Laboratory diagnosis of respiratory tract infections

Dr. Shler Ghafour Raheem

BSc., MSc., PhD Medical Microbiology

shler.ghafour@tis.edu.iq

Infections to be diagnosed

• Upper respiratory tract :- Pharyngitis/Tonsillitis

- Diphtheria

- Rhinosinusitis and otitis media

• Lower respiratory tract :- Pneumonia

- Tuberculosis

Common pathogenic bacteria

- Β-haemolytic streptococci group A

- Streptococcus pneumoniae

- Klebsiella pneumonia

- Corynebacterium diphtheriae

- Bordetella pertussis

- Mycobacterium tuberculosis

Sample collection

- Throat swab (posterior pharyngeal swab)

- Nasopharyngeal swab

- Ear swab

- Sputum

Collection of sputum

• Early morning sputum is preferred because they containpooled overnight secretion in which, pathogenic bacteria are morelikely to be concentrated.

Sample collection for tuberculosis

Fresh sputum

Gastric Lavage

Urine

Blood

Other body fluids ( Spinal, Pleural, pus )

Tissue Biopsy

• Gram stain

• Albert stain (Corynebacterium diphtheriae)

• Ziehl–Neelsen stain

1- Microscopically

Ziehl-Neelsen stain

• Acid-fast stain (Ziehl-Neelsen method) is a usefuldifferential staining procedure that specifically stains allmembers of the genera mycobacteria. Gold standard for TB andLeprosy.

• Carbol fuschin

• Acid (such as sulfuric acid or hydrochloric acid)

• Methylene blue

Streptococcus pyogenes in gram stain

Diagnostic characteristics :

- Gram positive.

- Cocci.

- In chain.

This bacteria cause Pharyngitis or Tonsillitis

in upper respiratory tract

Klebsiella pneumoniae

Diagnostic characteristics :

- Gram-negative.

- Encapsulated.

- Rod-shaped.

This bacteria cause Pneumonia

in lower respiratory tract

Corynebacterium diphtheriae

Gram stain Albert stain

Streptococcus pneumonia

Diagnostic characteristics :

-Streptococcus pneumonia in sputum

smear stained with gram stain.

- Gram positive.

- Diplococci or short chain.

- Encapsulated.

This bacteria cause Pneumonia

in lower respiratory tract

Identification of M. tuberculosis in clinical specimens

• Microscopic search for acid-fast bacilli using:-

• Ziehl-Neelsen stain most rapid test

– M. tuberculosis not be reliably distinguished

• Small amount of bacilli in sample give negative

results

• Low sensitivity.

• Therefore definitive identification only be obtained

by:-

• culturing organism (The Löwenstein–Jensen medium)`

• molecular methods (PCR).

• Blood agar

• Chocolate agar (5-10% CO2)

• Tellurite blood agar (Corynebacterium spp).

• Lowenstein Jensen medium (pulmonary tuberculosis)

2- Cultivation

Purpose of a clinical specimen culture:

• To identify the specific organisms that cause infection.

• To test the antibiotics for their effectiveness in treating different infections.

Antibiotic sensitivity assay

Streptococcus pyogenes in blood agar culture

Group A (beta-hemolytic) streptococcus

klebsiella pneumoniae in sputum in MacConkey agar

Haemophilus influenzae on chocolate agar enriched with (factor X & V).

Streptococcus pneumonia on blood agar

- Disadvantage: slow growth of about 4 to 6 weeks.

- Advantage: more sensitive than direct microscopy

When grown on LJ medium, M. tuberculosisappears as brown, granular colonies (sometimescalled "buff, rough and tough")

• Cultures detect small Number of organisms in

sample.

Mycobacterium tuberculosis on lowenstein jensen medium

Tuberculin test (tuberculosis skin test)

• A tuberculin skin test to see if you have ever been

exposed to tuberculosis.

• The TB antigens used in a tuberculin skin test are called

purified protein derivative (PPD).

• A tuberculin skin test cannot tell how long you have been

infected with TB. It also cannot tell if the infection

is latent(inactive) or is active and can be passed to

others.

• The skin test reaction should be read between 48

and 72 hours after administration

• The reaction should be measured in millimeters

of the induration (palpable, raised, hardened area

or swelling)

About 10 mm positive

Below 5 mm negative

Nucleic acid amplification (Molecular test)

• Polymerase chain reaction (PCR) :

• Amplifies a small portion of a predetermined target region of M.

tuberculosis DNA.

• Shorten time required to detect and identify M. tuberculosis in clinical

specimens.

• Sensitivity of test ranges from 75 %to 100 %

• Specificity of 95% to 100 %

References

• Luis M. de la Maza, Marie T. Pezzlo, Cassiana E. Bittencourt,Ellena M. Peterson. 2020. Color Atlas of MedicalBacteriology. (2020, Wiley) - libgen.lc.

• Robert W. Bauman, Todd P. Primm. 2018. Microbiology withDiseases by Body System. Fifth edition, Pearson

• Gary W. Procop,Deirdre L. Church , et al. 2017.Koneman'sColor Atlas and Textbook of DiagnosticMicrobiology.7th Edition. Jones & Bartlett Learning