Purpose The value of SARS-CoV-2 monitoring in urban wastewater samples (WWS) for surveillance of virus spread at a population-wide level has been largely demonstrated. Aim of this study was to optimize an analytical workflow to detect SARS-CoV-2 RNA in WWS and to monitor SARS-CoV-2 spread during the first wave of COVID-19 epidemic (March–June 2020) in Lombardy, northern Italy. Methods & Materials The workflow consisted in WWS concentration by using PEG-8000 precipitation, a modified RNA extraction (QIAamp MinElute Virus Spin Kit; QIAGEN) and a one-step real-time RT-PCR detecting two portions of the N gene of SARS-CoV-2. Composite 24-hour WWS were collected once a week at the inlet of 8 wastewater treatment plants (WWTPs) with an overall catchment of 2,276,000 inhabitants, located in representative COVID-19 hotspots in Lombardy, from the end of March to mid-June 2020. 107 WWS were obtained and analysed. SARS-CoV-2 RNA copies/L/WWS were multiplied by the flow rate of each WWTP (m3/day) and the obtained load (copies/day/1,000 people) was normalized to the number of inhabitants served by WWTPs. Results The optimized workflow allowed to identify 1E+3 copies/mL of SARS-CoV-2 in concentrated WWS with a turnaround time of 8 hours. Overall, the presence of SARS-CoV-2 RNA was identified in 65/107 WWS (61%). The highest rate of positive WWS (78.7%; 26/33) was identified in the Bergamo province, that was the epicentre during the first wave of COVID-19 epidemic (March-June 2020) in Lombardy. The highest amount of SARS-CoV-2 RNA was identified in late March/early April, when the overall viral load reflecting the number of individuals shedding the virus ranged from 9.3E+10 copies/day/1,000 people to 8.2E+8 copies/day/1,000 people. Since the end of May, WWS tested negative to SARS-CoV-2 detection. Conclusion According to the epidemiological features of the first wave of SARS-CoV-2 epidemic in Lombardy, the highest amount of SARS-CoV-2 RNA was detected in WWS collected in the areas most affected by COVID-19 (i.e. Bergamo province). This optimized workflow of WWS surveillance can help assessing the real number of individuals – both symptomatic and asymptomatic – able to spread the virus and appraising the effect of preventive measures.

Purpose: To report a descriptive analysis of the virological results of Influenza-like illness (ILI) surveillance, comparing the frequency of detection of respiratory viruses during the pre-pandemic (2018-2019) and SARS-CoV-2 pandemic (2020-2021) winter seasons in Lombardy (Northern Italy).Methods & Materials: From week 46 to week 17 of the following year, nasal-pharyngeal swabs (NPS) collected from ILIs were tested for influenza viruses (IV), RSV, rhinovirus and enterovirus with specific real-time RT-PCR assays.In 2020-2021, SARS-CoV-2 molecular detection was also included.Results: 464 and 446 NPS were collected in 2018-2019 and 2020-2021, respectively.Sex distribution was similar between these two seasons (males: 51.5% vs 47%, P = 0.165), while mean age was statistically higher in 2020-2021 (28.2 years [IQR: 40.5] vs. 43.6 years [IQR: 32.6], P < 0.0 0 01).ILI incidence was significantly higher in 2018/19 compared with 2020/21 season season (5.4/1,0 0 0 inhabitants vs. 1.9/1,0 0 0 inhabitants, P < 0.0 0 01.ILI cases < 15 years were less reported in 2020-2021 than in 2018-2019 season (P < 0.0 0 01).Contrary, those > 15 years were more reported in 2020-2021 than in 2018-2019 (15-44 years, P = 0.008; 45-64 years, P = 0.0 02; ≥65 years, P < 0.0 0 01).In 2018-2019, IV and RSV accounted for 55% and 10% of ILIs, whereas in 2020-2021 no circulation of these viruses was observed.Enterovirus detections decreased from 3.4% in 2018-2019 to 0.4% in 2020-2021 (P = 0.01), while rhinoviruses were detected at higher frequency in the latter season (8.6% vs. 11.9%,P = 0.07).SARS-CoV-2 was detected in 25.3% of NPS in 2020-2021.Conclusion: Since the beginning of the pandemic, nonpharmaceutical measures, including use of face-masks, was massively applied to contrast SARS-CoV-2 spread.This dramatically reduced the incidence of ILI, the circulation of SARS-CoV-2 and other respiratory viruses (such as IV and RSV) transmitted by droplets, but did not reduce rhinovirus detection, which continued to circulate in 2020-2021 season.ILIs surveillance has demonstrated to be able to capture changes in the epidemiology of respiratory viruses and it should be sustained and improved to increase the capture of ILI remained with unknown etiology.

