|New Tool Helps Identify Mysterious Viruses that
Caused New York Respiratory Illnesses in 2004
A fast, sensitive and inexpensive diagnostic tool called MassTag
PCR has been developed that can identify the specific pathogen
that causes a particular case of respiratory infection, according
to a new study funded by the National Institute of Allergy and
Infectious Diseases (NIAID), one of the National Institutes of
Health (NIH). The research team — headed by scientists in
the Jerome L. and Dawn Greene Infectious Disease Laboratory at
the Columbia University Mailman School of Public Health and their
colleagues at the Wadsworth Center of the New York State Department
of Health — used MassTag PCR to identify previously undiagnosed
pathogens that caused influenza-like illnesses in New York State
during the winter of 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 the November 15, 2006 issue of The Journal
of Infectious Diseases.
“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.
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
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
“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
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
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.
NIAID is a component of the National Institutes of Health.
NIAID supports basic and applied research to prevent, diagnose
and treat infectious diseases such as HIV/AIDS and other sexually
transmitted infections, influenza, tuberculosis, malaria and
illness from potential agents of bioterrorism. NIAID also supports
research on basic immunology, transplantation and immune-related
disorders, including autoimmune diseases, asthma and allergies.
News releases, fact sheets and other NIAID-related materials
are available on the NIAID Web site at http://www.niaid.nih.gov.
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