Classical homocystinuria (HCY) due to cystathionine beta-synthase (CBS) deficiency is included in many newborn screening programs, which traditionally use dried blood spot (DBS) methionine as the primary biomarker. However, some patients have normal or only mildly elevated methionine in the neonatal period, leading to false-negative results. We report a four-month-old male infant referred for sagittal craniosynostosis, whose metabolic evaluation revealed markedly elevated plasma methionine and total homocysteine concentrations. Genetic analysis identified a homozygous, pathogenic variant (c.1007G>A) in the CBS gene, confirming HCY. On retrospective review, the DBS sample had been collected at 48 h of life, and methionine was within the reference range. Consequently, the screening algorithm had not triggered second-tier total homocysteine testing. Sanger sequencing of the original DBS sample confirmed the same homozygous CBS variant, ruling out sample swap and establishing the case as a true false-negative result. Treatment with pyridoxine and folic acid rapidly normalized biochemical parameters, consistent with a pyridoxine-responsive phenotype. This case illustrates that normal neonatal methionine does not exclude CBS deficiency and that genomic newborn screening approaches could detect this disease more reliably.
Newborn screening (NBS) is a critical component of the three-tiered prevention strategy for birth defects, reducing congenital disorder burden and improving long-term child health outcomes. Over the past 45 years, NBS in China has evolved into a nationwide quality-controlled network driven by policy support and technological advances. Currently, phenylketonuria and congenital hypothyroidism are included in universal NBS across the country, and several provinces have expanded to encompass congenital adrenal hyperplasia, glucose-6-phosphate dehydrogenase deficiency, and additional inherited metabolic disorders identified through tandem mass spectrometry. The application of next-generation sequencing and other technologies has further enhanced detection capacity and expanded detectable disease spectra. Meanwhile, the National Quality Management System for NBS (QMS-NBS) has realized the visualization and standardization of screening quality and performance. Despite these advances, challenges remain, including regional disparities, inadequate follow-up, and long-term management. This review summarizes the historical evolution and policy framework of NBS in China, outlines the development of screening institutions, the spectrum and incidence of screened disorders, advances in detection technologies, and the establishment of QMS-NBS. It also highlights future priorities: expanding screened conditions, strengthening follow-up and long-term care, promoting regional equity, and advancing novel technologies to improve child health and foster precision public health.
Newborn screening (NBS) facilitates the presymptomatic diagnosis and early treatment of spinal muscular atrophy (SMA). An analytical study was conducted to evaluate the performance and limitations of a real-time polymerase chain reaction-based SMA-NBS assay. Dried blood spot specimens collected from 21 newborns and three patients with genetically confirmed SMA were analyzed. The SMA controls included two patients with homozygous SMN1 deletion and one with compound heterozygous SMN1 variants. SMN1 exon 7 copy numbers were quantified and interpreted. All newborn participants had SMN1 copy numbers above the screening cutoff value of 668 copies/µL and were classified as screening-negative. The two SMA controls with homozygous SMN1 deletion showed no detectable SMN1 amplification, which is consistent with positive screening results. In contrast, the compound heterozygous SMA control exhibited a mean SMN1 value of 7330 copies/µL, which was more than tenfold above the screening cutoff and would therefore have been classified as screening-negative; this value also fell within the range observed in the newborn cohort. This patient had a negative NBS result at birth despite subsequently developing genetically confirmed SMA. The SMA-NBS assay accurately detected patients with homozygous SMN1 deletion. However, it did not identify patients with compound heterozygous SMN1 variants. In conclusion, deletion-based SMA-NBS has an inherent false-negative limitation, and a negative screening result does not completely exclude an SMA diagnosis.
