Sensitive Commercial NASBA Assay for the Detection of Respiratory Syncytial Virus in Clinical Specimen

The aim of the study was to evaluate the usability of three diagnostic procedures for the detection of respiratory syncytial virus in clinical samples. Therefore, the FDA cleared CE marked NOW® RSV ELISA, the NucliSENS® EasyQ RSV A+B NASBA, and a literature based inhouse RT-PCR protocol were compared for their relative sensitivities. Thereby, NASBA turned out to be the most sensitive method with a total number of 80 RSV positive samples out of a cohort of 251 nasopharyngeal washings from patients suffering from clinical symptoms, followed by the inhouse RT-PCR (62/251) and ELISA (52/251). Thus, NASBA may serve as a rapid and highly sensitive alternative for RSV diagnostics

Respiratory Infections by HMPV and RSV Are Clinically Indistinguishable but Induce Different Host Response in Aged Individuals

Background: Human metapneumovirus and respiratory syncytial virus can cause severe respiratory diseases, especially in infants, young children, and the elderly. So far it remains unclear why infections in the elderly become life threatening despite the presence of neutralizing antibodies in the serum, and to which extent double infections worsen the clinical course. Methods: Young and aged BALB/c-mice were infected with RSV or/and HMPV. Appearance of the mice was observed during course of infection. On day 5 p.i. animals were dispatched by cervical dislocation and levels of TNF-a and NF-kB were determined. Results: The observation of activity, weight and appearance of the different mice showed no differences among the tested groups. Despite this, the immunologic response depends on the animals ’ age and the virus they were infected with. In young animals, NF-kB levels were elevated if infected with HMPV and HMPV/RSV but remained low in RSV infections, whereas in aged animals the opposite was observed: solely RSV-infected animals showed elevated levels of NF-kB. TNF-a was slightly elevated in HMPV-infected young and old animals, but only in young animals this elevation was significant. Conclusions: Contrary to other studies, no weight loss or change in activity despite productive lung infection with the different viruses were observed. This may be due to the weaker anaesthesia or the lesser volume of virus solution used

Detection of Head-to-Tail DNA Sequences of Human Bocavirus in Clinical Samples

Parvoviruses are single stranded DNA viruses that replicate in a so called “rolling-hairpin” mechanism, a variant of the rolling circle replication known for bacteriophages like ϕX174. The replication intermediates of parvoviruses thus are concatemers of head-to-head or tail-to-tail structure. Surprisingly, in case of the novel human bocavirus, neither head-to-head nor tail-to-tail DNA sequences were detected in clinical isolates; in contrast head-to-tail DNA sequences were identified by PCR and sequencing. Thereby, the head-to-tail sequences were linked by a novel sequence of 54 bp of which 20 bp also occur as conserved structures of the palindromic ends of parvovirus MVC which in turn is a close relative to human bocavirus

The Human Bocavirus Is Associated with Some Lung and Colorectal Cancers and Persists in Solid Tumors.

Human bocavirus is the second autonomous human parvovirus with assumed pathogenic potential. Other parvoviruses are known to persist and even integrate into the host genome, eventually contributing to the multi-step development of cancer. Human bocavirus also persists in an unknown percentage of clinically asymptomatic patients in addition to those with primary infection. The aim of the present study was to analyze the role of Human bocavirus in lung and colorectal cancers. Therefore, formalin-fixed, paraffin-embedded, archived tumor samples were screened for Human bocavirus DNA by PCR, Southern blotting, and sequencing. Positive tissues were further subjected to fluorescence in situ hybridization analysis to specifically detect human bocavirus DNA in the infected cells. In total, 11 of the 60 (18.3%) lung and 9 of the 44 (20.5%) colorectal tumors tested positive for human bocavirus DNA by PCR and were confirmed by sequencing and fluorescence in situ hybridization analysis. Thus, human bocavirus DNA is present in the nuclei of infected cells, in either single or multiple copies, and appears to form concatemers. The occurrence of these human bocavirus DNA structures supports the existence of the postulated σ- or rolling-hairpin replication mechanism. Moreover, the fluorescence in situ hybridization patterns inspired the hypothesis that human bocavirus DNA either persists as cccDNA or is integrated into the host genome. This finding suggests that this virus may indirectly contribute to the development of some colorectal and lung cancers, as do other DNA viruses, such as the human hepatitis B virus, or may play an active role in cancer by interacting with the host genome

Utility of the QIAGEN/artus PCR assays and Meningofinder (TM) for the detection of human herpes virus genomes in formalin-fixed paraffin-embedded clinical samples

Aim: The aim of the study was to analyze whether human herpes viruses can be detected from formalin-fixed paraffin-embedded (FFPE) tissues with two commercial methods. Materials & methods: In total, 91 clinical FFPE samples were analyzed with QIAGEN/ artus (Hilden, Germany) qPCRs and the Meningofinder (TM) assay from PathoFinder (Maastricht, The Netherlands). Results: A total of 27 out of 91 samples were tested for HSV-1 and HSV-2. Three of these 27 samples (11.1%) were positive for HSV-1 and one sample was positive for HSV-2. Out of 91 samples, 50 were tested for CMV, of which six samples were positive (12%). In total, 12 out of 91 samples were tested for the presence of Varizella zoster virus DNA, revealing one as positive (8%). Finally, two samples were tested negative for EBV. Conclusion: Minor differences were identified between both assays, making them a reliable tool for detection of herpes viruses from FFPE material, although this is an off-label use for both assays

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

Detailed overview on the mutations detected by and the sensitivity of the GeneReader NGS sequencing platform

This article presents additional next generation data from our preclinical validation study. In total 121 samples (clinical specimen and interlaboratory test samples) were tested successfully with next generation sequencing. 38 different mutations in six different genes were detected. Next to the detection of different mutations, the reproducibility of the NGS test was analyzed. Three samples were analyzed five times and the results were compared. Several mutations classified as non-pathogenic so far, have been detected repeatedly. (C) 2018 The Authors. Published by Elsevier Inc

Comparison of molecular multiplex assays for the detection of human papillomaviruses from clinical gynecological tissue samples

Papillomavirus-associated cervical cancer remains a major disease burden for young women. Therefore, it plays an important role in the pathological diagnosis. Besides cytological and classical pathological methods, molecular biology tests are used for the analysis of HPV status. Here we compare three PCR assays for the detection of human papillomavirus DNA from 50 gynecological samples, namely the HPV Genotyping Kit HPVsign(R) (Qiagen, Germany), a novel HPV-genotyping kit based on pyrosequencing, the LCD array HPV Type 3.5 (Chipron, Germany) and an in-house PCR of the National Reference Center for Papilloma-and Polyomaviruses at the Institute for Virology of the University Cologne (Germany). The HPVsign assay mainly detected the assumed leading HPV type, while both the in-house PCR and the Chipron HPV Assay 3.5 reproducibly detected single and multiple infections