While avian and pandemic influenza receive terrifying media coverage, it is important to remember that the seasonal flu strains that reappear each winter can also cause more than simple respiratory infections. Flu (often complicated by pneumonia) kills about 36,000 people in the United States in a typical year, in addition to causing 200,000 inpatient admissions and costing $10.4 billion to treat. For the United States, the total economic damage due to influenza, including millions of lost workdays, is estimated at $87 billion per year.¹

The RNA viruses that cause influenza belong to the orthomyxovirus family. The viral nucleoprotein's immunoreactivity is used to identify the genus as A or B, and the ribonucleoprotein's lipid envelope has hemagglutinin (H) and neuraminidase (N) protein antigens used to narrow the identification further.

The Legacy of 1918

Much of today's knowledge of influenza is a direct inheritance from 1918, as are many of the laboratory procedures and public-health measures designed to cope with the disease. The 1918 flu pandemic (called the 3-day fever before it was mislabeled the Spanish flu) killed 650,000 US residents, reducing the average national longevity by 12 years. Today's best evidence implies that it began as a spontaneous mutation in an avian flu virus; it first infected soldiers in Kansas and was spread to the rest of the world when US troops arrived in Europe to fight in the First World War.

Before the pandemic ended, one third of all humans had been infected, and at least 50 million (and perhaps as many as 100 million) had died. On a single day in New York City, 851 flu victims died, and many areas fared so much worse that society ceased to function temporarily. For example, in Nome, Alaska, flu killed 59% of the Alaskan Native population.

In 2005, the first virus to be replicated in the laboratory after its natural extinction was that of the 1918 flu, which was completely decoded using lung tissue from an Alaskan body preserved in the permafrost since the pandemic. The virus was found to be an avian H1N1 strain that had mutated not only to move from birds to humans and from one human to another, but to multiply more rapidly and infect areas deeper in the lungs than previous seasonal flu strains could.

After further mutations changed it from H1N1 to other forms such as H1N2 and H3N2, it also became the source of all subsequent pandemic flu, along with nearly all the seasonal type-A flu strains that have circulated ever since. The parental H1N1 strain, after becoming steadily less destructive, became extinct in humans in the 1950s. It survived only in swine, but it was released again into the human population in 1977 (from a laboratory freezer in China) and continues to infect humans today.²

Indirectly, the 1918 pandemic is responsible for a considerable portion of today's laboratory activity: DNA was eventually discovered as a result of the flu/pneumonia research undertaken at that time.

Flu Tests

Before antivirals were available, laboratory testing for flu focused on identifying the circulating strains so that they could be represented in the vaccine for the following year. Once treatment reached the market, however, the focus of testing changed completely: Antivirals are useful only at the beginning of illness, so rapid results are needed to determine whether they can help. In the absence of those results, many physicians will choose to prescribe expensive antivirals anyway, and others will prescribe antibiotics if they do not suspect flu. Rapid tests not only improve patient care, but greatly reduce the cost of prescribing unnecessary drugs.

In addition to rapid antigen testing, influenza testing can involve viral culture, polymerase chain reaction (PCR), and immunofluorescence. Serology, although possible, is not recommended by the US Centers for Disease Control and Prevention (CDC). Although rapid tests have supplanted the older tests for most clinical purposes, other tests are still needed for disease surveillance (and to rule out possible inaccuracies in cases where clinical symptoms do not seem to agree with rapid-test results). Viral culture, for example, has higher specificity and sensitivity than rapid tests, and rapid tests do not always indicate flu subtypes. Rapid tests also become less useful as the infection progresses.

Some forms of rapid tests are intended for use in the clinical laboratory and designated moderately complex under the Clinical Laboratory Improvement Amendments of 1988 (CLIA), while others are CLIA waived for use in any medical setting. The CDC now recommends rapid tests whenever flu testing is conducted. Viral culture and PCR testing are used for surveillance and to confirm the results of rapid tests, but neither is likely to yield results in time to affect the use of antivirals. Immunofluorescence can be used if rapid tests are unavailable, however, since many laboratories can provide timely results.

Emergency hospital during 1918 influenza epidemic, Camp Funston, Kan. Courtesy of the National Museum of Health and Medicine, Armed Forces Institute of Pathology, Washington, DC, Image NCP 1603.

Directigen Flu A+B (BD, Franklin Lakes, NJ) requires only 15 minutes to detect influenza A and B and to differentiate between them. It is a rapid immunoassay that can use many types of specimens (nasopharyngeal wash, nasopharyngeal aspirate, nasopharyngeal swab, lower nasal swab, throat swab, and bronchoalveolar lavage fluid). Because nucleoproteins are the test's target, it is not affected by antigenic drift.

