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Genome sequences from evolving infectious pathogens allow quantification of case introductions and local transmission dynamics. We sequenced 11,357 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes from Switzerland in 2020—the sixth largest effort globally. Using a representative subset of these data, we estimated viral introductions to Switzerland and their persistence over the course of 2020. We contrasted these estimates with simple null models representing the absence of certain public health measures. We show that Switzerland’s border closures decoupled case introductions from incidence in neighboring countries. Under a simple model, we estimate an 86 to 98% reduction in introductions during Switzerland’s strictest border closures. Furthermore, the Swiss 2020 partial lockdown roughly halved the time for sampled introductions to die out. Last, we quantified local transmission dynamics once introductions into Switzerland occurred using a phylodynamic model. We found that transmission slowed 35 to 63% upon outbreak detection in summer 2020 but not in fall. This finding may indicate successful contact tracing over summer before overburdening in fall. The study highlights the added value of genome sequencing data for understanding transmission dynamics.

IntroductionCOVID-19 vaccines significantly reduce SARS-CoV-2 (SCoV2)-related hospitalization and mortality in randomized controlled clinical trials, as well as in real-world effectiveness against different circulating SCoV2-lineages. However, some vaccine recipients show breakthrough infection and it remains unknown, which host and viral factors contribute to this risk and how many resulted in severe outcomes. Our aim was to identify demographic and clinical risk factors for SCoV2 breakthrough infections and severe disease in fully vaccinated individuals and to compare patient characteristics in breakthrough infections caused by SCoV2 Alpha or Delta variant.MethodsWe conducted an exploratory retrospective case-control study from 28th of December to 25th of October 2021 dominated by the Delta SCoV2 variant. All cases of infection had to be reported by law to the local health authorities. Vaccine recipients’ data was anonymously available from the national Vaccination Monitoring Data Lake and the main local vaccine center. We compared anonymized patients’ characteristics of breakthrough infection (n=492) to two overlapping control groups including all vaccine recipients from the Canton of Basel-City (group 1 n=126’586 and group 2 n=109’382). We also compared patients with breakthrough infection caused by the Alpha to Delta variant. We used different multivariate generalized linear models (GLM).ResultsWe found only 492/126’586 (0.39%) vaccine recipients with a breakthrough infection after vaccination during the 10 months observational period. Most cases were asymptomatic or mild (478/492 97.2%) and only very few required hospitalization (14/492, 2.8%). The time to a positive SCoV2 test shows that most breakthrough infections occurred between a few days to about 170 days after full vaccination, with a median of 78 days (interquartile range, IQR 47-124 days). Factors associated with a lower odds for breakthrough infection were: age (OR 0.987, 95%CI 0.983-0.992), previous COVID-19 infection prior to vaccination (OR 0.296, 95%CI 0.117-0.606), and (self-declared) serious side-effects from previous vaccines (OR 0.289, 95%CI 0.033-1.035). Factors associated with a higher odds for breakthrough infection were: vaccination with the Pfizer/BioNTech vaccine (OR 1.459, 95%CI 1.238-1.612), chronic disease as vaccine indication (OR 2.109, 95%CI 1.692-2.620), and healthcare workers (OR 1.404, 95%CI 1.042-1.860). We did not observe a significantly increased risk for immunosuppressed patients (OR 1.248, 95% CI 0.806-1.849).ConclusionsOur study shows that breakthrough infections are rare and show mild illness, but that it occurs early after vaccination with more than 50% of cases within 70 to 80 days post-full vaccination. This clearly implies that boost vaccination should be much earlier initiated compared to the currently communicated 180-day threshold. This has important implications especially for risk groups associated with more frequent breakthrough infections such as healthcare workers, and people in high-risk care facilities. Due to changes in the epidemiological dynamic with new variants emerging, continuous monitoring of breakthrough infections is helpful to provide evidence on booster vaccines and patient groups at risk for potential complications.

Genome sequences from evolving infectious pathogens allow quantification of case introductions and local transmission dynamics. We sequenced 11,357 SARS-CoV-2 genomes from Switzerland in 2020 - the 6th largest effort globally. Using a representative subset of these data, we estimated viral introductions to Switzerland and their persistence over the course of 2020. We contrast these estimates with simple null models representing the absence of certain public health measures. We show that Switzerland’s border closures de-coupled case introductions from incidence in neighboring countries. Under a simple model, we estimate an 86 - 98% reduction in introductions during Switzerland’s strictest border closures. Furthermore, the Swiss 2020 partial lockdown roughly halved the time for sampled introductions to die out. Finally, we quantified local transmission dynamics once introductions into Switzerland occurred, using a novel phylodynamic model. We find that transmission slowed 35 – 63% upon outbreak detection in summer 2020, but not in fall. This finding may indicate successful contact tracing over summer before overburdening in fall. The study highlights the added value of genome sequencing data for understanding transmission dynamics.One Sentence SummaryPhylogenetic and phylodynamic methods quantify the drop in case introductions and local transmission with implementation of public health measures.

ZusammenfassungTraditionell werden pharmazeutische Zubereitungen der Weidenmistel häufig bei der Therapie von Blasen- und Nierenzellkarzinomen eingesetzt. Dennoch existieren bislang nur wenige wissenschaftliche Untersuchungen zu Wirkmechanismen von Weidenmistelpräparaten. Daher wurde mithilfe verschiedener Testverfahren die Hemmung der Viabilität von Tumorzellen durch den Weidenmistelextrakt Iscucin® Salicis bestimmt und die beteiligten Mechanismen untersucht. Des Weiteren wurde die Hemmung der Zellmigration analysiert. Ergebnisse Der Extrakt hemmte Zellwachstum und -migration in vitro, wobei die Sensitivität der einzelnen Tumorzelllinien variierte. Vor allem sprachen neuroepitheliale Tumorzellen und Bronchialkarzinomzellen gut an. Zudem wurden pro-apoptotische Marker aktiviert. Klinische Untersuchungen stehen aus, um zu klären, bei welchen Tumoren das größte Potenzial für eine Behandlung mit Iscucin® Salicis besteht.

