In developed countries, congenital cytomegalovirus (cCMV) infection is the most common congenital viral infection, representing the leading non-genetic cause of sensorineural hearing loss (HL). Diagnosis of cCMV infection can be performed by detection of CMV DNA in urine or saliva within 2–3 weeks after birth, or later in dried blood samples on the Guthrie card. Currently, there are many controversies regarding the preventive, diagnostic, and therapeutic approaches to cCMV infection. HL secondary to cCMV is highly variable in onset, side, degree, audiometric configuration, and threshold changes over time. Therefore, it is of paramount importance to perform a long and thorough audiological follow-up in children with cCMV infection to ensure early identification and prompt treatment of progressive and/or late-onset HL. Early cochlear implantation appears to be a valid solution not only for children with bilateral profound HL, but also for those with single-sided deafness, improving localization ability and understanding speech in noisy environments. Moreover, the decision to apply a unilateral cochlear implant in children with cCMV is strengthened by the non-negligible possibility of hearing deterioration of the contralateral ear over time.

This paper outlines the role of Lombardy’s regional influenza reference laboratory (Northern Italy) in the surveillance of influenza-like illnesses (ILIs) in monitoring SARS-CoV-2 circulation by analyzing 631 consecutive nasopharyngeal swabs (NPSs) collected from ILI outpatients by sentinel physicians during the 2019–2020 season. The samples were tested by specific real-time RT-PCRs targeting SARS-CoV-2, influenza viruses, and RSVs. Results: Of these NPSs, 31% tested positive for influenza viruses, 10% for SARS-CoV-2, and 7% for RSV. No coinfections were detected. Influenza viruses and RSVs circulated throughout the surveillance period until the end of February (week 9-2020), when they suddenly ceased to circulate seven weeks earlier than during the previous five influenza seasons. After the first detection of SARS-CoV-2 in our ILI outpatients at the beginning of March (week 10-2020), SARS-CoV-2 remained the only virus identified throughout the surveillance period. Patients ≥ 65 years had a 3.2-fold greater risk of being infected with SARS-CoV-2, while school-age children (5–14 years) and children < 5 years proved to be the age groups most at risk of contracting influenza viruses and RSV, respectively. Our experience demonstrates that laboratory-based ILI surveillance networks are essential for identifying SARS-CoV-2 cases that would otherwise remain undetected, in order to stop their spread within our communities.

Congenital Cytomegalovirus (cCMV) is the most common cause of non-genetic hearing loss in childhood. A newborn hearing screening program (NHSP) is currently running in Italy, but no universal cCMV nor statewide hearing-targeted CMV screening programs have been implemented yet. This observational monocentric study was aimed at estimating the rate of cCMV infections identified by CMV-DNA analysis on Dried Blood Spots (DBS) samples in deaf children identified via NHSP in Northern Italy in the period spanning from 2014 to 2018. Children with a confirmed diagnosis of deafness and investigated for CMV-DNA by nucleic acid extraction and in-house polymerase-chain reaction (PCR) on stored newborns screening cards (DBS-test) were included in this study. Deafness was defined by a hearing threshold ≥20 decibel (dB HL) by Auditory Brainstem Responses (ABR); all investigated DBS samples were collected within 3 days of life. Overall, 82 children were included (median age: 3.4 months; lower-upper quartiles: 2–5.3 months; males: 60.9%). Most of them (70.7%) presented bilateral hearing loss with a symmetrical pattern in 79.3% of the cases. ABR thresholds were ≥ 70 dB HL (severe/profound deafness) in 46.5% of children. Among all tested children, 6.1% resulted positive for cCMV. The rate of severe/profound deafness was statistically higher in children with cCMV infection. The addition of DBS-test to the NHSP allowed the identification, in their first months of life, of a cCMV infection in 6.1% of children who had failed NHS. The introduction of a targeted CMV screening strategy could help clinicians in the differential diagnosis and in the babies’ management. DBS samples can be considered a “universal newborns biobank”: their storage site and duration should be the subject of political decision-making.

(1) Background: The surveillance of SARS-CoV-2 RNA in urban wastewaters allows one to monitor the presence of the virus in a population, including asymptomatic and symptomatic individuals, capturing the real circulation of this pathogen. The aim of this study was to evaluate the performance of different pre-analytical and analytical methods for identifying the presence of SARS-CoV-2 in untreated municipal wastewaters samples by conducting an inter-laboratory proficiency test. (2) Methods: three methods of concentration, namely, (A) Dextran and PEG-6000 two-phase separation, (B) PEG-8000 precipitation without a chloroform purification step and (C) PEG-8000 precipitation with a chloroform purification step were combined with three different protocols of RNA extraction by using commercial kits and were tested by using two primers/probe sets in three different master mixes. (3) Results: PEG-8000 precipitation without chloroform treatment showed the best performance in the SARS-CoV-2 recovery; no major differences were observed among the protocol of RNA extraction and the one-step real-time RT-PCR master mix kits. The highest analytic sensitivity was observed by using primers/probe sets targeting the N1/N3 fragments of SARS-CoV-2. (4) Conclusions: PEG-8000 precipitation in combination with real-time RT-PCR targeting the N gene (two fragments) was the best performing workflow for the detection of SARS-CoV-2 RNA in municipal wastewaters.

Wastewater-based environmental surveillance enables the monitoring of SARS-CoV-2 dynamics within populations, offering critical epidemiological insights. Numerous workflows for tracking SARS-CoV-2 have been developed globally, underscoring the need for interlaboratory comparisons to ensure data consistency and comparability. An inter-calibration test was conducted among laboratories within the network monitoring SARS-CoV-2 in wastewater samples across the Lombardy region (Italy). The test aimed to evaluate data reliability and identify potential sources of variability using robust statistical approaches. Three wastewater samples were analyzed in parallel by four laboratories using identical pre-analytical (PEG-8000-based centrifugation) and analytical processes (qPCR targeting N1/N3 and Orf-1ab). A two-way ANOVA framework within Generalized Linear Models was applied, and multiple pairwise comparisons among laboratories were performed using the Bonferroni post hoc test. The statistical analysis revealed that the primary source of variability in the results was associated with the analytical phase. This variability was likely influenced by differences in the standard curves used by the laboratories to quantify SARS-CoV-2 concentrations, as well as the size of the wastewater treatment plants. The findings of this study highlight the importance of interlaboratory testing in verifying the consistency of analytical determinations and in identifying the key sources of variation.

Many congenitally cytomegalovirus-infected (cCMV) neonates are at risk for severe consequences, even if they are asymptomatic at birth. The assessment of the viral load in neonatal blood could help in identifying the babies at risk of sequelae. In the present study, we elaborated the results obtained on blood samples collected in the first two weeks of life from 22 symptomatic and 48 asymptomatic newborns with cCMV diagnosed through urine testing. We evaluated the performances of two quantitative methods (pp65 antigenemia test and plasma Real-time PCR) and the semi-quantitative results of dried blood sample (DBS) test in the aim of identifying a valid method for measuring viral load. Plasma qPCR and DBS tests were positive in 100% of cases, antigenemia in 81%. Only the latter test gave quantitatively different results in symptomatic versus asymptomatic children. qPCR values of 103 copies/ml were found in 52% of newborn. "Strong" DBS test positivity cases had higher median values of both pp65 positive PBL and DNA copies/ml than cases with a "weak" positivity. As expected antigenemia test was less sensitive than molecular tests and DBS test performed better on samples with higher rates of pp65 positive PBL and higher numbers of DNA copies/ml. The prognostic significance of the results of these tests will be evaluated on completion of the ongoing collection of follow-up data of these children.