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HHS Pandemic Influenza Plan Supplement 2 Laboratory Diagnostics

 

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Table of Contents

Summary of Roles and Responsibilities for Public Health and Clinical Laboratories In Laboratory Diagnostics

  1. S2-I.  Rationale
  2. S2-II.  Overview
  3. S2-III.  Recommendations For The Interpandemic and Pandemic Alert Periods
    1. Laboratory support for seasonal influenza surveillance
    2. Laboratory testing for novel influenza subytpes
      1. Testing for human cases of avian influenza
      2. Testing for human influenza strains with pandemic potential
    3. Surveillance for novel strains of influenza during the Pandemic Alert Period
      1. Detection and characterization of novel influenza strains
      2. Laboratory reporting
      3. Distribution of diagnostic reagents and test information
      4. Laboratory surge capacity planning
        1. Staffing and training
        2. Supplies and equipment
        3. Specimen management
      5. Partnerships with healthcare providers and clinical laboratories
  4. S2-IV.  Recommendations for The Pandemic Period
    1. Laboratory support for disease surveillance
    2. Laboratory support for clinicians
    3. Biocontainment procedures
    4. Occupational health issues for laboratory workers

Box 1. Use of diagnostic assays during an influenza pandemic
Box 2. Laboratory support for seasonal influenza surveillance
Box 3. Avian influenza strains with high and low pathogenicity

Appendix 1. Influenza diagnostic assays
Appendix 2. Interim recommendations: Enhanced U.S. surveillance and diagnostic evaluation to identify cases of human infection with avian influenza A (H5N1)
Appendix 3. Reference testing guidelines for potential pandemic strains of influenza
Appendix 4. Laboratory biosafety guidelines for handling and processing specimens or isolates of novel influenza strains
Appendix 5. Guidelines for collecting and shipping specimens for influenza diagnostics
Appendix 6. Rapid diagnostic testing for influenza
Appendix 7. Guidelines for medical surveillance of laboratory research personnel working with novel strains of influenza, including avian strains and other strains with pandemic potential



Summary of Roles and Responsibilities for Public Health and Clinical Laboratories In Laboratory Diagnostics

Interpandemic and Pandemic Alert Periods

Clinical and hospital laboratories:

State and local public health laboratories:

HHS responsibilities:

Pandemic Period

Clinical and hospital laboratories:

State and local public health laboratories:

HHS responsibilities:


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S2-I. Rationale

The goals of diagnostic testing during a pandemic are to:

Diagnostic testing for pandemic influenza virus may involve a range of laboratory assays, including rapid antigen tests, reverse-transcription polymerase chain reaction (RT-PCR), virus isolation, and immunofluorescence antibody (IFA) assays (see Box 1 and Appendix 1).

During the earliest stages of a pandemic, public health, hospital, and clinical laboratories might receive a large and potentially overwhelming volume of clinical specimens. Pre-pandemic planning is therefore essential to ensure the timeliness of diagnostic testing and the availability of diagnostic supplies and reagents, address staffing issues, and disseminate protocols for safe handling and shipping of specimens. Once a pandemic is underway, the need for laboratory confirmation of clinical diagnoses may decrease as the virus becomes widespread.


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S2-II. Overview

Supplement 2 provides recommendations to state and local public health partners and other laboratories on the use of diagnostic tests to detect, characterize, and monitor novel subtypes of influenza, including avian influenza A (H5N1) and other viruses with pandemic potential. The recommendations for the Interpandemic and Pandemic Alert Periods focus on laboratory testing in support of seasonal influenza surveillance, laboratory-based detection of novel influenza subtypes, and preparedness planning to support the laboratory component of the response to a pandemic (e.g., detection and characterization of viruses, case reporting, specimen management, surge capacity). The recommendations for the Pandemic Period focus on the provision of laboratory support for disease surveillance and to assist clinicians and hospitals. The recommendations also cover occupational health issues for laboratory workers.


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S2-III. Recommendations For The Interpandemic and Pandemic Alert Periods

  1. Laboratory support for seasonal influenza surveillance

    State and local public health laboratories and clinical laboratories (including hospital and private commercial laboratories) should continue to participate in laboratory-based surveillance for new subtypes of influenza through the U.S.-based laboratories in the World Health Organization (WHO) Global Influenza Surveillance Network and the National Respiratory and Enteric Virus Surveillance System (NREVSS). Additional information on seasonal influenza surveillance (including surveillance for influenza mortality and pediatric hospitalizations) is provided in Supplement 1. Information on the WHO Global Influenza Surveillance Network and NREVSS is provided in Box 2.

  2. Laboratory testing for novel influenza subtypes

    During the Pandemic Alert Period, state and local health departments, hospitals, and clinicians should enhance surveillance to identify patients who may present with possible cases of novel influenza (see Appendix 2). Health Alert Network (HAN) messages will be issued, as needed, to provide updates and guidance as new situations arise.

    State and local public health laboratories should be prepared to process and, in some instances, test—if they have the capability (see below)—specimens from suspected cases of infection with:

    • Avian influenza A (H5N1) and other avian influenza viruses
    • Other animal influenza viruses (e.g., swine influenza viruses)
    • New or re-emergent human influenza viruses (e.g., H2) with pandemic potential

    Clinicians should contact their state or local health departments if they suspect a human case of infection with any novel influenza A virus. State and local health departments, in turn, should contact CDC via the CDC Emergency Response Hotline: 770-488-7100.

    Guidelines on when to send specimens or isolates of suspected novel avian or human strains to CDC for reference testing are provided in Appendix 3.

    1. Testing for human cases of avian influenza

      Currently, avian influenza strains implicated in human disease (in addition to influenza A [H5N1]) include the highly pathogenic avian influenza (HPAI) strain H7N7 and the low pathogenic avian influenza (LPAI) strains H9N2, H7N2, and H7N3 (see Box 3). As of October 2005, no laboratory-confirmed cases of human infection with influenza A (H5N1) had been reported in the United States. However, CDC has confirmed two non-fatal cases of avian A (H7N2) influenza in Virginia and New York (Box 3). As new U.S. cases of human infection with avian influenza viruses are reported, they will be posted at: www.aphis.usda.gov/vs/birdbiosecurity/hpai.html and at: www.cdc.gov/flu.

      Recommendations on laboratory testing for human cases of avian influenza are as follows:

      • State public health laboratories may conduct testing to identify suspected subtypes of avian influenza, including H5 and H7, if appropriate laboratory capacity and biocontainment equipment are available. Because of the danger that HPAI strains present to the U.S. agricultural industry, U.S. Department of Agriculture (USDA) regulations require that HPAI strains such as H5N1 (which are classified as select agents) must be cultured using BSL-3 biocontainment conditions with enhancements (see Appendix 4).

      • Public health laboratories that lack BSL-3 facilities may use RT-PCR with BSL-2 containment to test clinical specimens from suspected human cases of avian influenza to identify and subtype influenza A viruses (e.g., H1, H3, H5, and H7; see S2-III.C). Or, they may send specimens to CDC, using the collection, handling, and shipping procedures described in Appendix 5.

      During the Pandemic Alert Period, specimens from suspected cases of human infection with novel influenza viruses should be sent for testing to public health laboratories with proper biocontainment facilities:

      • RT-PCR – BSL-2
      • Virus isolation – BSL-3 with enhancements

      The American Society for Microbiology maintains a list of emergency contacts in state public health laboratories, which is available at: www.asm.org/.

      If an avian influenza strain—or a human virus variant that evolves from it—causes an influenza pandemic, it might become necessary to re-evaluate biocontainment requirements and select agent registration requirements for laboratory testing. CDC and the Laboratory Response Network (LRN) will assist USDA, as requested, in making such a decision.

