Course:PATH417:2021W2/Case4

Path 417 Cases
Instructor
David Harris Agatha Jassem Ramon KleinGeltink Inna Sekirov
Case Projects
Case 1
Case 2
Case 3
Case 4

Case 4

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).

The Body System

i) Describe the signs and symptoms presented in the case. Are there any other signs or symptoms that could have been commented on but are not presented in the case? What are the key History of Presenting Illness elements presented? What laboratory samples are taken and why? What are the meanings of the laboratory results reported? (No need to describe physiology of the signs and symptoms and no need to describe the laboratory testing itself as these are the basis of other questions).

In Pulmonary TB, during the early primary infection stage, the patient is likely asymptomatic. As it progresses, the patient may experience fever and chills, pleuritic chest pains, difficulty to sleep, and unproductive coughs [1]. After primary TB infection, the bacteria remain latent and inactive. It may be reactivated when the patient's immune system is weakened and the infection becomes active again [2]. If the disease further develops into reactivation TB, which is usually 1-2 years after the initial infection, the patient may have symptoms like fever, night sweats, weight loss, and cough that produces bloody/purulent sputum that lasts for more than 2 weeks [3]. These symptoms are similar to pneumonia so TB should always be a differential diagnosis if the patient is suspected to have pneumonia. This is likely the stage of infection Robert is experiencing as he also has fever, night sweats, and productive cough.

Mycobacterium tuberculosis is a slow-growing aerobic bacterium with a complex cell wall made up of peptidoglycans and a large number of complex, long-chain lipids [4]. M. tuberculosis is a "acid-fast bacillus" because of the free lipids on the outer layer, which make it relatively hydrophobic and resistant to staining decolorization with acid solutions. M. tuberculosis, like other Mycobacterium species, may be isolated using appropriate solid agar medium or broth cultures.

The word "sign," which comes from the Latin word "signum," refers to a disease symptom that can be objectively detected by another person [5]. The word "symptom" comes from the Greek word "symptoma," and it refers to a subjective experience that can only be identified by the patient [5].

Signs of TB that Robert has presented within this case include a fever of 38.5°C.  In adults, temperatures above 37.6°C and 38.1°C in the axillary or oral temperatures and in the ear, respectively, is considered a fever [6].  Furthermore, Robert's family doctor conducted an auscultation of his lungs and found additional signs of TB, including crackles in the right lung and decreased breath sounds in the right lower lung field.  Chronic productive cough is a sign of the respiratory system infection, and night sweats is another sign that can be objectively detected by another individual.

Figure: the process of an auscultation.

Crackles are the clicking, rattling, or crackling noises emitted  during inhalation by one or both lungs of an individual with respiratory disease [7]. Excess fluid (secretions) in the airways, either an exudate or a transudate, causes crackles. Exudate occurs as a result of a lung infection (ex. pneumonia), whereas transudate occurs as a result of congestive heart failure [7]. In the case of Robert, his crackles are caused by exudates dislodged by the cough, and this is a common sign during early TB [8].

Reduced breath sounds can be caused by air or fluid in or around the lungs (such as pneumonia, heart failure, and pleural effusion), increased thickness of the chest wall, over-inflation of a part of the lungs (emphysema can cause this), or reduced airflow to part of the lungs [9]. In the case of Robert, his reduced breath sounds are most likely caused by fluid(secretions) in the lungs.

Symptoms of TB that Robert has noticed himself, presented in this case include chills, which cannot be determined or measured by anyone but himself.

Additional signs which Robert does not have, but patients with primary or secondary TB may develop include: tender or swollen lymph nodes within areas like the neck, weight loss and cough up blood [9];, as well as hemoptysis due to erosion of blood vessels [10].

Other common symptoms of TB are: fatigue, loss of appetite, and chest pain [9]; pain in the thoracic region that is due to inflammation in the pleura, difficulty in breathing [10].

The History of Present Illness (HPI) serves as the foundation for generating different diagnoses, guiding medical decision-making, investigating the patient's condition, and finally analyzing the patient's illness [11]. The fact that Robert, 53 years of age, had migrated from India in the past year.  Furthermore, Robert has been experiencing some of the above signs and symptoms for the past month now.  This may be crucial information because it could be high levels of stress that Robert may have experienced with his immigration which may play a role in increasing susceptibility to TB [12]. According to the World Health Organization (WHO), India has the world's greatest TB pandemic [13]. The WHO Global tuberculosis report 2021 indicates that India accounted for 26% of all incident tuberculosis cases worldwide. The incidence rate in India was 192 cases per 100,000 people. India was responsible for 34% of worldwide TB mortality among HIV-negative persons and 38% of the overall number of TB deaths among HIV-positive and HIV-negative people. In addition, India accounted for 24% of the global gap between projected TB incidence and the number of patients newly diagnosed with TB and reported in 2020 [11]. Knowing that India has the world's greatest TB pandemic and aligning this with Robert’s signs, symptoms and HPI, the doctor could quickly make an inference that Robert potentially has TB and assign relevant tests to confirm.

Laboratory symptoms that were taken in this case include a chest X-ray and 3 sterile containers.  A chest X-ray can be useful in helping to better understand the extent of TB in Robert [14]. Further X-rays may be conducted as well if it is suspected that there is spread to other organs. The three sterile containers, instructing Robert to generate three deep sputum samples can be useful for determining and confirming whether the patient has TB after examination under a microscope [15].  Furthermore, this can also be used to check if the treatment is effective.  In this case, the chest X-ray must have shown changes, abnormalities or spots on the patient's right lung as a result of TB and the sputum tests must have shown positive for the TB bacteria [16].

Robert was instructed to take three deep sputums over three mornings. Sputum (or phlegm) is mucus that comes up from deep within your lungs when you cough. It's frequently dark, viscous, and sticky [17]. Three samples are collected over the interval of three days for maximal accuracy [17]. Two tests will be done on the sputum: a smear and a culture.

In an acid-fast bacilli smear, the sputum is smeared onto a glass slide and a specific dye is used to make M. tuberculosis visible under a microscope [18]. It is the most effective method for detecting infectious TB sources and confirming the diagnosis in those who have been diagnosed with the disease [19]. However, Sputum smears have a sensitivity of 34-80% for diagnosing pulmonary TB. This could provide the first line of evidence that mycobacteria is present. On the other hand, a sputum culture is much more sensitive.

Acid-fast bacilli are counted when they are found in a smear. There is a system for reporting the number of acid-fast bacilli that are seen at a certain magnification. Smears are classed as 4+, 3+, 2+, or 1+ based on the quantity of acid-fast bacilli observed. The higher the number, the more contagious the patient is [20].

An acid-fast bacilli sputum culture is a test used to identify microorganisms that might cause illness (such as M. tuberculosis) and is the gold standard. Cultures have a high sensitivity of 80-93%, as well as a high specificity of 98% [19]. There are two sorts of methods: solid media and liquid media. Solid media have long been the gold standard for growing M. tuberculosis, however they are slower to develop than liquid media. A culture can turn positive in as little as four weeks, but it normally takes eight weeks of no development before it can be labelled as negative [19]. Liquid media can identify M. tuberculosis development within 2 weeks. For drug susceptibility testing, both liquid and solid media are employed, however only solid media is used for testing second-line drugs [19]. The findings of a sputum culture might take anywhere from 1 to 8 weeks.

The culture is negative if no bacteria develop. The culture is positive if bacteria exists. If M. tuberculosis proliferate, the individual has TB. The test can also reveal whether a lung infection is caused by a different type of bacteria [20].

Although finding AFB in a sputum smear is strong presumptive evidence of TB, definitive diagnosis requires a positive mycobacterial culture or nucleic acid amplification test (NAAT) [20]. NAAT amplify DNA and RNA segments in order to quickly identify the microorganisms in a sample. In comparison to culture, NAA testing may consistently detect M. tuberculosis bacteria in specimens within hours.

When the clinical suspicion of TB is moderate to high, a single negative NAA test result should not be taken as a decisive result to rule out TB. Rather, the negative NAA test result should be used as a supplement to clinical judgement, to speed testing for an alternative diagnosis, or to avoid needless TB disease therapy [20] For laboratory proof of TB disease, sputum culture remains the gold standard.

Bibliography

1. Loddenkemper R, Lipman M, Zumla A. Clinical aspects of adult tuberculosis. Cold Spring Harb Perspect Med [Internet]. 2015;6(1):a017848. Available from: http://dx.doi.org/10.1101/cshperspect.a017848
2. Schlossberg D. Acute tuberculosis. Infect Dis Clin North Am [Internet]. 2010 [cited 2022 Apr 8];24(1):139–46. Available from: https://pubmed.ncbi.nlm.nih.gov/20171549/
3. Heemskerk D, Caws M, Marais B, Farrar J. Clinical Manifestations. Berlin, Germany: Springer; 2015.
4. Antonini JM. Mycobacterium. In: Reference Module in Biomedical Sciences. Elsevier; 2014.
5. King LS. Signs and symptoms. JAMA [Internet]. 1968 [cited 2022 Apr 8];206(5):1063–5. Available from: https://pubmed.ncbi.nlm.nih.gov/4877912/
6. Body temperature [Internet]. Healthlinkbc.ca. [cited 2022 Apr 8]. Available from: https://www.healthlinkbc.ca/tests-treatments-medications/medical-tests/body-temperature
7. Auscultation [Internet]. Physiopedia. [cited 2022 Apr 8]. Available from: https://www.physio-pedia.com/Auscultation
8. Sarkar M, Madabhavi I, Niranjan N, Dogra M. Auscultation of the respiratory system. Ann Thorac Med [Internet]. 2015;10(3):158–68. Available from: http://dx.doi.org/10.4103/1817-1737.160831
9. Breath sounds [Internet]. Mount Sinai Health System. [cited 2022 Apr 8]. Available from: https://www.mountsinai.org/health-library/symptoms/breath-sounds
10. Vaishnav B, Bamanikar A, Rathore VS, Khemka VK. Fatal hemoptysis due to ruptured peripheral pulmonary artery pseudoaneurysm. Lung India [Internet]. 2017 [cited 2022 Apr 8];34(1):106–7. Available from: http://dx.doi.org/10.4103/0970-2113.197107
11. Skeff KM. Reassessing the HPI: The chronology of present illness (CPI). J Gen Intern Med [Internet]. 2014;29(1):13–5. Available from: http://dx.doi.org/10.1007/s11606-013-2573-3
12. Farah MG, Meyer HE, Selmer R, Heldal E, Bjune G. Long-term risk of tuberculosis among immigrants in Norway. Int J Epidemiol [Internet]. 2005 [cited 2022 Apr 8];34(5):1005–11. Available from: https://academic.oup.com/ije/article/34/5/1005/645880
13. Global tuberculosis report 2021 [Internet]. Who.int. [cited 2022 Apr 8]. Available from: https://www.who.int/teams/global-tuberculosis-programme/tb-reports/global-tuberculosis-report-2021
14. Pulmonary tuberculosis: TB causes, symptoms, & treatments [Internet]. Houstonmethodist.org. [cited 2022 Apr 8]. Available from: https://www.houstonmethodist.org/pulmonology/tuberculosis/
15. Rapid sputum tests for tuberculosis (TB) [Internet]. Alberta.ca. [cited 2022 Apr 8]. Available from: https://myhealth.alberta.ca/Health/pages/conditions.aspx?hwid=abk7483
16. TB (tuberculosis) tests [Internet]. WebMD. [cited 2022 Apr 8]. Available from: https://www.webmd.com/lung/tests-tuberculosis
17. Sputum testing for tuberculosis (TB) [Internet]. Healthlinkbc.ca. [cited 2022 Apr 8]. Available from: https://www.healthlinkbc.ca/healthlinkbc-files/sputum-testing-tuberculosis-tb
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19. Schaaf H. Spinal tuberculosis in childrenA report of a complicated case. In: Tuberculosis. Elsevier; 2009. p. 871–3.
20. Chapter 4 Diagnosis of Tuberculosis Disease [Internet]. CDC. [cited 2022 Apr 8]. Available from: https://www.cdc.gov/tb/education/corecurr/pdf/chapter4.pdf

TB generally affects the lungs, but other organ systems may also be affected such as the skin, liver, reproductive system, musculoskeletal system, gastrointestinal system and lymphoreticular system (1).  As such, the physiological functioning of the respiratory system would mostly be affected by the infection.  This may be the case because the lungs are exposed to the air as it functions in the gas exchange and Mycobacterium tuberculosis (M.tb) infection occurs due to airborne dried mucous droplets that are transmitted from another individual with active TB (2).  Further, this would include other compartments of the respiratory tract as well such as the nose, pharynx, larynx, trachea, bronchi, bronchioles and lungs (2).  

In its early stages, when TB just reached the alveoli, alveolar macrophages recognize the bacteria and initiates an inflammatory response (3).  This leads to formation of granuloma, which is a structure that contains many immune cells like macrophages, neutrophils, T and B cells (4). The granuloma surrounds caseous and necrotic alveolar macrophages that are infected by TB (5). While this is the host response to protect itself from TB attack, it causes host damage as well as the granulomas breakdown due to necrosis, leading to the formation of a cavity (6). Alternatively, it is also suggested that lipid pneumonia lesions formed by TB is the cause of caseous necrosis formation (7,8), where the alveolar cells and nearby vessels of bronchi are broken down. This is then coughed out by the patient, leaving a cavity where the necrotic tissue once was (8,9). Additionally, pulmonary fibrosis occurs due to the long-term tissue damage experienced in the lung. Extracellular matrix proteins deposit in the lungs and normal lung tissue is replaced with collagenous tissue, making the lung walls more thick and stiff (Ravimohan et al., 2018). Bronchiectasis may also develop and is characterized with dilation of the bronchi as well as bronchial wall thickening. The bronchial wall loses its elasticity and ability to contract as muscles are destroyed (4).

As a result, these changes in the respiratory system physiology, like inflammation within the airway which causes a reduction in the ability for air to be exchanged by the respiratory tract as the airways become narrower, can lead to airway obstruction (2).  The reason for this physiological change is to slow down the M.tb from moving into, accessing and damaging the healthy lungs of the individual (2).  Further, inflammation can cause disruptions and swelling of the mucous membranes which line the bronchi and bronchioles (2).  This manifests as symptoms such as inability to breath and reduced exercise capabilities. Additionally, cavities formed in the respiratory tract may distort the airways, further contributing to difficulties in breathing. Bronchiectasis also contributes to this as the bronchi is less elastic (Roberts et al., 2000). Fibrosis and nodular infiltrates or a combination of pulmonary diseases may also be present in the lungs of an individual (11).  This is also what contributes to the breathlessness, wheezing, chest pain or cough an individual may experience when infected with M.tb.  Infiltrates abnormally accumulate within the lungs or airways and cavities that are left untreated can ultimately lead to respiratory failure in the patient (12).

Moreover, the caseous necrotic tissue that was formed may rupture and allow the TB antigen to enter into the pleural space surrounding the lungs (13, 14). The pleural space contains pleural fluid in healthy patients and allows the lungs to inflate and deflate during respiration. However, the TB antigen stimulates an inflammatory response, which leads to increased permeability of neary capillaries to allow leukocytes to migrate and enter the infected tissue. This influx of proteins would lead to more pleural fluid production (15). This accumulation of fluid leads to pleural effusion and is responsible for symptoms such as inability to breathe, as well as pain during respiratory cycles (16).

Figure 1. TB is inhaled into the lungs and this may potentially be carried to other organs as well

When left untreated, TB may spread via hematogenous route, lymphatic route, descending direct spread or in rare cases via sexual transmission to infect the genital tract (17).  Thus, the reproductive system would be negatively impacted and infertility may occur if the female genital organs, like the fallopian tubes are affected by M.tb (17).  Additionally, the musculoskeletal system may be affected if treatment is delayed and spread occurs, causing spinal damage or damage to the joints and bones (18).  Finally, the gastrointestinal system may also be impacted and in such cases, patients can experience abdominal pain, fever and in rarer cases develop diarrhea (19).

Tuberculous lymph gland involvement can occur as a result of infection with M. tuberculosis or other nontuberculous mycobacteria (20). The anterior and posterior triangles of the neck, supraclavicular and axillary regions, as well as a number of additional nodal locations, have all been shown to have TB lymphadenitis. A single node site or many node sites might be used for presentation. The illness is often indolent, with the patient presenting with a single, unilateral, nontender neck mass. Scrofula is a name that has been used to denote tuberculous involvement of a cervical lymph node with the development of a sinus tract or ulceration of the surrounding skin in the past.

M. tuberculosis seeds the vascular renal cortex during original infection or during spread associated with reactivation (20). Healed granulomatous lesions in the glomeruli can burst into the renal tubule and become mechanically caught up at the loop of Henle, causing granulomatous progression, necrosis, and cavitation in the medullary part, which has inadequate host defense. Renal failure is unusual because, despite the fact that both kidneys are normally seeded, serious renal involvement is often asymmetric or unilateral (25 percent). The infundibulum, ureter, bladder, prostate, epididymis, and testes may then be affected by descending infection. The presence of both upper and lower respiratory tract illness is extremely indicative of tuberculosis. Granulomatous lesions, usually in the upper or lower third of the ureter, can cause narrowing of the collecting system and strictures that can progress despite treatment (20).

Figure 2: Shruti et al. Mechanisms and features associated with tuberculosis.

Miliary TB is a description of the clinical disease caused by the widespread hematogenous dissemination of bacteria to most organs of the body (20). Bacteria enter the circulation during initial infection before the host's immune system has fully reacted, or during latent infection reactivation. On a chest radiograph, the illness may appear as a miliary pattern, which is defined by 1-5 mm nodules.

TB can also travel to the central nervous system and lead to tuberculous meningitis. To do this, TB has to cross the blood-brain barrier and the blood-cerebrospinal fluid barrier (21). This is achieved by TB through rearrangement the actin filaments in brain endothelial cells so it can travel through and reach the brain from the brain capillaries (22). Additionally, TB can also enter by hijacking macrophages and neutrophils, and infecting and traveling in these cells that are able to cross the barriers (23). After entry to the brain, TB stimulates an immune response that leads to inflammation, which can contribute to brain damage. For example, TB can induce changes to neuroendocrine metabolism (More et al., 2017). This is likely because TB infection in the brain leads to disruption of basal structures that are in proximity to the pituitary gland, pituitary stalk, and hypothalamus. An exudate forms here and leads to edema and perivascular infiltration, resulting in encephalitis. This contributes to metabolic changes observed (24). This is confirmed in post-mortem studies of patients with tuberculous meningitis, where they found thick inflammatory exudate at the basal structures as well as subarachnoid spaces of the brain (25). The basal structures especially are important as it contains major cerebral vessels, an exudate there may block the circulation of cerebrospinal fluid, and the cranial nerves there are affected, which can lead to cranial nerve palsies (26).

In nearly half of all bone and joint TB cases, spinal or vertebral TB (Pott's disease) is found (20). Vertebral bodies have a high level of vascularity well into adulthood, which explains why bone and joint TB can develop here. Infection usually begins in the anterior-inferior portion of a vertebral body, spreads beneath the anterior longitudinal ligament, and spreads to neighbouring vertebral bodies, causing illness. In spinal tuberculosis, the lower thoracic and upper lumbar vertebrae are the most commonly afflicted. The majority of patients arrive with gradually worsening back pain. Except in the case of extraspinal or disseminated illness, fever and constitutional symptoms are uncommon. Paraspinous fluid collections, which appear fusiform on imaging and can proceed to psoas muscle abscesses, are one of the most common complications. Advanced illness might cause compression of the spinal cord or peripheral nerves, resulting in neurologic impairments (20).

Tuberculous arthritis commonly affects major, weight-bearing joints like the hip or knee as a monoarthritis. Swelling, discomfort, and loss of function are all possible symptoms. Late presentation has been linked to cartilage deterioration, deformity, and leaking sinuses. M. tuberculosis has also been linked to infections in prosthetic joints. Although osteomyelitis infecting other parts of the skeleton is rare, it has been documented (20).

The ileocecal, jejunoileal, or anorectal areas are the most common sites of gastrointestinal TB, however the esophagus, stomach, and duodenum have also been reported (20). Hepatosplenic, biliary tract, and pancreatic tuberculosis have all been documented, however they are quite uncommon. Patients with ileocecal TB may have clinical and radiological signs and symptoms that are similar to those of Crohn's disease, such as persistent abdominal pain (up to 90%), constitutional symptoms, and a right lower quadrant mass (25-50%).

The peritoneum becomes studded with tubercles that leak proteinaceous fluid, which is clinically referred to as ascites, in patients with predominantly peritoneal involvement. Late-stage TB peritonitis might manifest as "dry," with fibro-adhesive characteristics ("doughy abdomen") and little ascitic fluid (20).

References:

1. Jilani TN, Avula A, Zafar GA, Siddiqui AH. Active Tuberculosis. 2022 [cited 2022 Apr 2]; Available from: https://pubmed.ncbi.nlm.nih.gov/30020618/
2. Torrelles JB, Schlesinger LS. Integrating lung physiology, immunology, and tuberculosis. Trends Microbiol [Internet]. 2017 [cited 2022 Apr 2];25(8):688–97. Available from: http://dx.doi.org/10.1016/j.tim.2017.03.007
3. Dannenberg A, Rook G. Pathogenesis of Pulmonary Tuberculosis: an Interplay of Tissue-Damaging and Macrophage-Activating Immune Responses-Dual Mechanisms That Control Bacillary Multiplication. Tuberculosis. 2014;:459-483.
4. Ramakrishnan L. Revisiting the role of the granuloma in tuberculosis. Nature Reviews Immunology. 2012;12(5):352-366.
5. Dorhoi A, Kaufmann SH. Pathology and immune reactivity: understanding multidimensionality in pulmonary tuberculosis. Semin Immunopathol. 2016;38(2):153-66. doi: 10.1007/s00281-015-0531-3.
6. Helke KL, Mankowski JL, Manabe YC. Animal models of cavitation in pulmonary tuberculosis. Tuberculosis (Edinb). 2006;86(5):337-48. doi: 10.1016/j.tube.2005.09.001.
7. Hunter RL, Jagannath C, Actor JK. Pathology of postprimary tuberculosis in humans and mice: contradiction of long-held beliefs. Tuberculosis (Edinb). 2007;87(4):267-78. doi: 10.1016/j.tube.2006.11.003.
8. Hunter RL. Pathology of post primary tuberculosis of the lung: an illustrated critical review. Tuberculosis (Edinb). 2011;91(6):497-509. doi: 10.1016/j.tube.2011.03.007.
9. Grosset J. . Mycobacterium tuberculosis in the extracellular compartment: an underestimated adversary. Antimicrobial agents and chemotherapy, 2003;:47(3), 833–836. https://doi.org/10.1128/AAC.47.3.833-836.2003
10. Roberts HR, Wells AU, Milne DG, Rubens MB, Kolbe J, Cole PJ, Hansell DM. Airflow obstruction in bronchiectasis: correlation between computed tomography features and pulmonary function tests. Thorax. 2000;55(3):198-204. doi: 10.1136/thorax.55.3.198.
11. Ravimohan S, Kornfeld H, Weissman D, Bisson GP. Tuberculosis and lung damage: from epidemiology to pathophysiology. Eur Respir Rev [Internet]. 2018 [cited 2022 Apr 2];27(147):170077. Available from: https://err.ersjournals.com/content/27/147/170077
12. Nontuberculous Mycobacterial lung disease - NORD (national organization for rare disorders) [Internet]. NORD (National Organization for Rare Disorders). 2015 [cited 2022 Apr 2]. Available from: https://rarediseases.org/rare-diseases/nontuberculous-mycobacterial-lung-disease/
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14. STEAD WW, EICHENHOLZ A, STAUSS HK.  Operative and pathologic findings in twenty-four patients with syndrome of idiopathic pleurisy with effusion, presumably tuberculous. Am Rev Tuberc. 1955;71(4):473-502. doi: 10.1164/artpd.1955.71.4.473.
15. Light RW. Update on tuberculous pleural effusion. Respirology. 2010;15(3):451-8. doi: 10.1111/j.1440-1843.2010.01723.x.
16. Jany, B., & Welte, T. Pleural Effusion in Adults-Etiology, Diagnosis, and Treatment. Deutsches Arzteblatt international, 2019;116(21), 377–386. https://doi.org/10.3238/arztebl.2019.0377
17. Grace GA, Devaleenal DB, Natrajan M. Genital tuberculosis in females. Indian J Med Res [Internet]. 2017 [cited 2022 Apr 2];145(4):425–36. Available from: http://dx.doi.org/10.4103/ijmr.IJMR_1550_15
18. Ozdemir M, Ozdemir HG. Evaluation of patients admitted with musculoskeletal tuberculosis: sixteen years’ experience from a single center in Turkey. BMC Musculoskelet Disord [Internet]. 2021;22(1):542. Available from: http://dx.doi.org/10.1186/s12891-021-04426-y
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iii) What are treatments will be offered to Robert and how do these work? Representing mechanisms diagrammatically is helpful to demonstrate understanding (No need to include pictures of chemical structures).

