Ramaz Mitaishvili, MD
Glendale, CA

The PCDCP will provide 3 specimen collection kits and instructions to each of the sentinel providers to ensure the proper collection and transport of influenza specimens during the influenza season (October – March). These collections kits are prepackaged and shipped to the sentinel sites at the beginning of the influenza season, and are continually re-supplied to the sentinel provider as they submit specimens throughout the season. Also provided within each kit is a pre-addressed Federal Express (Fed Ex) label which allows the sentinel site to ship the specimens “postage paid” to the PCDCP.

Procedures

General

Sentinel laboratories should not inoculate specimens suspected of containing influenza A H5N1 virus into cell culture. Only laboratories capable of performing culture under BSL 3 conditions with enhancements should perform culture to evaluate a suspected influenza H5N1 case. If these criteria are met, and culture is performed, consultation with PCDCP and the state public health laboratory is recommended. The use of rapid antigen tests for influenza is increasing in laboratories, point of care locations, and in physicians’ offices. These tests are among the least reliable for diagnosis of influenza, and should not be used to rule out avian influenza in a suspect case, especially during the current pre-pandemic phase.
Although influenza A H5N1 is the agent of highest concern at this time, it is important to note that a pandemic could occur from other novel strains of influenza. For this reason, testing for H5N1 virus alone is not recommended and any unusual influenza viruses that cannot be subtyped should be referred to the PCDCP.

Precautions

Culture diagnosis of suspected avian influenza A H5N1 requires enhanced BSL 3 laboratory conditions. Enhancements include use of respirators, decontamination of all waste (solid and liquid), and showering of personnel before exiting. Molecular and rapid antigen testing can be performed on respiratory specimens under standard BSL 2 conditions in a Class II biological safety cabinet.  
Specimens
Respiratory specimens. Throat swabs and lower respiratory samples such as bronchoalveolar lavage and tracheal aspirates are the preferred specimens for detection of influenza A H5N1 virus. Nasal swabs and aspirates are acceptable, but may contain lower titers than throat swabs. This is an important distinction between H5N1 and seasonal, human adapted influenza A subtypes.
Collection of a nasopharyngeal swab and a throat swab from the same patient (can be submitted in the same tube of viral transport medium) would provide optimal specimens for both human adapted and avian influenza strains.
Rectal specimens. In contrast to seasonal human influenza, diarrhea is a common symptom of H5N1 infections. Influenza A H5N1 viral RNA has been detected in rectal swabs by RT-PCR. Additionally, rectal specimens are inappropriate for human-adapted influenza strains.
Serum. Influenza A H5N1-specific antibody can be detected in serum by the microneutralization assay. Paired specimens, the first collected during acute illness and the second collected 2-4 weeks later, are required for definitive diagnosis. Sentinel laboratories should contact their local or state public health laboratory, or the PCDCP for information on serologic testing for influenza A H5N1.

Specimen collection and handling

Detection of influenza A H5N1 is more likely from specimens collected within the first three days of illness onset. If possible, serial specimens should be collected over several days from the same patient to increase clinical sensitivity.
Dacron or rayon tipped swabs should be used for specimen collection, as other materials may inhibit RT-PCR. Rapid antigen detection kits provide or specify swab types. Swabs placed in viral transport medium are generally suitable for RT-PCR testing. Specific specimen requirements are provided by the testing laboratory.
The collection of lower respiratory specimens generates aerosols, and requires infection control precautions for influenza A H5N1, including the use of gloves, gown, eye protection, and a respirator rated at least N-95.
Specimens should be stored at refrigerated temperatures, unless specified otherwise by test procedures. For virus isolation, specimens should be stored at refrigerated temperatures no longer than 2 days, or frozen at ≤70°, and shipped on dry ice. Follow current regulations for packaging and shipping hazardous materials ASM Sentinel Laboratory Guidelines for Suspected Agents of Bioterrorism/Packing and Shipping Infectious Substances, Diagnostic Specimens, and Biological Agents
The Sentinel laboratory should contact the nearest designated LRN Reference laboratory prior to shipping specimens.