    2. Testing for human influenza strains with pandemic potential

      During the Pandemic Alert Period, diagnostic laboratories should be on the alert for new human subtypes of influenza that might have pandemic potential. Recommendations are as follows:

      • State and local public health laboratories that can detect human and avian influenza subtypes by RT-PCR should report all unusual subtypes to CDC via the Emergency Response Hotline (770-488-7100).
      • Public health laboratories that can detect human (but not avian) influenza subtypes by IFA staining or RT-PCR should send influenza A isolates that cannot be subtyped to CDC. (If an avian strain is suspected, virus isolation and IFA should be performed under BSL-3 conditions with enhancements.)
      • Public health laboratories should send specimens to CDC if a patient meets the clinical and epidemiologic criteria for infection with a novel influenza virus and:
        • Tests positive for influenza A by RT-PCR or by rapid diagnostic testing, or
        • Tests negative for influenza A by rapid diagnostic testing and/or RT-PCR testing for influenza is not available
      • Clinical laboratories that receive diagnostic specimens from patients with suspected novel influenza (based on clinical and epidemiologic data) should contact their state or local health departments.
      • If new or re-emergent human influenza strains with pandemic potential are suspected, laboratories should conduct RT-PCR only under BSL-2 containment conditions and viral culture only under BSL-3 conditions with enhancements (see Appendix 4).
  3. Laboratory planning to support the response to an influenza pandemic

    Advance planning is essential to anticipate adequate laboratory capacity to support medical and public health partners during an influenza pandemic. Some aspects of this planning, such as surge capacity planning, can be coordinated with bioterrorism preparedness planning.

    1. Detection and characterization of novel influenza strains

      • As of October 2005, about 48 state and large local public health laboratories have received training in RT-PCR protocols for molecular detection of H1, H3, H5, and H7 subtypes. These laboratories should incorporate RT-PCR testing into their standard influenza laboratory activities. Real-time RT-PCR protocols are available through the website of the Association of Public Health Laboratories (APHL) and will be updated as required to monitor the appearance and evolution of novel influenza viruses. A positive RT-PCR test result for a novel influenza strain should be considered presumptive, pending testing by a second reference laboratory.
      • State and local public health laboratories should provide hospitals and healthcare providers with information on how to contact the laboratory when a novel influenza subtype is suspected and how to handle, label, and ship clinical specimens for diagnostic evaluation.
      • State and local public health laboratories should contact laboratories in their jurisdictions that conduct RT-PCR influenza testing or that have BSL-3 containment facilities to remind them to notify the state health department if they receive specimens from suspected cases of novel influenza.

    2. Laboratory reporting

      State and local health departments that report laboratory-confirmed seasonal influenza cases to CDC use a variety of reporting mechanisms, including faxes, the Public Health Information System (PHLIS), and a web-based NREVSS data-entry system. Cases of novel influenza should be reported to CDC by the same mechanisms.

    3. Distribution of diagnostics reagents and test information

      CDC is working with USDA and the Food and Drug Administration (FDA) to address any regulatory barriers to emergency distribution and use of diagnostic tests and reagents during a pandemic. CDC will provide updated preparedness information regarding diagnostic tests and reagents to state and local public health partners via the LRN and HAN.

    4. Laboratory surge capacity planning

      Health departments should assess projected statewide needs for scaled-up diagnostic activity during the early stages of a pandemic, in terms of laboratory staffing, training, reporting, and supplies, and should develop strategies to address them.

      1. Staffing and training Laboratories should plan for increased staffing needs. Some strategies include:
        • Cross-training personnel during the regular influenza season in the use of rapid diagnostic tests and RT-PCR protocols and in reporting results through existing surveillance systems
        • Arranging to recruit and train temporary staff for employment during a pandemic
      2. Supplies and equipment

        Laboratories are likely to require additional diagnostic supplies and equipment to process large numbers of samples during the initial stages of a pandemic. Some preparedness strategies include:

        • Establishing the current level of diagnostic supplies, including personal protective equipment for laboratorians (e.g., gloves, masks)
        • Assessing anticipated equipment and supply needs, and determining a trigger point for ordering extra resources. Laboratories should also consider the need for back-up sources of supplies if most laboratories in a state or large city rely on the same manufacturer for particular supplies or equipment.
        • Determining how consumption of supplies will be tracked during a pandemic
      3. Specimen management

        State and local health departments should inform and educate public health staff (including laboratorians), local physicians, and hospital workers on safe and effective methods for specimen collection and management, making use of the guidelines in Appendix 5, Guidelines for Collecting and Shipping Specimens for Influenza Diagnostics. Safety issues related to specimen handling are also addressed in Supplement 4.

        Procedures for specimen collection, handling, and shipping during a pandemic will be the same as those used for seasonal disease surveillance. However, laboratory staff should anticipate shipping a much larger number of specimens in a very short time, especially during the early stages of a pandemic. Once the pandemic is underway and healthcare providers rely on clinical criteria and rapid test kits, more diagnostic activities may be conducted locally and fewer shipments may be needed.

  4. Partnerships with healthcare providers and clinical laboratories

    Good working relationships between healthcare providers and public health laboratories will facilitate diagnostic activities during a pandemic.

    • Public health laboratories should continue to build partnerships with healthcare providers in their jurisdictions, including physicians who participate in the Sentinel Provider Network (SPN) during the regular influenza season (see Supplement 1).
    • Public health laboratories should build partnerships with clinical laboratories and provide them with updated information and (if feasible) training in influenza diagnostics.

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S2-IV. Recommendations for The Pandemic Period

  1. Laboratory support for disease surveillance

  2. Laboratory support for clinicians

    • When a pandemic begins, public health and clinical laboratories will scale up to manage increased numbers of requests for influenza testing. As part of this effort, CDC will work with state and local public health laboratories and the LRN to provide clinical laboratories with guidelines for safe handling, processing, and rapid diagnostic testing of clinical specimens from patients who meet the case definition for pandemic influenza.

    If private laboratories perform RT-PCR testing during the early phase of an influenza pandemic, the results should be confirmed in consultation with the state public health laboratory.

    • State and local health laboratories should provide local healthcare providers with:
      • Specimen submission forms that specify the clinical and epidemiologic data that should accompany clinical specimens sent to state public health laboratories. (During the early stages of a pandemic, clinicians should include information on patients’ symptoms and risk factors, if known.)
      • Rapid communication of test results and reminders that a negative test result (especially by rapid diagnostic testing) might not rule out influenza and should not affect patient management or infection control decisions.
      • Guidance on the use of commercially available rapid diagnostic tests for the detection of influenza A. These tests may be used by physicians to supplement clinical diagnoses of pandemic influenza. Because the sensitivity of rapid diagnostic kits might not be optimal, physicians should take their positive and negative predictive values into consideration when interpreting test results (Appendix 6).
      • Guidance on which specimens to send to state public health laboratories as the pandemic continues.

  3. Biocontainment procedures

    During an influenza pandemic, laboratory procedures should be conducted under appropriate biosafety conditions:

    • Commercial antigen detection testing for influenza should be conducted using BSL-2 work practices.
    • Public health laboratories may conduct RT-PCR testing using BSL-2 work practices and virus isolation using BSL-3 practices with enhancements.

    Additional information on laboratory biocontainment is provided in Appendix 4.

  4. Occupational health issues for laboratory workers

    To protect the health of laboratory workers during a pandemic, public health, clinical, and hospital laboratories should maintain the safety practices used during the Interpandemic and Pandemic Alert Periods. These include:

    • Conducting laboratory procedures under appropriate biocontainment conditions
    • Encouraging routine vaccination of all eligible laboratory personnel who are exposed to specimens from patients with respiratory infections

    Guidelines for medical surveillance of laboratory personnel are provided in Appendix 7.


