Microcirculation-guided resuscitation in sepsis: the next frontier?

Microcirculatory dysfunction plays a key role in the pathogenesis of tissue dysoxia and organ failure in sepsis. Sublingual videomicroscopy techniques enable the real-time non-invasive assessment of microvascular blood flow. Alterations in sublingual microvascular perfusion were detected during sepsis and are associated with poor outcome. More importantly, sublingual videomicroscopy allowed to explore the effects of commonly applied resuscitative treatments in septic shock, such as fluids, vasopressors and inotropes, and showed that the optimization of macro-hemodynamic parameters may not be accompanied by an improvement in microvascular perfusion. This loss of "hemodynamic coherence," i.e., the concordance between the response of the macrocirculation and the microcirculation, advocates for the integration of microvascular monitoring in the management of septic patients. Nonetheless, important barriers remain for a widespread use of sublingual videomicroscopy in the clinical practice. In this review, we discuss the actual limitations of this technique and future developments that may allow an easier and faster evaluation of the microcirculation at the bedside, and propose a role for sublingual microvascular monitoring in guiding and titrating resuscitative therapies in sepsis.

Anesthetic management of patients with sepsis/septic shock.

Sepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection, while septic shock is a subset of sepsis with persistent hypotension requiring vasopressors to maintain a mean arterial pressure (MAP) of ≥65 mmHg and having a serum lactate level of >2 mmol/L, despite adequate volume resuscitation. Sepsis and septic shock are medical emergencies and time-dependent diseases with a high mortality rate for which early identification, early antibiotic therapy, and early source control are paramount for patient outcomes. The patient may require surgical intervention or an invasive procedure aiming to control the source of infection, and the anesthesiologist has a pivotal role in all phases of patient management. During the preoperative assessment, patients should be aware of all possible organ dysfunctions, and the severity of the disease combined with the patient's physiological reserve should be carefully assessed. All possible efforts should be made to optimize conditions before surgery, especially from a hemodynamic point of view. Anesthetic agents may worsen the hemodynamics of shock patients, and the anesthesiologist must know the properties of each anesthetic agent. All possible efforts should be made to maintain organ perfusion supporting hemodynamics with fluids, vasoactive agents, and inotropes if required.

Shock index as predictor of massive transfusion and mortality in patients with trauma: a systematic review and meta-analysis.

BACKGROUND: Management of bleeding trauma patients is still a difficult challenge. Massive transfusion (MT) requires resources to ensure the safety and timely delivery of blood products. Early prediction of MT need may be useful to shorten the time process of blood product preparation. The primary aim of this study was to assess the accuracy of shock index to predict the need for MT in adult patients with trauma. For the same population, we also assessed the accuracy of SI to predict mortality. METHODS: This systematic review and meta-analysis was performed in accordance with the PRISMA guidelines. We performed a systematic search on MEDLINE, Scopus, and Web of Science from inception to March 2022. Studies were included if they reported MT or mortality with SI recorded at arrival in the field or the emergency department. The risk of bias was assessed using the QUADAS-2. RESULTS: Thirty-five studies were included in the systematic review and meta-analysis, for a total of 670,728 patients. For MT the overall sensibility was 0.68 [0.57; 0.76], the overall specificity was 0.84 [0.79; 0.88] and the AUC was 0.85 [0.81; 0.88]. Positive and Negative Likelihood Ratio (LR+; LR-) were 4.24 [3.18-5.65] and 0.39 [0.29-0.52], respectively. For mortality the overall sensibility was 0.358 [0.238; 0.498] the overall specificity 0.742 [0.656; 0.813] and the AUC 0.553 (confidence region for sensitivity given specificity: [0.4014; 0.6759]; confidence region for specificity given sensitivity: [0.4799; 0.6332]). LR+ and LR- were 1.39 [1.36-1.42] and 0.87 [0.85-0.89], respectively. CONCLUSIONS: Our study demonstrated that SI may have a limited role as the sole tool to predict the need for MT in adult trauma patients. SI is not accurate to predict mortality but may have a role to identify patients with a low risk of mortality.

Too much tolerance for hyperoxemia in mechanically ventilated patients with SARS-CoV-2 pneumonia? Report from an Italian intensive care unit.

