Influenza Virus Type A and Type B, and Respiratory Syncytial Virus (RSV), Molecular Detection, PCR
Rapid and accurate detection of influenza A, influenza B, and respiratory syncytial virus in a single test
Clinical Information Discusses physiology, pathophysiology, and general clinical aspects, as they relate to a laboratory test
Influenza, otherwise known as the "flu," is an acute, contagious respiratory illness caused by influenza A, B, and C viruses. Of these, only influenza A and B are thought to cause significant disease, with infections due to influenza B usually being milder than infections with influenza A. Influenza A viruses are further categorized into subtypes based on the 2 major surface protein antigens: hemagglutinin (H) and neuraminidase (N).
Common symptoms of influenza infection include fever, chills, sore throat, muscle pains, severe headache, weakness/fatigue, and a nonproductive cough. Certain patients, including infants, the elderly, the immunocompromised, and those with impaired lung function, are at risk for serious complications. In the United States, influenza results in approximately 36,000 deaths and more than 200,000 hospitalizations each year.(1)
In the northern hemisphere, annual epidemics of influenza typically occur during the fall or winter months. However, the peak of influenza activity can occur as late as April or May, and the timing and duration of flu seasons vary. In 2009 to 2010, a novel influenza virus (called 2009 H1N1, previously "swine" flu) appeared in Mexico and quickly spread worldwide, causing the first influenza pandemic in more than 40 years. The resultant influenza season had an atypical distribution, with illness occurring during normally low-incidence months. The Centers for Disease Control and Prevention (CDC) now predicts that the influenza season will return to a winter distribution.(1)
Influenza infection may be treated with supportive therapy, as well as antiviral drugs such as the neuraminidase inhibitors, oseltamivir (TAMIFLU) and zanamivir (RELENZA), and the adamantanes, rimantadine and amantadine. These drugs are most effective when given within the first 48 hours of infection, so prompt diagnosis and treatment are essential for proper management.
Respiratory syncytial virus (RSV) is a respiratory virus that also infects the respiratory system and can cause an influenza-like illness. Most otherwise healthy people recover from RSV infection in 1 to 2 weeks. However, infection can be severe in infants, young children, and older adults. RSV is the most common cause of bronchiolitis (inflammation of the small airways in the lung) and pneumonia in children under 1 year of age in the United States, and is more frequently being recognized as an important cause of respiratory illness in older adults.
RSV and influenza virus RNA can be detected by PCR in respiratory secretions, including upper and lower respiratory specimens. Nasopharyngeal swabs or aspirates are the preferred specimen types for detection of RNA from influenza A, influenza B, and RSV. Nasal swabs have also been shown to provide equivalent yield to nasopharyngeal specimens for molecular detection of influenza A and B RNA, but not RSV RNA.(2,3) Tracheal aspirates are generally not acceptable for testing due to the viscous nature of these specimens.
Reference Values Describes reference intervals and additional information for interpretation of test results. May include intervals based on age and sex when appropriate. Intervals are Mayo-derived, unless otherwise designated. If an interpretive report is provided, the reference value field will state this.
A positive test result indicates that the patient is presumptively infected with the indicated virus. The test does not indicate the stage of infection. Rarely, more than 1 virus may be detected from the same patient specimen. Laboratory test results should always be considered in the context of clinical observations and epidemiologic data in making a final diagnosis.
Cautions Discusses conditions that may cause diagnostic confusion, including improper specimen collection and handling, inappropriate test selection, and interfering substances
Given that influenza A and B and respiratory syncytial virus (RSV) can cause identical appearing clinical illness, this test should typically be ordered as a panel. However, individual tests for influenza A and B (combined) or RSV may also be ordered.
This test has been designed to minimize the likelihood of false-positive test results. However, should false-positive results occur, risks to patients could include a recommendation for quarantine of household or other close contacts, a recommendation for patient isolation that might limit contact with family or friends, the ability to work, or the ability to receive certain medical care, prescription of an antiviral drug or other therapy, or other unintended adverse effects.
The sensitivity of the assay is very dependent upon the quality of the specimen submitted. Nasopharyngeal swabs or aspirates are the preferred specimen types and are optimal for detection of RSV RNA. However, the test is validated for use with most upper and lower respiratory specimens, including nasal swabs, throat swabs, bronchoalveolar lavage specimens, and bronchial brushings/washings. Tracheal aspirates are not acceptable for testing due to the viscous nature of these specimens.
This test should not be performed unless the patient meets clinical and epidemiologic criteria for testing.
The test is specific for influenza A, influenza B, and RSV, therefore, the results do not exclude the possibility of infection with other respiratory viruses. Influenza C virus is not detected by this assay.
