Dengue virus transmission risk in blood donation: Evidence from Thailand.

Individuals infected with dengue virus (DENV) often show no symptoms, which raises the risk of DENV transfusion transmission (TT-DENV) in areas where the virus is prevalent. This study aimed to determine the evidence of DENV infection in blood donors from different geographic regions of Thailand. A cross-sectional study was conducted on blood donor samples collected from the Thai Red Cross National Blood Center and four regional blood centers between March and September 2020. Screening for DENV nonstructural protein 1 (NS1), anti-DENV immunoglobulin G (IgG), and IgM antibodies was performed on residual blood from 1053 donors using enzyme-linked immunosorbent assay kits. Positive NS1 and IgM samples indicating acute infection were verified using four different techniques, including quantitative real-time (q) RT-PCR, nested PCR, virus isolation in C6/36 cells, and mosquito amplification. DENV IgG seropositivity was identified in 89% (938/1053) of blood donors. Additionally, 0.4% (4/1053) and 2.1% (22/1053) of Thai blood donors tested positive for NS1 and IgM, respectively. The presence of asymptomatic dengue virus infection in healthy blood donors suggests a potential risk of transmission through blood transfusion, posing a concern for blood safety.

Long-term kidney outcomes after leptospirosis: a prospective multicentre cohort study in Thailand.

BACKGROUND: Leptospirosis is one of the most important public-health zoonotic diseases in the tropics that can cause severe organ dysfunction and death. Currently there are insufficient data on long-term renal dysfunction in patients after leptospirosis infection. METHODS: A prospective multicentre cohort study was conducted at 15 hospitals in the Sisaket province of Thailand. Confirmed leptospirosis patients admitted from 1 December 2015 to 30 November 2018 were followed between 1 February 2020 and 31 October 2020 (median 4.1 years after hospital discharge). The primary outcome was a composite of major kidney adverse events (MAKEs) including all-cause mortality, dialysis and new-onset chronic kidney disease (CKD). RESULTS: Of the 217 confirmed leptospirosis cases enrolled, 32.7% were classified as having severe leptospirosis. Fifteen cases (6.9%) were deceased at the time of hospital admission. After a median follow-up time of 4.18 years, 30 patients had died and 33 patients developed CKD. Patients with severe leptospirosis had a significantly higher risk of MAKEs {adjusted hazard ratio 2.45 [95% confidence interval (CI) 1.44-4.18]}. Patients with intensive care unit admission, pulmonary haemorrhage and acute kidney injury also had a higher risk of MAKEs and all-cause mortality. Participants with severe leptospirosis in the follow-up cohort showed a higher risk of developing CKD compared with non-severe leptospirosis [adjusted odds ratio 3.22 (95% CI 1.04-9.96)], especially renal magnesium and phosphate wasting. CONCLUSION: Leptospirosis patients, especially severe leptospirosis, are associated with long-term kidney sequelae. Our finding reflects the importance of long-term follow-up and the urgent need for specific interventions.

Immunomodulation Effect of Regional Citrate Anticoagulation in Acute Kidney Injury Requiring Renal Replacement Therapy.

INTRODUCTION: Acute kidney injury (AKI) is a common syndrome in critically ill patients. Continuous renal replacement therapy (CRRT) is the standard treatment for patients with AKI. Research on the immunomodulating effects of regional citrate anticoagulation (RCA) remains limited in patients with AKI receiving CRRT. We aimed to evaluate the immunomodulating effects of RCA in patients with AKI receiving CRRT. METHODS: A randomized controlled trial study on critically ill adult patients with AKI undergoing CRRT was undertaken. Participants were randomized into either a regional citrate group or control group (either heparin anticoagulant or normal saline). Measurements were taken at baseline, 6 and 24 h after commencing CRRT for CD11b expression, C3a, C5a, and plasminogen activator inhibitor-1 (PAI-1) levels. Clinical outcomes assessed were 28-day survival rate, length of ICU stay, renal support duration, and renal function at discharge. RESULTS: Thirty patients were recruited and randomized into 2 groups of 15 subjects. Baseline demographic and clinical data were comparable between groups. In the citrate group, CD11b expression was significantly decreased at 24 h compared to the control group (1.84% [1.18-3.32] versus 4.92% [2.63-6.93], p < 0.01). The complement level, including c3b and c5a, was stable during CRRT. Additionally, the PAI-1 levels were significantly decreased at 24 h compared to the control group (114 ng/mL [19-193] versus 359 ng/mL [264-491], p < 0.01). No significant difference in survival rate was observed. CONCLUSIONS: RCA may have the potential to mitigate the inflammatory response by decreasing CD11b expression of neutrophil and improve fibrinolysis activity through a reduction of PAI-1 levels. Larger clinical trials are warranted to test this immunomodulation effect of RCA.

