Reassessing the Performance of the "Step-By-Step" Approach to Febrile Infants 90 Days of Age and Younger in the Context of the COVID-19 Pandemic: A Multicentric Retrospective Study.

BACKGROUND: Infants with COVID-19 can often present with fever without source, which is a challenging situation in infants <90 days old. The "step-by-step" algorithm has been proposed to identify children at high risk of bacterial infection. In the context of the COVID-19 pandemic, we aimed to reassess the diagnostic performance of this algorithm. METHODS: We performed a multicentric retrospective study in 3 French pediatric emergency departments between 2018 and 2020. We applied the "step-by-step" algorithm to 4 clinical entities: COVID-19, febrile urinary tract infections (FUTI), invasive bacterial infection (IBI), and enterovirus infections. The main outcome was the proportion of infants classified at high risk (ill-appearing, ≤21 days old, with leukocyturia or procalcitonin level ≥0.5 ng/mL). RESULTS: Among the 199 infants included, 40 had isolated COVID-19, 25 had IBI, 60 had FUTI, and 74 had enterovirus infection. All but 1 infant with bacterial infection were classified at high risk (96% for IBI and 100% for FUTI) as well as 95% with enterovirus and 82% with COVID-19. Infants with COVID-19 were classified at high risk because an ill-appearance (72%), an age ≤21 days (27%), or leukocyturia (19%). All these infants had procalcitonin values <0.5 ng/mL and only 1 had C-reactive protein level >20 mg/L. CONCLUSIONS: The "step-by-step" algorithm remains effective to identify infants with bacterial infection but misclassifies most infants with COVID-19 as at high risk of bacterial infection leading to unnecessary cares. An updated algorithm based adding viral testing may be needed to discriminate fever related to isolated COVID-19 in infants <90 days old.

A monocyte/dendritic cell molecular signature of SARS-CoV-2-related multisystem inflammatory syndrome in children with severe myocarditis.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in children is generally milder than in adults, but a proportion of cases result in hyperinflammatory conditions often including myocarditis. METHODS: To better understand these cases, we applied a multiparametric approach to the study of blood cells of 56 children hospitalized with suspicion of SARS-CoV-2 infection. Plasma cytokine and chemokine levels and blood cellular composition were measured, alongside gene expression at the bulk and single-cell levels. FINDINGS: The most severe forms of multisystem inflammatory syndrome in children (MIS-C) related to SARS-CoV-2 that resulted in myocarditis were characterized by elevated levels of pro-angiogenesis cytokines and several chemokines. Single-cell transcriptomics analyses identified a unique monocyte/dendritic cell gene signature that correlated with the occurrence of severe myocarditis characterized by sustained nuclear factor κB (NF-κB) activity and tumor necrosis factor alpha (TNF-α) signaling and associated with decreased gene expression of NF-κB inhibitors. We also found a weak response to type I and type II interferons, hyperinflammation, and response to oxidative stress related to increased HIF-1α and Vascular endothelial growth factor (VEGF) signaling. CONCLUSIONS: These results provide potential for a better understanding of disease pathophysiology. FUNDING: Agence National de la Recherche (Institut Hospitalo-Universitaire Imagine, grant ANR-10-IAHU-01; Recherche Hospitalo-Universitaire, grant ANR-18-RHUS-0010; Laboratoire d'Excellence ''Milieu Intérieur," grant ANR-10-LABX-69-01; ANR-flash Covid19 "AIROCovid" and "CoVarImm"), Institut National de la Santé et de la Recherche Médicale (INSERM), and the "URGENCE COVID-19" fundraising campaign of Institut Pasteur.

Imported Schistosomiasis in Children: Clinical, Diagnostic Aspects And Outcome in 5 Tertiary Hospitals in France.

The objective of this retrospective study is to describe imported schistosomiasis in children in the Paris region between 2010 and 2015. Forty children with a diagnosis of schistosomiasis were included. Thirty-seven (93%) had a chronic urinary form with hematuria. The lost-to-follow up rate for the second consultation was 25%. The diagnosis and management of imported schistosomiasis must be improved-notably by raising awareness among clinicians and providing families with more information.