Treatment for TB infection has an 85% success rate (1). Without appropriate treatment, however, there is a 60% mortality rate seen primarily in developing countries with a lack of medical resources (2). Another contributing factor to mortality is multidrug resistant strains of TB that emerged due to not completing the whole course of antibiotic treatment and the use of lower quality drugs (3, 4).  Swift diagnosis and treatment is necessary in localizing the infection and preventing spread to other organs and tissues (5).

Robert will be offered antituberculosis medication for his infection (6). For drug-susceptible tuberculosis, this refers to an initial (or induction) phase of the drugs rifampin, isoniazid, pyrazinamide, and ethambutol (7). The minimal period of treatment for completely susceptible illness is 6 months for rifampin-based regimens that include isoniazid and pyrazinamide in the intense phase of therapy [8]. For certain extrapulmonary TB, the duration may be longer, for example TB meningitis and TB affecting bones and joints may warrant treatment for up to 9 months (9). For TB meningitis and TB pericarditis, corticosteroids may also be administered for the first 1 to 2 months (10). There are also effective longer term treatments, but it has been shown that shorter treatments are less toxic and patients are more likely to follow through (11).

The standard treatment regimen includes ethambutol which is only used during the induction period and is added as protection for the case of unrecognized resistance to one of the three other drugs (7). If it becomes clear that the bacterium is susceptible to the effects of isoniazid, rifampin, and pyrazinamide, ethambutol can be discontinued (7). The first two months of the standard treatment with these first-line drugs leads to the destruction of bacteria in all growth stages (12). The following four months is treatment with only rifampin and isoniazid, in which rifampin focuses on eliminating any residual dormant bacilli and isoniazid ensures that rifampin-resistant mutants are killed (12).

The main goals of drug therapy for tuberculosis are to kill all actively metabolizing bacilli in the lungs and eliminate less actively replicating that can cause a relapse of the disease (12). It is important to note the chemical composition of the cell wall of Mycobacterium tuberculosis, as it contains peptidoglycans and lipids (particularly mycolic acids) which play a significant role in the virulence of the bacteria (12). The function of mycolic acid is to prevent chemical damage and dehydration of the cell wall of the bacteria (13). This also means that hydrophobic antibiotics will not be able to penetrate the cell wall due to this fatty acids’ presence. Mycolic acid gives the bacterium the ability to grow easily within macrophages without being destroyed by hiding it from the host’s immune system. Due to these important roles in the survival of the bacterium, mycolic acid is a strong target for drugs, and therefore, Isoniazid functions to block the production of this fatty acid (13).

Drug Action: Isoniazid (INH)

Isoniazid is able to suppress TB infection by attacking several bacterial targets. Isoniazid enters the bacteria through passive diffusion (14).  The prodrug of isoniazid circulates throughout the bloodstream and becomes activated into its acyl radical form via a bacterial catalase-peroxidase enzyme that is encoded by the KatG gene (12, 15). This enzyme is also known as a hemeB containing dimer, and only one domain is functional, and has peroxidase activities, which activates isoniazid by peroxidation ultimately producing reactive isoniazid-derived species that can damage the bacteria. Catalase-peroxidase functions to adapt to the low pH that is caused during the so-called oxidative burst, which occurs when the host cell phagocytes liberate oxygen radicals and convert them to peroxide as means to destroy pathogens. When this occurs, the pH drastically declines, which is when the enzyme helps the bacterium accustom to the new acidity. The enzyme does this by degrading the peroxide, ensuring that the pH levels do not reach lethal levels in their cells (12). In the context of the drug, this enzyme will catalyze the formation of the activated isoniazid intermediate from the prodrug, which will then bind to the InhA protein. The InhA protein is an enoyl-acyl carrier protein reductase encoded in the Mycobacterium gene and it functions to inhibit fatty acid synthesis by catalyzing the reduction of long-chain trans-2-enoyl-ACP in the type II fatty acid biosynthesis pathway of the bacteria (15). Inhibition of InhA disrupts the biosynthesis of the mycolic acid, thereby leaving the bacterium vulnerable to destruction by the immune system (12). This may also happen when isoniazid covalently binds to the cosubstrate of InhA NADH (or oxidation product NAD+), as InhA will again be inhibited (15).The the oxidation of NADH or isoniazid can also auto-oxidize to provide oxidants for katG peroxidase activity (16, 17, 18, 19).  Isoniazid must be taken with other drugs, as when used alone the bacterium may evolve resistance against specific targets of the drug (14).

Figure: Campbell, 2001. Mechanism of RNA polymerase inhibition by rifampicin

Rifampin, used in combination with Isoniazid, has the mechanism of action involving the inhibition of bacterial DNA-dependent RNA polymerase via the drug directly blocking elongation of RNA (20).  Rifampicin is a lipophilic, broad-spectrum antibiotic (12). It’s role is to bind to the B subunit of the DNA-dependent RNA polymerase, blocking RNA transcription and therefore bacterial RNA synthesis. This occurs specifically by steric occlusion such that the synthesis of short oligoribonucleotides to full-length transcripts is inhibited (12). Rifampicin has a high affinity for the DNA-dependent polymerase, and the dominant effect of this binding is a total blockage of either the second or third phosphodiester bond (21). There are a few important concepts tied to the function of Rifampicin: it does not interfere with substrate binding/catalytic activity, and if the RNA polymerase has already synthesized a transcript that has entered the elongation phase, it is completely resistant to the drug (21). These findings have helped characterize rifampicin’s mechanism of action, as it is clear that the drug inhibits the polymerase by simply blocking the path of the elongating RNA chain at the 5’ end (21).

Drug Action: Rifampicin (RIF)

This drug is used with isoniazid as it was discovered long ago that monotherapy (compared to combination therapy) resulted in short-lived improvements only and that the prevalence of drug-resistant bacteria seemed to increase (22).

Rifapentine prevents TB spread by inhibiting the bacterial DNA-dependent RNA polymerase. This prevents the replication of the bacteria and consequentially leads to cell death (23). Rifampin works through the same mechanism of action. The difference between rifapentine and rifampin is that rifampin has a shorter half-life and has a higher minimum inhibitory concentration (24). Rifampin is given once daily whereas rifapentine is given once weekly (24).

Drug Action: Pyrazinamide (PZN)

Pyrazinamide has a role in shortening the length of standard tuberculosis therapy, from 9-12 months to 6 months (25). Pyrazinamide functions as a sterilizing agent and can kill populations of the bacterium that may have been residually left by other drugs. Pyrazinamide, a prodrug, is converted to its active form, pyrazinoic acid, through nicotinamidase which is encoded by the pncA gene in M. tuberculosis (10).  In its active form, pyrazinoic acid is able to passively diffuse and exit the TB bacterium which can eventually re-enter the bacteria through acid facilitation (26). Pyrazinamide is unlike other drugs as it does not target growing bacteria and rather is active against nongrowing bacilli. This drug is also only active at an acidic pH (ie less than pH 5.5), and its activity increases as metabolic activity declines (25).  If the extracellular pH is acidic and pyrazinoic acid is outside the cell, a small amount of the drug will become protonated, which will easily permeate through the membrane. Eventually, the bacterial cells will have an accumulation of the drug, and the protonated drug will bring more protons into the cell, causing acidification of the bacteria such that the crucial enzymes for function will be inhibited (25). For example, the fatty synthase enzyme will be affected, inhibiting fatty acid synthesis and disrupting bacteria growth and replication (27). Pyrazinoic acid can also de-energize the membrane by damaging the proton motive force required for the membrane transport, thereby also inhibiting protein and RNA synthesis (25). Additionally, pyrazinoic acid is able to inhibit ribosomal protein S1, thus preventing trans-translation which is essential for bacterial growth (25).

Ethambutol works by reducing the production of lipoarabinomannan and arabinogalactan, which are bacterial cell wall components (28). The drug diffuses into the bacterial cell and inhibits arabinosyltransferases that are responsible for producing these molecules. This consequently prevents bacterial replication as it needs cell walls to divide (28). Additionally, lipoarabinomannan is responsible for TB interaction with host cells, and disrupting its production may interfere with this process as well (29).

It is important to keep in mind that the treatment of TB will take longer than other bacterial infections and infected individuals will only start feeling better from the administered medication after a few weeks of usage (30). Incorrect usage of medication or not using the prescribed medication to completion could lead to ineffective treatment as well as a higher chance for future complications (30). As with other bacterial species, incorrect usage of the antibacterial medicine in particular can lead to resistant forms of TB and reduced effectiveness of treatment in the future. During the process of medication treatment, it is recommended to stick to a routine in order to ensure it is taken every day (30).

References:

1. World Health Organization. Global Tuberculosis Report 2015. 20. Geneva: WHO; 2015.

2. Kaye, K., and T. R. Frieden. (1996). Tuberculosis control: the relevance of classic principles in an era of acquired immunodeficiency syndrome and multidrug resistance. Epidemiol. Rev. 18:52-63.

3. Smith I. (2003). Mycobacterium tuberculosis pathogenesis and molecular determinants of virulence. Clinical microbiology reviews, 16(3), 463–496. https://doi.org/10.1128/CMR.16.3.463-496.2003

4. O'Brien, R. J. (2001). Tuberculosis: scientific blueprint for tuberculosis drug development. Global Alliance for TB Drug Development, New York, N.Y.

5. Pulmonary tuberculosis: TB causes, symptoms, & treatments [Internet]. Houstonmethodist.org. Available from: https://www.houstonmethodist.org/pulmonology/tuberculosis/

6. Sterling, T., Njie, G., Zenner, D., Cohn, D., Reves, R., & Ahmed, A. et al. (2020). Guidelines for the Treatment of Latent Tuberculosis Infection: Recommendations from the National Tuberculosis Controllers Association and CDC, 2020. MMWR. Recommendations And Reports, 69(1), 1-11. https://doi.org/10.15585/mmwr.rr6901a1

7. Horsburgh Jr, C. R., Barry III, C. E., & Lange, C. (2015). Treatment of tuberculosis. New England Journal of Medicine, 373(22), 2149-2160.

8. Silva DR, Mello FC de Q, Migliori GB. Shortened tuberculosis treatment regimens: what is new? J Bras Pneumol [Internet]. 2020 [cited 2022 Apr 2];46(2):e20200009. Available from: http://dx.doi.org/10.36416/1806-3756/e20200009

9. Nahid P, Dorman SE, et al. (2016). Official American Thoracic Society/Centers for Disease Control and Prevention/Infectious Diseases Society of America Clinical Practice Guidelines: Treatment of Drug-Susceptible Tuberculosis. Clin Infect Dis, 63(7):e147-e195. doi: 10.1093/cid/ciw376.

10. Wiysonge CS, Ntsekhe M, Thabane L, Volmink J, Majombozi D, Gumedze F, Pandie S, Mayosi BM. (2017). Interventions for treating tuberculous pericarditis. Cochrane Database Syst Rev, 9(9):CD000526. doi: 10.1002/14651858.CD000526.pub2.

11. CDC: Treatment Regimens for Latent TB Infection (LTBI). CDC website. Reviewed February 13, 2020. Accessed March 31, 2022. https://www.cdc.gov/tb/topic/treatment/ltbi.htm

12. Rivers, E. C., & Mancera, R. L. (2008). New anti-tuberculosis drugs in clinical trials with novel mechanisms of action. Drug discovery today, 13(23-24), 1090-1098.

13. Somasundaram, S., Ram, A., & Sankaranarayanan, L. (2014). Isoniazid and rifampicin as therapeutic regimen in the current era: a review. Journal of Tuberculosis Research, 2014.

14. Bardou, F., Raynaud, C., Ramos, C., Laneelle, M.A., and Laneelle, G. (1998). Mechanism of isoniazid uptake in Mycobacterium tuberculosis. Microbiology 144 (Pt 9): 2539–2544.

15. He, X., Alian, A., & De Montellano, P. R. O. (2007). Inhibition of the Mycobacterium tuberculosis enoyl acyl carrier protein reductase InhA by arylamides. Bioorganic & medicinal chemistry, 15(21), 6649-6658.

16. Ghiladi, R.A., Medzihradszky, K.F., Rusnak, F.M., and Ortiz de Montellano, P.R. (2005) Correlation between isoniazid resistance and superoxide reactivity in Mycobacterium tuberculosis KatG. J Am Chem Soc 127: 13428–13442.

17. Zhao, X., Yu, H., Yu, S., Wang, F., Sacchettini, J.C., and Magliozzo, R.S. (2006) Hydrogen peroxide-mediated isoniazid activation catalyzed by Mycobacterium tuberculosis catalase-peroxidase (KatG) and its S315T mutant. Biochemistry 45: 4131–4140.

18. Wengenack, N.L., and Rusnak, F. (2001) Evidence for isoniazid-dependent free radical generation catalyzed by Mycobacterium tuberculosis KatG and the isoniazid-resistant mutant KatG (S315T). Biochemistry 40: 8990–8996.

19. Winder, F.G., and Denneny, J.M. (1959) Metal-catalysed auto-oxidation of isoniazid. Biochem J 73: 500–507.

20. Suresh AB, Rosani A, Wadhwa R. Rifampin. StatPearls Publishing; 2022.

21. Campbell, E. A., Korzheva, N., Mustaev, A., Murakami, K., Nair, S., Goldfarb, A., & Darst, S. A. (2001). Structural mechanism for rifampicin inhibition of bacterial RNA polymerase. Cell, 104(6), 901-912.

22. Rothstein, D. M. (2016). Rifamycins, alone and in combination. Cold Spring Harbor perspectives in medicine, 6(7), a027011.

23. Munsiff, S., Kambili, C., & Ahuja, S. (2006). Rifapentine for the Treatment of Pulmonary Tuberculosis. Clinical Infectious Diseases, 43(11), 1468-1475. https://doi.org/10.1086/508278

24. Alfarisi O, Alghamdi WA, Al-Shaer MH, Dooley KE, Peloquin CA. (2017). Rifampin vs. rifapentine: what is the preferred rifamycin for tuberculosis? Expert Rev Clin Pharmacol. 10(10):1027-1036. doi: 10.1080/17512433.2017.1366311.

25. Zhang, Y., Shi, W., Zhang, W., & Mitchison, D. (2014). Mechanisms of pyrazinamide action and resistance. Microbiology spectrum, 2(4), 2-4.

26. Zhang Y, Shi W, Zhang W, Mitchison D. Mechanisms of pyrazinamide action and resistance. Microbiol Spectr [Internet]. 2013 [cited 2022 Apr 2];2(4):1–12. Available from: http://dx.doi.org/10.1128/microbiolspec.MGM2-0023-2013

27. Boshoff HI, Mizrahi V, Barry CE 3rd. (2002). Effects of pyrazinamide on fatty acid synthesis by whole mycobacterial cells and purified fatty acid synthase I. J Bacteriol. 184(8):2167-72. doi: 10.1128/JB.184.8.2167-2172.2002.

28. Zhang L, Zhao Y, Gao Y, Wu L, Gao R, Zhang Q, Wang Y, Wu C, Wu F, Gurcha SS, Veerapen N, Batt SM, Zhao W, Qin L, Yang X, Wang M, Zhu Y, Zhang B, Bi L, Zhang X, Yang H, Guddat LW, Xu W, Wang Q, Li J, Besra GS, Rao Z. (2020). Structures of cell wall arabinosyltransferases with the anti-tuberculosis drug ethambutol. Science. 368(6496):1211-1219. doi: 10.1126/science.aba9102.

29. Goude R, Amin AG, Chatterjee D, Parish T. (2009). The arabinosyltransferase EmbC is inhibited by ethambutol in Mycobacterium tuberculosis. Antimicrob Agents Chemother. 53(10):4138-46. doi: 10.1128/AAC.00162-09.

30. Treating and managing tuberculosis [Internet]. Lung.org. Available from:https://www.lung.org/lung-health-diseases/lung-disease-lookup/tuberculosis/treating-and-managing

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

Tuberculosis kills more than three million people each year, making it a global public health concern, particularly in developing countries (1). TB is the second most common cause of mortality and affects up to millions around the world every year. Currently, 33% of the world’s population is infected with latent TB, and this number is growing every year (2). TB is especially a concern as it is highly transmissible through spread in the air. Individuals can contract the bacteria through breathing in air particles that contain the pathogen as an actively TB infected person is coughing, sneezing, or talking (2). To prevent and control the spread of TB, governments must identify and treat individuals that are actively infected with TB, as well as individuals that have a latent infection but are at risk of developing into active disease (2). Thus, in this case, it is critical that the Public Health unit is notified as soon as possible, ideally within twenty-four hours, so that further potential cases and outbreaks can be avoided or minimized. Disease transmission can essentially continue without proper notification, resulting in devastating outbreaks (3). Because Robert is an immigrant from India, it is critical to determine whether the infection occurred after he arrived in Canada or if it occurred while the patient was still in India, a country with one of the highest TB cases. By determining the source of the infection, an appropriate diagnosis and treatment for Robert can be provided. In British Columbia, reports to the Public Health unit would include active and latent TB surveillance, case definitions of active TB, site of disease, and latent Tb infection, as well as contact tracing reports (4). An essential approach to achieve this is case finding through contact investigation/tracing. This has been shown to be very effective in detecting potential cases of active TB and thus taking appropriate action to reduce the possibility of further spreading of the disease (5).

Laboratories, like healthcare providers who treat patients, should report suspected or confirmed cases of tuberculosis, including the date, results of tests performed in the lab, and the name and address of the physician who examined the patient (6). Failure to submit these reports may result in penalties such as citations and fines, especially if negative health outcomes result (6).

References:

1. Hershfield ES. Tuberculosis - Still a major health problem. Can J Infect Dis [Internet]. 1991 Winter [cited 2022 Apr 2];2(4):131–2. Available from: http://dx.doi.org/10.1155/1991/297605
2. Public Health Agency of Canada. Center for Communicable Diseases and Infection Control. (2014). TUBERCULOSIS PREVENTION AND CONTROL IN CANADA - A FEDERAL FRAMEWORK FOR ACTION. Public Health Agency of Canada.
3. Uplekar M, Atre S, Wells WA, Weil D, Lopez R, Migliori GB, et al. Mandatory tuberculosis case notification in high tuberculosis-incidence countries: policy and practice. Eur Respir J [Internet]. 2016 [cited 2022 Apr 2];48(6):1571–81. Available from: http://dx.doi.org/10.1183/13993003.00956-2016
4. Tuberculosis reports [Internet]. Bccdc.ca. [cited 2022 Apr 2]. Available from: http://www.bccdc.ca/health-professionals/data-reports/tuberculosis-reports
5. Behr MA, Hopewell PC, Paz EA, Kawamura LM, Schecter GF, et al. (1998) Predictive value of contact investigation for identifying recent transmission of Mycobacterium tuberculosis. American journal of respiratory and critical care medicine 158: 465–469.
6. Reporting cases of suspected or confirmed tuberculosis [Internet]. Sccgov.org. [cited 2022 Apr 2]. Available from: https://publichealthproviders.sccgov.org/diseases/tuberculosis-tb/reporting-cases-suspected-or-confirmed-tuberculosis

The Microbiology Laboratory

i) Including any stated bacteria, what are the most common bacterial pathogens associated with this type of clinical presentation?

The bacteria that may cause tuberculosis (TB) in our patient is from the Mycobacterium tuberculosis complex which umbrellas nine other species in the genus Mycobacterium, family Mycobactericidal and order Actinomycetales and these nine species may cause human TB or zootonic TB (1). M. tuberculosis was found in a complex in 2019 that has at least 9 members: M. tuberculosis sensu stricto, M. africanum, M. canetti, M. bovis, M. caprae, M. microti, M. pinnipedii, M. mungi, and M. orygis (1).

The species share a high similarity in their sequences, and it is hypothesized that they all originate from one ancestor (1). However, there are three types of Mycobacteria that cause human TB, which will be discussed. There are also three other types of Mycobacteria that cause massive lung infiltration and result in similar clinical presentations that Robert is experiencing (1).

Fig 1. Transmission of M. tuberculosis From: CDCTB. Tuberculosis (TB)- How TB Spreads [Internet]. Centers for Disease Control and Prevention. 2016 [cited 2022 Apr 7]. Available from: https://www.cdc.gov/tb/topic/basics/howtbspreads.htm

Mycobacterium tuberculosis sensu stricto

This bacterium is an aerobic, non-spore-forming, non-motile bacillus species with a high cell wall content. Interestingly, this bacterium has genes that encode enzymes for pathways of lipogenesis and lipolysis. This species causes most human TB disease, and humans are the only reservoir for this bacterium. But it must be noted that animals may still be infected (1).  Person-to-person transmission of M. tuberculosis occurs through the inhalation of M. tuberculosis-carrying aerosol droplets that are 1-5 microns in diameter (2) Infectious droplets are produced when individuals who have pulmonary or laryngeal TB cough, sneeze, shout, or sing (2). It should be noted that transmission of M. tuberculosis occurs solely through the air, and not through surface contact(3).

This bacterium is the most common cause of human TB and is most likely the bacteria Robert is infected with. The typical presentations of this bacteria include acute fever, cough and local pleuritic chest pain, night sweats, and chills which are Robert’s main symptoms (4).

This bacterium is first known to enter the alveoli by airborne transmission, colonize the lungs, and resist macrophage-mediated destruction. It can eventually enter the blood and cause systemic spread, allowing samples to be taken from more than one anatomical location (5).

The environment also plays a role in the transmission of M. tuberculosis, with specific environmental factors being the concentration of infectious aerosol droplets, air circulation, and ventilation (3). Lastly, person-to-person transmission is, of course, impacted by contact between individuals. More specifically, the duration, frequency, and physical proximity of contact between individuals dictates the likelihood of transmission (3). It should also be noted that individuals may have a latent tuberculosis infection (LTBI) or TB disease, and while individuals with the latter may spread it to other individuals, those with LTBI are unable to spread the infection to other people(3). Additionally, individuals with HIV are more likely to be infected by M. tuberculosis (3).

Mycobacterium bovis

This is a gram-positive, aerobic, nonmotile rod-shaped bacterium. This bacterium causing TB infection was more common in the Middle Ages when the ingestion of unpasteurized milk from cattle was more common (3). M. bovis is a zoonotic disease and it is commonly transmitted from animals to humans through the eating or drinking of contaminated unpasteurized dairy products. It can also be transmitted between animals and humans through direct contact with infected wounds, which may occur during slaughter or hunting(6).

This arises if dairy products are not appropriately treated and ingestion leads to direct gastrointestinal infection; in this case, a sputum sample would not be beneficial. It is unlikely that Robert is infected with this bacterium (5).

However, the clinical presentation of this infection includes fever and night sweats which are symptoms Robert is experiencing. Yet, he is not reporting abdominal pain and diarrhea, which are common symptoms of GI infection with this bacterium. It is to note that less than 2% of human TB cases are from this bacteria and have been significantly limited with disease control in cattle and routine pasteurization of cow’s milk (6).

Mycobacterium africanum

This bacterium causes half of the human TB cases in West Africa. This strain was first identified as an intermediate between M. tuberculosis and M. bovis and found that this culture grew much slower than the other mycobacterium (7). This strain has only been reported in West Africa and not in different regions in Africa, so it is unlikely that Robert has this bacterium unless he has recently travelled to these areas after immigrating to America (7). But, this strain has been identified in Germany, England, California, France and Spain sporadically from pulmonary and extra-pulmonary sources (7).

This bacterium is also known to cause pulmonary tuberculosis and at least half of the cases in West Africa. Symptoms of this bacterial infection causing TB may include coughing, chest pain, fever, night sweats, chills, fatigue, breathlessness, coughing of blood or phlegm, and general TB symptoms. Robert has not reported coughing of blood or phlegm yet, so this may not be the bacterium he is infected by and considering he has not travelled to West Africa recently.

