Newborn screening for aromatic l-amino acid decarboxylase deficiency - Strategies, results, and implication for prevalence calculations.

BACKGROUND: Aromatic l-amino acid decarboxylase deficiency (AADCD) is a rare, autosomal-recessive neurometabolic disorder caused by variants in dopa decarboxylase (DDC) gene, resulting in a severe combined deficiency of serotonin, dopamine, norepinephrine, and epinephrine. Birth prevalence of AADCD varies by population. In pilot studies, 3-O-methyldopa (3-OMD) was shown to be a reliable biomarker for AADCD in high-throughput newborn screening (NBS) allowing an early diagnosis and access to gene therapy. To evaluate the usefulness of this method for routine NBS, 3-OMD screening results from the largest three German NBS centers were analyzed. METHODS: A prospective, multicenter (n = 3) NBS pilot study evaluated screening for AADCD by quantifying 3-OMD in dried blood spots (DBS) using tandem mass spectrometry (MS/MS). RESULTS: In total, 766,660 neonates were screened from January 2021 until June 2023 with 766,647 with unremarkable AADCD NBS (766,443 by 1st-tier analysis and 204 by 2nd-tier analysis) and 13 with positive NBS result recalled for confirmatory diagnostics (recall-rate about 1:59,000). Molecular genetic analysis confirmed AADCD (c.79C > T p.[Arg27Cys] in Exon 2 und c.215 A > C p.[His72Pro] in Exon 3) in one infant. Another individual was highly suspected with AADCD but died before confirmation (overall positive predictive value 0.15). False-positive results were caused by maternal L-Dopa use (n = 2) and prematurity (30th and 36th week of gestation, n = 2). However, in 63% (n = 7) the underlying etiology for false positive results remained unexplained. Estimated birth prevalence (95% confidence interval) was 1:766,660 (95% CI 1:775,194; 1:769,231) to 1:383,330 (95% CI 1:384,615; 1:383,142). The identified child remained asymptomatic until last follow up at the age of 9 months. CONCLUSIONS: The proposed screening strategy with 3-OMD detection in DBS is feasible and effective to identify individuals with AADCD. The estimated birth prevalence supports earlier estimations and confirms AADCD as a very rare disorder. Pre-symptomatic identification by NBS allows a disease severity adapted drug support to diminish clinical complications until individuals are old enough for the application of the gene therapy.

High Throughput Newborn Screening for Sickle Cell Disease - Application of Two-Tiered Testing with a qPCR-Based Primary screen.

BACKGROUND: Sickle cell disease (SCD) is a group of hemoglobinopathies with a common point mutation causing the production of sickle cell hemoglobin (HbS). In high-throughput newborn screening (NBS) for SCD, a two-step procedure is suitable, in which qPCR first pre-selects relevant samples that are differentiated by a second method. METHODS: Three NBS centers using qPCR-based primary screening for SCD performed a laboratory comparison. Methods using tandem MS or HPLC were used for differentiation. RESULTS: In a benchmarking test, 450 dried blood samples were analyzed. Samples containing HbS were detected as reliably by qPCR as by methods established for hemoglobinopathy testing. In a two-step screening approach, the 2nd-tier-analyses have to distinguish the carrier status from pathological variants. In nine months of regular screening, a total of 353,219 samples were analyzed using two-stage NBS procedures. The 1st-tier screening by qPCR reduced the number of samples for subsequent differentiation by>99.5%. Cases with carrier status or other variants were identified as inconspicuous while 78 cases with SCD were revealed. The derived incidence of 1:4,773, is in good agreement with previously published incidences. CONCLUSION: In high-throughput NBS for SCD, qPCR is suitable to focus 2nd-tier analyses on samples containing HbS, while being unaffected by factors such as prematurity or transfusions. The substantial reduction of samples numbers positively impacts resource conservation, sustainability, and cost-effectiveness. No false negative cases came to attention.

Ten rapid antigen tests for SARS-CoV-2 widely differ in their ability to detect Omicron-BA.4 and -BA.5.

