Distinct and dynamic activation profiles of circulating dendritic cells and monocytes in mild COVID-19 and after yellow fever vaccination.

Dysregulation of the myeloid cell compartment is a feature of severe disease in hospitalized COVID-19 patients. Here, we investigated the response of circulating dendritic cell (DC) and monocyte subpopulations in SARS-CoV-2 infected outpatients with mild disease and compared it to the response of healthy individuals to yellow fever vaccine virus YF17D as a model of a well-coordinated response to viral infection. In SARS-CoV-2-infected outpatients circulating DCs were persistently reduced for several weeks whereas after YF17D vaccination DC numbers were decreased temporarily and rapidly replenished by increased proliferation until 14 days after vaccination. The majority of COVID-19 outpatients showed high expression of CD86 and PD-L1 in monocytes and DCs early on, resembling the dynamic after YF17D vaccination. In a subgroup of patients, low CD86 and high PD-L1 expression were detected in monocytes and DCs coinciding with symptoms, higher age, and lower lymphocyte counts. This phenotype was similar to that observed in severely ill COVID-19 patients, but less pronounced. Thus, prolonged reduction and dysregulated activation of blood DCs and monocytes were seen in a subgroup of symptomatic non-hospitalized COVID-19 patients while a transient coordinated activation was characteristic for the majority of patients with mild COVID-19 and the response to YF17D vaccination.

Refractivity corrected distance measurement using the intermode beats derived from a supercontinuum.

Simultaneous distance measurements on two or more optical wavelengths enable dispersion-based correction of deviations that result from insufficient knowledge of the refractive index along the signal propagation path. We demonstrate a supercontinuum-based approach for highly accurate distance measurements suitable for such an inline refractivity compensation. The distance is estimated from the phase delay observations on the intermode beats. We use a supercontinuum (SC) coherently broadened from a 780 nm frequency comb and spanning the spectral range of 570-970 nm. Experiments are performed on the 590 and 890 nm wavelength bands filtered from the SC spectrum. Results show distance measurements with standard deviations of around 0.01 mm at 50 m, and a distance-dependent component below 0.2 ppm on the individual spectral bands. Distance residuals compared to a reference interferometer are on the order of 0.1 ppm for displacements up to 50 m. Controlled pressure-induced refractivity variations are created over a length of 15 m, resulting in an optical path length change of 0.4 mm. Using the two-color method, we demonstrate refractivity-corrected distance measurement with a standard deviation of around 0.08 mm for a 60 s averaging window. The current experimental configuration can be easily extended to distance measurements on more than two wavelengths. The results highlight its potential for practical long-distance measurements through inline refractivity compensation.

Impact of the exposure of sublethal dose of mosquito coil on the development of insecticide resistance in Aedes aegypti (L.) (Diptera: Culicidae).

Mosquito coil is commonly used in many African households for protection against mosquito bites. The coil usually has semi-volatile pyrethroids as an active ingredient, which usually diffuse across open space, and the cloud either kills mosquitoes that are exposed, or mosquitoes can be exposed to sublethal doses of the insecticides. This study was conducted to assess the impact of sublethal doses of mosquito coil on the development of insecticide resistance in Aedes aegypti, a major vector for dengue fever and several other arboviral diseases. A laboratory colony of Ae. aegypti was exposed to sublethal doses of a meperfluthrin-based mosquito coil in a Peet-Grady chamber once per generation for 16 generations. The susceptibility of the exposed colony to a diagnostic dose of the mosquito coil as well as to three other insecticides was determined. Three different kdr mutations and five enzyme activities were evaluated in both the exposed and control colonies. After 16 generations of sublethal exposure to mosquito coils, the full diagnostic dose of the coil caused 68% mortality to the exposed colony compared to 100% mortality in the control colony. Mortality caused by deltamethrin (0.05%) was also significantly lower in the exposed colony. The frequency of 1016I kdr mutation as well as MFO and alpha esterase activities were higher in the exposed colony compared to the control colony. This study provides evidence of the development of pyrethroid resistance in an Ae. aegypti population due to sublethal exposure to mosquito coil for 16 generations. Given the large-scale use of mosquito coils in many African households, its role as a pyrethroid resistance selection source should be taken into consideration when designing resistance management strategies.

