TOPIC 8 MICROBIOLOGY OF RESPIRATORY NON-MOLECULAR METHODS FOR

SYSTEM (SPUTUM) DETECTION AND IDENTIFICATION OF

RESPIRATORY PATHOGENS
Common bacterial Common viral
pathogens pathogens →Electron microscopy (EM) is one of the
 Streptococcus • Rhinovirus oldest direct examination methods been
pyogene • Coronavirus implemented for clinical viral diagnosis and
 Hemophilus • Influenza virus study of viral ultrastructure and pathogenesis.
influenza • Respiratory
 Corynebacterium syncytial virus Disadvantage of using EM:
diphtheria • Parainfluenza virus
1) Expensive
 Streptococcus
2) Laborious
pneumoniae
3) Time-consuming
 Mycobacterium
tuberculosis 4) Insensitive when compared to other
 Bordetella diagnostic methods
pertussis CULTURE (FOR VIRUSES)
 Legionella
pneumoniae • Detection of viruses by observing the
 Mycoplasma cytopathic effect has been considered
pneumoniae the “gold standard” for diagnosis of
 Bacteroides sp. respiratory viral pathogens for
 Fusabacterium sp. decades.
• Viruses such as adenovirus, influenza
FUNGAL INFECTION A/B, RSV, and human parainfluenza
viruses are the most common
Commonly seen in patients with defective respiratory viruses that are isolated
immunity. and detected by cell culture.
Example of pathogens: Disadvantage;-
• Aspergillus fumigatus 1) Exhibit higher false negative rates
• Pneumocystis jirovecis 2) Longer turnaround times, making viral
culture less clinically relevant
Aspergillus fumigatus cause allergic
bronchopulmonary aspergillosis, aspergilloma ANTIGEN DETECTION ASSAYS
or dessiminated aspergillosis
→lateral flow devices= is the most versatile
and popular immune-chromatographic
method
Diagnostic test for viral infection
→RIAs are relatively inexpensive, easy to
1) Serology
perform
2) Detecting of antibody
3) Detection of viral antigen by NUCLEIC ACID AMPLIFICATION TESTS FOR
immunofluorescence or colorimetric DETECTION AND IDENTIFICATION OF
methods RESPIRATORY PATHOGENS
4) Virus isolation in cell culture
→Accuracy of respiratory virus detection by
molecular tests is not only dependent on their
specific assay chemistry, but is also critically
affected by the type, quantity, and quality of Mucoid (mild)→ blood
specimens collected. stained (severe)
Gram’s 1) To identify
staining morphology/shape
Molecular Test (Nucleic Acid Detection) 2) To determine gram’s
positive/ negative
1) Obtain specimen swab Culture 1) Blood agar
2) Extract RNA from specimen and Media 2) Chocolate agar
convert to DNA 3) MacConkey
3) Amplify by PCR with SARS-Cov-2 4)
specific primers
4) Interpret results: presence of viral RNA IDENTIFICATION OF STREPTOCOCCUS
indicates active SARS-CoV-2 infection. PNEUMONIAE

