Respiratory Module: Lab Diagnosis Overview

Respiratory Module: Laboratory Diagnosis

• Focus: Laboratory diagnosis of bacterial, viral, and fungal upper respiratory tract infections.
• Target Audience: Medical students.
• Instructor: Dr. Haneya Anani, Professor of Medical Microbiology and Immunology, Faculty of Medicine.

Learning Outcomes, Lab 1

• Streptococcus pyogenes: Students will learn about the morphology and culture characteristics of this bacteria.
• Acute Follicular Tonsillitis: The laboratory diagnosis of acute follicular tonsillitis will be covered.
• Diphtheria, Vincent's Angina, and Oral Candidiasis: The laboratory diagnosis of these conditions will be studied.
• Viruses Causing Respiratory Tract Infection: Laboratory diagnosis of viruses causing respiratory tract infections is included.

Bacteria Causing Upper Respiratory Tract Infections

• Acute follicular tonsilitis
• Scarlet fever
• Diphtheria
• Vincent's angina
• Otitis media

Viruses Causing Upper Respiratory Tract Infections

• Adenovirus
• Rhinoviruses
• Corona viruses
• Epstein-Barr Virus (EBV)
• Parainfluenza type 1 and 2
• Respiratory syncytial virus (adults and children)
• Herpes simplex

Hints about Streptococci

• Classification: Aerobic and facultative anaerobes are classified according to hemolysis on blood agar.
• Beta-hemolytic streptococci: Produce complete lysis of red blood cells, releasing hemoglobin due to streptolysin S.
• Alpha-hemolytic streptococci: Cause incomplete lysis, forming green pigments.
• Non-hemolytic streptococci: Not classified by hemolysis; differentiated by carbohydrate cell wall antigens (Lancefield groups A-U). Groups A, B, C, D, and G cause human disease.
• Lancefield Classification: Illustrates streptococcal classification.

Diagnosis of Streptococcus pyogenes Tonsilitis

• Specimens: Throat swab
• Gram stain: Gram-positive cocci in chains
• Culture: Best growth on blood agar and chocolate agar; complete (beta) hemolysis, clear zone.
• Biochemical identification: Catalase-negative; Bacitracin-sensitive.

Identification of Streptococcus pyogenes

• Group A Streptococcus: Beta-hemolytic, sensitive to bacitracin.
• Catalase test: Negative
• Gram Stain: Gram positive cocci forming chains.
• Bacitracin Sensitivity: Sensitive to bacitracin

Lancefield Grouping Test

• Beta hemolytic streptococci: Identified by clumping blue latex particles with antibodies.
• Agglutination: Agglutination occurs when antibodies extracted from a throat swab react.

Laboratory diagnosis of Scarlet fever

• Causative agent: Streptococcus pyogenes (erythrogenic toxin)
• Spot diagnosis: Tonsilitis, rash, and strawberry tongue.
• Specimen: Throat swab
• Rapid strep test
• Throat culture: Gram-positive cocci in chains on blood agar.
• Specific test: In vivo toxin neutralization test (Schultz-Charlton test), which causes disappearance of erythematous area after antitoxin injection.

Diagnosis of Diphtheria Case

• Specimen: Throat swabs from membrane.
• Direct Smears: Gram stain showing Gram-positive bacilli
• Cultures: Loffler's serum (identifies suspected colonies by morphology, gram stain, and methylene blue).
• Culture on Blood Agar: Excludes Streptococcus pyogenes tonsillitis.
• Culture on Blood Tellurite Agar: Identifies types (black colonies)
• Toxigenicity test: Elek's test
• ELISA: Detects diphtheria toxin.

Carrier Diagnosis

• Specimen: Throat/nasal swabs (diagnosed as in a case).

Elek's Test

• Toxigenic strain detection: Double immunodiffusion test, which shows a line of precipitation in the presence of toxic strain.

Vincent's Angina

• Cause: Two anaerobic organisms (Treponema/Borrelia vincentii or Fusobacterium ulcerance)
• Characteristics: Acute necrotizing ulcerative gingivitis with ulcerative lesions in the oral cavity and tonsillar area.

Laboratory Diagnosis of Vincent's Angina

• Smear: Pus cells, spirochaetes, and Gram-negative fusiform bacilli.
• Gram stain: Sufficient for diagnosis.

Laboratory Diagnosis of Oral Candidiasis (Oral Thrush)

• Growth Media: Nutrient, blood agar, Sabouraud dextrose agar.
• Specimen: Throat swab.
• Direct Microscopic Examination: Gram-positive budding yeast cells with pseudohyphae.
• Culture: Sabouraud's dextrose, blood, or nutrient agar. Colonies identified by:
  • Morphology
  • Germ tube test (Candida albicans).
  • Biochemical tests.

Laboratory Diagnosis of Viral Infections

• General methods: Virus isolation and identification, detection of viral antigens in specimens, detection of viral antibodies, electron microscopy, and molecular diagnosis.
• Cell culture systems: Tissue culture, chick embryo, and laboratory (animal) models are used.
• Cytopathic effect (CPE): Degenerative cellular changes produced by many viruses in susceptible cell cultures. Observations are often made through an ordinary light microscope.
• Inclusion bodies: Viral material or cell debris aggregates in the cells.

Laboratory Diagnosis of Adenovirus

• Specimen: Secretions
• Detection: Antigens (ELISA), PCR
• Cell culture: Cells from man/animals in artificial culture media (glass bottles). Monolayer is critical for viral replication.

Detection of Viral Growth in Tissue Culture (CPE)

• CPE (Cytopathic Effect): Degenerative cell changes in susceptible cell cultures, observed from light microscopy. Virally induced changes often identify the causative virus. A change may be the presence or absence of inclusion bodies.

