Laboratory diagnosis of lower respiratory tract infections

Respiratory tract infection is one of the commonest infection seen in the community. In hospital set up , respiratory tract infections, both upper & lower, are seen in a major number of indoor patients. Ventilator-associated pneumonia holds a high rank in healthcare-associated infections., This chapter intends to develop a gross & correlative perspective for LRTI.

 

Learning objectives

1. Normal immune mechanism involved in defence against LRTI

2. Different infectious conditions of respiratory tract & their etiologies

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

4. Staining methods and culture techniques for sample analyses.

5. Antibiotic selection for treatment.

Innate Immune Mechanisms

·       Nasal hairs

·       Convoluted passages

·       Secretory IgA

·       Cilia – propels liquid layer of mucus

·       Antimicrobials – lactoferrin(disrupts bacterial biofilm)

·       Reactive O₂ species – produce hypochlorous acid which is bactericidal.

·       Alveolar macrophages

Microbial factors

• Adherence – pili, fimbriae
• Evasion – capsule

   - intracellular growth

Infectious diseases of Lower Respiratory tract

1. Pneumonia - Lobar pneumonia, Bronchopneumonia, Atypical/Interstitial pneumonia

2. Lung abscess

3. Pleuritis, Empyema

4. Bronchiectasis

5. Bronchitis

6. Alveolitis

7. Tuberculosis

In addition to these, certain infectious disease may involve upper and lower respiratory tract simultaneously e.g. croup (laryngo-tracheo-bronchitis)

Symptomatology of LRTI

·        Coughing that may produce phlegm (mucus)

·        Fever, sweating or chills

·        Dyspnoea

·        Chest pain(pleurisy) that’s worse when one breathes or coughs

·        Feelings of tiredness or fatigue

·        Loss of appetite

·        Nausea or vomiting

• Children under 5 years old may have fast breathing or wheezing.
• Infants may appear to have no symptoms, but sometimes they may vomit, lack energy, or have trouble drinking or eating.
• Older people may have milder symptoms. They can also exhibit confusion or a lower than normal body temperature.

 

PNEUMONIA

This infection causes inflammation in the alveoli in the lungs. The alveoli get fill with fluid or pus, making it difficult to breathe. (Fig.1)

Fig. 1Inflammation & fluid collection in alveoli

 

Pneumonia can be encountered clinically in different forms; in terms of its origin, localization of infection & causative infectious agents. And based on all these factors the antibiotic selection is to be done, also taking into consideration the age, immune status & any underlying respiratory pathology or other co-morbidities. Hence, it is essential to understand these aspects of pneumonia.

Causative agents of Respiratory Tract Infections

Various microorganisms are found to cause infections of respiratory tract.

Table 1: Causative agents of LRTI

Infectious Agent Group	Name of Pathogen
Gram positive bacteria	Streptococcus pyogenes
	Streptococcus pneumoniae (Pneumococci)
	Staphylococcus aureus
	Bacillus anthracis(Anthrax)
	Nocardia sp.
Gram negative bacteria	Haemophilusinfluenzae
	Pseudomonas aeruginosa
	Klebsiella spp.
	Other enterobacteriacae
	Moraxella catarrhalis
	Acinetobacter sp.
	Yersinia pestis(Plague)
Miscellaneous bacteria	Mycobacterium tuberculosis(Tuberculosis)
	Mycoplasma pneumoniae(Atypical pneumonia)
	Legionella pneumoniae
Fungi	Candida sp.
	Aspergillus sp.
	Cryptococcus neoformans
	Histoplasmacapsulatum
	Blastomycesdermatitidis
	Pneumocystis jirovecii
Parasites	Paragonimuswestermanii
	Entamoebahistolytica
	Echinococcusgranulosus
Viruses	Orthomyxoviruses (Influenzae)
	Paramyxoviruses (parainfluenzae, respiratory syncitial virus etc.)
	Adenoviruses
	Herpesviruses (i.e. CMV)
	Enteroviruses
	Coronaviruses (SARS, MERS, SARS CoV2)

 

Normal flora of mouth and throat

            Oral cavity and throat have plenty of normal microbial flora which are commonly isolated from sputum sample. Few of them can also cause infections. Isolation of such organisms should be evaluated with clinical background of the patient. These are Staphylococcus spp., Streptococcus spp, Micrococci, Lactobacilli, Diphtheroids, Neisseria, Moraxella spp., Fusobacterium etc.

Lower respiratory tract however, is essentially sterile.

Ventilator associated Pneumonia

Ventilator-associated pneumonia is a lung infection that develops in a person who is on a ventilator. A ventilator is a machine that is used to help a patient breathe by giving oxygen through a tube placed in a patient’s mouth or nose, or through a hole in the front of the neck. An infection may occur if germs enter through the tube and get into the patient’s lungs.

Ventilator Associated Events

VAEs are identified by using a combination of objective criteria: deterioration in respiratory status after a period of stability or improvement on the ventilator, evidence of infection or inflammation, and laboratory evidence of respiratory infection.

Identification of the three stages of VAE:

1.  Ventilator Associated Condition(VAC)

o   Presence of mechanical ventilator at least for 2 calendar days.

