David Stubljar

Non-culture-based methods to diagnose bloodstream infection: Does it work?

Bloodstream infections are a major cause of morbidity and mortality worldwide. Molecular methods for the detection of pathogens in blood have been developed. The clinical utility of these methods and their integration into the clinical workflow is discussed.

Diagnostic and prognostic value of sCD14-ST—presepsin for patients admitted to hospital intensive care unit (ICU)

BackgroundSepsis is a serious problem in intensive care units all over the world. Biomarkers could be useful to identify patients at risk. We focused especially on the performance of presepsin (sCD14-ST), compared to C-reactive protein (CRP), procalcitonin (PCT) and CD64, to determine its diagnostic and prognostic indications.MethodsThe study was conducted on 47 hospitalized patients after procedures, who were divided into three groups; systemic inflammatory response (SIRS), sepsis and septic shock. Expression of CD64 on neutrophils presented as CD64 index, sCD14-ST, CRP and PCT were measured in whole blood or plasma samples. All patients had standard samples like urine, respiratory tract samples etc. taken for culturing. Blood cultures were drawn to confirm bloodstream infection.ResultsForty (85 %) patients had SIRS with bacterial infection and seven (15 %) patients had SIRS with no infection. All infections were confirmed with blood cultures. Biomarkers were evaluated in all patients. In patients with confirmed infection the values were high. The patients with bacterial infection showed statistical significance with CD64 index (p = 0.003), CRP (p = 0.049) and sCD14-ST (p = 0.026), but not with PCT (p = 1.000). The severity of diagnosed SIRS was significant only with PCT (p < 0.001).ConclusionCD64 index, CRP and sCD14-ST served as good parameters to determine possible infection in patients that needed intensive care after major procedures. Values of PCT were the only ones to predict SIRS severity and could distinguish between sepsis and severe sepsis or septic shock.

Diagnostic Accuracy of Presepsin (sCD14-ST) for Prediction of Bacterial Infection in Cerebrospinal Fluid Samples from Children with Suspected Bacterial Meningitis or Ventriculitis

ABSTRACT Children with temporary external ventricular drains (EVD) are prone to nosocomial infections. Diagnosis of bacterial meningitis and ventriculitis in these children is challenging due to frequent blood contamination of cerebrospinal fluid (CSF) and the presence of chemical ventriculitis. The aim of this study was to compare diagnostic accuracy of presepsin (sCD14-ST), a novel biomarker of bacterial infection in CSF, to predict bacterial infection in comparison to the accuracy of established biomarkers like those demonstrated in biochemical analysis of CSF. We conducted a prospective study with 18 children with suspected bacterial meningitis or ventriculitis who had 66 episodes of disease. CSF samples were taken from external ventricular drainage. We measured presepsin in CSF, as well as CSF leukocyte count, glucose, and proteins. CSF was also taken to prove bacterial infection with culture methods or with 16S rRNA gene broad-range PCR (SepsiTest; Molzym, Germany). Infection was clinically confirmed in 57 (86%) episodes of suspected meningitis or ventriculitis. Chemical ventriculitis was diagnosed in 9 (14%) episodes of suspected meningitis or ventriculitis. Diagnostic accuracies presented as area under the curve (AUC) for sCD14-ST, leukocytes, and proteins measured in CSF were 0.877 (95% confidence interval [CI], 0.793 to 0.961), 0.798 (95% CI, 0.677 to 0.920), and 0.857 (95% CI, 0.749 to 0.964), respectively. With CSF culture, we detected bacteria in 17 samples, compared to 37 detected with broad-range PCR. It was found that presepsin was present at a significantly higher level in children with clinically proven ventriculitis than in those without meningitis or ventriculitis. Diagnostic accuracies of presepsin were superior to those of leukocytes or proteins in CSF. Presepsin-guided 16S rRNA gene PCR could be used in everyday clinical practice to improve etiological diagnosis of meningitis and ventriculitis and to prescribe more appropriate antibiotics.

Expression of CD64 on neutrophils (CD64 index): diagnostic accuracy of CD64 index to predict sepsis in critically ill patients.

