INTRODUCTION
Sepsis has been ranked as one of the major public health concerns in intensive care units (ICUs) worldwide, causing an increasing number of deaths each year.[1] Early identification of responsible pathogens and prompt administration of specific antibiotics are crucial steps for patients’ prognosis.[2,3] Early antimicrobial treatment is recommended in current clinical instructions, specifically within one hour after the diagnosis of sepsis.[4] However, the majority of current therapies are empirical. Blood culture has been the gold standard for sepsis diagnosis, whereas obtaining final results of blood cultures can be time-consuming and some pathogenic microsomes are difficult to be cultured in vitro.[5] Additionally, prior usage of broad-spectrum antibiotics may confound the real specific diagnosis.[6,7] Polymerase chain reaction (PCR)-based techniques have been introduced for pathogen detection in recent decades.[8] Nevertheless, these techniques are limited by the collection of tissues and the lack of quantitative measurement of microorganisms load, and are also unable to detect the cause of antibiotic resistance.[9]
Despite the limitations, the techniques mentioned above have improved the accuracy and speed of clinical diagnoses of causative pathogens. However, some clinical concerns remain to be addressed. In our clinical practice, we found that many patients with bacterial-infected sepsis did not acquire satisfactory improvement regarding survival rate and ICU scores, even those treated by targeted antibiotic therapy based on blood culture. Traen et al[10] found that ICU patients with positive herpes simplex virus (HSV) type 1 (HSV-1) could acquire lower ICU mortality after the prophylactic use of acyclovir. A different study conducted by Schuierer et al[11] reported that in patients with ventilator-associated pneumonia and antibiotic treatment failure, acyclovir treatment was associated with significantly longer survival time in the ICU and improved circulatory and pulmonary functions. We hypothesized that concomitant viral infection could affect the survival of patients with sepsis.
Next-generation sequencing (NGS) has recently been proposed for critically ill patients who suffer from bloodstream infections.[12] NGS allows for an unbiased analysis of the bloodstream, including prompt diagnosis, stable results, and quantitative score.[13] This technology could be used in medical microbiology laboratories and useful for infection prevention measures.
Therefore, this study aims to investigate the performance of an NGS-based diagnostic technique for the identification of potential bacterial and viral infections, and effects of concomitant viral infection on the survival rate of sepsis patients in the ICU.
METHODS
Study design
This present study was approved by the Ethics Committee Board of our institution. Data were collected from all ICU patients who were admitted to our institution from February 1, 2018 to June 30, 2019.
The inclusion criteria for patients were as follows: (1) complete blood culture results and medical records; (2) aged over 18 years; (3) body temperature over 38 °C or below 36 °C; (4) simultaneously combined with one of the following items: (a) the definite invasive sites or migration foci; (b) systemic toxemic symptoms but no obvious infection foci; (c) unexplained rashes or bleeding spots, hepatosplenomegaly, or increased blood neutrophil counts; (d) systolic pressure less than 90 mmHg (1 mmHg=0.133 kPa) or decreased over 40 mmHg; (e) the diagnosis of pathogenic microorganisms via blood culture (if coagulase-negative Staphylococcus, Propionibacterium, and other common skin contaminants exist, the blood sample collection would be conducted at a different time point) or identification of antigen of responsible pathogens before recruitment.
The exclusion criteria for patients were as follows: (1) pregnant or breast feeding; (2) malignant tumor, blood-related diseases, or HIV infection; (3) severe organ dysfunction; and (4) chronic infection, such as chronic inflammatory bowel disease.
Thirteen patients were excluded from this study, including two patients because of poor sample quality and 11 because of incomplete medical records. Finally, a total of 74 patients were enrolled, and all the patients signed an informed consent form. If the patient was incapable of giving consent owing to the underlying severe infection, informed consent could be given by the patient’s legal guardian until the patients were informed.
Sample preparation and processing
A total of 118 venous blood samples (5 mL each) were collected in cell-free DNA (cfDNA) tubes (PAXgene Blood ccfDNA Tube, Feldbachstrasse 8634 Hombrechtikon, Switzerland) from the 74 patients with the blood infection. Blood samples were stored at 4 °C before plasma separation. Samples were collected when the patient’s body temperature was higher than 38 °C or below 36 °C. The tubes were spun down at 1,600g for 10 minutes at room temperature for plasma preparation. The cfDNA was extracted from plasma using QIAamp circulating nucleic acid kit (Qiagen, Valencia, USA) following the manufacturer’s instructions. The extracted DNA specimens were used to construct DNA libraries.
Sequencing
NGS libraries were prepared for sequencing. Negative controls (buffer only instead of plasma) and positive controls (healthy plasma spiked with a known mixture of microbial DNA fragments) were processed alongside patient samples in each batch. The quality of the DNA libraries was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, USA) to measure the adapters before sequencing. High throughput sequencing (MedcareDx Bio-Tech, Shanghai, China) was performed on the cfDNA sample using an Illumina NextSeq550Dx platform. On average, twenty-five million sequence reads were acquired for each sample.
Reference database and quality control
Reference genomes for Homo sapiens (National Center for Biotechnology Information [NCBI], National Library of Medicine [NLM]), and microorganisms (bacteria, viruses, fungi/molds, and other eukaryotic pathogens) were retrieved from NCBI ftp site (ftp://ftp.ncbi.nlm.nih.gov/genomes/). After elimination of the taxonomic mislabeling and sequence contamination in the reference genomes, the curated sequences were assembled into a single Fasta file and used to generate our microorganism reference database, which contained genomic sequences from 6,030 bacteria, 3,551 viruses, 185 fungi, and 87 parasites.
