Measurement of a Novel Biomarker, Secretory Phospholipase A2 Group IIA as a Marker of Sepsis: A Pilot Study.

INTRODUCTION: Early identification of sepsis is critical as early treatment improves outcomes. We sought to identify threshold values of secretory phospholipase A2 (sPLA2)-IIA that predict sepsis and bacterial infection compared to nonseptic controls in an emergency department (ED) population. MATERIALS AND METHODS: This is a prospective cohort of consenting adult patients who met two or more systemic inflammatory response syndrome (SIRS) criteria with clinical diagnosis of infectious source likely (septic patients). Controls were nonseptic consenting adults undergoing blood draw for other ED indications. Both groups had blood drawn, blind-coded, and sent to an outside laboratory for quantitative analysis of sPLA2-IIA levels. The study investigators reviewed patients' inpatient medical record for laboratory, imaging, and microbiology results, as well as clinical course. RESULTS: sPLA2-IIA levels were significantly lower in control patients as compared to septic patients (median = 0 ng/ml [interquartile range (IQR): 0-6.5] versus median = 123 ng/ml [IQR 44-507.75]; P < 0.0001). SPLA2-IIA levels were higher in patients with confirmed source (n = 28 patients, median = 186 ng/ml, 95% confidence interval = 115.1-516.8) as compared to those with no source identified or a viral source (n = 17, median = 68 ng/ml, 95% confidence interval = 38.1-122.7; P = 0.04). Using a cutoff value of 25 ng/ml, sPLA2-IIA had a sensitivity of 86.7% (confidence interval 72.5-94.5) and a specificity of 91.1% (confidence interval 77.9-97.1) in detecting sepsis. CONCLUSIONS: sPLA2-IIA shows potential as a biomarker distinguishing sepsis from other disease entities. Further study is warranted to identify predictive value of trends in sPLA-IIA during disease course in septic patients.

Distinguishing septic from normal donors by detection of sPLA2-IIA from human plasma using a microsieve-based immunoassay.

Bloodstream infections that progress to septic shock are responsible for hundreds of thousands of deaths each year, and are associated with significant healthcare costs. Recent studies have shown that a member of the secreted phospholipase protein family, termed sPLA2-IIA, may play a role during the innate immune response to bacterial infections, and is elevated in the plasma of septic patients. In this report, the feasibility of a simple microsieve-based sPLA2-IIA detection immunoassay was explored. Microsieves containing 5µm pores were covalently coupled with a sPLA2-IIA-specific monoclonal antibody at 0.1, 1.0, and 10µg/mL and then assayed with plasma-based positive and negative controls to determine the optimal coating concentration. Recombinant sPLA2-IIA was then serially diluted to a final concentration of 200, 100, 50, 25, 12.5, and 6.25ng/mL and tested alongside a non-spiked sample to estimate the detection limit of the prototype assay. Recombinant sPLA2-IIA was also spiked into serum, EDTA-plasma, and Lithium-Heparin plasma, in an effort to evaluate assay performance when analyzing these sample matrices. The preliminary limit of detection studies suggests that the microsieve assay is able to distinguish approximately 6-12ng/mL of sPLA2-IIA from a non-spiked sample. When compared to an immunoassay diluent, the microsieve assay also yielded acceptable percent recoveries for each of the three sample matrices spiked with clinically significant levels of sPLA2-IIA. The sPLA2-IIA microsieve assay prototype also clearly distinguished five samples from septic patients from five normal donor samples, and the results were in good agreement with a comparator ELISA test system (R2=0.9347).

Feasibility of a simple microsieve-based immunoassay platform.

The intrinsic properties of silicon microsieves, such as an optically flat surface, high overall porosity, and low flow resistance have led to an increasing number of biotechnology applications. In this report, the feasibility of creating a microsieve-based immunoassay platform was explored. Microsieves containing 5µm pores were coupled with poly-acrylic acid chains, and then mounted into a plastic holder to enable rapid reagent exchanges via a wicking mechanism. The mounted microsieves were coated with infectious disease-related antigens at [2.5 and 25µg/mL], [20 and 50µg/mL], and [20 and 100µg/mL] to facilitate detection of serum-derived human antibodies against Rubella (3-day measles), B. burgdorferi (Lyme disease), or T. pallidum (syphilis), respectively. The prototype microsieve-based immunoassay platform was able to distinguish positive control sera containing antibodies against Rubella, T. pallidum, and B. burgdorferi from negative control sera with similar qualitative results as FDA-approved ELISA tests. Testing of a WHO IgG syphilitic standard at 0.3, 0.15, 0.075, 0.0375, and 0.01875IU/mL demonstrated that the T. pallidum microsieve assay is able to distinguish disease-specific IgG signal from background signal at similar, and possibly lower, levels than the corresponding ELISA. The T. pallidum microsieve assay prototype also differentiated positive clinical serum samples from negative donor samples, and the results were in good agreement with ELISA (R(2)=0.9046). These feasibility studies demonstrate the potential for utilizing microsieves, along with a reagent wicking device, as a simple diagnostic immunoassay platform.

