key: cord-0704703-8h3ibufj authors: Olearo, Flaminia; Nörz, Dominik; Hoffman, Armin; Grunwald, Moritz; Gatzemeyer, Kimani; Christner, Martin; Both, Anna; Campos, Cristina Elena Belmar; Braun, Platon; Andersen, Gabriele; Pfefferle, Susanne; Zapf, Antonia; Aepfelbacher, Martin; Knobloch, Johannes K.M.; Lütgehetmann, Marc title: Clinical performance and accuracy of a qPCR-based SARS-CoV-2 mass-screening workflow for healthcare-worker surveillance using pooled self-sampled gargling solutions: a cross-sectional study date: 2021-09-06 journal: J Infect DOI: 10.1016/j.jinf.2021.08.047 sha: bf1445968d596db553f8572b9972ef9512423e9f doc_id: 704703 cord_uid: 8h3ibufj INTRODUCTION: The large number of asymptomatic SARS-CoV-2 infections necessitates general screening of employees. We evaluate the performance of a SARS-CoV-2 screening program in asymptomatic healthcare-workers (HCW), utilizing self-sampled gargling-solution and sample pooling for RT-qPCR. METHODS: We conducted a cross-sectional retrospective study to collect real-life data on the performance of a screening-workflow based on automated-pooling and high-throughput qPCR testing over a 3-month-period at the University Hospital Hamburg. RESULTS: Matrix validation reveals that lower limit of detection for SARS-CoV-2 RNA in gargling-solution was 180 copies/mL (5-sample-pool). A total of 55,122 self-collected gargle samples (=7,513 HCWs) was analyzed. The median time to result was 8.5 hours (IQR 7.2-10.8). Of 11,192 pools analyzed, 11,041 (98.7%) were negative, 69 (0.6%) were positive and 82 (0.7%) were invalid. Individual testing of pool participants revealed 57 SARS-CoV-2 previously unrecognized infections. All 57 HCWs were either pre-symptomatic or asymptomatic (prevalence 0.76%,CI95%0.58-0.98%). Accuracy based on HCWs with gargle-solution and NP-swab available within 3-day-interval (N=521) was 99.5% (CI95%98.3-99.9%), sensitivity 88.9% (CI95%65.3-98.6%) while specificity 99.8% (CI95%98.9-99.9). CONCLUSION: This workflow was highly effective in identifying SARS-CoV-2 positive HCWs, thereby lowering the potential of inter-HCW and HCW-patient transmissions. Automated-sample-pooling helped to conserve qPCR reagents and represents a promising alternative strategy to antigen testing in mass-screening programs. Healthcare workers (HCW) are on the frontlines of the current worldwide SARS-CoV-2 pandemic, placing themselves, their families and their patients at further risk of infection. Furthermore, their absence during periods of isolation for either suspected or proven infections places further strain on an already overburdened healthcare system. Because of the high number of asymptomatic SARS-CoV-2 infected individuals 1 , the focus of testing in HCWs should shift from a symptom-based approach to continuous screening of all those in contact with patients. However, regular mass screening by RT-qPCR using nasopharyngeal (NP) swabs is currently not feasible due to the scarcity of reagents, turnaround times and lack of trained personnel necessary to perform NP swabs. Similarly, rapid antigen tests have shown detection limits of 10 4 -10 5 SARS-CoV-2 RNA copies/mL 2 and lower sensitivity, particularly in asymptomatic individuals 3 . 6 A number of less invasive sampling methods for PCR tests have been explored in recent months. First studies have demonstrated acceptable performance using saliva samples 4 , which have also been proposed as a candidate for HCW mass screening schemes via sample pooling 5, 6 . However, "real-life" data on the performance of such methods in practice is still lacking at this point. Furthermore, pooling of these kinds of samples entails many technical challenges: e.g., the sample viscosity requires manual processing, which is both time consuming and risks errors and contaminations. Gargling samples represent another swab-independent sampling method which is both less invasive and offers good performance in detecting SARS-CoV-2 infections 7, 8 . In this study we report real-life performance data for an RT-qPCR-based mass screening approach in a large cohort of asymptomatic health care workers, utilizing pooled gargling solution as non-invasive sample type. Gargling-solution is not approved for use with the cobas SARS-CoV-2 PCR test by the manufacturer. To rule out substantial impairment of target detection due to the matrix, pooled SARS-CoV-2 negative gargling-solution was used to prepare a two-fold dilution series of SARS-CoV-2 reference material (Accuplex SARS-CoV-2, Infl-A/B, RSV) and subjected to testing on the system (9 dilution steps, 8 repeats each, 500 -2 cp/mL). Analytical LoD was assessed by probitanalysis. To test the viability of gargle-samples for SARS-CoV-2 detection, we asked SARS-CoV-2 positive HCWs and patients to self-collect gargle-samples in addition to a diagnostic NP sample performed by a medical doctor. NP flocked swabs were collected via the left or right naris using UTM based collection kits by Copan (Italy, Brescia). The gargle-sample collection procedure is described below in more detail. Before the introduction of gargle pooling in December 2020, HCWs at the University Hospital of Hamburg were instructed to get tested for SARS-CoV-2 at the occupational