Direct comparison of Altona-SARS-CoV-2 dual target RT-qPCR Assay with commercial LAMP Assay using throat washes in health care staff testing.

Background: Rapid molecular diagnostics by PCR has a crucial role in handling the global SARS-CoV-2 pandemic. As diagnoses are time-sensitive and global supply chains are susceptible to various factors alternative detection methods would be an important backup. Objectives: During the study the performance of a commercially available isothermal LAMP method for SARS-CoV-2 detection was compared to a IVD RT-PCR Assays using throat wash specimens that were routinely taken in our hospital setting. Study design: Throat wash specimens of hospital staff (n = 174) previously tested positive for SARS-CoV-2 by the Altona Diagnostics RealStar SARS-CoV-2 RT-PCR (Altona Diagnostics, Hamburg, Germany) was tested for SARS-CoV-2 also by the SARS-CoV-2 Rapid Colorimetric LAMP Assay (NEB Germany GmbH, Frankfurt a.M., Germany). Results: The sensitivity of the colorimetric LAMP Assay compared to RT-qPCR was 78.74%, and the specificity was determined to 88.24% with a positive predictive value of 0.986 and a negative predicitve value of 0.882. The positive and negative likelihood ratio for LAMP was 6.693 and 0.241, respectively, while the diagnostic odds ratio was 27.77. Conclusions: In times of limited PCR test ressources and in settings with limited PCR capacities, the colorimetric LAMP Assay could serve as an alternative, if a calculable loss of sensitivity is acceptable from the Public Health perspective in certain settings.

SARS-CoV-2, CT-Values, and Infectivity-Conclusions to Be Drawn from Side Observations.

In their recent article published in Viruses, Michel Drancourt and colleagues [...].

Renal transplantation after recovery from COVID-19 - a case report with implications for transplant programs in the face of the ongoing corona-pandemic.

BACKGROUND: The ongoing coronavirus pandemic has major impacts on both patients and healthcare systems worldwide, thus creating new realities. Patients on maintenance dialysis listed for renal transplantation are a vulnerable subgroup with many comorbidities and recurring contacts with the healthcare system. Due to the COVID-19 pandemic transplant numbers have dropped considerably, further increasing waiting times in this high-risk population. On the other hand, knowledge of the severity of SARS-CoV-2 infection in immunocompromised patients, development and persistence of neutralising antibodies in such patients is just emerging. It is unclear how best to address the dilemma of postponing the life-saving transplantation. CASE PRESENTATION: We present a case report of a successful kidney transplantation only 65 days after the recipient was hospitalized for treatment of COVID-19 pneumonia. In a follow up of 9 months, we observed no signs of recurrent disease and transplant function is excellent. Monitoring SARS-CoV-2 antibody response demonstrates stable IgG levels. CONCLUSION: This reassuring case provides guidance to transplant centers how to proceed with kidney transplantation safely during the pandemic. Careful consideration of risks and benefits of the organ offer, full recovery from COVID-19 symptoms and the presence of a positive SARS-CoV-2 IgG antibody test, qualifies for kidney transplantation.

Correction: Schildgen, V., et al. Human Bocavirus Infection of Permanent Cells Differentiated to Air-Liquid Interface Cultures Activates Transcription of Pathways Involved in Tumorigenesis. Cancers 2018, 10, 410.

The authors wish to make the following correction to this paper [...].

Comparison of Biocartis IDYLLA ™ cartridge assay with Qiagen GeneReader NGS for detection of targetable mutations in EGFR, KRAS/NRAS, and BRAF genes.

Lung and colorectal cancers (CRC) have two of the highest mortality rates among all cancer types, and their occurrence and the need for personalized diagnostics and subsequent therapy were not influenced by the COVID-19 pandemics. However, due to the disruption of established delivery chains, standard assays for in vitro diagnostics of those cancers were temporarily not available, forcing us to implement alternative testing methods that enabled at least basic therapy decision making. For this reason, we evaluated rapid testing on the Biocartis Idylla™ platform (Biocartis, Mechelen, Belgium) for four important genes commonly mutated in lung and colorectal cancers, namely EGFR, NRAS, KRAS, and BRAF. Clinical specimens from which the mutation status has previously been determined using Next Generation Sequencing (NGS), were retested to determine whether Idylla™ can offer accurate results. To compare the results, the sensitivity, specificity, positive predictive values (PPV) and negative predictive values (NPV) are calculated for each of the mutation types and then combined to determine the values of the Idylla™ system in total, while setting NGS as the gold-standard basis the assays were compared with. Idylla testing thereby displayed acceptable sensitivity and specificity and delivered reliable results for initial therapy decisions.

