway surface liquid (ASL), which contains mucus, immunoglobulin A and other innate immune factors [Q4-1 7]. ASL also contains secretions by AECs to recruit and activate phagocytes in the airways [Q4-1 7].

If M. tuberculosis is able to penetrate the epithelial layer of the respiratory mucosa, the first cells they encounter are AECs. AECs express pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs), Dectin-1, C-type lectin receptors (CLRs), and nucleotide-binding oligomerization domain-containing protein 2 (NOD2) [Q4-1 7]. The recognition of PAMPs (pathogen-associated molecular patterns) present on the surface of M. tuberculosis initiates the production of cytokines (TNF-a, IFN-g, GM-CSF, IL-6, IL-10, etc.), chemokines (IL-8, IP-10, IL-27, MCP-1, MIG) and other effector molecules to mount an innate immune response [Q4-1 7]. An inflammatory response is induced and chemokines (CXCL1, CSCL2, CXCL4) and pro-inflammatory cytokines (IL-1b, TNF-a) are expressed [Q4-1 7].

Phagocytosis in the Alveoli

If M. tuberculosis is able to successfully surpass the defence mechanisms of the upper airways, it will be able to enter the alveoli. There are many resident alveolar macrophages, DCs and neutrophils present to defend against infection and colonization. However, these cells are susceptible to infection after they phagocytose the pathogen [Q4-1 8]. Phagocytosis occurs when  surface receptors (PRRs - pattern recognition receptors) on phagocytes recognize PAMPs on M. tuberculosis and extend their plasma membrane to engulf the pathogen [Q4-1 9]. This internalizes them into the endosomes, which will fuse with lysosomes full of reactive oxygen species or reactive nitrogen species that will kill the pathogen [Q4-1 9]. Phagocytosis causes signalling pathways to increase the production of cytokines, chemokines, and antimicrobial peptides that mediate innate immunity and initiate a signal cascade to recruit more immune cells [Q4-1 8]. Recognition of the pathogens by TLRs also trigger IFN- α, β production, which will activate Natural Killer cells [Q4-1 9]. An important cytokine that is produced is Tumour necrosis factor (TNF), which is a type II transmembrane protein [Q4-1 10]. It is also responsible for activating macrophages and vascular epithelium to increase vascular permeability [Q4-1 10]. This allows for additional immune cells to access the infection site.


Macrophages are highly phagocytic and release signalling molecule TNF-a and cytokines to activate and recruit neutrophils and monocytes to the site of infection. Macrophages also express various virulence factors to work against infection with M. tuberculosis [Q4-1 7]. Some of these factors of macrophage-based immunity include the inhibition of intracellular trafficking, inhibition of autophagy, induction of (infected) host cell death, and neutral.

When macrophages are infected by M. tuberculosis, the balance of lipid mediators that are proapoptotic, such as prostaglandin E2, or pronecrotic, such as lipoxin A4, determines the outcome of the infection and immune response [Q4-1 8]. Apoptotic death of macrophages maintains an intact plasma membrane and therefore decreases pathogen viability while enhancing immunity [Q4-1 8]. Efferocytosis, the process by which apoptotic cells are removed by phagocytic cells, promotes compartmentalizing the pathogen as well as the apoptotic cell within another macrophage [Q4-1 8]. This allows the delivery of the pathogen into the lysosomal compartment for degradation [Q4-1 8]. Necrotic death would allow the pathogen to spread to surrounding cells as it involves the lysis of the macrophage membrane [Q4-1 8].

Autophagy is another important innate immune response mechanism in eliminating M. tuberculosis. Autophagy is a process in which infected organelles are encapsulated in a vesicle with a double membrane and transported to the lysosomes for degradation or recycling [Q4-1 11]. Although not much is known about this mechanism in human cells, it is proven that autophagy can be activated by IFN-γ, which will induce phagosome maturation and increase its acidification to degrade the bacterium [Q4-1 8].   There has also been research demonstrating that active forms of vitamin D3 induce autophagy in monocytes and macrophages [Q4-1 7]. Vitamin D3 expresses antimicrobial peptide cathelicidin, which will activate the genes encoding for autophagic mechanisms [Q4-1 7]. Furthermore, vitamin D is a key factor in containing M. tuberculosis within the macrophage, as the macrophages phagosome matures, and immune activity relies on the presence of Vitamin D [Q4-1 7]. Vitamin D3 disrupts the membranes of bacteria and allows for the localization of M. tuberculosis into autophagosomes [Q4-1 7]. This prevents the spread of M. tuberculosis into neighbouring cells [Q4-1 9]. Deficiencies in Vitamin D have been linked to increased productivity in M. tuberculosis replication [Q4-1 7]. Reactive oxygen species (ROSs) in macrophages also play a key role in the immune response to M. tuberculosis. Inside a macrophage that has phagocytosed a pathogen, the macrophages release an overload of Cu and Zn which are toxic to the pathogen [Q4-1 7].