Sepsis is still a serious problem and diagnosis should be available as fast as possible [1] . The biomarkers of bacterial infection can be helpful to make the correct diagnosis [2] . Procalcitonin (PCT) is the marker that has been used the most. However, it is also increased in cases of systemic inflammatory response syndrome (SIRS) due to non-infectious disease conditions, such as severe congestive heart failure, or in acute pancreatitis and viral infection [3] . We designed an observational retrospective study aimed at evaluating the diagnostic accuracy and the prognostic value of neutrophil CD64 expression in critically ill patients with possible systemic bacterial infection hospitalized at two intensive care units in comparison to PCT, C-reactive protein (CRP), white cells blood count and percentage of neutrophils to predict possible severe systemic bacterial infection in an observational retrospective study. The study took place at University Clinical Center Ljubljana, Slovenia. We included 88 adult patients who self-reported to have fever ≥ 38 ° C at least during the last 24 h and had at least two SIRS criteria set by Surviving Sepsis Campaign [1] . We excluded patients if they had taken antibiotics during the last 24 h. The Republic of Slovenia National Medical Ethics Committee approved the study. The following samples for culture were taken: urine, respiratory tract samples, samples from other sites when infection foci were unknown, blood filled in two pairs (4 bottles altogether) of blood cultures (aerobic and anaerobic) bottles (BacT/ALERT 3D, bioMerieux, France). We identified positive culture findings with VITEK ® automated analyzer (bioMerieux). We included the following biomarkers of infection in the study: the white blood cell count (WBC), the CRP (Siemens Healthcare Diagnostics, Germany), PCT (Brahms, Germany) and expression of CD64 on neutrophils presented as CD64 index measured on flow cytometer (Trillium Diagnostics, LCC, USA). After the end of the treatment two physicians retrospectively evaluated the patient ’ s data. They assigned the final diagnosis based on clinical, laboratory and microbiological data. Bacterial infection was confirmed if antibiotic therapy helped and if WBC and CRP normalized. The statistical analysis was performed by using Statistical Package for the Social Sciences 19.0 (SPSS, Chicago, IL, USA). A non-parametric Kruskal-Wallis test and χ 2 -test were used for comparing quantitative variables between four groups of diagnoses (SIRS without infection, sepsis, severe sepsis, septic shock) and between two groups of patients (one group patients without infection and the other patients with infection). A p-value < 0.05 were set as statistically significant. Areas under the curve (AUCs) with confidence intervals (CIs) calculations were also calculated. a Petra Rogina and David Stubljar contributed equally to the study. *Corresponding author: Miha Skvarc, Institute of Microbiology and Immunology, Faculty of Medicine, Zaloska 4, 1000 Ljubljana, Slovenia, Phone: + 38615437470, E-mail: miha.skvarc@mf.uni-lj.si Petra Rogina: General Hospital Novo mesto, Smihelska 1, Novo mesto, Slovenia David Stubljar: Institute of Microbiology and Immunology, Faculty of Medicine, Ljubljana, Slovenia Lejko-Zupanc T: Infectious Disease Department, University Medical Center, Ljubljana, Slovenia Josko Osredkar: Clinical Institute of Clinical Chemistry and Biochemistry, University Medical Centre Ljubljana, Ljubljana, Slovenia

Inhibition of cathepsin X enzyme influences the immune response of THP-1 cells and dendritic cells infected with Helicobacter pylori

Abstract Background. The immune response to Helicobacter pylori importantly determines the outcome of infection as well as the success of eradication therapy. We demonstrate the role of a cysteine protease cathepsin X in the immune response to H. pylori infection. Materials and methods. We analysed how the inhibition of cathepsin X influenced the immune response in experiments when THP-1 cells or dendritic cells isolated from patients were stimulated with 48 strains of H. pylori isolated from gastric biopsy samples of patients which had problems with the eradication of bacteria. Results. The experiments, performed with the help of a flow cytometer, showed that the expression of Toll-like receptors (TLRs), especially TLR-4 molecules, on the membranes of THP-1 cells or dendritic cells was higher when we stimulated cells with H. pylori together with inhibitor of cathepsin X 2F12 compared to THP-1 cells or dendritic cells stimulated with H. pylori only, and also in comparison with negative control samples. We also demonstrated that when we inhibited the action of cathepsin X in THP-1 cells, the concentrations of pro-inflammatory cytokines were lower than when THP-1 cell were stimulated with H. pylori only. Conclusions. We demonstrated that inhibition of cathepsin X influences the internalization of TLR-2 and TLR-4. TLR-2 and TLR-4 redistribution to intra-cytoplasmic compartments is hampered if cathepsin X is blocked. The beginning of a successful immune response against H. pylori in the case of inhibition of cathepsin X is delayed.

