Follow-up SARS-CoV-2 serological study of a health care worker cohort following COVID-19 booster vaccination.

BACKGROUND: Studies have shown that Omicron breakthrough infections can occur at higher SARS-CoV-2 antibody levels compared to previous variants. Estimating the magnitude of immunological protection induced from COVID-19 vaccination and previous infection remains important due to varying local pandemic dynamics and types of vaccination programmes, particularly among at-risk populations such as health care workers (HCWs). We analysed a follow-up SARS-CoV-2 serological survey of HCWs at a tertiary COVID-19 referral hospital in Germany following the onset of the Omicron variant. METHODS: The serological survey was conducted in January 2022, one year after previous surveys in 2020 and the availability of COVID-19 boosters including BNT162b2, ChAdOx1-S, and mRNA-1273. HCWs voluntarily provided blood for serology and completed a comprehensive questionnaire. SARS-CoV-2 serological analyses were performed using an Immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA). Antibody levels were reported according to HCW demographic and occupational characteristics, COVID-19 vaccination and SARS-CoV-2 infection history, and multivariate linear regression was used to evaluate these associations. RESULTS: In January 2022 (following the fourth COVID-19 wave in Germany including the onset of the Omicron variant), 1482/1517 (97.7%) HCWs tested SARS-CoV-2 seropositive, compared to 4.6% in December 2020 (second COVID-19 wave). Approximately 80% had received three COVID-19 vaccine doses and 15% reported a previous laboratory-confirmed SARS-CoV-2 infection. SARS-CoV-2 IgG geometric mean titres ranged from 335 (95% Confidence Intervals [CI]: 258-434) among those vaccinated twice and without previous infection to 2204 (95% CI: 1919-2531) among those vaccinated three times and with previous infection. Heterologous COVID-19 vaccination combinations including a mRNA-1273 booster were significantly associated with the highest IgG antibody levels compared to other schemes. There was an 8-to 10-fold increase in IgG antibody levels among 31 HCWs who reported a SARS-CoV-2 infection in May 2020 to January 2022 after COVID-19 booster vaccination. CONCLUSIONS: Our findings demonstrate the importance of ongoing COVID-19 booster vaccination strategies in the context of variants such as Omicron and despite hybrid immunity from previous SARS-CoV-2 infections, particularly for at-risk populations such as HCWs. Where feasible, effective types of booster vaccination, such as mRNA vaccines, and the appropriate timing of administration should be carefully considered.

Genome sequencing of the mpox virus 2022 outbreak with amplicon-based Oxford Nanopore MinION sequencing.

We present an amplicon-based assay for MinION Nanopore sequencing of mpox virus (MPXV) genomes from clinical specimens, obtaining high-quality results with an average genome coverage of 99% for Ct values of up to 25, and a genome coverage of 97.1% for Ct values from 25 to 30 which are challenging to sequence. This assay is easy to implement in PCR-based workflows and provides accurate genomic data within a short time.

A bead-based multiplex assay covering all coronaviruses pathogenic for humans for sensitive and specific surveillance of SARS-CoV-2 humoral immunity.

Serological assays measuring antibodies against SARS-CoV-2 are key to describe the epidemiology, pathobiology or induction of immunity after infection or vaccination. Of those, multiplex assays targeting multiple antigens are especially helpful as closely related coronaviruses or other antigens can be analysed simultaneously from small sample volumes, hereby shedding light on patterns in the immune response that would otherwise remain undetected. We established a bead-based 17-plex assay detecting antibodies targeting antigens from all coronaviruses pathogenic for humans: SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV strains 229E, OC43, HKU1, and NL63. The assay was validated against five commercial serological immunoassays, a commercial surrogate virus neutralisation test, and a virus neutralisation assay, all targeting SARS-CoV-2. It was found to be highly versatile as shown by antibody detection from both serum and dried blot spots and as shown in three case studies. First, we followed seroconversion for all four endemic HCoV strains and SARS-CoV-2 in an outbreak study in day-care centres for children. Second, we were able to link a more severe clinical course to a stronger IgG response with this 17-plex-assay, which was IgG1 and IgG3 dominated. Finally, our assay was able to discriminate recent from previous SARS-CoV-2 infections by calculating the IgG/IgM ratio on the N antigen targeting antibodies. In conclusion, due to the comprehensive method comparison, thorough validation, and the proven versatility, our multiplex assay is a valuable tool for studies on coronavirus serology.