Neutrophils are another important innate immune cell that may have both a protective and detrimental effect on the host cell [Q4-1 8]. It is shown in mouse models that respiratory failure and mortality is associated with increased levels of neutrophils in the blood [Q4-1 8]. However, neutrophils have the role of facilitating the activation of naive antigen-specific CD4+ T cells, which is required for the promotion of an adaptive immune response [Q4-1 8].  Neutrophils are recruited to the site of infection when IL-1b and TNF-a help assist the neutrophil to recognize ICAM-1 on the endothelial surface [Q4-1 12]. Neutrophils can form extracellular traps called NETs which capture pathogens [Q4-1 13]. They are also capable of degranulation which is the release of vasoactive and toxic molecules to help clear M. tuberculosis [Q4-1 13]. Specifically (within macrophages), neutrophil granules can fuse with phagosomes containing Mtb and can kill the bacterium [Q4-1 14]. They produce and secrete antimicrobial enzymes (a-defensins, matrix metalloproteases, lactoferrin, and lipocalin) which restricts mycobacteria growth within macrophages, promotes apoptosis of infected macrophages, and limits M. tuberculosis survival [Q4-1 4].

Natural Killer Cells

Natural killer cells are also involved in the elimination of M. tuberculosis, as they are recruited to the site of infection upon detecting infected macrophages via receptor molecules [Q4-1 7]. These innate immune cells amplify the antimicrobial defence to TB by lysing infected macrophages [Q4-1 7]. They are also responsible for producing IFN-γ to activate additional phagocytes to help clear the infection [Q4-1 7]. NK cells express  both NK and T cell markers and help protect humans from TB infection by producing IFN-g, TNF-a, IL-17, IL-2, and IL-21 [Q4-1 4]. These cells exist in CD56+ phenotype (cytotoxic) and CD56- phenotype (cytokine secreting) [Q4-1 14]. Components of the M. tuberculosis cell wall are recognized and bound by the NKp44 receptor of NK cells [Q4-1 14]. NK cells require an imbalance of activating signals compared to inhibitory signals in order to become activated and take on cytotoxic activity [Q4-1 13]. These signals are called “stress signals” and can be provided in the form of cytokine production from dendritic cells, macrophages, or from the altered presentation of foreign peptide on MHC molecules (compared to self-peptide) [Q4-1 13].

Bridging Innate and Adaptive Immunity

Complement Proteins

Complement Pathway

The role of the complement cascade in TB infection is not yet well defined [Q4-1 14]. C5 and C7 components commonly play a defensive role, however high expression of C1q has been correlated with more severe clinical conditions during M. tuberculosis  infection [Q4-1 14]. As a result, C1q levels have been used as a biomarker to discriminate between latent TB infection and active TB infection [Q4-1 14].

The complement system of immunity uses serum complement proteins (active or inactive) which act together through a series of reactions to kill extracellular pathogens [Q4-1 13]. Bactericidal factors include opsins and the membrane attack complex. A key protein activated in the complement pathway is the C3 convertase that cleaves C3 into C3a and C3b [Q4-1 13]. C3b is able to bind the pathogen's surface and recruit the C5 convertase [Q4-1 13]. The C5 convertase converts C5 to C5a and C5b (which can also bind the pathogens surface); C5a and C2b initiate the assembly of the membrane attack complex [Q4-1 13]. Complement can be activated by 1 of 3 ways:

1.     The Classical Pathway: Complement proteins are taken to their active form when antibodies made in a previous immune response bind to the pathogen surface via their complement specific Fc regions (on the antibody) [Q4-1 13]. Binding of the antibody to the pathogen activates early complement cascade forming the C3 convertase [Q4-1 13].

