Impaired humoral immunity to BQ.1.1 in convalescent and vaccinated patients.

Determining SARS-CoV-2 immunity is critical to assess COVID-19 risk and the need for prevention and mitigation strategies. We measured SARS-CoV-2 Spike/Nucleocapsid seroprevalence and serum neutralizing activity against Wu01, BA.4/5 and BQ.1.1 in a convenience sample of 1,411 patients receiving medical treatment in the emergency departments of five university hospitals in North Rhine-Westphalia, Germany, in August/September 2022. 62% reported underlying medical conditions and 67.7% were vaccinated according to German COVID-19 vaccination recommendations (13.9% fully vaccinated, 54.3% one booster, 23.4% two boosters). We detected Spike-IgG in 95.6%, Nucleocapsid-IgG in 24.0%, and neutralization against Wu01, BA.4/5 and BQ.1.1 in 94.4%, 85.0%, and 73.8% of participants, respectively. Neutralization against BA.4/5 and BQ.1.1 was 5.6- and 23.4-fold lower compared to Wu01. Accuracy of S-IgG detection for determination of neutralizing activity against BQ.1.1 was reduced substantially. We explored previous vaccinations and infections as correlates of BQ.1.1 neutralization using multivariable and Bayesian network analyses. Given a rather moderate adherence to COVID-19 vaccination recommendations, this analysis highlights the need to improve vaccine-uptake to reduce the COVID-19 risk of immune evasive variants. The study was registered as clinical trial (DRKS00029414).

Factors associated with vaccine-induced T cell immune responses against SARS-CoV-2 in kidney transplant recipients.

Vaccination against SARS-CoV-2 is an important prophylactic measure in kidney transplant recipients (KTRs), however, the immune response is often impaired. Here, we examined the T cell immune response against SARS-CoV-2 in 148 KTRs after three or four vaccine doses including 35 KTRs with subsequent SARS-CoV-2 infection. The frequency of spike-specific T cells was lower in KTRs compared to immunocompetent controls and correlated with the level of spike-specific antibodies. Positive predictors for detection of vaccine-induced T cells were detection of spike-specific antibodies, heterologous immunization with mRNA and a vector vaccine and longer time past transplant. In vaccinated KTRs with subsequent SARS-CoV-2 infection, the T-cell response was greatly enhanced and was significantly higher than in vaccinated KTRs without SARS-CoV-2 infection. Overall, the data show a correlation between impaired humoral and T-cell immunity to SARS-CoV-2 vaccination and provide evidence for greater robustness of hybrid immunity in KTRs.

Efficient virus detection utilizing chitin-immobilized nanobodies synthesized in Ustilago maydis.

The COVID-19 pandemic has greatly impacted the global economy and health care systems, illustrating the urgent need for timely and inexpensive responses to pandemic threats in the form of vaccines and antigen tests. Currently, antigen testing is mostly conducted by qualitative flow chromatography or via quantitative ELISA-type assays. The latter mostly utilize materials like protein-adhesive polymers and gold or latex particles. Here we present an alternative ELISA approach using inexpensive, biogenic materials and permitting quick detection based on components produced in the microbial model Ustilago maydis. In this fungus, heterologous proteins like biopharmaceuticals can be exported by fusion to unconventionally secreted chitinase Cts1. As a unique feature, the carrier chitinase binds to chitin allowing its additional use as a purification or immobilization tag. Recent work has demonstrated that nanobodies are suitable target proteins. These proteins represent a very versatile alternative antibody format and can quickly be adapted to detect novel antigens by camelidae immunization or synthetic libraries. In this study, we exemplarily produced different mono- and bivalent SARS-CoV-2 nanobodies directed against the spike protein receptor binding domain (RBD) as Cts1 fusions and screened their antigen binding affinity in vitro and in vivo. Functional nanobody-Cts1 fusions were immobilized on chitin forming an RBD tethering surface. This provides a solid base for future development of inexpensive antigen tests utilizing unconventionally secreted nanobodies as antigen trap and a matching ubiquitous and biogenic surface for immobilization.

