Int. J. Neonatal Screen., Volume 9, Issue 3 (September 2023) – 19 articles

The collection of dried blood spots (DBS) facilitates newborn screening for a variety of rare, but very serious conditions in healthcare systems around the world. Sub-punches of varying sizes (1.5–6 mm) can be taken from DBS specimens to use as inputs for a range of biochemical assays. Advances in DNA sequencing workflows allow whole-genome sequencing (WGS) libraries to be generated directly from inputs such as peripheral blood, saliva, and DBS. We compared WGS metrics obtained from libraries generated directly from DBS to those generated from DNA extracted from peripheral blood, the standard input for this type of assay. We explored the flexibility of DBS as an input for WGS by altering the punch number and size as inputs to the assay. We showed that WGS libraries can be successfully generated from a variety of DBS inputs, including a single 3 mm or 6 mm diameter punch, with equivalent data quality observed across a number of key metrics of importance in the detection of gene variants. We observed no difference in the performance of DBS and peripheral-blood-extracted DNA in the detection of likely pathogenic gene variants in samples taken from individuals with cystic fibrosis or phenylketonuria. WGS can be performed directly from DBS and is a powerful method for the rapid discovery of clinically relevant, disease-causing gene variants. Full article

Newborn screening (NBS) for X-linked adrenoleukodystrophy (ALD) can identify affected individuals before the onset of life-threatening manifestations. Some countries have decided to only screen boys (sex-specific screening). This study investigates the attitudes of individuals with ALD towards sex-specific NBS for ALD. A questionnaire was sent to all patients in the Dutch ALD cohort. Invitees were asked who they thought should be screened for ALD: only boys, both boys and girls or neither. The motives and background characteristics of respondents were compared between screening preferences. Out of 108 invitees, 66 participants (61%), 38 men and 28 women, participated in this study. The majority (n = 53, 80%) favored screening both newborn boys and girls for ALD, while 20% preferred boys only. None of the respondents felt that newborns should not be screened for ALD. There were no differences in the background characteristics of the respondents between screening preferences. Our study revealed a diverse range of motivations underlying respondents’ screening preferences. This study is one of the first to investigate the attitudes of patients towards sex-specific screening for ALD. The outcomes of this study can offer insights to stakeholders engaged in the implementation of NBS programs. ALD patients are important stakeholders who can provide valuable input in this process. Full article

Screening for congenital adrenal hyperplasia (CAH) remains heterogenous across geographies—we sought to determine the proportion of non-classical CAH (NCAH) detection by one vs. two newborn screens (NBS) in two U.S. regions. Data were collected at tertiary centers in Houston (HOU) and Los Angeles (LA) on 35 patients with NCAH, comparing patients identified via the NBS vs. during childhood, 17-hydroxyprogesterone (17-OHP) levels, genotype, and phenotype. The NBS filter-paper 17-OHP levels and daily cutoffs were recorded on initial and second screens. In all, 53% of patients with NCAH in the HOU cohort were identified as infants via the second NBS. Patients identified clinically later in childhood presented at a similar age (HOU: n = 9, 5.5 ± 3.1 years; LA: n = 18, 7.9 ± 4 years) with premature pubarche in almost all. Patients in LA had more virilized phenotypes involving clitoromegaly and precocious puberty and were older at treatment onset compared with those identified in HOU by the second NBS (HOU: 3.2 ± 3.9 years; LA: 7.9 ± 4.0 years, p = 0.02). We conclude that the early detection of NCAH could prevent hyperandrogenism and its adverse consequences, with half of the cases in HOU detected via a second NBS. Further studies of genotyping and costs are merited. Full article