The combination of RPA-CRISPR/Cas12a and Leptospira IgM RDT enhances the early detection of leptospirosis.

BACKGROUND: Lack of available sensitive point-of-care testing is one of the primary obstacles to the rapid diagnosis of leptospirosis. The purpose of this study was to test the performance of two point-of-care tests, a clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 12a (CRISPR/Cas12a) fluorescence-based diagnostic assay (FBDA), a Leptospira immunoglobulin M (IgM) rapid diagnostic test (RDT), and the two tests combined. METHODOLOGY/PRINCIPAL FINDINGS: For the diagnosis of 171 clinical samples, a recombinase polymerase amplification (RPA)-CRISPR/Cas12a FBDA for whole blood and Leptospira IgM RDT (Medical Science Public Health, Thailand) for serum were used. The confirmed cases were determined by using any positive qPCR, microscopic agglutination test (MAT), and culture results. Diagnostic accuracy was assessed on the first day of enrollment and stratified by the day after symptom onset. The overall sensitivity of the Leptospira IgM RDT and RPA-CRISPR/Cas12a FBDA was 55.66% and 60.38%, respectively. When the two tests were combined, the sensitivity rose to 84.91%. The specificity of each test was 63.08% and 100%, respectively, and 63.08% when combined. The sensitivity of the Leptospira IgM RDT rose on days 4-6 after the onset of fever, while the RPA-CRISPR/Cas12a FBDA continued to decrease. When the two tests were combined, the sensitivity was over 80% at different days post-onset of fever. CONCLUSIONS/SIGNIFICANCE: The combination of Leptospira IgM RDT and RPA-CRISPR/Cas12 FBDA exhibited significant sensitivity for the detection of leptospires at various days after the onset of fever, thereby reducing the likelihood of misdiagnosis. The combination of these assays may be suitable for early leptospirosis screening in situations with limited resources.

Comparison of circulating bacterial profiles between mild and severe COVID-19 patients.

Recent reports revealed that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected patients can develop bacteremia; however, the circulating bacterial profile is not well studied. Therefore, this study has aimed to investigate circulating bacterial profile in mild (n = 15) and severe (n = 13) SARS-CoV-2-infected patients as well as healthy controls (n = 10), using 16S rDNA (V4) sequencing approach. The alpha diversity indexes and Bray-Curtis dissimilarity matrix revealed that the bacterial profiles between the two conditions are significantly different. Correspondingly, the relative abundance indicates that the predominant bacterial phylum in both conditions was Proteobacteria. At genus level, the dominant bacterial genera in the mild patients belonged to Sphingomonas, Stenotrophomonas, and Achromobacter, while bacterial genera belonging to Enhydrobacter, Comamonas, and Acinetobacter were dominant in the severe patients. Furthermore, Linear discriminant analysis (LDA) Effect Size (LEfSe). revealed that Stenotrophomonas, Delftia, Achromobacter, and Neisseria were enriched in the mild condition, while Agrobacterium, Comamonas, Pseudomonas, Corynebacterium, Alkaliphilus, and Kocuria were enriched in the severe patients. These results revealed a distinct circulating bacterial profile in the mild and severe SARS-CoV-2-infected patients, which may provide an insight for further therapeutic strategy.

Seroprevalence of leptospirosis among blood donors in an endemic area.