Other species within the Mycobacterium tuberculosis complex:

Similar to M. bovis, M. caprae is another species that is part of the Mycobacterium tuberculosis complex and is known to cause disease in mammals other than humans. Cattle, in particular, are a known reservoir of M. caprae (8). M. pinnipedii and M. microti, which cause disease in seals and rodents respectively, have also been reported to result in zoonotic TB in humans (1). Lastly, the M. canettii strains are extremely rare and a limited number of human TB infections resulting from this particular species have been reported (9).

Non-Tuberculosis Mycobacteria

In this subsection, lower respiratory illnesses may resemble tuberculosis and could be a suspected potential illness Robert could be suffering from before diagnosis with TB. These bacteria cannot be transmitted between humans but are instead picked up from soil or water.  

Mycobacterium kansaii

This is a gram-positive, non-motile and non-spore-forming bacterium (10). This bacterium is an environmental bacterium known to cause opportunistic infection within humans. Commonly found in tap water, swimming pools, brackish water and seawater. However, infection is mainly done through aerosol transmission, and human-to-human transmission is hypothesized not to be a mode of transmission (6).

Common symptoms of infection with this bacterium include fever, chills, and night sweats which are characteristics of the case. But a key factor is that this bacterium causes a non-productive cough or a productive cough, whereas Robert is only experiencing a productive cough. It has been noted that this lung disease is clinically indistinguishable from TB, and symptoms may be more severe or chronic (10). Therefore, further laboratory testing must be done for diagnosis.

Fig 2. Diagram of Mycobacterium that make up MAC From: Osmosis - A Better Way To Learn - Mycobacterium avium complex (NORD): Video & Anatomy [Internet]. Osmosis. [cited 2022 Apr 7]. Available from: https://www.osmosis.org/learn/Mycobacterium_avium_complex_(NORD)

Mycobacterium avium-complex (MAC)

This is a non-motile, gram-positive non-spore-forming group of an opportunistic bacterium closely related to tuberculosis (11). This is more common in immunocompromised patients, such as those diagnosed with AIDS, as dissemination occurs in these cases. This bacterium is also known to infect the lungs after inhalation or ingestion and infect the GI tract (7). This bacterium adheres to the mucosal lining of the tract and can spread to other regions, including the liver, spleen, lymph nodes and bone marrow. This bacterium is also a collective term for Mycobacterium avium and Mycobacterium intracellular. These are both acid-fast bacilli that are more commonly found in atypical tuberculosis and may cause diarrheal symptoms and abdominal pain, which is not reported in the case (12). M. avium subspecies Avium (two strains) are responsible for pulmonary infections (13). Mycobacterial infections including M. avium complex infections can be categorized into several clinical patterns including pulmonary disease, skin, and soft tissue infections, musculoskeletal infections, disseminated disease, catheter-associated disease, and lymphadenitis (13). Pulmonary disease is the most common presentation (13). Mycobacterium avium complex infections occur in both immunocompetent and in immunosuppressed patients (13). M. avium is the most frequent organism in HIV, and immunosuppressed patients, about 40% of pulmonary infections in immunocompetent patients, can be due to M. intracellulare (4). Despite the ubiquitous nature of MAC organisms in the environment, relatively few of those who are infected develop disease (14). Thus, some degree of susceptibility due to either underlying lung disease or immunosuppression is often required (14). Organisms belonging to MAC are commonly isolated from water, house dust, and soil (14). MAC organisms have been isolated from both natural and treated water and presumably enter drinking water treatment plants via their attachment to soil particles in surface water sources (14). Because of their lipid-rich outer membranes, they are relatively resistant to chemical disinfection by chlorine, chloramines, and ozone (5). It is hypothesized that humans become infected with MAC through exposure to environmental sources in the home and elsewhere(14). Inhalation of infectious aerosols from the environment is likely the primary mode of transmission and results in pulmonary disease (14).

It is to note that this type of infection is not contagious and includes common symptoms pointed out in the case, such as cough, night sweats, and fatigue (11). However, this may also cause belly pain, diarrhea, weight loss and swollen glands not pointed out in the case (15). This bacterium naturally resides in soil and water; thereby, stirring soil can make the bacteria airborne and inhaled (16).  This causes non-tuberculosis mycobacteria infection (NTM). This bacterium can also cause two types of MAC lung disease: nodular bronchiectasis and fibro cavitary disease.

Mycobacterium scrofulaceum

This is a gram-positive, rod-shaped bacterium. Infection with this bacterium is more common in children’s cases of cervical lymphadenitis and is rarely encountered in adults, so it is unlikely Robert is infected with this bacterium. However, there have been adult cases if there was a prior structural lung disease before infection (17).

Bacterium That May Cause Similar Symptoms

Fig 3. Symptoms of Pneumonia, similar to Robert’s symptoms From: What Is Double Pneumonia? [Internet]. Verywell Health. [cited 2022 Apr 7]. Available from: https://www.verywellhealth.com/double-pneumonia-5179463

Bacterial Pneumonia

• Streptococcus pneumonia is a gram-positive, lancet-shaped, facultatively anaerobic bacteria with a multitude of serotypes (18). This bacterium can cause pneumococcal disease by infecting the lungs and cause chest pain, stiff neck, increased sensitivity to light, joint pain, ear pain and irritability, which are not described in the case (19). However, it may have similar symptoms presented in the case, such as chills and fever, fatigue, and cough. This bacterium is spread from person to person by inhalation or direct exposure to droplets of this bacterium through coughing or sneezing via human transmission (20). But, it can also enter the lungs via the bloodstream or lymphatics if the bacteria have colonized elsewhere. This bacterium is found more commonly in low or middle-income countries with less pneumococcal vaccine distribution (20). Since Robert immigrated from India less than a year ago, it may be likely that he has not received this vaccine due to India being a lower-middle-income country. Therefore, infection with this bacterium is possible.

• Staphylococcus aureus is a gram-positive round-shaped bacilli bacteria. This pathogen is very contagious and can be passed from breathing in droplets of an infected person or by touching a person carrying this bacteria or contaminated surfaces or invasion via a cut (21). This pathogen is spread systemically through blood circulation, where it will reach the lungs, which can cause pneumonia. Symptoms of infection with this bacterium include difficulty breathing, fever, and chills which are a part of the symptoms Robert reports. As well, it may cause malaise (21).

• H. influenzae is a bacteria characterized as a small, facultatively anaerobic, pleomorphic, and capnophilic gram-negative coccobacillus of the family Pasteurellaceae (22). This gram-negative coccobacillus facultative anaerobe bacterium Hemophilus influenzae usually is present as a commensal bacterium in the nasopharynx of healthy adults. Still, it can invade and spread into the respiratory tract, causing infection and spreading systemically (23). Physicians consider it to infect the blood or fluid surrounding the lungs, causing bronchopneumonia or segmental pneumonia, which contains similar symptoms to what Robert is experiencing, such as fever and cough (23). However, patients with segmental pneumonia present fever and pleuritic chest pain, which Robert is experiencing, but it also presents a sore throat not noted in the case. Bronchopneumonia can present with tachypnea, not shown in the case but present Robert’s mild fever symptom. Haemophilus influenza is more commonly seen as bronchopneumonia (24).

• P. aeruginosa is a gram-negative, aerobic, non-spore forming rod that is capable of causing a variety of infections in both immunocompetent and immunocompromised hosts(22). Pseudomonas aeruginosa is commonly found in the environment, particularly in freshwater. Reservoirs in urban communities include hot tubs, jacuzzis, and swimming pools (22).

Brucellosis

Brucellosis is an infectious disease caused by Brucella species of bacteria (25). It is known by many other names, including remitting fever, undulant fever, Mediterranean fever, Maltese fever, Gibraltar fever, Crimean fever, goat fever, and Bang disease(25). The disease is transmitted from animals to humans by consuming unpasteurized milk and dairy products, consuming undercooked meat, or skin penetration of those in contact with livestock (25). Brucella organisms are small aerobic intracellular coccobacilli (25). These are found in the reproductive organs of host animals, causing abortions and sterility (25). They are shed in urine, milk, placental fluid, and other fluids of the animals (25). Many species have been identified, but four have moderate-to-significant human pathogenicity: Brucella melitensis (from sheep), Brucella suis (from pigs), Brucella abortus (from cattle), and Brucella canis (from dogs) (25). Brucella melitensis and suis have the highest pathogenicity while Brucella abortus and canis have moderate pathogenicity (10). All Brucella species are gram-negative, nonmotile, facultative intracellular coccobacilli (10). Brucella species do not form spores or toxins (25). It is one of the most common laboratory-acquired bacterial infections in the United States (25). It also is transmitted by inhalation of contaminated aerosols, conjunctival inoculation, blood transfusions, transplacentally from mother to fetus, and rarely from person to person (25)h. The brucella causes more than 500,000 infections per year worldwide (25). Symptoms of brucellosis include a headache, cyclical fever, migratory arthralgia (joint stiffness), myalgia, asthenia, weight loss, fatigue, malaise, weakness, sweating, vomiting, diarrhea, abdominal pain, and miscarriage (25). There is a clear overlap between brucellosis and tuberculosis in terms of clinical presentation especially with respect to cough, fever, night sweats, fatigue, and weight loss (25).

Pertussis

Pertussis, a respiratory illness commonly known as whooping cough, is a very contagious disease caused by a Bordetella pertussis or Bordetella parapertussis (26). These bacteria attach to the cilia (tiny, hair-like extensions) that line part of the upper respiratory system (26). The bacteria release toxins (poisons), which damage the cilia and cause airways to swell (26). Pertussis is a very contagious disease only found in humans (26). Pertussis spreads from person to person (26). People with pertussis usually spread the disease to another person by coughing or sneezing or when spending a lot of time near one another where you share breathing space (50). Many babies who get pertussis are infected by older siblings, parents, or caregivers who might not even know they have the disease (26). Traditional symptoms of pertussis may appear and include: paroxysms (fits) of many, rapid coughs followed by a high-pitched “whoop” sound, vomiting (throwing up) during or after coughing fits, exhaustion (very tired) after coughing fits – all of which parallel TB symptoms (26). Bordetella is a gram-negative coccobacillus that adheres to ciliated respiratory epithelial cells (27). Local inflammatory changes occur in the mucosal lining of the respiratory tract (27). Released toxins (pertussis toxin, dermonecrotic toxin, adenylate cyclase toxin, and tracheal cytotoxin) act locally and systemically, although the organism itself does not fully penetrate the respiratory tract, and almost never is found in blood cultures (27).

Legionnaire’s disease

Clinical manifestations of Legionnaire’s disease that are similar to that of TB include fever, coughing up blood, shortness of breath, chest pain, nausea and vomiting(28). It is a form of atypical pneumonia that is caused by different species of Legionella bacteria, the most common one being Legionella pneumophila. L. pneumophila is an aerobic, flagellated, gram-negative bacteria. It is transmitted through inhalation of contaminated aerosols, either those of water or through direct contact with surgical wounds(28)c. Contact with contaminated solid are also sources of several cases of Legionnaire’s disease. Legionnaire’s disease accounts for 2%-9% of CAP worldwide(28).

References

1.         Steere AC. MANDELL, DOUGLAS, AND BENNETT’S PRINCIPLES AND PRACTICE OF INFECTIOUS DISEASES. 9th edition. Philedelphia: Elsevier; 2019. 2911-2922.e2 p.

2.         Smith I. Mycobacterium tuberculosis Pathogenesis and Molecular Determinants of Virulence. Clin Microbiol Rev. 2003 Jul;16(3):463–96.

3.         chapter4.pdf [Internet]. [cited 2022 Mar 28]. Available from: https://www.cdc.gov/tb/education/corecurr/pdf/chapter4.pdf

4.         Heemskerk D, Caws M, Marais B, Farrar J. Clinical Manifestations [Internet]. Tuberculosis in Adults and Children. Springer; 2015 [cited 2022 Mar 29]. Available from: https://www.ncbi.nlm.nih.gov/books/NBK344404/

5.         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 2022 Mar 28]. Available from: http://www.ncbi.nlm.nih.gov/books/NBK7812/

6.         Fact Sheets | General | Mycobacterium bovis (Bovine Tuberculosis) in Humans | TB | CDC [Internet]. 2021 [cited 2022 Mar 29]. Available from: https://www.cdc.gov/tb/publications/factsheets/general/mbovis.htm

7.         de Jong BC, Antonio M, Gagneux S. Mycobacterium africanum—Review of an Important Cause of Human Tuberculosis in West Africa. PLoS Negl Trop Dis. 2010 Sep 28;4(9):e744.

8.         Milner DA, Pecora N, Solomon I, Soong TR, editors. Mycobacterium Tuberculosis Complex Infections. In: Diagnostic Pathology: Infectious Diseases [Internet]. Philadelphia: Elsevier; 2015 [cited 2022 Apr 7]. p. II-2–60. (Diagnostic Pathology). Available from: https://www.sciencedirect.com/science/article/pii/B9780323376778500607

9.         Supply P, Brosch R. The Biology and Epidemiology of Mycobacterium canettii. In: Gagneux S, editor. Strain Variation in the Mycobacterium tuberculosis Complex: Its Role in Biology, Epidemiology and Control [Internet]. Cham: Springer International Publishing; 2017 [cited 2022 Apr 7]. p. 27–41. Available from: https://doi.org/10.1007/978-3-319-64371-7_2

10.       Akram SM, Rawla P. Mycobacterium Kansasii. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022 [cited 2022 Mar 30]. Available from: http://www.ncbi.nlm.nih.gov/books/NBK430906/

11.       What Is Mycobacterium Avium Complex? [Internet]. WebMD. [cited 2022 Apr 2]. Available from: https://www.webmd.com/hiv-aids/guide/aids-hiv-opportunistic-infections-mycobacterium-avium-complex

12.       Mycobacterium Intracellulare - an overview | ScienceDirect Topics [Internet]. [cited 2022 Mar 30]. Available from: https://www.sciencedirect.com/topics/immunology-and-microbiology/mycobacterium-intracellulare

13.       Akram SM, Attia FN. Mycobacterium Avium Intracellulare. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022 [cited 2022 Apr 1]. Available from: http://www.ncbi.nlm.nih.gov/books/NBK431110/

14.       Daley CL. Mycobacterium avium Complex Disease. Microbiol Spectr. 2017 Apr 21;5(2):5.2.29.

15.       MAC Lung Disease [Internet]. [cited 2022 Mar 30]. Available from: https://www.lung.org/lung-health-diseases/lung-disease-lookup/mac-lung-disease

16.       MAC Lung Disease: Causes, Symptoms and Treatment [Internet]. Cleveland Clinic. [cited 2022 Mar 30]. Available from: https://my.clevelandclinic.org/health/diseases/22256-mac-lung-disease

17.       Wilson JW, Jagtiani AC, Wengenack NL. Mycobacterium scrofulaceum disease: experience from a tertiary medical centre and review of the literature. Infect Dis. 2019 Aug 3;51(8):602–9.

18.       Streptococcus pneumoniae: Information for Clinicians | CDC [Internet]. 2022 [cited 2022 Mar 29]. Available from: https://www.cdc.gov/pneumococcal/clinicians/streptococcus-pneumoniae.html

19.       Pneumococcal Disease (Streptococcus pneumoniae) | Disease Directory | Travelers’ Health | CDC [Internet]. [cited 2022 Mar 29]. Available from: https://wwwnc.cdc.gov/travel/diseases/pneumococcal-disease-streptococcus-pneumoniae

20.       Streptococcus pneumoniae [Internet]. [cited 2022 Mar 29]. Available from: https://idph.iowa.gov/cade/disease-information/streptococcus-pneumoniae

21.       About Staphylococcus aureus - Minnesota Dept. of Health [Internet]. [cited 2022 Mar 29]. Available from: https://www.health.state.mn.us/diseases/staph/basics.html

22.       Khattak ZE, Anjum F. Haemophilus Influenzae. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022 [cited 2022 Apr 7]. Available from: http://www.ncbi.nlm.nih.gov/books/NBK562176/

23.       King P. Haemophilus influenzae and the lung (Haemophilus and the lung). Clin Transl Med. 2012 Jun 14;1:10.

24.       VisualDx - Haemophilus influenzae pneumonia [Internet]. VisualDx. [cited 2022 Mar 29]. Available from: https://www.visualdx.com/visualdx/diagnosis/haemophilus+influenzae+pneumonia?diagnosisId=53815&moduleId=25

25.       Hayoun MA, Muco E, Shorman M. Brucellosis. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022 [cited 2022 Apr 7]. Available from: http://www.ncbi.nlm.nih.gov/books/NBK441831/

26.       Causes and Transmission of Whooping Cough (Pertussis) | CDC [Internet]. 2021 [cited 2022 Apr 7]. Available from: https://www.cdc.gov/pertussis/about/causes-transmission.html

27.       Lauria AM, Zabbo CP. Pertussis. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022 [cited 2022 Apr 7]. Available from: http://www.ncbi.nlm.nih.gov/books/NBK519008/

28.       Cunha BA, Burillo A, Bouza E. Legionnaires’ disease. Lancet Lond Engl. 2016 Jan 23;387(10016):376–85.

ii) What samples could be taken for laboratory testing and how does the Microbiology Laboratory help in the diagnosis of this bacterial disease?

In the diagnosis of this bacterial disease, the physician begins by assessing the lymph nodes for swelling, as well as listening to the sounds produced by an individual's lungs during exhalation and inhalation (2). Following this, the most common test utilized in the diagnosis of TB disease is a skin test; however, blood test-based diagnosis is becoming increasingly popular (2). In addition to this, the physician may order chest X-rays, as seen in Robert's case, which show white spots in the lungs as a sign of TB, following which, the physician collects sputum samples for further testing (2).

Figure 1A. Biological specimen used in the diagnosis of tuberculosis (1)

Figure 1B. Biological specimen used in the diagnosis of tuberculosis (1)

Respiratory Samples:

(i) Sputum:

Because M. tuberculosis is primarily involved in lung infections, the primary specimen collected for the diagnosis of pulmonary tuberculosis is sputum (3). Sputum (phlegm) is a thick mucus that is made in the lungs and coughed up from the lower airways, including the trachea and bronchi (3). Sputum samples are used to detect active TB infections, since it is likely to contain the microorganism involved in infection. There are 4 main methods used to collect sputum: coughing, sputum induction, bronchoscopy, and gastric aspiration.

Coughing is the most commonly used method to collect sputum; however, it should be noted that sputum should be brought up from the lungs (3). Mucus from the nose or saliva from the mouth is not appropriate for use as samples in this case (3). This is because neither mucus nor saliva are appropriate representations of microorganisms within the lungs, and as such, they are subject to false-negative test results (3). Purulent and mucopurulent sputum is considered optimal for testing, while mucoid, mucosalivary, and salivary samples are categorized as suboptimal (3). Additionally, the sputum sample should not contain food or other contaminants; however, a slight amount of blood is acceptable (3). Lastly, it is recommended that this method be supervised by a healthcare worker wearing PPE to ensure it is being collected correctly (4). This is the most inexpensive and simple method of sputum collection.

Sputum induction may be used to assist sputum production in individuals who are unable to cough an adequate amount on their own (3). This is usually done through the inhalation of aerosolized warm hypertonic saline (3-5%), which liquefies airway secretions and promotes coughing (3). This, in turn, promotes the expectoration of sputum (3). This is an easy method but not as productive and may cause bronchospasm (4). Because the sputum produced through this method is usually saliva-like in its consistency, great care must be taken to ensure proper labeling of the container as “induced sputum” so that the sample isn’t discarded due to consistency issues (3). Bronchoscopy, on the other hand, is utilized when sputum induction is ineffective. Bronchoscopy is a semi-invasive and expensive method visualizing the respiratory tract, and it may also be used to extract sputum through bronchial washing, brushing, or biopsying (3). Bronchoscopy is also used to collect bronchoalveolar lavage (BAL) fluid, if pulmonary TB is suspected (3). It is recommended that this method be avoided, if at all possible, since it is quite expensive and requires a specialist, as well as the need for anesthesia (4). Lastly, gastric aspiration is a procedure used to obtain sputum if the patient cannot cough enough sputum (4). A tube is inserted through the mouth or nose into the stomach to recover sputum coughed into the throat but swallowed. Since M. tuberculosis-containing mucus is present in the stomach until the stomach empties, it is best to perform sputum collection using this technique early in the morning, prior to eating (3). The sputum specimen must be transported to the lab immediately for neutralization after.

Figure 2. Methods of Obtaining a Sputum Specimen (4)

Sputum collection is usually done under the supervision of a healthcare worker wearing personal protective equipment (3). In addition to this, sputum collection is performed in well-ventilated areas in order to minimize the risk of nosocomial infections resulting from M. tuberculosis-containing aerosolized droplets (3).  Sputum is collected in a 50-ml plastic, screw-capped container which acts as a secure containment unit for the sample (3). Moreover, the container must be transparent, which allows for the sample to be visually inspected quite easily, in order to judge its quality and quantity (3). A minimum of 5 ml of sample is needed for testing, and any volume less than this negatively impacts the sensitivity of the test (3). Additional labeling of the container with the name of the patient and date of collection is also necessary (3). The samples must be stored at 2 to 8°C until they can be shipped to a laboratory. Lastly, a minimum of two samples is necessary for testing, although three is recommended (3). It is also recommended that at least one, if not all, of the samples be produced early morning on an empty stomach, at 8 to 24 hour intervals (1,5). Lastly, sputum can be used for a variety of tests, including microscopy, culture, and NAAT (1). Bronchoalveolar lavage (BAL) fluid can be used for the tests previously mentioned for sputum, in addition to its usage in the interferon gamma release assay (1).

Non-Respiratory Samples:

Clinical samples can be used to determine whether or not the infection has spread past the respiratory system, or has, alternatively, originated from a different bodily system. Samples collected for these purposes include urine, blood, bone marrow, cerebrospinal fluid (CSF), stool, pleural fluid, pericardial fluid, joint fluid, lymph node biopsy, and skin biopsy (1). These samples are usually collected when a case of extrapulmonary TB is suspected, which is why these were not obtained in Robert’s case.

(i) Urine:

Urine is usually collected and tested if the individual is suspected to have renal TB, and a minimum of 30-40 ml of fluid must be collected to ensure testing accuracy (1,6). Urine samples can be used in the following tests: microscopy, culture, and NAAT (1). Because organisms are thought to accumulate in the bladder throughout the night, it is recommended that the specimen be collected during the first void in the morning, and this is to be done for a total of 3 samples collected on 3 consecutive days (6). The urine accumulated overnight in the bladder is more concentrated, thus provides an insight into the kidneys’ concentrating capacities and allows for the detection of trace amounts of substances that may not be present in more diluted samples (7).

There are two methods to obtain a urine specimen: non-invasive and invasive techniques. Spontaneous voiding is the main non-invasive technique, although other strategies may be used in children who cannot yet control their voiding (i.e., bag urine) (7). In contrast, urethral catheterization and suprapubic bladder puncture are the two invasive procedures described to date (7). The fundamental principle of either technique is to obtain a specimen without external contamination (7).

(a) Non-invasive Techniques:

Spontaneous voiding is the simplest and most commonly used method in clinical practice (7). Before collecting the sample, health personnel should be provided clear instructions to patients in order to minimize the chance of contamination from penile/vaginal microbiota (“clean-catch” method) (7). Most urine collection kits include a sterile container with a lid and sterile moist towels to wipe the urethral area before collection; if not, cotton wool or toilet tissue and/or tap water with soap may be used (7). Traditionally, male patients are instructed to retract the foreskin and clean the glans of the penis before urinating (7). Consequently, females should clean the labia and urethral meatus as well before collection (7). Subsequently, the patient should first void a small amount of urine into the toilet and afterward position the container mid-stream in the flow of urine (7). Approximately, only 15 mL to 30 mL of urine is sufficient for accurate analysis, so, in most cases, patients should be advised not to fill the containers to their full capacity (7). Finally, the container is closed with careful precautions not to contaminate its lid or rim, and the patient may finish urinating in the toilet, bedpan, etc. The sample must be labeled before or immediately following collection, and it should not be on the lid (7).

