Analysis of Inactivation of SARS-CoV-2 by Specimen Transport Media, Nucleic Acid Extraction Reagents, Detergents, and Fixatives.

The COVID-19 pandemic has necessitated a multifaceted rapid response by the scientific community, bringing researchers, health officials, and industry together to address the ongoing public health emergency. To meet this challenge, participants need an informed approach for working safely with the etiological agent, the novel human coronavirus SARS-CoV-2. Work with infectious SARS-CoV-2 is currently restricted to high-containment laboratories, but material can be handled at a lower containment level after inactivation. Given the wide array of inactivation reagents that are being used in laboratories during this pandemic, it is vital that their effectiveness is thoroughly investigated. Here, we evaluated a total of 23 commercial reagents designed for clinical sample transportation, nucleic acid extraction, and virus inactivation for their ability to inactivate SARS-CoV-2, as well as seven other common chemicals, including detergents and fixatives. As part of this study, we have also tested five filtration matrices for their effectiveness at removing the cytotoxic elements of each reagent, permitting accurate determination of levels of infectious virus remaining following treatment. In addition to providing critical data informing inactivation methods and risk assessments for diagnostic and research laboratories working with SARS-CoV-2, these data provide a framework for other laboratories to validate their inactivation processes and to guide similar studies for other pathogens.

Laboratory Findings, Compassionate Use of Favipiravir, and Outcome in Patients With Ebola Virus Disease, Guinea, 2015-A Retrospective Observational Study.

BACKGROUND: In 2015, the laboratory at the Ebola treatment center in Coyah, Guinea, confirmed Ebola virus disease (EVD) in 286 patients. The cycle threshold (Ct) of an Ebola virus-specific reverse transcription-polymerase chain reaction assay and 13 blood chemistry parameters were measured on admission and during hospitalization. Favipiravir treatment was offered to patients with EVD on a compassionate-use basis. METHODS: To reduce biases in the raw field data, we carefully selected 163 of 286 patients with EVD for a retrospective study to assess associations between potential risk factors, alterations in blood chemistry findings, favipiravir treatment, and outcome. RESULTS: The case-fatality rate in favipiravir-treated patients was lower than in untreated patients (42.5% [31 of 73] vs 57.8% [52 of 90]; P = .053 by univariate analysis). In multivariate regression analysis, a higher Ct and a younger age were associated with survival (P < .001), while favipiravir treatment showed no statistically significant effect (P = .11). However, Kaplan-Meier analysis indicated a longer survival time in the favipiravir-treated group (P = .015). The study also showed characteristic changes in blood chemistry findings in patients who died, compared with survivors. CONCLUSIONS: Consistent with the JIKI trial, this retrospective study revealed a trend toward improved survival in favipiravir- treated patients; however, the effect of treatment was not statistically significant, except for its influence on survival time.

The RNA Helicase DDX6 Associates with RIG-I to Augment Induction of Antiviral Signaling.

Virus infections induce sensitive antiviral responses within the host cell. The RNA helicase retinoic acid-inducible gene I (RIG-I) is a key sensor of influenza virus RNA that induces the expression of antiviral type I interferons. Recent evidence suggests a complex pattern of RIG-I regulation involving multiple interactions and cellular sites. In an approach employing affinity purification and quantitative mass spectrometry, we identified proteins with increased binding to RIG-I in response to influenza B virus infection. Among them was the RIG-I related RNA helicase DEAD box helicase 6 (DDX6), a known component of cytoplasmic mRNA-ribonucleoprotein (mRNP) granules like P-bodies and stress granules (SGs). RIG-I and DDX6 both localized to the cytosol and were detected in virus-induced SGs. Coimmunoprecipitation assays detected a basal level of complexes harboring RIG-I and DDX6 that increased after infection. Functionally, DDX6 augmented RIG-I mediated induction of interferon (IFN)-ß expression. Notably, DDX6 was found to bind viral RNA capable to stimulate RIG-I. These findings imply a novel function for DDX6 as an RNA co-sensor and signaling enhancer for RIG-I.

