The rapid spread of SARS-COV-2 Omicron variant in Italy reflected early through wastewater surveillance

The SARS-CoV-2 Omicron variant emerged in South Africa in November 2021, and has later been identified worldwide, raising serious concerns. A real-time RT-PCR assay was designed for the rapid screening of the Omicron variant, targeting characteristic mutations of the spike gene. The assay was used to test 737 sewage samples collected throughout Italy (19/21 Regions) between 11 November and 25 December 2021, with the aim of assessing the spread of the Omicron variant in the country. Positive samples were also tested with a real-time RT-PCR developed by the European Commission, Joint Research Centre (JRC), and through nested RT-PCR followed by Sanger sequencing. Overall, 115 samples tested positive for Omicron SARS-CoV-2 variant. The first occurrence was detected on 7 December, in Veneto, North Italy. Later on, the variant spread extremely fast in three weeks, with prevalence of positive wastewater samples rising from 1.0% (1/104 samples) in the week 5–11 December, to 17.5% (25/143 samples) in the week 12–18, to 65.9% (89/135 samples) in the week 19–25, in line with the increase in cases of infection with the Omicron variant observed during December in Italy. Similarly, the number of Regions/Autonomous Provinces in which the variant was detected increased from one in the first week, to 11 in the second, and to 17 in the last one. The presence of the Omicron variant was confirmed by the JRC real-time RT-PCR in 79.1% (91/115) of the positive samples, and by Sanger sequencing in 66% (64/97) of PCR amplicons. In conclusion, we designed an RT-qPCR assay capable to detect the Omicron variant, which can be successfully used for the purpose of wastewater-based epidemiology. We also described the history of the introduction and diffusion of the Omicron variant in the Italian population and territory, confirming the effectiveness of sewage monitoring as a powerful surveillance tool

The rapid spread of SARS-COV-2 Omicron variant in Italy reflected early through wastewater surveillance

The SARS-CoV-2 Omicron variant emerged in South Africa in November 2021, and has later been identified worldwide, raising serious concerns. A real-time RT-PCR assay was designed for the rapid screening of the Omicron variant, targeting characteristic mutations of the spike gene. The assay was used to test 737 sewage samples collected throughout Italy (19/21 Regions) between 11 November and 25 December 2021, with the aim of assessing the spread of the Omicron variant in the country. Positive samples were also tested with a real-time RT-PCR developed by the European Commission, Joint Research Centre (JRC), and through nested RT-PCR followed by Sanger sequencing. Overall, 115 samples tested positive for Omicron SARS-CoV-2 variant. The first occurrence was detected on 7 December, in Veneto, North Italy. Later on, the variant spread extremely fast in three weeks, with prevalence of positive wastewater samples rising from 1.0% (1/104 samples) in the week 5–11 December, to 17.5% (25/143 samples) in the week 12–18, to 65.9% (89/135 samples) in the week 19–25, in line with the increase in cases of infection with the Omicron variant observed during December in Italy. Similarly, the number of Regions/Autonomous Provinces in which the variant was detected increased fromone in the first week, to 11 in the second, and to 17 in the last one. The presence of the Omicron variant was confirmed by the JRC real-time RT-PCR in 79.1% (91/115) of the positive samples, and by Sanger sequencing in 66% (64/97) of PCR amplicons

The rapid spread of SARS-COV-2 Omicron variant in Italy reflected early through wastewater surveillance

The SARS-CoV-2 Omicron variant emerged in South Africa in November 2021, and has later been identified worldwide, raising serious concerns. A real-time RT-PCR assay was designed for the rapid screening of the Omicron variant, targeting characteristic mutations of the spike gene. The assay was used to test 737 sewage samples collected throughout Italy (19/21 Regions) between 11 November and 25 December 2021, with the aim of assessing the spread of the Omicron variant in the country. Positive samples were also tested with a real-time RT-PCR developed by the European Commission, Joint Research Centre (JRC), and through nested RT-PCR followed by Sanger sequencing. Overall, 115 samples tested positive for Omicron SARS-CoV-2 variant. The first occurrence was detected on 7 December, in Veneto, North Italy. Later on, the variant spread extremely fast in three weeks, with prevalence of positive wastewater samples rising from 1.0% (1/104 samples) in the week 5-11 December, to 17.5% (25/143 samples) in the week 12-18, to 65.9% (89/135 samples) in the week 19-25, in line with the increase in cases of infection with the Omicron variant observed during December in Italy. Similarly, the number of Regions/Autonomous Provinces in which the variant was detected increased from one in the first week, to 11 in the second, and to 17 in the last one. The presence of the Omicron variant was confirmed by the JRC real-time RT-PCR in 79.1% (91/115) of the positive samples, and by Sanger sequencing in 66% (64/97) of PCR amplicons. In conclusion, we designed an RT-qPCR assay capable to detect the Omicron variant, which can be successfully used for the purpose of wastewater-based epidemiology. We also described the history of the introduction and diffusion of the Omicron variant in the Italian population and territory, confirming the effectiveness of sewage monitoring as a powerful surveillance tool