Thailand is known to be endemic for leptospirosis. This bacterium may pose a potential risk to transfusion safety. This study was a cross-sectional study examining the seroprevalence of leptospirosis among Thai blood donors. A total of 1053 serum specimens collected from blood donors residing in 5 regions of Thailand during March to September 2020 were included in this study. All samples were tested for the presence of antibodies to 22 leptospiral serovars using the microscopic agglutination test (MAT) and anti-Leptospira IgG antibodies using commercially available enzyme immunoassay. We found no evidence of recent exposure to Leptospira spp. in sera of healthy Thai blood donors by MAT, including those in higher-risk areas. However, in this same group, we did find small numbers of past exposure (1.7%) to Leptospira spp. by IgG ELISA. According to the findings of this study, there is currently no evidence for implementing new blood banking procedures to identify possible carriers in Thailand, however these should be continually monitored and revised according to the infectious disease burden in each country. It should be noted that there was a difference in the occupation rate between the general population reported in Thailand and blood donors in this study; it may not reflect the actual situation in the country.

Serum Cortisol as a Biomarker of Severe Dengue.

Dengue infection presents a wide range of clinical symptoms. Serum cortisol is known as a severity predictor of serious infection but is not yet clearly understood in dengue infection. We aimed to investigate the pattern of cortisol response after dengue infection and evaluate the possibility of using serum cortisol as the biomarker to predict the severity of dengue infection. This prospective study was conducted in Thailand during 2018. Serum cortisol and other laboratory tests were collected at four time points: day 1 at hospital admission, day 3, day of defervescence (DFV) (4-7 days post-fever onset), and day of discharge (DC). The study recruited 265 patients (median age (IQR) 17 (13, 27.5)). Approximately 10% presented severe dengue infection. Serum cortisol levels were highest on the day of admission and day 3. The best cut-off value of serum cortisol level for predicting severe dengue was 18.2 mcg/dL with an AUC of 0.62 (95% CI, 0.51, 0.74). The sensitivity, specificity, PPV and NPV were 65.4, 62.3, 16 and 94%, respectively. When we combined serum cortisol with persistent vomiting and day of fever, the AUC increased to 0.76. In summary, serum cortisol at day of admission was likely to be associated with dengue severity. Further studies may focus on the possibility of using serum cortisol as one of the biomarkers for dengue severity.

Citrate pharmacokinetics in critically ill liver failure patients receiving CRRT.

Citrate has been proposed as anticoagulation of choice in continuous renal replacement therapy (CRRT). However, little is known about the pharmacokinetics (PK) and metabolism of citrate in liver failure patients who require CRRT with regional citrate anticoagulation (RCA). This prospective clinical PK study was conducted at King Chulalongkorn Memorial Hospital between July 2019 to April 2021, evaluating seven acute liver failure (ALF) and seven acute-on-chronic liver failure (ACLF) patients who received CRRT support utilizing RCA as an anticoagulant at a citrate dose of 3 mmol/L. For evaluation of the citrate PK, we delivered citrate for 120 min and then stopped for a further 120 min. Total body clearance of citrate was 152.5 ± 50.9 and 195.6 ± 174.3 mL/min in ALF and ACLF, respectively. The ionized calcium, ionized magnesium, and pH slightly decreased after starting citrate infusion and gradually increased to baseline after stopping citrate infusion. Two of the ACLF patients displayed citrate toxicity during citrate infusion, while, no ALF patient had citrate toxicity. In summary, citrate clearance was significantly decreased in critically ill ALF and ACLF patients receiving CRRT. Citrate use as an anticoagulation in these patients is of concern for the risk of citrate toxicity.

Rapid and sensitive point-of-care detection of Leptospira by RPA-CRISPR/Cas12a targeting lipL32.

