key: cord-0289074-l0z13nen authors: Riis, A. G.; Rossland, T. M.; Löhr, I. H.; Dalen, I.; Kleppe, L. K.; Sundal, J.; Berg, A.; Vadla, M. S.; Lenning, O. B.; Syre, H. title: Clinical validation of 3D-printed nasopharyngeal and oropharyngeal swabs for SARS-CoV-2 RT-PCR date: 2022-05-19 journal: nan DOI: 10.1101/2022.05.16.22274315 sha: 098ebaab89e08bb98c2bb809e7f4c65c38f981d4 doc_id: 289074 cord_uid: l0z13nen Due to limited access to commercially available flocked nasopharyngeal (NP) and oropharyngeal (OP) swabs during the SARS-COV-2 pandemic, we have evaluated the sensitivity of 3D-printed swabs compared to commercial swabs in a clinical setting. We included 35 subjects with known exposure to SARS-CoV-2. Participants were tested with commercial and prototype NP/OP swab pairs 8 and 22 days after exposure. At day 8, the sensitivity of the prototype was 96% for NP-samples (CI 81-99%) and 91% for OP-samples (CI 72-97%). The sensitivity of the commercial swab was 92% for NP-samples (CI 76-98%) and 91% for OP-samples (CI 72-97%). At day 22, the sensitivities of the commercial swab were 100% for NP-samples (CI 82-100%) and OP-samples (CI 77-100%), whereas sensitivity of the prototype was 61% for NP-samples (CI 39-80%) and 54% for OP-samples (CI 29-77%). In conclusion, the prototype might be an alternative to commercial swabs when used early in the course of infection. Coronavirus disease 2019 is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and was first described late 2019. The virus has spread widely throughout the world, and on March 11 2020 -the World Health Organization (WHO) declared a pandemic. Large scale testing is a crucial step in controlling the ongoing COVID-19 pandemic, to identify infected individuals and for surveillance of novel virus variants [1] . National testing guidelines for COVID-19 varies between countries. The Infectious Disease Society of America (IDSA) recommends testing for all individuals with symptoms suspicious for COVID-19 [2] . One of the recommended methods for initial diagnostic testing for SARS-CoV-2 infection is nucleic acid amplification tests (NAAT) [3] from samples collected from an upper respiratory specimen [4] . Multiple Real Time reverse transcriptase (RT)-PCR assays targeting different genes of the SARS-CoV-2 genome have been developed during the course of the COVID-19 pandemic [5, 6] . WHO recommends using a method for detection of two different targets, including one target specific for COVID-19 or SARS-like coronavirus [7] . The cycle threshold (Ct) value obtained by RT-PCR represents the number of cycles needed in the PCR reaction to surpass the threshold for positive test results and is inversely related to the viral load [8] . Thus, the Ct value can potentially be used as an indirect estimate of the viral RNA copy number in a sample [8] . In a clinical setting SARS-CoV-2 RT-PCR test results are normally qualitatively reported as negative or positive without reporting the Ct value. There is no perfect standard for the evaluation of the clinical sensitivity of COVID-19 diagnostic tests. A meta-analysis, suggested that Nasopharyngeal (NP) swabs, or pooled nasal and oropharyngeal (OP) swabs, offer the best diagnostic performance among samples collected in the upper respiratory tract [9] . The clinical sensitivity may be influenced by several factors, including the prevalence in the population tested, the analytic sensitivity of the test assay, and the quality of the swab used. Hence, the false negative rates in samples collected from the nasopharynx varies from 1.8 to 33% in a systematic review, with an unexplained heterogeneity in the proportion of false negative RT-PCR results [10] . The increasing demand for SARS-CoV-2 testing during the pandemic placed pressure on supply chains. During the initial phase of the pandemic, limited access to diagnostic equipment, including commercially available flocked NP and OP swabs, was a well-known concern [11] , 3D printing of NP All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. and OP swabs could possibly reduce the problem. Callahan et al [12] evaluated 160 3D-printed NP swab designs from 24 different manufacturers. Four 3D-printed prototypes, including a prototype from HP inc, passed initial testing and completed a clinical trial. Reference and prototype swab pairs were collected from outpatients who came to a test station with COVID-19 suspicious symptoms. All four prototypes exhibited a high degree of concordance with the reference swab. During a period of limited access to commercial flocked NP and OP swabs, and lack of 3D-printed swabs with CE approval in our region, we wanted to evaluate the utility and sensitivity of 3D-printed NP and OP swabs from a local producer. 3D-printed swabs were compared to commercial flocked NP and OP swabs used in our outpatient test stations. We included both symptomatic and asymptomatic patients with known exposure to SARS-CoV-2. Related to a bus tour for senior citizens in the South of Norway mid-September 2020, an outbreak of COVID-19 occurred. Seven days after departure, one of 40 passengers developed symptoms and tested positive for SARS-CoV-2, this was defined as day 0. The following seven days, the remaining 39 passengers underwent repeated testing with commercial OP, and for some cases with commercial NP swabs. In total 38 of 40 passengers tested positive for SARS-CoV-2. Two passengers remained PCRnegative. Thirty-five of the 40 passengers were included in the study. Participants gave informed consent. Day 0-7 was defined as baseline. At baseline, the study population consisted of 33 PCR-positive and 2 PCR-negative individuals. The two PCR-negative individuals remained PCR-negative throughout the follow-up period, and did