The influenza viruses, influenza A (Flu A) and influenza B (Flu B), are major causes of respiratory
infections in the U.S. and worldwide. With winter epidemics occurring annually, these viruses
represent a significant diagnostic challenge to virology laboratories, causing increased demand for
rapid testing and upping workloads in diagnostic virology facilities. Virologists grapple with the issues
of balancing appropriate rapid testing with meeting clinical need and make decisions based on test
volume, physician demand, technologist availability and skill levels, and cost of testing. These
decisions are difficult at best. In 2001, with the reintroduction of anthrax into the diagnostic arena, the
need for rapid influenza testing–to differentiate influenza infections from clinically-similar early anthrax
symptoms–re-focused attention on the rapid influenza testing issue.
Flu A and Flu B viruses cause systemic illness characterized by abrupt onset of fever, cough,
pharyngitis, prostration, myalgia, and headache, with disease severity more prominent with Flu A than
Flu B. Amantadine and Rimantadine are FDA-approved drugs for Flu A prevention and are reported
to be 70-90% effective in preventing illness when administered for 3-5 days following identification of
an outbreak in a community. Zanamivir and Oseltamivir, FDA approved for treatment of both Flu A
and Flu B, have been shown to reduce flu symptoms if taken at onset. Rapid detection of influenza
virus infections is required if antivirals are to be applied optimally. Likewise, differentiation of
influenza infections from infections due to other viruses or to bacteria are important in the selection of
appropriate therapy.
For many years, rapid detection of the influenza viruses relied upon immunofluorescence (IF)
staining for viral antigen, usually by indirect immunofluorescence (IIF). Although the IIF methods
were both sensitive and specific for detection of both Flu A and Flu B, the processing/staining
protocol required nearly 2.5 hours to complete, and mandated considerable expertise on the part of
the virologist performing/interpreting the test result. Providing the IIF tests during non-routine hours
(evenings/overnights/weekends/holidays) presented a significant staffing problem in virology
laboratories, preventing around-the-clock availability of this testing. Approximately 5 years ago, a
non-IF influenza detection method was made available. This was a membrane based enzyme
immunoassay (EIA) in a cassette-type format. This provided the first viable alternative to IF for rapid
Flu A detection, requiring 15-25 minutes to perform and very little expertise on the part of the
technologist. Although this test yielded lower sensitivity and specificity that IIF and was more costly,
the demand for rapid Flu A diagnosis made this test desirable.
Recently, more rapid influenza antigen assays have arrived on the market. The IIF was joined by
direct immunofluorescence (DIF) procedures, requiring only 30 minutes of staining time and providing
both sensitive and specific detection. DIF reagents are available from several major vendors. New
non-IF products also became available. A cassette-type membrane EIA, featuring two test ports,
allows specific detection/differentiation of Flu A and Flu B. The other new rapid tests detect both Flu
A and Flu B but do not differentiate the two. One optical immunoassay depends on viral antigen
binding to thicken the mirror-like reaction surface, which changes the optical properties of the surface
and results in a visible change in surface appearance.
Another method is an immunochromatographic method in which patients’ samples are mixed in a
reaction tube, and then components migrate up a test strip to produce colored bands. Another assay
is not an antigen detection test. This assay signals that presence of influenza virus via detection of
viral neuraminidase activity. The physical appearance of this product is very similar to the other
cassette-type membrane EIAs. None of the rapid non-IF methods is a sensitive as the IF methods.
However, sensitivity improves if the test sample is a nasal wash or aspirate rather than a
nasopharyngeal swab.
Despite all available options for rapid influenza detection, there is no standard protocol that is
embraced by all laboratories concerning which tests to offer, when to offer them, and what the
specimen requirements should be. Many laboratories use IIF or DIF stains during routine day shift
hours and then use the non-IF tests during evening/overnights/weekends/holidays when the virology
laboratory is not fully staffed. Many smaller laboratories and emergency centers that do not have a
staff of well-trained virologists may use the rapid non-IF tests at all times.
With the frightening news that inhalation anthrax had caused the death of a U.S. citizen, the
problems of rapid diagnosis of influenza was exacerbated. Why? As the new media were quick to
point out to the public, clinical signs and symptoms in the early stages of inhalation anthrax are
similar to those of influenza and other respiratory viral illnesses. Although anthrax may cause profuse
sweating (drenching), shortness of breath, abdominal pain, and elevated transaminases, which are
not typical in influenza and influenza infections are characterized by sore throat and rhinorrhea which
are not typical in anthrax, the widely dispersed information that anthrax “starts out like a common cold
or flu,” sent many patients with respiratory disease to their doctors seeking antibiotics effective
against anthrax. For these patients, a rapid diagnosis of influenza (or other viral respiratory infection)
would facilitate not only appropriate therapy but also reduce anxiety levels that were high at the time.
How did these events affect rapid influenza testing in virology laboratories across the U.S.? In
view of the lack of uniformity of influenza rapid testing prior to the anthrax scare, it is not unexpected
that each virology laboratory and health care facility seemed to react/respond to the anthrax scare in
its own way.
Although this episode had the potential to precipitate dramatic changes, there were several
circumstances that helped avert crises in the virology laboratory. One very helpful factor was that
influenza activity in the U.S. remained low from October through mid-January. For this reason,
clinicians were not faced with large number of influenza sufferers during the October/November
period when the anthrax problem was at its height. In many situations, virology laboratories were
contacted in October and asked what could be done to augment rapid influenza testing. Fortunately,
by late January, when influenza became prevalent, much of the hysteria surrounding the anthrax
episodes had disappeared.
Dr. Marie Landry of Yale/New Haven reports that, in response to the anthrax cases in nearby
New York City and Oxford, CT, her lab, which routinely offers respiratory viral antigen DIF 18 hours
each day in January and February, added a Flu A and B membrane.