Serological methods for the detection of antibodies against monkeypox virus applicable for laboratories with different biosafety levels.

The monkeypox virus (MPXV) outbreak in 2022 has renewed interest in the detection of antibodies against orthopox viruses (OPXV) and MPXV, as serological methods can aid diagnostics and are key to epidemiological studies. Here three complementary serological methods are described with different strengths to aid the development and evaluation of in-house assays: An immunofluorescence assay (IFA) for specific detection of IgG and IgM, an enzyme-linked immunosorbent assay for higher sample throughput to aid epidemiological studies and a neutralization test to detect virus neutralizing antibodies. As implementation of MPXV-specific diagnostics is often hampered by the requirement for a dedicated biosafety level 3 laboratory (BSL-3), the focus of this study is on biosafety aspects to facilitate safe testing also under BSL-2 conditions. To this aim, it was analyzed whether OPXV, which can be handled under BSL-2 conditions, could be used as less virulent alternatives to MPXV. Furthermore, an inactivation method was established to remove up to five log-steps of infectious virus particles from viraemic sera without compromising antibody detection. The results show that immunological cross-reactivity between OPXV provides an opportunity for the interchangeable usage of different OPXV species in serological assays, enabling MPXV serology outside of BSL-3 facilities.

Results of German external quality assessment schemes for SARS-CoV-2 antigen detection.

The COVID-19 pandemic illustrated the important role of diagnostic tests, including lateral flow tests (LFTs), in identifying patients and their contacts to slow the spread of infections. INSTAND performed external quality assessments (EQA) for SARS-CoV-2 antigen detection with lyophilized and chemically inactivated cell culture supernatant of SARS-CoV-2 infected Vero cells. A pre-study demonstrated the suitability of the material. Participants reported qualitative and/or quantitative antigen results using either LFTs or automated immunoassays for five EQA samples per survey. 711 data sets were reported for LFT detection in three surveys in 2021. This evaluation focused on the analytical sensitivity of different LFTs and automated immunoassays. The inter-laboratory results showed at least 94% correct results for non-variant of concern (VOC) SARS-CoV-2 antigen detection for viral loads of ≥ 4.75 × 106 copies/mL and SARS-CoV-2 negative samples. Up to 85% had success for a non-VOC viral load of ~ 1.60 × 106 copies/mL. A viral load of ~ 1.42 × 107 copies/mL of the Delta VOC was reported positive in > 96% of results. A high specificity was found with almost 100% negative SARS-CoV-2 antigen results for HCoV 229E and HCoV NL63 positive samples. Quantitative results correlated with increasing SARS-CoV-2 viral load but showed a broad scatter. This study shows promising SARS-CoV-2 antigen test performance of the participating laboratories, but further investigations with the now predominant Omicron VOC are needed.

Flexible upscaling of laboratory PCR testing capacity at the Robert Koch Institute during the SARS-CoV-2 pandemic.