doctor's practice for free at their own discretion, in case they experienced any symptoms. Testing was carried out by RT-qPCR using NP swabs. Following an institutional agreement, all HCWs were invited to self-collect gargle samples weekly or twice weekly, depending on their area of work, for general SARS-CoV-2 screening. This scheme was limited to asymptomatic employees with direct contact with patients; those experiencing symptoms continued to receive standard testing by NP swab. We performed a cross-sectional retrospective study from 1 st December 2020 to 28 th of February 2021 in a large tertiary care hospital in Germany. We compared the incidence of SARS-CoV-2 infections and proportion of positive tests in an occupational practice for HCWs with a gargling solution-pooling technique. The latter was also compared with the proportion of positive tests in Germany in the same period. The study was performed in compliance with the Helsinki declaration, ethics votum was waived since data were collected anonymously. As a consequence, demographic data and clinical characteristics were not accessible. Users were instructed (with flyers and instruction videos) to gargle at home with 5-7 mL of tap water for 30 seconds and then spit into an empty 10 mL sterile polypropylene container using a straw (step I in the Figure 1 ). The tube was then labeled by the user with an individualized barcode tag and sent to the microbiology department for analysis (step II, Figure 1 ). After reception and registration, 1mL of cobas PCR Media (<= 40% Guanidine hydrochloride solution in Tris-HCL) was added to each gargling-sample using a Hamilton Starlet IVD liquid handler (step III, Figure 1 ). Pools of 5 (300µL per sample for 1500µL total volume) were generated using IVD pooling software (Hamilton STARlet IVD, Hamilton company, Reno, Nevada, step IV, Figure 1) and pooling protocols released to the LIS (laboratory information system) for result allocation (step IV, Figure 1 ). The sample pools were consecutively analyzed using the cobas SARS-CoV-2 IVD test for the cobas6800 system (Roche, Mannheim, Germany). Target-1 (ORF1ab) and Target-2 (E gene) results were analyzed separately, though the entire assay was deemed positive as long as one target returned a positive result (step V Figure 1 Sensitivity, specificity and overall accuracy including two-sided 95% Wald confidence intervals (CI95%) were assessed for those patients for whom a gargle sample and NP swab as gold standard were performed within a 3 day-interval. Invalid samples (NP swabs or gargles) were excluded from the analysis. A follow-up of positive gargle solutions was performed in order to assess a confirmation of the positive results by NP swabs or by serology beyond 3 days, although these samples were not included in the accuracy analysis. We calculated the incidence rate per 100´000 days at risk considering the time-atrisk started with 1 st of December 2020 and ended the 31th of January 2021. All statistical analyses were performed using GraphPad Prism version 9 (GraphPad Software, San Diego, CA) and STATA 15. For this study we received no funding. Analytical LoD for the cobas SARS-CoV-2 IVD test in gargling solution was determined as 36 copies/mL (CI95% 27.78 -58.81 copies/mL; see also Appendix Figure 1 ). This corresponds to an LoD of 180 copies/mL (CI95% 138.9 -294.05 copies/mL) for one clinical sample in a pool of five. To compare SARS-CoV-2 RNA detection in self-sampled gargling solution and professionally performed NP swab, we analyzed 21 sample pairs obtained from SARS-CoV-2 positive HCWs (n=7) and patients (n=14). Viral RNA ranged from 6.76x10 2 -3.98x 10 8 copies/mL and 5.37x10 2 -6.17x10 6 copies/mL in NP swab and gargling solution respectively. Overall qualitative agreement was 21/21 (100%) while viral loads were on average 1.43 log10 copies/mL lower in self-collected gargling solution (see Appendix Figure 2 ). Interestingly, quantitative difference between NP swab and gargling was smaller in HCW indicating that correct gargling procedure and the ability to hold breath for 30s, particularly difficult for critical ill patients, is important for successful self-sampling. Clinical departments were added successively to the general screening program as outlined above. As a result, sample numbers increased approximately 10fold over the course of the observation period (Appendix Figure 3) . A total of 55,122 self-collected gargle-samples were processed, representing 7,513 individual asymptomatic healthcare-workers ( Figure 2 ). The median number of samples submitted per HCW was 4 (range 1-18). Of the submitted gargle- 13 samples, 2.8% (1,554/55,122) had to be rejected due to improper preparation (e. g. not closed properly, too full, etc.). The remaining 52,715 samples were used to generate a total of 11,192 pools, which were consecutively analyzed by PCR. The median time to result was 8.5 hours (IQR 7.2-10.8). The overall prevalence of SARS-CoV-2 infections in asymptomatic HCWs was 0.76% (CI95% 0.58-0.98%). Incidence rates per 100,000 days at risk over the trial period can be seen in Appendix Table 1 , as reported by 10 In this study we report real-life data of a large-scale screening