Bacterial ligands as flexible and sensitive detectors in rapid tests for antibodies to SARS-CoV-2.

Lateral flow immunoassay (LFIA) is widely employed as point-of-care tests (POCT) for the diagnosis of infectious diseases. The accuracy of LFIA largely depends on the quality of the immunoreagents used. Typical LFIAs to reveal the immune response to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) employ anti-human immunoglobulin (hIG) antibodies and recombinant viral antigens, which usually are unstable and poorly soluble. Broad selective bacterial proteins, such as Staphylococcal protein A (SpA) and Streptococcal protein G (SpG) can be considered alternatives to anti-hIG to increase versatility and sensitivity of serological LFIAs because of their high binding capacity, interspecies reactivity, and robustness. We developed two colorimetric LFA devices including SpA and SpG linked to gold nanoparticles (GNP) as detectors and explored the use of a specific, stable, and soluble immunodominant fraction of the nucleocapsid protein from SARS-CoV-2 as the capturing agent. The optimal amount of SpA-GNP and SpG-GNP conjugates and the protein-to-GNP ratios were defined through a full factorial experimental design to maximize the diagnostic sensitivity of the LFIAs. The new LFA devices were applied to analyze 105 human serum samples (69 positive and 36 negatives according to reference molecular diagnostic methods). The results showed higher sensitivity (89.9%, 95% CI 82.7-97.0) and selectivity (91.7%, 82.6-100) for the SpA-based compared to the SpG-based LFA. In addition, 18 serum samples from cats and dogs living with COVID-19 patients were analyzed and 14 showed detectable levels of anti-SARS-CoV-2 antibodies, thus illustrating the flexibility of the SpA- and SpG-based LFAs.

Antimicrobial stewardship experience in paediatrics: first-year activity report.

Antimicrobial resistance is one of the most relevant threats in public health worldwide. Strategies as antimicrobial stewardship programs, aiming to preserve our antibiotic armamentarium, have been implemented since 2007 in adult and paediatric patients. We aim to describe the first experience of a paediatric antimicrobial stewardship program. We conducted a retrospective observational study in a tertiary care children's hospital. A team composed of a microbiologist, an infectious diseases physician, and a paediatrician led the project. All positive blood and cerebrospinal fluid cultures and other biological samples yielding multi-drug-resistant bacteria were collected and reviewed through a prospective-audit-with-feedback strategy. We recorded patient characteristics and worth monitoring prescribed antibiotics. The antimicrobial stewardship audit could end in intervention (step-up/step-down and broadening/narrowing) or recommendation(s). We then checked out wards staff compliance. The team performed 192 interventions out of 584 reviews, mostly suggesting discontinuation of antibiotics (in 76.0% of cases and 39.7% of running molecules). The antibiotic spectrum was more likely tapered than expanded (p < 0.0001), and we ordered more narrow-spectrum antibiotic molecules than local medical staff straightaway did (p = 0.0113). Interventions were most likely needed in case of documented infections (p < 0.0001) and in surgical patients (p = 0.0002). In 85.9% of interventions, ward teams fully agreed with our argument. This study demonstrated an antimicrobial stewardship program to be a suitable method for improving the appropriateness of antimicrobial use in hospitalized children.

COVID-19 in Children: Expressions of Type I/II/III Interferons, TRIM28, SETDB1, and Endogenous Retroviruses in Mild and Severe Cases.