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Box 1. Use of Diagnostic Assays during an Influenza Pandemic

Public health and clinical laboratories will use different types of diagnostic tests for influenza at different stages of a pandemic. Each of the tests discussed below is described in detail in Appendix 1.

Virus Isolation
Virus isolation—growing the viral strain in cell culture—is the “gold standard” for influenza diagnostics because it confirms that the virus is infectious. During a pandemic, virus isolation followed by antigenic and genetic (sequencing) analysis will be used to characterize the earliest pandemic isolates, as well as to monitor their evolution during the pandemic. Laboratories that participate in the WHO Global Influenza Surveillance Network typically use virus isolation followed by hemagglutination inhibition (HAI), IFA staining, or RT-PCR to monitor circulating seasonal strains of influenza. If clinical and epidemiologic data suggest that a human case of influenza might be due to infection with avian influenza A (H5N1) or another highly pathogenic avian influenza strain (see Box 3), the virus should not be cultured except under BSL-3 conditions with enhancements. Laboratories that lack BSL-3 enhanced facilities may either perform RT-PCR subtyping using BSL-2 containment procedures or send the specimen to CDC for isolation and characterization.

Immunofluorescence Antibody Staining
IFA staining following virus isolation can be used to identify influenza types (A, B) and influenza A HA subtypes using a panel of specific antisera. In some cases, IFA can be used for direct testing of cells pelleted from original clinical samples. CDC’s Influenza Branch produces and distributes a reagent kit to WHO collaborating laboratories that includes monoclonal antibodies for typing and subtyping currently circulating influenza viruses by IFA. Many laboratories use commercially available reagents to type influenza viruses by direct immunofluorescence tests (DFA).

RT-PCR Subtyping
Influenza specimens may also be typed and subtyped using RT-PCR, which does not require in vitro growth or isolation of virus. As of October 2005, CDC has trained scientists from 48 states to use RT-PCR subtyping to identify human and avian HA subtypes of public health concern. APHL members can access protocols and sequences of primers and probes that can be used for typing and subtyping on the APHL website.

Serologic Tests
Tests based on detection of antibodies in patient sera—e.g., enzyme-linked immunosorbent assay (ELISA), HAI, and microneutralization assay—can be used to retrospectively confirm influenza infection. Although microneutralization assay is the most comprehensive test for detection in humans of antibodies to avian influenza viruses, it is available in only a few state public health laboratories.

Rapid Diagnostic Tests
Several rapid diagnostic test kits based on antigen detection are commercially available for influenza. Laboratories in outpatient settings and hospitals can use these tests to detect influenza viruses within 30 minutes. Some tests can detect influenza A viruses (including avian strains); others can detect influenza A and B viruses without distinguishing between them, and some can distinguish between influenza A and B viruses. The type of specimens used in these tests (i.e., nasal wash/aspirate, nasopharyngeal swabs, or nasal swab or throat swab) may also vary. Like RT-PCR, rapid diagnostic tests do not require in vitro growth or isolation of virus. During a pandemic, rapid diagnostic tests will be widely used to distinguish influenza A from other respiratory illnesses. See Appendix 6 for additional information.


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Box 2. Laboratory Support for Seasonal Influenza Surveillance

U.S. Collaborating Laboratories of the WHO Global Influenza Surveillance Network
All state and several large local public health laboratories, as well as about 25 tertiary-care hospital and academic center laboratories, participate as U.S. collaborating laboratories in the WHO Global Influenza Surveillance Network, which collects worldwide data on circulating strains of influenza viruses. These data are used to develop recommendations for the formulation of each year’s influenza vaccines, as well as to detect new human influenza viruses that might have pandemic potential. CDC’s Influenza Laboratory serves as the WHO Collaborating Center for Surveillance, Epidemiology, and Control of Influenza, along with the WHO Collaborating Centers for Reference and Research on Influenza in Australia, Japan, and the United Kingdom.

The U.S.-based WHO collaborating laboratories provide CDC with weekly reports of laboratory-confirmed cases of influenza A and B viruses, by age group. These laboratories typically use virus isolation followed by antigenic testing with IFA staining or HAI—or by molecular testing with RT-PCR—to identify known subtypes of human influenza viruses. If unusual subtypes are detected, or if the specimens cannot be subtyped using available techniques, the specimens are sent to CDC for further testing.

NREVSS Collaborating Laboratories
The National Respiratory and Enteric Virus Surveillance System (NREVSS;
http://www.cdc.gov/ncidod/dvrd/revb/nrevss/) includes more than 90 laboratories throughout the country, including many hospital laboratories, some state public health laboratories, and a few private commercial laboratories. About 40 of the NERVSS laboratories are also WHO collaborating laboratories.

Like the WHO collaborating laboratories, NREVSS laboratories provide CDC with weekly reports of laboratory-confirmed cases of influenza A and B viruses. These laboratories typically test respiratory specimens with commercially available rapid diagnostic tests. Several NREVSS laboratories also perform virus isolation followed by rapid diagnostic tests or antigenic typing by IFA. If untypable viruses or unusual subtypes are detected, the specimens are sent to the state public health laboratory or to CDC for further testing.


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Box 3. Avian Influenza Strains with High and Low Pathogenicity

The U.S. Department of Agriculture (USDA) classifies avian influenza viruses as low pathogenic avian influenza (LPAI) viruses or highly pathogenic avian influenza (HPAI) viruses, based on characteristics of a virus’ hemagglutinin cleavage site or its virulence in birds, as determined by laboratory testing. LPAI strains are endemic in wild birds worldwide and are responsible for most avian influenza outbreaks in poultry. LPAI strains with H5 and H7 subtypes sometimes evolve into highly pathogenic forms. HPAI strains are extremely contagious and cause severe illness and high mortality rates in poultry.

LPAI strains include:

HPAI strains include:

The 2004 outbreak in Texas was the first HPAI outbreak in the United States since a previous outbreak of H5N2 in 1983-84 in the northeastern United States. The 1983-84 disease control effort involved the destruction of approximately 17 million birds and cost more than $70 million.

Although avian influenza A viruses do not usually infect humans, several instances of human infections of avian influenza have been reported since 1997. Cases of avian influenza infection in humans are apparently caused by contact with infected poultry or with surfaces contaminated with avian influenza viruses.

LPAI strains associated with human infection include:

HPAI viruses associated with human infection include:


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Appendix 1. Influenza Diagnostic Assays

Among the several types of assays used to detect influenza, rapid antigen tests, reverse-transcription polymerase chain reaction (RT-PCR), viral isolation, immunofluorescence assays (IFA), and serology are the most commonly used. The sensitivity and specificity of any test for influenza will vary by the laboratory that performs the test, the type of test used, and the type of specimen tested. A chart that lists influenza diagnostic procedures and commercially available rapid diagnostic tests follows more detailed descriptions provided below.

Virus Isolation
Biocontainment level: Interpandemic and Pandemic Alert Periods – BSL-3 with enhancements; Pandemic Period – BSL-2

Virus isolation is a highly sensitive and very useful technique when the clinical specimens are of good quality and have been collected in a timely manner (optimally within 3 days of the start of illness). Isolation of a virus in cell culture along with the subsequent identification of the virus by immunologic or genetic techniques are standard methods for virus diagnosis. Virus isolation amplifies the amount of virus from the original specimen, making a sufficient quantity of virus available for further antigenic and genetic characterization and for drug-susceptibility testing if required. Virus isolation is considered the “gold standard” for diagnosis of influenza virus infections.