2.     The Lectin Pathway: Mannose binding lectin and ficolins are able to recognize pathogen associated molecular patterns forming an active C3 convertase [Q4-1 13].

3.     The Alternative Pathway: C3 self-hydrolyzes to C3(H2O). This form of C3 can bind Factor B which changes conformation and can now be cleaved by serum proteases factor D generating Ba and Bb fragments [Q4-1 15]. Bb fragments remain associated with the complex and then can cleave C3 molecules producing C3b [Q4-1 15]. Once C3b is made it can associate with factor B and generate more C3 convertase [Q4-1 15].


Opsonization refers to the alteration of a pathogens surface making it more easily phagocytosed [Q4-1 13]. This is done through the binding of C3b to the pathogens surface. All phagocytic cells have receptors capable of binding to C3b on a pathogens surface, facilitating pathogen engulfment [Q4-1 13].

Dendritic cells

Dendritic cells play a key role due to antigen presentation, co-stimulatory activity, and large cytokine producing activity [Q4-1 14]. Evidence has shown that M. tuberculosis are capable of dendritic cell manipulation which can be used as a tool to hinder specific T-cell responses, and to use dendritic cells as niche [Q4-1 14]. Specifically, CD209 on the dendritic cell is coupled with lipoarabinomannan mannose of the M. tuberculosis that penetrates the cell [Q4-1 14]. This penetration leads to dendritic cell release of IL-10 (anti-inflammatory cytokine), which reduces the production of IL-12 ultimately suppressing T lymphocyte activity [Q4-1 14].

Bridge Between Innate and Adaptive Immunity
Adaptive Immunity

Adaptive immunity is the third line of defense which is specific, has memory, yet is slow to start. Also referred to as humoral immunity, this form of immunity is defined by the use of type-specific antibodies which help tackle an infection. The adaptive immune response is usually activated within 10 to 14 days of infection and is highly specific [Q4-1 8]. However, in regards to TB, adaptive immunity is not activated until 5 to 6 weeks after infection [Q4-1 16]. It generates a memory response, which will result in a faster response upon subsequent exposures [Q4-1 8]. Major cells in adaptive immunity include B cells, T cells, and antigen-presenting cells (APCs), such as dendritic cells and macrophages [Q4-1 8]. Humoral components include antibodies and cytokines [Q4-1 8].  Adaptive immunity can be separated into a cell mediated response and a humoral response.

Cell-Mediated Response

Firstly, antigen presenting cells (APCs) such as dendritic cells mature with the onset of an infection and can phagocytose Mtb in order to present their bacterial peptides on MHC classes I and II. After this presentation, DCs are activated and can migrate to the lymph nodes under the influence of IL-12, CCL19 and CCL21 [Q4-1 8].  The activated DCs have the ability to activate naive T cells to become mature cytotoxic T cells or helper T cells depending on whether or not it binds to a CD8+ receptor or a CD4+ receptor [Q4-1 8]. MHC class I molecules will activate CD8+ T cells while MHC class II molecules will activate CD4+ T cells.

T cell activation requires two signals. Firstly, signal 1 is marked by the T cell receptor recognizing its antigen bound to the correct class of MHC on a dendritic cell [Q4-1 17]. Co-receptors CD4 or CD8 bind MHC on the dendritic cell to enhance the strength of the interaction, and CD3 initiates a signaling complex, delivering signal 1 to the nucleus [Q4-1 17]. Signal 2 is termed “co-stimulation” in that co-stimulatory molecule “B7” on the dendritic cell binds CD28 on the T Cell, delivering signal 2 to the nucleus. This B7/CD28 co-stimulation induces cytokine production (i.e. IL-2) which stimulates T cell proliferation [Q4-1 17]. T-cells can be dangerous to the body, therefore two signals are required to activate T-cells as a safety mechanism [Q4-1 17].