CD64 index on neutrophils can diagnose sepsis and predict 30-day survival in subjects after ventilator-associated pneumonia.

INTRODUCTION Sepsis associated with ventilator-associated pneumonia (VAP) causes mortality in intensive care unit (ICU) patients. The time of diagnosis is crucial, and microbiological cultures take time. In this study, the diagnostic accuracy of CD64 index to predict VAP-induced sepsis and survival time in subjects requiring mechanical ventilation were evaluated and compared to conventional biomarkers and culturing methods. METHODOLOGY A total of 32 subjects with VAP were included. Sepsis after VAP was diagnosed in 25 (78.1%) patients according to clinical signs, radiographic examination, and samples of blood and trachea taken for culturing. Simultaneously with cultures, CD64 index on neutrophils, C-reactive protein (CRP), procalcitonin (PCT), and count of leucocytes and neutrophils were determined. RESULTS Biomarker values were evaluated in both groups of subjects (with and without sepsis after VAP). The values of CD64 index and CRP were significantly higher in the sepsis group. Receiver operating characteristic (ROC) curve analysis revealed an area under curve (AUC) of 0.929 for CD64 index in differentiating subjects with VAP-induced sepsis from those without sepsis. The biomarkers CRP and PCT showed comparable results (AUC of 0.869 and 0.909, respectively). Blood cultures were positive in 12 subjects, endotracheal aspirate in 19. CD64 index and isolation of pathogen with positive blood cultures or from endotracheal aspirate (positive in 24 cases) could predict survival time before application of more targeted antibiotic therapy. CONCLUSIONS CD64 index may be used as a useful diagnostic tool to recognize VAP-induced sepsis; moreover, accompanied with an identified pathogen, can predict survival for ICU patients.

Dynamics of inflammation biomarkers C-reactive protein, leukocytes, neutrophils, and CD64 on neutrophils before and after major surgical procedures to recognize potential postoperative infection

Abstract Background. Major trauma and soft tissue injuries result in a substantial activation of systemic immune response and post-traumatic complications such as postoperative infections. The aim was to assess the dynamics of expressed inflammatory biomarkers after surgery and to detect possible postoperative infection. Methods. A total of 229 patients were included and separated into three different groups, depending on the procedure they underwent (colorectal, maxillofacial, open heart surgery). Biomarkers CD64 on neutrophils, C-reactive protein (CRP), count of leucocytes and neutrophils were measured to detect postoperative infection. Results. The values of all biomarkers after surgery were generally elevated and had then dropped 48 h after the procedure. The levels were dependent on the type of operation and showed higher levels after more serious procedures. In the patients with postoperative infections the values were considerably higher. Moreover, biomarkers’ cut-off values for positive infection were higher from patients who underwent surgery, compared to the cut-off values from patients with no surgical procedure. CD64 index was the only biomarker that could predict postoperative infection (p < 0.001). Other biomarkers could not statistically predict the infection. Conclusions. Newly acquired postoperative infection is difficult to diagnose using just biomarkers due to the strong activation of immune response. CD64 index with its slightly higher cut-off (> 1.27) is the only biomarker that could be used as a diagnostic tool to rapidly detect postoperative bacterial infection.

