Diagnosing congenital Cytomegalovirus infection: Don't get rid of dried blood spots

Background: Congenital Cytomegalovirus (cCMV) is a serious global public health issue that can cause irreversible fetal and neonatal congenital defects in symptomatic or asymptomatic newborns at birth. In absence of universal cCMV screening, the retrospective diagnosis of cCMV infection in children is only possible by examining Dried Blood Spot (DBS) samples routinely collected at birth and stored for different time spans depending on the newborn screening regulations in force in different countries. In this article, we summarize the arguments in favor of long-term DBS sample storage for detecting cCMV infection. Main text: CMV infection is the most common cause of congenital infection resulting in severe defects and anomalies that can be apparent at birth or develop in early childhood. Sensorineural hearing loss is the most frequent consequence of cCMV infection and may have a late onset and progress in the first years of life. The virological diagnosis of cCMV is essential for clinical research and public health practices. In fact, in order to assess the natural history of CMV infection and distinguish between congenital or acquired infection, children should be diagnosed early by analyzing biological samples collected in the first weeks of life (3 weeks by using viral culture and 2 weeks by molecular assays), which, unfortunately, are not always available for asymptomatic or mildly symptomatic children. It now seems possible to overcome this problem since the CMV-DNA present in the blood of congenitally infected newborns can be easily retrieved from the DBS samples on the Guthrie cards routinely collected and stored within 3 days from birth in the neonatal screening program for genetic and congenital diseases. Early collection and long-term storage are inexpensive methods for long-term bio-banking and are the key points of DBS testing for the detection of cCMV. Conclusion: DBS sampling is a reliable and inexpensive method for long-term bio-banking, which enables to diagnose known infectious diseases - including cCMV - as well as diseases not jet recognized, therefore their storage sites and long-term storage conditions and durations should be the subject of political decision-making

Heterogeneity of covid-19 outbreak in italy

An outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) started in December 2019 in China and was declared a pandemic on 11.03.2020 by WHO. Italy is one of the most afflicted Country by this epidemic with 136,110 confirmed cases and 16,654 deaths on 9.4.2020 (at the same date, the Ministry of Health was reporting 143,626 cases). During these few months the National Health Service have made a great effort to cope with the increasing request of intensive care beds and all the elective activities in hospital have been suspended. Data from the different Italian regions shows different patterns of positive and dead for this syndrome. Moreover, striking differences of the observed lethality of the infections among different areas were immediately evident from the epidemic reports. It will be of critical relevance to understand the expected evolution of the first lock-down phase, driving the exhaustion of the Covid-19 outbreak

Molecular characterization and phylogenetic analysis of human influenza A viruses in three consecutive seasons with different epidemiological profiles

INTRODUCTION: Influenza activity and influenza virus circulation were observed in Lombardy (northern Italy) during three consecutive seasons and the molecular characteristics of circulating viruses analysed to control for introduction of new variants. METHODS: The molecular characterization of 38 isolates, namely 20 A/H3N2 and 18 A/H1N1 influenza strains from the 2005/06, 2006/07 and 2007/08 seasons, was performed by sequence analysis of the globular head region of the HA protein (HA1 subunit), specific for influenza virus A/H3 and A/H1. RESULTS AND DISCUSSION: The last three influenza seasons in the study region were characterized by medium-low activity. A typical co-circulation of several variants was shown for A/H3 viruses for approximately two years and were subsequently almost entirely substituted by new emerging variants. Vice versa, A/H1 viruses had a more homogeneous circulation with a single lineage clearly dominating each season. The HA sequences of the A/H3 and the A/H1 viruses isolated in the last three seasons fell into 4 and 3 principal phylogenetic groups, respectively. No evidence of positive or negative selection in the sequence alignments was observed. CONCLUSIONS: Molecular characterization of the influenza viruses in three consecutive seasons highlighted considerable heterogeneity in their HA sequences. A careful surveillance of genetic changes in the HA1 domain during seasonal influenza epidemics may reveal immune escape and provide early information on newly emerging strains with epidemiologic inference

Contribution of respiratory syncytial virus (RSV) among patients <15 years hospitalized with severe acute respiratory infection (SARI) in Milan, 2014-2017

