Molecular Detection of Feline Hemotropic Mycoplasma from Blood Samples of Sick Cats: A Comparative Study Between a Commercial Nucleic Acid Test Card and Conventional Polymerase Chain Reaction

Feline hemotropic mycoplasmosis presents a diagnostic challenge in clinical practice because of the variable pathogenicity of Mycoplasma species and the limited sensitivity of conventional blood smear examination, highlighting the need for rapid and accurate point-of-care molecular diagnosis. This study aimed to evaluate the diagnostic performance of the Pluslife® Feline Hemotropic Mycoplasma (FHM) nucleic acid test card and to determine the detection frequency of the pathogen among clinically ill cats presented to a veterinary teaching hospital in Bangkok, Thailand. Whole blood samples were collected from 114 clinically ill cats presented to Kasetsart University Veterinary Teaching Hospital between September 2025 and May 2026. The nucleic acid test card assay was evaluated against conventional PCR targeting the 16S rRNA gene as the reference method. PCR identified ‘Candidatus Mycoplasma haemominutum’ (CMhm) as the predominant Mycoplasma species (45.61%), followed by Mycoplasma haemofelis (Mhf; 5.26%) and ‘Candidatus Mycoplasma turicensis’ (CMt; 0.88%). Compared with conventional PCR, the nucleic acid test card assay demonstrated a sensitivity of 93.22%, specificity of 96.36%, overall accuracy of 94.74%, and precision of 96.49%, with results available within 30 min. Sequence analysis of discordant samples confirmed CMhm in four false-negative specimens, suggesting that low bacterial burdens or other assay-related factors may have contributed to the discordant results. Overall, the Pluslife® FHM assay demonstrated high diagnostic performance and may provide a practical rapid molecular point-of-care tool to support timely diagnosis and clinical decision-making in cats with suspected hemotropic mycoplasmosis.