Biotin labelled DNA-probe based detection of nucleic acid in reverse transcription-LAMP amplified oropharyngeal viral swab samples via a lateral flow assay (preprint)

This study focuses on three key aspects: a) crude throat swab sample as 30 template for RT-LAMP reaction, b) biotinylated DNA probes enhanced specificity for LFA readout and c) digital semi-quantification of LFA readout. The throat swab samples from SARS-CoV-2 positive and negative patients have been used in their crude (no cleaning or pre-treatment) form for the RT-LAMP reaction. The samples were heat-inactivated but not treated for any kind of nucleic acid extraction or purification. RT-LAMP (20 min processing time) result was readout out via a LFA approach, using two labels: FITC and Biotin. FITC was enzymatically incorporated into the RT-LAMP amplicon with LF-LAMP-primer and biotin was later introduced using biotinylated DNA probes specific for the amplicon region. DNA probes based LFA readout of RT-LAMP amplicon was 97.73% sensitive and 93.10% specific. The LFA result was further analysed via a smartphone based IVD-device wherein the test line intensity was recorded. The LFA test line intensity was then correlated with the qRT-PCR CT-value of the positive swab samples. A digital semi-quantification of RT-LAMP-LFA is reported with the correlation coefficient R2= 0.7. The overall RT-LAMP-LFA assay time was recorded to be 35 min with the LoD of 4,000 RNA copies/ml.

How an ACE2 mimicking epitope-MIP nanofilm recognizes template-related peptides and the receptor binding domain of SARS-CoV-2.

Here we aim to gain a mechanistic understanding of the formation of epitope-imprinted polymer nanofilms using a non-terminal peptide sequence, i.e. the peptide GFNCYFP (G485 to P491) of the SARS-CoV-2 receptor binding domain (RBD). This epitope is chemisorbed on the gold surface through the central cysteine 488 followed by the electrosynthesis of a ∼5 nm thick polyscopoletin film around the surface confined templates. The interaction of peptides and the parent RBD and spike protein with the imprinted polyscopoletin nanofilm was followed by electrochemical redox marker gating, surface enhanced infrared absorption spectroscopy and conductive AFM. Because the use of non-terminal epitopes is especially intricate, here we characterize the binding pockets through their interaction with 5 peptides rationally derived from the template sequence, i.e. implementing central single amino acid mismatch as well as elongations and truncations at its C- and N- termini. Already a single amino acid mismatch, i.e. the central Cys488 substituted by a serine, results in ca. 15-fold lower affinity. Further truncation of the peptides to tetrapeptide (EGFN) and hexapeptide (YFPLQS) results also in a significantly lower affinity. We concluded that the affinity towards the different peptides is mainly determined by the four amino acid motif CYFP present in the sequence of the template peptide. A higher affinity than that for the peptides is found for the parent proteins RBD and spike protein, which seems to be due to out of cavity effects caused by their larger footprint on the nanofilm surface.

Septic arthritis caused by Brucella melitensis - An orthopedic chal-lenge (11194)

