Comparative study of a broad qPCR panel and centrifugal flotation for detection of gastrointestinal parasites in fecal samples from dogs and cats in the United States
Authors: Leutenegger Christian M. , Lozoya Cecilia E. , Tereski Jeffrey , Andrews Jan , Mitchell Kelly D. , Meeks Cathy , Willcox Jennifer L. , Freeman Gregory , Richmond Holly L. , Savard Christian , Evason Michelle D. 
Published: August 2023

Abstract
Background
For decades, zinc sulfate centrifugal fecal flotation microscopy (ZCF) has been the mainstay technique for gastrointestinal (GI) parasite screening at veterinary clinics and laboratories. Elsewhere, PCR has replaced microscopy because of generally increased sensitivity and detection capabilities; however, until recently it has been unavailable commercially. Therefore, the primary aim of this study was to compare the performance of real-time PCR (qPCR) and ZCF for fecal parasite screening. Secondary aims included further characterization of markers for hookworm treatment resistance and Giardia spp. assemblages with zoonotic potential and qPCR optimization.

Methods
A convenience sampling of 931 canine/feline fecal samples submitted to a veterinary reference laboratory for routine ZCF from the Northeast US (11/2022) was subsequently evaluated by a broad qPCR panel following retention release. Detection frequency and agreement (kappa statistics) were evaluated between ZCF and qPCR for seven GI parasites [hookworm/(Ancylostoma spp.), roundworm/(Toxocara spp.), whipworm/(Trichuris spp.), Giardiaduodenalis, Cystoisospora spp., Toxoplasmagondii, and Tritrichomonasblagburni] and detections per sample. Total detection frequencies were compared using a paired t-test; positive sample and co-infection frequencies were compared using Pearson’s chi-squared test (p ≤ 0.05 significant) and qPCR frequency for hookworm benzimidazole (BZ) resistance (F167Y) and zoonotic Giardia spp. assemblage markers calculated. Confirmatory testing, characterization, and qPCR optimization were carried out with Sanger sequencing.

Results
qPCR detected a significantly higher overall parasite frequency (n = 679) compared to ZCF (n = 437) [p =  < 0.0001, t = 14.38, degrees-of-freedom (df) = 930] and 2.6 × the co-infections [qPCR (n = 172) vs. ZCF (n = 66)], which was also significant (p =  < 0.0001, X2 = 279.49; df = 1). While overall agreement of parasite detection was substantial [kappa = 0.74; (0.69–0.78], ZCF-undetected parasites reduced agreement for individual and co-infected samples. qPCR detected markers for Ancylostomacaninum BZ resistance (n = 5, 16.1%) and Giardia with zoonotic potential (n = 22, 9.1%) as well as two parasites undetected by ZCF (T.gondii/T.blagburni). Sanger sequencing detected novel roundworm species, and qPCR optimization provided detection beyond ZCF.

Conclusions
These results demonstrate the statistically significant detection frequency advantage offered by qPCR compared to routine ZCF for both single and co-infections. While overall agreement was excellent, this rapid, commercially available qPCR panel offers benefits beyond ZCF with detection of markers for Giardia assemblages with zoonotic potential and hookworm (A.caninum) BZ resistance.

Graphical Abstract