Children with the new coronavirus disease 2019 (COVID-19) have milder symptoms and a better prognosis than adult patients. Several investigations assessed type I, II, and III interferon (IFN) signatures in SARS-CoV-2 infected adults, however no data are available for pediatric patients. TRIM28 and SETDB1 regulate the transcription of multiple genes involved in the immune response as well as of human endogenous retroviruses (HERVs). Exogenous viral infections can trigger the activation of HERVs, which in turn can induce inflammatory and immune reactions. Despite the potential cross-talks between SARS-CoV-2 infection and TRIM28, SETDB1, and HERVs, information on their expressions in COVID-19 patients is lacking. We assessed, through a PCR real time Taqman amplification assay, the transcription levels of six IFN-I stimulated genes, IFN-II and three of its sensitive genes, three IFN-lIIs, as well as of TRIM28, SETDB1, pol genes of HERV-H, -K, and -W families, and of env genes of Syncytin (SYN)1, SYN2, and multiple sclerosis-associated retrovirus (MRSV) in peripheral blood from COVID-19 children and in control uninfected subjects. Higher expression levels of IFN-I and IFN-II inducible genes were observed in 36 COVID-19 children with mild or moderate disease as compared to uninfected controls, whereas their concentrations decreased in 17 children with severe disease and in 11 with multisystem inflammatory syndrome (MIS-C). Similar findings were found for the expression of TRIM-28, SETDB1, and every HERV gene. Positive correlations emerged between the transcriptional levels of type I and II IFNs, TRIM28, SETDB1, and HERVs in COVID-19 patients. IFN-III expressions were comparable in each group of subjects. This preserved induction of IFN-λs could contribute to the better control of the infection in children as compared to adults, in whom IFN-III deficiency has been reported. The upregulation of IFN-I, IFN-II, TRIM28, SETDB1, and HERVs in children with mild symptoms, their declines in severe cases or with MIS-C, and the positive correlations of their transcription in SARS-CoV-2-infected children suggest that they may play important roles in conditioning the evolution of the infection.

Detection of faecal SARS-CoV-2 RNA in a prospective cohort of children with multisystem inflammatory syndrome (MIS-C).

BACKGROUND: Multisystem Inflammatory Syndrome in Children (MIS-C) is a rare but severe illness associated with SARS-CoV-2 infection. A dysregulated immune response is recognized as the main pathogenic mechanism. Previous studies demonstrated the presence of SARS-CoV-2 RNA in faeces of almost one-third of patients with COVID-19, while data are currently missing about MIS-C. OBJECTIVES: to evaluate faecal sample positivity to SARS-CoV-2 in MIS-C and to compare the positivity rate between MIS-C and COVID-19 hospitalised children.  DESIGN: observational descriptive study with prospective patient enrollment. SETTING AND PARTICIPANTS: the SARS-CoV-2 positivity was evaluated in stool samples obtained in a prospective series of 63 paediatric patients admitted to Regina Margherita Children's Hospital (Azienda Ospedaliero Universitaria - Città della Salute e della Scienza, Turin, Northern Italy) with diagnosis of MIS-C (N. 31) or COVID-19 (N. 32), during the first year of pandemic emergency. The real-time reverse transcription polymerase chain reaction (real-time RT-PCR), was performed using a validated kit measuring 3 target SARS-CoV-2 genes: E gene, N gene, and ORF1ab gene MAIN OUTCOME MEASURES: SARS-CoV-2 stool positivity and concomitant gastrointestinal symptoms. RESULTS: overall, 16/63 (25%) stool samples revealed the presence of SARS-CoV-2 mRNA. In patients with COVID-19, faecal samples were collected 8 days as median (IQR 7) after the presumed viral exposure and were positive in 12/31 (39%; 95%CI 23.2-56.2); among children with MIS-C, stools were collected 27.5 days as median (IQR 26.25) after presumed contact and the positivity rate was 12.5% (95%CI 4.4-27.0) (4/32). More than 80% of the children with MIS-C presented gastrointestinal symptoms, but the frequency of gastrointestinal symptoms in patients with positive stools for SARS-CoV-2 RNA is not higher than patients tested negative (p=0.092). CONCLUSIONS: MIS-C patients frequently experienced gastrointestinal symptoms, confirming the intestinal involvement in MIS-C already described in the literature. The presence of SARS-CoV-2 mRNA in faecal samples is confirmed in more than 10% of MIS-C patients and stool positivity was also detected many days after presumed first contact with the virus. This data suggests the possibility of tracing SARS-COV-2 also in faeces for a better description of its circulation and spread in the environment.

Multicentre Italian study of SARS-CoV-2 infection in children and adolescents, preliminary data as at 10 April 2020.

Data on features of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in children and adolescents are scarce. We report preliminary results of an Italian multicentre study comprising 168 laboratory-confirmed paediatric cases (median: 2.3 years, range: 1 day-17.7 years, 55.9% males), of which 67.9% were hospitalised and 19.6% had comorbidities. Fever was the most common symptom, gastrointestinal manifestations were frequent; two children required intensive care, five had seizures, 49 received experimental treatments and all recovered.

