Validation of a multilocus genotyping scheme for subtyping Cryptosporidium parvum for epidemiological purposes Robinson, Guy Pérez Cordón, Gregorio Cryptosporidium parvum MLVA scheme Multilocus Outbreaks Subtype Tandem repeat Validation We would like to thank Prof. Giovanni Widmer and Dr. Willie Weir for their helpful advice on data analysis and Dr. Harriet Risby for her assistance with the manuscript formatting and additional laboratory testing. This work was part funded by the European Union Seventh Framework Programme ( [FP7/2007-2013] [FP7/2007-2011] under Grant agreement no: 311846) . Subtyping Cryptosporidium parvum for outbreak investigations or epidemiological surveillance usually relies on DNA sequence analysis of a gene coding for a 60 KDa glycoprotein (gp60). Although gp60 can be useful for allelic discrimination and to help investigate sources and routes of transmission, the presence of common subtypes and recombination during the parasite's sexual life-cycle demand a multilocus-based method for more discriminatory genotyping. While whole genome sequencing would provide the ultimate approach, it is a time consuming and expensive option for faecal parasites such as Cryptosporidium that occur at low density and are difficult to propagate routinely. In this study, we selected and evaluated a panel of previously identified variable-number tandem-repeat (VNTR) markers, to establish a multilocus genotyping scheme based on fragment sizing, appropriate for inter-laboratory surveillance and outbreak investigations. Seven VNTR markers were validated in vitro and demonstrated typeability of 0.85 and discriminatory power of 0.99. The discriminatory power was much greater than the currently used gp60 sequencing (0.74), which identified 26 subtypes, compared to 100 different MLVA profiles within the same sample set. The assay was robust, with repeatable results and reproducibility across three laboratories demonstrating the scheme was suitable for inter-laboratory comparison of C. parvum subtypes. As the majority of genotypes (79%) were unique among epidemiologically unrelated samples, there was efficiency to infer linkage, and epidemiological concordance was observed in historical outbreaks. We propose that the multilocus variable number of tandem repeats analysis scheme is suitable to assist outbreak investigations. 2022-06-07T06:53:53Z 2022-06-07T06:53:53Z 2022-04-11 info:eu-repo/semantics/article Guy Robinson... [et al.]. Validation of a multilocus genotyping scheme for subtyping Cryptosporidium parvum for epidemiological purposes, Food and Waterborne Parasitology, Volume 27, 2022, e00151, ISSN 2405-6766, [https://doi.org/10.1016/j.fawpar.2022.e00151] http://hdl.handle.net/10481/75292 10.1016/j.fawpar.2022.e00151 eng info:eu-repo/grantAgreement/EC/FP7/311846 http://creativecommons.org/licenses/by-nc-nd/3.0/es/ info:eu-repo/semantics/openAccess Atribución-NoComercial-SinDerivadas 3.0 España Elsevier