WEEK 1: Microbiology of Mycobacterium Tuberculosis

Question 1

TB accounts for how many % of adult deaths & how many % of deaths of people living with HIV/AIDS?

TB burden uneven globally: primarily affects what 2 continents?

Where is the HIV/TB co-infection highest?

In 2018, TB leading cause of deaths amongst people with HIV-infection. Overall, no. of deaths reduced with better access to treatment.

Botswana is endemic for TB & HIV: TB incidence rate of 326 / 100,000 &

How many % of TB patients co-infected with HIV/AIDS?

Answer 1

TB accounts for ~13% of adult deaths & ~40% of deaths of people living with HIV/AIDS

TB burden uneven globally: primarily affects South east Asia & Africa

HIV/TB co-infection highest in Africa
In 2018, TB leading cause of deaths amongst people with HIV-infection. Overall, no. of deaths reduced with better access to treatment

Botswana is endemic for TB & HIV: TB incidence rate of 326 / 100,000 & ~70% of TB patients co-infected with HIV/AIDS. MDR Mtb. a big concern

Question 2

What is mycobacterium complex?

Answer 2

M. tuberculosis complex (MTC): group of genetically related mycobacteria with 99.9% nucleotide similarity & identical 16S rRNA sequences.

All can cause TB, a chronic granulomatous disease affecting humans & many other mammals.

Question 3

State the 8 components of the mycobacterium complex.

Answer 3

M. tuberculosis (Mtb.)
Primary cause of TB in humans worldwide (can infect several animal species but humans principal hosts)

*M. bovis
Common cause of TB in cattle (& other mammals)
Attenuation of laboratory strain of M. bovis led to development of BCG vaccine (1921)

M. africanum
Mainly in equatorial Africa. Type 1 common cause of TB in humans West-Africa; type 2 mainly in east Africa

M. caprae
Mainly in central Europe, isolated from livestock i.e. goats, cattle, pigs. Rarely isolated from humans: contact with livestock likely means of transmission.

M. Canetti
First isolated by G. Canetti in 1969 from a French farmer with pulmonary tuberculosis. Since then, it has been infrequently isolated from patients in East Africa

M. microti
Typically causes disease in voles, wood mice & shrews rarely isolated from other animals & humans.
M. pinnipedii
Primarily isolated from marine animals (seals)

M. mungi
Novel pathogen (2010) in mongooses in Chobe district, Botswana

Question 4

M. Complex different by host tropisms, phenotypes & pathogenicity.

State those which are exclusively human pathogens.

Answer 4

Different by: host tropisms, phenotypes & pathogenicity i.e. some exclusively human pathogens (M. tuberculosis, M. africanum, M. canettii); rodent pathogens (M. microti); others have wider host spectrum (M. bovis)

Question 5

Define non-tuberculous Mycobacteria.

Answer 5

“Non-tuberculous mycobacteria (NTM)” are Mycobacteria spp. other than MTC (& M. leprae)

Question 6

Describe non-tuberculous Mycobacteria.

Answer 6

Ubiquitous in environ. & normally colonise body surfaces

But in immunocompromised patients can cause severe clinical syndromes

Question 7

Outline Severe clinical syndromes caused by NTM.

What is the leading NTM pathogens?

Answer 7

Progressive pulmonary disease

Disseminated disease.

Superficial (& cervical) lymphadenitis

Skin & soft tissue infections (SSTIs)

Bronchiectasis

M. aviumcomplex (MAC),M. Kansasii&M.abscessus (esp. older persons

Question 8

Classification by growth rate & assoc. infections.

How long do fast growers and slow growers take?

Review table on notes!!!!!

Answer 8

<7 days fast growers & >7 days slow growers (time for appearance of colonies on solid medium)

Question 9

Describe the morphology of Tuberculosis.

Describe the colonies in solid medium and in liquid culture.

Answer 9

Aerobic, non-motile, non-spore forming, non-encapsulated rods (pleomorphic i.e. rods or slightly curved)

Solid medium: tight & wrinkled colonies i.e. Löwenstein–Jensen agar & Middlebrook 7H10 agar
Due to high lipid content & hydrophobic nature of cell wall

In liquid culture: some virulent strains may appear as twisted rope-like ‘serpentine’ structures.

Question 10

The cell wall of mycobacterium tuberculosis IS unique lipid rich (60%) cell wall comprising 3 important macromolecules.

State the 3 macromolecules.

Answer 10

Unique lipid rich (60%) cell wall comprising 3 important macromolecules:

*Peptidoglycan:
-A polymer consisting of sugars (glycan chains) cross-linked by short peptides.
-It provides structural support to the bacterial cell wall.

*Arabinogalactan (D-arabinose & D-galactose) attached to peptidoglycan. This layer plays a role in the structural integrity of the cell wall.

*Mycolic acids (long chain fatty acids) linked to arabinogalactan
e.g. Trehalose 6,6′-dimycolate/ 6,6’-dimycoloyl trehalose.

