A rapid sporozoite viability assay identifies leads and targetable enzymatic activities against the zoonotic Cryptosporidium parvum.

Current phenotypic screens for anti-Cryptosporidium compounds typically quantify intracellular parasite growth in host cell cultures after two days of infection. Here, we developed a rapid, host cell-free phenotypic assay that directly measures compound-induced loss of viability in excysted Cryptosporidium parvum sporozoites, the invasive stage that initiates infection. We first compared qRT-PCR, luminescence ATP, and resazurin fluorescence readouts for detecting viable sporozoites. The luminescence ATP assay provided the best balance of linear dynamic range, assay time, parasite input, and cost, and was therefore adapted for high-throughput screening. Screening 5000 bioactive compounds at 40 μM identified 28 primary hits with ≥50% inhibition of sporozoite viability, including 14 with >60% inhibition. Secondary screening of these 14 compounds at 4 μM identified five hits retaining >50% inhibition: ZL0420, sulbactam, abexinostat, kojic acid, and SIB-1757. All five showed submicromolar to low-micromolar activity against free sporozoites, with EC