CONTRIBUTO AL PROGETTO OMS DI ERADICAZIONE DELLA POLIOMIELITE: RISULTATI DELLE ATTIVITÀ DI SORVEGLIANZA IN LOMBARDIA.

A contribution to the WHO global polio eradication initiative: the results of surveillance activities in Lombardy (Northern Italy), 2012-2015. Although in 2015 poliovirus (PV) transmission has been reported at the lowest levels ever recorded, the virus is still endemic in two countries \u2013 Afghanistan and Pakistan. Until PV transmission is interrupted in these countries, all countries remain at risk of virus importation - especially vulnerable countries with weak public health and immunization services and travel or trade links to endemic countries. The high levels of immigration flows across the Mediterranean Sea jeopardize Italy for PV reintroduction. The \u201cWHO Strategic Plan of the Global Polio Eradication Initiative\u201d indicates the nationwide surveillance of Acute Flaccid Paralysis (AFP) as the gold standard for detecting cases of poliomyelitis. In addition, systematic Environmental Surveillance (ES), seeking the presence of PV in sewage, is recognized as a powerful tool to confirm PV circulation in absence of AFP cases, especially in polio-free countries. In Italy, nationwide AFP surveillance has been set up in 1997, and ES has been established since 2005 in Milan and other five Italian cities (Bolzano, Parma, Sassari, Napoli and Palermo). This PhD project aimed at evaluating these two public health surveillance systems ongoing in Lombardy (Northern Italy) in the period spanning from 2012 to 2015, in order to improve their quality and efficiency, and to achieve the WHO criteria. The results of surveillance activities were described and the assessment of system attributes (including data quality, sensitivity and timeliness) were discussed. Additionally, in the framework of the ES, the circulation and characterization of non-polio Enterovirus (NPEV) was evaluated in the study population. The surveillance activities were carried out according to WHO guidelines from January 2012 to October 2015. For AFP surveillance, all children <15 years who met the WHO definition of AFP case were enrolled, and the collection of stool and serum samples from each case was requested. For ES, wastewater samples were collected twice a month at the intel of 3 wastewater treatment plants located in Milan. Stool specimens collected from AFP cases and wastewater samples were analyzed to detect PV and NPEVs by virus isolation in RD (human rhabdomyosarcoma) and L20B (murine transgenic L cells) cell cultures, and by PCR assay specific for the 5\u2032 noncoding region [5\u2032NCR] (nucleotide [nt]: 179-575). The identified viruses were genotyped by sequence analysis of the VP1 gene (nt. 2602-2977). In order to define the serological status/immunity against PV, serum samples collected from AFP cases were analyzed by microneutralization assay against PV1, PV2, and PV3. According to WHO, an antibody titre >1:8 was considered protective. From January 2012 to October 2015, 52 AFP cases were reported in Lombardy with an incidence rate of 1.04/100\u2019000 children <15 years of age. The median age of AFP cases was 5.8 years [inter-quartile range (IQR): 10.0 years]; no gender difference was observed. The annual incidence rates were 0.8/100\u2019000 in 2012, 1.5/100\u2019000 in 2013, 1.1/100\u2019000 in 2014, and 0.6/100\u2019000 in 2015 (preliminary data up to October). According to the WHO, the sensitivity of the AFP surveillance system is considered adequate when at least one case of AFP is detected annually per 100\u2019000 children aged less than 15 years. As in 2012 the sensitivity of the surveillance system did not met this criterion, several interventions were implemented to raise awareness among the parts involved in the surveillance. On purpose, reports on the AFP notified weekly by sentinel hospitals were sent out quarterly to all physicians involved in the surveillance system along with epidemiological alerts and bulletins on PV circulation and polio endgame. Phone and e-mail contacts were kept with physicians who reported AFP cases to guarantee the adequacy of notification. Moreover, a workshop on PV and its surveillance system was arranged in collaboration with regional and national (ISS) public health authorities. These actions allowed the sensitivity of the AFP surveillance system to meet the WHO criterion from 2013 onwards. The WHO recommends completing the case investigation by virological analysis of stool samples in at least 80% of AFP cases: during our study, this rate was achieved in 2014 and 2015 (85.7%), whereas in 2012 and 2013 the analysis was completed in 63.6% and 60% of stool samples, respectively. The improvement of virological investigation completeness of AFP cases since 2014 was probably due to the raised awareness of physicians involved in the surveillance. About 40% (21/52) of AFP cases were diagnosed as Guillain-Barr\ue9 syndrome, 23.1% (12/52) and 19.2% (10/52) were ascribed to genetic disease and myelitis, respectively. Most (35/52; 67%) of AFP cases were reported by pediatric wards during summer and winter. According to virological results, no AFP case was caused by a PV infection, even though one AFP case (that occurred in 2014 in a 6-month boy affected by Bruton disease who received the first dose of oral polio vaccine in Albania) was characterized as a vaccine associated paralytic paralysis (VAPP). NPEVs were detected in 6 AFP cases (10.5%): 2 were Echovirus-11, 1 was Echovirus-6, and the remaining 3 were not genotyped. Serological investigation was carried out in 48 (48/52: 92.3%) AFP cases and a protective antibody titre (neutralising antibodies titre 651:8) was detected in 94% (45/48) of individuals. In the framework of the ES, 273 wastewater samples were collected and no PVs were isolated. In contrast, NPEVs were detected in 65.2% (172/273) of tested samples. The proportion of NPEVs detected in sewage by year was 70% (42/60) in 2012, 56.9% (41/72) in 2013, 66.7% (48/72) in 2014 and 68.3% (41/60) in 2015 (up to October). In 2013 the rate of NPEVs detected was significantly lower (p< .05) than those recorded in the other years of study. The WHO declares that at least 30% of samples collected in the ES setting have to be positive for NPEV, thus our results demonstrated the good performance of the ongoing surveillance system in the whole study period. All NPEVs were characterized as EV belonging to species B: Echovirus-11 and Echovirus-6 were the most frequently detected viruses, being the 29.1% (41/141) and 20.6% (29/141) of genotyped NPEVs, respectively. No difference among the NPEV genotypes circulating in the three wastewater treatment plants was identified. It is important to strengthen surveillance of AFP cases at regional and national level in order to detect rapidly any virus importation or emergence and enable a prompt public health response. Although AFP surveillance remains the gold standard, systematic ES is a powerful tool to detect PV in the absence of polio cases, especially in polio-free countries. During our study, AFP surveillance met the WHO criteria for sensitivity from 2013 onwards and the level of completeness of case investigation improved significantly from 2014. Physicians involved in the activities have proved to give special attention to AFP surveillance thanks to several initiatives implemented since 2013, such as the sharing of surveillance reports, epidemiological alerts and updates on polio eradication progress. Epidemiological features of AFP cases were similar to those reported in the current scientific literature and our data confirm adequate levels of immunization in population as well as the absence of wild PV infections. ES was suitable to investigate EV circulation in the population and the high rate of NPEV detected underlines a massive virus circulation. No silent PV reintroduction was noted during ES. As long as in the current polio endgame PV outbreaks reflect serious gaps in immunity to PV due to the weakness of routine immunization coverage in otherwise polio-free countries, all countries should maintain uniformly high immunization coverage at the district level to minimize the consequences of any virus introduction. Keeping strong and encouraging both AFP surveillance and ES all over the world is crucial to ensure the PV will not return unnoticed and, finally, to achieve the global eradication goal