Two types of T-cells exist: Cytotoxic CD8 + T cells (MHC I expressing) release toxic molecules, inducing apoptosis of infected cells via unloading of toxic molecules directly into the cytoplasm of the infected cell, killing the infected cell and limiting the spread of infection [Q4-1 17]. CD8+ T Lymphocytes have been shown to produce IL-2, IFN-g and TNF-a during Mtb infection which all play a key role in controlling Mtb. CD8+ T cells also have cytolytic action against Mtb via perforin and granulysin [Q4-1 14]. CD4+ Helper T cells provide help through both cytokines and CD40L/CD40 interactions to CD8 cytotoxic T cells to enhance effector functions, macrophages to increase pathogen killing ability and B cells to make antibodies [Q4-1 17]. Alongside CD8+ T lymphocytes, CD4+ T (ab and gd subtypes) cells contribute to increased levels of IFN-g which induce the process of phagosome maturation in macrophages that have phagocytosed Mtb [Q4-1 14]. This is key in the immune response against Mtb as knockout mice for IFN-g have been shown to suffer from a more severe course of Mtb infection [Q4-1 14]. A subset of CD4+ T (Tfh) cells which have been activated by dendritic cells migrate to lymphoid follicles in the lymph node where they can interact with and activate B cells through interactions between CD40 and CD40L (ligand) [Q4-1 13]. Other T helper cells called Th1 cells find their way to the infection site due to the high expression of the chemokines CXCR3 and CCR5 as well as high levels of ligands for E-selectin and P-selectin [Q4-1 18]. These T cells secrete IFN-g, and once activated create the CD40L-CD40 complex with DCs and macrophages to secrete more IL-12 to enhance cytotoxic activity. These Th1 cells are key to controlling Mtb infection [Q4-1 19].

Memory B cells contribute to the memory to Mtb infection where a stronger and faster humoral mediated immune response will be elicited upon secondary infection with Mtb [Q4-1 13]. Plasma B cells are specialists at producing antibodies upon activation in lymphoid follicles (with the help of Tfh cells) [Q4-1 13]. B cells also regulate the level of granulomatous reaction, cytokine production and the T cell response during Mtb infection [Q4-1 20].

Humoral Response

The humoral response is centered around antibody production, consisting of neutralization, opsonization and complement activation. The role that humoral immunity plays in Mtb infection is uncertain as complement-mediated opsonization of Mtb does not alter survival [Q4-1 14]. In fact, high levels of antibodies correlate with more serious conditions of Mtb infection, and passive immunization with antibodies does not confer protection [Q4-1 14]. However, Fc portions in the immunoglobulin which can bind and activate various immune cells (NK cells, monocytes, neutrophils) have been shown to aid in clearance of Mtb [Q4-1 14]. Neutralization requires antigens binding to the TB bacterium and blocking its interaction with host cells [Q4-1 17]. Opsonization is when antibodies bind and coat the pathogen with their variable region, while their constant region is recognized by the macrophage through Fc receptors, increasing contact with phagocytic receptors and increasing the efficiency of phagocytosis [Q4-1 17]. Classical activation of the complement pathway requires antibodies (coupled with CI, C2, C4 and the pathogen) to activate C3 convertase [Q4-1 17]. Mast cells are also activated as they express Fc receptors, which binds IgE and causes cross-linking, resulting in mass cell degranulation and more release of histamine [Q4-1 17].

Antibodies are made by B cells. B cells are activated when the B cell first sees the antigen (signal 1), followed by T-Helper cells providing CD40L (signal 2) [Q4-1 17]. Some B cells become memory cells for future infections, while others become plasma cells and secrete antibodies in the lymph node (signal 2) [Q4-1 17].

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Question (ii)

(ii)  Host damage: what damage ensues to the host from the immune response?
Summary of respiratory damage caused by infection with Mtb. Ravimohan et al 2018 doi: 10.1183/16000617.0077-2017

With tuberculosis, tissue damage results from the toxic mediators released by lymphoid cells rather than from the bacterial toxins themselves [Q4-2 1]. Infection begins with intracellular multiplication of M. tuberculosis in alveolar macrophages, along with innate immune response recruitment of monocytes, neutrophils and their cytotoxic products [Q4-2 2]. Ultimately, extensive tissue destruction and necrosis, formation of cavities and fibrosis make up the majority of the damage caused by tuberculosis [Q4-2 2].