This assay detects influenza A virus RNA, but does not distinguish between the different subtypes of influenza A.
Negative results do not preclude infection with influenza A, influenza B, or RSV viruses and should not be used as the sole basis for treatment or other patient management decisions.
This assay detects both viable and nonviable virus. Test performance depends on viral load in the specimen and may not correlate with cell culture performed on the same specimen.
All testing was performed on the Roche LightCycler 480 instrument using the Prodesse ProFlu+ test for influenza A, influenza B, and respiratory syncytial virus (RSV).
Influenza A, 2009 H1N1
Twenty-eight clinical specimens were tested by this Prodesse ProFlu+ assay and a validated laboratory-developed test (LDT) for influenza A targeting the matrix gene. Eighteen positives and 8 negatives were detected by both methods. Two additional low-positive specimens were detected by the ProFlu+ that were not initially detected by the LDT, giving a sensitivity of 100% and specificity of 80%. These 2 specimens were detected by the LDT on repeat testing.
Spiked specimens were used to supplement influenza A clinical specimens. Sixty negative patient specimen matrices (30 swab/upper respiratory and 30 nonswab respiratory specimens) were spiked with dilutions of culture-positive 2009 H1N1 influenza A at the limit of detection and run in a blinded manner with 60 negative patient specimens. The 60 spiked specimens were positive and the 60 nonspiked specimens were negative (100% concordance).
Influenza A, H3N2
Thirty negative patient specimen matrices (swab/upper respiratory) were spiked with dilutions of culture-positive influenza A H3N2 at the limit of detection and run in a blinded manner with 30 negative specimens. The 30 spiked specimens were positive and the 30 nonspiked specimens were negative (100% concordance).
Influenza A, Seasonal H1N1
Thirty negative patient specimen matrices (swab/upper respiratory) were spiked with dilutions of culture-positive influenza A seasonal H1N1 at the limit of detection and run in a blinded manner with 30 negative specimens. The 30 spiked specimens were positive and the 30 nonspiked specimens were negative (100% concordance).
Sixty negative patient specimen matrices (30 swab/upper respiratory and 30 nonswab respiratory specimens) were spiked with dilutions of culture-positive influenza B virus at the limit of detection and run in a blinded manner with 60 negative specimens. Of the spiked specimens, 59 were positive and 1 was not detected. All 60 of nonspiked specimens were negative.
A total of 113 clinical specimens were tested by this Prodesse ProFlu+ Assay and the BinaxNow RSV antigen card test. Discrepant specimens were tested by a laboratory-developed RSV PCR assay targeting the nucleocapsid gene. Of these, 89 specimens were positive by both methods and 9 were negative by both. A total of 14 specimens were detected by the BinaxNow test that were not detected by the ProFlu+ PCR, but only 7 of these were positive by additional PCR testing (final sensitivity=93%). One specimen was detected by the ProFlu+ PCR and not the BinaxNow test.
-Qualitative intra-assay precision=100%; standard deviation <0.9 PCR cycles
-Qualitative interassay=98%; standard deviation <0.9 PCR cycles
Limit of detection (LoD):
-The LoD using a quantified plasmid standard was 10 targets/microliter for swab respiratory specimens, and 25 targets/microliter for nonswab specimens.
-The LoD using quantitated cells cultures (TCID50) was 1 TCID50/mL for influenza A, 1 TCID50/mL for influenza B, and 10 TCID50/mL for RSV.
No cross-reactivity observed using a panel of 34 viruses, bacteria, fungi, and mycobacteria that may be found in respiratory specimens.
No inhibition was seen in any of the extracted lower or upper respiratory specimens when tested at the LoD.
A review of the literature indicates that the reference range is "negative" for this assay. This assay is designed to detect influenza A/B or RSV and is to be used for patients with a clinical history and symptoms consistent with this disease. This test should not be used to screen healthy patients.
This is a qualitative assay and results are reported as negative or positive for influenza A, influenza B, or RSV.
Clinical Reference Provides recommendations for further in-depth reading of a clinical nature
1. Centers for Disease Contol and Prevention. Seasonal Influenza: http://www.cdc.gov/flu/; Novel H1N1 Flu (Swine Flu): Available from URL: http://www.cdc.gov
2. Meerhoff TJ, Houben ML, Coenjaerts FE, et al: Detection of multiple respiratory pathogens during primary respiratory infection: nasal swab versus nasopharyngeal aspirate using real-time polymerase chain reaction. Eur J Clin Microbiol Infect Dis 2010;29:365-371
3. Heikkinen T, Marttila J, Salmi AA, Ruuskanen O: Nasal swab versus nasopharyngeal aspirate for isolation of respiratory viruses. J Clin Microbiol 2002;40(11):4337-4339