WHO guidelines for the collection of human specimens for laboratory diagnosis of avian influenza infection

12 January 2005

General information

Respiratory virus diagnosis depends on the collection of high-quality specimens, their rapid transport to the laboratory and appropriate storage before laboratory testing. Virus is best detected in specimens containing infected cells and secretions. Specimens for the direct detection of viral antigens or nucleic acids and virus isolation in cell cultures should be taken preferably during the first 3 days after onset of clinical symptoms.

Type of specimens

A variety of specimens are suitable for the diagnosis of virus infections of the upper respiratory tract:

  • nasal swab
  • nasopharyngeal swab
  • nasopharyngeal aspirate
  • nasal wash
  • throat swab.

In addition to swabs from the upper respiratory tract, invasive procedures can be performed for the diagnosis of virus infections of the lower respiratory tract where clinically indicated:

  • transtracheal aspirate
  • bronchoalveolar lavage
  • lung biopsy
  • post-mortem lung or tracheal tissue.

Specimens for the laboratory diagnosis of avian influenza A should be collected in the following order of priority:

  • nasopharyngeal aspirate
  • acute serum
  • convalescent serum.

Specimens for direct detection of viral antigens by immunofluorescence staining of infected cells should be refrigerated and processed within 1–2 hours. Specimens for use with commercial near-patient tests should be stored in accordance with the manufacturer’s instructions. Specimens for virus isolation should be refrigerated immediately after collection and inoculated into susceptible cell cultures as soon as possible. If specimens cannot be processed within 48–72 hours, they should be kept frozen at or below –70 °C.

Respiratory specimens should be collected and transported in virus transport media. A number of media that are satisfactory for the recovery of a wide variety of viruses are commercially available.

Procedures for specimen collection

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 mediumfor use in collecting throat and nasal swabs

  • Add 10 g veal infusion broth and 2 g bovine albumin fraction V to sterile distilled water (to 400 ml).
  • Add 0.8 ml gentamicin sulfate solution (50 mg/ml) and 3.2 ml amphotericin B (250 μg/ml)
  • 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: general patient information, type of specimens, date of collection, and contact information of person completing the form, etc.

Standad precautions should always be followed, and barrier protections applied whenever samples are obtained from patients.

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.