Question 11

State other important macromolecules anchored in cytoplasmic membrane but extending across the cell wall.

Answer 11

Other important macromolecules anchored in cytoplasmic membrane but extending across the cell wall.

*Lipoarabinomannan-LAM & its precursor lipomannan
NB: Virulent Mtb. have ‘mannose capped’ LAM (Man LAM)

*Cytoplasmic membrane assoc. proteins e.g. 19-kDa:
Role not well understood but very antigenic.

& 27kDa lipoprotein

*Phosphatidyl-myo-inositol mannosidase-PIM

Question 12

Cell wall confers many of mycobacteria characteristic properties.

Answer 12

Cell wall confers many of mycobacteria characteristic properties i.e.:

Slow growth, acid-fastness

Certain cell wall macromolecules antigenic &/or significant virulence factors
Evasion of host immune response esp. macrophages

Resistance to common antibiotics, primarily due to lipid-rich cell wall
Lipophilic drugs i.e. fluoroquinolones or rifamycin’s more easily ‘diffuse’ through & thus more active against mycobacteria

Question 13

Describe the physiology of exposure to TB.

Answer 13

Upon inhalation, aerosols containing Mtb ‘arrive’ at the alveoli which are always exposed to airborne particulates & pathogens

Contain innate immune cells i.e. alveolar macrophages, one of the initial immune cells to encounter the Mtb.

Also there are dendritic cells found in the interstitial space

Upon inhalation, Mtb. are ingested by resident alveolar phagocytes & antigen presenting cells i.e. macrophages & dendritic cells

Macrophages=main niche for Mtb. replication. BUT Mtb. can also infect non phagocytic cells e.g. M-cells, alveolar endothelial cells & type 1 & 2 epithelial cells (pneumocytes)

Type II alveolar epithelial cells can also be infected by Mtb because these fail to control the infection, this route is thought to be another important way in which Mtb traverses the mucosa

Macrophages & dendritic cells vital against Mtb.
Macrophages have an array of receptors incl. specific Pattern Recognition Receptors (PRRs) e.g. TLR, complement receptor (CR3), mannose receptor, scavenger receptors & DC-SIGN, which bind to conserved motifs on microbial pathogensi.e. pathogen associated molecular patterns (PAMPs)

Specific PRRs binding to Mtb PAMPS triggers a series of immune responses that may lead to an early immune response that results in the clearance of Mtb. or granuloma formation

Other immune cells in 1st line of defense
…..‘The sentinels’…..

Neutrophils
Activated neutrophils kill Mtb. using antimicrobial molecules in their granules i.e. defensins, lactoferrin, cathelicidin & lysozyme

Activate macrophages by release of granule proteins & heat shock protein 72 (Hsp72) released from apoptotic neutrophils

Complement system & several types of innate immune cells i.e. neutrophils & natural killer (NK) cells vital against Mtb.

Natural Killer (NK) cells: granular lymphocytes activated via complex interactions between IL-12, IL-18, IFN-α

NK cells also lyse Mtb. infected macrophages: by production of perforin; granzyme or granulysin

So: a combination of antimicrobial activities & the regulation of inflammation is essential for the successful control of Mtb. Infection

Including robust macrophage-based control of bacterial replication by intracellular antimicrobial mechanisms

Cytokines i.e. GM-CSF produced by non-hematopoietic cells & IFN-γ produced by CD4 T cells promote the microbiocidal activities of macrophages

Question 14

State the 3 main outcomes of TB exposure

Answer 14

Outcomes
Clearance by innate immune system

Latent infection

Active disease

Question 15

Of those exposed:
How many % clear infection via innate immune response, showing no signs of disease or immunological memory against the pathogen?

Of those who become latently infected:
How many % progress to active TB in 5yr (rate much higher in HIV patients)?

How much remaining % control infection throughout lifetime, only progressing to active disease when immunocompromised i.e. HIV-infection, old age, treatment with immunosuppressive drugs or re-infection

Answer 15

Of those exposed:
50% clear infection via innate immune response, showing no signs of disease or immunological memory against the pathogen
Of those who become latently infected:
5% progress to active TB in 5yr (rate much higher in HIV patients)

Remaining 95% control infection throughout lifetime, only progressing to active disease when immunocompromised i.e. HIV-infection, old age, treatment with immunosuppressive drugs or re-infection

Question 16

Describe how uncontrolled infection come about.

Answer 16

In contrast, uncontrolled infection results from either a failure of antimicrobial control or imbalanced cytokine production

If antimicrobial mechanisms fail, the increased bacterial load triggers excessive production of inflammatory cytokines, leading to the recruitment of excessive neutrophils that may contribute to uncurbed inflammation

OR increased type I interferon production can block IL-1 signaling, leading to immune failure

Question 17

Macrophage polarization is a dynamic process, and macrophages can switch between M1 and M2 phenotypes based on the microenvironment and signals they receive.

This plasticity allows macrophages to adapt to different stages of the immune response, participating in both the induction and resolution of inflammation.