Since late 2021, the variant landscape of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been dominated by the variant of concern (VoC) Omicron and its sublineages. We and others have shown that the detection of Omicron-BA.1 and -BA.2-positive respiratory specimens by rapid antigen tests (RATs) is impaired compared to Delta VoC-containing samples. Here, in a single-center retrospective laboratory study, we evaluated the performance of ten most commonly used RATs for the detection of Omicron-BA.4 and -BA.5 infections. We used 171 respiratory swab specimens from SARS-CoV-2 RNA-positive patients, of which 71 were classified as BA.4 and 100 as BA.5. All swabs were collected between July and September 2022. 50 SARS-CoV-2 PCR-negative samples from healthy individuals, collected in October 2022, showed high specificity in 9 out of 10 RATs. When assessing analytical sensitivity using clinical specimens, the 50% limit of detection (LoD50) ranged from 7.6 × 104 to 3.3 × 106 RNA copies subjected to the RATs for BA.4 compared to 6.8 × 104 to 3.0 × 106 for BA.5. Overall, intra-assay differences for the detection of these two Omicron subvariants were not significant for both respiratory swabs and tissue culture-expanded virus isolates. In contrast, marked heterogeneity was observed among the ten RATs: to be positive in these point-of-care tests, up to 443-fold (BA.4) and up to 56-fold (BA.5) higher viral loads were required for the worst performing RAT compared to the best performing RAT. True-positive rates for Omicron-BA.4- or -BA.5-containing specimens in the highest viral load category (Ct values < 25) ranged from 94.3 to 34.3%, dropping to 25.6 to 0% for samples with intermediate Ct values (25-30). We conclude that the high heterogeneity in the performance of commonly used RATs remains a challenge for the general public to obtain reliable results in the evolving Omicron subvariant-driven pandemic.

Automated antigen assays display a high heterogeneity for the detection of SARS-CoV-2 variants of concern, including several Omicron sublineages.

Diagnostic tests for direct pathogen detection have been instrumental to contain the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) pandemic. Automated, quantitative, laboratory-based nucleocapsid antigen (Ag) tests for SARS-CoV-2 have been launched alongside nucleic acid-based test systems and point-of-care (POC) lateral-flow Ag tests. Here, we evaluated four commercial Ag tests on automated platforms for the detection of different sublineages of the SARS-CoV-2 Omicron variant of concern (VoC) (B.1.1.529) in comparison with "non-Omicron" VoCs. A total of 203 Omicron PCR-positive respiratory swabs (53 BA.1, 48 BA.2, 23 BQ.1, 39 XBB.1.5 and 40 other subvariants) from the period February to March 2022 and from March 2023 were examined. In addition, tissue culture-expanded clinical isolates of Delta (B.1.617.2), Omicron-BA.1, -BF.7, -BN.1 and -BQ.1 were studied. These results were compared to previously reported data from 107 clinical "non-Omicron" samples from the end of the second pandemic wave (February to March 2021) as well as cell culture-derived samples of wildtype (wt) EU-1 (B.1.177), Alpha VoC (B.1.1.7) and Beta VoC (B.1.351)). All four commercial Ag tests were able to detect at least 90.9% of Omicron-containing samples with high viral loads (Ct < 25). The rates of true-positive test results for BA.1/BA.2-positive samples with intermediate viral loads (Ct 25-30) ranged between 6.7% and 100.0%, while they dropped to 0 to 15.4% for samples with low Ct values (> 30). This heterogeneity was reflected also by the tests' 50%-limit of detection (LoD50) values ranging from 44,444 to 1,866,900 Geq/ml. Respiratory samples containing Omicron-BQ.1/XBB.1.5 or other Omicron subvariants that emerged in 2023 were detected with enormous heterogeneity (0 to 100%) for the intermediate and low viral load ranges with LoD50 values between 23,019 and 1,152,048 Geq/ml. In contrast, detection of "non-Omicron" samples was more sensitive, scoring positive in 35 to 100% for the intermediate and 1.3 to 32.9% of cases for the low viral loads, respectively, corresponding to LoD50 values ranging from 6181 to 749,792 Geq/ml. All four assays detected cell culture-expanded VoCs Alpha, Beta, Delta and Omicron subvariants carrying up to six amino acid mutations in the nucleocapsid protein with sensitivities comparable to the non-VoC EU-1. Overall, automated quantitative SARS-CoV-2 Ag assays are not more sensitive than standard rapid antigen tests used in POC settings and show a high heterogeneity in performance for VoC recognition. The best of these automated Ag tests may have the potential to complement nucleic acid-based assays for SARS-CoV-2 diagnostics in settings not primarily focused on the protection of vulnerable groups. In light of the constant emergence of new Omicron subvariants and recombinants, most recently the XBB lineage, these tests' performance must be regularly re-evaluated, especially when new VoCs carry mutations in the nucleocapsid protein or immunological and clinical parameters change.