Background: In COVID-19 patients requiring mechanical ventilation, the administration of high oxygen (O2) doses for prolonged time periods may be necessary. Although life-saving in most cases, O2 may exert deleterious effects if administered in excessive concentrations. We aimed to describe the prevalence of hyperoxemia and excessive O2 administration in mechanically ventilated patients with SARS-CoV-2 pneumonia and determine whether hyperoxemia is associated with mortality in the Intensive Care Unit (ICU) or the onset of ventilator-associated pneumonia (VAP). Materials and methods: Retrospective single-center study on adult patients with SARS-CoV-2 pneumonia requiring invasive mechanical ventilation for ≥48 h. Patients undergoing extracorporeal respiratory support were excluded. We calculated the excess O2 administered based on the ideal arterial O2 tension (PaO2) target of 55-80 mmHg. We defined hyperoxemia as PaO2 > 100 mmHg and hyperoxia + hyperoxemia as an inspired O2 fraction (FiO2) > 60% + PaO2 > 100 mmHg. Risk factors for ICU-mortality and VAP were assessed through multivariate analyses. Results: One hundred thirty-four patients were included. For each day of mechanical ventilation, each patient received a median excess O2 of 1,121 [829-1,449] L. Hyperoxemia was found in 38 [27-55]% of arterial blood gases, hyperoxia + hyperoxemia in 11 [5-18]% of cases. The FiO2 was not reduced in 69 [62-76]% of cases of hyperoxemia. Adjustments were made more frequently with higher PaO2 or initial FiO2 levels. ICU-mortality was 32%. VAP was diagnosed in 48.5% of patients. Hyperoxemia (OR 1.300 95% CI [1.097-1.542]), time of exposure to hyperoxemia (OR 2.758 [1.406-5.411]), hyperoxia + hyperoxemia (OR 1.144 [1.008-1.298]), and daily excess O2 (OR 1.003 [1.001-1.005]) were associated with higher risk for ICU-mortality, independently of age, Sequential Organ failure Assessment score at ICU-admission and mean PaO2/FiO2. Hyperoxemia (OR 1.033 [1.006-1.061]), time of exposure to hyperoxemia (OR 1.108 [1.018-1.206]), hyperoxia + hyperoxemia (OR 1.038 [1.003-1.075]), and daily excess O2 (OR 1.001 [1.000-1.001]) were identified as risk factors for VAP, independently of body mass index, blood transfusions, days of neuromuscular blocking agents (before VAP), prolonged prone positioning and mean PaO2/FiO2 before VAP. Conclusion: Excess O2 administration and hyperoxemia were common in mechanically ventilated patients with SARS-CoV-2 pneumonia. The exposure to hyperoxemia may be associated with ICU-mortality and greater risk for VAP.

Antibiotic Treatment of Acinetobacter baumannii Superinfection in Patients With SARS-CoV-2 Infection Admitted to Intensive Care Unit: An Observational Retrospective Study.

Introduction: In COVID-19 patients on mechanical ventilation, VAP from Acinetobacter baumannii remains a crucial risk factor for death. Antibiotic resistance represents an important problem in treating this infection. This study aims to describe the evolution of the superinfection from PDR Acinetobacter baumannii in patients with acute respiratory failure from SARS-CoV-2 infection admitted to ICU and compare the impact of two different antibiotic strategies on microbiological negativization. Methods: Single-center observational retrospective study, including patients admitted to our ICU from March 2020 to May 2021 for acute respiratory failure from SARS-CoV-2 infection who developed PDR Acinetobacter baumannii superinfection. Clinical data at ICU admission were collected, as well as the timing of isolation of Acinetobacter baumannii, its resistance profile, the site of infection, and the antibiotic therapy. Results: Of the 32 patients enrolled, 10 patients (31.2%) were treated with the combination of high-dose ampicillin/sulbactam, high-dose tigecycline, intravenous and inhaled colistin (Protocol), the other 22 (68.8%) were treated with the combination of two antibiotics (Control). Of the 10 patients in the Protocol group, 8 patients (80%) received also fosfomycin. All patients (100%) in the Protocol group had microbiological negativization, while in the Control group microbiological negativization was observed in 8 (36.4%) patients, p < 0.01. Conclusion: Our report shows microbiological negativization in all patients treated with the combination therapy of nebulized and intravenous colistin, high-dose tigecycline, and high-dose ampicillin/sulbactam. This combination of antibiotics seems to be a useful alternative when other treatments are not available or fail.

