key: cord-0754010-1h3uzj49
authors: Drew, Richard J.; O'Donnell, Sinéad; LeBlanc, David; McMahon, Mary; Natin, Dominic
title: The importance of cycle threshold values in interpreting molecular tests for SARS- CoV-2
date: 2020-07-10
journal: Diagn Microbiol Infect Dis
DOI: 10.1016/j.diagmicrobio.2020.115130
sha: fa4aa76c826ec872b13ea50ece037aeab0803d38
doc_id: 754010
cord_uid: 1h3uzj49

Abstract Using the Allplex™ 2019-nCoV assay (Seegene, South Korea) 285 samples were tested, 49 (17%) were positive for 3 genes, four (1.4%) samples were positive for two genes (all N gene and RdRP gene), eight (3%) samples positive for one gene (all N gene only) and 224 (78.5%) samples were negative.

SARS-CoV-2 is a novel coronavirus that has emerged in the last year, leading to a worldwide pandemic of Covid-19 disease. 1, 2 A significant volume of work has been invested in trying to provide rapid polymerase chain reaction (PCR) diagnostics to facilitate laboratory diagnosis of symptomatic patients, and to develop an understanding of viral load dynamics. 3, 4 A feature of this pandemic has been the high numbers of healthcare workers who have acquired the disease. 5 Identifying and testing these healthcare workers has been essential to prevent nosocomial transmission of Covid-19. 6 Using PCR as a way to screen staff to ensure they are no longer infectious has been proposed, but this is difficult as there can be variation in sampling technique, levels of detectable virus in the nasopharynx as the disease progresses and also it is unclear if the staff member remains infectious while low levels of virus are detected in the nasopharynx following clinical recovery. 7, 8 The aim of this retrospective study was to review SARS-CoV-2 PCR results of patients and staff using the Allplex™ 2019-nCoV assay (Seegene, South Korea) to increase knowledge around the expected Ct value for PCR testing. This was not a diagnostic accuracy or validation study. The clinical details were sought from patients and staff if their result was considered a weak positive i.e. only positive for one or two of the three genes tested.

This was a retrospective review of SARS-CoV-2 PCR tests performed in the Rotunda Hospital. Only tests from staff and adult patients at the Rotunda Hospital, and staff from Children's Health Ireland (CHI) at Temple Street taken between 24 th March 2020 and 15 th April 2020 were reviewed. All patients and staff were aged over 18 years. The Rotunda Hospital is a stand-alone tertiary level care maternity hospital in the centre of Dublin, Ireland, and the Children's Health Ireland at Temple Street is a stand-alone paediatric tertiary referral hospital. Testing was only performed on symptomatic patients and staff, at the discretion of either occupational health department or the patient's clinician. At this time there was no asymptomatic screening taking place for either staff or patients. As a consequence, a detected result at any cycle threshold value was considered to be positive, and the staff member was excluded for 14 days and contact tracing performed. Confirmation on a second platform was not performed. Only the first PCR test per adult patient was included, and all samples were combined nasopharyngeal/oropharyngeal swabs. The eNAT (Copan, USA) flocked swabs for collection and preservation of nucleic acids were used for sampling. Extraction was performed using the Nimbus platform (Hamilton,USA) and then PCR was performed using the Allplex™ 2019-nCoV assay (Seegene, South Korea) on the CFX96 (Bio-Rad,USA) in line with manufacturer's instructions. The gene targets for the PCR assay were the N gene, the E gene and the RdRP gene. The cycle threshold (Ct) value was recorded for each of the three genes. Samples with a detected result for all three genes ,or a single target detected just the RdRP or N gene, were interpreted as SARS Cov2 PCR positive in line with manufacturer's guidance. For each sample it was also recorded if the person was a patient or staff member, and for samples with only 1 or 2 of the genes detected clinical symptoms were also recorded. This was approved by the Ethics committees of the Rotunda Hospital and the CHI at Temple Street as a retrospective audit (Rotunda ethics approval #RAG-2020-009; CHI at Temple Street approval #20.025) J o u r n a l P r e -p r o o f

Over the three-week period, 358 samples from the Rotunda Hospital and CHI at Temple Street were tested. Ten duplicate samples and two paediatric samples were removed leaving a final list of 346 samples for analysis. Sixty-one (17%) were from adult patients with a median age of 35 years (range 15-52 years). The remaining 285 (83%) were from staff with a median age of 39 years (range 18-67 years). Of the 61 patients, 10 (16%) were positive for all three gene targets, and 51 (84%) were negative for all three gene targets. Of the 285 staff samples, 49 (17%) were positive for three gene targets, four (1.4%) samples were positive for two genes (all N gene and RdRP gene), eight (3%) positive for one gene (all N gene only) and 224 (78.5%) samples were negative. All positive healthcare workers were excluded from work for 14 days and contact tracing was done in line with national guidelines. The available clinical details of these staff with one or two genes positive is shown in table 1. The median Ct value for the N gene was lower in samples that were positive for all three genes (median Ct 24, range 12-36) than in samples that were positive for two genes (median Ct 36) and just one gene (median Ct 39), (figure 1). 

This study has shown that overall the positivity rate for SARS-CoV-2 by PCR in our population was 16% in patients and 17% in staff which is a similar level to a study on staff in the United Kingdom. 9 All the patients had Ct values <36 in all 3 genes, likely reflecting that they are true positives as they were symptomatic as well. However, of the 61 staff with a detectable gene in the assay 12 (20%) of them were positive in only one or two genes at a high Ct value. This 20% frequency of high Ct value results poses a difficulty when interpreting the potential infectivity of a staff member, and whether or not they can return to work safely. As testing expands to include asymptomatic people presurgery, consideration should be given to having an indeterminate cut-off, after which positive results should be confirmed on a second platform.

The reason for these high Ct values is not clear, but in our study we have detailed the symptoms recorded at the time of testing. For patients with two gene targets detected (N and RdRP genes), these seem to be clinically significant results as the patients were either known positives or symptomatic with a close contact history. For patients with just the N gene detectable, there appears to be two groups. The first have had symptoms for more than seven days and so the weak positive result may reflect degraded viral RNA and clearance of the virus. The second group were swabbed on the first or second day of their illness. Perhaps if they were retested two days later the viral load may be higher and they may have had a lower Ct value result for each of the three genes. Further work is needed to determine if virus detected at these high cycle threshold values is viable and can lead to cross-transmission. The interpretation of detected results with high Ct values needs to be done in the context of the clinical situation and timing of testing relative to symptoms or exposure.

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The authors would like to thank the occupational health nurses, laboratory scientists and the infection control midwives/nurses from the Rotunda Hospital and Temple Street for their help with data collection.