Pathogenic microorganisms cause a large burden of disease globally. Rapid and accurate detection of these pathogens is essential for treatment. The goal of our project is to evaluate the analytical and clinical sensitivity of Reverse Transcription quantitative Polymerase Chain Reaction (RT-qPCR) for detecting such microorganisms compared to conventional qPCR. qPCR targets and amplifies specific DNA sequences and is widely used in laboratories to detect these organisms. However, when there is only a single organism in a diagnostic sample, the DNA amplified from such a limited number of template copies may not be detectable by qPCR. RT-qPCR overcomes this by targeting ribosomal RNA (rRNA), which is in greater abundance compared to the rRNA-encoding DNA. rRNA-targeted assays can provide more sensitive detection because the target is more abundant. In addition, rRNA is relatively resistant to degradation, unlike more labile mRNA targets. To evaluate RT-qPCR, we selected important human pathogens, designed reagents for DNA dye based (LCGreen) or probe-based amplification/detection assays, and isolated total nucleic acids from samples by using a silica-based extraction instrument (bioMérieux). Nucleic acids from extracted samples were tested on a real-time PCR machine using with and without RT (+RT/-RT). We evaluated the relative increase in rRNA compared to coding DNA by comparing the +RT and -RT standard curves’ cycle thresholds (Ct). In all experiments, RT-qPCR detected the target organism earlier than qPCR although the relative enrichment of rRNA varied by organism. For instance, in Toxoplasma gondii, 18S rRNA was up to 500 times more abundant than the coding DNA. In contrast, in Cryptosporidium parvum rRNA was only modestly enriched at 50-fold increase compared to coding DNA. Our work will provide a survey of relative enrichment of rRNA and will evaluate whether this target should be widely pursued as an improved approach for pathogen detection.

Heparin is given to patients on Extracorporeal Membrane Oxygenation (ECMO) to prevent clot formation in the ECMO circuit. The anti-Xa assay is a chromogenic assay used to monitor heparin therapy by measuring the absorbance of a colored product generated from the reaction between reagent Factor Xa (FXa) and a chromogenic substrate to represent the heparin activity in plasma. Hemolysis and hyperbilirubinemia are mechanical complications of ECMO that interfere with heparin monitoring by anti-Xa assay by reporting falsely lower heparin activity measurements. There are three possible mechanisms for the apparent decrease in heparin activity due to hemoglobin and bilirubin: increased FXa activity, heparin neutralization, or spectrophotometer interference. The objective of this study is to investigate the mechanisms by which hemoglobin and bilirubin interfere with heparin monitoring by anti-Xa assay. The slope of absorbance change, heparin activity, and absorbance values reported between the measurement window were determined on the Stago STA Compact® analyzer for plasma samples spiked with heparin and varying dilutions of hemoglobin or bilirubin. The addition of hemoglobin and bilirubin to the assay resulted in an apparent decrease in the measured heparin activity that was proportional to the amount of hemoglobin and bilirubin added to the sample at 0, 0.3 and 0.6 U/mL heparin levels. The rate of the reaction increased proportionally to the amount of hemoglobin or bilirubin added and the relative increase in apparent reaction rate was independent of the heparin concentration in the reaction. Changes in absorbance values were linear between the measurement window for samples with a range of increasing initial absorbances. We concluded that the apparent decrease in heparin activity was a result of increased FXa activity caused by hemolysis and bilirubin, not heparin neutralization or spectrophotometer interference. This study improves the understanding of interferences in heparin monitoring by the anti-Xa assay in clinical laboratories.

In this method comparison, research was performed to determine if the polyspecific anti-human globulin (AHG) reagent from Immucor® was significantly better than the currently used Bio-Rad. Direct antiglobulin test (DAT) experiments were performed on 11 subject samples with strengths of strong positive, weakly positive, and negative. After reviewing the results obtained, it was observed that the currently used Bio-Rad polyspecific AHG reagent produced stronger results with regards to samples that were weakly positive when compared to Immucor®. Results that were 1+ or higher indicate that the two reagents express relatively consistent results, with Bio-Rad presenting stronger agglutination when compared with Immucor®. A comparison between using 1 drop vs 2 drops of Immucor®(Norcross, GA) polyspecific AHG was also performed to determine if there was a significant difference in results, as the package insert from Immucor® mentioned that 1 or 2 drops may be used, with 2 drops having the risk of diminishing results. The results after testing suggest that there is most likely no significant difference between the numbers of drops used.