This flexibility is vital for maintaining tissue homeostasis and responding appropriately to various challenges.

Compare the M1and M2 macrophages.

Answer 17

M1 Macrophages:

Induced by pro-inflammatory signals, such as IFN-γ, TNF-α, GM-CSF, and microbial products.

Microbiocidal and possess inflammatory properties, secreting cytokines like IL-1, IL-12, TNF-α, IL-23, and IL-6.

Express IL-1 receptor, MHC class II, TLR2, and TLR4.

Intracellularly express iNOS, reactive oxygen species (ROS), antimicrobial peptides, interferon-inducible GTPases, and participate in autophagy.

Effective against bacterial infections.

M2 Macrophages:

Induced by anti-inflammatory signals, such as IL-4, IL-13, and M-CSF.

Have regulatory properties and express arginase, IL-10, TGF-β, and other anti-inflammatory cytokines, contributing to immunosuppressive functions.

Play a role in building extracellular matrix, wound healing, and tissue repair.

Question 18

Mtb. can bind to diff. but specific PRRs which trigger phagocytosis.

Outline the different PRR.

Outline Mtb. PAMPs which bind to PRRs.

Answer 18

Mtb. can bind to diff. but specific PRRs which trigger phagocytosis:

TLR2, TLR4, C-type lectins incl. mannose receptors (MR), DC-SIGN, Dectin-1, Complement receptor (CR3), FcR (binds to IgG opsonised Mtb)

Mtb. PAMPs which bind to PRRs incl.:
19 & 27kDa lipoproteins
38 kDa glycoprotein
Lipomannan
Mannose-capped lipoarabinomannan

Question 19

After specific receptor-mediated phagocytosis occurs, “phagosomal maturation” takes place (series of fusion & fission events). Results in the “phagolysosome”

The phagolysosome is an effective microbiocidal structure in the macrophage.

Outline the different main antimicrobial mechanisms of mature macrophage phagolysosome.

Answer 19

Vacuolar H+-ATPases cause acidification/pH 4-4.5; activation of NADPH oxidase & inducible nitric oxide synthase (iNOS) generates reactive oxygen species (ROS) & reactive nitrogen intermediates (RNI)

Antimicrobial peptides & degradative proteases: scavenge nutrients to deprive pathogen or form pores in pathogen cell wall & membrane

Iron depriving mechanisms e.g. lactoferrin, Fe exporters (NRAMP1) remove Fe, essential for DNA synthesis & mitochodrial respiration )

Additional antimicrobial activities of macrophage-’self-destruction’

Autophagy: degradation of cellular components

Apoptosis/ programmed cell death i.e. destroys replication niche of Mtb.

Question 20

…..The later defenders……
Adaptive immunity

Answer 20

Infected dendritic cells, macrophages, inflammatory monocytes & migrate from the lung to draining lymph nodes

The infected macrophages & dendritic cells prime T cells via the process of antigen presentation, this activates T cells

The T cells migrate back to the lungs via blood, activate macrophages to kill their intracellular Mtb & participate in granuloma formation occurs

Adaptive immunity essential against Mtb.
Infected macrophages & dendritic cells present Mtb. antigens to CD4+ T helper cells via MHC-II

CD4+ Th1 cells immune response central to immunity against Mtb.:
*IL-12 & IFN-γ pathway stimulated by pro-inflammatory factors induced by Mtb. cell wall lipids essential for macrophage activation

*Required for ‘phagolysosomal maturation’ & Mtb. killing
BUT

Mtb. manipulates cytokine response inducing Th2 cytokines i.e. IL-4, IL-10 promoting anti-inflammatory macrophage response & hampers production of IL-12 & IL18. Hampers antigen presentation via MHC-II

Question 21

Describe granuloma formation process.

Answer 21

Activated macrophages also attract monocytes, lymphocytes & neutrophils via production of chemokines

Formation of granulomas facilitates containment of bacteria:
Comprises macrophage-derived giant cells & lymphocytes

Development of ‘caseous/ cheesy’ centre, surrounded by fibroblasts, extracellular matrix proteins, lymphocytes & monocytes

Hostile internal environment: acidic pH, low O2 & toxic fatty acids

Macrophages with intracellular bacteria largely killed

Question 22

What is latency?

Answer 22

Latency: asymptomatic & non transmissible stage.

In most infected immunocompetent individuals Mtb. seemingly contained, primary infection resolves
However
survival of Mtb. in granuloma occurs

In healthy individuals, risk of developing active TB after initial infection highest within 1st & 2nd yr (1% & 0.3% respectively), much lower in subsequent years

Question 23

Define Post-primary TB.

What are the risk factors?

Answer 23

Reactivation of dormant foci or exogenous re-infection

Risk factors: congenital or immunosuppression i.e. HIV infection

Question 24

Describe the pathogenesis of Post primary TB.

