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1.
Infection ; 2022 May 20.
Article in English | MEDLINE | ID: covidwho-1850466

ABSTRACT

PURPOSE: Omicron is rapidly spreading as a new SARS-CoV-2 variant of concern (VOC). The question whether this new variant has an impact on SARS-CoV-2 rapid antigen test (RAT) performance is of utmost importance. To obtain an initial estimate regarding differences of RATs in detecting omicron and delta, seven commonly used SARS-CoV-2 RATs from different manufacturers were analysed using cell culture supernatants and clinical specimens. METHODS: For this purpose, cell culture-expanded omicron and delta preparations were serially diluted in Dulbecco's modified Eagle's Medium (DMEM) and the Limit of Detection (LoD) for both VOCs was determined. Additionally, clinical specimens stored in viral transport media or saline (n = 51) were investigated to complement in vitro results with cell culture supernatants. Ct values and RNA concentrations were determined via quantitative reverse transcription polymerase chain reaction (RT-qPCR). RESULTS: The in vitro determination of the LoD showed no obvious differences in detection of omicron and delta for the RATs examined. The LoD in this study was at a dilution level of 1:1,000 (corresponding to 3.0-5.6 × 106 RNA copies/mL) for tests I-V and at a dilution level of 1:100 (corresponding to 3.7-4.9 × 107 RNA copies/mL) for tests VI and VII. Based on clinical specimens, no obvious differences were observed between RAT positivity rates when comparing omicron to delta in this study setting. Overall positivity rates varied between manufacturers with 30-81% for omicron and 42-71% for delta. Test VII was only conducted in vitro with cell culture supernatants for feasibility reasons. In the range of Ct < 23, positivity rates were 50-100% for omicron and 67-93% for delta. CONCLUSION: In this study, RATs from various manufacturers were investigated, which displayed no obvious differences in terms of analytical LoD in vitro and RAT positivity rates based on clinical samples comparing the VOCs omicron and delta. However, differences between tests produced by various manufacturers were detected. In terms of clinical samples, a focus of this study was on specimens with high virus concentrations. Further systematic, clinical and laboratory studies utilizing large datasets are urgently needed to confirm reliable performance in terms of sensitivity and specificity for all individual RATs and SARS-CoV-2 variants.

2.
Virol J ; 19(1): 76, 2022 04 26.
Article in English | MEDLINE | ID: covidwho-1817229

ABSTRACT

BACKGROUND: During the ongoing Covid-19 pandemic caused by the emerging virus SARS-CoV-2, research in the field of coronaviruses has expanded tremendously. The genome of SARS-CoV-2 has rapidly acquired numerous mutations, giving rise to several Variants of Concern (VOCs) with altered epidemiological, immunological, and pathogenic properties. METHODS: As cell culture models are important tools to study viruses, we investigated replication kinetics and infectivity of SARS-CoV-2 in the African Green Monkey-derived Vero E6 kidney cell line and the two human cell lines Caco-2, a colon epithelial carcinoma cell line, and the airway epithelial carcinoma cell line Calu-3. We assessed viral RNA copy numbers and infectivity of viral particles in cell culture supernatants at different time points ranging from 2 to 96 h post-infection. RESULTS: We here describe a systematic comparison of growth kinetics of the five SARS-CoV-2 VOCs Alpha/B.1.1.7, Beta/B.1.351, Gamma/P.1, Delta/B.1.617.2, and Omicron/B.1.1.529 and a non-VOC/B.1.1 strain on three different cell lines to provide profound information on the differential behaviour of VOCs in different cell lines for researchers worldwide. We show distinct differences in viral replication kinetics of the SARS-CoV-2 non-VOC and five VOCs on the three cell culture models Vero E6, Caco-2, and Calu-3. CONCLUSION: This is the first systematic comparison of all SARS-CoV-2 VOCs on three different cell culture models. This data provides support for researchers worldwide in their experimental design for work on SARS-CoV-2. It is recommended to perform virus isolation and propagation on Vero E6 while infection studies or drug screening and antibody-based assays should rather be conducted on the human cell lines Caco-2 and Calu-3.