The study objective was to identify communication messages that parents of children diagnosed with congenital cytomegalovirus (cCMV) infection reported as essential and helpful. We performed a secondary analysis of focus groups and interviews conducted with 41 parents of children with cCMV who had enrolled in a long-term follow-up cCMV study at an academic medical center. Three groups of parents who had children with cCMV participated in the study: parents with children symptomatic at birth, parents with children asymptomatic at birth who later developed sensorineural hearing loss, and parents with children asymptomatic at birth who remained asymptomatic into adulthood. Using a health marketing approach, we identified six general themes from the focus group sessions: initial diagnosis, likely health outcome(s), comfort and coping, symptom watch, resources, and prevention. Receiving the initial diagnosis was shocking for many parents, and they wanted to know how their child would or could be affected. They valued access to the information, follow-up visits for early detection of hearing loss and other developmental delays, and support from other parents. Parents wished to obtain this information from their pediatrician but felt that experts offered more up-to-date knowledge about prognosis, monitoring, and treatment. With more U.S. states implementing cCMV screening strategies which would lead to more infant diagnoses, it will be necessary for providers to meet parents’ expectations and communication needs. Full article

Long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) and mitochondrial trifunctional protein (MTP) deficiencies are rare fatal disorders of fatty acid β-oxidation with no apparent genotype–phenotype correlation. The measurement of acylcarnitines by MS/MS is a current diagnostic workup in these disorders. Nevertheless, false-positive and false-negative results have been reported, highlighting a necessity for more sensitive and specific biomarkers. This study included 54 patients with LCHAD/MTP deficiency that has been confirmed by biochemical and molecular methods. The analysis of acylcarnitines in dried blood spots was performed using ESI-MS/MS. The established “HADHA ratio” = (C16OH + C18OH + C18:1OH)/C0 was significantly elevated in all 54 affected individuals in comparison to the control group. Apart from 54 LCHAD deficiency patients, the “HADHA ratio” was calculated in 19 patients with very-long-chain acyl-CoA dehydrogenase (VLCAD) deficiency. As VLCAD-deficient patients did not show increased “HADHA ratio”, the results emphasized the high specificity of this new ratio. Therefore, the “HADHA ratio” was shown to be instrumental in improving the overall performance of MS/MS-based analysis of acylcarnitine levels in the diagnostics of LCHAD/MTP deficiencies. The ratio was demonstrated to increase the sensitivity and specificity of this method and reduce the chances of false-negative results. Full article

The aim of this study is to evaluate the strategy of the cystic fibrosis newborn screening (CFNBS) programme in Hungary based on the results of the first year of screening. A combined immunoreactive trypsinogen (IRT) and pancreatitis-associated protein (PAP) CFNBS protocol (IRT/IRT×PAP/IRT) was applied with an IRT-dependent safety net (SN). Out of 88,400 newborns, 256 were tested screen-positive. Fourteen cystic fibrosis (CF) and two cystic fibrosis-positive inconclusive diagnosis (CFSPID) cases were confirmed from the screen-positive cases, and two false-negative cases were diagnosed later. Based on the obtained results, a sensitivity of 88% and a positive predictive value (PPV) of 5.9% were calculated. Following the recognition of false-negative cases, the calculation method of the age-dependent cut-off was changed. In purely biochemical CFNBS protocols, a small protocol change, even after a short period, can have a significant positive impact on the performance. CFNBS should be monitored continuously in order to fine-tune the screening strategy and define the best local practices. Full article

A universal screening research study was conducted in six hospitals to identify the clinical sensitivity of polymerase chain reaction (PCR) testing on newborn dried blood spots (DBSs) versus saliva specimens for the diagnosis of congenital cytomegalovirus (cCMV). CMV DNA positive results from DBSs or saliva were confirmed with urine testing. Findings of several false-positive (FP) saliva PCR results prompted an examination of a possible association with donor milk. Documentation of the frequency of positive saliva results, including both true-positive (TP) and FP status from clinical confirmation, occurred. The frequency of donor milk use was compared for TP and FP cases. Of 22,079 participants tested between 2016 and 2022, 96 had positive saliva results, 15 were determined to be FP, 79 TP, and 2 were excluded for incomplete clinical evaluation. Newborn donor milk use was identified for 18 (19.14%) of all the positive saliva screens. Among the 15 FPs, 11 (73.33%) consumed donor milk compared to 7 of the 79 TPs (8.8%) (OR 28.29, 95% CI 7.10–112.73, p < 0.001). While milk bank Holder pasteurization inactivates CMV infectivity, CMV DNA may still be detectable. Due to this possible association, screening programs that undertake testing saliva for CMV DNA may benefit from documenting donor milk use as a potential increased risk for FP results. Full article