Mid-Regional Proadrenomedullin (MR-proADM) and Microcirculation in Monitoring Organ Dysfunction of Critical Care Patients With Infection: A Prospective Observational Pilot Study.

Introduction: Microvascular alterations are involved in the development of organ injury in critical care patients. Mid-regional proadrenomedullin (MR-proADM) may predict organ damage and its evolution. The main objective of this study was to assess the correlation between MR-proADM and microvascular flow index (MFI) in a small cohort of 20 adult critical care patients diagnosed with infection, sepsis, or septic shock. Further objectives were to evaluate the correlation between the clearance of MR-proADM and the variables of microcirculation and between MR-proADM and the Sequential Organ Failure Assessment (SOFA) score. Materials and Methods: This is a prospective observational pilot study. Inclusion criteria: consecutive adult patients admitted to intensive care unit (ICU) for or with infection-related illness. Daily measurement of MR-proADM and calculation of the SOFA score from admission in ICU to day 5. Repeated evaluations of sublingual microcirculation, collection of clinical data, and laboratory tests. Results: Primary outcome: MR-proADM was not significantly correlated to the MFI at admission in ICU. A clearance of MR-proADM of 20% or more in the first 24 h was related to the improvement of the MFIs and MFIt [percentual variation of the MFIs + 12.35 (6.01-14.59)% vs. +2.23 (-4.45-6.01)%, p = 0.005; MFIt +9.09 (4.53-16.26)% vs. -1.43 (-4.36-3.12)%, p = 0.002]. Conclusion: This study did not support a direct correlation of MR-proADM with the MFI at admission in ICU; however, it showed a good correlation between the clearance of MR-proADM, MFI, and other microvascular variables. This study also supported the prognostic value of the marker. Adequately powered studies should be performed to confirm the findings.

Effects of Normoxia, Hyperoxia, and Mild Hypoxia on Macro-Hemodynamics and the Skeletal Muscle Microcirculation in Anesthetised Rats.

Objectives: Excessive oxygen (O2) administration may have a negative impact on tissue perfusion by inducing vasoconstriction and oxidative stress. We aimed to evaluate the effects of different inhaled oxygen fractions (FiO2) on macro-hemodynamics and microvascular perfusion in a rat model. Methods: Isoflurane-anesthetised spontaneously breathing male Wistar rats were equipped with arterial (carotid artery) and venous (jugular vein) catheters and tracheotomy, and randomized into three groups: normoxia (FiO2 21%, n = 6), hyperoxia (FiO2 100%, n = 6) and mild hypoxia (FiO2 15%, n = 6). Euvolemia was maintained by infusing Lactate Ringer solution at 10 ml/kg/h. At hourly intervals for 4 h we collected measurements of: mean arterial pressure (MAP); stroke volume index (SVI), heart rate (HR), respiratory rate (by means of echocardiography); arterial and venous blood gases; microvascular density, and flow quality (by means of sidestream dark field videomicroscopy on the hindlimb skeletal muscle). Results: MAP and systemic vascular resistance index increased with hyperoxia and decreased with mild hypoxia (p < 0.001 in both cases, two-way analysis of variance). Hyperoxia induced a reduction in SVI, while this was increased in mild hypoxia (p = 0.002). The HR increased under hyperoxia (p < 0.05 vs. normoxia at 3 h). Cardiax index, as well as systemic O2 delivery, did not significantly vary in the three groups (p = 0.546 and p = 0.691, respectively). At 4 h, microvascular vessel surface (i.e., the percentage of tissue surface occupied by vessels) decreased by 29 ± 4% in the hyperoxia group and increased by 19 ± 7 % in mild hypoxia group (p < 0.001). Total vessel density and perfused vessel density showed similar tendencies (p = 0.003 and p = 0.005, respectively). Parameters of flow quality (microvascular flow index, percentage of perfused vessels, and flow heterogeneity index) remained stable and similar in the three groups. Conclusions: Hyperoxia induces vasoconstriction and reduction in skeletal muscle microvascular density, while mild hypoxia has an opposite effect.

Variation in the Outcome of Norepinephrine-Dependent Septic Patients After the Institution of a Patient-Tailored Therapy Protocol in an Italian Intensive Care Unit: Retrospective Observational Study.