Retinol Binding Protein (RBP) is a protein used by the body to transport Vitamin A and is often used to monitor short-term nutritional changes. Traditionally, RBP has been measured via a nephelometric immunoassay with a high sample requirement. In an attempt to reduce turnaround time and to reduce the volume of sample needed for testing, an in-house assay using liquid chromatography tandem-mass spectrometry (LC-MS/MS) technology is being developed.The method being developed is based on the Vitamin D Binding Protein assay which sets a precedent for LC-MS/MS methods targeted toward high-abundance globulins. This method consists of denaturation of RBP using heat and denaturation agents, then digested by trypsin into peptides which are detected by LC-MS/MS. Currently there are three peptides with multiple transitions being monitored for each (FSGTWYAMAK, YWGVASFLQK, and LIVHNGYC[+57]DGR).The assay is being developed and validated according to the document CLSI C-62A and the assay is currently in its pre-validation phase. Experiments done include standard curve verification and comparison to development data, simple repeatability 5x5, carryover and various troubleshooting experiments.Data analysis has exposed high control and internal standard variability in the assay which are being resolved, but progress looks promising.The RBP assay is still far from completion and further testing and troubleshooting needs to be done to reduce variability before it can enter the validation phase. Once optimized, the LC-MS/MS assay, while less automated than nephelometric platforms, should allow UW Medical Center to have better control over an in-house assay in terms of turnaround time, while also using much smaller volumes of sample. LC-MS/MS also has improved sensitivity when compared to the nephelometric assay. This increased performance may lead to assay utility with other conditions such as diabetes, liver disease and kidney disease.
 

Research has shown that chronic inflammation can facilitate the progression of certain diseases, including cancer and diabetes. One way to prevent inflammatory-linked diseases is to reduce the occurrences of inflammation. Since much of society uses nature-derived approaches of healing and disease prevention, we focus on how natural products (i.e. compounds from nature) alter cellular inflammatory status. Fungi and fungal extracts provide a rich source of novel natural products. Fungi have been shown to have beneficial health properties, exemplified by the antibiotic penicillin, derived from the fungus Penicillium chrysogenum. By studying many different fungal extracts and chemically separated fractions of fungal extracts, we hope to find novel anti-inflammatory compounds. In this study, novel fungal extracts and fractions were tested for anti-inflammatory activity against the cellular transcription factor, nuclear factor kappa B (NF-kB), a major mediator of cellular inflammation. Prior single-dose screening resulted in extracts and fractions that were shown to inhibit NF-kB activity. From these, eight were tested in dose-response assays that measured NF-kB activity and cytotoxicity. Human hepatoma Huh7.5.1 cells were transfected with a luciferase reporter gene under control of the NF-kB promoter. Twenty-four hours later, transfected cells were incubated with either extracts or fractions for 30 minutes prior to activation of inflammation by tumor necrosis factor-alpha, (TNF-α). NF-kB activity and cellular ATP were measured by luminescence 3.5 hours later. The ultimate goal is to identify extracts, fractions, and pure compounds that inhibit NF-kB without causing cytotoxicity. Such compounds will be advanced for further study and possible application in chronic inflammatory disease states.

We aim to find diversity in the HHV-6B genome via sequencing of the U90 gene. We have previously sequenced 120 whole genomes of HHV-6B from New York, Japan, and Fred Hutch chromosomally integrated (Ci-HHV6B) strains and found the U90 gene to encode the most diversity. Our hypothesis was that candidate U90 sequencing of clinical samples for which whole genome sequence could not be accomplished due to low viral concentrations would give the most amount of phylogenetic information of candidate loci available. Eleven excess serum samples from Seattle Cancer Care Alliance and Fred Hutch for which genome sequencing failed were amplified for the U90 locus using nested PCR, pooled, and Nextera/Illumina sequenced. Of those nine samples three had fewer than five changes across the 3kb locus, and one sample accounted for 95% of the diversity in the gene among these eleven samples. We found sequence clusters outside of sequences published to genbank, as well as clusters outside of the 120 whole genome sequences. Nine of the eleven samples clustered with the New York isolates. One shows significant genetic distance from the Japanese group, while another isolate shows similarity to the Z29 reference strain. This demonstrates an unexplored diversity in HHV6B genomes with much more diversity to be expected. Further U90 sequencing studies would expose more diversity in HHV-6B in a low cost, but effective, manner. 

Traditional gastrointestinal pathogen detection includes many bacterial, viral, and parasite tests that require clinicians to order many different tests that have various sensitivity and turn-around-time (TAT). As a result, pathogen detection can be delayed or missed, if the correct tests are not ordered. This ongoing study is evaluating the time-to-diagnosis of gastrointestinal infections using the FilmArray Gastrointestinal (GI) Panel test (BioFire Diagnostics, Salt Lake City, UT, USA) compared to conventional methods. The Filmarray GI test is a multiplex PCR test with targets for 22 gastrointestinal bacteria, viruses, and parasites. The study is also comparing the FilmArray GI test to conventional stool cultures, to evaluate the impact of the FilmArray GI test on clinical decision-making. In this interim analysis, the sample population was composed of 167 outpatient or recently admitted (<3 days) patients who had stool specimens submitted for the FilmArray GI test. The time from collection to result for the FilmArray GI test was compared to stool culture. Results from our interim analysis found that the mean time from collection to a FilmArray GI result was 9.8 hours, while the mean time from collection to first actionable stool culture result (if positive) was 62.9. The time from collection to final stool culture result was even longer (77.2 hours). We also found that the FilmArray GI test identified many more pathogens than stool culture: 38% of the samples were positive by FilmArray GI, while only 5% were positive by stool culture. Thus far, the FilmArray test has demonstrated more rapid results and higher sensitivity than stool culture. Ongoing work is evaluating the clinical impact of the FilmArray GI test, but this interim analysis shows that it is more rapid and sensitive than traditional stool culture. As such, it appears to be a promising test for promptly detecting gastrointestinal pathogens.

