RNA Sequencing of Sepsis Patients Informs Tests to Quickly Diagnose Pathogens and Resistance

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A faster diagnosis of the pathogen and resistance via RNA sequencing informed PCR will improve outcomes. We hypothesize that we can use RNA sequencing from patients with sepsis to identify novel targets for future nucleic acid-based tests. This cohort study of 46 sepsis patients admitted to the ICU with samples taken on days 0, 1, 3, and 7 with follow up through the hospital stay during 2021–2022. All patients had RNA sequencing, depth of > 100 million reads, conducted on days in the ICU from a single center medical intensive care unit. Patients were admitted to the ICU with a diagnosis of sepsis. Patients or surrogates were approached consecutively and those who consented were enrolled. RNA sequencing of peripheral blood was performed to identify RNA targets from pathogens. RNA sequencing data that did not map to the human genome was then aligned to resistance genes and pathogen genomes and used to design novel PCR tests. These tests were correlated with culture diagnosis and clinical outcomes. Forty-six patients (mean age 62.2, 48% female) were enrolled and samples from 87 time points were collected. These samples resulted in 8.6 billion RNA sequencing reads to identify pathogen RNA. PCR target discovery for this study focused on positive blood cultures (n = 40) due Escherichia coli (5), Staphylococcus aureus (6), and Pseudomonas aeruginosa (3) as well as identification of resistance genes. From these RNA sequencing reads, 40 targets were defined and tested by quantitative PCR. In cohort of patients (n = 9) the some of the proposed PCRs identified all cases of positive blood cultures (Pseudomonas aeruginosa and Staphylococcus aureus, Escherichia coli had no positive blood cultures in this cohort). RNA sequencing from patients with sepsis can identify RNA from pathogens causing the infection. This can be used to design PCR primers that can identify patients with positive blood cultures. Translation of these primers to clinical microbiology machines is the next step and will allow the diagnosis pathogen and resistance faster than blood culture. Biological sciences/Microbiology/Infectious-disease diagnostics Biological sciences/Biological techniques/Sequencing/RNA sequencing Health sciences/Diseases/Infectious diseases/Bacterial infection Health sciences/Medical research/Translational research Figures Figure 1 Introduction Sepsis is responsible for one out of five deaths in the world. 1 Prior to the COVID-19 pandemic, 1.7 million patients have sepsis and 270,000 deaths were seen in the United States. 2 Sepsis is defined by altered physiology leading to organ and immune system dysfunction due to an infection 3 and the care of sepsis is multifactorial with focus on early antimicrobial treatment, fluid resuscitation, and support with vasoactive medications. 4 The diagnosis of the infection is a prominent challenge to sepsis care. 5 The Surviving Sepsis Campaign has standardized treatment for sepsis that includes blood cultures before broad-spectrum antibiotics and initiation of antibiotics within one hour. 6 In multivariate analysis of factors impacting mortality in patients with septic shock, time to initiation of antibiotics was the most impactful variable; exemplified by 79.9% survival in septic shock patients with antibiotics in the first hour and a reduction in survival by 7.6% for every hour delay. 7 The importance of appropriate antibiotics is exemplified by the fact that treating five patients saves one life. 8 Sepsis not diagnosed on admission also has larger costs and utilization burden. 9 It is therefore important to obtain samples to diagnose the pathogens early and quickly. Antimicrobial resistance is another hurdle in sepsis management. Resistance is associated with approximately five million deaths each year in the world. 10 Infections in the ICU with resistant bacteria have a higher risk of in-hospital death. 11 Resistant E. coli , for example, causes 16.5 deaths per 100,000 annually in Europe. 12 Typical treatment of sepsis begins with broad-spectrum antibiotics that are deescalated once the pathogen and microbial sensitivities are determined. If a patient has a resistant pathogen, this empiric treatment may not be effective and would not change until the cultures mature. This could result in delaying appropriate treatment for days. While broad-spectrum antibiotics may provide appropriate empiric coverage, they are independently associated with an adjusted increased risk of mortality. 13 Other studies have shown that unnecessary use of broad-spectrum antibiotics increases the risk of in-hospital deaths 14 . This project seeks to advance the diagnosis of bacterial infections and associated antimicrobial resistance with RNA sequencing in order to improve patient outcomes. In the initial assessment of RNA sequencing data, the reads are aligned, or mapped, to the genome of the species from which the sample was derived. Typically, the unmapped reads account for 5-20% of the data and is typically discarded. In our mouse sepsis models and in humans with critical illness, approximately 30% of the reads are unmapped. 15,16 These unmapped reads represent a rich resource for pathogen discovery. In this study we identified bacterial RNA from unmapped reads in patients with bacteremia that was diagnosed by standard culture. This RNA sequencing data was used to develop targets for PCR-based diagnostics. Unlike conventional molecular microbiology tests like DNA-based PCR or cell-free DNA sequencing, RNA molecules are the target in these assays, thereby exploiting the inherent higher copy number of highly expressed genes or RNA molecules with structures resistant to degradation. Results Patients Forty-six patients enrolled in the study (Table 1), 87 distinct time point samples were collected while the patients were in the ICU. This resulted in over 8.6 billion data points from the RNA sequencing workflow that was used to develop the PCR probes (Figure 1). In this cohort of patients, 228 total cultures were sent and 114 were positive, 90 of these cultures were from the blood and 40 were positive. The most commonly found pathogens for positive blood culture in this cohort were analyzed to create PCR targets: Escherichia coli (5 patients) , Pseudomonas aeruginosa (3 patients) , Staphylococcus aureus (6 patients) . Alignment to pathogens/resistance genes and target creation Removing the RNA sequencing reads that mapped to the human genome left 2.13 billion unmapped reads of which ~1.2 million aligned to a pathogen (eTable 3). All the unmapped reads were aligned to each pathogen genome, therefore a single RNA read could map to multiple pathogens. As seen in Table 2 (and eTable 3) simply using the number of reads as a diagnostic tool is not adequate. Sequencing data from patients with