Role of TNF-α and TGF-β:

TNF-α is cytotoxic to epithelial cells, decreases production of surfactant protein by type II alveolar cells (which regulates normal lung function), increases fibroblast formation, enhances production of fibroblast collagenases, and promotes production of reactive oxygen intermediaries that are cytotoxic to tissues [Q4-2 2]. The host will experience decreased levels of oxygen and if this is prolonged, many host tissues will die. When there is an abundance of TNF-α (and other pro-inflammatory cytokines), there is a possibility that edema may result due to the influx of immune cells recruited to the site of infection and fluid leaking from the vascular system [Q4-2 2].

Simultaneously, TGF-β promotes extensive fibrosis and subsequent tissue damage [Q4-2 2]. It does this by strongly inhibiting epithelial and endothelial cell growth, while promoting production of the collagen matrix and macrophage collagenases, leading to tissue fibrosis [Q4-2 2].

Hallmark symptoms of tuberculosis such as fever, night sweats and weight loss indicate increased circulating levels of cytokines like TNF-α and TGF-β [Q4-2 2]. It has also been shown that these increased levels positively correlate with the extent of cavity formation and are also increased in bronchoalveolar fluid [Q4-2 2].

Role of B Cells:

B cells have been shown to regulate the level of granule release, cytokine production, and T cell response [Q4-2 3]. While experiments testing TB on B-cell-deficient mice show an increased pathology of TB, they also show decreased granuloma (small area of inflammation) formation in the lungs, lending evidence that B cells directly contribute to granuloma formation in the lungs [Q4-2 3]. Similar to the aforementioned cytokines, B cells are also shown to increase 8-fold during TB infections [Q4-2 3].

Overall damage to host:

The damage caused by cytokines and B cells manifests in patients as chest pain, dyspnea (shortness of breath), cough accompanied by purulent/bloody sputum, fever and chills. Many of the tell-tale signs and symptoms of tuberculosis (sweats and fever) are a result of excess circulating inflammatory cytokines [Q4-2 2]. These symptoms often mimic those of pneumonia, therefore differential diagnosis between both diseases is critical for effective treatment. Overall, lung damage is the most common form of damage from TB infection and presents as a result of lesions (Ghon complexes) or non-specific inflammation to destroy the infection which ultimately damages the host as well [Q4-2 4]. Excessive fibrosis (scarring of the lung), bronchiectasis (destruction of the elasticity and muscular components of the bronchial walls), caseous necrosis (lesions that can liquefy to cause abscesses breaching the bronchial walls as a result of tissue death), disfigured shape of the lungs as airway narrowing, cavitation, all lead towards a negative impact on the capacity of the lungs to allow sufficient oxygen exchange [Q4-2 4].

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Question (iii)

(iii)  Bacterial evasion: how do the bacteria attempt to evade these host response elements

M. tuberculosis possess a large array of structural and physiological properties that have been recognized not only to evade host immune responses, but also greatly enhance their pathogenesis in human hosts [1]. The following mechanisms are relevant to TB virulence:

M. tuberculosis is incredibly successful as a pathogen because of the various counter-strategies the bacillus has developed to counter the host defenses [2,3]. Below lists how Mtb evades the hosts’ immune system [3].

Host Defense Strategies Myobacterial Counterstrategies
Recruit microbicidal macrophages to infection
  • mask PAMPs with the cell surface lipids, avoiding TLR-signalled microbicidal macrophage recruitment
  • recruit growth-permissive macrophages by inducing the monocyte chemokine CCL2
Promote phagosome-lysosome fusion
  • Avoid/Tolerate phagosome-microbicidal lysosome fusion
Aggregate macrophages into epithelioid granulomas to restrict and contain mycobacteria
  • Use early granuloma for intracellular expansion via macrophage-to-macrophage spread
  • Activate granuloma-specific genes that help the mycobacteria survive in the granuloma
  • Vascularize the granuloma such that it is conducive to bacterial growth
Processing and presenting bacterial Ags
  • Inhibit MHC-II Ag processing and presentation
  • Divert secreted Ags from MHC II molecules to the export pathway, avoiding CD4 T cell recognition
  • Alter Ag expression, evading T cell recognition by inducing suboptimal activation of CD4 effector cells
  • Avoid focusing T cell immune responses to conserved/subdominant regions
Activation of T cells to control infection
  • Release PAMPs to inhibit TCR signaling and induce CD4+ T cell anergy
  • Delay DC migration to lymph nodes which delay the creation of effector T cells
Increase microbial capacity of the granuloma by recruiting effector T cells to it
  • Induce antigen-specific Treg cells to delay effector T cell priming and recruitment
  • Downregulate key mycobacterial antigens to render infected macrophages “invisible”
  • Induce suppressive factors (NO, IL-10, and TGFb) to restrict effector T cell function