Qualified sequencing data of each clinical sample were generated by removing adapter contamination, low-quality reads, duplicates, and short reads (length <35 bp) from the raw sequencing data, followed by excluding human host sequences mapped to human reference (GRCh38.p12) with the Burrows-Wheeler Alignment tool.[14] After removing low-complexity reads, the unmapped sequencing reads in each sample were retained for further analysis.
Determination of pathogens
To identify the pathogenic sequences, the remaining unmapped sequences were aligned to the curated microorganism reference database as described above. The strictly mapped sequencing reads were classified into bacterial, viral, fungal, and parasitic at the species level. Considering the confounding factors, such as the number of sequencing reads, the genome size, and coverage rate, the quantity for each microbe identified was expressed as the normalized number (NN) of DNA sequencing reads in plasma in terms of Langelier’s study.[15] Species with NN less than three were removed, whereas species with NN greater than ten were reported. For those species with NN between three and ten, the Basic Local Alignment Search Tool for nucleotide was implemented in the nucleotide database to verify the identification accuracy, and the verified species were reported.[16]
Statistical analysis
Statistical calculations were performed in R (version 3.5.0) with the following packages: exactRankTests, survival, survminer, ggplot2, and PMCMRplus.[12] Continuous data were presented as mean and standard deviation and were compared using Student’s t-test. Categorical data were compared using Fisher’s exact test or Chi-square test. Kaplan-Meier plots were used to visualize survival curves for a 90-day follow-up, where were compared using the log-rank test. We then applied univariate and multivariate Cox proportional hazards models to identify independent prognostic factors for 90-day mortality. All the variables (including gender, age, Acute Physiology and Chronic Health Evaluation II [APACHE II] score and Sequential Organ Failure Assessment [SOFA] scores, white blood cell [WBC], neutrophil, leukomonocyte, platelet counts, procalcitonin, C-reactive protein, and lactic acid levels) were considered in separate univariate Cox regression models and those with a P-value of 0.05 or less entered into a multivariate Cox regression model. Analyses of correlation between continuous variables were performed using linear regression with coefficient of determination (R2). A P-value of less than 0.05 was considered statistically significant.
RESULTS
Patient population
In total, 74 patients diagnosed with sepsis were included (Table 1). Patients were divided into the virus-positive group and the virus-negative group according to the presence or absence of concomitant viral infection confirmed by NGS. There were 27 male and 10 female patients in the virus-negative group, whereas there were 23 male and 14 female patients in the virus-positive group (P>0.05). The mean age of the virus-negative group (53.86±17.03 years) was lower than that of the virus-positive group (62.35±14.43 years) (P<0.05). Patients with concomitant viral infection experienced a longer period of ICU hospitalization than those in the virus-negative group (39.11±28.18 days vs. 24.62±28.05 days, P<0.05). Patients in the virus-positive group had higher APACHE II and SOFA scores than those in the virus-negative group (both P<0.01). However, no statistically significant differences were observed in the laboratory infection parameters.
Table 1 Demographic data and clinical characteristics of patients in this study
| Parameters | Virus-negative group (n=37) | Virus-positive group (n=37) | P-value |
|---|---|---|---|
| Demographic data | |||
| Gender (male/female) | 27/10 | 23/14 | 0.456 |
| Age (years) | 53.86±17.03 | 62.35±14.43 | 0.024 |
| Duration of ICU (days) | 24.62±28.05 | 39.11±28.18 | 0.030 |
| ICU scores at admission | |||
| APACHE II score | 16.51±6.93 | 22.22±5.75 | <0.001 |
| SOFA | 6.35±3.23 | 8.24±2.22 | 0.004 |
| Laboratory infection parameters | |||
| WBC (×109/L) | 14.69±6.85 | 12.54±7.18 | 0.191 |
| Neutrophil (%) | 85.97±6.82 | 82.75±17.67 | 0.304 |
| Leukomonocyte (%) | 8.14±4.20 | 7.93±5.57 | 0.857 |
| Platelet (×109/L) | 174.08±83.50 | 199.46±153.52 | 0.380 |
| Procalcitonin (ng/mL) | 5.12±9.31 | 4.85±13.67 | 0.924 |
| C-reactive protein (mg/L) | 118.85±159.95 | 128.10±114.75 | 0.776 |
| Lactic acid (g/L) | 1.03±1.45 | 1.10±2.39 | 0.889 |
ICU: intensive care unit; APACHE II: Acute Physiology and Chronic Health Evaluation II; SOFA: Sequential Organ Failure Assessment; WBC: white blood cell.
Top 20 positive pathogenic microorganisms detected by NGS
A total of 118 samples from 74 patients were tested by NGS, which yielded 88.98% positive results over the whole study period. Pseudomonas chlororaphis, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia were the most frequently detected pathogens after the exclusion of typical skin commensals such as coagulase-negative (CoN) Staphylococci. However, despite the current gold standard, blood cultures were unable to identify concomitant viral infection, whereas NGS could give a broad viral profile. Human herpesvirus 5 was the most frequently detected pathogen, present in approximately 29.73% (22 of 74) of our patients.
Correlation of viral load with sepsis severity identified by Cox regression analysis and survival time
Owing to the limited number of virus varieties detected in the patients, we did not perform a similar analysis. Instead, we employed Cox regression analysis. Seven variables (sex, WBC count, procalcitonin, lactic acid level, APACHE II score, SOFA score, and concomitant viral infection) were observed to correlate with the severity of sepsis based on univariate Cox regression analysis (Table 2). Further, to correct for possible confounding factors, data for these seven variables were adjusted based on the Cox multivariate regression model. In all patient groups, the concomitant virus infection was closely associated with an increased hazard ratio for sepsis severity (Figure 1). The impact of concomitant viral infection on the survival rate was significantly obvious (P<0.05; Figure 2). In addition, at 25 days after admission, the survival rate decreased continuously for those in the virus-positive group, whereas it did not change significantly in the virus-negative group. These results indicated that concomitant viral infection correlated closely with sepsis severity and had negative effects on the survival of patients with sepsis.