(b) Invasive Techniques:

Invasive urine collection is warranted when patients cannot cooperate, have urinary incontinence or external urethral ulceration that increases contamination risk (7). Both of these techniques pose a risk for the inoculation of pathogens, thus causing urinary tract infections (7).

Urethral catheterization involves a small French urinary catheter passed through the urethral meatus after the previous cleansing with proper equipment (7). Depending on the catheter, personnel may or may not need a sterile syringe (7). In cases where patients already have a urinary catheter placed, the specimen should never be taken from the catheter bag as it is considered contaminated (7).

Suprapubic needle aspiration of the bladder is both the most invasive and uncomfortable procedure of all previously mentioned and may generate false-positive results (protein, red and white cells) as a consequence of blood contamination (7). They are generally reserved for situations where samples may not be obtained or are persistently contaminated through previous methods, which usually occurs in small children (7). The main advantage is that, by bypassing the urethra, it minimizes the risk of obtaining a contaminated sample (7). Before the procedure, trained personnel must identify the bladder by examination (7). If not distinguished, it is recommended to hydrate the patient and wait until correct identification or use ultrasound guidance if available (7). After proper cleaning with an antiseptic solution and anesthetizing the skin located approximately 5 cm above the pubic symphysis, a small needle (i.e., 22-gauge spinal needle x 10 cm in adults) is inserted approximately at 60 degrees at the point identified previously (7). The needle is directed slightly caudal or cephalic in adults or children, respectively, according to the anatomic location (7). Usually, the needle will enter the abdominal bladder after advancing it approximately 5 cm in adults (7). Finally, attempt to aspirate using a sterile syringe (7). If a sample is not obtained, advance the needle applying continuous suction on the syringe (7). If unsuccessful after an additional 5 cm in adults, withdraw the needle and repeat the procedure (7). If unsuccessful, personnel should seek help from a specialist or use ultrasound guidance if not previously done (7).

(ii) Blood & Bone Marrow:

Both, blood and bone marrow, are collected only in immunosuppressed individuals (1). Usually, 5-10 ml of blood is collected in an SPS or heparin tube, and it is transported at room temperature (1). Blood is also usually collected if the physician suspects disseminated disease owing to MAC organisms (1). Both, blood and bone marrow, can be used in microscopy, culture, and NAAT. Blood can also be used in the interferon gamma release assay, while bone marrow can also be utilized in histopathology (1).

(iii) Cerebrospinal Fluid (CSF):

Central nervous system (CNS) infection due to TB includes three clinical categories: Meningitis, intracranial tuberculoma, and spinal involvement (8). All these forms of CNS infection are encountered frequently in regions where the incidence of TB is high (8). Central nervous system TB accounts for about 5% of all cases of TB (8). Among various forms of extrapulmonary TB, tuberculous meningitis is the most severe form and remains a major global health problem with a high mortality rate (8). Cerebrospinal fluid (CSF) is collected via a lumbar puncture/spinal tap.

During a spinal tap, hollow needle is inserted between the third and fourth lumbar vertebrae into the spinal canal (9). Between 5 to 20 ml of cerebrospinal fluid in 2 to 4 tubes is collected (9). CSF specimens should be transported to a microbiology laboratory as soon as possible (9). Specimens for culture should not be refrigerated or exposed to extreme cold, excessive heat, or sunlight (9). They should be transported at temperatures between 20°C and 35°C (9). For proper culture results, CSF specimens must be plated within 1 hour (9). If a delay of several hours in processing CSF specimens is anticipated and T-I medium is not available, incubating the specimens (with screwcap loosened) at 35-37°C with ~5% CO2 (or in a candle-jar) may improve bacterial survival (9). CSF can be utilized in the following tests: microscopy, culture, NAAT, and interferon gamma release assay (1).

(iv) Stool:

Like blood, stool is usually collected if the physician suspected disseminated disease caused by MAC organisms (10). In this case, a minimum volume of 5-10 ml will be collected, and it should be refrigerated if transported is delayed for longer than 1 hour (10). Stool samples are useful in the following tests: microscopy, culture, and NAAT (1).

(v) Pleural Fluid:

When a patient presents with new pleural effusion, the diagnosis of TB pleuritis should be considered (11). The patient is at risk for developing pulmonary or extrapulmonary TB if the diagnosis is not made (11). Between 3% and 25% of patients with TB will have TB pleuritis (11). The incidence of TB pleuritis is higher in patients who are human immunodeficiency virus (HIV)-positive (11). Pleural fluid is an exudate that usually has a predominance of lymphocytes (11).

Thoracentesis is a procedure that is performed to remove fluid or from the thoracic cavity for both diagnostic purposes (12). Thoracentesis is also known as thoracentesis, pleural tap, needle thoracostomy, or needle decompression (11). The preferred site for the procedure is on the affected side in either the midaxillary line if the procedure is being performed in the supine position or the posterior midscapular line if the procedure is being performed in the upright or seated position (11). After the local anesthetic is administered, use a larger 20 or 22 gauge needle to infiltrate the tissue around the rib, marching the needle tip just above the rib margin (11). Insert the needle, or catheter attached to a syringe, or the prepackaged catheter directly perpendicular to the skin (11). If using a catheter kit, it may be helpful to make a small nick in the skin using an 11-blade scalpel to be able to advance the catheter through the skin and soft tissue smoothly (11). Apply negative pressure to the syringe during needle or catheter insertion until a loss of resistance is felt and a steady flow of fluid is obtained (11). This is paramount to detect unwanted entry into a vessel or other structure (11). After you collect sufficient fluid in the syringe for fluid analysis, either remove the needle (if performing a diagnostic tap) or connect the collecting tubing to either the needle or the catheter's stopcock (11). Drain larger volumes of fluid into a plastic drainage bag using gravity feed or serial syringe draw with a three-way stop-cock (11). After you have drained the desired amount of fluid, remove the catheter, and hold pressure to stop any bleeding from the insertion site (11). Between 20 to 40 mL of pleural fluid is needed for a complete analysis (13). Fresh fluid should be promptly transported to the laboratory at ambient temperature (13). The maximum acceptable time delay before the processing of pleural fluid specimens in the laboratory is 2 hours (13). If a longer delay is expected, the specimen should be stored in the refrigerator at 4ºC, except for microbiological cultures (13).

(vi) Pericardial Fluid:

Tuberculous pericarditis occurs in approximately 1 to 2 percent of patients with pulmonary TB (14). Pericardial Fluid surrounding the heart can be examined for diagnosis. A small needle is inserted into the chest between the ribs into the thin sac that surrounds the heart (the pericardium) (14). A small amount of fluid is removed. Pericardial fluid samples should be sent to the laboratory as soon as possible in a fresh state or refrigerated at 2-8º C (14). The volume must be a minimum of 2 mL for each laboratory test (14).

(vii) Joint Fluid:

Synovial fluid, also known as joint fluid, is a thick liquid located between your joints (15). This sample can be used when knee joint involvement is suspected in TB. Knee-joint involvement in TB is extremely rare, comprising approximately 0.1% of all forms of tuberculosis (15). A sterile needle is inserted through the skin and into the joint space. Fluid is then drawn through the needle into a sterile syringe. Fluid sample should be sent to the laboratory as soon as possible in a fresh state or refrigerated at 2-8º C. Up to 1-2 mL can be collected (16).

(viii) Lymph Node Biopsy:

A lymph node biopsy is a useful method for diagnosis of tuberculous lymphadenitis. This may be performed via fine needle aspiration, core needle biopsy, or open biopsy (17). In fine needle aspiration, a thin needle is inserted into an area of abnormal-appearing tissue or body fluid (17). A core needle biopsy involves the same basic procedure as the fine needle aspiration, but uses a larger needle with a larger hollow center (17). With this needle, a small block of tissue is removed, which gives more information than you can get from fluid and cells (17). Open biopsy is the most invasive and cuts into your skin to remove all or part of a lymph node (17). The fresh specimen should reach the pathology laboratory with within 60 minutes of their removal (17).

(ix) Skin Biopsy:

A skin biopsy is a useful method for diagnosis of cutaneous tuberculosis. The three main types of skin biopsies (18). In a shave biopsy, a razor is used to remove a small section of the top layers of skin (epidermis and a portion of the dermis) (18). In a punch biopsy, a circular tool removes a small core of skin, including deeper layers (epidermis, dermis and superficial fat) (18). In an excisional biopsy, a small knife (scalpel) is used to remove an entire lump or an area of abnormal skin, including a portion of normal skin down to or through the fatty layer of skin (18).

References:

1.         Lange C, Mori T. Advances in the diagnosis of tuberculosis. Respirology. 2010;15(2):220–40.

2.         Tuberculosis - Diagnosis and treatment - Mayo Clinic [Internet]. [cited 2022 Apr 6]. Available from: https://www.mayoclinic.org/diseases-conditions/tuberculosis/diagnosis-treatment/drc-20351256

3.         Bayot ML, Mirza TM, Sharma S. Acid Fast Bacteria. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022 [cited 2022 Apr 1]. Available from: http://www.ncbi.nlm.nih.gov/books/NBK537121/

4.         chapter4.pdf [Internet]. [cited 2022 Apr 6]. Available from: https://www.cdc.gov/tb/education/corecurr/pdf/chapter4.pdf

5.         Bayot ML, Mirza TM, Sharma S. Acid Fast Bacteria. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022 [cited 2022 Apr 6]. Available from: http://www.ncbi.nlm.nih.gov/books/NBK537121/

6.         Mycobacterium – Urine [Internet]. Public Health Ontario. 2021 [cited 2022 Apr 1]. Available from: https://www.publichealthontario.ca/en/Laboratory-Services/Test-Information-Index/Mycobacterium-Urine

7.         Queremel Milani DA, Jialal I. Urinalysis. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022 [cited 2022 Apr 6]. Available from: http://www.ncbi.nlm.nih.gov/books/NBK557685/

8.         Shirani K, Talaei Z, Yaran M, Ataei B, Mehrabi-Koushki A, Khorvash F. Diagnosed tuberculous meningitis using cerebrospinal fluid polymerase chain reaction in patients hospitalized with the diagnosis of meningitis in referral hospitals in Isfahan. J Res Med Sci. 2015 Mar;20(3):224–7.

9.         Meningitis Lab Manual: Specimen Collection and Transport | CDC [Internet]. 2022 [cited 2022 Apr 6]. Available from: https://www.cdc.gov/meningitis/lab-manual/chpt05-collect-transport-specimens.html

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

11.       Extrapulmonary Tuberculosis (TB) - Infectious Diseases [Internet]. Merck Manuals Professional Edition. [cited 2022 Apr 6]. Available from: https://www.merckmanuals.com/en-ca/professional/infectious-diseases/mycobacteria/extrapulmonary-tuberculosis-tb

12.       Wiederhold BD, Amr O, Modi P, O’Rourke MC. Thoracentesis. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022 [cited 2022 Apr 6]. Available from: http://www.ncbi.nlm.nih.gov/books/NBK441866/

13.       Porcel JM. HANDLING PLEURAL FLUID SAMPLES FOR ROUTINE ANALYSES. Plev Bult. 2013 Jul 10;7(2):19–22.

14.       Pericardial fluid culture: MedlinePlus Medical Encyclopedia [Internet]. [cited 2022 Apr 6]. Available from: https://medlineplus.gov/ency/article/003720.htm

15.       Fujimoto N, Gemba K, Yao A, Ozaki S, Ono K, Wada S, et al. Tuberculosis diagnosed by PCR analysis of synovial fluid. J Infect Chemother. 2010 Feb;16(1):53–5.

16.       Synovial fluid – eClinpath [Internet]. [cited 2022 Apr 6]. Available from: https://eclinpath.com/cytology/synovial-fluid/

17.       What Are Lymph Node Biopsies? [Internet]. [cited 2022 Apr 6]. Available from: https://www.webmd.com/cancer/what-are-lymph-node-biopsies

18.       Skin biopsy - Mayo Clinic [Internet]. [cited 2022 Apr 6]. Available from: https://www.mayoclinic.org/tests-procedures/skin-biopsy/about/pac-20384634

Mantoux Tuberculin Skin Test (TST)

While this is not a microbiology laboratory test, it is a standard test used to detect if there is a TB infection. This is done by injecting 0.1 ml of tuberculin purified protein derivative to the inner forearm surface with the needle facing upward for the intradermal injection (10). The injection site should then have a pale elevation of the skin 6-10 mm in diameter if done correctly (10).

Acid Fast Staining

M. tuberculosis is considered an acid-fast bacteria (AFB) owing to its ability to resist decolorization after staining (2). As such, acid-fast staining allows for sensitive and specific detection of M. tuberculosis, and it is especially useful in low and middle-income countries (2)

The process of acid-fast staining begins with making a 3 cm by 2 cm smear on a slide using the sputum sample (2). Although sputum is commonly used, acid-fast staining can be done use other biological samples as well, such as pleural fluid, cerebrospinal fluid, urine, and bronchoalveolar lavage (BAL) fluid (4,13). Next, the smeared slide is heat-fixed prior to staining (2). The staining process may involve one of two methods: carbolfuchsin staining or the fluorochrome procedure (2).

Steps of Ziehl Neelsen stain

Carbolfuchsin

Carbolfuchsin staining comprises the Ziehl-Neelsen method and the Kinyoun method (2). In the Ziehl-Neelsen method, smeared slides are first stained with carbolfuchsin (CF). This is done by submerging the smear in a drop of carbolfuchsin and subsequently heating it using an alcohol lamp until steam can be seen rising (2). This facilitates the penetration of the stain inside each bacterium (2). Care must be taken that boiling does not occur as that may alter the results of the test (2). The stain and smear should remain in contact for approximately 10 minutes and be allowed to cool down thereafter, thus trapping the stain within the bacterial cell wall (2). These steps make the entire smear, including acid-fast bacilli, red (2). After stain fixation, the second step focuses on washing the excess stain off from the smear (2). This is done by gently washing the smear in a stream of water and then covering it with acid alcohol for 2 to 3 minutes (2). Acid alcohol has the ability to completely decolorize all non-acid-fast organisms, thus only leaving behind red-colored acid-fast organisms, like M. tuberculosis (2). The slides are then stained a second time with methylene blue that serves as a counterstain (2). The recommended time for stain to smear contact is 1 minute but is largely dependent on the quality of methylene blue (2). Counterstaining creates an effective visual contrast of red acid-fast bacilli during microscopy (2). The Ziehl-Neelsen method of staining is also called the hot method as it involves heating the carbolfuchsin stain (2). In contrast, the historic method of staining called the Kinyoun method does not involve heating and is hence known as the cold method (2). Currently, the cold method is already obsolete (2).

Fluorochrome

The fluorochrome procedure primarily utilizes one of two dyes, the auramine-O dye, or the auramine-rhodamine dye (2). Auramine-O is a hydrochloride dye that causes stained AFB to emit fluorescence (green or yellow) when viewed under a fluorescence microscope (2). Unlike the Ziehl-Neelsen method, heating is not required for the penetration of the stain into the bacteria (2). The stain to smear contact time, however, must be a minimum of 20 minutes for the acid-fast organisms to pick up the stain properly (2). After the auramine dye has fully stained the smear, a drop of acid alcohol is applied for one to two minutes to decolorize the smear (2). Methylene blue or potassium permanganate is used as a counterstain to provide background color (2). Potassium permanganate is preferred as it provides a darker background giving it a better contrast and sensitivity as compared to methylene blue (2). The last step is to gently wash the slide with slow running water and let it dry (2). Blotting the slide is avoided as it may damage the stained smear (2).

Nucleic Acid Amplification Testing (NAAT)

Nucleic acid amplification tests are used to amplify DNA and RNA segments to rapidly identify the microorganisms in a specimen (3). NAAT testing can reliably detect M. tuberculosis bacteria in pulmonary and extrapulmonary specimens within 24 hours as compared to 1 week or more for culture (3). It is commonly used to corroborate the results of a positive acid-fast staining test.

CDC recommends that NAAT testing be performed on at least one respiratory specimen from each patient with signs and symptoms of pulmonary TB for whom a diagnosis of TB is being considered but has not yet been established, and for whom the test result would alter case management or TB control activities, such as contact investigations (3).

Clinicians should interpret all laboratory results in the context of the clinical situation (3). A single negative NAAT result should not be used as a definitive result to exclude TB disease, especially when the clinical suspicion of disease is moderate to high (3). Rather, the negative NAAT result should be used as additional information in making clinical decisions, to expedite testing for an alternative diagnosis, or to prevent unnecessary TB disease treatment (3).

The Xpert MTB/RIF assay, in particular, has been FDA-approved for molecular tests aimed at detecting M. tuberculosis, and it is endorsed by the World Health Organization (WHO) (4). It has 89% sensitivity and 99% specificity when compared to the gold standard of testing, which is culture-based testing (4). The Xpert MTB/RIF assay can detect the presence of M. tuberculosis complex, as well as rifampin resistance within 2 hours, while standard culture experiments take 2 to 6 weeks to detect the presence of M. tuberculosis and approximately another 3 weeks to determine drug resistance (14). Given the importance of immediate treatment in TB infections, waiting weeks to make a diagnosis can be extremely detrimental to patient health.

Steps of polymerase chain reaction (PCR)

The most common form of NAAT is the polymerase chain reaction (PCR) (4). PCR is a laboratory-based method that is sued to amplify a target DNA sequence into millions of copies in a relatively short time period (Eisenach et al., 1991). PCR involves three main steps: denaturation, annealing, and extension (5). To start, heat and detergents are used to lyse the bacteria so that DNA may be extracted (5). This is followed by the first main step of PCR: denaturation (5). Denaturation is a process in which the bacterial DNA is heated to a temperature of 95°C for approximately 8 minutes, in an attempt to separate the double-stranded DNA into single strands (5). The next step is annealing, during which primers, which are short oligonucleotides that are complementary to DNA sequences flanking the target region, bind to the target DNA (5). Primers are usually 20-30 base pairs long, and they are essential for amplification of the target sequence because they provide a starting point for DNA synthesis (5). Annealing usually occurs at a temperature of 55°C (5). The last step of PCR is extension. During this step, Taq DNA polymerase, which is a thermostable form of DNA polymerase extracted from Thermus aquaticus, binds to the primer and begins to extend the primer by adding nucleotides in the 5’ to 3’ direction (5). This step is typically performed at a temperature of 72°C (5). This process is repeated approximately 20-40 times, resulting in amplification of the target DNA sequence (5). This mechanism is especially useful in the detection of M. tuberculosis, since it allows for the detection of DNA sequences that are conserved in this particular bacterium, and, therefore, for the indirect detection of the bacterium itself (5). In the past, a 123-base pair segment of IS6110 has been targeted given that it’s specific to the M. tuberculosis complex (6).

Culture

Culture is the gold standard for detecting mycobacteria in clinical specimens (8). Regardless of the results of an AFB smear or NAA testing, it is recommended by the Centre of Disease Control (14) that culture examinations be performed on all clinical samples (14). Culture sensitivity is much lower than its specificity, with 80-85% and 98.5% respectively in the pulmonary forms of TB (7). Culture can be performed on all sample types (7). A downside of culture tests is that results may take anywhere from 4 days to 12 weeks, depending on the specific method used and the speed at which the organism grows (14).

Samples of sputum or tissue require initial decontamination to remove fast-growing non-mycobacterial organisms and liquefaction to allow access of decontaminants to non-mycobacterial organisms and media nutrients to surviving mycobacteria (8). Decontamination-liquefaction is most commonly done using N -acetyl- l -cysteine as a mucolytic in 1% sodium hydroxide solution (8). Mycobacteria are relatively protected during this procedure by a fatty acid–rich cell wall (8). However, normally sterile tissues or fluids such as CSF or pleural fluid should not be decontaminated, because some loss of mycobacterial viability does occur (8). The sample is then neutralized and centrifuged, and the sediment is inoculated onto media (8).

M. tuberculosis growth in liquid broth takes an average of 10-20 days to confirm TB diagnosis, while growth on the aforementioned solid forms of media can take anywhere from 3 to 8 weeks (4). Liquid cultures are about 10% more sensitive than solid cultures, although more prone to contamination (7). Solid media allow examination of colony morphology, detection of mixed cultures, and quantification of growth (8). Additionally, specific strains of M. tuberculosis are unable to grow in liquid broth (9). As such, the use of both, liquid broth and solid growth media, is recommended in order to promote increased diagnostic accuracy (4). Three types of media may be used for culture of mycobacteria: solid egg-based media (e.g., Löwenstein-Jensen), solid agar-based media (e.g., Middlebrook 7H10/7H11), and liquid broth (e.g., Middlebrook 7H12) (8). Furthermore, occasional strains of mycobacteria may grow only on solid media (8).

Mycobacterium tuberculosis Culture

Liquid broth systems facilitate mycobacterial culture and monitor growth through CO2 production or O2 consumption (10). The growth is detected through radiometric, fluorometric or colorimetric indicators. However, the BACTEC Mycobacteria growth indicator tube system is more commonly used to detect growth in 1-3 weeks using a fluorometric method (10). Liquid broth culture takes a shorter amount of time, about 10 days of incubation if smear-positive and 20 days for smear-negative.

Interferon Gamma Release Assay

Interferon gamma release assay (IGRA) is an in-vitro assay that is usually done with sputum, blood, and CSF (11). It measures the response of T-cells in these clinical samples upon stimulation with M. tuberculosis-specific antigens through measurement of IFN-g release (11). Some antigens that are commonly used include the early secreted antigenic target 6 (ESAT-6) and culture filtrate protein 10 (CFP-10) (11).

IGRA is an enzyme-linked immunosorbent (ELISA)-based assay (11). Two of the most widely available commercial IGRAs are the QuantiFERON-TB Gold In-Tube (QFT) assay and the T-SPOT.TB assay (11).

IGRA-ELISA involves adding 1 ml of the collected blood sample to the following three tubes: T, N, and P tube (12). The T tube contains ESAT-6 and CFP-10 antigens (12). The N tube does not contain any antigens and is the negative control (12). The P tube is the positive control and, thus, contains phytohemagglutinin (12). All tubes are incubated at 37°C for up to 24 hours, prior to centrifugation. The resulting supernatant is added to wells. The plate is incubated once again at 37°C for 60 minutes, and following this, horseradish peroxidase-labeled anti-IFNg is added to the wells (12). Again, the plates are incubated at 37°C for 60 minutes (12). Next, the plates were washed prior to the addition of the substrate, after which the optical density is read at 450 nm for quantitative analysis of M. tuberculosis, if it is present at all (12).

LF-LAM Assay - Detection in Urine

Urine samples can be helpful in the diagnosis of tuberculosis in patients diagnosed with advanced AIDS. The method used is the lateral flow urine lipoarabinomannan (LF-LAM) assay (10). Studies found that it had a high specificity of 92% but lower sensitivity of 45% in HIV-infected patients, only taking 25 minutes to show results. The combination of LF-LAM and microscopy can be used as a rapid to diagnose HIV-infected patients (10).