Ceftazidime as a cephalosporin antibiotic has become much less effective against Gram-negative bacterial infections because of the appearance of multi-drug resistant, β-lactamase-producing strains. One of the newly FDA-approved drug inhibitor combinations, ceftazidime-avibactam, is a promising solution to rapidly decreasing susceptibilities to ceftazidime-resistant bacteria. E-test is a well-known manual antimicrobial susceptibility testing method widely used in clinical laboratories as an alternative for the gold standard. This study aims to validate ceftazidime E-tests with a selected panel of non-fastidious Gram negatives consisting of Escherichia coli, Stenotrophomonas maltophilia , Acinetobacter baumannii calcoaceticus complex, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella typhi, Citrobacter spp., Enterobacter cloacae, Klebsiella oxytoca and Proteus mirabillis, against gold standards including TREK Sensitre® micro-broth dilution and agar dilution while performing ceftazidime-avibactam E-tests to generate more data for future investigations on the novel antibacterial drug and its resistance profiles at University of Washington Medical Center. Ceftazidime and ceftazidime-avibactam E-tests were done on all selected patient isolates, and agar dilution was performed only on Stenotrophomonas maltophilia as the reference method instead of TREK Sensitre®. Both the accuracy and precision study results support ceftazidime E-test as a valid susceptibility test, and ceftazidime-avibactam was found to restore sensitivity in most of the resistant strains that were tested. We conclude that ceftazidime E-test is verified and the comparison of ceftazidime and ceftazidime-avibactam yields consistent outcomes as previous research.

Matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a method of organism identification that is currently used widely in clinical microbiology. Compared to traditional biochemical testing, it offers a faster turnaround time and provides a more cost-efficient way to identify clinical isolates. Currently at the University of Washington clinical microbiology laboratory, if a yeast grows from any media other than inhibitory mold agar (IMA) media, it is subcultured onto IMA and incubated to test by MALDI-TOF the following day. This validation study will allow medical laboratory scientists (MLS) to bypass the subculturing step, which will decrease the turnaround time by approximately 24 hours. We evaluated five different media types and incubation conditions, with a sample size of 51 organisms. Through this validation study, it was concluded that the various media and incubation conditions had little to no effect on yeast identification by MALDI-TOF. Because the different conditions tested had no effect on the MALDI-TOF MS score values, this successful validation will allow for shorter turn-around time for yeast identification, which in turn will allow for faster and more accurate patient care.

Impairments in the DNA mismatch repair system give rise to mutations in microsatellite loci, which are short, repetitive stretches of DNA. Also known as microsatellite instability (MSI), these mutations manifest as new alleles of variable length. MSI is an important marker of cancer outcomes, and moreover, can directly drive oncogenesis. Despite their clinical relevance, microsatellite loci are challenging to reliably sequence using existing high throughput sequencing methods. Here, we have performed exploratory analyses using high throughput next-generation sequencing (NGS) in conjunction with single molecule molecular inversion probes (smMIPS) to enable typing of large numbers of cancer-relevant microsatellite loci. smMIPs integrate multiplex targeted sequencing with molecular-tag-based error correction, resulting in a cost-effective method for large-scale, accurate genotyping of microsatellite alleles. smMIPs are 80bp oligonucleotides, each containing a random, unique molecular tag, that hybridize to flanking regions around microsatellite loci. The targeted sequence is copied into the probe through the action of DNA polymerase and ligase. Capture reactions are then exonuclease treated, PCR amplified, and sequenced using the Illumina NGS platform. Error correction is achieved through consensus mapping of probes associated with a unique molecular tag, eliminating analytic errors otherwise inherent to the process. We have developed a panel of probes for detecting microsatellite instability events in 150 cancer-linked loci. We have compared our data to conventional NGS methods and have demonstrated higher accuracy genotyping compared to existing methods. The combination of high-throughput sequencing and robust error correction suggests that further development of this technology will be well suited for clinical laboratories searching for streamlined and accurate approaches to MSI diagnostics. The intent of this project is to develop a smMIP panel that detects microsatellite instability in heterogeneous tumor samples and that offers other advantages over existing testing methods.