Convalescent human plasma candidate reference materials protect against Crimean-Congo haemorrhagic fever virus (CCHFV) challenge in an A129 mouse model.

Crimean-Congo Haemorrhagic Fever Virus (CCHFV) is spread by infected ticks or direct contact with blood, tissues and fluids from infected patients or livestock. Infection with CCHFV causes severe haemorrhagic fever in humans which is fatal in up to 83 % of cases. CCHFV is listed as a priority pathogen by the World Health Organization (WHO) and there are currently no widely-approved vaccines. Defining a serological correlate of protection against CCHFV infection would support the development of vaccines by providing a 'target threshold' for pre-clinical and clinical immunogenicity studies to achieve in subjects and potentially obviate the need for in vivo protection studies. We therefore sought to establish titratable protection against CCHFV using pooled human convalescent plasma, in a mouse model. Convalescent plasma collected from seven individuals with a known previous CCHFV virus infection were characterised using binding antibody and neutralisation assays. All plasma recognised nucleoprotein and the Gc glycoprotein, but some had a lower Gn glycoprotein response by ELISA. Pooled plasma and two individual donations from convalescent donors were administered intraperitoneally to A129 mice 24 h prior to intradermal challenge with CCHFV (strain IbAr10200). A partial protective effect was observed with all three convalescent plasmas characterised by longer survival post-challenge and reduced clinical score. These protective responses were titratable. Further characterisation of the serological reactivities within these samples will establish their value as reference materials to support assay harmonisation and accelerate vaccine development for CCHFV.

Virucidal efficacy of guanidine-free inactivants and rapid test buffers against SARS-CoV-2.

A pathogen inactivation step during collection or processing of clinical samples has the potential to reduce infectious risks associated with diagnostic procedures. It is essential that these inactivation methods are demonstrated to be effective, particularly for non-traditional inactivation reagents or for commercial products where the chemical composition is undisclosed. This study assessed inactivation effectiveness of twenty-four next-generation (guanidine-free) nucleic acid extraction lysis buffers and twelve rapid antigen test buffers against SARS-CoV-2, the causative agent of COVID-19. These data have significant safety implications for SARS-CoV-2 diagnostic testing and support the design and evidence-based risk assessment of these procedures.

"Kankasha" in Kassala: A prospective observational cohort study of the clinical characteristics, epidemiology, genetic origin, and chronic impact of the 2018 epidemic of Chikungunya virus infection in Kassala, Sudan.

BACKGROUND: The public health impact of Chikungunya virus (CHIKV) is often underestimated. Usually considered a mild condition of short duration, recent outbreaks have reported greater incidence of severe illness, fatality, and longer-term disability. In 2018/19, Eastern Sudan experienced the largest epidemic of CHIKV in Africa to date, affecting an estimated 487,600 people. Known locally as Kankasha, this study examines clinical characteristics, risk factors, and phylogenetics of the epidemic in Kassala City. METHODOLOGY/PRINCIPAL FINDINGS: A prospective cohort of 102 adults and 40 children presenting with chikungunya-like illness were enrolled at Kassala Teaching Hospital in October 2018. Clinical information, socio-demographic data, and sera samples were analysed to confirm diagnosis, characterise illness, and identify viral strain. CHIKV infection was confirmed by real-time reverse transcription-PCR in 84.5% (120/142) of participants. Nine (7.5%) CHIKV-positive participants had concurrent Dengue virus (DENV) infection; 34/118 participants (28.8%) had a positive Rapid Diagnostic Test for Plasmodium falciparum; six (5.0%) had haemorrhagic symptoms including two children with life-threatening bleeding. One CHIKV-positive participant died with acute renal injury. Age was not associated with severity of illness although CHIKV-infected participants were younger (p = 0.003). Two to four months post-illness, 63% of adults available for follow-up (30) were still experiencing arthralgia in one or more joints, and 11% remained moderately disabled on Rapid3 assessment. Phylogenetic analysis showed all CHIKV sequences from this study belonged to a single clade within the Indian Ocean Lineage (IOL) of the East/Central/South African (ECSA) genotype. History of contact with an infected person was the only factor associated with infection (p = 0.01), and likely related to being in the same vector environment. CONCLUSIONS/SIGNIFICANCE: Vulnerability to CHIKV remains in Kassala and elsewhere in Sudan due to widespread Aedes aegypti presence and mosquito-fostering household water storage methods. This study highlights the importance of increasing awareness of the severity and impact of CHIKV outbreaks, and the need for urgent actions to reduce transmission risk in households.

