Mycobacteria: Mycobacterium tuberculosis

Question 1

What are Mycobacteria?

Describe its properties?

Answer 1

• Unicellular rod-shaped micro-organisms
• obligate aerobes
• non-motile
• non-capsulated
• non-sporulating
• 2-4 μm length X 0.2-0.5 μm width
• Many species of Mycobacterium exist; only a few cause disease in humans
• Many important species are SLOW GROWING

(generation time: 15-20 hrs)

Cell wall comprises HIGH LIPID CONTENT (unlike Gram positive / negative bacteria)

Question 2

Describe a Gram positive cell wall

Answer 2

Cytoplasmic membrane followed by a thick layer of peptidoglycan leafleted with teichoic acid and lipoteichoic acid

Question 3

Describe a Gram negative cell wall

Answer 3

Outer membrane (with LPSand lipid A) and cytoplasmic membrane sandwiching a thin layer of peptidoglycan

Question 4

Mycobacteria: Describe its unique Cell Wall Structure

Answer 4

• Features of cell wall:
• (60% lipid)
• Inner membrane followed by a large periplasmic space leafleted with LAM and towards the end the LAM is anchored to a pepdioglycan layer
• cental component of the cell wall is thearabinogalactan-peptidoglycan layer
• High glycolipid content eg. MYCOLIC ACID (unique to mycobacteria) in the outer mebrane
• Other lipids include: surface acyl lipids; lipoarabinomannan (LAM); phosphatidy inositol mannosides (PIM); cord factor-Trehalose Dimycolate (TDM)(sugar with 2 mycolic acids)
• TDM aids intracellular survival of mycobacteria→stops lysosome from fusing with phagosome

Question 5

Why is the cell wall of myobacteria important medically?

Answer 5

• It promotes Intracellular survival (prevents phagosome fusing with lysosome)
• Confers resistance (eg. many antimicrobials, heat, chemicals, drying, STAINS)
• Mycobacteria are ACID-FAST
• Special stains required to visualise Mycobacteria

Question 6

What stain is used for acid-fast bacteria?

Answer 6

Ziehl-Neelson Stain

Question 7

Describe the Ziehl-Neelson Stain for Acid-Fast bacteria

Answer 7

1. Stain with hot concentrated carbol fuchsin and (all bacterial cells stained pink at this stage)
2. De-stain with 1% acid-alcohol (hydrochloric acid / ethanol) OR 20% H₂SO₄ (mycobacterial cells remain pink, all others colourless)
3. Counterstain with methylene blue (mycobacterial cells remain pink, all others bacteria and cells eg WBC stain blue)

Question 8

Infections Caused by Mycobacteria: Tuberculosis (TB)

What species of myobacterium is this disease caused by?

Answer 8

M. tuberculosis

Question 9

What are the symptoms of TB?

Answer 9

• Often asymptomatic
• pneumonia-like presentation (weight loss, temperature, cough, blood in sputum)

Question 10

Infections Caused by Mycobacteria: Tuberculosis (TB); acquired from consumption of unpasteurised milk

What species of myobacterium is this disease caused by?

Answer 10

This is caused by M. bovis

Question 11

Infections Caused by Mycobacteria:

Disseminated TB in immunocompromised patients eg. HIV

What species of myobacterium is this disease caused by?

Answer 11

M. avium intracellulare

Non-tuberculosis mircrobacterium (NTM)

Question 12

What the symptoms of Disseminated TB in immunocompromised patients eg. HIV?

Answer 12

Lung infection and spread to bones, joints, blood, renal system, meninges; frequently results in death

Question 13

Which disease is cauesd by M. leprae (Hansen’s bacillus)?

Answer 13

Leprosy

Question 14

What are the symptoms of leprosy?

Answer 14

Folded lesions on face and limbs

Disfiguration

Loss of peripheral nerves

Secondary infection

Question 15

Tuberculosis: Describe the beginnings

Answer 15

• 1720: Benjamin Marten described ‘Consumption’; wonderfully minute living creatures; A New Theory of Consumption published
• 1882: discovered the cause of ‘consumption’; caused by an infectious agent; Koch’s bacillus (M. tuberculosis)
• Granville’s Mummy (600BC): First ancient Egyptian mummy to be subjected to a scientific autopsy (1825); incorrect diagnosis-ovarian tumour, found mycolic acid to disprove this.
• 21st century (UCL); TB DNA identified (lung, femur, gall bladder) in Granville’s Mummy – ‘a wrong diagnosis’ by Granville

