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2.
Vaccines (Basel) ; 10(8)2022 Aug 19.
Article in English | MEDLINE | ID: covidwho-2024372

ABSTRACT

(1) Background: Over the last few years, there has been growing interest in the whole genome sequencing (WGS) of rapidly mutating pathogens, such as influenza viruses (IVs), which has led us to carry out in-depth studies on viral evolution in both research and diagnostic settings. We aimed at describing and determining the validity of a WGS protocol that can obtain the complete genome sequence of A(H3N2) IVs directly from clinical specimens. (2) Methods: RNA was extracted from 80 A(H3N2)-positive respiratory specimens. A one-step RT-PCR assay, based on the use of a single set of specific primers, was used to retro-transcribe and amplify the entire IV type A genome in a single reaction, thus avoiding additional enrichment approaches and host genome removal treatments. Purified DNA was quantified; genomic libraries were prepared and sequenced by using Illumina MiSeq platform. The obtained reads were evaluated for sequence quality and read-pair length. (3) Results: All of the study specimens were successfully amplified, and the purified DNA concentration proved to be suitable for NGS (at least 0.2 ng/µL). An acceptable coverage depth for all eight genes of influenza A(H3N2) virus was obtained for 90% (72/80) of the clinical samples with viral loads >105 genome copies/mL. The mean depth of sequencing ranged from 105 to 200 reads per position, with the majority of the mean depth values being above 103 reads per position. The total turnaround time per set of 20 samples was four working days, including sequence analysis. (4) Conclusions: This fast and reliable high-throughput sequencing protocol should be used for influenza surveillance and outbreak investigation.

3.
Diagnostics (Basel) ; 12(6)2022 Jun 16.
Article in English | MEDLINE | ID: covidwho-1911236

ABSTRACT

For diagnosing SARS-CoV-2 infection and for monitoring its spread, the implementation of external quality assessment (EQA) schemes is mandatory to assess and ensure a standard quality according to national and international guidelines. Here, we present the results of the 2020, 2021, 2022 EQA schemes in Lombardy region for assessing the quality of the diagnostic laboratories involved in SARS-CoV-2 diagnosis. In the framework of the Quality Assurance Programs (QAPs), the routinely EQA schemes are managed by the regional reference centre for diagnostic laboratories quality (RRC-EQA) of the Lombardy region and are carried out by all the diagnostic laboratories. Three EQA programs were organized: (1) EQA of SARS-CoV-2 nucleic acid detection; (2) EQA of anti-SARS-CoV-2-antibody testing; (3) EQA of SARS-CoV-2 direct antigens detection. The percentage of concordance of 1938 molecular tests carried out within the SARS-CoV-2 nucleic acid detection EQA was 97.7%. The overall concordance of 1875 tests carried out within the anti-SARS-CoV-2 antibody EQA was 93.9% (79.6% for IgM). The overall concordance of 1495 tests carried out within the SARS-CoV-2 direct antigens detection EQA was 85% and it was negatively impacted by the results obtained by the analysis of weak positive samples. In conclusion, the EQA schemes for assessing the accuracy of SARS-CoV-2 diagnosis in the Lombardy region highlighted a suitable reproducibility and reliability of diagnostic assays, despite the heterogeneous landscape of SARS-CoV-2 tests and methods. Laboratory testing based on the detection of viral RNA in respiratory samples can be considered the gold standard for SARS-CoV-2 diagnosis.

4.
Diagnostics ; 12(6):1483, 2022.
Article in English | MDPI | ID: covidwho-1894163

ABSTRACT

For diagnosing SARS-CoV-2 infection and for monitoring its spread, the implementation of external quality assessment (EQA) schemes is mandatory to assess and ensure a standard quality according to national and international guidelines. Here, we present the results of the 2020, 2021, 2022 EQA schemes in Lombardy region for assessing the quality of the diagnostic laboratories involved in SARS-CoV-2 diagnosis. In the framework of the Quality Assurance Programs (QAPs), the routinely EQA schemes are managed by the regional reference centre for diagnostic laboratories quality (RRC-EQA) of the Lombardy region and are carried out by all the diagnostic laboratories. Three EQA programs were organized: (1) EQA of SARS-CoV-2 nucleic acid detection;(2) EQA of anti-SARS-CoV-2-antibody testing;(3) EQA of SARS-CoV-2 direct antigens detection. The percentage of concordance of 1938 molecular tests carried out within the SARS-CoV-2 nucleic acid detection EQA was 97.7%. The overall concordance of 1875 tests carried out within the anti-SARS-CoV-2 antibody EQA was 93.9% (79.6% for IgM). The overall concordance of 1495 tests carried out within the SARS-CoV-2 direct antigens detection EQA was 85% and it was negatively impacted by the results obtained by the analysis of weak positive samples. In conclusion, the EQA schemes for assessing the accuracy of SARS-CoV-2 diagnosis in the Lombardy region highlighted a suitable reproducibility and reliability of diagnostic assays, despite the heterogeneous landscape of SARS-CoV-2 tests and methods. Laboratory testing based on the detection of viral RNA in respiratory samples can be considered the gold standard for SARS-CoV-2 diagnosis.