A high fidelity approach to assembling the complex Borrelia genome.

BACKGROUND: Bacteria of the Borrelia burgdorferi sensu lato (s.l.) complex can cause Lyme borreliosis. Different B. burgdorferi s.l. genospecies vary in their host and vector associations and human pathogenicity but the genetic basis for these adaptations is unresolved and requires completed and reliable genomes for comparative analyses. The de novo assembly of a complete Borrelia genome is challenging due to the high levels of complexity, represented by a high number of circular and linear plasmids that are dynamic, showing mosaic structure and sequence homology. Previous work demonstrated that even advanced approaches, such as a combination of short-read and long-read data, might lead to incomplete plasmid reconstruction. Here, using recently developed high-fidelity (HiFi) PacBio sequencing, we explored strategies to obtain gap-free, complete and high quality Borrelia genome assemblies. Optimizing genome assembly, quality control and refinement steps, we critically appraised existing techniques to create a workflow that lead to improved genome reconstruction. RESULTS: Despite the latest available technologies, stand-alone sequencing and assembly methods are insufficient for the generation of complete and high quality Borrelia genome assemblies. We developed a workflow pipeline for the de novo genome assembly for Borrelia using several types of sequence data and incorporating multiple assemblers to recover the complete genome including both circular and linear plasmid sequences. CONCLUSION: Our study demonstrates that, with HiFi data and an ensemble reconstruction pipeline with refinement steps, chromosomal and plasmid sequences can be fully resolved, even for complex genomes such as Borrelia. The presented pipeline may be of interest for the assembly of further complex microbial genomes.

Supercontinuum-based hyperspectral LiDAR for precision laser scanning.

Hyperspectral LiDAR enables non-contact mapping of the 3D surface geometry of an object along with its spectral reflectance signature and has proved to be effective for automated point cloud segmentation in various remote sensing applications. The established hyperspectral LiDAR methods offer a range precision of a few mm to a few cm for distances exceeding several meters. We propose a novel approach to hyperspectral LiDAR scanning based on a supercontinuum (SC) coherently broadened from a 780 nm frequency comb. It provides high precision distance measurements along with target reflectance over the 570-970 nm range of the SC output. The distance measurements are carried out by monitoring the differential phase delay of intermode beat notes generated by direct photodetection, while the backscattered light spectrum is acquired using a commercial CCD spectrometer with 0.16 nm resolution across the 400 nm bandwidth of the SC output. We demonstrate a measurement precision below 0.1 mm for a stand-off range up to 50 m on a diffuse target with around 89% reflectance. The measured relative accuracy as compared to a reference interferometer is on the order of 10-5 for distances up to 50 m. Initial results also indicate spectrum-based material classification within a 3D point cloud using a linear support vector machine. The results highlight the potential of this approach for joint high-precision laser scanning and automated material classification.

Variation of structured laser beam pattern and optimization for an alignment reference line creation.

The alignment of particle accelerators demands a dedicated measurement system based on a straight-line reference. This straight line can be provided by a laser beam. The alignment then involves accurately measuring the offset of accelerator components with respect to this light path. In order to be efficient, the laser beam needs to serve as a stable and straight reference for distances of several hundreds of meters. The attainable accuracy depends, among other parameters, on the laser spot size, which should ideally change very little over the distances at which the alignment system needs to operate. Due to the significant divergence of Gaussian laser beams, we propose using a structured laser beam (SLB) for alignment. Its transversal intensity profile is similar to a Bessel beam and consists of an intense inner core (IC) and concentric rings. The divergence of the IC, i.e., the growth of its size with distance, can be limited to 10µrad using a favorable generator configuration. Thus an SLB may be suitable as a straight-line reference for long-distance alignment applications. However, the SLB is distorted if obstructions cover parts of the outermost ring (OR) of the beam within, which should therefore also be small. In this paper, we investigate the relationship between the size of the IC and OR depending on the design parameters of the SLB generator. We use numerical simulations and experiments with different generators over distances up to 50 m to analyze the transversal intensity profile and wavefronts of different SLBs. The results indicate the general suitability of an SLB as a reference line for long-distance alignment but also expose tradeoffs between small IC and small OR. The findings outlined in the paper help to describe the optimal SLB parameters for given conditions.