Highly pathogenic avian influenza (HPAI) viruses are BSL-3 agents. During the Interpandemic and Pandemic Alert Periods, laboratories should attempt to culture HPAI viruses—as well as other influenza viruses with pandemic potential—only under BSL-3 conditions with enhancements in order to optimally reduce the risk of a novel influenza virus subtype spreading to persons or animals. During the Pandemic Period, biocontainment of BSL-2 is appropriate to prevent laboratory-acquired infection and the virus will already be widespread.

In recent years, the use of cell lines has surpassed the use of embryonated eggs for culturing of influenza viruses, although only viruses grown in embryonated eggs are used as seed viruses for vaccine production. Because standard isolation procedures require several days to yield results, they should be used in combination with the spin-amplification shell-vial method. The results of these assays can be obtained in 24–72 hours, compared to an average of 4.5 days using standard culture techniques. Spin-amplification should not be performed using 24-well plates because of increased risk of cross-contamination. The most effective combination of cell lines recommended for public health laboratories is primary rhesus monkey for standard culture, along with Madin Darby Canine Kidney (MDCK) in shell vial. The use of these two cell lines in combination has demonstrated maximum sensitivity over time for recovery of evolving influenza strains. Some clinical laboratories have recently reported good isolation rates using commercially available cell-line mixed-cell combinations; however, data are lacking on the performance of these mixed cells with new subtypes of Influenza A viruses.

Appropriate clinical specimens for virus isolation include nasal washes, nasopharyngeal aspirates, nasopharyngeal and throat swabs, tracheal aspirates, and bronchoalveolar lavage. Ideally, specimens should be collected within 72 hours of the onset of illness.

Viral culture isolates are used to provide specific information regarding circulating influenza subtypes and strains. This information is needed to compare current circulating influenza strains with vaccine strains, to guide decisions on influenza treatment and chemoprophylaxis, and to select vaccine strains for the coming year. Virus isolates also are needed to monitor the emergence of antiviral resistance and of novel influenza A subtypes that might pose a pandemic threat. During outbreaks of influenza-like illness, viral culture may help identify other causes of illness when influenza is not the etiology (except when using MDCK cells or the MDCK shell-vial technique).

Immunofluorescence Assays
Biocontainment level: BSL-2 when performed directly on clinical specimens; if used on cultures for earlier detection of virus, biocontainment recommendations for viral culture apply

Direct (DFA) or indirect (IFA) immunofluorescence antibody staining of virus-infected cells is a rapid and sensitive method for diagnosis of influenza and other viral infections. DFA and IFA can also be used to type and subtype influenza viruses using commercially available monoclonal antibodies specific for the influenza virus HA. The sensitivity of these methods is greatly influenced by the quality of the isolate, the specificity of the reagents used, and the experience of the person(s) performing, reading, and interpreting the test.

Although IFA can be used to stain smears of clinical specimens directly, when rapid diagnosis is needed it is preferable to first increase the amount of virus through growth in cell culture. For HPAI isolates, attempts to culture the virus should be made only under BSL-3 conditions with enhancements.

Reverse-Transcription Polymerase Chain Reaction (RT-PCR)
Biocontainment level: BSL-2

PCR can be used for rapid detection and subtyping of influenza viruses in respiratory specimens. Because the influenza genome consists of single-stranded RNA, a complementary DNA (cDNA) copy of the viral RNA must be synthesized using the reverse-transcriptase (RT) enzyme prior to the PCR reaction.

Laboratories can obtain CDC protocols and sequences of primers and probes for rapid RT-PCR detection of human and avian HA subtypes of current concern at the APHL website (available for members only). These protocols use real-time RT-PCR methods with fluorescent-labeled primers that allow automatic, semi-quantitative estimation of the input template. The RT-PCR results are analyzed and archived electronically, without the need for gel electrophoresis and photographic recording. A large number of samples may be analyzed at the same time, reducing the risk of carry-over contamination.

As with all PCR assays, interpretation of real-time RT-PCR tests must account for the possibility of false-negative and false-positive results. False-negative results can arise from poor sample collection or degradation of the viral RNA during shipping or storage. Application of appropriate assay controls that identify poor-quality samples (e.g., an extraction control and, if possible, an inhibition control) can help avoid most false-negative results.

The most common cause of false-positive results is contamination with previously amplified DNA. The use of real-time RT-PCR helps mitigate this problem by operating as a contained system. A more difficult problem is the cross-contamination that can occur between specimens during collection, shipping, and aliquoting in the laboratory. Use of multiple negative control samples in each assay and a well-designed plan for confirmatory testing can help ensure that laboratory contamination is detected and that negative specimens are not inappropriately identified as influenza-positive.

Specimens that test positive for a novel subtype of influenza virus should be forwarded to CDC for confirmatory testing. (Due to the possibility of contamination, it is important to provide original clinical material.) All laboratory results should be interpreted in the context of the clinical and epidemiologic information available on the patient.

Rapid Diagnostic Tests
Biocontainment level: BSL-2

Commercial rapid diagnostic tests can be used in outpatient settings to detect influenza viruses within 30 minutes. These rapid tests differ in the types of influenza viruses they can detect and in their ability to distinguish among influenza types. Different tests can 1) detect influenza A viruses only (including avian strains); 2) detect both influenza A and B viruses, without distinguishing between them; or 3) detect both influenza A and B viruses and distinguish between them.

The types of specimens acceptable for use (i.e., nasal wash/aspirate, nasopharyngeal swab, or nasal swab and throat swab) also vary by test. The specificity and, in particular, the sensitivity of rapid tests are lower than for viral culture and vary by test and specimen tested. The majority of rapid tests are >70% sensitive and >90% specific. Thus, as many as 30% of samples that would be positive for influenza by viral culture may give a negative rapid test result with these assays.

When interpreting results of a rapid influenza test, physicians should consider the level of influenza activity in the community. When influenza prevalence is low, positive rapid test results should be independently confirmed by culture or RT-PCR. When influenza is known to be circulating, clinicians should consider confirming negative tests with viral culture or other means because of the lower sensitivity of the rapid tests. Package inserts and the laboratory performing the test should be consulted for more details regarding use of rapid diagnostic tests. Additional information on diagnostic testing is provided at: http://www.cdc.gov/flu/professionals/labdiagnosis.htm. Detailed information on the use of rapid diagnostics tests is provided in Appendix 6.

Serologic Tests

Hemagglutination Inhibition (HAI)
Biocontainment level: BSL-2

Serologic testing can be used to identify recent infections with influenza viruses. It can be used when the direct identification of influenza viruses is not feasible or possible (e.g., because clinical specimens for virus isolation cannot be obtained, cases are identified after shedding of virus has stopped, or the laboratory does not have the resources or staff to perform virus isolation).

Since most human sera contain antibodies to influenza viruses, serologic diagnosis requires demonstration of a four-fold or greater rise in antibody titer using paired acute and convalescent serum samples. HAI is the preferred diagnostic test for determining antibody rises. In general, acute-phase sera should be collected within one week of illness onset, and convalescent sera should be collected 2–3 weeks later.

There are two exceptions in which the collection of single serum samples can be helpful in the diagnosis of influenza. In investigations of outbreaks due to novel viruses, testing of single serum samples has been used to identify antibody to the novel virus. In other outbreak investigations, antibody test results from single specimens collected from persons in the convalescent phase of illness have been compared with results either from age-matched persons in the acute phase of illness or from non-ill controls. In such situations, the geometric mean titers between the two groups to a single influenza virus type or subtype can be compared. In general, these approaches are not optimal, and paired sera should be collected whenever possible.

Because HAI titers of antibodies in humans infected with avian influenza viruses are usually very low or even undetectable, more sensitive serologic tests, such as microneutralization, may be needed.

