The document outlines the laboratory diagnosis of respiratory infections, detailing the common pathogens and methodologies for specimen collection and processing for both lower and upper respiratory tract infections. It emphasizes the importance of accurate specimen handling and the interpretation of results, highlighting specific tests like sputum cultures and blood cultures. Additionally, it discusses various causative agents, including bacteria, fungi, and viruses, along with their identification techniques.

1. Laboratory Diagnosis of
Respiratory Infections

2.  Respiratory infections are one of the most
common microbial infections.
 Frequent exposure of respiratory mucosa to
microbes inhaled with air.

3. Lower Respiratory Tract Infections
Major sections
– Clinical aspects of diseases of LRT
– Specimen collection
– Specimen processing
– Interpretation of bacterial cultures
– Most common pathogens

4. Categories of Lower Respiratory
Tract Infections
Acute bronchitis
Community acquired pneumonia
Hospital acquired pneumonia
Pneumonia in the immunocompromised
host.
Tuberculosis

5. Community Acquired Pneumonia
Etiologic Agents
Pathogen Frequency (%)
Streptococcus pneumoniae 66
Haemophilus influenzae 1-12
Moraxella catarrhalis 10
Legionella species 2-15
Mycoplasma pneumoniae 2-14
Klebsiella species 3-14
Enteric gram negative bacilli 6-9
Staphylococcus aureus 3-14
Chlamydia species 5-15
Influenza viruses 5-12
Other viruses <1-12
Unknown 23-49

6. Available Test Methodologies
Sputum Gram stain and culture
Blood cultures
Serological studies
Nucleic acid amplification tests

7. Sputum Gram Stain and Culture
Demonstration of predominant morphotype on
Gram stain guides therapy.
Accuracy is good when strict criteria are used
Cheap, rapid

8. Sputum Collection
Proper patient instruction
– The mouth should be rinsed with saline or water
– Patient should breathe and cough deeply
– Patient should expectorate into a sterile container
Transport container immediately to lab.
Perform Gram stain and culture specimen
as soon as possible.

9. Induced sputum
Patients unable to produce sputum may be
assisted by respiratory therapy technician.
Aerosol induced specimen: collected by allowing
the patient to breath aerosolized droplets of a
solution of 15% sodium chloride and 10%
glycerin for approximately 10 minute .
Avoid the need for invasive procedures such as
bronchoscopy or needle aspiration

10. Gastric aspiration
Used exclusively for isolation of acid-fast bacilli
and may be collected from patients who are
unable to produce sputum, particularly young
children.
The relative resistance of Mycobacteria allows
them to remain viable for a short period. Gastric
lavage must be delivered to the lab immediately
so that the acidity can be neutralized.

11. Respiratory Specimens
Bronchoalveolar Lavage (BAL)
– Samples large area of the lung
– Performed using a bronchoscope
– Saline injected
– Injected saline along with secretions is collected
by aspiration
Transthoracic Aspiration
– Involves percutaneous introduction of a needle.

12. Sputum Gram Stain
Unacceptable

13. Sputum Gram Stain
Good Quality

14. Sputum Gram Stain
Good Quality

15. Sputum Gram Stain
Good quality specimens
Quantify number and types of inflammatory cells
Concentrate on areas with WBCs when looking
for organisms
Determine if there is a predominant organism (>
10 per oil immersion field)
– If no predominant organism is present, report “mixed
gram positive and gram negative flora”

17. “ The culture of lower respiratory
specimens may result in more
unnecessary microbiologic effort
than any other type of specimen.”
Raymond C Bartlett
Routine culture

18. Routine culture:
Sputum sample should be satisfactory otherwise do
not process (Reject the sample).
Do not examine clear, watery saliva.
General guidelines for satisfactory sample:
>25 PMNL and <10 epithelial cells per low power
magnification.
Note: may not be applicable for pt. with
neutropenia.

19. Routine culture:
 Inoculation of sputum sample on suitable
culture media should be done as soon as
possible, otherwise bacteria like
Haemophilus, Pneumococci may die.
 Should not be refrigerated.

20. Routine culture
Routinely used media :
5% sheep blood agar
chocolate agar: Neisseria spp. and
Haemophilus spp.
MacConkey agar
SDA (if fungal infection is suspected)

21. Identification of the isolates:
Identification of bacteria can be done as per
standard methods of identification.
Should be able to differentiate normal flora and
pathogen.
Interpretation of report should be done carefully.

22. Blood culture
Bacteremia may occur in pt. with pneumonia.
Isolating organism from blood will provide strong
evidence rather than isolation from sputum due to
throat flora contamination.

23. Fungal RTI (Causative agents):
- Histoplasma capsulatum
- Coccidioides immitis
- Sporothrix schenckii
- Cryptococcus neoformans
- Aspergillus species
- Pneumocystis jiroveci.
- Candida species

24. Identification of fungi: (M/E: KoH mount, culture)
Colony morphology: Yeast colonies similar to bacteria
while filamentous fungi show cottony or velvety growth.
LPCB mount: fungal morphology.

25. Parasitic pneumonia
Protozoa: Entamoeba histolytica
Cestodes: Echinococcus granulosus
Trematodes: Paragonimus westermani
Nematodes: Ascaris lumbricoides
Hookworm
Strongyloides stercoralis
Diagnosed by Microscopy.

26. Viral pneumonia:
- Respiratory Syncytial virus
- Influenza, Parainfluenza virus
- Adenoviruses
- Cytomegalovirus
- Herpes simplex virus
- Severe acute respiratory syndrome virus
- Diagnosis may not be required as viral infections are
self limiting. Microscopy & cell culture.

27. Laboratory Diagnosis of
Upper Respiratory Tract
Infections

28. Type of specimens
A variety of specimens are suitable for the
diagnosis of virus infections of the upper
respiratory tract:
Nasal swab
Nasopharyngeal swab
Throat swab

29. THROAT SWAB FOR CULTURE
MATERIALS NEEDED:
For bacteria: Culture Swab
All others: Virus/Chlamydia/
Mycoplasma Collection Pack

30. METHOD:
1. With the patient’s head tilted back and the throat
well illuminated, depress the tongue so that the
back of the throat can be seen.
2. Rub the swab up and down the back of the throat
and against any white patches in the tonsillar area.
Avoid the tongue and the cheeks.
3. Replace the swab in the transport tube.

31. Nasal swab
A dry swab is inserted into the
nostril, parallel to the palate,
and left in place for a few
seconds.
It is then slowly withdrawn with
a rotating motion. Specimens
from both nostrils are obtained
with the same swab.