Variable detection of Omicron-BA.1 and -BA.2 by SARS-CoV-2 rapid antigen tests.

During 2022, the COVID-19 pandemic has been dominated by the variant of concern (VoC) Omicron (B.1.1.529) and its rapidly emerging subvariants, including Omicron-BA.1 and -BA.2. Rapid antigen tests (RATs) are part of national testing strategies to identify SARS-CoV-2 infections on site in a community setting or to support layman's diagnostics at home. We and others have recently demonstrated an impaired RAT detection of infections caused by Omicron-BA.1 compared to Delta. Here, we evaluated the performance of five SARS-CoV-2 RATs in a single-centre laboratory study examining a total of 140 SARS-CoV-2 PCR-positive respiratory swab samples, 70 Omicron-BA.1 and 70 Omicron-BA.2, as well as 52 SARS-CoV-2 PCR-negative swabs collected from March 8th until April 10th, 2022. One test did not meet minimal criteria for specificity. In an assessment of the analytical sensitivity in clinical specimen, the 50% limit of detection (LoD50) ranged from 4.2 × 104 to 9.2 × 105 RNA copies subjected to the RAT for Omicron-BA.1 compared to 1.3 × 105 to 1.5 × 106 for Omicron-BA.2. Overall, intra-assay differences for the detection of Omicron-BA.1-containing and Omicron-BA.2-containing samples were non-significant, while a marked overall heterogeneity among the five RATs was observed. To score positive in these point-of-care tests, up to 22-fold (LoD50) or 68-fold (LoD95) higher viral loads were required for the worst performing compared to the best performing RAT. The rates of true-positive test results for these Omicron subvariant-containing samples in the highest viral load category (Ct values < 25) ranged between 44.7 and 91.1%, while they dropped to 8.7 to 22.7% for samples with intermediate Ct values (25-30). In light of recent reports on the emergence of two novel Omicron-BA.2 subvariants, Omicron-BA.2.75 and BJ.1, awareness must be increased for the overall reduced detection rate and marked differences in RAT performance for these Omicron subvariants.

Newbornscreening SMA - From Pilot Project to Nationwide Screening in Germany.

Now that targeted therapies for spinal muscular atrophy are available, attempts are being made worldwide to include screening for spinal muscular atrophy in general newborn screening. In Germany, after pilot projects from 2018-2021, it was included in the general newborn screening from October 2021. To ensure a smooth transition, criteria for follow-up were developed together with key stakeholders. At the beginning of the transition to nationwide screening, false positive findings were reported in 3 patients. After optimization of the screening method in the laboratories concerned, all findings have been subsequently confirmed. On average, the first presentation to a neuromuscular center occurred on day 12 of life, and in patients with 2 or 3 SMN2 copies, therapy started on day 26 of life. Compared with the pilot project, there was no significant delay in timing.

Comparison of fecal calprotectin and pancreatic elastase assays based on proficiency testing results.

BACKGROUND: Fecal calprotectin and fecal pancreatic elastase assays are not standardized because of a lack of suitable reference material. Laboratories may have difficulty in switching assays because different manufacturers do not compare well with each other despite having similar reference intervals. Data from proficiency testing performed in Germany (Fecal Diagnostics 01 Survey, INSTAND eV) were investigated to understand how results differed across eight calprotectin and five pancreatic elastase manufacturers. METHODS: Data were collected from participating laboratories in external quality assessment schemes from 2015 to 2020 for calprotectin and 2017 to 2020 for pancreatic elastase. The manufacturer group mean values and standard deviations were calculated. Reference points were created for each external quality assessment scheme by calculating the average of all manufacturer group means. Deming regression analyses were used to observe the differences across manufacturers. RESULTS: The slopes of the Deming regression spanned 0.37-1.91 for calprotectin and 0.84-1.33 for pancreatic elastase. The calprotectin assays had a high degree of variability in quantitative results by manufacturer. However, pancreatic elastase assays appear to be harmonized across the different manufacturer when considering the qualitative interpretation. CONCLUSIONS: Both calprotectin and pancreatic elastase assays could be improved by standardization efforts. Given the clinical utility and our data demonstrating high inter-manufacturer variability, calprotectin should be prioritized over pancreatic elastase in standardization efforts.

