Background In December, 2019, the newly identified severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, China, causing COVID-19, a respiratory disease presenting with fever, cough, and often pneumonia. WHO has set the strategic objective to interrupt spread of SARS-CoV-2 worldwide. An outbreak in Bavaria, Germany, starting at the end of January, 2020, provided the opportunity to study transmission events, incubation period, and secondary attack rates.Methods A case was defined as a person with SARS-CoV-2 infection confirmed by RT-PCR. Case interviews were done to describe timing of onset and nature of symptoms and to identify and classify contacts as high risk (had cumulative face-to-face contact with a confirmed case for ≥15 min, direct contact with secretions or body fluids of a patient with confirmed COVID-19, or, in the case of health-care workers, had worked within 2 m of a patient with confirmed COVID-19 without personal protective equipment) or low risk (all other contacts). High-risk contacts were ordered to stay at home in quarantine for 14 days and were actively followed up and monitored for symptoms, and low-risk contacts were tested upon self-reporting of symptoms. We defined fever and cough as specific symptoms, and defined a prodromal phase as the presence of non-specific symptoms for at least 1 day before the onset of specific symptoms. Whole genome sequencing was used to confirm epidemiological links and clarify transmission events where contact histories were ambiguous; integration with epidemiological data enabled precise reconstruction of exposure events and incubation periods. Secondary attack rates were calculated as the number of cases divided by the number of contacts, using Fisher's exact test for the 95% CIs.Findings Patient 0 was a Chinese resident who visited Germany for professional reasons. 16 subsequent cases, often with mild and non-specific symptoms, emerged in four transmission generations. Signature mutations in the viral genome occurred upon foundation of generation 2, as well as in one case pertaining to generation 4. The median incubation period was 4•0 days (IQR 2•3-4•3) and the median serial interval was 4•0 days (3•0-5•0). Transmission events were likely to have occurred presymptomatically for one case (possibly five more), at the day of symptom onset for four cases (possibly five more), and the remainder after the day of symptom onset or unknown. One or two cases resulted from contact with a case during the prodromal phase. Secondary attack rates were 75•0% (95% CI 19•0-99•0; three of four people) among members of a household cluster in common isolation, 10•0% (1•2-32•0; two of 20) among household contacts only together until isolation of the patient, and 5•1% (2•6-8•9; 11 of 217) among non-household, high-risk contacts.Interpretation Although patients in our study presented with predominately mild, non-specific symptoms, infectiousness before or on the day of symptom onset was substantial. Additionally, the incubation period was often very short ...

The need for timely establishment of diagnostic assays arose when Germany was confronted with the first travel-associated outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Europe. We describe our laboratory experiences during a large contact tracing investigation, comparing previously published real-time RT-PCR assays in different PCR systems and a commercial kit. We found that assay performance using the same primers and probes with different PCR systems varied and the commercial kit performed well.At the end of December 2019, an outbreak caused by a novel coronavirus was announced in Wuhan, China. Since then, the number of cases has increased, especially in China but also in other countries, and public health authorities are in need to rapidly implement diagnostic tools. In this paper, we describe our laboratory experiences with the novel real-time RT-PCR assays comparing different one-step PCR systems and a commercial kit, using a Bio-Rad CFX 96 cycler.

Background Fast, reliable and easy to handle methods are required to facilitate urgently needed point-of-care testing (POCT) in the current coronavirus pandemic. Life-threatening severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly spread all over the world, infecting more than 33,500,000 people and killing over 1 million of them as of October 2020. Infected individuals without any symptoms might still transfer the virus to others underlining the extraordinary transmissibility of this new coronavirus. In order to identify early infections effectively, treat patients on time and control disease spreading, rapid, accurate and onsite testing methods are urgently required. Results Here we report the development of a loop-mediated isothermal amplification (LAMP) based method to detect SARS-CoV-2 genes ORF8 and N directly from pharyngeal swab samples. The established reverse transcription LAMP (RT-LAMP) assay detects SARS-CoV-2 directly from pharyngeal swab samples without previous time-consuming and laborious RNA extraction. The assay is sensitive and highly specific for SARS-CoV-2 detection, showing no cross reactivity when tested on 20 other respiratory pathogens. The assay is 12 times faster and 10 times cheaper than routine reverse transcription real-time polymerase chain reaction, depending on the assay used. Conclusion The fast and easy to handle RT-LAMP assay amplifying specifically the genomic regions ORF8 and N of SARS-CoV-2 is ideally suited for POCT at e.g. railway stations, airports or hospitals. Given the current pandemic situation, rapid, cost efficient and onsite methods like the here presented RT-LAMP assay are urgently needed to contain the viral spread.