Aim: Respiratory syncytial virus (RSV) infections may range from cold to severe acute respiratory infection (SARI) and are responsible for substantial pediatric morbidity. We describe the results of RSV molecular detection in respiratory samples collected from children <15 years hospitalized with SARI in Milan (Italy) during four consecutive years. Method: From January 1st, 2014, to December 31st, 2017, 3013 respiratory samples (2826 upper-respiratory-tract [UTR] and 187 lower-respiratory-tract [LTR] specimens) collected from as many children <15 years hospitalized with SARI at an University hospital in Milan were analysed. After DNA/RNA extraction, samples were tested by a multiplex real-time PCR to detect RSV and other respiratory viruses. Results: 571 (19%) respiratory samples tested RSV-positive. RSV-positivity rate by sample type was similar (URT vs LRT: 19.2% vs 14.4%; p=0.09). The median age of RSV-positive cases was 6.6 months (inter-quartile range: 17.2 months); 52.2% were males. 62.2% (355/571) of RSV-positive samples were identified in children <1 year and 12.4% (71/571) in those <1 month. RSV was detected throughout the study period; 59.2% (338/571) cases were identified during seasonal peaks (December-February). In 49.9% (285/571) of RSV-positive samples at least another virus (mainly Rhinovirus: 45.9%) was detected, particularly (60%; 171/285) in samples collected from children >1 year. Conclusions: Accordingly to other studies, RSV was detected in 19% of hospitalized-SARI cases <15 years, mainly in children <1 year and in December-February. Sampling of upper or lower airways resulted in similar RSV-positivity rate. Routine molecular testing to detect RSV is warranted to improve clinical management of pediatric patients

Analysis of a pandemic in the Italian newspapers : the A (H1N1) experience

Background: in 2009 a novel infective agent, A(H1N1), was recognized by the World Health Organization (WHO) as a pandemic virus. Like most European countries, Italy experienced a single pandemic wave during fall-winter 2009. The objective of our study was to evaluate the news reports and the representation of the A(H1N1) pandemic in the Italian newspapers both quantitatively and qualitatively. Methods: from April 24th, 2009 to February 28th, 2010, seven national newspapers were monitored for the quantitative reporting of A(H1N1). In a three month sample period, reports were evaluated qualitatively by considering their front page presence, tones used for headlines, and images and figures dedicated to the topic. Results: in a ten month window, a total of 1220 articles were published. The reporting period showed four peaks and one hollow, with a similar pattern for all the newspapers. During the three-month sample period, we found a total of 382 articles, 98.4% of which appeared on front pages, 33.8% of which contained headlines using alarming tones, and 47.8% which contained info-graphic elements. Conclusions: the A(H1N1) 2009 pandemic in Italy was mild; nonetheless, newspapers devoted great attention to the new influenza and used alarmist tones. In similar situations, there are several areas where scientists should play a greater role. Scientists should support journalists in understanding scientific issues and help them translate scientific information into news items. Scientists should also help to contain the anxiety aroused in lay people by a pandemic, and support vaccination efforts dedicated to it

Early co-circulation of different clades of influenza A/H1N1v pandemic virus in Northern Italy

Introduction. The spatial diffusion over time of pandemic influenza A/H1N1 virus (A/H1N1v) was surveyed in Northern Italy (nearly 10 million inhabitants) from April to December 2009, and the molecular characteristics of circulating viruses were analyzed to identify the appearance of drift variants. About 45% of analyzed samples were laboratory-confirmed cases of A/H1N1v. Sporadic cases occurred until the middle of June 2009, then, case numbers began to increase delineating distinct epidemiological phases of viral circulation. Methods. RNA was extracted using RNeasy Mini kit (QIAGEN GmbH, Germany). Virological diagnosis of A/H1N1v infection was carried out by real-time RT-PCR assay. Sequence analysis of hemagglutinin (HA) gene was performed through a RT-PCR assay specific for a 995 bp fragment (nt. 64-1,058) in the HA1 domain. The nucleotide sequences were obtained by automated DNA sequencing. The HA1 sequences were aligned with other sequences collected from GenBank database by ClustalX software. The multiple sequence alignment was used to perform a basic phylogenetic analysis and a phylogenetic tree from HA sequences was constructed. Results. The HA gene sequences of A/H1N1v analyzed segregated into three genetically distinct clades and were characterized by the appearance of amino acid variations that were progressively fixed in the field viral population under scrutiny. Conclusions. These data suggest an early co-circulation of genetically distinct A/H1N1v variants and emphasize the importance of a close molecular surveillance to detect rapidly the spread of new viral variants and to define their epidemiological impact

Genetic analysis of human and swine influenza A viruses isolated in Northern Italy during 2010–2015

Summary Influenza A virus (IAV) infection in swine plays an important role in the ecology of influenza viruses. The emergence of new IAVs comes through different mechanisms, with the genetic reassortment of genes between influenza viruses, also originating from different species, being common. We performed a genetic analysis on 179 IAV isolates from humans (n. 75) and pigs (n. 104) collected in Northern Italy between 2010 and 2015, to monitor the genetic exchange between human and swine IAVs. No cases of human infection with swine strains were noticed, but direct infections of swine with H1N1pdm09 strains were detected. Moreover, we pointed out a continuous circulation of H1N1pdm09 strains in swine populations evidenced by the introduction of internal genes of this subtype. These events contribute to generating new viral variants—possibly endowed with pandemic potential—and emphasize the importance of continuous surveillance at both animal and human level