positive blood cultures for E. coli (5) , P. aeruginosa (3) , S. aureus (6) was then utilized to identify unique regions of the genomes that were specific to patients with that specific bacteremia (Table 2). After identifying the unique RNA sequences, PCR primers were designed against these targets (eTable 4, 5, 6). These tables also list the gene to which those nucleic acid sequences align as well as the type of gene. Many fewer reads aligned to specific resistance genes, 46 total (Figure 1). From these reads, targets for PCR were identified from six genes. There were 11 regions across these genes that were targets, specifically 4 targets for TEM-1 and 3 targets for TEM-2. eTable 7 shows the nucleic acid sequences of the target and the gene it will identify. Results of novel PCR in patient samples A subset (n=9) of the patients initially included in the sequencing analysis had RNA sample available for testing by PCR (eTable 8). Three of the patients had S. aureus bacteremia, one had P. aeruginosa bacteremia, and none had E. coli bacteremia (Table 3). One target for S. aureus , SAOUSHC-00439, had a sensitivity of 33% and specificity of 100% while another, SAOUSHC-R0001,2, had a sensitivity of 100% and specificity of 66%. For P. aeruginosa, the target PA0668.4-2 had 100% sensitivity and specificity. One patient had an E. coli urinary tract infection and was the only patient with a PCR positive for yegP . Since there were no samples for patients with E. coli bacteremia, negative and positive results in this cohort can provide some insights. All patient samples were positive for rrIABCEH , rrIDG , and rrID , indicating lack of specificity for E. coli bacteremia. However, emrB , rapA-1 , yghQ , gdx were all negative and may therefore be diagnostic of E. coli bacteremia.PA0668.4-1 and PA4280.2 had low specificity for P. aeruginosa because they were positive in many samples. In contrast, rpoS , oprN , and ahpF could be used in combination with PA0668.4-2 to identify Pseudomonas bloodstream infections. Two primers, SAOUSHC-R0009,10 and SAOUSHC-R0006,7 also had low specificity for S. aureus due to many false positives in other specimens (eTable 8). The target SAOUSHC-R0008,9 provides an interesting result as the cycle counts needed for a patient with bacteremia are lower than other false positives. There was limited antimicrobial resistance identified by culture in this patient cohort. In the absence of phenotypic resistance, Mex-MexY , CTX-M-223 , and ompF could not be formally evaluated. Multiple specimens showed positive PCR results for TEM-1 and TEM-2 and more clinical correlation is needed. One patient’s blood was positive by PCR for NDM-1 even though no resistance identified in the clinical microbiology laboratory. This patient died in the hospital despite receiving broad spectrum antibiotics that did not include coverage for carbapenemases. Discussion Diagnosis of infections causing sepsis needs a faster solution so appropriate antibiotics can be administered and reduce mortality. Utilizing PCR tests targeting RNA from pathogens, informed from RNA sequencing data from patients with sepsis can identify bacteria and potential resistance directly from the blood, without the need for culture in approximately fours hours (Figure 1). This works shows that RNA sequencing data from patients (Table 2) with positive cultures can design primers for Escherichia coli , Pseudomonas aeruginosa , Staphylococcus aureus (eTable 4,5,6) that show promising results in a small cohort of patients (Table 3). Many molecular diagnostics for infectious disease focus on DNA. 17 This work focuses on RNA from pathogens as identified from RNA sequencing data (eTable3) of patients with sepsis. Since RNA is the focus, stabilization of RNA at collection is paramount for the success of this technique (use of PAXgene tubes). RNA as the target also allows for the PCR to occur directly from the blood with out the need to culture to increase the amount of pathogen DNA available since the RNA target is actively being transcribed. The half-life of most bacterial mRNA is less than an hour so if detected it is coming from an active infection 18 and if RNA is detected that gene (particularly resistance genes) is being expressed. It is important to note that the proposed nucleic acid target is RNA, when other current look at DNA. The RNA that is identified in the blood of patients with sepsis is RNA from the causative pathogen. The RNA that is identified from each pathogen with reads across multiple patients who are critically ill with that infection will become the target for the PCR test. This new PCR test will allow for faster diagnostic compared to the gold standard of blood culture. Target primers to resistance genes can be developed that are identified in the blood by RNA sequencing. The use of PCR in this work shows that it is more sensitive than RNA sequencing alone. All the PCR primers were designed from patients with positive blood cultures. The targets were then checked to ensure that they were not present in any of the samples without positive blood cultures and against the genomes of all the pathogens studied. However, some of the targets (eTable 8) were positive in all the samples. This increased sensitivity is important because the RNA from the pathogen and resistance genes will be rare in the samples. Identification of resistance is key to changing the treatment of patients with sepsis. Some research suggests using sequencing to identify resistance in clinical isolates 19 ; still requiring culture. Current techniques to use PCR on DNA targets does not identify a living cell or expression of that gene, so use with antimicrobial resistance is limited. 17 However, identifying RNA produced from the resistance gene suggests that the pathogen is producing the protein. Heteroresistance is a clinically significant problem and can explain why some patients have no resistance detected but do not respond to antibiotics and have poor clinical outcomes. 20 In the sample of patients only two had any resistance detected by culture, but one patient had a positive PCR for NDM-1 (Table 3). The identification of this resistance in this patient could have occurred on day 0 of their hospital stay and antibiotics could have been given to prevent this death. This PCR for resistance genes could be done daily to identify patients treated with culture supported efficacy that fail due to induction of resistance genes in the pathogen. 21 In the initial data questions arise as to why is there RNA from many pathogens detected in the blood. This was seen in previous work 15 and there is much research on the blood microbiome. 22 In addition, so as to not bias the results, all un-mapped reads were aligned to each individual pathogen and therefore a read could map to multiple pathogens when they are genetically similar. 