MYCOBACTERIUM Flashcards
Mycobacterium tuberculosis
Characteristics
• In tissue, thin, straight rods,
_____in tissue;
_____in length
_____in width
0.4 x 3 um
2-4 um
0.2-0.5 um
Mtb
On media,_____,_____,______; CSF
form small & (color) colonies
coccoid, filamentous, serpentine cord forms
buff colored or colorless
MTB
•______bacilli;
_______(macrophages) bacteria;
has a slow generation time,
_____- prevents immune recognition
Obligate aerobic
facultative intracellular
18H
MTB
Either GP or GN; very weakly Gram-positive or not at all _______
ghost cells
MTB
• strongly ________due to the____
acid-fast (Ziehl-Neelsen stain)
mycolic acid
MTB
•Resistant to…
dyes, antibacterial agents, acids, alkalies, quats; drying
T or F
•Survive in milk, organic materials, on pasture land
True
MTB
•Sensitive to UPFAG
UV light
pasteurization
formaldehyde
alcohol
glutaraldehyde
Strongly acid fast
Slightly acid fast
Non acid fast
Mycobacteria
Nocardia
Corynebacteria
Peptidoglycan Layer:
This is the basic part of the bacterial cell wall. It contains:
• N-acetylglucosamine (sugar)
• D-glutamic acid, m diaminopimelic acid, D- and L-alanine (amino acids)
Arabinogalactan Mycolate Layer:
This layer contains sugars:
• Connected to mycolic acids.
D-arabinose and D-galactose (sugars)
These are long fatty acids (C78-C90) that make the cell wall lipid-rich.
Mycolic Acids:
These lipids help make the bacteria acid-fast, meaning it doesn’t lose color in certain stains (like Ziehl-Neelsen). This makes it easy to identify under the microscope.
Mycolic acid
Proteins like ______ cause an immune response (the tuberculin reaction).
PPD (Purified Protein Derivative)
“Arabs & Myco Love Long Chains”
Arabinogalactan (D-arabinose & D-galactose)
Mycolic acids (C78-C90, long fatty acids)
Lipid-rich (acid-fast)
Layers (peptidoglycan, arabinogalactan)
prevent intracellular killing and digestion, inhibit phago-lysosome fusion
Mycolic acids, mycosides, sulfatides
complexed with mycolic acids cause granuloma formation
• Muramyl dipeptide (peptidoglycan)
- major virulence, inactivate macrophages and scavenge oxidative radicals (catalase, superoxide dismutase)
• Lipoarabinomannan (LAM)
serves as adjuvant;
with heat shock protein/tuberculin proteins cause hypersensitivity and form tubercle
Wax D
• cause caseous necrosis
Phospholipids
virulent strains form serpentine cords; inhibit phagocyte chemotaxis, activation, phagosome-lysosome fusions and digestion
Cord factor (trehalose-6,6”-dimycolate)
TUBERCULOSIS:
Predisposing factors
• Close contact with large populations of people, i.e., schools, nursing homes, dormitories, prisons, etc.
• Poor nutrition
• Pregnancy
• HIV infection
• IV drug use
• Alcoholism
In most cases, your body’s immune system controls the infection, leading to presumed sterilizing immunity. This means the bacteria are eliminated before they cause any symptoms.
In about____of people, the infection progresses quickly and leads to active, primary TB, causing symptoms like coughing, fever, and weight loss.
Self-Cure (90%)
Early Progression (5%)
In most cases (95%), the immune system contains the bacteria, but doesn’t kill them. This results in latent TB —you’re infected, but have no symptoms and can’t spread TB.
Later in life, 5% of people with latent TB may experience reactivation (the bacteria become active again) or get reinfected, leading to active TB symptoms.
Initial Containment (95%)
Reactivation or Reinfection
Pathogenesis os TB
Stage I: Inhalation
You inhale tiny droplet nuclei (SIZE) containing Mycobacterium tuberculosis (Mtb).
These droplets are expelled by someone with_____ tuberculosis through coughing, sneezing, or even talking.
Stage II: Primary Infection
Once inhaled, the Mtb bacteria reach the lungs and infect______ (cells in the lungs that normally destroy invaders).
