Interlaboratory evaluation of Mucorales PCR assays for testing serum specimens: A study by the fungal PCR Initiative and the Modimucor study group.

Interlaboratory evaluations of Mucorales qPCR assays were developed to assess the reproducibility and performance of methods currently used. The participants comprised 12 laboratories from French university hospitals (nine of them participating in the Modimucor study) and 11 laboratories participating in the Fungal PCR Initiative. For panel 1, three sera were each spiked with DNA from three different species (Rhizomucor pusillus, Lichtheimia corymbifera, Rhizopus oryzae). For panel 2, six sera with three concentrations of R. pusillus and L. corymbifera (1, 10, and 100 genomes/ml) were prepared. Each panel included a blind negative-control serum. A form was distributed with each panel to collect results and required technical information, including DNA extraction method, sample volume used, DNA elution volume, qPCR method, qPCR template input volume, qPCR total reaction volume, qPCR platform, and qPCR reagents used. For panel 1, assessing 18 different protocols, qualitative results (positive or negative) were correct in 97% of cases (70/72). A very low interlaboratory variability in Cq values (SD = 1.89 cycles) were observed. For panel 2 assessing 26 different protocols, the detection rates were high (77-100%) for 5/6 of spiked serum. There was a significant association between the qPCR platform and performance. However, certain technical steps and optimal combinations of factors may also impact performance. The good reproducibility and performance demonstrated in this study support the use of Mucorales qPCR as part of the diagnostic strategy for mucormycosis.

Antimicrobial efficacy and inactivation kinetics of a novel LED-based UV-irradiation technology.

BACKGROUND: Ultraviolet (UV)-light-emitting diodes (UV-LEDs) are energy efficient and of special interest for the inactivation of micro-organisms. In the context of the coronavirus disease 2019 pandemic, novel UV technologies can offer a powerful alternative for effective infection prevention and control. METHODS: This study assessed the antimicrobial efficacy of UV-C LEDs on Escherichia coli, Pseudomonas fluorescens and Listeria innocua, as well as severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), human immunodeficiency virus-1 (HIV-1) and murine norovirus (MNV), dried on inanimate surfaces, based on European Standard EN 17272. RESULTS: This study found 90% inactivation rates for the tested bacteria at mean UV-C doses, averaged over all three investigated UV-C wavelengths, of 1.7 mJ/cm2 for E. coli, 1.9 mJ/cm2 for P. fluorescens and 1.5 mJ/cm2 for L. innocua. For the tested viruses, UV doses <15 mJ/cm2 resulted in 90% inactivation at wavelengths of 255 and 265 nm. Exposure of viruses to longer UV wavelengths, such as 275 and 285 nm, required much higher doses (up to 120 mJ/cm2) for inactivation. Regarding inactivation, non-enveloped MNV required much higher UV doses for all tested wavelengths compared with SARS-CoV-2 or HIV-1. CONCLUSION: Overall, the results support the use of LEDs emitting at shorter wavelengths of the UV-C spectrum to inactivate bacteria as well as enveloped and non-enveloped viruses by exposure to the appropriate UV dose. However, low availability and excessive production costs of shortwave UV-C LEDs restricts implementation at present, and supports the use of longwave UV-C LEDs in combination with higher irradiation doses.

ColdZyme Maintains Integrity in SARS-CoV-2-Infected Airway Epithelia.