Do All Children Who Present With a Complex Febrile Seizure Need a Lumbar Puncture?

STUDY OBJECTIVE: We assess the prevalences of bacterial meningitis and herpes simplex virus meningoencephalitis (HSV-ME) in children with a complex febrile seizure and determine these prevalences in the subgroup of children with a clinical examination result not suggestive of meningitis or encephalitis. METHODS: This multicenter retrospective study was conducted in 7 pediatric emergency departments (EDs) in the region of Paris, France. Visits of patients aged 6 months to 5 years for a complex febrile seizure from January 2007 to December 2011 were analyzed. We defined a subgroup of patients whose clinical examination result was not suggestive of meningitis or encephalitis. Bacterial meningitis and HSV-ME were sequentially sought for by analyzing bacteriologic and viral data at the visit, looking for data from a second visit to the hospital after the index visit, and telephoning the child's parents. RESULTS: From a total of 1,183,487 visits in the 7 pediatric EDs, 839 patients presented for a complex febrile seizure, of whom 260 (31.0%) had a lumbar puncture. The outcomes bacterial meningitis and HSV-ME were ascertainable for 715 (85%) and 657 (78.3%) visits, respectively, and we found 5 cases of bacterial meningitis (0.7% [95% confidence interval [CI] 0.2% to 1.6%]) and no HSV-ME (0% [95% CI 0% to 0.6%]). Among the 630 visits of children with a clinical examination result not suggesting meningitis or encephalitis, we found no bacterial meningitis (0% [95% CI 0% to 0.7%]) and no HSV-ME (0% [95% CI 0% to 0.8%]). CONCLUSION: In children with a complex febrile seizure, bacterial meningitis and HSV-ME are unexpected events when the clinical examination after complex febrile seizure is not suggestive of meningitis or encephalitis.

Mosaic Tetrasomy 9p: A Mendelian Condition Associated With Pediatric-Onset Overlap Myositis.

Pediatric-onset inflammatory myositis (IM) and systemic lupus erythematosus (SLE) are rare inflammatory diseases. Both result from the complex interaction of genetic and environmental factors. An increasing number of Mendelian conditions predisposing to the development of SLE have been recently identified. These include monogenic conditions, referred to as the type I interferonopathies, associated with a primary upregulation of type I interferon (IFN), a key cytokine in the pathogenesis of SLE and some cases of IM. Here, we report on a pediatric-onset inflammatory overlap phenotype in a 6-year-old girl who was shown to carry mosaic tetrasomy 9p. The patient presented with myositis overlapping with lupuslike features. Myositis was characterized by a proximal muscular weakness and HLA class I antigen myofiber overexpression on muscle biopsy. Lupus-like manifestations consisted of pericarditis, pleuritis, and positive antinuclear and anti-SSA (Sjögren-syndrome A) antibodies. Complete remission was achieved with corticosteroids and mycophenolate mofetyl. Analysis of tetrasomy 9p showed mosaic tetrasomy in the 9p24.3q12 region, including the type I IFN cluster, and increased expression of IFN-stimulated genes. These data suggest that mosaic tetrasomy 9p can be associated with an upregulation of type I IFN signaling, predisposing to inflammatory myositis and lupus-like features. Thus, unexplained muscle or other organ involvement in patients carrying mosaic tetrasomy of the type IFN cluster of chromosome 9p should lead to the search for IM and/or lupuslike disease, and karyotype should be performed in patients with SLE or IM with mental retardation.

Clinical spectrum and long-term follow-up of 14 cases with G6PC3 mutations from the French Severe Congenital Neutropenia Registry.