The rapid spread of the SARS-CoV-2 lineages B.1.1.7 (N501Y.V1) throughout the UK, B.1.351 (N501Y.V2) in South Africa, and P.1 (B.1.1.28.1; N501Y.V3) in Brazil has led to the definition of variants of concern (VoCs) and recommendations for lineage specific surveillance. In Switzerland, during the last weeks of December 2020, we established a nationwide screening protocol across multiple laboratories, focusing first on epidemiological and microbiological definitions. In January 2021, we validated and implemented an N501Y-specific PCR to rapidly screen for VoCs, which are then confirmed using amplicon sequencing or whole genome sequencing (WGS). A total of 13,387 VoCs have been identified since the detection of the first Swiss case in October 2020, with 4194 being B.1.1.7, 172 B.1.351, and 7 P.1. The remaining 9014 cases of VoCs have been described without further lineage specification. Overall, all diagnostic centers reported a rapid increase of the percentage of detected VOCs, with a range of 6 to 46% between 25 to 31 of January 2021 increasing towards 41 to 82% between 22 to 28 of February. A total of 739 N501Y positive genomes were analysed and show a broad range of introduction events to Switzerland. In this paper, we describe the nationwide coordination and implementation process across laboratories, public health institutions, and researchers, the first results of our N501Y-specific variant screening, and the phylogenetic analysis of all available WGS data in Switzerland, that together identified the early introduction events and subsequent community spreading of the VoCs., Microorganisms, 9 (4)

BackgroundIn December 2020, the United Kingdom (UK) reported a SARS-CoV-2 Variant of Concern (VoC) which is now named B.1.1.7. Based on initial data from the UK and later data from other countries, this variant was estimated to have a transmission fitness advantage of around 40-80% [1, 2, 3].AimThis study aims to estimate the transmission fitness advantage and the effective reproductive number of B.1.1.7 through time based on data from Switzerland.MethodsWe generated whole genome sequences from 11.8% of all confirmed SARS-CoV-2 cases in Switzerland between 14 December 2020 and 11 March 2021. Based on these data, we determine the daily frequency of the B.1.1.7 variant and quantify the variant’s transmission fitness advantage on a national and a regional scale.ResultsWe estimate B.1.1.7 had a transmission fitness advantage of 43-52% compared to the other variants circulating in Switzerland during the study period. Further, we estimate B.1.1.7 had a reproductive number above 1 from 01 January 2021 until the end of the study period, compared to below 1 for the other variants. Specifically, we estimate the reproductive number for B.1.1.7 was 1.24 [1.07-1.41] from 01 January until 17 January 2021 and 1.18 [1.06-1.30] from 18 January until 01 March 2021 based on the whole genome sequencing data. From 10 March to 16 March 2021, once B.1.1.7 was dominant, we estimate the reproductive number was 1.14 [1.00-1.26] based on all confirmed cases. For reference, Switzerland applied more non-pharmaceutical interventions to combat SARS-CoV-2 on 18 January 2021 and lifted some measures again on 01 March 2021.ConclusionThe observed increase in B.1.1.7 frequency in Switzerland during the study period is as expected based on observations in the UK. In absolute numbers, B.1.1.7 increased exponentially with an estimated doubling time of around 2-3.5 weeks. To monitor the ongoing spread of B.1.1.7, our plots are available online.

The rapid spread of the SARS-CoV-2 lineages B.1.1.7 (N501Y.V1) throughout the UK, B.1.351 (N501Y.V2) in South Africa, and P.1 (B.1.1.28.1; N501Y.V3) in Brazil has led to the definition of variants of concern (VoCs) and recommendations by the European Center for Disease Prevention and Control (ECDC) and World Health Organization (WHO) for lineage specific surveillance. In Switzerland, during the last weeks of December 2020, we established a nationwide screening protocol across multiple laboratories, focusing first on epidemiological definitions based on travel history and the S gene dropout in certain diagnostic systems. In January 2021, we validated and implemented an N501Y-specific PCR to rapidly screen for VoCs, which are then confirmed using amplicon sequencing or whole genome sequencing (WGS). A total of 3492 VoCs have been identified since the detection of the first Swiss case in October 2020, with 1370 being B1.1.7, 61 B.1.351, and none P.1. The remaining 2061 cases of VoCs have been described without further lineage specification. In this paper, we describe the nationwide coordination and implementation process across laboratories, public health institutions, and researchers, the first results of our N501Y-specific variant screening, and the phylogenetic analysis of all available WGS data in Switzerland, that together identified the early introduction events and subsequent community spreading of the VoCs.

Pathogen genomes provide insights into their evolution and epidemic spread. We sequenced 1,439 SARS-CoV-2 genomes from Switzerland, representing 3-7% of all confirmed cases per week. Using these data, we demonstrate that no one lineage became dominant, pointing against evolution towards general lower virulence. On an epidemiological level, we report no evidence of cryptic transmission before the first confirmed case. We find many early viral introductions from Germany, France, and Italy and many recent introductions from Germany and France. Over the summer, we quantify the number of non-traceable infections stemming from introductions, quantify the effective reproductive number, and estimate the degree of undersampling. Our framework can be applied to quantify evolution and epidemiology in other locations or for other pathogens based on genomic data.One Sentence SummaryWe quantify SARS-CoV-2 spread in Switzerland based on genome sequences from our nation-wide sequencing effort.