5.
Water ; 14(5):833, 2022.
Article in English | MDPI | ID: covidwho-1732289

ABSTRACT

(1) Background: The surveillance of SARS-CoV-2 RNA in urban wastewaters allows one to monitor the presence of the virus in a population, including asymptomatic and symptomatic individuals, capturing the real circulation of this pathogen. The aim of this study was to evaluate the performance of different pre-analytical and analytical methods for identifying the presence of SARS-CoV-2 in untreated municipal wastewaters samples by conducting an inter-laboratory proficiency test. (2) Methods: three methods of concentration, namely, (A) Dextran and PEG-6000 two-phase separation, (B) PEG-8000 precipitation without a chloroform purification step and (C) PEG-8000 precipitation with a chloroform purification step were combined with three different protocols of RNA extraction by using commercial kits and were tested by using two primers/probe sets in three different master mixes. (3) Results: PEG-8000 precipitation without chloroform treatment showed the best performance in the SARS-CoV-2 recovery;no major differences were observed among the protocol of RNA extraction and the one-step real-time RT-PCR master mix kits. The highest analytic sensitivity was observed by using primers/probe sets targeting the N1/N3 fragments of SARS-CoV-2. (4) Conclusions: PEG-8000 precipitation in combination with real-time RT-PCR targeting the N gene (two fragments) was the best performing workflow for the detection of SARS-CoV-2 RNA in municipal wastewaters.

6.
2021.
Preprint in English | Other preprints | ID: ppcovidwho-294400

ABSTRACT

Wastewater-based viral surveillance is a promising approach to monitor the circulation of SARS-CoV-2 in the general population. The aim of this study was to develop an analytical method to detect SARS-CoV-2 RNA in urban wastewater, to be implemented in the framework of a surveillance network in the Lombardy region (Northern Italy). This area was the first hotspot of COVID-19 in Europe. Composite 24h samples were collected weekly in eight cities from end-March to mid-June 2020 (first peak of the epidemic). The method developed and optimized, involved virus concentration, using PEG centrifugation, and one-step real-time RT-PCR for analysis. SARS-CoV-2 RNA was identified in 65 (61%) out of 107 samples, and the viral concentrations (up to 2.1 E +05 copies/L) were highest in March-April. By mid-June, wastewater samples tested negative in all the cities. Viral loads were used for inter-city comparison and Brembate, Ranica and Lodi had the highest. The pattern of decrease of SARS-CoV-2 in wastewater was closely comparable to the decline of active COVID-19 cases in the population, reflecting the effect of lock-down. Wastewater surveillance of SARS-CoV-2 can integrate ongoing virological surveillance of COVID-19, providing information from both symptomatic and asymptomatic individuals, and monitoring the effect of health interventions.

7.
Sci Total Environ ; 806(Pt 4): 150816, 2022 Feb 01.
Article in English | MEDLINE | ID: covidwho-1458560

ABSTRACT

Wastewater-based viral surveillance was proposed as a promising approach to monitor the circulation of SARS-CoV-2 in the general population. The aim of this study was to develop an analytical method to detect SARS-CoV-2 RNA in urban wastewater, and apply it to follow the trends of epidemic in the framework of a surveillance network in the Lombardy region (Northern Italy). This area was the first hotspot of COVID-19 in Europe and was severely affected. Composite 24 h samples were collected weekly in eight cities from end-March to mid-June 2020 (first peak of the pandemic). The method developed and optimized, involved virus concentration using PEG centrifugation, and one-step real-time RT-PCR for analysis. SARS-CoV-2 RNA was identified in 65 (61%) out of 107 samples, and the viral concentrations (up to 2.1 E + 05 copies/L) were highest in March-April. By mid-June, wastewater samples tested negative in all the cities corresponding to the very low number of cases recorded in the same period. Viral loads were calculated considering the wastewater daily flow rate and the population served by each wastewater treatment plant, and were used for inter- city comparison. The highest viral loads were found in Brembate, Ranica and Lodi corresponding to the hotspots of the first peak of pandemic. The pattern of decrease of SARS-CoV-2 in wastewater was closely comparable to the decline of active COVID-19 cases in the population, reflecting the effect of lock-down. This study tested wastewater surveillance of SARS-CoV-2 to follow the pandemic trends in one of most affected areas worldwide, demonstrating that it can integrate ongoing virological surveillance of COVID-19, providing information from both symptomatic and asymptomatic individuals, and monitoring the effect of health interventions.