Introduction The zoonotic infection with Brucella melitensis can be acquired by inges-tion of unpasteurized goat's or sheep's milk. The infection is common in Eastern Mediterranean countries (EMC), but rare in western Europe (6 cases in Switzerland, 2021). When evaluating patients with symptoms of septic arthritis, brucellosis is not the foremost differential diagnosis. How-ever, with the increasing population of people from EMC in western Eu-rope, the incidence may be rising. We present a patient who was initially suspected to suffer from Long-COVID-Syndrome (LCS), which underscores the relevance of this case in a pandemic situation. Methods/Results A 58-year-old male patient was admitted to the emergency department with a painful right knee effusion after a minor trauma. Additionally, he suffered from fatigue, subfebrile temperatures, back pain and myalgias for more than two months. He was suspected to suffer from LCS after a mild COVID-19 three months earlier. The culture of the arthrocentesis (14.400 cells/mul with 61% polynuclear cells) unexpectedly turned positive for B. melitensis. The patient declared that he had been drinking three liters of unpasteurized goat's milk to cure the presumptive LCS. To ensure staff safety, arthroscopic lavage was postponed until brucella-active antibiotics had been administered for at least 24 hours. Surgery was performed under strict infection control measures to avoid generating aerosols. According to Duke, one major (continuous bacteremia over 14 days) and 2 minor criteria (fever, most probably septic embolic gonarthritis) were fulfilled. Therefore, possible endocarditis had to be assumed although transesophageal echocardiography was normal. Antibiotic treatment was escalated to a quadruple regimen (intravenous gentamicin for three weeks;as well as oral doxycycline, trimethoprim/sulfomethoxazole and rifampin for at least 3 months). The clinical recovery - still under treatment - is protracted with slowly improving knee pain and normalizing signs of inflammation. Conclusion Although B. melitensis is a rare pathogen in Switzerland, orthopedic sur-geons, rheumatologists and infectious disease specialists need to be aware of diseases with low incidence and non-specific symptoms espe-cially in times of a global pandemia. A high index of suspicion is needed in patients related to EMC. When brucellosis is confirmed, strict infection control measures to protect staff involved in aerosol generating proce-dures must be adopted.

Lab-on-a-Chip Immunoassay for Prediction of Severe COVID-19 Disease.

Most people infected by the SARS-CoV-2 virus which causes COVID-19 disease experience mild or no symptoms. Severe forms of the disease are often marked by a hyper-inflammatory response known as a cytokine storm. Thus, biomarker tests which can identify these patients and place them on the appropriate treatment regime at the earliest possible phase would help to improve outcomes. Here we describe an automated microarray-based immunoassay using the Fraunhofer lab-on-a-chip platform for analysis of C-reactive protein due to its role in the hyper-inflammatory response.

Impact of the SARS-CoV-2 pandemic on trauma care: a nationwide observational study.

PURPOSE: The SARS-CoV-2 pandemic severely disrupted society and the health care system. In addition to epidemiological changes, little is known about the pandemic's effects on the trauma care chain. Therefore, in addition to epidemiology and aetiology, this study aims to describe the impact of the SARS-CoV-2 pandemic on prehospital times, resource use and outcome. METHODS: A multicentre observational cohort study based on the Dutch Nationwide Trauma Registry was performed. Characteristics, resource usage, and outcomes of trauma patients treated at all trauma-receiving hospitals during the first (W1, March 12 through May 11) and second waves (W2, May 12 through September 23), as well as the interbellum period in between (INT, September 23 through December 31), were compared with those treated from the same periods in 2018 and 2019. RESULTS: The trauma caseload was reduced by 20% during the W1 period and 11% during the W2 period. The median length of stay was significantly shortened for hip fracture and major trauma patients (ISS ≥ 16). A 33% and 66% increase in the prevalence of minor self-harm-related injuries was recorded during the W1 and W2 periods, respectively, and a 36% increase in violence-related injuries was recorded during the INT. Mortality was significantly higher in the W1 (2.9% vs. 2.2%) and W2 (3.2% vs. 2.7%) periods. CONCLUSION: The imposed restrictions in response to the SARS-CoV-2 pandemic led to diminished numbers of acute trauma admissions in the Netherlands. The long-lasting pressing demand for resources, including ICU services, has negatively affected trauma care. Further caution is warranted regarding the increased incidence of injuries related to violence and self-harm.

Lateral flow-based nucleic acid detection of SARS-CoV-2 using enzymatic incorporation of biotin-labeled dUTP for POCT use.