SARS-CoV-2, the virus responsible for the current COVID-19 pandemic, is evolving into different genetic variants by accumulating mutations as it spreads globally. In addition to this diversity of consensus genomes across patients, RNA viruses can also display genetic diversity within individual hosts, and co-existing viral variants may affect disease progression and the success of medical interventions. To systematically examine the intra-patient genetic diversity of SARS-CoV-2, we processed a large cohort of 3939 publicly-available deeply sequenced genomes with specialised bioinformatics software, along with 749 recently sequenced samples from Switzerland. We found that the distribution of diversity across patients and across genomic loci is very unbalanced with a minority of hosts and positions accounting for much of the diversity. For example, the D614G variant in the Spike gene, which is present in the consensus sequences of 67.4% of patients, is also highly diverse within hosts, with 29.7% of the public cohort being affected by this coexistence and exhibiting different variants. We also investigated the impact of several technical and epidemiological parameters on genetic heterogeneity and found that age, which is known to be correlated with poor disease outcomes, is a significant predictor of viral genetic diversity.Author SummarySince it arose in late 2019, the new coronavirus (SARS-CoV-2) behind the COVID-19 pandemic has mutated and evolved during its global spread. Individual patients may host different versions, or variants, of the virus, hallmarked by different mutations. We examine the diversity of genetic variants coexisting within patients across a cohort of 3939 publicly accessible samples and 749 recently sequenced samples from Switzerland. We find that a small number of patients carry most of the diversity, and that patients with more diversity tend to be older. We also find that most of the diversity is concentrated in certain regions and positions of the virus genome. In particular, we find that a variant reported to increase infectivity is among the most diverse positions. Our study provides a large-scale survey of within-patient diversity of the SARS-CoV-2 genome.

ZusammenfassungDie Wurzel von Liebstöckel (Levisticum officinale Koch), der v. a. als Gewürzpflanze bekannt ist, wird seit Jahrhunderten zur Herstellung von Arzneimitteln eingesetzt. Neben der traditionellen Verwendung zur Durchspülungstherapie bei Nierenleiden findet in der anthroposophischen Medizin ein 5 %iger Auszug der getrockneten Wurzel in nativem Olivenöl Anwendung bei Mittelohrentzündung. Sicherheit und Wirkungen dieses Auszugs waren Ziele einer präklinischen und klinischen Untersuchung.Methoden: Präklinisch wurden die Effekte des 5 %igen Auszugs sowie des Auszugsöls auf die 5-Lipoxygenase und die Cyclooxygenase-2 und die von ihnen katalysierte Synthese von antiinflammatorischen Mediatoren, Leukotrien B4 und Prostaglandin E2 untersucht. Klinisch lieferte eine nichtinterventionelle Studie mit 85 Patienten Daten aus der ärztlichen Routineanwendung des Auszugs von Levistici radix bei Otitis media.Ergebnisse: Der 5 %ige Auszug von Levistici radix in nativem Olivenöl reduzierte deutlich die Leukotrien-B4- und weniger deutlich die Prostaglandin-E2-Synthese. Die Wirkung des parallel getesteten Olivenöls fiel wesentlich geringer aus. Die klinische Anwendung des Auszugs in Form von Ohrentropfen ergab eine als gut bis sehr gut beurteilte Wirkung und Verträglichkeit.Fazit: Die präklinische Untersuchung liefert eine rationale Basis für die lokale klinische Anwendung des 5 %igen Auszugs von Levistici radix in nativem Olivenöl bei Otitis media, die in der Praxis bestätigt wird.

Ethnopharmacological relevance Oak bark has been used since ancient times in Europaen ethnomedicine because of its adstringent, antimicrobial and hemostatic features, e.g. as a remedy for the treatment of wounds and skin diseases. Purpose Oak bark tannins are considered as bioactive natural products, interacting with surface proteins of mucous membranes and might be beneficial for the treatment of allergic diseases. This study investigated the effect of an oak bark decoction (OBD) and isolated tannin fractions on the degranulation capacity and cytokine/chemokine release from rat basophilic cells and human mast cells in vitro, which are essential for the initiation of early- and late-phase allergic reactions. Methods and methods By chromatographic separation on Sephadex® LH-20 high- and low-molecular weight tannins were separated from OBD and the tannin composition analyzed by HPLC(DAD)-MSn. Then, the OBD and its fractions were tested in degranulation (β-hexosaminidase activity) of allergen-specific-activated basophilic cells in a photometric assay. Results The OBD and the high-molecular tannin fraction showed a dose-dependent inhibition of cell degranulation. Furthermore, the OBD and particularly its high molecular weight tannin fraction exhibited an inhibitory activity on the IL-8-, IL-6- and TNF-α-secretion from stimulated human mast cells, detected and quantified by ELISA. Conclusion The OBD and its high-molecular weight tannins revealed an impact on allergic mediator release of basophilic cells and human mast cells and thereby provide a rationale for the topical treatment with OBD preparations.