FLU OIA (BioStar Inc, Boulder, Colo), a division of Inverness Medical Innovations, Inc, is a rapid test with the sensitivity of a viral culture for detecting influenza A or B without differentiation. It yields results in 15 minutes using specimens from nasal aspirates, nasopharyngeal swabs, throat swabs, or sputum. The test uses the company's proprietary Optical ImmunoAssay technology.

The Xpect Flu A&B Test Kit (Remel Inc, Lenexa, Kan) detects the H5N1 and H9N2 strains of avian flu, in addition to influenza A and B. This rapid test provides results in 15 minutes (with 100% specificity) and requires only a minute of hands-on staff time to perform. Sensitivity for influenza B is 97.8%; for influenza A, 92.2%.

The BinaxNOW Influenza A & B Test is available from Binax (Scarborough, Me), a division of Inverness Medical Innovations Inc. A one-step procedure allows differentiation between influenza A and B. This immunochromatographic assay detects nucleoprotein antigens and uses specimens from nasopharyngeal swabs or nasal wash/aspirates. The test's CLIA status is waived and moderately complex.

The CLIA-waived QuickVue Influenza A+B Test (Quidel, San Diego) differentiates between influenza A and B in 10 minutes, using a three-step process that calls for 90 seconds of hands-on time. Nasal-swab, nasopharyngeal-swab, nasal-wash, and nasal-aspirate specimens can be used. The economical, lateral-flow immunodiagnostic test can be stored at room temperature and has a 2-year shelf life. It uses a built-in control (a color change), and the test kit also incorporates external controls for the laboratory's use. Quidel is adding the MChip, developed jointly with the CDC to detect avian flu, to its tests.

ZstatFlu Rapid Test for Influenza A and B (ZymeTx Inc, Oklahoma City) is also CLIA waived and can be stored at room temperature. It can detect H5N1 avian flu and emerging flu strains, in addition to influenza A and B, using a throat-swab specimen. Results are available in 20 minutes, and hands-on time is 2 minutes. All A and B strains seen during the past 30 years are detected by this test with 99% specificity.

The OSOM Influenza A&B Test (Genzyme Diagnostics, Cambridge, Mass) uses a nasal-swab specimen and delivers results in 10 minutes. It can be stored at room temperature; the test differentiates between influenza A and B and requires less than a minute of hands-on time. For external quality control, each kit includes two additional test sticks.

An innovative test from Seegene (Rockwell, Md) called the Seeplex 18-plex Respiratory Test detects the most common respiratory pathogens, including influenza A and B. A single tube, with a specimen from nasopharyngeal aspirates, nasopharyngeal swabs, or bronchoalveolar lavage fluid, can detect two DNA viruses, five pneumonia bacteria, and 11 RNA viruses. When flu is suspected, this PCR test can indicate not only whether it is present but, when it is not, what organism is really causing symptoms. Bacterial superinfection in the presence of influenza or other viral infections can also be detected, allowing appropriate drug therapy. The pathogens detected are influenza A and B; respiratory syncytial virus A and B; parainfluenzae 1, 2, and 3; coronavirus 229E/NL63 and OC43/HKU1; rhinovirus; enterovirus; adenovirus; bocavirus; Legionella pneumoniae; Streptococcus pneumoniae; Chlamydophila pneumoniae; Haemophilus influenzae; and Mycoplasma pneumoniae.

Flu Vaccines

This year, the CDC's Advisory Committee on Immunization Practices (ACIP) is recommending vaccination for anyone who wants it, since there is no shortage of vaccine. While immunization of health care personnel has always been recommended (and often conducted within hospitals), this year's ACIP guidelines¹ are more adamant: Staff vaccination is to be considered an indicator of high-quality patient care, and any staff member who is not immunized (for example, due to egg allergy) should be required to sign a waiver.

Staff vaccination not only protects workers, but prevents them from spreading flu to vulnerable inpatients, among whom flu mortality rates can be high. Some laboratories have been securing vaccine supplies themselves and requiring employee immunization for years, since the possibility of exposure to infectious specimens is always present.

In February 2007, the World Health Organization recommended the strains to be included in vaccines for the 2007–2008 flu season, and the FDA chose the same strains soon afterward. This year's vaccines include an A/Solomon Islands/3/2006 (H1N1)-like virus, an A/Wisconsin/67/2005 (H3N2)-like virus, and a B/Malaysia/2506/2004-like virus.

