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.

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.

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.

Designer nuclease-mediated gene correction via homology-directed repair in an in vitro model of canine hemophilia B.

BACKGROUND: Gene correction at specific target loci provides a powerful strategy for overcoming genetic diseases. In the present study, we aimed to use an in vitro model for canine hemophilia B containing a single point mutation in the catalytic domain of the canine coagulation factor IX (cFIX) gene. To correct the defective gene via homology-directed repair (HDR), we designed transcription-activator like effector nucleases and clustered regularly interspaced short palindromic repeats including Cas9 (CRISPR/Cas9) for introduction of double-strand breaks at the mutation site. METHODS: To generate a stable cell line containing the mutated cFIX locus, a 2-kb genomic DNA fragment derived from a hemophilia B dog was amplified and integrated utilizing the phiC31 integrase system. Designer nucleases were assembled and cloned into vectors for constitutive and inducible expression. To detect mutations, insertions and deletions, and HDR events after nuclease treatment T7E1 assays, an amplification-refractory mutation system-quantitative polymerase chain reaction and pyrosequencing were performed. RESULTS: To perform HDR correction experiments, we established a cell line carrying the mutated cFIX locus. In HDR approaches we either explored a wild-type or an optimized cFIX sequence and we found that our modified HDR cassette showed higher gene correction efficiencies of up to 6.4%. Furthermore, we compared inducible and constitutive designer nuclease expression systems and found that the inducible system resulted in comparable HDR efficiencies. CONCLUSIONS: In conclusion, the present study demonstrates the potential of this strategy for gene therapeutic approaches in vitro and in a canine model for hemophilia B.

Microsatellite instability testing in colorectal cancer using the QiaXcel advanced platform.

BACKGROUND: Microsatellite instability (MSI) is a major predictive and diagnostic marker in several cancers including colorectal carcinomas. Diagnostic testing for microsatellites is generally performed using capillary sequencers, which requires expensive high-end equipment including expensive chemistry using fluorescent dyes labelling the PCR products of interest. In this study we have modified such a diagnostic protocol and established the microsatellite testing on the QiaXcel Advanced platform. METHODS: MSI testing was based on a previously established protocol describing a multiplex PCR followed by fluorescent detection of PCR products in a capillary sequencing device. Ten microsatellites were included in the new protocol: BAT25, BAT26, BAT40, D2s123, D10s197, D13s153, D17s250, D18s58, D5s346, and MycI. In this protocol the PCR was demultiplexed and established on the QiaXcel Advanced system (Qiagen, Hilden, Germany). RESULTS: Making use of a series of FFPE control samples with known MSI status including those with and without MSI a protocol for MSI testing was successfully established on the QiaXcel Advanced platform. CONCLUSIONS: MSI testing for human colorectal cancers using the QiaXcel Advanced system could serve as an economic acceptable tool for rapid diagnostics in laboratories that do not have access to a capillary sequencing unit.

Pre-clinical validation of a next generation sequencing testing panel.

OBJECTIVE: Next Generation Sequencing (NGS) has become a useful tool for gene mutation testing which is required for targeted therapies. The aim of this study was to validate the GeneRead QIAact Actionable Insights Tumor Panel (Qiagen) on the GeneReader System in a diagnostic laboratory setting. METHODS: The GeneRead QIAact Actionable Insights Tumor Panel allows the analysis of 773 variant positions in 12 genes (ALK, BRAF, EGFR, ERBB2, ERBB3, ESR1, KIT, KRAS, NRAS, PDGFRA, PIK3CA and RAF1). For the validation of the panel we used a commercial available multiplex reference standard carrying 11 mutations in defined positions, samples from interlaboratory tests, and FFPE tumor samples from patients which were tested previously for mutations in KRAS, NRAS, BRAF, EGFR, KIT, and/or PDGFRA with pyrosequencing. RESULTS: Among the 122 tested samples, 121 samples (99.2%) were successfully sequenced. The sensitivity and specificity for detecting variants was 100% and results proved to be reproducible and precise. 119 (98.3%) results were concordant to the expected results. The differences between NGS and pyrosequencing observed in two samples were due to a wrong analysis by the pyrosequencing software which did not cover the present mutations. CONCLUSION: Overall, the GeneRead QIAact Actionable Insights Tumor Panel was specific and sensitive for mutation analysis for targeted therapies and can be incorporated into laboratory diagnostics' daily practice.

Combined point mutation in KRAS or EGFR genes and EML4-ALK translocation in lung cancer patients.

