An Acute Flaccid Paralysis (AFP) surveillance system was set up in Lombardy (Northern Italy) in 1997 in the framework of the national AFP surveillance system, as part of the polio eradication initiative by the World Health Organization (WHO). This surveillance system can now be used to detect Poliovirus (PV) reintroductions from endemic countries. This study aimed at describing the results of the AFP surveillance in Lombardy, from 1997 to 2011. Overall, 131 AFP cases in Lombardy were reported with a mean annual incidence rate of 0.7/100?000 children<15 years of age (range: 0.3/100?000-1.1/100?000). The sensitivity of the surveillance system was optimal from 2001-2003. The monthly distribution of AFP cases was typical with peaks in November, in January, and in March. The major clinical diagnoses associated with AFP were Guillain-Barré Syndrome (GBS, 40%) and encephalomyelitis/myelitis (13%). According to the virological results, no poliomyelitis cases were caused by wild PV infections, but two Vaccine-Associated Paralytic Paralysis (VAPP) cases were reported in 1997 when the Sabin oral polio vaccine (OPV) was still being administered in Italy. Since a surveillance system is deemed sensitive if at least one case of AFP per 100,000 children<15 years of age is detected each year, our surveillance system needs some improvement and must be maintained until global poliovirus eradication will be declared.
As the regional influenza reference centre operating within the Italian network InfluNet, here we report data on virological and epidemiological surveillance of influenza, as well as on the vaccination coverage rates achieved in Lombardy (Northern Italy) over 10 consecutive winter seasons (2004-2014). Over the past 10 years, influenza vaccine coverage declined both in the general population (from 15.7% in 2004-2005 to 11.7% in 2013-2014) and in the vaccine-target population of individuals ?65-y-of-age (from 65.3% in 2004-2005 to 48.6% in 2013-2014) and is far below the minimum planned threshold level (75%). The highest influenza-like illness (ILI) rates were recorded during the 2004-2005 and 2009-2010 epidemics (peak incidence: 12.04‰ and 13.28‰, respectively). Both seasons were characterised by the introduction of novel viral strains: A/Fujian/411/2002(H3N2) (a drifted hemagglutinin variant) and A/California/7/2009(H1N1) pandemic virus (a swine origin quadruple reassortant), respectively. Because the antigenic match between vaccine and circulating strains was good in both of these seasons, a relevant proportion of cases may have been prevented by vaccination. A different situation was observed during the 2011-2012 season, when ILI morbidity rates in individuals ?65-y-of-age were 1.5-6-fold higher than those registered during the other epidemics under review. The higher morbidity resulted from the circulation during the 2011-2012 season of an A/Victoria/361/2011(H3N2)-like variant that presented a reduced genetic match with the A(H3N2) strain included in the 2011-2012 vaccine composition. The continuous surveillance of the characteristics of circulating viruses is an essential tool for monitoring their matching with seasonal vaccine strains. Strategies to increase coverage rates are warranted.
Measles and rubella are infectious diseases and humans are the only reservoir of these infections. Effective vaccines are available with the potential for measles (MV) and rubella (RuV) virus eradication. According to the World Health Organisation WHO guidelines, a national plan was approved in Italy in 2013 to achieve the MV/RuV elimination by 2015, and active MV/RuV integrated surveillance initiated. Towards this purpose, a regional laboratory centre was set up on 1 September 2013 in Lombardy, Northern Italy. This paper aimed at: (1) evaluating measles-mumps-rubella (MMR) vaccine coverage and MV/RuV notified cases retrospectively; and (2) presenting the results of MV/RuV integrated surveillance (laboratory confirmed and viral genetic profiles). The 95% target for MMR vaccine coverage was achieved in 2001, and coverage increased until 2007 (96.6%), but then a decreasing trend was observed. Since 2000 to 2014, 3,026 rubella cases were notified, with nearly 58% of them in the 2002 epidemic. From 2009, less than 45 RuV cases per year were reported. From 2000 to 2014, 5024 measles cases were notified. Since 2008, three large outbreaks (in 2008, 2011, and 2013) were observed. From data obtained during our surveillance activity, there were no rubella cases, and 57.5% (46/80) collected samples were MV-positive by real-time RT-PCR. A fragment of the MV N gene was sequenced from 37 MV-positive samples; D8, D9, and B3 genotypes were detected. Data obtained retrospectively and from active surveillance underline the necessity to achieve and maintain high vaccination coverage and to improve surveillance and the effectiveness of healthcare actions.