BACKGROUND: The purpose of this study was to describe the natural history of severe congenital neutropenia (SCN) in 14 patients with G6PC3 mutations and enrolled in the French SCN registry. METHODS: Among 605 patients included in the French SCN registry, we identified 8 pedigrees that included 14 patients with autosomal recessive G6PC3 mutations. RESULTS: Median age at the last visit was 22.4 years. All patients had developed various comordibities, including prominent veins (n = 12), cardiac malformations (n = 12), intellectual disability (n = 7), and myopathic syndrome with recurrent painful cramps (n = 1). Three patients developed Crohn's disease, and five had chronic diarrhea with steatorrhea. Neutropenia was profound (<0.5 × 109/l) in almost all cases at diagnosis and could marginally fluctuate. The bone marrow smears exhibited mild late-stage granulopoeitic defects. One patient developed myelodysplasia followed by acute myelogenous leukemia with translocation (18, 21) at age 14 years, cured by chemotherapy and hematopoietic stem cell transplantation. Four deaths occurred, including one from sepsis at age 5, one from pulmonary late-stage insufficiency at age 19, and two from sudden death, both at age 30 years. A new homozygous mutation (c.249G > A /p.Trp83*) was detected in one pedigree. CONCLUSIONS: Severe congenital neutropenia with autosomal recessive G6PC3 mutations is associated with considerable clinical heterogeneity. This series includes the first described case of malignancy in this neutropenia.

Anti-TNF-α therapy may cause neonatal neutropenia.

Although anti-tumor necrosis factor (anti-TNF) antibodies are associated with a clear risk of agranulocytosis in adults and are known to cross the placenta, monitoring of the absolute neutrophil count (ANC) in neonates born to mothers receiving these biological agents is not currently recommended. Here, we report on the first case series of 4 newborn patients with severe neutropenia born to mothers treated for ulcerative colitis with infliximab during pregnancy (including the third trimester). The newborns presented with severe neutropenia at birth, which was subsequently complicated by skin infections. The newborns' ANCs returned to the normal range within 8 to 14 weeks, at which time infliximab could not be detected in the blood. Anti-TNF agents probably exert a direct, toxic effect on the bone marrow. Furthermore, the detection of a CD16 autoantibody in 1 mother-newborn pair suggests that infliximab can induce autoimmune neutropenia. Abnormally high levels of the CD16 autoantibody in newborn serum or immaturity of the fetal bone marrow might explain why neutropenia was observed in the child but not in the mother. We recommend the systematic measurement of ANC on cord blood at birth and (in the event of an infection) in the weeks thereafter.

Multiple congenital anomalies-intellectual disability (MCA-ID) and neuroblastoma in a patient harboring a de novo 14q23.1q23.3 deletion.

Neuroblastoma is the most frequent extra cranial solid tumor in infants and children. Genetic predisposition to neuroblastoma has been suspected previously due to familial cases of sporadic NB and predisposition to NB in several syndromes. Here, we report on a de novo 14q23.1-q23.3 microdeletion in a male presenting with a neuroblastoma diagnosed at 9 months, and spherocytosis, congenital heart defect, cryptorchidism, hypoplasia of corpus callosum, epilepsy, and developmental delay. Myc-associated-factor X (MAX) haploinsufficiency could be regarded as the predisposing factor to NB. Indeed 14q deletion is a recurrent somatic rearrangement in NB and MAX somatic and germline loss of function mutation have recently been described in pheochromocytoma and paraganglioma. However, MAX was expressed in the tumor of the patient we report on and, accordingly, loss of heterozygosity, mutation, or promoter methylation were excluded. In addition, we discuss the potential involvement in the clinical spectrum presented by the patient of five of the deleted genes, namely DAAM1, PLEKHG3, SPTB, AKAP5, and ARID4A.

A mild form of SLC29A3 disorder: a frameshift deletion leads to the paradoxical translation of an otherwise noncoding mRNA splice variant.

We investigated two siblings with granulomatous histiocytosis prominent in the nasal area, mimicking rhinoscleroma and Rosai-Dorfman syndrome. Genome-wide linkage analysis and whole-exome sequencing identified a homozygous frameshift deletion in SLC29A3, which encodes human equilibrative nucleoside transporter-3 (hENT3). Germline mutations in SLC29A3 have been reported in rare patients with a wide range of overlapping clinical features and inherited disorders including H syndrome, pigmented hypertrichosis with insulin-dependent diabetes, and Faisalabad histiocytosis. With the exception of insulin-dependent diabetes and mild finger and toe contractures in one sibling, the two patients with nasal granulomatous histiocytosis studied here displayed none of the many SLC29A3-associated phenotypes. This mild clinical phenotype probably results from a remarkable genetic mechanism. The SLC29A3 frameshift deletion prevents the expression of the normally coding transcripts. It instead leads to the translation, expression, and function of an otherwise noncoding, out-of-frame mRNA splice variant lacking exon 3 that is eliminated by nonsense-mediated mRNA decay (NMD) in healthy individuals. The mutated isoform differs from the wild-type hENT3 by the modification of 20 residues in exon 2 and the removal of another 28 amino acids in exon 3, which include the second transmembrane domain. As a result, this new isoform displays some functional activity. This mechanism probably accounts for the narrow and mild clinical phenotype of the patients. This study highlights the 'rescue' role played by a normally noncoding mRNA splice variant of SLC29A3, uncovering a new mechanism by which frameshift mutations can be hypomorphic.