Differential cytology profiles in bronchoalveolar lavage (BAL) in COVID-19 patients: A descriptive observation and comparison with other corona viruses, Influenza virus, Haemophilus influenzae, and Pneumocystis jirovecii.

ABSTRACT: Brochoalvelolar lavages (BALs) from patients suffering from hospitalized infections with SARS-CoV-2, other corona viruses (human coronavirus (HCoV)-229E, HCoV-OC43, HCoV-NL63, and HCoV-HKU1), Influenza virus type A and B, Haemophilus influenzae and Pneumocystis jirovecii were compared cytopathologically.The aim of the study was to evaluate if the cellular profile detectable in BAL may be specific for the respective pathogens and could lead to diagnosis of COVID-19 even in the absence of PCR results.Differential cytology and flow cytometry datasets of 62 patients were observed and compared.We observed a significant association between individual cell pattern changes and the causing pathogen, but no general cell distribution pattern.The cytology pattern of the BAL fluid in COVID-19 is not specific enough to use it as a sole diagnostic criterion, although it may support clinical decision making.

Limits and Opportunities of SARS-CoV-2 Antigen Rapid Tests: An Experienced-Based Perspective.

BACKGROUND: Due to the steadily rising case numbers of SARS-CoV-2 infections worldwide, there is an increasing need for reliable rapid diagnostic devices in addition to existing gold standard PCR methods. Actually, public attention is focused on antigen assays including lateral flow tests (LFTs) as a diagnostic alternative. Therefore, different LFTs were analyzed regarding their performance in a clinical setting. MATERIAL AND METHODS: A pilot sample panel of 13 bronchoalveolar fluids (BALFs) and 60 throat washing (TW) samples with confirmed PCR results, as well as eight throat washes invalid by PCR, were tested with the BIOCREDIT test (RapiGEN), the PanbioTM assay (Abbott), and the SARS-CoV-2 rapid antigen test (Roche). CONCLUSION: The analyzed antigen test showed an interassay correlation of 27.4%, with overall specificities ranging from 19.4% to 87.1%, while sensitivities of the respective tests ranged between 33.3% and 88.1%. Because these assays did not entirely meet all high expectations, their benefit has to be carefully evaluated for the respective test strategy and setting.

Predictive molecular pathology in the time of coronavirus disease (COVID-19) in Europe.

AIMS: Lung cancer predictive biomarker testing is essential to select advanced-stage patients for targeted treatments and should be carried out without delays even during health emergencies, such as the coronavirus (COVID-19) outbreak. METHODS: Fifteen molecular laboratories from seven different European countries compared 4 weeks of national lockdown to a corresponding period in 2019, in terms of tissue and/or plasma-based molecular test workload, analytical platforms adopted, number of cases undergoing programmed death-ligand1 (PD-L1) expression assessment and DNA-based molecular tests turnaround time. RESULTS: In most laboratories (80.0%), tissue-based molecular test workload was reduced. In 40.0% of laboratories (6/15), the decrease was >25%, and in one, reduction was as high as 80.0%. In this instance, a concomitant increase in liquid biopsy was reported (60.0%). Remarkably, in 33.3% of the laboratories, real-time PCR (RT-PCR)-based methodologies increased, whereas highly multiplexing assays approaches decreased. Most laboratories (88.9%) did not report significant variations in PD-L1 volume testing. CONCLUSIONS: The workload of molecular testing for patients with advanced-stage lung cancer during the lockdown showed little variations. Local strategies to overcome health emergency-related issues included the preference for RT-PCR tissue-based testing methodologies and, occasionally, for liquid biopsy.

