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Sorveglianza delle Gastroenteriti Acute da Rotavirus in 3 province della Lombardia
No full textWorkshop
SORVEGLIANZA
DELLE PARALISI FLACCIDE ACUTE
E DELL’INFEZIONE DA ROTAVIRUS IN ITALI
Sorveglianza delle Gastroenteriti Acute da Rotavirus in Lombardia
No full textSorveglianza delle Gastroenteriti Acute da Rotavirus in Lombardi
CONTRIBUTO AL PROGETTO OMS DI ERADICAZIONE DELLA POLIOMIELITE: RISULTATI DELLE ATTIVITÀ DI SORVEGLIANZA IN LOMBARDIA.
Full text linkA 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 – 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 “WHO Strategic Plan of the Global Polio Eradication Initiative” 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′ noncoding region [5′NCR] (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 1.04/100’000 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 0.8/100’000 in 2012, 1.5/100’000 in 2013, 1.1/100’000 in 2014, and 0.6/100’000 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 100’000 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-Barré 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 ≥1:8) 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
Phylogenetic analysis of the hemagglutinin (HA) gene of A(H3N2) influenza viruses circulating in Northern Italy during the 2016-2017 influenza season
No full textBackground
A(H3N2) influenza virus predominated in Europe during the 2016-2017 season, characterised by an excess mortality in people >65 years concurrent with A(H3N2) circulation. A molecular and evolutionary characterisation of A(H3N2) detected in Lombardy (Northern Italy) within the Italian Influenza Surveillance Network (InfluNet) during 2016-2017 season was performed.
Methods
549 respiratory samples from ILI outpatients (525/549=95.6%) and SARI/ARDS inpatients (24/549=4.4%) were collected in Lombardy from week 46-2016 to week 17-2017. Influenza viruses were typed (A/B) and subtyped (H1pdm09/H3N2) by real-time RT-PCR. A(H3N2) HA complete gene (nt. 1-1778) was phylogenetically analysed.
Results
Influenza viruses were detected in 52.3% (287/549) specimens; 93.4% (268/287) were A(H3N2). 34% (91/268) was sequenced. All HA sequences clustered in the genetic group 3C, sub-group 3C.2a, which included the vaccine strain A/HongKong/4801/2014 (similarity: 98.3-99.4%). Most (78/91=85.7%) sequences were A/Bolzano/7/2016-like (similarity: 98.7-99.9%) and belonged to sub-clade 3C.2a1, characterised by amino acid (aa) substitutions N171K (in epitope D), I406V and G484E. 41% of these sequences had mutation T135 (loss of a glycosylation site). Two additional sub-clades were identified: 3C.2a2 (7/91=7.7%) and 3C.2a3 (4/91=4.4%), characterised by aa changes N121K/S144K (epitope A/D) and T131K/R142K (epitope A), respectively. 57 mutations in 54 aa positions were identified, many at single sequence level. Most (>80%) aa changes were detected in HA1 subunit and >50% occurred in epitope A or D. No signature substitutions were observed in HA sequences of A(H3N2) strains detected in SARI/ARDS cases or vaccinated individuals.
Conclusion
The majority of A(H3N2) viruses circulating in 2016-2017 season clustered in sub-clade 3C.2a1, molecularly and antigenically similar to A/HongKong/4801/2014 vaccine strain. Several HA variants were identified. Full-length sequencing will be useful to define the molecular and evolutionary characteristics of these A(H3N2) viruses
Introduzione di enterovirus neurotropi emergenti rilevati grazie all’attività di sorveglianza delle Paralisi Flaccide Acute, Lombardia, 2016
No full textIntroduzione. La sorveglianza delle Paralisi Flaccide Acute (PFA) è il gold-standard per individuare l’introduzione di poliovirus (PV) - selvaggi e vaccino-derivati - in paesi dichiarati polio-free. Le attività di sorveglianza permettono inoltre di monitorare la circolazione di enterovirus-non polio (EVNP) nella popolazione. In Italia la sorveglianza delle PFA è condotta su base regionale dal 1997. Di seguito i risultati dell’attività di sorveglianza ottenuti in Lombardia nel 2016.
