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Correspondence: Susanne Pfefferle (s.pfefferle@uke.de) Citation style for this article: Pfefferle Susanne , Reucher Svenja , Nörz Dominic , Lütgehetmann Marc . Evaluation of a quantitative RT-PCR assay for the detection of the emerging coronavirus SARS-CoV-2 using a high throughput system. Euro Surveill. 2020;25(9):pii=2000152. https://doi.

The extent of monkeypox virus environmental contamination of surfaces is unclear. We examined surfaces in rooms occupied by two monkeypox patients on their fourth hospitalisation day. Contamination with up to 105 viral copies/cm2 on inanimate surfaces was estimated by PCR and the virus was successfully isolated from surfaces with more than 106 copies. These data highlight the importance of strict adherence of hospital staff to recommended protective measures. If appropriate, pre-exposure or early post-exposure vaccination should be considered for individuals at risk.

Highlights Clinical performance of five different commercially available automated SARS-CoV-2 antibody tests. No overlap of “false” positive samples between different serology assays was observed. The ability to rule out acute SARS-CoV-2 infection at hospital admission with serology is limited.

Cholangiocarcinoma (CCA) is a rare, but devastating disease arising from the epithelium of intrahepatic and extrahepatic bile ducts. There are neither effective systemic therapies nor satisfying treatment options for inoperable CCA. Histopathological and biochemical studies of CCA show frequent dysregulation of the phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin (mTOR) pathway. Therefore, we investigated the efficacy of the mTOR inhibitor RAD001 and the impact of AKT signaling following mTOR inhibition in the treatment of CCA. RAD001 significantly inhibits proliferation of CCA cell lines, however, a concentration-dependent and isoform specific feedback activation of the three AKT isoforms (AKT1, AKT2 and AKT3) was observed after mTOR inhibition. As activation of AKT might limit the RAD001-mediated anti-tumor effect, the efficacy of combined mTOR and AKT inhibition was investigated using the allosteric AKT inhibitor MK-2206. Our results show that inhibition of AKT potentiates the efficacy of mTOR inhibition both in vitro and in a xenograft mouse model in vivo. Mechanistically, the antiproliferative effect of the pan-AKT inhibitor MK2206 in the CCA cell line TFK-1 was due to inhibition of AKT1 and AKT2, because knockdown of either AKT1 or AKT2, but not AKT3, showed a synergistic reduction of cell proliferation in combination with mTOR treatment. Finally, using an AKT isoform specific in vitro kinase assay, enzymatic activity of each of the three AKT isoforms was detected in all tissue samples from CCA patients, analyzed. In summary, our preclinical data suggest that combined targeting of mTOR and AKT using RAD001 and MK-2206 might be a new, effective strategy for the treatment of CCA.Cholangiocarcinoma (CCA) is a devastating disease that arises from the epithelium of intrahepatic and extrahepatic biliary ducts. Although CCA account for only 3% of all gastrointestinal cancers, CCA represent the second most frequent primary hepatic malignancy after hepatocellular carcinoma, and a progressively increasing incidence was observed worldwide in the last decades. 1,2 Surgical resection is the only chance for cure, but even after careful resection, local recurrence is frequent. 3 Yet, no systemic therapy was shown to be effective. Therefore, prognosis of patients with CCA is extremely dismal, with less than 5% of all patients, and only 22-36% of patients undergoing surgical resection surviving 5 years. 1,4 Thus, new and effective treatment options are urgently needed.The phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway plays a central role in regulation of tumor cell proliferation, migration, survival and angiogenesis. 5 A frequent dysregulation of AKT and mammalian target of rapamycin (mTOR) in up to 60% of intrahepatic and 80% of extrahepatic CCA has been reported, and a correlation between poor survival and phosphorylated AKT in patients with extrahepatic CCA was shown. 6,7 Therefore, the PI3K/AKT/mTOR pathway represents a promising target for new treatment strategies.

