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1.
biorxiv; 2021.
Preprint in English | bioRxiv | ID: ppzbmed-10.1101.2021.06.07.447338

ABSTRACT

A number of RT-qPCR assays for the detection of SARS-CoV-2 have been published and are listed by the WHO as recommended assays. Furthermore, numerous commercial assays with undisclosed primer and probe sequences are on the market. As the SARS-CoV-2 pandemic progresses, the virus accrues mutations, which in some cases - as seen with the B.1.1.7 variant - can outperform and push back other strains of SARS-CoV-2. If mutations occur in primer or probe binding sites, this can impact RT-qPCR results and impede SARS-CoV-2 diagnostics. Here we tested the effect of primer mismatches on RT-qPCR performance in vitro using synthetic mismatch in vitro transcripts. The effects of the mismatches ranged from a shift in ct values from -0.13 to +7.61. Crucially, we found that a mismatch in the forward primer has a more detrimental effect for PCR performance than a mismatch in the reverse primer. Furthermore, we compared the performance of the original Charite RdRP primer set, which has several ambiguities, with a primer version without ambiguities and found that without ambiguities the ct values are ca. 3 ct lower. Finally, we investigated the shift in ct values observed with the Seegene Allplex kit with the B.1.1.7 SARS-CoV-2 variant and found a three-nucleotide mismatch in the forward primer of the N target.

2.
medrxiv; 2021.
Preprint in English | medRxiv | ID: ppzbmed-10.1101.2021.04.27.21256133

ABSTRACT

Background: In the 2nd year of the Covid-19 pandemic, knowledge about the dynamics of the infection in the general population is still limited. Such information is essential for health planners, as many of those infected show no or only mild symptoms and thus, escape the surveillance system. We therefore aimed to describe the course of the pandemic in the Munich general population living in private households from April 2020 to January 2021. Methods: The KoCo19 baseline study took place from April to June 2020 including 5313 participants (age 14 years and above). From November 2020 to January 2021, we could again measure SARS-CoV-2 antibody status in 4,433 of the baseline participants (response 83%). Participants were offered a self-sampling kit to take a capillary blood sample (dry blood spot; DBS). Blood was analysed using the Elecsys(R) Anti-SARS-CoV-2 assay (Roche). Questionnaire information on socio-demographics and potential risk factors assessed at baseline was available for all participants. In addition, follow-up information on health-risk taking behaviour and number of personal contacts outside the household (N=2768) as well as leisure time activities (N=1263) were collected in summer 2020. Results: Weighted and adjusted (for specificity and sensitivity) SARS-CoV-2 sero-prevalence at follow-up was 3.6% (95% CI 2.9-4.3%) as compared to 1.8% (95% CI 1.3-3.4%) at baseline. 91% of those tested positive at baseline were also antibody-positive at follow-up. While sero-prevalence increased from early November 2021 to January 2021, no indication of geospatial clustering across the city of Munich was found, although cases clustered within households. Taking baseline result and time to follow-up into account, men and participants in the age group 20-34 years were at the highest risk of sero-positivity. In the sensitivity analyses, differences in health-risk taking behaviour, number of personal contacts and leisure time activities partly explained these differences. Conclusion: The number of citizens in Munich with SARS-CoV-2 antibodies was still below 5% during the 2nd wave of the pandemic. Antibodies remained present in the majority of baseline participants. Besides age and sex, potentially confounded by differences in behaviour, no major risk factors could be identified. Non-pharmaceutical public health measures are thus still important.


Subject(s)
COVID-19
3.
biorxiv; 2021.
Preprint in English | bioRxiv | ID: ppzbmed-10.1101.2021.02.05.429917

ABSTRACT

Controlling and monitoring the still ongoing SARS-CoV-2 pandemic regarding geographical distributions, evolution and emergence of new mutations of the SARS-CoV-2 virus is only possible due to continuous next-generation sequencing (NGS) and worldwide sequence data sharing. Efficient sequencing strategies enabling the retrieval of the maximum number of high quality, full-length genomes are hence indispensable. Here, we describe for the first time a combined approach of digital droplet PCR (ddPCR) and NGS to evaluate five commercially available sequence capture panels targeting SARS-CoV-2. In doing so, we were not only able to determine the most sensitive and specific capture panel, but to discriminate their mode of action and number of read pairs needed to recover a high quality full length genome. Thereby, we are providing essential information for all sequencing laboratories worldwide striving for maximizing the sequencing output and simultaneously minimizing time, costs and sequencing resources.

4.
medrxiv; 2021.
Preprint in English | medRxiv | ID: ppzbmed-10.1101.2021.01.13.21249735