Diagnostic Utility of Broad Range Bacterial 16S rRNA Gene PCR with Degradation of Human and Free Bacterial DNA in Bloodstream Infection Is More Sensitive Than an In-House Developed PCR without Degradation of Human and Free Bacterial DNA

We compared a commercial broad range 16S rRNA gene PCR assay (SepsiTest) to an in-house developed assay (IHP). We assessed whether CD64 index, a biomarker of bacterial infection, can be used to exclude patients with a low probability of systemic bacterial infection. From January to March 2010, 23 patients with suspected sepsis were enrolled. CD64 index, procalcitonin, and C-reactive protein were measured on admission. Broad range 16S rRNA gene PCR was performed from whole blood (SepsiTest) or blood plasma (IHP) and compared to blood culture results. Blood samples spiked with Staphylococcus aureus were used to assess sensitivity of the molecular assays in vitro. CD64 index was lower in patients where possible sepsis was excluded than in patients with microbiologically confirmed sepsis (P = 0.004). SepsiTest identified more relevant pathogens than blood cultures (P = 0.008); in three patients (13%) results from blood culture and SepsiTest were congruent, whereas in four cases (17.4%) relevant pathogens were detected by SepsiTest only. In vitro spiking experiments suggested equal sensitivity of SepsiTest and IHP. A diagnostic algorithm using CD64 index as a decision maker to perform SepsiTest shows improved detection of pathogens in patients with suspected blood stream infection and may enable earlier targeted antibiotic therapy.

The influence of cytokine gene polymorphisms on the risk of developing gastric cancer in patients with Helicobacter pylori infection

Abstract Background. Helicobacter pylori infection is the main cause of gastric cancer. The disease progression is influenced by the host inflammatory responses, and cytokine single nucleotide polymorphisms (SNPs) may have a role in the course of the disease. The aim of our study was to investigate proinflammatory cytokine polymorphisms, previously associated with the development of gastric cancer, in a Slovenian population. Patients and methods. In total 318 patients and controls were selected for the study and divided into three groups: (i) patients with gastric cancer (n = 58), (ii) patients with chronic gastritis (n = 60) and (iii) healthy control group (n = 200). H. pylori infection in patient groups was determined by serology, histology and culture. Four proinflammatory gene polymorphisms were determined (IL-1β, IL-1rα, TNF-α, TLR-4) in all subjects. Results. We found a statistically significant difference between males and females for the groups (p = 0.025). Odds ratio (OR) for gastric cancer risk for females was 0.557 (95% confidence interval [CI]: 0.233―1.329) and for chronic gastritis 2.073 (95% CI: 1.005―4.277). IL-1B-511*T/T homozygous allele for cancer group had OR = 2.349 (95% CI: 0.583―9.462), heterozygous IL-1B-511*T had OR = 1.470 (95% CI: 0.583―3.709) and heterozygotes in TNF-A-308 genotype for chronic gastritis had OR = 1.402 (95% CI: 0.626―3.139). Other alleles had OR less than 1. Conclusions. We could not prove association between gastric cancer and chronic gastritis due to H. pylori in any cytokine SNPs studied in Slovenian population. Other SNPs might be responsible besides infection with H. pylori for the progression from atrophy to neoplastic transformation.

Potential Immune Biomarkers in Diagnosis and Clinical Management for Systemic Lupus Erythematosus

Summary Background: There is still no reliable, specific biomarker for precision diagnosis and clinical monitoring of systemic lupus erythematosus. The aim of this study was to investigate the importance of the determination of immunofenotypic profiles (T, B lymphocytes and NK cells) and serum cytokine concentrations (IL-17 and IFN-alpha) as potential biomarkers for this disease. Methods: The study included 55 patients with SLE and 25 healthy controls. The proportion of T, B, NK cells were assessed in peripheral blood using flow cytometric assays while the serum cytokine concentration (IL-17 and IFNalpha) was determined by ELISA test. Results: ROC curve analysis showed good accuracy to distinguish between patients and healthy individuals for activated T cells (AUC=0.798; p<0.001), Treg (AUC= 0.651; p=0.036), and memory B cells (AUC=0.285; p=0.002). We found statistically significant difference (p=0.036) in the levels of serum IL-17 between patients with SLE (IL-17=49.27 pg/mL) and controls (IL-17= 28.64 pg/mL). Conclusions: Significant increase in the relative number of Treg lymphocytes, and decrease in memory B cells, as well as decrease level of IL-17, in SLE patients may be implicated in the pathogenesis of the disease. These parameters, as biomarkers, could distinguish SLE patients and no-SLE patients. Monitoring subpopulations of immune cells in peripheral blood using flow cytometry provides insight into abnormal T and B cell function in SLE. Progress in understanding the immunity at SLE, results in concrete benefits for the SLE patients, which include new clinical management and therapeutic strategies.