Dried blood spot (DBS) cards from newborn screening (NBS) programs represent a wealth of biological data. They can be stored easily for a long time, have the potential to support medical and public health research, and have secondary usages such as quality assurance and forensics, making it the ideal candidate for bio-banking. However, worldwide policies vary with regard to the duration of storage of DBS cards and how it can be used. Recent advances in genomics have also made it possible to perform extended genetic testing on DBS cards in the newborn period to diagnose both actionable and non-actionable childhood and adult diseases. Both storage and secondary uses of DBS cards raise many ethical, clinical, and social questions. The openness of the key stakeholders, namely, parents and healthcare providers (HCPs), to store the DBS cards, and for what duration and purposes, and to extended genetic testing is largely dependent on local cultural–social-specific factors. The study objective is to assess the parents’ and HCPs’ awareness and receptivity toward DBS retention, its secondary usage, and extended genetic testing. A cross-sectional, self-administrated survey was adopted at three hospitals, out of which two were public hospitals with maternity services, between June and December 2022. In total, 452 parents and 107 HCPs completed and returned the survey. Overall, both HCPs and parents were largely knowledgeable about the potential benefits of DBS card storage for a prolonged period and its secondary uses, and they supported extended genetic testing. Knowledge gaps were found in respondents with a lower education level who did not know that a DBS card could be stored for an extended period (p < 0.001), could support scientific research (p = 0.033), and could aid public health research, and future policy implementation (p = 0.030). Main concerns with regard to DBS card storage related to potential privacy breaches and anonymity (Parents 70%, HCPs 60%). More parents, compared to HCPs, believed that storing DBS cards for secondary research does not lead to a reciprocal benefit to the child (p < 0.005). Regarding extended genetic testing, both groups were receptive and wanted to know about actionable childhood- and adult-onset diseases. More parents (four-fifths) rather than HCPs (three-fifths) were interested in learning about a variant with unknown significance (p < 0.001). Our findings report positive support from both parents and HCPs toward the extended retention of DBS cards for secondary usage and for extended genetic testing. However, more efforts to raise awareness need to be undertaken in addition to addressing the ethical concerns of both parents and HCPs to pave the way forward toward policy-making for DBS bio-banking and extended genetic testing in Hong Kong. Full article

In this retrospective study, we aimed to evaluate the performance of dried-blood-spot (DBS) testing as a diagnostic method for the congenital cytomegalovirus (cCMV). We reviewed the medical records and DBS test results of 89 patients who had also undergone diagnostic cCMV testing within the first 21 days of life. The DBS test had a sensitivity of 83.9%, a specificity of 100%, a positive predictive value of 100%, and a negative predictive value of 73%. Patients with a true-positive DBS had a higher median level of CMV in blood according to PCR than those with a false-negative result. Additionally, all patients with cCMV and hearing loss had a positive DBS test, with higher median viremia levels observed in those with hearing loss compared to those without a CMV PCR blood test. These results suggest that DBS-based testing is useful in the diagnosis of cCMV, and its performance may be related to levels of CMV viremia. DBS testing accurately identified those patients with congenital/early onset hearing loss and those at risk of developing late-onset hearing loss. Full article