2.  Infection related Ventilator Associated Complication (IVAC)

o   Characteristics of VAC

o   Presence of any one of these: fever/hypothermia/leucocytosis/leucopaenia

o   Any new antibiotic started or continued for ≥ 4 days.

3. Possible Ventilator Associated Pneumonia (PVAP)

o   Characteristics of IVAC

o   Isolation of significant count of pathogens causing pneumonia from the respiratory specimens.

 

Laboratory diagnosis of Respiratory tract infection

Specimen collection:

1) Sputum:

Sputum is a good sample majorly for URTI. It may be collected for LRTI too, but is not an ideal sample. Still,itis the most commonly collected sample for bacteriological analaysis.

For method of Sputum sample collection, refer Chapter No…..(URTI)

 

2) Swab:

     Swab also is a common sampling method For URTI. In case of LRTI, bacteriological infectious agent  can’t be generally identified by swabs. However,  swabs are a major specimen when it comes to viral LRTI. Generally, nasal swabs, throat swabs & nasopharyngeal swabs are taken in decreasing order of preference when a  viral etiology is suspected for either URTI or LRTI.

 

3) Aspiration:

      a.Tracheal or endotracheal aspiration:

           This specimen isusually taken to diagnose LRTI, particularly in patients with endotracheal intubation or with tracheostomy. The specimen is aspirated with a  special

device known as a mucus extractor. One end of the device is inserted through the

endotracheal tube or tracheostomy site, the other end opens in a sterile container. Sample

is collected by creating negative pressure in the container with the helpof suction

machine(which is attached to the  container by another tube.)        

 

      b.Broncho alveolar lavage (BAL):

      This is a specimen collected at the time of bronchoscopy. Lavage is taken after saline wash

directly from infection site (bronchi, broncheoli). This is the most informative sample for

lower RTI as it completely bypasses the oral cavity & throat and hence, does not contain

the commensals. If intracellular bacteria are present in more than 25 % of inflammatory

cells; they serve as indicators of pneumonia.

 

      c.USG/CT guided percutaneous needle aspiration:

If the lesion is present at a distal site of the lungs, it is very difficult to extract specimen

even with bronchoscope or bronchial wash. In such cases, sample is taken by

     

4) Blood culture : Done in cases of bacteremia associated with pneumonia; e.g., S. pneumoniae.

 

5)Lung biopsy: This is an option in life threatening, difficult to diagnose infections like Herpes simplex pneumonia or Pneumocystis pneumonia.

 

Transport of specimen:

ü  Sample should be immediately transported to the laboratory.

ü  Special transport media are recommended in certain cases if transportation of the swab specimen   is expected to be delayed.

      e.g. 1. Streptococcal infection: Pike's medium

             2. Viral infection: Special viral transport media.

 

Gross examination of sample:

ü  Sputum samples may be purulent, muco-purulent, bloody or salivary. In pyogenic infections sputum will be purulent or muco-purulent. In case of tuberculosis blood may be present in sputum. Salivary samples are unsatisfactory for microbiological evaluation of RTI

     

Microscopic examination:

      1. Sputum / aspiration / lavage: Smear prepared from the specimen should be examined by

            a. Gram stain: To demonstrate and identify bacteria by their morphological study; i.e.,

gram  negative or gram positive; cocci or bacilli.

            b. Z N stain: To detect Acid Fast Bacilli.

            c. KOH wet mount preparation: To detect presence of fungi

            d. Giemsa stain: For cytological examination.

            e. Special fungal stains may be needed in suspected cases of fungal infections e.g. GMS

(GomoriMethnamine Silver) stain for Pneumocystis jirovecii.

     These preliminary examinations help clinicians to start or alter the empirical therapy according to the group of organisms involved; e.g., if KOH mount shows fungal hyphae in BAL, appropriate antifungals can be started immediately.

 

Culture examination:

 

Culture media:

Sample should be cultured on the following media

1. Simple media: Nutrient agar

2. Differential media: Mac Conkey agar

3. Enriched media: Blood agar, Chocolate agar

4. Selective media: e.g. For Tuberculosis: L J medium .

5. For suspected fungal etiology: SDA

Incubation of culture plates:

1. For bacterial cultures:

One set of inoculated plates should be incubated at 37° C temperature, aerobically & the second set of plates should be incubated at 37° C temperature, in presence of 5-10% CO₂ (i.e.candle jar / CO₂ incubator)

2. For fungal cultures:

One set of inoculated SDA should be incubated at 37° C temperature, and the other set should be incubated at 25° C temperature; so that any dimorphic fungi(e.g. Histoplasmacapsulatum) may also be diagnosed. These cultures have to be followed for at least a week.

 

On the next day(for bacterial cultures):

The growth observed on inoculated plate should be processed for

1. Identification of bacteria: By colony morphology, microscopic examination and biochemical         

reactions.

2. Antimicrobial sensitivity testing.

 

Exercise:

        A 4 years old male child presented with high grade fever and tachypnea. X-ray chest shows a consolidation in Right lung.

 

Q.1 Which different samples will you take for detection of etiological agent?

Q.2 Which are the staining techniques for demonstration of infecting bacteria?

Q.3 Which culture media should be seeded and how they should be incubated?

 Q.4 Which are the common bacteria responsible for such infectious disease?