Verification of lateral flow antigen tests for SARS-CoV-2 by qPCR directly from the test device.

OBJECTIVE: Fast and reliable detection of infection is a key to control the SARS-CoV-2 pandemic. Lateral flow antigen tests (LFATs) are inexpensive, easy to use, but have to be verified, as they are rather unspecific and can produce both, false positive and false negative results. Our objective was to combine the speed of LFAT for SARS-CoV-2 with the reliability of qPCR tests. METHODS: A serial dilution of a patient sample positive for SARS-CoV-2 was prepared and added to LFAT wells from two manufacturers. After evaluation, the devices were opened, the strips removed and extracted in a solution. Amplification was performed using point of care PCR systems (cobas® Liat®, ID NOW™) or on a LightCycler after extraction by MagNAPure 96. RESULTS: The nucleic acid amplification systems yielded higher sensitivity to LFAT. Thus, all samples determined positive by LFAT from the serial dilution were also positive in the subsequent amplification reactions. Sensitivity using extracted eluates was 10-100 times higher. SIGNIFICANCE: The usage of LFAT is highly recommended for single samples in emergency dental or emergency clinical settings, for smaller cohorts, or even for larger population screening, as it is inexpensive and fast. Positive results can be conveniently verified directly from the test devices using either point of care test equipment or more complex laboratory equipment thus making a major impact on efficient management of infections and isolations.

Direct and indirect eluates from bulk fill resin-based-composites.

OBJECTIVES: To compare elutable substances directly released from bulk-fill (BF) resin-based composites (RBCs) with indirect elution from teeth restored with a BF composite. In addition to (co)monomers, the analytical focus was on other potentially toxic ingredients or impurities. Furthermore, the barrier function of the residual dentin/adhesive layer was studied. METHODS: Six BF-RBC materials were studied. For each material subgroup, ten human third molar teeth with standard Class-I occlusal cavities were prepared and provided with a three-step adhesive system and the respective composite restoration (tooth groups). Same sized control specimens of the restorative material were prepared ('direct BF-RBC' groups). Each specimen was placed in an elution chamber such that the elution media (ethanol/water, 3:1) only contacted the tooth root or ¾ height of each specimen. They were incubated at 37 °C for up to 7 d. Samples of eluate were taken after 1, 2, 4 and 7 d and were analysed by high-temperature gas chromatography/mass spectrometry. RESULTS: (Co)monomers such as Bisphenol A ethoxylate dimethacrylate (bisEMA) or tetraethylene glycol dimethacrylate (TEEGDMA) were mostly found in the eluates of the 'direct BF-RBC' groups in statistically significantly greater amounts than in the eluates of the 'tooth groups'. The residual dentin and/or adhesive layers acted as a diffusion barrier for most of the substances except for triethylene glycol dimethacrylate (TEGDMA) or diethylene glycol dimethacrylate (DEGDMA). For TEGDMA up to 3 orders of magnitude more were found in the 'tooth groups' compared to the 'direct BF-RBC' groups, evidently released by the adhesive system. Substances of Very High Concern (SVHC) including TINUVIN® 328 and BPA were found mainly in the eluates of 'direct BF-RBC' groups. SIGNIFICANCE: For estimation of biocompatibility, a total system, specifically BF-RBC + adhesive, should always be investigated since individual considerations, such as only elution from a BF-RBC, do not correctly reflect the total clinical situation. The focus of elution tests should not only be on the co(monomers), but also on other ingredients or impurities that may be released.

Impaired detection of omicron by SARS-CoV-2 rapid antigen tests.