Newborn screening (NBS) is a cornerstone of preventive medicine, enabling early diagnosis and treatment of severe congenital disorders. In Spain, despite the existence of a Basic Common Portfolio, the absence of a harmonized national panel has led to significant inter-regional variability, affecting equity in access to early diagnosis. The Spanish Uniform Screening Panel (SUSP) was developed through a structured nationwide consensus process involving all NBS centers in Spain, with additional input from Portugal. The process included a comprehensive survey of current practices, expert workshops, multiple consensus rounds, and predefined inclusion criteria. Disease nomenclature was standardized using OMIM and ORPHAcode identifiers. Conditions were classified as primary screening targets or secondary findings, and consensus was reached on confirmatory biochemical and genetic testing pathways. The operational dataset comprised 48 biomarker-based screening entries. Because some conditions can be detected through multiple primary markers, duplicate analytical routes were retained but counted once, resulting in a total 189 unique clinical conditions, including 87 primary and 102 secondary conditions. The SUSP represents the first nationwide harmonized framework for newborn screening in Spain and constitutes a major step toward equitable and standardized implementation. It provides a scalable model that may inform other countries facing similar disparities.
Informed consent is fundamental to Australian newborn bloodspot screening (NBS), but emerging genomic screening technologies pose new challenges to clinical care and research. Optimizing consent processes is necessary to support ethical practice and maintain public trust as NBS evolves. This study aimed to identify gaps and opportunities to improve NBS consent processes in Queensland, Australia, while also exploring preliminary insights into the evolving complexity of consent in the context of genomic NBS (gNBS) and NBS-related research. A qualitative study design was used, with two facilitated interest-holder workshops (a total of 12 h) involving 86 participants (healthcare professionals, policy-makers, researchers, genomics experts, and consumer representatives). Workshop 1 (n = 25) was held virtually, and Workshop 2 (n = 61) was held in person. Thematic analysis was used to identify practical recommendations and ethical considerations. Participants identified three priority domains for improving consent in Queensland’s NBS program: (1) revision of the Guthrie Card and consent statement, (2) development of consistent, antenatal information resources across healthcare providers, and (3) standardized consent delivery training for healthcare staff. Discussions also highlighted tensions around information requirements for informed consent, revealing growing complexities regarding layered consent models. While recommendations on research consent were not fully developed at the workshop, insights highlighted the growing complexity and divergence of views on layered consent models. Findings suggest that improving NBS consent requires both operational reform and reassessment of ethical standards, alongside broader interest-holder engagement and feasible, scalable models also suited to genomic technologies. A nationally consistent framework is needed.
5q spinal muscular atrophy (5q-SMA) is a leading genetic cause of infant mortality. Presymptomatic intervention with disease-modifying therapies significantly improves motor outcomes, but effectiveness depends on early detection through newborn screening (NBS). Despite global 5q-SMA NBS expansion and recent Brazilian federal legislation, regional disparities and a lack of systematic monitoring hinder access to timely diagnosis and care. This study addresses these gaps by evaluating a statewide pilot program in São Paulo. We used multiplex real-time PCR to detect SMN1 exon 7 deletions in dried blood spots, confirming SMN1/2 copy numbers via MLPA in positive cases. Under real-world conditions, timeliness key performance indicators were evaluated to assess operational efficiency. 194,714 newborns were screened with 14 positive cases, yielding a prevalence of 1:13,908. First-tier results and treatment initiation occurred at a median of 10.8 and 28 days of life, respectively. Notably, 78.6% of patients had two SMN2 copies, of which approximately half were symptomatic by the first evaluation, highlighting the critical need for rapid screening to prevent irreversible motor decline. Screening achieved 100% specificity. This pilot demonstrates the feasibility of 5q-SMA NBS within the Brazilian public health system, providing essential evidence to overcome logistical and socioeconomic barriers and support nationwide expansion.
Newborn screening initiatives have the potential to mitigate childhood morbidity in Africa, but they also have special ethical, legal, and social implications (ELSI) that are influenced by issues with the health system, cultural diversity, and limited resources. This scoping review explores the ELSI of newborn screening across Africa to identify key challenges, gaps, and future research needs. A systematic search identified 27 peer-reviewed studies published between 2008 and 2025, covering 12 African countries. Data were extracted on study characteristics, disease types, and ELSI dimensions from African Journals Online (AJOL), Scopus, PubMed, Web of Science, and BMJ Journals. Thematic analysis mapped recurring ethical, legal, and social concerns. Most studies examined ethical and social dimensions, while legal frameworks were rarely addressed. South Africa, Tanzania, and Ghana contributed the largest number of publications. Sickle cell disease (52%) and hearing screening (30%) were the dominant foci. Common ethical issues included informed consent, privacy, and justice; legal gaps centered on the absence of data protection and frameworks; and social concerns involved stigma, awareness, and cultural perceptions of hereditary disease. Ethical and social issues dominate NBS discourse in Africa, whereas legal oversight remains limited. To guarantee fair, reliable, and long-lasting newborn screening programs, national policy guidelines, community involvement, and context-specific ethical frameworks must be strengthened.
