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Laboratory diagnosis of respiratory tract infections
Dr. Shler Ghafour Raheem
BSc., MSc., PhD Medical Microbiology
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