References

1. CDCTB. Tuberculosis (TB) Fact Sheets- Tuberculin Skin Testing [Internet]. Centers for Disease Control and Prevention. 2020 [cited 2022 Apr 1]. Available from: https://www.cdc.gov/tb/publications/factsheets/testing/skintesting.htm
2. Bayot, M. L., Mirza, T. M., & Sharma, S. (2022). Acid Fast Bacteria. In StatPearls. StatPearls Publishing. http://www.ncbi.nlm.nih.gov/books/NBK537121/
3. Fact sheets | testing and diagnosis | fact sheets - diagnosis of tuberculosis disease | tb | cdc [Internet]. 2020 [cited 2022 Mar 31]. Available from: https://www.cdc.gov/tb/publications/factsheets/testing/diagnosis.htm
4. Bennet, J., Dolin, R., & Blaser, M. (2020, March 31). Mycobacterium tuberculosis—ClinicalKey. https://www-clinicalkey-com.ezproxy.library.ubc.ca/#!/content/book/3-s2.0-B9780323482554002496?scrollTo=%23hl0001320
5. Lo, Y. M. D., & Chan, K. C. A. (2006). Introduction to the Polymerase Chain Reaction. In Y. M. D. Lo, R. W. K. Chiu, & K. C. A. Chan (Eds.), Clinical Applications of PCR (pp. 1–10). Humana Press. https://doi.org/10.1385/1-59745-074-X:1
6. Eisenach, K. D., Sifford, M. D., Cave, M. D., Bates, J. H., & Crawford, J. T. (1991). Detection of Mycobacterium tuberculosis in Sputum Samples Using a Polymerase Chain Reaction. American Review of Respiratory Disease, 144(5), 1160–1163. https://doi.org/10.1164/ajrccm/144.5.1160
7. Canadian Lung Association, Canadian Thoracic Society, Public Health Agency of Canada, Centre for Communicable Diseases and Infection Control (Canada). Canadian tuberculosis standards. 2014.
8. Narvskaya O, Otten T, Limeschenko E, Sapozhnikova N, Graschenkova O, Steklova L, et al. Nosocomial outbreak of multidrug-resistant tuberculosis caused by a strain of mycobacterium tuberculosis w-beijing family in st. Petersburg, russia. Eur J Clin Microbiol Infect Dis [Internet]. 2002 Aug [cited 2022 Apr 2];21(8):596–602. Available from: http://link.springer.com/10.1007/s10096-002-0775-4
9. Akram SM, Attia FN. Mycobacterium avium intracellulare. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022 [cited 2022 Mar 30]. Available from: http://www.ncbi.nlm.nih.gov/books/NBK431110/
10. Steere AC. MANDELL, DOUGLAS, AND BENNETT’S PRINCIPLES AND PRACTICE OF INFECTIOUS DISEASES. 9th edition. Philedelphia: Elsevier; 2019. 2911-2922.e2 p.
11. Pai, M., Denkinger, C. M., Kik, S. V., Rangaka, M. X., Zwerling, A., Oxlade, O., Metcalfe, J. Z., Cattamanchi, A., Dowdy, D. W., Dheda, K., & Banaei, N. (2014). Gamma Interferon Release Assays for Detection of Mycobacterium tuberculosis Infection. Clinical Microbiology Reviews, 27(1), 3–20. https://doi.org/10.1128/CMR.00034-13
12. Wang, L., Tian, X., Yu, Y., & Chen, W. (2018). Evaluation of the performance of two tuberculosis interferon gamma release assays (IGRA-ELISA and T-SPOT.TB) for diagnosing Mycobacterium tuberculosis infection. Clinica Chimica Acta, 479, 74–78. https://doi.org/10.1016/j.cca.2018.01.014
13. Lange, C., & Mori, T. (2010). Advances in the diagnosis of tuberculosis. Respirology, 15(2), 220–240. https://doi.org/10.1111/j.1440-1843.2009.01692.x
14. Centers for Disease Control and Prevention. (n.d.). Centers for Disease Control and Prevention. Retrieved Mar 28, 2022, from https://www.cdc.gov/

iv) What are the results expected from the tests that might allow the identification of different bacteria causing these symptoms, including any stated bacteria above

Mantoux TB Skin Test

Example of TB Skin Test

Since our patient is diagnosed with TB, we would expect that after 48-72 hours, the patient will return to be evaluated. The forearm reaction is measured in millimetres of firm swelling (induration) and not measured based on the redness (1)

It is to note that people who have received the bacilli Calmette-Guerin vaccine (BCG) may undergo a TB skin test to test for infection, but BCG vaccination may cause a positive reaction to the test (2). Therefore, it is not indicated if the positive test is due to the vaccine or infection with TB bacteria. For those who have gotten the BCG vaccine, a TB blood test is recommended as it is not affected by vaccination, therefore are no false-positive results (2).

Blood Test (IGRA)

If positive, the blood test (IGRA) will mean that the individual is infected with Mycobacterium tuberculosis complex bacteria, and additional testing may be required to determine if it is latent TB infection or TB disease (30). If the blood test comes negative for TB, the person’s blood did not react with the test, and TB disease or TB latent infection is not likely. If Robert took a blood test, it would likely come back positive (2).

Usually, TB blood tests are preferred if the patient has received the BCG vaccine or if the patient cannot return to a second appointment to see the reaction after a TB skin test (2).

However, it must be checked if the patient, Robert, is immunocompromised as a negative IGRA test may not completely exclude TB (1). This test is insensitive in immunocompromised patients, such as HIV patients with low CD4+ T cell count. It is recommended to do culture in conjunction (3).

Culture

Image of TB colonies on LJ medium

Culture on media is a huge advantage that allows us to visualize colony morphology and pigmentation, which can diagnose colonies of M. tuberculosis from nontuberculous mycobacteria mentioned in question i. (4). If the bacterium is M. tuberculosis, then on either of the two mediums selected, we would see small colonies with a buff colour, and it would take around 3-8 weeks to grow this bacterium. Since Robert is most likely infected with M. tuberculosis, we would look for this colony characteristic (4). Each bacterium will have different morphologies and characteristics in culture, and this is the best method to rule out bacteria that may be suspected to cause illness.

It is also beneficial due to it allows the visualization of colony morphology and pigmentation to distinguish M. tuberculosis complex colonies from non-tuberculosis mycobacteria (4).

Acid-Fast Staining

Visualization of Acid-Fast Staining

Acid-fast staining is beneficial in separating bacteria into an acid-fast group and a non-acid fast group (5). This is particularly helpful if gram staining cannot distinguish a microorganism; we see this particularly in mycobacterium as they are species that are resistant to a gram or simple staining and can only be visualized by acid-fast staining (5). If the bacteria is acid-fast, in the Ziehl–Neelsen method it will be a bright red to intensive purple shade with straight/slightly curved rods, which may be present in small groups or separately (5). If non-acid fast, they will be blue/green, which can help differentiate whether the bacteria causing Robert’s illness is a mycobacterium or a different bacteria mentioned earlier in question i. As well, if the Truant method is used the acid-fast bacteria will be a bright yellow or orange fluorescence against the background. Usually, Mycobacteria are acid-fast and can be distinguished from the non-acid-fast bacteria, which include Streptococcus pneumonia, Staphylococcus aureus, and Haemophilus species.

It is of note that positive sputum smears are thought to indicate M. tuberculosis over MAC in areas where both bacterial infections are common. But, a secondary test such as a culture or NAAT can confirm the results and distinction between the bacteria(3).

Nucleic Acid Amplification Testing (NAAT):

The results of NAA tests should be considered in conjunction with results of the previously discussed acid-fast staining test. For example, a single, negative NAA test, without the results of an acid-fast staining test, can’t be used to rule out TB disease(6).

The sensitivity of the NAA test in individuals with positive AFB sputum smears is greater than 95% (6). A positive NAA test can be used to confirm a diagnosis of TB in individuals with a positive acid-fast staining test (6)A negative NAA test in these individuals is also quite informative in that it is strongly suggestive of the causative agent being an acid-fast bacilli other than M. tuberculosis, such as the aforementioned species that are part of the M. avium complex (6).

The sensitivity of the NAA test in individuals with a negative AFB sputum smear varies greatly (6). A negative NAA test, in these cases, can’t be used to rule out TB disease; however, a positive NAA test is indicative of TB disease with a specificity up to 97-98% (6).

Although NAA tests are useful in the diagnosis of TB disease, they do have some limitations, with one of them being the possible presence of inhibitors in clinical samples which prevent or reduce amplification, leading to false-negative results (7)However, false-negatives due to the presence of inhibitors are rarely (< 3% of all samples) observed in conjunction with positive acid-fast staining tests(7).

Additionally, there is currently no standardized protocol involving a universally accepted amplification target, which leads to heterogenous diagnostic accuracy (6). However, there has been a correlation observed between the use of IS6110 as an amplification target and a higher diagnostic accuracy (6). Furthermore, NAA tests are not entirely reliable, since false positives have been reported in individuals with a medical history involving TB disease and/or bronchogenic carcinoma (6).

Interferon Gamma Release Assay:

As mentioned in the previous section, the IGRA-ELISA involves the use of three tubes: T, N, and P tube (8). It is important to remember that the solution in these tubes was subsequently added to plates prior to incubation. Because the N tube, the negative control, did not have any antigens, no stimulation of T cells, and, thus, no production of IFN-g is expected. In the absence of IFN-g, the anti-IFN-g will be washed away, and there will be no horseradish peroxidase to facilitate a color change of the substrate (8). As such, no reaction is expected in this solution. Phytohemagglutinin, which was added to the P-tube, stimulates IFN-g production by T-cells, and so, anti-IFN-g will bind to IFN-g and remain in the on the plate even after washing. Subsequent addition of the substrate of horseradish peroxidase will result in a color change (8). A color change seen in the solution originating from the T tube is indicative of IFN-g release due to the presence T-cells that were stimulated against M. tuberculosis antigens. This is indicative of a positive result, while a lack of color change reflects a negative result (8).

References

1.          CDCTB. Tuberculosis (TB) Fact Sheets- Tuberculin Skin Testing [Internet]. Centers for Disease Control and Prevention. 2020 [cited 2022 Apr 1]. Available from: https://www.cdc.gov/tb/publications/factsheets/testing/skintesting.htm

2.          CDCTB. Tuberculosis (TB) - Testing in BCG-Vaccinated Persons [Internet]. Centers for Disease Control and Prevention. 2016 [cited 2022 Mar 28]. Available from: https://www.cdc.gov/tb/topic/testing/testingbcgvaccinated.htm

3.          Steere AC. MANDELL, DOUGLAS, AND BENNETT’S PRINCIPLES AND PRACTICE OF INFECTIOUS DISEASES. 9th edition. Philedelphia: Elsevier; 2019. 2911-2922.e2 p.

4.          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 2022 Mar 28]. Available from: http://www.ncbi.nlm.nih.gov/books/NBK7812/

5.          Aryal S. Acid-Fast Stain- Principle, Procedure, Interpretation and Examples [Internet]. Microbiology Info.com. 2015 [cited 2022 Mar 31]. Available from: https://microbiologyinfo.com/acid-fast-stain-principle-procedure-interpretation-and-examples/

6.          Lange C, Mori T. Advances in the diagnosis of tuberculosis. Respirology. 2010;15(2):220–40.

7.          Considerations | Nucleic Acid Amplification | Lab Info | TB | CDC [Internet]. 2020 [cited 2022 Apr 8]. Available from: https://www.cdc.gov/tb/publications/guidelines/amplification_tests/considerations.htm

8.          Wang L, Tian X, Yu Y, Chen W. Evaluation of the performance of two tuberculosis interferon gamma release assays (IGRA-ELISA and T-SPOT.TB) for diagnosing Mycobacterium tuberculosis infection. Clinica Chimica Acta. 2018 Apr 1;479:74–8.

Bacterial Pathogenesis

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?

Host

TB is spread from person to person through the air. The dots in the air represent droplet nuclei containing Mycobacterium tuberculosis. Infection occurs when a person inhales droplet nuclei containing Mycobacterium tuberculosis that reach the alveoli of the lungs (29).

Tuberculosis (TB) in humans is primarily caused by Mycobacterium tuberculosis (Mtb) bacteria (1). However, the genus Mycobacterium has several TB causing species aside from M. tuberculosis, including M. bovis, M. caprae, M. cannettii, M. africanum, M. pinnipedii, and M. microti (1).  M. bovis primarily infects livestock but can also infect other species of mammals (rarely humans) (2). M. bovis was once a common causative agent of TB, but cases have significantly been reduced by introducing pasteurized milk (3, 4). M. caprae, is a TB strain adapted to goats, but it can also affect livestock (5).  M. africanum, has solely been found in human populations in Africa (it has much lower pathogenicity compared to Mtb), although it is not believed that humans are the reservoir (6). The hosts of M. microti are rodents and insectivores, and it only causes TB in these species (1). M. pinnipedii causes TB in pinnipeds (marine animals with front and rear flippers, like seals) (1). Mycobacterium tuberculosis is expelled from infected persons (though coughing, sneezing, shouting, etc), spread through airborne particles, following which they may infect others and adhere to respiratory epithelial cells (7; 8). Those droplets are about 1 to 5 microns in diameter (9). Depending on the environment, M. tuberculosis can remain suspended in the air for several hours. People who breathe in the air containing the bacteria can become infected (10). Due to the lungs’ constant exposure to the air and high surface area, they are susceptible to these airborne droplets, of which virtually all successful tuberculosis infections arise (11). Mycobacterium species are obligate aerobes, which make them well suited to mammalian hosts as they enter into the lungs which are an oxygenated environment (12). When the bacteria enters the host as air droplets by being inhaled in, larger particles may be trapped in the upper airway to cause TB in the oropharynx or cervical lymph nodes, whereas smaller particles may proceed onto the lower respiratory tract (13). Inside the host, Mtb is active and infects the lungs in 90% of cases, particularly an area called Ghon focus, which is either in the upper part of lower lobe or lower part of upper lobe. More specifically, the bacteria usually resides intracellularly in lung alveoli – epithelial cells, macrophages, dendritic cells, and neutrophils (14). However, Mtb may also spread to the pleura, central nervous system, lymphatic system (scrofula in neck), genitourinary system, bones, and joints, causing additional complications, swelling, and bleeding (15). In severe cases, patients may develop disseminated tuberculosis or military tuberculosis, which is when Mtb is widespread in various organs, including lungs, liver, and spleen, also impacting pancreas and adrenal glands (15). Latent TB infection may also occur, in which macrophages surround the bacteria to form a granuloma, which keeps the bacteria contained and allows for prolonged residence in the host (16).

Geography

World Map of Countries by Tuberculosis Incidence (28).

While currently, we see cases of tuberculosis worldwide, it is one of the oldest known human pathogens. Dating back to 2400 BC, Egyptian mummies reveal skeletal deformities typical of tuberculosis (17). In fact, evidence of the oldest confirmed human case of tuberculosis dates back to 9,000 years ago, while statistical models suggest the disease evolved 40,000 years ago, following the expansion and out-ward migration of human populations outside of Africa (18). Geographically, M. tuberculosis is distributed all around the world (19). TB became much more common in developed countries such as the United States in the 1980s following a sharp rise in HIV infections (20). HIV is one of the most important risk factors in the development of TB from a latent M. tuberculosis infection, which explains the increased TB rates (20, 21). TB incidence rates in the U.S. have since declined to reach the lowest in history in 2016, largely as a result of intensified diagnostic, treatment, and prevention efforts and antiretroviral therapy to treat HIV (20). Today, Tuberculosis (TB) is a disease that primarily affects low-income countries.  In 2020, the largest number of new TB cases occurred in the WHO South-East Asian Region, with 43% of new cases, followed by the WHO African Region, with 25% of new cases and the WHO Western Pacific with 18% (22). The World Health Organization (WHO) identifies 22 high-burden countries for TB, most notably, China and India, which account for over one-third of new cases of TB in 2009 (23). TB remains a leading cause of morbidity and mortality in developing countries presumably because more people in these regions live and work in poorly ventilated and overcrowded conditions, which provide ideal conditions for M. tuberculosis to spread in the air. Also, with more people suffering from malnutrition and disease, particularly HIV, the weak immune system reduces resistance to TB (24). Furthermore, due to the limited access to healthcare and poor medical knowledge of TB, just one person with untreated infectious TB can pass the illness on to 10-15 people annually (24). In addition, a 2019 review article conducted on the impacts of temperature and altitude differences on TB rates of transmission, found that higher temperature and lower altitude were more conducive to TB transmission (25). The reasoning behind these conclusions is speculative as higher temperature and lower altitude may impact social context (more overcrowding) and medical conditions which increases rates of TB transmission, and consequently Mtb population persistence (25).

Robert

Thus, Robert’s immigration from India is the likely place of tuberculosis exposure for this patient. The patient, Robert, may have encountered Mtb in India, where he immigrated from a year ago. This is a reasonable prediction as India is one of the countries with the highest cases of TB in the world. Additionally, Mtb is capable of surviving in the host as a latent infection with no clinical symptoms that can later progress to an active disease (26).  Since M. tuberculosis is spread in nearly all cases by aerosolized particles, Robert likely came in contact with the bacteria by breathing in aerosolized droplets with M. tuberculosis, released by someone infected with pulmonary tuberculosis (27). It is likely that Robert was infected with Mtb in India and was asymptomatic for a year before feeling sick because he had a latent TB infection (M. tuberculosis was not active in his body), then an event resulting in a weakened immune response allowed the bacteria to be reactivated and cause active TB . India is currently classified by the World Health Organization as a high-burden country for TB and has a higher prevalence of TB than Canada (19). This suggests that Robert was likely infected more than a year ago when he was in India, rather than in Canada where TB cases are rare.

References:

1. Cassidy JP. The pathogenesis and pathology of bovine tuberculosis with insights from studies of tuberculosis in humans and laboratory animal models. Vet Microbiol [Internet]. 2006;112(2–4):151–61. Available from: https://www.sciencedirect.com/science/article/pii/S0378113505003895
2. Ciaravino G, Vidal E, Cortey M, Martín M, Sanz A, Mercader I, et al. Phylogenetic relationships investigation of Mycobacterium caprae strains from sympatric wild boar and goats based on whole genome sequencing. Transbound Emerg Dis [Internet]. 2021;68(3):1476–86. Available from: http://dx.doi.org/10.1111/tbed.13816
3. Van Soolingen D, Hoogenboezem T, De Haas PEW, Hermans PWM, Koedam MA, Teppema KS, et al. A novel pathogenic taxon of the mycobacterium tuberculosis complex, canetti: characterization of an exceptional isolate from africa. International Journal of Systematic and Evolutionary Microbiology [Internet]. [cited 2022 Mar 30];47(4):1236–45. Available from: https://www.microbiologyresearch.org/content/journal/ijsem/10.1099/00207713-47-4-1236
4. Thoen C, LoBue P, de Kantor I. The importance of Mycobacterium bovis as a zoonosis. Veterinary Microbiology [Internet]. 2006 Feb 25 [cited 2022 Mar 30];112(2):339–45. Available from: https://www.sciencedirect.com/science/article/pii/S0378113505004086
5. Ciaravino G, Vidal E, Cortey M, Martín M, Sanz A, Mercader I, et al. Phylogenetic relationships investigation of Mycobacterium caprae strains from sympatric wild boar and goats based on whole genome sequencing. Transbound Emerg Dis [Internet]. 2021;68(3):1476–86. Available from: http://dx.doi.org/10.1111/tbed.13816
6. de Jong BC, Antonio M, Gagneux S. Mycobacterium africanum--review of an important cause of human tuberculosis in West Africa. PLoS Negl Trop Dis [Internet]. 2010 [cited 2022 Apr 2];4(9):e744. Available from: http://dx.doi.org/10.1371/journal.pntd.0000744
7. Tuberculosis (TB) program dispensary pre-authorization for Non-Routine TB Services [Internet]. Wisconsin Department of Health Services. 2016 [cited 2022Apr1]. Available from: https://www.dhs.wisconsin.gov/library/f-01459.htm
8. Hall-Stoodley L, Watts G, Crowther JE, Balagopal A, Torrelles JB, Robison-Cox J, et al. Mycobacterium tuberculosis binding to human surfactant proteins A and D, fibronectin, and small airway epithelial cells under shear conditions [Internet]. Infection and immunity. American Society for Microbiology; 2006 [cited 2022Apr1]. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1479241/#:~:text=tuberculosis%20adhees%20to%20respiratory%20epithelial,7%2C%208%2C%2030).
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11. Torrelles JB, Schlesinger LS. Integrating lung physiology, immunology, and tuberculosis [Internet]. Trends in microbiology. U.S. National Library of Medicine; 2017 [cited 2022Apr1]. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5522344/
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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).

Structure and cellular constituents of tuberculous granuloma. Many other cell types also populate the granuloma, such as neutrophils, dendritic cells, B and T cells, natural killer (NK) cells, fibroblasts and cells that secrete extracellular matrix components (15).

Physiologically, M. tuberculosis enters human hosts almost exclusively through the respiratory system following the inhalation of airborne, aerosolized droplet nuclei containing the bacteria (1). Usually, prolonged exposure is required and several nuclei need to be inhaled to successfully establish an infection (2). Droplet nuclei that are small enough travel through the mouth or nasal passages, upper respiratory tract, and bronchi to be deposited in the alveoli of the lungs (1,5). Larger droplets cannot reach the distal airways due to their size, but can be deposited in the oropharynx to cause oropharyngeal tuberculosis (1). This is comparatively rare and tuberculosis is almost always caused by the deposition of M. tuberculosis aerosol particles in the distal airways, which will be the focus of this journal (1). However, the ability of small droplets to pass through the respiratory tract is a key factor that determines which host cells the bacteria will adhere to and infect.

Cellularly, the adherence of M. tuberculosis to host cells plays a major role in establishing infection and involves interactions between both bacterial cells and host cells (3). The alveoli consists of type I and II epithelial cells in addition to phagocytic cells: macrophages, dendritic cells, and neutrophils (1). M. tuberculosis is capable of infecting epithelial cells, as observed in vitro and in post-mortem studies, possibly resulting in limited early bacterial growth (1,4). However, M. tuberculosis is mainly characterized by its infection of phagocytic cells including macrophages, dendritic cells, and neutrophils in the alveoli (1).

List of currently known M. tuberculosis adhesins (6)

Molecularly, adherence involves the binding of bacterial adhesins with host receptors to promote bacterial colonization and infection (3). Host cell surface receptors that mediate this interaction include toll-like receptors (ex. TLR2), C-type lectin receptors (ex. ICAM-3), scavenger receptors (ex. CD36), complement receptors (ex. CR3), and Fc receptors (2,3). In addition to these surface receptors, ECM components such as fibronectin, collagen, elastin, and laminin also serve as binding sites for M. tuberculosis adhesins (3). These host factors bind bacterial adhesins, resulting in the adhesion of M. tuberculosis to host cells, the induction of an immune response, and the pathogenesis of tuberculosis (3). These host receptors and ECM components interact with bacterial cell surface adhesins such as heparin-binding hemagglutin (HbhA), fibronectin-binding protein A (FbpA), and PE-PGRS family (5,6). HbhA is a bacterial surface protein that binds epithelial cells and FbpA is a protein that binds fibronectin in the host extracellular matrix – possibly playing a role in the extrapulmonary spread of the bacteria (5). PE-PGRS is a family of glycine-rich proteins, many of which are known or putative adhesins as observed in Figures 1 and 2 (6). Additionally, a network of coiled, aggregated fibers known as M. tuberculosis pilli (MTP) have also been identified, which bind to laminin in the extracellular matrix (5). MTP are produced in vivo during infection, suggesting that they are an adhesin important in M. tuberculosis adherence during TB infection (5). The combination of these bacterial factors and host factors help determine site specificity of M. tuberculosis infection (5).

List of computationally-determined putative M. tuberculosis adhesins (6)

References

1. Bussi C, Gutierrez MG. Mycobacterium tuberculosis infection of host cells in space and time. FEMS Microbiol Rev. 2019 Mar 27;43(4):341–61.
2. Mycobacterium tuberculosis - ClinicalKey [Internet]. [cited 2022 Mar 30]. Available from: https://www-clinicalkey-com.ezproxy.library.ubc.ca/#!/content/book/3-s2.0-B9780323482554002496?indexOverride=GLOBAL
3. Vinod V, Vijayrajratnam S, Vasudevan AK, Biswas R. The cell surface adhesins of Mycobacterium tuberculosis. Microbiol Res. 2020 Feb 1;232:126392.
4. Stek C, Allwood B, Walker NF, Wilkinson RJ, Lynen L, Meintjes G. The Immune Mechanisms of Lung Parenchymal Damage in Tuberculosis and the Role of Host-Directed Therapy. Front Microbiol [Internet]. 2018 [cited 2022 Mar 31];9. Available from: https://www.frontiersin.org/article/10.3389/fmicb.2018.02603
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6. Ramsugit S, Pillay M. Identification of Mycobacterium tuberculosis adherence-mediating components: a review of key methods to confirm adhesin function. Iran J Basic Med Sci. 2016 Jun;19(6):579–84.
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iii) Multiplication and Spread: does the organism remain extracellular or do they enter into cells and what are the molecular and cellular determinants of these events. Do the bacteria remain at the entry site or do they spread beyond the initial site i.e. are there secondary sites of infection and why do the bacteria hone in on these particular secondary sites.

Mycobacterium tuberculosis, otherwise known as Tubercle bacilli, multiply in the alveoli inside the lungs after infection (19).

