Gonococcal Infection and Ventriculoperitoneal Shunts.

ABSTRACT: Neisseria gonorrhoea e and Chlamydia trachomatis are pathogens commonly isolated in pelvic inflammatory disease. Neisseria gonorrhoea e may uncommonly spread outside the urogenital tract to cause complications. We present 2 cases of adolescents with ventriculoperitoneal shunt infection due to N. gonorrhoea e, requiring shunt externalization.

Asthma as a risk factor for hospitalization in children with COVID-19: A nested case-control study.

BACKGROUND: Most pediatric studies of asthma and COVID-19 to date have been ecological, which offer limited insight. We evaluated the association between asthma and COVID-19 at an individual level. METHODS: Using data from prospective clinical registries, we conducted a nested case-control study comparing three groups: children with COVID-19 and underlying asthma ("A+C" cases); children with COVID-19 without underlying disease ("C+" controls); and children with asthma without COVID-19 ("A+" controls). RESULTS: The cohort included 142 A+C cases, 1110 C+ controls, and 140 A+ controls. A+C cases were more likely than C+ controls to present with dyspnea and wheezing, to receive pharmacologic treatment including systemic steroids (all p < .01), and to be hospitalized (4.9% vs. 1.7%, p = .01). In the adjusted analysis, A+C cases were nearly 4 times more likely to be hospitalized than C+ controls (adjusted OR = 3.95 [95%CI = 1.4-10.9]); however, length of stay and respiratory support level did not differ between groups. Among A+C cases, 8.5% presented with an asthma exacerbation and another 6.3% developed acute exacerbation symptoms shortly after testing positive for SARS-CoV-2. Compared to historic A+ controls, A+C cases had less severe asthma, were less likely to be on controller medications, and had better asthma symptom control (all p < .01). CONCLUSIONS: In our cohort, asthma was a risk factor for hospitalization in children with COVID-19, but not for worse COVID-19 outcomes. SARS-CoV-2 does not seem to be a strong trigger for pediatric asthma exacerbations. Asthma severity was not associated with higher risk of COVID-19.

Emerging arboviruses and implications for pediatric transplantation: A review.

Recent years have brought a rise in newly emergent viral infections, primarily in the form of previously known arthropod-transmitted viruses that have increased significantly in both incidence and geographical range. Of particular note are DENV, CHIKV, and ZIKV, which are transmitted mostly by Aedes species of mosquitoes that exhibit a wide and increasing global distribution. Being important pathogens for the general population, these viruses have the potential to be devastating in the international transplant community, with graft rejection and death as possible outcomes of infection. In this review, we discuss the current state of knowledge for these viruses as well as repercussions of infection in the solid organ and HSCT population, with a focus, when possible, on pediatric patients.

Coronavirus replicase-reporter fusions provide quantitative analysis of replication and replication complex formation.

UNLABELLED: The replication of coronaviruses occurs in association with multiple virus-induced membrane structures that evolve during the course of infection; however, the dynamics of this process remain poorly understood. Previous studies of coronavirus replication complex organization and protein interactions have utilized protein overexpression studies and immunofluorescence of fixed cells. Additionally, live-imaging studies of coronavirus replicase proteins have used fluorescent reporter molecules fused to replicase proteins, but expressed from nonnative locations, mostly late-transcribed subgenomic mRNAs, in the presence or absence of the native protein. Thus, the timing and targeting of native replicase proteins expressed in real time from native locations in the genome remain unknown. In this study, we tested whether reporter molecules could be expressed from the replicase polyprotein of murine hepatitis virus as fusions with nonstructural protein 2 or 3 and whether such reporters could define the targeting and activity of replicase proteins during infection. We demonstrate that the fusion of green fluorescent protein and firefly luciferase with either nonstructural protein 2 or 3 is tolerated and that these reporter-replicase fusions can be used to quantitate replication complex formation and virus replication. The results show that the replicase gene has flexibility to accommodate a foreign gene addition and can be used directly to study replicase complex formation and evolution during infection as well as to provide highly sensitive and specific markers for protein translation and genome replication. IMPORTANCE: Coronaviruses are a family of enveloped, positive-sense RNA viruses that are important agents of disease, including severe acute respiratory syndrome coronavirus and Middle East respiratory syndrome coronavirus. Replication is associated with multiple virus-induced membrane structures that evolve during infection; however, the dynamics of this process remain poorly understood. In this study, we tested whether reporter molecules expressed from native locations within the replicase polyprotein of murine hepatitis virus as fusions with nonstructural proteins could define the expression and targeting of replicase proteins during infection in live cells. We demonstrate that the replicase gene tolerates the introduction of green fluorescent protein or firefly luciferase as fusions with replicase proteins. These viruses allow early quantitation of virus replication as well as real-time measurement of replication complexes.