Microneutralization Assay
Biocontainment level: Interpandemic and Pandemic Alert Periods – BSL-3 with enhancements; Pandemic Period – BSL-2

The virus neutralization test is a highly sensitive and specific assay for detecting virus-specific antibody in animals and humans. The neutralization test is performed in two steps: 1) a virus-antibody reaction step, in which the virus is mixed with antibody reagents, and 2) an inoculation step, in which the mixture is inoculated into a host system (e.g. cell cultures, embryonated eggs, or animals). The absence of infectivity constitutes a positive neutralization reaction and indicates the presence of virus-specific antibodies in human or animal sera.

The virus neutralization test gives the most precise answer to the question of whether or not a person has antibodies that can neutralize the infectivity of a given virus strain. The neutralization test has several additional advantages for detecting antibody to influenza virus. First, the assay primarily detects antibodies to the influenza virus HA and thus can identify functional, strain-specific antibodies in animal and human serum. Second, since infectious virus is used, the assay can be developed quickly upon recognition of a novel virus and before suitable purified viral proteins become available for use in other assays.

The microneutralization test is a sensitive and specific assay for detecting virus-specific antibody to avian influenza A (H5N1) in human serum and potentially for detecting antibody to other avian subtypes. Microneutralization can detect H5-specific antibody in human serum at titers that cannot be detected by HAI. Because antibody to avian influenza subtypes is presumably low or absent in most human populations, single serum samples can be used to screen for the prevalence of antibody to avian viruses. However, if infection of humans with avian viruses is suspected, the testing of paired acute and convalescent sera in the microneutralization test would provide a more definitive answer regarding the occurrence of infection. Conventional neutralization tests for influenza viruses based on the inhibition of cytopathogenic effect (CPE)-formation in MDCK cell cultures are laborious and rather slow, but in combination with rapid culture assay principles the neutralization test can yield results within 2 days. For HPAI viruses, neutralization tests should be performed at BSL-3 enhanced conditions.

Quick Reference Chart of Influenza Diagnostic Tests(1) (From: Prevention and Control of Influenza: Recommendations of the Advisory Committee on Immunization Practices (ACIP). MMWR 2004;53(RR-6):1-40.)

Procedure

Influenza Types Detected

Acceptable Specimens

Time for Results

Rapid result available

Viral culture

A and B

nasal wash/aspirate, NP swab,2 nasal aspirate, nasal swab and throat swab, sputum

5–10 days(3)

No

Immunofluorescence Antibody Staining

A and B

nasal wash/aspirate, NP swab,2 nasal aspirate, nasal swab and throat swab, sputum

2–4 hours

No

RT-PCR(5)

A and B

nasal wash/aspirate, NP swab,2 nasal aspirate, throat swab, bronchial wash, nasal aspirate, sputum

Hours

No

Serology

A and B

paired acute/convalescent serum samples(6)

>2 weeks

No

Rapid Diagnostic Tests

 

 

 

 

Directigen Flu A7 (Becton-Dickinson)

A

NP swab,2 throat swab, nasal wash, nasal aspirate

See insert

Yes

Directigen Flu A+B7, 9 (Becton-Dickinson)

A and B

NP swab,2 throat swab, nasal wash, nasal aspirate

See insert

Yes

FLU OIA7 (Thermo Electron)

A and B4

NP swab,2 throat swab, nasal aspirate, sputum

See insert

Yes

FLU OIA A/B7, 9 (Thermo Electron)

A and B

NP swab,2 throat swab, nasal aspirate, sputum

See insert

Yes

XPECT Flu A/B7, 9 (Remel)

A and B

Nasal wash, NP swab,2 throat swab

See insert

Yes

NOW Flu A Test7, 9 NOW Flu B Test7, 9 (Binax)

A B

Nasal wash, NP swab2 Nasal wash, NP swab2

See insert

Yes Yes

QuickVue Influenza Test8 (Quidel)

A and B4

NP swab,2 nasal wash, nasal aspirate

See insert

Yes

QuickVue Influenza A+B Test8 (Quidel)

A and B9

NP swab,2 nasal wash, nasal aspirate

See insert

Yes

AS Influenza A7, 9 SAS Influenza B7, 9

A B

NP wash,2 NP aspirate2 NP wash,2 NP aspirate2

See insert

Yes Yes

ZstatFlu8 (ZymeTx)

A and B4

throat swab

See insert

Yes

1   The list might not include all FDA-approved test kits.
2   NP = nasopharyngeal
3   Shell-vial culture, if available, may reduce time for results to 2 days.
4   Does not distinguish between influenza A and B virus infections.
5   RT-PCR = reverse-transcription polymerase chain reaction
6   A fourfold or greater rise in antibody titer from the acute- (collected within the first week of illness) to the convalescent-phase sample (collected 2–4 weeks after the acute sample) indicates recent infection.
7   Moderately complex test that requires specific laboratory certification
8   CLIA-waived test. Can be used in any office setting. Requires a certificate of waiver or higher laboratory certification
9   Distinguishes between influenza A and B virus infections.

Disclaimer: Use of trade names or commercial sources is for identification only and does not imply endorsement by the Centers for Disease Control and Prevention or the Department of Health and Human Services.


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Appendix 2. Interim Recommendations: Enhanced U.S. Surveillance and Diagnostic Evaluation to Identify Cases of Human Infection with Avian Influenza A (H5N1)

NOTE: This guidance pertains to the avian influenza A (H5N1) situation in October 2005. CDC will provide updated guidance for avian influenza A (H5N1) and for new situations, as needed, through the Health Alert Network (HAN).

Enhanced surveillance efforts by state and local health departments, hospitals, and clinicians are needed to identify patients at increased risk for influenza A (H5N1). Interim recommendations include the following:

Testing for avian influenza A (H5N1) is indicated for hospitalized patients with:

Testing for avian influenza A (H5N1) should be considered on a case-by-case basis in consultation with state and local health departments for hospitalized or ambulatory patients with:

  • Documented temperature of >100.4°F (>38°C), and
  • One or more of the following: cough, sore throat, or shortness of breath, and
  • History of close contact either with poultry (e.g., visited a poultry farm, a household raising poultry, or a bird market) in an H5N1-affected country, or with a known or suspected human case of influenza A (H5N1) within 10 days prior to onset of symptoms.

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Appendix 3. Reference Testing Guidelines for Potential Pandemic Strains of Influenza

State and local laboratories may conduct initial testing on patient specimens for influenza A or potential highly pathogenic strains, if laboratory capacity is available. Due to the spread of avian influenza A (H5N1) in poultry in Asia, laboratories should be on the alert for avian and human H5 viruses. Procedures for diagnosis of human cases of influenza A (H5N1) are provided in Appendix 2. Influenza A viruses other than currently circulating H1 and H3 subtypes should also be considered as potentially pandemic if detected in humans.

*Because the sensitivity of commercially available rapid diagnostic tests for influenza may not always be optimal, CDC will also accept specimens taken from persons who meet the clinical and epidemiological criteria even if they test negative by influenza rapid diagnostic testing—if PCR assays are not available at the state laboratory.


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Appendix 4. Laboratory Biosafety Guidelines for Handling and Processing Specimens or Isolates of Novel Influenza Strains

Key Messages


  • State and local public health laboratories may test clinical specimens from suspected novel influenza cases by RT-PCR using standard BSL-2 work practices in a Class II biological safety cabinet. Commercial rapid antigen detection testing may also be conducted under BSL-2 biocontainment conditions.

  • Highly pathogenic avian influenza A (H5) and A (H7) viruses are classified as select agents. USDA regulations require that these viruses (as well as exotic low pathogenic avian influenza viruses) be handled under BSL-3 laboratory containment conditions, with enhancements (i.e., controlled-access double-door entry with change room and shower, use of respirators, decontamination of all wastes, and showering of all personnel). Laboratories that work with these viruses must be certified by USDA.

