Tuberculosis: Mycobacterium & Acid-Fast Staining

Tuberculosis (TB) Overview

• Tuberculosis poses a chronic infectious disease
• Tuberculosis is caused by Mycobacterium tuberculosis
• It primarily affects the lungs (pulmonary TB)
• Tuberculosis can also affect other organs (extra-pulmonary TB)

Mycobacterium tuberculosis Characteristics

• Mycobacterium tuberculosis is aerobic, acid-fast bacillus
• It also grows slowly and is non-spore-forming

Mycobacterium tuberculosis Cell Wall

• The Mycobacterium tuberculosis cell wall contains a high amount of mycolic acids
• Mycolic acids are long-chain fatty acids
• These acids create a waxy, hydrophobic (water-resistant) layer around the bacteria
• The waxy, hydrophobic layer makes the Mycobacterium tuberculosis resistant to conventional staining methods

Term "Acid-Fast" Definition

• During the Ziehl-Neelsen stain procedure, bacterial cells are exposed to a strong acidic solution after being stained with a red dye (carbol fuchsin)
• Mycobacterium tuberculosis retains the dye and appears bright red under the microscope
• Mycobacterium tuberculosis can resist acid decolorization
• Because of this ability to resist acid decolorization, Mycobacterium tuberculosis is termed "acid-fast"

Pathophysiology of Tuberculosis

• Mtb, Mycobacterium tuberculosis, enters the lungs and is engulfed by alveolar macrophages
• Mtb survives and replicates inside macrophages by inhibiting the fusion of lysosomes with phagosomes
• Humans are the only reservoir for Mycobacterium tuberculosis
• Alveolar macrophages and neutrophils are the target cells
• Mycobacterium tuberculosis can cause granulomas in the lungs

Granuloma Formation

• Granulomas are small, localized clusters of immune cells that form in response to chronic inflammation
• Granulomas develop when the immune system attempts to isolate substances it cannot easily eliminate
• Examples of substances that are difficult to eliminate include infections (e.g., tuberculosis), foreign materials, or autoimmune conditions
• Fibrotic granuloma involves the formation of scar tissue
• Caseous granuloma is characterized by a cheese-like appearance of the tissue at the center of the granuloma
• Necrotizing granuloma, a part of the granuloma often at its core, experiences cell death due to inflammation and/or infection

Spread of Tuberculosis in Body

• Local spread involves macrophages tracking the infection
• Lymphatic spread involves tuberculous lymphadenitis
• Blood spread occurs as an extension of lymphatic involvement
• Extra pulmonary TB can affect the lungs, spleen, liver, kidneys, and central nervous system (CNS), potentially leading to tuberculous meningitis

Symptoms of Tuberculosis

• A persistent cough lasting more than 3 weeks may be a symptom
• Hemoptysis (coughing up blood) may be another symptom
• Night sweats, fever, and weight loss are classic signs of systemic symptoms
• Fatigue and anorexia can also be symptoms

Laboratory Diagnosis of Tuberculosis

• Microscopy involves Acid-Fast Staining of specimen
• Culturing aims to isolate Tuberculosis bacilli in the culture
• In Vivo tests involve infecting experimental animals
• Hypersensitivity testing assesses the reaction to TB proteins using a Tuberculin skin test
• Interferon Gamma Release Assay (IGRA) is used for detection of latent infections in asymptomatic individuals
• DNA detection, using PCR, is the most specific method
• Chest X-ray, is the first imaging study, used to detect lung cavitation, infiltrates, and granulomas

Treatment for Tuberculosis

• First-line drugs for active TB include Isoniazid (INH), Rifampin (RIF), Pyrazinamide (PZA), and Ethambutol (EMB)
• Isoniazid (INH) inhibits bacterial cell wall synthesis
• Rifampin (RIF) inhibits bacterial RNA synthesis
• Pyrazinamide (PZA) lowers the pH in the bacteria's environment, enhancing the activity of the other drugs
• Ethambutol (EMB) inhibits the synthesis of the bacterial cell wall and is used to treat TB caused by resistant strains
• Surgery may be required for hydrocephalus
• About 25% of patients have residual sequelae
• Second-line drugs, including fluoroquinolones and aminoglycosides, may be used
• Drug susceptibility testing is essential for determining which second-line drugs will be effective

Sputum Collection Steps

• Proper sputum collection requires patient preparation and specific collection instructions
• Ensure the patient washes their hands and mouth
• Provide a sterile sputum container with a secure lid
• Collect sputum early in the morning for concentration
• Instruct the patient to take a deep breath, hold it, and cough forcefully
• Ask the patient to spit directly into the container, avoiding contact with the mouth
• It is important to avoid contamination
• Do not touch the container's rim or inside with hands
• Label the container with the patient's details
• Store sputum samples in a cool, dry place (4°C) if not immediately sent to the lab
• Transport samples to the laboratory for testing promptly, especially for acid-fast bacilli (AFB) smear microscopy
• Proper sputum collection is critical for accurate diagnosis of TB and the identification of drug-resistant strains

Acid-Fast Stain (Ziehl-Neelsen stain)

• Members of the genus are resistant to simple and gram staining
• Members of the genus Mycobacterium are difficult to penetrate the dyes due to cell walls
• Once the dye has penetrated, it cannot be removed, and these organisms are called acid-fast
• The stain binds to the mycobacterial cell wall

Acid-Fast Stain Solution

• After staining, an acid decolorizing solution is applied
• The acid decolorizing solution removes the red dye from background cells, tissue fibers, and organisms except mycobacteria, referred to as acid-fast bacilli (AFB)
• Following decolorization, the sputum smear is counterstained with malachite green or methylene blue
• The counterstain stains the background material
• The counterstain provides a contrast color against the red AFB

Acid-Fast Stain Procedure

• Prepare a smear of the microorganism on a clean slide or collect sputum and make the sputum smear
• Put the slide over a steam bath and flood the smear with strong carbol fuchsin for 5 minutes, preventing stain from drying or boiling
• Allow to cool and then wash with tap water
• Decolourize with acid alcohol until the effluent runs almost clear with a slight red tinge
• Wash with tap water and counterstain with methylene blue for 2 minutes
• Wash smear with tap water, blot dry, and examine with a microscope
• Acid-fast bacteria appear red, while non-acid-fast bacteria appear blue

Results of Acid-Fast Staining

• Use carbol fushin with heat

  • Acid fast: Red (hot pink)
  • Non-acid fast: Red (hot pink)

• Use Acid alcohol

  • Acid fast: Red
  • Non-acid fast: Colorless

• Use Methylene Blue/Malachite Green

  • Acid fast: Red
  • Non-acid fast: Blue (Green)

Explore Tuberculosis (TB), a chronic infectious disease caused by Mycobacterium tuberculosis, focusing on its characteristics as an aerobic, acid-fast bacillus with a unique cell wall. Learn about its impact on lungs and other organs. Understand the acid-fast characteristic of Mycobacterium tuberculosis through Ziehl-Neelsen staining.