Contemporary enterovirus-D68 isolates infect human spinal cord organoids.

Enterovirus D68 (EV-D68) is a nonpolio enterovirus associated with severe respiratory illness and acute flaccid myelitis (AFM), a polio-like illness causing paralysis in children. AFM outbreaks have been associated with increased circulation and genetic diversity of EV-D68 since 2014, although the virus was discovered in the 1960s. The mechanisms by which EV-D68 targets the central nervous system are unknown. Since enteroviruses are human pathogens that do not routinely infect other animal species, establishment of a human model of the central nervous system is essential for understanding pathogenesis. Here, we describe two human spinal cord organoid (hSCO)-based models for EV-D68 infection derived from induced, pluripotent stem cell (iPSC) lines. One hSCO model consists primarily of spinal motor neurons, while the another model comprises multiple neuronal cell lineages, including motor neurons, interneurons, and glial cells. These hSCOs can be productively infected with contemporary strains, but not a historic strain, of EV-D68 and produce extracellular virus for at least 2 weeks without appreciable cytopathic effect. By comparison, infection with hSCO with another enterovirus, echovirus 11, causes significant structural destruction and apoptosis. Together, these findings suggest that EV-D68 infection is not the sole mediator of neuronal cell death in the spinal cord in those with AFM and that secondary injury from the immune response likely contributes to pathogenesis. IMPORTANCE AFM is a rare condition that causes significant morbidity in affected children, often contributing to life-long sequelae. It is unknown how EV-D68 causes paralysis in children, and effective therapeutic and preventative strategies are not available. Mice are not native hosts for EV-D68, and thus, existing mouse models use immunosuppressed or neonatal mice, mouse-adapted viruses, or intracranial inoculations. To complement existing models, we report two hSCO models for EV-D68 infection. These three-dimensional, multicellular models comprised human cells and include multiple neural lineages, including motor neurons, interneurons, and glial cells. These new hSCO models for EV-D68 infection will contribute to understanding how EV-D68 damages the human spinal cord, which could lead to new therapeutic and prophylactic strategies for this virus.

Setting the Stage for the Next Great Vaccine Success Story.

In this Commentary, the article by Rosenfeld et al. "Cross-Reactive Antibody Responses against Nonpoliovirus Enteroviruses" is put into context of the historic poliovirus epidemics and resultant vaccination success story as it compares to the current state of acute flaccid myelitis; the relationship to nonpoliovirus enteroviruses (EVs), in particular EV-D68 and EV-A71; and the potential for successful vaccination strategies. The discovery of cross-protective antibody neutralization among polio and nonpolio enteroviruses, specifically EV-D68, opens future questions about EV-D68 vaccination strategies, circulation patterns of nonpolio enteroviruses, and the interpretation of EV-D68 serostudies.

Performance of the Sofia SARS-CoV-2 Rapid Antigen Test in Symptomatic and Asymptomatic Pediatric Patients.

The sensitivity and specificity of SARS-CoV-2 antigen tests have not been widely assessed in children. We evaluated children presenting to outpatient care with Quidel Sofia SARS-CoV-2 antigen test (Sofia-Ag-RDT) compared against Cepheid Xpert Xpress SARS-CoV-2/Flu/RSV reverse transcriptase-polymerase chain reaction test from November 2020 to April 2021. Sofia-Ag-RDT had the highest sensitivity in symptomatic (82%; 95% confidence interval, 68%-91%) children.

Neonatal Toxic Shock Syndrome-Like Exanthematous Disease in North America.

Neonatal toxic shock syndrome (TSS)-like exanthematous disease (NTED) is a syndrome first reported in Japan. Neonates develop systemic exanthema, thrombocytopenia, and fever usually during the first week of life. The disease is distinguished from frank TSS because affected infants are not severely ill and do not meet TSS criteria. Most infants are confirmed to be colonized with TSST-1 producing strains of S. aureus. Suggested diagnostic criteria for NTED include a skin rash with generalized macular erythema and one of the following symptoms: fever >38.0°C, thrombocytopenia <150 x103uL, or low positive C-reactive protein (1-5 mg/dL) in the absence of another known disease process. NTED is common in Japanese NICUs, but outside Japan, only one case has been reported in France. We describe the first case of NTED reported in North America.