Inside these macrophages, the bacteria start to multiply freely, as the macrophages are not yet able to stop them.
less than 25 micrometers
cavitary TB
non-immune alveolar macrophages
Pathogenesis of TB
Stage III: T-Cell Response
_______(a type of immune cell) recognize the Mtb antigens (MTB Ag) and begin to move into the infected area.
These T-cells release______, which activates macrophages and helps destroy the Mtb bacteria.
Stage IV: Unactivated Macrophages
Despite some macrophages being activated by the T-cells, many other macrophages remain unactivated or poorly activated.
Because of this, Mtb bacteria continue to replicate, and the infected area (???) grows larger.
Stage V: Caseation and Cavity Formation
The infected tubercles become______ and start to liquefy.
This liquefaction creates an ideal environment for Mtb to grow rapidly.
Eventually, this leads to_____ formation, which allows the bacteria to spread into the airways and potentially infect other parts of the lungs, or be coughed out, further spreading the infection.
T lymphocytes
gamma interferon (IFN)
tubercles
caseous (cheese-like in appearance)
cavity
In this form, the body’s immune response causes exudation, or the release of fluid, similar to pneumonia.
The ______will be positive, meaning the person has been exposed to TB, but the infection doesn’t progress.
This type resembles an early, non-specific immune response to Mtb.
tuberculin skin test (TST)
This type leads to the formation of chronic granulomas—clusters of immune cells that wall off the bacteria.
These granulomas contain the infection, but the bacteria may remain dormant inside.
The immune system produces these to control the infection and prevent it from spreading, resulting in a chronic, contained state
Productive Type (chronic granuloma)
________
Seen in primary tuberculosis (PTB).
It involves the initial lung lesion and affected lymph nodes.
If untreated, the lesion may become fibrotic (scar tissue forms) as the body tries to heal.
_________
This is the calcified version of a Ghon lesion.
It indicates that the body has controlled the TB infection by hardening (calcifying) the Ghon lesion and the nearby mediastinal lymph nodes.
_______
Occurs when TB spreads to the upper part (apex) of the lung.
It forms a small nodule (focus) at the lung’s apex, typically seen in reactivated TB or secondary TB.
- Ghon Complex
- Ranke Complex
- Simon Focus
[primary lesion]
- granuloma + draining lymph nodes
Ghon complex
A granuloma in tuberculosis is a structured immune response designed to contain the infection. It has three distinct zones:
_____
Contains multinucleated giant cells, which are large immune cells formed by the fusion of several macrophages.
These cells attempt to trap and destroy the TB bacteria at the core.
_________
Filled with pale epithelioid cells , which are activated macrophages that look like epithelial cells.
They form a barrier around the central area to further contain the infection.
_________
The outermost layer, which includes lymphocytes (a type of white blood cell) and other immune cells like fibroblasts.
These cells coordinate the immune response and help maintain the structure of the granuloma.
Central Area
Mid Zone
Peripheral Zone:
Clinical Features of Tuberculosis
- Chronic cough, dyspnea, fever, hemoptysis, night sweats, weight loss
• Two patterns:
Primary - childhood, disseminated (poor CMI response);
Secondary - hypersensitive tissue destructive response
Pulmonary infection
Extrapulmonary TB affects areas outside the lungs.
1. \_\_\_\_\_\_\_\_: • TB infection in the lining of the lungs (pleura) causing fluid buildup (effusion). • Symptoms may include chest pain, shortness of breath, and fever. 2. \_\_\_\_\_\_\_\_\_: • TB infects the lining of the brain and spinal cord (meninges). • Symptoms include severe headache, fever, stiff neck, and neurological symptoms (confusion, seizures). 3. \_\_\_\_\_\_\_\_: • A rare form where TB bacteria spread through the bloodstream, affecting multiple organs. • Symptoms are non-specific and may include fever, weakness, weight loss, and difficulty breathing. 4. \_\_\_\_\_\_\_: • TB infection in the kidneys and urinary tract. • Symptoms can include blood in the urine, frequent urination, or lower back pain. 5. \_\_\_\_\_\_\_: • TB infects the bones and joints, commonly the spine (called Pott’s disease). • Symptoms include pain, swelling, and difficulty moving affected joints. 6. \_\_\_\_\_\_\_\_\_: • TB infection of the intestinal tract. • Symptoms may include abdominal pain, diarrhea, weight loss, and fever.