Following adhesion, the M. tuberculosis organisms are ingested by alveolar macrophages (8). This process is initiated by contact between the bacterium and macrophage mannose or complement receptors. Mannose receptor activity is upregulated by surfactant protein A, a glycoprotein present on the alveolar surface (9). Furthermore, one of the virulence factors macrophages are able to recognize is phthiocerol dimycocerosate (DIM) in the bacterial cell wall which facilitates phagocytosis (12). The consequent hostile environment includes acidic pH levels, reactive oxygen intermediates (which, experimentally, are the most significant players against M. tuberculosis), lysosomal enzymes, and peptides toxic to the bacterium (9). This immune response is suppressed by the inhibition of phagocytosis maturation through changing its composition, mediated by protein tyrosine kinase A (10). Mtb can evade being killed in lysosomes by preventing the phagosome maturation process as it secretes Zmp1 protein which suppresses caspase-1 preventing maturation to phagolysosome stage (13). Mtb expression of nuoG and secA2 genes can also inhibit apoptosis in macrophages by inhibiting TNF-alpha mediated apoptosis pathways (14). Surviving mycobacterium multiplies intracellularly within the macrophages, and upon macrophage death and lysis, M. tuberculosis is released (8). TB infection officially begins when Mtb reaches the alveolar air sacs in the lungs. There, Mtb will invade and replicate inside alveolar macrophages (3). The primary site of infection and replication in the lungs is the Ghon focus. As aforementioned, this area is either the upper part of lower lobe or lower part of upper lobe; however, if Mtb was delivered to lungs via blood stream, it may be Simon focus, which is infection in the top area of the lung (4, 5).

Multiplication occurs inside human cells prior to dissemination (2). Although there are still many aspects in Mtb replication that are unknown such as specific locations or conditions to be met as it is difficult to observe these processes live, it is still however clear that Mtb can potentially replicate in different cellular compartments, including lysosomes as well as resting, activated, and necrotic human macrophages (2, 6). Research also indicates that the interactions involved in Mtb replication are transient and dynamic (2). Also, Mtb has been observed to successfully replicate in both resting and IFN-gamma-activated GM-CSF- and M-CSF-differentiated human macrophages (6). It is believed that the GM-CSF phenotype of alveolar macrophages allow Mtb to replicate, whereas M-CSF macrophages and IGN-gamma promotes replication (6). Moreover, Mtb replication was continued to be observed even if macrophage lost its plasma membrane (PM) integrity. Altogether, this means that Mtb replication continues regardless of macrophage morphology and condition, whether it is resting, activated, necrotic, or leaky. This suggests that Mtb replication is not dependent on the macrophage’s outer structure, activation, or whether it is live. However, there has been additional research conducted to indicate that inhibiting host cell necrosis limits Mtb replication. This implies that although Mtb replication may not necessarily be dependent on whether the macrophage is live or not, it may be further promoted in necrotic macrophages due to factors that are only present (or absent) in necrotic macrophages.

Unbalanced immune system in TB patients results in the development of diverse diseases such as pulmonary, systematic, metabolic and gut diseases (18).

In terms of the process, Mtb replicates asexually via binary fission or budding (7). It contains all the genetic material to replicate; thus, likely does not need to use host’s mechanisms despite only replicating intracellularly (7). The rate of replication is also relatively slow, dividing once every 16-20 hours, whereas most other bacteria divides within minutes or hours (7).

Dissemination may occur via lymphatic system or bloodstream, though the Mtb will remain inside human cells (eg. macrophages). If there is an area of damaged tissue, Mtb can enter the bloodstream. The initial, or primary, infection with M. tuberculosis involves replication of the organism at the initial pulmonary site of infection, later the bacteria can spread to local lymph nodes within the lung, and eventual dissemination of infection to remote sites in the body like kidney, spine, and brain (15). TB in a body part other than the lungs is called extrapulmonary TB (EPTB) (16). This represents approximately 15% of all TB infections (17). For example, lymph node TB can develop when M. tuberculosis reach the lymph nodes through hematogenous spread and lodge in the sinuses, where they are phagocytosed by inactive macrophages. It may potentially also infect heart, skeletal muscles, pancreas, and thyroid, but these cases are far rarer, although the rationale is unknown (8). Extrapulmonary tuberculosis is overrepresented by those within more vulnerable populations, such as the elderly, children, and those who are immunocompromised or malnourished (11).

References

1. Mahamed D, Boulle M, Ganga Y, Mc Arthur C, Skroch S, Oom L, et al. Intracellular growth of Mycobacterium tuberculosis after macrophage cell death leads to serial killing of host cells. eLife [Internet]. [cited 2022 Apr 1];6:e22028. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5319838/

2. Bussi C, Gutierrez MG. Mycobacterium tuberculosis infection of host cells in space and time. FEMS Microbiol Rev [Internet]. 2019 Mar 27 [cited 2022 Mar 30];43(4):341–61. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6606852/

3. Houben EN, Nguyen L, Pieters J. Interaction of pathogenic mycobacteria with the host immune system. Current Opinion in Microbiology [Internet]. 2006 Feb 1 [cited 2022 Apr 1];9(1):76–85. Available from: https://www.sciencedirect.com/science/article/pii/S1369527405002080

4. Kumar V, Robbins SL. Robbins basic pathology. Philadelphia, PA: Saunders/Elsevier; 2007. 3. Khan MR. Essence of pediatrics. Elsevier India; 2011. 561 p.

5. Lerner TR, Borel S, Greenwood DJ, Repnik U, Russell MRG, Herbst S, et al. Mycobacterium tuberculosis replicates within necrotic human macrophages. J Cell Biol [Internet]. 2017 Mar 6 [cited 2022 Apr 1];216(3):583–94. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5350509/

6. Markova N, Slavchev G, Michailova L. Unique biological properties of Mycobacterium tuberculosis L-form variants: impact for survival under stress. International Microbiology [Internet]. 2012 [cited 2022 Apr 1];15:61-8. Available from: http://citeseerx.ist.psu.edu/viewdoc/download?doi=10.1.1.1064.7413&rep=rep1&type=pdf

7. R, Malhotra P, Awasthi A, Kakkar N, Gupta D. Tuberculous dilated cardiomyopathy: an under-recognized entity? BMC Infect Dis [Internet]. 2005 Apr 27 [cited 2022 Apr 1];5:29. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1090580

8. Chapter 2 transmission and pathogenesis of tuberculosis [Internet]. [cited 2022 Apr 2]. Available from: https://www.cdc.gov/tb/education/corecurr/pdf/chapter2.pdf

9. Smith I. Mycobacterium tuberculosis pathogenesis and molecular determinants of virulence [Internet]. Clinical microbiology reviews. American Society for Microbiology; 2003 [cited 2022 Apr1]. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC164219/

10. Zhai W, Wu F, Zhang Y, Fu Y, Liu Z. The immune escape mechanisms of mycobacterium tuberculosis [Internet]. International journal of molecular sciences. MDPI; 2019 [cited 2022 Apr1]. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6359177/

11. Moule MG, Cirillo JD. mycobacterium tuberculosis dissemination plays a critical role in pathogenesis [Internet]. Frontiers in cellular and infection microbiology. Frontiers Media S.A.; 2020 [cited 2022Apr1]. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7053427/

12. Augenstreich J, Haanappel E, Ferré G, Czaplicki G, Jolibois F, Destainville N, et al. The conical shape of DIM lipids promotes Mycobacterium tuberculosis infection of macrophages. Proc Natl Acad Sci U S A [Internet]. 2019;116(51):25649–58. Available from: http://dx.doi.org/10.1073/pnas.1910368116

13. Ferraris D, Miggiano R, Rossi F, Rizzi M. Mycobacterium tuberculosis molecular determinants of infection, survival strategies, and vulnerable targets. Pathogens [Internet]. 2018;7(1):17. Available from: http://dx.doi.org/10.3390/pathogens7010017

14. Velmurugan K, Chen B, Miller JL, Azogue S, Gurses S, Hsu T, et al. Mycobacterium tuberculosis nuoG is a virulence gene that inhibits apoptosis of infected host cells. PLoS Pathog [Internet]. 2007 [cited 2022 Apr 2];3(7):e110. Available from: http://dx.doi.org/10.1371/journal.ppat.0030110

15. Glickman MS, Jacobs WR. Microbial pathogenesis of Mycobacterium tuberculosis: dawn of a discipline. Cell. 2001 Feb 23;104(4):477-85

16. Tuberculosis: General Information [Internet]. Centers For Disease Control and Prevention. 2021 [cited 2 April 2022]. Available from:     https://www.cdc.gov/tb/publications/factsheets/general/tb.htm

17. Rodriguez-Takeuchi SY, Renjifo ME, Medina FJ. Extrapulmonary tuberculosis: pathophysiology and imaging findings. Radiographics. 2019 Nov;39(7):2023-37

18. Chai Q, Zhang Y, Liu CH. Mycobacterium tuberculosis: an adaptable pathogen associated with multiple human diseases. Frontiers in cellular and infection microbiology. 2018 May 15;8:158.

19. TB education:Transmission and Pathogenesis of Tuberculosis [Internet]. CDC. [cited 8 April 2022]. Available from: https://www.cdc.gov/tb/education/corecurr/pdf/chapter2.pdf

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? Can bacterial damage be paradoxically enhanced by giving antibiotics?

Summary of mechanisms of actions of current TB drugs. Thioamides, Nitroimidazoles, Ethambutol, and Cycloserine act on cell wall synthesis. Diarylquinoline inhibits ATP synthase. PAS, Fluoroquinolones, Cyclic Peptides and Aminoglycosides act on the DNA (11).

The host immune response does most of the damage caused by active TB infection, but Mtb can cause direct damage to the host (1). Under the burden and strain of bacterial survival and subsequent rapid multiplication, the granuloma may fail its role as a barrier, and lyse (2). In such a scenario, the released M. tuberculosis particles multiply and directly damage the host by contributing to the destruction of lung tissue cells (3, 4). This occurs through the actions of bacterial protease, which results in caseous necrosis, in conjunction with TNF-α release by macrophages and apoptosis via cytotoxic T cells (2). This compromises the integrity of the lungs, and, in turn, may contribute to the respiratory signs and symptoms of Robert’s case, such as his chronic productive cough, or the crackles in the right lung and decreased sounds in the right lower lung field. More specifically, pulmonary fibrosis, or, the scarring of the lung, may occur following lung tissue necrosis, impairing gas exchange, causing crackles and decreased respiration.

Additionally, Mtb causes upregulation of matrix malloproteinases (MMPs) in Mtb-infected cells, which results in degradation of extracellular matrix components, this causes lung tissue destruction and pulmonary impairment in TB patients (1). This may explain why Robert had decreased breath sounds, because his lung tissue is being compromised and this may impact the ability of his lungs to properly fill with air.  Mtb can also induce macrophage cell death as the ESX-1 protein allows Mtb to escape the endosome and enter the cytosol, where it can induce host cell death (5). This destruction of immune cells may cause the infection to be more difficult to clear and result in more profound bacterial damage before a sufficient inflammatory response can be mounted.

Administering antibiotics does not enhance bacterial damage in most cases, so cases of TB are often treated with an antibiotic regimen (6). Physicians typically prescribe antibiotics for upwards of 6 months to fight Mtb infection (7). Treatment varies between latent and active tuberculosis, however, depending on patient risk assessment, doctors may prescribe both (7). The current treatment options for latent tuberculosis includes isoniazid, rifampin, and rifapentine, with the latter two prescribed when harmful side effects to isoniazid are present. For active tuberculosis, ethambutol, isoniazid, pyrazinamide, or rifampin may be prescribed. Each antibiotic has potential for the emergence of specific resistant strains of M. tuberculosis, enhancing immune evasion and pathogenesis (8). This in part due to the long period of administration that may lead to patient non-adherence to their medication, which allows the bacteria greater opportunities for mutations or horizontal gene transfer (8). M. tuberculosis is also capable of developing biofilms, which affords the bacterium protection against antibiotics, leading to treatment failure (9).

Multidrug-resistant TB (MDR TB) is caused by organisms resistant to the most effective anti-TB drugs, isoniazid and rifampin. Drug-resistant TB is transmitted in the same way as drug-susceptible TB, and is no more infectious than drug-susceptible TB (12).* Often resistant to additional drugs ** Resistant to any fluoroquinolone and at least one of three injectable second-line drugs (i.e., amikacin, kanamycin, or capreomycin)

Antibiotic resistance has been a significant challenge in treating Mtb (10). Multidrug-resistant TB (MDR-TB) are Mtb that are resistant to at minimum the two most effective TB drugs (isoniazid and rifampin) (10). Bactericidal anti-TB drugs have been demonstrated to induce mitochondrial dysfunction and oxidative damage in host cells causing apoptosis, so in MDR-TB they can increase the host cell damage (10). Additionally, rifampin has been shown to have an immunosuppressive effect by suppressing T cells, so in MDR-TB Mtb effects would be exacerbated because there would be a hindered immune response (10).

References

1. Ravimohan S, Kornfeld H, Weissman D, Bisson GP. Tuberculosis and lung damage: from epidemiology to pathophysiology. Eur Respir Rev [Internet]. 2018;27(147):170077. Available from: https://err.ersjournals.com/content/errev/27/147/170077.full.pdf

2. McMurray DN. Mycobacteria and Nocardia [Internet]. Medical Microbiology. 4th edition. U.S. National Library of Medicine; 1996 [cited 2022Apr1]. Available from: https://www.ncbi.nlm.nih.gov/books/NBK7812/

3. Smith I. Mycobacterium tuberculosis pathogenesis and molecular determinants of virulence [Internet]. Clinical microbiology reviews. American Society for Microbiology; 2003 [cited 2022Apr1]. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC164219/

4. Ollinger J, O'Malley T, Kesicki EA, Odingo J, Parish T. Validation of the essential CLPP protease in mycobacterium tuberculosis as a novel drug target. Journal of Bacteriology. 2012;194(3):663–8.

5. Simeone R, Bobard A, Lippmann J, Bitter W, Majlessi L, Brosch R, et al. Phagosomal rupture by Mycobacterium tuberculosis results in toxicity and host cell death. PLoS Pathog [Internet]. 2012 [cited 2022 Apr 2];8(2):e1002507. Available from: http://dx.doi.org/10.1371/journal.ppat.1002507

6. Todar K, Madison, WI. Tuberculosis [Internet]. Textbookofbacteriology.net. [cited 2022 Apr 8]. Available from: http://textbookofbacteriology.net/tuberculosis_2.html

7. Tuberculosis (TB) treatment after exposure: Medications used [Internet]. WebMD. WebMD; [cited 2022Apr1]. Available from: https://www.webmd.com/lung/understanding-tuberculosis-treatment

8. Nguyen L. Antibiotic resistance mechanisms in M. tuberculosis: An update [Internet]. Archives of toxicology. U.S. National Library of Medicine; 2016 [cited 2022Apr1]. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4988520/

9. Esteban J, García-Coca M. Mycobacterium biofilms. Frontiers in Microbiology. 2018;8.

10. Park H-E, Lee W, Shin M-K, Shin SJ. Understanding the reciprocal interplay between antibiotics and host immune system: How can we improve the anti-Mycobacterial activity of current drugs to better control tuberculosis? Front Immunol [Internet]. 2021;12:703060. Available from: http://dx.doi.org/10.3389/fimmu.2021.703060

11. Tuberculosis Drugs and Mechanisms of Action [Internet]. National Institude Of Allergy And Infectious Diseases. 2016 [cited 2 April 2022]. Available from: https://www.niaid.nih.gov/diseases-conditions/tbdrugs

12. TB education:Transmission and Pathogenesis of Tuberculosis [Internet]. CDC. [cited 8 April 2022]. Available from: https://www.cdc.gov/tb/education/corecurr/pdf/chapter2.pdf

The Host Immune Response

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

Tuberculosis is a chronic and life-threatening infectious disease caused by Mycobacterium tuberculosis complex. It is one of the leading causes of mortalities worldwide and has caused 1.4 million deaths globally in 2015 (1). The disease is transmitted via respiratory droplets, usually when a person infected with Mtb in the lungs coughs, speaks, or sings (2). Upon inhalation, larger droplets containing the pathogen are lodged in the upper respiratory tract and thus unlikely to lead to infection; however, smaller droplets can be deposited in the terminal bronchioles and distal alveoli to establish infection. Infections mostly affect the lungs; however, they can also spread to secondary sites such as the lymph nodes, bones, and joints (3).

Interestingly, only a portion of Mtb-infected individuals develop active TB, which occurs when the host’s immune system is unsuccessful in controlling the infection and the bacterium is able to remain active and multiply within the host (4). These individuals will exhibit the symptoms that Robert does and can spread the infection to others. In many cases, the host immune system will be successful in fighting off Mtb, resulting in latent TB infection (LTBI). In LTBI, the bacterium remains dormant for a lifetime within the body without replicating or causing symptoms, though approximately 1 in 10 patients will develop active TB if left untreated (5). This can occur when the host immune system is weakened, such as when a person begins taking immunosuppressive medications or in individuals with chronic health conditions such as HIV, cancer, chronic kidney disease, or diabetes (6). Due to the unique ability of Mtb infection to resolve into latency, it is extremely prevalent in the general population. In fact, it is estimated that one-third of the world is infected with Mtb, with more than 9 million new cases being reported each year (7).

Both the innate and adaptive immune responses are involved in defending the host against this infection; the corresponding elements are described below.

Innate Response

The innate immune response towards this infection involves various effector cells, complement, antimicrobial substances, and soluble inflammatory mediators.

Main effector cells of the innate response that are involved in fighting against Mycobacterium tuberculosis (Mtb) infection are macrophages, dendritic cells, natural killer cells, and neutrophils. The pathogen is detected by pattern recognition receptors (PRRs), such as Nod-like receptors (NLR) and Toll-like receptors (TLR) found on these immune cells, and various immune defence-associated cellular mechanisms are initiated by pathogen recognition. For instance, TLR2 can recognize Mtb lipoproteins encoded by ipqH.  At the same time, other immune cells are recruited and activated along with an increased cytokine production (1). Several cells that could also initiate innate immune responses are involved in the host defence against Mtb as well, including non-conventional T cells such as mucosal-associated invariant T (MAIT) cells, CD1-restricted lymphocytes and NKT cells (1). Furthermore, other cell types, such as airway epithelial cells and mast cells, which are not classically defined as immune cells, have also been shown to contribute to early immune responses against Mtb (1).

Following inhalation, the first line of defence against Mtb are the airway epithelial cells (AECs) (8). Airway epithelial cells function not only as a physical barrier against the external environment, but also play a role in initial recognition of Mtb. Although AECs are not usually categorized as innate immune cells, they are one of the first cells to encounter Mtb and serve important immunological functions through their PRRs. They are able to perceive the presence of Mtb via PRRs, which recognize pathogen-associated molecular patterns (PAMPs) on the surface of the bacterium (8). In mouse models, the Toll-like receptors TLR2 and TLR9, which recognize lipoproteins and unmethylated CpG DNA respectively, have been identified as key players in the defence against Mtb infection (9). Upon recognition via TLR2 or TLR9, the innate immune response is initiated and inflammatory cytokines such as interleukin-12 (IL-12) are produced. These inflammatory cytokines are necessary for stimulating the IFN-g-producing T cells which are critical in controlling Mtb infection (9).

Figure 1. Overview of the cellular immune response to Mtb. Reprinted from O’Garra et al. (10).

Cellular Mechanisms & Effector Cells

Upon recognizing Mtb, immune cells initiate various cellular mechanisms to control the infection.

Macrophages

Resident alveolar macrophages are the next line of defence against Mtb and are able to recruit different types of macrophages, such as monocyte-derived macrophages, during early infection (1).  However, the macrophage response has been shown to be unable to prevent establishment of Mtb infection in the absence of an adaptive immune response (9). This has been proven in both mouse and nonhuman primate (NHP) models, where Mtb is able to replicate uncontrollably in macrophages up until an adequate CD4 T cell response can be mounted (9). When the infection cannot be contained by this initial response, macrophages then become a niche in which Mtb may begin replicating within, providing protection against other host immune responses and allowing for persistence of Mtb in the host during latent TB.

The role of macrophages in a typical innate immune response is to recognize and engulf the pathogen, secreting antimicrobial peptides (AMPs) and producing inflammatory cytokines. They possess great capacity for phagocytosis and express a large array of pattern recognition receptors (PRR), including Toll-like receptors (TLRs), C-type lectin receptors (CLRs), and Nod-like receptors (NLRs), all of which have been shown to participate in Mycobacterium tuberculosis (Mtb) recognition.  Macrophages can eliminate Mtb through multiple mechanisms, such as the production of reactive oxygen and nitrogen species and cytokines, phagosome acidification and autophagy of intracellular Mtb. (1)

In in vitro studies, complement receptor 3 (CR3) has been implicated as one of the main receptors expressed by macrophages for the engulfment of Mtb (11). However, macrophages also trigger phagocytosis upon recognition of PAMPs via PRRs such as TLR2 against lipomannans (LM), lipoarabinomannan (LAM), and phosphatidyl-myo-inositolmannoside (PIM) (12). Other receptors which recognize Mtb-associated PAMPs include various C-type lectin receptors, Fc receptors, scavenger receptors, and cytosolic DNA sensors (13). Upon PAMP recognition by a PRR, a variety of cellular processes are triggered; a few examples are discussed below.

Following phagocytosis, Mtb is localized within cellular phagosomes, which typically undergo maturation – that is, fusion with acidic AMP-containing vesicles resulting in an increasingly hostile environment in the phagosome (14). This maturation process is highly regulated and involves the recruitment of specific Rab GTPases to facilitate the formation of mature phagolysosomes, which contain hydrolytic enzymes and AMPs (14). The interior of the phagolysosome also decreases in pH as the expression of vesicular proton-pump ATPases shuttle protons into the vesicle. This acidic environment is not only required for optimal enzymatic activity of hydrolases in the phagolysosome but is also ideal for the production of ROS (14). It was found that patients with mutations in the catalytic subunit of NADPH-oxidase 2 on the phagolysosomal membrane, which is involved in ROS production, are more susceptible to Mtb infection (8).

PRR interactions with PAMPs also activate genes that regulate cytokine production, and therefore lead to increased production of inflammatory cytokines, such as IFN-γ, IL-12, and IL-1β (1). These cytokines then recruit other immune cells. Recognition of Mtb via TLR2 and TLR9 also induces a myeloid differentiation primary response protein 88 (MyD88)-dependent signaling cascade, which activates the macrophage and causes translocation of nuclear transcription factors (such as NF-kB) into the nucleus to stimulate production of pro-inflammatory cytokines (in particular, TNF-a, which has been shown to be critical in controlling Mtb infection) (13).

TNF-α-mediated apoptosis in infected macrophages is a major defence mechanism, where TNF-α production is stimulated by the binding of macrophages to Mtb. For naive macrophages that are not yet sensitive to TNF-α-mediated apoptosis, intracellular Mtb will activate the TNFR1 death pathway and prime these macrophages. Through apoptosis of infected cells, potential sites of Mtb replication are eliminated and bacterial load in the host is reduced (15).

In addition, intracellular bacteria such as Mtb can be targeted by the autosomal pathway, which sequesters cytosolic components into an autophagosome to be delivered to a lysosome for degradation (10). This process, termed autophagy, can be activated in macrophages by IFN-g. Autophagy in macrophages aids in immune defence through the production of antimicrobial peptides and enhancement of phagosomal acidification (1).

Once activation has been sufficiently elicited, AMs in tuberculosis (TB) patients can produce nitric oxide and reactive oxygen species, two antimycobacterial effector molecules that are able to kill Mtb (16). In addition, M1 and M2 Macrophages have been shown to play important roles in maintaining the balance between exacerbated pathology and control of mycobacterial growth (1).

In Mtb infection, the pathogen expresses a variety of virulence factors which allow it to circumvent or neutralize these cellular weapons (8,13). These will be discussed in section iii).

Dendritic cells

(DCs) serve the essential function of bridging the innate and adaptive immune responses, possessing the ability to migrate from sites of infection to secondary lymphoid tissues (9). Typically, they accomplish this by performing antigen processing and presentation, co-stimulating lymphocytes, and producing large amounts of cytokines (8). Monocyte-derived human DCs express mannose receptors (MRs) and DC-specific ICAM-grabbing non integrin (DC-SIGN), which are capable of recognizing Mtb ligands, such as Mtb lipoprotein lprG and hexa mannosylated phosphatidylinositol mannosides (PIMs) (1). They also express high levels of MHC-II, CD80, and CD86 and are primarily located in the lung parenchyma, rather than the lung vasculature, suggesting that these cells have potent antigen-presentation capacity (16).