Table 2 Univariate Cox proportional hazards analysis for the 90-day mortality
| Parameters | β HR | 95% CI for HR | P-value |
|---|---|---|---|
| Age | 0.023 | 1.00 (1.00-1.00) | 0.077 |
| Sex | -0.831 | 0.44 (0.21-0.89) | 0.023 |
| WBC | 0.054 | 1.10 (1.00-1.10) | 0.028 |
| Neutrophil | -0.002 | 1.00 (0.97-1.00) | 0.910 |
| Leukomonocyte | -0.052 | 0.95 (0.87-1.00) | 0.230 |
| Platelet | -0.001 | 1.00 (1.00-1.00) | 0.380 |
| Procalcitonin | -0.001 | 1.00 (1.00-1.00) | 0.220 |
| C-reactive protein | 0.033 | 1.00 (1.00-1.10) | 0.033 |
| Lactic acid | 0.211 | 1.20 (1.00-1.50) | 0.023 |
| APACHE II score | 0.162 | 1.20 (1.10-1.20) | <0.001 |
| SOFA | 0.203 | 1.20 (1.10-1.40) | <0.001 |
| Concomitant viral infection | 0.981 | 2.70 (1.20-5.80) | 0.014 |
HR: hazard ratio; CI: confidence interval; WBC: white blood cell; APACHE II: Acute Physiology and Chronic Health Evaluation II; SOFA: Sequential Organ Failure Assessment.
Figure 1.
Figure 1.
Forest plots of multivariable Cox model adjusted hazard ratios with 95% confidence interval for the 90-day mortality. The multivariable Cox model was adjusted for sex, WBC count, SOFA, lactic acid, PCT, and concomitant virus infection at admission. WBC: white blood cell; SOFA: Sequential Organ Failure Assessment; PCT: procalcitonin.
Figure 2.
Figure 2.
Kaplan-Meier plots of survival curves for ICU sepsis patients with and without virus. ICU: intensive care unit.
No significant correlation between bacteria and sepsis severity
As the primary cause of sepsis, pathogenic bacteria play a crucial role. Therefore, we also investigated the correlation between the bacteria varieties and sepsis severity. No significant correlation was observed between the bacteria varieties and sepsis severity in either laboratory results or ICU scores for patients with less than ten types of bacteria (R2=0.15, P=0.20), and for patients with more than ten types of bacteria (R2=0.02, P=0.88). These results indicated that bacteria varieties did not correlate with the severity of sepsis.
DISCUSSION
Sepsis has a significant impact on the critically ill population. Rapid diagnosis is thus important not only to enhance patients’ survival rate but also to promote reasonable antibiotic use. Nevertheless, an accurate and comprehensive identification of pathogenic microorganisms is still challenging. In addition, patients’ clinical symptoms and laboratory results are sometimes nonspecific.[17] In recent years, whether the concomitant viral infection is a potential causative agent in failed sepsis treatment has emerged as a subject of active debate.[18] Increasing evidence has suggested a close correlation between viral load and poor prognosis.[19,20,21] A prospective study with 329 patients enrolled for septic shock reported that 112 (34.04%) patients who had multiple concurrent viremia events suffered higher risk of mortality.[22] These results suggested that concurrent viral load could not be ignored. Therefore, a timely and accurate identification of viral load could provide valuable information for later reasonable therapy in critically ill patients.
NGS-based approaches could provide a rapid and comprehensive detection of bacterial, fungal, and viral infections in a single assay.[12] To the best of our knowledge, few published studies have investigated the effect of concomitant viral infection on the survival of patients with sepsis.[23,24] Therefore, we investigated the performance of NGS-based diagnosis technique for the effect of concomitant viral infection on the survival rate in ICU septic patients.
Pathogenic bacteria are crucial for the initiation and development of sepsis. We found that NGS exhibited the satisfactory performance in detecting potential bacterial infections, including certain bacteria that were particularly difficult to be cultured, which was consistent with the results of a previous study.[13] Furthermore, NGS-based diagnosis had a higher sensitivity than blood culture, independent of antimicrobial treatment (data not shown). In this present study, we analyzed the effect of bacteria varieties on sepsis severity. However, the results revealed no significant correlation between the bacteria varieties and sepsis severity. Therefore, the concurrent viral load became our focus.
We divided patients into a virus-positive group and a virus-negative group according to the presence or absence of the viral load. Patients with viral load were older and experienced a longer period of ICU hospitalization, with higher ICU scores (including APACHE II score and SOFA score) compared to those with bacterial infection only. These results suggested that patients with concurrent viral load may suffer more severe sepsis. Human herpesvirus 5 was the most frequently detected pathogen, occurring in approximately 29.73% (22 of 74) of the patients included in our study. This was in line with previous studies, which found that treating documented HSV patients with acyclovir could improve outcomes.[10] Cox regression analysis results showed an obvious impact of concurrent viral load on the survival rate. Interestingly, at 25 days after admission, the survival rate in the virus-negative group did not change significantly, whereas the survival rate decreased continuously in the virus-positive group. These results indicated that concomitant viral infection may have a negative impact on the survival of patients with sepsis.
Previous studies have shown a close correlation between infection with the virus HSV-1 and the therapeutic effect of ICU sepsis treatment. Traen et al[10] reported that acyclovir treatment was positively linked to shorter in-hospital stay and ICU stay, and lower mortality. Schuierer et al[11] divided patients into three groups: untreated group, acyclovir-treated patients with high (>105 HSV copies/mL) and low (103-105 HSV copies/mL) viral load groups, and found that prophylactic usage of acyclovir improved median ICU survival time (8 days vs. 22 days, P<0.05) with significantly improved circulatory and pulmonary function. Notably, because the prolonged ICU stay would accompany increasing complications, the long-term therapeutic benefit resulting from acyclovir treatment still required further study. Nevertheless, these potential complications could be prevented by proper patient management. Overall, we believe that prophylactic administration of antiviral drugs combined with antibiotics may improve the efficacy of treatment of sepsis patients compared with antibiotics alone. However, the exact mechanism of the effect of viral load on the survival of sepsis patients remains elusive.