Background Healthcare professionals might play a significant role in tobacco control. The aims of this study were to investigate tobacco cigarette and electronic cigarette (e-cigarette) consumption among university students enrolled in courses of the healthcare area, and to understand whether training in healthcare could induce to smoking cessation. Study design Cross-sectional survey using a self-administered, structured questionnaire. Methods Tobacco smoking habits of 560 students of four different medical area courses at the University of Milan, enrolled both in the first and in the last course year during the 2017-2018 academic year, were collected. Results The prevalence of smokers was 34.8%, almost the same for males and females, and higher in Italian students compared to foreigners. Smoking prevalence was higher among employed (46.9%) than unemployed (35.1%) students, without differences between healthcare and non-healthcare workers. The influence of family, and particularly friends, is confirmed to be relevant. About 25% of respondents tried e-cigarettes, mainly smokers (44.6%) and former smokers (38.6%) with the goal of quitting smoking. More than 44% were dual users of both tobacco cigarettes and e-cigarettes. Comparing smoking habits between first-year and final-year students, only students of healthcare assistance course showed a significant drop in smoking during the university studies. Conclusions The implementation of specific educational curricula on smoking dangers and on smoking cessation techniques might have the double effect of supporting students in quitting smoking and of properly preparing them for their future task of helping people to quit smoking.

A previously healthy 6-year-old boy was admitted to hospital with hypotonia and hyposthenia of lower limbs. Electromyography and slow motor nerve conduction velocity test identified a lower limb acute motor axonal neuropathy. Brain and spinal cord magnetic resonance imaging demonstrated multifocal cortical gray matter lesions in both cerebral hemispheres consistent with gray matter acute disseminated encephalitis otherwise with viral/Mycoplasma pneumoniae encephalitis, and signs of involvement of anterior nerve roots of the cauda equina consistent with Guillain-Barré syndrome. The patient resulted negative to routinely bacterial and viral investigations but positive to human parechovirus that sequence analyses confirmed as type 6. Intravenous immunoglobulins and methylprednisolone treatment were administered but did not relieve the symptoms of Guillain-Barré syndrome. The disease improved gradually over the next 3-month follow-up with a complete remission of both central and peripheral nervous system symptoms.

As the regional reference laboratory for acute flaccid paralysis (AFP) surveillance (Lombardy, northern Italy), a case of acute flaccid myelitis (AFM) came to our attention at the end of July 2016 when a previously healthy 4-year-old child with febrile (body temperature > 38 °C) respiratory illness and headache was hospitalised at a hospital in our region. The child’s condition suddenly worsened with the occurrence of severe neurological manifestations such as stiff neck, flaccid limb weakness associated with hyporeflexia, and bulbar muscle weakness, requiring intensive care unit care.

Purpose: As the regional reference laboratory for Lombardy (a region of Northern Italy) we participate to the RotaNet-Italy Study Group, in the framework of the EuroRotaNet surveillance network. In this study, group A rotaviruses (RVA) from cases of acute gastroenteritis (AGE) were investigated with the aim of evaluating the co-circulation of different strains and the emergence of uncommon or unusual rotavirus genotypes. Although most of RVA infections are supported by common genotypes G1–G4, and G9 associated with either P[8] or P[4], an unexpected spreading of uncommon genotypes could occur, suggesting gene reassortment events between strains of different origin. Methods & Materials: During the first part of 2015-2016 rotavirus season, 38 positive-RVA stool samples were collected from as many children (median age: 27 months; IQR: 38.9 months) with AGE admitted to ‘ASST-CREMONA’ hospital in Cremona, a city of Lombardy region accounting for nearly 71,000 inhabitants. Molecular characterization of RVA, based on EuroRotaNet methods and algorithms, was performed by the regional reference laboratory located at the University of Milan in collaboration with the Istituto Superiore di Sanità, which coordinates the project at national level. Results: A single rotavirus infection was recorded in 97.4% (38/39) of AGE and a double rotavirus infection was detected in one out 38 children (2.6%). According to G/P-type RVA characterization, the most predominant combination was the uncommon G12P[8] strain, being present in 39.5% (15/38) of children. G1P[8] was detected in 26.3% (10/38) of samples and G9P[8] and G4P[8] were identified at the same extent (6/38:15.8%); one (1/38:2.6%) RVA was G2P[4] and no G3P[8] combination was detected during the study period. Conclusion: This study reports for the first time a predominant and overwhelming introduction of the uncommon genotype G12P[8] RVA in children hospitalized for AGE in a small area of Northern Italy. Continuation of RVA monitoring activities by the RotaNet-Italy can help defining a baseline of circulating RVA genotypes before mass vaccination and assess possible genotype replacement once rotavirus vaccination will be introduced.

JC virus (JCV) is a double-stranded DNA virus belonging to family Polyomaviridae. It causes progressive multifocal leukoencephalopathy (PML), mainly in immunosuppressed people. JCV had been shown to require the serotonin 2A receptor for host cell entry. We report a case of clinical, neuroradiological and virological response of biopsy-proven PML in a 33-year-old comatose woman after treatment with the anti-psychotic drug risperidone. Since risperidone is the tightest binding of current drugs to this receptor we think this may have blocked JCV entry in our patient, allowing her immune recovery and viral clearance.

