The following CBME core competencies are covered in this chapter. EN2.9: Choose correctly and interpret microbiological investigations relevant to the ENT disorders.

Microbiology in ENT

Introduction

Microbiological examination forms the backbone of diagnosing and managing infectious ENT diseases. Therefore, choosing the right sample, collecting it correctly, and interpreting the laboratory report accurately can save lives, especially in conditions like rhino-cerebral mucormycosis or lateral sinus thrombophlebitis. This chapter provides a complete, standalone guide to mastering ENT microbiology for theory, practical, and PG entrance exams.

Common ENT Infections

1. Pharyngitis and Tonsillitis. Most pharyngitis cases are viral. However, the most common bacterial agent is Group A beta-hemolytic Streptococcus pyogenes. Therefore, a throat swab for culture is essential to diagnose streptococcal sore throat, diphtheria, gonococcal pharyngitis, or candidal thrush. In viral pharyngitis, culture remains negative.
Advanced Diagnosis – Multiplex PCR for Pharyngitis: When a viral or atypical bacterial aetiology is suspected, a single nasopharyngeal swab can detect adenovirus, coronaviruses (including SARS-CoV-2), influenza A/B, RSV, parainfluenza, Bordetella pertussis, Mycoplasma pneumoniae, and Chlamydia pneumoniae via multiplex PCR. However, its high cost limits routine use.

2. Sinusitis. Most acute sinusitis is viral. Nevertheless, community-acquired acute purulent sinusitis most commonly involves Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, and Streptococcus pyogenes. In chronic sinusitis, the main isolates shift to Staphylococcus aureus and anaerobic bacteria.
For nosocomial sinusitis or in immunocompromised hosts, consider Pseudomonas aeruginosa and other Gram-negative bacilli. In neutropenic patients, fungi (especially Aspergillus and Mucorales) and Pseudomonas are common.
Life-Saving Tip: In an immunocompromised patient with facial swelling, black eschar, and nasal discharge, immediately perform a KOH mount of the nasal secretion. Rapid diagnosis of Mucor or Rhizopus (aseptate hyphae) allows early surgical debridement and amphotericin B, which is lifesaving.

3. Otitis Media. In acute otitis media (AOM), the three most frequent pathogens are Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis. In older children, Streptococcus pyogenes is also possible, while Chlamydia pneumoniae affects younger children. Therefore, antibiotics must cover these organisms.

4. Lateral Sinus Thrombophlebitis. This is an intracranial complication of otitis media. Therefore, you must order a blood culture (collected during fever spikes) in addition to an ear swab culture. The common pathogen is Streptococcus or Staphylococcus. Remember, a negative blood culture does not rule out the condition.

Types of ENT Samples for Microbiological Examination

You can send various clinical samples from ENT patients for microbiological analysis. These include pus or secretions on swabs, fine-needle aspirates, washings from sinuses or middle ear, tissue biopsies, blood, and cerebrospinal fluid (CSF). Consequently, the choice of sample directly affects the diagnostic yield.

Table: ENT Samples and Their Clinical Applications

Sample Type	Common ENT Conditions	Key Note
Throat swab	Pharyngitis, tonsillitis, diphtheria	Avoid touching the tongue/cheeks
Nasal swab/discharge	Sinusitis, rhinitis, COVID-19	Use a nasal endoscope for the middle meatus
Ear discharge swab	Otitis media, otitis externa	Clean the external canal first
Pus from an abscess	Peritonsillar abscess, neck abscess	Aspirate preferred over swab
Tissue biopsy	Fungal sinusitis, TB lymphadenitis	Send in saline, not formalin
Blood	Lateral sinus thrombophlebitis, sepsis	Collect during a fever spike
Cerebrospinal fluid (CSF)	Meningitis, intracranial complications	Collect by lumbar puncture

Practical Tip for Viva: Always label the sample with the exact anatomical site (e.g., “right middle meatus swab”, not just “nasal swab”). Consequently, the lab can use the appropriate culture media.

Sample Collection Techniques: A Step-by-Step Guide

First, use a sterile cotton swab or a nylon brush (specifically for fungal aetiology). Then, collect the sample from the active margin of the lesion, not from crusts or dried pus. Crucially, avoid touching any other surface (cheek, tongue, or glove) with the swab tip. Finally, immediately place the swab into a sterile transport tube and seal it.

For nasal samples, a nasal endoscope helps obtain a representative sample from the middle meatus or the sinus ostia. Similarly, for chronic ear discharge, clean the external canal thoroughly first; then collect the sample from the middle ear under microscopic or endoscopic guidance. For anaerobic infections, you must use a special anaerobic transport vial. Remember, a tissue biopsy in saline is far superior to a swab for diagnosing fungal or tubercular infections.

Culture and Sensitivity: The Gold Standard

Culture identifies the causative organism, and sensitivity testing guides effective antibiotic therapy. Therefore, performing culture and sensitivity (C&S) prevents empirical treatment failures and reduces antibiotic resistance. The average turnaround time for routine aerobic C&S is 48–72 hours.