To investigate COVID-19 surveillance among pregnant women, the California Genetic Disease Screening Program conducted a screening performance and seroprevalence evaluation of maternal SARS-CoV-2 antibodies detected in banked newborn dried blood spots (DBS). We obtained seropositive results for 2890 newborn DBS from cohorts in 2020 and 2021 using Enable Bioscience’s Antibody Detection by Agglutination-PCR (ADAP) assay for SARS-CoV-2 antibodies. To infer maternal infection, we linked 312 women with a known laboratory-confirmed COVID-19 episode with their newborn’s DBS SARS-CoV-2 antibody result. Among 2890 newborns, we detected 453 (15.7%) with SARS-CoV-2 antibodies in their DBS. Monthly snapshot statewide seroprevalence among neonates was 12.2% (95% CI 10.3–14.1%, n =1156) in December 2020 and 33.3% (95% CI 29.1–37.4%, n = 26) in March 2021. The longest time recorded from COVID-19 infection to a seropositive neonatal result was 11.7 months among the 312 mothers who had an available SARS-CoV-2 PCR test result. Approximately 94% (153/163) of DBS were seropositive when a known maternal infection occurred earlier than 19 days before birth. The estimated relative sensitivity of DBS to identify prevalent maternal infection was 85.1%, specificity 98.5% and PPV 99.2% (n = 312); the sensitivity was lowest during the December 2021 surge when many infections occurred within 19 days of birth. Fifty pre-pandemic specimens (100% seronegative) and 23 twin-pair results (100% concordant) support an intrinsic specificity and PPV of ADAP approaching 100%. Maternal infection surveillance is limited by a time lag prior to delivery, especially during pandemic surges. Full article

Spinal muscular atrophy (SMA) is a progressive neuromuscular disease caused by biallelic pathogenic/likely pathogenic variants of the survival motor neuron 1 (SMN1) gene. Early diagnosis via newborn screening (NBS) and pre-symptomatic treatment are essential to optimize health outcomes for affected individuals. We developed a multiplex quantitative polymerase chain reaction (qPCR) assay using dried blood spot (DBS) samples for the detection of homozygous absence of exon 7 of the SMN1 gene. Newborns who screened positive were seen urgently for clinical evaluation. Confirmatory testing by multiplex ligation-dependent probe amplification (MLPA) revealed SMN1 and SMN2 gene copy numbers. Six newborns had abnormal screen results among 47,005 newborns screened during the first year and five were subsequently confirmed to have SMA. Four of the infants received SMN1 gene replacement therapy under 30 days of age. One infant received an SMN2 splicing modulator due to high maternally transferred AAV9 neutralizing antibodies (NAb), followed by gene therapy at 3 months of age when the NAb returned negative in the infant. Early data show that all five infants made excellent developmental progress. Based on one year of data, the incidence of SMA in Alberta was estimated to be 1 per 9401 live births. Full article

The sweat test (ST) is the current diagnostic gold standard for cystic fibrosis (CF). Many CF centres have switched from the Gibson–Cooke method to the Macroduct system-based method. We used these methods simultaneously to compare CF screening outcomes. STs using both methods were performed simultaneously between March and December 2022 at CF Centre in Florence. We included newborns who underwent newborn bloodspot screening (NBS), newborns undergoing transfusion immediately after birth, and children with CF screen-positive, inconclusive diagnosis (CFSPID). We assessed 72 subjects (median age 4.4 months; range 0–76.7): 30 (41.7%) NBS-positive, 18 (25.0%) newborns who underwent transfusion, and 24 (33.3%) children with CFSPID. No significant differences were found between valid sample numbers, by patient ages and groups (p = 0.10) and between chloride concentrations (p = 0.13), except for sweat chloride (SC) measured by the Gibson–Cooke and Macroduct methods in CFSPID group (29.0, IQR: 20.0–48.0 and 22.5, IQR: 15.5–30.8, respectively; p = 0.01). The Macroduct and Gibson–Cooke methods showed substantial agreement with the SC values, except for CFSPID, whose result may depend on the method of sweat collection. In case of invalid values with Macroduct, the test should be repeated with Gibson–Cooke method. Full article

