Influenza and RSV: know the diagnostic options

Nov. 18, 2012

The respiratory tract is one of the most common sites for infections because it comes into contact with pathogens frequently. While infection may be caused by a number of different viruses and bacteria, influenza A and B viruses and RSV are collectively responsible for a majority of respiratory illnesses and cause significant morbidity and mortality.1-4

Infections with influenza A and B viruses often result in the respiratory illness commonly referred to as the “flu.” Flu is highly contagious, and, according to the Centers for Disease Control and Prevention (CDC), 5% to 20% of the population contracts the flu each year. Flu viruses are believed to be transmitted primarily via small droplets when individuals with the flu cough or sneeze. Typically, healthy adults are capable of infecting others from one day before symptoms are present to five to seven days after the onset of symptoms. Because of the ease of transmission and high infectivity of flu particles, flu epidemics and even pandemics are always a potential for seasonal flu strains. Each year, more than 200,000 people are hospitalized and between 3,000 and 49,000 people die of complications, typically pulmonary, depending on the severity of the season.5 Symptoms include fever, cough, headache, body aches, congestion, and fatigue.

The elderly, very young, and immunocompromised are at an increased risk of serious complications from the flu, as the virus can potentially spread to the lower respiratory tract, causing pneumonia. Flu can lead to other serious complications such as bronchitis, sinus infections, and a general worsening of chronic conditions.6 These viral infections can also decrease the immune system’s line of defense against many bacteria. Secondary bacterial pneumonias, caused by a range of bacteria including Staphylococcus aureus and Strepococcus pneumonia, become a potential threat that physicians must monitor closely until the illness has cleared.

Respiratory Syncytial Virus (RSV) infection is the most common cause of bronchiolitis and pneumonia in children under one year of age in the United States. Each year 75,000 to 125,000 children in this age group are hospitalized due to RSV infection. Symptoms include coughing, sneezing, runny nose, fever, and decrease in appetite. RSV is also recognized as a serious contributor to respiratory ailments in the aged and immunocompromised demographic. Flu and RSV occur as seasonal outbreaks in the United States, generally starting as early as October or November and ending as late as April or May.7

Viral culture is a sensitive and specific method for detecting influenza and RSV, and is widely considered to be the gold standard. Culture involves inoculating cell monolayers with a patient sample and inspecting for cytopathic effect after one to 14 days of incubation. The two most common viral cell culture techniques are the standard tube cell culture and shell vial culture system. Viral cultures are essential for determining subtypes of influenza A and circulating strains of influenza A and B, which is necessary for epidemiology and surveillance purposes during an outbreak. While this method remains one of the most accurate ways to test for influenza and RSV, slow turnaround times, the need for highly trained staff, and the high cost of maintaining cell lines have caused most labs to abandon viral culture testing in favor of other methods.

Enzyme immunoassays (EIAs) or rapid influenza diagnostic tests (RIDTs) have become much more popular methods for detection of influenza and RSV. EIAs use antibodies tagged with chromatographic or fluorescently dyed particles to capture influenza or RSV antigens in a patient sample. Certain EIAs can distinguish between influenza A and B infections; however, none of these tests can identify influenza A subtypes or specific strains of influenza A or B. Results can be read with the naked eye in less than 30 minutes with little hands-on time and low cost. The reported sensitivity of these tests can range from 60% to 90%, while specificity ranges from 65% to 100%.8 However, other peer-reviewed studies have reported sensitivities as low as 9.6% for influenza and 10% for RSV.9,10

A more accurate and increasingly popular method for influenza and RSV detection is reverse-transcription polymerase chain reaction (RT-PCR). In RT-PCR, viral RNA from a patient sample is enzymatically copied for an exponential reproduction of certain highly conserved gene sequences. These sequences are then detected through nucleic acid hybridization of complementary sequences bound to reporter probes (usually fluorophores). Certain molecular assays are not only able to detect influenza A and B but also accurately identify influenza A hemagglutinin genes, making it possible to determine influenza A subtypes. RT-PCR assays are extremely sensitive and specific, and have even been shown to demonstrate higher sensitivity than viral culture.9

With hospitals focused on controlling costs now more than ever, recent studies have underscored the importance of accuracy in testing for respiratory viruses. Mahoney and colleagues found that inaccurate results (false negatives and false positives) lead to unnecessary additional testing and increased lengths of stay.4 These factors can add an average of more than $2,000 to the cost of care for inpatients.

While RT-PCR testing is more expensive than EIA testing and generally involves longer turnaround times, recent advances in automation have closed these gaps significantly. New CLIA Moderate Complexity testing platforms are capable of reporting highly accurate results within a couple of hours with less than five minutes of hands-on time. Self-contained, disposable test consumables arrive pre-loaded with all of the wet chemistry needed for test processing, allowing users to simply transfer a patient sample directly from a primary sample tube into a test consumable. These consumables are then loaded onto an instrument for automated, walk-away test processing from sample to result. Since run controls are built into test consumables, these tests can be run on-demand as patient samples come into the lab, further reducing turnaround times for physicians and patients and enabling the use of RT-PCR 24/7 in near-patient settings.

Diagnostic options for influenza and RSV, the viruses collectively responsible for a majority of respiratory illness, come in all different shapes and sizes. These methods vary widely in accuracy, turnaround time, complexity, and cost. As technological innovation continues to deliver improved automation and reduced complexity, laboratories will have an increasingly wide range of options for the accurate and timely detection of these two important viruses.

Zack Crowther is the product manager for microbiology at Northbrook, Illinois-based Nanosphere, provider of the sample-to-result Verigene System and its FDA-cleared multiplexed assays for respiratory viruses and Gram-positive blood cultures. Zack’s background in biomedical engineering led him to the molecular diagnostics industry, in which he has worked for the past six years.


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