Other components produced by M. tuberculosis that are used to help successful growth and evasion of host defenses include:

Special mechanisms for cell entry:

The tubercle bacillus of TB can directly bind mannose receptors on macrophages (via cell wall mannosylated glycolipids on M.tb cell surface) or can indirectly bind various complement/Fc receptors – all of which lead to TB entry into immune cells [1]. Engaging with phagocytic receptors allows entry and intracellular survival of TB via activation of specific signaling pathways within the host cell [4]. The presence of mannosylated glycolipids on M. tb (i.e. ManLAM or mannose-capped lipoarabinomannan) on M.tb’s surface greatly facilitates interactions between the bacterium and macrophages, allowing for further virulence mechanisms post-entry [4].

Intracellular growth:

An effective way that TB evades the immune system is by growing within host cells, evading actions by antibodies and complement that target external pathogens [1]. Once M. tb. Is phagocytosed, it inhibits phagosome-lysosome fusion via secretion of a protein that changes the phagosome membrane [1]. It does this via M. tuberculosis phagosome maturation arrest, where the secreted bacterial factors (LAM, SapM, PtpA) disrupt Rab GTPases – proteins that drive phagosome progression from early to later stages of maturation [5]. This allows M.tb. to either remain inside the phagosome or escape into the cytoplasm – both of which provide protective environments for bacterial growth [1].

Detoxification of oxygen radicals:

During normal phagocytosis, reactive oxygen intermediates (ROI) are produced which are cytotoxic to engulfed bacteria. M.tb. interferes with ROI production firstly by producing products such as glycolipids, sulfatides and LAM that down regulate the oxidative cytotoxic mechanism [1]. These compounds act as potent scavengers of oxygen radicals [6]. Additionally, due to the inhibition of phagosome maturation (where pH normally drops from neutral to 5 throughout the process) the acidic pH is not present, which is essential for optimal activity of lysosomal digestive enzymes to produce the ROIs [5]. As a result, significantly less ROIs are produced, protecting the integrity of the bacterium. M. tb. also produces enzymes that counteract the oxidative burst, for example catalase and superoxide dismutase enzymes [1]. Finally, macrophage uptake (through complement receptor activation) may bypass the activation of a respiratory burst altogether [1].

Antigen 85 Complex:

A complex composed of a group of proteins secreted by M. tb. that bind fibronectin, aid in detaching the bacteria from immune system cells and facilitate tubercle formation [1]. Tubercles are the nodules of the bacterium that contain necrotic factors. The complex is coded by FbpA, FbpB and FbpC2 genes [7].

Slow generation time:

M. tb. have a slow generation time, meaning that they don’t express virulence quickly after infection, but rather grow and manifest slowly. This allows them to remain undetected by the immune system, or ability to circumvent a greatly diminished immune response [1]. M. tb. are then able to grow and replicate enough to create a large virulence response before the immune system has time to control the infection.

High lipid concentration in cell wall:

Over 60% of the mycobacterial cell wall is lipid mycolic acid, along with glucolipids, LAM, sulfolipids and other lipids [8]. This accounts for impermeability and resistance to antimicrobial agents, ability to resist actions of acidic and alkaline compounds from both intra- and extracellular origin, and resistance to osmotic lysis (normally done via complement deposition and lysosomal attack) [1]. The pathogen is also able to alter the metabolism of its fatty acid to survive conditions in the host, reflected subsequently by changes in the cell wall lipid composition [8].

Cord factor:

Cord factor is a cell wall glycolipid found in mycobacteria that causes the cells to grow in snake-like cords [1]. Cord factor is toxic to mammalian cells, and further inhibits polymorphonuclear leukocytes from migrating to the site of infection [1]. Cord factor is only toxic on lipid surfaces and are directly involves with caseating granulomas in the lung, a hallmark symptom of tuberculosis [9].