Germline gain-of-function mutations of ALK disrupt central nervous system development.

Neuroblastoma (NB) is a frequent embryonal tumor of sympathetic ganglia and adrenals with extremely variable outcome. Recently, somatic amplification and gain-of-function mutations of the anaplastic lymphoma receptor tyrosine kinase (ALK) gene, either somatic or germline, were identified in a significant proportion of NB cases. Here we report a novel syndromic presentation associating congenital NB with severe encephalopathy and abnormal shape of the brainstem on brain MRI in two unrelated sporadic cases harboring de novo, germline, heterozygous ALK gene mutations. Both mutations are gain-of-function mutations that have been reported in NB and NB cell lines. These observations further illustrate the role of oncogenes in both tumour predisposition and normal development, and shed light on the pleiotropic and activity-dependent role of ALK in humans. More generally, missing germline mutations relative to the spectrum of somatic mutations reported for a given oncogene may be a reflection of severe effects during embryonic development, and may prompt mutation screening in patients with extreme phenotypes.

Dissection of the MYCN locus in Feingold syndrome and isolated oesophageal atresia.

Feingold syndrome (FS) is a syndromic microcephaly entity for which MYCN is the major disease-causing gene. We studied the expression pattern of MYCN at different stages of human embryonic development and collected a series of 17 FS and 12 isolated oesophageal atresia (IOA) cases. An MYCN gene deletion/mutation was identified in 47% of FS cases exclusively. We hypothesized that mutations or deletions of highly conserved non-coding elements (HCNEs) at the MYCN locus could lead to its misregulation and thereby to FS and/or IOA. We subsequently sequenced five HCNEs at the MYCN locus and designed a high-density tiling path comparative genomic hybridization array of 3.3 Mb at the MYCN locus. We found no mutations or deletions in this region, supporting the hypothesis of genetic heterogeneity in FS.

Differential contributions of rare and common, coding and noncoding Ret mutations to multifactorial Hirschsprung disease liability.

The major gene for Hirschsprung disease (HSCR) encodes the receptor tyrosine kinase RET. In a study of 690 European- and 192 Chinese-descent probands and their parents or controls, we demonstrate the ubiquity of a >4-fold susceptibility from a C-->T allele (rs2435357: p = 3.9 x 10(-43) in European ancestry; p = 1.1 x 10(-21) in Chinese samples) that probably arose once within the intronic RET enhancer MCS+9.7. With in vitro assays, we now show that the T variant disrupts a SOX10 binding site within MCS+9.7 that compromises RET transactivation. The T allele, with a control frequency of 20%-30%/47% and case frequency of 54%-62%/88% in European/Chinese-ancestry individuals, is involved in all forms of HSCR. It is marginally associated with proband gender (p = 0.13) and significantly so with length of aganglionosis (p = 7.6 x 10(-5)) and familiality (p = 6.2 x 10(-4)). The enhancer variant is more frequent in the common forms of male, short-segment, and simplex families whereas multiple, rare, coding mutations are the norm in the less common and more severe forms of female, long-segment, and multiplex families. The T variant also increases penetrance in patients with rare RET coding mutations. Thus, both rare and common mutations, individually and together, make contributions to the risk of HSCR. The distribution of RET variants in diverse HSCR patients suggests a "cellular-recessive" genetic model where both RET alleles' function is compromised. The RET allelic series, and its genotype-phenotype correlations, shows that success in variant identification in complex disorders may strongly depend on which patients are studied.

