Hemodiafiltration Decreases Serum Levels of Inflammatory Mediators in Severe Leptospirosis: A Prospective Study

Background Leptospirosis is a health problem worldwide. Its most severe form is a classic model of sepsis, provoking acute respiratory distress syndrome (ARDS) and acute kidney injury (AKI), with associated mortality that remains unacceptably high. We previously demonstrated that early initiation of sustained low-efficiency dialysis (SLED) followed by daily SLED significantly decreases mortality. However, the mode of clearance can also affect dialysis patient outcomes. Therefore, the objective of this study was to compare the effects of SLED with traditional (diffusive) clearance, via hemodialysis, and SLED with convective clearance, via hemodiafiltration (SLEDf), in patients with severe leptospirosis. Methods In this prospective study, conducted in the intensive care unit (ICU) from 2009 through 2012, we compared two groups—SLED (n = 19) and SLEDf (n = 20)—evaluating demographic, clinical, and biochemical parameters, as well as serum levels of interleukins, up to the third day after admission. All patients received dialysis early and daily thereafter. Results During the study period, 138 patients were admitted to our ICU with a diagnosis of leptospirosis; 39 (36 males/3 females) met the criteria for ARDS and AKI. All patients were on mechanical ventilation and were comparable in terms of respiratory parameters. Mortality did not differ between the SLEDf and SLED groups. However, post-admission decreases in the serum levels of interleukin (IL)-17, IL-7, and monocyte chemoattractant protein-1 were significantly greater in the SLEDf group. Direct bilirubin and the arterial oxygen tension/fraction of inspired oxygen ratio were significantly higher in the SLED group. We identified the following risk factors (sensitivities/specificities) for mortality in severe leptospirosis: age ≥ 55 years (67%/91%); serum urea ≥ 204 mg/dl (100%/70%); creatinine ≥ 5.2 mg/dl (100%/58%); Acute Physiology and Chronic Health Evaluation II score ≥ 39.5 (67%/88%); Sequential Organ Failure Assessment score ≥ 20.5 (67%/85%); and inspiratory pressure ≥ 31 mmHg (84%/85%). Conclusions The mode of dialysis clearance might not affect outcomes in severe leptospirosis.


Results
During the study period, 138 patients were admitted to our ICU with a diagnosis of leptospirosis; 39 (36 males/3 females) met the criteria for ARDS and AKI. All patients were on mechanical ventilation and were comparable in terms of respiratory parameters. Mortality did not differ between the SLEDf and SLED groups. However, post-admission decreases in the serum levels of interleukin (IL)-17, IL-7, and monocyte chemoattractant protein-1 were significantly greater in the SLEDf group. Direct bilirubin and the arterial oxygen tension/fraction of inspired oxygen ratio were significantly higher in the SLED group. We identified the following risk factors (sensitivities/specificities) for mortality in severe leptospirosis: age 55 years (67%/91%); serum urea 204 mg/dl (100%/70%); creatinine 5.2 mg/dl (100%/ 58%); Acute Physiology and Chronic Health Evaluation II score 39.5 (67%/88%); sustained low-efficiency dialysis (SLED, via hemodialysis; n = 19); and those undergoing extended SLED via hemodiafiltration (SLEDf; n = 20). We evaluated demographic, clinical, and biochemical parameters, including the serum interleukin (IL) levels. The patients in both groups received prompt, frequent (daily) dialysis. We also compared all survivors and nonsurvivors, regardless of which type of dialysis they underwent.