A total of three cases with novel constellations regarding mutation patterns in non-small-cell lung cancer (NSCLC) are reported. The mutation patterns that are observed are novel and unexpected. First, a combined simultaneous KRAS mutation and EML4-ALK translocation, both in the main tumor and a bone metastasis, were observed, these mutations are assumed to mutually exclude each other. A further two cases include a father and a daughter, both of whom are suffering from NSCLC with different EGFR mutation patterns. A common cause was assumed; however, could not be deduced to mutations in the KRAS, BRAF and EGFR genes. The aforementioned cases are important, as it must be taken into account that mutations previously assumed to be exclusive can occur in combination, may influence the clinical outcome and may require different therapy compared with single mutated tumors. It has to be discussed whether diagnostic algorithms need to be adapted. The cases of father and daughter show that further unknown factors can influence development of NSCLC.

Human Metapneumovirus: lessons learned over the first decade.

It has been 10 years since human metapneumovirus (HMPV) was identified as a causative agent of respiratory illness in humans. Since then, numerous studies have contributed to a substantial body of knowledge on many aspects of HMPV. This review summarizes our current knowledge on HMPV, HMPV disease pathogenesis, and disease intervention strategies and identifies a number of areas with key questions to be addressed in the future.

Seroprevalence of human respiratory syncytial virus and human metapneumovirus in healthy population analyzed by recombinant fusion protein-based enzyme linked immunosorbent assay.

BACKGROUND: Human respiratory syncytial virus (hRSV) and human metapneumovirus (hMPV) are two of the most frequent respiratory pathogens that circulate worldwide. Infection with either virus can lead to hospitalization of young children, immunocompromised people and the elderly.A better understanding of the epidemiological aspects, such as prevalence of these viruses in the population will be of significant importance to the scientific community. The aim of this study was to gain some detailed knowledge on the humoral immune response to both viruses in different populations of individuals. FINDINGS: The fusion protein (F) of hRSV and hMPV was expressed in the baculovirus and Escherichia coli systems, respectively, and used as antigen in two independent enzyme-linked immunosorbent assays (ELISAs) for detection of specific antibodies in human sera. The seroprevalence of each virus in a large cohort of individuals with ages ranging from 0 to 89 years old was determined. Although the general distribution of the antibody response to each virus in the different age group was similar, the prevalence of hRSV appeared to be higher than that of hMPV in most of them. The group of children with ages between 0 and 2 showed the highest seronegative rates. After this age, an increase in the antibody response was observed, most likely as the result of new infections or even due to reinfections. CONCLUSIONS: The use of these specific F-ELISAs in seroepidemiological studies might be helpful for a better understanding of the human antibody response to these viruses.

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.

Does human bocavirus infection depend on helper viruses? A challenging case report.

A case of severe diarrhoea associated with synergistic human bocavirus type 1 (HBoV) and human herpes virus type 6 (HHV6) is reported. The case supports the hypotheses that HBoV infection under clinical conditions may depend on helper viruses, or that HBoV replicates by a mechanism that is atypical for parvoviruses, or that HBoV infection can be specifically treated with cidofovir.

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.

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 [...].

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.

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 [...].

Novel mutation in YMDD motif and direct neighbourhood in a child with chronic HBV-infection and clinical lamivudine and adefovir resistance - a scholarly case.

CONTEXT: Chronic HBV infection is a major cause of hepatocellular carcinoma (HCC) which meanwhile has become the 5th most reason for a fatal outcome of cancer. Worldwide, approximately 350 million people are chronically HBV infected and as such of risk to develop HCC, of those an estimated high rate of children. Treatment of chronic infection is sufficient to reduce the rate of HCC but the rate of sustained virological response remains to low, not at least due to emergence of resistant virus strains. Less is known on HBV infection in children despite the extremely high rate of chronicity. OBJECTIVE, DESIGN, SETTING, AND PATIENT: The case of a nine years old male with a 6 year history of chronic HBV infection, of those 5 years with antiviral treatment is described. INTERVENTIONS AND MAIN OUTCOME MEASURE(S): Before our lab was consulted, the patient was unsuccessfully treated with interferon, an obscure drug named Hepon, which should activate antiviral immune response, and Lamivudine, the latter most likely becoming ineffective due to the mergence of resistant subpopulations (rtL180 M, rtV207 M, two strains with stop codons at position rt188 and rt198, rtM204V (YVDD), rtM204K (YKDD)). Replacement of Lamivudine by adefovir displayed no advantage despite the lack of resistance mutations, thus no decrease in viremia was observed under adefovir treatment. RESULTS AND CONCLUSIONS: Novel mutations in the YMDD motif and its direct neighbourhood were observed, both being compatible with Lamivudine resistance. No mutations were found that are associated with ADF resistance. Both, the clinical course of treatment and the genotypic resistance profile emphasize the need for systematic analyses of the HBV resistance mechanisms and structured therapy concept also for children chronically infected with HBV.

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.