Approach Considerations
The gold standard for confirming influenza virus infection is reverse transcription-polymerase chain reaction (RT-PCR) or viral culture of nasopharyngeal or throat secretions. Rapid diagnostic tests for influenza are available and are becoming more widely used. These tests have high specificity but only moderate sensitivity.
Findings of standard laboratory studies, such as a complete blood count (CBC) and electrolyte levels, are nonspecific but helpful in the workup of influenza. Leukopenia and relative lymphopenia are typical findings. Thrombocytopenia may be present. In severe cases of influenza, the patient is likely to have hypoxemia, and the alveolar-arterial (A-a) gradient may be increased (>35 mm Hg). Patients with physical examination findings compatible with meningitis should undergo lumbar puncture.
Rapid Diagnostic Tests
The FDA waived federal Clinical Laboratories Improvement Act (CLIA) requirements and cleared for marketing seven rapid influenza diagnostic tests that directly detect influenza A or B virus–associated antigens or enzyme in throat swabs, nasal swabs, or nasal washes. These tests can produce results within 30 minutes. [33]
A meta-analysis examining the accuracy of rapid influenza diagnostic tests found a pooled sensitivity of 62% and specificity of 98%. [34] The tests tended to be more sensitive in children (67%) than in adults (54%) and better at detecting influenza A (65%) than influenza B (52%).
The accuracy of these tests depends in part on the collection technique and skill of the person performing the test. Nasal swabs must be deeply inserted and then swirled to attach the influenza virus.
The following 3 rapid diagnostic tests are considered of low complexity and may be used in physicians’ offices:
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QuickVue Influenza A+B test (Quidel)
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ZstatFlu (ZymeTx)
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QuickVue Influenza test (Quidel)
The QuickVue tests provide results in 10 minutes or less; the ZstatFlu test provides results in 20 minutes. Genetic and antigenic changes in the virus can adversely affect diagnostic test performance; thus, these tests should be monitored annually. [35]
In June 2014, the FDA also approved the Alere i Influenza A & B Test, a new point-of-care influenza test that delivers highly accurate molecular results in less than 15 minutes. [36] The test, which extracts and analyzes DNA and RNA strands to detect sequences associated with bacterial and viral infections, has a sensitivity of greater than 90% for both influenza A and B. Although other influenza detection tests that produce results in about 15 minutes are already on the market, those tests rely on antigen detection and their sensitivity ranges from 50% to 70%. [36]
Viral Culture and Polymerase Chain Reaction Testing
Viral Culture and Polymerase Chain Reaction Testing
RT-PCR testing or viral culture of nasopharyngeal or throat secretions is the gold standard for confirming influenza virus infection. Culture may require 3 to 7 days, yielding results long after the patient has left the clinic, office, or emergency department and well past the time when drug therapy could be efficacious.
Viral Culture
For viral culture, nasopharyngeal samples are obtained with Dacron swabs and sent in appropriate viral transport media (eg, multimicrobe [M4] transport media) to the laboratory to be cultured in several lines of cells. A laboratory diagnosis of influenza is established once specific cytopathic effect is observed or hemadsorption testing findings are positive. For example, staining the infected cultured cell lines with fluorescent antibody confirms the diagnosis.
Polymerase Chain Reaction Tests
Most laboratories and hospitals now offer nucleic acid (PCR)-based studies. A nasal swab is submitted in special transport media to the laboratory, and results can be available within 24 hours. Sensitivity for influenza is greater than 90%. These tests may be offered as respiratory panels that provide information on the presence of other viruses, such as respiratory syncytial virus (RSV) and adenovirus.
The FDA has approved an influenza RT-PCR test developed by the US Centers for Disease Control and Prevention (CDC) that can provide results within 4 hours. It is the only in vitro diagnostic test for influenza that is cleared by the FDA for use with lower respiratory tract specimens. [10] Consisting of 3 modules, the kit can do the following:
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Identify and distinguish between influenza A and B viruses
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Classify influenza A viruses by subtype
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Detect highly pathogenic avian influenza A (H5N1) virus infection in human respiratory tract specimens
Direct Immunofluorescent Tests and Serologic Testing
Some laboratories offer direct immunofluorescent tests on fresh specimens, but these tests are labor- and personnel-intensive and are less sensitive than culture methods. To overcome the expensive and time-consuming obstacle of culturing, several serologic tests have become available. Many of these are not actually bedside tests; generally, 30 to 60 minutes are required to perform the tests’ multiple steps. Test sensitivities generally range from 60-70%.
A study by Haran et al suggests that cytokine markers may help distinguish influenza from bacterial pneumonia or other viral respiratory infections. In this study, differences were observed between the bacterial pneumonia group, on one hand, and all other viral infections grouped together, on the other, with regard to interleukin (IL)-4, IL-5, IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), and interferon gamma levels. However, IL-10 concentrations were uniquely elevated in patients with influenza (88.69 pg/mL) as compared with all other groups combined (39.19 pg/mL; P = .003). [37]
Testing for Avian Influenza
Testing for Avian Influenza
The standard commercially available rapid influenza A tests do not detect H5N1 avian influenza. [38] A rapid test from nasopharyngeal swab specific to H5N1 influenza (Arbor Vita Corporation) was approved by the FDA in 2009. [39]
A CBC may be more clinically useful in avian influenza than in seasonal influenza. Leukopenia (WBC count 454-4900/µL), especially lymphopenia, is common and is observed in 50-80% of patients. [40] In at least one study, lymphopenia at presentation (absolute lymphocyte count < 1500/µL) was a significant predictor of the progression to acute respiratory distress syndrome. [41] More than half of patients will have mild-to-moderate thrombocytopenia.
