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dysuria, loin pain; loin/suprapubic tenderness. A culture count of 103 microorgansims may be considered significant if obtained from a suprapubic puncture or in the presence of an antibiotic. It should be noted that many critical care patients may not be able to communicate their symptoms and steroids may results in failure to exhibit fever; therefore, culture counts of 105 microorgansims in a urine culture obtained from an indwelling Foley catheter were considered to be urinary tract infections. Other infections of the urinary tract include presence of an abscess by direct observation during a surgical procedure.

Sinusitis was defined by organisms cultured from the sinus cavity with the • presence of radiographic changes. In addition, one or more of the following symptoms with no other recognized cause may be present: fever ( 38◦C), pain or tenderness over the involved sinus, headache, purulent exudate, or nasal obstruction.

A Cardiovascular system infection –VASC (formerly catheter-related • infection) was defined as at least 15 colonies by semi-quatitative culture from an intravascular cannula tip in the presence of fever ( 38◦C), pain, erythema, or heat at involved vascular size regardless of blood culture results.

Additionally, a catheter related infection can be diagnosed if purulent drainage was present at the vascular site. It should be noted that many critical care patients may not be able to communicate their symptoms and steroids may results in failure to exhibit fever.

In addition to infections that are definitive (evidence by positive cultures), it is possible that a patient may have had an infection that is not detected by culture. All patients were followed prospectively. In the absence of positive cultures, a high degree of clinical suspicion of infection which is treated and improves with an antibiotic was recorded. New fever in the ICU can be infectious or non-infectious, thus it is standard practice that any new fever is carefully investigated by the critical care team.54 All potentially non-definitive infections were reviewed with a critical care clinician. Primary analysis will include only definitive infections.

Invasive Devices

The presence of invasive devices was recorded daily through ICU discharge (or up to 28 days for participants with a prolonged ICU stay). Invasive devices include endotracheal tubes, central venous catheters, peripheral intravenous catheters, arterial catheters, chest tubes, nasal feeding tubes, Foley catheters, and other drains or catheters that have been inserted. A total daily invasive devise score as well as a cumulative daily score will be calculated for each patient. Each invasive devise will be given a score of 1.

For example, if a patient has three invasive devices for days 1-5, then only one device for days 6-14, then their daily score will be 3 for the first five days and 1 for subsequent days. On day 14, the cumulative score will be (3*5 + 1*9) = 24. A cumulative score for each day of the study will provide an estimate of their total exposure to invasive devices at any given time during the study period. This is a novel way to examine invasive devices and may be important since infections that occur early (day 5) will have less exposure to invasive devices than those that occur later. The invasive device score to be used in the regression model will be the invasive device score at the time of first HAI. In patients who do not develop any HAIs, the total ICU stay (up to day 28) invasive device score will be used in the regression model.

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Universal precautions were used during specimen collection, processing, and analysis. All specimens were collected at study entry.

Plasma Processing Blood was collected on each participant at baseline in 5 ml ACD tubes. The samples were placed on ice immediately and transported to the laboratory for processing as quickly as possible. A refrigerated centrifuge was used in the hospital Core Lab to spin the samples for 15 minutes at 2000G to separate the plasma from other blood components. Each plasma samples was then aliquoted into 2 equal portions and stored at

-80 degrees until batch analysis. Samples were placed on dry ice when moving across campus prior to storage in the Crowe Building freezer, where all analyses were to be performed.