Answer 24

Breakdown of granuloma/ Ghon focus
Tends to occur in upper lobes of lungs, ideal for bacterial replication

Extensive tissue destruction: cavity formation

Large nos. of bacilli gain access to sputum & possibly circulatory system

Patient infectious, wasting & fevers characteristic of disease

Question 25

In immunocompromised people formation of granulomas does not effectively occur, leading to quicker spread of Mtb.

State 2 ways the HIV-1 infection might diminish granuloma function.

Answer 25

Formation of granulomas does not effectively occur, leading to quicker spread of Mtb.

E.g. it’s likely that HIV-1 infection might diminish granuloma function via two ways:

Impact of HIV-1 Infection on Granuloma Function:

*HIV-1 infection may lead to the systemic depletion of mononuclear cells necessary for the development, maintenance, and functioning of granulomas.

The reduction in these immune cells hampers the body’s ability to form effective granulomas, which are essential for containing and controlling Mtb infection.

*HIV-1-infected cells, such as lymphocytes or macrophages, may infiltrate the granuloma, potentially disrupting its structure and function.

This infiltration can compromise the integrity of the granuloma, leading to a less effective containment of Mtb.

Question 26

Describe how systemic manifestations and extrapulmonary TB spread.

Answer 26

Systemic Manifestations and Extra-Pulmonary TB:

The compromised granuloma function can result in the spread of Mtb beyond the lungs, leading to systemic manifestations of tuberculosis.

Extra-pulmonary TB may manifest in various organs, such as the meninges (meningitis), kidneys, spine (Pott’s disease), bones, and joints.

Dissemination of Bacilli:
If granulomas are unable to effectively contain Mtb, foci within the granulomas may rupture into blood vessels.

This can result in the dissemination of Mtb throughout the body, leading to the formation of numerous granulomata in various organs.

The widespread dissemination may give rise to millet seed-like lesions, a condition known as “miliary tuberculosis.”

Question 27

Extrapulmonary TB: “unfamiliar presentations of a familiar disease”

RECALL: Mtb. is primarily a respiratory pathogen

Define active and latent TB.

But TB may also present as “Extrapulmonary infections”

Why does extrapulmonary TB pose diagnostic challenges?

Answer 27

Active TB:
Typically pulmonary infection presenting with: cough (lasting >few weeks), often assoc. with bloody sputum; chills, fever, wt. loss & night sweats

Latent TB:
Usually no clinical symptoms, patients may never know they were infected until reactivation.

But TB may also present as “Extrapulmonary infections”

Pose diagnostic challenges as does not necessarily test +ve with “sputum smear” (standard TB test)

Question 28

Describe the signs and symptoms of extrapulmonary TB.

Answer 28

Lymphadenitis (esp. cervical lymph nodes): most common extrapulmonary TB: swelling of lymph nodes

Pott’s disease: distinctive curvature of spine & lower limbs palsy (assoc. with abscess between 1vertebrae)

Musculoskeletal TB: Mtb can infect a variety of bones & joints, i.e. articular TB (TB of hips or knee joints)

Pleural TB: infection of membranes lining the lungs & build-up of fluid (pleural effusions) between the membranes & lung

Gastrointestinal tract infection caused more by M. bovis than Mtb.

CNS infections: less common but serious i.e. meningitis, encephalitis, or abscess or tuberculoma

Systemic infection: hematogenous spread of Mtb in entire body, resulting in numerous small lesions ‘resembling millet seeds’ in any tissue i.e “miliary TB”.

Esp. in highly vascularised organs e.g. lungs, liver, spleen, kidney & bone marrow

Question 29

Unlike many bacteria, Mtb. do not produce any toxins, or spores, or capsules

What is its main weapon?

Answer 29

Unlike many bacteria, Mtb. do not produce any toxins, or spores, or capsules

Main weapon

• ability to survive & multiply within macrophages

-Slow growing & tolerate intracellular environment, remaining metabolically inert for years before reactivation

-Remain in endocytic vaculole & hinder fusion of phagosome with lysosome

NB. Prevention of acidification of the phagosome that necessary for effective killing of microbes by lysosomal enzymes.

Question 29

Who is more vulnerable to TB?

Answer 29

Vulnerable pop.s:
HIV infection
Children & individuals with malnutrition

Question 30

Discuss the cell wall associated virulence factors of mtb.

Answer 30

Lipoarabinomannan
*Binding of macrophage mannose receptors to man-LAM assoc. with anti-inflammatory response i.e. shifts cytokine response from ‘pro- to anti-inflammatory’ pathway. *inhibits IL-12 production

*Inhibits phagosome maturation & eliminates cytotoxic oxygen-free radicals in phagolysozyme

Cording factor (Trehalose 6,6′-dimycolate/ 6,6’-dimycoloyl trehalose) confer branching, filamentous/ cord-like morphology

*Mycolic acids attached to trehalose inhibit fusion of phagocytic vesicles with lysosomes in macrophages inhibiting bacteria degradation

*Facilitates long-term survival of bacteria in host cells

3. 19-kDa lipoprotein assoc. with switching of ‘pro-inflammatory’ pathway to ‘anti-inflammatory’. During latent phase of infection

