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New tool helps identify mysterious viruses that caused New York respiratory illnesses in 2004

That year, scores of New Yorkers with respiratory infections had respiratory swabs taken and sent to the New York State Department of Health to identify the exact cause of their illnesses, but in many cases no diagnoses could be made because the swabs tested negative with existing assays for known respiratory pathogens.

Nobody knew what had caused these mysterious illnesses until the Columbia team, led by Ian Lipkin, M.D., reanalyzed the samples and detected, in approximately 30 percent of the cases, nine previously undiagnosed pathogens, including six viruses and three bacteria. Among the viruses were many strains of rhinoviruses, which was unexpected because rhinoviruses usually cause mild respiratory diseases such as the common cold. Dr. Lipkin and his colleagues also discovered that some of the New Yorkers had been infected with an unusual and previously unknown type of rhinovirus, which they describe in a paper in the November 15, 2006 issue of The Journal of Infectious Diseases that is now available online.

"Being able to accurately detect the exact cause of an individual's influenza-like illness is important because it helps doctors make appropriate treatment decisions," says NIAID Director Anthony S. Fauci, M.D.

Adds Dr. Lipkin, "Had MassTag PCR been available to physicians caring for these patients it might have made a difference in disease management and outcome."

The diagnosis and treatment of influenza and other respiratory illnesses is confounded by the numerous pathogens that can cause the same symptoms. Generally, when someone presents with a high fever and a cough or sore throat, their condition is defined non-specifically as an "influenza-like illness," unless a definitive cause can be identified.

The standard technique for identifying the specific pathogen causing the illness is to collect a respiratory specimen and analyze it with a culture test (positive if viruses or bacteria caught in the swab grow in the laboratory), an antigen test, (positive if proteins or other pieces of the pathogen are detected) or a polymerase chain reaction (PCR) test (positive if pieces of the pathogen's genome are detected).

These tests are conclusive when they work, but not every pathogen that causes an influenza-like illness will grow in culture or be detected with PCR or an antigen test. As a result, says Karen Lacourciere, Ph.D., NIAID influenza program officer, "Many respiratory infections go undiagnosed--even for people with all the classic symptoms of influenza."

"In New York state in the winter of 2004, we identified a cluster of undiagnosed influenza-like illnesses in a period of several weeks from October to December 2004," says Kirsten St. George, Ph.D., director of the Clinical Virology Program at the Wadsworth Center. Concerned that some new pathogen might have caused the cluster, Dr. St. George contacted Dr. Lipkin. She asked that he reanalyze the samples with MassTag PCR, which he had developed as a cheap and sensitive test for analyzing samples taken from people with hemorrhagic fevers like Ebola and Marburg. They knew that MassTag PCR could identify a broader spectrum of viruses as well as bacteria.

To detect pathogens, MassTag PCR uses small chemicals tags. Genetic material from a throat swab or other sample is first extracted and then mixed with PCR primers--short pieces of DNA that recognize specific DNA sequences within the genomes of the target viruses or bacteria. If a throat swab contains pathogens with nucleic acid sequences that match those of the primers, then the primers will copy the target DNA multiple times. When the target DNA is amplified, chemical tags attached to the primers are also amplified. The tags can then be purified, stored, shipped and easily identified with mass spectrometry, a technology that separates and identifies molecules based on their masses.

To see if MassTag PCR could identify the mysterious cause of the unidentified respiratory illnesses in New York state, Dr. Lipkin and his colleagues designed PCR primers to look for various viruses and bacteria that cause respiratory disease. Scientists at Columbia University and the Wadsworth Center then used the new respiratory MassTag PCR assay to analyze 151 specimens taken from New Yorkers ranging in age from 4 months to 98 years (median age 25 years) during the 2004-05 winter season. Of these specimens, about half (72) had previously tested positive for some known infectious agent--mostly influenza A or B. Tests on the remaining 79 samples had failed to detect anything.

In 33 percent (26/79) of the swabs that lacked a positive diagnosis, Dr. Lipkin's team identified a number of infectious agents, many of which were rhinoviruses--indicating that rhinoviruses were a major cause of influenza-like illness in New York state in 2004.

Moreover, eight of these specimens tested positive for rhinoviruses that are unlike any known rhinovirus--the longest genome portion analyzed to date is only 50 to 60 percent similar to the genomes of other known rhinoviruses. Unsure of the significance of this new virus, Dr. Lipkin and his colleagues are now looking at other samples taken from patients around the world to see if the same rhinoviruses caused infections in other countries.

The analysis also picked up nine people with coinfections and four people infected with at least three pathogens--findings that could have benefited those people in 2004, says Dr. Lipkin. More accurate diagnoses would help reduce inappropriate prescriptions of antibiotics and slow the spread of antibiotic resistance, he notes.

MassTag PCR, say the authors, compares favorably to existing methods of diagnosis. While the mass spectrometry instrument needed to analyze the samples is expensive, costing around $100,000, testing one specimen is relatively inexpensive--about $12 to look for 20 different pathogens at a time. This compares with about $30 per sample per pathogen for conventional PCR. The technique is also rapid--screens can be done in a single day while culturing pathogens from a specimen may take days to weeks. And the method is more sensitive than antigen tests, says Dr. Lipkin.

Jason Socrates Bardi | EurekAlert!
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