What is the purpose of Shiga toxin gene PCR (Polymerase Chain Reaction) in patient (Pt) diagnosis?

Shiga Toxin Gene PCR: Purpose in Patient Diagnosis

Shiga toxin gene PCR is a molecular diagnostic test used to detect the presence of stx1 and stx2 genes in patient specimens for the diagnosis of Shiga toxin-producing Escherichia coli (STEC) infections, which can cause severe complications including hemolytic uremic syndrome and death. 1

Test Characteristics and Clinical Application

Technical Aspects

• The test detects the genes encoding Shiga toxins (stx1 and stx2) through polymerase chain reaction (PCR)
• PCR assays can distinguish between stx1 and stx2 genes depending on the primers used 1
• Most PCR assays are designed for testing isolated colonies from plated media, though some are validated for testing stool specimens subcultured to enrichment broth (18-24 hours incubation) 1
• Testing time ranges from 3 hours (for isolated colonies) to 24-36 hours (for specimens requiring enrichment) 1

Clinical Significance

• STEC detection is critical as these pathogens can cause:
  • Hemorrhagic colitis
  • Hemolytic uremic syndrome (HUS)
  • Significant morbidity and mortality, especially in children and the elderly

Advantages Over Other Methods

• PCR can detect both O157 and non-O157 STEC strains, which is important as non-O157 strains account for a significant portion of STEC infections 2
• More sensitive than traditional culture methods for detecting STEC
• Can distinguish between stx1 and stx2 genes, with stx2 being associated with more severe disease outcomes 1
• Faster than traditional culture methods which may take several days

Important Limitations and Considerations

Regulatory Status

• DNA-based Shiga toxin gene detection is not FDA-approved for diagnosis of human STEC infections by clinical laboratories 1
• Public health laboratories may use this technique for confirmatory testing after internal validation 1
• Clinical laboratories using PCR assays must:
  • Establish performance specifications as required by CLIA
  • Include disclaimers on reports stating the test is not FDA-approved 1

Technical Limitations

• PCR on DNA extracted directly from whole stool specimens is not recommended due to low sensitivity 1
• No commercially available proficiency testing programs exist in the US for PCR assays targeting Shiga toxin genes 1
• False negatives may occur with low bacterial counts in specimens 3

Best Practices for STEC Testing

Recommended Testing Algorithm

1. Culture stool specimens on selective media AND perform Shiga toxin testing
2. For PCR testing:
   • Use isolated colonies or enrichment broth cultures (not direct stool)
   • Ensure the assay can detect both stx1 and stx2 genes 1
   • Forward positive specimens to public health laboratories for confirmation and further characterization

Critical Actions

• Immediately report positive results to the treating clinician and public health officials 1
• Forward positive specimens or enrichment broths to public health laboratories for isolation and additional characterization 1
• Ensure proper specimen collection and handling to maximize detection

Emerging Technologies

• Newer multiplex PCR assays can simultaneously detect:
  • stx1 and stx2 genes
  • eae gene (encoding intimin)
  • O-antigen specific genes for serogroup identification 4
• Immuno-PCR methods combining antibody capture and DNA amplification can detect as little as 10 pg/ml of purified Stx2, compared to 1 ng/ml by conventional EIA 3
• Quantitative PCR (qPCR) assays have been developed that can detect all known stx gene subtypes in a single reaction 5

By implementing appropriate molecular diagnostic testing for STEC, clinicians can rapidly identify infected patients, initiate appropriate management, and prevent complications that contribute to significant morbidity and mortality.