Interim Guidance for Laboratory Testing of Persons with Suspected Infection with Highly Pathogenic Avian Influenza A (H5N1) Virus in the United States

This document provides revised interim guidance for testing of suspected human cases of highly pathogenic avian influenza (HPAI) A (H5N1) virus infection in the United States and is based on current knowledge regarding human infection with highly pathogenic avian influenza (HPAI) A (H5N1) virus.

The epidemiology of human infections with HPAI H5N1 virus has not changed significantly since the re-emergence of human cases of HPAI H5N1 virus infection in 2003. CDC continues to recommend that surveillance for suspected human cases of HPAI H5N1 in the United States remain at the enhanced level. To date, there have not been any reports of HPAI H5N1 virus infections among poultry, wild birds, other animals or humans in the United States. Therefore, successful identification of cases will depend upon health care providers obtaining information from patients who present with respiratory illness about their recent travel outside of the U.S. and their activities while traveling. Clinicians should consider HPAI H5N1 along with seasonal influenza virus infection in returning travelers presenting with respiratory illness. CDC recommends that health care providers consider H5N1 testing for persons that have both a clinical syndrome consistent with HPAI H5N1 disease and relevant exposure to HPAI H5N1 virus.

This guidance will be updated if the epidemiology of human infection with HPAI H5N1 virus changes.

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Reporting and Testing Guidelines

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 HPAI H5N1 virus infection. Clinicians should notify their state health department immediately when they decide to test a patient for HPAI H5N1 virus infection so that appropriate testing and follow up of contacts is initiated (See Interim Guidance for Follow-up of Contacts of Persons with Suspected Infection with Highly Pathogenic Avian Influenza A (H5N1) Virus ) CDC should be notified immediately in the event that any clinical specimens from suspected cases test positive for H5N1 virus. Human infection with a novel influenza A virus is a nationally notifiable condition.

Testing for HPAI H5N1 virus infection is recommended for a patient who:

1. Has an illness that requires hospitalization or is fatal;
   AND
2. Has or had a documented temperature of ≥38°C (≥100.4° F) in the past 24 hours OR has a history of feverishness in the past 24 hours;
   AND
3. Has radiographically confirmed pneumonia, acute respiratory distress syndrome (ARDS), or other severe respiratory illness;
   AND
4. Has at least one of the following potential exposures within 7 days of symptom onset:
   1. History of travel to a country where HPAI H5N1 virus has been documented in poultry, wild birds, and/or humans,† AND had at least one of the following potential exposures during travel:
      1. Direct contact with (e.g. handling, slaughtering, defeathering, butchering, preparation for consumption) well-appearing, sick or dead poultry or wild birds;
      2. Direct contact with surfaces contaminated with poultry feces or poultry parts (carcasses, internal organs, etc.) that might contain HPAI H5N1 virus;
      3. Consumption of raw or incompletely cooked poultry or poultry products;
      4. Close contact (approach within about 6 feet)* with a confirmed HPAI H5N1-infected animal other than poultry or wild birds (e.g.cat or dog);
      5. Close contact (approach within about 6 feet)* with a person who was hospitalized or died due to a severe unexplained respiratory illness;
      6. Visiting a market where live poultry are sold or slaughtered;
      7. Handling samples (animal or human) suspected of containing HPAI H5N1 virus in a laboratory or other setting.
   2. Close contact (approach within about 6 feet)* with an ill person with confirmed HPAI H5N1 virus infection;
   3. Close contact (approach within about 6 feet)* with an ill person who was under investigation for possible HPAI H5N1 virus infection;
   4. Working with live HPAI H5N1 virus in a laboratory.

In addition, testing for HPAI H5N1 virus infection can be considered on a case by case basis, in consultation with local and state health departments, for:

1. A patient with mild or atypical disease‡ (hospitalized or ambulatory) who has one of the exposures listed above (criteria 4.a, b, c or d); OR
2. A patient with severe or fatal respiratory disease whose epidemiological information is uncertain, unavailable, or otherwise suspicious but does not meet the criteria above. An example would include an ill returned traveler that visited a country where HPAI H5N1 virus has been documented or is highly suspected in birds.