23 In order to create primers unique for the pathogen, this study utilizes samples from Escherichia coli (5) , Pseudomonas aeruginosa (3) , and Staphylococcus aureus (6) positive blood stream infections but only tests on a sample of nine patients. Future testing of these targets on more samples, particularly those not used to design the primers, is needed and planned. Since the target is RNA, these samples must be collected in a way that the RNA is immediately stabilized. More samples will also allow for expansion of the pathogens and resistance genes targeted. In order to design a primer for a pathogen, patients with positive infections will need to be sequenced to allow for identification of unique RNA. In addition, patients with resistance will need to be sequenced as well, particularly for common pathogens like MRSA (no patients in this cohort had MRSA bacteremia). The focus of this study was also patients with bacteremia, but sepsis can be due to infections anywhere in the body. Future work will need to cluster the patients based on site and previous work suggests that the targets for a specific pathogen will differ based upon the site of infection. 24 Translation of the workflow from RNA isolation to the PCR reactions done on research machines in this study to machines approved for in-vitro diagnostics will need to be done. There is also potential to use digital PCR (dPCR) as it would be more sensitive as well as the ability to quantify levels and monitor treatment overtime. 25 Methods This is a single center, prospective study of critically ill patients with sepsis and their pathogen profile. The patients were enrolled from Rhode Island Hospital Medical Intensive Care Unit (MICU), all of whom or their appropriate surrogate provided informed consent as approved by the Rhode Island Hospital Institutional Review Board (411616, 205821). Clinical data was collected as previously described. 26 Whole blood samples were drawn in PAXgene tubes (Qiagen, Germantown, MD) to immediately stabilize the RNA and then sent to Genewiz (South Plainfield, NJ) for RNA extraction, human ribosomal RNA depletion and deep RNA sequencing. Of note, we did not select for polyadenylic acid (poly A) tails to include non-protein coding RNAs. The sequencing was performed on Illumina HiSeq machines to provide 150 base pair, paired-end reads and at least 100 million reads per sample. Raw sequencing data was returned on secure external hard drives 15 . The raw data was assessed for quality control with FastQC 27 then aligned to the most recent assembly (GCF_000001405.40) of Genome Reference Consortium Human Build 38 (GRCh38) 28 – the latest version of human reference genome – with the STAR aligner 29 . Reads that aligned to GRCh38 (“mapped” reads) were separated from “unmapped” reads. The unmapped reads representing non-human genome were then aligned to each reference genome of 28 pathogens (eTable 1) and an anti-microbial resistance (AMR) custom genome selected based on clinical utility. The pathogen reference genome was obtained from NCBI RefSeq database. The AMR custom genome was created by concatenating 25 common resistance genes in pathogens (eTable 2). After mapping the unmapped reads to each pathogen, the most clinically relevant pathogens (as defined by positive blood culture)– Escherichia coli , Pseudomonas aeruginosa , Staphylococcus aureus – and AMR genes were selected for primer design. For each pathogen, the reads of each patient sample with known bacteremia, as defined by positive blood culture, were compared to discover common sequences at least 100 base pairs (bp) long. This minimum bp criterion was chosen to provide sufficient length for primer design. To this end, each 150 bp read from each sample was searched in other samples with each pathogen-specific bacteremia. If a common 150 bp sequence was found in all bacteremia samples, then this sequence was selected as a primer target based on the following criteria: at least 85-90% hits unique to the specific pathogen on NCBI genome blast, a few to no hits for other samples without bacteremia, and no hits for samples mapped to other pathogens. These criteria were established to exclude common sequences that also aligned to related pathogens (e.g. E. coli and Shigella species) and to include sequences that are unique to sepsis. If there were no common sequences 150 bp long, then the search with shorter bp (e.g. 145, 140, 135) continued until there was a match among all samples. If there was no match above 100 bp among all samples with bacteremia, then sequences above 100 bp common to at least a few bacteremia samples were searched. If none were found, then a read that meets the aforementioned criteria from each sample was chosen. For the AMR genes, the same process was performed except the sequences did not require to have 85-90% hits unique to a specific pathogen, as AMR genes can be found in multiple pathogens. These computational analyses were performed on RStudio and command line using bash , awk , and grep scripts. Primers were designed to the targets described above. PCR primer sets were designed using NCBI's Primer-BLAST online software. Sequences derived from RNA-seq data were uploaded, specificity checking parameters were adjusted to Refseq RNA, and internal probe output was requested. Primer sets were chosen to have similar amplicon size while minimizing self-complementarity. They were ordered from IDT and tested against nucleic acids recovered from a small sample of sepsis patients that had RNA available for testing. Briefly the reverse transcription of RNA to cDNA was done with 100ng RNA input using the SuperScript IV First Strand Synthesis System Kit (ThermoFisher/Invitrogen, Waltham, MA). The cDNA was then used as template in the qPCR reaction and carried out with the Launa Universal Probe qPCR Master Mix kit(New England Biolabs, Ipswich, MA) and the custom primers/probes (IDT Coralville, IA). The qPCR reaction was setup at 1ul of Primer/probe(primer 400nM/probe 200nM);1ul of Template and 10ul of the Master mix. The amplification was run on qPCR instrument (BIO RAD CFX96 Real-Time System) with the thermocycling condition (95^C for 60 second; 95^C for 15 second, 60^C for 30 second) for 40cycles. Reactions were done in pairs with no reverse transcription controls. Positive results from the PCR were defined as greater than 2 cycle counts over the no reverse transcription controls or less than 40 cycle counts if the no reverse transcription controls was negative. References Rudd, K.E., et al. 