Mycobacteria (9-11)

Question 1

What makes slow-growing mycobacteria pathogenic?

Answer 1

Low infectious dose (1 cell)
Intracellular pathogens - in macrophage
Resistant cell wall
Subvert the human immune system → mycobacterial cell surface components stop phagosome killing
The immune response is integral to disease symptoms
Form characteristic granulomatous lesions → immune cell cluster limiting mycobacterial invasion which mycobacteria can survive within
Slow-growing

Question 2

What are slow-growing mycobacteria?

Answer 2

Their doubling time is no less than 16 hours
→ most are harmless environmental bacteria
→ a few have evolved as major human pathogens
e.g. M. tuberculosis → causes tuberculosis - the worlds deadliest infectious disease
M. laprae → causes leprosy - affects skin, nerves, eyes and respiratory tract, documented in ancient literature
M. ulcerans → causes Buruli ulcer - affects skin and sometimes bone, can lead to permanent disfigurement and long-term disability
→ all 3 are chronic due to the slow-growing nature of the mycobacteria
→ unique features: cell wall structure, slow doubling time, immune subversion

Question 3

What is a granuloma?

Answer 3

Cluster of immune cells - host immune cells high infection by clustering to form a granuloma
→ mycobacteria are inside the macrophages in the centre of the granuloma - contained but surviving, suppressing phagosome killing

Question 4

How is the mycobacterial cell wall

Answer 4

Neither Gram +/-ve

Thick, waxy hydrophobic cell envelope
→ thin peptidoglycan
→ arabinogalactan - provides structural support, attachment site for mycolic acids
→ mycolic acid - long-chain fatty acids, form an outer lipid layer giving the cell its impermeability and resistance to many chemical agents
→ acyl lipids
→ lipoarabinomannan (LAM) - glycolipids important for immune evasion
→ proteins (e.g. porins) - cell signalling

Question 5

What are the challenges in treating slow-growing mycobacteria?

Answer 5

Resilient to most antibiotics
→ drugs are available that treat mycobacterial infections
Resistance develops through mutation only
→ long term treatment with antibiotic mixtures necessary to overcome development of resistance
The vaccine BCG offers partial protection particularly to children
→ vaccines work poorly due to immune evasion strategies of mycobacteria

Question 6

What is leprosy (Hansen’s disease)?

Answer 6

Chronic infectious disease of the skin and nerves
→ main causative agent is Mycobacterium leprae
Symptoms: loss of sensation in hands and feed, leading to disability through injury, blindness
→ curable with multi drug treatment regimes

In 2008 another causative agent M. lepramatosis was discovered - not focused

One of the oldest and most stigmatised of diseases → stigma which can result in rejection and exclusion linked to widespread miss understanding about the disease
→ its not highly contagious - typically caught via close contact over an extended period, spread mainly through droplets from the nose and mouth by coughing and sneezing

Question 7

How is leprosy distributed?

Answer 7

Remains endemic in central Africa, Southeast Asia and South America
→ more than 200,000 new cases per year globally
→ 15,000 children diagnosed
→ 2-3 million people with leprosy
→ countries with the highest number of new diagnoses were India, Brazil and Indonesia
→ over half of all new cases diagnosed in India - home to 1/3 of the world’s poor, disproportionately affected by the disease

Question 8

How is leprosy transmitted?

Answer 8

Transmission routes are not fully clear, not everyone exposed develops disease - genetic susceptibility? immune response? play a role in disease progression

Known transmission → human-human, human-animals, animals-human (zoonotic transmission not frequent), human-nonhuman primates

Possible transmission → with vectors, environment-humans/animals (environment is a possible pool)

In USA nine-banded armadillos have been shown to transmit to humans → possible zoonotic reservoir
In UK red squirrels detected → no known squirrel-human transmission case, possible zoonotic reservoir

Question 9

What is the disease presentation of leprosy?