Integrated genomic surveillance enables tracing of person-to-person SARS-CoV-2 transmission chains during community transmission and reveals extensive onward transmission of travel-imported infections, Germany, June to July 2021.

BackgroundTracking person-to-person SARS-CoV-2 transmission in the population is important to understand the epidemiology of community transmission and may contribute to the containment of SARS-CoV-2. Neither contact tracing nor genomic surveillance alone, however, are typically sufficient to achieve this objective.AimWe demonstrate the successful application of the integrated genomic surveillance (IGS) system of the German city of Düsseldorf for tracing SARS-CoV-2 transmission chains in the population as well as detecting and investigating travel-associated SARS-CoV-2 infection clusters.MethodsGenomic surveillance, phylogenetic analysis, and structured case interviews were integrated to elucidate two genetically defined clusters of SARS-CoV-2 isolates detected by IGS in Düsseldorf in July 2021.ResultsCluster 1 (n = 67 Düsseldorf cases) and Cluster 2 (n = 36) were detected in a surveillance dataset of 518 high-quality SARS-CoV-2 genomes from Düsseldorf (53% of total cases, sampled mid-June to July 2021). Cluster 1 could be traced back to a complex pattern of transmission in nightlife venues following a putative importation by a SARS-CoV-2-infected return traveller (IP) in late June; 28 SARS-CoV-2 cases could be epidemiologically directly linked to IP. Supported by viral genome data from Spain, Cluster 2 was shown to represent multiple independent introduction events of a viral strain circulating in Catalonia and other European countries, followed by diffuse community transmission in Düsseldorf.ConclusionIGS enabled high-resolution tracing of SARS-CoV-2 transmission in an internationally connected city during community transmission and provided infection chain-level evidence of the downstream propagation of travel-imported SARS-CoV-2 cases.

Adjusted COVID-19 booster schedules balance age-dependent differences in antibody titers benefitting risk populations

We provide follow-up data on the humoral immune response after COVID-19 vaccinations of two distinct cohorts aged below 60 and over 80 years to screen for age-related differences in the longevity and magnitude of the induction of the antibody responses post booster-vaccinations. While anti-SARS-CoV-2 spike-specific IgG and neutralization capacity waned rapidly after the initial vaccination schedule, additional boosters highly benefitted the humoral immune responses especially in the elderly cohort, including the neutralization of Omikron variants. Thus, adjusted COVID-19 booster vaccination schedules are an appropriate tool to overcome limitations in the success of vaccinations.

Immune response to third SARS-CoV-2 vaccination in seronegative kidney transplant recipients: Possible improvement by mycophenolate mofetil reduction.

Modification of vaccination strategies is necessary to improve the immune response to SARS-CoV-2 vaccination in kidney transplant recipients (KTRs). This multicenter observational study analyzed the effects of the third SARS-CoV-2 vaccination in previously seronegative KTRs with the focus on temporary mycophenolate mofetil (MMF) dose reduction within propensity matched KTRs. 56 out of 174 (32%) previously seronegative KTRs became seropositive after the third vaccination with only three KTRs developing neutralizing antibodies against the omicron variant. Multivariate logistic regression revealed that initial antibody levels, graft function, time after transplantation and MMF trough levels had an influence on seroconversion (P < .05). After controlling for confounders, the effect of MMF dose reduction before the third vaccination was calculated using propensity score matching. KTRs with a dose reduction of ≥33% showed a significant decrease in MMF trough levels to 1.8 (1.2-2.5) µg/ml and were more likely to seroconvert than matched controls (P = .02). Therefore, a MMF dose reduction of 33% or more before vaccination is a promising approach to improve success of SARS-CoV-2 vaccination in KTRs.

Comparison of commercial SARS-CoV-2 surrogate neutralization assays with a full virus endpoint dilution neutralization test in two different cohorts.

