Tuberculosis and Mycobacteria Quiz

Mycobacteria Overview

• Mycobacteria are a genus of bacteria.
• They are characterized by their waxy cell walls.
• This unique feature makes them resistant to many disinfectants.
• Their appearance under a microscope is distinctive.

Mycobacteria Classification

• Mycobacteria are classified based on their habitat and host.
• Some species are responsible for tuberculosis in humans and animals.
• The Mycobacterium tuberculosis complex (MTC) is a group of closely related bacteria causing tuberculosis (TB)
• Includes: M. tuberculosis, M. bovis, and M. africanum.
• Atypical mycobacteria (non-tuberculous mycobacteria (NTM)) are a diverse group.
• Often found in the environment
• Can cause infections in humans.
• Particularly problematic in those with compromised immune systems.
• M. leprae is a unique species of mycobacteria that cannot be cultured in artificial media.

Mycobacteria Transmission and Pathogenesis

• Mycobacterium tuberculosis (MTB) spreads through airborne droplets from infected individuals.
• Infection occurs through inhalation.
• Droplet nuclei (1-5 micrometers in diameter) remain suspended in the air.
• Procedures involving infected lesions or clinical specimens can generate these droplets.

Mycobacteria Clinical Features

• Cough, fever, night sweats, and weight loss are common clinical features of tuberculosis.
• Tuberculosis (TB) can affect various parts of the body, primarily the lungs (pulmonary TB).
• It can also affect other organs (extrapulmonary TB) such as lymph nodes, brain, kidneys, and bones.

Mycobacteria Diagnosis

• Diagnosis involves microscopic examination of sputum, culture, indirect and molecular methods.

Mycobacteria Definition (Structure)

• Rod-shaped aerobic bacteria
• Possess a unique cell wall structure, resisting staining and disinfection.
• The waxy cell wall gives them characteristic acid-fastness.
• The cell wall also makes them difficult to treat contributing to their ability to persist in the environment and within host cells.

Classification Based on Habitat

• Obligate pathogens thrive exclusively in a living host.
• Facultative/Opportunistic pathogens can exist independently but pathogenic under specific conditions. An example is the M. avium complex. (which is a group of related mycobacteria)
• Free-living saprophytes primarily exist in the environment (soil, water, or decaying matter). They typically do not infect humans, unless the bacteria enter the body through a wound.

Classification Based on Host

• M. tuberculosis (MTB) primarily infects humans and causes most globally occurring TB cases.
• M. bovis primarily found in cattle, and is a rare cause of TB in humans.
• M. leprae is the causative agent of leprosy. It mainly affects the skin, peripheral nerves, upper respiratory tract, and eyes.

Mycobacterium Tuberculosis Complex

• A group of closely related bacteria causing TB
• The primary cause of tuberculosis.

Route of Transmission

• Mycobacterium tuberculosis is primarily transmitted through the air via droplet nuclei.
• Droplets are released into the air when an infected individual coughs, sneezes, speaks, or sings.
• Droplet nuclei can remain suspended in the air for extended periods, posing risks to healthcare professionals handling infected materials.

Pathogenesis (Primary and Post-primary)

• Initial infection with M. tuberculosis is known as a primary infection.
• Subsequent disease in a previously sensitized person, either from an exogenous source or by reactivation of a primary infection, is known as post-primary tuberculosis.
• Primary and post-primary TB exhibit different pathological features.

Clinical Features of TB

• TB can affect various body parts, usually the lungs (pulmonary TB).
• Extrapulmonary TB can affect other organs, including lymph nodes, brain, kidneys, and bones.

Symptoms of Tuberculosis

• Common symptoms include cough (dry or productive), shortness of breath, fever and night sweats, and chest pain.

Extrapulmonary TB

• When M. tuberculosis spreads beyond the lungs, it affects various organs and systems of the body.
• This widespread form of TB is called extrapulmonary tuberculosis.

Diagnosis of TB Infection

• Non-Specific Tests: Employ general tests such as elevated erythrocyte sedimentation rate (ESR) and complete blood count (CBC) with monocytosis and neutrophilic leukocytosis.
• Radiological Examination: Chest X-ray plays a crucial diagnostic role. It can reveal characteristic patterns of lung involvement in TB infection.
• Hypersensitivity Tests: Tuberculin skin test (TST) and QuantiFERON-TB Gold (QFT) are used to detect TB infection (identify immune response).
• Histopathological Examination: Lymph node biopsy can show granulomatous inflammation with caseation necrosis, indicative of TB infection.
• Bacteriological Examination: Direct smear microscopy (e.g., Ziehl-Neelsen stain) and culture, are necessary for confirming TB infection.

Bacteriologic Methods

• Sputum, blood, urine and biopsy samples are typically used for analysis.
• Sputum is usually used to diagnose pulmonary TB.
• Blood cultures can assist in detecting tuberculosis infections when they have spread throughout the body.
• Urine samples are commonly useful to ascertain whether the infection has reached the kidneys.
• Biopsies of affected tissues like lungs, lymph nodes, etc. help confirm the presence of TB bacteria.

Examination of Direct Smears (e.g., Ziehl-Neelsen Stain)

• Ziehl-Neelsen stain is used to identify acid-fast bacilli (AFB) in a sample.
• AFB are characterized by their resistance to decolorization with acid alcohol.
• The resistance is due to the presence of mycolic acids within the cell wall of AFB.
• Important advantage: Rapid, Specific, Inexpensive.
• Important disadvantage: Less sensitive than other methods, and often needs large amounts of bacteria (10,000+ organisms/mL) to be effective.