Types of Cultures in ENT

1. Aerobic Culture and Sensitivity: This is the most commonly ordered test for pus, throat swabs, and ear discharge. Consequently, it identifies pyogenic bacteria like Streptococcus pyogenes and Staphylococcus aureus.
2. Anaerobic Culture: Although rare, anaerobes like Peptostreptococcus and Fusobacterium can cause chronic sinusitis or deep neck space infections. However, clinicians rarely order anaerobic cultures because they are technically demanding and slow. Therefore, antibiotic cover for anaerobes (e.g., metronidazole) is usually given empirically.
3. Fungal Culture: Always send tissue or aspirate in sterile saline (never formalin) for fungal culture. Turnaround time varies from 24 hours for yeasts like Candida to 15 days for moulds like Aspergillus. Sensitivity for antifungals, if required, must be requested separately.

Table: Culture Media in ENT Microbiology

Medium	Type	Specific Use in ENT
Nutrient agar	Solid (general)	Routine growth of non-fastidious bacteria
Blood agar	Solid (general)	Most common; detects hemolysis (e.g., beta-hemolytic streptococci)
Chocolate agar	Solid (enriched)	Haemophilus influenzae, Neisseria gonorrhoeae
Lowenstein-Jensen (LJ) medium	Solid (specific)	Mycobacterium tuberculosis (Lymphadenitis, laryngeal TB)
Peptone water	Liquid (general)	Sugar fermentation test
Robertson’s cooked meat medium	Liquid (special)	Anaerobic and aerobic bacteria together
Semisolid medium	Semisolid	Motility testing and transport media

Mnemonic for Media: No Body Can Live Purely Raw – Nutrient agar, Blood agar, Chocolate agar, LJ medium, Peptone water, Robertson’s medium.

Direct Microscopy: Rapid Diagnosis at Bedside

While culture takes days, direct microscopy gives results within hours. Therefore, it is invaluable for initiating life-saving treatment.

1. Gram Stain: Directly performed on pus, CSF, or swab. Turnaround time is 6–8 hours. It shows Gram-positive cocci (Streptococci, Staphylococci), Gram-negative bacilli (e.g., Klebsiella), and budding yeast cells. However, the result may be inconclusive if the patient has already taken antibiotics.
2. KOH Mount (Potassium Hydroxide): Perform this at the bedside for rapid diagnosis of fungal elements. A 10% KOH preparation dissolves keratin and cellular debris, revealing fungal hyphae. For example, broad, aseptate, right-angled branching hyphae indicate Rhizopus or Mucor (mucormycosis). Conversely, septate, acute-angle branching suggests Aspergillus.
3. Albert Stain: Specifically used to detect Corynebacterium diphtheriae (diphtheria). It stains the metachromatic granules (volutin granules) at the poles of the bacilli. However, with good vaccine coverage, this test is now rare.

Molecular and Rapid Diagnostic Tests in ENT

1. RT-PCR (Reverse Transcription Polymerase Chain Reaction): This is the gold standard for detecting RNA viruses like SARS-CoV-2, influenza, and RSV. A nasopharyngeal swab is collected in viral transport media. Real-time RT-PCR amplifies and detects viral RNA within hours, providing highly accurate results. For a COVID-19 diagnosis, it remains the most reliable method.
2. Rapid Antigen Test (RAT): This test directly detects viral antigens from a nasopharyngeal swab. It gives results in 5–30 minutes, requires minimal training, and is cheap. However, its sensitivity and specificity are lower than RT-PCR. Therefore, a negative RAT in a symptomatic patient should be confirmed by RT-PCR. Clinically, use RAT as a screening test in high-prevalence settings.

Table 3: Comparison of RT-PCR vs Rapid Antigen Test

Feature	RT-PCR	Rapid Antigen Test
Target	Viral RNA	Viral protein (antigen)
Turnaround time	2–4 hours	5–30 minutes
Sensitivity	Very high (gold standard)	Moderate (lower for low viral load)
Specificity	Very high	High but false negatives occur
Cost	Expensive	Cheap
Best use	Confirmatory diagnosis	Screening during outbreaks

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High-Yield Points for Quick Revision

1. Culture and sensitivity identify organisms and antibiotic susceptibility.
2. Proper sample collection decides diagnostic accuracy.
3. Throat swab culture is mainly useful for streptococcal pharyngitis, diphtheria, candida, and the carrier state.
4. Blood agar is a general medium; chocolate agar isolates H. influenzae.
5. Lowenstein–Jensen medium is used for Mycobacterium tuberculosis.
6. KOH mount is the fastest test for mucormycosis and fungal sinusitis.
7. Gram stain gives early clues, but commensals can confuse results.
8. Anaerobic infections occur in chronic sinusitis and deep neck infections.
9. RT-PCR is the gold standard for SARS-CoV-2 detection.
10. A rapid antigen test is quick but less sensitive than PCR.
11. Acute sinusitis bacteria: S. pneumoniae, H. influenzae, Moraxella.
12. Acute otitis media bacteria: S. pneumoniae, H. influenzae, Moraxella

