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

Interpandemic and Pandemic Alert Periods

Clinical and hospital laboratories:

• Work with state and local health departments to address laboratory surge capacity issues and train personnel in management of respiratory specimens during an influenza pandemic.

• Send clearly labeled specimens from patients with suspected novel influenza to state or local health departments. Hospital labs should NOT attempt to isolate influenza viruses from patients with suspected novel influenza virus infection.

• Institute surveillance for influenza-like illnesses (ILI) among laboratory personnel working with novel influenza viruses.

State and local public health laboratories:

• Work with federal partners to enhance laboratory-based monitoring of seasonal influenza virus subtypes, as described in Supplement 1.

• Conduct testing for novel subtypes of influenza viruses only if BSL-3 biocontainment conditions with enhancements are available.

• Institute surveillance for ILI among laboratory personnel.

• Conduct preparedness planning to support the response to an influenza pandemic.

HHS responsibilities:

• Monitor preparedness and laboratory capacity for seasonal influenza and assess surge capacity.

• Provide technical support to the WHO Influenza Network and ministries of health and agriculture, as requested, in analyzing novel influenza virus subtypes—including avian isolates and human isolates with pandemic potential—in terms of antigenicity, RNA sequence, and drug sensitivities.

• Work with state and local public health laboratories to ensure that diagnostics for identifying "pandemic alert" strains are available and are used safely and effectively.

• Provide guidance on biosafety and safe handling of respiratory specimens from potential cases of pandemic influenza.

Pandemic Period

Clinical and hospital laboratories:

• Scale up to manage increased numbers of requests for influenza testing.

• Send selected specimens from possible pandemic influenza patients to state or local health departments.

State and local public health laboratories:

• Scale up to manage increased numbers of requests for influenza testing.

• Work with federal partners to provide healthcare providers and clinical laboratories with guidelines on all aspects of specimen management and diagnostic testing.

• Work with federal partners to monitor the pandemic virus and conduct special studies related to vaccine development, or other aspects of the response.

HHS responsibilities:

• Work with U.S. and global partners to characterize new pandemic viruses in terms of antigenicity, RNA sequence, and drug sensitivities, and to monitor changes over time.

• Work with state and local public health laboratories to ensure the availability and the safe and effective use of diagnostic tests and reagents.

• Conduct reference testing of positive samples, and perform viral isolation, especially at the beginning of a pandemic.

• Provide laboratory support for the selection of seed strains to be used in a vaccine against the pandemic virus.

S2-I. Rationale

The goals of diagnostic testing during a pandemic are to:

• Identify the earliest U.S. cases of pandemic influenza (whether the pandemic begins in the United States or elsewhere).

• Support disease surveillance to monitor the pandemic’s geographic spread and impact of interventions.

• Facilitate clinical treatment by distinguishing patients with influenza from those with other respiratory illnesses.

• Monitor circulating viruses for antiviral resistance.

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.

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.

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

S2-IV. Recommendations for The Pandemic Period

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.

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.

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:

• H5N2, the cause of poultry outbreaks in New York, Maine, and California in 2002

• H7N2, the cause of poultry outbreaks in Delaware, Maryland, and New Jersey in 2004

HPAI strains include:

• H5N1, the cause of major poultry outbreaks in Southeast Asia

• H7N7, the cause of a 2003 outbreak in the Netherlands

• H7N3, the cause of a 2004 outbreak in British Columbia

• H5N2, the cause of a 2004 outbreak in poultry in Texas

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:

• H9N2, which caused three cases of influenza-like illness in Hong Kong between 1999 and 2003, and other cases in China in 1998 and 1999

• H7N2, which was detected by serology in one person involved in the culling of sick chickens during the response to a poultry outbreak in Virginia in 2002, and was isolated from a New York resident in 2003 (unknown source of the infection)

HPAI viruses associated with human infection include:

• H5N1, which caused 51 deaths in Southeast Asia between January 2004 and April 2005

• H7N7, which caused the death of a veterinarian as well as 83 cases of mild human disease (including conjunctivitis) during the 2003 poultry outbreak in the Netherlands.

• H7N3, which caused 2 cases of very mild human disease (conjunctivitis, headache) in persons culling sick poultry in British Columbia in 2004

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.

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:

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.

• State/local laboratories should send specimens to CDC if:





• A sample tested by the state or local laboratory is positive for H5 or another novel subtype;

• Note: A laboratory should test for influenza A (H5) only if it is able to do so by PCR or has a BSL-3-enhanced facility for influenza A(H5) viral culture.

or

• A sample from a patient who meets the clinical and epidemiologic criteria for possible infection with a potentially pandemic virus is positive for influenza A by RT-PCR or rapid antigen detection,* is negative for influenza A(H1) and A(H3), and the referring jurisdiction is not equipped to test for specific strains;

or

• The referring jurisdiction is not equipped to test samples for novel influenza viruses by RT-PCR and is requesting testing at CDC.

Shipping procedures for potential pandemic strains of influenza are provided in Appendix 5.

*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.

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

Key Messages

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

Key Messages

• Appropriate specimens for influenza testing vary by type of test.

• Before collecting specimens, review the infection control precautions are described in Supplement 3.

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.

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

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.