Patients
Patients were selected from among those suspected of having severe leptospirosis and admitted to the ICU of the Emílio Ribas Institute of Infectology, a referral center for leptospirosis located in the city of Sao Paulo, Brazil. We included only those patients diagnosed with AKI and ARDS on ICU admission. The criteria for a diagnosis of ARDS were being intubated (receiving positive-pressure ventilation), having a partial pressure of arterial oxygen/fraction of inspired oxygen (PaO 2 /FiO 2 ) ratio < 200, and presenting radiologic evidence of bilateral infiltrates consistent with pulmonary edema or pulmonary hemorrhage. In ARDS patients, dialysis was started if serum creatinine was 2.0 mg/dl and one-hour urine volume was < 100 ml for three consecutive hours. In a previous study of ARDS patients with severe leptospirosis [5], we evaluated patients on mechanical ventilation who underwent dialysis on the first day of their ICU stay. We found that such preemptive dialysis decreased mortality in that population. Therefore, in the present study, our criteria for when dialysis was indicated were less stringent than those set forth in the Kidney Disease: Improving Global Outcomes guidelines. The patients were allocated to the SLED group or to the SLEDf group depending on whether they were admitted on an odd or even day, respectively. The study was approved by the Ethics in Research Committee of the Emílio Ribas Institute of Infectology. Written informed consent was obtained from the legal guardians of all of the patients evaluated.

Hemodialysis and hemodiafiltration
The patients in the SLED group underwent daily dialysis with SLED, which was performed with a volumetrically controlled machine (4008S; Fresenius Medical Care, Bad Homburg, Germany). The bicarbonate dialysate flow rate was 300-500 ml/min, and the blood flow rate was 180-250 ml/min. The patients in the SLEDf group underwent daily dialysis with SLEDf, which was performed using a volumetrically controlled machine (4008S On-Line HDF, Fresenius Medical Care, Bad Homburg, Germany). The bicarbonate dialysate flow rate was 300-500 ml/ min, and the blood flow rate was 180-250 ml/min. The convection dose was of 50-60 ml/min. We employed only first-use, synthetic (polysulfone) dialyzer membranes (F8 and F80 for SLED and SLEDf, respectively; Fresenius).
Each dialysis session lasted 8-10 hours. A dual-lumen catheter (Arrow International, Reading, PA, USA) was inserted into the femoral or jugular vein for vascular access. No anticoagulants were administered.

Serological diagnosis and treatment regimens
The diagnosis of leptospirosis was confirmed by serology with immunoglobulin M enzymelinked immunosorbent assay for Leptospira (PanBio, Brisbane, Australia) and the microscopic agglutination test (MAT). For each patient, the MAT was conducted with a single sample, and a sample with a titer 1:800 was considered positive for a diagnosis of leptospirosis. Using the MAT, with Ellinghausen McCullough Johnson Harris medium (Difco, Sparks, MD, USA) and incubation at 28-30°C, we tested the main Leptospira serovars isolated in the city of São Paulo: Australis, Autumnalis, Bataviae, Canicola, Castellonis, Copenhageni, Cynopteri, Djasiman, Grippotyphosa, Hebdomadis, Icterohaemorrhagiae, Javanica, Panama, Patoc, Pomona, Hardjo, Sejroe, Pyrogenes, Tarassovi, and Wolfii. The diagnosis of leptospirosis was made on the basis of clinical characteristics suggestive of the disease, the patient having been admitted during the rainy season (when leptospirosis is most prevalent), positive serology on an immunoglobulin M enzyme-linked immunosorbent assay, and a MAT titer 1:800.
All leptospirosis patients were initially treated with penicillin or ceftriaxone. In some cases, nosocomial infection occurred and the treatment regimens were subsequently modified on the basis of the culture results.

Clinical and biochemical variables
The severity of illness was determined on the basis of the Acute Physiology and Chronic Health Evaluation II (APACHE II) and Sequential Organ Failure Assessment (SOFA) scores obtained on admission. Biochemical variables were also evaluated on admission, including serum levels of bilirubin, urea, creatinine, pH, bicarbonate, creatine phosphokinase, sodium, potassium, calcium, and magnesium, as well as hemoglobin levels, leukocyte counts, platelet counts, and levels of liver transaminases (aspartate aminotransferase and alanine aminotransferase). We also collected blood samples on the first 3 days after ICU admission (before each dialysis session) and stored the serum at −70°C for further cytokine analysis. Net fluid intake per day was compared between the SLED and SLEDf groups on the first 3 days in the ICU. Intradialysis episodes of hypotension, defined as a mean arterial pressure of 70 mmHg or the need for intervention, were analyzed for the first three dialysis sessions. Patients were treated and monitored according to accepted intensive care practices. None of the patients were receiving parenteral nutrition. Recovery of renal function was defined as no more need for RRT and a 24-hour urine volume > 1000 ml.