Since autumn 2020, rapid antigen tests (RATs) have been implemented in several countries as an important pillar of the national testing strategy to rapidly screen for infections on site during the SARS-CoV-2 pandemic. The current surge in infection rates around the globe is driven by the variant of concern (VoC) omicron (B.1.1.529). Here, we evaluated the performance of nine SARS-CoV-2 RATs in a single-centre laboratory study. We examined a total of 115 SARS-CoV-2 PCR-negative and 166 SARS-CoV-2 PCR-positive respiratory swab samples (101 omicron, 65 delta (B.1.617.2)) collected from October 2021 until January 2022 as well as cell culture-expanded clinical isolates of both VoCs. In an assessment of the analytical sensitivity in clinical specimen, the 50% limit of detection (LoD50) ranged from 1.77 × 106 to 7.03 × 107 RNA copies subjected to the RAT for omicron compared to 1.32 × 105 to 2.05 × 106 for delta. To score positive in these point-of-care tests, up to 10-fold (LoD50) or 101-fold (LoD95) higher virus loads were required for omicron- compared to delta-containing samples. The rates of true positive test results for omicron samples in the highest virus load category (Ct values < 25) ranged between 31.4 and 77.8%, while they dropped to 0-8.3% for samples with intermediate Ct values (25-30). Of note, testing of expanded virus stocks suggested a comparable RAT sensitivity of both VoCs, questioning the predictive value of this type of in vitro-studies for clinical performance. Given their importance for national test strategies in the current omicron wave, awareness must be increased for the reduced detection rate of omicron infections by RATs and a short list of suitable RATs that fulfill the minimal requirements of performance should be rapidly disclosed.

Special Teaching Formats during the COVID-19 Pandemic-A Survey with Implications for a Crisis-Proof Education.

Modern teaching formats have not been considered necessary during the COVID-19 pandemic with uncertain acceptance by students. The study's aim was to describe and evaluate all measures undertaken for theoretical and practical knowledge/skill transfer, which included objective structured practical examinations (OSPEs) covering a communication skills training. The students' performance in the OSPE as well as the theoretical knowledge level were assessed, of which the latter was compared with previous terms. In conservative dentistry and periodontology (4th and 5th year courses), theoretical teaching formats were provided online and completed by a multiple-choice test. Practical education continued without patients in small groups using the phantom-head, 3D printed teeth, and objective structured practical examinations (OSPEs) including communication skills training. Formats were evaluated by a questionnaire. The organization was rated as very good/good (88.6%), besides poor Internet connection (22.8%) and Zoom® (14.2%) causing problems. Lectures with audio were best approved (1.48), followed by practical videos (1.54), live stream lectures (1.81), treatment checklists (1.81), and virtual problem-based learning (2.1). Lectures such as .pdf files without audio, articles, or scripts were rated worse (2.15-2.30). Phantom-heads were considered the best substitute for patient treatment (59.5%), while additional methodical efforts for more realistic settings led to increased appraisal. However, students performed significantly worse in the multiple-choice test compared to the previous terms (p < 0.0001) and the OSPEs revealed deficits in the students' communication skills. In the future, permanent available lectures with audio and efforts toward realistic treatment settings in the case of suspended patient treatment will be pursued.

Head-to-head evaluation of seven different seroassays including direct viral neutralisation in a representative cohort for SARS-CoV-2.

A number of seroassays are available for SARS-CoV-2 testing; yet, head-to-head evaluations of different testing principles are limited, especially using raw values rather than categorical data. In addition, identifying correlates of protection is of utmost importance, and comparisons of available testing systems with functional assays, such as direct viral neutralisation, are needed.We analysed 6658 samples consisting of true-positives (n=193), true-negatives (n=1091), and specimens of unknown status (n=5374). For primary testing, we used Euroimmun-Anti-SARS-CoV-2-ELISA-IgA/IgG and Roche-Elecsys-Anti-SARS-CoV-2. Subsequently virus-neutralisation, GeneScriptcPass, VIRAMED-SARS-CoV-2-ViraChip, and Mikrogen-recomLine-SARS-CoV-2-IgG were applied for confirmatory testing. Statistical modelling generated optimised assay cut-off thresholds. Sensitivity of Euroimmun-anti-S1-IgA was 64.8%, specificity 93.3% (manufacturer's cut-off); for Euroimmun-anti-S1-IgG, sensitivity was 77.2/79.8% (manufacturer's/optimised cut-offs), specificity 98.0/97.8%; Roche-anti-N sensitivity was 85.5/88.6%, specificity 99.8/99.7%. In true-positives, mean and median Euroimmun-anti-S1-IgA and -IgG titres decreased 30/90 days after RT-PCR-positivity, Roche-anti-N titres decreased significantly later. Virus-neutralisation was 80.6% sensitive, 100.0% specific (≥1:5 dilution). Neutralisation surrogate tests (GeneScriptcPass, Mikrogen-recomLine-RBD) were >94.9% sensitive and >98.1% specific. Optimised cut-offs improved test performances of several tests. Confirmatory testing with virus-neutralisation might be complemented with GeneScriptcPassTM or recomLine-RBD for certain applications. Head-to-head comparisons given here aim to contribute to the refinement of testing strategies for individual and public health use.

