Miha Skvarc

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.

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.

Anticryptococcal cytotoxicity of murine nonadherent cells is perforin and nonperforin mediated

The encapsulated fungal pathogen Cryptococcus neoformans is a significant agent of life-threatening infections, particularly in people with suppressed cell-mediated immunity. The cellular cytotoxicity against C. neoformans infection is mainly mediated by NK and T cells, but effector mechanisms are not well understood. The objective of this study was (i) to determine whether prior exposure to the cryptococcal antigens enhances anticryptococcal activity of cytotoxic cells in mice and (ii) the contribution of perforin- and nonperforin-mediated cytotoxicity of NK and T cells in growth inhibition of C. neoformans. Our data showed that in vitro exposure of nonadherent (NA) spleen mononuclear cells from nonimmunized mice to heat-killed C. neoformans strain Cap67 unencapsulated mutant of B3501 (Ag1) or its supernatant (Ag2) demonstrated higher anticryptococcal activity. This effector mechanism can be enhanced further after immunization with either Ag1 or Ag2. There is a synergistic effect of immunization and in vitro incubation of the NA cells with the same antigens. Concanamycin A (CMA) and strontium chloride (SrCl2) inhibition assays were performed to clarify the contribution of perforin- and nonperforin-mediated anticryptococcal cytotoxicity of NA cells in these events. Treatment with these inhibitors demonstrated that anticryptococcal cytotoxicity of non-primed NA cells was primarily perforin mediated. Anticryptococcal activity of the NA cells obtained from immunized mice after in vitro incubation with cryptococcal antigens was both perforin and non-perforin mediated. Taken together these data demonstrate that in mice a nonperforin-mediated pathway of anticryptococcal cytotoxicity can be induced by immunization. Further research is needed to examine their potential role for human vaccines strategies and/or therapies.

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.

Differences in the Antigens of Helicobacter pylori Strains Influence on the Innate Immune Response in the In Vitro Experiments

The immune response to Helicobacter pylori importantly determines the pathogenesis of infection as well as the success of antibiotic eradication of the bacteria. Strains of H. pylori were gathered from 14 patients who failed to eradicate H. pylori infection with antibiotics—therapy resistant strains (TRS)—or from patients who were able to eradicate H. pylori infection—therapy susceptible strains (TSS). The THP-1 cells were stimulated with H. pylori antigens. Cathepsin X expression on THP-1 cells and concentration of cytokines in the supernatant of THP-1 cells were measured with a flow cytometer. TSS H. pylori antigens increased the proportion of cathepsin X positive cells compared to TRS H. pylori antigens. TSS H. pylori antigens induced higher secretion of IL-12 and IL-6 compared to TRS H. pylori antigens (P < 0.001; 0.02). Polymyxin B, a lipid A inhibitor, lowered the secretion of IL-12 and IL-6 in TRS and TSS. We demonstrated a H. pylori strain-dependent cathepsin X and cytokine expression that can be associated with H. pylori resistance to eradication due to lack of effective immune response. Differences in lipid A of H. pylori might have an influence on the insufficient immune response, especially on phagocytosis.

Helicobacter pylori Susceptible/Resistant to Antibiotic Eradication Therapy Differ in the Maturation and Activation of Dendritic Cells

The natural course of Helicobacter pylori infection, as well as the success of antibiotic eradication is determined by the immune response to bacteria. The aim of the study is to investigate how different Helicobacter pylori isolates influence the dendritic cells maturation and antigen‐presenting function in order to elucidate the differences between Helicobacter pylori strains, isolated from the patients with successful antibiotic eradication therapy or repeated eradication failure.