BACKGROUND: Over the course of the COVID-19 pandemic, laboratories worldwide have been facing an unprecedented increase in demand for PCR testing because of the high importance of diagnostics for prevention and control of virus spread. Moreover, testing demand has been varying considerably over time, depending on the epidemiological situation, rendering efficient resource allocation difficult. Here, we present a scalable workflow which we implemented in our laboratory to increase PCR testing capacity while maintaining high flexibility regarding the number of samples to be processed. METHODS: We compared the performance of five automated extraction instruments, using dilutions of SARS-CoV-2 cell culture supernatant as well as clinical samples. To increase PCR throughput, we combined the two duplex PCR reactions of our previously published SARS-CoV-2 PCR assay into one quadruplex reaction and compared their limit of detection as well as their performance on the detection of low viral loads in clinical samples. Furthermore, we developed a sample pooling protocol with either two or four samples per pool, combined with a specifically adapted SARS-CoV-2 quadruplex PCR assay, and compared the diagnostic sensitivity of pooled testing and individual testing. RESULTS: All tested automated extraction instruments yielded comparable results regarding the subsequent sensitivity of SARS-CoV-2 detection by PCR. While the limit of detection of the quadruplex SARS-CoV-2 PCR assay (E-Gene assay: 28.7 genome equivalents (ge)/reaction, orf1ab assay: 32.0 ge/reaction) was slightly higher than that of our previously published duplex PCR assays (E-Gene assay: 9.8 ge/reaction, orf1ab assay: 6.6 ge/reaction), the rate of correctly identified positive patient samples was comparable for both assays. Sample pooling with optimized downstream quadruplex PCR showed no loss in diagnostic sensitivity compared to individual testing. CONCLUSION: Specific adaptation of PCR assays can help overcome the potential loss of sensitivity due to higher levels of PCR multiplexing or sample dilution in pooled testing. Combining these adapted PCR assays with different sample processing strategies provides a simple and highly adjustable workflow for resource-efficient SARS-CoV-2 diagnostics. The presented principles can easily be adopted in a variety of laboratory settings as well as be adapted to pathogens other than SARS-CoV-2, making it feasible for any laboratory that conducts PCR diagnostics.

Direct comparison of clinical diagnostic sensitivity of saliva from buccal swabs versus combined oro-/nasopharyngeal swabs in the detection of SARS-CoV-2 B.1.1.529 Omicron.

BACKGROUND/PURPOSE: While current guidelines recommend the use of respiratory tract specimens for the direct detection of SARS-CoV-2 infection, saliva has recently been suggested as preferred sample type for the sensitive detection of SARS-CoV-2 B.1.1.529 (Omicron). By comparing saliva collected using buccal swabs and oro-/nasopharyngeal swabs from patients hospitalized due to COVID-19, we aimed at identifying potential differences in virus detection sensitivity between these sample types. METHODS: We compare the clinical diagnostic sensitivity of paired buccal swabs and combined oro-/nasopharyngeal swabs from hospitalized, symptomatic COVID-19 patients collected at median six days after symptom onset by real-time polymerase chain reaction (PCR) and antigen test. RESULTS: Of the tested SARS-CoV-2 positive sample pairs, 55.8% were identified as SARS-CoV-2 Omicron BA.1 and 44.2% as Omicron BA.2. Real-time PCR from buccal swabs generated significantly higher quantification cycle (Cq) values compared to those from matched combined oro-/nasopharyngeal swabs and resulted in an increased number of false-negative PCR results. Reduced diagnostic sensitivity of buccal swabs by real-time PCR was observed already at day one after symptom onset. Similarly, antigen test detection rates were reduced in buccal swabs compared to combined oro-/nasopharyngeal swabs. CONCLUSION: Our results suggest reduced clinical diagnostic sensitivity of saliva collected using buccal swabs when compared to combined oro-/nasopharyngeal swabs in the detection of SARS-CoV-2 Omicron in symptomatic individuals.

Extensive ITR expansion of the 2022 Mpox virus genome through gene duplication and gene loss.

Poxviruses are known to evolve slower than RNA viruses with only 1-2 mutations/genome/year. Rather than single mutations, rearrangements such as gene gain and loss, which have been discussed as a possible driver for host adaption, were described in poxviruses. In 2022 and 2023 the world is being challenged by the largest global outbreak so far of Mpox virus, and the virus seems to have established itself in the human community for an extended period of time. Here, we report five Mpox virus genomes from Germany with extensive gene duplication and loss, leading to the expansion of the ITR regions from 6400 to up to 24,600 bp. We describe duplications of up to 18,200 bp to the opposed genome end, and deletions at the site of insertion of up to 16,900 bp. Deletions and duplications of genes with functions of supposed immune modulation, virulence and host adaption as B19R, B21R, B22R and D10L are described. In summary, we highlight the need for monitoring rearrangements of the Mpox virus genome rather than for monitoring single mutations only.

Performance of 20 rapid antigen detection tests to detect SARS-CoV-2 B.1.617.2 (Delta) and B.1.1.529 (Omicron) variants using a clinical specimen panel from January 2022, Berlin, Germany.