SARS-CoV-2 and regular patient treatment - from the use of rapid antigen testing up to treatment specific precaution measures.

INTRODUCTION: The COVID-19 pandemic poses a continued challenge for all parties involved especially for the dentist as routine operation must be resumed. Rapid Antigen Tests (RATs) are actually recommended to identify and minimize infectious risks. However, there is still no guideline on the implementation of RATs in a dental or medical setting. METHODS: Based on data and an extensive literature research regarding rapid antigen testing and reflecting the recommendations given by the various professional societies a task force was formed to determine a specific testing and treatment strategy. RESULTS: A comprehensive test and treatment strategy and risk analysis was developed with practical suggestions for a wide range of typical activities in dental and medical offices. The transmission of SARS-CoV-2 and its variants via aerosols and droplets as well as the difficulties to maintain the minimum distance form special challenges to the dental routine. RATs might in addition to optimal and necessary hygienic standards in combination with the use of adequate personal protection equipment be an important instrument in managing the challenges. CONCLUSIONS: The present work gives recommendations for dental routine operation (dental practices, outpatient clinics) to provide the necessary dental care for the population while protecting the doctor, practice team and patient at the same time.

Direct and indirect monomer elution from an RBC product family.

OBJECTIVE: To develop a model for quantitative comparison of elutable substances by direct elution from resin-bonded composite (RBC) test specimens versus indirect elutability of substances from RBC-restored teeth. Furthermore, it was to be investigated whether the different composites of the Tetric® RBC product family release different types and amounts of substances. METHODS: Four different composite materials from the Tetric® product family were studied. For each material subgroup ten human third molar teeth were prepared with standard Class-I occlusal cavities. These 'tooth group' specimens were provided with a three-step adhesive system (incorporating TEGDMA) and the respective composite restoration. Same sized control specimens, of each RBC restorative material, were prepared ('direct RBC' groups). All specimens were placed in individual elution chambers such that the elution media (ethanol/water, 3:1) only came into contact with either the tooth root or ¾ height of the 'direct RBC' materials. They were incubated at 37 °C for up to 7 d. Samples of the eluant were taken after 1, 2, 4 and 7 d and were analysed by high-temperature gas chromatography/mass spectrometry. RESULTS: Bisphenol A ethoxylate dimethacrylate (bisEMA), bisphenol A glycidyldimethacrylate (bisGMA), tetraethylene glycol dimethacrylate (TEEGDMA), decan-1,10-diol dimethacrylate (DDDMA) were mostly found in the eluates of the 'direct RBC' groups in statistically significantly greater amounts than in the eluates of the 'tooth groups'. Such quantitative differences were also the case with eluates containing bisphenol A (BPA), dicyclohexyl phthalate (DCHP) and drometrizole, which are common in the environment. In contrast to the behavior found with all the other monomers, up to 3 orders of magnitude more triethylene glycol dimethacrylate (TEGDMA) was found in the 'tooth groups' compared to the 'direct RBC' groups, evidently released by the adhesive system. SIGNIFICANCE: The release of most of the substances was clearly delayed in the 'tooth groups' indicative of their chronic, rather than acute, elution to the oral environment. A barrier function of the residual dentin layer and the adhesion layer can be inferred. The different release patterns of substances from the various composites of the RBC product family is a manifestation of their different and indication-specific compositions. Consideration of an overall restorative care (RBC plus adhesive) system, when assessing the total amount of released substances, is emphasized.