not develop SARS-CoV-2 specific antibodies. The proportion of asymptomatic, PCR-positive individuals was 24% (8/33), while 76% (25/33) developed symptomatic COVID- All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The 35 study participants underwent repeated testing with NP and OP swabs on day 8 -10, and on day 22-24 (from now on referred to as day 8, and day 22). Commercial (FLOQSwabs, Copan, Italy) and prototype (3D-printed; HP inc, Norway) NP and OP swab pairs were collected from each participant in a random order. Each swab was placed in 3 ml sterile universal transport medium (UTM; Copan). Due to discomfort, some of the participants opposed repeated testing with NP swabs. An experienced general practitioner or infectious disease doctor performed sampling. The specimens were transported at room temperature to the laboratory and were stored at 4°C for up to 4 days prior to RT-PCR. All samples were analyzed at Stavanger University Hospital, Norway. NP and OP swabs, both commercial and prototypes, collected the same day for each patient, were processed and analyzed within the same batch for comparison. Viral RNA extraction was performed using the RNAdvance Viral Rea- (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. pre-test probability (known outbreak in a defined group), the gold standard was defined as any positive or weak positive test result in either 3D printed or commercially available swabs for the respective sample sites and time points. For data presentation, weak positive samples without a Ct value, but a ΔRn above 0.05 were assigned a Ct value of 40 (n=28; 16 commercial and 12 prototype) and negative samples were assigned a Ct value of 45 (n=110; 31 commercial and 79 prototype). Diagnostic categories were compared between prototype swabs and commercial swabs by cross-tabulation. Chance-adjusted agreement was estimated as quadratically weighted Cohen's Kappa (K) and Gwet's AC2 coefficient and reported with t-distribution based 95% confidence intervals (CI). Gwet's AC2 is often preferred in situations with uneven marginals, where Cohen's kappa may give paradoxical results [13] . Differences between swabs were tested with McNemar's test for paired data. Ct values are presented as medians and interquartile ranges (IQR), and compared between swab types for a given lo- We collected and analyzed 124 prototype and commercial swab pairs (60 NP and 64 OP) from 35 participants. Twenty-eight NP and 30 OP swab pairs were collected on day 8; remaining pairs were collected on day 22. Figure 1 shows comparison of performances of NP and OP swabs at day 8 and day 22. Overall, when comparing performances of the two swab types with the gold standard, the prototype failed to detect 16 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Ct values were significantly higher for the prototype compared to commercial swabs at both day 8 and day 22 and for both OP and NP samples (p values from 0.002 to 0.017) (Table 1, Figure 2 ). Furthermore, Ct values were significantly higher at day 22 compared to day 8 ( Table 2 ). The COVID-19 pandemic has resulted in severe shortages of medical diagnostic equipment; and in a period of limited access to commercially available flocked NP and OP swabs, we wanted to test the performance of a 3D-printed NP and OP swab for collection of samples for RT-PCR-analysis. This study shows that the 3D-printed prototype was non-inferior to the commercial swab (FLOQSwabs, Copan) in diagnosing infections with SARS-CoV-2 at day 8, and overall in samples with high viral loads. Ct values for the prototype were higher than for the commercial swab at day 8, but there was no difference in the rate of false negatives between the groups. At day 22, the prototype detected substantially less gold-standard positive samples, than the commercial swab. Most samples had low viral loads at day 22. The results from day 8 are consistent with a previous clinical trial of outpatients with COVID-19 suspicious symptoms [12] . But in contrast to Callahan et al. [12] we found significantly higher Ct values for All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. the prototype compared to the commercial swab, for all samples, including those collected at day 8, i.e. shortly after exposure to SARS-CoV-2 (P-values from 0.002 to 0.017). In patients infected with SARS-CoV-2, viral loads are reported to peak at symptom onset or during the first week of illness [14, 15] , followed by a gradual decline [14] [15] [16] . Mean duration of SARS-CoV-2 RNA shedding from the upper respiratory tract is reported to be around 17 days in patients with mild to moderate illness [16, 17] , and 19.8 days in severely ill patients [17] . According to a systematic review, live virus from respiratory samples could not be isolated beyond nine days of illness [16] . Isolation of live virus from respiratory specimens has been described beyond this period, and is probably more common in patients with severe illness [17, 18] . A sensitive test during the period where the viral load is at highest is critical to reduce further transmission of the virus. Nevertheless, there is potential harm in a false negative test, also later in the course of infection. This could affect COVID-19 public health management strategies in terms of case identification and contact tracing [19] . We collected the first swab-pairs at day 8, and have not evaluated the sensitivity of the prototype the first days after SARS-CoV-2 exposure. However, viral loads in the upper respiratory tract are reported to gradually increase towards symptom onset [16] . A study from 2020 suggest that the false negative rate of RT-PCR based tests decreases from time of exposure, and is lowest 8 days after exposure [20] . Thus, we consider