Large-scale molecular epidemiological survey of <i>Giardia</i> and <i>Cryptosporidium</i> in Victoria, Australia (2020–2024), reveals novel subtypes and outbreak-associated lineages

<i>Cryptosporidium</i> and <i>Giardia</i> are major causes of gastrointestinal illness globally. In Australia, cryptosporidiosis is nationally notifiable, yet molecular characterization is not routinely undertaken, limiting outbreak detection, source attribution, and zoonotic risk assessment. This limitation became apparent in 2024, when Australia experienced a 273% increase in cryptosporidiosis notifications, the third-highest rise globally. We conducted a molecular epidemiological investigation of <i>Cryptosporidium</i> and <i>Giardia</i> in fecal samples from humans with gastroenteritis in Victoria, Australia, between 2020 and 2024. Positive samples were sequenced using <i>SSU</i> and <i>gp60</i> for <i>Cryptosporidium</i> and <i>tpi</i> for <i>Giardia</i>, and parasite load was estimated. Of the 2,330 samples tested, 225 were positive for <i>Cryptosporidium</i> and nine for <i>Giardia</i>. One <i>Giardia</i> sample was identified as sub-assemblage AI, two as AII, and six as assemblage B. Seven <i>Cryptosporidium</i> species and 24 subtypes were identified, including eight novel subtypes. <i>Cryptosporidium hominis</i> predominated (85%), followed by <i>C. parvum</i> and <i>C. meleagridis</i>. Six <i>C. hominis</i> subtypes were detected, three associated with 11 recreational water-linked outbreaks in 2024. Subtypes IaA12R3 and IeA11G3T3 were most frequent, in line with global trends; concordance analysis indicated that at least 52 additional cases were likely linked to outbreaks. Several <i>C. parvum</i> subtypes were identified, including two linked to childcare and camp outbreaks. Novel human-infective subtypes of <i>C. occultus</i>, <i>C. fayeri</i>, and <i>C. meleagridis</i> were also detected. These findings reveal extensive <i>Cryptosporidium</i> diversity in Victoria and highlight the value of molecular surveillance for resolving transmission pathways and informing public health responses.IMPORTANCE<i>Cryptosporidium</i> is a nationally notifiable pathogen in Australia, yet routine subtyping is not performed, limiting the detection of both outbreak-related and sporadic transmission and constraining source attribution. This study represents the first large-scale molecular surveillance of human <i>Cryptosporidium</i> and <i>Giardia</i> infections in southern Australia, including the first molecular epidemiological exploration of the marked increase in cryptosporidiosis observed in 2024. By integrating species, subtype, or assemblage information with epidemiological metadata, we demonstrate substantial <i>Cryptosporidium</i> lineage diversity in symptomatic patients, including multiple novel subtypes and zoonotic transmission. These findings demonstrate the critical value of molecular tools for uncovering hidden transmission, resolving pathways contributing to both sporadic disease and outbreak, and informing targeted public health responses. The findings provide a strong foundation for incorporating routine genotyping into national surveillance systems for parasitic eukaryotic enteropathogens, and the accompanying international comparison of case trends suggests broader transmission dynamics of significant public health relevance.