key: cord-0803623-ywr6plsh authors: Sirivongrangson, P.; Kulvichit, W.; Payungporn, S.; Pisitkun, T.; Chindamporn, A.; Peerapornratana, S.; Pisitkun, P.; Chitcharoen, S.; Sawaswong, V.; Worasilchai, N.; Kampunya, S.; Putcharoen, O.; Tawitsri, T.; Leelayuwatanakul, N.; Kongpolprom, N.; Phoophiboon, V.; Sriprasart, T.; Samransamruajkit, R.; Tungsanga, S.; Tiankanon, K.; Lumlertgul, N.; Leelahavanichkul, A.; Sriphojanart, T.; Tantawichien, T.; Thisyakorn, U.; Chirathaworn, C.; Praditpornsilpa, K.; Tungsanga, K.; Eiam-Ong, S.; Sitprija, V.; Kellum, J. A.; Srisawat, N. title: Endotoxemia and circulating bacteriome in severe COVID-19 patients date: 2020-06-02 journal: nan DOI: 10.1101/2020.05.29.20109785 sha: bf0e339d85079e33aae0ec9f6fa02abab17f2515 doc_id: 803623 cord_uid: ywr6plsh Background: When severe, COVID-19 shares many clinical features with bacterial sepsis. Yet, secondary bacterial infection is uncommon. However, as epithelium are injured and barrier function is lost, bacterial products entering the circulation might contribute to the pathophysiology of COVID-19. Methods: We studied 19 adults, severely ill patients with COVID-19 infection, who were admitted to King Chulalongkorn Memorial Hospital, Bangkok, Thailand, between 13th March and 17th April 2020. Blood samples on day 1, 3, and 7 of enrollment were analyzed for endotoxin activity assay (EAA), Beta-D-Glucan (BG), and 16S rRNA gene sequencing to determine the circulating bacteriome. Findings: Of the 19 patients, 14 were in intensive care and 10 patients received mechanical ventilation. We found 8 patients with high EAA ([≥] 0.6) and about half of the patients had high serum BG levels which tended to be higher in later in the illness. Although only 1 patient had a positive blood culture, 18 of 19 patients were positive for 16S rRNA gene amplification. Proteobacteria was the most abundant phylum. The diversity of bacterial genera was decreased overtime. Interpretation: Bacterial DNA and toxins were discovered in virtual all severely ill COVID-19 pneumonia patients. This raises a previously unrecognized concern for significant contribution of bacterial products in the pathogenesis of this disease. ( COVID-19) are mild, severe COVID-19 pneumonia can occur with a mortality rate as high as 50% [1] . I t i s u n c l e a r w h y s o m e p a t i e n t s d e v e l o p clinical features of sepsis/septic shock with multiple organ dysfunction [2] . The majority of bacterial cultures from severe COVID-19 patients are negative, [3] and although empiric antibiotics are commonly used, [3] [4] [5] t h e y a r e n o t r e c o m m e n d e d [ 6 ] . However, while the respiratory tract is the principle site of infection for COVID-19, the disease has been shown to involve the GI tract as well and symptoms such as diarrhea are reported in about a third of cases [7] . Enterocytes in ileum and colon express the ACE2 receptor and virus has been detected in stool. Thus, there is a possibility that bacterial translocation from the GI tract might complicate severe COVID- Endotoxin, a part of the cell wall of Gram-negative bacteria, has been extensively investigated and acknowledged as one of the key triggers of lethal shock during severe sepsis and also one of the primary drivers of the cytokine storm [9-11]. Serum Thus, we designed this investigation to determine whether bacterial products were present in the blood of severe COVID-19 pneumonia patients and whether their source was likely to be the gut as evidenced by serum BG. We also sought to characterize the circulating bacteriome in COVID-19 pneumonia. This was a prospective observational study in COVID-19 pneumonia patients admitted to King Chulalongkorn Memorial Hospital, Bangkok, Thailand, between 13 th March and 17 th April 2020. Our inclusion criteria included ( 1) age >18 years, ( 2) confirmed COVID-19 pneumonia, and ( 3) had leftover blood samples. The first day of enrollment was the day that patients fulfilled inclusion criteria. The study was reviewed and approved by Faculty of Medicine, Chulalongkorn University ethics committee consent was waived due to the observational nature of the study. The study was designed and conducted according to the STROBE guideline [13] . We obtained demographic data, information on clinical presentations, laboratory investigations, and radiography at the time of presentation, and during intensive care unit (ICU) admission. We collected blood samples that were left