Cytokine Analysis

The cytokines for IL-6 and IL-10 were measured (pg/ml) in duplicate by Luminex (Luminex Corporation, Austin, TX) using Human Cytokine/ Chemokine Multiplex Kits (Millipore Corporation, Billerica, MA) according to manufactures instructions. In brief, previously frozen plasma samples were prepared by vortexing and certifuging to remove particulates before performing the assay. Beads were mixed according to instructions. A standard curve was prepared with the concentrations of 3.2 pg/ml, 16 pg/ml, 80pg/ml, 400 pg/ml, 2000 pg/ml, and 10,000 pg/ml. The 96 well plate was prepared by adding 200 μl of wash buffer to each well, shaking at room temperature for 10 minutes, and then vacuumed. Next, 25 μl of standard each curve concentration, each control, and assay buffer were added to each well in duplicate as indicated. The plasma samples were then added to the appropriate sample wells (25 μl) and serum matrix (25 μl) added to each standard curve and control well. Next, 25 μl of mixed beads were added to each well. The plate sealed and incubated overnight on the shaker at 4°C. The next day, fluid was removed by vacuum and the plate was washed twice with 200 μl of wash buffer and vacuumed. Next, 25 μl of detection antibodies were added and the plate was placed on the shaker for one hour at room temperature. Next, 25 μl of Streptavidin-Phycoerythrin was added to each well. The plate was sealed and placed on the shaker at room temperature for 30 minutes. The plate was then vacummed and washed twice with 200 μl of wash buffer. The beads were then resuspended in 150 μl of sheath fluid and analyzed on the Luminex machine.

Luminex software provides an automated data interpretation report with standard curves. It provide several report, including a bead count report, median florescent intensity (MIF), results, and average results for duplicates. Luminex software calculates the results by extrapolating MIF values on the standard curve. No reading can be accurately measured if below the lowest point on the standard curve (3.2 pg/ml) or above the highest point on the curve (10,000 pg/ml). When results occur outside of these detectable limits, the lowest or highest detectable value is substituted for statistical purposes. Results for each well were reviewed. According to the manufacturer instruction, data resulting from a cell with a bead count of at least 50 beads provides reliable results. In cases where the bead count was less than 50 or when the duplicate results were greater than 10 percent difference, the assay was repeated to increase the accuracy of the measurement. A total of four were batches run. Batch one (samples 1

- 39), batch two (samples 1 - 39), batch three samples 40 - 78), and batch four (repeat samples from batches two and three).

It was initially anticipated that only two kits would be needed to complete these measurements and two kits were ordered. There were multiple issues with batch one due to wells not vacumming and low bead counts. It was considered a test batch and none of the results were used. Two additional kits were ordered to assure that the same lot number was used, and the prior additional kit was retained to run repeat samples as needed. Batch 4 included duplicate samples from batch two and three that were needed to clarify the results as well as several additional duplicate samples to test reproducibility.

Before the analysis was complete, the bead counts became extremely low and it was assumed that the Luminex needle was clogged. The system was flushed to assure that the needle was working properly. The plate was vacuumed and beads were resuspended with 150 μl of sheath fluid, and the analysis for batch four was repeated. This time the bead counts for the first few wells (which previously had sufficient bead counts) were extremely low and the batch was discontinued. Limited results from batch four were available. Results and bead counts were carefully reviewed. A total of five cytokines were actually tested but only two are included in this dissertation study. It was a puzzling finding that some wells (from batches 2 and 3) which had sufficient bead counts for one cytokine but not another could have different results when retested. Each batch contained its own standard curve and controls. Perhaps this could be explained by the fact that the kit used for batch four had a different lot number and samples 1 - 39 had undergone one freeze thaw cycle. Most samples were measured in duplicate and averaged. Due to the inconsistent results of batch four, results from batch 2 and 3 were used primarily. A small number of values are based on single and not duplicate values.

Reliability and Validity of Cytokine Measures

The literature describes a high degree of variability among earlier generation cytokine results obtained from different manufacturers. Studies from the early 1990’s found great variably in cytokine measurements depending on the type of fluid being analyzed and the assay used.113 Fahey et al. compared laboratories testing for cytokines and found both intra and inter laboratory variability. Several problems were identified before uniform results were obtained.114 The World Health Organization established cytokine standards to facilitate development of cytokine kits in research, and resulted in a dramatic reduction of the variation.115 It is recommended that when measuring cytokines, the same manufacturer’s kit, methods, and lot number are used to enhance the internal validity of the study. Millipore reports precision percentage for inter-assay is 3.7 - 17.2 and intra-assay is 4.6 - 13.8.

