Patients hear the words “flu” or “virus,” and they may think they are no big deal, but respiratory viruses, including influenza, are a major cause of morbidity and mortality in the United States. Every winter, the two major strains of influenza viruses,types A and B, circulate and cause illness that results in an average of 36,000 deaths each year, according to the Centers for Disease Control and Prevention(CDC).

Other statistics do nothing to alleviate national concerns over public health issues caused by respiratory viruses, including the danger of a pandemic (worldwide outbreak of disease) caused by the avian bird flu. The leaders in diagnostic testing for respiratory viruses are hoping their cutting-edge technologies and new products will help temper these concerns.

“The question is not if there will be a pandemic,” says Roxanne Shively of the Department of Health and Human Services (HHS). “The question is when.”

Indeed, the United States government is so concerned that it formed the Biomedical Advanced Research and Development Authority (BARDA) as part of HHS 1½ years ago to work with industry to bring products to market so they can be stockpiled for the protection of the United States, says HHS Acting Director Mike Perdue.

Influenza and “Bird Flu” Are Major Concerns

The CDC estimates that each year in the United States, approximately 5% to 20% of the population contracts one of the two types of seasonal flu, and more than 200,000 are hospitalized from flu complications. In addition, influenza viruses are constantly changing through a process called antigenic drift, which causes loss of immunity. Another type of influenza virus, type C, causes a mild respiratory illness and is not thought to cause epidemics on the scale of types A and B.

Laboratorians are being called upon to perform more testing to differentiate influenza from other respiratory pathogens.

Influenza A viruses are divided into subtypes based on two proteins on the surface of the virus: hemagglutinin (H) and neuraminidase (N). There are 16 different hemagglutinin subtypes and nine different neuraminidase subtypes. Influenza A viruses can be further broken down into different strains; the current subtypes of influenza A viruses found in people are H1N1, H1N2, H3N2, and H7N2. Influenza B viruses are not divided into subtypes, but can be further broken down into different strains.

A staggering health concern to HHS and the CDC is the avian bird flu—a flu caused by influenza A viruses that occurs naturally among birds. There are different subtypes of these viruses because of changes in certain proteins (hemagglutinin and neuraminidase) on the surface of the influenza A virus and the way the proteins combine. Each combination represents a different subtype, and all known subtypes of influenza A viruses can be found in birds. According to Shively, project officer in the Division of Influenza and Emerging Diseases in HHS, the avian flu currently of concern is the H5N1 subtype.

While the risk from avian influenza is generally low to most people because the viruses do not usually infect humans, H5N1 has crossed the species barrier to infect humans, and it is the most serious of those that have crossed the barrier. If a person walks around with the undetected H5N1 virus, it could be deadly.

According to the CDC, since 2003, the highly pathogenic avian influenza H5N1 has infected 382 people and resulted in 241 deaths. Although the majority of cases have been caused by direct contact with infected poultry, there have been a few cases of human-to-human transmission, and experts fear the virus will mutate into a form that will be easily passed from person to person.

In the outbreaks in Asia, Europe, and Africa, more than half of those infected with the H5N1 virus, mainly previously healthy children and young adults, have died. However, according to the CDC,it is possible that the only cases currently being reported are those in the most severely ill people, and that the full range of illness caused by the H5N1 virus has not yet been defined.

Symptoms of avian influenza in humans have ranged from typical human influenza-like symptoms—fever, cough, sore throat, and muscle aches—to eye infections, pneumonia, severe respiratory diseases (such as acute respiratory distress), and other severe and life-threatening complications. The symptoms of avian influenza may depend on which virus caused the infection.

Because these viruses do not commonly infect humans, there is little or no immune protection against them in the human population. If H5N1 virus were to gain the capacity to spread easily from person to person, a pandemic could begin, although no one can predict when this might occur, as Shively says.

“The early detection of emerging pandemic influenza is critical to the nation’s pandemic response,” says Mike Leavitt, HHS secretary.

Non-Flu Viruses Are Also a Seasonal Threat

In addition to the flu virus, several other respiratory viruses also can circulate during the flu season, causing symptoms and illnesses similar to those seen with flu infection and threatening public health. The CDC estimates that combined together, respiratory viral infections are the seventh leading cause of death in the United States, claiming the lives of 60,000 annually. Respiratory viruses also are responsible for 75% to 80% of physician visits, according to the National Center for Health Statistics.

Non-flu viruses include rhinovirus (one cause of the “common cold”) and respiratory syncytial virus (RSV), which is the most common cause of severe respiratory illness in young children, as well as a leading cause of death from respiratory illness in those aged 65 years and older.

New rapid tests are being developed as part of the CDC’s preparations for potential epidemics and pandemics.

RSV is the most common cause of bronchiolitis and pneumonia among infants and children younger than 1 year, with the majority of children hospitalized for RSV infections younger than 6 months. During their first RSV infection, between 25% and 40% of infants and young children have signs or symptoms of bronchiolitis or pneumonia and 0.5% to 2% require hospitalization. RSV also causes repeated infections throughout life, usually associated with moderate to severe cold-like symptoms. However, severe lower respiratory tract disease may occur at any age, especially among the elderly or among those with compromised cardiac, pulmonary, or immune systems.

Parainfluenza virus, one in a group of four RNA viruses, ranks second only to RSV as a common cause of lower respiratory tract disease in young children. Like RSV, human parainfluenza viruses (HPIVs) can cause repeated infections throughout life and are usually manifested by an upper respiratory tract illness, such as a cold or sore throat. HPIVs also can cause serious lower respiratory tract disease with repeat infection, including pneumonia, bronchitis, and bronchiolitis, especially among the elderly, and among patients with compromised immune systems.