Rejection criteria

Rejection criteria include lack of patient identification on the specimen, incomplete documentation on the requisition form, and improper specimen type or handling. Exceptions may be made, at the discretion of the laboratory director, out of public health or medical necessity. Due to the potential for false positive results and public health panic, specimens may also be rejected for H5N1-specific testing if the patient does not meet clinical case or risk assessment criteria.

Testing

Rapid antigen tests

Because rapid influenza antigen tests provide a result in 30 minutes or less, they significantly impact patient treatment and management. These tests are widely used for diagnosis of influenza in central, point-of-care, and physician office laboratories. Several rapid antigen tests are commercially available, some of which are able to distinguish between influenza A and B types. Some of these tests are CLIA waived even in the United States, therefore it can be waived for local facilities in Abkhazia too. Rapid antigen tests are less sensitive than culture or RT-PCR. Manufacturer claimed sensitivity ranges from 40 to100% and specificity from 52-100% compared to viral culture. Because of the varied specificity, the positive predictive value of rapid influenza tests is often reduced when disease prevalence is low. Therefore, positive results outside of the influenza “season” should be interpreted with caution, and confirmed by additional tests.
While rapid antigen capture assays may detect avian influenza subtypes, including H5N1, currently available tests are not capable of distinguishing specific influenza A subtypes. Recent evidence indicates that currently available rapid antigen tests are extremely insensitive for H5N1, and should not be used to rule out avian influenza in a suspect case, especially during the current pre-pandemic phase. Rapid antigen testing can be performed on respiratory specimens from suspected avian influenza cases under standard BSL 2 conditions in a Class II biological safety cabinet.

Fluorescent antibody staining of antigens

The staining of influenza antigens with fluorescent antibody is an additional rapid test. When performed directly on cells from respiratory specimens, this method can provide results in less than an hour. Availability of fluorescent antibody staining is restricted to laboratories with immunofluorescent microscopes and trained technologists able to accurately interpret fluorescent staining patterns. Since this is not a point-of-care test, the factors with the most significant impact on turnaround time of test results are specimen transport to the testing laboratory, and batching of specimens. Fluorescently-labeled antibodies specific for influenza A and B viruses are available. Some commercially available influenza antibodies are provided in pools with antibodies to other common respiratory viruses. Fluorescent antibody staining is generally considered to be slightly more sensitive than rapid antigen tests. Specificity is high, but depends on well trained, experienced technologists.
Fluorescent antibody staining reagents specific for influenza A virus will detect avian influenza A H5N1. When this guideline was written there was no H5N1-specific fluorescent antibody reagent commercially available. Fluorescent antibody staining can be performed on respiratory specimens from suspected avian influenza cases under BSL 2 conditions in a Class II biological safety cabinet.

Nucleic acid amplification

Nucleic acid amplification methods such as RT-PCR and nucleic acid sequence-based amplification (NASBA) are becoming more commonly used for detection of influenza virus and other respiratory viruses. Using real-time, fluorescent detection of amplified product, laboratories are able to perform molecular tests in less than 3 hours. These are consistently the most sensitive methods for detection of influenza virus, including H5N1. High specificity requires judicious selection of primers and probes, optimization of amplification conditions, and interpretation of results. Continuous adherence to laboratory protocol is essential to avoid false positives due to carry-over contamination. Currently, the only Food and Drug Administration-cleared nucleic acid amplification test for influenza viruses, including H5N1, is the RT-PCR method used by LRN Reference laboratories. In addition to LRN Reference laboratories, some commercial and hospital laboratories offer nucleic acid amplification testing for influenza A H5N1. Unlike the LRN method, these tests are developed and validated in-house by each laboratory. As such, the performance characteristics of the tests may vary between laboratories. So, in this case one of the partners in proposed project must be LRN in some instances.
RT-PCR testing only for the H5N1 subtype is not recommended. Specimens from suspect cases should be tested for both influenza A and B, and currently circulating influenza A subtypes in addition to H5N1.
Initial specimen processing, including addition of lysis buffer can be performed within a biological safety cabinet in a BSL 2 laboratory. Some specimen lysis buffers do not inactivate viruses. If lysis buffer that is known to inactivate the virus is used, further processing can be performed outside the biological safety cabinet.