Respiratory tract infection (RTI) involves a variety of viruses and bacteria, which can be conveniently detected by multiplex nucleic acid amplification testing (NAT). To compare the novel Luminex-based NxTAG-Respiratory Pathogen Panel (NxTAG-RPP) with the routine multiplex-ligation-NAT based RespiFinder-22® (RF-22), 282 respiratory specimens including nasopharyngeal swabs (71%), broncho-alveolar lavage (27%), throat swabs, tracheal secretions, and sputum (2%) from 116 children and 155 adults were extracted using a Corbett CAS1200 (Qiagen), and analyzed in parallel by the routine RF-22 and NxTAG-RPP. Concordant results were obtained in 263 (93.3%) cases consisting of concordant positives in 167 (59.2%) and concordant negatives in 96 (34%). Results were discordant in 19 (6.7%) consisting of 15 positive:negative, and 4 negative:positive results by NxTAG-RPP versus RF-22, respectively. Co-infections were observed in 10.3% with NxTAG-RPP and in 5.9% with RF-22. Most additional viral pathogens identified by the NxTAG-RPP involved dual infections with rhinovirus and RSV. Discordant samples were mainly due to low genome signals of Ct less than 36, when retested by QNAT suggesting a higher sensitivity of the NxTAG-RPP, also when detecting multiple infections. Hands-on time after extraction for 24 and 96 samples was 0.25 and

Objectives While Taraxacum officinale (dandelion) extracts showed antitumor effects on adult cancer cells, effects on pediatric tumor cells as a single agent or in combination with mistletoe extracts are hitherto unknown. Material and methods The anti-proliferative effects of an aqueous fermented Taraxacum officinale extract (Taraxacum) on a pediatric cancer cell line panel were assessed by cell viability assays (MTT). In two neuroblastoma cell lines, SH-SY5Y and Kelly, the effects on cell cycle distribution (PI staining), mitochondrial integrity (MitoTracker staining), invasion (Boyden chamber assay) and migration (Scratch-assay) as well as the synergistic effects of the co-treatment of Taraxacum and mistletoe preparations (Iscucin® Tiliae or Iscucin® Pini) were investigated. Results All tested cancer cell lines were more susceptible to Taraxacum than the normal human fibroblast cell line, NHDF-C. In neuroblastoma cell lines Taraxacum caused apoptosis and loss of mitochondrial integrity as well as an inhibition of invasion and migration. The simultaneous therapy of Taraxacum and the mistletoe extracts revealed synergistic effects. Conclusion This preclinical data support the use of Taraxacum as a potential adjuvant application in pediatric oncology.

Gastrointestinal infections are caused by a broad spectrum of pathogens. Conventional diagnostic procedures are resource and time consuming due to single pathogen testing, often in different laboratories.We analyzed 312 consecutive stool samples from pediatric patients (n = 127) with gastroenteritis or from adult travelers returning from the tropics with suspected parasite infestation (n = 185) using commercial multiplex nucleic acid amplification testing (NAT) (xTAG gastrointestinal pathogen panel, Luminex) covering 15 diarrhea-causing pathogens. The results of the positive samples and a representative number of negative samples were compared to standard methods, including NAT, direct antigen detection (DAD), bacterial culture and microscopy.Of the 185 samples from adult travelers, 21 (11 %) were multiplexNAT-positive, with enterotoxigenic Escherichia coli (4 %) being the predominant pathogen. Microscopic examination revealed Blastocystis hominis in 23 % not covered by the panel. MultiplexNAT scored positive in 66 pediatric samples (52 %), with rotavirus (27 %) being the most prevalent. All adenovirus-, rotavirus-, Clostridium difficile- and Cryptosporidium-positive samples were confirmed in external laboratories, but only 40 % of norovirus- and 29 % of Giardia-positive samples. Analysis of frozen specimens by bacterial culture showed the highest discrepancies with the multiplexNAT.Our study demonstrates broad detection of relevant gastroenteritis pathogens by multiplexNAT with a short turnaround time. This is important for diagnosis, infection control and empiric management of gastroenteritis patients, but may be selectively complemented by bacterial culture and resistance testing.

Das Waldbingelkraut (Mercurialis perennis L.) - eine Pflanze aus der Familie der Wolfsmilchgewachse (Euphorbiaceae) - war bereits in vorchristlicher Zeit und im Mittelalter als probates Mittel zur Behandlung von eitrigen Wunden, Ekzemen und Geschwuren bekannt. Auch als starkes Abfuhrmittel und bei Menstruationsbeschwerden wurde Mercurialis verwendet. Hydroalkoholische Auszuge des Krautes und wassrig-fermentierte Extrakte finden heute insbesondere in der Komplementarmedizin Anwendung. Indikationen solcher Mercurialis-Zubereitungen sind z.B. schlecht heilende Wunden, Entzundungen, Verbrennungen, Hamorrhoiden sowie trockene Bindehautentzundung des Auges (Konjunktivitis). Die Inhaltsstoffe von M. perennis und deren Pharmakologie galten bislang als wenig erforscht. Neue phytochemische Untersuchungen zeigen, dass die Pflanze Alkaloide, atherisches Ol, n-Alkylresorcine sowie Depside und Flavonoide enthalt. In einem In-vitro-Entzundungsmodell an isolierten menschlichen Monozyten wurde nun erstmals die immunmodulatorische Wirkung der fermentierten Mercurialis-Urtinktur nachgewiesen - ein Befund, der mit einem beschleunigten Wundheilungsprozess nach Anwendung von Mercurialis-Urtinkturpraparaten im Einklang steht. Der Beitrag halt eine Ruckschau auf altere Literatur zur Pharmakognosie von M. perennis und stellt Daten aus der aktuellen Forschung vor.

Respiratory tract infection (RTI) involves a variety of viruses and bacteria, which can be conveniently detected by multiplex nucleic acid amplification testing (NAT). To compare the novel Luminex‐based NxTAG‐Respiratory Pathogen Panel (NxTAG‐RPP) with the routine multiplex‐ligation‐NAT based RespiFinder‐22® (RF‐22), 282 respiratory specimens including nasopharyngeal swabs (71%), broncho‐alveolar lavage (27%), throat swabs, tracheal secretions, and sputum (2%) from 116 children and 155 adults were extracted using a Corbett CAS1200 (Qiagen), and analyzed in parallel by the routine RF‐22 and NxTAG‐RPP. Concordant results were obtained in 263 (93.3%) cases consisting of concordant positives in 167 (59.2%) and concordant negatives in 96 (34%). Results were discordant in 19 (6.7%) consisting of 15 positive:negative, and 4 negative:positive results by NxTAG‐RPP versus RF‐22, respectively. Co‐infections were observed in 10.3% with NxTAG‐RPP and in 5.9% with RF‐22. Most additional viral pathogens identified by the NxTAG‐RPP involved dual infections with rhinovirus and RSV. Discordant samples were mainly due to low genome signals of Ct less than 36, when retested by QNAT suggesting a higher sensitivity of the NxTAG‐RPP, also when detecting multiple infections. Hands‐on time after extraction for 24 and 96 samples was 0.25 and