ABSTRACT

BackgroundSerosurveys are essential to understand SARS-CoV-2 exposure and enable population-level surveillance, but currently available tests need further in-depth evaluation. We aimed to identify testing-strategies by comparing seven seroassays in a population-based cohort. MethodsWe analysed 6,658 samples consisting of true-positives (n=193), true-negatives (n=1,091), and specimens of unknown status (n=5,374). For primary testing, we used Euroimmun-Anti-SARS-CoV-2-ELISA-IgA/IgG and Roche-Elecsys-Anti-SARS-CoV-2; and virus-neutralisation, GeneScript(R)cPass, VIRAMED-SARS-CoV-2-ViraChip(R), and Mikrogen-recomLine-SARS-CoV-2-IgG, including common-cold CoVs, for confirmatory testing. Statistical modelling generated optimised assay cut-off-thresholds. FindingsSensitivity of Euroimmun-anti-S1-IgA was 64.8%, specificity 93.3%; for Euroimmun-anti-S1-IgG, sensitivity was 77.2/79.8% (manufacturers/optimised cut-offs), specificity 98.0/97.8%; Roche-anti-N sensitivity was 85.5/88.6%, specificity 99.8/99.7%. In true-positives, mean and median titres remained stable for at least 90-120 days after RT-PCR-positivity. Of true-positives with positive RT-PCR (<30 days), 6.7% did not mount detectable seroresponses. Virus-neutralisation was 73.8% sensitive, 100.0% specific (1:10 dilution). Neutralisation surrogate tests (GeneScript(R)cPass, Mikrogen-recomLine-RBD) were >94.9% sensitive, >98.1% specific. Seasonality had limited effects; cross-reactivity with common-cold CoVs 229E and NL63 in SARS-CoV-2 true-positives was significant. ConclusionOptimised cut-offs improved test performances of several tests. Non-reactive serology in true-positives was uncommon. For epidemiological purposes, confirmatory testing with virus-neutralisation may be replaced with GeneScript(R)cPass or recomLine-RBD. Head-to-head comparisons given here aim to contribute to the refinement of testing-strategies for individual and public health use.


Subject(s)
COVID-19 , Severe Acute Respiratory Syndrome
5.
medrxiv; 2021.
Preprint in English | medRxiv | ID: ppzbmed-10.1101.2021.01.10.20248871

ABSTRACT

Prolonged shedding of infectious SARS-CoV-2 has recently been reported in a number of immunosuppressed individuals with COVID-19. Here, we describe the detection of high levels of replication-competent SARS-CoV-2 in specimens taken from the respiratory tract of a B-cell depleted patient up to 154 days after initial COVID-19 diagnosis concomitant with the development of high mutation rate. In this patient, a total of 11 nonsynonymous mutations were detected in addition to the Y144 deletion in the spike protein of SARS-CoV-2. Virus evolution studies revealed a dramatic diversification in viral population coinciding with treatment with convalescent plasma and clinical respiratory deterioration. Our findings highlight the urgent need for continuous real-time surveillance of genetic changes of SARS-CoV-2 adaptation alongside immunological investigations in patients with severely compromised humoral responses who may shed infectious virus over prolonged periods of time.


Subject(s)
COVID-19
6.
medrxiv; 2020.
Preprint in English | medRxiv | ID: ppzbmed-10.1101.2020.09.23.20165639

ABSTRACT

We are currently facing a pandemic of COVID-19, caused by a spillover from an animal-originating coronavirus to humans occuring in the Wuhan region, China, in December 2019. From China the virus has spread to 188 countries and regions worldwide, reaching the Sahel region on the 2nd of March 2020. Since whole genome sequencing (WGS) data is very crucial to understand the spreading dynamics of the ongoing pandemic, but only limited sequence data is available from the Sahel region to date, we have focused our efforts on generating the first Malian sequencing data available. Screening of 217 Malian patient samples for the presence of SARS-CoV-2 resulted in 38 positive isolates from which 21 whole genome sequences were generated. Our analysis shows that both, the early A (19B) and the fast evolving B (20A/C) clade, are present in Mali indicating multiple and independent introductions of the SARS-CoV-2 to the Sahel region.


Subject(s)
COVID-19
7.
medrxiv; 2020.
Preprint in English | medRxiv | ID: ppzbmed-10.1101.2020.07.29.20154104

ABSTRACT

In the current pandemic of SARS-CoV-2, rapid identification of infected individuals is crucial for management and control of the outbreak. However, transport of samples, sample processing and RT-qPCR analysis in laboratories are time-consuming. Here we present a nucleic acid-based test format - pulse controlled amplification - that allows detection of SARS-CoV-2 directly from up to eight swab samples simultaneously without the need for RNA extraction within 20 min in a point-of-care setting.

8.
medrxiv; 2020.
Preprint in English | medRxiv | ID: ppzbmed-10.1101.2020.03.05.20030502

ABSTRACT

Background: In coronavirus disease 2019 (COVID-19), current case definitions presume mainly lower respiratory tract infection. However, cases seen outside the epicenter of the epidemic may differ in their overall clinical appearance due to more sensitive case finding. Methods: We studied viral load courses by RT-PCR in oro- and nasopharyngeal swabs, sputum, stool, blood, and urine in nine hospitalized cases. Infectious virus was detected by cell culture. Active replication was demonstrated by analysis of viral subgenomic replicative intermediates. Serology including neutralization testing was done to characterize immune response. Results: Seven cases had upper respiratory tract disease. Lower respiratory tract symptoms seen in two cases were limited. Clinical sensitivity of RT-PCR on swabs taken on days 1-5 of symptoms was 100%, with no differences comparing swab and sputum samples taken simultaneously. Average viral load was 6.76x10E5 copies per swab during the first 5 days. Live virus isolates were obtained from swabs during the first week of illness. Proof of active viral replication in upper respiratory tract tissues was obtained by detection of subgenomic viral RNA. Shedding of viral RNA from sputum outlasted the end of symptoms. Seroconversion occurred after about one week. Conclusions: The present study shows that COVID-19 can often present as a common cold-like illness. SARS-CoV-2 can actively replicate in the upper respiratory tract, and is shed for a prolonged time after symptoms end, including in stool. These findings suggest adjustments of current case definitions and re-evaluation of the prospects of outbreak containment.


Subject(s)
Respiratory Tract Infections , COVID-19
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