program in asymptomatic healthcare workers using self-collected gargling solution as sampling method with pooled RT-qPCR for SARS-CoV-2 detection. Preliminary validation showed that expected viral concentrations are systematically lower in gargling solution but without a substantial loss in overall positive Furthermore, this workflow of gargling-solution tested via pooled qPCR were not compared with other screening strategies, e. g. by rapid antigen test. SARS-CoV-2 outbreaks in healthcare facilities have been a major concern during the ongoing pandemic, as these institutions concentrate some of the most vulnerable members of the population. In an environment of broad community spread, infections may be carried into hospitals in many different ways, including infected patients, healthcare workers, visitors and others 11 . Given the wide spectrum of potential courses of the disease, infected individuals may actively spread the virus before becoming symptomatic or even without ever developing any symptoms that would help identify the risk 12 . As a result, general SARS-CoV-2 screening, regardless of symptoms, is necessary to control transmission within hospitals. Rapid antigen tests have been proposed for such screening schemes, as they provide fast results and are widely available in large quantities. However, their accuracy is imperfect, particularly in asymptomatic patients 13 , thus carrying the risk to miss cases, some of which would then lead to outbreaks. Using mathematical modeling approaches, there have been multiple studies suggesting that increased testing frequency can mitigate lower sensitivity in individual tests, however, there is currently no real-life data available to support this concept in practice 14 . This would also exacerbate the impact of sporadic false positives. There have been recent reports of screening approaches using pooled RT-qPCR 15, 16 , sacrificing some sensitivity to vastly increase capacity for PCR testing. These have shown success in practice, but still rely on conventional nasopharyngeal swabs as material. Nasopharyngeal swabs have become the mainstay for SARS-CoV-2 detection as they were shown to yield the most amount of virus compared to other modes of sampling 17 . Notably, rapid antigen tests often rely on them for optimal performance. However, NP swabs have inherent disadvantages as they are fairly invasive and uncomfortable to perform and usually require a dedicated operator for collection. A number of alternative sampling methods have been proposed in recent months, including saliva and gargling solution 4, 5, 18 . Our data shows that screening by self-sampled gargling-solution, tested via pooled qPCR was highly effective for identifying SARS-CoV-2 RNA positive HCWs. Expanding the described screening scheme to two-times per week would, in all likelihood, effectively eliminate the potential for large outbreaks with a reasonable margin of safety. This is feasible due to the high accuracy of the RT-qPCR based approach and the unmatched resource efficiency of sample pooling strategies. It thus represents a promising alternative to rapid antigen testing. There are no funding source for this study. COVID-19: the case for health-care worker screening to prevent hospital transmission Scaling up COVID-19 rapid antigen tests: promises and challenges Comparison of seven commercial SARS-CoV-2 rapid point-ofcare antigen tests: a single-centre laboratory evaluation study Saliva or Nasopharyngeal Swab Specimens for Detection of SARS-CoV-2 Pooled saliva samples for COVID-19 surveillance programme Pooling of samples for testing for SARS-CoV-2 in asymptomatic people Gargle-Direct: Extraction-Free Detection of SARS-CoV-2 using Real-time PCR (RT-qPCR) of Saline Gargle Rinse Samples Pharynx gargle samples are suitable for SARS-CoV-2 diagnostic use and save personal protective equipment and swabs Pushing beyond specifications: Evaluation of linearity and clinical performance of the cobas 6800/8800 SARS-CoV-2 RT-PCR assay for reliable quantification in blood and other materials outside recommendations Antibody Status and Incidence of SARS-CoV-2 Infection in Health Care Workers Nosocomial Transmission of Coronavirus Disease 2019: A Retrospective Study of 66 Hospital-acquired Cases in a London Teaching Hospital Clinical Course and Molecular Viral Shedding Among Asymptomatic and Symptomatic Patients With SARS-CoV-2 Infection in a Community Treatment Center in the Republic of Korea Performance and operational feasibility of antigen and antibody rapid diagnostic tests for COVID-19 in symptomatic and asymptomatic patients in Cameroon: a clinical, prospective, diagnostic accuracy study Rethinking Covid-19 Test Sensitivity -A Strategy for Containment Swab pooling: A new method for large-scale RT-qPCR screening of SARS-CoV-2 avoiding sample dilution Hebrew University-Hadassah COVID-19 Diagnosis Team. Lessons from applied large-scale pooling of 133,816 SARS-CoV-2 RT-PCR tests Performance of Oropharyngeal Swab Testing Compared With Nasopharyngeal Swab Testing for Diagnosis of Coronavirus Disease 2019-United States Saliva is more sensitive than nasopharyngeal or nasal swabs for diagnosis of asymptomatic and mild COVID-19 infection