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FastQC: a quality control tool for high throughput sequence data. (2010). Schneider, V.A., et al. Evaluation of GRCh38 and de novo haploid genome assemblies demonstrates the enduring quality of the reference assembly. Genome Res 27 , 849–864 (2017). Dobin, A., et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics (Oxford, England) 29 , 15–21 (2013). Tables Table 1 Baseline Characteristics of ICU Patients with Sepsis with RNA Sequencing Data Table 1 N=46 patients Age, mean, years 62.17 Female (%) 34 (74) Black Race (%) 3 (7) Other Race (%) 8 (17) Hypertension (%) 18 (39) Cardiovascular Disease (%) 9 (20) Congestive Heart Failure (%) 2 (4) Diabetes (%) 7 (15) Chronic Obstructive Pulmonary Disease (COPD) (%) 6 (13) Renal Failure (%) 4 (9) Malignancy (%) 1 (2) Immunocompromised (%) 1 (2) Mortality (%) 17 (37) Shock (%) 25 (54) Intensive Care Unit Length of Stay, average, days 5.6 Hospital Length of Stay, average, days 11.7 Sequential Organ Failure Assessment (SOFA) Score, average 5.7 Table 2 Patients with Positive Blood Cultures and Number of Unmapped Reads that Align to Each Pathogen Table 3 Cohort of Patients that PCRs were Tested On with Clinical Data and Relevant PCR Results Additional Declarations Yes there is potential Competing Interest. A.M.F, S.F.M., and G.J.N. are co-inventors on a provisional patent using RNA-seq data to design diagnostic primers. S.F.M. is Founder of Alcini, LLC. Supplementary Files Onlinematerial.docx Online tables Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5454900","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":380192052,"identity":"ea9a05cf-223a-44b5-b06d-7314d9b6e867","order_by":0,"name":"Sean 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University","correspondingAuthor":false,"prefix":"","firstName":"Alger","middleName":"","lastName":"Fredericks","suffix":""}],"badges":[],"createdAt":"2024-11-14 15:06:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5454900/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5454900/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":69830793,"identity":"b82fb1fb-496e-424d-a406-12aeaa8f23d4","added_by":"auto","created_at":"2024-11-25 15:36:57","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":44452,"visible":true,"origin":"","legend":"\u003cp\u003eWork Flow for the Development and Testing of PCR Primers for Pathogens and Resistance from RNA Sequencing Data of Sepsis Patients\u003c/p\u003e","description":"","filename":"Figure1DevelopmentWorkFlow.png","url":"https://assets-eu.researchsquare.com/files/rs-5454900/v1/5042492f20d8d10f5bc816b7.png"},{"id":69832370,"identity":"87c7bf78-d040-4ec6-a1a1-0df950561540","added_by":"auto","created_at":"2024-11-25 15:44:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":455760,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5454900/v1/35a1923d-ec62-442b-9614-fd4c6549855b.pdf"},{"id":69830795,"identity":"370e09b5-b831-44e4-b94e-c5f9bce204c5","added_by":"auto","created_at":"2024-11-25 15:36:57","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":82768,"visible":true,"origin":"","legend":"Online tables","description":"","filename":"Onlinematerial.docx","url":"https://assets-eu.researchsquare.com/files/rs-5454900/v1/6e7ff27b2eeedfaf79a2107d.docx"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e there is potential Competing Interest.\nA.M.F, S.F.M., and G.J.N. are co-inventors on a provisional patent using RNA-seq\r\ndata to design diagnostic primers. S.F.M. is Founder of Alcini, LLC.","formattedTitle":"RNA Sequencing of Sepsis Patients Informs Tests to Quickly Diagnose Pathogens and Resistance","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSepsis is responsible for one out of five deaths in the world.\u003csup\u003e1\u003c/sup\u003e Prior to the COVID-19 pandemic, \u0026nbsp;1.7 million patients have sepsis and 270,000 deaths were seen in the United States.\u003csup\u003e2\u003c/sup\u003e\u0026nbsp; Sepsis is defined by altered physiology leading to organ and immune system dysfunction due to an infection\u0026nbsp;\u003csup\u003e3\u003c/sup\u003e and the care of sepsis is multifactorial with focus on early antimicrobial treatment, fluid resuscitation, and support with vasoactive medications.\u003csup\u003e4\u003c/sup\u003e \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe diagnosis of the infection is a prominent challenge to sepsis care.\u003csup\u003e5\u003c/sup\u003e The Surviving Sepsis Campaign has standardized treatment for sepsis that includes blood cultures before broad-spectrum antibiotics and initiation of antibiotics within one hour.\u003csup\u003e6\u003c/sup\u003e In multivariate analysis of factors impacting mortality in patients with septic shock, time to initiation of antibiotics was the most impactful variable; exemplified by 79.9% survival in septic shock patients with antibiotics in the first hour and a reduction in survival by 7.6% for every hour delay.\u003csup\u003e7\u003c/sup\u003e The importance of appropriate antibiotics is exemplified by the fact that treating five patients saves one life.\u003csup\u003e8\u003c/sup\u003e Sepsis not diagnosed on admission also has larger costs and utilization burden.\u003csup\u003e9\u003c/sup\u003e It is therefore important to obtain samples to diagnose the pathogens early and quickly.\u003c/p\u003e\n\u003cp\u003eAntimicrobial resistance is another hurdle in sepsis management. \u0026nbsp;Resistance is associated with approximately five million deaths each year in the world.\u003csup\u003e10\u003c/sup\u003e Infections in the ICU with resistant bacteria have a higher risk of in-hospital death.\u003csup\u003e11\u003c/sup\u003e Resistant \u003cem\u003eE. coli\u003c/em\u003e, for example, causes 16.5 deaths per 100,000 annually in Europe.\u003csup\u003e12\u003c/sup\u003e Typical treatment of sepsis begins with broad-spectrum antibiotics that are deescalated once the pathogen and microbial sensitivities are determined. If a patient has a resistant pathogen, this empiric treatment may not be effective and would not change until the cultures mature. \u0026nbsp;This could result in delaying appropriate treatment for days. \u0026nbsp;While broad-spectrum antibiotics may provide appropriate empiric coverage, they are independently associated with an adjusted increased risk of mortality.\u003csup\u003e13\u003c/sup\u003e Other studies have shown that unnecessary use of broad-spectrum antibiotics increases the risk of in-hospital deaths\u003csup\u003e14\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis project seeks to advance the diagnosis of bacterial infections and associated antimicrobial resistance with RNA sequencing in order to improve patient outcomes. \u0026nbsp;In the initial assessment of RNA sequencing data, the reads are aligned, or mapped, to the genome of the species from which the sample was derived. Typically, the unmapped reads account for 5-20% of the data and is typically discarded. \u0026nbsp;In our mouse sepsis models and in humans with critical illness, approximately 30% of the reads are unmapped.\u003csup\u003e15,16\u003c/sup\u003e These unmapped reads represent a rich resource for pathogen discovery.