This study aimed to investigate the immunomodulatory and antiallergic properties of preparations from lemon, Citrus medica L. (citrus), and quince, Cydonia oblonga Mill. (Cydonia), which are used in pharmaceutical products to treat patients suffering from allergic disorders. Preparations were analyzed with respect to their impact on the degranulation capacity from basophilic cells as well as mediator release from activated human mast cells in vitro, including IL-8 and TNF- α secretion. The results show that the degranulation of basophilic cells was diminished only in the presence of Citrus, and this effect was compared to the synthetic drug azelastine. Furthermore, Citrus and Cydonia both inhibited the production of IL-8 and TNF- α from human mast cells, and at low concentrations additive effects were observed. As a positive inhibition control, dexamethasone was used. LC-MS analyses showed that the major phenolic components in extracts from Citrus and Cydonia are eriocitrin and neochlorogenic acid, respectively. Nevertheless, these compounds do not show biological effects at concentration levels detected in their corresponding extracts. In conclusion, the present data provide a rational base for the use of the single pharmaceutical preparations from Citrus and Cydonia in a differentiated treatment of allergic disorders in part by the regulation of soluble allergic mediators from basophilic cells and mast cells.

Surveillance of cytomegalovirus (CMV) replication in transplant patients is crucial for the success of transplantation. To compare a CMV pp65 antigenemia (pp65Ag) and a quantitative real-time PCR targeting the CMV-UL111a (UL111aPCR), all whole blood samples taken between July 2008 and October 2009 were identified which had been analyzed prospectively by both assays in parallel. Discordant results were re-analyzed using a published CMV duplex PCR targeting regions UL55 and UL123exon4. Of 720 samples from 81 transplant patients, CMV replication was detected in 244 specimens (34%) by the UL111aPCR (median, 1,019 geq/ml), compared to 113 (16%) detected by the pp65Ag (median, 2/250,000 leukocytes). Concordant UL111aPCR/pp65Ag results were obtained in 561 (78%) samples, being positive in 99 (14%), and negative in 462 (64%). As a rule of thumb, 1 pp65Ag-positive cell per 250,000 leukocytes corresponded to 1,000 geq/ml CMV DNA of whole blood. Discordant results were found in 159 samples (22%), being UL111aPCR-positive/pp65Ag-negative in 145 (91%; median, 650 geq/ml), or UL111aPCR-negative/pp65Ag-positive in 14 (9%; median, 1/250,000 cells). Using the duplex PCR targeting the CMV UL55 and the UL123-exon4 genes, 131 of 139 (94%) discordant UL111aPCR-positives (median UL111aPCR, 639 geq/ml; median UL55PCR, 715 geq/ml; median UL123PCR, 1,103 geq/ml) were confirmed. Of 14 discordant pp65Ag-positives, duplex PCR was also negative in 8, and of low copy number in 6. Thus, CMV UL111aPCR provides more sensitive quantitation of CMV replication than pp65Ag, however, discordant results can occur at very low viral loads.

Highlights • Rapid isothermal NAT for influenza reveals improved sensitivity and specificity compared to direct antigen testing. • Simple sample processing and short turn-around time render isothermal NAT suitable for sequential near-patient testing. • The turn-around time advantage is lost by the single-test format precluding sample cohorting and testing of larger series., Background Rapid diagnosis of influenza is important for controlling outbreaks and starting antiviral therapy. Direct antigen detection (DAD) is rapid, but lacks sensitivity, whereas nucleic acid amplification testing (NAT) is more sensitive, but also more time-consuming. Objectives To evaluate the performance of a rapid isothermal NAT and two DADs. Study design During February–May 2014, we tested 211 consecutive patients with influenza-like illness using a commercial isothermal NAT (Alere™ Influenza A&B) as well as the DAD Sofia® Influenza A + B and BinaxNOW® Influenza A&B for detection of influenza-A and -B virus. RespiFinder-22® a commercial multiplex NAT served as reference test. Serial 10-fold dilutions of influenza-A and -B cell culture supernatants were examined. Another 225 patient samples were tested during December 2014–February 2015. Results Compared to RespiFinder-22®, the isothermal NAT Alere™ Influenza A&B, and the DAD Sofia® Influenza A + B and BinaxNOW® Influenza A&B had sensitivities of 77.8%, 59.3% and 29.6%, and specificities of 99.5%, 98.9% and 100%, respectively, for the first 211 patient samples. Alere™ Influenza A&B showed 85.7% sensitivity and 100% specificity in the second cohort. Isothermal NAT was 10-100-fold more sensitive compared to DAD for influenza virus culture supernatants with a lower limit of detection of 5000–50,000 copies/mL. The average turn-around time (TAT) of isothermal NAT and DADs was 15 min, but increased to 110 min for Alere™ Influenza A&B, 30 min for BinaxNOW® Influenza A&B, and 45 min for Sofia® Influenza A + B, when analyzing batches of 6 samples. Conclusion Simple sample processing and a TAT of 15 min render isothermal NAT Alere™ Influenza A&B suitable for sequential near-patient testing, but the TAT advantage is lost when testing of larger series.