Congenital Cytomegalovirus (cCMV) infection is the leading non-genetic cause of sensorineural hearing loss in newborns. Systematic nationwide screening programmes are lacking. Antiviral valganciclovir therapy could improve auditory outcomes if initiated within the first 30 days of life, making timely diagnosis crucial. To address this, we investigated whether a hearing screening-based protocol is suitable. Between 2015 and 2019, newborns, aged ≤21 days, with repeated abnormal newborn hearing screening (NHS) were prospectively enrolled at University Hospital Frankfurt. Oral mucosal swabs were tested for CMV DNA by real-time PCR, with confirmatory urine and blood diagnostics in positive cases. Of 2741 infants presenting for repeat NHS, 2059 (75.1%) showed normal bilateral findings. Of the 682 (24.9%) with abnormal results, 575 (84.3%) were aged >21 days and thus ineligible. A total of 107 infants (3.9%) met both criteria-abnormal NHS and aged ≤21 days-of whom 100 entered per-protocol analysis. Two (2%) were confirmed as cCMV-positive and received valganciclovir. Among the 48 infants who additionally underwent DBS testing, diagnostic sensitivity and specificity were 100%. The presented NHS-driven cCMV protocol reliably identified cCMV-infected newborns to offer timely antiviral therapy. In the absence of universal cCMV screening, this targeted approach offers a challenging but WHO screening-criteria-compliant strategy to enable timely antiviral intervention.
Congenital heart disease is the most common birth defect worldwide. Perinatal detection, either in utero or prior to hospital discharge is important for planning delivery, ordering adjunct testing, and planning for surgical or medical care. In countries with access to ultrasound screening, prenatal detection is highly accurate though with variable sensitivity and specificity around the world. Pulse oximetry screening, in some cases coupled with murmur auscultation in the nursery can also reach very high degrees of sensitivity for critical congenital heart disease. Use of the modalities per guidelines and recommendations is an important aspect of decreasing infant mortality and morbidity worldwide.
The early detection of Severe Combined Immunodeficiency (SCID) and X-linked agammaglobulinemia (XLA) prevents fatal outcomes. This study presents the first pilot TREC/KREC newborn screening (NBS) program in southern Kazakhstan, a high-birth-rate region, to establish local reference ranges and assess operational viability. A multiplex real-time PCR assay was used to quantify T-cell (TREC) and kappa-deleting recombination excision circles (KRECs) from dried blood spots of 5000 unselected neonates. Biomarkers were normalized to copies per 106 cells using albumin as a diploid reference gene. Regional 0.5th percentile cut-offs were established (TREC < 3165 copies/106 cells and KREC < 2554 copies/106 cells), and gender and gestational age did not significantly impact biomarker levels. While a low birth weight (≤2500 g) significantly reduced KREC levels, the extreme lower distribution tails remained unaffected, validating the use of universal, unstratified thresholds. Applying these cut-offs yielded an optimal 1.0% initial recall rate. Consistent with global incidence rates, no true positive cases were identified. The established assay and universal percentile cut-offs demonstrate high levels of analytical reliability and demographic stability. This pilot confirms the regional pediatric healthcare infrastructure’s readiness for a routine, population-based NBS program without the need for complex algorithms.