Determination of neutralizing antibody titers is still considered the gold standard for infection protection. A full virus neutralization test (VNT) with replication-competent, infectious SARS-CoV-2, is labor-intensive and requires Biosafety Level 3 certified laboratories. Therefore, several commercial SARS-CoV-2 surrogate virus neutralization tests (sVNTs) have been developed that aim to detect neutralizing antibodies targeting the receptor binding domain (RBD) of the viral spike glycoprotein (S). Neutralizing antibodies to the RBD block its interaction with the angiotensin-converting enzyme 2 (ACE2) receptor protein. Here, we compared a full virus neutralization test (VNT) with two SARS-CoV-2 surrogate virus neutralization tests (sVNT) and validated them in two cohorts of i) convalescent SARS-CoV-2-infected individuals and ii) COVID vaccinated individuals. The sVNTs showed highly different results both, compared to the VNT-titers and also between the two cohorts. This indicates that currently, sVNT provide a qualitative instead of a quantitative measurement of neutralizing antibodies. The findings in this work show that the cutoff levels for sVNTs might need to be readjusted for convalescent and vaccinated individuals.

CT Findings in Patients with COVID-19-Compatible Symptoms but Initially Negative qPCR Test.

PURPOSE: To assess whether it is possible to reliably detect patients with strong suspicion of COVID-19 despite initially negative quantitative polymerase-chain-reaction (qPCR) tests by means of computed tomography (CT). MATERIALS AND METHODS: 437 patients with suspected COVID-19 but initially negative qPCR and subsequent chest CT between March 13 and November 30, 2020 were included in this retrospective study. CT findings were compared to results of successive qPCR tests (minimum of 3 qPCR tests if CT suggested infection) to determine the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of CT for diagnosing COVID-19. RESULTS: COVID-19 was diagnosed correctly with a sensitivity of 100 % [95 % confidence interval (CI): 65-100] and a specificity of 88 % [95 % CI: 84-90]. A PPV of 12 % [95 % CI: 6-22] and an NPV of 100 % [95 % CI: 99-100] were determined. CONCLUSION: CT is able to detect COVID-19 before qPCR in initially negative patients in this special study setting. Similar CT findings in COVID-19 and other atypical pneumonias can lead to high numbers of false-positive patients, reducing the specificity of CT. KEY POINTS: · Low-dose chest CT is able to diagnose COVID-19 in symptomatic patients even in cases of an initially negative quantitative PCR result and therefore is a fast support method to detect COVID-19, especially in early disease.. · Low-dose chest CT can reliably exclude COVID-19 in a pandemic setting.. · CT does not always ensure a reliable differentiation from other viral diseases.. CITATION FORMAT: · Valentin B, Steuwe A, Wienemann T, et al. CT Findings in Patients with COVID-19-Compatible Symptoms but Initially Negative qPCR Test. Fortschr Röntgenstr 2022; 194: 1110 - 1118.

Impaired Immune Response to SARS-CoV-2 Vaccination in Dialysis Patients and in Kidney Transplant Recipients.

Background: Patients with kidney failure on dialysis or after renal transplantation have a high risk for severe COVID-19 infection, and vaccination against SARS-CoV-2 is the only expedient prophylaxis. Generally, immune responses are attenuated in patients with kidney failure, however, systematic analyses of immune responses to SARS-CoV-2 vaccination in patients on dialysis and in kidney transplant recipients (KTRs) are still needed. Methods: In this prospective, multicentric cohort study, antibody responses to COVID-19 mRNA vaccines (BNT162b2 [BioNTech/Pfizer] or mRNA-1273 [Moderna]) were measured in 32 patients on dialysis and in 28 KTRs. SARS-CoV-2-specific antibodies and neutralization capacity were evaluated and compared with controls (n=78) of a similar age range. Results: After the first vaccination, SARS-CoV-2-specific antibodies were nearly undetectable in patients with kidney failure. After the second vaccination, 93% of the controls and 88% of patients on dialysis but only 37% of KTRs developed SARS-CoV-2-specific IgG above cutoff. Moreover, mean IgG levels were significantly lower in KTRs (54±93 BAU/ml) compared with patients on dialysis (503±481 BAU/ml; P<0.01). Both KTRs and patients on dialysis had significantly lower IgG levels compared with controls (1992±2485 BAU/ml; P<0.001 and P<0.01, respectively). Importantly, compared with controls, neutralizing antibody titers were significantly lower in KTRs and patients on dialysis. After the second vaccination, 76% of KTRs did not show any neutralization capacity against SARS-CoV-2, suggesting impaired seroprotection. Conclusions: Patients with kidney failure show a significantly weaker antibody response compared with controls. Most strikingly, only one out of four KTRs developed neutralizing antibodies against SARS-CoV-2 after two doses of vaccine. These data suggest that vaccination strategies need modification in KTRs and patients on dialysis.Clinical Trial registry name and registration number: Vaccination Against COVID-19 in Chronic Kidney Disease, NCT04743947.