Subject(s)
COVID-19 , Carcinoma , Caco-2 Cells , Cell Culture Techniques , Chlorocebus aethiops , Humans , Kinetics , Pandemics , SARS-CoV-2/genetics
3.
Infection ; 50(3): 761-766, 2022 Jun.
Article in English | MEDLINE | ID: covidwho-1712370

ABSTRACT

BACKGROUND: Five SARS-CoV-2 variants are currently considered as variants of concern (VOC). Omicron was declared a VOC at the end of November 2021. Based on different diagnostic methods, the occurrence of Omicron was reported by 52 countries worldwide on December 7 2021. First notified by South Africa with alarming reports on increasing infection rates, this new variant was soon suspected to replace the currently pre-dominating Delta variant leading to further infection waves worldwide. METHODS: Using VOC PCR screening and Next Generation Sequencing (NGS) analysis of selected samples, we investigated the circulation of Omicron in the German federal state Bavaria. For this, we analyzed SARS-CoV-2 surveillance data from our laboratory generated from calendar week (CW) 01 to 49/2021. RESULTS: So far, we have detected 69 Omicron cases in our laboratory from CW 47-49/2021 using RT-qPCR followed by melting curve analysis. The first 16 cases were analyzed by NGS and all were confirmed as Omicron. CONCLUSION: Our data strongly support no circulation of the new Omicron variant before CW 47/2021.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/diagnosis , COVID-19/epidemiology , High-Throughput Nucleotide Sequencing , Humans , Real-Time Polymerase Chain Reaction , SARS-CoV-2/genetics
5.
Epidemiol Infect ; 149: e226, 2021 10 26.
Article in English | MEDLINE | ID: covidwho-1537267

ABSTRACT

The corona virus disease-2019 (COVID-19) pandemic began in Wuhan, China, and quickly spread around the world. The pandemic overlapped with two consecutive influenza seasons (2019/2020 and 2020/2021). This provided the opportunity to study community circulation of influenza viruses and severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) in outpatients with acute respiratory infections during these two seasons within the Bavarian Influenza Sentinel (BIS) in Bavaria, Germany. From September to March, oropharyngeal swabs collected at BIS were analysed for influenza viruses and SARS-CoV-2 by real-time polymerase chain reaction. In BIS 2019/2020, 1376 swabs were tested for influenza viruses. The average positive rate was 37.6%, with a maximum of over 60% (in January). The predominant influenza viruses were Influenza A(H1N1)pdm09 (n = 202), Influenza A(H3N2) (n = 144) and Influenza B Victoria lineage (n = 129). In all, 610 of these BIS swabs contained sufficient material to retrospectively test for SARS-CoV-2. SARS-CoV-2 RNA was not detectable in any of these swabs. In BIS 2020/2021, 470 swabs were tested for influenza viruses and 457 for SARS-CoV-2. Only three swabs (0.6%) were positive for Influenza, while SARS-CoV-2 was found in 30 swabs (6.6%). We showed that no circulation of SARS-CoV-2 was detectable in BIS during the 2019/2020 influenza season, while virtually no influenza viruses were found in BIS 2020/2021 during the COVID-19 pandemic.


Subject(s)
COVID-19/epidemiology , Influenza, Human/epidemiology , Sentinel Surveillance , COVID-19/diagnosis , Germany/epidemiology , Humans , Incidence , Influenza, Human/diagnosis , Oropharynx/virology , Orthomyxoviridae/classification , Orthomyxoviridae/genetics , Orthomyxoviridae/isolation & purification , RNA, Viral/genetics , Retrospective Studies , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Seasons
6.
Microorganisms ; 9(9)2021 Sep 16.
Article in English | MEDLINE | ID: covidwho-1410330

ABSTRACT

Rapid antigen tests (RATs) are an integral part of SARS-CoV-2 containment strategies. As emerging variants of concern (VOCs) displace the initially circulating strains, it is crucial that RATs do not fail to detect these new variants. In this study, four RATs for nasal swab testing were investigated using cultured strains of B.1.1 (non-VOC), B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma), and B.1.617.2 (Delta). Based on dilution series in cell culture medium and pooled saliva, the limit of detection of these RATs was determined in a laboratory setting. Further investigations on cross-reactivity were conducted using recombinant N-protein from seasonal human coronaviruses (hCoVs). RATs evaluated showed an overall comparable performance with cultured strains of the non-VOC B.1.1 and the VOCs Alpha, Beta, Gamma, and Delta. No cross-reactivity was detected with recombinant N-protein of the hCoV strains HKU1, OC43, NL63, and 229E. A continuous evaluation of SARS-CoV-2 RAT performance is required, especially with regard to evolving mutations. Moreover, cross-reactivity and interference with pathogens and other substances on the test performance of RATs should be consistently investigated to ensure suitability in the context of SARS-CoV-2 containment.

7.
Euro Surveill ; 26(30)2021 07.
Article in English | MEDLINE | ID: covidwho-1334902

ABSTRACT

A breakthrough infection occurred in a fully Comirnaty (BNT162b2) vaccinated healthcare worker with high levels of neutralising antibodies with the SARS-CoV-2 B.1.351 (Beta) variant in February 2021. The infection was subsequently transmitted to their unvaccinated spouse. Sequencing revealed an identical virus in both spouses, with a match of all nine single nucleotide polymorphisms typical for B.1.351. To the best of our knowledge, no transmission of any variant of SARS-CoV-2 from a fully vaccinated person has been described before.