MCQs 

1. A 25-year-old presents with severe sore throat, fever, and thick exudate over tonsils. Throat swab Gram stain shows Gram-positive cocci in chains. The most likely organism is: A. Staphylococcus aureus B. Streptococcus pyogenes C. Streptococcus pneumoniae D. Neisseria gonorrhoeae.
2. Which culture medium is specifically used for isolating Haemophilus influenzae from a case of acute sinusitis? A. Blood agar B. Chocolate agar C. MacConkey agar D. Lowenstein-Jensen medium.
3. A diabetic patient presents with facial swelling, black eschar over the nose, and ptosis. Which rapid bedside test will give immediate diagnosis? A. Albert stain B. India ink preparation C. 10% KOH mount D. Gram stain.
4. The most common causative organism for lateral sinus thrombophlebitis is identified by: A. Throat swab culture B. Blood culture C. CSF culture D. Ear swab culture alone.
5. All are common pathogens in acute otitis media EXCEPT: A. Moraxella catarrhalis B. Streptococcus pneumoniae C. Pseudomonas aeruginosa D. Haemophilus influenzae.
6. For diagnosing tubercular cervical lymphadenitis, the sample should be sent to the laboratory in: A. 10% formalin B. Sterile saline C. Absolute alcohol D. Viral transport media.
7. The gold standard confirmatory test for SARS-CoV-2 infection in a patient with anosmia is: A. Rapid antigen test B. CT chest C. RT-PCR from nasopharyngeal swab D. IgM antibody test.
8. In chronic sinusitis, which group of organisms becomes more important? A. Viruses and Mycoplasma B. Anaerobes and Staphylococcus aureus C. Only Gram-negative bacilli D. Only Streptococcus pyogenes.
9. A throat swab from a child with a greyish-white membrane shows bacilli with metachromatic granules on Albert stain. The diagnosis is: A. Vincent’s angina B. Diphtheria C. Streptococcal pharyngitis D. Infectious mononucleosis.
10. The average turnaround time for aerobic culture and sensitivity from an ear swab is: A. 6–8 hours B. 24 hours C. 48–72 hours D. 15 days.

MCQ Answers: 1: B. 2: B. 3: C. 4: B. 5: C. 6: B. 7: C. 8: B. 9: B. 10: C.

Frequently Asked Questions in Viva

• What is the most common organism in acute bacterial pharyngitis? The most common bacterial organism is Group A beta-hemolytic Streptococcus pyogenes, although most pharyngitis cases are viral.
• How do you collect a sample for fungal culture from the nose? Use a sterile nylon brush (not cotton) under endoscopic guidance from the middle meatus, and place the sample immediately into sterile saline, not formalin.
• Why is chocolate agar called “chocolate” agar? It is called chocolate agar because heated blood (lysed red blood cells) gives the medium a brown color, and it provides factors X and V for Haemophilus influenzae growth.
• What is the difference between RT-PCR and a rapid antigen test for COVID-19? RT-PCR detects viral RNA and is the highly sensitive gold standard, while a rapid antigen test detects viral proteins, providing quick results but with lower sensitivity.
• Which ENT condition requires mandatory blood culture? Lateral sinus thrombophlebitis, an intracranial complication of otitis media, requires mandatory blood culture in addition to ear swab culture.
• What does a positive KOH mount in sinusitis indicate? A positive KOH mount indicates the presence of fungal hyphae, suggesting fungal sinusitis, which requires immediate antifungal therapy and surgical debridement in immunocompromised patients.
• Can a throat swab detect diphtheria? Yes, a throat swab stained with Albert stain shows characteristic metachromatic granules, and culture on Löffler’s medium confirms Corynebacterium diphtheriae.

———— End ————

Reference Textbooks.

• Scott-Brown, Textbook of Otorhinolaryngology-Head and Neck Surgery.
• Cummings, Otolaryngology-Head and Neck Surgery.
• Stell and Maran’s, Textbook of Head and Neck Surgery and Oncology.
• Ballenger’s, Otorhinolaryngology Head And Neck Surgery
• Susan Standring, Gray’s Anatomy.
• Frank H. Netter, Atlas of Human Anatomy.
• B.D. Chaurasiya, Human Anatomy.
• P L Dhingra, Textbook of Diseases of Ear, Nose and Throat.
• Hazarika P, Textbook of Ear Nose Throat And Head Neck Surgery Clinical Practical.
• Mohan Bansal, Textbook of Diseases of Ear, Nose and Throat Head and Neck Surgery.
• Hans Behrbohm, Textbook of Ear, Nose, and Throat Diseases With Head and Neck Surgery.
• Logan Turner, Textbook of Diseases of The Nose, Throat and Ear Head And Neck Surgery.
• Arnold, U. Ganzer, Textbook of  Otorhinolaryngology, Head and Neck Surgery.
• Ganong’s Review of Medical Physiology.
• Guyton & Hall Textbook of Medical Physiology.

Author:

Dr. Rahul Bagla
MBBS (MAMC, Delhi) MS ENT (UCMS, Delhi)
Fellow Rhinoplasty & Facial Plastic Surgery.
Renowned Teaching Faculty
Mail: msrahulbagla@gmail.com
India

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