Magicplex(TM) Sepsis Real-Time test to improve bloodstream infection diagnostics in children.

UNLABELLED: Sepsis is the major cause of morbidity and mortality in children, especially in immunocompromised patients, and a rapid identification of causative pathogen is strongly related with a better outcome. This prospective study analyzes the role of a multiplex real-time polymerase chain reaction in sepsis' etiological diagnosis. Magicplex(TM) Sepsis Real-Time tests were performed in tertiary Regina Margherita Children's Hospital (Turin, Italy), and the medical records of children who underwent a Magicplex test were prospectively evaluated. Results of the Magicplex test were compared with those of blood culture collected at a close time point. One hundred fifty Magicplex tests were collected from 89 patients (54 males and 35 females, age interquartile range: 2.6-12.1 years). Etiological definition was achieved in 60 bloodstream infection cases (40 %). In 32 episodes, Magicplex test alone gave a positive result, and blood culture alone permitted the etiological diagnosis in 5 septic episodes. Magicplex test allowed a 143 % increase in the diagnostic value of blood cultures. CONCLUSION: These results suggest that molecular biology can be useful for rapid pathogen's identification also in children. WHAT IS KNOWN: • Sepsis represents a major cause of morbidity and mortality in children. • Sepsis outcome is strongly related to rapid microbiological identification and prompt initiation of an appropriate chemotherapy. What is New: • This manuscript is the first that describes the use of Magicplex (TM) Sepsis Real-Time test in children. • The results suggest that molecular biology can be useful for rapid pathogen's identification also in children.

Red Code Management in a Pediatric Emergency Department: A Retrospective Study.

The "red code" (RC) represents the highest level of emergency in the emergency department (ED). This study retrospectively analyzed RCs in the Regina Margherita Children's Hospital ED, a regional referral center in north Italy, between 1 July 2020 and 30 June 2023. The aim was to describe RC characteristics and to identify significant correlations between presenting complaints and clinical management. The study includes 934 RCs (0.9% of overall ED admissions); 64% were assigned based on the Pediatric Assessment Triangle alteration. Most patients, 86.5%, followed the medical pathway, while 13.5% were surgical cases. Admission complaints were respiratory (46.9%), neuropsychiatric (26.7%), traumatic (11.8%), cardiologic (9.3%), metabolic (3.8%), and surgical (1.5%). Seventy-six percent of patients received vascular access, and intraosseous access was obtained in 2.2% of them. In one-third of RCs, an urgent critical care evaluation was necessary, and 19% of cases required admission to the intensive care unit. The overall mortality rate was 3.4% (0.4% in ED setting). The study identified six distinct diagnostic pathways, each associated with specific characteristics in clinical presentation, management, therapeutic interventions, and outcomes. Our findings underscore the need for a systematic approach in pediatric emergency settings, supported by international and national guidelines but also by clearly defined diagnostic pathways, aiming to enhance the quality of care and patient outcomes.

Long COVID-19/post-COVID condition in children: do we all speak the same language?

Post-COVID condition is a new and highly debated entity that is still to be outlined in its complexity, especially in the pediatric population. In response to the article by Trapani and colleagues, we report the results of a long-term follow-up conducted in the outpatient clinic of the Pediatric Infectious Diseases Unit on children admitted to our hospital with SARS-CoV-2 infection.

A Neuropsychiatric Assessment of Children with Previous SARS-CoV-2 Infection.

AIM: Concerns have been raised about possible neuropsychiatric sequelae of COVID-19. The objective of this study was to examine the plausibility of long-term mental health consequences of COVID-19 by assessing a sample of children after the resolution of the acute SARS-CoV-2 infection. METHOD: As part of a systematic follow-up assessment of pediatric patients with COVID-19 conducted at two university children's hospitals, 50 children (56% males) aged 8 to 17 years (median 11.5), 26% with previous multisystem inflammatory syndrome in children (MIS-C), without a prior history of neuropsychiatric disorders, received a battery of clinical neuropsychiatric and neuropsychological rating scales that included the Pediatric Migraine Disability Assessment (PedMIDAS), Sleep Disturbance Scale for Children (SDSC), Multidimensional Anxiety Scale for Children (MASC-2), Child Depression Inventory (CDI-2), Child Behavior Checklist (CBCL), and the NEPSY II (Neuropsychological Assessment, Second Edition). The assessments were conducted between 1 and 18 months (median 8 months) after the acute infection. RESULTS: The CBCL internalizing symptoms score was in the clinical range for 40% of the participants (vs. a population expected rate of about 10%, p < 0.001). A sleep disturbance was detected in 28%, clinically significant anxiety in 48%, and depressive symptoms in 16%. The NEPSY II scores showed impairment in attention and other executive functions in 52%, and memory deficits in 40% of the children. CONCLUSIONS: These data from direct assessment of a sample of children who had SARS-CoV-2 infection show higher than expected rates of neuropsychiatric symptoms, thus supporting the possibility that COVID-19 may have mental health sequelae long after the resolution of the acute infection.

