COVID-19 outbreak in Europe: Issues on PCR diagnostics for SARS-nCoV2 in unprepared landscape and immediate field implementations of practical measures
Authors: Martina Mrkvicova, Petr Müller, Lenka Zdrazilova-Dubska, Roman Hrstka, Lukas Uhrik, Monika Dolejska, Marek Svoboda, Dalibor Valik
Affiliations: Masaryk Memorial Cancer Institute, Member of OECI (www.mou.cz, Biobanking and Biomolecular Resources Research Infrastructure, Czech Republic, BBMRI-CZ (www.bbmri.cz )
Masaryk Memorial Cancer Institute (MMCI) in Brno, Czech Republic, is a tertiary cancer care-oriented hospital accredited by OECI (www.oeci.eu) as Clinical Cancer Centre integrating comprehensive cancer care with cancer research (RECAMO) and Europe-wide research infrastructure for biobanking (BBMRI-CZ). Continuum in cancer care during COVID-19 outbreak is a must while ensuring safety for both hospital personnel and cancer patients who are at higher risk for severe clinical course of COVID-19. Therefore, we are facing the need for availability of SARS-nCov2 PCR diagnostic testing in biological specimens with short turn-around time that would fit into daily practice of cancer patient management.
Current (mid/late March 2020) COVID-19 challenges leading to bottlenecks in massive PCR SARS-nCov2 testing in the Czech Republic but also elsewhere are 1/lack of nasopharynx sampling swabs suitable for PCR detection of coronavirus and 2/high virulence of SARS-nCov2 requiring BSL-3 safety measures for handling including RNA isolation from biological specimens. This is in striking contrast with low capacity of PCR testing capabilities in medical diagnostic labs for clinical microbiology equipped with BSL-3 vs. abundant PCR testing capacity in academia-based labs without BSL-3 safety level infrastructure, furthermore complicated by COVID-19 pandemia-induced lack of personal protective equipment.
Obstacle 1: First, we have been working out (research, clinical laboratory, pharmacy) to deal with lack of sampling swab suitable for nasopharynx sampling that are compatible with PCR testing (no wood, no cotton). We designed 3D printer drawing of nasopharyngeal (sampling swabs) and printed the plastic base of the sampling stick on 3D printer from PETG material. After sterilization with ethanol-based disinfection, sampling stick were furnished with polyester wadding and subjected to sterilization at 75°C for 30 min. Our swab stick is flexible (Figure 1) enabling mucus collection from nasal cavity without hurting patients. Having this option in place we overcome the imminent lack of commercially available supplies we are now facing.
The obstacle 2: The oropharyngeal and nasopharyngeal swabs are collected by physicians under full BSL-3 PPE in out-patient settings or from hospitalized patients. The collected specimen is then immediately inactivated at the sampling site by its short incubation in a denaturing/inactivation medium. Therefore, the swab specimen is not contagious anymore when entering a laboratory and requires BSL-2 only conditions for RNA isolation and further manipulation. The inactivation medium is based on a strong chaotropic agent 4 M guanidium thiocyanate for protein denaturation, citrate for ion chelation and subsequent RNase inactivation, sarcosyl detergents and finally 2-mercaptoethanol in a concentration above 20 mM for degradation of mucus and virucidal action for coronavirus. This approach requires the RNA isolation being started within 24 h from swab sampling and the denaturation media being prepared fresh before sampling, which is manageable under MMCI conditions and assumingly other local conditions as well. Subsequently, both oropharyngeal and nasopharyngeal swabs are pooled together for RNA isolation according WHO guidelines. Applicability of both innovations, home-made sampling swabs and denaturation/inactivation media, and their suitability for SARS-nCov2 detection in biological specimen has been tested and verified (Figure 2).
Samples were taken from patients A and B using two types of sampling swabs either commercially available FLOQSwabs (Coban) or MMCI-made swabs. Positive control specimen was collected by commercially available swab into cultivation medium without immediate inactivation. Detection of SARS-nCov2 was performed using RT PCR with OneStep RT-qPCR MasterMix (Generi Biotech) according to the recommended WHO protocol on LightCycler 480 (Roche). (A) RT-PCR detection of SARS-nCov2 – control of RNA extraction is shown on internal control (IC) amplification serving as a control of RNA isolation from the clinical sample using the target amplification of human RNase P. (B) RT-PCR detection of SARS-nCoV-2 – viral gene E (E). In this reaction a target sequence of viral E gene is detected showing the presence of SARS-nCov2 in B sample.
As for PCR detection of SARS-nCov2, we are using the mastermix from a Czech (i.e. local) company under a reassurance that this mastermix will be available on a long-time basis and not preferentially diverged to countries with much larger markets. We use primers and probes according WHO recommendation detecting gene E, gene N, gene RdRp.
As for the lab processes, we incorporated currently available hands-on capacities of researchers into an accredited diagnostic clinical laboratory workflow. In practice, i) the RNA isolation has been developed and is currently provided by MMCI research group and ii) the amplification, data interpretation and result report are provided by the qualified staff of the clinical laboratory. We provide two testing batches per day. The turn-around time of the result is 5 h from completing of a sampled cohort. The RNA samples (positive and negative for SARS-nCov2) are collected into biobank repository in a manner that keeps their traceability to the source patient. Taken together, within 2 weeks we were able to set up the complete diagnostic procedure from specimen collection, PCR analytical procedure for SARS-nCov2 detection to qualified result reporting, fulfilling the criteria of the Czech National Reference Laboratory. Most importantly, our SARS-nCov2 PCR diagnostic process does not require BSL-3 setting in the testing laboratory and appears robust enough to withstand current uncertainties in material supplies during the course of COVID-19 pandemia that may render laboratories dependent rather on local than global supplies.
COVID-19 challenge for biobanking: Currently, there is an absence of validated assays for assessment of antibody-based immune response to the SARS-nCov2 infection. A vast array of “rapid tests” based on immunochromatography using colloidal gold particles have been put on the market usually (due to objective reasons) lacking thorough validation studies. To meet the need for current validation/verification procedures for blood-based assays we have implemented measures to keep all patient-positive biological material in biobanking repositories. Currently, we encourage the laboratories to store multiple sample threads from PCR positive and clinically apparent cases to have at least a paired sequential specimen of plasma/serum from each patient. Furthermore, isolated nucleic acid is kept frozen along with the blood specimens. Although a mounting evidence is being collected on the virological positivity of stool specimens we have not yet considered to add stool sample to the patients data/sample set. We are aware, though, that this specimen category may be of special importance in the specific cohorts such as children where most of the COVID-19 clinical courses appear mild and asymptomatic carriers may also occur and go unrecognized.
Working summary: We have proposed and tested under the field conditions a set of procedures enabling to increase the number of tests of individuals with suspected COVID-19. We set up a procedure linked to clinical laboratory workflows enabling preservation of patient-derived clinical material for imminent purposes of method validation/verification and future research as well.