Answer 9

Disease affecting skin and nerves

M. leprae is an intracellular parasite of: keratinocytes, nerve cells, dendritic cells, macrophages

Symptoms: skin lesions, loss of sensation in hands and feet - leading to disability through injury, blindness (nerve damage), nasal septum damage

Question 10

Why is there limited understanding of the pathologenesis of leprosy?

Answer 10

Inability to culture the bacterium in vitro because its intracellular → limits understanding of its pathogenesis and interaction with the human host

Its a chronic granulomatous disease effecting skin and peripheral nerves

Question 11

How does leprosy disease progress?

Answer 11

95% of people show no symptoms and clear the disease → heritable - genetic links
5% who show symptoms there are a range of symptoms and severities
→ two main types: tuberculoid leprosy (paucibacillary), lepromatous leprosy (multibacillary)

Question 12

What is the spectrum from lepromatous to tuberculoid leprosy?

Answer 12

Lepromatous leprosy → characterised by a Th2 T-cell immune response
→ antibody complex formation, absence of granulomas, failure to restrain M. leprae growth, robust antibody formation but its not protective, cell-mediated immunity is conspicuously absent (e.g. activation of macrophages)

Tuberculoid leprosy → characterised by Th1 T-cell cytokine response
→ vigorous T-cell responses to M. leprae antigen, containment of the infection in well-formed granulomas

Borderline forms represent transitional stages between these two extremes
→ tuberculoid (TT), borderline tuberculoid (BT), borderline borderline (BB), borderline lepromatous (BL), lepromatous (LL)

Question 13

How does M. leprae interact with nerve cells?

Answer 13

Is an obligate intracellular pathogen with a distinct preference for Schwann cells of the peripheral nervous system and for macrophages
Schwann cells → produce myelin sheath around neuronal axons

Mechanism for nerve fame is unclear - possible mechanisms - inflammatory mediators and T-cell mediated cytolysis leads to:
→ ischemia - restricted blood flow
→ apoptosis - programmed cell death
→ demyelination - loss of myelin sheath

Question 14

How did M. leprae co-evolve with humans?

Answer 14

An obligate parasite whose primary host is humans → likely that leprosy bacilli started parasitic evolution in humans or early hominoids millions of years ago

Reductive evolution → loss of functional genes
→ increasingly parasitic lifestyle, only able to grow within host cells
→ small genome (3.27 Mb) smaller than that of related Mycobacterium tuberculosis
→ contained around 1,600 pseudogenes (considered molecular fossils) with loss of ~50% of the ancestral genes

Susceptibility to leprosy varies, with protection heritable
→ multiple single nucleotide polymorphisms (SNPs) involving many genes have been found to be associated with increased or decreased protection, region specific prevalences
→ e.g. Tol-like receptor 1 (TLR1) mutation from isoleucine to serine at AA 602, homozygous individuals protected against leprosy - TLR1 (involved in immune response/signalling in macrophage) part of pathogenesis mechanism

Question 15

How is leprosy diagnosed?

Answer 15

At least one of:
1. Definite loss of sensation in a pale (hpopigmented) or reddish skin patch
2. Thickened or enlarged peripheral nerve, with loss of sensation and/or weakness of the muscles supplied by that nerve
3. Microscopic detection of bacilli in a slit-skin smear

Classification:
Paucibacillary → 1 to 5 skin lesions, without demonstrated presence of bacilli in a skin smear
Multibacillary → more than 5 skin lesions, nerve involvement (pure neuritis or any number of skin lesions and neuritis), demonstrated presence of bacilli in a slit-skin smear, irrespective of the number of skin lesions

Question 16

How is leprosy treated?

Answer 16

In 1981 WHO recommended multi drug therapy of: dapsone, clofazimine, rifampin
→ 6 months for paucibacillary, 12 months for multibacillary
→ early diagnosis with prompt treatment can help prevent disabilities
→ WHO provide the multi drug therapy free of cost - donated through an agreement with Novartis

Problems of resistance emerging to all 3 drugs
→ dapsone has been used since 1945 - most resistance
→ some cases relapse several years after therapy completed
→ second line drugs are available if resistance to MDT arises

Question 17

What is dapsone?