SARS-CoV-2 infection causing the COVID-19 pandemic calls for immediate interventions to avoid viral transmission, disease progression, and subsequent excessive inflammation and tissue destruction. Primary normal human bronchial epithelial cells are among the first targets of SARS-CoV-2 infection. Here, we show that ColdZyme medical device mouth spray efficiently protected against virus entry, excessive inflammation, and tissue damage. Applying ColdZyme to fully differentiated, polarized human epithelium cultured at an air-liquid interphase (ALI) completely blocked binding of SARS-CoV-2 and increased local complement activation mediated by the virus as well as productive infection of the tissue model. While SARS-CoV-2 infection resulted in exaggerated intracellular complement activation immediately following infection and a drop in transepithelial resistance, these parameters were bypassed by single pretreatment of the tissues with ColdZyme mouth spray. Crucially, our study highlights the importance of testing already evaluated and safe drugs such as ColdZyme mouth spray for maintaining epithelial integrity and hindering SARS-CoV-2 entry within standardized three-dimensional (3D) in vitro models mimicking the in vivo human airway epithelium.IMPORTANCE Although our understanding of COVID-19 continuously progresses, essential questions regarding prophylaxis and treatment remain open. A hallmark of severe SARS-CoV-2 infection is a hitherto-undescribed mechanism leading to excessive inflammation and tissue destruction associated with enhanced pathogenicity and mortality. To tackle the problem at the source, transfer of SARS-CoV-2, subsequent binding, infection, and inflammatory responses have to be avoided. In this study, we used fully differentiated, mucus-producing, and ciliated human airway epithelial cultures to test the efficacy of ColdZyme medical device mouth spray in terms of protection from SARS-CoV-2 infection. Importantly, we found that pretreatment of the in vitro airway cultures using ColdZyme mouth spray resulted in significantly shielding the epithelial integrity, hindering virus binding and infection, and blocking excessive intrinsic complement activation within the airway cultures. Our in vitro data suggest that ColdZyme mouth spray may have an impact in prevention of COVID-19.

Cluedo - Source identification in a case of septicemia fatality caused by Capnocytophaga canimorsus.

The Gram negative pathogen Capnocytophaga canimorsus is a frequent commensal in the oral cavity of cats and dogs. Although the bacterium is generally considered harmless, infections in humans can occur displaying a broad spectrum of clinical syndromes. This makes a clinical diagnosis difficult. The patient in the present case was 67 years old and presented to the emergency room (ER) with pain in the upper right abdomen and clinical signs of a feverish infection. The only noticeable record in the patient´s medical history was a splenectomy in childhood. The anamnesis revealed that the patient was the owner of two dogs. After a suspected diagnosis of sepsis the patient was transferred to the intensive care unit (ICU), where his medical condition deteriorated rapidly. Despite intensive care measures as well as the fast initialization of a broad-spectrum antibiotic therapy, the patient died 37 h after his presentation in the ER. The search for the causative pathogen turned out to be challenging. Eventually, molecular biological methods assisted in solving the puzzle. It could be demonstrated that the pathogen, found in the patient´s blood, was also present in one of his dogs' oral cavity.

Combined effects of exposure to engineered silver nanoparticles and the water-soluble fraction of crude oil in the marine copepod Calanus finmarchicus.

While it is likely that ENPs may occur together with other contaminants in nature, the combined effects of exposure to both ENPs and environmental contaminants are not studied sufficiently. In this study, we investigated the acute and sublethal toxicity of PVP coated silver nanoparticles (AgNP) and ionic silver (Ag+; administered as AgNO3) to the marine copepod Calanus finmarchicus. We further studied effects of single exposures to AgNPs (nominal concentrations: low 15 µg L-1 NPL, high 150 µg L-1 NPH) or Ag+ (60 µg L-1), and effects of co-exposure to AgNPs, Ag+ and the water-soluble fraction (WSF; 100 µg L-1) of a crude oil (AgNP + WSF; Ag++WSF). The gene expression and the activity of antioxidant defense enzymes SOD, CAT and GST, as well as the gene expression of HSP90 and CYP330A1 were determined as sublethal endpoints. Results show that Ag+ was more acutely toxic compared to AgNPs, with 96 h LC50 concentrations of 403 µg L-1 for AgNPs, and 147 µg L-1 for Ag+. Organismal uptake of Ag following exposure was similar for AgNP and Ag+, and was not significantly different when co-exposed to WSF. Exposure to AgNPs alone caused increases in gene expressions of GST and SOD, whereas WSF exposure caused an induction in SOD. Responses in enzyme activities were generally low, with significant effects observed only on SOD activity in NPL and WSF exposures and on GST activity in NPL and NPH exposures. Combined AgNP and WSF exposures caused slightly altered responses in expression of SOD, GST and CYP330A1 genes compared to the single exposures of either AgNPs or WSF. However, there was no clear pattern of cumulative effects caused by co-exposures of AgNPs and WSF. The present study indicates that the exposure to AgNPs, Ag+, and to a lesser degree WSF cause an oxidative stress response in C. finmarchicus, which was slightly, but mostly not significantly altered in combined exposures. This indicated that the combined effects between Ag and WSF are relatively limited, at least with regard to oxidative stress.