INTRODUCTION:Enterovirus (EV) and parechovirus (PeV) can either infect humans asymptomatically or can cause gastroenteritis, respiratory symptoms and, sometimes, severe disease. As the number of newly identified EV and PeV genotypes keeps increasing, diagnostic methods need to be updated. To this end, we described a novel multiplex one-step real-time RT-PCR to detect EV and human PeV (HPeV) simultaneously in fecal samples collected from children with rotavirus group A (RV-A)-related gastroenteritis. METHODS:The specificity and sensitivity of the EV/HPeV realtime RT-PCR were evaluated with two 2011 Quality Control for Molecular Diagnostics (QCMD) panels for EV and HPeV detection. RNA was extracted from 111 RV-A-positive fecal samples collected from children up to 5 years of age who had been hospitalized for gastroenteritis from September 2010 to August 2011. RESULTS:The EV/HPeV real-time RT-PCR showed a 100% sensitivity and specificity for EV and 91% and 91.7% for HPeV, respectively. Of the 111 RV-A-positive stool specimens, 28 (25.2%) were EV-positive and 7 (6.3%) were HPeV-positive. No clinical differences between children with single or double infections were observed. DISCUSSION:In our study, the frequency of EV and HPeV infections was surprisingly high, thus underlining the importance of including EV and HPeV detection in diagnostic panels. The multiplex real-time RT-PCR presented in this paper can therefore be a useful method in a diagnostic setting.

Sewage surveillance in seven Italian cities between 2005 and 2008, after the introduction of inactivated poliovirus vaccination (IPV) in 2002, showed rare polioviruses, none that were wild-type or circulating vaccine-derived poliovirus (cVDPV), and many other enteroviruses among 1,392 samples analyzed. Two of five polioviruses (PV) detected were Sabin-like PV2 and three PV3, based on enzyme-linked immunosorbent assay (ELISA) and PCR results. Neurovirulence-related mutations were found in the 5'noncoding region (5'NCR) of all strains and, for a PV2, also in VP1 region 143 (Ile>Thr). Intertypic recombination in the 3D region was detected in a second PV2 (Sabin 2/Sabin 1) and a PV3 (Sabin 3/Sabin 2). The low mutation rate in VP1 for all PVs suggests limited interhuman virus passages, consistent with efficient polio immunization in Italy. Nonetheless, these findings highlight the risk of wild or Sabin poliovirus reintroduction from abroad. Non-polio enteroviruses (NPEVs) were detected, 448 of which were coxsackievirus B (CVB) and 294 of which were echoviruses (Echo). Fifty-six NPEVs failing serological typing were characterized by sequencing the VP1 region (nucleotides [nt] 2628 to 2976). A total of 448 CVB and 294 Echo strains were identified; among those strains, CVB2, CVB5, and Echo 11 predominated. Environmental CVB5 and CVB2 strains from this study showed high sequence identity with GenBank global strains. The high similarity between environmental NPEVs and clinical strains from the same areas of Italy and the same periods indicates that environmental strains reflect the viruses circulating in the population and highlights the potential risk of inefficient wastewater treatments. This study confirmed that sewage surveillance can be more sensitive than acute flaccid paralysis (AFP) surveillance in monitoring silent poliovirus circulation in the population as well as the suitability of molecular approaches to enterovirus typing.

AimsHuman Enteroviruses (HEVs) infections have a significant impact on public health, being implicated in outbreaks of meningitis, encephalitis, hand-foot-mouth disease and other acute and chronic manifestation. In the strategic plan for poliomyelitis eradication, the environmental surveillance of poliovirus (PV) has been identified by the World Health Organization (WHO) as an activity that can complement the surveillance of polio. Having wastewater samples available for PV surveillance allows us to study nonpolio enteroviruses (NPEVs) circulating in the study population, which are widely spread.Methods and ResultsThis study was carried out according to the WHO guidelines for environmental surveillance of PV and analysed the circulation of PV and NPEVs through the isolation of viruses in cell cultures in Milan area; from 2006 to 2010, 321 wastewater samples were collected, regularly over time, at the inlet of three diverse waste water treatment plants (WWTPs). Culturable HEVs were isolated in 80% of sewage samples: all isolates belonged to the HEV-B group and those circulating more intensely were CVB5 and Echo 6, while CVB4 was the predominant serotype found in 2010. In this study, two type 2 PVs were isolated, both characterized as Sabin like.ConclusionEnvironmental monitoring of HEVs in Milan has proved to be an interesting tool to investigate the circulation and distribution of viruses.Significance and Impact of the StudyThe detection of PV and other NPEV could be predictive of possible re-emergence of these viruses with an impact on public health. NPEV monitoring could also be a powerful public health tool to investigate the possible role of NPEV in different clinical manifestations.