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Improved sensitivity, safety, and rapidity of COVID-19 tests by replacing viral storage solution with lysis buffer.

ABSTRACT: Conducting numerous, rapid, and reliable PCR tests for SARS-CoV-2 is essential for our ability to monitor and control the current COVID-19 pandemic. Here, we tested the sensitivity and efficiency of SARS-CoV-2 detection in clinical samples collected directly into a mix of lysis buffer and RNA preservative, thus inactivating the virus immediately after sampling. We tested 79 COVID-19 patients and 20 healthy controls. We collected two samples (nasopharyngeal swabs) from each participant: one swab was inserted into a test tube with Viral Transport Medium (VTM), following the standard guideline used as the recommended method for sample collection; the other swab was inserted into a lysis buffer supplemented with nucleic acid stabilization mix (coined NSLB). We found that RT-qPCR tests of patients were significantly more sensitive with NSLB sampling, reaching detection threshold 2.1±0.6 (Mean±SE) PCR cycles earlier then VTM samples from the same patient. We show that this improvement is most likely since NSLB samples are not diluted in lysis buffer before RNA extraction. Re-extracting RNA from NSLB samples after 72 hours at room temperature did not affect the sensitivity of detection, demonstrating that NSLB allows for long periods of sample preservation without special cooling equipment. We also show that swirling the swab in NSLB and discarding it did not reduce sensitivity compared to retaining the swab in the tube, thus allowing improved automation of COVID-19 tests. Overall, we show that using NSLB instead of VTM can improve the sensitivity, safety, and rapidity of COVID-19 tests at a time most needed.

SUBMITTER: Erster O

PROVIDER: S-EPMC8009371 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundThe detection of direct oral anticoagulants (DOACs) is still challenging but important in emergency patients. We recently demonstrated that modified thromboelastometry can detect rivaroxaban and dabigatran. Data on the detection rates of modified compared to standard thromboelastometric tests of apixaban and edoxaban, are missing. The aim of this in-vitro dose-effect-study was to add data on these DOACs and to evaluate thromboelastometric tests in-vitro using data of both studies.MethodsThe study was approved by the Ludwig-Maximilians-University ethics committee (No 17-525-2). Written informed consent was obtained from all individuals. Blood samples of healthy volunteers and samples of 10 volunteers for each DOAC were used. Blood samples were spiked with six different concentrations of edoxaban and apixaban (0ng/ml; 31.25ng/ml; 62.5ng/ml; 125ng/ml; 250 ng/ml; 500ng/ml). Modified tests (low-tissue-factor test TFTEM and ecarin-based test ECATEM) as well as standard tests (e.g. FIBTEM) analyzing extrinsic pathway of coagulation were used. Receiver operating characteristics analyzes were performed as well as regression analyzes.ResultsTFTEM CT correlated well with anti-Xa levels of apixaban and edoxaban (apixaban: r2 = 0.8064 p < 0.0001; edoxaban: r2 = 0.8603; p < 0.0001). The detection of direct FXa inhibitors (> 30 ng/mL) was successful with FIBTEM CT with a sensitivity and specificity of 81% and 90%, respectively. As expected, ECATEM CT was not prolonged by direct FXa-inhibitors due to its specificity for direct thrombin inhibitors. Again, TFTEM CT provided the highest sensitivity and specificity for the detection of direct FXa inhibitors with 96% and 95%, respectively. ECATEM test showed 100% sensitivity and 100% specificity for the detection of dabigatran.ConclusionsOur study presents modified thromboelastometric tests with improved detection of even low DOAC concentrations > 30 ng/mL, including apixaban in-vitro. The study thus complements the previously published data on dabigatran and rivaroxaban. Validation studies must confirm the results due to the explanatory design of this study.

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Improved sensitivity, safety, and rapidity of COVID-19 tests by replacing viral storage solution with lysis buffer.

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