key: cord-0746350-pn954c0b authors: Maes, M.; Higginson, E.; Pereira Dias, J.; Curran, M. D.; Parmar, S.; Khokhar, F.; Cuchet-Lourenco, D.; Lux, J.; Sharma-Hajela, S.; Ravenhill, B.; Mahroof, R.; Solderholm, A.; Forrest, S.; Sridhar, S.; Brown, N. M.; Baker, S.; Navapurkar, V.; Dougan, G.; Bartholdson Scott, J.; Conway Morris, A. title: Secondary pneumonia in critically ill ventilated patients with COVID-19 date: 2020-06-28 journal: nan DOI: 10.1101/2020.06.26.20139873 sha: 038c26ffc03221c90df13408f1591c0bfa09f23c doc_id: 746350 cord_uid: pn954c0b Background Pandemic COVID-19 caused by the coronavirus SARS-CoV-2 has a high incidence of patients with severe acute respiratory syndrome (SARS). Many of these patients require admission to an intensive care unit (ICU) for invasive artificial ventilation and are at significant risk of developing a secondary, ventilator-associated pneumonia (VAP). Objectives To study the incidence of VAP, as well as differences in secondary infections, and bacterial lung microbiome composition of ventilated COVID-19 and non-COVID-19 patients. Methods In this prospective observational study, we compared the incidence of VAP and secondary infections using a combination of a TaqMan multi-pathogen array and microbial culture. In addition, we determined the lung microbime composition using 16S RNA analyisis. The study involved eighteen COVID-19 and seven non-COVID-19 patients receiving invasive ventilation in three ICUs located in a single University teaching hospital between April 13th 2020 and May 7th 2020. Results We observed a higher percentage of confirmed VAP in COVID-19 patients. However, there was no statistical difference in the detected organisms or pulmonary microbiome when compared to non-COVID-19 patients. Conclusion COVID-19 makes people more susceptible to developing VAP, partly but not entirely due to the increased duration of ventilation. The pulmonary dysbiosis caused by COVID-19, and the array of secondary infections observed are similar to that seen in critically ill patients ventilated for other reasons. . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 28, 2020. . https://doi.org/10.1101/2020. 06.26.20139873 doi: medRxiv preprint Methods In this prospective observational study, we compared the incidence of VAP and secondary 48 infections using a combination of a TaqMan multi-pathogen array and microbial culture. In addition, 49 we determined the lung microbime composition using 16S RNA analyisis. The study involved 50 eighteen COVID-19 and seven non-COVID-19 patients receiving invasive ventilation in three ICUs 51 located in a single University teaching hospital between April 13 th 2020 and May 7 th 2020. 52 Results We observed a higher percentage of confirmed VAP in COVID-19 patients. However, there 53 was no statistical difference in the detected organisms or pulmonary microbiome when compared to 54 non-COVID-19 patients. 55 Conclusion COVID-19 makes people more susceptible to developing VAP, partly but not entirely 56 due to the increased duration of ventilation. The pulmonary dysbiosis caused by COVID-19, and the 57 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 28, 2020. CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 28, 2020. Additionally, the choice of diagnostic sample is critical and directed bronchoscopy can limit 85 contamination from the proximal airway [12] . An observation that the rate of VAP amongst patients 86 with COVID-19 appeared to be higher than our background rate led to the institution of a minimally-87 aerosol generating bronchoscopic sampling procedure to seek to minimise over-diagnosis inherent in 88 endo-tracheal aspirate-based sampling [12] . 89 90 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted June 28, 2020. Samples for routine microbiology were processed according to the UK Standards for Microbiology 116 Investigations [13] . Any significant growth with a CFU of >10 4 /mL was identified by MALDI-ToF 117 mass spectrometry. 118 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 28, 2020. . https://doi.org/10.1101/2020.06.26.20139873 doi: medRxiv preprint RNA/DNA extraction and SARS-CoV-2 qPCR 120 500µl of BAL was subjected to RNA/DNA extraction following an existing method [14] . Viscous 121 samples were first treated with 10% v/v mucolysin, before 500µl lysis buffer (25mM Tris-HCL+ 4M 122 Guanidine thiocyanate with 0.5% b-mercaptoethanol) and glass beads were added to each sample. 123 Tubes were vortexed, and 100% analytical grade ethanol was added to a final concentration of 50%. 124 After a 10 min incubation, 860µl of lysis buffer (containing MS2 as an internal extraction and 125 amplification control) was added. This was then run over an RNA spin column as previously 126 described [14] . SARS-CoV-2 specific real-time RT-PCR was performed and interpreted as 127 previously described [14] . Table 1 ; five patients were sampled on two occasions and two patients sampled on 174 three occasions over the study period. Nineteen of these samples were SARS-CoV-2 positive by RT-175 PCR, with a mean CT of 28.3. Fifteen samples tested negative for SARS-CoV-2; however, six of 176 these samples came from patients previously diagnosed with COVID-19 by RT-PCR (Table 2) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 28, 2020. . https://doi.org/10.1101/2020.06.26.20139873 doi: medRxiv preprint or conventional microbiology were abundantly identified in samples by 16S sequencing (Figure 1) . 200 When comparing COVID-19 positive to COVID-19 negative patients, there was no specific taxon that 201 was more prevalent in either group. Additionally, the microbiomes of COVID-19 positive patients 202 were not significantly different in either the species richness (alpha diversity) or the microbial 203 composition (beta diversity) to those of COVID-19 negative patients. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 28, 2020. problems of quantity, we did not find evidence in this report of a qualitative difference. Indeed, the 254 microbial profiles of ventilated patients with active SARS-CoV-2, those who had cleared SARS-CoV-255 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 28, 2020. The TaqMan multi-pathogen array has been adopted as a routine clinical service in our institution 274 following a previous evaluation study. The as Biological Safety Officer to ensure we work in a safe environment. We also thank Estée Török and 283 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 28, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted June 28, 2020. CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted June 28, 2020. . https://doi.org/10.1101/2020.06.26.20139873 doi: medRxiv preprint CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted June 28, 2020. . https://doi.org/10.1101/2020.06.26.20139873 doi: medRxiv preprint Clinical Characteristics of COVID-19 in is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)The copyright holder for this preprint this version posted June 28, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 28, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 28, 2020. Tables 414 Table 1 . Clinical and demographic features of reported population. CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 28, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 28, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 28, 2020.