key: cord-0707520-fgzwr4wx authors: Schmidt, Michael; Hoehl, Sebastian; Berger, Annemarie; Zeichhardt, Heinz; Hourfar, Kai; Ciesek, Sandra; Seifried, Erhard title: Novel multiple swab method enables high efficiency in SARS‐CoV‐2 screenings without loss of sensitivity for screening of a complete population date: 2020-07-06 journal: Transfusion DOI: 10.1111/trf.15973 sha: 3ed90c7a74a7578c4d533588955c2359167e6080 doc_id: 707520 cord_uid: fgzwr4wx BACKGROUND: In the pandemic, testing for SARS‐CoV‐2 by RT‐PCR is one of the pillars on which countermeasures are based. Factors limiting the output of laboratories interfere with the effectiveness of public health measures. Conserving reagents by pooling samples in low‐probability settings is proposed, but may cause dilution and loss of sensitivity. Blood transfusion services had experience in performance of high throughput NAT analysis and can support the national health system by screening of the inhabitants for SARS‐COV‐2. METHODS: We evaluated a new approach of a multiple swab method by simultaneously incubating multiple respiratory swabs in a single tube. Analytical sensitivity was constant up to a total number of 50 swabs. It was consequently applied in the testing of 50 symptomatic patients (five‐sample pools) as well as 100 asymptomatic residents of a nursing home (ten‐sample pools). RESULTS: The novel method did not cause false negative results with non‐significantly differing Ct values between single swab and multiple swab NAT. In two routine applications, all mini pools containing positive patient samples were correctly identified. CONCLUSIONS: The new method enables countries to increase the total number of testing significantly. The multiple swab method is able to screen system relevant groups of employees frequently. Secondly the example in Germany shows that blood transfusion services can support general health systems with their experience in NAT and their high throughput instruments. Screening of a huge number of inhabitants is currently the only option to prevent a second infection wave and enable exit strategies in many countries. This article is protected by copyright. All rights reserved. SARS-CoV-2 is the causative agent of the novel lung disease COVID-19. With more than 1.3 million cases and almost 80 thousand deaths recorded worldwide by April 8 th 2020, 1 cases are still rising sharply in many parts of the world. Nations throughout the world are attempting to slow down the surge in cases by putting extensive countermeasures in place. Infection may remain asymptomatic or pass with only minor symptoms, making a clinical diagnosis impossible in many cases. [2] [3] [4] High viral titers in the upper airways during the first week of symptoms 5, 6 and presymptomatic transmission 7 likely contributes to the difficulty containing the pandemic. In the struggle against the pandemic, the WHO recently urged nations to "test, test, test". 8 Detection of SARS-CoV-2 by nucleic amplification technologies (NAT), such as PCR, in a nasopharyngeal or throat swab and/or lower respiratory specimen is the preferred method as recommended by the WHO. 9 The unprecedented demand for NAT reagents and test kits has already led to shortages, obstructing the efforts to combat COVID-19. Another factor limiting the output of laboratories is the availability of qualified staff. Furthermore, especially in low-income settings, where the threat by COVID-19 is no less imminent, cases may go undetected when tests are too expensive. Blood donor services have been asked in many regions to make their high throughput NAT testing systems available to support patient and population testing for SARS-Co-V 2, in addition to donor screening. To make testing for SARS-CoV-2 more efficient, sample pooling has been proposed, and recently applied in a retrospective analysis. 10 Dilution effects leading to a loss in diagnostic sensitivity is a concern in this strategy, when sample solutions are pooled. Here, we evaluated a new alternate multiple swab NAT protocol (FACT-method for Frankfurt adjusted COVID-19 testingmethod) without any volume dilutions in the pooling process. This article is protected by copyright. All rights reserved. We applied a novel multiple swab protocol to NAT testing of respiratory swabs for SARS-CoV-2: The new method was tested for following dry swabs (dry swab, Roche, Mannheim, Germany, uni swab sample; dry swab, Sarstedt, Nümbrecht, Germany, neutral swabs and dry swab Copan, CA, USA, Classiq swab TM dry swab). Other dry swabs are possible but currently not tested by the authors. It is recommended in Germany to take specimens first from the pharyngeal region followed by the nasal region. Samples can be taken with two swabs or with one swab. Due to experimental data (not shown) the time between sample collection and performing NAT should be not longer than 48h. Swabs should be protected from UV-light. Respiratory swabs were first incubated in a reference tube containing 4.3 ml of guanidinium hydrochloride buffer (Roche cobas medium) solution for 5 minutes with constant agitation. Consequently, all swabs used for the multiple swab method are removed and collectively placed in one new single media tube containing 2 ml of guanidinium hydrochlorid buffer, the multiple swab tube, under constant agitation for 5 minutes (Figure 1 ). Up to ten swabs can be placed at one time point into the multiple swab tube. Other buffer reagents e.g. guanidinium thiocyanate mixed with PBS (1:1) is in addition possible. All swabs were performed in a laminar flow hood. Before transferring the swabs into a new tube they were wiped softly off the tube wall. The swabs are