Novel Chimeric Gene Therapy Vectors Based on Adeno-Associated Virus and Four Different Mammalian Bocaviruses.

Parvoviruses are highly attractive templates for the engineering of safe, efficient, and specific gene therapy vectors, as best exemplified by adeno-associated virus (AAV). Another candidate that currently garners increasing attention is human bocavirus 1 (HBoV1). Notably, HBoV1 capsids can cross-package recombinant (r)AAV2 genomes, yielding rAAV2/HBoV1 chimeras that specifically transduce polarized human airway epithelia (pHAEs). Here, we largely expanded the repertoire of rAAV/BoV chimeras, by assembling packaging plasmids encoding the capsid genes of four additional primate bocaviruses, HBoV2-4 and GBoV (Gorilla BoV). Capsid protein expression and efficient rAAV cross-packaging were validated by immunoblotting and qPCR, respectively. Interestingly, not only HBoV1 but also HBoV4 and GBoV transduced pHAEs as well as primary human lung organoids. Flow cytometry analysis of pHAEs revealed distinct cellular specificities between the BoV isolates, with HBoV1 targeting ciliated, club, and KRT5+ basal cells, whereas HBoV4 showed a preference for KRT5+ basal cells. Surprisingly, primary human hepatocytes, skeletal muscle cells, and T cells were also highly amenable to rAAV/BoV transduction. Finally, we adapted our pipeline for AAV capsid gene shuffling to all five BoV isolates. Collectively, our chimeric rAAV/BoV vectors and bocaviral capsid library represent valuable new resources to dissect BoV biology and to breed unique gene therapy vectors.

Oncotype DX Breast Cancer recurrence score resists inter-assay reproducibility with RT2-Profiler Multiplex RT-PCR.

The Oncotype Dx assay is frequently used to test if breast cancer patients can be spared from chemotherapy without negative effects for their future clinical course. However, due to conflicting data on the assay utility, in the recent past its reimbursement situation in Germany was revised; due to continued requests by clinicians for predictive values, our group decided to implement an Oncotype Dx like alternative assay with the objective of obtaining quality and cost optimization. Customized RT2-Profiler assays covering the 21 gene panel of the Oncotype Dx assay were applied to a pilot cohort of breast cancer patients with known Oncotype Dx Recurrence Score (RS). The Ct values obtained with RT2-Profiler-assays were used to calculate the unscaled Recurrence Score (RSu) values and the thereon based RS according to the Oncotype DX assay rules if available. Despite consistent assay performance it was impossible to establish correlations between RT2-Profiler recurrence scores with the respective Oncotype DX values not to mention exact matches. By following the Oncotype DX assay and its interpretation as close as possible we faced several obstructions such as lack of information on RNA amount used, missing units in the single gene expression report, missing references cited in the original study that should explain the determination of the recurrence score formula, and vague information on the normalization of the gene expression impeding the reproduction of Oncotype Dx results in other laboratories. Unfortunately, the Oncotype Dx assay cannot be confirmed by the customized RT2-profiler assay, not least because of the fact that the individual gene measurements are not provided in the medical report, although these are mandatory for the RS calculation. In fact, the "single gene report" only contains unscaled scores of the ER, PR, and Her2 genes without any internationally accepted unit used to describe a transcript quantity. Therefore a direct comparison with the in-house measurement to evaluate its performance is impossible. With regard to our findings and the fact that the Oncotype RS represents a likelihood of the risk of relapse it thus remains impossible to assess the clinical necessity of this assay.

Human bocavirus is detected in human placenta and aborted tissues.

BACKGROUND: To date, four human bocaviruses (HBoV) have been described. The most closely related viruses (bovine and canine parvoviruses) are associated with miscarriage in their hosts. The objective of this retrospective study was to determine the frequency of HBoV DNA in miscarriage. STUDY DESIGN: Tissue samples from 172 patients, in which miscarriage occurred, were included and tested with a published qPCR protocol. Positive PCRs were mutually confirmed by sequencing. RESULTS: 43 patients (25%) were positive for HBoV DNA. Of those, the majority of HBoV-positive samples were tissues from miscarriage (placenta: 6; aborted tissue products of conception: 37 specimens). The samples were not paired; either placental or aborted tissue was available. CONCLUSIONS: The results show that, as long as no animal model is available, the role of HBoV in the occurrence of miscarriage requires additional prospective studies in order to investigate its significance and causal involvements of this pathogen.