Question 16

State 21^st Century Tuberculosis figures

Answer 16

Worldwide Figures

• 1/3 of world population predicted to have TB
• 8.4 million new cases per year
• 1.5 million deaths per year

Question 17

The Fall and Rise of TB on the Rise in UK:

State UK historical trends and annual figures

Answer 17

1950: 50,000

mid-1980s: 6,000

1987-1990: 7,000

2010: 9,000

now: 6000

Question 18

State some predisposing factors of TB infection

Answer 18

• HIV infection is the MAIN predisposing factor for TB infection. 10 percent of all HIV-positive individuals have TB (400-times the rate associated with the general public)
• Close contact with large populations of people, i.e., schools, nursing homes, dormitories, prisons, etc
• Poor nutrition
• iv drug use
• Alcoholism

Question 19

TB Infection: Descibe the properties of droplet Nuclei

What releases droplet nuclei?

Answer 19

• TB spreads via Droplet nuclei (5mm, approx 3 bacilli- minimum infective dose)
• Aerosolised:

Talking for 5 min: 3,000

Coughing: 3,000

Sneezing: 3,000 up to 10 feet away

(a) remain suspended for hours; environment is infectious after infected individual has left
(b) small size allows for bypass of mucociliary lining
* Coughing, talking, sneezing releases DN

Question 20

Explain the stages of TB Infection

Answer 20

Stage 1: Droplet nuclei inhaled

Stage 2: (7-21 days) Microbacterium tuberculosis-MTB multiplies within (alveolar) macrophages (INTRACELLULAR); macrophages secrete IL-12 and present MTB antigen on their surface; eventually burst liberating MTB

Stage 3: IL-12 stimulates T-lymphocytes to infiltrate; recognise MTB antigen; become activated (sensitized) and start to release inflammatory factors (eg. gamma IFN); TUBERCLE formation (PRIMARY LESION or granuloma) provides protection of the host from MTB and MTB from the host. MTB can secrete immunomodulatory and antiinflammatory factors

Stage 4: MTB continues to multiply within unactivated / poorly activated macrophages and tubercle expands

Stage 5: Primary lesion heals: GHON FOCUS (dormant lesion; contains MTB; may re-activate) formed in the lung, isolation site if MTB

NB. IT IS THE CELL MEDIATED RESPONSE (CMI) IN ‘HEALTHY’ INDIVDUALS THAT ‘HEALS’ THE PRIMARY LESION

Question 21

Describe the TB Granuloma

Answer 21

Once the immune system has successfully encapsulated the infection

• Necrotic infected macrophages in the center that has burst and release the bacteria
• Te bacteria use the nutrients from the necrotic macrophages for their own metabolism but are under hypoxic conditions
• Surrounding this are macrophages that have become epithelioid (outside of bacteria, interior of granuloma)
• T and B cell that form the wider protective barrier

Question 22

Draw a diagram to show the stages of TB Infection

Answer 22

1. Exposure to source
2. Aerosolisation of TB
3. Inhalation of bacteria
4. Bacteria reach lungs + enter macrophages (25-50%)
5. Bacteria multiply in macrophages
6. Granulomatous lesions begin to form (caseous necrosis)
7. Latent or active infection

Question 23

Compare latent to active TB infection

Answer 23

After granulomatous lesions begin to form, bacteria can cease to grow and lesion calcifies (90%)→ LATNET TB INFECTION

Alternatively, the lesion liquefies and bacteria can spread to blood/organs→ACTIVE TB INFECTION Bacteria may be coughed up in sputum. This can lead to death

Question 24

TRUE or ALSE: 90% of individuals with TB are asymptomatic

Answer 24

TRUE

Healthy cell mediated immune response (CMI) keeps infection under control

Question 25

10% of individuals with TB develop primary tuberculosis and experience what symptoms?

Answer 25

**Lower respiratory tract infection:** * cough (sputum with blood) * weight loss * night sweats * fatigue * fever **May spread to other body parts:** * meningitis * blood poisoning * kidney infection * joint infection Patients are hyper-sensitised ie. cell mediated immunity (T-lymphocytes) ready to attack re-exposure to M. tuberculosis

Question 26

What are the symptoms of secondary tuberculosis?