BACKGROUND: One of the key barriers preventing rapid diagnosis of leptospirosis is the lack of available sensitive point-of-care testing. This study aimed to develop and validate a clustered regularly-interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 12a (CRISPR/Cas12a) platform combined with isothermal amplification to detect leptospires from extracted patient DNA samples. METHODOLOGY/PRINCIPAL FINDINGS: A Recombinase Polymerase Amplification (RPA)-CRISPR/Cas12a-fluorescence assay was designed to detect the lipL32 gene of pathogenic Leptospira spp. The assays demonstrated a limit of detection (LOD) of 100 cells/mL, with no cross-reactivity against several other acute febrile illnesses. The clinical performance of the assay was validated with DNA extracted from 110 clinical specimens and then compared to results from qPCR detection of Leptospira spp. The RPA-CRISPR/Cas12a assay showed 85.2% sensitivity, 100% specificity, and 92.7% accuracy. The sensitivity increased on days 4-6 after the fever onset and decreased after day 7. The specificity was consistent for several days after the onset of fever. The overall performance of the RPA-CRISPR/Cas12a platform was better than the commercial rapid diagnostic test (RDT). We also developed a lateral flow detection assay (LFDA) combined with RPA-CRISPR/Cas12a to make the test more accessible and easier to interpret. The combined LFDA showed a similar LOD of 100 cells/mL and could correctly distinguish between known positive and negative clinical samples in a pilot study. CONCLUSIONS/SIGNIFICANCE: The RPA-CRISPR/Cas12 targeting the lipL32 gene demonstrated acceptable sensitivity and excellent specificity for detection of leptospires. This assay might be an appropriate test for acute leptospirosis screening in limited-resource settings.

Discovery and validation of circulating miRNAs for the clinical prognosis of severe dengue.

BACKGROUND: Early prognostic markers of severe dengue may improve case management and reduce dengue-related mortalities. This study aimed to identify circulating microRNAs (miRNAs) as biomarkers for predicting severe dengue. METHODOLOGY: Serum samples from dengue-infected patients were collected on the first day of admission. Patients were followed up for 14 days after admission to determine the final diagnosis. Participants were divided into non-severe and severe dengue, as defined by WHO 2009 criteria. Circulating microtranscriptome analysis was performed using NanoString miRNA Expression Assay. The expression level of candidate miRNAs were then validated by quantitative reverse transcription-PCR method. PRINCIPAL FINDINGS: The discovery cohort (N = 19) lead to the identification of 37 differentially expressed miRNAs between the two groups. Six up-regulated candidate miRNAs were selected and further validated in the larger cohort (N = 135). MiR574-5p and miR1246 displayed the highest diagnostic performance in discriminating between severe from non-severe dengue (ROC-AUC = 0.83). Additionally, miR574-5p and miR1246 had high sensitivity and high negative predictive value for detecting severe dengue. Multivariate analysis suggested that serum miR574-5p was an independent predictor of severe dengue (odds ratio 3.30, 95% CI 1.81-6.04; p<0.001). CONCLUSION: Our study indicated that circulating miRNAs, especially miR-574-5p and miR-1246, might be a promising diagnostic and prognostic biomarker for severe dengue upon hospital admission, especially when using these biomarkers on days 1 to 2 before the onset of severe dengue complications.

A prospective study to evaluate the accuracy of rapid diagnostic tests for diagnosis of human leptospirosis: Result from THAI-LEPTO AKI study.

BACKGROUND: Rapid diagnostic tests (RDTs) have become widely used in low-resource settings for leptospirosis diagnostic. This study aims to evaluate the diagnostic performance of the five commercially available RDTs to detect human IgM against Leptospira spp. in Thai population. METHODOLOGY/PRINCIPAL FINDINGS: Ninety-nine serum samples from Leptospirosis suspicious patients were tested with five RDTs, including Medical Science Public Health, Leptocheck-WB, SD bioline, TRUSTline, and J.Mitra. The case definition was based on MAT, qPCR, and culture results. Diagnostic accuracy was determined based on the first day of enrollment in an overall analysis and stratified according to days post-onset of fever. The five RDTs had overall sensitivity ranging from 1.8% to 75% and specificity ranging from 52.3% to 97.7%. Leptocheck-WB had high sensitivity of 75.0%. The sensitivity of five RDTs increased on days 4-6 post-onset of fever, while the specificity of all tests remained relatively stable at different days post-onset of fever. CONCLUSIONS/SIGNIFICANCE: The tested RDTs showed low sensitivity. Therefore, based on the present study, five commercially available RDTs might not be an appropriate test for acute leptospirosis screening in the Thai population.