The Importance of RSV Epidemiological Surveillance: A Multicenter Observational Study of RSV Infection during the COVID-19 Pandemic.

The restrictive measures adopted worldwide against SARS-CoV-2 produced a drastic reduction in respiratory pathogens, including RSV, but a dramatic rebound was thereafter reported. In this multicenter retrospective observational study in 15 Pediatric Emergency Departments, all children <3 years old with RSV infection admitted between 1 September and 31 December 2021 were included and compared to those admitted in the same period of 2020 and 2019. The primary aim was to evaluate RSV epidemiology during and after the COVID-19 pandemic peak. The secondary aims were to evaluate the clinical features of children with RSV infection. Overall, 1015 children were enrolled: 100 in 2019, 3 in 2020 and 912 in 2021. In 2019, the peak was recorded in December, and in 2021, it was recorded in November. Comparing 2019 to 2021, in 2021 the median age was significantly higher and the age group 2-3 years was more affected. Admissions were significantly higher in 2021 than in 2020 and 2019, and the per-year hospitalization rate was lower in 2021 (84% vs. 93% in 2019), while the duration of admissions was similar. No difference was found in severity between 2019-2020-2021. In conclusion, after the COVID-19 pandemic, an increase in RSV cases in 2021 exceeding the median seasonal peak was detected, with the involvement of older children, while no difference was found in severity.

Can we trust in Sars-CoV-2 rapid antigen testing? Preliminary results from a paediatric cohort in the emergency department.

BACKGROUND: Rapid identification of Covid-19 in the paediatric emergency department is critical; Antigen tests are fast but poorly investigated in children. AIMS: To investigate Sars-CoV-2 antigen rapid test in children. METHODS: We compare the performance of LumiraDx with molecular tests in a paediatric emergency department. RESULTS: A retrospective cohort of 191 patients with AT and PCR tests performed in the same episode was analysed; 16% resulted positive for Sars-CoV-2. Using the PCR test as the gold standard, we calculated antigen testing overall sensitivity of 94.1%, specificity of 91.9%, and NPV of 99.4%. Only one false-negative test was found. CONCLUSIONS: AT may be helpful in the initial screening of patients at PED.

Imported malaria in children: A 13 years retrospective study.

Malaria is not endemic in Italy, however every year about 600-700 imported cases are detected in people born or living in Italy who return from a stay in their country of origin (visiting friends and relatives - VFR). Children account for 20% of this population and they have an higher risk of severe disease. Socio-economic problems and deficiencies in the doctor-patient relationship often lead to a lack of awareness of the importance of prophylaxis, making the category of VFRs at increased risk of disease. The aim of this study is to analyze the characteristics of pediatric imported malaria, with a specific focus on prevention and risk factors for severe malaria. All malaria cases diagnosed from 2007 to 2019 in Ospedale Infantile Regina Margherita in Turin were retrospectively observed. Epidemiological and clinical data were described. A total of 72 patients were reported: 98.6% had African origins and 73.6% traveled as VFRs. Plasmodium falciparum was the species most commonly isolated (94.4%). Twenty-four patients (33.3%) underwent chemoprophylaxis and never appropriately. Patients not undergoing prophylaxis and those undergoing incomplete prophylaxis showed not statistically significant difference in term of disease severity (p = 0.26). Nineteen cases were considered severe, including 3 with cerebral malaria. High levels of parasitemia were statistically significantly correlated with severe anemia (p = 0.049) and severe thrombocytopenia (p = 0.036). In 25% of cases the first diagnosis was incorrect. The therapeutic use of artemisinin derivatives has resulted in a significant shortening of the parasitemia clearance time compared to the use of other drugs (p < 0.001). Families have to be educated about the serious implications of a malaria infection and the importance of a correct and complete prophylaxis. Clinicians should always consider malaria in the differential diagnoses in patients with fever and a history of a recent travel to an endemic area. Prompt diagnosis and use of appropriate drugs, according to the latest guidelines, could guarantee a better outcome for patients.