The degree of detrimental effects inflicted on mankind by the COVID-19 pandemic increased the need to develop ASSURED (Affordable, Sensitive, Specific, User-friendly, Rapid and Robust, Equipment-free, and Deliverable) POCT (point of care testing) to overcome the current and any future pandemics. Much effort in research and development is currently advancing the progress to overcome the diagnostic pressure built up by emerging new pathogens. LAMP (loop-mediated isothermal amplification) is a well-researched isothermal technique for specific nucleic acid amplification which can be combined with a highly sensitive immunochromatographic readout via lateral flow assays (LFA). Here we discuss LAMP-LFA robustness, sensitivity, and specificity for SARS-CoV-2 N-gene detection in cDNA and clinical swab-extracted RNA samples. The LFA readout is designed to produce highly specific results by incorporation of biotin and FITC labels to 11-dUTP and LF (loop forming forward) primer, respectively. The LAMP-LFA assay was established using cDNA for N-gene with an accuracy of 95.65%. To validate the study, 82 SARS-CoV-2-positive RNA samples were tested. Reverse transcriptase (RT)-LAMP-LFA was positive for the RNA samples with an accuracy of 81.66%; SARS-CoV-2 viral RNA was detected by RT-LAMP-LFA for as low as CT-33. Our method reduced the detection time to 15 min and indicates therefore that RT-LAMP in combination with LFA represents a promising nucleic acid biosensing POCT platform that combines with smartphone based semi-quantitative data analysis.

Peptide epitope-imprinted polymer microarrays for selective protein recognition. Application for SARS-CoV-2 RBD protein.

We introduce a practically generic approach for the generation of epitope-imprinted polymer-based microarrays for protein recognition on surface plasmon resonance imaging (SPRi) chips. The SPRi platform allows the subsequent rapid screening of target binding kinetics in a multiplexed and label-free manner. The versatility of such microarrays, both as synthetic and screening platform, is demonstrated through developing highly affine molecularly imprinted polymers (MIPs) for the recognition of the receptor binding domain (RBD) of SARS-CoV-2 spike protein. A characteristic nonapeptide GFNCYFPLQ from the RBD and other control peptides were microspotted onto gold SPRi chips followed by the electrosynthesis of a polyscopoletin nanofilm to generate in one step MIP arrays. A single chip screening of essential synthesis parameters, including the surface density of the template peptide and its sequence led to MIPs with dissociation constants (K D) in the lower nanomolar range for RBD, which exceeds the affinity of RBD for its natural target, angiotensin-convertase 2 enzyme. Remarkably, the same MIPs bound SARS-CoV-2 virus like particles with even higher affinity along with excellent discrimination of influenza A (H3N2) virus. While MIPs prepared with a truncated heptapeptide template GFNCYFP showed only a slightly decreased affinity for RBD, a single mismatch in the amino acid sequence of the template, i.e. the substitution of the central cysteine with a serine, fully suppressed the RBD binding.

Peptide microarray-based analysis of antibody responses to SARS-CoV-2 identifies unique epitopes with potential for diagnostic test development.

Humoral immunity to the Severe Adult Respiratory Syndrome (SARS) Coronavirus (CoV)-2 is not fully understood yet but is a crucial factor of immune protection. The possibility of antibody cross-reactivity between SARS-CoV-2 and other human coronaviruses (HCoVs) would have important implications for immune protection but also for the development of specific diagnostic ELISA tests. Using peptide microarrays, n = 24 patient samples and n = 12 control samples were screened for antibodies against the entire SARS-CoV-2 proteome as well as the Spike (S), Nucleocapsid (N), VME1 (V), R1ab, and Protein 3a (AP3A) of the HCoV strains SARS, MERS, OC43, and 229E. While widespread cross-reactivity was revealed across several immunodominant regions of S and N, IgG binding to several SARS-CoV-2-derived peptides provided statistically significant discrimination between COVID-19 patients and controls. Selected target peptides may serve as capture antigens for future, highly COVID-19-specific diagnostic antibody tests.

Peptide microarray based detection of antibody responses against SARS-CoV-2 species-specific epitopes in spike and nucleocapsid proteins with potential for diagnostic test development (preprint)

Humoral immunity to the Severe Adult Respiratory Syndrome (SARS) Coronavirus (CoV)-2 is not well understood but may be a crucial factor of immune protection. The possibility of antibody cross-reactivity between SARS-CoV-2 and other human coronaviruses (HCoVs) would have important implications for immune protection but also for the development of specific diagnostic ELISA tests. Using peptide microarrays, n=24 patient samples and n=12 control samples were screened for antibodies against the entire SARS-CoV-2 proteome as well as the Spike (S), Nucleocapsid (N), VME1 (V), R1ab, and Protein 3a (AP3A) of the HCoV strains SARS, MERS, UC43 and 229E. While widespread cross-reactivity was revealed across several immune dominant regions of S and N, IgG binding to several SARS-CoV-2-derived peptides provided statistically significant discrimination between COVID-19 patients and controls. Selected target peptides may serve as capture antigens for future, highly COVID-19-specific diagnostic antibody tests.