Objective: To evaluate the outcome of patients with septic shock after the institution of a patient tailored therapy protocol in our Intensive Care Unit (ICU). Methods: Single-center retrospective observational study including 100 consecutive septic patients (≥ 16 years) requiring norepinephrine infusion, admitted to our ICU between 2018 and 2019 after the institution of a patient-tailored therapy protocol, compared with a historical control group of 100 patients admitted between 2010 and 2013 (historical controls). The patient-tailored therapy protocol included the use of IgM-enriched immunoglobulins for patients with low plasma IgM levels, blood purification strategies for patients with high plasma levels of cytokines or endotoxin, albumin correction and modulation of vasoactive agents. Clinical and therapeutic parameters were noted at the time of initiation of norepinephrine infusion and for the 1st 24 h. The primary outcome was ICU mortality. Results: ICU-mortality was lower in the patient-tailored therapy cohort as compared to historical controls (32 vs. 57%, p < 0.001). Patient-tailored therapy was associated with a lower risk of ICU-mortality even after adjusting for the main clinical severity indices (adjusted odds ratio 0.331 [95% confidence interval 0.166-0.658], p = 0.002). After propensity score matching, 48 patients in historical control group and 48 patients in the patient-tailored therapy cohort with similar general characteristics were selected. ICU-mortality was lower in the patient-tailored therapy matched subgroup as compared to historical controls (40 vs. 60%, p = 0.037). Conclusions: An individualized therapeutic approach in septic patients may be associated with a survival benefit. However, the use of an historical control group of patients admitted between 2010 and 2013 may introduce substantial bias. Further adequately designed studies are needed to demonstrate the impact of patient-tailored therapy on outcome.

Sublingual microcirculation in patients with SARS-CoV-2 undergoing veno-venous extracorporeal membrane oxygenation.

Veno-Venous Extracorporeal Membrane Oxygenation (VV-ECMO) is a rescue treatment for severe acute respiratory failure refractory to conventional ventilation. We examined the alterations of sublingual microcirculation in patients with SARS-CoV-2 during VV-ECMO treatment and assessed the relationship between microvascular parameters and ventilation, hemodynamics, and laboratory tests. Nine patients were included in the study and the following microcirculatory parameters were estimated: TVD 16.81 (14.46-18.6) mm/mm2; PVD 15.3 (14.09-17.96) mm/mm2; PPV 94.85% (93.82%-97.79%); MFI 2.5 (2.5-2.92); HI 0.4 (0.18-0.4). TVD and PVD were inversely related to D-dimer levels (rho = -0.667, p = 0.05 and rho = -0.733, p = 0.025 respectively), aspartate aminotransferase (AST) (rho = -0.886, p = 0.019 and rho = -0.886, p = 0.019 respectively) and alanine aminotransferase (ALT) (rho = -0.829, p = 0.042 and rho = -0.829, p = 0.042 respectively). Our results showed an altered sublingual microcirculation in patients receiving VV-ECMO for severe SARS-CoV-2 and suggest a potential contribution of endothelia dysfunction to determine microvascular alteration.

Relationship between norepinephrine dose, tachycardia and outcome in septic shock: A multicentre evaluation.

PURPOSE: Septic shock is associated with massive release of endogenous catecholamines. Adrenergic agents may exacerbate catecholamine toxicity and contribute to poor outcomes. We sought to determine whether an association existed between tachycardia and mortality in septic shock patients requiring norepinephrine for more than 6 h despite adequate volume resuscitation. MATERIALS AND METHODS: Multicentre retrospective observational study on 730 adult patients in septic shock consecutively admitted to eight European ICUs between 2011 and 2013. Three timepoints were selected: T1 (first hour of infusion of norepinephrine), Tpeak (time of highest dose during the first 24 h of treatment), and T24 (24-h post-T1). Binary logistic regression models were constructed for the three time-points. RESULTS: Overall ICU mortality was 38.4%. Mortality was higher in those requiring high-dose (≥0.3 mcg/kg/min) versus low-dose (<0.3 mcg/kg/min) norepinephrine at T1 (53.4% vs 30.6%; p < 0.001) and T24 (61.4% vs 20.4%; p < 0.0001). Patients requiring high-dose with concurrent tachycardia had higher mortality at T1; in the low-dose group tachycardia was not associated with mortality. Resolving tachycardia (from T1 to T24) was associated with lower mortality compared to patients where tachycardia persisted (27.8% vs 46.4%; p = 0.001). CONCLUSIONS: Use of high-dose norepinephrine and concurrent tachycardia are associated with poor outcomes in septic shock.

