Respiratory viruses constitute a major health issue in the United States. The Centers for Disease Control and Prevention (CDC) estimates that 5% to 20% of the population gets the flu every year, accounting for more than 200,000 hospitalizations. The flu, caused by influenza viruses A and B, kills approximately 36,000 people every year, mainly children younger than 2 years of age, those 65 and older, and individuals with medical conditions such as congestive heart failure, asthma, or diabetes that can be exacerbated by flu.

In addition to the flu, respiratory viruses are responsible for other threats to public health. The respiratory syncytial viruses (RSV A and B) are the most common cause of severe lower respiratory disease in infants, and they are responsible for a significant number of bronchiolitis and bronchopneumonia cases. The CDC cites RSV as “the single most important respiratory pathogen of infancy and early childhood worldwide.”

The parainfluenza viruses (parainfluenza 1, 2, and 3) most frequently cause laryngotracheobronchitis, commonly referred to as croup. This disease, characterized by its harsh “barking” cough and fever, typically strikes infants and young children 3 months to 5 years old. Other conditions that can be caused by parainfluenza viruses include bronchiolitis, pneumonia, tracheobronchitis, and Corza-like illnesses. These conditions, while not typically fatal, nevertheless contribute to the national health care burden and cause suffering for children and parents alike.

Because many of the symptoms and signs of respiratory-virus infections are similar and overlap, accurate diagnosis is critical for correct treatment and intervention. In particular, according to Yi-Wei Tang, associate professor of medicine and pathology at Vanderbilt University, “RSV, parainfluenza viruses, and other respiratory pathogens can be overlooked if the focus is only on flu.”

Issues of Underdiagnosis
Recent evidence indicates that flu is frequently undiagnosed. A study published in The New England Journal of Medicine found that “most influenza infections in children were not diagnosed clinically.”1 In the study, headed by Katherine A. Poehling, MD, MPH, of Vanderbilt University Medical Center, investigators conducted surveillance diagnosis on inpatients and outpatients reporting respiratory symptoms at hospitals in Nashville, Tenn, Rochester, NY, and Cincinnati, as part of the CDC-sponsored project New Vaccine Surveillance Network (NVSD). The surveillance diagnosis, including laboratory confirmation of pathogen presence, was conducted on all patients with symptoms, independent of the clinical diagnosis and care administered by attending physicians. The study found that only one third of the hospitalized children the surveillance team identified as flu-positive had been tested for flu as part of their clinical care. Of the children with surveillance-confirmed infections, only 28% of inpatients and 17% of outpatients had received clinical diagnoses of influenza. The remaining patients were given diagnoses of various other conditions, including asthma, pneumonia, or a “nonspecific diagnosis of viral infection,” when in fact they were flu-positive.

As the authors state in their discussion, “surveillance that relies on data from physician-directed testing alone substantially underestimates the influenza burden,” reflecting a “lack of recognition of influenza during most visits.”

Furthermore, the NVSD study found that 22 of 160 hospitalized children that tested positive for influenza also had other viral coinfections, mostly RSV and a small number of adenovirus and parainfluenza infections. According to Tang, RSV is “a big issue … because it is a pathogen that can spread through a ward …  where it can cause serious problems for immunocompromised children, such as those receiving a bone marrow transplant.”

Current Testing Methods
At the front line of diagnostic testing for flu and other respiratory viruses are the rapid diagnostic tests based on immuno-detection methods. Because they can deliver results in as few as 30 minutes, these tests are typically the first administered when flu is suspected. The drawback of these tests, however, is that they are not very sensitive and not always specific. According to the CDC and the US Food and Drug Administration (FDA), depending upon conditions, the tests can display sensitivities as low as 60% or 70%. This lack of sensitivity leads to a large number of false-negative results, which must be backed up with additional testing. Alternative immunological methods, such as enzyme immunoassay (EIA) and direct fluorescence assay (DFA), typically deliver results in several hours with greater sensitivity, but also generate relatively large numbers of false negatives.

The high rates of false-negative results necessitate follow-up on all negative samples by alternate methods. Several options exist for this back-up testing. Traditional viral culture, while highly accurate, can take from days to longer than a week. The drawback to culture is that during this time, patients are not treated or are frequently given antibiotics, which do not address the true problem and furthermore contribute to issues of antibiotic resistance in the population.

Molecular testing methods based on polymerase chain reaction (PCR) have been developed in the last few years that are both highly sensitive and specific, and offer results fairly quickly. Most laboratories can turn around PCR-based assays in a day. This rapid turnaround time can greatly facilitate the accurate diagnosis and timely treatment of respiratory viruses. According to Tang, the development of molecular testing “was very much needed.”

Multiplexed Molecular Testing
Nanogen, an advanced diagnostics company headquartered in San Diego, offers multiplexed analyte-specific reagents (ASRs) that CLIA-certified high complexity laboratories can use to develop tests for the detection of genetic sequences associated with influenza A and B; parainfluenza 1, 2, and 3; and RSV A and B.

Following reverse transcription of samples, in which the RNA-based genetic material of viruses is converted to DNA, 13 PCR primers can amplify specific sequences associated with the seven viruses. This approach is very cost-efficient, because up to seven sequences can be detected and amplified from a single RT-PCR reaction.

A mix of discriminators, molecules that hybridize to both an amplified product and a reporter probe, target regions within the amplicons of influenza A and B; parainfluenza 1, 2, and 3; and RSV A and B. Red or green fluorescently labeled reporter probes anneal to the discriminators, and when used with an appropriate fluorescence-detection platform, provide a clear measure of the presence or absence of amplicons from the targeted regions.

The Nanogen reagents offer the clinical laboratory the opportunity to create a quick and simple solution that takes advantage of molecular methods for the detection of multiple respiratory pathogens from a single sample. CLIA laboratories concerned with issues regarding reimbursement on multiplexed assays if physicians request only one or two detections can optimize assay reporting to match test requests, and the company has developed a new pricing schedule that will help labs with this issue.

Suzanne Clancy, PhD, is director of corporate communications, Nanogen Inc.

1. Poehling KA, Edwards KM, Weinberg GA, et al. The underrecognized burden of influenza in young children. New Engl J Med. 2006;355:31–40.