Welcome, Orientation, Language Training: a project at the Charité for new international medical students.

Objective: A comprehensive, integrated support programme for new international students of medicine has been developed, implemented and evaluated at the Charité. The objectives of the programme were improved social integration, orientation on the study program and Charité campus, as well as qualification in medical specialist language. Project outline: The "Charité Orientation Module for International Students" (ChOIS) was designed by a working group with a variety of expertise in the field of international students. The programme has three stages: Recruitment (specific invitation on matriculation); Orientation week before semester start; and Parallel events during the first semester. ChoOIS was piloted in the Winter Semester 2015/16 and, following evaluation, continued in a modified form in the Summer Semester 2016. Key features were: Welcome and social integration by faculty welcome-events and student group activities; Orientation on the study program, on teaching infrastructures at the Charité and on student life in Berlin by senior medical students; and Training in language for medical communication and bedside teaching by professional lecturers. Results: Results of evaluations conducted after the orientation weeks, at the end of the semester and retrospectively in the 3rd semester produced high approval ratings of the individual features of the ChOIS-programme and of the programme as a whole by participating students. Discussion: A comprehensive, integrated support programme for new international students of medicine has been developed and implemented. The ChOIS-programme can serve as a practice model to guide other medical faculties. In future, a programme that goes beyond the start of the course and includes more involvement by senior students would be desirable.

In vitro-cultivation of human periosteum derived cells in bioresorbable polymer-TCP-composites.

Bone replacement materials for reconstruction of bone defects must be biocompatible and biodegradable and must have osteoconductive or even osteogenic potential. Ideally, their shape should also be adaptable to the defect and they should possess long-term adaptability to the biomechanical situation at the implantation site. Human mesenchymal stem cells of the cambium layer of the periosteum were cultivated, placed in a fibrin suspension on a preformed carrier structure (PGLA polymer + beta-TCP), and cultivated under conditions of osteogenic differentiation. After 10, 20, 30, and 40 days, histological examination was performed, alkaline phosphatase activity and levels of osteocalcin, DNA, and collagen were determined, and the influence of addition of TGF-beta1 at a concentration of 5 ng/ml to the culture medium was investigated. Demonstration of bone-specific marker proteins indicated that the in vitro combination of mesenchymal stem cells, PGLA polymer, beta-TCP, and fibrin resulted in de-novo synthesis of human preosseous tissue, while addition of TGF-beta1 resulted in greater new bone formation with significantly higher concentrations of marker proteins. Histological examination showed the presence of newly formed bone at the surface of the implant. As compared with the use of structured TCP or hydroxyapatite implants as in earlier works, use of a combination of autologous cell material, PGLA polymer, and beta-TCP results in a malleable, vital implant that is adaptable to the bone defect. This combination thus may represent a new option for the treatment of bone defects.

Optimization of bone engineering by means of growth factors in a three-dimensional matrix.