Thoracic continuous spinal anesthesia for high-risk comorbid older patients undergoing major abdominal surgery: one-year experience of an Italian geriatric hospital.

BACKGROUND: General anesthesia is associated with high morbidity/mortality in comorbid older adults. Thoracic continuous spinal anesthesia/analgesia (TCSA) may be an alternative for major abdominal surgery. We report a one-year experience of the use of TCSA in an Italian geriatric center. METHODS: Retrospective review of case notes of high-risk older patients (ASA class ≥III) who underwent TCSA for major abdominal surgery between May 2017-May 2018. TCSA was performed with a 21-gauge Tuohy-shaped spinal needle and a 24-gauge catheter (level of insertion between T6-7 and T10-11). Hyperbaric bupivacaine or levobupivacaine (two boluses of 2.5 mg) plus fentanyl (10-25 µg) were injected before incision, followed by additional doses if needed. Intrathecal levobupivacaine plus fentanyl were infused for 72 hours after surgery. We described the anesthesiologic management and evaluated the incidence of postoperative complications. RESULTS: Ninety patients (age 84.4±6.9 years, 53.3% male) were enrolled. High comorbidity, according to the Geriatric Index of Comorbidity, was found in 64.4% of the patients. Mean Metabolic Equivalents were 2.58±0.99. Switching to general anesthesia was required in one case. Intraoperative noradrenaline (0.17±0.12 mcg/kg/min) was required in 70.6%. Additional intravenous paracetamol or weak opioids were required in 23% and 2.2% of patients, respectively. No direct complications of TCSA were reported. Cardiac, respiratory and surgical complications occurred in 15.6%, 13.3% and 13.3% of patients, respectively. Delirium occurred in 14.4%. Hospital mortality was 5.6%. CONCLUSIONS: TCSA may be a valid alternative to general anesthesia in high-risk older patients undergoing major abdominal surgery. Further studies are needed to confirm its safety and benefits.

Changes in Cytokines, Haemodynamics and Microcirculation in Patients with Sepsis/Septic Shock Undergoing Continuous Renal Replacement Therapy and Blood Purification with CytoSorb.

BACKGROUND: Extracorporeal blood purification therapies have been proposed as a strategy to remove inflammatory mediators during sepsis, thus improving outcome. OBJECTIVES: We aimed to evaluate changes in cytokines, haemodynamics and microcirculation during blood purification with Cytosorb adsorber in septic patients. METHODS: Prospective observational study on critically ill adult patients with sepsis/septic shock underwent renal replacement therapy (RRT) for acute renal failure and haemoadsorption with Cytosorb as adjunctive therapy for 24 h. Measurements were taken at baseline, after 6 and 24 h: haemodynamic parameters, arterial and central venous blood gases, plasma levels of tumour necrosis factor alpha, interleukin (IL) 1-beta, IL-6, IL-8 and IL-10. The sublingual microcirculation was assessed with sidestream dark field videomicroscopy to evaluate the perfused vessel density (PVD) and microvascular flow quality. Tissue oxygenation and microvascular reactivity were assessed with thenar near infrared spectroscopy (NIRS) with a vascular occlusion test. RESULTS: Nine patients; plasma levels of IL-8 decreased at 24 h (p < 0.05 versus 6 h); no significant variation was found for other cytokines. Haemodynamic remained stable throughout the observation. Microvascular perfusion improved over time, with an increase in PVDs at 6 and 24 h (from 13.9 [13.3-16.4] to 15.7 [15-17.3] and 17 [14.8-18.6] mm/mm2 respectively, p = 0.003) and total vessel densities at 24 h (14.9 [13.9-16.9] vs. 17.9 [15.3-20], p = 0.0015). No significant variation was detected in NIRS-derived parameters. The Sequential Organ Failure Assessment score decreased from 12 ± 3 to 10 ± 1 at 24 h (p = 0.039). CONCLUSIONS: In septic patients undergoing RRT, haemoadsorption with Cytosorb seems to determine a decreasing in plasma levels of IL-8, although levels of other cytokines did not vary significantly, and an improvement of microcirculation despite no significant variation in macro-haemodynamics.

