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France's Institut Pasteur Develops Resequencing Array for Pathogen Identification


By Justin Petrone

Researchers at the Institut Pasteur's Laboratory for Urgent Response to Biological Threats in Paris have developed a new resequencing microarray for use in public health emergencies triggered by exposure to pathogens.

The investigators validated the chip in a recent study by evaluating its ability to detect re-assorted strains of swine-origin H1N1 influenza A virus.

In the paper, the authors described how they used random non-PCR–based nucleic-acid amplification and high-density resequencing DNA microarrays to rapidly identify re-assortant influenza A virus strains of swine origin.

The array, called PathogenID, contains 126 viral sequences from a range of viruses. It is engineered to detect and describe four genes in order to type and subtype influenza viruses from a large diversity of natural and permanent hosts, such as humans, birds, horses, and pigs.

As part of the study, the researchers used PathogenID to test six clinical specimens. The researchers also mixed two separate sequences to create an artificial scenario of co-infection in a sample that could lead to a new virus. In their study, they showed that PathogenID was capable of identifying the sequence in such a situation.

Institut Pasteur's Jean-Claude Manuguerra told BioArray News this week that the array was designed with assistance from Affymetrix for the "broad detection of bacteria and pathogens," but was particularly assessed in this new study for its ability to detect new strains of swine-origin H1N1.

According to Manuguerra, the institute is currently evaluating the chip, called PathogenID, for detecting other pathogens. If all validations go as planned, other researchers will be able to order PathogenID directly from Affy within the next year.

"We decided to develop the array for broad detection of bacteria and pathogens" about 4 years ago, Manuguerra said. "We first started with threat agents and progressed to natural epidemics as they are more frequent.

"The approach of the chip was not to specialize in any one kind of virus or bacteria and to have a multi-parametric system," he said. "We have worked on different viruses to try to develop the array to maximize viral species we can detect while not increasing the number of probes on the array."

According to Manuguerra, his lab does not currently use arrays as a routine tool for identifying influenza viruses, typically using quantitative real-time PCR instead. However, the firm uses the array platform in situations where the specific sequence of the pathogen is unknown. "The idea was to have a system that could be used no matter what happened," he said.

There are currently a number of assays available for labs that need to identify various strains of flu. The majority of the assays authorized for emergency use by the US Food and Drug Administration are RT-PCR based, but Potomac Falls, Va.-based TessArae in December won emergency use authorization for its Resequencing Influenza A Microarray Detection Panel which, like PathogenID, is manufactured by Affy (see BAN 12/22/2009).

Other firms to develop array-based approaches to identifying various flu strains include Sunnyvale, Calif.-based Arrayit, Boulder, Colo.-based InDevr, Mukilteo, Wash.-based CombiMatrix, Singapore-based Veredus Laboratories, and Hyderabad, India-based Ocimum Biosolutions.

Manuguerra said that one advantage of Institut Pasteur's approach is that it does not use an RT-PCR-based amplification step. "Our experience is that if you do RT-PCR on a virus, it will affect the signal on the chip," Manuguerra said. "So we work with non-PCR-based amplification, which has very good output in terms of production of DNA."

In the paper, the authors highlighted other advantages over existing tests, primarily those that are RT-PCR based. According to the authors, PathogenID is able to "generate sequences that are not already tiled" and is ready for use and can be implemented immediately, as there would be "no lag time between public health authorities issuing an alert and laboratories around the world implementing tests."

"The idea was to have a system that could be used no matter what happened," Manuguerra stated. "Indeed, when [an RT-PCR test] was made available to everyone around the world by the CDC, we had to not only order primers but the probes," he said. This need to order primers and probes "prevented immediate implementation of specific H1N1 assays in many centers across the world."

Manuguerra also sees PathogenID as an alternative to high-throughput sequencing-based approaches. "We can have the sequence within 24 hours," Manuguerra said. "This is compatible with an emergency situation when the authorities want to know if a virus has arrived on territory or not. My lab works with public health emergencies we have to have tool that is very versatile."

In the paper, Manuguerra and colleagues wrote that the "cost of sequencing instrumentation and reagents, as well as the delay in interpreting the data, makes these systems less useful to public health authorities seeking the first occurrence of a novel virus in a given country.

"Although the sequence information provided by resequencing microarrays are more limited than those produced by high-throughput pyrosequencing technologies, their ability to generate results as early as 24 [hours] after the beginning of the experiment is a substantial advantage."

Despite these limitations, Manuguerra's lab is using both arrays and sequencing. Still, he said that deep sequencing requires an investment in bioinformatics that results in a "big delay in obtaining what you need in terms of time and cost for the time being." The cost of a PathogenID chip is about €300 ($419) per array.

As Institut Pasteur validates PathogenID for further implementation, Manuguerra's lab is also working with other researchers to develop a new version of the array that will include viruses found in other countries, making the chip more useful on a global scale. Specifically, he said that he and fellow researchers may collaborate with the 29 institutions within the Institut Pasteur International Network, which employs a staff of 9,500 spread over five continents.

"This work is important for viruses that we cannot find in temperate climate like France that are more specific to other regions," Manuguerra said.

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