*It’s been suggested that 19-kDa is an agonist of TLR2, facilitates prolonged TLR2 signaling, inhibits effective MHC-II expression & processing of antigens by the macrophage

19-kDa protein assoc. hampered response of macrophages to IFN- & inhibition of antigen presentation

4. Secreted antigenic proteins (ESAT-6/ CFP-10 system (early secreted antigenic target &10-kDa culture filtrate protein) are assoc. with:

*Inhibiting Tcell IFN-γ production & reducing IL-12 secretion by antigen presenting cells

*Inhibiting H+ATPases, lysosomal hydrolases resulting in lack of acidification in macrophages

*Lysis of alveolar epithelial cells & macrophages, enabling Mtb. spread from macrophage-to-macrophage

Question 31

State other survival mechanisms of mtb.

Answer 31

Mtb manipulates the phagocyte
i) makes environ. more hospitable
ii) escapes the phagolysosome & replicates in cytosol

Inhibits phagosomal maturation

Deactivates reactive O2 species: Catalase peroxidase (KatG)

Scavenges reactive nitrogen species: by a truncated haemaglobin

Question 32

Sputum collection for lab diagnosis.

Smear positive patients are a high risk of transmission in community. Diagnosing & treating them is highest priority

Describe sputum collecting process for TB diagnosis.

State tests which are done using sputum.

Answer 32

Sputum collection MUST include “spot & early morning (& spot)”

One “spot” specimen is submitted under HCW supervision during the 1st interview

Second “early morning” collected by patient, next morning, before cleaning mouth or eating. NB. has highest yield of Mtb.
(Spot when patient returns with 2nd specimen)

NB. For in-patients 3 consecutive early morning sputum samples
Sputum must be delivered quickly to the lab

Sputum for sputum smear microscopy &/ or culture

Question 33

Identification of Mtb. in sputum & other body fluids or tissues.

Discuss the different tests done on the sputum.

Answer 33

1. Sputum smear microscopy (Performed at the facility level)

Cost-effective & rapid test but sensitivity variable

Must be done in a ventilated lab

Processing of sputum in a Biosafety level-2 cabinet with BSL-3 safety equipment & work practices

2. PCR / Gene Xpert (facility level & NTRL): Rapid & highly sensitive. Detects Rifampicin resistance for MDR-TB
3. Culture (performed at the National TB Reference Lab-NTRL)
   -Gold standard
   -High sensitivity
   -Must be done in a containment laboratory (BSL-3) with double door entry
4. Drug Susceptibility Testing at NTRL
   Performed only after a culture is positive

Question 34

Sputum smear staining

NB: Sputum smear is a ‘presumptive diagnosis’ of TB infection. What does that mean?

State the common staining methods.

Answer 34

NB: Sputum smear is a ‘presumptive diagnosis’ of TB infection

A presumptive diagnosis suggests a high suspicion of TB infection, but further confirmatory tests may be required for a conclusive diagnosis.

Common staining methods:

*Acid Fast Staining
Ziehl-Neelsen (hot method) & Kinyoun method (cold method- elimination of heat & use of higher concentration carbolfuchsin primary stain)

*Fluorescent microscopy i.e. with auramine-rhodamine fluorochrome - advantageous due to speed & simplicity

Question 35

Describe the Acid Fast Ziehl-Neelsen staining process.

Answer 35

Step 1. Prepare a smear & heat fix

Step 2. Carefully place slide over beaker of boiling water

Step 3. Addition of Carbolfuchsin (5mins do not let it dry), carefully remove slide & with H2O)
Heat & longer time facilitates penetration of the stain through lipid wall & into cytoplasm. All cells stained red

Step 4. Decolourise with acid-alcohol (1min, rinse with H2O)
Acid-fast cells will retain primary stain; non-acid fast cells lose the primary stain

Step 5. Counterstain with methylene blue
(30secs-1min, then rinse with H2O)

Finally, viewing under the microscope with the oil immersion lens
Acid fast cells stain red with blue background, whereas non-acid fast cells stain blue

Question 36

Quantification of AFB is important.

No. of AFB in a smear reflects the patient’s infectivity.

Question 37

NB. Sensitivity of sputum smear microscopy is variable:

State the 2 patient groups whom the test can be negative. WHY??

So, negative result does not mean patient not infected.

What is the Gold standard for lab TB diagnosis?

Answer 37

NB. Sensitivity of sputum smear microscopy is variable:

NEGATIVE IN HIV PATIENTS & PAEDIATRIC CASES

HIV-Infected Individuals:

*HIV infection can lead to immunosuppression, affecting the ability of the immune system to mount an effective response against Mycobacterium tuberculosis.

In HIV-positive patients co-infected with TB, there may be a reduced bacterial load in the sputum, resulting in a lower sensitivity of smear microscopy.

Additionally, extrapulmonary TB is more common in HIV-positive individuals, and sputum smear microscopy primarily focuses on pulmonary TB.