Serum cytokine levels
We submitted samples to multiplex cytokine assay (Bio-Plex Rat Cytokine Group 9-Plex Assay; Bio-Rad, Hercules, CA, USA) in order to determine the serum levels of IL-1α, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, eotaxin, granulocyte colony-stimulating factor (G-CSF), interferon gamma, tumor necrosis factor alpha, monocyte chemoattractant protein-1 (MCP-1), platelet-derived growth factor, monocyte inflammatory protein-1 beta, chemokine CCL5, vascular endothelial growth factor, and interferon-gamma-inducible protein-10. The assay was read on the Bio-Plex suspension array system, and the data were analyzed with Bio-Plex Manager software, version 4.0. We compared the serum levels of cytokines between the SLED and SLEDf groups. We also compared the two groups in terms of the differences in the serum level of each cytokine between the first and second day (Δ1) and between the second and third day (Δ2). We obtained the Δ1 values by subtracting the day 2 levels from the day 1 levels and the Δ2 values by subtracting the day 3 levels from the day 2 levels. Therefore, a positive Δ1 or Δ2 value would indicate that there was no decrease in the serum levels between the two time points, whereas a negative Δ1 or Δ2 value would indicate that there was such a decrease.

Statistical analysis
Nonparametric analysis was performed to assess quantitative (continuous) variables. Data are expressed as mean ± standard deviation, and values of p 0.05 were considered statistically significant. The power of the predicted values to discriminate between positive and negative outcomes in the model was quantified by receiver operating characteristic analysis.

Characteristics of the study population
During the study period, 138 patients were admitted to our ICU with a diagnosis of leptospirosis; 39 patients (36 males and 3 females) met the criteria for ARDS and AKI. All 39 of those patients tested positive for Leptospira interrogans, and 32 (82.1%) presented the serovar Icterohaemorrhagiae.
At baseline, the SLED and SLEDf groups were similar with respect to demographic characteristics, serum urea, serum creatinine, liver enzymes, electrolytes, and acidosis (S1 Table). The severity of illness, according to the APACHE II and SOFA scores, was also similar between the two groups. All of the patients were on mechanical ventilation and were comparable in terms of respiratory parameters. None of the patients were receiving diuretics at the time of RRT initiation. The time from ICU admission to the initiation of dialysis (door-to-dialysis time) was similar between the groups.

Outcome data
Complete outcome data are shown in S2 Table. Of the 19 SLED group patients, 3 died in the ICU, as did 3 of the 20 patients in the SLEDf group. Among the nonsurvivors in the SLED group, death occurred at a mean of 3.0 ± 2.6 days after ICU admission, compared with 8.3 ± 6.8 days among those in the SLEDf group. Although that difference was not significant, it indicated a trend toward better survival in the SLEDf group. In the sample as a whole, the number of dialysis sessions, days to renal recovery of renal function, and days on mechanical ventilation were similar between the two groups, as were the length of ICU stay and overall length of hospital stay. In addition, during the first 3 days after ICU admission, ultrafiltration for each dialysis session (net fluid intake) and mean arterial pressure during dialysis were also similar between the two groups (S2 Table). On the first day in the ICU, 17 (89.5%) of the 19 SLED group patients were receiving norepinephrine, as were all 20 of the SLEDf group patients. On the second and third days, norepinephrine was used in 18 (94.7%) and 19 (95.0%) of the SLED and SLEDf group patients, respectively. However, the difference between the two groups was not significant on any of those days.