Targeted screening for congenital CMV infection (cCMV), which entails CMV testing of infants who fail newborn hearing screening (NBHS), has become common practice. However, this strategy misses nearly all infected infants with normal hearing at birth who are nonetheless at high risk of subsequent hearing loss and would benefit from timely cCMV diagnosis. The objective of this study was to identify expanded criteria predictive of cCMV to increase the scope and utility of targeted newborn CMV screening. In this retrospective study, 465 newborns were tested for cCMV at a single tertiary care center with a targeted screening program between 2014 and 2018. Twenty-two infants were diagnosed with cCMV, representing 0.2% of the 12,189 births over this period and 4.7% of the infants tested. The highest prevalence of cCMV infection was among infants tested because of primary maternal CMV infection (8/42, 19%), followed by failed initial NBHS (10/88, 11.4%), maternal HIV infection (3/137, 2.2%), and clinical suspicion alone (5/232, 2.2%). The symptoms with the highest prevalence of infection among all infants tested included an enlarged liver and/or spleen (33.3%) (3/9), followed by petechiae (33.3%), microcephaly (9.4%), direct hyperbilirubinemia (7.7%), thrombocytopenia (6%), and growth impairment (4.3%). In addition to CMV screening of newborns who fail the NBHS, these data suggest that certain clinical signs of cCMV—in particular: thrombocytopenia, growth impairment, and HIV exposure in pregnancy—should be additional criteria for expanded targeted newborn CMV screening, where universal screening is not yet the standard of care. Full article

Whether or not conditions should be included in publicly funded newborn screening (NBS) programs should be discussed according to objective and transparent criteria. Certain criteria have been developed for the introduction of NBS programs in the context of individual countries; however, there are no standard selection criteria for NBS programs in Japan. This study aimed to develop a quantitative scoring model to assess newborn screening that incorporates the views of a variety of stakeholders in Japan. The five recommended eligibility criteria for NBS were stratified based on previous studies and expert opinions, using the analytic hierarchy process. We conducted a cross-sectional, web-based questionnaire targeting a wide range of people involved in NBS to investigate pairwise comparisons of the evaluation items between February and April of 2022. There were 143 respondents. Most of our respondents (44.1%) were physicians. Fifty-eight respondents (40.6%) had been engaged in NBS-related research or work for more than 10 years. The distribution of allocation points was the highest for ‘intervention’, ‘screening test’, ‘follow-up setting’, ’economic evaluation’, and ’disease/condition’, in that order. The algorithm in this study will guide decision makers in collecting and evaluating objective data, thus enabling transparent discussions to occur. Full article

The assumption of this study is strictly connected to the need to focus and to know more about the impact on the psychological state of the parents whose newborn babies get a positive result at Expanded Newborn Screening (ENS). As clinical experience shows us, this aspect seems to have a potentially lasting resonance on the way the disease will be managed and handled in the family, leading to potential negative effects and repercussions on the child’s wellbeing and on the quality of life within the family. On the basis of this and on the evidence emerging from a review of the literature, this study aims to investigate and objectify possible distress indicators elicited at the moment of the communication of a positive result at ENS. Questionnaires containing the Beck Depression Inventory-II, the State-Trait Anxiety Inventory-Y, and the Short Form 36 Health Survey tests were administered to the parents of 87 newborns who received positive results at ENS. The parents of 32 babies expressed the presence of discomfort potentially related to the communication of a positive result at ENS. Full article

Cytomegalovirus (CMV) infection during pregnancy may result in long-term health problems for children with congenital CMV (cCMV). Currently, no prevention or treatment interventions are approved by the Food and Drug Administration for a cCMV indication. Healthcare provider and public awareness is low, and formal clinical practice guidelines and local practice patterns vary. A pilot study of eight cCMV experts was performed using qualitative semi-structured interviews to better understand clinical practice guidelines and patterns in the United States. Results from participant interviews highlighted the need for better prenatal diagnostic techniques, broader neonatal screening opportunities, and more robust evidence supporting intervention strategies. Healthcare provider and public partnerships are essential for advancing cCMV guidelines and improving care delivery. Our results provide a preliminary knowledge base and framework for developing a consensus cCMV research agenda to address evidence gaps that limit the revision of clinical practice guidelines. The changes in clinical practice patterns that may arise as a result of further research have the potential to reduce risk during pregnancy and improve care for children with cCMV infection. Full article