The direction and acceleration of differentiation by administering growth factors is one of the ways of optimizing bone engineering. The present study considered the influence of the growth factors factor XIII, TGF-beta 1, and b-FGF on the proliferation and osteogenic differentiation of porcine periosteal cells in a three-dimensional carrier matrix (bead), consisting of a fibrin-alginate-hydroxyapatite composite. F XIII, TGF-beta 1, and b-FGF were added to the culture medium of monolayer culture and fibrin beads in different concentrations. The monolayer culture was assessed on the basis of cell counts, while DNA, osteocalcin, osteonectin, and collagen content and alkaline phosphatase activity were determined, and microscopic and immunohistologic evaluations were performed for the beads. In the monolayer, the addition of b-FGF led to a significantly shorter time up to the confluence of the cells. In the bead, cell proliferation was accelerated by b-FGF and TGF-beta 1. With regard to alkaline phosphatase activity, factor XIII led to significantly higher values, while b-FGF and TGF-beta 1 resulted in lower activities. Osteocalcin content was significantly increased by the application of b-FGF. For the osteonectin content the addition of growth factors did not produce any changes. The application of TGF-beta 1 during the monolayer culture significantly increased the primary collagen content of the beads. The administration of different growth factors opens up new ways of optimizing cell growth in vitro and of directing the osteogenic differentiation of periosteal cells, without one universally applicable factor having been demonstrated. It will be the task of further studies to analyze the interaction of individual factors and the chronologic dependency of the action, on the way to in vitro bone generation.

Iridoid and phenolic glycosides from Wulfenia carinthiaca.

Two new phenylpropanoid glycosides (2'-O-acetylplantamajoside and 2'-O, 6"-O-diacetylplantamajoside), a new iridoid glycoside (10-O-(cinnamoyl)-6'-O-(desacetylalpinosidyl)-catalpol), the two known iridoid glycosides globularin and isoscrophularioside, and the known phenylpropanoid glycoside platamajoside were isolated from the methanolic extract of the underground parts of Wulfenia carinthiaca. Structure elucidations were based on high-resolution mass spectrometry and extensive 1-D and 2-D NMR spectroscopy.

Wulfenia carinthiaca Jacq., antioxidant and pharmacological activities.

Relative antioxidant activities of a methanolic extract of three phenylpropanoid glycosides and three iridoid glycosides from Wulfenia carinthiaca were evaluated using the Briggs-Rauscher (BR) reaction method. This method is based on the inhibitory effects by antioxidants on oscillations of the BR reaction. The total extract showed a certain antioxidant activity with respect to resorcinol chosen as standard. The three phenylpropanoid glycosides showed a very high relative antioxidant activity while iridoid glycosides had practically no activity. These experimental results were confirmed by empirical calculations based on the BDE (Bond Dissociation Enthalpy) theory. The total phenolic content was also measured for the phenylpropanoid glycosides using the Folin-Ciocalteu reagent. The obtained values as gallic acid equivalents were in perfect agreement with the relative antioxidant activities. From a pharmacological point of view the results obtained demonstrate that the methanolic extract of W. carinthiaca have antinociceptive and antiedematogenic effects in the different models adopted. The plant extract produced a significant inhibition, dose related, of the rat paw edema induced by carrageenin. The anti-inflammatory activity is probably due to the phenylpropanoid compounds present in the plant. The histological sections of paw tissue in animals treated with Wulfenia carinthiaca extract confirmed the anti-inflammatory effects. The results of the antinociceptive assay indicated a significant reduction on the number of abdominal writhes of mice, induced by acetic acid.

Chondrocytes suspended in modified fibrin glue for vocal fold augmentation: An in vitro study in a porcine larynx model.

BACKGROUND: Injection laryngoplasty is an option for treatment of dysphonia following vocal fold paralysis. Modified fibrin glue with suspended chondrocytes might be a favorable cell-based material for permanent vocal fold medialization. METHODS: We compared fibrin glue with suspended chondrocytes to collagen and hyaluronic acid gels concerning alteration of vocal fold vibration and correct intralaryngeal placement after intralaryngeal injection into porcine larynges. Viscoelastic properties of the materials were analyzed by means of a parallel plate rheometer. RESULTS: Fibrin glue with cells was comparable to collagen and hyaluronic acid with respect to amplitudes, symmetry, and periodicity of vocal fold vibration. Application and positioning of fibrin glue with suspended chondrocytes were technically undemanding and comparable with controls. Complex stress modulus of fibrin glue with suspended cells was comparable to that of collagen gel. CONCLUSIONS: Fibrin glue with suspended chondrocytes seems suitable for the indication of injection laryngoplasty and holds promise for permanent vocal fold medialization.