The lonely driver or the orchestra of mutations? How next generation sequencing datasets contradict the concept of single driver checkpoint mutations in solid tumours - NSCLC as a scholarly example.

Driver mutations are considered to be responsible for the majority of cancers and several of those mutations provide targets in order to set up personalized therapies. So far the generally accepted opinion had been that driver mutations occur as stand-alone factors, but novel sequencing technologies induced an essential rethink. Next generation sequencing approaches have shown that double, triple or multiple concurrent mutations could occur within the same tumour and may by interaction influence sensitivity to anticancer drugs and therapy success. This review focusses on this novel concept and discusses the challenges for molecular pathology and laboratory diagnostics while providing putative solutions to overcome the present pitfalls, thereby taking NSCLC as an example.

Association of the Human Bocavirus With Tonsil Squamous Cell Carcinomas.

Background: The human bocavirus (HBoV) is known to persist latently in the infected host cells and seems to replicate its DNA via the DNA damage response system, which is frequently defect in tumors and correlates with microsatellite instability (MSI). Because HBoV is able to persist in the infected tissues, induces pro-fibrotic and pro- cancerogenic cytokines in vivo and in vitro, and is detected in colorectal and lung tumors, the virus may be involved in cancerogenesis at least as a cofactor. Recently it was shown that the adenotonsillar tissue is an important site of HBoV1 persistence and replication. Considering the background that approximately 60% of oropharyngeal cancers were thought to be attributable to a HPV infection, a co-participation of HBoV in terms of a chronic virus infection might play a role in the cancerogenesis of tonsil tumors. Methods: Formalin-fixed, paraffin-embedded tonsil tumor samples were screened for HBoV and HPV DNA. Positive tissue sections were afterward subjected to fluorescence in situ hybridization (FISH) analysis to identify HBoV and HPV infected cells. By use of an in vitro cell culture model with primary tonsil fibroblasts, keratinocytes, and lymphocytes infected by HBoV we tried to find the target cells of virus replication. MSI testing was based on a previously published protocol using a de-multiplexed PCR followed by fluorescent detection of PCR products in a capillary sequencing device. Results: In total 62 of 103 (60, 19%) of the tonsil squamous cell carcinomas tested positive for HBoV DNA and 66 of 103 (66%) samples were identified as HPV positive. The FISH analysis revealed both double infection of HPV and HBoV in the same cells as well as single infections of both viruses within the tumor tissue. Twenty-two of 62 HBoV positive tumors tested HPV negative, 40 of 62 tissue sections were HBoV and HPV positive. We analyzed 21 out of the 62 HBoV positive tumors for MSI. Of those four tonsils displayed MSI in at least 1 of 10 microsatellite markers. Conclusion: Our findings support the hypothesis that human bocavirus infections as a cofactor may have an impact on tumor development in tonsils, although it still remains possible that HBoV solely displays a tumor tropism.

Human Bocavirus Infection of Permanent Cells Differentiated to Air-Liquid Interface Cultures Activates Transcription of Pathways Involved in Tumorigenesis.

The parvoviral human bocavirus (HBoV) is a respiratory pathogen, able to persist in infected cells. The viral DNA has been identified in colorectal and lung tumors and thus it was postulated that the virus could be associated with tumorigenesis. This assumption was supported by the fact that in HBoV-infected patients and in an in vitro cell culture system, pro-cancerogenic and -fibrotic cytokines were expressed. In this work, it is shown by a whole transcriptome analysis that, also at the mRNA level, several pathways leading to neoplasia and tumorigenesis are significantly upregulated. In total, a set of 54 transcripts are specifically regulated by HBoV, of which the majority affects canonical pathways that may lead to tumor development if they become deregulated. Moreover, pathways leading to necrosis, apoptosis and cell death are downregulated, supporting the hypothesis that HBoV might contribute to the development of some kinds of cancer.