Subject(s)
COVID-19 , Waste Water , Communicable Disease Control , Humans , Pandemics , RNA, Viral , SARS-CoV-2 , Wastewater-Based Epidemiological Monitoring
8.
Viruses ; 13(4)2021 04 16.
Article in English | MEDLINE | ID: covidwho-1194708

ABSTRACT

This paper outlines the role of Lombardy's regional influenza reference laboratory (Northern Italy) in the surveillance of influenza-like illnesses (ILIs) in monitoring SARS-CoV-2 circulation by analyzing 631 consecutive nasopharyngeal swabs (NPSs) collected from ILI outpatients by sentinel physicians during the 2019-2020 season. The samples were tested by specific real-time RT-PCRs targeting SARS-CoV-2, influenza viruses, and RSVs. Results: Of these NPSs, 31% tested positive for influenza viruses, 10% for SARS-CoV-2, and 7% for RSV. No coinfections were detected. Influenza viruses and RSVs circulated throughout the surveillance period until the end of February (week 9-2020), when they suddenly ceased to circulate seven weeks earlier than during the previous five influenza seasons. After the first detection of SARS-CoV-2 in our ILI outpatients at the beginning of March (week 10-2020), SARS-CoV-2 remained the only virus identified throughout the surveillance period. Patients ≥ 65 years had a 3.2-fold greater risk of being infected with SARS-CoV-2, while school-age children (5-14 years) and children < 5 years proved to be the age groups most at risk of contracting influenza viruses and RSV, respectively. Our experience demonstrates that laboratory-based ILI surveillance networks are essential for identifying SARS-CoV-2 cases that would otherwise remain undetected, in order to stop their spread within our communities.


Subject(s)
COVID-19/epidemiology , Coinfection/epidemiology , Influenza, Human/epidemiology , Adolescent , Adult , Aged , Aged, 80 and over , COVID-19/diagnosis , COVID-19/virology , Child , Child, Preschool , Coinfection/virology , Epidemiological Monitoring , Female , Humans , Infant , Infant, Newborn , Influenza A virus/isolation & purification , Influenza, Human/diagnosis , Influenza, Human/virology , Italy/epidemiology , Male , Middle Aged , Molecular Epidemiology , Pandemics , Population Surveillance , SARS-CoV-2/isolation & purification , Seasons , Sentinel Surveillance , Young Adult
10.
J Infect Public Health ; 14(5): 674-680, 2021 May.
Article in English | MEDLINE | ID: covidwho-1096081

ABSTRACT

INTRODUCTION: In Lombardy, the influenza surveillance system relies on sentinel physicians that weekly report data on the number of Influenza-Like Illness (ILI) and a part of them also collect nasopharyngeal samples for virologic analyses. This study aims at comparing the ILI incidence of 2019-2020 influenza season with the incidence of COVID-19 cases in order to better understand the current epidemic and to evaluate whether the implementation of ILI surveillance system could succeed in early detection and monitoring of COVID-19 diffusion. METHODS: The distribution of ILI cases in the seasons 2017-2018, 2018-2019 and 2019-2020 was taken in consideration and the curve trends were compared and analyzed according to geographical areas, age groups and time differences. RESULTS: The curve trends presented a similar pattern up to the 9th week; in fact, a reduction in the ILI incidence rate was observed in the 2017-2018 and 2018-2019 season but in the 2019-2020 an increase in the reported ILI emerged. The relation between the numbers reported by 2019-2020 ILI surveillance and those reported for COVID-19 is supported by the curve trends, the correspondence between age groups, the correspondence by geographical location, and also by the results of the nasopharyngeal swab tests performed. DISCUSSION: The influenza surveillance system is an effective tool for early detection of COVID-19. It may provide timely and high-quality data evaluating the SARS-CoV-2 burden among population with ILI. Implementation of the system has to be prioritized in order to identify any future novel respiratory pathogen with pandemic potential.


Subject(s)
COVID-19 , Influenza, Human , Humans , Incidence , Influenza, Human/epidemiology , Italy/epidemiology , Laboratories , SARS-CoV-2 , Seasons , Sentinel Surveillance
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