BackgroundThere are conflicting reports on the performance of rapid antigen detection tests (RDT) in the detection of the SARS-CoV-2 Omicron (B.1.1.529) variant; however, these tests continue to be used frequently to detect potentially contagious individuals with high viral loads.AimThe aim of this study was to investigate comparative detection of the Delta (B.1.617.2) and Omicron variants by using a selection of 20 RDT and a limited panel of pooled combined oro- and nasopharyngeal clinical Delta and Omicron specimens.MethodsWe tested 20 CE-marked RDT for their performance to detect SARS-CoV-2 Delta and Omicron by using a panel of pooled clinical specimens collected in January 2022 in Berlin, Germany.ResultsWe observed equivalent detection performance for Delta and Omicron for most RDT, and sensitivity was widely in line with our previous pre-Delta/Omicron evaluation. Some variation for individual RDT was observed either for Delta vs Omicron detection, or when compared with the previous evaluation, which may be explained both by different panel sizes resulting in different data robustness and potential limitation of batch-to-batch consistency. Additional experiments with three RDT using non-pooled routine clinical samples confirmed comparable performance to detect Delta vs Omicron. Overall, RDT that were previously positively evaluated retained good performance also for Delta and Omicron variants.ConclusionOur findings suggest that currently available RDT are sufficient for the detection of SARS-CoV-2 Delta and Omicron variants.

Results of the first German external quality assessment scheme for the detection of monkeypox virus DNA.

BACKGROUND: In May 2022, the monkeypox virus (MPXV) spread into non-endemic countries and the global community was quick to test the lessons learned from the SARS-CoV-2 pandemic. Due to its symptomatic resemblance to other diseases, like the non-pox virus varicella zoster (chickenpox), polymerase chain reaction methods play an important role in correctly diagnosing the rash-causing pathogen. INSTAND quickly established a new external quality assessment (EQA) scheme for MPXV and orthopoxvirus (OPXV) DNA detection to assess the current performance quality of the laboratory tests. METHODS: We analyzed quantitative and qualitative data of the first German EQA for MPXV and OPXV DNA detection. The survey included one negative and three MPXV-positive samples with different MPX viral loads. The threshold cycle (Ct) or other measures defining the quantification cycle (Cq) were analyzed in an assay-specific manner. A Passing Bablok fit was used to investigate the performance at laboratory level. RESULTS: 141 qualitative datasets were reported by 131 laboratories for MPXV detection and 68 qualitative datasets by 65 laboratories for OPXV detection. More than 96% of the results were correctly identified as negative and more than 97% correctly identified as positive. An analysis of the reported Ct/Cq values showed a large spread of these values of up to 12 Ct/Cq. Nevertheless, there is a good correlation of results for the different MPXV concentrations at laboratory level. Only a few quantitative results in copies/mL were reported (MPXV: N = 5; OPXV: N = 2), but the results correlated well with the concentration differences between the EQA samples, which were to a power of ten each. CONCLUSION: The EQA results show that laboratories performed well in detecting both MPXV and OPXV. However, Ct/Cq values should be interpreted with caution when conclusions are drawn about the viral load as long as metrological traceability is not granted.

Vaccine effectiveness against severe COVID-19 during the Omicron wave in Germany: results from the COViK study.

PURPOSE: COViK, a prospective hospital-based multicenter case-control study in Germany, aims to assess the effectiveness of COVID-19 vaccines against severe disease. Here, we report vaccine effectiveness (VE) against COVID-19-caused hospitalization and intensive care treatment during the Omicron wave. METHODS: We analyzed data from 276 cases with COVID-19 and 494 control patients recruited in 13 hospitals from 1 December 2021 to 5 September 2022. We calculated crude and confounder-adjusted VE estimates. RESULTS: 21% of cases (57/276) were not vaccinated, compared to 5% of controls (26/494; p < 0.001). Confounder-adjusted VE against COVID-19-caused hospitalization was 55.4% (95% CI: 12-78%), 81.5% (95% CI: 68-90%) and 95.6% (95%CI: 88-99%) after two, three and four vaccine doses, respectively. VE against hospitalization due to COVID-19 remained stable up to one year after three vaccine doses. CONCLUSION: Three vaccine doses remained highly effective in preventing severe disease and this protection was sustained; a fourth dose further increased protection.