Sewage monitoring for pathogenic microrganisms detection by using a low density microarray

Aims. Sewages are highly contaminated by numerous biological agents: bacteria, protozoa, fungi and viruses. Some of them can be very resistant to treatments and remain at high concentration in the effluent, thus representing an hazard especially in the case of water reuse for agriculture. Molecular methods are almost an important way to detect these pathogens, in particular the most applied techniques are based on protocols of nucleic acid amplification and quantification, of which the quantitative polymerase chain reaction (qPCR) is rapidly becoming established in the environmental sector for its higher sensitivity in comparison with cultural assays. Moreover the possibility in using molecular methods for simultaneous and rapid multiple detection of pathogens could be useful for risk assessment or safety purposes. This approach could be now possible using the technological advances of the DNA microarray, born for clinical analysis and based on the simultaneous qualitative analysis for multiple genera, species and strains. The large number of DNA sequences that can be spotted on a microarray together with the high specificity of binding to the immobilised sequence targets, allows the detection of a large range of microorganisms with high discriminatory ability. Nevertheless the application of this technology requires a deep study of its sensitivity and specificity. In this work, an environmental monitoring of sewage, sampled from a Waste Water Treatment Plant (WWTP), was performed in order to detect some different viruses and bacteria frequently present: Human Adenovirus (HAdV), Norovirus GGII (NV), Hepatitis A Virus (HAV), Enterovirus (EV), Rotavirus (RV), Enterohemorrhagic E. coli (EHEC), Salmonella enterica (SE) using a low density microarray containing specific oligonucleotides sequences for each of them. Methods. In the first phase of the study, a series of trials were performed to choose the sample treatment that was able to increase the sensitivity of molecular method. In particular 10 sewage samples (10 L) were treated with a first step based on tangential ultrafiltration followed by a second step in which the obtained eluates were further concentrated by ultracentrifugation until a final volume of 1 ml. After each step, the obtained eluates were treated with commercial kit (QIAgen) to extract viral and bacteria nucleic acids (NA) that were quantified by separate and specific qPCR reactions (Genomic Copies - GC) according to published protocols. In the second phase, an environmental monitoring was performed for 1 year from June 2013 to June 2014 by monthly sampling of 10 L of sewage from a WWTP in Northern Italy (13 samples). The concentrated samples, obtained by the previous phase chosen method, were treated to extract NA that were subsequently concentrated by speed VAC, labelled and overnight hybridised on the microarray slides. After that, a scanner read the positive signal. Parallel, aliquots of extracted NA of each samples were quantify (GC/10 L) by qPCR reactions. Results. The data of the trials revealed that the combination of ultrafiltration-ultracentrifugation permitted to increase the mean concentration of target microorganisms of 1-2 Log in comparison with the use of only ultrafiltration method. The microarray analysis revealed the presence of HAdv, EV, EHEC and SE in tested samples, while no NV, HAV and RV were detected. In particular, 78% (10/13) of samples resulted positive for HAdV, 30% (4/13) for EV, 84% (11/13) for EHEH and 61% (8/13) for SE. The simultaneous presence in the same sample of all these 4 target was detected in 2 samples (15%), of three target in 6 samples (46%) and of two target in 2 of samples (15%). The mean concentration of positive target estimated by qPCR were 2 x 108 GC/10 L for HAdV, 4 x 108 CG/10 L for EV, 107 GC/10 L for EHEC and 3 x 105 GC/10 L for SE. These data are in according of sewage microbial concentration published by several authors (Figure 1). The absence of positive samples for NV, HAV and RV was probably due by epidemiological situation in the monitored region. The data, moreover, underlined a limit of sensitivity of the test: samples with a target concentration lower than 2,8 x 103 GC/10 L resulted negative to microarray. Conclusion. The results of environmental monitoring were very promising for a multiple detection of pathogens in sewage confirming the possible use of microarray as a tool for screening. The major limitation of this technique was the scarce sensitivity that can be improved with specific sample treatments permitting also the purification of samples, as made in this study, by the combination of ultrafiltration and ultracentrifugation

A biological filter for the treatment of waste water from wineries : the experience of the Autonomous Province of Trento

