Immunomagnetic separation (IMS) followed by viability PCR (vPCR) as a tool to determine "real" clinical cases of bacterial gastroenteritis in the culture-independent diagnostic era.

As laboratories embrace culture-independent diagnostic tests (CIDTs), microbiologists can be left to ask: are live bacteria present? When a patient is suspected of having bacterial gastroenteritis (nausea, vomiting, diarrhea), their specimen is genetically screened for pathogens. By reducing turnaround time and lowering the limit of detection, CIDTs have increased diagnostic efficiency and sensitivity. However, CIDT results can be discordant with the gold standard: despite genetic detection, culture can be unsuccessful. Some bacteria are difficult to culture and free-DNA (released from dead cells) can cause false-positive CIDT results leading to incorrect diagnoses. To resolve these issues, I propose a novel combination of immunomagnetic separation (IMS) and viability PCR (vPCR) as a way to exclusively detect intact cells directly from specimens. IMS involves antibody-coated magnetic beads, which bind organisms of interest before being recovered (with the bound-cells) by a magnet. I will then use vPCR to address non-specific binding. When activated by light, propidium monoazide (PMA) can irreversibly bind any free-DNA carried by the beads, ensuring that subsequent real-time PCR only detects DNA from intact cells. Though I will use shiga toxin-producing Escherichia coli (STEC), a common cause of gastroenteritis, this protocol will be compatible with most bacteria. Culture is used to characterize bacteria for surveillance purposes. In an outbreak, non-culturable specimens can delay investigations and worsen patient outcomes. The method proposed can be implemented to help confirm if live bacteria are present in cases of CIDT and culture discordance. This will help guide clinicians' decisions regarding patient therapy based on whether live bacteria are present or not.