Extrapulmonary TB can affect different parts of the body and may not always have typical lung-related symptoms.
TB Pleural Effusion
Tuberculous Meningitis
Miliary TB
Renal and Urogenital TB
Bone and Joint TB
TB Enteritis
___________:
• Also known as the(3), this is used to detect TB infection.
• Inject_____of_____ of PPD-S or 2 TU of PPD RT under the skin.
• A positive result shows as a raised bump at the injection site after____ hours, indicating TB exposure.
Skin Test (Tuberculin Skin Test, TST)
Mantoux, Pirquet, or PPD test
0.1 mL
5 TU (Tuberculin Units)
48-72
Culture and Sensitivity Testing:
• Culture the bacteria from samples (e.g.,).
• Test the bacteria for drug sensitivity to determine the best treatment options.
sputum, pleural effusion, urine, stool, cerebrospinal fluid [CSF], gastric aspirate, blood
(most common sample for pulmonary TB)
(for tuberculous
Gastric aspirate (for children or cold abscesses)
(for miliary or hematogenous TB)
Sputum
CSF
Blood
Laboratory Investigation of Tuberculosis
Specimens:
• fresh sputum (_____), gastric washings, urine, pleural fluid, CSF, joint fluid, biopsy material, or blood
•_____ - homogenized by grinding
• Urine (_____), pleural fluid, CSF, joint fluid, blood, or other fluids - centrifuge (to concentrate mycobacteria set at 3000x g, at least 20 minutes) and the deposits examined
x3
Tissue biopsies
x3
Decontamination of non-sterile specimens
_________
• NALC is used to liquefy the specimen (such as sputum), breaking down mucus so that TB bacteria can be better isolated.
• The solution also contains______, which kills many other bacteria and fungi, helping to clean up the specimen.
__________:
• After liquefaction and decontamination, the mixture is neutralized with a buffer to stop the action of the NaOH and prevent further damage to the TB bacteria.
Concentration by Centrifugation:
• The neutralized sample is then centrifuged (spun at high speeds), which concentrates the bacteria at the bottom of the tube, making it easier to detect TB in the sample.
Liquefaction with N-acetyl-L-cysteine (NALC)
2% sodium hydroxide (NaOH)
Neutralization
Ziehl-Neelsen Technique (Hot Stain):
Components:
This stains the acid-fast bacteria (AFB) red.
Removes the stain from non-acid-fast bacteria.
Stains non-acid-fast bacteria blue.
• Procedure:
• The slide is____ to allow the carbolfuchsin to penetrate the waxy cell wall of the AFB.
• Results:
• AFB stain____.
• Non-AFB stain____.
Carbolfuchsin (primary stain):
Acid alcohol (decolorizer):
Methylene blue (counterstain):
steamed
red ; blue
Kinyoun Stain (Cold Stain):
• Similar to Ziehl-Neelsen but doesn’t require heating. • \_\_\_\_\_is used as a mordant (wetting agent) to help the primary stain penetrate. • Components: • \_\_\_\_\_\_without heating • \_\_\_\_\_\_\_\_\_\_as a decolorizer. • Malachite green (counterstain) stains non-AFB green. • Results: • AFB stain\_\_\_\_. • Non-AFB stain\_\_\_\_.
Tergitol
Carbolfuchsin (primary stain)
95% ethyl alcohol + 3% hydrochloric acid (acid alcohol)
red
green
Fluorochrome Stains (More Sensitive):
• Used for greater sensitivity in detecting AFB, viewed under UV light. • Components: • \_\_\_\_\_\_\_\_\_\_\_ Stain AFB for 15 minutes. •. \_\_\_\_\_\_\_\_\_\_: Decolorizer, removes the stain from non-AFB. • Potassium permanganate or acridine orange: Counterstains the background, making AFB stand out. • Results: • AFB stain \_\_\_\_\_\_ or\_\_\_\_\_\_under UV light. • Non-AFB (tissue and debris) are non-fluorescent.
Auramine O and rhodamine B:
Acid alcohol
yellow-green or reddish-orange
Ziehl-Neelsen stained acid-fast bacilli
Reading
Interpretation (2N stain, WHO)
0
n+
1 +
2+
3+
No acid fast bacilli seen in 300 fields.