then removed from the multiple swab tube, which proceeds to NAT testing. The complete process of performing multiple swab tubes was controlled by an inhouse IT software. In brief, all original swabs and the archive tubes were labelled with a primary barcode sticker and scanned after labelling. By transferring the swabs into the archive tubes all barcodes were scanned a second time. Mixed up errors were detected by comparing the barcodes from the swab and the archive tube. In the next step the multiple swab tube is labelled with MS-T sticker. By transferring the swabs from the archive tube to the multiple swab tube all archive tubes as well as the multiple swab tube are scanned again. The original swabs were discarded after This article is protected by copyright. All rights reserved. Accepted Article inoculation of the multiple swab tube. If the multiple swab tube showed a positive or invalid test result further testing was performed from the archive tube. Archive tubes are stored at 2-8°C until NAT analysis from the multiple swab tube is completed. In case of a negative NAT result in the multiple swab tube, each sample in the multiple swab tube receives a negative result. If the NAT result of the multiple swab tube is positive, individual SARS-CoV-2 NATs are carried out from the archive tubes. NAT was performed by Roche cobas SARS-CoV-2 on the Roche Cobas ® 6800 or Roche Cobas ® 8800 instrument. The sample input volume was 400µl. Amplification was done in a multiplex CE certified assay in the ORF 1a/b region as well as in the E-gene. All samples were tested in accordance to the instruction for use from the manufacturer (Roche Diagnostics, Mannheim, Germany). For the first series of experiments an inactivated SARS CoV-2 standard was used with a final concentration of 10 4 copies/ml. The standard was quantified with a quantitative realtime PCR described in detail be Toptan et al. 11 Evaluation This article is protected by copyright. All rights reserved. The concept was assessed in five setups, and the diagnostic value was evaluated in practical application in symptomatic patients as well as in a screening procedure in asymptomatic employees. In Germany each screening laboratory must participate in official proficiency panel tests to demonstrate correct testing. Therefore, unknown samples were send to each laboratory. These samples were solved in sterile water. In the next step sterile swabs were placed into each sample and tested by the individual swab method as well as by the multiple swab In these test series different numbers of multiple swab testing were evaluated against individual swab testing. Therefore dry swabs were incubated into an inactivated SARS CoV-2 standard solution (10 3 copies/ml) for 1 minute. Thereafter the swab were placed into archive tube (one swab per tube). After five minutes different multiple swab pools (pools with 10 swabs, pools with 20 swabs, pools with 30 swabs, pools with 40 swabs and pools with 50 swabs) were performed. In each pool one swab which was contaminated with SARS CoV-2 standard material was taken and combined with the outstanding number of swabs without SARS CoV-2 (with 9 negative swabs for pools of 10, with 19 negative swabs for pools of 20, with 29 negative swabs for pools of 30, with 39 negative swabs for pools of 40 and with 49 negative swabs for pools of 50). In the next step SARS CoV-2 NAT was performed for the archive samples as well as for the different multiple swab samples. Table 5 shows the CT-values of the individual testing of the archive sample as well as the CT-values of the different multiple swab samples. Paired T-Tests were calculated for Ct values between the single swab method and the multiple swab method. P-values below 0.05 were found to be statistically significant First, to evaluate for suitability of different mini-pool sizes, swabs were contaminated with a defined SARS-CoV-2 virus concentration of 1x10 4 copies/ml, and then placed in a series of 10 tubes with lysis buffer for 5 minutes each. Ct values in each tube were determined, which is proportional to copy numbers. The results were examined for significant increase in Ct values in the succession of tubes, which would signify loss of sensitivity. We did not observe a significant difference in the semi-quantitative viral load between the first tube (representing individual sample testing) and the tenth tube. The largest observed difference in Ct value was 1.73 and 2.23 for ORF 1a-and E-gene, respectively (table 1) Accepted Article correctly identified with the multiple swab method. Multiple swab tubes containing no positive sample were also correctly identified to be negative in multiple swab tubes of five swabs. Table 3 shows the comparative presentation of the Ct values from both methods. P-value for individual sample and multiple swab tube NAT was 0.299 and 0.354 for the ORF region and E-gene, respectively, which we consider not statistically significant. In a second real-life application, 100 samples from asymptomatic residents of a nursing home were randomly assigned to ten multiple swab tubes containing ten swabs each. All five multiple swab tubes containing a total of eight positive swabs were correctly identified. All five multiple swab tubes containing no positive swab sample were also true negative. Ctvalues did not differ significantly between multiple swab tubes and the single swab tubes testing (p-value for the ORF region and E gene were 0.44 and 0.46, respectively) (table 4). In a fourth evaluation the multiple swab method was tested for screening of 3110 asymptomatic employees. All samples were investigated in multiple swab tubes containing 10 swabs