Subject(s)
COVID-19 , Vaccines , COVID-19 Vaccines , Germany/epidemiology , Humans , SARS-CoV-2
8.
Emerg Infect Dis ; 27(7): 1974-1976, 2021 07.
Article in English | MEDLINE | ID: covidwho-1278359

ABSTRACT

We report a therapy cat in a nursing home in Germany infected with severe acute respiratory syndrome coronavirus 2 during a cluster outbreak in the home residents. Although we confirmed prolonged presence of virus RNA in the asymptomatic cat, genome sequencing showed no further role of the cat in human infections on site.


Subject(s)
COVID-19 , SARS-CoV-2 , Animals , Cats , Disease Outbreaks , Germany , Humans , RNA, Viral/genetics , Retirement
9.
Clin Microbiol Infect ; 27(9): 1353.e1-1353.e5, 2021 Sep.
Article in English | MEDLINE | ID: covidwho-1240261

ABSTRACT

OBJECTIVES: Detection and surveillance of SARS-CoV-2 is of eminent importance, particularly due to the rapid emergence of variants of concern (VOCs). In this study we evaluated if a commercially available quantitative real-time PCR (qRT-PCR) assay can identify SARS-CoV-2 B.1.1.7 lineage samples by a specific N gene dropout or Ct value shift compared with the S or RdRp gene. METHODS: VOC B.1.1.7 and non-B.1.1.7 SARS-CoV-2-positive patient samples were identified via whole-genome sequencing and variant-specific PCR. Confirmed B.1.1.7 (n = 48) and non-B.1.1.7 samples (n = 58) were analysed using the Allplex™ SARS-CoV-2/FluA/FluB/RSV™ PCR assay for presence of SARS-CoV-2 S, RdRp and N genes. The N gene coding sequence of SARS-CoV-2 with and without the D3L mutation (specific for B.1.1.7) was cloned into pCR™II-TOPO™ vectors to validate polymorphism-dependent N gene dropout with the Allplex™ SARS-CoV-2/FluA/FluB/RSV™ PCR assay. RESULTS: All studied B.1.1.7-positive patient samples showed significantly higher Ct values in qRT-PCR (Δ6-10, N gene dropout on Ct values > 29) of N gene than the corresponding values of S (p ≤ 0.0001) and RdRp (p ≤ 0.0001) genes. The assay reliably discriminated B.1.1.7 and non-B.1.1.7 positive samples (area under the curve = 1) in a receiver operating characteristic curve analysis. Identical Ct value shifts (Δ7-10) were detected in reverse genetic experiments, using isolated plasmids containing N gene coding sequences corresponding to D3 or 3L variants. DISCUSSION: An N gene dropout or Ct value shift is shown for B.1.1.7-positive samples in the Allplex™ SARS-CoV-2/FluA/FluB/RSV™ PCR assay. This approach can be used as a rapid tool for B.1.1.7 detection in single assay high throughput diagnostics.


Subject(s)
COVID-19/diagnosis , Coronavirus Nucleocapsid Proteins/genetics , SARS-CoV-2/classification , Whole Genome Sequencing/methods , COVID-19 Nucleic Acid Testing , Genome, Viral , High-Throughput Nucleotide Sequencing , Humans , Multiplex Polymerase Chain Reaction , Mutation , ROC Curve , SARS-CoV-2/genetics , Sensitivity and Specificity
10.
Euro Surveill ; 26(16)2021 04.
Article in English | MEDLINE | ID: covidwho-1200054

ABSTRACT

SARS-CoV-2 variants of concern (VOC) should not escape molecular surveillance. We investigated if SARS-CoV-2 rapid antigen tests (RATs) could detect B.1.1.7 and B.1.351 VOCs in certain laboratory conditions. Infectious cell culture supernatants containing B.1.1.7, B.1.351 or non-VOC SARS-CoV-2 were respectively diluted both in DMEM and saliva. Dilutions were analysed with Roche, Siemens, Abbott, nal von minden and RapiGEN RATs. While further studies with appropriate real-life clinical samples are warranted, all RATs detected B.1.1.7 and B.1.351, generally comparable to non-VOC strain.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19 Serological Testing , Germany , Humans
12.
Lancet Infect Dis ; 20(8): 920-928, 2020 08.
Article in English | MEDLINE | ID: covidwho-276988