Tissue oxygen saturation changes and postoperative complications in cardiac surgery: a prospective observational study.

BACKGROUND: Cardiac surgery with extracorporeal circulation (ECC) can induce microvascular dysfunction and tissue hypoperfusion. We hypothesized that the alterations in near-infrared spectroscopy (NIRS)-derived parameters would be associated with post-operative complications in cardiac surgery patients. METHODS: Prospective observational study performed at two University Hospitals. Ninety patients undergoing cardiac surgery with ECC were enrolled. The NIRS sensor was applied on the thenar eminence. A vascular occlusion test (VOT, 3-min ischemia) was performed at baseline (t0), at Intensive Care Unit (ICU) admission (t1), 3 (t2) and 6 (t3) hours later. Baseline tissue oxygen saturation (StO2), oxygen extraction rate and microvascular reactivity indices were calculated. RESULTS: In the first hours after cardiac surgery, StO2 tended to increase (86% [80-89] at T3 versus 82% [79-86] at T0, p = ns), while both tissue oxygen extraction and microvascular reactivity tended to decrease, as indicated by increasing occlusion slope (- 8.1%/min [- 11.2 to - 7] at T3 versus - 11.2%/min [- 13.9 to - 7.9] at T0, p = ns) and decreasing recovery slope (1.9%/sec [1.1-2.9] at T3 versus 3.1%/sec [2.3-3.9] at T0, p = ns). No substantial differences were found in NIRS-derived variables and their changes over time between patients with complications and those without complications. CONCLUSIONS: Peripheral tissue oxygen extraction and microvascular reactivity were reduced during the first hours after cardiac surgery. NIRS-derived parameters were not able to predict complications in this population of cardiac surgery patients.

IgM-enriched immunoglobulins (Pentaglobin) may improve the microcirculation in sepsis: a pilot randomized trial.

BACKGROUND: Polyclonal or IgM-enriched immunoglobulins may be beneficial during sepsis as an adjuvant immunomodulatory therapy. We aimed to test whether the infusion of IgM-enriched immunoglobulins improves microvascular perfusion during sepsis. METHODS: Single-centre, randomized, double-blind, placebo-controlled phase II trial including adult patients with a diagnosis of sepsis or septic shock for less than 24 h. Patients received an intravenous infusion of 250 mg/kg (5 mL/kg) per day of IgM-enriched immunoglobulins (Pentaglobin, n = 10) for 72 h or placebo (NaCl 0.9%, n = 9). At baseline and after 24 and 72 h of infusion, the sublingual microcirculation was assessed with Incident Dark Field videomicroscopy. Thenar near-infrared spectroscopy (NIRS) was applied with a vascular occlusion test to assess tissue oxygenation and microvascular reactivity. Levels of interleukin (IL) 1-beta, IL-6, IL-8, IL-10 and tumour necrosis factor alpha were measured in the serum. RESULTS: The perfused vessel density (PVD) for small vessels (diameter < 20 micron) increased in the Pentaglobin group (from 21.7 ± 4.7 to 25.5 ± 5.1 mm/mm2) and decreased in the placebo group (from 25 ± 5.8 to 20.7 ± 4.1 mm/mm2, p for interaction < 0.001, two-way analysis of variance). The absolute between-group difference at 72 h was 4.77 (standard error 2.34), p = 0.140. The microvascular flow index for small vessels increased at 24 h in the Pentaglobin group (from 2.68 [2.38-2.78] to 2.93 [2.82-3], p < 0.01) and decreased at 72 h in the placebo group (from 2.83 [2.60-2.97] to 2.67 [2.48-2.73], p < 0.05). Changes in general parameters, cytokines and NIRS-derived parameters were similar between the two groups, except for IL-6 and IL-10 that significantly decreased at 72 h only in the Pentaglobin group. CONCLUSIONS: A 72-h infusion of IgM-enriched immunoglobulins (Pentaglobin) in patients with sepsis or septic shock may be associated with an increase in sublingual microvascular perfusion. Further studies are needed to confirm our findings. Trial registration NCT02655133, www.ClinicalTrials.gov, date of registration 7th January 2016, https://www.clinicaltrials.gov/ct2/show/NCT02655133.