November 15th, 2006 11:23 EST
Inexpensive Test Detects H5N1 Infections Quickly, Accurately
Washington -- Scientists from the University of Colorado-Boulder and the U.S. Centers for Disease Control and Prevention (CDC) have developed an inexpensive “gene chip” test based on a single influenza virus gene that could let scientists quickly identify flu viruses, including H5N1 avian influenza, or bird flu.
A gene chip, also called a DNA microarray, is a group of microscopic DNA spots attached to a small piece (chip) of glass, plastic or silicon. This DNA array can be used to create a profile of the different proteins that the genes produce, or express.
Such gene chips can monitor expression levels for thousands of genes at once.
The advance was announced as the total number of human cases of H5N1 avian influenza rose to 258, with the confirmation November 13 by the Ministry of Health in Indonesia of two new cases there.
A 35-year-old female from Tangerang in Banten Province developed symptoms November 7 and was hospitalized November 10, according to a World Health Organization statement. She remains hospitalized and the source of her exposure is under investigation.
The second case occurred in a 30-month-old boy from Karawang in West Java Province. He developed symptoms November 5, was hospitalized November 10 and died November 13. An initial investigation found reports of chicken deaths near his home in the days before his illness.
Of 74 cases confirmed in Indonesia, 56 have been fatal. The total number of human deaths due to bird flu is 153.
NEW DIAGNOSTIC TEST
For the new diagnostic test, the researchers used the microarray, called the Mchip, to detect H5N1 in samples collected over three years from people and animals in a geographically diverse group of places, according to a November 14 press release from the National Institute of Allergy and Infectious Diseases (NIAID), part of the National Institutes of Health.
In tests on 24 H5N1 viral samples, the chip gave complete information about virus type and subtype in 21 cases and produced no false-positive results, the scientists reported, meaning the chip never indicated the presence of H5N1 when none was present.
The research was led by University of Colorado scientist Kathy Rowlen and funded by NIAID.
The MChip offers an advantage over available tests because it is based on a single gene segment that mutates less often than the flu genes typically used in diagnostic tests. As a result, the MChip might not need to be updated as often to keep up with the changing virus.
“Concerns about a possible influenza pandemic,” said NIAID Director Dr. Anthony Fauci in a statement, “make it imperative that we continue to devise reliable and easy-to-use diagnostic tests for H5N1 that can be employed on site where outbreaks are suspected.”
The MChip could be a valuable tool in global influenza surveillance efforts, he added.
The raw materials for the MChip cost less than $10 dollars, Rowlen said, and discussions are under way to commercialize its manufacture.
The MChip has several advantages over the FluChip, a flu diagnostic developed by the same research team, Rowlen said.
The FluChip is based on three flu genes -- hemagglutinin (HA), neuraminidase (NA) and matrix (M) -- and the MChip is based on only one gene segment.
There are 16 HA subtypes and nine NA subtypes. The letters H and N in subtype names like H5N1 refer to these genes. The H5N1 subtype, for example, has an HA 5 (H5) gene and an NA 1 (N1) gene.
But unlike HA and NA, which mutate constantly and so are technically difficult to use to develop gene chip diagnostic tests, the M gene segment mutates much less rapidly.
“The M gene segment is much less of a moving target than the HA or NA gene,” Rowlen said. “We believe that a test based on this relatively unchanging gene segment will be more robust because it will continue to provide accurate results even as the HA and NA genes mutate over time.”
There are three kinds of flu viruses -- influenza types A, B and C. Influenza A viruses mutate much more rapidly than influenza types B and C. Influenza A viruses infect humans, birds, pigs, horses, seals, whales and other animals, but wild birds are their natural hosts.
Current tests give information about the type (A or B) in a sample, but more tests must be run to determine the subtype (like H5N1).
The MChip will be able to screen large numbers of flu samples simultaneously to learn the virus type and subtype.
Working in labs enhanced to biosafety level 3 in Atlanta, CDC scientists extracted H5N1 genetic material from virus samples taken from human, cat and many bird hosts, including geese, chickens and ducks.
CDC has established four levels of precautions for biological agents: biosafety level 1 for work involving well-characterized agents not known to cause disease consistently in healthy adults; level 2 for work with agents of moderate potential hazard to people and the environment; level 3 for work with agents that might cause serious or potentially lethal disease; and level 4 for work with dangerous and exotic agents that pose a high individual risk of aerosol-transmitted laboratory infections and life-threatening disease.
The samples used at the Atlanta lab represented infections that occurred between 2003 and 2006 over a large geographic area, including Vietnam, Nigeria, Indonesia and Kazakhstan. Six of the human viral samples were taken from an Indonesian family in which human-to-human H5N1 virus transmission was suspected.
The diversity in the viral samples is important, Rowlen said, because any diagnostic tool designed for eventual use on a rapidly changing virus like H5N1 must be able to detect as many virus variants as possible.
Rowlen and colleagues tested the ability of the MChip to identify 24 different H5N1 viral samples and distinguish those from seven non-H5N1 samples.
The MChip identified and gave complete subtype information (identifying the samples as H5N1) for 21 of 24 H5N1strains and gave no false positives.
“This new technology, once manufactured and distributed, could have the potential to revolutionize the way laboratories test for influenza,” said Nancy Cox, director of the CDC influenza division.
For more information on U.S. and international efforts to combat avian influenza, see Bird Flu (Avian Influenza).
(The Washington File is a product of the Bureau of International Information Programs, U.S. Department of State. Web site: http://usinfo.state.gov)