Interim Guidance for Specimen Collection, Processing, and Testing for Patients with Suspected Infection with Novel Influenza A Viruses Associated with Severe Disease in Humans
Novel influenza A viruses are influenza viruses that are different from currently circulating human influenza A virus subtypes and include influenza viruses from predominantly avian or swine origin. In recent years, human infections with highly pathogenic avian influenza A (H5N1) virus in several Asian countries and Egypt, and avian influenza A (H7N9) virus in China, have been reported. The clinical presentation of human infection with avian influenza A viruses varies considerably: from mild illness, including conjunctivitis, fever, and cough; to severe illness, including fulminant pneumonia and acute respiratory distress syndrome (ARDS) leading to hospitalization or death. As of January 2014, the novel influenza A viruses most commonly associated with severe disease in humans are low pathogenic avian influenza (LPAI) A (H7N9) and highly pathogenic avian influenza (HPAI) A (H5N1) viruses.
This document provides interim guidance for clinicians and public health professionals in the United States on appropriate specimen collection, storage, processing, and testing for patients who may be infected with novel influenza A viruses that are known to cause severe disease in humans, including avian influenza A (H7N9) and HPAI (H5N1) viruses. Current influenza A (H7N9) case definitions and recommendations for patient testing; and influenza A (H5N1) case definitions and recommendations for patient testing should also be consulted.
CDC recommends maintaining the enhanced surveillance efforts practiced currently by state and local health departments, hospitals, and clinicians to identify patients at increased risk for novel influenza A virus infection. Clinicians should notify their state health department immediately when they decide to test a patient for novel influenza A virus infection so that appropriate testing and follow up of contacts is initiated. CDC should be notified immediately in the event that any clinical specimens from suspected cases test positive for novel influenza A virus. Human infection with a novel influenza A virus is a nationally notifiable condition.
The duration of shedding of novel influenza A viruses in humans is largely unknown, and there are currently limited data describing prolonged shedding of infected individuals with these viruses. Therefore, the estimated duration of viral shedding is based upon seasonal influenza virus infection. Specimens should be obtained for novel influenza A virus testing as soon as possible after illness onset, ideally within 7 days of illness onset. However, as some persons who are infected with seasonal influenza viruses are known to shed virus for longer periods (e.g., children and immunocompromised persons), specimens should be tested for novel influenza A virus even if obtained after 7 days from illness onset. Prolonged influenza viral shedding in the lower respiratory tract has been documented for critically ill patients with HPAI H5N1 virus and LPAI H7N9 virus infections.
Standard, contact, and airborne precautions are recommended for management of patients who may be infected with avian influenza A (H7N9) or avian influenza A (H5N1) viruses; this includes collection of respiratory specimens. See Interim Guidance for Infection Control Within Healthcare Settings When Caring for Confirmed Cases, Probable Cases, and Cases Under Investigation for Infection with Novel Influenza A Viruses Associated with Severe Disease for more information and consult CDC for specific case-by-case infection control recommendations if needed.
The following should be collected as soon as possible after illness onset: (i) a nasopharyngeal swab, or (ii) a nasal aspirate or wash, or (iii) two swabs combined into one viral transport media vial (e.g., nasal or nasopharyngeal swab combined with an oropharyngeal swab). If these specimens cannot be collected, a single nasal, or oropharyngeal swab is acceptable. For patients with lower respiratory tract illness, a lower respiratory tract specimen (e.g., an endotracheal aspirate or bronchoalveolar lavage fluid) is preferred because these specimens have a higher yield for detecting avian influenza H5N1 and H7N9 viruses. Specimens should be placed into sterile viral transport media and immediately placed on refrigerant gel-packs or at 4°C (refrigerator) for transport to the laboratory.
If possible, in order to increase the potential for H7N9 or H5N1 virus detection, multiple respiratory specimens from different sites should be obtained from the same patient on at least two consecutive days.
Swab specimens should be collected using swabs with a synthetic tip (e.g., polyester or Dacron®) and an aluminum or plastic shaft. Swabs with cotton tips and wooden shafts are not recommended. Specimens collected with swabs made of calcium alginate are not acceptable. The swab specimen collection vials should contain 1-3ml of viral transport medium (e.g., containing protein stabilizer, antibiotics to discourage bacterial and fungal growth, and buffer solution).