\u003c/p\u003e\n\u003cp\u003eIn this study we identified bacterial RNA from unmapped reads in patients with bacteremia that was diagnosed by standard culture. \u0026nbsp;This RNA sequencing data was used to develop targets for PCR-based diagnostics. \u0026nbsp;Unlike conventional molecular microbiology tests like DNA-based PCR or cell-free DNA sequencing, RNA molecules are the target in these assays, thereby exploiting the inherent higher copy number of highly expressed genes or RNA molecules with structures resistant to degradation.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cem\u003ePatients\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eForty-six patients enrolled in the study (Table 1), 87 distinct time point samples were collected while the patients were in the ICU. This resulted in over 8.6 billion data points from the RNA sequencing workflow that was used to develop the PCR probes (Figure 1). In this cohort of patients, 228 total cultures were sent and 114 were positive, 90 of these cultures were from the blood and 40 were positive. The most commonly found pathogens for positive blood culture in this cohort were analyzed to create PCR targets: \u003cem\u003eEscherichia coli (5 patients)\u003c/em\u003e, \u003cem\u003ePseudomonas aeruginosa (3 patients)\u003c/em\u003e, \u003cem\u003eStaphylococcus aureus (6 patients)\u003c/em\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAlignment to pathogens/resistance genes and target creation\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eRemoving the RNA sequencing reads that mapped to the human genome left 2.13 billion unmapped reads of which ~1.2 million aligned to a pathogen (eTable 3). All the unmapped reads were aligned to each pathogen genome, therefore a single RNA read could map to multiple pathogens. \u0026nbsp;As seen in Table 2 (and eTable 3) simply using the number of reads as a diagnostic tool is not adequate. Sequencing data from patients with positive blood cultures for \u003cem\u003eE. coli (5)\u003c/em\u003e, \u003cem\u003eP. aeruginosa (3)\u003c/em\u003e, \u003cem\u003eS. aureus (6)\u003c/em\u003e was then utilized to identify unique regions of the genomes that were specific to patients with that specific bacteremia (Table 2). \u0026nbsp; After identifying the unique RNA sequences, PCR primers were designed against these targets (eTable 4, 5, 6). These tables also list the gene to which those nucleic acid sequences align as well as the type of gene.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMany fewer reads aligned to specific resistance genes, 46 total (Figure 1). From these reads, targets for PCR were identified from six genes. There were 11 regions across these genes that were targets, specifically 4 targets for TEM-1 and 3 targets for TEM-2. eTable 7 shows the nucleic acid sequences of the target and the gene it will identify.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eResults of novel PCR in patient samples\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA subset (n=9) of the patients initially included in the sequencing analysis had RNA sample available for testing by PCR (eTable 8). Three of the patients had \u003cem\u003eS. aureus\u0026nbsp;\u003c/em\u003ebacteremia, one had \u003cem\u003eP. aeruginosa\u003c/em\u003e bacteremia, and none had \u003cem\u003eE. coli\u0026nbsp;\u003c/em\u003ebacteremia (Table 3). One target for \u003cem\u003eS. aureus\u003c/em\u003e, SAOUSHC-00439, had a sensitivity of 33% and specificity of 100% while another, SAOUSHC-R0001,2, had a sensitivity of 100% and specificity of 66%. For \u003cem\u003eP. aeruginosa,\u0026nbsp;\u003c/em\u003ethe target PA0668.4-2 had 100% sensitivity and specificity. One patient had an \u003cem\u003eE. coli\u0026nbsp;\u003c/em\u003eurinary tract infection and was the only patient with a PCR positive for \u003cem\u003eyegP\u003c/em\u003e. \u0026nbsp;Since there were no samples for patients with \u003cem\u003eE. coli\u0026nbsp;\u003c/em\u003ebacteremia, negative and positive results in this cohort can provide some insights. All patient samples were positive for \u003cem\u003errIABCEH\u003c/em\u003e,\u0026nbsp;\u003cem\u003errIDG\u003c/em\u003e, and \u003cem\u003errID\u003c/em\u003e, indicating lack of specificity for \u003cem\u003eE. coli\u003c/em\u003e bacteremia. \u0026nbsp;However, \u003cem\u003eemrB\u003c/em\u003e, \u003cem\u003erapA-1\u003c/em\u003e, \u003cem\u003eyghQ\u003c/em\u003e,\u0026nbsp;\u003cem\u003egdx\u003c/em\u003e were all negative and may therefore be diagnostic of\u0026nbsp;\u003cem\u003eE. coli\u003c/em\u003e bacteremia.PA0668.4-1 and PA4280.2 had low specificity\u0026nbsp;for \u003cem\u003eP. aeruginosa\u003c/em\u003e because they were positive in many samples. \u0026nbsp;In contrast,\u0026nbsp;\u003cem\u003erpoS\u003c/em\u003e, \u003cem\u003eoprN\u003c/em\u003e, and \u003cem\u003eahpF\u003c/em\u003e could be used in combination with PA0668.4-2 to identify \u003cem\u003ePseudomonas\u003c/em\u003e bloodstream infections. Two primers, SAOUSHC-R0009,10 and SAOUSHC-R0006,7 also had low specificity for\u0026nbsp;\u003cem\u003eS. aureus\u003c/em\u003e due to many false positives in other specimens (eTable 8). The target\u0026nbsp;SAOUSHC-R0008,9 provides an interesting result as the cycle counts needed for a patient with bacteremia are lower than other false positives.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThere was limited antimicrobial resistance identified by culture in this patient cohort. In the absence of phenotypic resistance, \u003cem\u003eMex-MexY\u003c/em\u003e, \u003cem\u003eCTX-M-223\u003c/em\u003e, and \u003cem\u003eompF\u003c/em\u003e could not be formally evaluated. Multiple specimens showed positive PCR results for TEM-1 and TEM-2 and more clinical correlation is needed. One patient\u0026rsquo;s blood was positive by PCR for NDM-1 even though no resistance identified in the clinical microbiology laboratory. \u0026nbsp;This patient died in the hospital despite receiving broad spectrum antibiotics that did not include coverage for carbapenemases.\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eDiagnosis of infections causing sepsis needs a faster solution so appropriate antibiotics can be administered and reduce mortality. Utilizing PCR tests targeting RNA from pathogens, informed from RNA sequencing data from patients with sepsis can identify bacteria and potential resistance directly from the blood, without the need for culture in approximately fours hours (Figure 1). \u0026nbsp;This works shows that RNA sequencing data from patients (Table 2) with positive cultures can design primers for \u003cem\u003eEscherichia coli\u003c/em\u003e, \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e, \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (eTable 4,5,6) that show promising results in a small cohort of patients (Table 3).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMany molecular diagnostics for infectious disease focus on DNA.