Extracorporeal membrane oxygenation (ECMO) is a miniaturized cardiopulmonary support system for neonatal, pediatric, and adult patients, representing an effective therapy for life-threatening heart or lung failure. Because of technological improvements and a significant reduction in potential complications, ECMO has become a standard procedure in modern intensive care treatment. For approximately a decade, ECMO has been established at our institute as a life-saving therapy option with steadily increasing case numbers and growing importance. However, nosocomial infections are a major clinical complication for critically ill ECMO patients, and the incidence of infections increases with the duration of ECMO support.1–8 The high risk of infection, especially bloodstream infection (BSI), is attributed to the multiple entry points within the ECMO circuit (including intravascular cannulas for ECMO), alterations in host immune function, and additional invasive devices during ECMO support, including indwelling central venous and arterial catheters, urinary catheters, and endotracheal tubes.1,3,6 Furthermore, device-associated infections caused by microbial adhesion and colonization present an ongoing medical problem.9 In ECMO treatment, blood comes in contact with a large extracorporeal surface, and the membrane oxygenator (MO) represents an area with low-flow velocities in the ECMO circuit. Knowing the possibility of implant-associated infections, we hypothesize that the artificial surfaces of the ECMO circuit, particularly the MO, could be the target of microbial adhesion and colonization favoring the development of septicemia. At present, only one study has addressed this issue.10 In our pilot study, we used a universal rDNA polymerase chain reaction (PCR) test to investigate the potential microbiological colonization of MOs on the membrane surfaces.
Materials and Methods
Patient Population and ECMO Support
From November 2009 to August 2010, the MOs of 20 patients (female/male ratio: 8/12; mean age: 41 years; age range: 17–71 years) with severe acute lung or heart failure treated with venovenous (VV) or venoarterial (VA) ECMO devices were analyzed for bacteria and fungi. The patient population consisted of 11 VV ECMO and 9 VA ECMO supports. The indication for ECMO support was either cardiac or respiratory failure with beginning or already existing end-organ failure. For ECMO support, a centrifugal pump (Maquet Rotaflow-RF-32; MAQUET Cardiovascular, Fairfield, NJ) and a MO (Quadrox-i Adult; MAQUET Cardiovascular) were used. The MO has a gas exchange surface of 1.8 m2, and the maximum gas flow is 15 L/min. The pressure gradient across the MO is 25 mm Hg at 2.5 L/min blood flow. The cannulas used on the arterial side were selected according to the patient’s body surface area (15F–17F cannulas; Novalung, Hechingen, Germany). For venous drainage, Heartport (Johnson & Johnson, New Brunswick, NJ) cannulas sized 20F to 24F were used. Cannulation was carried out in all cases using the Seldinger technique. The femoral vein and the femoral artery (VA ECMO) or jugular vein (VV ECMO) were selected as the implantation site. Anticoagulation was conducted using intravenous heparin, aiming for an activated clotting time between 160 and 180 seconds during the time of support. No antibiotic or antifungal prophylaxis was routinely used, but all patients were treated with antibiotics and, in some cases, with antifungals during ECMO therapy, based on culture results and clinical situations. Thirteen patients had already been on antibiotic treatment before ECMO initiation.
Preparation and Molecular Analysis of Membrane Oxygenators
After the successful weaning from the extracorporeal device, the death of the ECMO patient, or change-out of MO due to reduced MO performance, the MO was immediately prepared for molecular analysis. First, the MO was rinsed through the tube system with sterile physiological saline solution (0.9% w/v NaCl) to remove blood and was subsequently opened under sterile conditions. Using a sterile swab, a hollow fiber membrane smear was taken from the opened MO, and the sample was subsequently aseptically cut into small pieces. Swab samples were extracted according to the protocol supplied by the manufacturer (UMD Universal CE-IVD; Molzym, Bremen, Germany). Universal rRNA gene PCR assays for bacterial and fungal DNA extracted from swab samples were carried out in a Mastercycler gradient (Eppendorf, Hamburg, Germany). DNA extraction and PCR preparation were performed separately under an ultraviolet-decontaminated laminar flow and a PCR cabinet with standard precautions to avoid DNA contamination, respectively. Positive and negative PCR controls run with each experimental series as advised by the UMD Universal manual were as expected. Polymerase chain reaction products from positive reactions were sequenced and analyzed.11 The MO sample processing is shown in Figure 1. During ECMO treatment and within 7 days before initiation and after weaning, blood culture results and culture results from other sample materials collected from the ECMO patients served as reference points for molecular analysis. Cultivation and identification of microorganisms were carried out according to standard operating procedures in our microbiological laboratory (Vitek 2; bioMérieux, Marcy l’Etoile, France).