  • Laboratories should not perform virus isolation on respiratory specimens from patients who may be infected with an avian influenza virus unless stringent BSL-3 enhanced containment conditions can be met and diagnostic work can be kept separate from studies with other human influenza A viruses (i.e., H1 or H3). Therefore, respiratory virus cultures should not be performed in most clinical laboratories. Cultures for patients suspected of having influenza A (H5N1) infection should be sent only to state laboratories with appropriate BSL-3 with enhancement containment facilities or to CDC.


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Appendix 5. Guidelines for Collecting and Shipping Specimens for Influenza Diagnostics

Key Messages


I. Respiratory Specimens

Eight types of respiratory specimens may be collected for viral and/or bacterial diagnostics: 1) nasopharyngeal wash/aspirates, 2) nasopharyngeal swabs, 3) oropharyngeal swabs, 4) broncheoalveolar lavage, 5) tracheal aspirate, 6) pleural fluid tap, 7) sputum, and 8) autopsy specimens. Nasopharyngeal wash/aspirates are the specimen of choice for detection of most respiratory viruses and are the preferred specimen type for children aged <2 years.

Respiratory specimens for detection of most respiratory pathogens, and influenza in particular, are optimally collected within the first 3 days of the onset of illness. Before collecting specimens, review the infection control precautions in Supplement 4.

  1. Collecting specimens from the upper respiratory tract

    1. Nasopharyngeal wash/aspirate

      • Have the patient sit with head tilted slightly backward.
      • Instill 1 ml–1.5 ml of nonbacteriostatic saline (pH 7.0) into one nostril. Flush a plastic catheter or tubing with 2 ml–3 ml of saline. Insert the tubing into the nostril parallel to the palate. Aspirate nasopharyngeal secretions. Repeat this procedure for the other nostril.
      • Collect the specimens in sterile vials. Label each specimen container with the patient’s ID number and the date collected.
      • If shipping domestically, use cold packs to keep the sample at 4°C. If shipping internationally, pack in dry ice (see shipping instructions below).

    2. Nasopharyngeal or oropharyngeal swabs

      • Use only sterile dacron or rayon swabs with plastic shafts. Do not use calcium alginate swabs or swabs with wooden sticks, as they may contain substances that inactivate some viruses and inhibit PCR testing.
      • To obtain a nasopharyngeal swab, insert a swab into the nostril parallel to the palate. Leave the swab in place for a few seconds to absorb secretions. Swab both nostrils.
      • To obtain an oropharyngeal swab, swab the posterior pharynx and tonsillar areas, avoiding the tongue.
      • Place the swabs immediately into sterile vials containing 2 ml of viral transport media. Break the applicator sticks off near the tip to permit tightening of the cap. Label each specimen container with the patient’s ID number and the date the sample was collected.
      • If shipping domestically, use cold packs to keep the sample at 4°C. If shipping internationally, pack in dry ice (see shipping instructions below).
  2. Collecting specimens from the lower respiratory tract

    1. Broncheoalveolar lavage, tracheal aspirate, or pleural fluid tap

      • During bronchoalveolar lavage or tracheal aspirate, use a double-tube system to maximum shielding from oropharyngeal secretions.
      • Centrifuge half of the specimen, and fix the cell pellet in formalin. Place the remaining unspun fluid in sterile vials with external caps and internal O-ring seals. If there is no internal O-ring seal, then seal tightly with the available cap and secure with Parafilm®. Label each specimen container with the patient’s ID number and the date the sample was collected.
      • If shipping domestically, use cold packs to keep the sample at 4°C. If shipping internationally, ship fixed cells at room temperature and unfixed cells frozen (see shipping instructions below).

    2. Sputum

      • Educate the patient about the difference between sputum and oral secretions.
      • Have the patient rinse the mouth with water and then expectorate deep cough sputum directly into a sterile screw-cap sputum collection cup or sterile dry container.
      • If shipping domestically, use cold packs to keep the sample at 4°C. If shipping internationally, pack in dry ice (see shipping instructions below).

II.  Blood Components

Both acute and convalescent serum specimens should be collected for antibody testing. Collect convalescent serum specimens 2–4 weeks after the onset of illness. To collect serum for antibody testing:

  • Collect 5 ml–10 ml of whole blood in a serum separator tube. Allow the blood to clot, centrifuge briefly, and collect all resulting sera in vials with external caps and internal O-ring seals. If there is no internal O-ring seal, then seal tightly with the available cap and secure with Parafilm®.
  • The minimum amount of serum preferred for each test is 200 microliters, which can easily be obtained from 5 ml of whole blood. A minimum of 1 cc of whole blood is needed for testing of pediatric patients. If possible, collect 1 cc in an EDTA tube and in a clotting tube. If only 1cc can be obtained, use a clotting tube.
  • Label each specimen container with the patient’s ID number and the date the specimen was collected.
  • If unfrozen and transported domestically, ship with cold packs to keep the sample at 4°C. If frozen or transported internationally, ship on dry ice.

III.  Autopsy Specimens

CDC can perform immunohistochemical (IHC) staining for influenza A (H5) viruses on autopsy specimens. 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 IHC staining. Collection of the appropriate tissues ensures the best chance of detecting the virus by (IHC) stains.

  • If influenza is suspected, a minimum total of 8 blocks or fixed-tissue specimens representing samples from each of the following sites should be obtained and submitted for evaluation:
    • Central (hilar) lung with segmental bronchi
    • Right and left primary bronchi
    • Trachea (proximal and distal)
    • Representative pulmonary parenchyma from right and left lung

In addition, representative tissues from major organs should be submitted for evaluation. In particular, for patients with suspected myocarditis or encephalitis, specimens should include myocardium (right and left ventricle) and CNS (cerebral cortex, basal ganglia, pons, medulla, and cerebellum). Specimens should be included from any other organ showing significant gross or microscopic pathology.

  • Specimens may be submitted as:
    • Fixed, unprocessed tissue in 10% neutral buffered formalin, or
    • Tissue blocks containing formalin-fixed, paraffin-embedded specimens, or
    • Unstained sections cut at 3 microns placed on charged glass slides (10 slides per specimen)
  • Specimens should be sent at room temperature (NOT FROZEN).
  • Fresh-frozen unfixed tissue specimens may be submitted for RT-PCR.
  • Include a copy of the autopsy report (preliminary, or final if available), and a cover letter outlining a brief clinical history and the submitter’s full name, title, complete mailing address, phone, and fax numbers, in the event that CDC pathologists require further information. Referring pathologists may direct specific questions to CDC pathologists. The contact number for the Infectious Disease Pathology Activity is 404-639-3133, or the pathologists can be contacted 24 hours a day, 7 days a week through the CDC Emergency Response Hotline at 770-488-7100.

IV.  Shipping Instructions

  • State and local health departments should call the CDC Emergency Response Hotline (770-488-7100) before sending specimens for influenza A reference testing. This number is available 24 hours a day, 7 days a week. Hotline staff will notify a member of the Influenza Branch who will contact the health department to answer questions and provide guidance. In some cases, the state health department may arrange for a clinical laboratory to send samples directly to CDC.
  • Specimens should be sent by Priority Overnight Shipping for receipt within 24 hours. Samples (such as fresh-frozen autopsy samples for RT-PCR or other clinical materials) may be frozen at –70 if the package cannot be shipped within a specified time (e.g., if the specimen is collected on a Friday but cannot be shipped until Monday).
  • When sending clinical specimens, include the specimen inventory sheet (see below), include the assigned CDC case ID number, and note “Influenza surveillance” on all materials and specimens sent.

