Detection of Streptococcus equi subsp. equi in guttural pouch lavage samples using a loop-mediated isothermal nucleic acid amplification microfluidic device.

Abstract

BACKGROUND: Rapid point-of-care (POC) detection of Streptococcus equi subsp. equi (S. equi) would theoretically reduce the spread of strangles by identifying index and carrier horses. HYPOTHESIS: That the eqbE isothermal amplification (LAMP) assay, and the same eqbE LAMP assay tested in a microfluidic device format, are comparable to a triplex real-time quantitative polymerase chain reaction (qPCR) assay that is commonly used in diagnostic labs. SAMPLES: Sixty-eight guttural pouch lavage (GPL) specimens from horses recovering from strangles. METHODS: Guttural pouch lavage specimens were tested for S. equi retrospectively using the benchtop eqbE LAMP, the eqbE LAMP microfluidic device, and compared to the triplex qPCR, that detects 2 S. equi-specific genes, eqbE and SEQ2190, as the reference standard using the receiver operating characteristic area under the curve (ROC). RESULTS: The 27/68 specimens were positive by benchtop eqbE LAMP, 31/64 by eqbE LAMP microfluidic device, and 12/67 by triplex qPCR. Using the triplex PCR as the reference, the benchtop eqbE LAMP showed excellent discrimination (ROC Area = 0.813, 95% confidence interval [CI] = 0.711-0.915) as did the LAMP microfluidic device (ROC Area = 0.811, 95% CI = 0.529-0.782). There was no significant difference between the benchtop LAMP and LAMP microfluidic device (ROC Area 0.813 ± 0.055 vs 0.811 ± 0.034, P = .97). CONCLUSIONS: The eqbE LAMP microfluidic device detected S. equi in GPL specimens from convalescent horses. This assay shows potential for development as a POC device for rapid, sensitive, accurate, and cost-efficient detection of S. equi.