per tube (311 tubes). In total two multiple swab tubes achieved a SARS-CoV-2 positive NAT screening result. By testing the archive tubes two asymptomatic employees were identified as SARS-CoV-2 positive. Ct-values were between 36 and 37 and represent a low virus concentration. Ct values were identical for the multiple swab tube and for the archive tube. Finally the multiple swab method was extended to 50 swabs per tube. As shown in table 5 Ct-values were comparable between the single swab tubes and the multiple swab tubes up 50 swabs per tubes. Increased test efficiency is eagerly awaited for SARS-CoV-2, as effective strategies to slow down the pandemic depend on early detection of cases, while a finite supply of reagents, qualified personnel and high costs interfere. To preserve reagents, reduce hands-on time and expenses, sample pooling is being proposed for settings with a low pre-test probability. 10, 12 This pooling strategy was implemented in blood donor screening for transfusion transmitted virus infections like HCV or HIV-1 worldwide [13] [14] [15] . For virus infection with a very high doubling time like HCV the loss of the analytical sensitivity is low and acceptable for blood components. The mini-pool NAT strategy is a success full story that improves blood safety to a maximum 16, 17 . For symptomatic SARS-CoV-2 patients the virus load is usual high which enables the option to implement a mini-pool method with dilution of sample volume or extracted volume. But the implementation of screening of asymptomatic persons will be a challenge that on the one hand side a method with a very high diagnostic sensitivity is needed and on the other side an easy high throughput system should be present to screen a very high amount of samples. Both criteria are fulfilled by using the multiple swab method. Here, we present a novel alternate multiple swab protocol that is based on incubation of a respiratory swab first in a single sample tube, and then again in a multiple swab tube. We detected no significant difference in the amount of virus detectable by NAT in the single sample and multiple swab tube. Therefore, by applying this protocol in the diagnostic process, no loss of diagnostic or analytical sensitivity would be observed, dismissing a main concern that might hinder implementation. We presume that our multiple swab method can be implemented for all NAT methods and all dry swabs. We applied the protocol in two routine scenarios, where the novel protocol was able to reduce to total number of required NAT tests by up to 80%, without loss of diagnostic sensitivity. By putting this method into practice in the current SARS-CoV-2 pandemic, the number of samples that can be tested with a given amount of NAT reagents could immediately be This article is protected by copyright. All rights reserved. Accepted Article increased in a sub-cohort with a low pretest probability, when it is not likely that pools must be resolved and samples tested individually, which would void the initial benefit. This could be especially useful when screening professional groups that are exposed to the virus while also posing a risk of spreading it, such as health care workers and emergency responders, or groups at risk, such as the elderly. This approach would not be efficient in a setting with high pre-test probability, where it would be likely that the individual samples would have to be retested. Here, a single sample test, or a smaller pool size would be advisable. This article is protected by copyright. All rights reserved. Swabs were incubated first in a reference tube followed by a 5 minute incubation in the mini-pool tube. SARS CoV-2 virus concentration did not differ significantly between both samples. This article is protected by copyright. All rights reserved. Tables: Table 1 : Incubation of a SARS CoV-2 contaminated swab with 10 4 copies/ml sequentially into ten sample tubes with lysis buffer ORF region E-gene This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. Coronavirus disease (COVID-19) outbreak situation Transmission of 2019-nCoV Infection from an Asymptomatic Contact in Germany Evidence of SARS-CoV-2 Infection in Returning Travelers from Wuhan, China Covid-19 in South Korea -Challenges of Subclinical Manifestations SARS-CoV-2 Viral Load in Upper Respiratory Specimens of Infected Patients Virological assessment of hospitalized patients with COVID-2019 Presymptomatic Transmission of SARS-CoV-2 -Singapore WHO Director-General's opening remarks at the media briefing on COVID-19 -16 Laboratory testing for coronavirus disease (COVID-19) in suspected human cases: Interim guidance Sample Pooling as a Strategy to Detect Community Transmission of SARS-CoV-2 Optimized qRT-PCR approach for the detection of intra-and extra-cellular SARS-CoV-2 RNAs Boosting test-efficiency by pooled testing strategies for SARS-CoV-2 NAT and viral safety in blood transfusion History and Future of Nucleic Acid Amplification Technology Blood Donor Testing NAT screening of blood donors for severe acute respiratory syndrome coronavirus can potentially prevent transfusion associated transmissions Experience of German Red Cross blood donor services with nucleic acid testing: results of screening more than 30 million blood donations for human immunodeficiency virus-1, hepatitis C virus, and hepatitis B virus International survey on NAT testing of blood donations: expanding implementation and yield from 1999 to This article is protected by copyright. All rights reserved This article is protected by copyright. All rights reserved.Accepted Article This article is protected by copyright. All rights reserved.Accepted Article