In addition to thrombocytopenia, some patients with severe disease will develop disseminated intravascular coagulation (DIC), as shown in coagulation studies. [13]
Liver function tests (LFTs) may be useful in differentiating avian influenza from other febrile tropical diseases. Aminotransferase levels are elevated in more than half of all patients with H5N1 infection. [13]
A basic metabolic panel is generally required in the care of all seriously or critically ill patients. Abnormalities in renal function may herald the progression to organ failure.
According to CDC recommendations, [30] clinicians should attempt to specifically identify avian H5N1 influenza in patients with all of the following characteristics:
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Severe illness that necessitates hospitalization or is fatal
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Documented temperature of 38°C (100.4°F) or higher in the past 24 hours or a history of feverishness during that time
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Radiographically confirmed pneumonia, acute respiratory distress syndrome (ARDS), or other severe respiratory illness for which an alternative diagnosis has not been established
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At least one potential exposure within 7 days of symptom onset
Testing may be considered in discussion with public health authorities in patients who have only some of these characteristics; all testing should be discussed with local public health departments.
The CDC defines potential exposure as follows:
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History of travel to a country where highly pathogenic avian influenza (HPAI) H5N1 virus has been documented in poultry, wild birds, or humans, with potential exposure to the virus during the visit
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Close contact (approach within ~6 ft) with an ill person who was under investigation for possible HPAI H5N1 virus infection
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Working with live HPAI H5N1 virus in a laboratory
If avian influenza is suspected, cultures should not be ordered without guidance from a public health laboratory. Many laboratories are not equipped to deal with the isolation needed to safely contain avian influenza (biosafety category 3+ containment, which is higher than that used for HIV). If a sample is accidentally handled, the laboratory may have to be shut down for decontamination.
Samples from patients with suspected avian influenza should be sent to a dedicated central reference laboratory, such as that at the CDC. The CDC laboratory can perform antiviral sensitivity testing, as well as subtyping of the virus.
The best specimens are material collected with oropharyngeal swabs, material from bronchoalveolar washes, or tracheal aspirates. Specimens from nasopharyngeal swabs are acceptable, but they may contain a low quantity of the virus. The CDC recommends obtaining multiple respiratory specimens from different sites on at least 2 consecutive days, as soon as possible after illness onset—ideally, within the first 7 days. [30]
Pneumatic tubing is not recommended for transport; hand transport using a leakproof specimen bag is preferred. The specimen should be clearly labeled as “suspected AI,” and the person who transports the specimen should use appropriate protective equipment. [30]
Testing for H7N9
In a June 7, 2013 Health Update, the CDC recommended that only patients who meet specific exposure criteria and have respiratory illness severe enough to require hospitalization should be tested for avian influenza A (H7N9). The recommendations include the following [42, 43] :
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Clinicians should consider H7N9 testing by reverse-transcription polymerase chain reaction (RT-PCR) assay for patients who meet both clinical criteria (new onset of acute respiratory infection that is severe enough to require hospitalization, and lack of identification of an alternative infectious etiology) and exposure criteria (travel within 10 days of symptom onset to areas with known human cases of H7N9 infection or to areas where H7N9 viruses are circulating in animals, or close contact with confirmed human cases of H7N9 infection)
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In cases in which human H7N9 infection is suspected on the basis of current screening recommendations, respiratory specimens should be collected using infection precautions for novel virulent influenza viruses; the swab or aspirate should be placed in viral transport medium, and the state or local health department should be contacted to arrange transport to the appropriate health department for testing (viral culture should not be performed in these cases)
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Rapid influenza diagnostic tests may not identify H7N9 in respiratory specimens, and a negative test result does not exclude H7N9 infection; in addition, a positive test result for influenza A is unable to distinguish between influenza A virus subtypes, so it cannot confirm avian influenza virus infection; respiratory specimens should be obtained and sent for RT-PCR assay at a state public health laboratory when rapid influenza diagnostic tests are positive for influenza in patients suspected of having novel influenza A virus infection
Chest Radiography
In elderly or high-risk patients with pulmonary symptoms, chest radiography is indicated to exclude pneumonia. Early radiographic findings include no or minimal bilateral symmetrical interstitial infiltrates. Later, bilateral symmetrical patch infiltrates become visible. Focal infiltrates indicate superimposed bacterial pneumonia.
With avian influenza, pulmonary infiltrates are seen in almost all patients. The widely varied radiographic characteristics range from diffuse or patchy infiltrates to lobar multilobar consolidation. Effusions and lymphadenopathy are also observed, as well as cystic changes.
In avian influenza, the severity of radiologically apparent disease is a good predictor of mortality, including findings consistent with ARDS, such as a diffuse, bilateral ground-glass appearance.
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Countries where avian influenza has been reported. Image courtesy of the World Health Organization.
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Colorized transmission electron micrograph shows avian influenza A H5N1 viruses (gold) grown in MDCK cells (green). Image courtesy of Centers for Disease Control and Prevention.
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Transmission electron micrograph (original magnification 150,000X) shows ultrastructural details of an avian influenza A (H5N1) virion, a subtype of avian influenza A. Note the stippled appearance of the roughened surface of the proteinaceous coat encasing the virion. Image courtesy of Centers for Disease Control and Prevention.
