Anti-SARS-CoV-2 antibodies in breast milk during lactation after infection or vaccination: A cohort study.

Breast milk is a pivotal source to provide passive immunity in newborns over the first few months of life. Very little is known about the antibody transfer levels over the period of breastfeeding. We conducted a prospective study in which we evaluated concentrations of anti-SARS-CoV-2 Spike IgA and RBD IgG/M/A antibodies in maternal serum and breast milk over a duration of up to 6 months after delivery. We compared antibody levels in women with confirmed COVID-19 infection during pregnancy (n = 16) to women with prenatal SARS-CoV-2 vaccination (n = 5). Among the recovered women, n = 7 (44%) had been vaccinated during the lactation period as well. We observed intraindividual moderate positive correlations between antibody levels in maternal serum and breast milk (r = 0.73, p-value<0.0001), whereupon the median levels were generally higher in serum. Anti-RBD IgA/M/G transfer into breast milk was significantly higher in women recovered from COVID-19 and vaccinated during lactation (35.15 AU/ml; IQR 21.96-66.89 AU/ml) compared to the nonvaccinated recovered group (1.26 AU/ml; IQR 0.49-3.81 AU/ml), as well as in the vaccinated only group (4.52 AU/ml; IQR 3.19-6.23 AU/ml). Notably, the antibody level in breast milk post SARS-CoV-2 infection sharply increased following a single dose of vaccine. Breast milk antibodies in all groups showed neutralization capacities against an early pandemic SARS-CoV-2 isolate (HH-1) and moreover, also against the Omicron variant, although with lower antibody titer. Our findings highlight the importance of booster vaccinations especially after SARS-CoV-2 infection in pregnancy in order to optimize protection in mother and newborn.

The prevalence of early- and late-onset bacterial, viral, and fungal respiratory superinfections in invasively ventilated COVID-19 patients.

The role of respiratory superinfections in patients with coronavirus disease 2019 (COVID-19) pneumonia remains unclear. We investigated the prevalence of early- and late-onset superinfections in invasively ventilated patients with COVID-19 pneumonia admitted to our department of intensive care medicine between March 2020 and November 2020. Of the 102 cases, 74 (72.5%) received invasive ventilation and were tested for viral, bacterial, and fungal pathogens on Days 0-7, 8-14, and 15-21 after the initiation of mechanical ventilation. Approximately 45% developed one or more respiratory superinfections. There was a clear correlation between the duration of invasive ventilation and the prevalence of coinfecting pathogens. Male patients with obesity and those suffering from chronic obstructive pulmonary disease and/or diabetes mellitus had a significantly higher probability to develop a respiratory superinfection. The prevalence of viral coinfections was high, with a predominance of the herpes simplex virus (HSV), followed by cytomegalovirus. No respiratory viruses or intracellular bacteria were detected in our cohort. We observed a high coincidence between Aspergillus fumigatus and HSV infection. Gram-negative bacteria were the most frequent pathogen group. Klebsiella aerogenes was detected early after intubation, while Klebsiella pneumoniae and Pseudomonas aeruginosa were related to a prolonged respiratory weaning.

SARS-CoV-2 Blood RNA Load Predicts Outcome in Critically Ill COVID-19 Patients.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA loads in patient specimens may act as a clinical outcome predictor in critically ill patients with coronavirus disease 2019 (COVID-19). METHODS: We evaluated the predictive value of viral RNA loads and courses in the blood compared with the upper and lower respiratory tract loads of critically ill COVID-19 patients. Daily specimen collection and viral RNA quantification by reverse transcription quantitative polymerase chain reaction were performed in all consecutive 170 COVID-19 patients between March 2020 and February 2021 during the entire intensive care unit (ICU) stay (4145 samples analyzed). Patients were grouped according to their 90-day outcome as survivors (n=100) or nonsurvivors (n=70). RESULTS: In nonsurvivors, blood SARS-CoV-2 RNA loads were significantly higher at the time of admission to the ICU (P=.0009). Failure of blood RNA clearance was observed in 33/50 (66%) of the nonsurvivors compared with 12/64 (19%) survivors (P<.0001). As determined by multivariate analysis, taking sociodemographic and clinical parameters into account, blood SARS-CoV-2 RNA load represents a valid and independent predictor of outcome in critically ill COVID-19 patients (odds ratio [OR; log10], 0.23; 95% CI, 0.12-0.42; P<.0001), with a significantly higher effect for survival compared with respiratory tract SARS-CoV-2 RNA loads (OR [log10], 0.75; 95% CI, 0.66-0.85; P<.0001). Blood RNA loads exceeding 2.51×103 SARS-CoV-2 RNA copies/mL were found to indicate a 50% probability of death. Consistently, 29/33 (88%) nonsurvivors with failure of virus clearance exceeded this cutoff value constantly. CONCLUSIONS: Blood SARS-CoV-2 load is an important independent outcome predictor and should be further evaluated for treatment allocation and patient monitoring.