ABSTRACT

BACKGROUND: In December, 2019, the newly identified severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, China, causing COVID-19, a respiratory disease presenting with fever, cough, and often pneumonia. WHO has set the strategic objective to interrupt spread of SARS-CoV-2 worldwide. An outbreak in Bavaria, Germany, starting at the end of January, 2020, provided the opportunity to study transmission events, incubation period, and secondary attack rates. METHODS: A case was defined as a person with SARS-CoV-2 infection confirmed by RT-PCR. Case interviews were done to describe timing of onset and nature of symptoms and to identify and classify contacts as high risk (had cumulative face-to-face contact with a confirmed case for ≥15 min, direct contact with secretions or body fluids of a patient with confirmed COVID-19, or, in the case of health-care workers, had worked within 2 m of a patient with confirmed COVID-19 without personal protective equipment) or low risk (all other contacts). High-risk contacts were ordered to stay at home in quarantine for 14 days and were actively followed up and monitored for symptoms, and low-risk contacts were tested upon self-reporting of symptoms. We defined fever and cough as specific symptoms, and defined a prodromal phase as the presence of non-specific symptoms for at least 1 day before the onset of specific symptoms. Whole genome sequencing was used to confirm epidemiological links and clarify transmission events where contact histories were ambiguous; integration with epidemiological data enabled precise reconstruction of exposure events and incubation periods. Secondary attack rates were calculated as the number of cases divided by the number of contacts, using Fisher's exact test for the 95% CIs. FINDINGS: Patient 0 was a Chinese resident who visited Germany for professional reasons. 16 subsequent cases, often with mild and non-specific symptoms, emerged in four transmission generations. Signature mutations in the viral genome occurred upon foundation of generation 2, as well as in one case pertaining to generation 4. The median incubation period was 4·0 days (IQR 2·3-4·3) and the median serial interval was 4·0 days (3·0-5·0). Transmission events were likely to have occurred presymptomatically for one case (possibly five more), at the day of symptom onset for four cases (possibly five more), and the remainder after the day of symptom onset or unknown. One or two cases resulted from contact with a case during the prodromal phase. Secondary attack rates were 75·0% (95% CI 19·0-99·0; three of four people) among members of a household cluster in common isolation, 10·0% (1·2-32·0; two of 20) among household contacts only together until isolation of the patient, and 5·1% (2·6-8·9; 11 of 217) among non-household, high-risk contacts. INTERPRETATION: Although patients in our study presented with predominately mild, non-specific symptoms, infectiousness before or on the day of symptom onset was substantial. Additionally, the incubation period was often very short and false-negative tests occurred. These results suggest that although the outbreak was controlled, successful long-term and global containment of COVID-19 could be difficult to achieve. FUNDING: All authors are employed and all expenses covered by governmental, federal state, or other publicly funded institutions.


Subject(s)
Betacoronavirus/isolation & purification , Communicable Diseases, Imported/transmission , Coronavirus Infections/transmission , Disease Outbreaks , Disease Transmission, Infectious , Pneumonia, Viral/transmission , Travel-Related Illness , Adolescent , Adult , Betacoronavirus/classification , Betacoronavirus/genetics , COVID-19 , Child , Child, Preschool , China , Communicable Diseases, Imported/epidemiology , Communicable Diseases, Imported/pathology , Communicable Diseases, Imported/virology , Coronavirus Infections/epidemiology , Germany/epidemiology , Humans , Interviews as Topic , Middle Aged , Mutation , Pandemics , Pneumonia, Viral/epidemiology , RNA, Viral/genetics , Reverse Transcriptase Polymerase Chain Reaction , Risk Assessment , SARS-CoV-2 , Travel , Young Adult
13.
Euro Surveill ; 25(9)2020 03.
Article in English | MEDLINE | ID: covidwho-4532

ABSTRACT

The need for timely establishment of diagnostic assays arose when Germany was confronted with the first travel-associated outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Europe. We describe our laboratory experiences during a large contact tracing investigation, comparing previously published real-time RT-PCR assays in different PCR systems and a commercial kit. We found that assay performance using the same primers and probes with different PCR systems varied and the commercial kit performed well.


Subject(s)
Betacoronavirus , Clinical Laboratory Techniques , Coronavirus Infections , Pneumonia, Viral , Real-Time Polymerase Chain Reaction/methods , Betacoronavirus/genetics , Betacoronavirus/isolation & purification , COVID-19 , COVID-19 Testing , COVID-19 Vaccines , Coronavirus Infections/diagnosis , Coronavirus Infections/genetics , Germany , Humans , Oligonucleotide Array Sequence Analysis , Pneumonia, Viral/diagnosis , Pneumonia, Viral/genetics , SARS-CoV-2 , Sensitivity and Specificity , Time Factors , Viral Envelope Proteins/analysis , Viral Envelope Proteins/genetics , Workflow
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