Buccal Swab Collection for DNA

The Catch-All™ Sample Collection Swabs from EPICENTRE Biotechnologies were used to collect buccal/cheek swabs. Specimens were collected by swabbing the inner aspect of the cheek and retaining the sample until ready for DNA extraction. All specimens were collected without the presence of tea or coffee for at least one hour.

Yield is directly correlated with the starting amount of buccal cells; therefore, swabs were collected in duplicate due to the concern of low yield among patients receiving mechanical ventilation in which access to the mouth is obscured by a large bite block.

Specimens were allowed to dry at room temperature, returned to the collection sleeve, temporarily stored at room temperature, and then transferring to a -20 degree freezer in the Crowe building. The protocol allows for storage at room temperature for up to one week. Several specimens were stored in a locked cabinet for 24 to 48 hours prior to freezing.

DNA Extraction

The BuccalAmp™ Rapid DNA Extraction Kit was used to extract DNA according to the manufactures instructions and to prepare DNA for PCR amplification assays. In brief, frozen buccal swabs were allowed to thaw at room temperature. Tubes containing Quick DNA Extraction Solution (stored at -20 degrees) was allowed to thaw at room temperature. One of these tubes was then labeled for each patient. The tip of each buccal swab was gently rotated 10 times in a tube containing Quick DNA Extraction Solution. The swab was carefully removed while rotating and pressing on the side of the tube to prevent solution loss. The top was secured tightly on each tube, and then the tubes were vortexed for 10 seconds, incubated at 65°C for 1 minute, vortexed for 15 seconds, incubated at 98°C for 2 minutes, and then vortexed for 15 seconds. This process was done in batches as samples accumulated. A random sample (n = 6) was tested by spectrophotometry to assure the presence of DNA by examining 260/280 ratios, and then samples were stored at -70 degrees until ready for genetic testing. In the random sample 5/6 samples contained DNA. All samples were taken to the MRC to test the DNA quantity by NanoDrop but after checking several samples it was discovered that the proprietary reagents in the rapid extraction kits interfered with these results. According to manufactures instructions, SNP analysis can be performed without quantization or purification using this method.

DNA Analysis

SNP analysis was performed by real-time polymerase chain reaction (PCR) using the LightCycler® 480 Instrument, located in UTHSC’s Molecular Resource Center (MRC). TaqMan® SNP Genotyping Assays, Human, SM (40X) for rs1800795 and rs1800896 from Applied Biosystems (Life Technologies, United States, 2006) contained florescent reporter tags (VIC and FAM) to determine alleles 1 and 2, respectively. Table 3-2 includes SNP primer details. A context sequence is given for proprietary primers.

Table 3-2. SNP Primer and Reporter Details for Genotyping.

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* Roche LightCycler®480 Software reports describe alleles as Allele X, Allele Y, or Both Alleles. Allele X binds to fluorescent probes FAM at 483-533 nm, Allele Y binds to fluorescent probes VIC/HEX/Yellow555 at 523-568 nm.

Roche LightCycler® 480 Probe Master Mix and PCR plates were obtained from the MRC. A Master Mix was specifically made for each analysis which contained the Genotype Assay, Probe Master Mix, and PCR-grade water. The Genoype Assay was adjusted to a 20x concentration in the Master Mix. The total volume of each Master Mix depended on the number of reactions. The final reactions volume was 10 μl and contained 1 μl of unpurified DNA, 0.25 μl of the Genotype Assay, 5.0 μl TaqMan Master Mix, and 3.75 μl PCR-grade water. Real-time PCR was performed according to manufacturer’s instructions. Standard PCR methods were used. A negative control using PCR-grade water rather than DNA was included in each experiment. PCR included one preincubation period (1 cycle at 95°C), incubation (45 cycles at 95°C, 62°, and 72°C), and cooling period (1 cycle at 40°C). The primer target temperature is typically set approximately 5°C below the primer melting temperature (Tm); however, the Applied Biosystem’s TaqMan® SNP Genotyping Assays are all optimized for 60°C. If additional optimization is required, the manufacturer recommends either shortening cycle time to 40 or increasing the temperature to 62°C.

Statistical Analysis

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