Rapid Automated Screening for SARS-CoV-2 B.1.617 Lineage Variants (Delta/Kappa) through a Versatile Toolset of qPCR-Based SNP Detection.

BACKGROUND: The recent emergence of distinct and highly successful SARS-CoV-2 lineages has substantial implications for individual patients and public health measures. While next-generation-sequencing is routinely performed for surveillance purposes, RT-qPCR can be used to rapidly rule-in or rule-out relevant variants, e.g., in outbreak scenarios. The objective of this study was to create an adaptable and comprehensive toolset for multiplexed Spike-gene SNP detection, which was applied to screen for SARS-CoV-2 B.1.617 lineage variants. METHODS: We created a broad set of single nucleotide polymorphism (SNP)-assays including del-Y144/145, E484K, E484Q, P681H, P681R, L452R, and V1176F based on a highly specific multi-LNA (locked nucleic acid)-probe design to maximize mismatch discrimination. As proof-of-concept, a multiplex-test was compiled and validated (SCOV2-617VOC-UCT) including SNP-detection for L452R, P681R, E484K, and E484Q to provide rapid screening capabilities for the novel B.1.617 lineages. RESULTS: For the multiplex-test (SCOV2-617VOC-UCT), the analytic lower limit of detection was determined as 182 IU/mL for L452R, 144 IU/mL for P681R, and 79 IU/mL for E484Q. A total of 233 clinical samples were tested with the assay, including various on-target and off-target sequences. All SNPs (179/179 positive) were correctly identified as determined by SARS-CoV-2 whole genome sequencing. CONCLUSION: The recurrence of SNP locations and flexibility of methodology presented in this study allows for rapid adaptation to current and future variants. Furthermore, the ability to multiplex various SNP-assays into screening panels improves speed and efficiency for variant testing. We show 100% concordance with whole genome sequencing for a B.1.617.2 screening assay on the cobas6800 high-throughput system.

Clinical performance and accuracy of a qPCR-based SARS-CoV-2 mass-screening workflow for healthcare-worker surveillance using pooled self-sampled gargling solutions: A cross-sectional study.

INTRODUCTION: The large number of asymptomatic SARS-CoV-2 infections necessitates general screening of employees. We evaluate the performance of a SARS-CoV-2 screening program in asymptomatic healthcare-workers (HCW), utilizing self-sampled gargling-solution and sample pooling for RT-qPCR. METHODS: We conducted a cross-sectional retrospective study to collect real-life data on the performance of a screening-workflow based on automated-pooling and high-throughput qPCR testing over a 3-month-period at the University Hospital Hamburg. RESULTS: Matrix validation reveals that lower limit of detection for SARS-CoV-2 RNA in gargling-solution was 180 copies/mL (5-sample-pool). A total of 55,122 self-collected gargle samples (= 7513 HCWs) was analyzed. The median time to result was 8.5 hours (IQR 7.2-10.8). Of 11,192 pools analyzed, 11,041 (98.7%) were negative, 69 (0.6%) were positive and 82 (0.7%) were invalid. Individual testing of pool participants revealed 57 SARS-CoV-2 previously unrecognized infections. All 57 HCWs were either pre-symptomatic or asymptomatic (prevalence 0.76%,CI95%0.58-0.98%). Accuracy based on HCWs with gargle-solution and NP-swab available within 3-day-interval (N = 521) was 99.5% (CI95%98.3-99.9%), sensitivity 88.9% (CI95%65.3-98.6%) while specificity 99.8% (CI95%98.9-99.9). CONCLUSION: This workflow was highly effective in identifying SARS-CoV-2 positive HCWs, thereby lowering the potential of inter-HCW and HCW-patient transmissions. Automated-sample-pooling helped to conserve qPCR reagents and represents a promising alternative strategy to antigen testing in mass-screening programs.

Multicenter Evaluation of a Fully Automated High-Throughput SARS-CoV-2 Antigen Immunoassay.