The role of leptospiremia and specific immune response in severe leptospirosis.

Leptospirosis can cause a high mortality rate, especially in severe cases. This multicenter cross-sectional study aimed to examine both host and pathogen factors that might contribute to the disease severity. A total of 217 leptospirosis patients were recruited and divided into two groups of non-severe and severe. Severe leptospirosis was defined by a modified sequential organ failure assessment (mSOFA) score of more than two or needed for mechanical ventilation support or had pulmonary hemorrhage or death. We found that leptospiremia, plasma neutrophil gelatinase-associated lipocalin (pNGAL), and interleukin 6 (IL-6) at the first day of enrollment (day 1) and microscopic agglutination test (MAT) titer at 7 days after enrollment (days 7) were significantly higher in the severe group than in the non-severe group. After adjustment for age, gender, and the days of fever, there were statistically significant associations of baseline leptospiremia level (OR 1.70, 95% CI 1.23-2.34, p = 0.001), pNGAL (OR 9.46, 95% CI 4.20-21.33, p < 0.001), and IL-6 (OR 2.82, 95% CI 1.96-4.07, p < 0.001) with the severity. In conclusion, a high leptospiremia, pNGAL, and IL-6 level at baseline were associated with severe leptospirosis.

Dengue pre-vaccination screening test evaluation for the use of dengue vaccine in an endemic area.

BACKGROUND: The dengue vaccine (Dengvaxia) is only recommended for individuals with prior dengue infection (PDI). This study aimed to perform a serosurvey to inform decision-making for vaccine introduction and identify appropriate target populations. We also evaluated the performance of the serological tests using plaque reduction neutralization test (PRNT) as a reference test in identifying PDI to determine suitability for pre-vaccination screening. METHODS: We enrolled 115 healthy individuals between 10 and 22 years of age living in the Ratchaburi province of Thailand. The serum samples were tested by PRNT to measure the prevalence and concentration of serotype-specific neutralizing antibodies. The performance of the IgG rapid diagnostic test (RDT, SD Bioline, Korea) and IgG enzyme-linked immunosorbent assay (ELISA, EUROIMMUN, Germany) in identifying PDI were evaluated by using PRNT as a reference method. RESULTS: Ninety-four (81.7%) individuals neutralized one or more dengue serotypes at a titer threshold greater than or equal to 10. Multitypic profiles were observed in 70.4% of the samples which increased to 91.9% in subjects aged 19-22. Among monotypic samples, the highest proportion was reactive against DENV-1 followed by DENV-2, DENV-3, and DENV-4. The highest anti-dengue antibody titers were recorded against DENV-1 and increased with age to a geometric mean NT50 titer (GMT) of 188.6 in the 19-22 age group. While both RDT and ELISA exhibited 100% specificity, RDT demonstrated low sensitivity (35%) with ELISA displaying much greater sensitivity (87%). CONCLUSIONS: Almost 80% of adolescents and youth in Ratchaburi province had already been exposed to one or more of the dengue virus serotypes. The dengue IgG RDT displayed low sensitivity and is likely not be suitable for dengue pre-vaccination screening. These results support the use of IgG ELISA test for dengue vaccination in endemic areas.

Circulating microtranscriptome profiles reveal distinct expression of microRNAs in severe leptospirosis.