the timing of testing at day 8 appropriate for comparing the performance of the swabs at the expected peak of viral load. As for day 22, it is possible that the prototype would underperform compared to the commercial swab also during the first days after SARS-CoV-2 exposure and/or before symptom onset, when the viral load is expected to be low. The proportion of asymptomatic individuals in our study was 24%. Viral loads in samples collected from the upper respiratory tract inferred from Ct values, are reported to be similar [21, 22] or lower [23] in asymptomatic cases. Furthermore, for asymptomatic cases, the period of viral shedding is reported to be shorter [24, 25] , whereas in other studies statistically significant differences were not found [21, 23] . All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. This study has some limitations. Testing was conducted on a low number of participants, and almost all the participants were SARS-CoV-2 positive, thus the study was not suitable for evaluating the specificity of the prototype. Furthermore, the usability of the prototype was not assessed. Thus, further testing is needed to determine the test-accuracy of 3D-printed swabs. One strength of the study is that we repeated testing with both swab types shortly after exposure and later in the course of infection; this enabled us to evaluate the performance of the prototype on samples with different viral loads. Because our study population consisted of a defined group with known exposure to SARS-CoV-2, we were able to include both symptomatic and asymptomatic individuals. Finally, sample collection techniques can affect the clinical sensitivity of the test. In our study, experienced personnel performed sampling, which reduced the risk of false negative results. To prevent swab shortages during future pandemics, or seasonal epidemics, caused by influenza or other viruses, 3D printing of swabs might help to maintain rapid, large scale diagnostic testing. In summary, this study shows that the performance of the 3D-printed prototype was non-inferior to the commercial swab eight days after exposure to SARS-CoV-2. In a shortage situation, the prototype might be an alternative to a commercial swab when used early in the course of infection or within eight days after known exposure. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The authors confirm contribution to the paper as follows: study conception and design: IHL, HS; data collection: ÅGR, TMR, JS, ÅB, MSV, OBL, HS; analysis and interpretation of results: ÅGR, TMR, ID, IHL, HS; draft manuscript preparation: ÅGR, TMR. All authors reviewed the results and approved the final version of the manuscript. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Lines of equality have been added to all plots. Each dot may represent several observations. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Multiplex qPCR discriminates variants of concern to enhance global surveillance of SARS-CoV-2 Infectious Diseases Society of America Guidelines on the Diagnosis of COVID-19 Overview of Testing for SARS-CoV-2, the virus that causes COVID-19 Interim Guidelines for Collecting and Handling of Clinical Specimens for COVID-19 Testing Laboratory diagnosis of SARS-CoV-2 -A review of current methods Diagnostic approaches in COVID-19: clinical updates Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Euro surveillance : bulletin Europeen sur les maladies transmissibles = European communicable disease bulletin A Systematic Review of the Clinical Utility of Cycle Threshold Values in the Context of COVID-19 Diagnostic performance of different sampling approaches for SARS-CoV-2 RT-PCR testing: a systematic review and meta-analysis. The Lancet Infectious Diseases FALSE-NEGATIVE RESULTS OF INITIAL RT-PCR ASSAYS FOR COVID-19: A SYSTEMATIC REVIEW Comparison of Copan ESwab and FLOQSwab for COVID-19 Diagnosis: Working around a Supply Shortage Open Development and Clinical Validation of Multiple 3D-Printed Sample-Collection Swabs: Rapid Resolution of a Critical COVID-19 Testing Bottleneck How Robust Are Multirater Interrater Reliability Indices to Changes in Frequency Distribution? The American Statistician Temporal dynamics in viral shedding and transmissibility of COVID-19 Virological assessment of hospitalized patients with COVID-2019 SARS-CoV-2, SARS-CoV, and MERS-CoV viral load dynamics, duration of viral shedding, and infectiousness: a systematic review and meta-analysis Understanding viral shedding of severe acute respiratory coronavirus virus 2 (SARS-CoV-2): Review of current literature Duration of Culturable SARS-CoV-2 in Hospitalized Patients with Covid-19 Considerations for quarantine of contacts of COVID-19 cases: interim guidance Variation in False-Negative Rate of Reverse Transcriptase Polymerase Chain Reaction-Based SARS-CoV-2 Tests by Time Since Exposure 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 Upper respiratory viral load in asymptomatic individuals and mildly symptomatic patients with SARS-CoV-2 infection Viral dynamics in asymptomatic patients with COVID-19 Asymptomatic infection and atypical manifestations of COVID-19: Comparison of viral shedding duration Comparison of Clinical Characteristics of Patients with Asymptomatic vs Symptomatic Coronavirus Disease We want to thank Hans Petter Torvik for recruiting participants, and contributing in conduction of the clinical trial. Jorunn Nilsen, Sanna Palmqvist, Siri Øksnevad, and Linda Gloppen for their assistance in collection of patient samples. Hanne Røland Hagland who introduced us to the manufacturer of the 3Dprinted swabs. The Regional Committee for Medical Research Ethics Western Norway (REK West) gave ethical approval for this work (reference no. 118664).