Answer 26

* (a) associated with any impairment of cell mediated immunity-CMI (steroid therapy, immunosuppressive drugs, cancer chemotherapy, old age, HIV) * (b) dormant PRIMARY focus becomes necrotic (cheesy) and liquefies distributing 1000s M. tuberculosis into the lungs * (c) widespread distribution of M. tuberculosis in the lungs (and possibly throughout the body)

Question 27

What is the appropriate clinical sample in laboratory Diagnosis of Pulmonary (respiratory) TB?

Answer 27

Sputum (or bowel) sample ## Footnote Sputum is processed in a class 3 laboratory under a class 1 safety cabinet

Question 28

TRUE or FALSE: M. tuberculosis is a category 2 microorganism

Answer 28

FALSE M. tuberculosis is a category **3** microorganism

Question 29

What is a category 3 microorganism?

Answer 29

A biological agent that can cause severe human disease; serious hazard to employees; risk of spread in community; effective treatment / prophylaxis

Question 30

Describe the microscopy of Sputum

Answer 30

* Microscopy of smears of sputum stained by ZN (light microscopy) * View using x100 objective (=1000x magnification) * Examine for acid-fast (ZN positive bacteria) * Need approx 10,000/mL (high bacterial load) of sputum to visualise one organism

Question 31

Describe how to culture sputum

Answer 31

* Culture sputum on specialised agar: **Lowenstein-Jensen** (LJ slopes) –different from other selective microbiological agars * Contains: egg, glycerol, minerals, potato flour, antibiotics and malachite green * Incubate at 37^oC for up to 12 weeks * Examine for LJ slopes for typical colonies of M. tuberculosis (wrinkly/crusty orange colonies)

Question 32

Describe Mycobacterium tuberculosis colonies

Answer 32

* Generally grows in 4-6 weeks * Yellow / buff coloured colonies;irregular in shape * Colonies are ZN positive * Form distinctive _serpentine cords_ when stained (caused by TDM). This observation was first made by Robert Koch (Cord factor: trehalose 6, 6' dimycolate - a glycolipid found in the cell walls of mycobacteria - causes the cells to grow in ‘serpentine’ cords * **Cord factor**: Virulence factor; toxic to mammalian cells and inhibits white blood cell migration

Question 33

Explain TB treatment How does it differ for drug sensitive and drug resistant TB?

Answer 33

TB is difficult to eradicate; six month treatment regime (for drug sesnsitve TB); toxic anti-mycobacterial drugs Intravenous antibiotics for drug resistant TB (for up to 18 months) Combination therapy: - Isoniazid - Rifampicin (stains sweat and urine red) - Pyrazinamide - Ethambutol All taken for the first 2 months (initiation phase) For the remaining 4 months (continuation phase) only Isoniazid and Rifampicin are taken

Question 34

What are the main side effects of drugs used to treat TB?

Answer 34

Side effects include: hepatitis (inflammation of the liver); gastrointestinal disturbances

Question 35

Describe the methods of TB prevention

Answer 35

BCG Vaccination (1953) (Bacille Calmette- Guerin): * M. bovis BCG is a live ‘attenuated’ (reduced) form of M. bovis obtained by serial passage (230 passages over glycerinated bile potato medium) in vitro untill it lost the ability to infect a host * 50-80% effective; lasts 10-15 years (RD1 region was mutated sufficiently so no longer active in the genome) * 1953-2005: BCG universal vaccination programme delivered through schools 2005-present: Targeted vaccination scheme introduced aimed at high risk individualseg. babies born in areas of UK where TB prevalence is high eg. London and ‘at risk’ employees eg. healthcare workers

Question 36

What does Tuberculin Skin Testing (TST) / (Mantoux Test) demonstrate?

Answer 36

* Effective vaccination (BCG) * Previous exposure to TB / infection with TB

Question 37

Explain the principle of Tuberculin Skin Testing (TST) / (Mantoux Test)

Answer 37

TST detects delayed type 4 hypersensitivity reaction. If a previous TB infection has occurred sensitized T- lymphocytes circulate that respond to re-exposure to TB antigen to produce an injuration (indicative of a local inflammatory response)

Question 38

Explain the procedure of Tuberculin Skin Testing (TST) / (Mantoux Test)

Answer 38

* Inject 0.1 ml of tuberculin **purified protein derivative** (PPD) into the inner surface of the forearm (intradermal injection). * The injection should produce a pale elevation of the skin (wheal) 6 to 10 mm diameter

Question 39

Explain the interpretation of Tuberculin Skin Testing (TST) / (Mantoux Test)

Answer 39

Examined between 48 and 72 hours; measure induration (mm); positive if the induration is \>10mm. NB. BCG vaccination generates a positive response

Question 40

Generally, persons at high risk for developing TB disease fall into which two categories?