The present study has several limitations. Firstly, we only investigated the relationship between concomitant viral infection and the sepsis severity and survival rate of ICU sepsis patients, and did not study the effect of combined prophylactic antiviral and antibiotic treatments on the prognosis of ICU sepsis patients. Given the current lack of direct evidence supporting the use of prophylactic antiviral drugs to increase survival rate, no attending physicians would make this treatment decision aggressively. However, considering the current literature and our study results, we believe that sepsis patients in the ICU would benefit greatly from combined prophylactic antiviral and antibiotic treatments. Secondly, owing to the study period, our sample size was relatively small, so the generalization of the results from this study may not be appropriate. Therefore, further studies with a larger sample size and longer duration are required. Thirdly, it is not possible to conclude from the results of this study whether a causative relationship exists between concurrent viral infection and sepsis severity; however, this possibility seems plausible. As indicated in this study, all patients with viral load had higher ICU scores and lower survival rate compared with patients without concurrent viral infection. Fourthly, during the process of this study, the blood sample was tested in batches. In addition, the majority of the patients enrolled were discharged after their metagenomic next-generation sequencing (mNGS) results were obtained. As a result, we cannot obtain further blood samples from these patients, and a confirmatory PCR cannot be performed to confirm the viral DNA. However, previous studies have indicated relatively high consistency between mNGS and PCR.[25,26]
CONCLUSIONS
The present study reveals that the concurrent viral load correlates closely with sepsis severity and the survival rate of sepsis patients in the ICU. Therefore, prophylactic administration of antiviral drugs combined with antibiotics may provide several benefits to ICU sepsis patients, including fewer complications, better prognosis, and higher survival rate.
ACKNOWLEDGMENTS
We are grateful to Doctor Kai-qiang Sun for his help in embellishing the manuscript regarding the spell and grammar errors.
Funding: The study was supported by grants from Science and Technology Committee of Shanghai (18411951400); Key Clinical Medical Specialties Project in Shanghai Pudong New Area (PWZzk2017-22); Science and Technology Action Plan (19495810200); Leading Talent Project in Shanghai Pudong New Area Health System (PWRl2018-08).
Ethical approval: This present study was approved by the Ethics Committee Board of our institution.
Conflicts of interest: The authors confirm that no conflict of interest or any financial relationship that relates to the content of the manuscript has been associated with this publication.
Contributors: LWD, JLQ, and JW contributed equally to this study. All authors read and approved the final version.
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BACKGROUND: The aim of this study is to investigate the diagnostic and prognostic value of neutrophil CD64 (nCD64) as a novel biomarker in sepsis patients. METHODS: One hundred fifty-one adult patients diagnosed with sepsis and 20 age-matched healthy controls were enrolled in the study. Patients with sepsis were further subdivided into a sepsis group and a septic shock group. nCD64 expression, serum procalcitonin (PCT) level, C-reactive protein (CRP) level, and white blood cell (WBC) count were obtained for each patient, and Sequential Organ Failure Assessment (SOFA) scores were calculated. RESULTS: nCD64 expression was higher in the sepsis group with confirmed infection than in the control group. The receiver operating characteristic (ROC) curve of nCD64 was higher than those of SOFA score, PCT, CRP and WBC for diagnosing infection. The area under the curve (AUC) of nCD64 combined with SOFA score was the highest for all parameters. The AUC of nCD64 for predicting 28-day mortality in sepsis was significantly higher than those of PCT, CRP, and WBC, but slightly lower than that of SOFA score. The AUC of nCD64 or PCT combined with SOFA score was significantly higher than that of any single parameter for predicting 28-day mortality. CONCLUSION: nCD64 expression and SOFA score are valuable parameters for early diagnosis of infection and prognostic evaluation of sepsis patients.
Empiric antibiotic treatment reduces mortality in severe sepsis and septic shock from the first hour: results from a guideline-based performance improvement program
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OBJECTIVES: Compelling evidence has shown that aggressive resuscitation bundles, adequate source control, appropriate antibiotic therapy, and organ support are cornerstone for the success in the treatment of patients with sepsis. Delay in the initiation of appropriate antibiotic therapy has been recognized as a risk factor for mortality. To perform a retrospective analysis on the Surviving Sepsis Campaign database to evaluate the relationship between timing of antibiotic administration and mortality. DESIGN: Retrospective analysis of a large dataset collected prospectively for the Surviving Sepsis Campaign. SETTING: One hundred sixty-five ICUs in Europe, the United States, and South America. PATIENTS: A total of 28,150 patients with severe sepsis and septic shock, from January 2005 through February 2010, were evaluated. INTERVENTIONS: Antibiotic administration and hospital mortality. MEASUREMENTS AND MAIN RESULTS: A total of 17,990 patients received antibiotics after sepsis identification and were included in the analysis. In-hospital mortality was 29.7% for the cohort as a whole. There was a statically significant increase in the probability of death associated with the number of hours of delay for first antibiotic administration. Hospital mortality adjusted for severity (sepsis severity score), ICU admission source (emergency department, ward, vs ICU), and geographic region increased steadily after 1 hour of time to antibiotic administration. Results were similar in patients with severe sepsis and septic shock, regardless of the number of organ failure. CONCLUSIONS: The results of the analysis of this large population of patients with severe sepsis and septic shock demonstrate that delay in first antibiotic administration was associated with increased in-hospital mortality. In addition, there was a linear increase in the risk of mortality for each hour delay in antibiotic administration. These results underscore the importance of early identification and treatment of septic patients in the hospital setting.