This paper describes the work carried out by the Autonomous Province of Trento to develop immersed adhesive biomass technology for the pre-treatment of winery wastewater before its discharge into the sewer system within the limits laid down by current Italian law : BOD5 < 250 mg/l COD < 500 mg/l, suspended solids < 200 mg/l. The tests were carried out on a pilot plant which had a reactor volume of 3.3 m3. After an initial activation period, a stable balanced biomass was established and measurement of the main chemical and physical parameters began. These measurements showed that the performance of the type of installation tested esable to guarantee that the limits imposed by Italian law regarding discharges into the public sewer system are respected, provided that the maximum load conditions do not exceed 3 KgCOD/m3 per day. This result, if compared with standard traditional technologies, demonstrates that the use of immersed adhesive biomass filters allow the volume of the reactors to be 2 to 3 times smaller, with obvious economic advantages. / L'objet de l'expérience réalisée par la Province Autonome de Trente a consisté à développer la technologie à biomasse adhérente immergée, en vue d'une première évacuation des écoulements des caves à vins pour que ces flux soient acheminés dans les égouts, conformément à la loi en vigueur. Nous faisons, tout particulièrement, référence à un BOD5 < 250 mg/l. Les test ont été réalisés sur une installation pilote, dont le réacteur avait un volume utile de 3,3 m3. A l'issue d'une première phase de mise en route, nous avons pu obtenir un équilibre stable de la biomasse. Une fois que nous avons atteint ce stade, nous avons fait des prélèvements pour identifier les principaux paramètres chimiques et physiques. Ces analyses indiquent que le rendement d'épuration sur le type d'installation testée peut garantir le respect des limites envisagées par la réglementation italienne en matière d'évacuation dans les égouts publics, pour des conditions de charge maximale de 3 KgCOD/m3 par jour. Si on confronte ce résultat aux standards des technologies traditionnelles, on pourra prouver que l'utilisation de filtres à biomasse adhérente immergée permet de réduire de 2 à 3 fois le volume des réacteurs, ce qui comporte, naturellement, un gros avantage sur le plan économique

Ricerca di microrganismi patogeni enterici in matrici idriche mediante l'utilizzo della tecnologia del microarray

Obiettivi. Il monitoraggio ambientale basato sulla ricerca di indicatori rappresenta un importante strumento per la valutazione del rischio biologico nelle matrici idriche. Tuttavia per la scarsa correlabilità tra questi ed alcuni patogeni, soprattutto di natura virale, è stata suggerita anche a livello internazionale (OMS, EPA), la ricerca di 'patogeni indice' soprattutto per alcune matrici come ad esempio le acque di sorgente, quelle reflue trattate riemesse nell'ambiente e quelle destinate ad un riutilizzo agricolo o industriale. Uno dei problemi più rilevanti per tale scopo è la possibilità di avere un elevato numero di microrganismi patogeni e la difficoltà di eseguire dei test che permettano di identificarli simultaneamente. Questo approccio è ora possibile grazie ai progressi nel campo della biologia molecolare, che permettono un'analisi microbiologica più sofisticata riuscendo a risolvere molti problemi associati anche alla non colturabilità. In particolare, la "nanotecnologia" del DNA microarray, potrebbe essere applicata per valutare il rischio biologico nei campioni di acqua. L'elevato numero di sequenze sonde di DNA che possono essere inserite unitamente alla specificità di legame tra i target e queste, permettono il rilevamento simultaneo di una vasta gamma di generi, specie e ceppi microbici con elevata capacità discriminatoria. Tuttavia l'applicazione di questa tecnologia in campo ambientale richiede uno studio approfondito della sua sensibilità e specificità, nonché il confronto con le tecniche tradizionali. In questo lavoro, è stato sviluppato un oligonucleotide microarray per lo screening di microrganismi enterici (virus e batteri) legati all'inquinamento idrico: adenovirus, norovirus GGII, virus dell'epatite A, enterovirus, rotavirus, E.coli O157H7 e Salmonella enterica . Metodi. Dopo una prima fase, dove sono state identificate per ciascun microrganismo le regioni genomiche più conservate per la sintesi del microarray, sono stati allestiti una serie di test per verificare la specificità con campioni ad alto titolo per ciascun target. Quindi la metodica è stata testata con campioni di acqua reflua contaminata artificialmente per verificare la sensibilità con matrici simili a quelli "naturali". Dopo concentrazione, gli acidi nucleici sono stati contemporaneamente quantificati, mediante real time PCR, per stimare la contaminazione (copie genomiche/μl) e analizzati con la piattaforma microarray. Conoscendo la concentrazione iniziale di ciascun microrganismo ed il volume dei campioni depositato è stato possibile avere una stima della sensibilità. Risultati. I risultati dei test su campioni ad alto titolo hanno confermato la specificità della piattaforma microarray: ciascun microrganismo bersaglio è stato identificato. Il test sui campioni artificialmente contaminati hanno rivelato un limite di sensibilità media per ciascun target microbico di 1000-10000 copie genomiche/μl, corrispondente a 10^9-10^10 copie genomiche/L. Conclusioni. I dati ottenuti sono stati molto promettenti per l'applicazione della tecnica su matrici idriche dove la problematica correlazione indicatori-patogeni è ben nota e dove la concentrazione dei patogeni che si raggiunge è costantemente conforme al livello di sensibilità ottenuta, come nei reflui. Tuttavia per poterne auspicarne una sua applicazione in altre matrici come acque superficiali o potabili dove la contaminazione è molto più bassa, risulta necessario effettuare ulteriori studi per implementarne la sensibilità soprattutto nella fase di concentrazione e purificazione del campione dalla presenza di inibitori