Clinicians should notify their state health department immediately when they wish to test a patient for suspected HPAI H5N1 virus infection. Influenza H5N1-specific reverse-transcription polymerase chain reaction (RT-PCR) testing conducted under Biosafety Level 2 (BSL2) conditions is the preferred method for laboratory diagnosis. All state public health laboratories and several local public health laboratories are able to perform influenza HPAI H5N1 RT-PCR testing, and are the recommended sites for initial diagnosis. Emphasis should be placed upon proper infection control precautions and use of personal protective equipment (PPE) for contact with suspected cases, collection of the proper respiratory specimens, and testing of specimens using the instructions provided in the CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel package insert. Recommended infection control precautions (standard, contact, droplet, and airborne) and PPE (gown, gloves, eye protection, N95 fit-tested respirator or higher level of respiratory protection) for health care personnel providing direct care of patients with suspected or confirmed HPAI H5N1 virus infection can be found in the Department of Health and Human Services (HHS) Pandemic Plan (Supplement 4: Infection Control) [173 KB, 22 pages]. Clinicians can consult CDC for specific case-by-case infection control recommendations.

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Specimen Collection and Testing Guidelines for Clinicians

1. Respiratory Specimen Collection and Testing
   1. Empiric treatment with a neuraminidase inhibitor antiviral drug (oral oseltamivir or inhaled zanamivir) should be administered immediately to a patient with suspected HPAI H5N1 virus infection and not delayed pending collection of respiratory specimens or waiting for H5N1 laboratory testing results. Patients with severe disease should be treated with oseltamivir. See WHO Rapid Advice Guidelines on Pharmacological Management of Humans Infected with Avian Influenza A (H5N1) Virus for more information.
      For information on clinical management of HPAI H5N1 patients, see WHO Clinical Management of Human Infections with Avian Influenza A (H5N1) Virus.
   2. Appropriate PPE is recommended for any direct and close contact with a suspected or confirmed HPAI H5N1 patient in the outpatient and inpatient setting, and especially when entering a room where aerosol-generating procedures in such patients are being performed. In general, PPE should include respiratory protection (a particulate respirator that fits well and has undergone a fit check), at least as protective as a National Institute of Occupational Safety and Health (NIOSH)-approved N-95 filtering face piece, goggles, a face shield, non-sterile latex gloves, a gown and a head covering. If a particulate respirator is not available, other NIOSH-certified N-, R-, or P-class respirators should be used. Aerosol-generating procedures should only be performed if using an N-95 particulate respirator or higher level of respiratory protection. Powered air purifying respirators (PAPRs) are more protective than N-95 respirators and may be considered for certain workers and tasks (e.g., high-risk activities) if available.
      1. Bronchoalveolar lavage is considered to be a high-risk aerosol-generating procedure. Therefore, infection control precautions should include the use of gloves, gown, goggles and face shield, and a fit-tested respirator with an N-95 respirator or higher rated filter. A loose-fitting powered air-purifying respirator (PAPR) may be used if fit-testing is not possible (for example, if the person has a beard).
      
      See the Department of Health and Human Services (HHS) Pandemic Plan (Supplement 4: Infection Control) [173 KB, 22 pages] for more information about infection control. Clinicians can consult CDC for specific case-by-case infection control recommendations.
   3. Oropharyngeal (throat) swab specimens and, if available, lower respiratory tract specimens (e.g., bronchoalveolar lavage or endo-tracheal aspirate) are preferred for patients with severe lower respiratory tract disease because they have a higher yield for detecting HPAI H5N1 virus compared to nasopharyngeal and nasal specimens. Nasal or nasopharyngeal swab specimens may contain less HPAI H5N1 virus but are useful for detection of human influenza A and B viruses. Therefore, nasal or nasopharyngeal and throat swab specimens, as well as lower respiratory specimens (if available) should all be collected.
   4. If possible, in order to increase the potential for HPAI H5N1 virus detection, multiple respiratory specimens from different sites should be obtained from the same patient on at least two consecutive days. Ideally specimens should be collected as soon as possible and within the first 7 days after illness onset.
   5. Swabs used for specimen collection should have a Dacron tip and an aluminum or plastic shaft. Swabs with calcium alginate or cotton tips and wooden shafts are not recommended. § Specimens should be placed immediately into sterile viral transport medium and stored at 4°C until testing.
   6. Use of commercially available rapid influenza diagnostic tests for the purpose of detecting HPAI H5N1 virus infection is not recommended. Clinicians should be aware that these tests have very low sensitivities to detect HPAI H5N1 virus compared to other methods, and a negative result does not exclude a diagnosis of HPAI H5N1 virus infection. In addition, a positive result does not distinguish between infection with human and animal influenza A viruses.
   7. Clinicians should always consider diagnostic testing for other pathogens that can cause acute febrile respiratory illness since HPAI H5N1 virus infection of humans is very rare, even in exposed persons.

