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Cartridge developed by Infectio Diagnostic Inc. is an example of new technology being introduced for rapid identification of microbial pathogens.

Recent genome-related jubilation notwithstanding, many researchers, clinical investigators, and others continue to urge that the biological whole not be overlooked amid this current intensive focus on its genetic parts. Two rapid analytic technologies—one used for quickly identifying microbial pathogens and some of their medically important traits, and the other for assessing a wide range of cellular physiologic activities in a single step—offer some help on this score.

The members of a research and business team at Infectio Diagnostic Inc. (IDI) in Quebec City, Quebec, Canada, have been working for more than five years to develop a nucleic acid-based system for rapidly identifying microbial pathogens and some of their clinically important traits, particularly resistance to specific antibiotics, according to Jacque Milette, who is the company’s vice president for sales and marketing. In May, the company announced it was forming a jointly owned partnership with Cepheid, a specialty biotechnology instrument and technology development company headquartered in Sunnyvale, Calif. The partnership, called Aridia for "Automated Rapid Identification and Detection of Infectious Agents," is based in Halifax, Nova Scotia, Canada. It will soon be offering "turn-key products that are extremely simple to use" in medical diagnostic settings such as hospital laboratories, he says.

The first of those products will be designed to identify Group B streptococci in pregnant women, according to Milette. For instance, analysis of vaginal swab specimens, obtained when women are about to deliver their babies, will provide results to physicians within about an hour, thereby alerting them to potential infections involving newborns. The instruments, designed to do rapid-PCR-based (or alternative nucleic acid amplification-based) tests, will eventually be equipped with a wide range of cartridges capable of detecting many other pathogens with medically important antibiotic resistance traits. The next two in the queue are for analyzing specimens that may contain vancomycin-resistant enterococci and methicillin-resistant Staphylococcus aureus strains, he says.

The species- and trait-specific identifications of microbial pathogens are based on DNA segments that are "extremely specific to each bacteria," Milette says. Thus, the company has developed sets of probes that "target a few different genes in cells," adds his colleague Christian Menard. That approach was developed initially by Michel Bergeron and his collaborators at Universite Laval in nearby Ste-Foy, Quebec. IDI has also developed specific sample preparation techniques suitable for particular fluid specimens, such as blood and urine, or swabs of specific anatomic sites, Menard notes.

Typically, four separate gene targets are used to identify a particular bacterial species, according to Menard. One such target, for example, is a unique segment of the gene encoding a specific elongation factor, he says. "We can really identify the species…in the same way as the 16S [ribosomal] RNA can; this has the same potential for identification." Appropriate probes aimed at additional target sites can be used to identify antibiotic resistance markers. IDI has several patents covering many of these molecular targets and the broader technology used for identifying them for diagnostic purposes, Milette points out.

Meanwhile, members of a product research and development team at Biolog, Inc. in Hayward, Calif., have developed a technology, called Phenotype MicroArrays (PMs) and arranged in 96-well microtiter plates, for broadly analyzing cell phenotypes. With this technology, thousands of phenotypes can be tested simultaneously, according to Barry Bochner, who is chairman and vice president for research and development at the company. The current PM technology was developed specifically for use with microbial cells, but is being adapted for use in analyzing other cell types.

The PMs consists of sets containing different cell culture media, each designed to test a specific phenotype or cell function. In a typical analysis, an investigator adds a cell suspension to the array, incubates the mixtures for 24 to 48 hours, and then reads the set of phenotypic outcomes colorimetrically—by means of a specialized redox chemistry that integrates the respiration of the cells that occurs in each test well during the course of the incubation. In practical terms, the reaction wells range from dark purple, if they grow fully and respire, to colorless, if they fail to grow on the nutrients or reactants that are provided.

The system enables investigators to evaluate a wide array of potential phenotypes, encompassing many types of cell function, according to Bochner. Measurable phenotypes include: cell surface binding and transport functions; catabolism of carbon-, nitrogen-, phosphorus-, and sulfur-containing compounds; biosynthesis of small molecules; biosynthesis of polymeric macromolecules; formation of cellular structures; cellular respiratory functions; and stress and repair functions. He sees the principal uses of the technology as testing specific cell types with genetic differences or that are exposed to drugs or other chemicals.

Like DNA microarrays, PM arrays can provide a global view of thousands of cellular properties and can also be automated for high-throughput testing in research and diagnostic applications. However, DNA microarrays measure thousands of genes under one cellular condition, whereas PM arrays measure activities of one gene product under thousands of cellular conditions, Bochner points out. Thus, the system can help investigators who have already identified genes of interest but are seeking to understand more fully how those genes operate at the cellular level.

Jeffrey L. Fox