Humoral response to SARS-CoV-2 and seasonal coronaviruses in COVID-19 patients.

We used enzyme-linked immunoassay methods to measure the prevalence and the levels of antibody responses to the nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and four seasonal human coronaviruses (HCoV-OC43, HCoV-HKU1, HCoV 229E, and HCoV-NL63) in a cohort of 115 convalescent plasma donors infected with SARS-CoV-2 (1-61 days after symptom onset) compared to antibody levels in 114 individuals with no evidence of a recent infection with SARS-CoV-2. In the humoral response to the four seasonal coronaviruses, only HCoV-HKU1- and HCoV-229E-assays showed slightly elevated antibody levels in the COVID group compared to the control group. While in the COVID-group the levels of SARS-CoV-2 antibodies correlated significantly with disease severity, no association was found in the levels of antibodies against the seasonal coronaviruses. The most striking result in both groups was that the levels of antibodies against all tested coronaviruses, including the new SARS-CoV-2 showed a highly significant correlation with each other. There seems to be an individual predisposition to a weaker or stronger humoral immune response against all known seasonal human coronaviruses including the new SARS-CoV-2, which could lead to a definition of low and high responders against human coronaviruses with potential impact on the assessment of postinfection antibody levels and protection.

Applicability of CO-RADS in an Anonymized Cohort Including Early and Advanced Stages of COVID-19 in Comparison to the Recommendations of the German Radiological Society and Radiological Society of North America.

PURPOSE: Classifications were created to facilitate radiological evaluation of the novel coronavirus disease 2019 (COVID-19) on computed tomography (CT) images. The categorical CT assessment scheme (CO-RADS) categorizes lung parenchymal changes according to their likelihood of being caused by SARS-CoV-2 infection. This study investigates the diagnostic accuracy of diagnosing COVID-19 with CO-RADS compared to the Thoracic Imaging Section of the German Radiological Society (DRG) classification and Radiological Society of North America (RSNA) classification in an anonymized patient cohort. To mimic advanced disease stages, follow-up examinations were included as well. METHOD: This study includes all patients undergoing chest CT in the case of a suspected SARS-CoV-2 infection or an already confirmed infection between March 13 and November 30, 2020. During the study period, two regional lockdowns occurred due to high incidence values, increasing the pre-test probability of COVID-19. Anonymized CT images were reviewed retrospectively and in consensus by two radiologists applying CO-RADS, DRG, and RSNA classification. Afterwards, CT findings were compared to results of sequential real-time reverse transcriptase polymerase chain reaction (qPCR) test performed during hospitalization to determine statistical analysis for diagnosing COVID-19. RESULTS: 536 CT examinations were included. CO-RADS, DRG and RSNA achieved an NPV of 96 %/94 %/95 % (CO-RADS/DRG/RSNA), PPV of 83 %/80 %/88 %, sensitivity of 86 %/76 %/80 %, and specificity of 96 %/95 %/97 %. The disease prevalence was 20 %. CONCLUSION: All applied classifications can reliably exclude a SARS-CoV-2 infection even in an anonymous setting. Nevertheless, pre-test probability was high in our study setting and has a great influence on the classifications. Therefore, the applicability of the individual classifications will become apparent in the future with lower prevalence and incidence of COVID-19. KEY POINTS: · CO-RADS, DRG, and RSNA classifications help to reliably detect infected patients in an anonymized setting. · Pre-test probability has a great influence on the individual classifications. · Difficulties in an anonymized study setting are severe pulmonary changes and residuals.. CITATION FORMAT: · Valentin B, Steuwe A, Wienemann T et al. Applicability of CO-RADS in an Anonymized Cohort Including Early and Advanced Stages of COVID-19 in Comparison to the Recommendations of the German Radiological Society and Radiological Society of North America. Fortschr Röntgenstr 2022; 194: 862 - 872.