Increase in newly diagnosed type 1 diabetes and serological evidence of recent SARS-CoV-2 infection: Is there a connection?

Several studies have investigated the correlation between the COVID-19 pandemic and the onset of type 1 diabetes (T1D) in children, reporting an increased incidence of T1D and severe diabetic ketoacidosis (DKA). This study aimed to investigate the infection by SARS-CoV-2 in children with newly-diagnosed T1D to explore a possible link between SARS-CoV-2 infection, T1D and DKA. Thirty-nine children with a T1D new onset between October 15, 2020, and April 15, 2021, were enrolled. SARS-CoV-2 infection was investigated through a polymerase chain reaction on the nasal swab, dosage of specific antibodies, and an anamnestic question form. Nine (23%) of them had antibodies directed toward SARS-CoV-2, and five (12%) had a history of recent SARS-CoV-2 infection in themselves or in their family. No molecular swabs were positive. Compared to the general pediatric population, the overall incidence of COVID-19 was 5.6 times higher in the T1D patients' group (p < 0.00001). Referring only to the cases in the metropolitan area, we find a net increase in the incidence of T1D compared to the 5 years preceding our study, by 50% compared to the same months in 2016/2017 and 2017/2018, by 69% compared to 2018/2019 and by 77% compared to 2019/2020. The same trend was observed regarding DKA cases. The attributable risk of the pandemic cohort compared to the previous year is 44%. The abnormal disproportion of SARS-CoV-2 infection between children with T1D and the pediatric reference population, with a ratio of 5.6, appears to support the causative role of SARS-CoV-2 in triggering the immune response underlying diabetes, as often described for other viral infections. The difficulty accessing care services during the pandemic, with a consequent diagnosis delay, does not justify the increase in observed T1D cases, which could to be directly linked to the pandemic. The acceleration of the immune process provoked by SARS-CoV-2 may play a suggestive role in the development of T1D with DKA. Multicenter studies are needed to deepen and fully understand the pathophysiological link between SARS-CoV-2 and the onset of T1D in children.

Validation of the Giannella Risk Score for the Prediction of Infection by Carbapenemase-producing Enterobacteriaceae in the Pediatric Population.

BACKGROUND: Despite efforts made to prevent the spread of multi-drug-resistant bacteria, carbapenemase-producing Enterobacteriaceae (CPE) has become one of the most dangerous threat worldwide. However, data on the epidemiology of CPE and on the correlation between CPE colonization and infection are scanty. The objectives of this study were first to describe the epidemiologic characteristics of colonizations and invasive CPE infections in the pediatric population, and second, to apply the Giannella Risk Score (GRS) to the pediatric population for the assessment of the risk of invasive CPE infection in patients with already known colonization. METHODS: Pediatric patients with evidence of colonization by CPE were retrospectively enrolled. For each colonized patient, the subsequent development of an infection by CPE was then assessed for a 90-day period after the first CPE isolation; GRSs were compared between patients who had developed any type of CPE infection and those without infection. RESULTS: A total of 215 patients (113 males and 102 females) with at least 1 isolation of CPE during hospitalization were analyzed. Median age was 5.6 years [interquartile range (IQR), 1.89-12.2 years]. Overall, 28 CPE infections (13%) were documented: 23 blood stream infections and 5 complicated urinary tract infections. The 30-day mortality of invasive CPE infections was 34.8%. The GRS values in patients with any CPE infection were statistically higher than in noninfected patients: median GRS 9 (IQR, 4-12.5) versus 4 (IQR, 2-4), respectively; P < 0.0001. The analysis of the receiver operating characteristic curves identified a GRS cut-off value ≥8 as the best predictor of CPE infection. The likelihood ratio of the results was <2 and the informedness of the test had a value <0.50. CONCLUSIONS: Our study confirms that the spread of CPE is an impelling problem also in the pediatric population, with a high mortality rate of invasive infections. However, the application of the GRS appears to be poorly informative in the pediatric setting; it might sometimes help to identify patients at very low-risk of CPE infection, in whom it is reasonable to spare targeted antimicrobial treatments.