Studying temporal titre evolution of commercial SARS-CoV-2 assays reveals significant shortcomings of using BAU standardization for comparison.

BACKGROUND: Measuring specific anti-SARS-CoV-2 antibodies has become one of the main epidemiological tools to survey the ongoing SARS-CoV-2 pandemic, but also vaccination response. The WHO made available a set of well-characterized samples derived from recovered individuals to allow normalization between different quantitative anti-Spike assays to defined Binding Antibody Units (BAU). METHODS: To assess sero-responses longitudinally, a cohort of ninety-nine SARS-CoV-2 RT-PCR positive subjects was followed up together with forty-five vaccinees without previous infection but with two vaccinations. Sero-responses were evaluated using a total of six different assays: four measuring anti-Spike proteins (converted to BAU), one measuring anti-Nucleocapsid proteins and one SARS-CoV-2 surrogate virus neutralization. Both cohorts were evaluated using the Euroimmun Anti-SARS-CoV-2-ELISA anti-S1 IgG and the Roche Elecsys Anti-SARS-CoV-2 anti-S1 assay. RESULTS: In SARS-CoV-2-convalesce subjects, the BAU-sero-responses of Euroimmun Anti-SARS-CoV-2-ELISA anti-S1 IgG and Roche Elecsys Anti-SARS-CoV-2 anti-S1 peaked both at 47 (43-51) days, the first assay followed by a slow decay thereafter (> 208 days), while the second assay not presenting any decay within one year. Both assay values in BAUs are only equivalent a few months after infection, elsewhere correction factors up to 10 are necessary. In contrast, in infection-naive vaccinees the assays perform similarly. CONCLUSION: The results of our study suggest that the establishment of a protective correlate or vaccination booster recommendation based on different assays, although BAU-standardised, is still challenging. At the moment the characteristics of the available assays used are not related, and the BAU-standardisation is unable to correct for that.

Protection of biomanufacturing processes from virus contamination through upstream virus filtration of cell culture media.

Cell-based manufacturing processes have occasionally been exposed to adventitious viruses, leading to manufacturing interruptions and unstable supply situations. The rapid progress of advanced therapy medicinal products needs innovative approaches to avoid any unwelcome reminder of the universal presence of viruses. Here, we investigated upstream virus filtration as a clearance step for any product too complex for downstream interventions. Culture media virus filtration was investigated with respect to virus clearance capacities under extreme conditions such as high process feed loading (up to ~19,000 L/m²), long duration (up to 34 days), and multiple process interruptions (up to 21 h). The small nonenveloped Minute virus of mice was used as relevant target virus, and as worse-case challenge for the investigated virus filters with a stipulated pore-size of about 20 nm. Certain filters-especially of the newer second generation-were capable of effective virus clearance despite the harsh regimen they were subjected to. The biochemical parameters for un-spiked control runs showed the filters to have no measurable impact on the composition of the culture media. Based on these findings, this technology seems to be quite feasible for large volume premanufacturing process culture media preparations.

The representative COVID-19 cohort Munich (KoCo19): from the beginning of the pandemic to the Delta virus variant.

BACKGROUND: Population-based serological studies allow to estimate prevalence of SARS-CoV-2 infections despite a substantial number of mild or asymptomatic disease courses. This became even more relevant for decision making after vaccination started. The KoCo19 cohort tracks the pandemic progress in the Munich general population for over two years, setting it apart in Europe. METHODS: Recruitment occurred during the initial pandemic wave, including 5313 participants above 13 years from private households in Munich. Four follow-ups were held at crucial times of the pandemic, with response rates of at least 70%. Participants filled questionnaires on socio-demographics and potential risk factors of infection. From Follow-up 2, information on SARS-CoV-2 vaccination was added. SARS-CoV-2 antibody status was measured using the Roche Elecsys® Anti-SARS-CoV-2 anti-N assay (indicating previous infection) and the Roche Elecsys® Anti-SARS-CoV-2 anti-S assay (indicating previous infection and/or vaccination). This allowed us to distinguish between sources of acquired antibodies. RESULTS: The SARS-CoV-2 estimated cumulative sero-prevalence increased from 1.6% (1.1-2.1%) in May 2020 to 14.5% (12.7-16.2%) in November 2021. Underreporting with respect to official numbers fluctuated with testing policies and capacities, becoming a factor of more than two during the second half of 2021. Simultaneously, the vaccination campaign against the SARS-CoV-2 virus increased the percentage of the Munich population having antibodies, with 86.8% (85.5-87.9%) having developed anti-S and/or anti-N in November 2021. Incidence rates for infections after (BTI) and without previous vaccination (INS) differed (ratio INS/BTI of 2.1, 0.7-3.6). However, the prevalence of infections was higher in the non-vaccinated population than in the vaccinated one. Considering the whole follow-up time, being born outside Germany, working in a high-risk job and living area per inhabitant were identified as risk factors for infection, while other socio-demographic and health-related variables were not. Although we obtained significant within-household clustering of SARS-CoV-2 cases, no further geospatial clustering was found. CONCLUSIONS: Vaccination increased the coverage of the Munich population presenting SARS-CoV-2 antibodies, but breakthrough infections contribute to community spread. As underreporting stays relevant over time, infections can go undetected, so non-pharmaceutical measures are crucial, particularly for highly contagious strains like Omicron.

