Infection (either community acquired or nosocomial) is a major cause of morbidity and mortality in critical care medicine. Sepsis is present in up to 30% of all ICU patients. A large fraction of sepsis cases is driven by severe community acquired pneumonia (sCAP), which incidence has dramatically increased during COVID-19 pandemics. A frequent complication of ICU patients is ventilator associated pneumonia (VAP), which affects 10-25% of all ventilated patients, and bloodstream infections (BSIs), affecting about 10% of patients. Management of these severe infections poses several challenges, including early diagnosis, severity stratification, prognosis assessment or treatment guidance. Digital PCR (dPCR) is a next-generation PCR method that offers a number of technical advantages to face these challenges: it is less affected than real time PCR by the presence of PCR inhibitors leading to higher sensitivity. In addition, dPCR offers high reproducibility, and provides absolute quantification without the need for a standard curve. In this article we reviewed the existing evidence on the applications of dPCR to the management of infection in critical care medicine. We included thirty-two articles involving critically ill patients. Twenty-three articles focused on the amplification of microbial genes: (1) four articles approached bacterial identification in blood or plasma; (2) one article used dPCR for fungal identification in blood; (3) another article focused on bacterial and fungal identification in other clinical samples; (4) three articles used dPCR for viral identification; (5) twelve articles quantified microbial burden by dPCR to assess severity, prognosis and treatment guidance; (6) two articles used dPCR to determine microbial ecology in ICU patients. The remaining nine articles used dPCR to profile host responses to infection, two of them for severity stratification in sepsis, four focused to improve diagnosis of this disease, one for detecting sCAP, one for detecting VAP, and finally one aimed to predict progression of COVID-19. This review evidences the potential of dPCR as a useful tool that could contribute to improve the detection and clinical management of infection in critical care medicine. 

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Digital PCR applications for the diagnosis and management of infection in critical care medicine

Droplet digital PCR (ddPCR) has emerged as a promising tool of pathogen detection in bloodstream infections (BSIs) in critical care medicine. However, different ddPCR platforms have variable sensitivity and specificity for diverse microorganisms at various infection sites. There is still a lack of prospective clinical studies aimed at validating and interpreting the discrepant ddPCR results for diagnosing BSI in intensive care unit (ICU) practice.A prospective diagnostic study of multiplex ddPCR panels was conducted in a general ICU from May 21, 2021, to December 22, 2021. Paired blood cultures (BCs) and ddPCRs (2.5 h) were obtained synchronously to detect the 12 most common BSI pathogens and three antimicrobial resistance (AMR) genes. Firstly, ddPCR performance was compared to definite BSI. Secondly, clinical validation of ddPCR was compared to composite clinical diagnosis. Sensitivity, specificity, and positive and negative predictive values were calculated. Thirdly, the positive rate of AMR genes and related analysis was presented.A total of 438 episodes of suspected BSIs occurring in 150 critical patients were enrolled. BC and ddPCR were positive for targeted bacteria in 40 (9.1%) and 180 (41.1%) cases, respectively. There were 280 concordant and 158 discordant. In comparison with BCs, the sensitivity of ddPCR ranged from 58.8 to 86.7% with an aggregate of 72.5% in different species, with corresponding specificity ranging from 73.5 to 92.2% with an aggregate of 63.1%. Furthermore, the rate of ddPCR+/BC- results was 33.6% (147/438) with 87.1% (128 of 147) cases was associated with probable (n = 108) or possible (n = 20) BSIs. When clinically diagnosed BSI was used as true positive, the final sensitivity and specificity of ddPCR increased to 84.9% and 92.5%, respectively. In addition, 40 blaKPC, 3blaNDM, and 38 mecA genes were detected, among which 90.5% were definitely positive for blaKPC. Further, 65.8% specimens were predicted to be mecA-positive in Staphylococcus sp. according to all microbiological analysis.The multiplexed ddPCR is a flexible and universal platform, which can be used as an add-on complementary to conventional BC. When combined with clinical infection evidence, ddPCR shows potential advantages for rapidly diagnosing suspected BSIs and AMR genes in ICU practice. 