Genomic sequencing offers an unprecedented opportunity to detect inherited variants that are implicated in rare Mendelian disorders, yet there are many challenges to overcome before this technology can routinely be applied in the healthy population. The age-based genomic screening (ABGS) approach is a novel alternative to genome-scale sequencing at birth that aims to provide highly actionable genetic information to parents over the course of their child’s routine health care. ABGS utilizes an established metric to identify conditions with high clinical actionability and incorporates information about the age of onset and age of intervention to determine the optimal time to screen for any given condition. Ongoing partnerships with parents and providers are instrumental to the co-creation of educational resources and strategies to address potential implementation barriers. Implementation science frameworks and informative empirical data are used to evaluate strategies to establish this unique clinical application of targeted genomic sequencing. Ultimately, a pilot project conducted in primary care pediatrics clinics will assess patient and implementation outcomes, parent and provider perspectives, and the feasibility of ABGS. A validated, stakeholder-informed, and practical ABGS program will include hundreds of conditions that are actionable during infancy and childhood, setting the stage for a longitudinal implementation that can assess clinical and health economic outcomes. Full article

Newborn screening using dried blood spots (NBS) is widely acknowledged as a highly successful procedure in secondary prevention. For a number of congenital disorders, severe disability or death are impressively prevented by early detection and early treatment through NBS. However, as with any other screening, NBS can also cause harm, and the principle that “the overall benefits of screening should outweigh the harms” must be considered when introducing and implementing NBS programmes. This publication compiles the results of a systematic literature research on requirements for NBS infrastructure and procedures which was conducted as part of a research project on the quality and shortcomings of the NBS pathway in Germany. The compilation contains the requirements and recommendations for realising the principle of “maximise benefits and minimise harms” in relevant NBS pathway components such as parental education and information, coverage, timeliness, laboratory quality assurance, follow-up of abnormal results, confirmatory diagnostics, documentation, and evaluation. The results reflect the complexity of NBS infrastructure, and thus, they illustrate the importance of considering and implementing NBS as a well-coordinated public health programme with continuous quality management. Special attention should be paid to the perspectives of parents and families. Some NBS issues can substantially benefit from digital instruments or international cooperation. The literature review presented here has contributed to a concept of proposals for the advancement of NBS in Germany, and despite different settings, it may as well be of interest for other countries to achieve the best possible course and outcome of NBS for each child. Full article

Residual heel prick Dried Blood Spots (DBS) are valuable samples for retrospective investigation of inborn metabolic diseases (IMD) and biomarker analyses. Because many metabolites suffer time-dependent decay, we investigated the five-year stability of amino acids (AA) in residual heel prick DBS. In 2019/2020, we analyzed 23 AAs in 2170 residual heel prick DBS from the Dutch neonatal screening program, stored from 2013–2017 (one year at +4 °C and four years at room temperature), using liquid chromatography mass-spectrometry. Stability was assessed by AA changes over the five years. Hydroxyproline could not be measured accurately and was not further assessed. Concentrations of 19 out of the remaining 22 AAs degraded significantly, ranked from most to least stable: aspartate, isoleucine, proline, valine, leucine, tyrosine, alanine, phenylalanine, threonine, citrulline, glutamate, serine, ornithine, glycine, asparagine, lysine, taurine, tryptophan and glutamine. Arginine, histidine and methionine concentrations were below the limit of detection and were likely to have been degraded within the first year of storage. AAs in residual heel prick DBS stored at room temperature are subject to substantial degradation, which may cause incorrect interpretation of test results for retrospective biomarker studies and IMD diagnostics. Therefore, retrospective analysis of heel prick blood should be done in comparison to similarly stored heel prick blood from controls. Full article