Background: Newborn blood spot screening (NBS) for cystic fibrosis (CF) increasingly identifies infants with inconclusive results, classified as CF screen–positive inconclusive diagnosis (CFSPID). However, long-term outcome data remain limited.

Objective: To analyze the clinical and biochemical course of children with CFSPID.

Methods: We conducted a bi-center observational cohort study including all children designated as CFSPID through the Madrid NBS program from July 2009 through June 2024. Follow-up assessments included serial sweat chloride (SC) testing, respiratory and GI evaluations, spirometry from age 5 or older, nasopharyngeal cultures, and fecal elastase measurements.

Results: A total of 100 children were enrolled. After a median follow-up of 3.59 years [IQR, 1.73–5.43], 25% were reclassified as unaffected carriers following CFTR variant reinterpretation. The remaining 75 were categorized into 3 genotype groups: 66 with CF-causing (CFc)/Variant of Varying Clinical Consequence (VVCC), 4 with VVCC/VVCC, and 5 with CFc/Variant of Uncertain Significance (VUS). By the end of follow-up, 42.67% developed at least 1 intermediate or positive SC value. Three children (4%), all carrying the CFc/VVCC genotype, converted to cystic fibrosis (mean age at conversion, 4.23 years), and 1 child (1.3%) developed a CFTR-related disorder. Clinical signs were mild, with normal spirometry and full pancreatic sufficiency. No Pseudomonas aeruginosa isolates were detected.

Conclusions: Conversion from CFSPID to CF was rare (4%), but abnormal SC values were frequent (42.67%), supporting the need for structured, long-term monitoring. Continued follow-up in specialized CF centers is essential for early detection of disease progression.