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.

Characterization of a novel DNA polymerase activity assay enabling sensitive, quantitative and universal detection of viable microbes.

During the past 50 years, in vitro measurement of DNA polymerase activity has become an essential molecular biology tool. Traditional methods used to measure DNA polymerase activity in vitro are undesirable due to the usage of radionucleotides. Fluorescence-based DNA polymerase assays have been developed; however, they also suffer from various limitations. Herein we present a rapid, highly sensitive and quantitative assay capable of measuring DNA polymerase extension activity from purified enzymes or directly from microbial lysates. When tested with purified DNA polymerase, the assay detected as little as 2 × 10(-11)U of enzyme (∼ 50 molecules), while demonstrating excellent linearity (R(2)=0.992). The assay was also able to detect endogenous DNA polymerase extension activity down to less than 10 colony forming units (cfu) of input Gram-positive or Gram-negative bacteria when coupled to bead mill lysis while maintaining an R(2)=0.999. Furthermore, preliminary evidence presented here suggests that DNA polymerase extension activity is an indicator of microbial viability, as demonstrated by the reproducibly strong concordance between assay signal and bacterial colony formation. Together, the innovative methodology described here represents a significant advancement toward sensitive detection of potentially any microorganism containing active DNA polymerase within a given sample matrix.

Feasibility study demonstrating that enzymatic template generation and amplification can be employed as a novel method for molecular antimicrobial susceptibility testing.

BACKGROUND: Antimicrobial Susceptibility Testing (AST) is a methodology in which the sensitivity of a microorganism is determined via its inability to proliferate in the presence of an antimicrobial agent. Results are reported as minimum inhibitory concentrations (MICs). The present study demonstrates that measurement of DNA polymerase activity via Enzymatic Template Generation and Amplification (ETGA) can be used as a novel means of determining the MIC of a microbe to an antibiotic agent much sooner than the current standardized method. METHODS: Time course analysis of ETGA is presented from bacterial cultures containing antibiotic agents and compared to the end-point results of standard macrobroth method AST. RESULTS: MIC determinations from ETGA results at 4, 6, and 22 hours are compared to the MICs from the standard method and the results are shown to be in agreement. Additionally, reliable AST analysis using ETGA can be performed on bacteria harvested directly from spiked blood cultures. CONCLUSIONS: AST analysis with ETGA is shown to be equivalent to AST analysis using gene-specific qPCR assays against the measured microbe. Future development of this novel method for performing AST in a clinical setting is discussed.

Systematic determination of ion score cutoffs based on calculated false positive rates: application for identifying ubiquitinated proteins by tandem mass spectrometry.

We report a simple approach for determining ion score cutoffs that permit the confident identification of ubiquitinated proteins by tandem mass spectrometry (MS/MS). Initial experiments involving the analysis of gel bands containing multi-Ubiquitin chains with quadrupole time-of-flight and quadrupole ion trap mass spectrometers revealed that standard ion score cutoffs used for database searching were not sufficiently stringent. We also found that false positive and false negative rates (FPR and FNR) varied significantly depending on the cutoff scores used and that appropriate cutoffs could only be determined following a systematic evaluation of false positive rates. When standard cutoff scores were used for the analysis of complex mixtures of ubiquitinated proteins, unacceptably high FPR were observed. Finally, we found that FPR for ubiquitinated proteins are affected by the size of the protein database that is searched. These observations may be applicable for the study of other post-translational modifications.

The c-myc gene is a direct target of mammalian SWI/SNF-related complexes during differentiation-associated cell cycle arrest.

The activity of mammalian SWI/SNF-related chromatin remodeling complexes is crucial for differentiation, development, and tumor suppression. Cell cycle-regulating activities dependent on the complexes include induction of the p21(WAF1/CIP1) kinase inhibitor and repression of E2F-responsive promoters. These responses are linked through effects on pRb phosphorylation, but the direct role of the SWI/SNF-related complexes in their regulation is not fully understood. Results presented here reveal that the complexes are required for regulation of a distinct pathway of proliferation control involving repression of c-myc expression in differentiating cells. This involves direct promoter targeting of the c-myc gene by the complexes. Induction of p21(WAF1/CIP1) is specifically dependent on prior repression of c-myc, but repression of E2F-responsive genes is dissociable from the regulation of c-myc and p21(WAF1/CIP1).

Global gene expression profiling of circulating tumor cells.

Metastases from primary tumors are responsible for most cancer deaths. It has been shown that circulating tumor cells (CTCs) can be detected in the peripheral blood of patients with a variety of metastatic cancers and that the presence of these cells is associated with poor clinical outcomes. Characterization of CTCs in metastatic cancer patients could provide additional information to augment management of the disease. Here, we describe a novel approach for the identification of molecular markers to detect and characterize CTCs in peripheral blood. Using an integrated platform to immunomagnetically isolate and immunofluorescently detect CTCs, we obtained blood containing > or = 100 CTCs from one metastatic colorectal, one metastatic prostate, and one metastatic breast cancer patient. Using the RNA extracted from the CTC-enriched portion of the sample and comparing it with the RNA extracted from the corresponding CTC-depleted portion, for the first time, global gene expression profiles from CTCs were generated and a list of cancer-specific, CTC-specific genes was obtained. Subsequently, samples immunomagnetically enriched for CTCs from 74 metastatic cancer patients and 50 normal donors were used to confirm by quantitative real-time reverse transcription-PCR CTC-specific expression of selected genes and to show that gene expression profiles for CTCs may be used to distinguish normal donors from advanced cancer patients as well as to differentiate among the three different metastatic cancers. Genes such as AGR2, S100A14, S100A16, FABP1, and others were found useful for detection of CTCs in peripheral blood of advanced cancer 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.