Pediatric Cases:

*Children, especially younger ones, may have difficulty producing an adequate sputum sample for testing.

*Pediatric TB often presents with paucibacillary disease, meaning there may be a lower concentration of bacteria in the sputum, making it challenging to detect by smear microscopy.

*Similar to HIV-infected individuals, extrapulmonary TB is more common in children, and sputum smear microscopy may not be as sensitive for these cases.

Culture is the Gold standard for lab TB diagnosis

Question 38

Most sensitive phenotypic test for TB

Detects more cases than smear microscopy

Required for drug susceptibility testing (DST)

Name the test described above.

Answer 38

Sputum culture

Question 39

Sputum culture is slow & costly, so not routinely done for all patients BUT essential for certain patient groups.

Outline them.

Answer 39

*Populations assoc. low sensitivity of the sputum smear i.e. HIV PATIENTS & PAEDIATRIC CASES

*Patients from areas with endemic high rates of Mtb. drug-resistance

Question 40

Mycobacterial Culture Botswana National TB Ref. Lab

Culture is the gold standard but costly & time-consuming

Needs specialised media & lab expertise. Due to these constraints, in Botswana culture not done for “all cases” of routine case detection.

Outline cases that qualify for culture.

Answer 40

BUT there are stipulations, including:
*Patients with previous anti-TB treatment history

*Those who are sputum smear-positive on completion of TB treatment

*Symptomatic individuals at high risk of MDR-TB e.g. lab staff, MDR-TB patient’s contacts & HCWs of TB patients

*Children suspected to have drug-resistant TB, complicated cases or uncertain diagnosis

*Fluids suspected to be infected by Mtb, e.g., abscesses, pleural fluid, cerebrospinal fluid & urine

Question 41

State the 3 different types of media for TB

State the 2 types of convectionally culture media..

What are their advantages?

Answer 41

*Growth on solid medium - clinical specimens i.e. sputum & tissue

*Conventionally culture media: 2 types (optimal temp: 35-37C)

-Lowerstein-Jensen medium: coagulated egg base with malachite green to inhibit growth of contaminant bacteria

-Middlebrook media: agar base

Advantages:
-Relatively cheap
-Simple

*New - TK medium:
-new differential medium TB vs. non-TB bacteria)

Question 42

State the disadvantages of Mtb culture.

Answer 42

*Very slow growth of colonies:

-On Lowenstein-Jensen (L-J) media, growth of Mycobacteria is ~3 weeks. No growth by 8 weeks, result considered neg..

-On Middlebrook agar-17-21 days & 3-6 wk for drug sensitivity testing

*Culture is not sensitive for extrapulmonary TB, unless fluids attained from other body sites

*In certain cases: even in active pulmonary infection, Mtb. may not be sufficiently present in sputum, so may not grow in culture

Question 43

State the 4r drugs drug sensitivity is done on for all positive cultures.

Answer 43

Drug sensitivity testing performed at NTRL on all positive cultures:

-Isoniazid
-Rifampicin
-Ethambutol
-Streptomycin

Question 44

Discuss Molecular Methods: PCR

Answer 44

Detection of Mtb. DNA from sputum or body fluids

Highly sensitive & rapid 2hr

In Botswana: GeneXpert commonly used for sputum

Identifies both Mtb & resistance to rifampicin (MDR marker)

Question 45

Describe Automated Mycobacterial growth & detection systems.

Answer 45

*BD BACTEC MGIT system
Automated mycobacterial detection system & susceptibility testing.

Diagnosis 7days for sputum positive
(longer for sputum negative)
8 – 12 days for drug sensitivity testing

MB/ BacT system

-Also used for 1st & 2nd- line detection of drug resistance

Diagnosis 17days (range 7- 40)

8 – 12 days for drug sensitivity testing)

Question 46

Outline Alternative tests for TB.

Answer 46

*Serological methods: enzyme-linked immunoassays

Detection of antibodies to lipoproteins & lipoarabinomannan (LAM)
But
Sensitivity questionable: antibody levels often low in culture-negative individuals

*Urine - detection of LAM

Question 47

Describe the detection test for latent TB.

Answer 47

Latent TB infection can usually be detected by the PPD skin test.

Purified protein derivative (PPD) is a soluble protein preparation derived from M.Tuberculosis cultures. A very small amount (0.1 mg or 5 tuberculin units) is injected into the skin. This produces a delayed type hypersensitivity response which is viewed 48-72 hours after administration.

The result is quantified by measuring the width of the induration response produced – a red, slightly swollen firm lesion indicates a positive result.

A positive result indicates exposure to M. tuberculosis with some level of infection, which may be controlled or may be dormant, or exposure to BCG vaccination – see below- or exposure to other forms of mycobacterium.

But note that infected individuals with HIV infection and hence reduced numbers of CD4+ve T-cells may give a negative response.

Question 48

Which statement is correct regarding mycobacterium tuberculosis?

A. This bacterium is an anaerobic type of bacteria.
B. It is an alkali bacterium that stains bright red during an acid-fast smear test.
C. It is known as being an aerobic type of bacteria.
D. It’s an acid-fact bacterium that stains bright green during an acid-fast smear test.

