Use of a TaqMan Array Card for identification of enterotoxins and colonization factors directly from stool samples in an enterotoxigenic E. coli vaccine study

• DOI: 10.1128/spectrum.01870-24
• Publication Date: 2025-02-11T14:44:01Z
• Authors: Jie Liu; T. Sakari Jokiranta; Nils Carlin; Suzanne Stroup; Jixian Zhang; Bjorn Sjostrand; Ann-Mari Svennerholm; Eric R. Houpt; Anu Kantele
• Keywords: enterotoxigenic E. coli; ETVAX; colonization factor; TaqMan Array Card; Amplidiag; enterotoxin; Microbiology; QR1-502

ABSTRACT

Enterotoxigenic Escherichia coli (ETEC) is a leading cause of childhood and travelers’ diarrhea. The vaccine candidate ETVAX encompasses several ETEC colonization factors (CFs) with a hybrid LT (heat-labile toxin)/cholera toxin B subunit adjuvanted with a double-mutant LT. Stool samples from a Phase 2b ETVAX trial were tested by a PCR-based customized TaqMan Array Card (TAC), including three ETEC toxin genes (LT and heat-stable toxins, STh and STp) and 18 ETEC CFs. Stool samples were also tested with the molecular platform Amplidiag and culture, followed by GM1-enzyme-linked immunosorbent assay (ELISA) and inhibition GM1-ELISA for LT and ST and dot blot for CFs of ETECs identified among six culture isolates (maximum). Compared with Amplidiag, TAC yielded 89.4% sensitivity (320/358) and 96.4% specificity (405/420) for ETEC detection. The two methods demonstrated a good quantitative correlation (quantification cycle R 2 = 0.827, P < 0.05). Compared with culture, TAC and Amplidiag each exhibited 96.8% (184/190) sensitivity and identified an additional of 151 and 174 PCR positives in 588 culture-negative stools, respectively. The concordance of stool TAC versus ELISA of ETEC colonies for LT and STh/STp was 85.5% (165/193). TAC demonstrated 98% sensitivity and 92% specificity versus the dot blot results of 793 colonies for the ETVAX CFs CFA/I, CS3, CS5, and CS6. Overall ETEC was detected by TAC in 335 (43.1%) and by Amplidiag in 358 (46.0%) of specimens compared to 190 (24.4%) by culture. We conclude that molecular diagnostic approaches of TAC or Amplidiag increase the detection of ETEC compared with culture, and TAC can also provide vaccine-subtype ETEC data.

CLINICAL TRIALS

This study was registered with ClinicalTrials.gov as NCT03729219

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IMPORTANCE

Enterotoxigenic Escherichia coli (ETEC) is an important cause of childhood and travelers’ diarrhea. Vaccines in development utilize specific toxins and colonization factors (CFs) as antigens. Therefore, clinical microbiologic diagnostic methods are needed to discriminate specific toxins and CFs, both for vaccine trials and to guide epidemiology. In this work, we assessed the diagnostic performance of several methods for ETEC: a PCR-based customized TaqMan Array Card (TAC) and the molecular platform Amplidiag on stool and E. coli culture, followed by GM1-enzyme-linked immunosorbent assay for toxins and dot blot for CFs. Stool samples from a Phase 2b ETEC vaccine trial were used. Overall, ETEC was detected by TAC in 335 (43.1%) and by Amplidiag in 358 samples (46.0%) compared to 190 (24.4%) by culture. TAC additionally provided CF data with 98% sensitivity and 92% specificity. We conclude that the molecular diagnostic approaches of TAC or Amplidiag increase the detection of ETEC compared with culture.