Unexpected False-Positive Rates in Pediatric SARS-CoV-2 Serology Using the EUROIMMUN Anti-SARS-CoV-2 ELISA IgG Assay.

OBJECTIVES: Serologic assay performance studies for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-​2) in pediatric populations are lacking, and few seroprevalence studies have routinely incorporated orthogonal testing to improve accuracy. METHODS: Remnant serum samples for routine bloodwork from 2,338 pediatric patients at UPMC Children's Hospital of Pittsburgh were assessed using the EUROIMMUN Anti-SARS-CoV-2 ELISA IgG (EuroIGG) assay. Reactive cases with sufficient volume were also tested using 3 additional commercial assays. RESULTS: Eighty-five specimens were reactive according to the EuroIGG, yielding 3.64% (95% confidence interval [CI], 2.91%-4.48%) seropositivity, of which 73 specimens had sufficient remaining volume for confirmation by orthogonal testing. Overall, 19.18% (95% CI, 10.18%-28.18%) of samples were positive on a second and/or third orthogonal assay. This 80.82% false positivity rate is disproportionate to the expected false positivity rate of 50% given our pediatric population prevalence and assay performance. CONCLUSIONS: In pediatric populations, false-positive SARS-CoV-2 serology may be more common than assay and prevalence parameters would predict, and further studies are needed to establish the performance of SARS-CoV-2 serology in children.

Immunocompromised Seroprevalence and Course of Illness of SARS-CoV-2 in One Pediatric Quaternary Care Center.

BACKGROUND: The burden of coronavirus disease 2019 (COVID-19) is poorly understood in pediatric patients due to frequent asymptomatic and mild presentations. Additionally, the disease prevalence in pediatric immunocompromised patients remains unknown. METHODS: This cross-sectional study tested convenience samples from pediatric patients who had clinically indicated lab work collected and an immunocompromising condition, including oncologic diagnoses, solid organ transplant (SOT), bone marrow transplant, primary immunodeficiency, and rheumatologic conditions or inflammatory bowel disease on systemic immunosuppression, for the presence of antibodies to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). RESULTS: We tested sera from 485 children and observed SARS-CoV-2 seroprevalence of 1.0% (Confidence Interval [CI] 95%: 0.3%-2.4%). Two patients were positive by nasopharyngeal (NP) swab Reverse transcriptase polymerase chain reaction (RT-PCR), but only 1 seroconverted. Patients with oncologic diagnoses or SOT were most likely to be tested for COVID-19 when presenting with respiratory illness as compared with other groups. CONCLUSIONS: Seroprevalence of antibodies to SARS-CoV-2 in immunocompromised children was similar to that of an immunocompetent pediatric population (0.6%, CI 95%: 0.3%-1.1%), suggesting an adequate antibody response. However, none of the patients who tested positive for antibodies or via NP RT-PCR had more than a mild illness course and 2 patients did not have any reported illness, suggesting that SARS-CoV-2 may not cause a worse clinical outcome in immunosuppressed children, in contrast to immunocompromised adults.

Pediatric SARS-CoV-2 seroprevalence during mitigation procedures in Southwestern Pennsylvania.

Background: Children infected with SARS-CoV-2 are often asymptomatic or have only mild symptoms, leading to underestimation of disease prevalence in symptom-based testing strategies. Objectives: This study sought to determine pediatric SARS-CoV-2 disease burden during local mitigation efforts by using antibody testing to compare seroprevalence estimates to cumulative PCR prevalence estimates. Study design: In this cross-sectional study, we collected 1142 strict phase and 1196 relaxed phase remnant blood specimens from patients less than 19-years-old in southwestern Pennsylvania (SWPA). Patients were excluded if their residential zip code was outside the region of interest, if they were under 6-months-old, or they had recently received antibody-modifying treatments. Demographic, encounter, and laboratory electronic medical record information was extracted. Samples were tested for SARS-CoV-2 spike protein IgG using an EUA ELISA, and PCR results were recorded from county health department data. Seroprevalence and Clopper-Pearson exact 95% confidence intervals were calculated. Results: The observed seroprevalence of SARS-CoV-2 spike protein antibodies in children during strictest mitigation was 0.53% (95% CI 0.19, 1.14) and 0.92% (95% CI 0.46,1.64) during moderately relaxed. Strict and relaxed phase PCR-based prevalence were significantly higher, 2.87% (95% CI 1.95, 4.08) and 3.64 (95% CI 3.01, 4.38), respectively. Conclusions: Estimates of pediatric seroprevalence were significantly lower than cumulative PCR prevalence estimates, and less than adult seroprevalence estimates, potentially due to biological, population, or sampling differences. Biological differences in pediatric immune responses to SARS-CoV-2 may make serosurvey interpretation challenging and these differences warrant further study.