We report 2 neonates with human parechoviruses type 3 encephalitis. Both newborns presented with fever, irritability and seizures. Cerebrospinal fluid analyses were normal, but magnetic resonance imaging revealed white matter damage, suggesting human parechoviruse infection. Human parechoviruses type 3-RNA was detected in cerebrospinal fluid samples and in blood, stool, urine and respiratory samples, indicating the dissemination of the virus.
Respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infections in infants and young children. RSV is characterised by high variability, especially in the G glycoprotein, which may play a significant role in RSV pathogenicity by allowing immune evasion. To reconstruct the origin and phylodynamic history of RSV, we evaluated the genetic diversity and evolutionary dynamics of RSV A and RSV B isolated from children under 3 years old infected in Italy from 2006 to 2012. Phylogenetic analysis revealed that most of the RSV A sequences clustered with the NA1 genotype, and RSV B sequences were included in the Buenos Aires genotype. The mean evolutionary rates for RSV A and RSV B were estimated to be 2.1 × 10(-3) substitutions (subs)/site/year and 3.03 × 10(-3) subs/site/year, respectively. The time of most recent common ancestor for the tree root went back to the 1940s (95% highest posterior density-HPD: 1927-1951) for RSV A and the 1950s (95%HPD: 1951-1960) for RSV B. The RSV A Bayesian skyline plot (BSP) showed a decrease in transmission events ending in about 2005, when a sharp growth restored the original viral population size. RSV B BSP showed a similar trend. Site-specific selection analysis identified 10 codons under positive selection in RSV A sequences and only one site in RSV B sequences. Although RSV remains difficult to control due to its antigenic diversity, it is important to monitor changes in its coding sequences, to permit the identification of future epidemic strains and to implement vaccine and therapy strategies.
Since 2009 pandemic, international health authorities recommended monitoring severe and complicated cases of respiratory disease, that is, severe acute respiratory infection (SARI) and acute respiratory distress syndrome (ARDS). We evaluated the proportion of SARI/ARDS cases and deaths due to influenza A(H1N1)pdm09 infection and the impact of other respiratory viruses during pandemic and postpandemic period (2009-2011) in northern Italy; additionally we searched for unknown viruses in those cases for which diagnosis remained negative. 206 respiratory samples were collected from SARI/ARDS cases and analyzed by real-time RT-PCR/PCR to investigate influenza viruses and other common respiratory pathogens; also, a virus discovery technique (VIDISCA-454) was applied on those samples tested negative to all pathogens. Influenza A(H1N1)pdm09 virus was detected in 58.3% of specimens, with a case fatality rate of 11.3%. The impact of other respiratory viruses was 19.4%, and the most commonly detected viruses were human rhinovirus/enterovirus and influenza A(H3N2). VIDISCA-454 enabled the identification of one previously undiagnosed measles infection. Nearly 22% of SARI/ARDS cases did not obtain a definite diagnosis. In clinical practice, great efforts should be dedicated to improving the diagnosis of severe respiratory disease; the introduction of innovative molecular technologies, as VIDISCA-454, will certainly help in reducing such "diagnostic gap."