1-9 AFB/100 fields
10-99 AFB/100 fields
1-10 AFB per field in at least 50 fields
More than 10 AFB per field in at least 20 fields
Confirmatory Tests: characteristic growth and biochemical tests
• Gold standard:
Culture & susceptibility Testing
• Culture media (________ should be inoculated in parallel with selective broth media cultures (______, most sensitive).
(Lowenstein-Jensen or Middlebrook 7H10/7H11 biplate with antibiotics)
(Middlebrook 7H12, most sensitive)
• Incubate at______ in_____ for up to____ weeks; for slow growers NTM:
lower temp.______,___ weeks. 2 sets: ________ conditions
35-37°C; 5%-10% carbon dioxide; 8
24-35°C; 12; light and dark
• Conventional ID method:
rate of growth, colony morphology, pigmentation, and biochemical profiles (6-8 weeks)
= rapid growers;
produced pigment in light but not in darkness =
produced pigment in the dark =
nonpigmented/light tan/buff-colored colonies =
• Growth ≤ 7 days
photochromogens
scotochromogens
nonchromogens
Culture Media - Nonselective
• primary isolation from specimens, widely used
• ______medium;______ (inhibit growth of contaminating bacteria and provide contrasting color for mycobacteria)
• Löwenstein-Jensen (LJ) or Ogawa slants
Egg-glycerol
malachite green
MTB in LJ
• Mtb colonies
while M. bovis -
• fails to grow at____ or ____
off-white (buff) color, dry breadcrumb-like appearance, heaped up and luxuriant or “eugenic”
small, flat ‘dysgonic’ colonies
25°C or 41°C
• All mycobacteria are ______while M. bovis and M. africanum are____.
obligate aerobes
microaerophilic
Transparent Media
Growth detected In_____
7H10:____
7H11:____
10-12 days
dextrose
casein hydrolysate
(fastidious Mtb)
Liquid based:
7H12 broth
Middlebrook 7H9
BACTEC 12B Medium
• 7H12 broth
•_____ isolation
•____________-based medium
10 days
14C-labeled palmitic acid
Used for
• Drug susceptibility testing
BACTEC 12B Medium
• 7H12 broth
Used for growth inhibition on LJ or
Middlebrook media or on liquid media
BACTEC 12B Medium
• 7H12 broth
Middlebrook 7H9
contains glycerol
oleic acid
albumin
dextrose
is inhibited by glycerol.
• Mycobacterium bovis
Middlebrook 7H9
• Cultures should be read within_______ after inoculation and once a week thereafter for up to____ weeks.
5-7 days
8
Recent Methods for Diagnosis
• Specimens cultured in Middlebrook 7H9 broth containing C14-labeled palmitic acid & PANTA antibiotie mixture (polymyxin B, amphotericin B, nalidixic acid, trimethoprim, azlocillin - inhibits contaminating bacteria)
BACTEC 460 (rapid radiometric culture system)
PANTA antibiotie mixture
(polymyxin
B, amphotericin B, nalidixic acid, trimethoprim, azlocillin - inhibits contaminating bacteria)
BACTEC 460 (rapid radiometric culture system)
• Growing mycobacteria utilize acids, releasing_______, which is measured as growth index in the BACTEC instrument.
radioactive CO2
BACTEC 460 (rapid radiometric culture system)
Adv: rapid detection within_____, differentiates TB complex from NTM by____ test, high specificity
12 days
NAP test
BACTEC 460 (rapid radiometric culture system)
• Disadv:
expensive, hazardous use of radioactive material
More sophisticated equipment
Faster detection of growth
Higher sensitivity than solid media
Can also be used for drug-susceptibility testing
Liquid media
Recent Methods for Diagnosis
• Members of TB complex do not grow in the presence of________
BACTEC NAP Test
p-nitro-alpha-acetyl-amino-beta-hydroxypropriophenone (NAP)
Recent Methods for Diagnosis
BACTEC NAP Test
•______of NAP is added to actively growing culture in 12B medium vial.