Comb-based multispectral LiDAR providing reflectance and distance spectra.

Multispectral LiDAR enables joint observations of the 3D geometry and material properties of natural targets by combining ToF-based distance measurements with remote spectroscopy. Established multispectral LiDAR solutions provide mm-level range resolution and reflectance estimates of the target material over some tens of spectral channels. We propose a novel multispectral LiDAR approach based on an ultra-broadband frequency comb that enables enhanced remote spectroscopy by resolving relative delays in addition to reflectance. The spectrally-resolved delay and power measurements are transformed into distance and reflectance spectra by differential observations to a common reference object and adequate system calibration. These distance and reflectance spectra encode material information related to the surface and sub-surface composition and small-scale geometry. We develop the proposed comb-based multispectral LiDAR on an implementation covering the spectral range between 580 nm and 900 nm on 2 different spectral configurations with 7 and 33 channels of different spectral width. The performance assessment of the implemented system demonstrates a distance measurement precision better than 0.1 mm on most channels. Table-top probing results on five material specimens show that both the distance and the reflectance spectra alone enable discrimination of material specimens, while the novel distance signature particularly complements reflectance and increases classification accuracy when the material surface exhibits significant reflectance inhomogeneity. Material classification results using a support vector machine with radial basis function kernel demonstrate the potential of this approach for enhanced material classification by combining both signature dimensions.

Head-to-head evaluation of seven different seroassays including direct viral neutralisation in a representative cohort for SARS-CoV-2.

A number of seroassays are available for SARS-CoV-2 testing; yet, head-to-head evaluations of different testing principles are limited, especially using raw values rather than categorical data. In addition, identifying correlates of protection is of utmost importance, and comparisons of available testing systems with functional assays, such as direct viral neutralisation, are needed.We analysed 6658 samples consisting of true-positives (n=193), true-negatives (n=1091), and specimens of unknown status (n=5374). For primary testing, we used Euroimmun-Anti-SARS-CoV-2-ELISA-IgA/IgG and Roche-Elecsys-Anti-SARS-CoV-2. Subsequently virus-neutralisation, GeneScriptcPass, VIRAMED-SARS-CoV-2-ViraChip, and Mikrogen-recomLine-SARS-CoV-2-IgG were applied for confirmatory testing. Statistical modelling generated optimised assay cut-off thresholds. Sensitivity of Euroimmun-anti-S1-IgA was 64.8%, specificity 93.3% (manufacturer's cut-off); for Euroimmun-anti-S1-IgG, sensitivity was 77.2/79.8% (manufacturer's/optimised cut-offs), specificity 98.0/97.8%; Roche-anti-N sensitivity was 85.5/88.6%, specificity 99.8/99.7%. In true-positives, mean and median Euroimmun-anti-S1-IgA and -IgG titres decreased 30/90 days after RT-PCR-positivity, Roche-anti-N titres decreased significantly later. Virus-neutralisation was 80.6% sensitive, 100.0% specific (≥1:5 dilution). Neutralisation surrogate tests (GeneScriptcPass, Mikrogen-recomLine-RBD) were >94.9% sensitive and >98.1% specific. Optimised cut-offs improved test performances of several tests. Confirmatory testing with virus-neutralisation might be complemented with GeneScriptcPassTM or recomLine-RBD for certain applications. Head-to-head comparisons given here aim to contribute to the refinement of testing strategies for individual and public health use.

