Reply to Bouva et al. Comment on "Dijkstra et al. A False-Negative Newborn Screen for Tyrosinemia Type 1-Need for Re-Evaluation of Newborn Screening with Succinylacetone. Int. J. Neonatal Screen. 2023, 9, 66".

We thank the authors for their comments [...].

A False-Negative Newborn Screen for Tyrosinemia Type 1-Need for Re-Evaluation of Newborn Screening with Succinylacetone.

Undiagnosed and untreated tyrosinemia type 1 (TT1) individuals carry a significant risk for developing liver fibrosis, cirrhosis and hepatocellular carcinoma (HCC). Elevated succinylacetone (SA) is pathognomonic for TT1 and therefore often used as marker for TT1 newborn screening (NBS). While SA was long considered to be elevated in every TT1 patient, here we present a recent false-negative SA TT1 screen. A nine-year-old boy presented with HCC in a cirrhotic liver. Additional tests for the underlying cause unexpectedly revealed TT1. Nine years prior, the patient was screened for TT1 via SA NBS with a negative result: SA 1.08 µmol/L, NBS cut-off 1.20 µmol/L. To our knowledge, this report is the first to describe a false-negative result from the TT1 NBS using SA. False-negative TT1 NBS results may be caused by milder TT1 variants with lower SA excretion. Such patients are more likely to be missed in NBS programs and can be asymptomatic for years. Based on our case, we advise TT1 to be considered in patients with otherwise unexplained liver pathology, including fibrosis, cirrhosis and HCC, despite a previous negative TT1 NBS status. Moreover, because the NBS SA concentration of this patient fell below the Dutch cut-off value (1.20 µmol/L at that time), as well as below the range of cut-off values used in other countries (1.29-10 µmol/L), it is likely that false-negative screening results for TT1 may also be occurring internationally. This underscores the need to re-evaluate TT1 SA NBS programs.

Severe Immune-Related Enteritis after In Utero Exposure to Pembrolizumab.

Immune checkpoint blockade has become standard treatment for many types of cancer. Such therapy is indicated most often in patients with advanced or metastatic disease but has been increasingly used as adjuvant therapy in those with early-stage disease. Adverse events include immune-related organ inflammation resembling autoimmune diseases. We describe a case of severe immune-related gastroenterocolitis in a 4-month-old infant who presented with intractable diarrhea and failure to thrive after in utero exposure to pembrolizumab. Known causes of the symptoms were ruled out, and the diagnosis of pembrolizumab-induced immune-related gastroenterocolitis was supported by the results of histopathological assays, immunophenotyping, and analysis of the level of antibodies against programmed cell death protein 1 (PD-1). The infant's condition was successfully treated with prednisolone and infliximab.

Non-invasive diagnostics of pathogenic bacteria using a breath sampler in children with cystic fibrosis.

Cystic fibrosis (CF) is a common autosomal recessive disease causing thick, viscous secretions leading to pulmonary infections with pathogenic bacteria. As part of routine patient care, colonization and infection with these bacteria is monitored with cough swab or sputum cultures and sometimes bronchoalveolar lavage. In this cross-sectional proof-of-concept study in a cohort of CF patients we collected swabs or sputa and exhaled breath samples with the modular breath sampler (MBS), a newly developed two-way non-rebreathing sampling device. Pathogen specific polymerase chain reactions (PCRs) were performed on the MBS samples and compared with the results obtained with conventional diagnostics (i.e. culturing of swabs and sputa). A control group of stable asthma patients was used as negative control for the MBS measurements. The pathogens detected using MBS and conventional culturing differed:S. aureuswas found more often in swab or sputum samples whereasPseudomonas aeruginosaandS. pneumoniaewere found more often in MBS samples. We hypothesize that this is due to sampling of different compartments, MBS samples are derived from the lower respiratory tract while cultures from cough swabs and sputa are dominated by pathogens residing in the upper respiratory tract. Another important difference is the readout, i.e. culture versus PCR. The majority of CF patients in whomP. aeruginosawas found did not have recent positive cultures suggesting higher sensitivity of MBS-based than conventional diagnostics. The majority of parents/patients found the MBS easy to use and less of a burden than respiratory sampling.

Raltegravir-based Postnatal HIV Prophylaxis Therapy in a Neonate After in Utero Dolutegravir Exposure.

We present a case report of a neonate receiving raltegravir-based postnatal HIV prophylaxis after in utero dolutegravir exposure. High levels of raltegravir and dolutegravir can potentially cause bilirubin toxicity as they compete for albumin binding and follow the same metabolic pathway through UGT1A1. This case suggests delaying initiation of raltegravir-based postnatal prophylaxis by 24-48 hours after in utero dolutegravir exposure.

Respiratory Tract Infection Management and Antibiotic Prescription in Children: A Unique Study Comparing Three Levels of Healthcare in The Netherlands.

BACKGROUND: Respiratory tract infections (RTIs) are common in children with febrile illness visiting the general practitioner (GP) or emergency department. We studied the management of children with fever and RTI at 3 different levels of healthcare in The Netherlands, focusing on antibiotic prescription. METHODS: This prospective observational study is part of the Management and Outcome of Febrile children in Europe study. Data were used from face-to-face patient contacts of children with febrile illness in three healthcare settings in Nijmegen, The Netherlands during 2017. These settings were primary (GP), secondary (general hospital) and tertiary care (university hospital). RESULTS: Of 892 cases with RTI without complex comorbidities, overall antibiotic prescription rates were 29% with no differences between the 3 levels of healthcare, leading to an absolute number of 5031 prescriptions per 100,000 children per year in primary care compared with 146 in secondary and tertiary care combined. The prescription rate in otitis media was similar in all levels: 60%. In cases with lower RTI who received nebulizations prescription rates varied between 19% and 55%. CONCLUSIONS: Antibiotic prescription rates for RTIs in children were comparable between the 3 levels of healthcare, thus leading to a majority of antibiotics being prescribed in primary care. Relatively high prescription rates for all foci of RTIs were found, which was not in agreement with the national guidelines. Antibiotic stewardship needs improvement at all 3 levels of healthcare. Guidelines to prescribe small spectrum antibiotics for RTIs need to be better implemented in hospital care settings.