Pseudomonas aeruginosa airway infections are a major cause of morbidity and mortality in patients with cystic fibrosis. Treatment of established infections is difficult, even with microbiologically active agents. Thus, prevention of infection is an important goal of management. Isolates from cystic fibrosis patients appear to originate from the environment but adapt to the milieu of the airway of the cystic fibrosis patient and evolve toward a common phenotype. Identification of the antigens expressed early in infection may lead to novel targets for vaccine development. Immunogenic peptides were identified in a J404 random nonapeptide phage display library with serum from cystic fibrosis patients obtained within the first year of P. aeruginosa infection. One hundred sixty-five reactive clones were verified by plaque lift assays, and their inserts were sequenced. The sequenced nonapeptides were compared with the published sequence of strain PAO1, identifying homologies to 76 genes encoding outer membrane and secreted proteins. The majority of these were proteins involved in small-molecule transport, membrane structural proteins, and secreted factors. An in silico analysis was performed that suggested that the occurrence of multiple matches to predominantly outer membrane and secreted proteins was not attributable to random chance. Finally, gene expression array data from early isolates of P. aeruginosa from cystic fibrosis patients was compared with the results from phage display analysis. Eleven outer membrane and secreted proteins were common between the two data sets. These included genes involved in iron acquisition, antibiotic efflux, fimbrial biogenesis, and pyocin synthesis. These results demonstrate the feasibility and validity of this novel approach and suggest potential targets for future development.

Ziel: Nach jungeren Schatzungen leiden in westlichen Industrienationen 1,3–4,8% der Bevolkerung an einer Fibromyalgie. 80–90% der Betroffenen sind Frauen. Dieses deutliche Uberwiegen von Frauen, zumeist im reproduktionsfahigen Alter, lasst an einen Zusammenhang zwischen Fibromyalgie und Sexualhormonen denken. Methoden: Im Rahmen einer zweistufigen, bevolkerungsbezogenen Querschnittstudie bei 3174 Frauen zwischen 35 und 74 Jahren in Bad Sackingen wurde eine stratifizierte Zufallsstichprobe klinisch untersucht und befragt (n=653). Von diesen Frauen erfullten 36 alle Kriterien einer Fibromyalgie. 44 Frauen litten unter chronischen, ausgebreiteten Schmerzen, aber keiner Fibromyalgie (chronische Schmerzprobanden), 408 Frauen hatten keine chronischen, ausgebreiteten Schmerzen (Kontrollen). Ergebnisse: Fibromyalgie-Probanden haben gegenuber Kontrollen das 3,6fache, und gegenuber chronischen Schmerzprobanden das 3,9fache Risiko, einem niedrigeren sozialen Status anzugehoren. Fibromyalgie-Probanden trinken sowohl im Vergleich zu Kontrollen als auch chronischen Schmerzprobanden seltener Alkohol. Von den in der gynakologischen Anamnese erhobenen Daten zeigte sich in multivariaten Analysen, dass nach Adjustierung fur Alter die Fibromyalgie-Probanden gegenuber Kontrollen eine signifikant spatere Menarche hatten (OR=2,2 fur >14 Jahre) und nie schwanger waren (OR=0,3). Beim Vergleich chronischer Schmerzprobanden mit den Kontrollen zeigten sich diese Assoziationen nicht. Folgerungen: Die bei den Fibromyalgie-Probanden gefundenen Zusammenhange mit niedrigem Sozialstatus, niedrigem Alkoholkonsum, spatem Menarchenalter und seltenen Schwangerschaften sind spezifisch fur Fibromyalgie. Dies unterscheidet die Fibromyalgie-Probanden sowohl von den Kontrollen als auch den chronischen Schmerzprobanden. Die gleichen hormonellen Faktoren, die zu einer verzogerten Menarche und einer verminderten Fertilitat fuhren, scheinen bei der Entwicklung einer Fibromyalgie relevant zu sein.

We report on a 65-year-old male patient with a Shiga toxin-producing Escherichia coli O51:H49 gastrointestinal infection and sepsis associated with hemolytic uremic syndrome (HUS) with a fatal outcome. The strains isolated harbored stx 2e and eae , a very unusual and new virulence profile for an HUS-associated enterohemorrhagic E. coli .

Short courses of antibiotic therapy are recommended for treatment of pertussis. We report 2 young unvaccinated infants with persistently positive Bordetella pertussis by polymerase chain reaction from nasopharyngeal swabs despite 7 days of clarithromycin (15 mg/kg/d) therapy. In 1 patient, quantitative polymerase chain reaction was 7.02 (log GEq/mL) at the onset of treatment, 6.26 at the end of treatment and remained positive with 2.64 and 2.69 during and after a second 7-day course, respectively. The generally believed assumption that contagiousness of pertussis is terminated after 5 days of antibiotic treatment should be challenged, at least in young infants.

Background In Germany, Iscucin® Populi (IP), a preparation from mistletoe growing on the poplar tree, is used in cancer therapy while Viscum Mali e planta tota (VM), a preparation from mistletoe growing on the apple tree, is used in patients with osteoarthritis. Since mistletoe preparations are suspected to induce production of potentially tumor promoting cytokines like interleukin (IL)-6, further studies on the immunological effects are of interest. Methods In this 3-armed randomized, double blind clinical trial healthy volunteers received increasing doses of either IP (strength F, 0.0125%, G, 0.25% and H, 5%, each for 4 weeks), or VM (1:1000 [D3], 1:100 [D2] and 2% each for 4 weeks) or placebo (isotonic solution) subcutaneously twice per week over a period of 12 weeks. Physical examination was performed weekly. Routine laboratory parameters and immunological parameters (C-reactive protein (CRP), differential blood count, lymphocyte subsets, immunoglobulins, IL-6 and tumor necrosis factor (TNF)-α) were analysed every 4 weeks. Results 71 subjects were included in the study (IP = 30, VM = 21, placebo = 20) of whom 69 concluded it according to protocol. Application of IP strengths G and H caused strong local reactions at the site of injection. In parallel, a distinct eosinophilia (p < 0.001 compared to placebo) occurred. Furthermore, application of all IP concentrations resulted in an increase of CD4 cell counts (p < 0.05) compared to placebo. Stimulation of IL-6 production, CRP or relevant deviations in other laboratory parameters were not observed. Because of local reactions, IP strengths G and H were considered less tolerable than placebo. VM 2% was slightly less tolerable than placebo, caused only mild local reactions and an only small increase in eosinophile counts. Conclusion Treatment with IP results in eosinophilia and an increase of CD4 cells but not in an increase of IL-6 or CRP. No safety concerns regarding the two mistletoe preparations have been raised by this study. EudraCT-Number 2007-002166-35. Trial registration ClinicalTrials.gov: NCT01378702

