• According to WHO pneumonia kills more Children than any other illness- >AIDS, Malaria and Measles combined.
It includes a number of infectious agents like viruses, bacteria and fungi. The most common are:
1. Streptococcus pneumoniae: most common cause of bacterial pneumonia in children;
2. Haemophilus influenzae type b (Hib) –2nd most common cause of bacterial pneumonia
3. Respiratory syncytial virus (RSV): Most common viral cause of pneumonia
• Diseases:- Pneumonia, Bacteremia, sepsis, Meningitis, Otitis media and Sinusitis
• Present in Respiratory tract of 5-10% of adults (carriage rate up to 60% in closed population)
• Gram positive lanceolate shaped cocci arranged in pairs (diplococci) or short chains
• Produces alpha hemolysis
• Bile soluble
• Possess Polysaccharide capsule of >85 antigenically distinct types,
• Relatively few of these are responsible for most serious disease.
• Antibody to the capsular polysaccharide confers serotype specific protection against disease
• Quellung reactions is useful to identify the type.
Pathogenesis: Virulence factors
Ability to resist opsonization, phagocytosis, and intracellular killing by phagocytic cells.
• Capsular polysaccharide: Antiphagocytic
• Lipoteichoic acid: Activates complement and induces inflammatory cytokine production
• Pneumolysin: Cytotoxic- lyse cells/Activate classical complement pathway. In vitro:-causes alpha hemolysis
• IgA protease: Enhances organism’s ability to colonize the mucosa of the URT
• Others: Autolysins, Hyaluronidase, Neuraminidase (NanA and NanB), etc.
Factors predisposing to pneumococcal infection
1. Age: infants younger than 3 years and adults older than 65 years.
2. Underlying bronchopulmonary disease and compromised humoral immunity.
3. Absence of specific anticapsular antibody.
4. Depress cough reflex and increase aspiration of secretions.
5. Abnormality of the respiratory tract that affects mucociliary clearance mechanisms.
6. Abnormal circulatory dynamics.
7. Splenectomy etc.
• Pneumococci multiply in tissues and cause inflammation.
• In alveoli, there is outpouring of fluid and red and white blood cells, resulting in consolidation of lung.
• During recovery, pneumococci are phagocytized, mononuclear cells ingest debris, and the consolidation resolves.
• Important cause of URT infections:- otitis media, sinusitis, and epiglottitis
• Causes pneumonia in adults, in those with chronic obstructive lung disease
• Leading cause of meningitis in young children
• Small gram negative coccobacilli with polysaccharide capsule
• Major virulence factor IgA protease
• Do not grow in Sheep blood agar
• Grows on – Chocolate agar (heated and lysed blood agar) or – Blood agar supplemented with hemin (X factor) and nicotinamide-adenine-dinucleotide (NAD; V factor)
• Definitive identification by Quellung reaction.
Gram Negative Pleomorphic Coccobacilli
Chocolate Agar: Growth of Haemophilus Influenzae
Laboratory Diagnosis of Lower Respiratory Tract Infections
• Primary means of determining the causes of bacterial pneumonia
• Give the patient a clean, dry, wide-necked, leak-proof container
• Instruct patients to provide a deep-coughed specimen
• Minimize contamination with saliva.
• Transport to laboratory immediately. Problems
• Sputum contaminated with commensal of upper respiratory tract
Collection of Sputum
• To prevent the spread of infectious organism
• Avoid contaminating outside of the container
• Use phenol containing disinfectant to wipe the outside of the container after collecting the specimen.
• Via respiratory therapy technician (Postural drainage and thoracic percussion).
• Aerosol induced specimen (using 10-15% aerosolized saline solution). • Appear watery resembling saliva
• Acceptance without pre-screening
– Endotracheal or Tracheostomy Suction specimens
– Trans tracheal aspirates
– Other invasive procedures: Thoracentesis
Laboratory examination of sputum
• Deliver sample to laboratory as soon as possible
• H. influenzae and Streptococcus pneumoniae do not survive well in sputum – Never refrigerate the sample
In Microbiology Lab: Day 1
Report whether specimen:
1. Purulent, mucopurulent, Mucoid, salivary
2. Contains blood
1. Gram Smear
2. Zn Smear (AFB staining)
3. Giemsa smear
4. KOH preparation
Normal microbial flora of URT may be seen in gram stain of sputum.
• Gram positive diplococci (capsulated): could be S. pneumoniae
• Gram positive cocci in groups: could be S. aureus
• Gram negative rods and coccobacilli: could be H. influenzae
• Gram negative capsulated rods: K. pneumoniae
• Gram negative diplococci in and between pus cells: M. catarrhalis
Gram Positive diplococci
Day 1: Culture
1. 5% Sheep Blood agar: Add optochin disc in the middle of secondary streaking
2. Chocolate agar: For Haemophilus and Neisseria spp.
3. MacConkey Agar: Isolation and differentiation of Gram negative bacilli
Plate 1 & 2 are incubated in atmosphere containing extra CO2 at 35-36oC for 24 hours.
• Report cultural characteristics
– Colony characteristics
– Gram Staining
• Biochemical tests
– Optochin test
– Bile solubility test etc.
Laboratory diagnosis of S. pneumoniae
• Lancet-shaped gram positive diplococci
• Alpha hemolysis in Blood Agar (5% Sheep blood agar) and Chocolate Agar
• Colonies are bile soluble (colonies are lysed by bile)
• Growth is inhibited by Optochin
• Quellung reaction with multiple serotypes
Blood cultures are positive in 15-25% of pneumococcal infections.