Respiratory and intestinal epithelial cells exhibit differential susceptibility and innate immune responses to contemporary EV-D68 isolates.

Enterovirus D68 (EV-D68) has been implicated in outbreaks of severe respiratory illness and is associated with acute flaccid myelitis (AFM). EV-D68 is often detected in patient respiratory samples but has also been detected in stool and wastewater, suggesting the potential for both respiratory and enteric routes of transmission. Here, we used a panel of EV-D68 isolates, including a historical pre-2014 isolate and multiple contemporary isolates from AFM outbreak years, to define the dynamics of viral replication and the host response to infection in primary human airway cells and stem cell-derived enteroids. We show that some recent EV-D68 isolates have decreased sensitivity to acid and temperature compared with earlier isolates and that the respiratory, but not intestinal, epithelium induces a robust type III interferon response that restricts infection. Our findings define the differential responses of the respiratory and intestinal epithelium to contemporary EV-D68 isolates and suggest that a subset of isolates have the potential to target both the human airway and gastrointestinal tracts.

A Comprehensive Clinical Description of Pediatric SARS-CoV-2 Infection in Western Pennsylvania.

Objective: We sought to characterize clinical presentation and healthcare utilization for pediatric COVID-19 in Western Pennsylvania (PA). Methods: We established and analyzed a registry of pediatric COVID-19 in Western PA that includes cases in patients <22 years of age cared for by the pediatric quaternary medical center in the area and its associated pediatric primary care network from March 11 through August 20, 2020. Results: Our cohort included 424 pediatric COVID-19 cases (mean age 12.5 years, 47.4% female); 65% reported exposure and 79% presented with symptoms. The most common initial healthcare contact was through telehealth (45%). Most cases were followed as outpatients, but twenty-two patients (4.5%) were hospitalized: 19 with acute COVID-19 disease, and three for multisystem inflammatory syndrome of children (MIS-C). Admitted patients were younger (p<0.001) and more likely to have pre-existing conditions (p<0.001). Black/Hispanic patients were 5.8 times more likely to be hospitalized than white patients (p=0.012). Five patients (1.2%) were admitted to the PICU, including all three MIS-C cases; two required BiPAP and one mechanical ventilation. All patients survived. Conclusions: We provide a comprehensive snapshot of pediatric COVID-19 disease in an area with low to moderate incidence. In this cohort, COVID-19 was generally a mild disease; however, ~5% of children were hospitalized. Pediatric patients can be critically ill with this infection, including those presenting with MIS-C.

Coronaviruses induce entry-independent, continuous macropinocytosis.

Macropinocytosis is exploited by many pathogens for entry into cells. Coronaviruses (CoVs) such as severe acute respiratory syndrome (SARS) CoV and Middle East respiratory syndrome CoV are important human pathogens; however, macropinocytosis during CoV infection has not been investigated. We demonstrate that the CoVs SARS CoV and murine hepatitis virus (MHV) induce macropinocytosis, which occurs late during infection, is continuous, and is not associated with virus entry. MHV-induced macropinocytosis results in vesicle internalization, as well as extended filopodia capable of fusing with distant cells. MHV-induced macropinocytosis requires fusogenic spike protein on the cell surface and is dependent on epidermal growth factor receptor activation. Inhibition of macropinocytosis reduces supernatant viral titers and syncytia but not intracellular virus titers. These results indicate that macropinocytosis likely facilitates CoV infection through enhanced cell-to-cell spreading. Our studies are the first to demonstrate virus use of macropinocytosis for a role other than entry and suggest a much broader potential exploitation of macropinocytosis in virus replication and host interactions. Importance: Coronaviruses (CoVs), including severe acute respiratory syndrome (SARS) CoV and Middle East respiratory syndrome CoV, are critical emerging human pathogens. Macropinocytosis is induced by many pathogens to enter host cells, but other functions for macropinocytosis in virus replication are unknown. In this work, we show that CoVs induce a macropinocytosis late in infection that is continuous, independent from cell entry, and associated with increased virus titers and cell fusion. Murine hepatitis virus macropinocytosis requires a fusogenic virus spike protein and signals through the epidermal growth factor receptor and the classical macropinocytosis pathway. These studies demonstrate CoV induction of macropinocytosis for a purpose other than entry and indicate that viruses likely exploit macropinocytosis at multiple steps in replication and pathogenesis.