Pooled Nucleic Acid Amplification Test for Screening of Stool Specimens for Shiga Toxin-Producing Escherichia coli

Abstract

c Shiga toxin-producing Escherichia coli (STEC)-associated enteric illness is attributed to O157 and non-O157 serotypes; however, traditional culture-based methods underdetect non-O157 STEC. Labor and cost of consumables are major barriers to implementation of the CDC recommendation to test all stools for both O157 and non-O157 serotypes. We evaluated the feasibility of a pooled nucleic acid amplification test (NAAT) as an approach for screening stool specimens for STEC. For retrospective evaluation, 300 stool specimens were used to create pools of 10 samples each. The sensitivity was 83% for the preenrichment pooling strategy and 100% for the postenrichment pooling strategy compared with those for individual NAAT results. The difference in cycle threshold (C T ) between individual and pooled NAAT results for specimens was significantly lower and more consistent for postenrichment pooling (stx 1 mean ‫؍‬ 3.90, stx 2 mean ‫؍‬ 4.28) than those for preenrichment pooling (excluding undetected specimens; stx 1 mean ‫؍‬ 9.34, stx 2 mean ‫؍‬ 8.96) (P < 0.0013). Cost of consumables and labor time savings of 48 to 81% and 6 to 66%, respectively, were estimated for the testing of 90 specimens by the postenrichment pooled NAAT strategy on the basis of an expected 1 to 2% positivity rate. A 30-day prospective head-to-head clinical trial involving 512 specimens confirmed the sensitivity and labor savings associated with the postenrichment pooled NAAT strategy. The postenrichment pooled NAAT strategy described here is suitable for efficient large-scale surveillance of all STEC serotypes. Comprehensive detection of STEC will result in accurate estimation of STEC burden and, consequently, appropriate public health interventions. Shiga toxin-producing Escherichia coli (STEC) is a major cause of sporadic and outbreak-associated enteric illness worldwide. While more than 100 STEC serotypes can cause illness in humans (1), traditional testing methods focus primarily on the most common serotype, O157:H7, owing to the ease of its detection using culture-based media. These methods underdetect non-O157 serogroups, and as such, their clinical burden is not well understood (2-4). Moreover, recent evidence suggests that infections attributed to non-O157 STEC may be more prevalent than those attributed to O157 (1, 5), with common serogroups being O26, O45, O103, O111, O121, and O145 in the United States (6, 7) and in other countries (1). Although non-O157 STEC serotypes are generally associated with milder disease than O157 STEC, infections caused by non-O157 STEC can also lead to hemolytic uremic syndrome (1) and have been associated with major outbreaks, most notably a 2011 outbreak in Germany that involved 3,816 cases and 54 deaths (8). Enhanced surveillance evidence is needed to determine the true burden of non-O157 STEC for public health investigations of potential exposure sources. Such efforts are ongoing in Canada (9) but require improved and comprehensive screening methods.The Centers for Disease Control and Prevention (CDC) recom...

CPHLN recommendations for the laboratory detection of Shiga toxin-producing Escherichia coli (O157 and non‑O157)

“…As for the detection of Shiga toxins by EIA, both microwell and lateral flow formats are available. The sensitivity of EIAs is lower for direct stool testing; however, overnight enrichment in MacConkey broth (15) or other suitable broth may provide sensitivity approaching that of NAATs (13,16). Both in-house and commercial NAATs have equivalent sensitivities and are the most sensitive methods available (15,17).…”