Kupffer cell activation by different microbial lysates: Toll-like receptor-2 plays pivotal role on thromboxane A2 production in mice and humans.

Thromboxane (TX) A2 has been identified as an important intrahepatic vasoconstrictor upon Kupffer cell (KC) activation during infections such as spontaneous bacterial peritonitis (SBP). The study aimed to investigate the role of TLRs in the TXA2 increase in liver nonparenchymal cells and their related mechanisms. Here, we identified TLR-2 as a common pathway for different microbials: microbial lysates including Gram-positive bacteria, Gram-negative bacteria, and fungi all increased TXA2 secretion via activation of TLR-2 in human KCs, accompanied by increased expression and phosphorylation of Myd88-related pathway. Of all TLR agonists, only TLR-1, -2, and -4 agonists increased TXA2 in human KCs. These results were further confirmed by mouse liver nonparenchymal cells. Comparing the effects of TLR-1, -2, and -4 antagonists, only TLR-2 antagonist showed inhibitory effects with all tested microbial lysates. Pretreatment with TLR-2 antagonist in human KCs blocked the secretion of IL-10, CXCL-10, TNF-α, and IL-6 induced by Gram-positive and Gram-negative bacterial stimulation. IL-23 and IL-1ß were only induced by Gram-negative bacteria. Thus, TLR-2 might be a potential marker and an attractive target for future treatment of patients with SBP. In addition, IL-23 and IL-1ß might distinguish early between Gram-positive and Gram-negative SBP.

Carriage of ESBL-producing Enterobacterales in wastewater treatment plant workers and surrounding residents - the AWARE Study.

To investigate whether wastewater treatment plant (WWTP) workers and residents living in close proximity to a WWTP have elevated carriage rates of ESBL-producing Enterobacterales, as compared to the general population. From 2018 to 2020, we carried out a cross-sectional study in Germany, the Netherlands, and Romania among WWTP workers (N = 344), nearby residents (living ≤ 300 m away from WWTPs; N = 431) and distant residents (living ≥ 1000 m away = reference group; N = 1165). We collected information on potential confounders via questionnaire. Culture of participants' stool samples was performed with ChromID®-ESBL agar plates and species identification with MALDI-TOF-MS. We used logistic regression to estimate the odds ratio (OR) for carrying ESBL-producing E. coli (ESBL-EC). Sensitivity analyses included stratification by country and interaction models using country as secondary exposure. Prevalence of ESBL-EC was 11% (workers), 29% (nearby residents), and 7% (distant residents), and higher in Romania (28%) than in Germany (7%) and the Netherlands (6%). Models stratified by country showed that within the Romanian population, WWTP workers are about twice as likely (aOR = 2.34, 95% CI: 1.22-4.50) and nearby residents about three times as likely (aOR = 3.17, 95% CI: 1.80-5.59) to be ESBL-EC carriers, when compared with distant residents. In stratified analyses by country, we found an increased risk for carriage of ESBL-EC in Romanian workers and nearby residents. This effect was higher for nearby residents than for workers, which suggests that, for nearby residents, factors other than the local WWTP could contribute to the increased carriage.

Rapid prototyping vaccine approach in mice against multi-drug resistant Gram-negative organisms from clinical isolates based on outer membrane vesicles.

Hospital-acquired infections due to multi-drug resistant Gram-negative organisms (MDRGNO) pose a major threat to global health. A vaccine preventing colonization and consecutive infection with MDRGNO could be particularly valuable, as therapeutic options become increasingly limited. Outer membrane vesicles (OMV) of Escherichia coli strain CFT073 as well as three MDRGNO strains that had caused severe infections in humans were administered intranasally to mice, with and without cholera toxin as an adjuvant. The humoral immune responses were comparatively matched with the sera of patients, who had suffered an infection caused by the respective bacterium. Additionally, systemic and local toxicity was evaluated. Intranasal vaccination with OMV could elicit solid humoral immune responses (total IgM and IgG), specific for the respective MDRGNO in mice; decoration of vital bacterial membranes with antibodies was comparable to patients who had survived systemic infection with the respective bacterial isolate. After intranasal vaccination of mice with OMV no signs of local or systemic toxicity were observed. Intranasal vaccination with OMV may open up a rapid vaccine approach to prevent colonization and/or infection with pathogenic MDRGNOs, especially in an outbreak setting within a hospital. It may also be an option for patients who have to undergo elective interventions in centers with a high risk of infection for certain common MDRGNO. Future studies need to include challenge experiments as well as phase I trials in humans.