Answer 17

A sulfone antibiotic → inhibitor in the folate synthesising enzyme system, bacteriostatic
→ folic acid is necessary for bacteria and humans alike, essential for bacterial growth and replication - disrupts production of nucleic acids and proteins leading to bacterial death

Resistance can arise through point mutations on gene (folP1) for target enzyme

Question 18

What is rifampicin?

Answer 18

Ansamycin class of antibiotics → semisynthetic as its modified from the natural product rifamycin
RNA polymerase inhibitor → blocks bacterial translation
Side effects common → turns you organs, only take one a month

Resistance occurs through mutations at binding site of rifampicin on RNA pol

Question 19

What is clofazimine?

Answer 19

Synthesised for 70 years, exerts antibacterial effects
→ molecular mode of action against mycobacteria unclear
→ anti-inflammatory

Resistance can occur through over expression of transporters

Question 20

How is leprosy prevented/controlled?

Answer 20

Community awareness is key
Case detection and treatment with MDT
→ WHO recommends tracing household contacts along with neighbourhoods and social contacts of each patient
→ administration of a single dose of rifampicin as preventative chemotherapy to close contacts
BCG vaccine given primarily for TB, may offer some protection against leprosy

Question 21

What is the stigma associated with leprosy?

Answer 21

Many people living with leprosy are unable to work → disability caused by the illness, or may face stigma that prevents them from working
In some countries, the law allows a person to legally divorce a spouse because they are affected by the disease
Stigma affects the physical, psychological, social and economic well-being of those with leprosy → contributing to the cycle of poverty in affected regions

Question 22

What are the future directions for leprosy?

Answer 22

Eradication → as a curable disease there in an aim to eradicate it within the human population through interruption of transmission
The Global Leprosy Strategy 2021-2030 ‘Towards zero leprosy’:
1. Implement integrated, country-owned zero leprosy road maps in all endemic countries
2. Scale up leprosy prevention alongside integrated active case detection
3. Manage leprosy and its complications and prevent new disability
4. Combat stigma and ensure human rights are respected

Question 23

What is tuberculosis?

Answer 23

An infectious air borne disease → main causal agent Mycobacterium tuberculosis (Mtb)
→ immune response can contain Mtb as latent form of disease
→ in active form its contagious and causes disease symptoms - primarily a pulmonary disease, can cause disease in most parts of the body
In many low and middle income countries TB is major cause of morbidity and mortality
Curable with prolonged multi drug treatment regimes → increasing problems with drug resistance making some effectively incurable
BCG vaccine offers some protection - prevents severe forms
Other mycobacteria can also cause TB from the Mycobacterium tuberculosis complex (MTBC) inc. M. bovis

Question 24

How is tuberculosis distributed?

Answer 24

Particularly prevalent in subsaharan Africa and Asia

Co-infection with HIV also prevalent

Question 25

How is tuberculosis transmitted?

Answer 25

Primarily an airborne disease
→ not highly infectious
→ long period of infectiousness (without treatment) average >1yr

Question 26

What is the immune response to tuberculosis infection?

Answer 26

The innate and adaptive responses allow most healthy people (>90%) to control the growth
→ although they harbour latent infection (it is not known whether host immune responses can eliminate infection)
→ some individuals, particularly with impaired T cell function develop active TB, either as primary progression or as a reactivation
Mtb is phagocytosed by APCs (macrophages, monocytes and DCs), and survives in phagosomes
→ ligans inc. lipoproteins and glycolipids are recognised in APCs by TLRs and nucleotide-binding oligomerisation domain (NOD) protein - resulting in the secretion of inflammatory cytokines and chemokines
→ infected APCs migrate to regional lymphoid tissues, where adaptive immunity develops through antigen presentation to naive T cells
APCs process Mtb antigens by intravacuolar proteolysis to produce peptides that binds to MHC class II molecules - mediate presentation to CD4+ T cells
→ also presented by MHC class I molecules to CD8+ T cells
→ effector memory T cells migrate back to the sites of infection to control growth
→ granulomas develop through secretion of tumour necrosis factor and other effector cytokines

Question 27

What is the disease progression for tuberculosis?