Effect of a multifaceted educational intervention for anti-infectious measures on sepsis mortality: a cluster randomized trial
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PURPOSE: Guidelines recommend administering antibiotics within 1 h of sepsis recognition but this recommendation remains untested by randomized trials. This trial was set up to investigate whether survival is improved by reducing the time before initiation of antimicrobial therapy by means of a multifaceted intervention in compliance with guideline recommendations. METHODS: The MEDUSA study, a prospective multicenter cluster-randomized trial, was conducted from July 2011 to July 2013 in 40 German hospitals. Hospitals were randomly allocated to receive conventional continuous medical education (CME) measures (control group) or multifaceted interventions including local quality improvement teams, educational outreach, audit, feedback, and reminders. We included 4183 patients with severe sepsis or septic shock in an intention-to-treat analysis comparing the multifaceted intervention (n = 2596) with conventional CME (n = 1587). The primary outcome was 28-day mortality. RESULTS: The 28-day mortality was 35.1% (883 of 2596 patients) in the intervention group and 26.7% (403 of 1587 patients; p = 0.01) in the control group. The intervention was not a risk factor for mortality, since this difference was present from the beginning of the study and remained unaffected by the intervention. Median time to antimicrobial therapy was 1.5 h (interquartile range 0.1-4.9 h) in the intervention group and 2.0 h (0.4-5.9 h; p = 0.41) in the control group. The risk of death increased by 2% per hour delay of antimicrobial therapy and 1% per hour delay of source control, independent of group assignment. CONCLUSIONS: Delay in antimicrobial therapy and source control was associated with increased mortality but the multifaceted approach was unable to change time to antimicrobial therapy in this setting and did not affect survival.
Surviving sepsis campaign: international guidelines for management of sepsis and septic shock: 2016
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The era of molecular and other non-culture-based methods in diagnosis of sepsis
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Sepsis, a leading cause of morbidity and mortality throughout the world, is a clinical syndrome with signs and symptoms relating to an infectious event and the consequent important inflammatory response. From a clinical point of view, sepsis is a continuous process ranging from systemic inflammatory response syndrome (SIRS) to multiple-organ-dysfunction syndrome (MODS). Blood cultures are the current
Systematic review and meta-analysis of the efficacy of appropriate empiric antibiotic therapy for sepsis
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An alternate pathophysiologic paradigm of sepsis and septic shock: implications for optimizing antimicrobial therapy
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The advent of modern antimicrobial therapy following the discovery of penicillin during the 1940s yielded remarkable improvements in case fatality rate of serious infections including septic shock. Since then, pathogens have continuously evolved under selective antimicrobial pressure resulting in a lack of significant improvement in clinical effectiveness in the antimicrobial therapy of septic shock despite ever more broad-spectrum and potent drugs. In addition, although substantial effort and money has been expended on the development novel non-antimicrobial therapies of sepsis in the past 30 years, clinical progress in this regard has been limited. This review explores the possibility that the current pathophysiologic paradigm of septic shock fails to appropriately consider the primacy of the microbial burden of infection as the primary driver of septic organ dysfunction. An alternate paradigm is offered that suggests that has substantial implications for optimizing antimicrobial therapy in septic shock. This model of disease progression suggests the key to significant improvement in the outcome of septic shock may lie, in great part, with improvements in delivery of existing antimicrobials and other anti-infectious strategies. Recognition of the role of delays in administration of antimicrobial therapy in the poor outcomes of septic shock is central to this effort. However, therapeutic strategies that improve the degree of antimicrobial cidality likely also have a crucial role.
Use of PCR coupled with electrospray ionization mass spectrometry for rapid identification of bacterial and yeast bloodstream pathogens from blood culture bottles
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Actionable diagnosis of neuroleptospirosis by next-generation sequencing
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A 14-year-old boy with severe combined immunodeficiency presented three times to a medical facility over a period of 4 months with fever and headache that progressed to hydrocephalus and status epilepticus necessitating a medically induced coma. Diagnostic workup including brain biopsy was unrevealing. Unbiased next-generation sequencing of the cerebrospinal fluid identified 475 of 3,063,784 sequence reads (0.016%) corresponding to leptospira infection. Clinical assays for leptospirosis were negative. Targeted antimicrobial agents were administered, and the patient was discharged home 32 days later with a status close to his premorbid condition. Polymerase-chain-reaction (PCR) and serologic testing at the Centers for Disease Control and Prevention (CDC) subsequently confirmed evidence of Leptospira santarosai infection.
Is acyclovir effective among critically ill patients with herpes simplex in the respiratory tract?
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Impact of acyclovir use on survival of patients with ventilator-associated pneumonia and high load herpes simplex virus replication
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BACKGROUND: Herpes simplex virus (HSV) replication can be detected in the respiratory secretions of a high proportion of ventilated intensive care unit (ICU) patients. However, the clinical significance remains poorly defined. We investigated whether patients with ventilator-associated pneumonia not responding to antibiotics and in whom high levels of HSV could be detected in respiratory secretions benefit from acyclovir treatment. METHODS: Respiratory secretions (bronchoalveolar lavage fluid or tracheal aspirates) were tested for HSV replication by quantitative real-time PCR. ICU survival times, clinical parameters, and radiographic findings were retrospectively compared between untreated and acyclovir treated patients with high (> 10(5) HSV copies/mL) and low (10(3)-10(5) HSV copies/mL) viral load. RESULTS: Fifty-seven low and 69 high viral load patients were identified. Fewer patients with high viral load responded to antibiotic treatment (12% compared to 40% of low load patients, p = 0.001). Acyclovir improved median ICU survival (8 vs 22 days, p = 0.014) and was associated with a significantly reduced hazard ratio for ICU death (HR = 0.31, 95% CI 0.11-0.92, p = 0.035) in high load patients only. Moreover, circulatory and pulmonary oxygenation function of high load patients improved significantly over the course of acyclovir treatment: mean norepinephrine doses decreased from 0.05 to 0.02 mug/kg body weight/min between days 0 and 6 of treatment (p = 0.049), and median PaO2/FiO2 ratio increased from 187 to 241 between day 3 and day 7 of treatment (p = 0.02). Chest radiographic findings also improved significantly (p < 0.001). CONCLUSIONS: In patients with ventilator-associated pneumonia, antibiotic treatment failure, and high levels of HSV replication, acyclovir treatment was associated with a significantly longer time to death in the ICU and improved circulatory and pulmonary function. This suggests a causative role for HSV in this highly selected group of patients.