Characterization of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Infection Clusters Based on Integrated Genomic Surveillance, Outbreak Analysis and Contact Tracing in an Urban Setting.

BACKGROUND: Tracing of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission chains is still a major challenge for public health authorities, when incidental contacts are not recalled or are not perceived as potential risk contacts. Viral sequencing can address key questions about SARS-CoV-2 evolution and may support reconstruction of viral transmission networks by integration of molecular epidemiology into classical contact tracing. METHODS: In collaboration with local public health authorities, we set up an integrated system of genomic surveillance in an urban setting, combining a) viral surveillance sequencing, b) genetically based identification of infection clusters in the population, c) integration of public health authority contact tracing data, and d) a user-friendly dashboard application as a central data analysis platform. RESULTS: Application of the integrated system from August to December 2020 enabled a characterization of viral population structure, analysis of 4 outbreaks at a maximum care hospital, and genetically based identification of 5 putative population infection clusters, all of which were confirmed by contact tracing. The system contributed to the development of improved hospital infection control and prevention measures and enabled the identification of previously unrecognized transmission chains, involving a martial arts gym and establishing a link between the hospital to the local population. CONCLUSIONS: Integrated systems of genomic surveillance could contribute to the monitoring and, potentially, improved management of SARS-CoV-2 transmission in the population.

Age-dependent Immune Response to the Biontech/Pfizer BNT162b2 Coronavirus Disease 2019 Vaccination.

BACKGROUND: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has led to the development of various vaccines. Real-life data on immune responses elicited in the most vulnerable group of vaccinees older than age 80 years old are still underrepresented despite the prioritization of the elderly in vaccination campaigns. METHODS: We conducted a cohort study with 2 age groups, young vaccinees below the age of 60 years and elderly vaccinees over the age of 80 years, to compare their antibody responses to the first and second dose of the BNT162b2 coronavirus disease 2019 vaccination. RESULTS: Although the majority of participants in both groups produced specific immunoglobulin G antibody titers against SARS-CoV-2 spike protein, titers were significantly lower in elderly participants. Although the increment of antibody levels after the second immunization was higher in elderly participants, the absolute mean titer of this group remained lower than the <60 years of age group. After the second vaccination, 31.3% of the elderly had no detectable neutralizing antibodies in contrast to the younger group, in which only 2.2% had no detectable neutralizing antibodies. CONCLUSIONS: Our data showed differences between the antibody responses raised after the first and second BNT162b2 vaccination, in particular lower frequencies of neutralizing antibodies in the elderly group. This suggests that this population needs to be closely monitored and may require earlier revaccination and/or an increased vaccine dose to ensure stronger long-lasting immunity and protection against infection.

SARS-CoV-2 Infection in Fully Vaccinated Individuals of Old Age Strongly Boosts the Humoral Immune Response.

Prophylactic vaccination against SARS-CoV-2 is one of the most important measures to contain the COVID-19 pandemic. Recently, break-through infections following vaccination against this virus have been reported. Here, we describe the humoral immune response of break-through infections in fully vaccinated individuals of old age from an outbreak in a nursing home. In cooperation with the local health authority, blood samples from fully vaccinated and infected as well as fully vaccinated and uninfected residents of the nursing home were collected 4 weeks after the onset of the outbreak. The humoral immune response was determined in a neutralisation assay with replication-competent virus isolates and by a quantitative ELISA. In this outbreak a total of 23 residents and four health care workers were tested positive for SARS-CoV-2. Four residents were unvaccinated, including one with a severe course of disease who later severe disease course who later succumbed to infection. Despite their old age, all vaccinated residents showed no or only mild disease. Comparison of the humoral immune response revealed significantly higher antibody levels in fully vaccinated infected individuals compared to fully vaccinated uninfected individuals (p < 0.001). Notably, although only a minority of the vaccinated uninfected group showed neutralisation capacity against SARS-CoV-2, all vaccinated and infected individuals showed high-titre neutralisation of SARS-CoV-2 including the alpha and beta variant. Large SARS-CoV-2 outbreaks can occur in fully vaccinated populations, but seem to associate with mild disease. SARS-CoV-2 infection in fully vaccinated individuals is a strong booster of the humoral immune response providing enhanced neutralisation capacity against immune evasion variants.