Answer 27

Can be eliminated
Latent → strong cell mediated response
Active → weak cell mediated response, granulomas burst in lungs

Question 28

What is the life cycle for M. tuberculosis?

Answer 28

Mtb bacilli are inhaled and phagocytosed by resident alveolar macrophages
→ pro inflammatory response triggers the infected cells to invade by subtending epithelium
→ recruitment of monocytes from the circulation, extensive neovascularisation of the infection site
→ the macrophages form the granulomas along with epithelia cells, multinucleate giant cells and foamy cells filled with lipid droplets, a fibrous cuff of extracellular matrix material may form
→ progression towards disease is characterised by the loss of vascularisation, increased necrosis and the accumulation of caesium in the granuloma centre

Question 29

What are the tuberculosis disease symptoms?

Answer 29

Pulmonary presentation → a bad cough that lasts 3 weeks or longer, pain in the chest, coughing up blood or sputum (phlegm from deep inside the lungs) - lungs become less efficient
Other symptoms: weakness or fatigue, weight loss, no appetite, chills, fever, sweating at night - depend on the part affected

Question 30

How is tuberculosis diagnosed?

Answer 30

Physiology → night sweats, temperature, persistent cough, weight loss, fatigue
Smear test → microscopic visualisation of acid-fast bacilli in the sputum
X-ray → of the chest to detect patches on the lungs
Cultivation of septum → using highly growth sensitive systems such as the MGIT
Tuberculin skin test (Mantoux test) → for hypersensitivity to antigens of Mtb
IFN-gamma release assay
T-SPOT.TB assay → for activated TB-specific effector T cells (TB ELISpot)
Detection of Mtb specific antigens in the blood
Detection of specific genes → using qPCR GeneXpert targets Mtb using rpoB, also will detect rifampicin resistance

Question 31

What is the time scale for diagnosis of tuberculosis using smear microscopy?

Answer 31

The threshold for disability of AFB by smear microscopy isn’t until 5th month - by then too weak to work
→ commonly used in low and middle income countries - relatively fast, inexpensive and specific for tuberculosis
→ involves smears of unconcentrated sputum being used with Ziehl-Neelsen staining
→ notoriously inaccurate - low and variable sensitivity of 20-60% - implications to lag in diagnosis

Question 32

What is the recommended treatment regime for tuberculosis?

Answer 32

Multidrug treatment:
Standard regimen → rifampicin, isoniazid, pyrazinamide, ethambutol
Intermittent regimen → rifampicin, isoniazid, pyrazinamide

After 2 months there is a reduction, but still considerable disease

MDR-TB is resistant to at least isoniazid and rifampicin → the two most important first-line drugs used for treatment of TB
→ this may result from either primary infection with drug-resistant bacteria, or develop in the course of a patient’s treatment with non-optimal treatment duration or regimens are used
Cure rates for MDR-TB are lower → 50-70%

XDR-TB is resistant to isoniazid and rifampicin, as well as any fluoroquinolone and any of the second-line anti-TB injectable drugs
→ very high mortality rates

Question 33

What is latent tuberculosis?

Answer 33

Asymptomatic and non infectious → arises upon immune restriction of the growth of Mtb in hosts
→ hypoxia has been shown to induce non-replicative bacterial phenotypes, leading to tolerance towards certain drugs like cell-wall inhibitors such as isoniazid
→ lack of potent drug activity on these bacterial phenotypes may be responsible for prolonging the TB treatment duration

Question 34

What are the strategies for fighting tuberculosis drug resistance?

Answer 34

1. Change exist drugs to prevent resistance → alter the MPC (mutants prevention concentration) e.g. C8-methoxy fluroquinolones
2. Discover new drugs → design more drugs to attack targets such as mycolic acid synthesis mechanisms e.g. bedaquiline and pretomanid
3. Multiple drug therapy - altnerate drugs

Question 35

How is tuberculosis prevented/controlled?