Next-generation sequencing diagnostics of bacteremia in sepsis (Next GeneSiS-Trial): study protocol of a prospective, observational, noninterventional, multicenter, clinical trial
Enhanced performance of next-generation sequencing diagnostics compared with standard of care microbiological diagnostics in patients suffering from septic shock
DOI:10.1097/CCM.0000000000003658
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PMID:30720537
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OBJECTIVES: Culture-based diagnostics represent the standard of care in septic patients, but are highly insensitive and in many cases unspecific. We recently demonstrated the general feasibility of next-generation sequencing-based diagnostics using free circulating nucleic acids (cell-free DNA) in plasma samples of septic patients. Within the presented investigation, higher performance of next-generation sequencing-based diagnostics was validated by comparison to matched blood cultures. DESIGN: A secondary analysis of a prospective, observational, single-center study. SETTING: Surgical ICU of a university hospital and research laboratory. PATIENTS: Fifty patients with septic shock, 20 uninfected patients with elective surgery as control cohort. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: From 256 plasma samples of 48 septic patients at up to seven consecutive time points within the 28-day observation period, cell-free DNA was isolated and analyzed by next-generation sequencing and relevance scoring. In parallel, results from culture-based diagnostics (e.g., blood culture) were obtained. Plausibility of blood culture and next-generation sequencing results as well as adequacy of antibiotic therapy was evaluated by an independent expert panel. In contrast to blood culture with a positivity rate of 33% at sepsis onset, the positivity rate for next-generation sequencing-based pathogen identification was 72%. Over the whole study period, blood culture positivity was 11%, and next-generation sequencing positivity was 71%. Ninety-six percent of positive next-generation sequencing results for acute sepsis time points were plausible and would have led to a change to a more adequate therapy in 53% of cases as assessed by the expert evaluation. CONCLUSIONS: Our results show that next-generation sequencing-based analyses of bloodstream infections provide a valuable diagnostic platform for the identification of clinically relevant pathogens with higher sensitivity and specificity than blood culture, indicating that patients might benefit from a more appropriate therapy based on next-generation sequencing-based diagnosis.
Fast and accurate short read alignment with Burrows-Wheeler transform
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PMID:19451168
[Cited within: 1]
MOTIVATION: The enormous amount of short reads generated by the new DNA sequencing technologies call for the development of fast and accurate read alignment programs. A first generation of hash table-based methods has been developed, including MAQ, which is accurate, feature rich and fast enough to align short reads from a single individual. However, MAQ does not support gapped alignment for single-end reads, which makes it unsuitable for alignment of longer reads where indels may occur frequently. The speed of MAQ is also a concern when the alignment is scaled up to the resequencing of hundreds of individuals. RESULTS: We implemented Burrows-Wheeler Alignment tool (BWA), a new read alignment package that is based on backward search with Burrows-Wheeler Transform (BWT), to efficiently align short sequencing reads against a large reference sequence such as the human genome, allowing mismatches and gaps. BWA supports both base space reads, e.g. from Illumina sequencing machines, and color space reads from AB SOLiD machines. Evaluations on both simulated and real data suggest that BWA is approximately 10-20x faster than MAQ, while achieving similar accuracy. In addition, BWA outputs alignment in the new standard SAM (Sequence Alignment/Map) format. Variant calling and other downstream analyses after the alignment can be achieved with the open source SAMtools software package. AVAILABILITY: http://maq.sourceforge.net.
Metagenomic sequencing detects respiratory pathogens in hematopoietic cellular transplant patients
DOI:10.1164/rccm.201706-1097LE URL PMID:28686513 [Cited within: 1]
Basic local alignment search tool
DOI:10.1016/S0022-2836(05)80360-2
URL
PMID:2231712
[Cited within: 1]
A new approach to rapid sequence comparison, basic local alignment search tool (BLAST), directly approximates alignments that optimize a measure of local similarity, the maximal segment pair (MSP) score. Recent mathematical results on the stochastic properties of MSP scores allow an analysis of the performance of this method as well as the statistical significance of alignments it generates. The basic algorithm is simple and robust; it can be implemented in a number of ways and applied in a variety of contexts including straightforward DNA and protein sequence database searches, motif searches, gene identification searches, and in the analysis of multiple regions of similarity in long DNA sequences. In addition to its flexibility and tractability to mathematical analysis, BLAST is an order of magnitude faster than existing sequence comparison tools of comparable sensitivity.