Respiratory specimens should be kept at 4°C for no longer than 3 days. Specimens can alternatively be frozen at ≤-70°C. Avoid freezing and thawing specimens if at all possible.
Clinical specimens sent to state public health laboratories should be shipped in the appropriate packaging and according to instructions by the laboratory. If clinical specimens will be examined within 72 hours after collection, keep the specimen at 4°C (2-8°C) and ship on refrigerant gel-packs, otherwise store frozen at ≤-70°C and ship on dry ice. Avoid freezing and thawing specimens. Viability of some pathogens from specimens that were frozen and then thawed is greatly diminished. All specimens should be labeled clearly and include information requested by your state public health laboratory.
The performance of current Food and Drug Administration (FDA) cleared diagnostic tests for influenza has been demonstrated for seasonal human influenza viruses as described by the manufacturer package insert. Performance has not been demonstrated with novel influenza A viruses. However, some diagnostic assays may detect the presence of novel influenza A viruses.
Existing FDA-cleared molecular assays (e.g., rRT-PCR) may detect novel influenza A viruses with results that indicate “influenza A positive”, but with subtype undetected. For these assays a novel influenza A virus may give an influenza A “unsubtypable” result. Clinicians and laboratorians using molecular assays that are capable of detecting all currently circulating influenza A subtypes who identify an unsubtypable result should contact CDC and their state or local public health laboratory for additional testing (see below).
Rapid influenza diagnostic tests (RIDTs) and immunofluorescence assays are antigen detection tests that also have unknown sensitivity and specificity to detect human infection with novel influenza A virus in clinical specimens. Some studies suggest that antigen detection tests have low sensitivity to detect H5N1 viruses. Therefore, negative results from either type of test do not exclude influenza virus infection, especially in patients with signs and symptoms suggestive of influenza. Therefore, a negative test result could be a false negative and should not be used as a final diagnostic test for influenza, including novel influenza A virus infection. These tests may give a positive influenza A result for a specimen containing novel influenza A virus, but cannot identify the subtype and cannot distinguish a novel influenza A virus from a seasonal influenza A virus. Therefore, testing by rRT-PCR is recommended at state health laboratories for any patient with suspected novel influenza A virus infection.
Clinicians should always consider diagnostic testing for other pathogens that can cause acute febrile respiratory illness since H5N1 and H7N9 virus infections of humans are very rare, even in exposed persons.
Clinicians should notify their state health department immediately when they wish to test a patient for suspected H7N9 or H5N1 virus infection. Specimens to be tested for novel influenza A virus should be sent first to the state or local public health laboratory.
All state public health laboratories and several local public health laboratories are able to perform testing for seasonal influenza and for influenza H7N9 or H5N1 RT-PCR testing using the CDC Human Influenza Real-Time (real-time reverse-transcription polymerase chain reaction) rRT-PCR Diagnostic Panel (CDC Flu rRT-PCR Dx Panel), and are the recommended sites for immediate testing. Testing can be performed by public health laboratories on a portion of the specimen, while a portion of the sample should be reserved in case there is a need to ship it to CDC. CDC should be notified immediately in the event that any clinical specimens from suspected cases test positive for any non-seasonal influenza virus such as H7N9 or H5N1 virus, and clinical specimens should be shipped to CDC for confirmatory testing.
CDC Flu rRT-PCR Dx Panel testing algorithms should be used as described in the package insert to rule out seasonal influenza virus infection. Public Health officials should contact CDC immediately if they obtain unsubtypable results when testing an influenza specimen.
Testing with the CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel-Influenza A/H7 (Eurasian Lineage) Assay or the CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel-Influenza A/H5 Assay should not be performed unless the patient meets the currently recommended clinical and epidemiologic criteria for testing. These two assays can be performed for the presumptive identification of virus in patients who may be infected with influenza A/H7 (Eurasian Lineage) (this includes the avian influenza A (H7N9) virus first identified in China in 2013) and influenza A/H5, respectively.
Specimens that are unsubtypable or that are presumptive positive for novel influenza A should be sent to CDC, Influenza Division, Virology Surveillance and Diagnosis Branch Laboratory for confirmatory testing. Laboratories should not attempt diagnosis of patients who may be infected with avian influenza A (H7N9) virus or highly pathogenic avian influenza A (H5N1) virus using viral culture. Any work with live wild-type highly pathogenic avian influenza H5N1 viruses must be conducted in a USDA-approved Biosafety Level 3 enhanced containment facility. Please see the Biosafety in Microbiological and Biomedical Laboratories (BMBL) for more information about procedures and facilities recommended for manipulating highly pathogenic avian influenza viruses.