The peripheral perfusion index (PPI) is a non-invasive indicator of peripheral tissue perfusion in neonates. However, reference values for the PPI at different postnatal time points in healthy neonates residing at high altitudes, as well as early perfusion characteristics in infants with congenital heart disease (CHD) in these settings, remain inadequately defined. The primary aim was to determine the dynamic changes in and percentile-based reference values of the PPI in healthy neonates during the first 6-72 h after birth in the high-altitude region of Tibet. A secondary, exploratory aim was to observe PPI patterns in neonates with CHD born during the same period and to compare the two groups, given the limited number of CHD cases enrolled. A longitudinal cohort study was conducted among consecutively born neonates in a high-altitude region of Tibet. The PPI was measured in the right hand (pre-ductal) and either foot (post-ductal) at 6, ~12, ~24, ~48, and ~72 h after birth. Generalized Additive Models for Location, Scale and Shape (GAMLSS) were used to construct percentile reference curves for healthy neonates. Generalized Estimating Equations (GEE) were applied to evaluate differences in the PPI across time points, measurement sites, and between groups (healthy vs. CHD). A total of 1043 neonates were enrolled, including 1034 in the healthy group and only 9 CHD cases. In healthy neonates, the PPI increased progressively within the first 72 h, with a rapid rise from 6 to 24 h, a slower increase from 24 to 48 h, and stabilization between 48 and 72 h. At all time points, the lower-limb PPI was significantly higher than the upper-limb PPI (p < 0.001). The 50th percentile (P50) values for the upper-limb PPI at 6, 12, 24, 48, and 72 h were 2.32, 2.46, 2.62, 2.63, and 2.77, respectively; corresponding values for the lower-limb PPI were 2.53, 2.64, 2.76, 2.77, and 2.90. Compared to the healthy group, neonates with CHD at high altitudes had similar PPI at 6 h. However, their PPI was significantly lower between 12 and 48 h. At 72 h, no statistically significant difference was found between the groups, likely due to the small sample size of CHD infants. Conclusions: This study provides a reference for routine perfusion assessment in stable neonates born at high altitudes. Due to the limited sample size (only 9 CHD cases), our findings on PPI changes in neonates with CHD remain preliminary. The normative PPI percentile curves established in this study offer objective baseline perfusion data for stable neonates living in high-altitude areas. Future large-scale cohort studies are still needed to validate the clinical value of the PPI as a perfusion monitoring tool in infants with CHD.
Newborn screening benefits children with rare diseases by enabling early detection and intervention, which improves health outcomes. False-positive results are one harm of screening, with infants subject to further testing to resolve a positive screen and parents susceptible to psychosocial distress while awaiting the results. Newborn bloodspot screening (NBS) for severe combined immunodeficiency (SCID) using quantitative polymerase chain reaction (qPCR) and cystic fibrosis (CF) screening using immunoreactive trypsinogen (IRT) have high false-positive rates, with more than five infants screening positive and unaffected, for each child diagnosed. We used first-tier targeted gene sequencing (TGS) to prospectively screen 3025 newborns for CF, SCID and spinal muscular atrophy (SMA) and compared the results to current screening protocols using multiplex qPCR and IRT. TGS identified one infant with CF, missed by the IRT assay, and detected an infant with SMA, who screened positive with multiplex qPCR. TGS had zero false-positive results for SCID, CF and SMA. By contrast, multiplex qPCR and IRT screening protocols had approximately nine false positives for each child diagnosed with CF (positive predictive value, PPV 10.00%) and SCID (PPV 10.13%). Replacing current SCID and CF screening protocols with TGS would reduce NBS false positives and may result in a higher sensitivity for CF. TGS sensitivity and specificity would be comparable to qPCR for SMA. A larger trial is required to determine TGS sensitivity and cost-effectiveness.
Pulse oximetry screening (POS) is a simple non-invasive tool which enables the early detection of critical congenital heart defects (CCHDs). POS has moderate sensitivity and high specificity; it is cost-effective, readily accepted by parents and healthcare professionals, and its introduction significantly reduces mortality from CCHDs. As a result, most high-income countries and many middle-income countries have introduced, or are considering introducing, POS. Despite the international uptake of POS, there is still no consensus regarding the most appropriate screening algorithm, although several have been described. In addition, this review will consider the variations—and their relative advantages and disadvantages—between POS algorithms and also explore how modifications to existing algorithms—such as the addition of a perfusion index or the incorporation of machine learning—may have the potential to improve future detection of CCHDs.
