| Summary: | The major route for <i>Toxoplasma gondii</i> (<i>T. gondii</i>) infection is through the ingestion of foods contaminated with oocyst from cat faeces. The microscopic detection of <i>T. gondii</i> oocysts in cat faeces is challenging, which contributes to the failure of detecting or differentiating it from other related coccidian parasites. This study aims to detect <i>T. gondii</i> oocysts in cat faeces using two multicopy-target PCR assays and to evaluate their genetic diversity. Cat faecal (200) samples were collected from pet cats (PCs; 100) and free-roaming cats (FRCs; 100) within Klang Valley, Malaysia, and screened for coccidian oocysts by microscopy using Sheather’s sucrose floatation. PCR assays were performed on each faecal sample, targeting a B1 gene and a repetitive element (REP) gene to confirm <i>T. gondii</i> oocysts. Additionally, the PCR amplicons from the REP gene were sequenced to further confirm <i>T. gondii</i>-positive samples for phylogenetic analysis. Microscopy detected 7/200 (3.5%) <i>T. gondii</i>-like oocysts, while both the B1 gene and the REP gene detected 17/200 (8.5%) samples positive for <i>T. gondii</i>. All samples that were microscopically positive for <i>T. gondii</i>-like oocysts were also shown to be positive by both B1 and REP genes. The BLAST results sequenced for 16/200 (8.0%) PCR-positive <i>T. gondii</i> samples revealed homology and genetic heterogeneity with <i>T. gondii</i> strains in the GenBank, except for only one positive sample that did not show a result. There was almost perfect agreement (k = 0.145) between the two PCR assays targeting the B1 gene and the REP gene. This is the first report on microscopic, molecular detection and genetic diversity of <i>T. gondii</i> from cat faecal samples in Malaysia. In addition, the sensitivities of either the B1 gene or REP gene multicopy-target PCR assays are suitable for the accurate detection of <i>T. gondii</i> from cat faeces.
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