Include the CDC case ID number on all materials forwarded to CDC. Protocols for standard interstate shipment of etiologic agents should be followed, and are available at http://www.cdc.gov/od/ohs/biosfty/shipregs.htm. All shipments must comply with current DOT/IATA shipping regulations.

V.  Influenza Specimen Inventory Sheet

CDC Case ID:

List specimens sent to the CDC

Select a SOURCE* from the following list for each specimen: Serum (acute), serum (convalescent), NP swab, NP aspirate/wash, broncheoalveolar lavage specimen (BAL), OP swab, tracheal aspirate, or tissue.

Specimen Type #1:

  • Clinical Material
  • Extracted RNA
  • Virus Isolate

Source*:
__________________

Collected : __ __ / __ __ / __ __ __ __ (mm/dd/yyyy)

Date Sent: __ __ / __ __ / __ __ __ __(mm/dd/yyyy)

Specimen Type #2:

  • Clinical Material
  • Extracted RNA
  • Virus Isolate

Source*:
__________________

Collected : __ __ / __ __ / __ __ __ __(mm/dd/yyyy)
Date Sent: __ __ / __ __ / __ __ __ __(mm/dd/yyyy)

Specimen Type #3:

  • Clinical Material
  • Extracted RNA
  • Virus Isolate

Source*:
__________________

Collected : __ __ / __ __ / __ __ __ __(mm/dd/yyyy)
Date Sent: __ __ / __ __ / __ __ __ __(mm/dd/yyyy)

Specimen Type #4:

  • Clinical Material
  • Extracted RNA
  • Virus Isolate

Source*:
__________________

Collected : __ __ / __ __ / __ __ __ __(mm/dd/yyyy)
Date Sent: __ __ / __ __ / __ __ __ __(mm/dd/yyyy)

Specimen Type #5:

  • Clinical Material
  • Extracted RNA
  • Virus Isolate

Source*:
__________________

Collected : __ __ / __ __ / __ __ __ __(mm/dd/yyyy)
Date Sent: __ __ / __ __ / __ __ __ __(mm/dd/yyyy)

Carrier:

Tracking #:


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Appendix 6. Rapid Diagnostic Testing for Influenza

The following information in this appendix is designed to assist clinicians and clinical laboratory directors in the use of rapid diagnostic tests during interpandemic influenza seasons. During an influenza pandemic, one or more of these tests may be sensitive and specific enough to be used by clinicians to supplement clinical diagnoses of pandemic influenza. However, clinicians should be reminded that a negative test result might not rule out pandemic influenza and should not affect patient management or infection control decisions.

I.  INFORMATION FOR CLINICIANS

  1. Background

    Rapid diagnostic tests for influenza can help in the diagnosis and management of patients who present with signs and symptoms compatible with influenza. They also are useful for helping to determine whether institutional outbreaks of respiratory disease might be due to influenza. In general, rapid diagnostic testing for influenza should be done when the results will affect a clinical decision. Rapid diagnostic testing can provide results within 30 minutes.

  2. Reliability and interpretation of rapid test results

    The reliability of rapid diagnostic tests depends largely on the conditions under which they are used. Understanding some basic considerations can minimize being misled by false-positive or false-negative results.

    Median sensitivities of rapid diagnostic tests are generally ~70%–75% when compared with viral culture, but median specificities of rapid diagnostic tests for influenza are approximately 90%–95%. False-positive (and true negative) results are more likely to occur when disease prevalence in the community is low, which is generally at the beginning and end of the influenza season. False-negative (and true positive) results are more likely to occur when disease prevalence is high in the community, which is typically at the height of the influenza season.

  3. Minimizing the occurrence of false results
    • Use rapid diagnostic tests that have high sensitivity and specificity.
    • Collect specimens as early in the illness as possible (within 4–5 days of symptom onset).
    • Follow the manufacturer’s instructions, including those for handling of specimens.
    • Consider sending specimens for viral culture when:
    • Community prevalence of influenza is low and the rapid diagnostic test result is positive, or
    • Disease prevalence is high but the rapid diagnostic test result is negative.

    (Contact your local or state health department for information about influenza activity.)

  4. For further information

II.  Information For Clinical Laboratory Directors

  1. Background

    Rapid diagnostic tests for influenza are screening tests for influenza virus infection; they can provide results within 30 minutes. The use of commercial influenza rapid diagnostic tests by laboratories and clinics has increased substantially in recent years. At least ten rapid influenza tests have been approved by the U.S. Food and Drug Administration (FDA) (see Appendix 1).

    Rapid tests differ in some important respects. Some can identify influenza A and B viruses and distinguish between them; some can identify influenza A and B viruses but cannot distinguish between them. Some tests are waived from requirements under the Clinical Laboratory Improvement Amendments of 1988 (CLIA). Most tests can be used with a variety of specimen types, but sensitivity and specificity can vary with specimen type. FDA approval is based upon specific specimen types.

    Rapid tests vary in terms of sensitivity and specificity when compared with viral culture. Product insert information and research publications indicate that median sensitivities are approximately 70%–75% and median specificities are approximately 90%–95%.

    Specimens to be used with rapid tests generally should be collected as close as possible to the start of symptoms and usually no more than 4–5 days later in adults. In very young children, influenza viruses can be shed for longer periods; therefore, in some instances, testing for a few days after this period may still be useful. Test sensitivity will be greatest in children, who generally have higher viral titers, if the specimen is obtained during the first 2 days of illness, and if the clinician or laboratory has more experience performing the test. The quality of the specimen tested also is critical for test sensitivity.

  2. Accuracy depends on disease prevalence

    The positive and negative predictive values of rapid tests vary considerably depending on the prevalence of influenza in the community. False-positive (and true negative) influenza test results are more likely to occur when disease prevalence is low, which is generally at the beginning and end of the influenza season. False-negative (and true positive) influenza test results are more likely to occur when disease prevalence is high, which is typically at the height of the influenza season.

    1. Clinical considerations when influenza prevalence is low

      When disease prevalence is low, the positive-predictive value (PPV) is low and false-positive test results are more likely. By contrast, the negative-predictive value (NPV) is high when disease prevalence is low, and negative results are more likely to be truly negative (see Graphs 1 and 2).

      If flu prevalence is...

      and specificity is...

      then PPV is...

      false-positive rate is...

      VERY LOW (2.5%)

      POOR (80%)

      V POOR (6%–12%)

      V. HIGH (88%–94%)

      VERY LOW (2.5%)

      GOOD (98%)

      POOR (39%–56%)

      HIGH (44%–61%)

      MODERATE (20%)

      POOR (80%)

      POOR (38%–56%)

      HIGH (44%–62%)

      MODERATE (20%)

      GOOD (98%)

      GOOD (86%–93%)

      LOW (7%–14%)

      Interpretation of positive results should take into account the clinical characteristics of the case-patient. If an important clinical decision is affected by the test result, the rapid test result should be confirmed by another test, such as viral culture or PCR.

    2. Clinical considerations when influenza prevalence is high

      When disease prevalence is relatively high, the NPV is low and false-negative test results are more likely. By contrast, when disease prevalence is high, the PPV is high and positive results are more likely to be true (see Graph 2).

      If flu prevalence is…

      and sensitivity is…

      then NPV is…

      false-negative rate is…

      MODERATE (20%)

      POOR (50%)

      MODERATE (86%–89%)

      MODERATE (11%–14%)

      MODERATE (20%)

      HIGH (90%)

      V. GOOD (97%–99%)

      V. LOW (2%–3%)

      HIGH (40%)

      POOR (50%)

      MODERATE (70%–75%)

      MODERATE (25%–30%)

      HIGH (40%)

      HIGH (90%)

      V. GOOD (93%–94%)

      LOW (6%–7%)

      Interpretation of negative results should take into account the clinical characteristics of the case-patient. If an important clinical decision is affected by the test result, the rapid test result should be confirmed by another test, such as viral culture or PCR.