Culture

Culture provides highly specific laboratory diagnosis of influenza, but requires fresh, refrigerated specimens for optimal sensitivity. Specimens in viral transport medium must be kept at 2-8°C and processed within 48 hours to avoid excessive decrease in viral titer. With proper specimen handling, culture is more sensitive than antigen detection methods. Historically, culture methods have been considered too slow to impact patient management. Incubation for at least 5 days is generally required to detect influenza virus in tubes cultures. Tubes are generally held for 14 days prior to reporting a negative result. Influenza virus is detected in tube cultures by the presence of cytopathic effect (CPE), adsorption or agglutination of red blood cells, and fluorescently-labeled antibodies specific for influenza A and B viruses. Spin-amplified shell vial cultures have reduced the time to detection to 1-3 days. However, this is considered by many to be too slow to impact patient treatment or isolation decisions. Culture is essential for detecting influenza infection missed by rapid testing, confirmation of non-culture results when disease prevalence is low, and to obtain isolates for characterization and surveillance.
Influenza A H5N1 will grow in cell lines commonly used for isolation of human-adapted influenza virus, including primary monkey kidney, Madin Darby canine kidney, A549, and others. Sentinel laboratories must be cognizant that H5N1 and other highly pathogenic novel subtypes can be cultivated unknowingly from unrecognized human cases of avian influenza. Do not perform culture on specimens if avian influenza A H5N1 is suspected, unless performed under enhanced BSL 3 laboratory conditions. Contact the United States LRN Reference laboratory for instructions on handling and shipping of specimens from suspected avian influenza cases.

• Instructions for sentinel laboratories that set up viral cultures under BSL2 conditions on a specimen or patient later determined to be positive for influenza A H5N1:
• Consult with the public health laboratory or PCDCP. These laboratories may request specimens or cultures.
• Isolate specimens, cell culture vessels and supplies potentially contaminated with the influenza A H5N1 virus. Sterilize by autoclaving, and discard.
• Disinfect work area.
• Monitor potentially exposed staff for symptoms. Public health or medical experts may recommend quarantine and prophylactic treatment.
• Documentation of transfer or destruction must be maintained for three years.

Serology

Serologic test methods to detect influenza virus-specific antibodies are available, and generally performed in reference and public health laboratories. These methods include indirect fluorescent antibody (IFA), complement fixation (CF), hemagglutination inhibition (HI) and neutralization. HI and neutralization are often used with specific antigens to identify influenza virus type and subtype-specific antibody titers. The diagnostic utility of serology is limited by the need, generally, to collect both acute and convalescent sera to identify either seroconversion or a four-fold rise in antibody titer. As such, serologic methods that detect IgG responses have relatively little impact on patient management. IFA and other tests that detect IgM antibodies can detect acute infection, but sensitivity is reduced because serum IgM levels are often low due to repeated exposure to vaccine or circulating virus.

Interpretation/Reporting/Action

• When a patient presents with suspected avian influenza, communication with the public health department is essential.
• Specimens from suspected avian influenza cases should be referred to the local or state public health laboratory for instructions on handling and shipping of specimens from suspected avian influenza cases.
• In addition to sentinel site surveillance, any primary care physician that suspects avian flu or has presumptively identified a suspect cluster of influenza-like illness (ILI), may make a request through PCDCP  to submit influenza specimens. If sufficient justification exists, contact PCDCP for lab kit and instruction.
• Currently, specimens are received, accessioned, inoculated onto cell culture, incubated for 2-3 days, and if cytopathic effect is evident, a DFA (Direct fluorescent antibody test by microscopy) is performed to determine if the virus is influenza type A or type B. Cell culture is the laboratory method necessary to isolate viable virus to both confirm live virus in the clinical sample and to provide further strain characterization. If the specimen is negative, no further workup is necessary. If the specimen is type B, conduct no further testing. If the specimen is Type A, an IFA (Indirect fluorescent antibody test by microscopy) will be performed using WHO reagents to determine if the virus is H3 or H1, the current circulating strains. If a sample is identified as influenza A but cannot be subtyped as H1 or H3 by rRT-PCR, an immediate investigation would ensue with the support of PCDCP to determine the possibility of infection by a new subtype of influenza.