Diagnosing congenital Cytomegalovirus infection: Don&apos;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

Contribution of respiratory syncytial virus (RSV) among patients &lt;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 &lt;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 &lt;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 &lt;1 year and 12.4% (71/571) in those &lt;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 &gt;1 year. Conclusions: Accordingly to other studies, RSV was detected in 19% of hospitalized-SARI cases &lt;15 years, mainly in children &lt;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

Epidemiological and molecular characteristics of HPEV infection in children&lt;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 &lt;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 &lt;3 months and 20/27 (74.1%) &lt;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 &lt;6 months with symptoms of sepsis-like illness. Almost all HPeV infections were detected in children &lt;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

Influenza Vaccination in Italian Healthcare Workers (2018-2019 Season): Strengths and Weaknesses. Results of a Cohort Study in Two Large Italian Hospitals

Background: Annual vaccination is the most effective way to combat influenza. As influenza viruses evolve, seasonal vaccines are updated annually. Within the European project Development of Robust and Innovative Vaccine Effectiveness (DRIVE), a cohort study involving Italian healthcare workers (HCWs) was carried out during the 2018-2019 season. Two aims were defined: to measure influenza vaccine effectiveness (IVE) against laboratory-confirmed influenza cases and to conduct an awareness-raising campaign to increase vaccination coverage. Methods: Each subject enrolled was followed up from enrollment to the end of the study. Each HCW who developed ILI was swabbed for laboratory confirmation of influenza. Influenza viruses were identified by molecular assays. A Cox regression analysis, crude and adjusted for confounding variables, was performed to estimate the IVE. Results: Among the 4483 HCWs enrolled, vaccination coverage was 32.5%, and 308 ILI cases were collected: 23.4% were positive for influenza (54.2% A(H1N1) pdm09; 45.8% A(H3N2)). No influenza B viruses were detected. No overall IVE was observed. Analyzing the subtypes of influenza A viruses, the IVE was estimated as 45% (95% CI: -59 to 81) for A(H1N1) pdm09. Conclusions: Vaccination coverage among HCWs increased. Study difficulties and the circulation of drifted variants of A(H3N2) could partly explain the observed IVE

