Real‐time fluorometric isothermal assays for detection of <i>Streptococcus equi</i> subspecies <i>equi</i> and <i>Streptococcus equi</i> subspecies <i>zooepidemicus</i> in horses: Validation, comparison and evaluation of their clinical application

Infectious diseases significantly impact equine health and welfare, causing illness and death, and loss of productivity globally. One such disease is 'strangles', a highly contagious upper respiratory condition in horses caused by Streptococcus equi subspecies equi (SEE). Diagnostic methods for this pathogen include sensitive molecular assays and less reliable bacterial isolation and biochemical testing. However, the presence of closely related streptococci, such as Streptococcus equi subspecies zooepidemicus (SZOO), may confound diagnosis. Rapid assays for SEE are crucial for outbreak control. This study aimed to validate species-specific loop-mediated isothermal amplification (LAMP) assays for SEE and SZOO using a portable real-time fluorometer performed in a clinical setting and to compare their performance with laboratory-based conventional API20 Strep strip and qPCR assays. Rapid sample processing methods were also evaluated with a range of clinical samples, including nasopharyngeal and guttural pouch lavage, purulent exudate, mucosal swabs and bacterial isolates. The LAMP assays demonstrated an analytical sensitivity of 50 genome copies per reaction and showed high congruence with external diagnostic methods. For SEE, the agreement was 96.55% (Kappa 0.88), while for SZOO, it was 94.87% (Kappa 0.87). When applied to rapidly processed clinical samples, SEE and SZOO LAMPs achieved agreements of 97.01% (Kappa 0.70) and 94.94% (Kappa 0.87), respectively, compared with qPCR assays. Testing in an equine clinical setting revealed a 93.84% agreement between LAMP and qPCR results, and 86.96% between LAMP and API20 Strep assays. This study highlights LAMP assays as effective, rapid diagnostic tools, offering reliable options for clinical settings and enabling appropriate and more timely biosecurity interventions.