Interleukin levels
Over the first 3 ICU days, serum levels of interleukins were analyzed daily in blood samples collected prior to the dialysis sessions. The serum levels of all of the interleukins evaluated are presented in Table 1, and Fig 1 depicts the Δ1 and Δ2 values of selected interleukins. There was a statistically significant difference between the groups in terms of the Δ1 and Δ2 values for IL-7, IL-17, and MCP-1. In addition, there was a trend toward Δ2 values for IL-6, IL-15, and G-CSF being more often negative in the SLEDf group (Fig 1).

Survivors versus nonsurvivors
When we compared all survivors with all nonsurvivors, regardless of the mode of dialysis clearance employed, we identified significant differences between the two in terms of age; serum levels of urea, creatinine, and direct bilirubin, as well as the PaO 2 /FiO 2 ratio. As expected, APACHE II and SOFA scores also differed between the survivors and nonsurvivors ( Table 2). Logically (because death cut short the ICU stays of nonsurvivors), there were also differences between survivors and nonsurvivors in terms of the number of dialysis sessions and the time on mechanical ventilation, as well as the length of the ICU stay and overall hospital stay (S3 Table).

Discussion
In the present study, we have demonstrated that the mode of dialysis clearance has no apparent effect on mortality in patients with severe leptospirosis. We also demonstrated significant differences between survivors and nonsurvivors in terms of age; serum levels of urea, creatinine and direct bilirubin; and PaO 2 /FiO 2 . One interesting finding was that the decrease over time in the serum levels of IL-6, IL-7, IL-15, IL-17, G-CSF, and MCP-1 was greater in the SLEDf group than in the SLED group. In a previous study of patients with severe leptospirosis, we showed that there was a clinically relevant difference in mortality between the patients receiving daily dialysis and those receiving alternate-day dialysis [5]. That suggests that performing dialysis more frequently can decrease the risk of fatal complications in such patients. We therefore hypothesized that changing the type of dialysis could also improve survival in severe leptospirosis.
Although there is empirical evidence that continuous RRT (CRRT) induces less hemodynamic instability in critically ill patients than does intermittent hemodialysis, there is as yet no consensus in the literature regarding that point. In a randomized crossover trial comparing CRRT (24-hour continuous arteriovenous hemofiltration) and intermittent hemodialysis (a Table 2. Differences between survivors (n = 33) and nonsurvivors (n = 6). 24-hour period encompassing a 4-hour intermittent hemodialysis session) in 27 critically ill patients, no difference was found between the two methods in terms of the incidence of blood pressure drops and vasopressor requirement [8]. Various studies have attempted to address the question of whether the choice of RRT modality affects patient outcomes. A meta-analysis including 1400 patients treated with either intermittent hemodialysis or CRRT found no difference in mortality between the two modalities [9]. Increased concentrations of inflammatory mediators appear to be involved in the pathogenesis of severe leptospirosis [10][11][12][13][14][15][16]. On the basis of the humoral theory of sepsis, we can suggest that hemofiltration, which has been shown to have certain beneficial effects in severe sepsisimproving hemodynamics and unselectively removing pro-and anti-inflammatory mediators [17][18][19]-is a potentially useful therapeutic approach. Hoffmann et al. demonstrated that the ultrafiltrate from patients with sepsis contains compounds with significant immunomodulatory qualities [20]. The authors found that, in such patients, hemofiltration removed significant quantities of peripheral blood mononuclear cells and monocyte-derived tumor necrosis factor, although not of lymphocyte-derived IL-2 or IL-6 [20]. In addition, the use of specific types of dialyzer membranes has been shown to have beneficial effects on immune cell function and to improve survival in animal models of sepsis [21]. Various authors have reported that CRRT can eliminate inflammatory mediators [22]. In a randomized trial involving 425 critically ill patients with AKI treated with continuous hemofiltration [23], the authors compared three different ultrafiltration rates: 20, 35, and 45 ml/kg of body weight per hour. Survival was found to be significantly lower in the patients treated at 20 ml/kg of body weight per hour (41%) than in those treated at either of the higher rates (57% and 58%, respectively).