Primary ChAdOx1 vaccination does not reactivate pre-existing, cross-reactive immunity.

Currently available COVID-19 vaccines include inactivated virus, live attenuated virus, mRNA-based, viral vectored and adjuvanted protein-subunit-based vaccines. All of them contain the spike glycoprotein as the main immunogen and result in reduced disease severity upon SARS-CoV-2 infection. While we and others have shown that mRNA-based vaccination reactivates pre-existing, cross-reactive immunity, the effect of vector vaccines in this regard is unknown. Here, we studied cellular and humoral responses in heterologous adenovirus-vector-based ChAdOx1 nCOV-19 (AZ; Vaxzeria, AstraZeneca) and mRNA-based BNT162b2 (BNT; Comirnaty, BioNTech/Pfizer) vaccination and compared it to a homologous BNT vaccination regimen. AZ primary vaccination did not lead to measurable reactivation of cross-reactive cellular and humoral immunity compared to BNT primary vaccination. Moreover, humoral immunity induced by primary vaccination with AZ displayed differences in linear spike peptide epitope coverage and a lack of anti-S2 IgG antibodies. Contrary to primary AZ vaccination, secondary vaccination with BNT reactivated pre-existing, cross-reactive immunity, comparable to homologous primary and secondary mRNA vaccination. While induced anti-S1 IgG antibody titers were higher after heterologous vaccination, induced CD4+ T cell responses were highest in homologous vaccinated. However, the overall TCR repertoire breadth was comparable between heterologous AZ-BNT-vaccinated and homologous BNT-BNT-vaccinated individuals, matching TCR repertoire breadths after SARS-CoV-2 infection, too. The reasons why AZ and BNT primary vaccination elicits different immune response patterns to essentially the same antigen, and the associated benefits and risks, need further investigation to inform vaccine and vaccination schedule development.

Visualization of SARS-CoV-2 particles in naso/oropharyngeal swabs by thin section electron microscopy.

BACKGROUND: SARS-CoV-2 replicates efficiently in the upper airways of humans and produces high loads of virus RNA and, at least in the initial phase after infection, many infectious virus particles. Studying virus ultrastructure, such as particle integrity or presence of spike proteins, and effects on their host cells in patient samples is important to understand the pathogenicity of SARS-CoV-2. METHODS: Suspensions from swab samples with a high load of virus RNA (Ct < 20) were sedimented by desktop ultracentrifugation and prepared for thin section electron microscopy using a novel method which is described in detail. Embedding was performed in Epon or in LR White resin using standard or rapid protocols. Thin sections were examined using transmission electron microscopy. RESULTS: Virus particles could be regularly detected in the extracellular space, embedded in a background of heterogenous material (e.g. vesicles and needle-like crystals), and within ciliated cells. Morphology (i.e. shape, size, spike density) of virus particles in the swab samples was very similar to particle morphology in cell culture. However, in some of the samples the virus particles hardly revealed spikes. Infected ciliated cells occasionally showed replication organelles, such as double-membrane vesicles. The most common cells in all samples were keratinocytes from the mucosa and bacteria. CONCLUSIONS: The new method allows the ultrastructural visualization and analysis of coronavirus particles and of infected host cells from easy to collect naso/oropharyngeal patient swab samples.

Effectiveness of vaccines in preventing hospitalization due to COVID-19: A multicenter hospital-based case-control study, Germany, June 2021 to January 2022.

We included 852 patients in a prospectively recruiting multicenter matched case-control study in Germany to assess vaccine effectiveness (VE) in preventing COVID-19-associated hospitalization during the Delta-variant dominance. The two-dose VE was 89 % (95 % CI 84-93 %) overall, 79 % in patients with more than two comorbidities and 77 % in adults aged 60-75 years. A third dose increased the VE to more than 93 % in all patient-subgroups.

SARS-CoV-2 viral clearance and viral load kinetics in young children (1-6 years) compared to adults: Results of a longitudinal study in Germany.