The molecular characterization of circulating influenza A viruses is crucial to detect mutations potentially involved in increased virulence, drug resistance and immune escape. A molecular and phylogenetic analysis of A(H1N1)pdm09 neuraminidase (NA) gene sequences from different patient categories defined according to the severity of influenza infection were analyzed. A total of 126 influenza A(H1N1)pdm09 positive samples from patients with severe infections in comparison with those with moderate and mild infections was performed in Lombardy (Northern Italy, nearly 10 million inhabitants) during the 2010-2011 season. NA sequences included in this study segregated into five distinct clusters. Nineteen amino acid substitutions were detected exclusively in NA sequences of viruses identified in patients with severe or moderate influenza infection. Three of them (F74S, S79P, E287K) were observed in virus strains with the 222G/N hemagglutinin mutation. None of NA sequences under study had mutations related to the resistance to the NA inhibitors. Four out of 126 (3.2%) NA sequences from patients with severe infection lost a N-linked glycosylation site due to the change from N to K at residue 386. Two additional N-linked glycosylation sites in the NA stalk region (residues 42 and 44) were found in 12 (9.5%) NA sequences. Sporadic NA mutations were detected in NA viral sequences from critically ill patients, and no variants with reduced sensitivity to NA inhibitors were observed either in treated or untreated patients.
The activity and circulation of influenza viruses in Lombardy - Northern Italy - (a region with nearly 10 out of the 60 million inhabitants of Italy) were investigated during two consecutive seasons (2010-2011 and 2011-2012), as part of the Italian Influenza Surveillance Network. The molecular characteristics of the hemagglutinin (HA) sequence of circulating viruses were analyzed to investigate the emergence of influenza viral variants. In the surveyed area, the influenza activity of these two post-pandemic seasons was similar in terms of both time frame and impact. The timing of the influenza epidemics was similar to the timing seen prior to the emergence of the pandemic A(H1N1) virus in 2009. A(H1N1)pdm09 was the predominant virus circulating during the 2010-2011 post-pandemic season and then-unexpectedly-almost disappeared. The HA sequences of these A(H1N1)pdm09 viruses segregated in a different genetic group with respect to those identified during the 2009 pandemic, although they were still closely related to the vaccine viral strain A/California/07/2009. Influenza A(H3N2) viruses were the predominant viruses circulating during the 2011-2012 season, accounting for nearly 88% of influenza viruses identified. All HA sequences of the A(H3N2) viruses isolated in the 2011-2012 season fell into the A/Victoria/208/2009 genetic clade (although the A/Perth/16/2009 virus was the reference vaccine strain). B viruses presented with a mixed circulation of viral variants during these two seasons: viruses belonging to both B/Victoria and B/Yamagata lineages co-circulated in different proportions, with a notable rise in the proportion of B/Yamagata viruses (B/Wisconsin/1/2010-like) during the 2011-2012 epidemic. In conclusion, the continuous monitoring of the characteristics of circulating viruses is an essential tool for understanding the epidemiological and virological features of influenza viruses, for monitoring their matching with seasonal vaccine strains, and for tuning vaccination strategies.
Few data are available on the safety and long-term immunogenicity of A/H1N1 pandemic influenza vaccines for HIV-infected pediatric patients. We performed a randomized controlled trial to evaluate the safety and long-term immunogenicity of 1 versus 2 doses of the 2009 monovalent pandemic influenza A/H1N1 MF59-adjuvanted vaccine (PV) coadministered with the seasonal 2009-2010 trivalent nonadjuvanted influenza vaccine (SV) to HIV-infected children, adolescents, and young adults. A total of 66 HIV-infected patients aged 9 to 26 years were randomized to receive one (group 1) or two (group 2) doses of PV coadministered with 1 dose of SV. The main outcome was the seroconversion rate for PV at 1 month. Secondary outcomes were the geometric mean titer ratios and the seroprotection rates at 1 month for all vaccines, seroconversion rates at 1 month for SV, and longitudinal changes of antibody titers (ABTs) at 1, 2, 6, and 12 months for all vaccines. Groups 1 and 2 had similar CD4 counts and HIV RNA levels during the study. The seroconversion rate for PV was 100% at 1 month in both groups. ABTs for PV were high during the first 6 months and declined below seroprotection levels thereafter. Longitudinal changes in ABTs were similar in groups 1 and 2 for both PV and SV. The side effects of vaccination were mild and mostly local. In HIV-infected children, adolescents, and young adults, the immune response triggered by a single dose of PV was similar to that obtained with a double dose and was associated with long-term antibody response.