Ratanarat et al. demonstrated that pulse high-volume hemofiltration improves hemodynamics and survival in severe sepsis [19]. Although we did not use CRRT in the present study, we did use hemodiafiltration (60 ml/min). It has been postulated that effective removal of inflammatory mediators is only possible when a highly permeable membrane is employed and the ultrafiltration rate is high (> 2 L/hour). In a study comparing high-volume hemofiltration (6 L/hour) with standard continuous hemofiltration (1 L/hour) in patients with septic shock and multiple organ failure, the former was associated with a greater (temporary) decrease in vasopressor requirements and both therapies were associated with a temporary reduction in serum concentrations of the complements C3a and C5a [24]. However, in a study involving critically ill patients with AKI, intensive renal support was not found to decrease mortality, whereas mortality rates did decrease among the patients receiving less-intensive therapy with a defined dose of intermittent hemodialysis three times per week and among those receiving CRRT at 20 ml/kg of body weight per hour [25]. In a randomized controlled trial involving 1508 critically ill patients, the authors found that increasing the intensity of CRRT from 25 to 40 ml/kg of body weight per hour did not reduce mortality or the rate of dependence on dialysis [26]. In the present study, the dialysis dose, in terms of diffusive clearance, was the same for both groups. However, the convective clearance in the SLEDf group resulted in the overall dialysis dose being higher in that group.
To our knowledge, there have been no studies indicating which mediators should be removed at which point (i.e., in which phase of sepsis). It should be borne in mind that nutrients, hormones, and antibiotics are also removed during hemofiltration [27]. In the present study, the decreases in the levels of certain inflammatory mediators over time were greater in the SLEDf group than in the SLED group, although mortality did not differ between the two groups.
Our study has certain limitations. Because CRRT is not available in our ICU, we performed SLED or SLEDf. It is possible that the comparison between hemodialysis and hemodiafiltration would have been more definitive had we used CRRT. However, our data are relevant for the treatment of patients in the myriad ICUs where CRRT is not an option. Another limitation is that we did not compare the two groups in terms of the norepinephrine dose levels. Nevertheless, we did evaluate the need for (use vs. non-use of) norepinephrine.
Leptospirosis affects vulnerable populations, such as rural subsistence farmers and urban slum dwellers [28]. Costa et al. demonstrated that the risk of leptospirosis is greater among adult males than among children and females, being highest among males between 20 and 29 years of age [29]. The authors also showed that the risk of death from leptospirosis is highest among males between 50 and 59 years of age [29]. In our study, being 50 years of age or older was found to be a predictor of mortality in severe leptospirosis, as were serum urea 204 mg/ dl, serum creatinine 5.2 mg/dl, APACHE II score 39.5, SOFA score 20.5, and inspiratory pressure 31 mmHg. In another study conducted in our ICU, Marotto et al. identified three factors associated with mortality early in the course of severe respiratory failure in leptospirosis patients [30]: hemodynamic disturbance; serum creatinine > 265.2 mmol/L; and serum potassium 4.0 mEq/L. The patients evaluated in the present study had the more severe form of the disease; most were under treatment with vasoactive drugs, and all of them needed dialysis. The serum level of potassium is no longer a factor associated with mortality in our ICU. We believe that is because our routine protocol now includes the prompt initiation of dialysis.
In conclusion, SLED and SLEDf seem to be equally effective in decreasing serum levels of cytokines in patients with severe leptospirosis, with no significant difference in patient survival. The door-to-dialysis time continues to be more important than is the dialysis modality in leptospirosis-associated AKI.
Supporting Information S1 Table. Characteristics of patients with severe leptospirosis treated with sustained lowefficiency dialysis (n = 19) or with sustained low-efficiency dialysis with convective clearance, via hemodiafiltration (n = 20), on ICU admission. (DOCX) S2 Table. Outcome measures among patients with severe leptospirosis, by the type of renal replacement therapy performed. (DOCX) S3 Table. Differences between survivors and nonsurvivors in terms of the number of dialysis sessions and the time on mechanical ventilation, as well as the length of the intensive care unit stay and the overall hospital stay. (DOCX)