key: cord-0895321-cf5h1smy authors: Hamed, Islam; Shaban, Nesreen; Nassar, Marwan; Cayir, Dilek; Love, Sam; Curran, Martin D.; Webb, Stephen; Yang, Huina; Watson, Katherine; Rostron, Anthony; Navapurkar, Vilas; Mahroof, Razeen; Morris, Andrew Conway title: Paired nasopharyngeal and deep lung testing for SARS-CoV2 reveals a viral gradient in critically ill patients: a multi-centre study date: 2020-10-15 journal: Chest DOI: 10.1016/j.chest.2020.10.017 sha: 8f27d2c9685cc63ed56ec4a1cc2bf793cd6f7776 doc_id: 895321 cord_uid: cf5h1smy nan Since the start of the COVID19 pandemic, arising from SARS-CoV-2 viral infection, 1 approximately 13000 patients have been admitted to critical care in the United 2 Kingdom, the majority have required advanced respiratory support 1 . Samples for SARS-CoV-2 detection can be obtained from the upper 5 (nasopharyngeal/oropharyngeal swabs) or lower respiratory tract 6 (sputum/endotracheal aspirate/broncho-alveolar lavage (BAL)) 2 . Viral ribonucleic acid 7 (RNA) is detected using reverse transcriptase polymerase chain reaction (RT-PCR). The 8 Cycle threshold (Ct) has a simple negative linear correlation with the logarithm of the 9 number of gene copies in the original sample and thus can be used to provide a semi-10 quantitative estimate of the viral RNA in a specimen 3 . It has been suggested that SARS-CoV-2 is predominantly shed from upper respiratory 13 tract, distinguishing it from SARS-CoV-1, where replication occurs mainly in the lower 14 respiratory tract. 4 There was a significant gradient between NP and deep lung viral loads ( Figure 1A) between symptom onset and Ct value in NP swabs, deep lung samples tended towards 56 higher Ct values the later they were taken ( figure 2 A and B) . Consequently the NP-lung 57 gradient was smaller in patients sampled later in their disease course (2C). ECMO 58 patients tended to be sampled later after symptom onset than invasive and non-59 invasively ventilated patients (median duration from symptom onset to test 12 days for 60 ECMO, 7 for IMV and 8 for NIV/oxygen). Discussion 63 In our case series of critically ill patients with COVID19, NP swabs were relatively 64 insensitive for detection of SARS-CoV-2: 67% of NP samples detected viral RNA 65 compared to 96% of deep respiratory samples. There was also a clear viral gradient with 66 a median 5 cycle (9 cycles for mechanically ventilated) lower Ct value in the lungs. To 67 the best of our knowledge, this is the first report of paired respiratory samples from 68 critically ill patients with COVID19. In SARS, arising from SARS-CoV-1, there was a substantial rate of false negative 71 nasopharyngeal swabs 6 leading to the suggestion that SARS-CoV-1, unlike SARS-CoV-2, 72 had a predilection for lower airways 4 . Our work challenges this assumption, 73 demonstrating significantly higher viral loads in the lower respiratory tract amongst 74 critically ill patients. It is possible that the higher viral load in the lungs may contribute 75 to the harmful inflammatory response that constitutes the pathology of SARS. 76 Interestingly, Lucas and colleagues report that patients with severe covid-19 did not 77 clear their nasopharyngeal carriage which is consistent with our findings (Figure 2A ) 7 . 78 They also found that initial NP viral load did not correlate with severity of illness 7 , 79 however they did not report lung viral loads. Our finding of lower lung viral loads at 80 later timepoints, especially in patients with the most severe respiratory failure requiring 81 ECMO, suggests that viral clearance is not sufficient to ameliorate the pulmonary 82 inflammatory response. This may explain why later (>7 days after symptom onset) 83 corticosteroids improve pulmonary function in severely affected COVID-19 patients 8 . Although there is understandable concern about the risk of deep lung sampling such as 86 BAL or endotracheal aspirate generating aerosol and increasing risk of healthcare 87 worker infection, our experience during the pandemic is that such procedures can be 88 performed safely. Use of enhanced aerosol protecting personal protective equipment, 89 respiratory isolation and closed/semi-closed respiratory circuits combine to increase the 90 safety of this process. This study reports from five units using two different molecular tests, increasing the 93 generalisability of our findings. The retrospective nature of the data collection may 94 introduce a source of bias against NP testing, which is often used as the first line test, 95 and that deep respiratory sampling may have only been undertaken if RNA was not 96 detected from NP swabs. However, during the first wave of the pandemic, the turn-97 around time for PCR was generally greater than 24 hours, and it is unlikely that negative 98 NP swabs will have influenced the decision to obtain paired samples within the same 24 99 hour period. Although all NP swabs were taken by nurses appropriately trained in viral 100 sample acquisition, we cannot be certain that the patient's nasopharynx was correctly 101 sampled in all cases, however it does reflect the real-world experience of virological 102 sampling. It is possible that the gradient found reflects technical rather than biological 103 factors, as both tracheal washing and broncho-alveolar lavage will sample a much larger 104 surface area than an NP swab, although both these former techniques will dilute 105 respiratory lining fluid which may have the opposite effect. The finding of a temporal 106 relationship in the gradient noted supports our contention that this is genuine biological 107 finding. Irrespective of this, the clinical implication that deep lung samples are more 108 sensitive amongst critically ill patients remains a key finding. 109 110 In conclusion, we have found that critically ill patients with COVID19 demonstrate a 111 significant viral gradient from the upper to the lower respiratory tract, which may have 112 diagnostic and pathophysiological importance. We conclude that, in the absence of an Intensive Care National Audit and Research Network COVID19 Detection of SARS-CoV-2 in Different Types of Clinical Specimens Review of Viral Testing 3-(Polymerase Chain Reaction) and Antibody/Serology Testing for Severe Acute Respiratory Syndrome-Coronavirus-2 for the Intensivist Asymptomatic Transmission, the Achilles' Heel of Current Strategies to Control Covid-19 Virological assessment of 5-hospitalized patients with COVID-2019 Detection of SARS Coronavirus in Patients with Severe Acute Respiratory Syndrome by Conventional and Real-Time Quantitative Reverse Transcription-PCR Assays Longitudinal analyses reveal immunological misfiring in 7 severe COVID-19 Dexamethasone in Hospitalized Patients with Covid-19 -Preliminary Report Guarantor -ACM acts as guarantor for the content of this article, its data and analysis Authorship contribution: IH-investigation, analysis, writing (original draft). NS-conception, investigation, writing (review and editing), MN, DC, SL,-investigation, writing (review and editing), MDCinvestigation, provision of materials, writing (review and editing), SW-conception, project administration, provision of materials, writing (review and editing), HYinvestigation, project administration, writing (review and editing), KW-investigation, writing (review and editing), AR-conception, project administration, provision of materials, writing (review and editing), VN-provision of materials, writing (review and editing), RM-conception, supervision, project administration, writing (review and editing), ACM-Conception, analysis, supervision, project administration, writing (original draft). The funders had no role in the drafting or revision of the manuscript or decision to publish.