What Specimens Should Be Collected for Testing?
Testing for Avian H5N1 Influenza
Testing for avian H5N1 influenza may only be carried out in a reference laboratory (e.g., authorized state or local public health laboratory or CDC). Testing may be done on respiratory samples by reverse transcriptase polymerase chain reaction (RT-PCR) testing (see below), and serological confirmation may also be carried out. In addition, post-mortem testing on various tissues is also available. Viral culture is not recommended on specimens of any kind from patients with suspected avian H5N1 influenza due to biosafety concerns. Culturing avian H5N1 influenza viruses may only occur in a Biosafety Level 3+ laboratory, which excludes all New York City (NYC) hospital and commercial laboratories.
The following respiratory specimens should be collected and stored at 4oC for both RT-PCR and viral culture testing. All suspected cases should be reported to the New York City Health Department. After arrangements have been made with the New York Ciy Health Department, a Health Department courier will transport specimens to the Public Health Laboratory.
At least one nasopharyngeal (NP) specimen AND one oropharyngeal (OP) swab should be collected as follows:
- Collect specimen with a sterile Dacron/nylon swab with a non-wooden shaft (Do NOT use calcium alginate swabs or swabs with wooden sticks). For NP specimens, this must be an NP swab (small head, flexible wire shaft). Rigid shafts are good for OP only.
- For NP swab, insert swab into nostril parallel to the palate until the tip reaches the nasopharynx, and leave in place for a few seconds to absorb secretions. For OP swab, swab the posterior pharynx and tonsillar areas, avoiding the tongue.
- Transport medium: Place swab immediately into sterile vial containing 3 ml of viral transport media. Label each specimen container with patient's FIRST AND LAST NAME, date of birth, medical record number, date of collection, and specimen type.
- Place specimen vial onto ice or in refrigerator prior to and during transport to laboratory. Do not freeze.
- Avoid delay in transport and testing.
Nasopharyngeal wash/aspirates may also be used for diagnosis of avian H5N1 influenza. It is recommended that nasal wash/aspirate be collected as follows:
- Have the patient sit with head tilted slightly backward. Instill several drops of bacteriostatic saline (pH 7.0) into one nostril.
- Insert the tubing into the nostril parallel to the palate.
- Aspirate nasopharyngeal secretions. Repeat this procedure for the other nostril.
- Rinse syringe or bulb into viral transport medium or aspirate viral transport medium through catheter into collection trap.
- Place specimen onto ice or in refrigerator prior to and during transport to laboratory. Do not freeze.
- Avoid delay in transport and testing.
An acute serum sample should also be obtained from all suspect cases as follows:
- 5-10 ml whole blood in a serum separator or red top tube.
- Spin down and refrigerate at 4oC.
- Label each specimen container with patient's FIRST AND LAST NAME, medical record number, date of birth, specimen type, and collection date.
All information requested on the "Virus Detection Laboratory Submission Form" (Word) should be completely filled out and the form should be included with the specimen shipment.
In fatal cases associated with suspected avian H5N1 influenza infection, DOHMH personnel will help make arrangements for autopsy and coordinate specimen shipping and testing with the NYC Office of the Chief Medical Examiner and CDC.
Rapid Testing for Avian H5N1 Influenza
Rapid influenza diagnostic tests and immunofluorescence (indirect fluorescent antibody staining [IFA] or direct fluorescent antibody staining [DFA]) may be used to detect seasonal human strains of influenza, but should not be used to confirm or exclude avian H5N1 influenza. Rapid influenza tests have relatively low sensitivity for detecting seasonal influenza, and their ability to detect avian H5N1 influenza is unknown. The sensitivity of rapid diagnostic tests will likely be higher in specimens collected within two days of illness onset, in children, and when tested in clinical laboratories that perform a high volume of testing. Such tests can identify influenza A viruses but cannot distinguish between human infection with seasonal and avian H5N1 influenza viruses. A negative rapid influenza test result does not exclude human infection with either seasonal or avian H5N1 influenza viruses. A positive rapid influenza test result could be a false positive or represent infection with either seasonal or avian H5N1 influenza viruses. Therefore, both negative and positive rapid influenza test results and immunofluoresence results should be interpreted with caution, and RT-PCR testing for suspected avian H5N1 influenza virus should be performed in a reference laboratory, as outlined above.