Materiali e metodi. Tutti i casi di PFA che rispondevano ai criteri di inclusione dell’OMS sono stati notificati al Centro Regionale di Riferimento (CRR) lombardo dai medici sentinella. In accordo con i protocolli OMS, campioni fecali (e, se disponibili, altri campioni biologici) sono stati raccolti entro 14 giorni dall’esordio della PFA e inviati al CRR. Per ogni caso di PFA sono state condotte le seguenti analisi virologiche: I) isolamento di PV e EVNP in colture cellulari RD e L20b; II) identificazione molecolare di PV e EVNP mediante real-time RT-PCR; III) caratterizzazione molecolare e analisi filogenetica delle sequenze del gene VP1 (nt. 2602-2977) di PV e EVNP.
Risultati. Nel 2016 in Lombardia sono stati notificati 15 casi di PFA con un’incidenza di 1,07/100000 <15 anni d’età, rispondente ai criteri OMS (almeno 1/100000). L’età media dei casi era di 3,6 anni, nel 93% maschi; i casi erano associati a diversi quadri clinici e il 54% è stato notificato tra maggio e settembre. Nessun PV è stato identificato. Tre (20%) casi di PFA sono risultati positivi per EVNP; la caratterizzazione molecolare e l’analisi filogenetica degli EVNP ha permesso di individuare un Echovirus-25, un EVD68 (lineaggio B3) e un EVA71 (subgenogruppo C1).
Conclusioni. I risultati derivati dalla sorveglianza delle PFA in Lombardia nel 2016 hanno confermato l’assenza di circolazione di PV. L’attività si è dimostrata utile nel valutare la circolazione di EVNP e idonea nell’individuare l’introduzione di enterovirus neurotropi quali EVD68 ed EVA71, ritenuti ceppi emergenti e responsabili di recenti epidemie in Europa. Il monitoraggio di EVNP mediante la sorveglianza delle PFA è uno strumento indispensabile per pianificare future azioni e decisioni in materia di sanità pubblica
Enterovirus D68 and A71 recognized by the Acute Flaccid Paralyses (AFP) Surveillance in Northern Italy in 2016
No full textAcute Flaccid Paralyses Surveillance (AFPS) is the strategic system to monitor the emergence of polioviruses (wild and vaccine-derived) in polio-free countries and it is a sensitive case-based scheme to recognize non-polio enteroviruses (NPEVs). We report the findings of AFPS activities in Lombardy (Northern Italy) during 2016 in the framework of the Italian nationwide AFPS.
Methods
Physicians enrolled children <15 years who met the WHO definition of AFP case. Clinical samples were collected for virological investigations and analysed by virus isolation in RD and L20B cell cultures, and by RTPCR assay specific for the 5′ noncoding region (nt. 179-575) of EVs. EVpositive specimens were further analysed by sequencing a fragment of the EV-VP1 (nt. 2602-2977) gene.
Results
In 2016, 15 AFP cases were reported in Lombardy with an incidence of 1.07/100’000 children 98.6% with sequences of B3 lineage identified in the 2016 Dutch outbreak) and EV-A71 (similarity >98.4% with sequences of subgenogroup C1 detected in the 2015 German epidemic).
Conclusion
The results of 2016 AFPS confirmed the absence of poliovirus in the surveyed area, and provided valuable and up-to-date information on the occurrence of NPEVs of public health concern in Northern Italy. The unpredicted detection of EV-D68 and EV-A71, sharing high nucleotide similarity with viruses involved in the latest European outbreaks, emphasises the importance of monitoring NPEVs through AFPS to help public health response
Sorveglianza virologica dell'influenza in Lombardia: caratterizzazione molecolare dei virus influenzali A(H3N2) circolati durante la stagione 2016-2017 in Lombardia
No full textINTRODUZIONE:
La stagione 2016-2017 si è contraddistinta per un avvio anticipato, un impatto di media entità in termini di casi gravi e decessi, e dalla predominante circolazione di virus influenzali A(H3N2). Obiettivo: Caratterizzazione molecolare dei virus influenzali A(H3N2) identificati in Lombardia durante la stagione 2016-2017 nell’ambito delle attività di InfluNet.