Retrospective analysis of clinical data associated with patients enrolled in a molecular diagnostic feasibility study highlights the potential utility for rapid detection of bloodstream infection.

BACKGROUND: Measurement of pathogen DNA polymerase activity by enzymatic template generation and amplification (ETGA) has shown promise in detecting pathogens in bloodstream infection (BSI). We perform an in-depth analysis of patients with clinical BSI enrolled in ETGA feasibility experiments. METHODS: In addition to hospital blood cultures, 1 study aerobic culture bottle was drawn from patients with suspected BSI. The study bottle was split into 2 bottles and was additionally subjected to ETGA analysis. Enzymatic template generation and amplification sensitivity/specificity for BSI detection was determined against the Centers for Disease Control BSI definition. When split cultures were both positive, time course analysis was performed to determine time to detection. The records of patients with BSI were reviewed for presence of systemic inflammatory response syndrome, antibiotic timing and appropriateness, and organism identification. RESULTS: Of 307 enrollees, 38 met the Centers for Disease Control BSI definition. Seventy-four percent met systemic inflammatory response syndrome criteria on admission. Antibiotic coverage was adequate in 76% of patients. Antibiotics were more often delayed in afebrile patients (odds ratio, 5). Twenty-seven of the split study culture bottles were positive in at least 1 sample, and ETGA detected microbes within all samples (sensitivity/specificity, 70.3%/99.3%). Of these, 22 were culture positive in both split study bottles and underwent ETGA time course analysis. Enzymatic template generation and amplification detected microbes within these 3-fold faster than culture. CONCLUSIONS: Patients with BSI often have diagnostic and treatment delays. Enzymatic template generation and amplification provides clinically meaningful data more rapidly than cultures. Future development should focus on real-time application of assays that detect microbes at the molecular level.

A novel approach for rapid detection of bacterially contaminated platelet concentrates via sensitive measurement of microbial DNA polymerase activity.

BACKGROUND: Transfusion of bacterially contaminated platelet concentrates (PCs) can result in serious health consequences for the affected patient. Before being released from blood banking facilities, PCs are routinely screened for bacterial contamination by culture-based tests. However, culture-based PC screening methods require extended holding and incubation periods and are prone to false-negative results due to sampling error. Screening PCs closer to the time of transfusion using rapid point-of-issue tests represents an alternative approach; however, FDA-approved assays generally suffer from a lack of sensitivity. STUDY DESIGN AND METHODS: Presented herein is the feasibility of a novel approach toward rapid, sensitive, and universal detection of bacterially contaminated PCs via selective measurement of microbial DNA polymerase activity. This approach is achieved using a differential cell lysis procedure in combination with enzymatic template generation and amplification (termed ETGA-PC assay). RESULTS: Serial dilution spiking experiments revealed an approximate sensitivity of 30 to 200 colony-forming units (CFUs)/mL (mean, 85 CFUs/mL) for Staphylococcus epidermidis, Staphylococcus aureus, Escherichia coli, and Klebsiella pneumoniae. An additional 22 clinically relevant strains of bacteria were also detected below 200 CFUs/mL after spiking into PC aliquots. Furthermore, the ETGA-PC assay was able to accurately monitor the presence and growth of seven clinically relevant bacterial species that were spiked into PC units. CONCLUSION: Together, the data presented here demonstrate that the ETGA-PC assay is a feasible approach for rapid and sensitive detection of bacterially contaminated PCs. Experiments, aimed at simplification and/or automation of the assay procedure, are under way.

Measurement of microbial DNA polymerase activity enables detection and growth monitoring of microbes from clinical blood cultures.

