Development and Validation of Shiga Toxin-Producing Escherichia coli Immunodiagnostic Assay.

Miriam Silva,Anna Santos,Roxane Piazza,Luanda Santos,Denise Horton,Bruna Caetano,Juliana Polatto,Thais Mitsunari,Beatriz Guth,Luís Dos Santos,Leticia Rocha

doi:10.3390/microorganisms7090276

Abstract

Shiga toxin (Stx)–producing Escherichia coli (STEC) and its subgroup enterohemorrhagic E. coli are important pathogens involved in diarrhea, which may be complicated by hemorrhagic colitis and hemolytic uremic syndrome, the leading cause of acute renal failure in children. Early diagnosis is essential for clinical management, as an antibiotic treatment in STEC infections is not recommended. Previously obtained antibodies against Stx1 and Stx2 toxins were employed to evaluate the sensitivity and specificity of the latex Agglutination test (LAT), lateral flow assay (LFA), and capture ELISA (cEIA) for STEC detection. The LAT (mAb Stx1 plus mAb stx2) showed 99% sensitivity and 97% specificity. Individually, Stx1 antibodies showed 95.5% and 94% sensitivity and a specificity of 97% and 99% in the cEIA and LFA assay, respectively. Stx2 antibodies showed a sensitivity of 92% in both assays and a specificity of 100% and 98% in the cEIA and LFA assay, respectively. These results allow us to conclude that we have robust tools for the diagnosis of STEC infections.

Highlights

Among the E. coli human pathogens, the Shiga toxin (Stx)–producing Escherichia coli (STEC) and its subgroup enterohemorrhagic E. coli (EHEC) have gained importance in the three last decades due to their involvement in diarrhea [1], that may be complicated by hemorrhagic colitis (HC) [2] and the hemolytic uremic syndrome (HUS) [3,4], the foremost cause of acute renal failure in children [1] due to action of the two major types of the phage-encoded Stxs, Stx1 and/or Stx2
We included for the ELISA (EIA) cut-off definition and specificity of the latex agglutination (LA) and lateral flow assay (LFA), 12 typical enteropathogenic E. coli [36,37], 11 atypical enteropathogenic E. coli [38], 45 enterotoxigenic E. coli (ETEC) [39,40], nine enteroaggregative E. coli (EAEC) [41], eight enteroinvasive E. coli (EIEC) [42], 14 diffusely-adherent E. coli (DAEC) [42], three fecal E. coli negative for DEC virulence factors (NVF E. coli), four microbiota E. coli isolates and 19 Enterobacteriaceae isolates
For capture ELISA and latex agglutination test (LAT) bacterial culture was lysed with Triton X-100 or polymyxin sulfate B for the lateral flow assay (LFA), since the detergent presence impaired the sample flow in LFA

Summary

Introduction

Among the E. coli human pathogens, the Shiga toxin (Stx)–producing Escherichia coli (STEC) and its subgroup enterohemorrhagic E. coli (EHEC) have gained importance in the three last decades due to their involvement in diarrhea [1], that may be complicated by hemorrhagic colitis (HC) [2] and the hemolytic uremic syndrome (HUS) [3,4], the foremost cause of acute renal failure in children [1] due to action of the two major types of the phage-encoded Stxs, Stx and/or Stx. A large fraction of the reported STEC infections is due to E. coli O157:H7, the most involved serotype in complicated cases, which often evolve into HUS [8]. In the Latin American countries, human infections by STEC are endemic in Argentina and are mainly linked to O157 strains. In other Latin American countries STEC (O157 and non-O157) causes sporadic cases of diarrhea, bloody diarrhea, hemolytic anemia and HUS [11,12,13,14,15,16,17]. It is noteworthy that some relevant serotypes linked to human infections such as O103:H2 and O157:H7 have been recovered from the feces of sheep and cattle [18,28]

Methods

Results

Discussion

Conclusion