Mortality related to severe sepsis and septic shock among critically ill patients in Australia and New Zealand, 2000-2012
DOI:10.1001/jama.2014.2637
URL
PMID:24638143
[Cited within: 1]
IMPORTANCE: Severe sepsis and septic shock are major causes of mortality in intensive care unit (ICU) patients. It is unknown whether progress has been made in decreasing their mortality rate. OBJECTIVE: To describe changes in mortality for severe sepsis with and without shock in ICU patients. DESIGN, SETTING, AND PARTICIPANTS: Retrospective, observational study from 2000 to 2012 including 101,064 patients with severe sepsis from 171 ICUs with various patient case mix in Australia and New Zealand. MAIN OUTCOMES AND MEASURES: Hospital outcome (mortality and discharge to home, to other hospital, or to rehabilitation). RESULTS: Absolute mortality in severe sepsis decreased from 35.0% (95% CI, 33.2%-36.8%; 949/2708) to 18.4% (95% CI, 17.8%-19.0%; 2300/12,512; P < .001), representing an overall decrease of 16.7% (95% CI, 14.8%-18.6%), an annual rate of absolute decrease of 1.3%, and a relative risk reduction of 47.5% (95% CI, 44.1%-50.8%). After adjusted analysis, mortality decreased throughout the study period with an odds ratio (OR) of 0.49 (95% CI, 0.46-0.52) in 2012, using the year 2000 as the reference (P < .001). The annual decline in mortality did not differ significantly between patients with severe sepsis and those with all other diagnoses (OR, 0.94 [95% CI, 0.94-0.95] vs 0.94 [95% CI, 0.94-0.94]; P = .37). The annual increase in rates of discharge to home was significantly greater in patients with severe sepsis compared with all other diagnoses (OR, 1.03 [95% CI, 1.02-1.03] vs 1.01 [95% CI, 1.01-1.01]; P < .001). Conversely, the annual increase in the rate of patients discharged to rehabilitation facilities was significantly less in severe sepsis compared with all other diagnoses (OR, 1.08 [95% CI, 1.07-1.09] vs 1.09 [95% CI, 1.09-1.10]; P < .001). In the absence of comorbidities and older age, mortality was less than 5%. CONCLUSIONS AND RELEVANCE: In critically ill patients in Australia and New Zealand with severe sepsis with and without shock, there was a decrease in mortality from 2000 to 2012. These findings were accompanied by changes in the patterns of discharge to home, rehabilitation, and other hospitals.
Herpes simplex virus infections
URL PMID:11377626 [Cited within: 1]
Herpes simplex virus lung infection in patients undergoing prolonged mechanical ventilation
DOI:10.1164/rccm.200609-1322OC
URL
PMID:17234903
[Cited within: 1]
RATIONALE: It is not known whether the isolation of herpes simplex virus (HSV) from lower respiratory tract samples of nonimmunocompromised ventilated patients corresponds to bronchial contamination from the mouth and/or throat, local tracheobronchial excretion of HSV, or true HSV lung involvement (bronchopneumonitis) with its own morbidity/mortality. OBJECTIVES: This prospective, single-center, observational study was conducted to define the frequency, risk factors, and relevance of HSV bronchopneumonitis. METHODS: All consecutive nonimmunocompromised patients receiving mechanical ventilation for 5 days or more were evaluated. Bronchoalveolar lavage, oropharyngeal swabs, and bronchial biopsies (presence of macroscopic bronchial lesions) were obtained for all who deteriorated clinically with suspected lung infection. HSV bronchopneumonitis was defined as this deterioration, associated with HSV in bronchoalveolar lavage and HSV-specific nuclear inclusions in cells recovered during lavage or bronchial biopsies. MEASUREMENTS AND MAIN RESULTS: HSV bronchopneumonitis was diagnosed in 42 (21%) of the 201 patients who deteriorated clinically, with a mean mechanical ventilation duration before diagnosis of 14 +/- 6 days. Risk factors associated with HSV bronchopneumonitis were oral-labial lesions, HSV in the throat, and macroscopic bronchial lesions seen during bronchoscopy. Patients with HSV bronchopneumonitis were comparable to those without at admission, but their courses were more complicated, with longer duration of mechanical ventilation and intensive care unit stays. CONCLUSIONS: HSV bronchopneumonitis is common in nonimmunocompromised patients with prolonged mechanical ventilation, is associated with HSV reactivation or infection of the mouth and/or throat, and seems to be associated with poorer outcome.
Clinical relevance of and risk factors for HSV-related tracheobronchitis or pneumonia: results of an outbreak investigation
DOI:10.1186/cc6175
URL
PMID:17996032
[Cited within: 1]
INTRODUCTION: Herpes simplex virus (HSV) type 1 was identified in respiratory specimens from a cluster of eight patients on a surgical intensive care unit within 8 weeks. Six of these patients suffered from HSV-related tracheobronchitis and one from HSV-related pneumonia only. Our outbreak investigation aimed to determine the clinical relevance of and risk factors associated with HSV-related tracheobronchitis or pneumonia in critically ill patients, and to investigate whether the cluster was caused by nosocomial transmission. METHODS: A retrospective cohort study was performed to identify risk factors for the outcomes of HSV-related tracheobronchitis or pneumonia and death using univariable analysis as well as logistic regression analysis. Viruses were typed by molecular analysis of a fragment of the HSV type 1 glycoprotein G. RESULTS: The cohort of patients covering the outbreak period comprised 53 patients, including six patients with HSV-related tracheobronchitis and one patient with pneumonia only. HSV-related tracheobronchitis or pneumonia was associated with increased mortality (100% in patients with versus 17.8% in patients without HSV-related tracheobronchitis or pneumonia; P < 0.0001). The interaction of longer duration of ventilation and tracheotomy was associated with HSV-related tracheobronchitis or pneumonia in multivariable analysis. Identical HSV type 1 glycoprotein G sequences were found in three patients and in two patients. The group of three identical viral sequences belonged to a widely circulating strain. The two identical viral sequences were recovered from bronchoalveolar lavages of one patient with HSV-related tracheobronchitis and of one patient without clinical symptoms. These viral sequences showed unique polymorphisms, indicating probable nosocomial transmission. CONCLUSION: HSV-related tracheobronchitis or pneumonia is associated with increased mortality in critically ill patients. Care should be taken to avoid nosocomial transmission and early diagnosis should be attempted.