______is inhibited while_____ do not demonstrate significant inhibition.
5 ug
TB complex
NTM/MOTT
BACTE NAP TEST
Other tests:
Molecular based - PCR and Gene probe
• Tube contains modified Middlebrook 7H9 broth base with OADC (oleic acid, albumin, dextrose, catalase) enrichment and PANTA antibiotic mixture.
Mycobacteria Growth Indicator Tube (MGIT)
Mycobacteria Growth Indicator Tube (MGIT)
• Tube contains modified Middlebrook 7H9 broth base with_______ enrichment and_______ antibiotic mixture.
OADC (oleic acid, albumin, dextrose, catalase)
PANTA (polymyxin B, amphotericin B, nalidixic acid, trimethoprim, and azlocillin)
• All types of clinical specimens can be cultured on this medium.
Mycobacteria Growth Indicator Tube (MGIT)
• A fluorescent compound is embedded in silicone at the bottom to the tube. Actively growing MTB consume oxygen & allow fluorescence to be observed using UV trans-illuminator lamp.
Mycobacteria Growth Indicator Tube (MGIT)
Recent Methods for Diagnosis
a. QIAGEN TB blood test: QuantiFERON-TB Gold In-Tube Test.
ELISA that detects interferon gamma in whole blood
b. T-SPOT-TB
ELISA ImmunoSpot assay that uses purified peripheral blood mononuclear cells
Interferon-Gamma Release Assays (IGRA)
Recent Methods for Diagnosis
Interferon-Gamma Release Assays (IGRA)
a.________: QuantiFERON-TB Gold In-Tube Test.
ELISA that detects interferon gamma in whole blood
b. ________ELISA ImmunoSpot assay that uses purified peripheral blood mononuclear cells
QIAGEN TB blood test
T-SPOT-TB
Interferon-Gamma Release Assays (IGRA)
- based on host’s immune responses to specific Mtb antigens:
ESAT-6 (early secretory antigenic target-6), CFP-10 (culture filtrate protein-10), and TB7.7, which are *absent from most________ - Interferon gamma are released by sensitized CD4 T cells.
- Disadvantage: Cannot be used in patients who are immunosuppressed.
Non-Tuberculous Mycobacteria (NTM) and Bacillus Calmette-Guérin (BCG)
As with other nucleic acid amplification tests, the Xpert MTE.RIF
assay should be interpreted along with clinical, radiographic, and other
GENE XPERT MTB-RIF
Conventional biochemical tests -
M. tuberculosis
•______ for niacin accumulation
•______ reduction of nitrate to nitrite
•______ production of catalase which is destroyed after heating (heat stable catalase negative) [30% H202]
•______ to thiophene-2-carboxylic acid hydrazide (T2H) [M. bovis____]
•______ to pyrazinamidase activity [M. bovis naturally_______]
Positive
Positive
Positive
Resistant; susceptible
Susceptible; resistant
• Assign a treatment partner/center to ensure compliance and motivational supervision to TB patients
• Effective regimens for the treatment of TB must contain multiple drugs to which the organisms are susceptible
Directly Observed Treatment Short-course for Tuberculosis (DOTS-TB)
• New patients presumed to have drug-susceptible TB:
2 months______, 4 months____
• In tuberculous meningitis, ethambutol(E) replaced by streptomycin (S)
HRZE
HR
H- - Inhibits cell wall synthesis
R- - inhibits RNA synthesis
Z- - destroys plasma membrane, inhibits metabolic processes
E- - obstructs formation of cell wall
ISONIAZID (300mg)
RIFAMPICIN (450mg)
PYRAZINAMIDE (1g)
ETHAMBUTOL (800mg)
•______ to thiophene-2-carboxylic acid hydrazide (T2H) [M. bovis____]
•______ to pyrazinamidase activity [M. bovis naturally_______]
R; S
S; R
BCG (Bacillus of Calmette and Guerin) Vaccine.
• consists of a live attenuated strain derived from______
Mycobacterium bovis.
• The vaccine is not 100% effective. Studies suggest a _____effective rate in children.
o It cannot circumvent disease reactivation in previously exposed individuals. It does not prevent infection, only disease.
o It may complicate the way the tuberculin skin test is read.
60- 80%
M. tuberculosis Complex
TuLABU
M. tuberculosis
M. leprae
M. africanum
M. bovis
M. ulcerans