Group B streptococci (GBS) are a major cause of pneumonia, sepsis, and meningitis in newborns and infants. GBS initiate infection of the lung by colonizing mucosal surfaces of the respiratory tract; adherence of the bacteria to host cells is presumed to be the initial step in and prerequisite for successful colonization (G. S. Tamura, J. M. Kuypers, S. Smith, H. Raff, and C. E. Rubens, Infect. Immun. 62:2450-2458, 1994). We have performed a genome-wide screen to identify novel genes of GBS that mediate adherence to fibronectin. A shotgun phage display library was constructed from chromosomal DNA of a serotype Ia GBS strain and affinity selected on immobilized fibronectin. DNA sequence analysis of different clones identified 19 genes with homology to known bacterial adhesin genes, virulence genes, genes involved in transport or metabolic processes, and genes with yet-unknown function. One of the isolated phagemid clones showed significant homology to the gene (scpB) for the GBS C5a peptidase, a surface-associated serine protease that specifically cleaves the complement component C5a, a chemotaxin for polymorphonuclear leukocytes. In this work we have demonstrated that affinity-purified recombinant ScpB and a peptide ScpB fragment (ScpB-PDF), similar to the peptide identified in the phagemid, bound fibronectin in a concentration-dependent manner. Adherence assays to fibronectin were performed, comparing an isogenicscpBmutant to the wild-type strain. Approximately 50% less binding was observed with the mutant than with the wild-type strain. The mutant phenotype could be fully restored by intranscomplementation of the mutant with the cloned wild-typescpBgene, providing further evidence for the role of ScpB in fibronectin adherence. Our results suggest that C5a peptidase is a bifunctional protein, which enzymatically cleaves C5a and mediates adherence to fibronectin. Since binding of fibronectin has been implicated in attachment and invasion of eukaryotic cells by streptococci, our results may imply a second important role for this surface protein in the pathogenesis of GBS infections.

We have previously described a monoclonal antibody (mAb), 10C3, directed against the gene-3 protein (g3p) of filamentous phage M13, which was produced to study g3p fusion protein expression in Escherichia coli and its incorporation in the phage capsid [Tesar, M., Beckmann, C., Rottgen, P., Haase, B., Faude, U., Timmis, K., 1995. Monoclonal antibody against pIII of filamentous phage: an immunological tool to study pIII fusion protein expression in phage display systems. Immunology 1, 53-54]. In this study we report mapping of the antigenic epitope of the mAb 10C3, by means of short overlapping peptide-sequences [Frank, R., Overwin, H., 1996. Spot synthesis. In: Morris, G.E. (Ed.), Methods in Molecular Biology, Vol. 66: Epitope Mapping Protocols. Humana Press, Totowa, NJ, pp. 149-169.] comprising the C-terminal half of the g3-protein. A minimal recognizable peptide was found which is represented in the 11 amino acid sequence from positions 292 to 302 of g3p [Wezenbeek van, P.M.G.P., Hulsebos, T.J.M., Schoenmakers, J.G.G., 1980. Nucleotide sequence of the filamentous bacteriophage M13 DNA genome: comparison with phage fd. Gene 11, 129-148]. In order to use the antibody also for detection and purification of recombinant proteins, such as single chain antibodies, the epitope was introduced as a tag sequence into the phagemid pHEN1 [Hoogenboom, H.R., Griffith, A.D., Johnson, K., Chiswell, D.J., Hudson, P., Winter, G., 1991. Multi-subunit proteins on the surface of the filamentous phage: methodologies for displaying antibody (Fab) heavy and light chains. Nucleic Acid Res. 19, 4133-4137; Nissim, A., Hoogenboom, H.R., Tomlinson, I.M., Flynn, G., Midgley, C., Lane, D., Winter, G., 1994. Antibody fragments from a single pot phage display library as immunochemical reagents. EMBO J. 13 (3) 692-698]. Purified single chain antibodies containing this tag were detectable down to a concentration of 2 ng ml(-1) under non-denaturing conditions (ELISA) or 4 ng per lane on immunoblots. The high sensitivity of the antibody for the peptide tag was reflected in the antibody affinity constant K(D) of 6.80 x 10(-10) M, which was determined by real time biomolecular interaction analysis (BIA) based on surface plasmon resonance (SPR) [Karlsson, R., Falt, A., 1997. Experimental design for kinetic analysis of protein-protein interactions with surface plasmon resonance biosensors. J. Immunol. Methods 200, 121-133]. Finally, recombinant proteins in E. coli periplasmic extracts could be purified in a single step by affinity purification using immobilized mAb 10C3. These studies demonstrated that the new peptide-tag and its corresponding mAb represents a versatile tool for the detection of recombinant proteins selected by phage display technology.

