Microbiology: Mycobacterium tuberculosis Quiz

Mycobacteria Overview

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

Classification of Mycobacteria

• Mycobacteria are classified based on habitat, host, and biochemical properties.
• Mycobacterium tuberculosis (MTB) is spread through airborne droplets.
• Infection occurs via inhalation.
• Clinical features of tuberculosis (TB) include cough, fever, night sweats, and weight loss.

Lab Diagnosis

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

Definition of Mycobacteria

• Rod-shaped aerobic bacteria.
• Possessing a unique cell wall structure yielding acid fastness.
• This structure makes them resistant to staining and disinfection.
• This structure also makes them difficult to treat and contributes to their persistence in the environment.

Classification of Mycobacteria Complex

• Mycobacterium tuberculosis complex (MTC) includes several species (M. tuberculosis, M. bovis, and M. africanum).
• These species cause TB in humans and animals.
• Atypical Mycobacteria (non-tuberculous mycobacteria, NTM) are diverse and often found in the environment, causing infections in people with impaired immune systems.
• M. leprae, a non-cultivable species, is responsible for leprosy.

Classification based on Habitat

• Obligate pathogens thrive exclusively in living hosts, relying on the host for resources. M. tuberculosis and M. leprae are examples.
• Facultative/opportunistic pathogens can exist independently or become pathogenic depending on host conditions (e.g., weakened immune system). M. avium complex is an example.
• Free-living saprophytes primarily live in the environment (soil, water, decaying matter).

Classification based on Host

• Tubercle Bacilli (M. tuberculosis) primarily infect humans, causing the most common type of TB worldwide.
• Bovine Bacilli (M. bovis) primarily infect cattle and is a less frequent cause of TB in humans.
• Lepra Bacilli (M. leprae) is the causative agent of leprosy primarily affecting skin, peripheral nerves, upper respiratory tract, and eyes.

Mycobacterium Tuberculosis Complex

• A group of closely related bacteria that cause tuberculosis.

M. tuberculosis Characteristics

• Morphology: slightly curved, rod-shaped bacillus, 0.2-0.5 microns in diameter and 2-4 microns in length. Acid-fast.
• Growth characteristics: slow, with a generation time of approximately 18-24 hours, impacting the length of TB treatment.
• Cell wall composition: thick, waxy lipid wall resists environmental factors, critical for virulence and survival.

Route of Transmission

• Primarily transmitted through airborne droplet nuclei (1-5 micrometers).
• Transmission occurs during coughing, sneezing, speaking, and singing.
• Droplets stay suspended in air for extended periods, posing a risk to those inhaling them.

Pathogenesis

• Primary infection with M. tuberculosis, often referred to as primary TB, is the initial infection.
• Post-primary tuberculosis is a subsequent disease in individuals with previous sensitization (likely due to prior infection). This can manifest from reactivation of a primary infection or exposure to a new infection.
• Both types exhibit different pathological features.

Clinical Features

• Tuberculosis (TB) can affect various body parts.
• Pulmonary TB, the most common form, affects the lungs.
• Extrapulmonary TB affects organs like lymph nodes, brain, kidneys, and bones.

Symptoms and Signs of Tuberculosis

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

Extrapulmonary Tuberculosis

• TB that spreads beyond the lungs, affecting various organ systems.
• Common sites of extrapulmonary TB include the genitourinary system, meninges, gastrointestinal tract, skin, lymph nodes, bone marrow, spine, and joints.
• Symptoms depend on the affected organ.

Diagnosis of TB Infection

• Non-specific tests (ESR, CBC) detect immune response.
• Radiological examination (chest X-ray) identifies characteristic patterns of lung involvement.
• Histopathological examination (lymph node biopsy) reveals hallmark TB features.
• Hypersensitivity tests (TST, QFT) detect specific TB antigens.
• Bacteriological examination (direct smear, culture) confirms the presence of AFB (Acid Fast Bacilli).

Bacteriological Examination

• Sputum is the most common specimen for diagnosing pulmonary TB.
• Blood cultures are used to detect TB in cases of disseminated TB or when the infection has spread to the bloodstream.
• Urine may be needed if kidney involvement suspected.
• Biopsies of affected tissues (lung, lymph nodes, etc.) are important in diagnosing extrapulmonary TB.

Examination of Direct Smears

• Ziehl-Neelsen stain is a rapid, specific, and inexpensive acid-fast staining technique for identifying Acid Fast Bacilli (AFB) in a sample.
• AFB resist decolorization because of mycolic acids in their cell walls.

Culture Advantages and Limitations

• Sensitivity: Culture is highly sensitive, detecting low bacterial loads, including cases of limited bacilli.
• Specificity: Culture is highly specific, accurately identifying Mycobacterium species, crucial for accurate diagnosis & treatment.
• Time Consuming: Culturing takes several weeks delaying diagnosis & impacting patient care.