Seasonal influenza viruses and respiratory syncytial virus (RSV) are primary causes of acute respiratory tract infections (ARTIs) in children. New respiratory viruses including human metapneumovirus (hMPV), human bocavirus (hBoV), and influenza 2009 A(H1N1) virus have a strong impact on the pediatric population.
The discovery of human Metapneumovirus (hMPV) and human Bocavirus (hBoV) identified the etiological causes of several cases of acute respiratory tract infections in children. This report describes the molecular epidemiology of hMPV and hBoV infections observed following viral surveillance of children hospitalized for acute respiratory tract infections in Milan, Italy. Pharyngeal swabs were collected from 240 children ?3 years of age (130 males, 110 females; median age, 5.0 months; IQR, 2.0-12.5 months) and tested for respiratory viruses, including hMPV and hBoV, by molecular methods. hMPV-RNA and hBoV-DNA positive samples were characterized molecularly and a phylogenetical analysis was performed. PCR analysis identified 131/240 (54.6%) samples positive for at least one virus. The frequency of hMPV and hBoV infections was similar (8.3% and 12.1%, respectively). Both infections were associated with lower respiratory tract infections: hMPV was present as a single infectious agent in 7.2% of children with bronchiolitis, hBoV was associated with 18.5% of pediatric pneumonias and identified frequently as a single etiological agent. Genetically distinct hMPV and hBoV strains were identified in children examined with respiratory tract infections. Phylogenetic analysis showed an increased prevalence of hMPV genotype A (A2b sublineage) compared to genotype B (80% vs. 20%, respectively) and of the hBoV genotype St2 compared to genotype St1 (71.4% vs. 28.6%, respectively). Interestingly, a shift in hMPV infections resulting from A2 strains has been observed in recent years. In addition, the occurrence of recombination events between two hBoV strains with a breakpoint located in the VP1/VP2 region was identified.
During the 2006-2007 season, 19 HIV-uninfected and 33 HIV-infected children and adolescents with full immunovirologic response to HAART were immunized against influenza and subsequently followed up. One month post-immunization all subjects had protective antibodies titres which persisted for the whole influenza season. Seven vaccinees (four HIV-infected and three HIV-uninfected) were found to be infected by influenza viruses during the epidemic, but disease was lab-confirmed only in two HIV-infected subjects. Both presented a benign clinical course and were infected by an A/Brisbane/10/07-H3N2-like virus. These data indicate that HIV-infected subjects benefit from routine seasonal influenza vaccination.
Human bocavirus (HBoV) is a linear single-stranded DNA virus belonging to the Parvoviridae family that has recently been isolated from the upper respiratory tract of children with acute respiratory infection. All of the strains observed so far segregate into two genotypes (1 and 2) with a low level of polymorphism. Given the recent description of the infection and the lack of epidemiological and molecular data, we estimated the viruss rates of molecular evolution and population dynamics. A dataset of forty-nine dated VP2 sequences, including also eight new isolates obtained from pharyngeal swabs of Italian patients with acute respiratory tract infections, was submitted to phylogenetic analysis. The model parameters, evolutionary rates and population dynamics were co-estimated using a Bayesian Markov Chain Monte Carlo approach, and site-specific positive and negative selection was also investigated. Recombination was investigated by seven different methods and one suspected recombinant strain was excluded from further analysis. The estimated mean evolutionary rate of HBoV was 8.6x10(-4)subs/site/year, and that of the 1st+2nd codon positions was more than 15 times less than that of the 3rd codon position. Viral population dynamics analysis revealed that the two known genotypes diverged recently (mean tMRCA: 24 years), and that the epidemic due to HBoV genotype 2 grew exponentially at a rate of 1.01year(-1). Selection analysis of the partial VP2 showed that 8.5% of sites were under significant negative pressure and the absence of positive selection. Our results show that, like other parvoviruses, HBoV is characterised by a rapid evolution. The low level of polymorphism is probably due to a relatively recent divergence between the circulating genotypes and strong purifying selection acting on viral antigens.