Biomarkers to predict the severity of leptospirosis are still lacking. This study aimed to identify and validate microRNAs in patients with severe leptospirosis, that could potentially be used as biomarkers for predicting an unfavorable outcome. Serum samples were collected from participants with definite diagnosis of leptospirosis. The participants were divided into two groups, non-severe and severe leptospirosis, as defined by the Specific Organ Sequential Organ Failure (SOFA) Score of more than two in any organ. Microtranscriptome analysis was performed using the NanoString miRNA Expression Assay. The expression level of candidate miRNAs was then validated by quantitative RT-PCR. Based on the NanoString, the microtranscriptome profile of the severe group was significantly different from that of the non-severe group. Upregulation of miR155-5p, miR362-3p, miR502-5p, miR601, miR1323, and miR630 in the severe group were identified, and further investigated. A total of 119 participants were enrolled in the validation cohort. Serum miR155-5p and miR630 levels were significantly higher in the severe group compared to the non-severe group. The combined use of miR155-5p or miR-630 with serum bicarbonate levels had an AUC of 0.79 (95%CI; 0.69-0.89, p<0.001) in identifying the severity of the disease. This data provides the first evidence that the microtranscriptome profiles of patients with severe leptospirosis were different from the non-severe group. Serum miR155-5p and miR630 levels might be novel biomarkers for identifying severe leptospirosis.

Defective Neutrophil Function in Patients with Sepsis Is Mostly Restored by ex vivo Ascorbate Incubation.

BACKGROUND: Neutrophil function is essential for effective defence against bacterial infections but is defective in patients with sepsis. Ascorbate or vitamin C, which is low in the plasma of patients with sepsis, is stored inside human neutrophils and is essential for their normal function. OBJECTIVE: This study aimed to determine if ascorbate treatment ex vivo improved neutrophil function in patients with sepsis. PATIENTS AND METHODS: Human blood neutrophils were isolated from 20 patients with sepsis and 20 healthy age-matched controls. Neutrophils were incubated with or without ascorbate (1, 5, 10, 20 and 40 mM) for periods up to 2h. Chemotaxis was evaluated using a chemotactic chamber in response to the chemoattractant, fMLP. Phagocytosis (uptake of pHrodo red stained S. aureus) and apoptosis (annexin-V/propidium iodide staining) were measured by flow cytometry. Neutrophil extracellular trap (NET) formation was detected and quantified using DAPI, anti-myeloperoxidase and anti-neutrophil elastase immuno-fluorescence staining. Quantifluor detected the amount of dsDNA in NET supernatants, while quantitative PCR identified changes in expression of PADI4 gene. RESULTS: Chemotactic and phagocytic activities were decreased in patients with sepsis but increased after treatment with the high concentrations of ascorbate. Apoptosis was increased in the sepsis patients but not altered by ascorbate treatment. Spontaneous NET formation was observed in patients with sepsis. A quantity of 1mM ascorbate decreased spontaneous NETosis to that of normal, healthy neutrophils, while high concentrations of ascorbate (>10mM) further promoted NET formation. CONCLUSION: Dysregulated neutrophil function was observed in patients with sepsis which could contribute to disease pathology and outcomes. Exposure to ascorbate could reverse some of these changes in function. These novel discoveries raise the possibility that ascorbate treatment could be used as an adjunctive therapy that could result in improved neutrophil function during sepsis.

Urine Neutrophil Gelatinase-associated Lipocalin (NGAL) for Prediction of Persistent AKI and Major Adverse Kidney Events.

We aimed to determine whether urinary neutrophil gelatinase-associated lipocalin (uNGAL) can accurately predict persistent AKI, major adverse kidney events at 30 days (MAKE30) and 365 days (MAKE365) in hospitalized AKI patients. This is a retrospective study of adult patients who were admitted at King Chulalongkorn Memorial Hospital. We performed multivariable logistic regression for persistent AKI, MAKE30, and MAKE365. We developed equations for predicting MAKE30 and MAKE365 and divided the dataset into derivation and validation cohorts. uNGAL performance and predictive models were assessed using the area under the receiver operating characteristic curve (AROC). Among 1,322 patients with AKI, 76.9%, 45.1%, and 61.7% had persistent AKI, MAKE30, and MAKE365. The AROC were 0.75 (95% confidence interval[CI] 0.70-0.80), 0.66 (95%CI 0.61-0.71), and 0.64 (95%CI 0.59-0.70) for prediction of persistent AKI, MAKE30, and MAKE365 by uNGAL. The AROC in the validation dataset combining uNGAL with clinical covariates were 0.74 (95%CI 0.69-0.79) and 0.72 (95%CI 0.67-0.77) for MAKE30 and MAKE365. We demonstrated an association between uNGAL and persistent AKI, MAKE30, and MAKE365. Prediction models combining uNGAL can modestly predict MAKE30 and MAKE365. Therefore, uNGAL is a useful tool for improving AKI risk stratification.

