Improving isolate recovery and identification of the Shiga toxin type in Shiga toxin nucleic acid test-positive feces.

Abstract

UNLABELLED: Infections caused by Shiga toxin-producing Escherichia coli (STEC) strains carrying Shiga toxin 2 (stx2) are more likely to result in severe complications; however, most nucleic acid amplification tests used for STEC diagnosis do not differentiate between stx1 and stx2. We therefore sought to optimize stx typing and isolate recovery methods to guide clinical and public health management. stx polymerase chain reaction (PCR)-positive feces were cultured using CHROMagar STEC and gram-negative broth, with Stx1 and/or Stx2 antigen detection by enzyme immunoassay (EIA) on colony growth or turbid broth. When cultures were EIA-negative, growth from MacConkey agar (MAC), Trypticase soy broth, and the gram-negative broth was then tested using a lab-developed typing PCR for stx1 and stx2. Colonies were isolated on CHROMagar STEC or MAC and identified using the typing PCR. Using both EIA and typing PCR, the stx types were identified in 96.0% of cases (381/397). In 65.2% (259/397) of cases, culture was EIA-positive, of which 34.0% (87/256) were Stx2-positive. Among cultures that were EIA-negative but typing PCR-positive, 64.8% (79/122) were stx2-positive (P < 0.0001 compared to EIA-positive). Using both EIA and typing PCR resulted in 72.6% (286/394) of cases with successful attempts at isolate recovery, compared to 60.7% (239/394) with EIA alone. E. coli O157 was recovered from more EIA-positive cases (19.3%, 46/239) than EIA-negative ones (4.3%, 2/47) (P = 0.0097). Typing PCR on cultures improves stx typing (particularly stx2) and isolate detection compared to EIA alone. Screening BD Max PCR and subsequent typing PCR results showed excellent concordance. IMPORTANCE: Escherichia coli strains with one or both types of Shiga toxins (stx1 and stx2) are a common cause of bacterial diarrhea and can lead to serious complications such as kidney failure, especially in children. Infection by stx2-positive strains is more likely to do so. Therefore, knowing whether the infection is caused by a strain carrying stx2 is important for risk assessment and case follow-up. The conventional way to diagnose these infections is to grow the bacteria from stool, but most laboratories currently use nucleic acid detection (e.g., bacterial DNA detection by polymerase chain reaction [PCR]), and these assays do not differentiate between the two toxin genes. Culture is therefore required to determine toxin type, as well as for public health outbreak investigations, which require an isolate for whole-genome sequencing for serotyping and cluster analysis. We identified culture media and a PCR-based method to detect stx2 in culture that improved the detection of stx2 and isolate recovery. Our findings provide more accurate results for clinicians to improve patient care and tools for public health teams to control and prevent outbreaks.