Association of Ct Values from Real-Time PCR with Culture in Microbiological Clearance Samples for Shiga Toxin-Producing Escherichia coli (STEC)

Abstract

Shiga toxin-producing Escherichia coli (STEC) are associated with acute gastroenteritis worldwide, which induces a high economic burden on both healthcare and individuals. Culture-independent diagnostic tests (CIDT) in frontline microbiology laboratories have been implemented in Alberta since 2019. The objectives of this study were to determine the association between gene detection and culture positivity over time using STEC microbiological clearance samples and also to establish the frequency of specimen submission. Both stx genes' amplification by real-time PCR was performed with DNA extracted from stool samples using the easyMAG system. Stools were inoculated onto chromogenic agar for culture. An association between gene detection and culture positivity was found to be independent of which stx gene was present. CIDT can provide rapid reporting with less hands-on time and technical expertise. However, culture is still important for surveillance and early cluster detection. In addition, stool submissions could be reduced from daily to every 3-5 days until a sample is negative by culture.

Keywords: CIDT; STEC; real-time PCR; shedding/clearance.

Figure 1

Clearance curves based on the Turnbull algorithm. The black line indicates the proportion of cases who had not yet cleared their STEC infection according to the given method, by day. The gray shaded regions are the Turnbull intervals during which clearance could have occurred. The light gray lines indicate the 95% confidence intervals.

Figure 2

Determination of growth and presence of stx genes during clearance of STEC. (A) Boxplots showing the Ct of stx₁, stx₂, and lowest stx gene detected by quadrant for each analyzed specimen. stx detection was done by DNA extraction from the stool using easyMAG and analyzed with real-time PCR. The Ct value shown is a real-time PCR run from easyMAG extracted DNA, averaged from 3 independent assays. Quadrant negative (NEG) stools are defined as having no mauve colonies. Ct values were averaged from 3 independent assays. (B) Representative images for stx STEC stools. Growth determination on CHROMagar™ plates was done directly from the stool.