Intensity of mycophenolate mofetil treatment is associated with an impaired immune response to SARS-CoV-2 vaccination in kidney transplant recipients.

Kidney transplant recipients (KTRs) are extremely vulnerable to SARS-CoV-2 infection and show an impaired immune response to SARS-CoV-2 vaccination. We analyzed factors related to vaccination efficiency in KTRs. In a multicenter prospective observational study (NCT04743947), IgG antibodies levels against SARS-CoV-2 spike S1 subunit and their neutralization capacity after SARS-CoV-2 vaccination were analyzed in 225 KTRs and compared to 176 controls. After the vaccination, 56 (24.9%) KTRs became seropositive of whom 68% had neutralizing antibodies. This immune response was significantly lower compared to controls (239 [78-519] BAU/ml versus 1826 [560-3180] BAU/ml for KTRs and controls, p < .0001). The strongest predictor for an impaired response was mycophenolate mofetil (MMF) treatment. Multivariate regression analysis revealed that MMF-free regimen was highly associated with seroconversion (OR 13.25, 95% CI 3.22-54.6; p < .001). In contrast, other immunosuppressive drugs had no significant influence. 187 out of 225 KTRs were treated with MMF of whom 26 (13.9%) developed antibodies. 23 of these seropositive KTRs had a daily MMF dose ≤1 g. Furthermore, higher trough MMF concentrations correlated with lower antibody titers (R -0.354, p < .001) supporting a dose-dependent unfavorable effect of MMF. Our data indicate that MMF dose modification could lead to an improved immune response.

Sensitivity of anti-SARS-CoV-2 serological assays in a high-prevalence setting.

Evaluation and power of seroprevalence studies depend on the performed serological assays. The aim of this study was to assess four commercial serological tests from EUROIMMUN, DiaSorin, Abbott, and Roche as well as an in-house immunofluorescence and neutralization test for their capability to identify SARS-CoV-2 seropositive individuals in a high-prevalence setting. Therefore, 42 social and working contacts of a German super-spreader were tested. Consistent with a high-prevalence setting, 26 of 42 were SARS-CoV-2 seropositive by neutralization test (NT), and immunofluorescence test (IFT) confirmed 23 of these 26 positive test results (NT 61.9% and IFT 54.8% seroprevalence). Four commercial assays detected anti-SARS-CoV-2 antibodies in 33.3-40.5% individuals. Besides an overall discrepancy between the NT and the commercial assays regarding their sensitivity, this study revealed that commercial SARS-CoV-2 spike-based assays are better to predict the neutralization titer than nucleoprotein-based assays are.

SARS-CoV-2 targets neurons of 3D human brain organoids.

COVID-19 pandemic caused by SARS-CoV-2 infection is a public health emergency. COVID-19 typically exhibits respiratory illness. Unexpectedly, emerging clinical reports indicate that neurological symptoms continue to rise, suggesting detrimental effects of SARS-CoV-2 on the central nervous system (CNS). Here, we show that a Düsseldorf isolate of SARS-CoV-2 enters 3D human brain organoids within 2 days of exposure. We identified that SARS-CoV-2 preferably targets neurons of brain organoids. Imaging neurons of organoids reveal that SARS-CoV-2 exposure is associated with altered distribution of Tau from axons to soma, hyperphosphorylation, and apparent neuronal death. Our studies, therefore, provide initial insights into the potential neurotoxic effect of SARS-CoV-2 and emphasize that brain organoids could model CNS pathologies of COVID-19.

Extraction-free SARS-CoV-2 detection by rapid RT-qPCR universal for all primary respiratory materials.

