Assistant Professor Sophie Arbefeville

Lab Medicine & Pathology
Medical School
Twin Cities
Project Title: 
Bacterial and Fungal Identification by RNA Genes Sequencing / Implementation of Next-Generation Sequencing for the Detection of Microbiological Pathogens in Cerebrospinal Fluid Specimens

One of the essential roles of the Clinical Microbiology Laboratory is to provide rapid and accurate identification of bacteria and fungi isolated from patient specimens. The conventional methods used in the clinical laboratory to identify organisms rely on phenotypic characteristics such as microscopic morphology and Gram reaction, growth requirements and rate, colony morphology, and biochemical characteristics. However, some organisms are slow-growing, fastidious, or express biochemical characteristics that do not adequately fit into the pattern of a known genus or species. To identify such an isolate, a laboratory may need to resort to long incubation times, rarely used media, or methods that are not routinely available in most laboratories. Advances in the development of genetic methods provide the clinical microbiology laboratory with additional tools for bacterial identification that supplement conventional phenotypic testing. Of these new methods of identification, the most widely used are the rRNA genes sequencing for the identification of bacteria and fungi.

The 16S rRNA gene (about 1,500 base pairs long) is universal in all bacteria, highly conserved within living organisms of the same genus and species, but with enough variation between species of the same genus to be used to identify bacteria to the genus and species level. It has been demonstrated that the sequence information obtained from the initial 500 base pair of the 5’ region of the 16S rRNA gene often contains sufficient sequence variations to differentiate bacteria at the genus level and usually at the species level.

These researchers are in the process of evaluating the accuracy of multiple public databases to correctly give the identification of bacteria and fungi using the RNA genes.

A related project investigates new methods to determine the etiology of meningitis and encephalitis. Meningitis is an infection of the membranes (meninges) surrounding the brain and the spinal cord, and encephalitis is infection of the brain parenchyma. The infectious agent can be bacterial, viral, or fungal. Both entities are relatively rare but the morbidity, mortality, and costs associated with both are substantial in the United States and throughout the world. Rapid and accurate identification of the underlying etiological agents is directly linked to patient survival and outcome. Clinicians depend on diagnostic testing to determine the underlying etiology to guide therapy, but up to 50% remain undiagnosed due to suboptimal testing and lack of diagnostic tests for all pathogens. Direct sequencing of the PCR-amplified bacterial 16S rRNA gene is one of the molecular identification methods that has greatly improved our ability to identify infectious organisms, and particularly in patients who had received prior antibacterial therapy affecting the growth of bacteria.

However, it has not significantly contributed to the identification of etiologic agents of unexplained, culture-negative illnesses. Also, the 16S rRNA gene cannot be applied to polymicrobial specimens like brain abscesses (the presence of multiple templates results in superimposed Sanger reads that are generally uninterpretable) and has a low sensitivity when applied to direct specimen testing due to the mixture with human DNA. Despite such advances, the incidence of culture-negative meningitis/encephalitis remains high; in large studies more than 50% of encephalitis cases typically remain without an identified etiology. Better diagnostic technologies like next–generation sequencing that are impartial to the type of pathogens and can be potentially used to identify all possible etiologic pathogens need to be explored to define the etiologic agents responsible for these unexplained culture-negative infections. The efficiency and the speed of sequencing are increasing; with the possibility to sequence a microbial genome in just hours permits the technology to be introduced into diagnostic microbiology.

Next-generation sequencing technologies have gained increasing attention in the field of clinical microbiology and are about to revolutionize the way the clinical microbiology laboratory detects and identifies pathogens. Using next-generation sequencing directly from clinical specimens will provide timely information needed to optimize clinical management of patients.

Project Investigators

Assistant Professor Sophie Arbefeville
Dr. Christy Henzler
Todd Knutson
 
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