There is such a large plethora of methods and genes expressed within M. tuberculosis strains at the ready which help it escape destruction via our immune responses. There are various contradicting methods of immune evasion expressed, which are all dependent on the environment and genetic makeup of the M. tuberculosis strain.

To date, the understanding of the mechanism of immune escape by M. tuberculosis remains limited. Over the last fifty years, numerous studies have investigated the pathogenic mechanisms of M. tuberculosis  and the immune response [10]. However, tuberculosis caused by M. tuberculosis still endangers health worldwide. This is mainly due to its immune escape mechanisms, which greatly enhance its survival in the host. Aspects of M. tuberculosis infection and the immune escape mechanisms provide a basis for the future treatment of tuberculosis. By suppressing macrophage maturation and lysosomal acidification as well as inhibiting oxidative stress, apoptosis and autophagy, M. tuberculosis capable of remaining latent in the host [10].  Besides, iron, Ca2+ and H+ also function in the immune escape of M. tuberculosis, this process is achieved via various proteins and genes. Therefore, this work opens up a new avenue for drug research to treat tuberculosis.


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  3. Gupta A, Kaul A, Tsolaki AG, Kishore U, Bhakta S. Mycobacterium tuberculosis: Immune evasion, latency and reactivation. Immunobiology 2012;217:363–74.– Meeting report. Vaccine 2017;35:3433–40.
  4. Kang PB, Azad AK, Torrelles JB, et al. The human macrophage mannose receptor directs Mycobacterium tuberculosis lipoarabinomannan-mediated phagosome biogenesis. J Exp Med. 2005;202(7):987–999. doi:10.1084/jem.20051239
  5. O'Garra, A. et al. (2013). The immune response to Tuberculosis. Annual Reviews Immunology. 31:475-527. doi:10.1146/annurev-immunol-032712-095939
  6. Peterson JW. Bacterial Pathogenesis. In: Baron S, editor. Medical Microbiology. 4th edition. Galveston (TX): University of Texas Medical Branch at Galveston; 1996. Chapter 7. Available from:
  7. Kremer, L., Maughan, W., Wilson, R., Dover, L., & Besra, G. (2002). The M. tuberculosis antigen 85 complex and mycolyltransferase activity. Letters in Applied Microbiology, 34(4), 233–237. doi: 10.1046/j.1472-765x.2002.01091.x
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  10. Cooper AM. Cell mediated immune responses in Tuberculosis. Annu Rev Immunol. 2009; 27: 393–422. doi: 10.1146/annurev.immunol.021908.132703

Question (iv)

(iv) Outcome: is the bacteria completely removed, does the patient recover fully and is there immunity to future infections with this infectious agent?
TB Treatment:

Many individuals that suffer from TB, can be treated successfully with a rigorous antibiotic regimen lasting many months and can recover fully (1). To completely remove the bacteria, it is important that patients finish their medication as prescribed in order to fully recover (2). If treatment is not taken correctly, TB will become more drug resistant. Several drugs can be used to treat tuberculosis, and these drugs take six to nine months to fully clear the pathogen. Some common antibiotics include isoniazid, rifampin, ethambutol, and pyrazinamide (3). Patients who are concurrently HIV-positive are recommended to have treatment for nine-twelve months.

It is extremely important that Robert adheres strictly to his prescribed drug regimen to decrease the risk of anti-TB drug resistance (4). For example, if patients do not complete the full course of treatment, the wrong treatment/dosage is prescribed, when the supply of drugs is not always available, or when the drugs are of poor quality, these actions may lead to multidrug-resistant TB (i.e. TB resistant to two of the best anti-TB drugs – isoniazid and rifampicin), or extensively drug resistant TB (i.e. TB resistant to isoniazid, rifampin, fluoroquinolone and at least one of three injectable second-line drugs). In these cases, patients are left with less effective treatment options, and therefore likely have worse disease outcomes.