Limitations of Ziehl-Neilsen Stain

• Requires a high concentration of acid-fast bacteria (AFB) for a positive result.

Mycobacterial Culture Methods

• Specific media (e.g., Lowenstein-Jensen, Middlebrook) are essential for culturing Mycobacteria.
• Sensitivity: Highly sensitive, enabling detection of low bacterial loads in cases such as limited bacilli.
• Specificity: Highly specific, enabling accurate identification of Mycobacterium species.
• Time Consuming: Culture typically requires several weeks for visible growth.

Culture Characteristics

• Growth: M. tuberculosis grows slowly on specific media, e.g., Lowenstein-Jensen. Needs a 5-10% CO2 atmosphere. Takes 2-8 weeks to visually identify colonies.
• Virulence factor: Ability to survive within macrophages inside the host makes it challenging to eliminate. Form serpentine cord factor that arranges AFB into chains to protect their structural integrity.

Middlebrook Media

• Commonly used for culturing mycobacteria.
• Enriched medium (agar based) containing various nutrients (oleic acid, albumin, dextrose, catalase, glycerol, and malachite green) for mycobacterial growth.
• Often used in combination with other diagnostic tests, e.g., Ziehl-Neelsen stain to help confirm tuberculosis diagnosis.
• Advantages: Rapid results enabling identification of causative organism and medication sensitivity testing.
• Disadvantages: Expensive, and susceptible to contamination during laboratory processes.

BACTEC MGIT (Mycobacteria Growth Indicator Tube)

• A rapid and automated method for detecting mycobacteria.
• Utilizes modified Middlebrook OADC medium with key nutrients for mycobacterial growth, and a silicon film that measures oxygen consumption enabling rapid detection.
• Advantage: Can detect growth in as little as 5 days, a key advantage compared to traditional culture methods.

Molecular Techniques

• Molecular Probes (NAP): Technique used for identification of Mycobacterial growth from solid or liquid media through specific DNA sequence detection and identification.
• PCR (Polymerase Chain Reaction): Powerful tool for detecting M. tuberculosis directly in clinical samples, even before culture results available. Amplifies specific DNA sequences for rapid and sensitive pathogen detection. Can evaluate drug resistance mutations.

Sample Collection and Handling

• Use sterile technique for sample collection to avoid contamination.
• Select appropriate specimen (sputum, blood, urine, or tissue) based on suspected infection location.
• Label samples clearly with relevant information (patient details, collection date).
• Use specialized transport media.
• Refrigerate samples promptly.

Cultivation of Mycobacteria

• Media Selection: Use media specifically designed for mycobacterial growth, like Löwenstein-Jensen or Middlebrook 7H10.
• Incubation: Incubate the samples at 35-37°C and 5-10% CO2 atmosphere
• Growth Time: Mycobacteria grow slowly, requiring 2-8 weeks for visible colonies to form.
• Safety Precautions: Handle mycobacterium cultures in a BSL-3 (biosafety level 3) laboratory.

Growth Requirements

• Temperature: Typically thrive between 35-37°C.
• Oxygen: Most mycobacteria are aerobic and require oxygen for growth
• Nutrients: Require specific nutrients such as glycerol, fatty acids, and amino acids
• Time: Slow growth rates, cultures need to be incubated for several weeks, sometimes months.

M. tuberculosis Characteristics

• Morphology: Slightly curved, rod-shaped bacillus. About 0.2-0.5 microns wide and 2-4 microns in length.
• Acid fast: Resist decolorization with acidified alcohol after basic fuchsin staining.
• Growth Characteristics: M. tuberculosis multiplies slowly, usually 18-24 hours a generation.
• Cell Wall Composition: Thick, waxy lipid cell wall that provides resistance to environmental factors helping in host survival.

Non-Specific Tests (TB Screening)

• Non-specific tests are used for tuberculosis screening. They measure immune response to M. Tuberculosis, not directly measure the bacteria itself. Examples are tuberculin tests, like the Mantoux test or PPD, which are based on delayed-type hypersensitivity.

Tuberculin Skin Test (TST) Interpretation

• TST assess individual's immune response to Mtb antigen.
• Positive reaction (induration >10mm) suggests exposure to Mtb.
• Negative reaction (<10mm) suggests no prior Mtb infection.
• Other factors (BCG vaccination and allergies) can influence TST results.

Interferon Gamma Release Assays (IGRAs)

• IGRAs are blood tests (Quantiferon-GOLD) for detecting latent TB infection.
• Advantages: Rapid results (within 24 hours), no need for a second visit, less risk of false positives.
• Measures immune response to specific TB antigens in a blood sample.

BCG Vaccine

• Live attenuated vaccine derived from Mycobacterium bovis.
• Given via intradermal injection.
• Compulsory for newborns in some regions.
• Helps prevent miliary and disseminated TB.
• Does not prevent all TB infections.
• Not suitable for individuals with impaired immunity.

Summary Questions (with answers)

• Question: How long can the MGIT system take to detect mycobacterial growth?
  • Answer: 5 days.
• Question: What is a primary advantage of using culture over direct smear examination?
  • Answer: Culture can identify low bacterial loads effectively.
• Question: Is smear microscopy effective with low microbial densities?
  • Answer: No. (Smear microscopy requires a high concentration of bacteria to be effective)
• Question: What types of specimen are used to support TB diagnosis?
  • Answer Typical specimens for bacterial analysis include sputum, blood, urine, or tissue depending on the suspected infection site.

Test your knowledge on the characteristics of Mycobacteria, including their transmission, clinical features, and diagnostic methods. This quiz covers essential aspects of tuberculosis and extrapulmonary infections, including staining techniques and culture methods.