Anal intraepithelial neoplasia and anal cancer are closely related to infection from high-risk Human Papilloma Virus (HPV) genotypes. Since HPVs involved in disease progression are reported to vary by geographical regions, this study focuses on HPV genotypes spectrum in 289 males attending a Sexual Transmitted Diseases (STD) unit according to their nationality. Anal cytology, Digene Hybrid Capture Assay (HC2) and HPV genotyping were evaluated in 226 Italian (IT) and 63 foreign born (FB) subjects, recruited between January 2003 and December 2006. FB people were younger (median 32y-IQR 27-35 vs 36y-IQR 31-43, respectively; Mann-Whitney test p<0.0001) and had a higher rate of abnormal results (>or=atypical squamous cells of undetermined significance (ASCUS)) on anal cytology (95.0% vs 84.04%) (p=0.032; OR 3.61; 95% CI 1.04-1.23). HPV-16 is by far the most common genotype found in anal cytological samples independently from nationality while differences in distribution of other HPV genotypes were observed. The probability of infection from high-risk HPVs was higher in FB (OR 1.69; 95% CI 1.07-2.68) and is due to a higher rate of HPV-58 (OR 4.98; 95% CI 2.06-12.04), to a lower rate of HPV-11 (OR 0.35; 95% CI 0.16-0.77), to the presence of other high-risk genotypes (HPV-45, HPV-66, HPV-69). Multiple infections rate was high and comparable between IT and FB people. The relative contribution of each HPV genotype in the development of pre-neoplastic disease to an early age in the FB group cannot be argued by this study and more extensive epidemiological evaluations are needed to define the influence of each genotype and the association with the most prevalent high-risk HPVs on cytological intraepithelial lesions development.
A cross-sectional study was carried out to improve the state of evidence regarding the spectrum of HPV types and HPV-16 LCR variants circulating among men and women infected with HIV-1 in Italy. This study, conducted in 518 HIV-positive subjects (346 males and 172 females), showed a high prevalence of HPV anal infections (88.7%) in men and of cervical infections (65.1%) in women. A wide spectrum of HPV genotypes has been observed, as both single and multiple infections. Low-risk HPV types 6, 11 and 61 were frequently detected. HPV-16 was the prevalent high-risk type. Fourteen different HPV-16 LCR variants were found. Ten belonged to the European lineage (78.7% were detected in Italian subjects and 21.3% in foreign-born, all homo/bisexual men), two to the Asiatic lineage and two to the African-2 lineage. This study underlines the great genotypic heterogeneity characterizing anal and cervical HPV infections and the marked polymorphism of the predominant HPV-16 in this high-risk population in Italy.
To assess the safety and immunogenicity of influenza vaccination, patients with cirrhosis undergoing treatment or not and liver transplant recipients under standard immunosuppression were vaccinated and followed up for 6 months. One month after vaccination, seroprotection rates and antibody GMTs against the three vaccine antigens were higher than baseline levels in all three patients groups. No differences in seroconversion and seroprotection rates were found within groups, but antibody GMTs against A/H1N1 and A/H3N2 strains were lower in liver transplant recipients than in patients with cirrhosis without treatment. No serious adverse events and no alteration of the liver function tests were observed. Patients with cirrhosis, including those under treatment, and liver transplant recipients benefit from influenza vaccination and can be safely immunized.
Morbidity and mortality related to influenza are highest among people aged 65 and above, and among people of any age who have medical conditions that place them at increased risk of influenza complications, including hospitalisation and death. Influenza vaccination is the most effective tool for preventing influenza both in terms of cost-effectiveness and benefit-cost ratios. Given the health benefits of influenza vaccination in preventing the disease and its potentially severe complications, recommendations that focus on providing annual vaccination targeted to people at increased risk have been implemented, but coverage among these groups is still well below the Italian health authorities goals.