BACKGROUND: Fast and reliable detection of SARS-CoV-2 is crucial for efficient control of the COVID-19 pandemic. Due to the high demand for SARS-CoV-2 testing there is a worldwide shortage of RNA extraction reagents. Therefore, extraction-free RT-qPCR protocols are urgently needed. OBJECTIVES: To establish a rapid RT-qPCR protocol for the detection of SARS-CoV-2 without the need of RNA extraction suitable for all respiratory materials. MATERIAL AND METHODS: Different SARS-CoV-2 positive respiratory materials from our routine laboratory were used as crude material after heat inactivation in direct RT-qPCR with the PrimeDirect™ Probe RT-qPCR Mix (TaKaRa). SARS-CoV-2 was detected using novel primers targeted to the E-gene. RESULTS: The protocol for the detection of SARS-CoV-2 in crude material used a prepared frozen-PCR mix with optimized primers and 5 µl of fresh, undiluted and pre-analytically heat inactivated respiratory material. For validation, 91 respiratory samples were analyzed in direct comparison to classical RNA-based RT-qPCR. Overall 81.3 % of the samples were detected in both assays with a strong correlation between both Ct values (r = 0.8492, p < 0.0001). The SARS-CoV-2 detection rate by direct RT-qPCR was 95.8 % for Ct values <35. All negative samples were characterized by low viral loads (Ct >35) and/or long storage times before sample processing. CONCLUSION: Direct RT-qPCR is a suitable alternative to classical RNA RT-qPCR, provided that only fresh samples (storage <1 week) are used. RNA extraction should be considered if samples have longer storage times or if PCR inhibition is observed. In summary, this protocol is fast, inexpensive and suitable for all respiratory materials.

Genetic structure of SARS-CoV-2 reflects clonal superspreading and multiple independent introduction events, North-Rhine Westphalia, Germany, February and March 2020.

We whole-genome sequenced 55 SARS-CoV-2 isolates from Germany to investigate SARS-CoV-2 outbreaks in 2020 in the Heinsberg district and Düsseldorf. While the genetic structure of the Heinsberg outbreak indicates a clonal origin, reflecting superspreading dynamics from mid-February during the carnival season, distinct viral strains were circulating in Düsseldorf in March, reflecting the city's international links. Limited detection of Heinsberg strains in the Düsseldorf area despite geographical proximity may reflect efficient containment and contact-tracing efforts.

Assessment of SARS-CoV-2 in human semen-a cohort study.

OBJECTIVE: To investigate the presence of viral RNA in human semen of patients with severe acute-respiratory syndrome coronavirus 2 (SARS-CoV-2) and to evaluate its presence and relevance in semen parameters. DESIGN: Pilot cohort study. SETTING: University hospital. PATIENT(S): Thirty-four men were distributed as: 1) patients in convalescence (patients with confirmed SARS-CoV-2 infection in pharyngeal swab according to reverse-transcription polymerase chain reaction [RT-PCR] or antibodies); 2) negative control group (no antibodies); and 3) patients with an acute infection (detection of SARS-CoV-2 in pharyngeal swab). INTERVENTION: Semen and a blood sample were collected from each individual. MAIN OUTCOME MEASURE(S): Analysis of semen quality according to the World Health Organization standards. Detection of SARS-CoV-2 by RT-PCR in the native semen sample and after density gradient preparation. Confirmation of immunoglobulin (Ig) A und IgG antibodies in the blood. RESULT(S): Eighteen semen samples from recovered men were obtained 8-54 days after absence of symptoms, 14 from control subjects, and 2 from patients with an active COVID-19 infection. No RNA was detected by means of RT-PCR in the semen, including semen samples from two patients with an acute COVID-19 infection. Subjects with a moderate infection showed an impairment of sperm quality. CONCLUSION(S): A mild COVID-19 infection is not likely to affect testis and epididymis function, whereas semen parameters did seem impaired after a moderate infection. SARS-CoV-2 RNA could not be detected in semen of recovered and acute COVID-19-positive men. This suggests no viral transmission during sexual contact and assisted reproductive techniques, although further data need to be obtained.