MATERIALI E METODI:
Sono stati raccolti 549 tamponi respiratori da individui con sindrome simil-influenzale (ILI) (95.6%) o forme respiratorie severe (SARI/ARDS) (4.4%). L’identificazione dei virus è stata eseguita con real-time RT-PCR. L’analisi filogenetica dei virus A(H3N2) è stata condotta sul gene completo dell’emoagglutinina (HA).
RISULTATI:
Il 51.9% dei campioni è risultato positivo per virus influenzale, di cui il 94% per A(H3N2), lo 0.7% per A(H1N1)pdm09 e il 4.9% per B/Yamagata. Il 34% dei ceppi A(H3N2) è stato analizzato molecolarmente: tutte le sequenze appartenevano al gruppo genetico 3C, sottogruppo 3C.2a, che include il ceppo vaccinale A/Hong Kong/4801/2014 (similarità: 98,3-99,4%). L’85.7% delle sequenze di questo sottogruppo era A/Bolzano/7/2016-like (similarità: 98,7-99,9%) nel sub-clade 3C.2a1, definito dalle sostituzioni aminoacidiche N171K (epitopo D), I406V e G484E. Il 7.7% delle sequenze apparteneva al sub-clade 3C.2a2, caratterizzato dalle sostituzioni aminoacidiche N121K e S144K (epitopo D e A), e il 4.4% al 3C.2a3, contraddistinto dalle mutazioni T131K e R142K (entrambe epitopo A). Complessivamente, sono state individuate 57 mutazioni in 54 aminoacidi. Più dell’80% localizzate nella subunità HA1 e più della metà nell’epitopo A o D. Nessuna mutazione significativa è stata osservata nel gene HA dei ceppi A(H3N2) rilevati nei casi gravi (SARI/ARDS) o vaccinati.
CONCLUSIONI:
La maggior parte dei virus A(H3N2) identificati nella stagione 2016-2017 apparteneva al sub-clade 3C.2a1, antigenicamente e molecolarmente simile al ceppo vaccinale A/Hong Kong/4801/2014. Sono state individuate numerose sostituzioni aminoacidiche nel gene HA, il cui impatto sulle caratteristiche antigeniche virali necessita approfondimenti. Le sequenze del gene HA ottenute da ceppi virali di soggetti vaccinati non ha però evidenziato la presenza di particolari varianti
Genetic changes of influenza A(H1N1)pdm09 viruses circulating in 2018/2019 season affected subtyping of influenza virus by real-time RT-PCR assay
No full textAim: Reduced performance of real-time RT-PCR (rRT-PCR) for influenza A(H1N1)pdm09 virus subtyping was observed during the 2018/2019 season. We analysed and compared the primers/probe binding regions in the hemagglutinin (HA) gene to HA sequence of circulating strains. Method: 713 respiratory samples collected from in/outpatients with influenza illness in Lombardy (Northern Italy) during the 2018/2019 season were analysed. Influenza viruses were typed (A/B) and subtyped (H1N1pdm09/H3N2) by rRT-PCR targeting matrix/nucleoprotein and HA gene, respectively, according to international protocols. The expected difference between Ct values obtained from A-typing and H1N1pdm09-subtyping assays [∆(H1-A)Ct] is <5. Full-length HA gene (nt. 1-1778) sequence of A(H1N1)pdm09 circulating strains were obtained and compared to primers/probe sequences used in subtyping rRT-PCR assay. Results: Influenza A viruses were detected in 390/713 (54.7%) specimens, 48.5% (189/390) were A(H1N1)pdm09. 52/189 (27.5%) A(H1N1)pdm09-positive samples showed a ∆(H1-A)Ct≥5 (range: 5.00-14.95). 24/52 A(H1N1)pdm09 with ∆(H1-A)Ct≥5 and 71/137 with ∆(H1-A)Ct<5 were sequenced. Four nucleotide mismatches (C861T+C867T in the forward primer and A897G+A905C in the probe) were detected in 17/24 (70.8%) HA sequences of A(H1N1)pdm09 with ∆(H1-A)Ct≥5, all had a ∆(H1-A)Ct>8. No mismatches were observed among HA sequences of A(H1N1)pdm09 with ∆(H1-A)Ct<5. Conclusions: Four nucleotide mismatches in primers/probe of subtyping rRT-PCR assay were observed in nearly 30% of A(H1N1)pdm09 viruses circulating in Lombardy during the 2018/2019 season. These point mutations reduced the primer/probe binding efficiency affecting the performance of rRT-PCR assay for subtyping. These results emphasised the need of continuously updating the molecular assays for influenza detection considering the constant evolution of influenza viruses