From first to second wave: follow-up of the prospective COVID-19 cohort (KoCo19) in Munich (Germany).

BACKGROUND: In the 2nd year of the COVID-19 pandemic, knowledge about the dynamics of the infection in the general population is still limited. Such information is essential for health planners, as many of those infected show no or only mild symptoms and thus, escape the surveillance system. We therefore aimed to describe the course of the pandemic in the Munich general population living in private households from April 2020 to January 2021. METHODS: The KoCo19 baseline study took place from April to June 2020 including 5313 participants (age 14 years and above). From November 2020 to January 2021, we could again measure SARS-CoV-2 antibody status in 4433 of the baseline participants (response 83%). Participants were offered a self-sampling kit to take a capillary blood sample (dry blood spot; DBS). Blood was analysed using the Elecsys® Anti-SARS-CoV-2 assay (Roche). Questionnaire information on socio-demographics and potential risk factors assessed at baseline was available for all participants. In addition, follow-up information on health-risk taking behaviour and number of personal contacts outside the household (N = 2768) as well as leisure time activities (N = 1263) were collected in summer 2020. RESULTS: Weighted and adjusted (for specificity and sensitivity) SARS-CoV-2 sero-prevalence at follow-up was 3.6% (95% CI 2.9-4.3%) as compared to 1.8% (95% CI 1.3-3.4%) at baseline. 91% of those tested positive at baseline were also antibody-positive at follow-up. While sero-prevalence increased from early November 2020 to January 2021, no indication of geospatial clustering across the city of Munich was found, although cases clustered within households. Taking baseline result and time to follow-up into account, men and participants in the age group 20-34 years were at the highest risk of sero-positivity. In the sensitivity analyses, differences in health-risk taking behaviour, number of personal contacts and leisure time activities partly explained these differences. CONCLUSION: The number of citizens in Munich with SARS-CoV-2 antibodies was still below 5% during the 2nd wave of the pandemic. Antibodies remained present in the majority of SARS-CoV-2 sero-positive baseline participants. Besides age and sex, potentially confounded by differences in behaviour, no major risk factors could be identified. Non-pharmaceutical public health measures are thus still important.

Vaccine breakthrough infection and onward transmission of SARS-CoV-2 Beta (B.1.351) variant, Bavaria, Germany, February to March 2021.

A breakthrough infection occurred in a fully Comirnaty (BNT162b2) vaccinated healthcare worker with high levels of neutralising antibodies with the SARS-CoV-2 B.1.351 (Beta) variant in February 2021. The infection was subsequently transmitted to their unvaccinated spouse. Sequencing revealed an identical virus in both spouses, with a match of all nine single nucleotide polymorphisms typical for B.1.351. To the best of our knowledge, no transmission of any variant of SARS-CoV-2 from a fully vaccinated person has been described before.

Patients with cirrhosis and SBP: Increase in multidrug-resistant organisms and complications.

BACKGROUND: Spontaneous bacterial peritonitis (SBP) is a serious complication in patients with liver cirrhosis. In recent years, it has been postulated that the rate of multidrug-resistant organisms (MDROs) is increasing, especially in nosocomial SBP patients. Aim of the present work was to investigate this hypothesis and its possible clinical consequences. MATERIALS AND METHODS: One hundred and three culture-positive patients between 2007 and 2014 were compared with 81 patients between 2015 and 2017, to study the change of microbiological profiles and their clinical consequences. The cirrhosis patients with bacterascites requiring treatment were included as well. RESULTS: The most prevalent Gram-negative bacteria isolated from ascites were Enterobacterales (31.6%) and in Gram-positive pathogens Staphylococci (22.8%). There was a significant increase in MDROs (22.3% ICU 40.7%, P = .048), accompanied by an increased incidence of sepsis (from 21.4% to 37.0%, P = .021), hepatorenal syndrome (from 40.8% to 58.0%, P = .007) and the need of catecholamine therapy (from 21.4% to 38.8%, P = .036). Nosocomial origin correlated with higher MDRO proportion, more complications and lower antimicrobial susceptibility rates in 12 commonly used antibiotics. MDROs were confirmed as an isolated predictor for inpatient mortality and complications in multivariable logistic regression. CONCLUSIONS: The feeling in clinical practice that MDROs have increased in the last 11 years could be confirmed in our study in Munich, Germany. Nosocomial SBP correlated with significantly higher MDRO rates (nearly 50%) and complication rates. In our opinion, an antibiotic combination with comprehensive effect should be taken into account in nosocomial SBP patients in this region.