Objective: To investigate SARS-COV-2 viral clearance and viral load kinetics in the course of infection in children aged 1-6 years in comparison with adults. Methods: Prospective cohort study of infected daycare children and staff and their close contacts in households from 11/2020 to 06/2021. Adult participants took upper respiratory tract specimen from themselves and/or their children, for PCR tests on SARS-CoV-2. Data on symptoms and exposure were used to determine the date of probable infection for each participant. We determined (a) viral clearance, and (b) viral load dynamics over time. Samples were taken from day 4-6 to day 16-18 after diagnosis of the index case in the respective daycare group (5 samples per participant). Results: We included 40 children (1-6 years) and 67 adults (18-77 years) with SARS-CoV-2 infection. Samples were available at a mean of 4.3 points of time per participant. Among the participants, the 12-day study period fell in different periods within the individual course of infection, ranging from day 5-17 to day 15-26 after assumed infection.Children reached viral clearance at a median of 20 days after assumed infection (95% CI 17-21 days, Kaplan-Meier Analysis), adults at 23 days (95% CI 20-25 days, difference not significant). In both children and adults, viral load decreased over time with trajectories of the mean viral load not being statistically different between groups. Kaplan-Meier calculations show that from day 15 (95% CI 13-15), 50% of all participants had a viral load <1 million copies/ml, i.e. were no longer infectious or negative. Conclusion: Children aged 1-6 and adults infected with SARS-CoV-2 (wild type and Alpha variant) did not differ significantly in terms of viral load kinetics and time needed to clear the virus. Therefore, containment measures are important also in the daycare settings as long as the pandemic continues.

Nationally representative results on SARS-CoV-2 seroprevalence and testing in Germany at the end of 2020.

Pre-vaccine SARS-CoV-2 seroprevalence data from Germany are scarce outside hotspots, and socioeconomic disparities remained largely unexplored. The nationwide representative RKI-SOEP study (15,122 participants, 18-99 years, 54% women) investigated seroprevalence and testing in a supplementary wave of the Socio-Economic-Panel conducted predominantly in October-November 2020. Self-collected oral-nasal swabs were PCR-positive in 0.4% and Euroimmun anti-SARS-CoV-2-S1-IgG ELISA from dry-capillary-blood antibody-positive in 1.3% (95% CI 0.9-1.7%, population-weighted, corrected for sensitivity = 0.811, specificity = 0.997). Seroprevalence was 1.7% (95% CI 1.2-2.3%) when additionally correcting for antibody decay. Overall infection prevalence including self-reports was 2.1%. We estimate 45% (95% CI 21-60%) undetected cases and lower detection in socioeconomically deprived districts. Prior SARS-CoV-2 testing was reported by 18% from the lower educational group vs. 25% and 26% from the medium and high educational group (p < 0.001, global test over three categories). Symptom-triggered test frequency was similar across educational groups. Routine testing was more common in low-educated adults, whereas travel-related testing and testing after contact with infected persons was more common in highly educated groups. This countrywide very low pre-vaccine seroprevalence in Germany at the end of 2020 can serve to evaluate the containment strategy. Our findings on social disparities indicate improvement potential in pandemic planning for people in socially disadvantaged circumstances.

Analysis of two choir outbreaks acting in concert to characterize long- range transmission risks through SARS-CoV-2, Berlin, Germany, 2020.

BACKGROUND: Superspreading events are important drivers of the SARS-CoV-2 pandemic and long-range (LR) transmission is believed to play a major role. We investigated two choir outbreaks with different attack rates (AR) to analyze the contribution of LR transmission and highlight important measures for prevention. METHODS: We conducted two retrospective cohort studies and obtained demographic, clinical, laboratory and contact data, performed SARS-CoV-2 serology, whole genome sequencing (WGS), calculated LR transmission probabilities, measured particle emissions of selected choir members, and calculated particle air concentrations and inhalation doses. RESULTS: We included 65 (84%) and 42 (100%) members of choirs 1 and 2, respectively, of whom 58 (89%) and 10 (24%) became cases. WGS confirmed strain identity in both choirs. Both primary cases transmitted presymptomatically. Particle emission rate when singing was 7 times higher compared to talking. In choir 1, the median concentration of primary cases' emitted particles in the room was estimated to be 8 times higher, exposure at least 30 minutes longer and room volume smaller than in choir 2, resulting in markedly different estimated probabilities for LR transmission (mode: 90% vs. 16%, 95% CI: 80-95% vs. 6-36%). According to a risk model, the first transmission in choir 1 occurred likely after 8 minutes of singing. CONCLUSIONS: The attack rate of the two choirs differed significantly reflecting the differences in LR transmission risks. The pooled proportion of cases due to LR transmission was substantial (81%; 55/68 cases) and was facilitated by likely highly infectious primary cases, high particle emission rates, and indoor rehearsing for an extended time. Even in large rooms, singing of an infectious person may lead to secondary infections through LR exposure within minutes. In the context of indoor gatherings without mask-wearing and waning or insufficient immunity, these results highlight the ongoing importance of non-pharmaceutical interventions wherever aerosols can accumulate.

