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Title: A Multiple Protocol to Improve Diagnosis and Isolation of Shiga Toxin-Producing Escherichia coli (STEC) from Human Stool Specimens

Author
item Shelton, Daniel
item Karns, Jeffrey
item PARK, CHOONG - INOVA FAIRFAX HOSPITAL,VA

Submitted to: Diagnostic Microbiology and Infectious Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/1/2008
Publication Date: 6/12/2008
Citation: Shelton, D.R., Karns, J.S., Park, C.H. 2008. A multiple protocol to improve diagnosis and isolation of shiga toxin-producing escherichia coli (stec) from human stool specimens. Diagnostic Microbiology and Infectious Disease. http://dx.DOI.org/10.1016/j.diagmicrobio.2008.05.001.

Interpretive Summary: Shiga toxin-producing E. coli (STEC) are serious gastrointestinal pathogens that are typically transmitted to humans via contaminated meat or produce. For example, an outbreak of STEC occurred in 2006 due to the inadvertent contamination of spinach, resulting in several hundred illness and several deaths. The identification of such produce-borne outbreaks requires several independent steps: (i) patients who seek medical care are properly diagnosed; (ii) clinical laboratories isolate the responsible STEC strains from patients stool; and (iii) STEC strains are transmitted to state labs and the Centers for Disease Control in a timely manner for characterization and comparison. This process typically requires 2-3 weeks. This is problematic for produce-borne outbreaks (particularly leafy greens) because the shelf life is typically a few days. Consequently, more rapid diagnosis and preliminary strain characterization are required by clinical laboratories to detect incipient STEC outbreaks. A collaborative study was conducted with the INOVA Fairfax Hospital to identify the most rapid and efficient protocols for the detection, isolation and/or characterization of STEC strains from human stool. The results of this study indicated that the routine use of Shiga toxin immunoassays and genetic characterization (PCR) would allow for more rapid diagnosis. In addition, PCR profiles of enriched stool samples would allow for strain comparisons, and hence more rapid detection of outbreaks.

Technical Abstract: Many enterohemorrhaegic colitis infections caused by Shiga toxin-producing are undiagnosed, particularly those belonging to non-O157 STEC serogroups. We evaluated the use of a multiple protocol approach to improve diagnosis, isolation and characterization of STEC strains from human stool specimens. Among 18 presumptive STEC-positive stool samples received by the INOVA Fairfax Hospital (IFH) in the spring/summer of 2006 for analysis, 16 were Shiga toxin-positive. These 16 samples yielded 8 sorbitol-negative O157:H7 and 5 sorbitol-positive, non-O157 STEC isolates from direct plating onto Sorbitol MacConkey Agar (SMAC). The remaining 5 stool samples required enrichment due to a failure to isolate STEC from SMAC. All 5 enriched samples were toxin-positive (including the two samples that were previously toxin-negative from direct stool). Two O157:H7 and one non-O157 STEC were subsequently isolated. The two remaining enriched samples did not yield isolates; however, based on PCR analysis both enriched samples contained STEC genes at extremely low concentrations. Based on PCR analysis of non-O157 strains, 3 strain types were identified. Samples from 3 patients, which were received within 2 days of one another, had a similar gene profile--- eae- and stx1-negative, and stx2-positive. The uniqueness of this gene profile suggests that these patients were most likely infected with the same strain. Our results indicate that a multiple protocol approach is necessary to reliably diagnose and isolate STEC strains, and that PCR profiling of strains could allow for more rapid identification of localized outbreaks.