APPLIED MICROBIOLOGY

LABORATORY DIAGNOSIS OF ACUTE RESPIRATORY INFECTIONS (ARI)

Respiratory tract infections are one of the most frequently encountered diseases of childhood and
consist largely of acute infections.

Causative Agents

Bacterial:

• Streptococcus pneumoniae
• Haemophilus influenzae
• Staphylococcus aureus'
• Corynebacterium diphtheriae
• Bordetella pertussis

Viral:

• Influenza A, B, C
• Parainfluenza types 1-4 Respiratory
• syncytial virus (RSV)
• Adenoviruses
• Rhinoviruses
• Enteroviruses (some strains only)

Collection, Storage and Transport of Samples

Samples should be collected before the antibiotic therapy is started and should be collected with care.
The specimens for bacterial isolations are:

a. Nasal swab
b. Per-nasal swab
c. Throat swab
d. Nasopharyngeal aspirate
e. Transtracheal aspirate and lung aspirate
f. Sputum
g. CSF
h. Blood
i. Middle ear aspirate

Samples should be processed without delay. The two media commonly used for transport purpose, if
rquired are Stuart's medium and Arnie's medium. For transportation of swabs of respiratory
secretions or discharge, Arnie's transport medium is preferable to Stuart's.

The specimens for virus isolation are:

a. Nasopharyngeal specimen: nasopharyngeal aspirate, nasopharyngeal swab and nasopharyngeal

wash
b. Bronchoscopic aspirate, lung aspirate or pleural fluid are rarely collected.

During transport, samples are held at 4°C on wet ice in an insulated, well-sealed container to protect
them from direct sunlight and extreme temperature.
Diagnosis of Bacterial Pathogens

Gram Staining

It is particularly rewarding when the samples are from areas of the body which are normally sterile,
such as the lungs, middle ear and meninges.

Culture

For initial isolation the samples are primarily inoculated on blood agar, MacConkey's agar, and
chocolate agar, at a temperature of 35-36°C for 24-28 hrs. The growth of most of the respiratory
bacterial pathogens is better in the presence of 5-10 percent C02 which is achieved by candle jar. A
relative humidity of 70-90 percent is desirable which is achieved by keeping an open water reservoir
at the bottom of the incubator.

Identification of Bacterial Isolates: Streptococcus pneumoniae

Pneumococci typically produce an alpha-haemolytic low convex draughtsman shaped colonies which
may be entire or undulate. The confirmation of the isolates is done by: (a) optochin sensitivity test, (b)
mouse inoculation test, (c) quellung reaction, and (d) serotyping the isolate.

Corynebacterium diphtheriae:

Culture is done on Loeffler's serum slope and blood tellurite agar. Colonies develop very fast in
Loeffler's serum slope and also morphology is best in this medium. The characteristics of organism
are (a) thin, slender, Gram positive bacilli, (b) presence of metachromatic granules, (c) clubbing of the
bacteria, and (d) chinese letter arrangement of the bacteria.

Haemophilus influenzae:

Is a fastidious organism requiring media containing haemin (X-factor) and nicotinamide adenine
dinucleotide (V-factor). Growth on blood agar is poor, very discrete and may be alpha-haemolytic.
Luxurious growth is obtained on chocolate agar. The identification is done by following tests: (a)
Satellitism test, (b) X and V factor growth tests. H. influenzae is catalase positive, usually non-
haemolytic and requires both X and V factors. After biochemical identification, the serotyping is done
using type specific antisera.

Bordetella pertussis:

Fluorescent antibody technique offers a promising mean of rapid and early diagnosis and a greater
number of positive results, than with culture. Bordet-Gengou medium is used and the recommended
procedure of inoculation is cough-plate method which is a bed side procedure. Plates are incubated
in a moist chamber containing carbon dioxide enriched atmosphere for upto 7 days and examined
each day for growth of Bordetella. B. pertussis can be distinguished from B. parapertussis by its
inability to grow on nutrient agar, chocolate agar and urease negativity.

Staphylococcus. aureus:

On blood agar usually beta-haemolytic, typically golden opaque colonies are produced. Haemolysis
and pigmentation are not satisfactory for differentiating Staph. aureus from Staph. epidermidis.
Production of coagulase by Staph. aureus and its nonproduction by Staph. epidermidis is probably
the most accurate and convenient method of differentiating the two.
Rests of the organisms are also to be identified as per the routine tests.

Antibiotic Sensitivity Test

Stoke's method, wherein control strains are used, should be preferred method of sensitivity testing.
Combination drugs should be tested separately, e.g. sulfamethoxazole and trimethoprim testing in
cotrimoxazole should be done separately.

Blood Culture

This gives very reliable results about the invasion by the microrganisms, especially when it is done
before the antibiotic therapy is initiated.

Detection of Bacterial Antigens

The detection of bacterial antigen in the body fluids is done even:

a. When the organism is dead before the specimen is subjected to culture,
b. When the patient has already received antibiotic therapy, and
c. Microorganism has a long replication time.
Various recommended techniques are:
a. Counterimmunoelectrophoresis (CIEP)
b. Coagglutination
c. Latex agglutination
d. Enzyme linked immunosorbent assay (ELISA)

Isolation of Viruses Cell

Culture Techniques

The cell lines that are employed for the isolation of respiratory viruses as per recommendation of
WHO are:

Cell line Virus

Primary monkey kidney cell Influenza A and B

Parainfluenza
Mumps
HeLa or HEp2 cell line RSV
Adenoviruses
Enteroviruses
Human lung fibroblast cells Rhinoviruses
Herpesviruses

Each virus can be isolated in more than one cell line with varying sensitivities. The most sensitive cell
cultures for isolation of human viruses are primary human embryonic kidney (HEK) and primary
monkey kidney (PMK).

Developing Chick Embryo

Influenza viruses can be isolated by inoculating nasopharyngeal washings into the amniotic cavity of
11-13 days old eggs. Amniotic and allantoic fluids are harvested after incubating eggs at 35°C for 3
days. The presence of virus is identified by haemagglutination using guinea pig and fowl cells.
Detection of Viruses

Various techniques are now available for diagnosis like:

• Latex agglutination
• Co agglutination
• Counterimmunoelectrophoresis
• ELISA
• Immunofluorescence
• Immunoperoxidase test
• Dot-blot hybridization
• PCR or gene amplification.