Other methods for detecting viral growth in tissue culture:

• Haemagglutination: Some viruses cause haemagglutination when mixed with erythrocytes; this can be a method to detect those viruses.

Viral Haemangglutination tests (Influenza and Parainfluenza viruses)

• Titer detection: The titer is the inversion of the dilution in the last well, which includes small clumps of erythrocytes.

Epstein-Barr Virus (EBV) Case

• Presentation: Fever, sore throat, enlarged lymph nodes.
• Lymphocyte infection: B lymphocytes through CD21. Infected cells are then destroyed by CD8 lymphocytes that produce atypical lymphocytes.
• Laboratory Diagnosis:
  • Atypical T lymphocytes in blood picture
  • EBV nucleic acid detection by PCR
  • Detection of heterophile antibodies (Paul Bunnell test or monospot test)

Laboratory findings of infectious mononucleosis/EBV viral infection

• Monospot test: EBV antibodies agglutinate sheep red blood cells in a slide-based or tube-based technique.

Lab 2: Lower Respiratory Tract Infections

• Focus: Laboratory diagnosis of selected pathogens causing lower respiratory tract infections.
• Learning Outcomes:
  • Morphology and culture characteristics of Streptococcus pneumoniae.
  • Laboratory diagnosis of Pseudomonas, Klebsiella infections, pulmonary anthrax, and Mycobacterial tuberculosis.
  • Laboratory diagnosis of fungal pulmonary infections.

Mycobacterium tuberculosis

• Morphology: Thin straight or curved rods, acid-fast (not Gram-positive or Gram-negative).
• Culture: Obligate aerobe. Grows only on selective media like Lowenstein-Jensen medium. Slow growth rate (2-8 weeks).
• Specimen: Sputum (3 consecutive morning samples)
• Isolation: Direct smears on sputum, staining with Ziehl-Neelsen stain. Decontamination and concentration of the sputum.

Rapid Diagnosis by Culture on Fluid Media

• Growth: Mycobacteria grow more rapidly and reliably in liquid cultures than solid cultures.
• Direct Visualization (e.g., smear): Visualization of red-pink long or slightly curved bacilli often demonstrates the causative agent.

Rapid Diagnosis by Culture (C.BACTEC and Mycobacteria indicator tube)

• C.BACTEC systems: Culture media utilizing palmitic acid. Growing bacteria utilize the palmitic acid. Released radioactive CO2 detected by machine.
• Mycobacterium indicator tube: Fluid media containing a fluorescent sensor, enabling rapid identification when mycobacteria consume O2. Fluorescence is detected by UV light.

Laboratory Diagnosis of Fungal Pulmonary Infection (Aspergillosis):

• Specimens: Bronchial lavage, sputum, biopsy.
• Direct examination: Septate hyphae and characteristic Aspergillus heads (Lactophenol cotton blue stain).
• Culture: SDA medium, observing surface colonies (green velvety, powdery) and reverse color (cream/tan).

Laboratory Diagnosis of Coccidioidomycosis:

• Causative fungus: Coccidioides immitis
• Specimen: Sputum or tissue biopsy
• Microscopy: Microscopic examination displays spherules containing endospores.
• Culture: Culture on SDA medium.
• Serology: Latex agglutination test is best for diagnosis.

Laboratory Diagnosis of Lobar Pneumonia , (caused by Streptococcus pneumoniae)

• Specimen: Sputum
• Microscopy: Gram-positive diplococci with capsules.
• Culture: Alpha hemolysis on blood agar. Colonies identified through Gram-stained films showing gram-positive diplococci with capsules.
• Biochemical identification: Catalase-negative; Quellung test positive; differentiation from Streptococcus viridans.

Laboratory Diagnosis of Atypical Pneumonia (caused by Mycoplasma pneumoniae)

• Specimen: Throat swabs, respiratory secretions, sputum
• Direct Detection: PCR;
• Culture: Enriched media containing serum displaying Fried egg appearance after 3-10 days: grows better at 10% CO2.

Laboratory Diagnosis of Chlamydia Infection:

• Methods: PCR, direct microscopic examination (Giemsa stain), direct immunofluorescence, detection of chlamydial antigens, isolation in tissue cultures.

Laboratory Diagnosis of Acute Epiglottitis (by Haemophilus influenzae):

• Specimens: Sputum
• Microscopy: Gram-negative coccobacilli;
• Capsule detection: Quellung reaction (swelling of capsule after antiserum addition).
• Culture: Chocolate agar, 5-10% CO2 at 37°C.

Satellite Phenomenon:

• Growth: Haemophilus grow near colonies of Staphylococcus on blood agar.
• Identification: Haemophilus does not grow on blood agar alone.

Laboratory Diagnosis of Pulmonary Anthrax:

• Specimen: Sputum
• Microscopy: Gram-positive large bacilli (with square ends), arranged in a “bamboo-stalk” pattern, have capsules .
• Staining: Demonstration through polychrome methylene blue stain.
• Cultures: Non-haemolytic reactions with Medusa head colonies often visualized on blood agar.
• Medium: Gelatin medium produce inverted “fire tree” appearance (due to slow gelatin liquefiers).

This quiz covers the laboratory diagnosis of various bacterial, viral, and fungal infections affecting the upper respiratory tract. Medical students will explore specific pathogens such as Streptococcus pyogenes and conditions like acute follicular tonsillitis, as well as viral causes of respiratory infections. This is an essential assessment for understanding clinical microbiology and immunology in respiratory diseases.