Answer 35

Diagnosis and treatment of active TB cases → identify and test close contacts
Identifying high risk latent TB cases and giving preventative treatment
Manage environment → good ventilation, natural light (UV kills bacteria), good hygiene
Improve conditions → healthy immune system
BCG vaccination campaigns → varying efficacy

Question 36

What are the future directions for tuberculosis?

Answer 36

Overcoming drug resistance → new drugs, alter existing drugs, phage therapy (XTR -B - can treat with phage therapy as a last resort)

TNC207 is in phase IIb trails for MDR-TB and in phase IIa trials for DS-TB
The structure of AZD-5847 has not been disclosed

Question 37

What are environmental or non-tuberculous mycobacteria (NTM)?

Answer 37

Ubiquitous in the environment → saprophytic organisms
Typically not pathogenic → cause disease infrequently - opportunistic pathogens
In immunocompetent and immunocompromised people
No person-person transmission → pick up from the environment
Pulmonary disease is most common especially with those who have pre-existing lung disease - also localised infections in: lymph nodes, skin, soft tissue, rarely bones
Often misdiagnosed as tuberculosis

Question 38

What are the risk factors of NTM disease?

Answer 38

Combined host, environmental and organism

Host → disseminated ATM infection: primary immunodeficiency - mendelian susceptibility to mycobacterial disease (MSMD), severe combined immunodeficiency (SCID), acquired immunodeficiency - HIV/AIDS, haematological malignancy, auto-antibodies
→ pulmonary NTM infection: structural/function lung compromise - genetic (CF), acquired (COPD), Lady Windemere syndrome, drug induced, other
Environment → natural: tropical climates and geographic area, soil characteristics, animal reservoirs, water reservoirs
→ man made: domestic - hot tubs, potting soil, domestic animals, shower aerosol, healthcare - contaminated water sources and equipment
Organism → lipid rich cell wall: survival and growth in diverse habitats, biofilm formation and persistence, aerosolisation, antibiotic and disinfectant resistance
→ growth requirements: low doubling time in culture media reduces ability to identify these organisms in clinical and environmental samples, special media required to grow any of them, therefore not identified in routine lab cultures

Question 39

What are the complications and economic burdens of NTM?

Answer 39

Prolonged multidrug therapy for pulmonary NTM (12-24 months)
High rates of re-infection (rather than recurrence)
Frequent adverse drug reactions
20-40% of MAC-lung disease patients initially fail to respond
Drug resistance
Co-morbidity

Question 40

What are buruli ulcers?

Answer 40

Caused by Mycobacterium ulcerans → an indolent necrotising disease of the skin, subcutaneous tissue and bone
→ presently the third most common mycobacterial disease of humans after tuberculosis and leprosy and the least understood
Characterised by painless necrotic skin lesions
Closely related phylogenetically to M. tuberculosis but causes a very different disease
Replication and persistence of M. ulcerans in BU lesions is profoundly influenced by production of mycolactane → a cytotoxin that has immunosuppressant properties

Question 41

How is M. ulcerans transmitted?

Answer 41

Little evidence of human to human transmission → humans are infected from the environment
In endemic regions the disease is highly focal and usually associated with wetlands or coastal regions
PCR testing of environmental samples, such as water, aquatic plants, soil and detritus from swamps has found evidence of the mycobacteria
Increasing evidence that insects can harbour the disease → insects such as mosquitos and water-residing biting arthropods have been associated with it epidemiologically and via PCR testing - proposed vectors for transmission
In Australis evidence suggests that native possums might be involved in transmission

Question 42

What is the main difference between endemic and non-endemic areas of M. ulcerans?

Answer 42

Key difference concerned human activity
Despite their close proximity
non-endemic area → largely unaffected by human activity
endemic areas → the bank of the Nyong River has been deforested for agricultural and fishing activity
→ interventions disrupting the environment have been identified in several studies as factors potentially favouring the establishment of it

Question 43

What is the proposed transmission pathways of M. ulcerans between the environment, mosquitoes, possums and humans?