Surveillance of bloodstream infections (BSI) is a high priority within the hospital setting. Broth-based blood cultures are the current gold standard for detecting BSI, however they can require lengthy incubation periods prior to detection of positive samples. We set out to demonstrate the feasibility of using enzymatic template generation and amplification (ETGA)-mediated measurement of DNA polymerase activity to detect microbes from clinical blood cultures. In addition to routine-collected hospital blood cultures, one parallel aerobic blood culture was collected and immediately refrigerated until being transported for ETGA analysis. After refrigeration holding and transport, parallel-collected cultures were placed into a BACTEC incubator and ETGA time-course analysis was performed. Of the 308 clinical blood cultures received, 22 were BACTEC positive, and thus were initially selected for ETGA time course analysis. The ETGA assay detected microbial growth in all 22 parallel-positive blood cultures in less time than a BACTEC incubator and also yielded genomic DNA for qPCR-based organism identification. In summary, feasibility of detecting microbes from clinical blood culture samples using the ETGA blood culture assay was demonstrated. Additional studies are being considered towards development of clinically beneficial versions of this methodology.

Multiplex immunoassay for Lyme disease using VlsE1-IgG and pepC10-IgM antibodies: improving test performance through bioinformatics.

The Centers for Disease Control and Prevention currently recommends a 2-tier serologic approach to Lyme disease laboratory diagnosis, comprised of an initial serum enzyme immunoassay (EIA) for antibody to Borrelia burgdorferi followed by supplementary IgG and IgM Western blotting of EIA-positive or -equivocal samples. Western blot accuracy is limited by subjective interpretation of weakly positive bands, false-positive IgM immunoblots, and low sensitivity for detection of early disease. We developed an objective alternative second-tier immunoassay using a multiplex microsphere system that measures VlsE1-IgG and pepC10-IgM antibodies simultaneously in the same sample. Our study population comprised 79 patients with early acute Lyme disease, 82 patients with early-convalescent-phase disease, 47 patients with stage II and III disease, 34 patients post-antibiotic treatment, and 794 controls. A bioinformatic technique called partial receiver-operator characteristic (ROC) regression was used to combine individual antibody levels into a single diagnostic score with a single cutoff; this technique enhances test performance when a high specificity is required (e.g., ≥ 95%). Compared to Western blotting, the multiplex assay was equally specific (95.6%) but 20.7% more sensitive for early-convalescent-phase disease (89.0% versus 68.3%, respectively; 95% confidence interval [95% CI] for difference, 12.1% to 30.9%) and 12.5% more sensitive overall (75.0% versus 62.5%, respectively; 95% CI for difference, 8.1% to 17.1%). As a second-tier test, a multiplex assay for VlsE1-IgG and pepC10-IgM antibodies performed as well as or better than Western blotting for Lyme disease diagnosis. Prospective validation studies appear to be warranted.

Comparison of a multiplex, bead-based fluorescent assay and immunofluorescence methods for the detection of ANA and ANCA autoantibodies in human serum.

Detection of antinuclear (ANA) and antineutrophil cytoplasmic (ANCA) antibodies is extensively used for establishing a diagnosis in patients with clinical features suggestive of autoimmune disorders. The most common methods for the identification of positive patients' sera for ANA or ANCA are indirect immunofluorescence (IIF) and ELISA-based procedures. Considerable effort has been made in developing simpler automated assays for routine laboratory use. Recently a commercially available microsphere-based fluorescent assay has been introduced for the detection of ANA and ANCA. The aim of this study was to compare this technology with routinely used IIF and ELISA procedures, in patients with a suggested autoimmune disorder. A highly significant correlation between ELISA procedures for specific antibodies and the microsphere-based assays were obtained for both ANA and ANCA as well as for extractable nuclear antigens ELISA screening, indicating that multiplex technology could replace individual ELISA tests for the measurement of specific autoantibodies. However, a low sensitivity for identifying IIF-positive cases was obtained for both ANA (58.0%) and ANCA (59.1%), although there was a significant correlation between the assays. In conclusion, our data show that a microsphere-based fluorescent assay may be a valid platform for the simultaneous determination of circulating individual ANA and ANCA autoantibodies. Furthermore, multiplexing technology offers several advantages that will probably make it an attractive tool in the future. Nevertheless, until further studies are conducted that determine the clinical performance of the multiplex technology, the initial screening of patients for autoantibodies with IIF is still considered necessary.