During Mtb infection, however, the role of DCs is controversial. Activated DCs are known to secrete IL-12, which is required for induction of the protective IFN-g T cell response (10). They also contribute to TNF-a production, which serves to induce fever, cell apoptosis, and inflammation in the Mtb immune response. However, like other phagocytic cells such as macrophages and neutrophils, DCs can be invaded by Mtb (10). Thus, it is unclear whether this subset of immune cells plays a role in strengthening immune response to Mtb or not.

Neutrophils

Neutrophils are the first cells to infiltrate the lungs after Mtb infection and are the most abundant cell type appearing in the bronchoalveolar lavage and the sputum of the active pulmonary TB patients (16). There is a known correlation between decreased numbers of neutrophils in an individual’s peripheral blood and likelihood of developing TB after contact with an infected person (8). Potential antimycobacterial effector functions of neutrophils involve production of TNF-α, reactive oxygen species, antimicrobial peptides, extracellular traps (NETs), efferocytosis of infected myeloid cells, and boosting the ability of DCs to prime T-cell responses (16). The factors released by neutrophils during respiratory bursts, such as elastase, collagenase and myeloperoxidase, indiscriminately damage bacterial and host cells (1). They are also implicated in the production of a variety of cytokines such as IL-8, IL-1-b, and IFN-g during Mtb infection, which function to activate T lymphocytes, dendritic cells, and macrophages (17). Thus, neutrophils constitute a potent population of effector cells that can mediate both anti-mycobacterial activity and immunopathology during Mtb infection (1).

After phagocytosis, neutrophils exhibit pathogen-killing responses via oxidative bursts (generation of ROS and proteolytic enzymes, followed by release from the cell in the form of granules). The potential of neutrophils to release enzymes that lead to the destruction of pulmonary parenchyma and contribute to the immunopathology observed in patients, such as arginase and matrix metalloproteinase-9 (MMP-9 and gelatinase B), is shared by other innate immune cells and epithelial cells affected by Mtb (1).

Neutrophils are also able to release extracellular traps (NETs) to trap and kill microbes, minimizing damage to host tissues. These NETs are released when neutrophils undergo cell death and are composed of chromatin and granule proteins (17). However, the role of NETs in controlling Mtb infection has been disputed, with some studies showing that Mtb is able to be trapped by NETs but not killed (18).

Interestingly, neutrophils and macrophages are known to cooperate during Mtb infection. Since neutrophils are typically the first cells to arrive at the site of infection, they often engulf the pathogen in large numbers. This may pose a problem, however, as neutrophil contents are highly toxic and they are quite fragile and short-lived, resulting in apoptosis or necrosis after a short period of time (18). The phagocytosis of apoptotic neutrophils by macrophages not only removes the dead cells and prevents the toxic intracellular contents from damaging surrounding tissues, but also allows the macrophage to hijack the neutrophil-derived granule proteins for pathogen killing (i.e. the antimicrobial peptides contained within these neutrophils can then inhibit Mtb replication in the macrophages (1, 17)).

Natural killer cells

Natural killer cells are one of the immune cells that get involved early on during an infection, and they lyse autologous infected cells and tumor cells without prior sensitization (19). NK cells act early during infection and are not MHC-restricted (1). NK cells express natural cytotoxicity receptor (NCR) NKp44, which binds components of the Mtb cell wall (such as mycolic acids) (1). One of the most important cytotoxic mechanisms is the production of cytoplasmic granules, which contain perforin, granzyme, and granulysin that mediate apoptosis of infected cells.

NK cells contribute to the immune response both directly through the lysis of infected monocytes and cytotoxic mechanisms, and indirectly through the production of cytokines that stimulate other immune components, such as promoting macrophage activation, T cell proliferation, and enhancing phagolysosomal fusion in infected cells. In order to accomplish this, NK cells also secrete IFN-gamma to activate macrophages to kill viruses and intracellular parasites (19). As an important early source of IFN-gamma, NK cells are critical for activation of macrophages, and they may directly present antigen to T cells (19). Furthermore, NK cells can lyse M.tuberculosis-infected monocytes to a greater extent than uninfected monocytes (19). Lysis of infected monocytes was associated with increased expression of mRNA for the NKp46 receptor instead of the NKp44 receptors, and antisera to NKp46 markedly inhibited lysis of infected monocytes (19).

Overall, NK cell levels are inversely correlated with the transition from LTBI to active TB in humans (9). They are also inversely correlated with lung inflammation in TB patients, along with being correlated to disease progression (9).

The complement system

Finally, the effects of complement proteins on Mtb infection are currently being investigated. The complement system is made of numerous plasma proteins that are believed to play a key role during the early innate response in clearing the Mtb infection.

Figure 2. Main components of the innate response against Mtb infection, including effector cells, PRRs, and cellular processes. Reprinted from Liu et al (1).

Interactions between the pathogen and airway surface components induce the alveolar cells to produce various proteins involved in the alternative and classical complement pathways, and these proteins can be detected in the bronchoalveolar lavage fluid. Upon activation of the alternative or classical pathway, the C3 protein binds the bacterium through covalent linkages between reactive thiolester and a target containing a hydroxyl or amine group; this opsonization then enhances phagocytosis by the alveolar macrophages through a C3-dependent entry pathway (20). It is also thought that the components C5 and C7 play a role in antimicrobial defence against Mtb (8). Complement system activity is slightly reduced in the lungs relative to the serum; however, it is still detectable in the lungs.

Figure 2 provides an overview of the innate immune response to Mtb.

Adaptive immune responses

The adaptive immune response, which is mediated by B and T lymphocytes, is mounted against Mtb starting at secondary lymphoid organs. After the bacterial pathogen is recognized via PAMP/PRR signalling, professional antigen-presenting cells (APCs) such as dendritic cells phagocytose Mtb, breaking it down and processing bacterial peptides to be presented to T lymphocytes in the lymph node (8). During the process of antigen processing and presentation, the dendritic cell undergoes a maturation process, becoming less phagocytic and upregulating expression of certain cell markers while migrating from the site of infection to the lymph node. Once at the lymph node, dendritic cells present bacterial peptides to naïve T lymphocytes in order to activate them, kick-starting the adaptive immune response (Figure 3).

Figure 3. Antigen presentation pathways during Mtb infection. Reprinted from Kaufmann (21).

Adaptive immune responses mediated by T cells play a vital role in the elimination of M. tuberculosis (19). Cytotoxicity and cytokine production are the two major effector mechanisms utilized by T cells against intracellular pathogens (19). The onset of the adaptive immune response in TB is considered delayed relative to other infections, allowing the bacteria population in the lungs to replicate uncontrollably and spread to the spleen before a strong CD4+ T cell response can be mounted (22,23).  It may take up to 12 weeks for a cellular immune response to become detectable, and it is delayed even more in the lungs, which allows the infection to become more established (24). This delayed T cell response (compared to other pathogens such as Salmonella or Listeria) has been hypothesized to occur due to a variety of mechanisms and regulatory pathways. For instance, Mtb’s inhibition of apoptosis in macrophages and neutrophils delays antigen presentation by DCs (10,25). It is also hypothesized that dendritic cells within the alveolar tissues may have limited abilities for migration compared to the dendritic cells in the airway tissues (26). Production of IL-10, a cytokine whose major role is to suppress macrophage and DC functions, also serves to inhibit and delay production of IFN-g and IL-17 by CD4+ T cells (27). Finally, activation of pathogen-specific Foxp3+ regulatory cells (a subset of CD4+ T cells) prevents and delays the migration of CD4 effector cells to the lungs (28).

Cell-mediated immunity

Figure 4. Mechanisms of cell-mediated immunity. Reprinted from Lazarevic et al (29).

Figure 5. Naïve T cell activation in the draining lymph nodes. Reprinted from Cooper (26).

CD4+ T cells play a striking role in Mtb cell-mediated immunity (Figure 4). This is most evident in HIV+ patients (CD4+-depleted), who experience increased susceptibility to TB (7,30). During Mtb infection, initial activation of CD4+ T cells in the mediastinal lymph nodes adjacent to the lungs occurs 7-11 days after inhalation of the pathogen (10,22). Activation is triggered when an Mtb-derived peptide is presented to a naïve CD4+ T cell on an MHC class II molecule by a professional antigen-presenting cell (APC) such as a DC, with the cell-to-cell contact allowing for costimulatory signals to be received. Following activation, a rapid increase of CD4+ T cells is seen as they replicate and proliferate, whereupon they travel to the lungs in large numbers. Upon recognition of Mtb antigens, activated T cells produce pro-inflammatory (IL-6, IL-21, IL1b, IL-12p40, IL-17, IL-21, and IL-22) and anti-inflammatory (TGF-b) cytokines (23). In particular, the cytokines IL-17 and IL-21 produced by Th17 cells are key to the protective immune response against Mtb (23).

Importantly, CD4+ T cells are critical in the control of TB infections in humans because of the production of interferon γ (IFN-γ), which is required for resistance to fatal infection (31). One mechanism by which this is thought to occur is by IFN-g facilitating the process of phagosome maturation in macrophages (8). The cytokine TNF-a is also produced by T cells, serving to activate macrophages and induce production of chemokines as part of the protective immune response against Mtb (10).

In addition, CD8+ T cells – which recognize antigens through MHC-I – may contribute to the control of M. tuberculosis infection through four mechanisms: cytokine release, cytotoxicity via granule-dependent exocytosis pathway, cytotoxicity mediated through Fas/Fas ligand interaction, and direct microbicidal activity (29).

Both CD4+ and CD8+ T cells are important producers of Th1-type cytokines, such as IFN-γ and TNF-α, which act synergistically to activate M. tuberculosis–harboring macrophages (29). Activation of macrophages results in upregulation of inducible nitric oxide synthase, which leads to the production of reactive nitrogen intermediates, such as nitric oxide (NO) (29). Reactive nitrogen intermediates, together with reactive oxygen intermediates (O2), exert antimycobacterial effects, which lead to the reduction in viable mycobacteria (29).  CD8+ T cells also produce IL-2 and IFN-α which enhance the inflammatory response.

On recognition of M. tuberculosis–infected cells, CD8+ T cells release perforin-containing granules (29). Perforin polymerizes on the cell membrane of the target cells allowing the entry of effector molecules such as granzyme A and granzyme B which are serine proteases, leading to apoptosis or lysis of the target cells (29). The lysis of unresponsive macrophages infected with M. tuberculosis releases the pathogen into the extracellular environment to be taken up by freshly activated macrophages (29).

Cross-linking of Fas ligand (FasL) – which is expressed on activated CD8+ T cells – and Fas – which is expressed on target cell – leads to recruitment of Fas–associated death domain and activation of caspase 8 leading to apoptosis of target cells (29).

Finally, granules within human CD8+ T cells contain a newly identified molecule, granulysin, which has direct microbicidal effect on intracellular bacteria (29). Only in the presence of perforin, granulysin dramatically decreased the viability of intracellular M. tuberculosis (29).

However, it is important to note that despite all of its microbicidal capacity, CD8+ T cells also produce IL-10, which favors Mtb growth and replication (8).

Humoral immunity

Figure 6. Mechanisms by which B cells shape the immune response to M. tuberculosis. Reprinted from How B cells shape the immune (34).

The humoral response involves the activation and differentiation of B lymphocytes, culminating in the production of protective antibodies. In the case of Mtb infection, previous research has been unable to discern if B cells play a protective role (23). It was previously widely accepted that antibody-mediated immune response does not help in controlling Mtb infection. This is because the bacterium is an intracellular pathogen; it can also resist complement killing due to the high concentration of lipids in the bacterial cell wall. Therefore, passive immunization does not provide good protection, and patients with defective antibody-production mechanisms are not at higher risk of Mtb infection (32).

Recent studies, however, have suggested that B cells likely provide a greater contribution to the protective immune response than previously thought. Researchers have identified activated B cells within the granulomas of Mtb-infected NHPs, indicating a potential role in granuloma formation and function (33). Further studies have also shown that B cells can modulate the host response against M. tuberculosis in a variety of ways. Upon acute infection, selective engagement of stimulatory Fcγ receptors by antibody complexes heightens the Th1 response and promotes mycobacterial containment with minimal inflammation (34). On the contrary, engagement of inhibitory FcγRIIB increases IL-10 production and compromises immunity against M. tuberculosis (34).

Table 1. Overview of the effector functions of cells involved in the innate and adaptive response. Adapted from (23).

Additionally, B cells could regulate the host response to M. tuberculosis by functioning as antigen presenting cells to interact with T cells (35). The primary site of this reaction is at the germinal center (GC). The interaction between GC B cells and Tfh (T follicular helper cells) culminates in B cell expansion, somatic hypermutation, affinity maturation, class switching, and the development of memory B cells and antibody-producing plasma cells (35). Furthermore, B cells can produce cytokines that modulate responding immune cells including influencing the differentiation of T cells and hence have effector functions (35). The antibodies (Ab) produced could modulate multiple aspects of both the innate and adaptive immune response (35). M. tuberculosis-specific antibodies can opsonize extracellular bacilli, form immune complexes that fix complements, and engage Fc receptors of effector cells (35). Antibodies can also modulate the GC reactions as well as inflammation in infected tissues (35).

Table 1 provides an overview of the effector functions of cells involved in the innate and adaptive response. Adapted from (23).

Development of Latent Mtb infection

As adaptive immune response occurs, another complex immune mechanism also takes place. This process is characterized by granuloma formation, and occurs in at least 90% of patients. The inflammatory response triggered at the beginning of Mtb infection is thought to contribute to granuloma formation (8).

Figure 7. Mtb granuloma structure, with macrophages in the center and lymphocytes on the periphery, along with other cells such as neutrophils, dendritic cells, and natural killer cells. Reprinted from Ndlovu and Marakalala (36).

A granuloma is an organized structure composed of aggregates of immune cells, and is a characteristic feature of Mtb infections. Granulomas have been shown to be composed of a central mass of infected macrophages, stimulated macrophages possessing multiple nuclei, macrophages loaded with lipid droplets, neutrophils, and cells resembling epithelial cells (10). Surrounding this centre are lymphocytes – largely CD4+ T cells, but also consisting of CD8+ T cells and B cells – and fibroblasts. This outer layer of fibroblasts is what forms the fibrous capsule of the granuloma. A granuloma’s purpose is to both limit the growth of the pathogen and serve as a protective environment for pathogen survival.

Antigen presentation activates T cells that induce the killing of infected macrophages, and the cell-mediated adaptive immune response is thus activated. T lymphocytes secrete various cytokines such as IL-2 and IFN-γ that aid in the maintenance of the granuloma, while B lymphocytes accumulate in follicles and regulate both the cytokine and inflammatory response within the lesions.

Figure 8. Different outcomes of Mtb infection. Reprinted from Kaufmann (21).

Long-term persistence of granulomas requires immunomodulation of the inflammatory response, leading to decreased inflammation in the lungs (8). This decrease in inflammation is known to be triggered by Mtb virulence factors, leading to secretion of IL-10 by macrophages.

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36. Ndlovu H, Marakalala MJ. Granulomas and inflammation: host-directed therapies for tuberculosis. Front Immunol [Internet]. 2016 Oct 24 [cited 2022 Mar 30];7:434. Available from: https://www.frontiersin.org/articles/10.3389/fimmu.2016.00434/full

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

The host’s immune response to Mtb within the pulmonary airways may lead to exacerbated inflammation and eventually lung damage (6). About 50% of individuals who have recovered from Mtb infection will develop pulmonary impairment after tuberculosis (PIAT) (6). It has multiple clinical conditions as a result of chronic inflammation such as disrupted pulmonary structure/function and lesions formed in the parenchymal, airway, vascular, and mediastinal regions (6).

Oxidative Burst

Neutrophils play an important role in Mtb infection, but may have pathogenic effects through different functions. One of which is oxidative burst (6). This is a process that releases reactive oxygen species (ROS) and is mediated by nicotinamide adenine dinucleotide phosphate (NADP). It is a phagocytic process done by neutrophils and macrophages (6). This process is done by a myeloperoxidase system composed of NADPH2, reduced glutathione (GSH), azide, cyanide, thiocyanate, Tapazole, thiourea, cysteine, ergothioneine, thiosulfate, NADH2, and tyrosine (6). Tuberculosis patients seem to have lower GSH levels compared to healthy individuals. An increase in GSH level is associated with enhanced T cell activity for inhibiting Mtb growth within macrophages (6). ROS production by neutrophils promotes Mtb-induced necrosis which helps Mtb growth (6).

Neutrophil Extracellular Traps (NET)

Neutrophil extracellular traps occur when neutrophils are activated by liposaccaride (LPS), IL-8, or phorbol myristate acetate (PMA) (6). This results in the release of cell components and forms an extracellular fibril matrix. NET’s are usually composed of proteins, DNA, and chromatin-derived fibers. This process is called NETosis and is a crucial component to neutrophil mediated responses to pathogen infection. However, it may be harmful during an inflammatory disease (6). NETs can cause unwanted immune reactions and trigger tissue injury (6). It has been observed that NETs are capable of trapping Mtb but do not kill the bacteria. This might be due to enzymes involved in inflammatory pathways being suppressed (6). Macrophages can also be activated by Mtb-induced NETs which can lead to inflammatory lung damage in infected patients (6). NETs have been linked to lung damage in individuals infected with Mtb (6).

Figure 1. Different types of NETs by neutrophils

Metalloproteinases

Matrix metalloproteinases (MMPs) have been observed to be associated with excessive lung inflammation (6). MMPs are a family of 25 potent proteases that degrade extracellular matrix components and are likely central to TB-associated lung injury (8). MMP-9 has been secreted by neutrophils after proinflammatory stimulus and is thought to facilitate transmembrane neutrophil migration (6). An increase in MMP-8 has also been observed in ATB patients which has been previously shown to correlate with pulmonary tissue damage (6). MMP-14 promotes collagen degradation and regulates monocyte migration in infected patients (6). Whie the results are not conclusive and the exact mechanisms remain unknown, it has been thought that MMPs may promote tissue injury after being infected with Mtb (6).

Fever and Amyloidosis

Beyond granuloma generation the immune response to Mtb infections involves fever generation and amyloidosis, both of which damage the host inadvertently. Fever is produced as a result of the release of endogenous pyrogens, a set of cytokines produced during the inflammatory response by macrophages15. Endogenous pyrogens include IL-1, IL-6 and IFN-γ. These cytokines are able to access the temperature-setting region of the body, the hypothalamus, through the organum vasculum (OVLT) because the OVLT lacks the blood-brain barrier. These pyrogens will encourage the release of prostaglandin E2 (PGE2)15. Downstream signalling of PGE2 mediates fever generation across the body. Fevers, of course, are not exclusive to Mtb infection, as any infection which produces inflammation can cause fever. In Mtb infections fever development is associated with direct cellular damage and inflammation. Prolonged fever is directly cytotoxic, affecting membrane stability and transmembrane transport protein function16. Acute-phase inflammatory reactants, such as SAA, IL-1, IL-6 and IFN-γ are released upon exposure to hyperthermia, which can propagate a disadvantageous inflammatory response16. Extracellular Heat-Shock Proteins (HSP), which are expressed in response to fever, may induce further cytokine release upon their expression16. Tuberculosis is thought to be a significant potential cause of Fevers with Unknown Origin (FUO)17

Amyloidosis is the abnormal deposition of fibres of insoluble amyloid proteins in the extracellular space of various tissues and organs. During the inflammatory response, serum amyloid A protein (SAA) production is increased by the liver. SAA has immunological functions, such as degrading the extracellular matrix (for ease of immune cell access to infection sites) and recruiting immune cells to inflammatory sites18. Its buildup and change from soluble to insoluble forms, however, causes amyloidosis18. Amyloidosis in response to chronic inflammation is referred to as AA amyloidosis. Symptoms of AA amyloidosis include numbness, changes to skin texture, shortness of breath, and general fatigue and weakness19. Amyloidosis is most prevalent in Active TB, as inflammatory responses are stronger when the infection moves outside of the lung.

Granuloma

Granuloma formation is the hallmark for tuberculosis. A granuloma is an organized structure consisting of immune cell types (such as NK cells, neutrophils, macrophages, T- and B- Cells) surrounding a caseous necrotic core of TB-infected alveolar macrophages (5). They are a small area of inflammation that are involved in surveillance and direct immune responses. It occurs when alveolar macrophages release cytokines to recruit cells. Some immune cells that are associated with granulomas include CD4 T cells, CD8 T cells, B cells, and macrophages. Granulomas are crucial to the immune response, as it helps restrict and eliminate bacterial spread (7). TNF-α and IFN-γ are crucial for granuloma formation, while IL-10 negatively regulates the response (7). It is composed mostly of macrophages, epithelioid cells, and multinucleated giant cells (Langhans giant cells). The bacteria can persist within the granuloma in a latent state for decades (7). Infected cell death can form a necrotic zone within the granuloma which eventually disintegrates and releases the bacteria into the host environment (7). This results in a granulomatous inflammation that can have fibrotic and necrotic reactions that will harm the host (7). In more severe cases, they can lead to the erosion of blood vessels, which will then lead to patients coughing up blood.

Should the bacteria spread beyond the initial granulomas in the lungs, as it does in Active TB, granulomas will develop in other organ systems. In roughly 10% of patients, the bacteria will reactivate and escape the initial granulomas13. It is not fully known what factors contribute to this spread, however, T lymphocytes are thought to play a role, as immunocompromised individuals, such as those with HIV, have a much greater likelihood of developing Active TB from Latent TB13. It is known that CD4+ T cells encapsulating the granuloma exhibit cytotoxic activity against Mtb-infected macrophages that is at least partly mediated by the FAS/FASL pathway, which would contribute to the apoptosis of macrophages, and by extension, bacterial clearance13. Furthermore, CD4+CD25+FoxP3+ T regulatory cells (Tregs) have been shown to accumulate in the lung granulomas. Given their regulatory immune role, they are thought to “balance out” pro-inflammatory mediators within the granuloma, leading to the establishment of a persistent infection13.

The generation of granulomas, though intended to be protective, can ultimately contribute to organ dysfunction. If the bacteria spreads beyond its initial granulomas, more granulomas will be generated at secondary and tertiary sites of infection, causing widespread physiological dysfunction, leading to the pathological manifestations of Active Mtb infection14. In the lungs, the development of granulomas may cause extensive scarring, leading to dry/bloody cough and impaired respiration (apnea). Other sites of granuloma generation include the bones and spine (Pott disease), the lymphatic system, the central nervous system (tuberculosis meningitis), and in the genitourinary system14. Dysfunction in these systems is related to increased pressure and tissue necrosis arising from granuloma development. It has also been suggested that granulomas coalesce and breakdown via liquefactive necrosis, leaving behind a cavity during active disease, but human studies imply that cavities actually originate from lipid pneumonia during post-primary TB (5). As a result of caseous necrosis, the lipid pneumonia lesions may develop into areas of caseous pneumonia. Alveolar cells are destroyed during caseous necrosis alongside structures nearby such as vessels and bronchi (5). Necrotic tissue begins to soften and fissure to be eventually coughed out, and gas-filled spaces that are surrounded by a collagen capsule replace normal lung tissue following cavitations (5).

Figure 2. Cartoon depiction of a granuloma

Lung function deficits

Airflow obstruction is associated with decreased capacity to completely expel air out of the lungs and is likely due to inflammation-induced narrowing of airways (1). Restriction could be linked to the reduced ability to inhale fully resulting from extensive fibrosis and stiffening of lung parenchyma (1). Lung manifestations of pneumonia are also mediated by immune mechanisms (1). Airflow obstruction have associated symptoms of dyspnea, reduced exercise capacity an chronic bronchitis (1). Pulmonary cavitation may distort or obliterate airways which leads to obstruction of airflow in the lungs (1). Patients with cavities had significantly lower forced expiratory volume (FEV) and bronchogenic spread is also a hallmark of pulmonary TB where disintegration of cavities releases caseous material which passes through bronchial walls (2). Bronchiectasis may also occur due to the destruction of elastic and muscular components of the bronchial walls that contributes to airflow obstructed (2). Bronchiectasis is permanent distortion of airways that predisposes to lifelong morbidity with recurrent episodes of purulent sputum production, hemoptysis, and sometimes progresses to pneumonia (2).

