New Methods For Identification and Detection of Microbial Pathogens

Traditional Methods For Pathogen Identification

• Cultivation of organism in laboratory, and subsequent characterization.
• Detection of host immune response (e.g., antibodies) to known organism.
• Visual detection of "typical" microbial form in host.

Problems With Traditional Methods

• Cultivation-based methods insensitive for detecting some organisms.
• Cultivation-based methods limited to pathogens with known growth requirements.
• Poor discrimination between microbes with common behavioral features.
• Failure to detect infections caused by uncultivated (e.g., novel) organisms, or organisms that fail to elicit a detectable host immune response.
• Visual appearance of microorganisms is nonspecific.

Examples of Failures With Traditional Approaches

• Detection and speciation of slow-growing organisms takes weeks (e.g., M. tuberculosis).
• A number of visible microorganisms cannot be cultivated (e.g., Whipple bacillus).
• Diseases presumed to be infectious remain ill-defined with no detected microorganism (e.g., abrupt fever after tick bite).

New Molecular Methods

• Use of genetic (DNA or RNA) sequences to identify microorganisms plus DNA amplification procedures (e.g., polymerase chain reaction, PCR) leads to development of specific and consensus PCR methods.
• Other experimental sequence-based molecular methods for detection of novel microorganisms (e.g., "representation difference analysis").

Advantages

• Rapid (hours).
• Specific (can distinguish among different strains of same species).
• Sensitive (can detect one microorganism in some cases).
• Can be automated.

Examples of Successes With New Methods

Identification and characterization of...

• Agents of bacillary angiomatosis and cat scratch disease.
• Agents of human ehrlichiosis.
• Agent of Whipple's disease.
• Hantaviruses associated with human pulmonary disease (e.g., Sin Nombre Virus).
• Agent of Kaposi's sarcoma (Human Herpesvirus 8).
• Novel Babesia-like piroplasm (tick-borne protozoan).
• Novel viral hepatitis agents.

Future Applications

• Rapid, automated identification of known microbial pathogens on routine basis.
• More rapid and sensitive detection and identification of emerging, novel microbial pathogens (via active surveillance network?).

Research Priorities

• Optimize methods for extracting and amplifying DNA directly from clinical samples.
• Expand sequence databases.
• Look for microbial DNA/RNA in various chronic inflammatory diseases of unclear cause.

May 1996