Cholinergic switch associated with morphological differentiation in neuroblastoma.

The morphology of malignant cells distinguishes between undifferentiated, poorly differentiated and differentiating neuroblastomas and constitutes a strong prognostic factor. Spontaneous or treatment-induced maturation characterizes a subset of neuroblastomas. It constitutes the basis of retinoic acid treatment to improve survival in aggressive neuroblastomas. However, the molecular events that drive differentiation are poorly understood. In the present study we have investigated the relationships between gene expression profiles and differentiation criteria in stroma-poor neuroblastomas. This study included three undifferentiated (UN), 20 poorly differentiated (PDN) and 11 differentiating (DN) neuroblastomas. These groups could be clearly separated using unsupervised clustering methods, which further enabled a major classification impact of genes involved in neural development, differentiation and function to be identified. UNs are characterized by high ASCL1, high PHOX2B, low GATA2, low TH and low DBH expressions. Most PDNs harbour a clear adrenergic phenotype, even in the presence of missense PHOX2B mutations. Finally, all DN tumours demonstrate cholinergic features. Depending upon their association with adrenergic characteristics, this enables dual 'cholinergic/adrenergic' and 'fully cholinergic' neuroblastomas to be defined. This suggests that the cholinergic switch, a final specification process that occurs physiologically in a minority of sympathetic neurons, is a critical step of differentiation in some neuroblastic tumours. This switch is associated with a down regulation of DBH that is apparently not strictly dependent upon PHOX2B. Conversely, GATA2 and TFAP2B may play critical roles in maintaining adrenergic features in poorly differentiated tumours.

Epistasis between RET and BBS mutations modulates enteric innervation and causes syndromic Hirschsprung disease.

Hirschsprung disease (HSCR) is a common, multigenic neurocristopathy characterized by incomplete innervation along a variable length of the gut. The pivotal gene in isolated HSCR cases, either sporadic or familial, is RET. HSCR also presents in various syndromes, including Shah-Waardenburg syndrome (WS), Down (DS), and Bardet-Biedl (BBS). Here, we report 3 families with BBS and HSCR with concomitant mutations in BBS genes and regulatory RET elements, whose functionality is tested in physiologically relevant assays. Our data suggest that BBS mutations can potentiate HSCR predisposing RET alleles, which by themselves are insufficient to cause disease. We also demonstrate that these genes interact genetically in vivo to modulate gut innervation, and that this interaction likely occurs through complementary, yet independent, pathways that converge on the same biological process.

Somatic and germline activating mutations of the ALK kinase receptor in neuroblastoma.

Neuroblastoma, a tumour derived from the peripheral sympathetic nervous system, is one of the most frequent solid tumours in childhood. It usually occurs sporadically but familial cases are observed, with a subset of cases occurring in association with congenital malformations of the neural crest being linked to germline mutations of the PHOX2B gene. Here we conducted genome-wide comparative genomic hybridization analysis on a large series of neuroblastomas. Copy number increase at the locus encoding the anaplastic lymphoma kinase (ALK) tyrosine kinase receptor was observed recurrently. One particularly informative case presented a high-level gene amplification that was strictly limited to ALK, indicating that this gene may contribute on its own to neuroblastoma development. Through subsequent direct sequencing of cell lines and primary tumour DNAs we identified somatic mutations of the ALK kinase domain that mainly clustered in two hotspots. Germline mutations were observed in two neuroblastoma families, indicating that ALK is a neuroblastoma predisposition gene. Mutated ALK proteins were overexpressed, hyperphosphorylated and showed constitutive kinase activity. The knockdown of ALK expression in ALK-mutated cells, but also in cell lines overexpressing a wild-type ALK, led to a marked decrease of cell proliferation. Altogether, these data identify ALK as a critical player in neuroblastoma development that may hence represent a very attractive therapeutic target in this disease that is still frequently fatal with current treatments.

Rhinoscleroma: a French national retrospective study of epidemiological and clinical features.