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Clinical validation of a multiplex droplet digital PCR for diagnosing suspected bloodstream infections in ICU practice: a promising diagnostic tool

Abstract Background and objectives: Bloodstream infection (BSI) is a life-threatening condition in critically ill patients, but pathogen quantification techniques during treatment are laborious. This study aimed to explore the impact of monitoring pathogen DNA load changes and polymicrobial infection in blood by droplet digital polymerase chain reaction (ddPCR) on the prognosis of patients with BSIs. Methods This prospective case series study was conducted in the general intensive care unit of the Zhejiang Provincial People’s Hospital and included patients with BSIs from May 2020 to January 2021. Pathogens DNA load and presence of polymicrobial BSIs were dynamically monitored by ddPCR. Results Sixteen patients with BSIs proven by blood culture were recruited (87.5% men; mean age, 69.3 ± 13.7 years). All pathogens identified by blood culture were Gram-negative bacteria, among which seven were multidrug-resistant strains. The 28-day mortality rate was 62.5%. Compared to the 28-day survivors, the non-survivors were older ( P = 0.04), had higher pathogen DNA load on the second (day 3–4) and third (day 6–7) ddPCR assay ( P &lt; 0.01 in both cases). In addition, the changes of pathogen DNA load in the 28-day survivors had a downward trend in the first three ddPCR assay, whereas stable load or an upward trend was observed in the 28-day non-survivors. Moreover, the number of pathogen species in patients with BSIs in the 28-day survivors decreased during the period of effective antibiotic treatment. Conclusion The changes of pathogen DNA load and species monitored in blood by ddPCR may be used to determine antibiotic efficacy and make a more accurate prognostic assessment in patients with BSIs. 

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Pathogen load and species monitored by droplet digital PCR in patients with bloodstream infections: A prospective case series study

High-dose ascorbate confers tubular mitophagy responsible for septic acute kidney injury (AKI) amelioration, yet its biological roles in immune regulation remain poorly understood. Methods: The role of tubular mitophagy in macrophage polarization upon high-dose ascorbate treatment was assessed by fluorescence-activated cell sorter analysis (FACS) in vitro and by immunofluorescence in AKI models of LPS-induced endotoxemia (LIE) from Pax8-cre; Atg7flox/flox mice. The underlying mechanisms were revealed by RNA-sequencing, gene set enrichment analysis (GSEA), luciferase reporter, chromatin immunoprecipitation (ChIP) and adeno-associated viral vector serotype 9 (AAV9) delivery assays. Results: High-dose ascorbate enables conversion of macrophages from a pro-inflammatory M1 subtype to an anti-inflammatory M2 subtype in murine AKI models of LIE, leading to decreased renal IL-1β and IL-18 production, reduced mortality and alleviated tubulotoxicity. Blockade of tubular mitophagy abrogates anti-inflammatory macrophages polarization under the high-dose ascorbate-exposed coculture systems. Similar abrogations are verified in LIE mice with tubular epithelium-specific ablation of Atg7, where the high-dose ascorbate-inducible renal protection and survival improvement are substantially weaker than their control littermates. Mechanistically, high-dose ascorbate stimulates tubular secretion of serpin family G member 1 (SerpinG1) through maintenance of mitophagy, for which nuclear factor-erythroid 2 related factor 2 (NRF2) transactivation is required. SerpinG1 perpetuates anti-inflammatory macrophages to prevent septic AKI, while kidney-specific disruption of SerpinG1 by adeno-associated viral vector serotype 9 (AAV9)-short hairpin RNA (shRNA) delivery thwarts the anti-inflammatory macrophages polarization and anti-septic AKI efficacy of high-dose ascorbate. Conclusion: Our study identifies SerpinG1 as an intermediate of tubular mitophagy-orchestrated myeloid function during septic AKI and reveals a novel rationale for ascorbate-based therapy.

Tubule-mitophagic secretion of SerpinG1 reprograms macrophages to instruct anti-septic acute kidney injury efficacy of high-dose ascorbate mediated by NRF2 transactivation