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Testing Guidelines for Laboratory Staff

Real-time RT-PCR is the preferred assay for H5N1 testing under BSL2 conditions. CDC has made H5-specific primers and probes available to state health department laboratories. In addition, the U.S. Food and Drug Administration (FDA) cleared a real-time RT-PCR devicedeveloped by CDC for the presumptive identification of virus in patients who may be infected with influenza A subtype A/H5. CDC should be notified immediately in the event that any clinical specimens from suspected cases test positive for HPAI H5N1 virus, and clinical specimens should be shipped to CDC for confirmatory testing. Human infection with a novel influenza A virus is a nationally notifiable condition and should be reported immediately and confirmed at CDC. Viral culture should NOT be attempted on specimens from patients suspected to have HPAI H5N1 virus infection, and generally should not done except at CDC, unless conducted under Biosafety Level 3 (BSL3) enhanced conditions.

1. H5-specific RT-PCR
   Nucleic acid extraction lysis buffer can be added to specimens (for virus inactivation and RNA stabilization), after which specimens can be stored and shipped at 4°C. Otherwise, specimens should be frozen at or below -70°C and shipped on dry ice. Avoid repeated freeze/thaw cycles if at all possible.
2. Viral culture
   Isolation of HPAI H5N1 virus should not be performed except at the Influenza Division, CDC, unless enhanced BSL3 conditions are available. For viral culture, nucleic acid extraction lysis buffer should not be added to specimens. Clinical specimens from H5 positive patients should be shipped to CDC for confirmatory testing. If specimens are not expected to be inoculated into culture within 2 days, they should be frozen at or below -70°C and shipped on dry ice. Avoid repeated freeze/thaw cycles.

Collection and Testing of Other Clinical Specimens

Serologic testing for H5N1-specific antibody, using appropriately timed specimens, can be considered if other HPAI H5N1 diagnostic testing methods are unavailable (for example, due to delays in respiratory specimen collection). Serologic testing should only be performed at CDC. The Influenza Division, CDC, should be contacted if serological testing is being considered.

• Serological Testing
  1. Paired serum specimens from the same patient should be collected for HPAI H5N1 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-specific antibody level is required for a diagnosis of HPAI H5N1 virus infection. Currently, the microneutralization assay, which requires live HPAI H5N1 virus, is the recommended test for measuring H5N1-specific antibody.
  Any work with live wild-type HPAI H5N1 viruses must be conducted in a USDA-approved Biosafety Level 3 enhanced containment facility. See the Biosafety in Microbiological and Biomedical Laboratories (BMBL) for more information about procedures and facilities recommended for manipulating HPAI viruses.

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When to Contact CDC

The Influenza Division, CDC, should be contacted immediately for any positive results of HPAI H5N1 virus testing, or at any time for any questions related to HPAI H5N1 and specimen collection, laboratory testing, antiviral treatment, or infection control. Laboratory results for human clinical specimens that test positive for HPAI H5N1 virus by RT-PCR at a laboratory in the United States should be shipped as soon as possible for confirmation at the Influenza Division, National Center for Immunization and Respiratory Diseases, CDC, a designated WHO H5 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).

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Travel Health Notice

Currently, CDC does not recommend any travel restrictions to any of the countries affected by HPAI H5N1 virus in poultry. Six countries are currently considered endemic for HPAI H5N1 virus among poultry (Bangladesh, China, Egypt, India, Indonesia, and Vietnam). Sporadic HPAI H5N1 virus outbreaks occur among poultry in other countries. More information is available on the OIE web site and the FAO web site.

CDC does recommend that travelers to HPAI H5N1-endemic countries and those countries experiencing HPAI H5N1 poultry outbreaks observe the following:

• Avoid visiting poultry farms, bird markets and other places where live poultry are raised, kept, or sold
• Avoid preparing or eating raw or undercooked poultry products

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*Influenza is thought to be primarily transmitted from person-to-person via virus-laden droplets that are generated when infected persons cough or sneeze; these droplets can then settle on the mucosal surfaces of the upper respiratory tracts of susceptible persons who are near (e.g., within about 6 feet) infected persons. Three feet has often been used by infection control professionals to define close contact and is based on studies of respiratory infections; however, for practical purposes, this distance may range up to 6 feet. The World Health Organization defines close contact as "approximately 1 meter;" the U.S. Occupational Safety and Health Administration uses "within 6 feet." For consistency with these estimates, this document defines close contact as a distance of up to approximately 6 feet.

† For a listing of influenza H5N1-affected countries: visit the OIE website, the FAO website and the WHO website.

‡ For example, a patient with respiratory illness and fever who does not require hospitalization, or a patient with significant neurologic or gastrointestinal symptoms in the absence of respiratory disease.

§ Specimens can be transported in viral transport media, Hanks balanced salt solution, cell culture medium, tryptose-phosphate broth, veal infusion broth, or sucrose-phosphate buffer. Transport media should be supplemented with protein, such as bovine serum albumin or gelatin, to a concentration of 0.5% to 1%.

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