over on day 1, day 3, and day 7 after enrollment. All laboratory tests and radiologic assessments, including standard chest radiographs and chest computed tomography, were performed at the discretion of the treating physician. Endotoxin activity assay ( EAA), cytokines, and serum BG were measured on day 1, 3, and 7 of enrollment. We assessed clinical outcomes on day 28 after enrollment, including mechanical ventilation, ventilator-free day, vasopressor, prone position, extracorporeal membrane oxygenation ( ECMO), acute kidney injury ( AKI), renal replacement therapy ( RRT), and successful extubation. A confirmed case of COVID-19 was defined by a positive result of a reverse-transcriptase-polymerasechain-reaction ( RT-PCR) assay of a specimen collected from a nasopharyngeal swab. We defined COVID-19 pneumonia as a COVID-19 case who showed the evidence of pulmonary infiltration from chest radiography or chest computer tomography. We defined severe COVID-19 as a COVID-19 case who was admitted in ICU. lopinavir/ritonavir or darunavir, 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 copyright holder for this preprint this version posted June 2, 2020. We performed the chemiluminescent-based endotoxin activity assay assay detects BG through the activation of factor G, a protease zymogen which activates a second protease zymogen, pro-clotting enzyme, that cleaves a chromophore from a chromogenic peptide resulting in light absorbance at 405 nm. Serum BG >60 pg/mL was used as positive cut-off [16] . BG values at <7.8 pg/mL and >523.4 pg/mL were recorded as 0 and 523 pg/mL, respectively. 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 copyright holder for this preprint this version posted June 2, 2020. . Raw sequencing data were demultiplexed by MiSeq reporter software (which was not certified by peer review) 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 2, 2020. . data. All statistical analyses were performed using Stata version 15.1 ( STATA Corp, TX). P value of less than 0.05 was considered as statistical significance for all tests performed. A total of 147 patients were recruited. Of these, 53 ( Table 1 ). We showed detailed clinical data and outcomes of 19 patients in Table 2 and Table S1 . Overall median (which was not certified by peer review) 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 2, 2020. . ( Table S1) . Comparing clinical features between patients with high ( ≥ 0.6 on day 1) and low EAA ( < 0.6 on day 1) revealed that those with high EAA had lower median age, and higher severity scores ( Regarding clinical outcomes, patients with high EAA were more likely to develop subsequent bacterial infection within 28 days after enrollment ( 38% vs 18%). Patients with high EAA tended to need more mechanical ventilation support than the low EAA group, although the statistical significance was not attained ( 62.5% vs 45.5%, P=0.65). proportion of patients requiring vasopressors, prone position, and ECMO did not differ between EAA groups ( Table 2) . Twenty-eight-day outcomes are shown in Figure 6 . The overall rate of AKI In this cohort of COVID-19 pneumonia, we show that nearly 90% and 40% of patients had endotoxemia ( defined as a moderate to high level of EAA), and high levels of BG, a measure of gut permeability ( Figure 2 ). Using Next Generation Sequencing ( NGS), we could also demonstrate the dominant 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 copyright holder for this preprint this version posted June 2, 2020. . With NGS, a high sensitive technology, we could detect bacteria which were unable to grow using standard culture methods. From previous reports, bacterial DNA and RNA were discovered between 4% to 100% in blood of healthy individual [27] [28] [29] [30] [31] . Our data agree with Gosiewski et al. which showed increased abundance of proteobacteria in sepsis patients compared to the healthy population ( 60.1% vs 16.4%) [32] . This raises the possibility that COVID-19 could cause sepsis like syndrome with the same dominant bacteria phylum as in sepsis patients. This phylum contains many genera of bacteria. The most predominant bacteria were Sphingomonas, Bradyrhizobium, and Enhydrobacter. Sphingomonas paucimobilis is an opportunistic pathogen that can cause hospital associated infections from environmental exposure [33] . Bradyrhizobium enterica were found in patients with colitis [34] . Enhydrobacter aerosaccus can be detected from a patient with Hemophagocytic lymphohistiocytosis ( HLH) with concomitant corticosteroid use [35] . This is particularly of interest due to the growing evidence of HLH syndrome in severe COVID-19 patients [36] . Patients with COVID-19 have been found to have high levels of proinflammatory cytokines such as IL-6, IL-1β, IP10, and MCP-1 [4] . Similar findings were also reported in patients with SARS [37] and MERS-CoV 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 copyright holder for this preprint this version posted June 2, 2020. . 