Antibody-enhanced hepatitis E virus nanofiltration during the manufacture of human immunoglobulin.

BACKGROUND: Circulation of hepatitis E virus (HEV) in areas where plasma is sourced for the manufacture of plasma-derived medicinal products (PDMPs) has prompted verification of HEV clearance. HEV exists as quasi lipid-enveloped (LE) and non-lipid-enveloped (NLE) forms, which might be of relevance for HEV clearance from manufacturing processes of antibody-containing PDMPs with solvent/detergent (S/D) treatment upstream of further clearance steps. STUDY DESIGN AND METHODS: Presence of different HEV particles in stocks used in clearance studies was investigated, with nanofilters graded around the assumed HEV particle sizes and by gradient centrifugation. HEV removal by 35-nm nanofiltration was investigated in the presence or absence of HEV antibodies, in buffer as well as in immunoglobulin (IG) manufacturing process intermediates. RESULTS: HEV particles consistent with LE, NLE, and an "intermediate" (IM) phenotype, obtained after S/D treatment, were seen in different HEV stocks. In the absence of HEV antibodies, log reduction factors (LRFs) of 4.0 and 2.5 were obtained by 35-nm nanofiltration of LE and IM HEV, consistent with the larger and smaller sizes of these phenotypes. Addition of HEV antibodies enhanced IM HEV removal around 1000-fold (LRF, 5.6). Effective (LRF, >4.8 and >4.0) HEV removal was obtained for the nanofiltration processing step for IG intermediates with varying HEV antibody content. CONCLUSION: HEV spikes used in clearance studies should be carefully selected, as differences in physicochemical properties might affect HEV clearance. Antibody-mediated enhancement of HEV nanofiltration was demonstrated in IG process intermediates even at low HEV antibody concentration, illustrating the robustness of this manufacturing step.

Nanofiltration as a robust method contributing to viral safety of plasma-derived therapeutics: 20 years' experience of the plasma protein manufacturers.

BACKGROUND: Nanofiltration entails the filtering of protein solutions through membranes with pores of nanometric sizes that have the capability to effectively retain a wide range of viruses. STUDY DESIGN AND METHODS: Data were collected from 754 virus validation studies (individual data points) by Plasma Protein Therapeutics Association member companies and analyzed for the capacity of a range of nanofilters to remove viruses with different physicochemical properties and sizes. Different plasma product intermediates were spiked with viruses and filtered through nanofilters with different pore sizes using either tangential or dead-end mode under constant pressure or constant flow. Filtration was performed according to validated scaled-down laboratory conditions reflecting manufacturing processes. Effectiveness of viral removal was assessed using cell culture infectivity assays or polymerase chain reaction (PCR). RESULTS: The nanofiltration process demonstrated a high efficacy and robustness for virus removal. The main factors affecting nanofiltration efficacy are nanofilter pore size and virus size. The capacity of nanofilters to remove smaller, nonenveloped viruses was dependent on filter pore size and whether the nanofiltration process was integrated and designed with the intention to provide effective parvovirus retention. Volume filtered, operating pressure, and total protein concentration did not have a significant impact on the effectiveness of virus removal capacity within the investigated ranges. CONCLUSIONS: The largest and most diverse nanofiltration data collection to date substantiates the effectiveness and robustness of nanofiltration in virus removal under manufacturing conditions of different plasma-derived proteins. Nanofiltration can enhance product safety by providing very high removal capacity of viruses including small non-enveloped viruses.

Publisher Correction to: Protocol of a population-based prospective COVID-19 cohort study Munich, Germany (KoCo19).

An amendment to this paper has been published and can be accessed via the original article.