Feasibility of a novel approach for rapid detection of simulated bloodstream infections via enzymatic template generation and amplification (ETGA)-mediated measurement of microbial DNA Polymerase activity.

Bloodstream infections (BSIs) caused by bacteria and fungi are associated with significant morbidity and mortality. Currently, blood culture is the gold standard for confirming a suspected BSI, but has the drawback of lengthy time-to-detection (TTD) required for indicating the presence of microbes. Detection of conserved microbial nucleic acid sequences within blood culture samples via PCR has been demonstrated to offer potential for reducing the TTD of BSI; however, these approaches have various other limitations. We report a novel approach toward rapid detection of microbes from simulated BSI via differential hematopoietic cell lysis followed by enzymatic template generation and amplification (ETGA)-mediated measurement of microbial DNA polymerase extension activity. The differential cell lysis procedure effectively reduced the level of detectable DNA polymerase extension activity associated with human-derived hematopoietic cells present in blood culture samples taken from healthy donors. After treatment with the differential cell lysis procedure, the ETGA assay detected a panel of clinically prevalent bacteria and Candida albicans from spiked blood culture samples. The ETGA blood culture method also reduced by threefold the TTD required for simulated BSI, compared with a continuous-monitoring blood culture instrument. In summary, these findings demonstrate the feasibility of an innovative approach toward a rapid, sensitive, and universal screen for microbes within blood culture samples. Potential for clinical application and automation are also addressed.

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.

Physiological stress induces the metastasis marker AGR2 in breast cancer cells.

As an approach to understanding the factors that activate expression of tumor progression genes, the role of physiological stress in the activation of a panel of tumor cell markers was investigated. These studies identify the developmental gene product, anterior gradient 2 (AGR2) as a cancer cell marker specifically up-regulated in response to depletion of serum and oxygen. AGR2 has been identified as a tumor marker in primary and secondary cancer lesions, and as a marker for detection of circulating tumor cells (CTCs). Elevated levels of AGR2 are known to increase the metastatic potential of cancer cells, but conditions leading to increased expression of AGR2 are not well understood. The present results identify novel physiological parameters likely to contribute to AGR2 induction in situ.

The proteomic reactor facilitates the analysis of affinity-purified proteins by mass spectrometry: application for identifying ubiquitinated proteins in human cells.

Mass spectrometry (MS) coupled to affinity purification is a powerful approach for identifying protein-protein interactions and for mapping post-translational modifications. Prior to MS analysis, affinity-purified proteins are typically separated by gel electrophoresis, visualized with a protein stain, excised, and subjected to in-gel digestion. An inherent limitation of this series of steps is the loss of protein sample that occurs during gel processing. Although methods employing in-solution digestion have been reported, they generally suffer from poor reaction kinetics. In the present study, we demonstrate an application of a microfluidic processing device, termed the Proteomic Reactor, for enzymatic digestion of affinity-purified proteins for liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. Use of the Proteomic Reactor enabled the identification of numerous ubiquitinated proteins in a human cell line expressing reduced amounts of the ubiquitin-dependent chaperone, valosin-containing protein (VCP). The Proteomic Reactor is a novel technology that facilitates the analysis of affinity-purified proteins and has the potential to aid future biological studies.

Multigene reverse transcription-PCR profiling of circulating tumor cells in hormone-refractory prostate cancer.

BACKGROUND: Circulating tumor cells (CTCs) represent a surrogate source of tissue and conceptually represent a "real-time" biopsy. We previously reported that the number of CTCs mirrors disease progression in hormone-refractory prostate cancer (HRPC). To improve characterization of CTCs we further investigated whether in vitro transcription-based multigene reverse transcription-PCR expression profiles could be obtained from CTCs in HRPC. METHODS: We evaluated the expression of 37 genes with potential utility for epithelial cell characterization from antisense RNA libraries constructed from immunomagnetically enriched CTCs from 7.5-mL blood samples from healthy donors and patients with HRPC. RESULTS: In the control group 13 of 37 genes were not expressed. The most notable of the genes expressed in CTCs of 23 blood specimens drawn from 9 patients with metastatic prostate cancer were prostate-specific antigen (20 of 23; 87%), prostate-specific membrane antigen (17 of 23; 74%), androgen receptor (16 of 23; 70%), human glandular kallikrein 2 (7 of 23; 30%), epidermal growth factor receptor (4 of 23; 17%), and prostate-specific gene with homology to G protein receptor (2 of 23; 9%). The number of CTCs in these samples ranged from 4 to 283 in 7.5 mL of blood (mean, 87; median, 89). Expression of some of the genes was low in the control samples and higher in the patient samples. In all 23 samples, cytokeratin 19, epithelial cell adhesion molecule, or mucin 1 was expressed. Because of background expression in the controls, expression of 13 of the 37 genes, including HER-2, p53, and BCL-2, could not be measured in CTCs. CONCLUSION: Antisense RNA libraries can be constructed from CTCs and gene expression profiles of CTCs obtained from patients with HRPC. This could enhance the characterization of HRPC and facilitate the development of more effective therapies.