Answer 43

Possums ingest M. ulcerans from the environment and/or infected by an insect vector
Possums amplify and shed it into the environment
Insect vectors become contaminated with it from the environment and/or from contact with infected possums
Transmitted to humans via insect vector and/or direct contact with contaminated environment

Question 44

Where are M. ulcerans skin lesions on the body?

Answer 44

Most likely lower leg regions
→ also on upper leg and elbows

Question 45

What features of M. ulcerans promote pathogenesis of BU?

Answer 45

1. The low optimal temperature of growth (29-30C) → makes the skin its preferential target
2. Behaves as an intracellular pathogen triggering inflammatory cell responses and cell mediated immunity
3. Unusual toxin production → mycolactone associated with high cytotoxicity that contrasts with the intracellular traits of its mycobacterial nature, protects from infiltrating leukocytes

Question 46

What is mycolactone?

Answer 46

A polypeptide cytotoxin that has immunosuppressive properties
→ precise cellular targets unknown
→ induces apoptosis of infected hist cells, inhibits production of the pro inflammatory cytokine TNF-alpha by macrophages, suppresses DC cell priming of T cells
Can diffuse from infected skin tissue to lymphoid organs within mononuclear cells - may exert some of the immune suppressive functions
Damages nerve cells → contributes to the painlessness of BU
Genes required to make it are carried on a virulence plasmid, pMUM001 → acquired by HGT from an unknown source

Question 47

What is involved in granuloma formation of Buruli ulcers?

Answer 47

M. ulcerans and mycolactone in the centre → surrounded by neutrophils (apoptotic and debris), macrophages, B and T cells
Early immune response may in many cases be capable of clearing initial infection
High conc of mycolactone in the lesion core cause apoptosis of both resident skin cells and infiltrating leukocytes → chronic necrotic lesions develop with destruction of subcutaneous tissue

Question 48

How does disease progression of BU occur?

Answer 48

Pre-ulcerative → low bacterial load, low tissue necrosis, big local inflammation and IFN response
Ulcerative lesion → growing bacterial load and tissue necrosis, decreasing local immune response driven by immunosuppressant properties of mycolactone
Spontaneous healing/treatment → reduced bacterial load and tissue necrosis, increased local immune response

Question 49

How is BU diagnosed?

Answer 49

Clinical diagnosis
Microscopy - smear
Culture
IS2404 PCR-based tested, DNA based - fast

Question 50

How is BU treated?

Answer 50

Multi drug therapy to reduce selection of drug-resistance strains
→ combination of rifampicin and clarithromycin WHO recommended treatment
Surgery and hospital treatment of wounds for skin grafts
→ in well developed lesions, with ongoing necrosis due to effects of high mycolactone toxin. removal of tissue necessary
Physiotherpay and rehabilitation → overcome limitations due to tissue damage

Wound management → vital but often goes neglected, healing time can be month or years even with antibiotics, improved wound dressing emerging from other ulcer diseases e.g. diabetes

Question 51

How can BU be prevented/controlled?

Answer 51

Active surveillance, case management, treatment
e.g. Republic of Congo - identification of this zone as a high-risk area for BU helped the Ministry of Health improve → early detection, biological conformation, treatment programs

Vaccines → some protection seen with BCG, but inconsistent
→ specific vaccine to M. ulcerans targeting a mycolyl transferase (antigen 85A) is being tested

Avoid risky behaviours in endemic areas → swimming, fishing, agricultural works
→ mosquito nets may help, good pre-existing wound care, protective clothing

Question 52

What are the future directions for BU?

Answer 52

Rapid diagnostic tools → fast identification of infecting species e.g. ELISA
Simple and cheap screening tool to identify patients
Faster initiation of appropriate treatment and ultimately superior care

Efforts to shorten duration of treatment
→ e.g. new anti-tuberculosis drug showed extreme potency to M. ulcerans
→ clinical trial comparing standard treatment of the combination drug for 8 weeks, or with amoxicillin/clavulanate for 4 weeks