  3. Selecting tests

    Selection of a test should take into consideration several factors, such as the types of specimens that are considered optimal for that test. Also, tests with high sensitivity and specificity will provide better positive and negative predictive values. Information about test characteristics is provided in product inserts and scientific articles and by the manufacturer.

  4. Changes in recommended procedures can affect test results

    Modification by the user can affect test performances and increase false-positive and/or false-negative rates. Such modifications include using specimens for which the test is not optimized or using swabs that did not come with the rapid test kit (unless recommended).

  5. When are rapid diagnostic tests beneficial?

    Use of rapid diagnostic tests are beneficial in these situations:

    • To test cases during an outbreak of acute respiratory disease to determine if influenza is the cause, or
    • To test selected patients during the influenza season, or
    • In the fall or winter, to test selected patients presenting with respiratory illnesses compatible with influenza to help establish whether influenza is present in a specific population and to guide healthcare providers in diagnosing and treating respiratory illnesses.

    In general, the exclusive use of rapid tests does not address the public health need for obtaining viral isolates so that influenza virus strain subtyping and characterization can be conducted to monitor antigenic and genetic changes.

    During an influenza pandemic, some rapid diagnostic tests may be able to detect the pandemic strain with adequate sensitivity and specificity. Rapid tests can be used by physicians to supplement clinical diagnoses of pandemic influenza.

    Physicians should be reminded that a negative test result might not rule out influenza and should not affect patient management or infection control decisions.

  6. For further information

    Information on influenza diagnostics is provided on the CDC website at: http://www.cdc.gov/flu/professionals/labdiagnosis.htm.

Impact of Prevalence, Sensitivity and Specificity on Positive Predictive Value Graph: Image

Impact of Prevalence, Sensitivity and Specificity on Negative Predictive Value Graph: Image


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Appendix 7. Guidelines for Medical Surveillance of Laboratory Research Personnel Working with Novel Strains of Influenza, including Avian Strains and Other Strains with Pandemic Potential

Key Messages

  • Laboratory workers should receive training on the appropriate biosafety level for the type of work being performed.

  • Before working with avian influenza A viruses, including highly pathogenic strains, laboratory workers should have a baseline serum sample obtained and stored for future reference.

  • Workers in laboratories that contain avian influenza A viruses should report any fever or lower respiratory symptoms to their supervisors. Workers should be evaluated for possible exposures, and the clinical features and course of the illness should be closely monitored.

  • Laboratory workers who are believed to have had a laboratory exposure to an avian influenza A virus or other highly pathogenic strain should be evaluated, counseled about the risk of transmission to others, and monitored for fever or lower respiratory symptoms as well as for any of the following: sore throat, rhinorrhea, chills, rigors, myalgia, headache, diarrhea.

  • Local and/or state public health departments should be notified promptly of laboratory exposures and illnesses in exposed laboratory workers.

Medical surveillance of laboratory personnel can help to ensure that workers who are at risk of occupational exposure to avian influenza viruses or other novel animal or human influenza strains and who develop symptoms of illness receive appropriate medical evaluation and treatment, both for the benefit of their health and to prevent further transmission.

I.  Prerequisites For Working WithH Novel Avian or Human Influenza Viruses

  1. Baseline serum samples

    Before working with novel avian or human influenza viruses, laboratory workers should have a baseline serum sample obtained and stored for future reference.

  2. Influenza vaccine

    Laboratories should offer the current inactivated influenza vaccine to laboratory personnel. Its use is especially encouraged for personnel working with avian viruses in BSL-3 enhanced laboratory conditions and for those who may be exposed to these viruses in the field. Immunization might reduce the chance of illness from exposure to human influenza viruses currently circulating in the community that could lead to confusion in monitoring for avian influenza A infection. Vaccines against novel influenza A viruses (e.g., H5N1) are undergoing clinical trials and might be available in the future.

  3. Oseltamivir prophylaxis
    • It is not necessary to require oseltamivir for laboratory research personnel working with highly pathogenic influenza strains, but encourage it for those doing animal experiments only for the time they are working with animals and especially while working with ferrets.
    • When considering oseltamivir prophylaxis, be sure to evaluate appropriate candidates for contraindications, answer their questions, review adverse effects, and explain the benefits.
    • Maintain a log of persons on oseltamivir, persons evaluated and not on oseltamivir, doses dispensed, and adverse effects.
    • Periodically evaluate and update oseltamivir policies and procedures.
  4. Post-exposure prophylaxis

    Conditions for use of oseltamivir for post-exposure prophylaxis include a known or suspected laboratory exposure to live avian influenza virus, including highly pathogenic strains, for a person not on oseltamivir. Appropriate healthcare personnel should be available to evaluate immediately and dispense oseltamivir if the exposure occurs during working hours. If exposure occurs after working hours, an exposed laboratory person should present to the Emergency Department and, after evaluation, communicate with CDC for recommendations.

II.  Management of Influenza-Like Illness In Personnel With Possible Exposure to Novel Avian or Human Influenza Viruses

  1. General procedures

    • Maintain a daily sign-in/out sheet to record name, date, time in/out, use of oseltamivir, and brief description of job tasks. This record will facilitate retrospective documentation if an illness occurs.
    • Workers should report any influenza-like illness and any potential laboratory exposures to the supervisor (see also Supplement 4).

  2. Evaluation and treatment

    1. During regular working hours

      • The affected employee should notify the supervisor. The supervisor should immediately contact the appropriate healthcare personnel and facility contacts (e.g., occupational health, infection control, or designee).
      • Upon arrival at the designated clinic, the employee should be placed in a private room for isolation where a healthcare provider can provide consultation and evaluation.
      • The healthcare provider should obtain a respiratory specimen (e.g. nasopharyngeal swab or aspirate) for viral culture. A rapid antigen test with the ability to differentiate between influenza A and B should be used for initial diagnosis, followed by virus isolation.
      • Based on: 1) the rapid test result (if influenza A positive), 2) the status of oseltamivir prophylaxis, and 3) the clinical evaluation, the healthcare provider should determine whether the patient will return to work, be sent home, or be sent to an infectious disease consultant.

    2. During working hours when the employee calls from home

      • The employee should notify the supervisor. The supervisor should discuss the situation with the appropriate healthcare personnel and determine where and by whom the employee will be evaluated and specimens for viral culture will be obtained.
      • The employee may come to an on-site clinic for evaluation or may elect to see a personal physician. If the employee chooses to see a personal physician, the on-site clinician should discuss with the personal physician the likelihood of a laboratory-acquired infection. The personal physician should be asked to collect specimens for antigen detection and viral culture.
      • An employee who is not sick enough to be admitted to a hospital should remain at home under the care of a personal physician, pending results from the viral culture. If influenza A (H3N2) or A (H1N1) is identified, the employee should be advised and can resume normal activities as soon as symptoms subside.
      • If avian influenza A (e.g., H5, H7, H9) is identified, the family and other contacts should be monitored for illness.
      • Local public health officials should be notified about any confirmed avian influenza infections.

    3. After working hours

      • The employee should notify the supervisor. The supervisor should inform other persons as the situation dictates.
      • If the employee is acutely ill with symptoms consistent with influenza, the employee and/or supervisor should contact the appropriate healthcare provider for instructions. The healthcare provider should conduct the initial evaluation and patient management.
      • The supervisor should immediately ask the healthcare provider to collect specimens for rapid testing and viral culture.
      • The employee should follow the advice of the healthcare provider with regard to further evaluation/treatment.

 


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