Real-time fluorometric and end-point colorimetric isothermal assays for detection of equine pathogens C. psittaci and equine herpes virus 1: validation, comparison and application at the point of care

Abstract

Background: C. psittaci has recently emerged as an equine abortigenic pathogen causing significant losses to the Australian Thoroughbred industry, while Equine herpesvirus-1 (EHV-1) is a well-recognized abortigenic agent. Diagnosis of these agents is based on molecular assays in diagnostic laboratories. In this study, we validated C. psittaci and newly developed EHV-1 Loop Mediated Isothermal Amplification (LAMP) assays performed in a real-time fluorometer (rtLAMP) against the reference diagnostic assays. We also evaluated isothermal amplification using commercially available colorimetric mix (cLAMP), and SYBR Green DNA binding dye (sgLAMP) for “naked eye” end-point detection when testing ‘real-world’ clinical samples. Finally, we applied the C. psittaci LAMP assays in two pilot Point-of-Care (POC) studies in an equine hospital. Results: The analytical sensitivity of C. psittaci and EHV-1 rt-, and colorimetric LAMPs was determined as one and 10 genome equivalents per reaction, respectively. Compared to reference diagnostic qPCR assays, the C. psittaci rtLAMP showed sensitivity of 100%, specificity of 97.5, and 98.86% agreement, while EHV-1 rtLAMP showed 86.96% sensitivity, 100% specificity, and 91.43% agreement. When testing rapidly processed clinical samples, all three C. psittaci rt-, c-, sg-LAMP assays were highly congruent with each other, with Kappa values of 0. 906 for sgLAMP and 0. 821 for cLAMP when compared to rtLAMP. EHV-1 testing also revealed high congruence between the assays, with Kappa values of 0.784 for cLAMP and 0.638 for sgLAMP when compared to rtLAMP. The congruence between LAMP assays and the C. psittaci or EHV-1 qPCR assays was high, with agreements ranging from 94.12 to 100% for C. psittaci, and 88.24 to 94.12% for EHV-1, respectively. At the POC, the C. psittaci rt- and c-LAMP assays using rapidly processed swabs were performed by technicians with no prior molecular experience, and the overall congruence between the POC C. psittaci LAMPs and the qPCR assays ranged between 90.91–100%. Conclusions: This study describes reliable POC options for the detection of the equine pathogens: C. psittaci and EHV-1. Testing ‘real-world’ samples in equine clinical setting, represents a proof-of-concept that POC isothermal diagnostics can be applied to rapid disease screening in the equine industry.