\u003csup\u003e17\u003c/sup\u003e This work focuses on RNA from pathogens as identified from RNA sequencing data (eTable3) of patients with sepsis. Since RNA is the focus, stabilization of RNA at collection is paramount for the success of this technique (use of PAXgene tubes). RNA as the target also allows for the PCR to occur directly from the blood with out the need to culture to increase the amount of pathogen DNA available since the RNA target is actively being transcribed. The half-life of most bacterial mRNA is less than an hour so if detected it is coming from an active infection\u003csup\u003e18\u003c/sup\u003e and if RNA is detected that gene (particularly resistance genes) is being expressed.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIt is important to note that the proposed nucleic acid target is RNA, when other current look at DNA. The RNA that is identified in the blood of patients with sepsis is RNA from the causative pathogen. The RNA that is identified from each pathogen with reads across multiple patients who are critically ill with that infection will become the target for the PCR test. This new PCR test will allow for faster diagnostic compared to the gold standard of blood culture. Target primers to resistance genes can be developed that are identified in the blood by RNA sequencing.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe use of PCR in this work shows that it is more sensitive than RNA sequencing alone. All the PCR primers were designed from patients with positive blood cultures. The targets were then checked to ensure that they were not present in any of the samples without positive blood cultures and against the genomes of all the pathogens studied. However, some of the targets (eTable 8) were positive in all the samples. This increased sensitivity is important because the RNA from the pathogen and resistance genes will be rare in the samples.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIdentification of resistance is key to changing the treatment of patients with sepsis. Some research suggests using sequencing to identify resistance in clinical isolates\u003csup\u003e19\u003c/sup\u003e; still requiring culture. Current techniques to use PCR on DNA targets does not identify a living cell or expression of that gene, so use with antimicrobial resistance is limited.\u003csup\u003e17\u003c/sup\u003e However, identifying RNA produced from the resistance gene suggests that the pathogen is producing the protein.\u0026nbsp;Heteroresistance is a clinically significant problem and can explain why some patients have no resistance detected but do not respond to antibiotics and have poor clinical outcomes.\u003csup\u003e20\u003c/sup\u003e In the sample of patients only two had any resistance detected by culture, but one patient had a positive PCR for NDM-1 (Table 3). The identification of this resistance in this patient could have occurred on day 0 of their hospital stay and antibiotics could have been given to prevent this death. This PCR for resistance genes could be done daily to identify\u0026nbsp;patients treated with culture supported efficacy that fail due to induction of resistance genes in the pathogen.\u003csup\u003e21\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eIn the initial data questions arise as to why is there RNA from many pathogens detected in the blood. This was seen in previous work\u003csup\u003e15\u003c/sup\u003e and there is much research on the blood microbiome.\u003csup\u003e22\u003c/sup\u003e In addition, so as to not bias the results, all un-mapped reads were aligned to each individual \u0026nbsp;pathogen and therefore a read could map to multiple pathogens when they are genetically similar.\u003csup\u003e23\u003c/sup\u003e In order to create primers unique for the pathogen, this study utilizes samples from \u003cem\u003eEscherichia coli (5)\u003c/em\u003e, \u003cem\u003ePseudomonas aeruginosa (3)\u003c/em\u003e, and\u003cem\u003e\u0026nbsp;Staphylococcus aureus (6)\u003c/em\u003e positive blood stream infections but only tests on a sample of nine patients. Future testing of these targets on more samples, particularly those not used to design the primers, is needed and planned. Since the target is RNA, these samples must be collected in a way that the RNA is immediately stabilized. More samples will also allow for expansion of the pathogens and resistance genes targeted. In order to design a primer for a pathogen, patients with positive infections will need to be sequenced to allow for identification of unique RNA. In addition, patients with resistance will need to be sequenced as well, particularly for common pathogens like MRSA (no patients in this cohort had MRSA bacteremia).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe focus of this study was also patients with bacteremia, but sepsis can be due to infections anywhere in the body. Future work will need to cluster the patients based on site and previous work suggests that the targets for a specific pathogen will differ based upon the site of infection.\u003csup\u003e24\u003c/sup\u003e\u0026nbsp; Translation of the workflow from RNA isolation to the PCR reactions done on research machines in this study to machines approved for in-vitro diagnostics will need to be done. There is also potential to use digital PCR (dPCR) as it would be more sensitive as well as the ability to quantify levels and monitor treatment overtime.\u003csup\u003e25\u003c/sup\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eThis is a single center, prospective study of critically ill patients with sepsis and their pathogen profile. The patients were enrolled from Rhode Island Hospital Medical Intensive Care Unit (MICU), all of whom or their appropriate surrogate provided informed consent as approved by the Rhode Island Hospital Institutional Review Board (411616, 205821). Clinical data was collected as previously described.\u003csup\u003e26\u003c/sup\u003e Whole blood samples were drawn in PAXgene tubes (Qiagen, Germantown, MD) to immediately stabilize the RNA and then sent to Genewiz (South Plainfield, NJ) for RNA extraction, human ribosomal RNA depletion and deep RNA sequencing.\u0026nbsp;Of note, we did not select for polyadenylic acid (poly A) tails to include non-protein coding RNAs.\u0026nbsp;The sequencing was performed on Illumina HiSeq machines to provide 150 base pair, paired-end reads and at least 100 million reads per sample. Raw sequencing data was returned on secure external hard drives\u0026nbsp;\u003csup\u003e15\u003c/sup\u003e. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe raw data was assessed for quality control with FastQC\u0026nbsp;\u003csup\u003e27\u003c/sup\u003e then aligned to the most recent assembly (GCF_000001405.40) of Genome Reference Consortium Human Build 38 (GRCh38)\u0026nbsp;\u003csup\u003e28\u003c/sup\u003e \u0026ndash; the latest version of human reference genome \u0026ndash; with the STAR aligner\u0026nbsp;\u003csup\u003e29\u003c/sup\u003e. Reads that aligned to GRCh38 (\u0026ldquo;mapped\u0026rdquo; reads) were separated from \u0026ldquo;unmapped\u0026rdquo; reads. The unmapped reads representing non-human genome were then aligned to each reference genome of 28 pathogens (eTable 1) and an anti-microbial resistance (AMR) custom genome selected based on clinical utility. The pathogen reference genome was obtained from NCBI RefSeq database. The AMR custom genome was created by concatenating 25 common resistance genes in pathogens (eTable 2). After mapping the unmapped reads to each pathogen, the most clinically relevant pathogens (as defined by positive blood culture)\u0026ndash; \u003cem\u003eEscherichia coli\u003c/em\u003e, \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e, \u003cem\u003eStaphylococcus aureus\u003c/em\u003e \u0026ndash; and AMR genes were selected for primer design.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor each pathogen, the reads of each patient sample with known bacteremia, as defined by positive blood culture, were compared to discover common sequences at least 100 base pairs (bp) long. This minimum bp criterion was chosen to provide sufficient length for primer design. To this end, each 150 bp read from each sample was searched in other samples with each pathogen-specific bacteremia. If a common 150 bp sequence was found in all bacteremia samples, then this sequence was selected as a primer target based on the following criteria: at least 85-90% hits unique to the specific pathogen on NCBI genome blast, a few to no hits for other samples without bacteremia, and no hits for samples mapped to other pathogens. These criteria were established to exclude common sequences that also aligned to related pathogens (e.g. \u003cem\u003eE. coli and Shigella species)\u003c/em\u003e and to include sequences that are unique to sepsis. If there were no common sequences 150 bp long, then the search with shorter bp (e.g. 145, 140, 135) continued until there was a match among all samples. If there was no match above 100 bp among all samples with bacteremia, then sequences above 100 bp common to at least a few bacteremia samples were searched. If none were found, then a read that meets the aforementioned criteria from each sample was chosen. For the AMR genes, the same process was performed except the sequences did not require to have 85-90% hits unique to a specific pathogen, as AMR genes can be found in multiple pathogens. These computational analyses were performed on RStudio and command line using \u003cem\u003ebash\u003c/em\u003e, \u003cem\u003eawk\u003c/em\u003e, and \u003cem\u003egrep\u003c/em\u003e scripts.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePrimers were designed to the targets described above. PCR primer sets were designed using NCBI\u0026apos;s Primer-BLAST online software. \u0026nbsp;Sequences derived from RNA-seq data were uploaded, specificity checking parameters were adjusted to Refseq RNA, and internal probe output was requested. \u0026nbsp;Primer sets were chosen to have similar amplicon size while minimizing self-complementarity. They were ordered from IDT and tested against nucleic acids recovered from a small sample of sepsis patients that had RNA available for testing. Briefly the\u0026nbsp;reverse transcription of RNA to cDNA was done with 100ng RNA input using the SuperScript IV First Strand Synthesis System Kit (ThermoFisher/Invitrogen, Waltham, MA). \u0026nbsp;The cDNA was then used as template in the qPCR reaction and carried out with the Launa Universal Probe qPCR Master Mix kit(New England Biolabs, Ipswich, MA) and the custom primers/probes (IDT Coralville, IA). The qPCR reaction was setup at 1ul of Primer/probe(primer 400nM/probe 200nM);1ul of Template and 10ul of the Master mix. The amplification was run on qPCR instrument (BIO RAD CFX96 Real-Time System) with the thermocycling condition (95^C for 60 second; 95^C for 15 second, 60^C for 30 second) for 40cycles. Reactions were done in pairs with no reverse transcription controls. Positive results from the PCR were defined as greater than 2 cycle counts over the no reverse transcription controls or less than 40 cycle counts if the no reverse transcription controls was negative.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003e Rudd, K.E., \u003cem\u003eet al.\u003c/em\u003e Global, regional, and national sepsis incidence and mortality, 1990\u0026ndash;2017: analysis for the Global Burden of Disease Study. \u003cem\u003eLancet (London, England)\u003c/em\u003e \u003cb\u003e395\u003c/b\u003e, 200\u0026ndash;211 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Rhee, C., \u003cem\u003eet al.\u003c/em\u003e Incidence and Trends of Sepsis in US Hospitals Using Clinical vs Claims Data, 2009\u0026ndash;2014. \u003cem\u003eJama\u003c/em\u003e \u003cb\u003e318\u003c/b\u003e, 1241\u0026ndash;1249 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Singer, M., \u003cem\u003eet al.\u003c/em\u003e The Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3). \u003cem\u003eJama\u003c/em\u003e \u003cb\u003e315\u003c/b\u003e, 801\u0026ndash;810 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Rhodes, A., \u003cem\u003eet al.\u003c/em\u003e Surviving Sepsis Campaign: International Guidelines for Management of Sepsis and Septic Shock: 2016. \u003cem\u003eIntensive care medicine\u003c/em\u003e \u003cb\u003e43\u003c/b\u003e, 304\u0026ndash;377 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Duncan, C.F., Youngstein, T., Kirrane, M.D. \u0026amp; Lonsdale, D.O. 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(2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Schneider, V.A., \u003cem\u003eet al.\u003c/em\u003e Evaluation of GRCh38 and de novo haploid genome assemblies demonstrates the enduring quality of the reference assembly. \u003cem\u003eGenome Res\u003c/em\u003e \u003cb\u003e27\u003c/b\u003e, 849\u0026ndash;864 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Dobin, A., \u003cem\u003eet al.\u003c/em\u003e STAR: ultrafast universal RNA-seq aligner. \u003cem\u003eBioinformatics (Oxford, England)\u003c/em\u003e \u003cb\u003e29\u003c/b\u003e, 15\u0026ndash;21 (2013).