In vitro evaluation of the effect of mutations in primer binding sites on detection of SARS-CoV-2 by RT-qPCR

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.

Establishment of an evaluation panel for the decentralized technical evaluation of the sensitivity of 31 rapid detection tests for SARS-CoV-2 diagnostics

BackgroundThe detection of SARS-CoV-2 with rapid diagnostic tests has become an important tool to identify infected people and break infection chains. These rapid diagnostic tests are usually based on antigen detection in a lateral flow approach. Aims & MethodsWhile for PCR diagnostics the validation of a PCR assay is well established, for antigen tests e.g. rapid diagnostic tests there is no common validation strategy. Here we present the establishment of a panel of 50 pooled clinical specimens that cover a SARS-CoV-2 concentration range from approximately 1.1 x 109 to 420 genome copies per mL of specimen. The panel was used to evaluate 31 rapid diagnostic tests in up to 6 laboratories. ResultsOur results show that there is significant variation in the detection limits and the clinical sensitivity of different rapid diagnostic tests. We conclude that the best rapid diagnostic tests can be applied to reliably identify infectious individuals who are presenting with SARS-CoV-2 loads correlated to 106 genome copies per mL of specimen. Infected individuals displaying SARS-CoV-2 genome loads corresponding to less than 106 genome copies per mL will be identified by only some rapid diagnostics tests, while many tests miss these viral loads to a large extent. ConclusionsSensitive RDTs can be applied to identify infectious individuals with high viral loads, but not to identify infected individuals.

From first to second wave: follow-up of the prospective Covid-19 cohort (KoCo19) in Munich (Germany)

BackgroundIn 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. MethodsThe 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. ResultsWeighted 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. ConclusionThe 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.

Catching SARS-CoV-2 by sequence hybridization: a comparative analysis

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.

In search for the SARS-CoV-2 protection correlate: A head-to-head comparison of two quantitative S1 assays in a group of pre-characterized oligo-/asymptomatic patients

BackgroundQuantitative serological assays detecting response to SARS-CoV-2 infection are urgently needed to quantify immunity. This study analyzed the performance and correlation of two independent quantitative anti-S1 assays in oligo-/asymptomatic individuals from a previously characterized population-based cohort. MethodsA total of 362 samples included 108 from individuals who had viral RNA detected in pharyngeal swabs, 111 negative controls and 143 samples with positive serology but not confirmed by RT-PCR. Blood plasma was tested with quantitative assays Euroimmun Anti-SARS-CoV-2 QuantiVac ELISA (IgG) (EI-S1-IgG-quant) and Roche Elecsys(R) Anti-SARS-CoV-2 CoV-2 S (Ro-RBD-Ig-quant), which were compared with each other and with confirmatory tests, including wild-type virus micro-neutralization (NT) and GenScript(R)cPass. Results were analyzed using square roots R of coefficients of determination for association among continuous variables and non-parametric tests for paired comparisons. ResultsQuantitative anti-S1 serology correlated well with each other (96%/97% for true-positives and true-negatives, respectively). Antibody titers decreased over time (from <30 days to >240 days after initial positive RT-PCR). Agreement with GenScript-cPass was 96%/99% for true-positives and true-negatives, respectively, for Ro-RBD-Ig-quant and 93%/97% for EI-S1-IgG-quant. Ro-RBD-Ig-quant allowed a distinct separation between positive and negative values, and less non-specific reactivity compared with EI-S1-IgG-quant. Raw values (with 95% CI) [≥]28.7 U/mL (22.6-36.4) for Ro-RBD-Ig-quant and [≥]49.8 U/mL (43.4-57.1) for EI-S1-IgG-quant predicted virus neutralization >1:5 in 95% of cases. ConclusionsBoth quantitative anti-S1 assays, Ro-RBD-Ig-quant and EI-S1-IgG-quant, may replace direct neutralization assays in quantitative measurement of immune protection against SARS-CoV-2 in certain circumstances in the future. Key pointsTwo quantitative anti-S1 assays showed similar performance and a high level of agreement with direct virus neutralization and surrogate neutralization tests, arguing for their utility in quantifying immune protection against SARS-CoV-2.

A Serology Strategy for Epidemiological Studies Based on the Comparison of the Performance of Seven Different Test Systems - The Representative COVID-19 Cohort Munich

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.

Emergence of multiple SARS-CoV-2 mutations in an immunocompromised host

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.

First phylogenetic analysis of Malian SARS-CoV-2 sequences provide molecular insights into the genomic diversity of the Sahel region

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.

Rapid detection of SARS-CoV-2 by pulse-controlled amplification (PCA)

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.

Clinical presentation and virological assessment of hospitalized cases of coronavirus disease 2019 in a travel-associated transmission cluster

Coronavirus disease 2019 (COVID-19) is an acute respiratory tract infection that emerged in late 20191,2. Initial outbreaks in China involved 13.8% cases with severe-, and 6.1% with critical courses3. This severe presentation corresponds to the usage of a virus receptor that is expressed predominantly in the lung2,4. By causing an early onset of severe symptoms, this same receptor tropism is thought to have determined pathogenicity but also aided the control of severe acute respiratory syndrome (SARS) in 20035. However, there are reports of COVID-19 cases with mild upper respiratory tract symptoms, suggesting a potential for pre- or oligosymptomatic transmission6-8. There is an urgent need for information on body site - specific virus replication, immunity, and infectivity. Here we provide a detailed virological analysis of nine cases, providing proof of active virus replication in upper respiratory tract tissues. Pharyngeal virus shedding was very high during the first week of symptoms (peak at 7.11 x 108 RNA copies per throat swab, day 4). Infectious virus was readily isolated from throat- and lung-derived samples, but not from stool samples in spite of high virus RNA concentration. Blood and urine never yielded virus. Active replication in the throat was confirmed by viral replicative RNA intermediates in throat samples. Sequence-distinct virus populations were consistently detected in throat- and lung samples of one same patient. Shedding of viral RNA from sputum outlasted the end of symptoms. Seroconversion occurred after 6-12 days, but was not followed by a rapid decline of viral loads. COVID-19 can present as a mild upper respiratory tract illness. Active virus replication in the upper respiratory tract puts prospects of COVID-19 containment in perspective.