BACKGROUND: Rhinoscleroma (RS) is a rare, chronic, granulomatous disease of the upper respiratory tract that is associated with infection with Klebsiella rhinoscleromatis. RS is more common in certain geographic regions than in others, but other risk factors and the pathogenesis of RS remain unclear. METHODS: We sent a standardized questionnaire to all pathologists and otolaryngology specialists in French University Hospitals and asked whether they had seen patients with RS in the previous 16 years (1990-2005). We then retrospectively reviewed the files of all patients identified. RESULTS: We collected 11 cases of RS, with a median patient age at diagnosis of 35.7 years (range, 5-72 years). The 3 patients with a familial history of RS presented with early-onset forms of RS; 1 had an uncommon aggressive presentation of the disease with ischemic stroke. Two unrelated consanguineous families were identified, 1 of which included 2 affected siblings. Two patients with sporadic disease were positive for HIV infection. All patients were living in France, but most were immigrants from areas where RS is endemic (North Africa, 3 of the 11 patients; West Africa, 4 patients; and Turkey, 1 patient). The probable duration of exposure to K. rhinoscleromatis in endemic areas varied widely: 0-28 years. Clinical features and outcome also varied considerably among cases. Biopsies had been performed for all patients and revealed granulomas containing Mikulicz cells. Cultures of biopsy tissue were positive for K. rhinoscleromatis in 5 of the 11 cases. Prolonged antibiotic treatment was administered to all patients, as follows: ciprofloxacin (7 patients), third-generation cephalosporins (2), tetracycline (2), and clofazimine (2). Eight of the 11 patients did not experience relapse during extended periods of follow-up (1.3-12 years). Relapses in 3 patients were confirmed by a second biopsy. CONCLUSIONS: The occurrence of early-onset RS in multiplex and/or consanguineous families suggests that genetic control of the host response to K. rhinoscleromatis may be involved in the pathogenesis of RS in endemic areas.

Delineation of late onset hypoventilation associated with hypothalamic dysfunction syndrome.

Late Onset Central Hypoventilation Syndrome associated with Hypothalamic Dysfunction (LO-CHS/HD) is a distinct entity among the clinical and genetic heterogeneous group of patients with late onset central hypoventilation. Here we report a series of 13 patients with LO-CHS/HD. Rapid onset obesity is the first symptom of HD followed by hypoventilation with a mean delay of 18 mos. The outcome remains poor for this group of patients and would benefit from early diagnosis to anticipate ventilation and possible metabolic disorders. Tumor predisposition is more frequent than initially suspected and as high as 40% in this series. These tumors of the sympathetic nervous system (TSNS) are usually differentiated and do not significantly worsen the prognosis. We report a familial case with recurrence in siblings. The cause underlying LO-CHS/HD remains poorly understood although recurrence in siblings argues for a monogenic disorder. We ruled out PHOX2B, ASCL1, and NECDIN as disease-causing genes by direct sequencing in our series of patients and discuss possible disease-causing mechanisms.

Methylation-associated PHOX2B gene silencing is a rare event in human neuroblastoma.

Neuroblastoma (NB), an embryonic tumour originating from neural crest cells, is one of the most common solid tumours in childhood. Although NB is characterised by numerous recurrent, large-scale chromosome rearrangements, the genes targeted by these imbalances have remained elusive. We recently identified the paired-like homeobox 2B (PHOX2B, MIM 603851) gene as disease-causing in dysautonomic disorders including Congenital Central Hypoventilation Syndrome (CCHS), Hirschsprung disease (HSCR) and NB in various combinations. Most patients with NB due to a germline heterozygous PHOX2B gene mutation are familial and/or syndromic. PHOX2B, at chromosome 4p12, does not lie in a commonly rearranged locus in NB. To evaluate the role of PHOX2B in sporadic, isolated NB, we analysed 13 NB cell lines and 45 tumours for expression, mutations of coding and promoter sequences, loss of heterozygosity (LOH), or aberrant hypermethylation of PHOX2B (13 cell lines and 18 tumours). We didn't identify any mutation but LOH in about 10% of the cases and aberrant CpG dinucleotide methylation of the 500 bp PHOX2B promoter region in 4/31 tumours and cell lines (12.9%). Altogether, both germinal and somatic anomalies at the PHOX2B locus are found in NB.