Parainfluenzas 1 and 2 are the culprits behind laryngotracheobronchitis, most commonly known as croup. HPIV-1 is the leading cause of croup in children, whereas HPIV-2 is less frequently detected. Both HPIV-1 and HPIV-2 can cause other upper and lower respiratory tract illnesses. Parainfluenza 3 is more often associated with bronchiolitis and pneumonia, and parainfluenza 4 is infrequently detected, possibly because it is less likely to cause severe disease.

Since there are so many viral pathogens circulating today that can cause severe respiratory distress, and especially with the danger of the avian flu, physicians need to know which of these are—or are not—infecting a patient in order to effectively prescribe treatment and to control the spread of disease.

Unfortunately, determining the source of a respiratory infection using traditional methods can be slow, inconclusive, and inaccurate, resulting in missed infections or misdiagnosis. Many physicians choose not to test and send patients home without treatment. Or they prescribe antibiotics, even though this may be ineffective and contribute to the overuse of antibiotics and the creation of antibiotic-resistant bacteria or superbugs.

Since many of these respiratory viruses exhibit similar and overlapping symptoms, effective diagnostics and tests are critical. For the government’s antiviral strategy, differentiating among the viruses through multiplex technology is key, in part because there are increasing therapeutic alternatives for influenza A but not for influenza B. From an epidemiological standpoint, tracking A is key because the avian strain is a subtype of A, and the CDC needs to know how often and where this strain is occurring.

By 2010, the worldwide influenza diagnostics market is likely to reach $200 million, according to a press release by Nanogen, a San Diego-based company awarded approximately $15 million in contracts from the United States government.

Preparing for a Pandemic—New Tests on the Horizon

In June 2008, the CDC, in partnership with BARDA, awarded $12.9 million to Nanogen and Meso Scale Diagnostics LLC, Gaithersburg, Md, for the development of a fast, low-cost multianalyte molecular diagnostic assay for influenza to detect and differentiate seasonal human influenza viruses from avian influenza. This follows close on the heels of a December 2006 $4.5 million contract to develop a unique multianalyte point-of-care diagnostic assay for influenza.

“Early detection will aid in improving patient survival, overall health outcomes, and use of containment measures in the event of an influenza pandemic,” Leavitt says. Currently,the testing process for H5N1 can take up to 24 hours to complete.

The awards will support advanced development of laboratory influenza tests, which could be performed in a hospital or a commercial laboratory, and would expedite the diagnosis of large numbers of patients. The expanded testing capability will significantly enhance the hospital laboratory-based pandemic and seasonal flu diagnostic capacity in the United States, Shively says.

“It is difficult to get approved for novel types of the influenza virus, and you need diagnostics to mitigate a potential pandemic,” Perdue says. “We needed a program for both lab tests and point-of-care tests that allows us to diagnose the individual at the earliest possible stage and also track the progression of certain subtypes across the country.”

“There are no tests than can recognize and differentiate certain subtypes of H5 and the seasonal flu,” Shively says. “We need rapid diagnostic tests that can differentiate between the two.”

Rapid diagnostic tests, with delivery of 30 minutes and based on immuno-detection methods, are not new, and are usually the first test administered when a patient goes to see their physician. However, their sensitivities are variable. According to the American Journal of Clinical Pathology, these tests can display sensitivity as low as 50%, depending on the assay method and the virus being tested.

Such low sensitivity can lead to a high rate of false-negative results, which requires further testing—either through traditional culture testing, which takes at least 2 days, leaving the patient to walk around undiagnosed,or through the cutting-edge molecular testing methods. While the molecular tests, which look directly for the presence of viral DNA, have a quicker turnaround time—usually 1 day—than the culture method, they are more expensive.

According to Robert Proulx, vice president of marketing and sales for Nanogen,the movement now is toward multiplex diagnostic methods, which can test for a whole slew of viruses rather than just one. Traditional testing for multiple viruses requires multiple individual diagnostic tests to be performed on a patient sample and can take several days to provide a thorough diagnosis.

Nasal swab samples yield antibodies to multiple pathogens.
Nasal swab samples yield antibodies to multiple pathogens.

Under the first CDC contract, Nanogen, in conjunction with HX Diagnostics Inc, Emeryville, Calif, is developing a multiplex rapid point-of-care lateral flow test based on detecting the presence of antibodies. The new test will be able to simultaneously detect influenza A, influenza B, seasonal flu, and avian flu in one assay. Proulx explains that the product also should detect a smaller quantity of the target antibody so that patients can be diagnosed earlier.

Graham Lidgard, vice president of research and development, says, “We are seeing up to a 100-fold more analytical sensitivity than other tests out there [in the prototype].” The fully integrated system has three simple-to-use components, provides test results in approximately 25 minutes, and can be used in any clinical setting, even those distinct from traditional lab settings.

“The question is how early in the process of manufacturing the antibody can it be detected?” Proulx explains.” The CDC wants to detect the virus earlier and improve or reduce the false/negative capabilities.”

Under the second contract,the company (also with HX Diagnostics) is developing a molecular diagnostic test that will be significantly more sensitive than current rapid flu tests and is expected to be conducted in less than half the time it takes to run current molecular tests. The molecular test will simultaneously detect and differentiate among influenza A, influenza B, seasonal flu, and RSV. The contract also provides for a secondary test for avian flu strains to be available for samples that are determined to be positive for influenza A, but negative for seasonal flu.

“The CDC first wanted a test with improved sensitivity that offered multiplexing subtyping, and then they wanted the same kind of test, but in a molecular form,” Proulx says.

Lidgard adds, “We hope that the tests will provide an enhanced tool for both screening symptomatic patients with an influenza-like illness and for providing important public health information through rapid identification of potentially pandemic influenza strains.

– Shannon Rose is a freelance health and medical writer based in Temecula, Calif.