BackgroundTreatment of breast cancer patients with distant metastases represents one of the biggest challenges in today’s gynecological oncology. Therefore, a better understanding of mechanisms promoting the development of metastases is of paramount importance. The serine/threonine kinase AKT was shown to drive cancer progression and metastasis. However, there is emerging data that single AKT isoforms (i.e. AKT1, AKT2 and AKT3) have different or even opposing functions in the regulation of cancer cell migration in vitro, giving rise to the hypothesis that inhibition of distinct AKT isoforms might have undesirable effects on cancer dissemination in vivo.MethodsThe triple negative breast cancer cell line MDA-MB-231 was used to investigate the functional roles of AKT in migration and metastasis. AKT single and double knockdown cells were generated using isoform specific shRNAs. Migration was analyzed using live cell imaging, chemotaxis and transwell assays. The metastatic potential of AKT isoform knockdown cells was evaluated in a subcutaneous xenograft mouse model in vivo.ResultsDepletion of AKT3, but not AKT1 or AKT2, resulted in increased migration in vitro. This effect was even more prominent in AKT2,3 double knockdown cells. Furthermore, combined downregulation of AKT2 and AKT3, as well as AKT1 and AKT3 significantly increased metastasis formation in vivo. Screening for promigratory proteins revealed that downregulation of AKT3 increases the expression of S100A4 protein. In accordance, depletion of S100A4 by siRNA approach reverses the increased migration induced by knockdown of AKT3.ConclusionsWe demonstrated that knockdown of AKT3 can increase the metastatic potential of triple negative breast cancer cells. Therefore, our results provide a rationale for the development of AKT isoform specific inhibitors.

IMPORTANCECurrent recommendations are to avoid tissue for corneal transplant from donors with coronavirus disease 2019 or those who were recently exposed to COVID-19 owing to the lack of knowledge about the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in corneal tissues. Evidence of SARS-CoV-2 in corneal tissue would seem to have clinical relevance for corneal transplant.OBJECTIVES To investigate the presence of viral SARS-CoV-2 RNA in corneal discs of deceased patients with confirmed COVID-19 and assess viral genomic and subgenomic RNA load, possible infectivity, and histologic abnormalities. DESIGN, SETTING, AND PARTICIPANTSA case series was conducted of 11 deceased patients with COVID-19 who underwent autopsy between March 20 and May 14, 2020. Eleven corneal discs (1 corneal disc per patient) were harvested for molecular detection of viral genomic and subgenomic RNA, virus isolation, and immunohistochemistry. The SARS-CoV-2 RNA loads were compared with RNA loads in the conjunctival and throat swab samples and aqueous humor, vitreous humor, and blood samples. MAIN OUTCOMES AND MEASURES Evidence of SARS-CoV-2 RNA in human corneas.RESULTS This study comprised 11 patients (6 women [55%]; mean [SD] age, 68.5 [18.8] years). In 6 of 11 eyes (55%), SARS-CoV-2 genomic RNA was detected in the cornea; subgenomic RNA was present in 4 of these 6 eyes (67%). Infectivity or the presence of viral structural proteins could not be confirmed in any eye. However, patients whose corneal disc was positive for SARS-CoV-2 RNA also had positive results for SARS-CoV-2 RNA in 4 of 6 conjunctival swab samples, 1 of 3 aqueous humor samples, 3 of 5 vitreous humor samples, and 4 of 5 blood samples. Overall, conjunctival swab samples had positive results for SARS-CoV-2 RNA in 5 of 11 cases. Postmortem SARS-CoV-2 viremia was detected in 5 of 9 patients.CONCLUSIONS AND RELEVANCE Viral genomic and subgenomic RNA of SARS-CoV-2 was detected in the cornea of patients with COVID-19 viremia. The risk of COVID-19 infection via corneal transplant is low even in donors with SARS-CoV-2 viremia, but further research is necessary to assess the rate of SARS-CoV-2 transmission via corneal transplant.

Clinical characterization of COVID-19 (Corona Virus Disease 2019) is being performed worldwide to gain insights into the pathogenesis and course of the disease. Little is known regarding morphological findings, which are essential to understanding the unique features and pathomechanisms of the disease, from which one can identify possible new treatments. It has been shown that diffuse alveolar damage, signifying acute respiratory distress syndrome, is present together with atypical multinucleated cells in reported cases of the disease by Tian et al. (J Thorac Oncol 15:700-704, 2020). Macroscopic morphological findings in COVID-19 remain elusive to this day. Here, we report the case of the first German to die due to COVID-19. A detailed examination consisting of full-body computed tomography, autopsy, histology assessment, and viral assessment has been performed. The lungs of the deceased contained high concentrations of SARS-CoV-2 RNA and displayed the typical radiological signatures of COVID-19. Furthermore, a morphological pattern was found displaying hyperaemic areas interspersed by normally perfused areas affecting the whole lung. We also report a finding suggestive of micro-thrombotic events in the lung, which is compatible with the recently described coagulation changes and increased incidence of pulmonary artery embolisms seen in COVID-19 patients as reported by Wichmann et al. (Ann Intern Med, 2020). A broader study is needed to confirm these findings.