Plasma miR-370-3P as a Biomarker of Sepsis-Associated Encephalopathy, the Transcriptomic Profiling Analysis of Microrna-Arrays From Mouse Brains.

The diagnosis of sepsis-associated encephalopathy (SAE), an alteration of conscious from sepsis, is difficult due to the similarity to altered states of conscious that occur from other causes. Transcriptomic analyses between mouse brains at 24 h after cecal ligation and puncture (CLP) (SAE brain as evaluated by SHIRPA score) and at 120 h post-CLP (survivor) were performed to discover the SAE biomarker. Then, candidate microRNAs were validated in mouse and patient samples.As such, increased miR-370-3p in SAE mouse-brains (compared with recovery phase) was demonstrated by transcriptomic miR-profiling and was highly expressed in brain (but not other organs) of 24 h post-CLP mice. Plasma miR-370-3p also increased in CLP but was non-detectable in bilateral-nephrectomy (BiNx, a representative model of acute uremic encephalopathy) despite blood brain barrier permeability defect (determined by plasma s100ß and Evan blue dye assay) in both conditions. In parallel, high plasma miR-370-3p was demonstrated in patients with SAE (but not sepsis alone or uremia) suggesting the specificity toward SAE. The association among TNF-α, miR-370-3p and brain apoptosis was demonstrated by high serum TNF-α and increased brain apoptosis in SAE mice, TNF-α (but not other cytokines) activated miR-370-3p expression in PC-12 neuron cell, and increased cell apoptosis in miR-370-3p transfected PC-12 after incubation with TNF-α.In conclusion, miR-370-3p increased in brain and plasma of SAE mice but not uremic encephalopathy. Perhaps, TNF-α enhances cell susceptibility toward brain apoptosis in SAE, in part, through miR-370-3p induction in neuron. Our pilot results in patients with SAE supported the possibility that plasma miR-370-3p is an interesting SAE biomarker candidate. Further studies are warranted.

Gastrointestinal Leakage Detected by Serum (1→3)-ß-D-Glucan in Mouse Models and a Pilot Study in Patients with Sepsis.

Gastrointestinal (GI) leakage is believed to exacerbate sepsis and new, validated markers of GI barrier performance might benefit clinical decision-making. Serum (1→3)-ß-D-glucan (BG) was evaluated as a potential GI leakage marker. Serum BG was tested in several mouse models of GI leakage, including dextran sulfate solution (DSS) administration, endotoxin (LPS) injection, and cecal ligation and puncture sepsis (CLP). Serum BG titer was also evaluated in patients with sepsis and septic shock, for comparison.With 0.75% DSS administration, BG increased only after oral administration of heat-killed C. albicans, but increased spontaneously with 1.5% DSS. In the LPS and CLP models, BG increased as early as 1 h and at 12 h after LPS administration and surgery, respectively. GI leakage was confirmed by orthogonal validation methods including FITC-dextran oral administration in the DSS, LPS, and CLP models and, in the DSS model, with urine sucralose after oral administration and serum endotoxemia. IL-6 increased in parallel with serum BG. Serum BG or IL-6, at 18 h, anticipated sepsis mortality in the CLP model.Analysis of serum BG from patients with febrile neutropenic sepsis (N = 49) and febrile non-neutropenic sepsis (N = 39) demonstrated BG elevation. Patients with bacterial septic shock had serum BG titers similar to levels observed in invasive fungal disease, regardless of febrile neutropenia. Serum BG was lower in less severe cases of bacterial sepsis. Elevated serum IL-6 was associated with GI leakage and elevated serum BG.Serum BG may have potential as a sepsis/septic shock biomarker and further study in this context is warranted.