Currently, no method allows direct and quantitative comparison of MHC-presented peptides in pairs of samples, such as transfected and untransfected, tumorous and normal or infected and uninfected tissues or cell lines. Here we introduce two approaches that use isotopically labeled reagents to quantify by mass spectrometry the ratio of peptides from each source. The first method involves acetylation and is both fast and simple. However, higher peptide recoveries and a finer sensitivity are achieved by the second method, which combines guanidination and nicotinylation, because the charge state of peptides can be maintained. Using differential acetylation, we identified a beta catenin-derived peptide in solid colon carcinoma overpresented on human leucocyte antigen-A (HLA-A)(*)6801. Guanidination/nicotinylation was applied to keratin 18-transfected cells and resulted in the characterization of the peptide RLASYLDRV (HLA-A(*)0201), exclusively presented on the transfectant. Thus, we demonstrate methods that enable a pairwise quantitative comparison leading to the identification of overpresented MHC ligands.

Probiotics are viable microorganisms that are increasingly used for treatment of a variety of diseases. Occasionally, however, probiotics may have adverse clinical effects, including septicemia. Here we examined the role of the intestinal microbiota and the adaptive immune system in preventing translocation of probiotics (e.g., Escherichia coli Nissle). We challenged C57BL/6J mice raised under germfree conditions (GF-raised C57BL/6J mice) and Rag1 ؊/؊ mice raised under germfree conditions (GF-raised Rag1 ؊/؊ mice) and under specific-pathogen-free conditions (SPF-raised Rag1 ؊/؊ mice) with probiotic E. coli strain Nissle 1917, strain Nissle 1917 mutants, the commensal strain E. coli mpk, or Bacteroides vulgatus mpk. Additionally, we reconstituted Rag1 ؊/؊ mice with CD4 ؉ T cells. E. coli translocation and dissemination and the mortality of mice were assessed. In GF-raised Rag1 ؊/؊ mice, but not in SPF-raised Rag1 ؊/؊ mice or GF-raised C57BL/6J mice, oral challenge with E. coli strain Nissle 1917, but not oral challenge with E. coli mpk, resulted in translocation and dissemination. The mortality rate was significantly higher for E. coli strain Nissle 1917-challenged GF-raised Rag1 ؊/؊ mice (100%; P < 0. 001) than for E. coli strain Nissle 1917-challenged SPF-raised Rag1 ؊/؊ mice (0%) and GF-raised C57BL/6J mice (0%). Translocation of and mortality due to strain E. coli Nissle 1917 in GF-raised Rag1؊/؊ mice were prevented when mice were reconstituted with T cells prior to strain E. coli Nissle 1917 challenge, but not when mice were reconstituted with T cells after E. coli strain Nissle 1917 challenge. Cocolonization experiments revealed that E. coli mpk could not prevent translocation of strain E. coli Nissle 1917. Moreover, we demonstrated that neither lipopolysaccharide structure nor flagella play a role in E. coli strain Nissle 1917 translocation and dissemination. Our results suggest that if both the microbiota and adaptive immunity are defective, translocation across the intestinal epithelium and dissemination of the probiotic E. coli strain Nissle 1917 may occur and have potentially severe adverse effects. Future work should define the possibly related molecular factors that promote probiotic functions, fitness, and facultative pathogenicity.