Influenza A(H3N2) virus is once again the predominant strain after the 2009 pandemic. Its molecular epidemiology and phylogeny were investigated during the 2011-2012 season in Northern Italy. The epidemiological and virological influenza surveillance was carried out within the framework of the Italian Influenza Surveillance Network. The hemagglutinin (HA) gene of the A(H3N2) viruses detected was analyzed by means of a time-scaled phylogenetic approach. In Northern Italy, the 2011-2012 epidemic wave was sustained almost exclusively by influenza A(H3N2) viruses (87.2% of total influenza virus detections). The consultation rates for influenza-like illness (ILI) in the age group ?65 years were 1.5 to 6-fold higher than those registered during the previous eight epidemics: A(H3N2) was the only virus identified in this group. The phylogenetic analysis of A(H3N2) viruses showed viruses belonging to the A/Victoria/208/2009 genetic clade, characterized by substitutions in HA antigenic sites with respect to the A/Perth/16/2009-like 2011-2012 vaccine strain. About one-third of analyzed sequences fell into group 6 and two thirds into group 3 (subdivided into 3A, 3B, and 3C). The time scale reconstruction of the phylogeny showed several independent introductions of A(H3N2) groups between summer and winter of 2011. However, the common origin of all the circulating A(H3N2) strains dated back to the 2009 pandemic period (November 2009). The time scale phylogenetic approach is of particular importance for the evaluation of the introduction and circulation of new variants in the area. Therefore, it should be implemented within the framework of influenza virological surveillance.
The aim of this study was to reconstruct the evolutionary dynamics of the A(H1N1)pdm09 influenza virus in Italy during two epidemic seasons (2009/2010 and 2010/2011) in the light of the forces driving the evolution of the virus. Nearly six thousands respiratory specimens were collected from patients with influenza-like illness within the framework of the Italian Influenza Surveillance Network, and the A(H1N1)pdm09 hemagglutinin (HA) gene was amplified and directly sequenced from 227 of these. Phylodynamic and phylogeographical analyses were made using a Bayesian Markov Chain Monte Carlo method, and codon-specific positive selection acting on the HA coding sequence was evaluated. The global and local phylogenetic analyses showed that all of the Italian sequences sampled in the post-pandemic (2010/2011) season grouped into at least four highly significant Italian clades, whereas those of the pandemic season (2009/2010) were interspersed with isolates from other countries at the tree root. The time of the most recent common ancestor of the strains circulating in the pandemic season in Italy was estimated to be between the spring and summer of 2009, whereas the Italian clades of the post-pandemic season originated in the spring of 2010 and showed radiation in the summer/autumn of the same year; this was confirmed by a Bayesian skyline plot showing the biphasic growth of the effective number of infections. The local phylogeography analysis showed that the first season of infection originated in Northern Italian localities with high density populations, whereas the second involved less densely populated localities, in line with a gravity-like model of geographical dispersion. Two HA sites, codons 97 and 222, were under positive selection. In conclusion, the A(H1N1)pdm09 virus was introduced into Italy in the spring of 2009 by means of multiple importations. This was followed by repeated founder effects in the post-pandemic period that originated specific Italian clades.
This study evaluated the immunogenicity, safety and tolerability of a single 0.5 mL dose of the seasonal virosomal subunit influenza vaccine (Inflexal V, Crucell, Switzerland) in 205 healthy, unprimed children aged at least 6 to <36 months, evaluated at four weeks post-vaccination and seven months from baseline. Of the enrolled children, 102 received one single 0.5 mL dose and 103 received the standard two 0.25 mL doses given four weeks apart. Both treatments evoked an immune response that satisfied the EMA/CHMP criteria for yearly vaccine licensing for all three vaccine strains. Exploratory analyses revealed no differences between the groups at four weeks post-vaccination. Furthermore, immunogenicity was maintained seven months after the first vaccination after both the 0.5 mL and standard two 0.25 mL doses. Adverse events were comparable between groups and were as expected according to the safety profile of the vaccine; overall, the vaccine was well tolerated. Our results show that a single 0.5 mL dose effectively and safely provided long-term immunogenicity to all three influenza strains in unprimed children aged at least 6 to <36 months.
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