Hospital discharges-based search of acute flaccid paralysis cases 2007-2016 in Italy and comparison with the National Surveillance System for monitoring the risk of polio reintroduction

Background: Acute flaccid paralysis (AFP) surveillance has been adopted globally as a key strategy for monitoring the progress of the polio eradication initiative. Hereby, to evaluate the completeness of the ascertainment of AFP cases in Italy, a hospital-discharges based search was carried out. Methods: AFP cases occurring between 2007 and 2016 among children under 15 years of age were searched in the Italian Hospital Discharge Records (HDR) database using specific ICD-9-CM diagnostic codes. AFP cases identified between 2015 and 2016 were then compared with those notified to the National Surveillance System (NSS). Results: Over a 10-year period, 4163 hospital discharges with diagnosis of AFP were reported in Italy. Among these, 956 (23.0%) were acute infective polyneuritis, 1803 (43.3%) myopathy, and 1408 (33.8%) encephalitis, myelitis and encephalomyelitis. During the study period, a decreasing trend was observed for all diagnoses and overall the annual incidence rate (IR) declined from 5.5 to 4.5 per 100,000 children. Comparing NSS with HDR data in 2015-2016, we found a remarkable underreporting, being AFP cases from NSS only 14% of those recorded in HDR. In particular, the acute infective polyneuritis cases reported to NSS accounted for 42.6% of those detected in HDR, while only 0.9% of myopathy cases and 13.1% of encephalitis/myelitis/encephalomyelitis cases have been notified to NSS. The highest AFP IRs per 100,000 children calculated on HDR data were identified in Liguria (17.4), Sicily (5.7), and Veneto (5.1) Regions; regarding the AFP notified to the NSS, 11 out of 21 Regions failed to reach the number of expected cases (based on 1/100,000 rate), and the highest discrepancies were observed in the Northern Regions. Overall, the national AFP rate was equal to 0.6, therefore did not reach the target value. Conclusions: AFP surveillance data are the final measure of a country's progress towards polio eradication. The historical data obtained by the HDR have been useful to assess the completeness of the notification data and to identify the Regions with a low AFP ascertainment rate in order to improve the national surveillance system

Gastroenteropancreatic High-Grade Neuroendocrine Neoplasms: Histology and Molecular Analysis, Two Sides of the Same Coin

Background: In gastroenteropancreatic (GEP) high-grade neuroendocrine neoplasms (H-NENs), Ki-67 threshold of 55% defines three prognosis subclasses: neuroendocrine tumor (NET) G3, neuroendocrine carcinoma (NEC) &lt;55%, and NEC ≥55%. We investigated whether the molecular profiling of H-NENs differs among these subcategories and evaluated potential therapeutic targets, including PD-L1. Methods: In GEP-NEN patients, we evaluated: (i) 55% threshold for Ki-67 labeling index for further stratifying NEC and (ii) immunoreactivity and gene mutations by immunohistochemistry and targeted next-generation sequencing (T-NGS). Results: Fifteen NETs G3 and 39 NECs were identified. Ki-67 labeling index was &lt;55% in 9 NECs and ≥55% in 30 NECs. Gene mutations by NGS (TP53, 32.9%; KRAS, 5.5%; BRAF, 4.1%) were detected in 46.6% NENs, significantly enriched in NEC ≥55% (76.7%) compared to NEC &lt;55% (55.6%) or NET (20.0%). PD-L1 staining in tumor-infiltrating lymphocytes was observed in NEC ≥55% (36.7%; p = 0.03). Median OS was 4.3 years in NET G3, 1.8 years in NEC &lt;55%, and 0.7 years in NEC ≥55% (p &lt;0.0001); it was 2.3 years with NGS wild-type, 0.7 years with ≥1 mutation (p &lt;0.0001), 0.8 years in PD-L1-positive patients, and 1.7 years in PD-L1-negative subjects (p = 0.0004). In multivariate analysis, only the proposed subclassification approach yielded statistically significant differences between groups (NEC &lt;55% vs. NET G3, HR 14.1, 95% CI 2.2-89.8, p = 0.005; NEC ≥55% vs. NET G3, HR 25.8, 95% CI 3.9-169, p = 0.0007). Conclusions: These findings identify NEC ≥55% as a biologically and prognostically distinct subtype and pave the way for more personalized treatment