COVID-19 in Infants Less than 3 Months: Severe or Not Severe Disease?

Compared to adults, severe or fatal COVID-19 disease is much less common in children. However, a higher risk for progression has been reported in infants. Different pediatric COVID-19 severity scores are reported in the literature. Methods: Subjects under 90 days of age admitted to 35 Italian institutions for COVID-19 were included. The severity of COVID-19 was scored as mild/moderate or severe/critical following the classification reported in the literature by Venturini, Dong, Kanburoglu, and Gale. To assess the diagnostic accuracy of each classification system, we stratified all enrolled patients developing a posteriori severity score based on clinical presentation and outcomes and then compared all different scores analyzed. Results: We included 216 infants below 90 days of age. The most common symptom was fever, followed by coryza, poor feeding, cough, and gastrointestinal manifestations. According to Venturini, Dong, Kanburoglu, and Gale's severity scores, 18%, 6%, 4.2%, and 29.6% of infants presented with severe/critical disease, respectively. A correlation analysis between these four scores and the a posteriori severity score assigned to all enrolled subjects was performed, and a crescent strength of correlation from Gale (R = 0.355, p < 0.001) to Venturini (R = 0.425, p < 0.001), Dong (R = 0.734, p < 0.001), and Kanburoglu (R = 0.859, p < 0.001) was observed. Conclusions: The percentage of infants with severe COVID-19 varies widely according to the score systems. A unique clinical score should be designed for neonates and infants with COVID-19.

Pediatric Use of Recombinant Human Nerve Growth Factor 20 µg/mL Eye Drops (Cenegermin) for Bilateral Neurotrophic Keratopathy in Congenital Corneal Anesthesia.

PURPOSE: This study aimed to present the efficacy and safety of cenegermin eye drop (Oxervate; Dompè Farmaceutici, Milan, Italy) treatment in a pediatric patient affected by neurotrophic keratopathy (NK) with Goldenhar syndrome. METHODS: This case reports an infant presenting ulceration and a small central opacity in the cornea of the right and left eyes, respectively. The NK bilaterally worsened despite the use of therapeutic contact lenses and temporary partial tarsorrhaphy. Magnetic resonance imaging showed absence and hypoplasia of the right and left trigeminal nerves, respectively. Cenegermin eye drops were administered 1 drop/each eye, 6 times daily for 8 weeks to promote corneal healing. RESULTS: Complete healing was achieved in both eyes after treatment. During the 16-month follow-up period, no epithelial defect, recurrence, or complications were noticed, whereas corneal opacities progressively became clearer, although insignificant improvements in corneal sensitivity or in the reflex tearing were observed. CONCLUSIONS: Cenegermin was effective in treating NK in an infant with Goldenhar syndrome.

A multi-target lateral flow immunoassay enabling the specific and sensitive detection of total antibodies to SARS COV-2.

A rapid test for detecting total immunoglobulins directed towards the nucleocapsid protein (N) of severe acute syndrome coronavirus 2 (SARS CoV-2) was developed, based on a multi-target lateral flow immunoassay comprising two test lines. Both test lines bound to several classes of immunoglobulins (G, M, and A). Specific anti-SARS immunoglobulins were revealed by a colorimetric probe formed by N and gold nanoparticles. Targeting the total antibodies response to infection enabled achieving 100% diagnostic specificity (95.75-100, C.I. 95%, n = 85 healthy and with other infections individuals) and 94.6% sensitivity (84.9-98.9, C.I. 95%, n = 62 SARS CoV-2 infected subjects) as early as 7 days post confirmation of positivity. Agreeing results with a reference serological ELISA were achieved, except for the earlier detection capability of the rapid test. Follow up of the three seroconverting patients endorsed the hypothesis of the random rise of the different immunoglobulins and strengthened the 'total antibodies' approach for the trustworthy detection of serological response to SARS CoV-2 infection.