A monoclonal antibody directed against the gene 3 product (pIII) of filamentous phage M13 was produced to study pIII-fusion protein expression in E. coli and its incorporation in the phage capsid. The protein was gel-purified from E. coli expression cultures harboring the genetic information of pill under the control of an inducible lac promoter. To study pIII-fusion protein expression, phage display systems were applied in which either the whole pIII or the C-terminal half was used (McCafferty et al. (1990) Nature (London) 348, 552–554; Szardenings and Collins (1990) Gene 94, 1–7; Barbas and Lerner (1991) In: Methods: Companion to Methods in Enzymology, Combinatorial Immunoglobulin Libraries on the Surface of Phage (Phabs): Rapid Selection of Antigen-Specific Fabs, Vol. 2, Academic Press, Orlando, pp. 119–124). In all cases, the monoclonal antibody was able to detect the native and the recombinant protein in E. coli and on the phage tip using non-denaturing (ELISA) and denaturing (SDS-PAGE, immunoblot analysis) conditions. All selected pIII-specific monoclonal antibodies were found to be directed against epitopes within amino acids 198 to 406 of pIII, which is necessary for capsid incorporation and therefore included in all pIII-mediated phage display designs.

The horizontal and spatial distribution of artificial and natural radionuclides in soils, and the vertical migration velocity of 0.8–2.0 cm per annum for caesium were examined in order to determine whether caesium is still in the root zone of major plants and, therefore, part of the food-chain. The vertical distribution patterns of caesium are dependent on the different physico-chemical soil properties. The soil samples were analysed with a pure Ge detector. In south-eastern Lower Saxony, the maximum gamma-activity for 137Cs of 33.3 kBq m–2 was found in a depth range of 0–20 cm (January 1, 1989). This was correlated with areas that had received heavy rainfall on May 4, 1986. On January 1, 1989 approximately 60% of the Chernobyl caesium was still in the top 0–2 cm of the topsoil.

Background Mistletoe preparations Iscucin ® Populi (IP) and Viscum Mali (VM, both WALA GmbH) are licensed in Germany as supportive cancer medication within the concept of Anthroposophical Medicine. Safety and immunological effects in humans of these preparations were for the first time investigated in this study. Methods A 3-armed randomized study in healthy volunteers was performed. The probands injected in increasing doses either IP (strength F, G and H, each for 4 weeks) or VM (strength D3, D2 and 2% each for 4 weeks) or placebo (isotonic solution) subcutaneously twice weekly for a total of 12 weeks. Clinical and safety controls were performed weekly. Immunological outcome parameters (differential blood count, lymphocyte differentiation, interleukin (IL)-6) were analysed every 4 weeks. Results A total of 71 probands were included (IP=30, VM=21, placebo=20), 69 finished the study regularly and were analysed. Application of IP strength G and H caused strong local reactions at the site of injection. In parallel they resulted in distinct eosinophilia ( p p Discussion and conclusion Eosinophilia as an effect of mistletoe-lectin (ML) containing mistletoe preparations is well known and was pronounced due to the high content of ML in IP strength G and H. Most of the probands did not tolerate the full dose of IP strength G and H due to local reactions. Repeated injections induced, however, a certain tolerance (smaller local reactions, less eosinophilia despite inreasing dosages), which was accompanied by induction of mistletoe lectin antibodies in all subjects receiving IP (data not shown). Therefore, IP strength G and H should in general be used only after pretreatment with lower dosages. For the first time there was an increase of T-helper cells during application of mistletoe preparations in a placebo-controlled study. This could be beneficial for the treatment of immunosuppressed cancer patients. Eosinophils play a role in host response against cancer and eosinophilia correlates with a better prognosis in a variety of cancer types. Whether eosinophilia during treatment with IP has a clinical benefit for cancer patients has to be further investigated. Both mistletoe preparations have been shown to be safe. VM induces only mild local reactions and is used as mild counter stimulant in patients with painful osteoarthritis.

Background: In December 2020, the United Kingdom (UK) reported a SARS-CoV-2 Variant of Concern (VoC) which is now named B.1.1.7. Based on initial data from the UK and later data from other countries, this variant was estimated to have a transmission fitness advantage of around 40–80 % (Volz et al., 2021; Leung et al., 2021; Davies et al., 2021). Aim: This study aims to estimate the transmission fitness advantage and the effective reproductive number of B.1.1.7 through time based on data from Switzerland. Methods: We generated whole genome sequences from 11.8 % of all confirmed SARS-CoV-2 cases in Switzerland between 14 December 2020 and 11 March 2021. Based on these data, we determine the daily frequency of the B.1.1.7 variant and quantify the variant's transmission fitness advantage on a national and a regional scale. Results: We estimate B.1.1.7 had a transmission fitness advantage of 43–52 % compared to the other variants circulating in Switzerland during the study period. Further, we estimate B.1.1.7 had a reproductive number above 1 from 01 January 2021 until the end of the study period, compared to below 1 for the other variants. Specifically, we estimate the reproductive number for B.1.1.7 was 1.24 [1.07–1.41] from 01 January until 17 January 2021 and 1.18 [1.06–1.30] from 18 January until 01 March 2021 based on the whole genome sequencing data. From 10 March to 16 March 2021, once B.1.1.7 was dominant, we estimate the reproductive number was 1.14 [1.00–1.26] based on all confirmed cases. For reference, Switzerland applied more non-pharmaceutical interventions to combat SARS-CoV-2 on 18 January 2021 and lifted some measures again on 01 March 2021. Conclusion: The observed increase in B.1.1.7 frequency in Switzerland during the study period is as expected based on observations in the UK. In absolute numbers, B.1.1.7 increased exponentially with an estimated doubling time of around 2–3.5 weeks. To monitor the ongoing spread of B.1.1.7, our plots are available online., Epidemics, 37, ISSN:1878-0067, ISSN:1755-4365