Additionally, restricted airflow symptoms commonly include chest pain, cough, and shortness of breath (3). These symptoms may be caused by structural changes in the lung which result from aberrant lung tissue repair, such as bronchovesicular distortion, fibrotic bands, and pleural thickening (3).

References:

1. Hunter R. Pathology of post primary tuberculosis of the lung: An illustrated critical review. Tuberculosis. 2011;91(6):497-509.
2. Roberts HR, Wells AU, Milne DG, et al. Airflow obstruction in bronchiectasis: correlation between computed tomography features and pulmonary function tests. Thorax 2000; 55: 198–204
3. Plit ML, Anderson R, Van Rensburg CE, et al. Influence of antimicrobial chemotherapy on spirometric parameters and pro-inflammatory indices in severe pulmonary tuberculosis. Eur Respir J 1998; 12: 351–356.
4. Ehlers S, Schaible U. The Granuloma in Tuberculosis: Dynamics of a Host–Pathogen Collusion. Frontiers in Immunology. 2013;3.
5. Ravimohan S, Kornfeld H, Weissman D, Bisson G. Tuberculosis and lung damage: from epidemiology to pathophysiology. European Respiratory Review. 2018;27(147):170077.
6. Muefong CN, Sutherland JS. Neutrophils in Tuberculosis-Associated Inflammation and Lung Pathology. Front Immunol. 2020 May 27;11:962.
7. Silva Miranda M, Breiman A, Allain S, Deknuydt F, Altare F. The Tuberculous Granuloma: An Unsuccessful Host Defence Mechanism Providing a Safety Shelter for the Bacteria? Clin Dev Immunol. 2012;2012:139127.

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

Mycobacterium tuberculosis complex is composed of pathogens that are resistant to acid, alkalis, as well as dehydration, due to their bacterial cell wall composition. Further, Mycobacterium tuberculosis is a very successful intracellular pathogen, where it can survive in mononuclear phagocytes and replicate quickly through the disruption of various host cellular processes and inhibition of the innate immune system (1). When extracellular, it can also evade host immune responses (2). Some of these mechanisms and characteristics are described below.

Expression of cell surface lipids and entry into permissive niches

In order to evade host immune responses, Mtb expresses a variety of virulence factors. First, establishment of infection within a host requires the pathogen to be phagocytosed by permissive macrophages in the initial stages of infection. To accomplish this, Mtb expresses phthiocerol dimycocerosate (PDIM, a cell surface lipid), which interacts with the macrophage to dampen TLR-related signaling and suppress recruitment of iNOS-positive macrophages (3). Mtb also expresses a related cell membrane lipid, phenolic glycolipids (PGL), which stimulates recruitment of iNOS-negative macrophages (which are not as microbicidal as iNOS-positive macrophages and are thus more permissive and desirable as niches) by inducing CCL2 signaling (4). Mtb can also directly inhibit PRR signaling by producing an antagonist glycolipid (1). SGLs are specific Mtb lipids and are competitive inhibitors of TLR2/TLR1 or TLR2/TLR6 heterodimers, inhibiting NF-κB activation, cytokine production, and costimulatory molecule expression (1). An Mmpl8 mutant which is unable to make Ac4SGL has shown an increase in TLR2 signaling in macrophages, resulting in higher cytokine production (1). A diacylated SLG is sufficient in inhibiting the response triggered by TLR2 agonists (1).

In addition, the composition of the bacterial cell wall with high lipid concentration also contributes to its impermeability to antimicrobial substances, resistance to acidic or alkaline compounds, as well as resistance to osmotic lysis due to complement deposition (5). Once inside the macrophage, the bacterium manipulates the intracellular environment to become more favourable for its replication. By sequestering itself inside phagocytes, Mtb also protects itself from other functions of the immune response, such as detection and opsonization by complement proteins and antibodies, or complement-mediated killing via the membrane attack complex (MAC). The intracellular phase of the Mtb life cycle thus serves as a strategy for immune evasion and disease pathogenesis.

Mtb also interferes with dendritic cell functioning. Similar to other pathogens, structural mutations allow Mtb to escape recognition by TLR and subsequent phagocytosis. As well, Mtb can suppress immune responses by interfering with mononuclear cells’ development into competent dendritic cells by targeting DC-SIGN. The bacterial cell wall-related alpha-glucan induces mononuclear cells to differentiate into dendritic cells that do not express CD1, which are glycoproteins involved in presenting lipid antigen to T cells, thereby reducing the activation of T cells and weakening the adaptive immune response (6).

Arrest of phagosome-lysosome fusion and phagosome maturation

Figure 1. The mechanism by which Mtb inhibits phagosome-lysosome fusion. Reprinted from Gupta et al (8).

Once inside the macrophage, Mtb can be found inside phagosomes. Typically, phagosomes will undergo a maturation process, decreasing in pH and acquiring antimicrobial factors such as hydrolases and reactive oxygen/nitrogen species (7). These phagosomes also fuse with lysosomes to form phagolysosomes, subjecting bacteria to a hostile intracellular environment which eventually kills it. The maturation process is complex and relies on the ordered recruitment of various components such as the GTPase Rab5, VPS34, EEA1, Rab7, and is facilitated by membrane-bound proteins called SNAREs (shown below in Figure 1.)

Mtb subverts this mechanism of bacterial killing by inhibiting phagosome maturation and fusion with lysosomes (9). To accomplish this, Mtb employs a variety of methods, explained briefly below.

It is believed that acidification of the phagosome is inhibited by preventing Mtb-containing vesicles from acquiring the ATPase (H+ V-ATPase) required for flooding the compartment with protons (10). The mycobacterial phosphatase PtpA has been shown to directly interact with subunits of the ATPase, preventing assembly on the vesicular membrane (11).

A much more well-studied method of phagosome disruption is the mechanism by which Mtb prevents fusion of the phagosome with lysosomes. This is believed to occur due to retention of Rab5, a marker found in early endosomes but absent in late endosomes (9). This causes a failure to recruit Rab7 (the late endosomal marker) and subsequent inhibition of lysosomal fusion. Mtb expresses multiple cell wall components believed to be involved in this dysregulation, including mannosylated lipoarabinomannan (ManLAM), which inhibits VPS34 and activation of EEA1 (8). EEA1 (early endosome autoantigen 1) is a membrane tethering molecule which interacts with SNARE proteins to facilitate endosome-endosome fusion (9). Thus, disruption of this process inhibits phagosome maturation in the early stages.

An additional strategy by which Mtb prevents phagosome-lysosome fusion is via the recruitment of Coronin1 (also known as TACO), which serves to activate calcium-calcineurin signalling to block fusion (11). Mtb also secretes a phosphatase known as SapM, which inhibits the activity of phosphatidylinositol 3-phosphate (PI3P) and prevents the phagosome from acquiring lysosomal contents and delivery of hydrolytic enzymes from the Golgi network (11).

Finally, Mtb expresses proteins such as early secretory antigen-6 and ATP1/2 which prevent the accumulation of vascular ATP and GTP enzymes on the membrane of the phagolysosome (12). Mtb infection results in lower biosynthesis of the toxin lipoarabinomannan (LAM) and production of P13P helps with inhibiting phagosome/lysosome fusion (12).

Manipulation of lysosomal components

Manipulation of host lysosomal systems is a common strategy used by intracellular pathogens in enhancing survival. Similarly, Mtb can alter both the lysosomal content and activity in macrophages due to their surface components; specifically, the bacterial cell wall-associated lipid sulfolipid-1. By reducing the trafficking of phagocytic cargo to lysosomes, Mtb can persist and replicate in macrophages for extended periods of time (13).

Cytosolic escape

In addition to preventing phagosome maturation, the pathogen sometimes escapes from the phagosomes and translocates into host cells’ cytoplasm. This is achieved through the activation of host cytosolic phospholipase A2, and usually occurs later on during an infection. The translocation is hypothesized to be more effective in suppressing autophagy, especially for Mtb strains with less capacity for tolerating phagosomal conditions (5).

Resistance against reactive nitrogen intermediates and nitric oxides

Figure 2. Diagram of RNS production and subversion by Mtb. Reprinted from Gupta et al. (8).

Another mechanism of macrophage-mediated killing typically employed is the production of reactive nitrogen species (RNS). During the inflammatory immune response, the cytokines TNF-a, IL-1b, and IFN-g from Th1 lymphocytes stimulate inducible nitric oxide synthase (iNOS) in macrophage phagosomes (8). iNOS is responsible for converting arginine to nitric oxide (an antimicrobial product which reacts with superoxide anion to form a powerful oxidizer, peroxynitrite). These reactive intermediates then go on to damage proteins, lipids, and DNA.

Mtb can employ various mechanisms to interfere with the ROS toxicity. For example, Mtb expresses AhpC, an alkyl hydroperoxide reductase subunit C, which catalyzes the degradation of peroxynitrite (Figure 2) (8). Another enzyme, MsrA (methionine sulfoxide reductase), converts the product of the reaction between peroxynitrite and methionine residues on proteins into harmless methionine (8). Other features that defend Mtb against ROS include the ability to directly scavenge the oxygen radicals due to the thick cell wall that contains lipoarabinomannan, a potent scavenger of ROS, and the production of ROS scavenging enzymes such as superoxide dismutases  (14).  

Apoptosis and Autophagy

Mtb virulence factors like miR-30A are able to regulate the macrophage apoptotic response (12). Overexpression of miR-30A has been shown to inhibit autophagy, which results in suppressing the elimination of intracellular Mtb (12). Mtb has shown to be able to inhibit the intrinsic host cell apoptosis pathway by regulating pro and anti-apoptotic proteins (15). During infection, it is able to upregulate anti-apoptotic genes mcl-1 and A1 which codes for Bcl-like proteins (15). The extrinsic pathway can also be regulated by altering the Fas receptor or sTNFR2 (15). A decrease in Fas expression was observed upon Mtb infection, which limits the Fas-FasL interaction, therefore inhibiting apoptosis (15). sTNFR2 has been shown to bind to TNFɑ which also results in preventing apoptosis (15).

Interestingly, less virulent strains of Mtb are capable of promoting apoptosis. The P19 lipoprotein found in Mtb strains promote CD4 T cell differentiation and also the production of various cytokines such as IL-6 and IL-12 (12).

In addition, Mtb can also modulate cell death in neutrophils. As discussed previously, neutrophils are one of the first immune cells recruited to the site of infection and aid in the clearance of intracellular pathogens. However, Mtb has also developed mechanisms to target neutrophil responses. For example, Mtb can secrete an ESAT-6 protein through the type VII secretion system, which induces intracellular Ca2+ overload in neutrophils. This overload subsequently leads to neutrophil necrosis (16).

Finally, Mtb can also exploit a mechanism called neddylation, which is a process essential to the maintenance of cellular homeostasis and regulation of various defence mechanisms involving dendritic cells. Mtb antigen Rv2463 alters the expression level of several proteins involved in the neddylation pathway, thereby interfering with oxidative burst, phagolysosome fusion, and other apoptosis-associated mechanisms in dendritic cells. This modulation therefore enhances Mtb long-term survival by delaying the adaptive immune response (17).

Inflammasome activation

An inflammasome response occurs when components of the innate immune response such as NLR3 and NLR4 assemble into a multimeric inflammasome complex with cytosolic DNA sensor AIM2. The complex subsequently induces the activation of inflammatory caspase-1, which in turn leads to the maturation of pro-inflammatory cytokines such as IL-18 and pyroptosis, a form of programmed cell death. Mtb on the other hand, can modulate this response through effector proteins such as Rv0198c (Zmp1), which prevent caspase-1 activation and the cytokine secretion that follows. Therefore, Mtb with mutations in this virulence factor is found to be cleared by macrophages much more quickly. It is for this reason that Zmp1 is currently being investigated as a potential drug target (16).

Evasion of complement-mediated lysis

Figure 3. Mtb evasion of the immune system through the suppression of macrophage and dendritic cell functioning. Reprinted from Jankovic et al (5).

Figure 4. Examples of Mtb components that can interfere with host cellular functions. These components can either activate or inhibit phagocytosis, autophagy, apoptosis, and inflammasome activation to evade the immune system. Reprinted from Liu et al (19).

During infection, it is well documented that Mtb and the related pathogen M. bovis BCG activate the classical and alternative complement pathways, resulting in opsonization of the bacterium. However, it appears as if Mtb infection is not eliminated by any of the three complement pathways, leading to speculation that Mtb’s ability to bind factor H prevents the formation of the membrane attack complex responsible for cell lysis (8). Factor H is a major regulatory molecule in the activation of the alternative complement pathway, which acts during the cleavage of the opsonin C3b to iC3b (18).

The following table is reprinted from Gupta et al. and describes the evasion mechanisms by which Mtb circumvents host immune responses (8). The most well-studied evasion mechanisms include Mtb-mediated arrest of phagosome-lysosome fusion, resistance against RNS, and interference with MHC class II antigen presentation. Figures 3 and 4 provide illustrative overviews of the above concepts.

Table 1. Evasion mechanisms of Mtb. Reprinted from Gupta et al. (8).

Figure 5. Pathway for the presentation of Mtb-derived peptides on MHC class II molecules. Reprinted from Gupta et al. (8).

Interference with antigen presentation

Mtb antigens can be processed and presented to activate cells of adaptive immunity via three different pathways: presentation of peptide antigens via MHC class I to CD8+ T cells, presentation of peptide antigens CD4+ T cells via MHC class II, and direct recognition of lipid or glycolipid antigens (such as ManLAM) by CD8+ T cells or NK cells. In the context of Mtb infection, presentation via MHC class II has been deemed to be most critical, as dysregulation leads to inhibition of the protective CD4+ T cell response which is required for protection.

In the MHC class II pathway, phagocytosed Mtb is degraded into peptide fragments within phagosomes or phagolysosomes, then trafficked to an endosomal vesicle along with HLA-DM (8). In parallel, MHC class II molecules are synthesized in the endoplasmic reticulum (stabilized by class II-associated invariant chain peptides, CLIP) and then trafficked towards the cell membrane in another vesicle, which fuses with the endosomes containing the antigenic peptides. In the fused vesicle, HLA-DM catalyzes removal of CLIP from the peptide binding cleft of the MHC class II molecule, and a peptide fragment is loaded in its place (8). MHC class II molecules are only considered ‘stable’ and viable for delivery to the cell surface if a peptide fragment is loaded onto the peptide binding cleft. This process is illustrated below, in figure 6. MHC class II molecules are found on the professional APCs which Mtb favours as its niche. Upon exposure to IFN-g, another key cytokine in the protective immune response against Mtb, MHC class II is noticeably upregulated (9). However, in the case of Mtb infection, this upregulation appears to be inhibited – perhaps due a variety of reasons ranging from improper sequestering of class II molecules, inhibition of IFN-g signaling pathways, and decreased expression of the class II transactivator, CIITA (9). Of various virulence factors expressed by Mtb, ManLAM and a 19-kDa lipoprotein have been shown to attenuate IFN-g-stimulated MHC class II presentation (20,21).

Cytokine Production & Other Virulence Factors

Cytokines and other substances secreted by immune effector cells play an essential role in controlling Mtb infections. However, the bacterium has also evolved to develop mechanisms that target host intracellular trafficking and cytokine production pathways.

For example, cytokine production in macrophages can be altered by Mtb through secreted proteins such as PtpA, which interferes with the downstream molecules in the TLR signalling pathways that are involved in inflammatory cytokine production (16). Furthermore, Mtb mannose-capped lipoarabinomannan (ManLAM) is found to induce IL-10 production through the ligation of Dendritic Cell-Specific Intercellular Adhesion Molecule-3-Grabbing Non-integrin (DC-SIGN), which interferes with dendritic cell maturation, as well as the expression of other co-stimulatory molecules. IL-10 is found to both favor Mtb replication and delay the initiation of adaptive immune responses (16).

Another virulence factor is associated with the inhibition of PMN migration. It is called Cord factor, which is a glycolipid in the bacterial cell wall. The Cord factor is toxic to mammalian cells, and causes the bacteria to grow in long, intertwined filaments which prevent leukocyte migration (5).

Finally, Mtb mycolic acid, lipopeptides, and early secretory antigen-6 are found to weaken phagocytosis and reduce antigen process capacity in foamy macrophages (16).

Granuloma formation

The presence of granulomas, though critical in the protective immune response against Mtb, also appears to contribute to its persistence within the host. This is thought to be due to the structure and organization of the niche, with CD4+ T cells sequestered to the outer layer and infected macrophages remaining in the centre. In between these two cell populations lies a wall of activated macrophages and giant multinucleated cells. This wall serves as a double-edged sword, preventing bacteria from re-entering the outside environment, but also preventing T cells from diffusing inward and activating the microbicidal capacities of the infected macrophages within (22). Granulomas and their role in the continued survival of Mtb within the host will be discussed in the next section.

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20.  Noss EH, Pai RK, Sellati TJ, Radolf JD, Belisle J, Golenbock DT, et al. Toll-Like Receptor 2-Dependent Inhibition of Macrophage Class II MHC Expression and Antigen Processing by 19-kDa Lipoprotein of Mycobacterium tuberculosis. J Immunol [Internet]. 2001 Jul 15 [cited 2022 Apr 1];167(2):910–8. Available from: https://www.jimmunol.org/content/167/2/910

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

Outcomes

Tuberculosis (TB) is caused by Mycobacterium tuberculosis that most often affect the lungs, and this disease is curable and preventable. (2) People infected with Mtb have a 5–10% risk of becoming ill with TB. (2) Those with compromised immune systems, such as people with HIV, malnutrition or diabetes, have a higher risk of becoming ill. (2) When a person develops active TB disease, the symptoms, such as cough, fever, night sweats, or weight loss, may be mild for many months. (2) This can lead to delays in seeking care, and results in transmission of the bacteria to others. (2) People with active TB can infect 5–15 other people through close contact over the course of a year. (2) Without proper treatment, 45% of HIV-negative people with TB on average and nearly all HIV-positive people with TB could die from the disease. (2)

Figure 1. Phenotypes in the progression of tuberculosis (TB) disease after exposure (3)

Early clearance (EC) is one of the several phenotypes in the progression to TB disease after exposure. (3) By definition, EC occurs before the development of an adaptive immune response and is likely to be due to innate factors, during Stage I or II. (3) If the pathogen evades EC mechanisms, an adaptive response develops (Stage III), measurable through a positive tuberculin skin test (TST) or interferon-γ release assay (IGRA). (3) Early evasion of both innate and adaptive responses results in primary progressive TB. (3) However, in the majority of infected individuals, M. tuberculosis is contained as latent TB infection with only 5% later reactivating disease. (3) Clearly, if an individual does not have a waning immune system and is treated with drugs, the bacteria can be removed, and in fact, the disease has a fairly good chance of around 90% to be cured. (4)

Up to half of TB survivors have some form of persistent pulmonary dysfunction despite microbiologic elimination of the bacteria (5). Furthermore, treated TB patients appear to contribute substantially to the growing worldwide burden of chronic obstructive pulmonary disease (COPD) (6). Cumulative respiratory impairment following tuberculosis infection is referred to as Pulmonary Impairment After Tuberculosis (PIAT), or Post-Tuberculosis Lung Disease (PTLD). PIAT/PTLD is an overlapping spectrum of disorders that affects large and small airways (bronchiectasis and obstructive lung disease), lung parenchyma, pulmonary vasculature, and pleura and may be complicated by coinfection and hemoptysis (7). Pulmonary fibrosis, bronchiectasis and nodular infiltrates left over from the Mtb infection are thought to contribute to PIAT/PTLD symptoms (8). One study found that PIAT/PTLD occurs in 59% of those with past Active TB cases, and 20% of those with Latent TB infections (8). For many persons with tuberculosis, a microbiological cure is the beginning, not the end, of their illness. However, lung damage in TB patients following treatment varies dramatically. Some patients show no pulmonary impairment at all (5). The reasons for this variability have not been fully elucidated, however, varying host–pathogen interactions and the diverse immunological events that can follow Mtb infection are thought to play a role (5). In more severe cases Active TB infections can cause long-term dysfunction beyond the pulmonary system. For instance, bacterial infiltration into the spine can cause Pott’s Disease, wherein granuloma development causes long-term kyphotic deformity of the spine, compression fractures and neurological insults (9). However, in Robert’s case, it appears that his infection is limited to pulmonary and fever-like symptoms, indicating that any long-term side-effects of his recovery will likely be associated with the lungs.

Immunity

The majority of individuals infected with M. tuberculosis develop an asymptomatic chronic tuberculosis (TB) infection known as latent TB. (1) In the United States and Canada, most cases of recurrent tuberculosis are due to latent infections; however, infections due to new Mtb strains are more common in other parts of the world (WHO, 2018). During latent infection, activated host immune cells result in arrest of mycobacterial growth and control of disease progression. (1) Most importantly, a hallmark of the adaptive immune system is its ability to “remember”. When memory T and B cells recognize antigen, they generate effector responses that are quantitatively and qualitatively superior to naive T or B cells. (1) Memory immune responses also occur more rapidly than naive responses, resulting in efficient reduction or prevention of disease upon a subsequent exposure to a pathogen. (1)

Following infection or immunization, an adaptive effector immune response develops and then resolves, leaving behind a pool of long-lived antigen-specific lymphocytes that can mediate heightened resistance to infection. (1) These long-lived cells, known as memory lymphocytes, form a heterogeneous population that is superior to naive cells. (1) Subsets of memory cells are strategically located in anatomical sites that are most likely to be exposed to infection, which, in the case of TB, is the lung. (1) Memory cells are able to respond quickly and produce appropriate effector molecules upon reinfection. (1)

However, insufficient antigen exposure, such as from suboptimal vaccination, may generate a very small clonal burst and thereby fail to generate sufficient numbers of memory cells. (1) Therefore, when attempting to generate effective immunological memory against TB through vaccination, it is important to consider the mechanisms that influence clonal burst size and cell death during the contraction phase. (1)

DNA fingerprinting has demonstrated that reinfection contributes substantially to TB infections, more so than previously thought (10). Tb recurrence could be due to reinfection or relapse (14).  Most recurrent episodes occur within the first three years after initial infection (14). From a clinical perspective, however, reinfection isn’t particularly significant, given that reinfection and relapse have virtually identical clinical qualities, and that the majority of recurrences arise as a result of relapse, not reinfection. Relapse refers to an existing TB reinfection reestablishing itself, and reinfection refers to infection by a separate strain of TB encountered in the environment. One study found that 12% of Mtb infection recurrences were due to reinfection, with the remaining 88% due to relapse (11), though other estimates have the reinfection proportion as high as 50%. Country of residence and being immunocompromised (i.e., HIV-positive), are thought to be risk factors which make an individual more prone to developing Mtb reinfection (10). Tuberculosis reinfection by way of relapse may be caused by an incomplete or unsuccessful antibiotic regimen. In these cases, the prognosis is significantly more negative, as relapse may indicate that there is a population of antibiotic-resistant bacteria. The immune response to reinfection is quite similar to the initial infection (12). While the immune response to Mtb is largely T-cell dependent, and memory T cells do exist in cases of reinfection, macrophage involvement and ensuing granuloma generation is still the prevailing feature of reinfection (10). Latent Mtb infections are thought to persist throughout one’s life, however, recent studies have shown that most people with TB immunoreactivity do not develop active TB upon immunosuppression, suggesting that they have cleared their infection while retaining immunological memory to it (13). Individuals that are immunocompromised or immunosuppressed at time of infection are not as able to develop a memory response, increasing susceptibility to reinfection.

References:

1. Kirman, J. R., Henao-Tamayo, M. I., & Agger, E. M. (2016). The Memory Immune Response to Tuberculosis. Microbiology spectrum, 4(6), 10.1128/microbiolspec.TBTB2-0009-2016. https://doi.org/10.1128/microbiolspec.TBTB2-0009-2016
2. World Health Organization. (n.d.). Tuberculosis (TB). World Health Organization. Retrieved March 31, 2022, from https://www.who.int/news-room/fact-sheets/detail/tuberculosis
3. Verrall, A. J., Netea, M. G., Alisjahbana, B., Hill, P. C., & van Crevel, R. (2014). Early clearance of Mycobacterium tuberculosis: a new frontier in prevention. Immunology, 141(4), 506–513. https://doi.org/10.1111/imm.12223
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