Has VZV epidemiology changed in Italy? Results of a seroprevalence study

The aim of the study was to evaluate if and how varicella prevalence has changed in Italy. In particular a seroprevalence study was performed, comparing it to similar surveys conducted in pre-immunization era. During 2013–2014, sera obtained from blood samples taken for diagnostic purposes or routine investigations were collected in collaboration with at least one laboratory/center for each region, following the approval of the Ethics Committee. Data were stratified by sex and age. All samples were processed in a national reference laboratory by an immunoassay with high sensitivity and specificity. Statutory notifications, national hospital discharge database and mortality data related to VZV infection were analyzed as well. A total of 3707 sera were collected and tested. In the studied period both incidence and hospitalization rates decreased and about 5 deaths per year have been registered. The seroprevalence decreased in the first year of life in subjects passively protected by their mother, followed by an increase in the following age classes. The overall antibody prevalence was 84%. The comparison with surveys conducted with the same methodology in 1996–1997 and 2003–2004 showed significant differences in age groups 1–19&nbsp;y. The study confirms that in Italy VZV infection typically occurs in children. The impact of varicella on Italian population is changing. The comparison between studies performed in different periods shows a significant increase of seropositivity in age class 1–4&nbsp;years, expression of vaccine interventions already adopted in some regions

Microenvironment and tumor inflammatory features improve prognostic prediction in gastro-entero-pancreatic neuroendocrine neoplasms

Microenvironment-related immune and inflammatory markers, when combined with established Ki-67 and morphology parameters, can improve prognostic prediction in gastro-entero-pancreatic neuroendocrine neoplasms (GEP-NENs). Therefore, we evaluated the prognostic value of microenvironment and tumor inflammatory features (MoTIFs) in GEP-NENs. For this purpose, formalin-fixed paraffin-embedded tissue sections from 350 patients were profiled by immunohistochemistry for immune, inflammatory, angiogenesis, proliferation, NEN-, and fibroblast-related markers. A total of 314 patients were used to generate overall survival (OS) and disease-free survival (DFS) MoTIFs prognostic indices (PIs). PIs and additional variables were assessed using Cox models to generate nomograms for predicting 5-year OS and DFS. A total of 36 patients were used for external validation of PIs and nomograms' prognostic segregations. From our analysis, G1/G2 versus G3 GEP-NENs showed phenotypic divergence with immune-inflammatory markers. HLA, CD3, CD8, and PD-1/PD-L1 IHC expression separated G3 into two sub-categories with high versus low adaptive immunity-related features. MoTIFs PI for OS based on COX-2Tumor(T) &gt; 4, PD-1Stromal(S) &gt; 0, CD8S &lt; 1, and HLA-IS &lt; 1 was associated with worst survival (hazard ratio [HR] 2.50; 95% confidence interval [CI], 2.12–2.96; p &lt; 0.0001). MoTIFs PI for DFS was based on COX-2T &gt; 4, PD-1S &gt; 4, HLA-IS &lt; 1, HLA-IT &lt; 2, HLA-DRS &lt; 6 (HR 1.77; 95% CI, 1.58–1.99; p &lt; 0.0001). Two nomograms were developed including morphology (HR 4.83; 95% CI, 2.30–10.15; p &lt; 0.001) and Ki-67 (HR 11.32; 95% CI, 5.28–24.24; p &lt; 0.001) for OS, and morphology (PI = 0: HR 10.23; 95% CI, 5.67–18.47; PI = 5: HR 2.87; 95% CI, 1.21–6.81; p &lt; 0.001) and MoTIFs PI for DFS in well-differentiated GEP-NENs (HR 6.21; 95% CI, 2.52–13.31; p &lt; 0.001). We conclude that G1/G2 to G3 transition is associated with immune-inflammatory profile changes; in fact, MoTIFs combined with morphology and Ki-67 improve 5-year DFS prediction in GEP-NENs. The immune context of a subset of G3 poorly differentiated tumors is consistent with activation of adaptive immunity, suggesting a potential for responsiveness to immunotherapy targeting immune checkpoints