Laboratory diagnosis of upper respiratory tract infections

 

Respiratory tract infection is one of the commonest infections seen in the community. Upper respiratory tract infection (URTI) specifically is noted to occur round the year through the seasonal changes, mostly with viral etiology. Infections of respiratory tract can have different types of presentations. Mainly, they are divided into Upper respiratory tract infections (URTI) & Lower respiratory tract infections (LRTI) based on the part of the respiratory system affected. Any respiratory infection above the level of glottis or vocal cord is URTI & below it is LRTI.

Learning objectives

·       Different infectious conditions of respiratory tract & their etiologies

·       Sample collection for evaluation of RTI - Sputum; lavage/swabs/aspiration from relevant sites etc.

·       Staining methods and culture technique for sample analyses.

Anatomy:

Part of respiratory tract above the glottis or vocal cord is called upper respiratory tract. The major passages and structures of the upper respiratory tract include the nose or nostrils, nasal cavity, mouth, throat (pharynx), and voice box (larynx). Upper respiratory tract provides innate protection from development of lower respiratory tract infections through (1) Filtration action of nasal hair, (2) Mucus secretion by mucosa which traps small particles, (3) Rich blood supply to nasal epithelium which warms and moisten the inhaled air, (4) Lymphatic tissue like tonsils and adenoids present in pharynx those protect against infection by releasing T & B lymphocytes & (5) Epiglottis of larynx that prevents food and liquid to enter in to lower respiratory tract.

 

Types of upper respiratory tract infection

 

1.    Local inflammation: i.e. Sore throat (Tonsilitis or Pharyngitis), Rhinitis, Epiglottitis, Sinusitis, Otitis media etc.

2.    Abscess formation: Faucial or Tonsilar

3.    Ulcer formation: mouth, throat etc.

4.    Diphtheria

5.    Pertussis

6.    Quinsy (Infection of retropharyngeal space)

 

1.    Causative agents of Upper respiratory Tract Infections

 

Rhinitis

Mostly caused by virus

·       Rhinovirus

·       Influenza virus

·       Parainfluenza virus

·       Respiratory syncitial virus

·       Coronavirus

·       Adenovirus

·       Human metapneumovirus

 

Sinusitis

1.    Acute

Bacteria

·       Streptococcus pneumoniae

·       Haemophilus influenzae

·       Moraxella catarrhalis

·       Pseudomonas species and other gram negative bacilli that can cause nosocomial sinusitis

Virus

·       Rhinoviruses

·       Influenza viruses

·       Parainfluenza viruses

 

2.    Chronic sinusitis

·       Obligate anaerobes

·       Staphylococcus aureus

·       Fungus like Aspergillus, Mucor

 

Pharyngitis and tonsillitis

Bacteria

·       Streptococcus pyogenes

·       Corynebacterium diphtheriae

·       Mycoplasma pneumoniae

·       Vincent angina: Treponema vincentii

 

Virus

·       Influenza viruses

·       Parainfluenza viruses

·       Coxsackie virus A

·       Rhinoviruses

·       Coronavirus

·       Epstein-Barr virus

·       Adenoviruses

Fugal

·       Candida albicans

 

Laryngitis

Mostly viral agent

·       Influenza viruses

·       Parainfluenza viruses

·       Adenovirus

·       Coronavirus

·       Human metapneumovirus

 

Exudative tonsillitis / Membrane or exudates present

·       Corynebacterium diphtheria

·       Streptococcus pyogenes

·       Ebstein-Barr virus

 

Epiglottis

·       Most common agent Haemophilus influenzae type B

 

Laryngo tracheobronchitis (Croup)

·       Parainfluenza virus

·       Influenza virus

·       Rspiratory syncytial virus

·       Adenovirus

 

2.    Clinical presentation:

Rhinitis

·       Running nose

 

Sinusitis

·       Headache

·       Thick nasal mucus

·       Plugged nose

 

Pharyngitis and tonsillitis

·       Sore throat

·       Difficulty in swallowing

 

Laryngitis

·       Hoarseness of voice

·       Lowering and deepening of voice

Epiglottis

·       Fever

·       Difficulty in swallowing

·       Inspiratory stridor

Laryngo tracheal bronchitis

·       Hoarseness of voice

·       Fever

·       Cough (Harsh or barking non productive cough)

·       Inspiratory stridor

 

 

 

3.    Laboratory diagnosis of Upper respiratory tract infection

 

Specimen collection:

·       Nasal / throat swab: two throat swab samples should be collected from affected part, one for direct examination and one for culture.

·       Part of membrane & swab from fragile margin of membrane

·       Nasopharyngeal swab or aspirate for viral diagnosis

 

Processing the specimen

 

Microscopy

 

Gram stain:

Bacteria can be identified based on their morphology and shape.

Interpretation: Bacteria demonstrated in gram stain examination should be carefully interpretated i.e. presence of gram positive cocci in chains suggestive of Streptococcus species which may be commensals (streptococcus viridians) or pathogenic (streptococcus pyogenes).

Albert stain:

To demonstrate the Corynebacterium diphtheriae

Interpretation: presence of bluish-black metachromatic granules either at one end or both ends and cuneiform arrangement (Chinese letter pattern arrangement) of bacilli suggestive of C.diphtheriae

Immunofuorescence microscopy:

To demonstrate presence of antigens of viruses causing URTI

 

Culture:

For bacteriological culture specimen should be inoculated onto following culture media

·       Blood agar        

·       Chocolate agar

·       MacConkey’s agar

·       Loeffler’s serum slope and potassium tellurite blood agar (for isolation of Corynebacterium diphtheria in suspected cases of Diphtheria).

·       Sabouraud dextrose agar (SDA) is used if fungal infection is suspected.

 

Interpretation:

-        Bacterial growth can be identified by colony morphology / pigment production / haemolysis on blood agar & / or colour of colony on Mac Conkey agar.

-        On Loeffler’s serum slope media C.diphtheriae produces small, circular glistening, white with yellowish tinged colonies within 6-8 hours while on potassium tellurite blood agar C.diphtheriae produces black colonies due to reduction of tellurite to tellurium after 48 hours. Tellurite blood agar commonly helps in isolation of C.diphtheriae from carrier.

-        On SDA: Candida species produces smooth, creamy, white, pasty colonies. Aspergillus / mucor produce growth of mycelium.

 

 

Molecular test

-        Polymerase chain reaction method is used for diagnosis of various virus infections i.e. Swine flu / bird flu

-        Multiplex PCR assay are available where multiple primers targeting the genes specific for each of the suspected agents of URTI are used.

 

 

Exercise:

 

Case study:

 

A 5-year old child with history of fever and throat pain for 3 days was brought to ENT OPD. On examination, he was febrile (101˚^F) and throat examination revealed pustules over tonsils. His throat swab was sent to the microbiology laboratory and was subjected to culture and sensitivity testing.

1.    What is your clinical diagnosis?

2.    What are etiological agents, pathogenesis and clinical features of this condition?

3.    Discuss your approach for laboratory diagnosis?