Answer 40

* Persons who have been recently infected with TB bacteria * Persons with medical conditions that weaken the immune system

Question 41

TB Risk Factors-Persons who have been Recently Infected with TB Bacteria This includes:

Answer 41

* Close contacts of a person with infectious TB disease * Persons who have immigrated from areas of the world with high rates of TB * Children less than 5 years of age who have a positive TB test * Groups with high rates of TB transmission, such as homeless persons, injection drug users, and persons with HIV infection * Persons who work or reside with people who are at high risk for TB in facilities or institutions such as hospitals, homeless shelters, correctional facilities, nursing homes, and residential homes for those with HIV

Question 42

TB Risk Factors Babies and young children often have weak immune systems. Other people can have weak immune systems, too, especially people with any of these conditions:

Answer 42

* HIV infection (the virus that causes AIDS) * Substance abuse * Silicosis * Diabetes mellitus * Severe kidney disease * Low body weight * Organ transplants * Head and neck cancer * Medical treatments such as corticosteroids or organ transplant * Specialized treatment for rheumatoid arthritis or Crohn’s disease

Question 43

TB comorbidities: TB and HIV

Answer 43

* People living with HIV are from _15-22_ times more likely to develop TB than persons without HIV. * TB is the most common presenting illness among people living with HIV, including among those taking antiretroviral treatment and it is the major cause of HIV-related death. * In 2018, there were an estimated 251 000 deaths from HIV-associated TB. * Sub-Saharan Africa bears the brunt of the dual epidemic, accounting for approximately 84% of all TB/HIV deaths in 2018.

Question 44

TB comorbidities: TB and diabetes

Answer 44

* The risk of TB among people with diabetes is _2-3_ times higher than among those without diabetes. * Diabetes can worsen the clinical course of TB, and TB can worsen glycaemic control in people with diabetes. * Individuals with both conditions thus require careful clinical management. * Strategies are needed to ensure that optimal care is provided to patients with both diseases. * Diabetes prevalence is increasing globally. * The most dramatic increase is in low- and middle income countries undergoing rapid economic, social, and lifestyle changes. * Further increase in the number of diabetes-associated TB cases risks jeopardizing progress that has been made in the global fight against TB. * Therefore, prevention and care of diabetes should be a priority not only for stakeholders involved in care and control of non-communicable diseases, but also for those working on TB care and prevention. * This should be part of broader actions on risk factors and social determinants.

Question 45

TB comorbidities: TB and nutrition

Answer 45

* Malnutrition increases the risk of TB and TB can lead to malnutrition. * Malnutrition is therefore often highly prevalent among people with TB. * While appropriate TB treatment often helps normalize nutritional status, many TB patients are still malnourished at the end of TB treatment. * Therefore, nutritional assessment and counselling, and management of malnutrition based on the nutritional status are an important part of the TB treatment package. * Undernutrition, and underlying food insecurity, are among the most important determinants of TB. * Improving nutritional status at population level is important for TB prevention. * This should be part of broader actions on social determinants.

Question 46

TB comorbidities: TB and tobacco smoking

Answer 46

* Tobacco smoking increases the risk of TB _2-3 fold_, and is associated with poor TB treatment results. * Smoking prevalence is often high among people with TB, and prevalence of other smoking-related conditions can be high as well. * People diagnosed with TB should be asked about smoking, and should be offered advice about smoking cessation. * This is part of the practical approach to lung health. * High smoking prevalence in the population is an important contributor to high TB burden. Public health and regulatory efforts to reduce smoking prevalence can have significant impact on TB incidence. * It is key that TB prevention efforts are linked up with efforts to reduce other smoking related conditions in the population.

Question 47

TB comorbidities: TB and harmful use of alcohol

Answer 47

* Harmful use of alcohol increases the risk of TB threefold, and is also a strong risk factor for poor TB treatment adherence. * In countries with high prevalence of alcohol use disorders, and especially in intermediate- and low-incidence countries where TB has become highly concentrated to certain vulnerable groups, harmful alcohol use can be an important population level risk factor for TB, and is often a common co-morbidity among TB patients. * As part of a comprehensive care package it is important, especially in those countries, to identify problem drinkers, diagnose alcohol use disorder, and refer for appropriate alcohol interventions. * A few countries have experimented with systematic screening for harmful alcohol use of all TB patients. * Screening and diagnosis of other mental health problems may also be warranted.