Metachromatic leukodystrophy (MLD) is a rare, autosomal recessive lysosomal storage disorder caused by a deficiency of the enzyme arylsulfatase A (ARSA, MIM #250100), which leads to progressive demyelination in the central and peripheral nervous systems, resulting in severe neurodegeneration and premature death. With the recent approval of the first ex vivo gene therapy for MLD, newborn screening (NBS) programs have begun to implement pilot studies for early detection, as identifying affected individuals during the pre-symptomatic therapeutic window is crucial. We present the analytical workflow and the NBS algorithm developed during the first year of MLD screening in Lombardy. The screening strategy comprised a first-tier test (1TT) using mass spectrometry to quantify sulfatide concentrations, a second-tier test (2TT) measuring ARSA enzymatic activity, and a third-tier test (3TT) based on whole-exome sequencing. A total of 16,130 newborns were screened for MLD. Of these, 6.41% required retesting (1TT), 1.53% underwent duplicate 2TT, and 0.36% proceeded to 3TT. No neonates were recalled, and no cases of MLD were identified through the screening program. The first-year implementation of MLD NBS in Lombardy demonstrates that a mass spectrometry-based sulfatide assay combined with a multi-tiered screening algorithm is feasible and reliable. Incorporating genetic analysis alongside expanded validation of additional sulfatide species may enhance specificity, reduce false positives, and facilitate timely identification of infants affected by MLD.
Congenital cytomegalovirus infection (cCMV) is the most common congenital infection and an important cause of sensorineural hearing loss and neurodevelopmental impairment, yet many affected infants remain undetected under selective screening approaches. We conducted a prospective national pilot study to evaluate the feasibility and diagnostic yield of universal dried blood spot (DBS)-based screening for cCMV within the Slovenian newborn screening program. DBS samples collected within 72 h of life were tested by polymerase chain reaction (PCR), and screen-positive newborns underwent confirmatory urine PCR within 21 days together with standardized clinical evaluation. Among 5556 screened newborns, 13 (0.23%) screened positive and cCMV was confirmed in 10, corresponding to a lower-bound birth prevalence of 1.80 per 1000 live births (95% confidence interval, 0.98-3.31), because confirmatory testing was limited to DBS-positive newborns. None of the confirmed cases were clinically suspected at birth, and all passed newborn hearing screening. Six infants met protocol-defined criteria for symptomatic cCMV and received valganciclovir. Historical registry-based clinical case ascertainment in Slovenia corresponded to 0.09 detected cases per 1000 live births. These findings demonstrate the feasibility of universal DBS-based cCMV screening within an established newborn screening infrastructure and suggest substantial under-ascertainment under selective clinical detection pathways.
Enzyme replacement therapy (ERT) for central nervous system symptoms and newborn screening (NBS) is available in Japan for patients with mucopolysaccharidosis type II (MPS II). Of 12 suspected cases identified through the NBS program, 3 patients were diagnosed with neuronopathic MPS II (2 directly from the screened cohort and 1 affected sibling). We reviewed the clinical courses of these patients, who were treated with intracerebroventricular (ICV) ERT using idursulfase beta (Hunterase®) followed by umbilical cord blood transplantation (CBT). Heparan sulfate (HS) in the cerebrospinal fluid (CSF) was longitudinally measured as a therapeutic biomarker, and developmental age was evaluated. All patients achieved successful engraftment with no severe complications, except for one patient with sinusoidal obstruction syndrome. The CSF HS concentration showed a temporary increase during the ERT discontinuation period, which can be attributed to CBT, and a subsequent reduction after the resumption of ICV-ERT. The patients exhibited age-appropriate development. The pattern of change in HS indicates the importance of continuing ICV-ERT even after hematopoietic stem cell transplantation. The study results demonstrate that the combination of ICV-ERT and CBT may yield promising outcomes in patients with neuronopathic MPS II, underscoring the importance of early intervention through NBS.