Answer 48

The answer is C. Mycobacterium tuberculosis is AEROBIC (it thrives in conditions that are high in oxygen), and it is an ACID-FAST bacterium, which means when it is stained during an acid-fast smear it will turn BRIGHT RED.

Question 49

Your patient is diagnosed with a latent tuberculosis infection. Select all the correct statements that reflect this condition:
A. “The patient will not need treatment unless it progresses to an active tuberculosis infection.”
B. “The patient is not contagious and will have no signs and symptoms.”
C. “The patient will have a positive tuberculin skin test or IGRA test.
D. “The patient will have an abnormal chest x-ray.”
E. “The patient’s sputum will test positive for mycobacterium tuberculosis.”

Answer 49

The answers are B and C. The patient WILL need medical treatment to prevent this case of LBTI from developing into an active TB infection later on. The patient will NOT have an abnormal chest x-ray or a positive sputum test. This is only in active TB.

Question 50

A 52-year old female patient is receiving medical treatment for a possible tuberculosis infection. The patient is a U.S. resident but grew-up in a foreign country. She reports that as a child she received the BCG vaccine (bacille Calmette-Guerin vaccine). Which physician’s order below would require the nurse to ask the doctor for an order clarification?
A. PPD (Mantoux test)
B. Chest X-ray
C. QuantiFERON-TB Gold (QFT)
D. Sputum culture

Answer 50

The answer is A. Patients who have received the BCG vaccine will have a false positive on a PPD (Mantoux test), which is the tuberculin skin test. The BCG vaccine is a vaccine to prevent TB. It is given in foreign countries to children to prevent TB. Therefore, the person has already been exposed to the bacteria via vaccine and will have a false positive. A QuantiFERON-TB Gold test is a better option for this patient. It is a blood test.

Question 51

You’re teaching a group of long-term care health givers about the signs and symptoms of tuberculosis. What signs and symptoms will you include in your education?
A. Cough for a minimum of 6 weeks
B. Night sweats
C. Weight gain
D. Hemoptysis
E. Chills
F. Fever
G. Chest pain

Answer 51

The answers are B, D, E, F, and G. Option A is wrong because a cough should be present for 3 weeks or more (NOT 6 weeks). Option C is wrong because the patient will experience weight LOSS (not gain).

Question 52

A patient has a positive PPD skin test that shows an 8 mm induration. As the nurse you know that:
A. The patient will need to immediately be placed in droplet precautions and started on a medication regime.
B. The patient will need a chest x-ray and sputum culture to confirm the test results before treatment is provided.
C. The patient will need an IGRA test to help differentiate between a latent tuberculosis infection versus an active tuberculosis infection.
D. The patient will need to repeat the skin test in 48-72 hours to confirm the results.

Answer 52

The answer is B. A positive PPD result does NOT necessarily mean the patient has an active infection of TB. The patient will need a chest x-ray and sputum culture to determine if mycobacterium tuberculosis is present and then treatment will be based on those results. The IGRA test does NOT differentiate between LTBI or an active TB infection. Patients are placed in airborne precautions (NOT droplet) if they have ACTIVE TB.

The interferon gamma release assay (IGRA) test is a blood test used to see whether a person has been exposed to the tuberculosis (TB) bacteria.

The IGRA test is used to diagnose TB infection. This is when the TB bacteria is in the body but the person is not experiencing any symptoms suggestive of TB disease.

Question 53

10. A patient has a PPD skin test (Mantoux test). As the nurse you tell the patient to report back to the office in _________ so the results can be interpreted?
    A. 24-48 hours
    B. 12-24 hours
    C. 48-72 hours
    D. 24-72 hours

Answer 53

The answer is C. The patient should report back in 48-72 hours. If they fail to, the test must be repeated.

Question 54

A 48-year old homeless man, who is living in a local homeless shelter and is an IV drug user, has arrived to the clinic to have his PPD skin test assessed. What is considered a positive result?
A. 5 mm induration
B. 15 mm induration
C. 9 mm induration
D. 10 mm induration

Answer 54

The answer is D. 15 mm induration is positive in ALL people regardless of health history or risk factors. However, for patients who are homeless (living in homeless shelter) and are IV drug users, a 10 mm or more is considered positive.

-15 millimeters (mm) or more: Positive in all persons (doesn’t matter if the person does not have any risk factors)
-10 mm or more: positive if the person is an immigrant, IV drug user, working or living in tight living quarters, child less than 4
-5 mm or more: positive i person have HIV, in contact with someone with TB, organ transplant patient, or immunosuppressed..

Question 55

The physician orders an acid-fast bacilli sputum culture smear on a patient with possible tuberculosis. How will you collect this?
A. Collect 2 different sputum specimens 12 hours apart
B. Collect 3 different sputum specimens (one in the morning, afternoon, and at night)
C. Collect 3 different sputum specimens on 3 different days
D. Collect 2 different sputum specimens on 2 different days

Answer 55

The answer is C. This is how an AFB sputum culture is collected.