Newborn screening for spinal muscular atrophy in Germany: clinical results after 2 years.

BACKGROUND: Spinal muscular atrophy (SMA) is the most common neurodegenerative disease in childhood. Since motor neuron injury is usually not reversible, early diagnosis and treatment are essential to prevent major disability. Our objective was to assess the impact of genetic newborn screening for SMA on outcome. METHODS: We provided clinical data from 43 SMA patients, identified via polymerase chain reaction of the SMN1 gene from dried blood spots between January 2018 and January 2020 in Germany. Follow-up included neurophysiological examinations and standardized physiotherapeutic testing. RESULTS: Detection of SMA with newborn screening was consistent with known incidence in Germany. Birth prevalence was 1:6910; 39.5% had 2 SMN2 copies, 23% had 3 SMN2 copies, 32.5% had 4 copies, and 4.5% had 5 copies of the SMN2 gene. Treatment with SMA-specific medication could be started at the age of 14-39 days in 21 patients. Pre-symptomatically treated patients remained throughout asymptomatic within the observation period. 47% of patients with 2 SMN2 copies showed early, presumably intrauterine onset of disease. These patients reached motor milestones with delay; none of them developed respiratory symptoms. Untreated children with 2 SMN2 copies died. Untreated children with 3 SMN2 copies developed proximal weakness in their first year. In patients with ≥ 4 SMN2 copies, a follow-up strategy of "watchful waiting" was applied despite the fact that one of them was treated from the age of 6 months. Two infant siblings with 4 SMN2 copies were identified with a missed diagnosis of SMA type 3. CONCLUSION: Identification of newborns with infantile SMA and prompt SMA-specific treatment substantially improves neurodevelopmental outcome, and we recommend implementation in the public newborn screening in countries where therapy is available. Electrophysiology is a relevant parameter to support the urgency of therapy. There has to be a short time interval between a positive screening result and referral to a therapy-ready specialized treatment center.

Fast and cost-effective screening for SARS-CoV-2 variants in a routine diagnostic setting.

OBJECTIVE: The aim is to recommend a fast and cost-effective screening procedure for UK/SA SARS-CoV-2 variants in a routing diagnostic setting. METHODS: A rapid procedure using qPCR is described to provide clinicians with information about the two currently most prevalent variants (B1.1.7 and B1.351) that harbour receptor binding domain mutation N501Y. The N501Y specific assay only delivers an amplification signal if the Y501 variant is present. RESULTS: 436 samples initially screened positive for SARS-CoV-2 were randomly selected. Only one of these samples showed a fluorescence signal increase indicative for the Y501 variant. The remaining 435 samples had a melting peak at 54 °C indicating the N501 wildtype. SIGNIFICANCE: The screening of a broad population base can still be performed with the established test system. In case of a positive test for SARS-CoV-2 and corresponding clinical and anamnestic indications, a second qPCR for the mutation N501Y can follow and deliver the result to public health authorities and to the treating physician within a few hours.

Infants Diagnosed with Spinal Muscular Atrophy and 4 SMN2 Copies through Newborn Screening - Opportunity or Burden?

Although the value of newborn screening (NBS) for early detection and treatment opportunity in SMA patients is generally accepted, there is still an ongoing discussion about the best strategy in children with 4 and more copies of the SMN2 gene. This gene is known to be the most important but not the only disease modifier.In our SMA-NBS pilot project in Germany comprising 278,970 infants screened between January 2018 and November 2019 were 38 positive cases with a homozygous SMN1 deletion. 40% of them had 4 or more SMN2 copies. The incidence for homozygous SMN1 deletion was 1 : 7350, which is within the known range of SMA incidence in Germany.Of the 15 SMA children with 4 SMN2 copies, one child developed physical signs of SMA by the age of 8 months. Reanalysis of the SMN2 copy number by a different test method revealed 3 copies. Two children had affected siblings with SMA Type III, who were diagnosed only after detection of the index patient in the NBS. One had a positive family history with an affected aunt (onset of disease at the age of 3 years). Three families were lost to medical follow up; two because of socioeconomic reasons and one to avoid the psychological stress associated with the appointments.Decisions on how to handle patients with 4 SMN2 copies are discussed in the light of the experience gathered from our NBS pilot SMA program.