Epidemiological and molecular characteristics of HPEV infection in children<6 months hospitalized with symptoms of sepsie-like illness, northen Italy, 2015-2018

BACKGROUND. Human parechoviruses (HPeVs) are widespread pathogens belonging to the Picornaviridae family and currently divided into 19 genotypes. HPeV infections can be associated with severe clinical manifestations, such as sepsis-like illness, particularly in youngest children. The epidemiological and molecular characteristics of HPeV infections observed in children <6 months hospitalized with symptoms of sepsis-like illness were investigated. METHODS. From January 1st, 2015, to December 31st, 2018, clinical samples (cerebrospinal fluid samples and/or blood samples) were collected for diagnosis of HPeV infection from 193 patients (median age: 21 days, range: 1 day - 6 months) hospitalized with symptoms of sepsis-like illness, in two hospitals of Northern Italy. HPeV-RNA was detected by real-time RT-PCR (target 5\u2019UTR) and a portion of HPeV VP3/VP1 junction (nt. 2159\u20132458) was sequenced for typing and molecular characterization. RESULTS. 14% (27/193) of patients with symptoms of sepsis-like illness tested HPeV-positive. 26/27 (96.3%) HPeV-cases were <3 months and 20/27 (74.1%) <1 month. HPeV-positive cases were detected throughout the study period, mainly (12/27; 44.4%) during the summertime (June-August). 17/27 (63%) HPeV-positive samples were molecularly characterized: 16 resulted HPeV-3 and 1 HPeV-5. CONCLUSIONS. HPeV infection was identified in 14% of children <6 months with symptoms of sepsis-like illness. Almost all HPeV infections were detected in children <3 months and mainly during the summertime; almost all molecularly characterized HPeV belonged to type 3. Including HPeV molecular detection in routine diagnostic tests would allow estimating the burden of HPeV infection and improving clinical management of pediatric patients

Circulation of SARS-CoV-2 Variants among Children from November 2020 to January 2022 in Trieste (Italy)

Introduction: The ongoing coronavirus disease 19 (COVID-19) outbreak involves the pediatric population, but to date, few reports have investigated the circulation of variants among children. Material and Methods: In this retrospective study, non-hospitalized pediatric patients with SARS-CoV-2-positive nasopharyngeal swabs (NPS) were enrolled at the Institute for Maternal and Child Health-IRCCS Burlo Garofolo, Trieste (Italy), from November 2020 to January 2022. SARS-CoV-2 variants were identified by in vitro viral isolation, amplification, automatic sequencing of the receptor binding domain (RBD) of the SARS-CoV-2 spike coding gene, and subsequent nextgeneration sequencing. The growth curves of the isolated strains were defined in vitro by infecting Vero-E6 cells and quantifying the viral load in the supernatants up to 72 h post-infection by qRT– PCR. The neutralization activity of sera obtained from a COVID-19 vaccinated subject, recovered (2020) patient, vaccinated and recovered (2021) patient, and seronegative subject was assessed by microneutralization assay against the different variants. Results: In total, 32 SARS-CoV-2-positive children, 16 (50%) females, with a median age of 1.4 years (range: 1 day–13 years), were enrolled. The D614G amino acid substitution was detected in all isolated and amplified viral strains. Of the 32 isolates, 4 (12.5%) carried a nonsynonymous nucleotide mutation leading to the N439K (3/4), lineage B.1.258 (∆H69/∆V70), and S477N (1/4) substitution. In 7/32 (21.8%) isolates, amino acid substitutions allowed the identification of a delta variant, lineage B.1.617.2-AY.43, and in 1/32 (3.1%), the Omicron strain (B.1.1.529.BA1) was identified. The growth curves of the B.1, B.1.258 (∆H69/∆V70), B.1.617.2-AY.43, and B.1.1.529.BA1 variants did not show any significant differences. A reduction in the serum neutralizing activity against B.1.258 (∆H69/∆V70) only in a vaccinated subject (1.7-fold difference), against B.1.617.2-AY.43 in a vaccinated subject and in recovered patients (12.7 and ≥2.5-fold differences, respectively), and against B.1.1.529.BA1 variant (57.6-and 1.4-fold differences in vaccinated and in vaccinated and recovered patients) were observed compared to the B.1 variant. Conclusions: SARS-CoV-2 variants carrying the B.1.258 (∆H69/∆V70) and S477N substitutions were reported here in a pediatric population for the first time. Although the growth rates of the isolated strains (B.1.258, B.1.617.2-AY.43, B.1.1.529.BA1) did not differ from the B.1 variant, neutralizing activity of the sera from vaccinated subjects significantly decreased against these variants. Attention should be devoted to the pediatric population to prevent the spread of new SARS-CoV-2 variants in an unvaccinated and predominantly naive population