Detection of Viable Streptococcus equi equi Using Propidium Monoazide Polymerase Chain Reaction.

Area of Science:

• Veterinary Microbiology
• Molecular Diagnostics
• Bacterial Pathogenesis

Background:

• The clinical relevance of low-level Streptococcus equi subsp. equi detection via quantitative real-time PCR (qPCR) is debated.
• Propidium monoazide (PMA) treatment is a method used with qPCR to differentiate DNA from viable and nonviable bacterial cells.

Purpose of the Study:

• To assess the efficacy of PMA-enhanced eqbE SEQ2190 triplex qPCR in differentiating viable from nonviable Streptococcus equi in clinical specimens.
• To evaluate the impact of specimen storage on PMA qPCR results for Streptococcus equi detection.

Main Methods:

• Fifty-seven stored clinical specimens (36 positive, 21 suspect positive for S. equi) were analyzed using eqbE SEQ2190 triplex qPCR with and without PMA pretreatment.
• Comparison of cycle threshold values and positive detection rates between PMA-treated and untreated samples.
• SeeI qPCR served as the gold standard for initial specimen classification.

Main Results:

• PMA pretreatment resulted in higher cycle thresholds, indicating the presence of both viable and heat-killed cells.
• Significant differences in S. equi detection were observed: eqbE +PMA (6/57) vs. eqbE -PMA (13/57) (p = .079); SEQ2190 +PMA (10/57) vs. SEQ2190 -PMA (53/57) (p < .0001).
• Mean cycle thresholds increased with PMA: eqbE (23.88 vs. 29.89, p = .04) and SEQ2190 (24.9 vs. 31.9, p < .0001).

Conclusions:

• PMA qPCR is a viable method for determining Streptococcus equi viability.
• The study highlights the critical need to use fresh clinical specimens for accurate PMA qPCR viability assessment, as storage affects results.