|  Annex
2 : Procedures for specimen collection from human cases
Materials required
 Sputum/mucus trap
Polyester fibre-tipped applicator
Plastic vials
Tongue depressor
15-ml conical centrifuge tubes
Specimen collection cup or Petri dishes
Transfer pipettes
Virus transport medium
(A)
Virus transportation medium for use in
collecting throat and nasal swabs
1.Add 10 g veal infusion
broth and 2 g bovine albumin fraction V to sterile distilled water (to 400
ml).
2.Add 0.8 ml gentamicin
sulfate solution (50 mg/ml) and 3.2 ml amphotericin B (250 μg/ml)
3.Sterilize by filtration.
(B) Nasal wash medium
1. Sterile saline (0.85% NaCl).
Preparing to collect specimens
Clinical specimens should be
collected as described below and added to transport medium. Nasal or
nasopharyngeal swabs can be combined in the same vial of virus transport
medium. When possible, the following information should be recorded on the
Field Data Collection Form (see attached form): general patient information,
type of specimens, date of collection, and contact information of person
completing the form, etc
Nasal swab
A dry polyester 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 It is then slowly withdrawn with a rotating motion.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. The tip of the swab is put
into a plastic vial containing 2–3 ml of virus transport medium and the
applicator stick is broken off.
Nasopharyngeal swab
A flexible, fine-shafted
polyester swab is inserted into the nostril and back to the nasopharynx and
left in place for a few seconds. It is then slowly withdrawn with a rotating
motion. A second swab should be used for the second nostril. The tip of the
swab is put into a vial containing 2–3 ml of virus transport medium and the
shaft cut.
Nasopharyngeal aspirate
Nasopharyngeal secretions are
aspirated through a catheter connected to a mucus trap and fitted to a vacuum
source. The catheter is inserted into the nostril parallel to the palate. The
vacuum is applied and the catheter is slowly withdrawn with a rotating
motion. Mucus from the other nostril is collected with the same catheter in a
similar manner. After mucus has been collected from both nostrils, the
catheter is flushed with 3 ml of transport medium.
Nasal wash
The patient sits in a
comfortable position with the head slightly tilted backward and is advised to
keep the pharynx closed by saying "K" while the washing fluid
(usually physiological saline) is applied to the nostril. With a transfer
pipette, 1–1.5 ml of washing fluid is instilled into one nostril at a time. The patient
then tilts the head forward and lets the washing fluid flow into a specimen
cup or a Petri dish. The process is repeated with alternate nostrils until a
total of 10–15 ml of washing fluid has been used. Dilute approximately 3 ml
of washing fluid 1:2 in transport medium.
Throat swab
Both tonsils and the posterior
pharynx are swabbed vigorously, and the swab is placed in transport medium as
described above.
Sera collection for influenza diagnosis
An acute-phase serum specimen
(3–5 ml of whole blood) should be taken soon after onset of clinical symptoms
and not later than 7 days after onset. A convalescent-phase serum specimen
should be collected 14 days after the onset of symptoms. Where patients are
near death, a second ante-mortem specimen should be collected.
Although single serum
specimens may not provide conclusive evidence in support of an individual
diagnosis, when taken more than 2 weeks after the onset of symptoms they can
be useful for detecting antibodies against avian influenza viruses in a neutralization
test.
Field Data Collection Form
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