Evaluation of 11 commercially available PCR kits for the detection of monkeypox virus DNA, Berlin, July to September 2022.

Before the international spread of monkeypox in May 2022, PCR kits for the detection of orthopoxviruses, and specifically monkeypox virus, were rarely available. Here we describe the evaluation of 11 recently developed commercially available PCR kits for the detection of monkeypox virus DNA. All tested kits are currently intended for research use only and clinical performance still needs to be assessed in more detail, but all were suitable for diagnostics of monkeypox virus, with variations in specificity rather than sensitivity.

RespiCoV: Simultaneous identification of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and 46 respiratory tract viruses and bacteria by amplicon-based Oxford-Nanopore MinION sequencing.

Since December 2019 the world has been facing the outbreak of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Identification of infected patients and discrimination from other respiratory infections have so far been accomplished by using highly specific real-time PCRs. Here we present a rapid multiplex approach (RespiCoV), combining highly multiplexed PCRs and MinION sequencing suitable for the simultaneous screening for 41 viral and five bacterial agents related to respiratory tract infections, including the human coronaviruses NL63, HKU1, OC43, 229E, Middle East respiratory syndrome coronavirus, SARS-CoV, and SARS-CoV-2. RespiCoV was applied to 150 patient samples with suspected SARS-CoV-2 infection and compared with specific real-time PCR. Additionally, several respiratory tract pathogens were identified in samples tested positive or negative for SARS-CoV-2. Finally, RespiCoV was experimentally compared to the commercial RespiFinder 2SMART multiplex screening assay (PathoFinder, The Netherlands).

RNA reference materials with defined viral RNA loads of SARS-CoV-2-A useful tool towards a better PCR assay harmonization.

SARS-CoV-2, the cause of COVID-19, requires reliable diagnostic methods to track the circulation of this virus. Following the development of RT-qPCR methods to meet this diagnostic need in January 2020, it became clear from interlaboratory studies that the reported Ct values obtained for the different laboratories showed high variability. Despite this the Ct values were explored as a quantitative cut off to aid clinical decisions based on viral load. Consequently, there was a need to introduce standards to support estimation of SARS-CoV-2 viral load in diagnostic specimens. In a collaborative study, INSTAND established two reference materials (RMs) containing heat-inactivated SARS-CoV-2 with SARS-CoV-2 RNA loads of ~107 copies/mL (RM 1) and ~106 copies/mL (RM 2), respectively. Quantification was performed by RT-qPCR using synthetic SARS-CoV-2 RNA standards and digital PCR. Between November 2020 and February 2021, German laboratories were invited to use the two RMs to anchor their Ct values measured in routine diagnostic specimens, with the Ct values of the two RMs. A total of 305 laboratories in Germany were supplied with RM 1 and RM 2. The laboratories were requested to report their measured Ct values together with details on the PCR method they used to INSTAND. This resultant 1,109 data sets were differentiated by test system and targeted gene region. Our findings demonstrate that an indispensable prerequisite for linking Ct values to SARS-CoV-2 viral loads is that they are treated as being unique to an individual laboratory. For this reason, clinical guidance based on viral loads should not cite Ct values. The RMs described were a suitable tool to determine the specific laboratory Ct for a given viral load. Furthermore, as Ct values can also vary between runs when using the same instrument, such RMs could be used as run controls to ensure reproducibility of the quantitative measurements.