Abkhazia Institute for Social and Economic Research

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.