Emerging Trichomonad Infections in Companion Animals: Rapid Visual Detection of <i>Pentatrichomonas hominis</i> and <i>Tritrichomonas foetus</i> Using an RPA‐CRISPR/Cas12a Assay

<i>Pentatrichomonas hominis (P. hominis)</i> and <i>Tritrichomonas foetus (T. foetus)</i> are prevalent intestinal protozoa. <i>P. hominis</i> is associated with chronic diarrhea in humans and animals, whereas <i>T. foetus</i> causes gastrointestinal disease in companion animals and reproductive-tract infection in cattle. Rapid and accurate identification of these infections at the point-of-care (POC) is crucial for the diagnosis and effective management of zoonotic diseases. In this study, we developed two novel recombinase polymerase amplification (RPA) assays coupled with CRISPR/Cas12a detection. The dual-species assay, using a lateral-flow format, targeted species-specific regions of the 18S rRNA gene of <i>P. hominis</i> and <i>T. foetus</i>, and under ideal conditions, delivered visual results within 40 min for a single sample at 37°C. <i>P. hominis</i>-specific assay: To differentiate <i>P. hominis</i> in mixed infections with <i>T. foetus</i>, a second assay targeted the highly conserved <i>Spo11-1</i> gene of <i>P. hominis</i>. Optimal crRNA-412 and RPA primers were selected for maximal Cas12a cleavage efficiency. Analytical sensitivity and specificity were compared with conventional nested polymerase chain reaction (PCR) and Sanger sequencing. The results showed that The dual-species assay detected as few as 50 DNA copies/µL of either parasite with no cross-reactivity to <i>Giardia lamblia</i>, <i>Cystoisospora canis</i>, <i>Cryptosporidium</i> spp., <i>Toxoplasma gondii</i>, <i>Toxocara canis</i>, and <i>Toxascaris leonina</i>. Among 70 fecal samples of companion animal (48 dogs and 22 cats), 14 (29.2%) dogs tested positive for <i>P. hominis</i>, and eight cats (36.4%) tested positive for <i>T. foetus</i> by nested PCR. Due to financial and logistical constraints, we selected a smaller subset for subsequent analysis with the RPA-CRISPR/Cas12a lateral-flow strip (LFS) assay, which showed 100% diagnostic concordance with PCR. The Spo11-1 assay achieved a limit of detection of 20 DNA copies/µL and specifically recognized <i>P. hominis</i> among a panel that included seven non-target protozoa and helminths. Validation on 10 additional canine and feline samples (four positives and six negatives) showed complete agreement with nested-PCR results. In conclusion, this CRISPR-based diagnostic approach significantly enhances the efficiency and accuracy of Trichomonads detection, offering a practical, cost-effective solution particularly suitable for veterinary and potentially human healthcare diagnostics in resource-limited settings.