Early diagnosis and potential therapeutic targets of sepsis-induced cardiomyopathy (SIC) remain challenges clinically. Circulating extracellular vesicles from immune cells carrying crucial injurious mediators, including miRNAs in sepsis. However, the impacts of neutrophil-derived extracellular vesicles and their miRNAs in the SIC development are unknown.The present study focused on the in-depth miRNA expression profiles of neutrophil-derived extracellular vesicles and explored the potential molecular biomarkers during the process of SIC.Neutrophil-derived extracellular vesicles were isolated from the blood samples in three sepsis patients with or without cardiomyopathy on day 1 and day 3 after ICU admission in comparison with three healthy controls. miRNAs were determined by RNA sequencing. The closely related differentially expressed miRNAs with SIC were further validated through qRT-PCR in the other cohorts of sepsis patients with (30 patients) or without cardiomyopathy (20 patients) and the association between miRNAs and the occurrence or disease severity of septic cardiomyopathy were stratified with logistic regression analysis.Sixty-eight miRNAs from neutrophil-derived extracellular vesicles were changed significantly between healthy controls and without septic cardiomyopathy patients (61 miRNAs upregulated and seven downregulated). Thirty-eight miRNAs were differentially expressed in the septic cardiomyopathy patients. 27 common differentially expressed miRNAs were found in both groups with similar kinetics (23 miRNAs upregulated and four downregulated). The enriched cellular signaling pathway mediated by miRNAs from sepsis to septic cardiomyopathy was the HIF-1 signaling system modulated septic inflammation. Using multivariate logistic regression analysis, miR-150-5p coupled with NT-pro BNP, LVEF, and SOFA score (AUC = 0.941) were found to be the independent predictors of septic cardiomyopathy.miRNAs derived from neutrophil-derived extracellular vesicles play an important role in septic disease severity development towards cardiomyopathy. miR-150-5p may be a predictor of sepsis severity development but warrants further study. 

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miR-150-5p in neutrophil-derived extracellular vesicles associated with sepsis-induced cardiomyopathy in septic patients

The relatively long turnaround time and low sensitivity of traditional blood culture‐based diagnosis may delay effective antibiotic therapy for patients with bloodstream infections (BSIs). A rapid and sensitive pathogen detection method is urgently required to reduce the morbidity and mortality associated with BSIs. Acinetobacter baumannii and Klebsiella pneumoniae are two major microorganisms that cause BSIs. Here we report a novel droplet digital polymerase chain reaction (ddPCR) assay that can detect A. baumannii and K. pneumoniae in blood samples within 4 h, with a specificity of 100% for each strain and a limit of detection at 0.93 copies/μl for A. baumannii and 0.27 copies/μl for K. pneumoniae. Clinical validation of 170 patients with suspected BSIs showed that compared to blood cultures that detected four (2.4%) A. baumannii cases and seven (4.1%) K. pneumoniae cases, ddPCR detected 23 (13.5%) A. baumannii cases, 26 (15.3%) K. pneumoniae cases, and four (2.4%) co‐infection cases, including the 11 cases detected via blood culture. In addition, patients who tested positive via ddPCR alone (n = 42) had significantly lower serum concentrations of procalcitonin and lactate, SOFA and APACHE II scores, and 28‐day mortality than those reported positive via both blood culture and ddPCR (n = 11), suggesting that patients with less severe symptoms can potentially benefit from ddPCR‐based diagnosis. In conclusion, our study suggests that ddPCR represents a sensitive and rapid method for identifying causal pathogens in blood samples and guiding treatment decisions in the early stages of BSIs.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/mbo3.1247

Development and clinical validation of a droplet digital PCR assay for detecting Acinetobacter baumannii and Klebsiella pneumoniae in patients with suspected bloodstream infections

The immunosuppressive, inflammatory microenvironment orchestrated by neutrophil extracellular traps (NETs) plays a principal role in pathogenesis of sepsis. Fibroblast growth factor-inducible molecule 14 (Fn14) has been established as a potential target for septic acute kidney injury (AKI), making further therapeutic benefits from combined NETs and Fn14 blockade possible. Methods: The concurrence of NETs and Fn14 in mice and patients with septic AKI were assessed by immunofluorescence, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA) and in silico studies. Survival, histopathological and biochemical analyses of wild-type and PAD4-deficient CMV-Cre; PAD4 fl/fl mice with septic AKI were applied to evaluate the efficacy of either pharmacological or genetic NETs interruption in combination with Fn14 blockade. Molecular mechanisms underlying such effects were determined by CRISPR technology, fluorescence-activated cell sorter analysis (FACS), cycloheximide (CHX) pulse-chase, luciferase reporter and chromatin immunoprecipitation (ChIP) assay. Results: NETs formation is concurred with Fn14 upregulation in murine AKI models of abdominal, endotoxemic, multidrug-resistant sepsis as well as in serum samples of patients with septic AKI. Pharmacological or genetic interruption of NETs formation synergizes with ITEM-2, a monoclonal antibody (mAb) of Fn14, to prolong mice survival and provide renal protection against abdominal sepsis, the effects that could be abrogated by elimination of macrophages. Interrupting NETs formation predominantly perpetuates infiltration and survival of efferocytic growth arrest-specific protein 6+ (GAS6+) macrophages in combination with ITEM-2 therapy and enhances transcription of tubular cell-intrinsic Fn14 in a DNA methyltransferase 3a (DNMT3a)-independent manner through dismantling the proteasomes-mediated turnover of homeobox protein Hox-A5 (HOXA5) upon abdominal sepsis challenge or LPS stimuli. Pharmacological NETs interruption potentiates the anti-septic AKI efficacy of ITEM-2 in murine models of endotoxemic and multidrug-resistant sepsis. Conclusion: Our preclinical data propose that interrupting NETs formation in combination with Fn14 mAb might be a feasible therapeutic strategy for septic AKI.

