Universal Newborn Screening for Congenital Cytomegalovirus Infection - From Infant to Maternal Infection: A Prospective Multicenter Study

Introduction: Most infants at risk for cytomegalovirus (CMV)-associated sensorineural hearing loss (SNHL) are unrecognized because of the absence of a universal neonatal CMV screening. The search of CMV-DNA by molecular methods in salivary swabs was demonstrated to be a reliable approach. This study describes the results obtained by carrying out a universal screening for congenital CMV (cCMV) infection including all live-born newborns in three Italian sites, as well as the therapeutic interventions and clinical outcome of the CMV-infected neonates. Moreover, CMV maternal infection's characteristics were evaluated. Methods: To confirm or exclude cCMV infection, a CMV-DNA-positive result on a first salivary swab was followed by repeated saliva and urine samples collected within 21 days of age. Breast milk samples were also collected. The search of CMV-DNA was performed with a single automated quantitative commercial real-time PCR assay, regardless of the type of samples used. Results: A total of 3,151 newborns were enrolled; 21 (0.66%) of them were congenitally infected (median saliva viral load at screening, 6.65 [range, 5.03-7.17] log10 IU/ml). Very low/low viral load in screening saliva samples (median value, 1.87 [range, 1.14-2.59] log10 IU/ml) was associated with false-positive results (n = 54; 1.7%). CMV-DNA was detected in almost half of the breast milk samples of mother-infant pairs with a false-positive result, suggesting that contamination from breast milk may not be the only explanation in the study population. cCMV infection confirmation with the search of CMV-DNA in a urine sample proved to be the gold standard strategy, since false-positive results were observed in 4/54 (7.5%) of the repeated saliva samples. Symptomatic cCMV infection was observed in 3/21 (14.3%) infants; notably, one (4.7%) developed moderate unilateral SNHL at 5 months after birth. Finally, two symptomatic cCMV infections were associated with primary maternal infection acquired in the first trimester of gestation; one newborn with severe cCMV symptoms was born to a mother with no CMV checkups in pregnancy. Conclusion: Without universal neonatal CMV screening, some infected infants who develop late neurological sequelae may not be recognized and, consequently, they are not able to benefit early from instrumental and therapeutic interventions to limit and/or treat CMV disease

Feasibility and Effectiveness Assessment of SARS-CoV-2 Antigenic Tests in Mass Screening of a Pediatric Population and Correlation with the Kinetics of Viral Loads

The gold standard for diagnosis of SARS-CoV-2 infection has been nucleic acid amplification tests (NAAT). However, rapid antigen detection kits (Ag-RDTs), may offer advantages over NAAT in mass screening, generating results in minutes, both as laboratory-based test or point-of-care (POC) use for clinicians, at a lower cost. We assessed two different POC Ag-RDTs in mass screening versus NAAT for SARS-CoV-2 in a cohort of pediatric patients admitted to the Pediatric Emergency Unit of IRCCS—Polyclinic of Sant’Orsola, Bologna (from November 2020 to April 2021). All patients were screened with nasopharyngeal swabs for the detection of SARS-CoV-2-RNA and for antigen tests. Results were obtained from 1146 patients. The COVID-19 Ag FIA kit showed a baseline sensitivity of 53.8% (CI 35.4–71.4%), baseline specificity 99.7% (CI 98.4–100%) and overall accuracy of 80% (95% CI 0.68–0.91); the AFIAS COVID-19 Ag kit, baseline sensitivity of 86.4% (CI 75.0–93.9%), baseline specificity 98.3% (CI 97.1–99.1%) and overall accuracy of 95.3% (95% CI 0.92–0.99). In both tests, some samples showed very low viral load and negative Ag-RDT. This disagreement may reflect the positive inability of Ag-RDTs of detecting antigen in late phase of infection. Among all cases with positive molecular test and negative antigen test, none showed viral loads > 106 copies/mL. Finally, we found one false Ag-RDTs negative result (low cycle thresholds; 9 × 105 copies/mL). Our results suggest that both Ag-RDTs showed good performances in detection of high viral load samples, making it a feasible and effective tool for mass screening in actively infected children

Fetal Brain Damage in Human Fetuses with Congenital Cytomegalovirus Infection: Histological Features and Viral Tropism

Human cytomegalovirus (HCMV) causes congenital neurological lifelong disabilities. To date, the neuropathogenesis of brain injury related to congenital HCMV (cCMV) infection is poorly understood. This study evaluates the characteristics and pathogenetic mechanisms of encephalic damage in cCMV infection. Ten HCMV-infected human fetuses at 21 weeks of gestation were examined. Specifically, tissues from different brain areas were analyzed by: (i) immunohistochemistry (IHC) to detect HCMV-infected cell distribution, (ii) hematoxylin-eosin staining to evaluate histological damage and (iii) real-time PCR to quantify tissue viral load (HCMV-DNA). The differentiation stage of HCMV-infected neural/neuronal cells was assessed by double IHC to detect simultaneously HCMV-antigens and neural/neuronal markers: nestin (a marker of neural stem/progenitor cells), doublecortin (DCX, marker of cells committed to the neuronal lineage) and neuronal nuclei (NeuN, identifying mature neurons). HCMV-positive cells and viral DNA were found in the brain of 8/10 (80%) fetuses. For these cases, brain damage was classified as mild (n = 4, 50%), moderate (n = 3, 37.5%) and severe (n = 1, 12.5%) based on presence and frequency of pathological findings (necrosis, microglial nodules, microglial activation, astrocytosis, and vascular changes). The highest median HCMV-DNA level was found in the hippocampus (212 copies/5 ng of human DNA [hDNA], range: 10-7,505) as well as the highest mean HCMV-infected cell value (2.9 cells, range: 0-23), followed by that detected in subventricular zone (1.7 cells, range: 0-19). These findings suggested a preferential viral tropism for both neural stem/progenitor cells and neuronal committed cells, residing in these regions, confirmed by the expression of DCX and nestin in 94% and 63.3% of HCMV-positive cells, respectively. NeuN was not found among HCMV-positive cells and was nearly absent in the brain with severe damage, suggesting HCMV does not infect mature neurons and immature neural/neuronal cells do not differentiate into neurons. This could lead to known structural and functional brain defects from cCMV infection