Parents' Perspectives and Societal Acceptance of Implementation of Newborn Screening for SCID in the Netherlands.

PURPOSE: While neonatal bloodspot screening (NBS) for severe combined immunodeficiency (SCID) has been introduced more than a decade ago, implementation in NBS programs remains challenging in many countries. Even if high-quality test methods and follow-up care are available, public uptake and parental acceptance are not guaranteed. The aim of this study was to describe the parental perspective on NBS for SCID in the context of an implementation pilot. Psychosocial aspects have never been studied before for NBS for SCID and are important for societal acceptance, a major criterion when introducing new disorders in NBS programs. METHODS: To evaluate the perspective of parents, interviews were conducted with parents of newborns with abnormal SCID screening results (N = 17). In addition, questionnaires about NBS for SCID were sent to 2000 parents of healthy newborns who either participated or declined participation in the SONNET-study that screened 140,593 newborns for SCID. RESULTS: Support for NBS for SCID was expressed by the majority of parents in questionnaires from both a public health perspective and a personal perspective. Parents emphasized the emotional impact of an abnormal screening result in interviews. (Long-term) stress and anxiety can be experienced during and after referral indicating the importance of uniform follow-up protocols and adequate information provision. CONCLUSION: The perspective of parents has led to several recommendations for NBS programs that are considering screening for SCID or other disorders. A close partnership of NBS programs' stakeholders, immunologists, geneticists, and pediatricians-immunologists in different countries is required for moving towards universal SCID screening for all infants.

Exome sequencing in routine diagnostics: a generic test for 254 patients with primary immunodeficiencies.

BACKGROUND: Diagnosis of primary immunodeficiencies (PIDs) is complex and cumbersome yet important for the clinical management of the disease. Exome sequencing may provide a genetic diagnosis in a significant number of patients in a single genetic test. METHODS: In May 2013, we implemented exome sequencing in routine diagnostics for patients suffering from PIDs. This study reports the clinical utility and diagnostic yield for a heterogeneous group of 254 consecutively referred PID patients from 249 families. For the majority of patients, the clinical diagnosis was based on clinical criteria including rare and/or unusual severe bacterial, viral, or fungal infections, sometimes accompanied by autoimmune manifestations. Functional immune defects were interpreted in the context of aberrant immune cell populations, aberrant antibody levels, or combinations of these factors. RESULTS: For 62 patients (24%), exome sequencing identified pathogenic variants in well-established PID genes. An exome-wide analysis diagnosed 10 additional patients (4%), providing diagnoses for 72 patients (28%) from 68 families altogether. The genetic diagnosis directly indicated novel treatment options for 25 patients that received a diagnosis (34%). CONCLUSION: Exome sequencing as a first-tier test for PIDs granted a diagnosis for 28% of patients. Importantly, molecularly defined diagnoses indicated altered therapeutic options in 34% of cases. In addition, exome sequencing harbors advantages over gene panels as a truly generic test for all genetic diseases, including in silico extension of existing gene lists and re-analysis of existing data.

Impact of bacterial colonization on exhaled inflammatory markers in wheezing preschool children.

Wheeze is a common symptom in preschool children. The role of bacteria, regulatory T (T(reg)) cells and their association with airway inflammation in preschool wheeze is largely unknown. We evaluated inflammatory markers in exhaled breath condensate (EBC), bacterial colonization and circulating T(reg) cells in preschool children with and without recurrent wheeze. We recruited 252 children (aged two to four years) with (N = 202) and without (N = 50) recurrent wheeze. EBC was collected using an efficient closed glass condenser. Inflammatory markers in EBC (Interleukin(IL)-2, IL-4, IL-8, IL-10, IL-13) were assessed using multiplex immunoassay. Nasal and throat swabs were analysed for presence of Streptococcus pneumoniae, Haemophilus (para)influenzae and Staphylococcus aureus. Proportions of T(reg) cells (CD4(+)CD25(high)CD127(-)) were quantified by flow cytometry. Recurrent wheezing children had elevated EBC levels of IL-2, IL-4, IL-10 and IL-13 compared to non-wheezers (odds ratio (95% confidence interval): 1.67 (1.23-2.27): 1.58 (1.15-2.18): 1.47 (1.14-1.90): 1.55 (1.16-2.06), p <0.05, respectively). Bacteria were frequently present in children with and without wheeze, with no difference in prevalence (16-52% versus 16-50%, respectively). Moreover, the proportion of T(reg) cells did not differ between both groups. Wheezing children with bacterial colonization did not significantly differ in exhaled levels of inflammatory markers or proportion of T(reg) cells compared to wheezing children without colonization. The analysis of EBC might serve as a helpful non-invasive tool to early assess airway inflammation in wheezing children. The various elevated exhaled inflammatory markers indicate increased airway inflammation in wheezing preschool children. In the presence of wheeze, we found no evidence for bacterial induced airway inflammation.