Levosimendan for resuscitating the microcirculation in patients with septic shock: a randomized controlled study

After approval by the local institutional ethics committee, the study was performed in an 18-bed multidisciplinary intensive care unit (ICU) at the Department of Anesthesiology and Intensive Care of the University of Rome 'La Sapienza'. Informed consent was obtained from the patients' next of kin. Enrolment of patients started in January 2008 and ended in April 2009. We enrolled patients who fulfilled the criteria of septic shock that required norepinephrine (NE) to maintain a mean arterial pressure (MAP) of at least 65 mm Hg despite appropriate volume resuscitation (pulmonary arterial occlusion pressure [PAOP] = 12 to 18 mm Hg and central venous pressure [CVP] = 8 to 12 mm Hg) [18]. Exclusion criteria of the study were age of less than 18 years, pregnancy, significant valvular heart disease, present or suspected acute coronary syndrome, and limitations to the use of inotropes (that is, ventricular outflow tract obstruction and mitral valve systolic anterior motion). All patients were sedated with sufentanil and midazolam and received mechanical ventilation using a volume-controlled mode.

Systemic hemodynamic monitoring of the patients included a pulmonary artery catheter (7.5-F; Edwards Lifesciences, Irvine, CA, USA) and a radial artery catheter. MAP, right atrial pressure, mean pulmonary arterial pressure, and PAOP were measured at end-expiration. Heart rate was analyzed from a continuous recording of electrocardiogram with ST segments monitored. Cardiac index (CI) was measured using the continuous thermodilution technique (Vigilance II; Edwards Lifesciences). Systemic vascular resistance index, pulmonary vascular resistance index, and left and right ventricular stroke work indices were calculated by means of standard equations. Arterial and mixed-venous blood samples were withdrawn to determine oxygen tensions and saturations as well as carbon dioxide tensions, standard bicarbonate, base excess, pH, and lactate concentrations. SvO₂ was measured discontinuously by intermittent mixed-venous blood gas analyses (Gem 4000 Premier; Instrumentation Laboratory Company, Bedford, MA, USA). Systemic oxygen delivery index (DO₂I), oxygen consumption index, and oxygen extraction ratio were calculated by means of standard formulae.

Microvascular blood flow was visualized by means of an SDF imaging device (MicroScan^®; MicroVision Medical, Amsterdam, The Netherlands) with a 5× magnification lens [17]. The optical probe was applied to the sublingual mucosa after gentle removal of saliva with a gauze swab. Three discrete fields were captured with precaution to minimize motion artifacts. Individual sequences of approximately 15 seconds were analyzed off-line with the aid of dedicated software (Automated Vascular Analysis 3.0; Academic Medical Center, University of Amsterdam, The Netherlands) in a randomized fashion by a single investigator who was unaware of the study protocol. Vessel density was automatically calculated from the software as the total vessel lengths of the small, medium, and large vessels, divided by the total area of the image [17]. The 'De Backer score' was calculated as described previously [17] and is based on the principle that density of the vessels is proportional to the number of vessels crossing arbitrary lines. In this score, three equidistant horizontal lines and three equidistant vertical lines are drawn on the screen, and then the De Backer score can be calculated as the number of small, medium, and large vessels crossing the lines, divided by the total length of the lines [17]. Vessel density was also calculated as the total vessel lengths divided by the total area of the image [17]. Both indices were automatically calculated by means of dedicated software (Automated Vascular Analysis 3.0). Perfusion was then categorized by eye as present (normal continuous flow for at least 15 seconds), sluggish (decreased but continuous flow for at least 15 seconds), absent (no flow for at least 50% of the time), or intermittent (no flow for less than 50% of the time) [17]. The proportion of perfused vessels (PPV) was calculated as follows: 100 × [(total number of vessels - [no flow + intermittent flow])/total number of vessels]. Perfused vessel density (PVD) was calculated by multiplying vessel density by the proportion of perfused vessels [17]. Microvascular flow index [17] was used to quantify microvascular blood flow. In this score, flow is characterized as absent (0), intermittent (1), sluggish (2), or normal (3) [17]. Since our investigation was focused on small and medium vessels, calculations were performed separately for vessels with diameters of smaller than 20 μm (MFIs) and of larger than 20 μm but smaller than 50 μm (MFIm). Vessel size was determined with the aid of a micrometer scale. For each patient, values obtained from the three mucosa fields were averaged [17]. To assess flow heterogeneity between the different areas investigated, we used the heterogeneity index. The latter was calculated as the highest site flow velocity minus the lowest site flow velocity, divided by the mean flow velocity of all sublingual sites [17]. Percentage changes from baseline for all variables were determined as dVariable = 100 × [(Value_{24 hours} /Value_BL) - 1] [19].