About 132 million vaccine doses will be available in the United States this season. Availability was brought to this record level with the fast-track FDA approval of Afluria (CSL Ltd, Parkville, Australia). Postvaccination studies will be conducted to verify the agent's effectiveness. It joins five other vaccines already on the market, including the Fluarix (GlaxoSmithKline Biologicals, Rixensart, Belgium) and Flulaval (ID Biomedical, Laval, Quebec).

US Army Camp Hospital No. 45, Aix-Les-Bains, France, Influenza Ward No. 1, 1918. Courtesy of the National Museum of Health and Medicine, Armed Forces Institute of Pathology, Washington, DC, Image Reeve 14682.

FluMist (MedImmune, Gaithersburg, Md) is a live, attenuated vaccine delivered intranasally. Fluzone (Sanofi Pasteur, Swiftwater, Pa) is delivered as a traditional intramuscular injection, as is Fluvirin (Novartis Vaccines, Cambridge, Mass). Novartis and the nonprofit Families Fighting Flu Inc have launched a campaign to improve awareness of flu vaccination and its importance (including its power to reduce flu transmission within families, schools, and workplaces). The effort, called "Spread the Word—NOT the Flu!" will reach students and their families through the Weekly Reader, an in-depth curriculum, a Web site, and contest promotions.

Avian/Pandemic Flu

Pandemics are possible whenever the population has little or no previous experience with a virus and, therefore, no immunity. Post-1918 pandemics have included the less devastating Asian flu of 1957–1958 and the Hong Kong flu of 1968–1969. Seasonal flu, in contrast, may involve different strains each year, but will not usually be the result of major viral mutations.

Today's avian flu, H5N1, has been sporadically killing humans in contact with infected birds for a decade, but has not yet spread from human to human on any major scale. It has caused serious concern, however, that a forthcoming mutation could confer that ability. Pandemic flu arising today, according to White House estimates, could cause a 30% infection rate and 2 million deaths in the United States.

Much research is now focused on improving vaccine production. Because it currently requires three chicken eggs per dose and takes 9 months to complete, vaccine production cannot respond quickly to any novel mutations in the flu virus.

Because vaccine creation is slow, outbreak detection must be rapid. The US Laboratory Response Network is supplied with H5N1 detection kits by the CDC. If an outbreak of avian flu is suspected, these kits will provide confirmation in about 4 hours. Tests for use in clinical laboratories are also being developed under contract.

In 2006, Cepheid (Sunnyvale, Calif) was awarded a CDC contract to develop a test for avian flu. The point-of-care test will use the company's GeneXpert system for real-time PCR and will also detect seasonal influenza A and B. It will be made available for screening use at US points of entry, as well as in conventional medical settings. Users will require no special training, as the test will be CLIA waived. Results will be available in about 30 minutes, so arriving passengers with suspicious symptoms can be tested for avian flu without long delays. The test system may also be deployed throughout high-risk Indonesia by the AlertAsia Foundation.

Under federal pandemic-preparedness and avian-flu directives, Arbor Vita Corp, Sunnyvale, Calif, is working with the Naval Health Research Center to develop a rapid avian-flu test using the company's PDZ technology. The test will be made available to the US military for surveillance use in its worldwide laboratory network, as well as to public and private health care providers. It will detect a nonmutating protein found in all avian influenza viruses, so it will be able to detect future strains, as well as H5N1.

Nanogen, San Diego, is also developing a 15-minute test for avian flu under a CDC contract. The point-of-care test will detect influenza A (H1N1 and H3N2) and influenza B, in addition to avian flu and emerging strains, and will provide high sensitivity at low cost. The portable, rapid immunoassay will use proprietary Nanogen technology and is described by the company as a third-generation, high-sensitivity, lateral-flow test.

Influenza has been described as humankind's worst enemy, both for its high death toll and for its inevitable return in newer, more dangerous forms. Our odds in this contest are improving, however, thanks to broader vaccine use, greater public awareness and preparedness, and the vigilance of laboratories that monitor this ever-changing threat in both individuals and whole populations.

Kris Kyes is technical editor of CLP.

References

1. CDC. Prevention and control of influenza: recommendations of the Advisory Committee on Immunization Practices (ACIP). MMWR Morb Mortal Wkly Rep. 2007;56:1-54.
2. Taubenberger JK, Morens DM. 1918 influenza: the mother of all pandemics. Emerg Infect Dis. 2006. Available at: www.cdc.gov/ncidod/EID/vol12no01/05-0979.htm. Accessed October 8, 2007.