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Doug Litwin

Advisor: Heidi Kaplan, Ph.D.

Sepsis is a serious medical condition resulting from the severe dysregulation of the immune response that is generally triggered by infection. It affects more than 1.1 million Americans, has an average mortality rate of 30%, and is estimated to cost $24.3 billion annually. Currently, blood culture followed by Gram-stain analysis is the gold standard for diagnosing bacterial infections associated with sepsis. This method generates a high rate of false negative results and, in general, requires 20 to 48 hr to provide results. Both of these problems are related to the requirement that the bacterial pathogens grow under defined laboratory conditions. This delay and lack of accuracy in diagnosis affects the administration of the correct antimicrobial therapy.

I have designed, developed, and begun to validate a rapid, sensitive, and specific DNA-based quantitative PCR (qPCR) assay, designated the Molecular Gran- stain (MGS) assay, to detect bacterial pathogens directly from septic patient blood samples. This assay also differentiates Gram-positive and Gram-negative pathogens. Importantly, results from this assay may be used to remove patients from unnecessary antimicrobial treatment at least 4 days earlier than is currently possible, because in less than 5 hr it can be determined that no bacterial pathogen is present in a blood sample. The use of this assay may provide for more appropriate antimicrobial administration and decrease antimicrobial resistance and the related costs.

The MGS assay was designed to include internal checks and balances to provide more accurate pathogen detection. The assay utilizes a dual amplicon approach with two probes per amplicon. The presence of any bacterial DNA in a sample is detected by its ability to hybridize to universally conserved regions of the 16S rRNA gene. Hybridization to regions specific to Gram-positive and Gram- negative bacterial DNA serves to differentiate these two groups.

Eighteen clinical blood samples from suspected sepsis patients were analyzed using the MGS assay and compared to results obtained from the Memorial Hermann Hospital Clinical Microbiology Laboratory’s blood culture and Gram- strain analysis. There was 100% agreement between the two positive blood cultures. There was 37% agreement between the 16 culture-negative samples and the MGS assay results. There was 62% non-agreement between the culture- negative samples and the MGS assay results. None of the MGS amplification- negative results were culture positive, supporting the use of this assay as a reliable means to make the call to remove patients from antimicrobial treatment. It is anticipated that the use of this MGS assay will provide an increase in the standard of septic patient care, resulting in better patient outcomes with more rapid tailored antimicrobial use for those with bone fide infections and removal of antimicrobial therapy from those without infection.

Research Info

Development of a molecular gram-stain assay for the diagnosis of blood stream infections associated with sepsis