Individuare Enterovirus neurotropi emergenti sorvegliando Poliovirus: risultati della sorveglianza delle PFA in Lombardia, triennio 2016-2018
No full textPoliomielite ed immigrazione
No full textLa campagna mondiale di eradicazione della polio lanciata dall’OMS nel 1988 prevede per ogni Stato la realizzazione ed il mantenimento di buone condizioni igienico-sanitarie, elevate coperture vaccinali e di attività di sorveglianza delle Paralisi Flaccide Acute (PFA) e sorveglianza ambientale. Ad oggi, l’obiettivo dell’eradicazione non è ancora stato raggiunto sebbene l’incidenza della polio sia diminuita del 99%, solo tre Paesi siano endemici (Nigeria, Pakistan e Afghanistan) e, nel 2015, poliovirus selvaggio di tipo 2 (WPV2) sia stato dichiarato eradicato.
Fintanto che esisteranno paesi endemici per polio, la re-introduzione del virus in popolazioni polio-free rimarrà possibile come già dimostrato. In Tagikistan -Regione Europea OMS polio-free dal 2002- un’ampia epidemia (560 casi) di PFA da infezione con WPV1 è stata registrata nel 2010; indagini di filogenesi hanno indicato che il ceppo virale responsabile provenisse dall’India tramite movimenti migratori. Nel 2013, epidemie di polio causate da reintroduzione di WPV1 sono state registrate in Somalia e Kenya dove la trasmissione del virus era assente da decenni. In Israele, nello stesso anno, WPV1 è stato ritrovato in campioni di liquami in assenza di casi clinici mentre nel 2013-2016 un’ampia circolazione virale è stata registrata in numerosi paesi considerati non-endemici tra cui Siria, Iraq, Madagascar, Somalia, Etiopia, Camerun, Guinea Equatoriale, Laos ed Ucraina.
Tutte le epidemie registrate in questi ultimi anni sono risultato di importazione mediata da flussi migratori, commercio, viaggi internazionali da Paesi endemici verso Paesi in cui l’instabilità politica, conflitti, povertà ed inadeguate condizioni igienico-sanitarie portano alla diminuzione delle coperture vaccinali e dell’efficienza dei sistemi di sorveglianza. Sebbene l’Italia abbia registrato l’ultimo caso autoctono di polio nel 1982 e da importazione nel 1988, reintroduzioni rimangono possibili in considerazione della situazione geopolitica mondiale e del flusso immigratorio che sta investendo l’Europa intera.
In accordo con il ‘31° meeting of the European Regional Certification Commission for poliomyelitis eradication (RCC), 2017’ nella Regione Europea OMS non vi è stata alcuna introduzione e trasmissione di polio durante il 2016. Ciononostante la Bosnia-Erzegovina, la Romania e l’Ucraina sono considerati Paesi ad alto rischio di trasmissione virale a causa di misure preventive non ottimali ed è stata espressa preoccupazione per il numero di Paesi, compresa l’Italia, dove la copertura vaccinale è in declino e la qualità delle sorveglianze non raggiunge gli standard. Mai come in questa ultima fase del progetto di eradicazione ed in considerazione del contesto mondiale è importante sia mantenere adeguati livelli di immunizzazione che efficienti sistemi di sorveglianza per individuare e rispondere prontamente ad eventuali reintroduzioni; contestualmente è necessario che la trasmissione di polio cessi nei paesi ancora endemici
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