Antiviral treatment should not be withheld or delayed pending collection of specimens or laboratory testing. Empiric treatment with a neuraminidase inhibitor antiviral drug (oral oseltamivir or inhaled zanamivir) should be administered immediately according to current guidelines. For more information, see CDC’s interim guidance on the use of antiviral agents for treatment of human infections with novel influenza A viruses.
Specimens to be tested for novel influenza A virus that are shipped from state public health laboratories to CDC should include all information required for seasonal influenza surveillance isolate or specimen submission. Before sending specimens, state and local health departments should contact the CDC Influenza Division Epidemiology and Prevention Branch at (404) 639-3747 (Monday – Friday, 8:30 AM - 5:00 PM or the on-call epidemiologist at (770) 488-7100 (all other times).
Ship specimens to CDC at the following address:
Virology, Surveillance and Diagnosis Branch
ATTN: Steve Lindstrom
Influenza Division, NCIRD
Centers for Disease Control and Prevention
1600 Clifton Road NE MS G-16
Atlanta, GA 30333
Serologic testing for H5N1- or H7N9-specific antibody, using appropriately timed specimens, can be considered if other H5N1 or H7N9 diagnostic testing methods are unavailable (for example, due to delays in respiratory specimen collection). Serologic testing should only be performed at CDC. CDC, Influenza Division, should be contacted if serological testing is being considered.
Paired serum specimens from the same patient should be collected for H5N1 or H7N9 serology if possible: the first sample should be collected within the first week of illness, and a second sample should be collected 2-4 weeks later. Serological testing of deceased patients with only a single serum specimen may be possible upon consultation with CDC. A demonstrated rise in the H5N1- or H7N9-specific antibody level is required for a diagnosis of H5N1 or H7N9 virus infection. Currently, the hemagglutination inhibition assay using horse erythrocytes or the microneutralization assay, which requires live H5N1 virus, are the recommended tests for measuring H5N1- or H7N9-specific antibodies.
Under certain circumstances, post mortem testing may be the only option available to confirm a diagnosis of novel influenza A virus infection. In some patients with fatal respiratory disease for whom appropriate respiratory specimens were not obtained prior to death, pathologic testing of autopsy specimens may be possible and may benefit public health authorities. For example, in a patient whose epidemiological information is uncertain, unavailable, or otherwise suspicious for infection with a novel influenza A virus, establishing a post mortem diagnosis may assist authorities conducting an urgent public health investigation of ill close contacts of the deceased.
Viral antigens may be focal and sparsely distributed in patients with influenza and are most frequently detected in respiratory epithelium of large airways. Larger airways (particularly primary and segmental bronchi) have the highest yield for detection of influenza viruses by immunohistochemistry (IHC) staining. Collection of the appropriate tissues ensures the best chance of detecting the virus by immunohistochemical stains. Performance of specific immunohistochemical, molecular, or other assays will be determined using clinical and epidemiologic information provided by the submitter and the histopathologic features identified in the submitted tissue specimens. Please see Specimen Submission Guidelines for Pathologic Evaluation of Influenza Virus Infections for more information.
CDC, Influenza Division, must be contacted if pathologic testing at CDC is being considered.
CDC, Influenza Division, should be contacted immediately for any positive results of H7N9 or H5N1 virus testing, or at any time for any questions related to H7N9 or H5N1 and specimen collection, laboratory testing, antiviral treatment, or infection control. Laboratory results for human clinical specimens that test positive for H7N9 or H5N1 virus by RT-PCR at a laboratory in the United States should be shipped as soon as possible for confirmation at CDC, Influenza Division, a designated WHO Reference Laboratory located in Atlanta, Georgia. Before sending specimens, state and local health departments should contact the CDC Influenza Division Epidemiology and Prevention Branch at (404) 639-3747 (Monday – Friday, 8:30 AM - 5:00 PM or the on-call epidemiologist at (770) 488-7100 (all other times).
CDC will continue to evaluate new information as it becomes available and will update this guidance as needed. For further assistance with these recommendations, please contact CDC at (770) 488-7100.