Screening for biotinidase deficiency started in 2009, and enzyme testing has been the first-tier analysis in the whole of the period, but the follow-up algorithm changed: from 2009 to 2018 all children with a positive initial screening test were contacted for confirmative testing (biotinidase activity determination and molecular genetic studies); after 2018 a second-tier testing on the initial filter paper blood spot sample with sequencing of the biotinidase (BTD) gene was introduced, and only children with two pathogenic or likely pathogenic variants were reported. This evidence concerns the gene BTD and biotinidase deficiency.