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 Baseline Characteristics of ICU Patients with Sepsis with RNA Sequencing Data\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 67.2727%;\"\u003e\n \u003cp\u003eTable 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 32.7273%;\"\u003e\n \u003cp\u003eN=46 patients\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 67.2727%;\"\u003e\n \u003cp\u003eAge, mean, years\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 32.7273%;\"\u003e\n \u003cp\u003e62.17\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 67.2727%;\"\u003e\n \u003cp\u003eFemale (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 32.7273%;\"\u003e\n \u003cp\u003e34 (74)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 67.2727%;\"\u003e\n \u003cp\u003eBlack Race (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 32.7273%;\"\u003e\n \u003cp\u003e3 (7)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 67.2727%;\"\u003e\n \u003cp\u003eOther Race (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 32.7273%;\"\u003e\n \u003cp\u003e8 (17)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 67.2727%;\"\u003e\n \u003cp\u003eHypertension (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 32.7273%;\"\u003e\n \u003cp\u003e18 (39)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 67.2727%;\"\u003e\n \u003cp\u003eCardiovascular Disease (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 32.7273%;\"\u003e\n \u003cp\u003e9 (20)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 67.2727%;\"\u003e\n \u003cp\u003eCongestive Heart Failure (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 32.7273%;\"\u003e\n \u003cp\u003e2 (4)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 67.2727%;\"\u003e\n \u003cp\u003eDiabetes (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 32.7273%;\"\u003e\n \u003cp\u003e7 (15)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 67.2727%;\"\u003e\n \u003cp\u003eChronic Obstructive Pulmonary Disease (COPD) (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 32.7273%;\"\u003e\n \u003cp\u003e6 (13)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 67.2727%;\"\u003e\n \u003cp\u003eRenal Failure (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 32.7273%;\"\u003e\n \u003cp\u003e4 (9)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 67.2727%;\"\u003e\n \u003cp\u003eMalignancy (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 32.7273%;\"\u003e\n \u003cp\u003e1 (2)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 67.2727%;\"\u003e\n \u003cp\u003eImmunocompromised (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 32.7273%;\"\u003e\n \u003cp\u003e1 (2)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 67.2727%;\"\u003e\n \u003cp\u003eMortality (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 32.7273%;\"\u003e\n \u003cp\u003e17 (37)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 67.2727%;\"\u003e\n \u003cp\u003eShock (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 32.7273%;\"\u003e\n \u003cp\u003e25 (54)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 67.2727%;\"\u003e\n \u003cp\u003eIntensive Care Unit Length of Stay, average, days\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 32.7273%;\"\u003e\n \u003cp\u003e5.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 67.2727%;\"\u003e\n \u003cp\u003eHospital Length of Stay, average, days\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 32.7273%;\"\u003e\n \u003cp\u003e11.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 67.2727%;\"\u003e\n \u003cp\u003eSequential Organ Failure Assessment (SOFA) Score, average\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 32.7273%;\"\u003e\n \u003cp\u003e5.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;Table 2 Patients with Positive Blood Cultures and Number of Unmapped Reads that Align to Each Pathogen\u003c/p\u003e\n\u003cp\u003e\u003cimg 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\"\u003e\u003c/p\u003e\n\u003cp\u003eTable 3 Cohort of Patients that PCRs were Tested On with Clinical Data and Relevant PCR Results\u003c/p\u003e\n\u003cp\u003e\u003cimg 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\"\u003e\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-5454900/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5454900/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDiagnosis of infection in patients with sepsis takes days via culture and appropriate treatment of pathogens are delayed awaiting results. A faster diagnosis of the pathogen and resistance via RNA sequencing informed PCR will improve outcomes. We hypothesize that we can use RNA sequencing from patients with sepsis to identify novel targets for future nucleic acid-based tests. This cohort study of 46 sepsis patients admitted to the ICU with samples taken on days 0, 1, 3, and 7 with follow up through the hospital stay during 2021\u0026ndash;2022. All patients had RNA sequencing, depth of \u0026gt;\u0026thinsp;100\u0026nbsp;million reads, conducted on days in the ICU from a single center medical intensive care unit. Patients were admitted to the ICU with a diagnosis of sepsis. Patients or surrogates were approached consecutively and those who consented were enrolled. RNA sequencing of peripheral blood was performed to identify RNA targets from pathogens. RNA sequencing data that did not map to the human genome was then aligned to resistance genes and pathogen genomes and used to design novel PCR tests. These tests were correlated with culture diagnosis and clinical outcomes. Forty-six patients (mean age 62.2, 48% female) were enrolled and samples from 87 time points were collected. These samples resulted in 8.6\u0026nbsp;billion RNA sequencing reads to identify pathogen RNA. PCR target discovery for this study focused on positive blood cultures (n\u0026thinsp;=\u0026thinsp;40) due Escherichia coli (5), Staphylococcus aureus (6), and Pseudomonas aeruginosa (3) as well as identification of resistance genes. From these RNA sequencing reads, 40 targets were defined and tested by quantitative PCR. In cohort of patients (n\u0026thinsp;=\u0026thinsp;9) the some of the proposed PCRs identified all cases of positive blood cultures (Pseudomonas aeruginosa and Staphylococcus aureus, Escherichia coli had no positive blood cultures in this cohort). RNA sequencing from patients with sepsis can identify RNA from pathogens causing the infection. This can be used to design PCR primers that can identify patients with positive blood cultures. Translation of these primers to clinical microbiology machines is the next step and will allow the diagnosis pathogen and resistance faster than blood culture.\u003c/p\u003e","manuscriptTitle":"RNA Sequencing of Sepsis Patients Informs Tests to Quickly Diagnose Pathogens and Resistance","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-25 15:36:52","doi":"10.21203/rs.3.rs-5454900/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"31cce77d-3643-4f79-a0de-1665921d3b3a","owner":[],"postedDate":"November 25th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":40491004,"name":"Biological sciences/Microbiology/Infectious-disease diagnostics"},{"id":40491005,"name":"Biological sciences/Biological techniques/Sequencing/RNA sequencing"},{"id":40491006,"name":"Health sciences/Diseases/Infectious diseases/Bacterial infection"},{"id":40491007,"name":"Health sciences/Medical research/Translational research"}],"tags":[],"updatedAt":"2024-11-25T15:36:52+00:00","versionOfRecord":[],"versionCreatedAt":"2024-11-25 15:36:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5454900","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5454900","identity":"rs-5454900","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}