High-quality endoscope reprocessing decreases endoscope contamination.

OBJECTIVES: Several outbreaks of severe infections due to contamination of gastrointestinal (GI) endoscopes, mainly duodenoscopes, have been described. The rate of microbial endoscope contamination varies dramatically in literature. The aim of this multicentre prospective study was to evaluate the hygiene quality of endoscopes and automated endoscope reprocessors (AERs) in Tyrol/Austria. METHODS: In 2015 and 2016, a total of 463 GI endoscopes and 105 AERs from 29 endoscopy centres were analysed by a routine (R) and a combined routine and advanced (CRA) sampling procedure and investigated for microbial contamination by culture-based and molecular-based analyses. RESULTS: The contamination rate of GI endoscopes was 1.3%-4.6% according to the national guideline, suggesting that 1.3-4.6 patients out of 100 could have had contacts with hygiene-relevant microorganisms through an endoscopic intervention. Comparison of R and CRA sampling showed 1.8% of R versus 4.6% of CRA failing the acceptance criteria in phase I and 1.3% of R versus 3.0% of CRA samples failing in phase II. The most commonly identified indicator organism was Pseudomonas spp., mainly Pseudomonas oleovorans. None of the tested viruses were detected in 40 samples. While AERs in phase I failed (n = 9, 17.6%) mainly due to technical faults, phase II revealed lapses (n = 6, 11.5%) only on account of microbial contamination of the last rinsing water, mainly with Pseudomonas spp. CONCLUSIONS: In the present study the contamination rate of endoscopes was low compared with results from other European countries, possibly due to the high quality of endoscope reprocessing, drying and storage.

Cross-sectional point prevalence survey to study the environmental contamination of nosocomial pathogens in intensive care units under real-life conditions.

BACKGROUND: In intensive care units (ICUs), inanimate surfaces and equipment may be contaminated by nosocomial pathogens, including multi-drug-resistant micro-organisms. AIMS: To assess the degree of environmental contamination close to and distant from patients, and contamination of healthcare workers' (HCWs) hands with nosocomial pathogens under real-life conditions and to investigate potential transmission events. METHODS: Over the course of three weeks, agar contact samples were taken close to and distant from patient areas and from HCWs' hands in eight ICUs of a tertiary care hospital in Innsbruck, Austria. Each ICU was visited once without announcement. Species identification and antimicrobial susceptibility testing were performed according to standard methods, and corresponding strains from patient, environment and hand samples were genotyped using pulsed-field gel electrophoresis. FINDINGS: Among 523 samples, HCWs' hands were most frequently contaminated with potentially pathogenic bacteria (15.2%), followed by areas close to patients (10.9%) and areas distant from patients (9.1%). Gram-positive bacteria were identified most often (67.8%), with Enterococcus spp. being the most prevalent species (70% vancomycin sensitive and 30% vancomycin resistant) followed by Staphylococcus aureus, of which 64% were classified as meticillin-resistant Staphylococcus aureus. Molecular typing documented identical strains among patient, environment and hand isolates. CONCLUSION: This study found widespread contamination of the ICU environment with clinically relevant pathogens, including multi-drug-resistant micro-organisms, despite cleaning and disinfection. The bioburden might not be restricted to areas close to patients. The role of extended environmental disinfection of areas distant from patients in order to improve infection prevention needs further discussion.

Fast-track development of an in vitro 3D lung/immune cell model to study Aspergillus infections.