INTRODUCTION: Molecular testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to suffer from delays and shortages. Antigen tests have recently emerged as a viable alternative to detect patients with high viral loads, associated with elevated risk of transmission. While rapid lateral flow tests greatly improved accessibility of SARS-CoV-2 detection in critical areas, their manual nature limits scalability and suitability for large-scale testing schemes. The Elecsys® SARS-CoV-2 Antigen assay allows antigen immunoassays to be carried out on fully automated high-throughput serology platforms. METHODS: A total of 3139 nasopharyngeal and oropharyngeal swabs were collected at 3 different testing sites in Germany. Swab samples were pre-characterized by reverse transcription real-time polymerase chain reaction (RT-qPCR) and consecutively subjected to the antigen immunoassay on either the cobas e 411 or cobas e 801 analyzer. RESULTS: Of the tested respiratory samples, 392 were PCR positive for SARS-CoV-2 RNA. Median concentration was 2.95 × 104 (interquartile range [IQR] 5.1 × 102-3.5 × 106) copies/ml. Overall sensitivity and specificity of the antigen immunoassay were 60.2% (95% confidence interval [CI] 55.2-65.1) and 99.9% (95% CI 99.6-100.0), respectively. A 93.7% (95% CI 89.7-96.5) sensitivity was achieved at a viral RNA concentration ≥ 104 copies/ml (~ cycle threshold [Ct] value < 29.9). CONCLUSION: The Elecsys SARS-CoV-2 Antigen assay reliably detected patient samples with viral loads ≥ 10,000 copies/ml. It thus represents a viable high-throughput alternative for screening of patients or in situations where PCR testing is not readily available.

Evaluation of a fully automated high-throughput SARS-CoV-2 multiplex qPCR assay with built-in screening functionality for del-HV69/70- and N501Y variants such as B.1.1.7.

BACKGROUND: New SARS-CoV-2 variants with increased transmissibility, like B.1.1.7, first detected in England or B.1.351, first detected in South Africa, have caused considerable concern worldwide. In order to contain the spread of these lineages, it is of utmost importance to have rapid, sensitive and high-throughput detection methods at hand. METHODS: A set of RT-qPCR assays was modified for a diagnostic SARS-CoV-2 multiplex assay including detection of the del-HV69/70 and N501Y mutations on the cobas6800 platform. Analytical sensitivity was assessed for both wild-type SARS-CoV-2 and B.1.1.7 lineage by serial dilution. For clinical performance, a total of 176 clinical samples were subjected to the test and results compared to a commercial manual typing-PCR assay and next generation sequencing as gold standard. RESULTS: The multiplex assay was highly sensitive for detection of SARS-CoV-2 RNA in clinical samples, with an LoD of 6.16 cp/ml (CI: 4.00-8.31). LoDs were slightly higher for detection of the HV69/70 deletion (85.92, CI: 61-194.41) and the N501Y SNP (105.99 cp/ml, CI: 81.59 - 183.66). A total of 176 clinical samples were tested with the assay, including 50 samples containing SARS-CoV-2 of the B.1.1.7 lineage, one containing B.1.351 and 85 non-B.1.1.7/B.1.351 lineage, of which three also harbored a HV69/70 deletion. All were correctly identified by the multiplex assay. CONCLUSION: We describe here a highly sensitive, fully automated multiplex PCR assay for the simultaneous detection of the del-HV69/70 and N501Y mutations that can distinguish between B.1.1.7 and other lineages. The assay allows for high-throughput screening for currently relevant variants in clinical samples prior to sequencing.

Handling and accuracy of four rapid antigen tests for the diagnosis of SARS-CoV-2 compared to RT-qPCR.

BACKGROUND: SARS-CoV-2 molecular diagnostics is facing material shortages and long turnaround times due to exponential increase of testing demand. OBJECTIVE: We evaluated the analytic performance and handling of four rapid Antigen Point of Care Tests (AgPOCTs) I-IV (Distributors: (I) Roche, (II) Abbott, (III) MEDsan and (IV) Siemens). METHODS: 100 RT-PCR negative and 84 RT-PCR positive oropharyngeal swabs were prospectively collected and used to determine performance and accuracy of these AgPOCTs. Handling was evaluated by 10 healthcare workers/users through a questionnaire. RESULTS: The median duration from symptom onset to sampling was 6 days (IQR 2-12 days). The overall respective sensitivity were 49.4 % (CI95 %: 38.9-59.9), 44.6 % (CI95 %: 34.3-55.3), 45.8 % (CI95 %: 35.5-56.5) and 54.9 % (CI95 %: 43.4-65.9) for tests I, II, III and IV, respectively. In the high viral load subgroup (containing >106 copies of SARS-CoV-2 /swab, n = 26), AgPOCTs reached sensitivities of 92.3 % or more (range 92.3 %-100 %). Specificity was 100 % for tests I, II (CI95 %: 96.3-100 for both tests) and IV (CI95 %: 96.3-100) and 97 % (CI95 %: 91.5-98.9) for test III. Regarding handling, test I obtained the overall highest scores, while test II was considered to have the most convenient components. Of note, users considered all assays, with the exception of test I, to pose a significant risk for contamination by drips or spills. DISCUSSION: Besides some differences in sensitivity and handling, all four AgPOCTs showed acceptable performance in high viral load samples. However, due to the significantly lower sensitivity compared to RT-qPCR, a careful consideration of pro and cons of AgPOCT has to be taken into account before clinical implementation.