The Ethics Session of the International Primary Immunodeficiency Congress (IPIC), held in November 2025 and organised by the International Patient Organisation for Primary Immunodeficiencies (IPOPI), examined one of the most challenging developments emerging from genomic-enhanced newborn screening: the identification of serious, non-treatable disorders as incidental detection in programmes originally designed to detect severe combined immunodeficiency (SCID). Using a fictionalised clinical scenario based on the recent literature, the session explored the early diagnosis of ataxia-telangiectasia (AT) detected through TREC-based screening. The discussion highlighted the clinical value and psychological risks associated with presymptomatic detection, the persistent shortcomings in parental consent processes, the systemic pressures created by expanding genomic testing, and the ethical challenges surrounding the reporting and management of incidental findings in screening. The debate underscored the need for internationally coordinated frameworks to guide the management of incidental detection in newborn screening as genomic technologies become more deeply embedded in routine public health practice.
Newborn screening in England is a national program with laboratories adhering to common screening algorithms. Until recently, screening for inherited metabolic disorders was provided by ten laboratories using laboratory-developed tests (LDTs) and three using commercial assays: harmonization of results proved challenging. Introduction of hereditary tyrosinemia type 1 screening meant LDTs required modification to include the measurement of succinylacetone, and subsequent re-validation. This provided an opportunity to implement a single commercial reagent kit in all laboratories. It was anticipated that this would improve analytical performance and harmonization. This study aimed to determine whether these goals were achieved. Verification across the 13 laboratories revealed that the commercial kit reduced inter-laboratory variation for all analytes demonstrating improved harmonization. However, this was achieved by applying instrument-specific correction factors to all analytes, the magnitude of which were significant, indicating a lack of standardization. Performance of succinylacetone was limited by instrument-dependent background interference from the methionine stable isotope label, underscoring the need to establish evidence-based screening cut-off values (COV) rather than adopting published thresholds. This study emphasizes the need for traceable reference materials to improve laboratory quality and the value of screening outcome data.
Lysosomal diseases (LDs, or Lysosomal Storage Disorders) have become increasingly visible in the newborn screening community, with the addition of mucopolysaccharidosis type II (MPS-II) into the Recommended Uniform Screening Panel in August 2022 and Infantile Krabbe disease in June 2024. As more LDs are expected to be considered for screening adoption, the ability to multiplex conditions and expand proficiency testing (PT) using quality control materials is essential. This study examines the use of recombinant enzymes to produce first-tier PT materials for mucopolysaccharidosis type I, MPS-II, Gaucher, Fabry, Krabbe, Pompe, and Niemann-Pick A/B (acid sphingomyelinase deficiency)-adding four disorders to the CDC's Newborn Screening Quality Assurance Program (NSQAP) LD PT panel. Through an iterative process that included two prototype phases, two pilot phases, and external testing by up to 31 external laboratories, a new manufacturing process was developed for producing high-performing dried blood spot-based LD PT specimens. Materials were evaluated using several methods commonly employed by newborn screening laboratories, including tandem mass spectrometry with flow injection and liquid chromatography, digital microfluidics, and fluorometric assays. This novel process for producing LD PT materials offers several advantages over previous manufacturing methods that relied on immortalized cell lines from affected patients. Improved scalability, for example, has enabled NSQAP to expand LD PT enrollment internationally. Furthermore, the new process makes it easier to support future expansions of the LD screening panel. The updated specimens and expanded program were launched in January 2025.
Urea cycle disorders (UCDs) are rare inherited metabolic diseases associated with toxic hyperammonemia, leading to severe neurological damage and early mortality. Early diagnosis of distal UCDs through newborn screening (NBS) enables presymptomatic intervention; however, comparative real-world outcome data remain limited. We conducted a retrospective, multicenter study using data from the Spanish UCD Registry to describe the clinical characteristics and compare health outcomes between patients diagnosed through NBS (n = 40) and those diagnosed after clinical presentation (n = 53). Patients identified by NBS showed a markedly more favorable clinical prognosis, with a mortality rate of 2.5% compared with 15.1% in the unscreened cohort, as well as significantly lower rates of neurological involvement, fewer hospital admissions due to metabolic decompensation, and a reduced need for liver transplantation. Screening also identified a high prevalence of argininosuccinate synthetase deficiency (ASS1D) cases with attenuated biochemical profiles, highlighting the relevance of sensitive screening cutoffs. These findings provide real-world evidence that presymptomatic diagnosis through NBS is associated with improved survival and long-term neurological outcomes in patients with distal UCDs.