Question 56

A patient receiving medical treatment for an active tuberculosis infection asks when she can starting going out in public again. You respond that she is no longer contagious when:
A. She has 3 negative sputum cultures
B. Her signs and symptoms improve
C. She has completed the full medication regime
D. Her chest x-ray is normal
E. She has been on tuberculosis medications for about 3 weeks

Answer 56

The answers are A, B, and E. These are all criteria for when a patient with active TB can return to public life (school, work, running errands). Until then they are still contagious and must stay home in isolation.

Question 57

Discuss TB vaccination.

Answer 57

Vaccination is carried out using BCG vaccination.

BCG (Bacillus Calmette Guerin) is an attenuated form of M. bovis developed by selection of weakened strains over ten year period.

It is thus a live attenuated vaccine.

BCG vaccine has a success rate which varies widely between populations, from 0 – 80%.

It is recommended for children in infected areas, to prevent the development of TB before the immune system is fully functional.

There is no evidence that it has much effect on prevention of adult cases of TB.

Question 58

Discuss challenges to TB treatment.

Answer 58

1. Multidrug Resistant Tuberculosis (MDR-TB):

*Definition: MDR-TB is characterized by resistance to two of the most potent first-line anti-tuberculosis drugs, Isoniazid (INH) and Rifampin.

*Significance: This form of TB represents a significant challenge to global health efforts due to its resistance to standard treatment regimens.

2. Extensively Drug Resistant Tuberculosis (XDR-TB):

*Definition: XDR-TB is defined as MDR-TB with additional resistance to any of the second-line anti-tuberculosis drugs. It was first reported in 2005.

Implications:
The treatment for MDR-TB and XDR-TB is less effective compared to that for drug-susceptible TB.
The management of these resistant forms of TB is more complex, requiring more resources and posing higher costs.

3. Total Drug Resistant Tuberculosis (TDR-TB):

Emergence: Total drug-resistant TB has been identified in the last 2-3 years, with the first cases reported in 2011.

Threat: TDR-TB represents a critical threat to current TB control programs due to its resistance to all available anti-tuberculosis drugs, severely limiting treatment options.

Question 59

State the risk factors for TB.

Answer 59

Coming into close contact with people with active infection
Prolonged residence in a country with high prevalence of TB
And those which involve immunosuppression:
HIV infection
Treatment with immunosuppressive agents, particularly anti-tumour necrosis factor drugs
Poor living conditions
Malnutrition

There is also a genetic element – some individuals are more genetically susceptible to TB than others. This may be because of the balance of Th1 and Th2 cells which they produce. Individuals who’s Th cell balance favours Th2 cells have a poorer prognosis when infected.

Question 60

What type of hypersensitivity is TB?

Answer 60

Tuberculosis (TB) is primarily associated with a Type IV hypersensitivity reaction, also known as delayed-type hypersensitivity (DTH).

This type of hypersensitivity involves the activation of T lymphocytes, particularly CD4+ T cells, which release cytokines that recruit and activate macrophages. This immune response leads to the formation of granulomas, which are characteristic of TB infection.

Initial Infection: TB is caused by the bacterium Mycobacterium tuberculosis. When M. tuberculosis enters the body, typically through inhalation of infectious aerosols, it is engulfed by macrophages in the lungs.

Presentation to T cells: Within the macrophages, M. tuberculosis antigens are processed and presented on the surface of the cells via major histocompatibility complex (MHC) molecules. These antigen-presenting cells (APCs) migrate to nearby lymph nodes, where they present the antigens to T cells.

Activation of CD4+ T cells: Antigen presentation to naive CD4+ T cells triggers their activation. The interaction between the T cell receptor (TCR) on CD4+ T cells and the MHC-antigen complex, along with co-stimulatory signals, leads to the activation and differentiation of CD4+ T cells into effector T cells, particularly Th1 cells.

Th1 response and cytokine production: Th1 cells secrete cytokines such as interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), and interleukin-2 (IL-2). These cytokines play crucial roles in orchestrating the immune response against M. tuberculosis.

Activation of macrophages and granuloma formation: IFN-γ, in particular, activates macrophages, enhancing their ability to phagocytose and kill M. tuberculosis. TNF-α also plays a role in activating macrophages and promoting inflammation. The activated macrophages, along with other immune cells, aggregate to form granulomas around infected macrophages. Granulomas are organized structures consisting of immune cells, fibroblasts, and central caseous necrosis.

Chronic inflammation and containment of infection: The granulomas serve to contain the infection, preventing the spread of M. tuberculosis to other parts of the body. However, the bacteria can persist within granulomas in a latent state, posing a risk of reactivation later in life.

Pathological consequences: While granulomas are essential for containing the infection, they can also lead to tissue damage and lung pathology, especially in cases of extensive or poorly controlled inflammation. In some individuals, the immune response may fail to contain the infection, leading to active TB disease.