1 1 [38] . This has prompted several authors to discuss so-called " Cytokine Storm" in viral respiratory infection that is a cause of multiple organ failure. However, our results suggest that bacterial products might be another possible contributor to the cytokine storm rather than only the virus itself. The source of bacterial toxin, and bacterial DNA in the blood of patients with COVID-19 pneumonia is unclear. It is possible that viremia results in capillary leakage syndrome similar to bacterial sepsis which causes interstitial edema and induces dysfunction of the lung and intestinal barrier which may facilitate bacterial toxin and live bacteria translocation into the circulation. These bacterial products then induce the release of proinflammatory cytokines. In our study, many patients investigation. Second, our study did not include respiratory tract or gastrointestinal specimens, so we cannot completely establish the source of bacterial products in our patients. Future study should explore the effect of organ crosstalk between the lung and the intestine during COVID-19 infection. 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 copyright holder for this preprint this version posted June 2, 2020. all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. All authors interpreted the findings, contributed to writing the manuscript, and approved the final version for publication. Toray Industries provided endotoxin activity assay kits for use in this study. The company had no influence on the study design or analysis or on the comment of this article. None of the other authors have any disclosures. We would like to thank the staff, fellows, nurses, and research coordinators from the Excellence Center for Critical Care Nephrology The investigator-initiated study was funded by the Excellence Center for Critical Care Nephrology, King Chulalongkorn Memorial Hospital. 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 copyright holder for this preprint this version posted June 2, 2020. 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 copyright holder for this preprint this version posted June 2, 2020. . (which was not certified by peer review) 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 2, 2020. 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 copyright holder for this preprint this version posted June 2, 2020. . https://doi.org/10.1101/2020.05.29.20109785 doi: medRxiv preprint ( IQR) APACHE II, Acute Physiology and Chronic Health Evaluation II; CRP, C-reactive protein; EAA, endotoxin activity assay; FiO 2 , fractional inspired oxygen; IL-6, interleukin-6; PaO 2 , partial pressure of arterial oxygen; SOFA, sequential organ failure assessment. ( IQR) AKI, acute kidney injury; EAA, endotoxin activity assay; ECMO, extracorporeal membrane oxygenation; RRT, renal replacement therapy. 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 copyright holder for this preprint this version posted June 2, 2020. . https://doi.org/10.1101/2020.05.29.20109785 doi: medRxiv preprint Proteobacteria were observed to be the dominant bacterial phyla followed by Bacteroidetes, Actinobacteria and Firmicutes. Hence, we propose the sources of endotoxin to be majorly from the gut and minorly from the secondary bacterial infection of the lungs. Abbreviations: COVID-19, the coronavirus disease 2019; SAR-CoV-2, severe acute respiratory syndrome coronavirus 2; ARDS, acute respiratory distress syndrome; ACE2, angiotensin converting enzyme 2; MOF, multiple organ failure 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 copyright holder for this preprint this version posted June 2, 2020. . High EAA at day 1 8 Figure 1 . Study cohort. onia criteria No evidence of pneumonia ow EAA at day 1 11 No leftover samples 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 copyright holder for this preprint this version posted June 2, 2020. . 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