Neurological emergencies associated with COVID-19: stroke and beyond.

Novel coronavirus disease (COVID-19) was declared a global pandemic on March 1, 2020. Neurological manifestations are now being reported worldwide, including emergent presentation with acute neurological changes as well as a comorbidity in hospitalized patients. There is limited knowledge on the neurologic manifestations of COVID-19 at present, with a wide array of neurological complications reported, ranging from ischemic stroke to acute demyelination and encephalitis. We report five cases of COVID-19 presenting to the ER with acute neurological symptoms, over the course of 1 month. This includes two cases of ischemic stroke, one with large-vessel occlusion and one with embolic infarcts. The remainders of the cases include acute tumefactive demyelination, isolated cytotoxic edema of the corpus callosum with subarachnoid hemorrhage, and posterior reversible encephalopathy syndrome (PRES).

A unifying structural and functional model of the coronavirus replication organelle: Tracking down RNA synthesis.

Zoonotic coronavirus (CoV) infections, such as those responsible for the current severe acute respiratory syndrome-CoV 2 (SARS-CoV-2) pandemic, cause grave international public health concern. In infected cells, the CoV RNA-synthesizing machinery associates with modified endoplasmic reticulum membranes that are transformed into the viral replication organelle (RO). Although double-membrane vesicles (DMVs) appear to be a pan-CoV RO element, studies to date describe an assortment of additional CoV-induced membrane structures. Despite much speculation, it remains unclear which RO element(s) accommodate viral RNA synthesis. Here we provide detailed 2D and 3D analyses of CoV ROs and show that diverse CoVs essentially induce the same membrane modifications, including the small open double-membrane spherules (DMSs) previously thought to be restricted to gamma- and delta-CoV infections and proposed as sites of replication. Metabolic labeling of newly synthesized viral RNA followed by quantitative electron microscopy (EM) autoradiography revealed abundant viral RNA synthesis associated with DMVs in cells infected with the beta-CoVs Middle East respiratory syndrome-CoV (MERS-CoV) and SARS-CoV and the gamma-CoV infectious bronchitis virus. RNA synthesis could not be linked to DMSs or any other cellular or virus-induced structure. Our results provide a unifying model of the CoV RO and clearly establish DMVs as the central hub for viral RNA synthesis and a potential drug target in CoV infection.

A unifying structural and functional model of the coronavirus replication organelle: tracking down RNA synthesis (preprint)

Zoonotic coronavirus (CoV) infections, like those responsible for the current SARS-CoV-2 epidemic, cause grave international public health concern. In infected cells, the CoV RNA-synthesizing machinery associates with modified endoplasmic reticulum membranes that are transformed into the viral replication organelle (RO). While double-membrane vesicles (DMVs) appear to be a pan -coronavirus RO element, studies to date describe an assortment of additional coronavirus-induced membrane structures. Despite much speculation, it remains unclear which RO element(s) accommodate viral RNA synthesis. Here we provide detailed 2D and 3D analyses of CoV ROs and show that diverse CoVs essentially induce the same membrane modifications, including the small open double-membrane spherules (DMSs) previously thought to be restricted to gamma- and delta-CoV infections and proposed as sites of replication. Metabolic labelling of newly-synthesized viral RNA followed by quantitative EM autoradiography revealed abundant viral RNA synthesis associated with DMVs in cells infected with the beta-CoVs MERS-CoV and SARS-CoV, and the gamma-CoV infectious bronchitis virus. RNA synthesis could not be linked to DMSs or any other cellular or virus-induced structure. Our results provide a unifying model of the CoV RO and clearly establish DMVs as the central hub for viral RNA synthesis and a potential drug target in coronavirus infection.