A Simple and Cost-Effective DNA Preparation Method Suitable for High-Throughput PCR Quantification of Hepatitis B Virus Genomes.

Hepatitis B virus (HBV) is a para-retrovirus that reverse transcribes its pregenomic RNA into relaxed circular DNA inside viral nucleocapsids. The number of HBV genomes produced in vitro is typically quantified using commercial silica-membrane-based nucleic acid purification kits to isolate total DNA followed by HBV-specific quantitative PCR (qPCR). However, despite the convenience of commercial kits, this procedure is costly and time-consuming due to multiple centrifugation steps, which produce unnecessary waste. Here, we report a rapid, cost-effective, and environmentally friendly total DNA preparation method. The assay is based on the simple incubation of detergent and proteinase K with cells or cell-free supernatants to permeabilize cells and disrupt viral particles. After heat inactivation and subsequent centrifugation to clear the lysates, DNA samples are directly subjected to qPCR to quantify HBV genomes. As a proof of concept, the assay was developed in 12-well plates to assess intra- and extracellular HBV genome equivalents (GEqs) of stably viral-replicating cell lines (e.g., HepAD38) and HBV-infected HepG2-NTCP cells, both treated with lamivudine (LMV), an HBV replication inhibitor. Viral DNA was also prepared from the serum of patients chronically infected with HBV. To validate the assay, a representative commercial DNA isolation kit was used side-by-side to isolate intra- and extracellular HBV DNA. Both methods yielded comparable amounts of HBV GEqs with comparable LMV 50% efficient concentration (EC50) values. The assay was subsequently adapted to 96- and 384-well microtiter plates using HepAD38 cells. The EC50 values were comparable to those obtained in 12-well plates. In addition, the calculated coefficient of variation, Z' values, and assay window demonstrated high reproducibility and quality. We devised a novel, robust, reproducible, high-throughput microtiter plate DNA preparation method suitable for quantifying HBV GEqs by qPCR analysis. This strategy enables rapid and convenient quantitative analysis of multiple viral DNA samples in parallel to investigate intracellular HBV replication and the secretion of DNA-containing viral particles.

Evaluation of an interdisciplinary palliative care inhouse training for professionals in gynecological oncology.

PURPOSE: The aim of this study was to evaluate the effect of a pilot interdisciplinary inhouse training in palliative care (PC) for gynecological oncologists. METHODS: Competencies of participants from a gynecological university department were evaluated taking part in an interdisciplinary PC course in a pre and post design. The multiprofessional course covered basic principles of PC, symptom management and communication taught by PC specialists. Competencies were evaluated using self-designed questionnaires before (ISPG-1), right after (ISPG-2), and 6 months after the training (ISPG-3) (inhouse seminar palliative care in gynecology: ISPG). RESULTS: 31 persons from the department of gynecology took part in the course, of which 27 answered the first questionnaire (seven nurses (26%), 19 doctors (71%), one profession not indicated (3%), median working experience in gynecological oncology: 5 years). Return rates were: ISPG-1 27/31 (87.1%), ISPG-2 20/31 (64.5%) and IPSG-3 14/31 (45.2%). A more positive attitude towards PC could be observed in the majority of participants after the course (ISPG-2 62%, ISPG-3 71%). They felt more competent in the care of palliative patients (46%). PC would be initiated earlier and the interaction with other disciplines was improved (ISPG-2 85%, ISPG-3 100%). The participants assessed a significant improvement of their skills in all palliative fields which were analyzed. CONCLUSION: PC inhouse training improves the understanding of PC and the interdisciplinary approach in the management of patients with advanced disease. It is a feasible and useful instrument to improve the competencies in generalist PC of specialists in gynecological oncology.