Types of Culture Media

• Lowenstein-Jensen media: An egg-based, traditional method for cultivating M. tuberculosis. Includes glycerol, asparagine, and malachite green.
• Middlebrook media: Available in agar and broth forms, designed for rapid M. tuberculosis growth, with consistent nutrient composition allowing selective agent use (e.g., pyrazinamide, ethambutol).
• BACTEC media: An automated radiometric system measuring CO2 production to detect M. tuberculosis growth rapidly.
• MGIT media: Automated non-radiometric based on fluorescence of oxygen consumption to detect Mycobacterial growth.

Culture Characteristics

• M. tuberculosis grows only on egg-selective media (Lowenstein-Jensen medium).
• Acid-fast bacilli can form serpentine chains called cord factor.
• Growth is slow, typically 2-8 weeks.

Middlebrook Media

• A solid medium enriched with a variety of nutrients (oleic acid, albumin, dextrose, catalase, glycerol, and malachite green) for mycobacterium growth.

Middlebrook Media Advantages & Disadvantages

• Advantages: Provides rapid results; helps identify the causative organism (important for targeted treatment); offers information about resistance patterns to different antibiotics.
• Disadvantages: Expensive; prone to contamination, making follow-up studies less suitable.

BACTEC MGIT

• A rapid, automated method for detecting mycobacteria using a modified Middlebrook OADC medium with a silicon film as a fluorescence indicator. Output of a sensor decreases with bacterial presence for rapid outcome measurement.

Molecular Techniques

• Molecular probes (NAP) help identify mycobacterial growth from both solid and liquid media.
• PCR: A powerful tool to detect M. tuberculosis even before culture results become available. Amplifies specific DNA and detects drug resistance mutations.

Sample Collection and Handling

• Sterile Technique: Use aseptic techniques to avoid contamination throughout sample collection.
• Specimen Selection: Choose appropriate specimen (e.g. sputum, blood, urine, tissue) based on suspected infection site.
• Proper Labeling: Label specimens clearly with patient information and collection date.
• Transport Media: Use specialized transport media to maintain viability.
• Refrigeration: Refrigerate collected samples promptly to preserve mycobacteria viability.

Cultivation of Mycobacteria

• Media Selection: Use selective media such as Löwenstein-Jensen or Middlebrook 7H10 media.
• Incubation: Incubate media at 35-37°C with 5-10% CO2.
• Growth Time: Mycobacteria can take 2-8 weeks to form visible colonies.
• Safety Precautions: Adhere to biosafety levels (BSL-3) in labs handling potential pathogens during handling.

Growth Requirements

• Temperature range: 35°C to 37°C
• Oxygen requirement: Aerobic, requiring oxygen for growth.
• Nutrients: Specific nutrients (e.g., glycerol, fatty acids, amino acids).
• Time to visible growth: Takes weeks to months due to slow growth.

Non-Specific Tests

• Tuberculin tests (e.g. Mantoux, PPD) screen for tuberculosis. Measure the immune response to M. tuberculosis, not the bacteria itself. Based on delayed hypersensitivity principle.

TST Interpretation

• A positive TST (induration >10 mm) suggests exposure to M. tuberculosis, possibly indicating latent TB infection.
• A negative TST (induration <10 mm) suggests no prior M. tuberculosis infection, though other factors influence results.

TST Advantages & Disadvantages

• Advantages: Easy to administer, safe, inexpensive, valuable for latent TB detection
• Disadvantages: Multiple patient visits; limited specificity due to cross-reactivity; potentially influenced by prior BCG vaccination; does not reliably differentiate between active and latent infection; high rate of false positives and negatives

Interferon Gamma Release Assays (IGRAs)

• QuantiFERON-GOLD: A blood test measuring immune response to specific M. tuberculosis antigens for rapid TB detection
• Advantages: Results within 24 hrs; Avoids second visit; Less risk of false positives compared to TST.

BCG Vaccine

• A live-attenuated vaccine derived from Mycobacterium bovis to prevent TB.
• Compulsory for newborn infants in many regions.
• Not a guarantee to prevent infection, but confers some immunity.
• Not recommended for immunocompromised individuals.

Tuberculosis Control

• Requires a multifaceted approach (prevention, early detection, effective treatment).
• The WHO’s "End TB Strategy" aims to reduce TB deaths and incidence.

Treatment of TB

• A long-term process requiring a combination of drugs like isoniazid, rifampin, pyrazinamide, and ethambutol.
• Targets different M. tuberculosis metabolic pathways
• Adherence to the prescribed medication regimen is crucial. Poor adherence leads to drug resistance.

Test your knowledge on the diagnosis and cultivation of Mycobacterium tuberculosis. This quiz covers key advantages and disadvantages of various techniques, including culture methods and staining procedures. Assess your understanding of the characteristics and requirements for handling mycobacterial cultures effectively.