TB Latent Infection:

TB can remain as a latent infection (and not show any symptoms), and TB recurrence can be caused by a relapse to original infection, or exogeneous infection by new TB strain (Figure 4). TB Bacteria can persist in granulomas for years afterwards and continue to be reactivated when the host is immunocompromised. Another factor that influences the reactivation of TB is age with elderly TB patients being more at risk of forming cavities in the lungs that could result in coughing, fever, and night sweats (5). However, the line between latent and reactivated infection versus resolved infection and novel re-infection is not always completely known. High HIV incidence can generate higher recurrence rates due to reinfection. High relapse rates could be due partially to unsuccessful treatments instead of TB transmission within the community (6). Additionally, some individuals who are infected, do not undergo treatment if their livers will not be able to tolerate the damaging antibiotic regimen (1).

Although the infection can resolve and TB can remain latent in the granulomas, the extent to which the TB bacteria exist in and outside these granulomas is still under debate. Some studies show some granulomas as sterile following treatment, while others suggest the TB progeny persist, allowing reinfection when immunity is compromised. TB can lay dormant in inactivated macrophages or dendritic cells for an extended period of time in its latent form and is readily activated by changes in the host environment (7). In these (protected) replication centers, TB infection can amplify without restriction until the cells burst (7). If left untreated, tubercle bacilli are able to spread via lymph fluid to regional lymph nodes, and subsequently to the bloodstream via erosion of a vein with a caseating tubercle (8). When caseating lesions discharge their contents into the bronchi, the TB bacteria are aspirated and spread to other parts of the lungs, or swallowed and passed to the digestive system (8). TB is then able to travel to secondary sites bacteremically, and affect other body systems such as neurons, adrenal glands, heart tissue and the GI tract.

When TB enters the bloodstream, the hematogenous spread resulting in more severe infection is called milliary tuberculosis (7). Milliary tuberculosis can cause secondary lesions at many anatomical sites in the body including bones, joints, lymph nodes, and the peritoneum. Exudative lesions result from an accumulation of polymorphonuclear cells around the TB bacteria. In exudative lesions, bacteria can replicate with little resistance and form a “soft tubercle”. Granulomatous lesions form when the host becomes hypersensitive to proteins secreted by the TB bacteria (tuberculoproteins). Granulomatous lesions result in the formation of a “hard tubercle”. If the TB infection persists to a more severe stage, the bacteria can in fact replicate extracellularly and form Ghon complexes. Ghon complexes are characterized by lesions that calcify and become fibrous and can be identified upon performing X-rays of the chest (7).

Figure 4: Characteristics of latent vs active TB.

File:TB 700pix.jpg

Generating Immunity to TB:

Memory T cells are generated in response to TB infection, and in re-infection these T cells expand rapidly, generating effector memory T cells (and secrete IFN-gamma and promote significant protection at early time points after infection). The CD4+ T cells activate macrophages and endow them with mycobacteriostatic capabilities, which limit replication of intracellular TB, while the CD8+ T cells attack infected macrophages with mycobacterial antigens (9). Additionally memory B cells generated can provide modest protection through antibodies to TB, and cytokines generated in response to TB can polarize macrophages to an anti-inflammatory phenotype (10). Acquired immunity following TB infection normally develops 4-6 weeks post infection and is accompanied by hypersensitivity to mycobacterial antigens (2). The antimycobacterial antibodies within their system, however, are unsuccessful at generating long term protection and allow for re-infection to occur, meaning previous exposure to TB does not prevent infection in the future (6).

Similarly, a vaccine exists for TB, named the BCG (bacillus Calmette-Guérin) vaccine, generated from a similar strain Mycobacteria bovis. However, this vaccine has been shown to have variable efficacy to pulmonary TB strains, and many argue that long-lived protection does not occur. This further strengthens the complexity of lasting immunity to TB (11).

References List:

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  8. Carroll. (2015). Jawetz, Melnick, & Adelbergs Medical Microbiology, 27e. McGraw-Hill.
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  10. Zong Z, Huo F, Shi J, et al. Relapse Versus Reinfection of Recurrent Tuberculosis Patients in a National Tuberculosis Specialized Hospital in Beijing, China. Front Microbiol. 2018;9:1858. Published 2018 Aug 14. doi:10.3389/fmicb.2018.01858
  11. Kirman, J., Henao-Tamayo, M., & Agger, E. (2016). The memory immune response to tuberculosis. Microbiology Spectrum, 4(6) doi:10.1128/microbiolspec.TBTB2-0009-201