Interruption of neutrophil extracellular traps formation dictates host defense and tubular HOXA5 stability to augment efficacy of anti-Fn14 therapy against septic AKI.

ABSTRACT Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, has affected millions of individuals with various implications. Consistent with the crucial role of the microbiome in determining health and disease in humans, various studies have investigated the gut and respiratory microbiome effect on the COVID-19. Microbiota dysbiosis might support the entry, replication, and establishment of SARS-CoV-2 infection by modulating various mechanisms. One of the main mechanisms that the modulation of respiratory microbiota composition during the COVID-19 infection affects the magnitude of the disease is changes in innate and acquired immune responses, including inflammatory markers and cytokines and B- and T-cells. The diversity of respiratory microbiota in COVID-19 patients is controversial; some studies reported low microbial diversity, while others found high diversity, suggesting the role of respiratory microbiota in this disease. Modulating microbiota diversity and profile by supplementations and nutrients can be applied prophylactic and therapeutic in combating COVID-19. Here, we discussed the lung microbiome dysbiosis during various lung diseases and its interaction with immune cells, focusing on COVID-19.

https://www.tandfonline.com/doi/pdf/10.1080/21505594.2022.2090071?needAccess=true

The impact of infection with COVID-19 on the respiratory microbiome: A narrative review

Background Carbapenem-resistant gram-negative bacteria (CR-GNB) are becoming increasingly important bacterial pathogens in critically ill patients. Several clinicians use Intravenous colistin sulfate to treat infections due to CR-GNB, although the clinical data is limited. The aim of our retrospective observational study was to evaluate the effectiveness and nephrotoxicity of intravenous colistin sulfate in the treatment of CR-GNB infections. Methods Fifty critically ill intensive care patients with infections due to CR-GNB were retrospectively enrolled between January 2020 and December 2021 in the Zhejiang Provincial People’s Hospital. Favorable clinical response rate, bacterial clearance rate, nephrotoxicity, and 28-day mortality were evaluated. Results The overall favorable clinical response rate was 58%, the bacterial clearance rate was 40%, and the 28-day all-cause mortality was 44%. Temperature, neutrophil count, C-reaction protein (CRP), procalcitonin (PCT), creatinine (Cr), and lactate levels were also significantly decreased (P<0.05). The major adverse reaction is nephrotoxicity, and renal function was evaluated on the day before and after treatment with colistin sulfate. Possible nephrotoxicity was observed in three patients (6%). Backward logistic regression was conducted to determine risk factors for the nephrotoxicity of colistin sulfate, the result showed there were no significant differences in the duration and dose of colistin sulfate. Conclusions Our results provide evidence for the positive clinical efficacy and safety of colistin sulfate. Appropriate use of colistin sulfate may be viable and safe in the treatment of severe infections caused by CR-GNB.

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Clinical efficacy and nephrotoxicity of intravenous colistin sulfate in the treatment of carbapenem-resistant gram-negative bacterial infections: a retrospective cohort study

Jun Jin

Papers

Development and clinical validation of a droplet digital PCR assay for detecting Acinetobacter baumannii and Klebsiella pneumoniae in patients with suspected bloodstream infections

Interruption of neutrophil extracellular traps formation dictates host defense and tubular HOXA5 stability to augment efficacy of anti-Fn14 therapy against septic AKI.

Pathogen load and species monitored by droplet digital PCR in patients with bloodstream infections: A prospective case series study

The impact of infection with COVID-19 on the respiratory microbiome: A narrative review

Clinical efficacy and nephrotoxicity of intravenous colistin sulfate in the treatment of carbapenem-resistant gram-negative bacterial infections: a retrospective cohort study