Monitoring of herpes simplex virus in the lower respiratory tract of critically ill patients using real-time PCR: a prospective study
URL PMID:19260875 [Cited within: 1]
Epidemiology of multiple herpes viremia in previously immunocompetent patients with septic shock
DOI:10.1093/cid/cix120
URL
PMID:28158551
[Cited within: 1]
Background: Systemic reactivations of herpesviruses may occur in intensive care unit (ICU) patients, even in those without prior immune deficiency. However, the clinical relevance of these events is uncertain. Methods: In this study we selected patients admitted with septic shock and treated for more than 4 days from a prospectively enrolled cohort of consecutive adults in the mixed ICUs of 2 tertiary care hospitals in the Netherlands. We excluded patients who had received antiviral treatment in the week before ICU admission and those with known immunodeficiency. We studied viremia episodes with cytomegalovirus (CMV), Epstein-Barr virus (EBV), human herpesvirus 6 (HHV-6), herpes simplex virus types 1 (HSV-1) and 2 (HSV-2), and varicella zoster virus (VZV) by weekly polymerase chain reaction in plasma. Results: Among 329 patients, we observed 399 viremia episodes in 223 (68%) patients. Viremia with CMV, EBV, HHV-6, HSV-1, HSV-2, and VZV was detected in 60 (18%), 157 (48%), 80 (24%), 87 (26%), 13 (4%), and 2 (0.6%) patients, respectively; 112 (34%) patients had multiple concurrent viremia events. Crude mortality in the ICU was 36% in this latter group compared to 19% in remaining patients (P < .01). After adjustment for potential confounders, time-dependent bias, and competing risks, only concurrent CMV and EBV reactivations remained independently associated with increased mortality (adjusted subdistribution hazard ratio, 3.17; 95% confidence interval, 1.41-7.13). Conclusions: Herpesvirus reactivations were documented in 68% of septic shock patients without prior immunodeficiency and frequently occurred simultaneously. Concurrent reactivations could be independently associated with mortality. Clinical Trials Registration: NCT01905033.
Next-generation sequencing diagnostics of bacteremia in septic patients
URL PMID:27368373 [Cited within: 1]
Diagnosis of sepsis with cell-free DNA by next-generation sequencing technology in ICU patients
URL PMID:27751370 [Cited within: 1]
Metagenomic next-generation sequencing for identification and quantitation of transplant-related DNA viruses
DOI:10.1128/JCM.01113-19
URL
PMID:31554674
[Cited within: 1]
Infections with DNA viruses are frequent causes of morbidity and mortality in transplant recipients. This study describes the analytical and clinical performance characteristics of the Arc Bio Galileo Pathogen Solution, an all-inclusive metagenomic next-generation sequencing (mNGS) reagent and bioinformatics pipeline that allows the simultaneous quantitation of 10 transplant-related double-stranded DNA (dsDNA) viruses (adenovirus [ADV], BK virus [BKV], cytomegalovirus [CMV], Epstein-Barr virus [EBV], human herpesvirus 6A [HHV-6A], HHV-6B, herpes simplex virus 1 [HSV-1], HSV-2, JC virus [JCV], and varicella-zoster virus [VZV]). The mNGS 95% limit of detection ranged from 14 copies/ml (HHV-6) to 191 copies/ml (BKV), and the lower limit of quantitation ranged from 442 international units (IU)/ml (EBV) to 661 copies/ml (VZV). An evaluation of 50 residual plasma samples with at least one DNA virus detected in prior clinical testing showed a total percent agreement of mNGS and quantitative PCR (qPCR) of 89.2% (306/343), with a kappa statistic of 0.725. The positive percent agreement was 84.9% (73/86), and the negative percent agreement was 90.7% (233/257). Furthermore, mNGS detected seven subsequently confirmed coinfections that were not initially requested by qPCR. Passing-Bablok regression revealed a regression line of y = 0.953x + 0.075 (95% confidence interval [CI] of the slope, 0.883 to 1.011; intercept, -0.100 to 0.299), and Bland-Altman analysis (mNGS - qPCR) showed a slight positive bias (0.28 log10 concentration; 95% limits of agreement, -0.62 to 1.18). In conclusion, the mNGS-based Galileo pipeline demonstrates analytical and clinical performance comparable to that of qPCR for transplant-related DNA viruses.
Analytical and clinical validation of a microbial cell-free DNA sequencing test for infectious disease
DOI:10.1038/s41564-018-0349-6
URL
PMID:30742071
[Cited within: 1]
Thousands of pathogens are known to infect humans, but only a fraction are readily identifiable using current diagnostic methods. Microbial cell-free DNA sequencing offers the potential to non-invasively identify a wide range of infections throughout the body, but the challenges of clinical-grade metagenomic testing must be addressed. Here we describe the analytical and clinical validation of a next-generation sequencing test that identifies and quantifies microbial cell-free DNA in plasma from 1,250 clinically relevant bacteria, DNA viruses, fungi and eukaryotic parasites. Test accuracy, precision, bias and robustness to a number of metagenomics-specific challenges were determined using a panel of 13 microorganisms that model key determinants of performance in 358 contrived plasma samples, as well as 2,625 infections simulated in silico and 580 clinical study samples. The test showed 93.7% agreement with blood culture in a cohort of 350 patients with a sepsis alert and identified an independently adjudicated cause of the sepsis alert more often than all of the microbiological testing combined (169 aetiological determinations versus 132). Among the 166 samples adjudicated to have no sepsis aetiology identified by any of the tested methods, sequencing identified microbial cell-free DNA in 62, likely derived from commensal organisms and incidental findings unrelated to the sepsis alert. Analysis of the first 2,000 patient samples tested in the CLIA laboratory showed that more than 85% of results were delivered the day after sample receipt, with 53.7% of reports identifying one or more microorganisms.
