Design
A secondary analysis of the PREVAIL study, a phase 2 randomized, multi-centre, double-blinded placebo controlled trial conducted in five Canadian tertiary ICUs studying the effect of lactoferrin on the acquisition of NIs [32]. The protocol for this study has been published and the trial was registered at www.clinicaltrials.gov on 18 November 2013 (registration number NCT01996579) [33].
Patients
Adult patients (≥ 18 years old) receiving invasive mechanical ventilation on ICU admission and who were expected to receive mechanical ventilation for > 72 hours were included in the original trial. Patients who met the following criteria were excluded:
- who were expected to be in the ICU for < 72 hours,
- immunocompromised patients including those post-organ transplant, patients with Acquired Immunodeficiency Syndrome (AIDS), neutropenia, use of glucocorticoids (> 20 mg/day of prednisone equivalent for more than 6 months), use of immunosuppressant medication (e.g. patients with rheumatological conditions on methotrexate, etc.)
- patients with end stage liver disease or fulminant liver failure
- pregnant or lactating patients
- patients with a life expectancy of less than six months due to pre-existing conditions
- enrollment in other interventional trials.
All patients were followed for the duration of their ICU or until day 28 for the acquisition of NIs. During the study, if there was a prescription of a new antibiotic or the patient was investigated for infection with the collection of microbial cultures, a suspicion of infection event was triggered; the attending physician was then asked to assess the probability of infection; definite, probable and possible which was then centrally adjudicated to ensure consistency [33]. The definitions used for each category of NI are outlined further in the supplemental digital content (Supplementary Digital Content 1: Definitions of nosocomial infections). These suspicions were then adjudicated by an assessor blinded to treatment allocation for the presence of infection. Discrepancies between the attending physician and adjudicator were resolved by consensus.
As part of the original protocol, laboratory investigations included measurement of the following cytokines on admission to the ICU, as well as on days 4 and 7, and then weekly until 28 days post-admission: IL-6, IL-10, and TNF-α. Immune function was measured over time by the levels of TNF-α in response to an ex-vivo LPS stimulation assay on those same days. The LPS stimulation assay was conducted by sampling the patient’s blood in sodium heparin tubes. Fifty (50) μl of whole blood was then pipetted into 500 μl of LPS stimulation solution, at a concentration of 500 pg/mL [34]. The time between collection of the blood and processing was less than 30 minutes. The samples were incubated at 37°C for 4 hours and then centrifuged. The supernatant was pipetted into the microcentrifuge tubes, stored at -80°C and then analyzed.
The primary outcome for this study was the occurrence of NIs acquired during the ICU admission in relation to the tertiles of delta and peak TNF-α levels post-LPS stimulation. NIs were defined as an infection occurring after 72 hours of ICU admission. For the purposes of this analysis we considered all categories of suspected infection including possible, probable and definite as positive [35]. Secondary outcomes included clinical outcomes and laboratory outcomes. Clinical outcomes included ICU and hospital length of stay (LOS) and ICU, hospital and 90-day mortality. Severity of illness was measured using the APACHE II and Sequential Organ Failure Assessment (SOFA) scores. The SOFA score was recorded throughout the ICU admission.
Statistical Analysis
Sample Size.
The sample size was based on the clinical study [33]. A total of 214 patients were enrolled; blood samples for analysis were available for analysis in 201. The PREVAIL study found no difference in outcomes based on allocation so both allocation groups were combined for this secondary analysis.
Statistical Methods
The primary variable of interest (TNF-α response) was represented in two ways: the maximum TNF-α level post-LPS challenge (peak TNF-α) and the change from pre-challenge to post-challenge levels (delta TNF-α). As done in prior studies by Heagy, W., et al. and Mózes, T et al, patients were divided into tertiles based on the level of TNF-α in response to ex-vivo LPS stimulation assay on admission to the ICU [31, 36]. Any patients without a peak TNF-α measure were excluded from both baseline analyses, while patients without a pre-challenge TNF-α measure reported were excluded from the delta TNF-α analysis.
Clinical outcomes represented as continuous variables, including the total number of adjudicated nosocomial infections and the rate of adjudicated nosocomial infections per patient, were compared between TNF-α tertiles using the Kruskal-Wallis test. Categorical and binary variables, including mortality and source of adjudicated infection, were compared using the Chi-Squared test. This comparison method was mirrored for all other variables that were compared, with one exception: primary diagnosis on admission was compared using Fisher’s exact test due to small cell sizes. Because the large number of diagnosis categories made this computationally prohibitive, a Monte Carlo simulation was employed.
Multivariable logistic regression models for each of peak TNF-α and delta TNF-α data were created using the occurrence of ever having a nosocomial infection during the hospital stay as the dependent variable (See Supplemental Content 2: Multivariable analysis). Age, sex, medical or surgical admission type, lactoferrin versus placebo arm, APACHE II score, and severe sepsis were included as covariates, in addition to either the peak or delta TNF-α tertile (using the highest tertile as the referent).
Multiple Time Point Analysis
Data on TNF-α response levels were collected on ICU days 0, 4, 7, 14, 21 and 28. Box plots of the distributions of peak TNF-α, delta TNF-α and baseline serum TNF-α were created. A panel of profile plots showing the change from baseline to peak TNF-α for each patient, on each day, was produced. A longitudinal plot showing the average baseline and peak TNF-α values on each day was also produced, as was a similar longitudinal plot of the change in TNF-α values for each patient on each day.