Patients were enrolled within the first 24 hours from the onset of septic shock after having established normovolemia (PAOP = 12 to 18 mm Hg and CVP = 8 to 12 mm Hg) [18] and an MAP of at least 65 mm Hg using norepinephrine, if needed. Packed red blood cells were transfused when hemoglobin concentrations decreased to below 7 g/dL [18] or if the patient exhibited clinical signs of inadequate systemic oxygen supply. Forty patients were randomly allocated to the treatment with either (a) intravenous levosimendan 0.2 μg/kg per minute (without a loading bolus dose) for 24 hours or (b) intravenous dobutamine 5 μg/kg per minute as active comparator (= control) in a double-blinded manner (each n = 20). The consort diagram is presented in Figure 1. Systemic and pulmonary hemodynamic variables, microcirculatory flow variables, blood gases, and norepinephrine requirements were determined at baseline and 24 hours after randomization. After the 24-hour intervention period, study drugs were discontinued and open-label dobutamine was started if judged as appropriate by the attending ICU physician.

An a priori analysis of sample size revealed that at least 17 patients per group were required to demonstrate a minimum difference of 20% between groups in the primary endpoint with an estimated standard deviation of 20%, a test power of 80%, and an alpha error of 5%. Data are expressed as median (25th; 75th percentile) if not otherwise specified. Sigma Stat 3.10 software (Systat Software, Inc., Chicago, IL, USA) was used for statistical analysis. Baseline and demographic data were compared with a Mann-Whitney rank sum test or chi-square test, as appropriate. Microvascular and hemodynamic variables were analyzed with a Mann-Whitney rank sum test. The correlation between systemic and microcirculatory flow variables within each group was tested by means of Spearman rank order correlation. A P value of less than 0.05 was considered statistically significant for all tests.

Baseline characteristics, including age, gender, body weight, and origin, as well as onset time of septic shock, Simplified Acute Physiology Score II (SAPS II), and mortality were not different among groups (Table 1). In addition, there was no significant difference between groups at baseline in any of the investigated hemodynamic or microcirculatory variables.

Systemic and pulmonary hemodynamic variables were comparable between groups. SvO₂ and arterial pH tended to be higher whereas NE requirements tended to be lower in the levosimendan group (Table 2). However, these differences did not reach statistical significance.

Microcirculatory data are presented in Figures 2, 3 and 4. MFIm and MFIs were significantly higher (MFIm 3.0 [3.0; 3.0] versus 2.9 [2.8; 3.0]; P = 0.02; MFIs 2.9 [2.9; 3.0] versus 2.7 [2.3; 2.8]; P < 0.001) and heterogenity index was lower after 24 hours of treatment with levosimendan versus dobutamine (heterogenity index 0.63 [0.44; 0.87] versus 0.26 [0.12; 0.51]; P = 0.001). Since baseline data varied (non-significantly) among groups, relative changes from baseline were calculated and compared between groups. Relative increases from baseline of MFIs, MFIm, PPV, and PVD (that is, dMFIs, dMFIm, dPPV, and dPVD) were significantly higher in the levosimendan group (Figure 3 and 4). In addition, the heterogeneity index decreased relative to baseline only in the levosimendan group. Correlation analyses (that is, DO₂I and CI versus MFIm and MFIs in each group) revealed no statistically significant results (each P > 0.05; Figure 5).