To study interactions of airborne pathogens, e.g. Aspergillus (A.) fumigatus with upper and lower respiratory tract epithelial and immune cells, we set up a perfused 3D human bronchial and small airway epithelial cell system. Culturing of normal human bronchial or small airway epithelial (NHBE, SAE) cells under air liquid interphase (ALI) and perfusion resulted in a significantly accelerated development of the lung epithelia associated with higher ciliogenesis, cilia movement, mucus-production and improved barrier function compared to growth under static conditions. Following the accelerated differentiation under perfusion, epithelial cells were transferred into static conditions and antigen-presenting cells (APCs) added to study their functionality upon infection with A. fumigatus. Fungi were efficiently sensed by apically applied macrophages or basolaterally adhered dendritic cells (DCs), as illustrated by phagocytosis, maturation and migration characteristics. We illustrate here that perfusion greatly improves differentiation of primary epithelial cells in vitro, which enables fast-track addition of primary immune cells and significant shortening of experimental procedures. Additionally, co-cultured primary DCs and macrophages were fully functional and fulfilled their tasks of sensing and sampling fungal pathogens present at the apical surface of epithelial cells, thereby promoting novel possibilities to study airborne infections under conditions mimicking the in vivo situation.

The lipid peroxidation product 4-hydroxynonenal is formed by--and is able to attract--rat neutrophils in vivo.

4-Hydroxynonenal (HNE), a major aldehydic product of lipid peroxidation, is a chemoattractant for neutrophilic polymorphonuclear granulocytes in vitro. The question was studied, whether HNE is formed during the ingress of neutrophils in the Sephadex model of inflammation. The polydextrane Sephadex G-200, which causes an acute aseptic traumatic inflammation, was injected subcutaneously into rats. The implants were excised 6-36 hours later, and the neutrophils separated from the exsudate by centrifugation. After extraction with dichloromethane HNE was identified in the exsudate by non-derivative reversed phase HPLC in combination with on-line uv-spectroscopy. The concentration of HNE in the inflammatory focus did not correlate with the number of neutrophils present. While the peak of HNE coincided with the time point of the highest turnover rate of neutrophils (0.13 microM at 6 hrs after implantation), the highest number of neutrophils (about 100 million cells) occurred not earlier than 18 hrs later (24 hrs after onset of inflammation). When neutrophils were isolated from the inflammatory focus and stimulated with Zymosan, they were able to produce HNE in vitro depending on the time of isolation. The highest production of HNE (0.17 microM) by phagocyting neutrophils was observed at the shortest inflammation time studied (3 hrs). In order to compare these results with the oxidative burst of neutrophils the formation of superoxide was also measured by the cytochrome c reduction assay in vitro. The maximum of the production rate of superoxide anion was observed at the same inflammation time (6 hrs), when the HNE maximum occurred.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantification of midodrine and its active metabolite in plasma using a high performance liquid chromatography column switching technique.

An automated column-switching HPLC system is described for the simultaneous determination of midodrine, an alpha-adrenergic stimulating drug, and its active metabolite, ST-1059. Serum or plasma (850 microliters) is directly injected onto a RP18 (30 micrograms particle size) pre-column (9 x 4 mm ID) which acts as an on-line liquid-solid extractor and analyte enrichment system. The injection is followed by washing steps. The fraction containing the analytes is transferred onto an analytical RP18 column via step gradient elution where the final analysis is performed. Fluorescence detection is used (lambda ex 290 nm and lambda em 322 nm), and method detection limits of 0.8 ng/mL plasma were reached. These were sufficiently low to determine the plasma concentration-time profiles for both compounds following oral administration of 2.5 mg and 5 mg midodrine hydrochloride. The assay in serum or plasma was linear in the range of 1 to 15 ng analyte/mL, the recovery was greater than 95%, and the reproducibility was sufficient. The assay was rugged and was maintained by routinely changing the home-made, dry packed pre-column every 20th serum injection.

[Analysis of residues of the fungicide cymoxanil in grapes using multicolumn HPLC]. / Rückstandsbestimmung des Fungicides Cymoxanil auf Weintrauben mittels Mehrsäulen-HPLC.

Residues of the contact fungicide Cymoxanil in grapes were determined by a multidimensional multicolumn high pressure liquid chromatography technique (MC-HPLC). The sample pretreatment is a simple water extraction. Further clean-up and trace enrichment of an aliquot of the aqueous acidic sample solution is performed on-line via an automatized microprocessor controlled MC-HPLC system. In the range of 0.05-2.0 mg/kg grapes good linearity is achieved. Recoveries of Cymoxanil added to untreated grapes range between 70% and 80% at 0.05 and 2.0 mg/kg values, respectively, with a reproductibility of s +/- 3% at 0.2 mg/kg (n = 5). The total time for analysis is approximately 30 min.