✓ Streptococcus pneumoniae
DIAGNOSTIC TESTS FOR BACTERIAL
➢ Gram positive – diplococci
RESPIRATORY TRACT INFECTIONS
➢ Catalase test – NEGATIVE because if positive
diagnostic technique for respiratory tract
its strep not staph
infections of bacterial cause is traditional
culture, followed by identification and ➢ Blood agar – α-hemolytic
antimicrobial susceptibility testing by various
manual or automated methods. ➢ Antibiotic disc – optochin sensitivity test
give positive result.
Diagnostic tests for bacterial respiratory tract
infections Standard process of culture and
susceptibility testing generally takes 2–3 days,
STREPTOCOCCUS ➢ Gram positive –
with at least 1 day for culture and a second day
PNEUMONIAE diplococci
for antimicrobial susceptibility testing.
➢ Catalase test –
NEGATIVE because
Laboratory if positive its strep
diagnosis not staph
Type of • Nose swab
samples: • Throat ➢ Blood agar – α-
• swab hemolytic
• Sputum
• Bronchial washing ➢ Antibiotic disc –
(lavage optochin sensitivity
test give positive
For any non-cultural bacteria, result.
viruses and fungi, they are
not directly diagnosed from HAEMOPHILUS SP. ➢ Gram-negative –
sputum but from serum bacilli/ coccobacilli
samples. (pleomorphic)
Appearance • Mucoid
of sputum • Mucopurulent ➢ Grow better on
• Purulent the chocolate agar
• Blood stained
 ➢ Confirmation test • Sputum processing: digestion,
– X and V test decontamination, and/or
✓ Use tryticase soy concentration of a primary patient
agar specimen prior to setting up culture
and smear
➢ H. influenza grow • Smear: A small amount of primary
around the XV disc patient specimen (direct or processed)
only is placed on a slide for the purpose of
microscopic examination
➢ H. parainfluenza • Positive AFB smear results provide a
grow around the V first indication of mycobacterial
and XV disc infection and potential TB disease

➢ H. ducreyi grow
around the X and
Smear Types
XV disc
KLEBSIELLA ➢ Gram-negative 1) Direct smear
PNEUMONIAE – rod shape
➢ Smear prepared directly from a patient
➢ Lactose- specimen prior to processing
fermenting 2) Concentrated smear
bacteria
➢ Smear prepared from a processed specimen
➢ Appear very after centrifugation is used to concentrate the
mucoid on the material
culture media
PSEUDOMONAS ➢ Gram-negative
AERUGINOSA – capsulate, rod Decontamination
shape
1) Specimen
2) NaOH will be added and left in RT for
➢ Non-lactose
20 min
fermenting,
3) Then add dH2O to dilute the NaOH and
oxidase positive
neutralize the pH
4) Centrifuge 3000 rpm, 10 min
ISOLATION AND IDENTIFICATION OF 5) Discard the supernatant
MYCOBACTERIUM TUBERCULOSIS 6) Re-suspend the pellet
7) AFB Staining or culture on LJ media
• AFB Smear Microscopy: Microscopic
examination of specially stained
smears to detect acid-fast bacilli
organisms such as Mycobacterium
tuberculosis and non-tuberculous
mycobacteria (NTM)
• Acid Fast Bacilli (AFB): resists
decolorization with acid alcohol due to
the lipid-rich mycolic acids in the cell
wall thereby retaining the primary
stain
CONCENTRATED SMEAR ➢ Local prevalence of
MTB and NTM
➔ A few ml of sputum is processed and
determine the predictive
concentrated by centrifugation values of a positive
Smears can be made smear for MTB

➢ Directly from processed pellet

Culture on Media
✓ May increase smear sensitivity
• The concentrated sediment from the
✓ However, may result in less material sputum sample is inoculated onto
available for NAA testing & culture appropriate culture media.
• The most commonly used culture
➢ From re-suspended pellet after the addition media for Mycobacterium tuberculosis
of buffer include ______________________.
✓ Re-suspended in 1~2 ml buffer; one drop for • These media contain specific nutrients
smear and inhibitors to support the growth of
mycobacteria while suppressing the
growth of other bacteria.
• Biochemical tests such as niacin
AFB Explain
MICROSCOPY accumulation, nitrate reduction, and
Technique Brightfield: carbol catalase activity can be performed
fuchsin staining
➢ Contrast red AFB on
blue or green
background

Fluorescence: Auramine
staining
➢ Also known as
Fluorochrome staining
➢ Contrast light & dark
Limitations of  Does not distinguish
AFB Microscopy between viable and
dead organisms
➢ Follow-up specimens
from patients on
treatment may be smear
positive yet culture
negative

 Limited specificity

➢ All mycobacteria are

acid fast bacilli
organisms
➢ Does not provide
species identification