A study population of 20 patients with severe respiratory or circulatory failure treated with a VV or VA ECMO device was examined. The clinical outcome data of the ECMO patients are shown in Table 1. The average duration of ECMO support was 17.1 days (interquartile range: 2–51 days); the average duration of system runtime was 6.1 days. Ten patients died while on extracorporeal support (ECMO-related mortality rate 50%), and two more patients died a few days after successful weaning from an extracorporeal device (no. 3 and 14). Thus, the overall in-hospital mortality rate was 60% in our population. For eight patients, ECMO therapy was successfully used as a bridge to recovery (four patients) or as a bridge to lung (three patients) or heart (one patient) transplantation. The mortality rate was 64% in the VV ECMO population and 56% in the VA ECMO population. Fifteen ECMO patients exhibited septic symptoms (75%, 15/20), resulting primarily in multiple organ failure and death (73%, 11/15). The American College of Chest Physicians/Society of Critical Care Medicine Consensus Conference criteria were used to define sepsis in patients receiving ECMO therapy. The antimicrobial therapy duration of the ECMO patients is shown in Table 1.
Molecular Analysis of Membrane Oxygenators
In total, molecular analysis of 20 MOs of 20 patients was carried out, and the PCR results were compared with blood culture results and microbiological findings from other cultured materials generated before, during, and after the ECMO treatment (Table 1). The results of PCR, blood cultures, and PCR-relevant culture results from other sample materials are listed in Table 1. The overall patient-based PCR positivity from MO analysis was 45% (9/20 PCR-positive ECMO patients). The PCR positivity was 56% in the VA ECMO population (5/9) and 36% in the VV ECMO population (4/11). Looking at the patient population with septic symptoms, the PCR positivity was 53% (8/15). Bacterial DNA was detected in eight of nine PCR-positive ECMO cases. In contrast, the detection of fungal DNA was demonstrated in only one case by PCR (no. 7, Candida albicans). Mixed bacterial strains were found by PCR analysis of the MOs of five ECMO patients (no. 5, 8, 12, 14, and 15). Regarding the blood culture results, 9 episodes of bacterial but none of fungal BSI were observed in 6 of 20 patients (30%) during the evaluation period. Of the nine episodes, five episodes were acquired during the period of ECMO support in four patients (20%). In three ECMO patients, the detection of bacteria by MO analysis and blood culture were consistent (no. 5 [Escherichia coli], no. 14 [Staphylococcus epidermidis ], and no. 18 [Enterococcus faecalis]). Moreover, the PCR results of three patients were consistent with additional microbiological findings (no. 5 [E. coli, bronchial secretion], no. 15 [Staphylococcus aureus, perfusate], and no. 18 [E. faecalis, midstream urine; Enterococcus spp., bronchial secretion]). Five patients were PCR positive (no. 1, 4, 7, 8, and 12) but blood culture negative, and no additional microbiological findings were coinciding. Gram-negative bacteria (no. 5, 12, and 15), excluding E. coli (no. 5), and the yeast C. albicans (no. 7), which were detected on membranes by molecular analysis were not detected by blood culture during the investigation period. Moreover, eight of nine PCR-positive patients developed septic symptoms (89%), and six of eight PCR-positive patients died while on ECMO or after successful weaning (78%).
In an overall consideration of the identified spectrum of microorganisms, a total of 14 pathogens (including ten different species) were detected in nine PCR-positive MOs (Table 2). Gram-positive bacteria (including staphylococci, enterococci, and streptococci) were by far the most abundant microorganisms in our population (71%, 10/14) and were found in eight of nine PCR-positive cases, followed by Gram-negative bacteria (17%, 2/12), which were detected in two of eight PCR-positive cases, and fungi (7%, 1/14) in one of nine PCR-positive cases. The most frequently detected pathogens were staphylococci (57%), and at the species level, the following pathogens were the most common: S. aureus, S. epidermidis, and E. coli.
Nosocomial infections acquired during ECMO support have been described in several retrospective studies as a serious complication increasing the risk of a fatal outcome in neonatal, pediatric, and adult patients.1,12–16 For instance, a single-center retrospective study reported an overall in-hospital mortality rate of 68.3% in a series of 334 patients receiving ECMO support. The death rate of patients with infections was 75.6% and that of patients with BSIs was 88.2%.8 Therefore, the early diagnosis of infections in critically ill patients on ECMO is of crucial importance for adequate and successful antimicrobial treatment and to improve patient outcome.
It is essential, for this purpose, to study the influence of synthetic ECMO surfaces on the development and promotion of BSI, which has been reported in retrospective studies as the most common infection occurring during ECMO support.2,4–6,8 In this pilot study, we investigated the potential colonization of MOs by microorganisms from patients undergoing ECMO using a universal rDNA PCR test, and we correlated the PCR results with microbiological findings from routine clinical diagnostics of the blood and other samples. To our knowledge, this was the first time a molecular diagnostic method was used to detect adherent microorganisms on the artificial surfaces of the ECMO setting.
In our study, bacteria and fungi were determined by the molecular test to occur on oxygenator membranes. Assuming that free pathogen DNA was eliminated during the extraction procedure of the samples,17 we concluded that the pathogens are part of a viable microbial colonization and an acute MO infection. These initial results confirm that microorganisms can potentially colonize the hollow fiber membranes of MOs during extracorporeal treatment and that these microorganisms can be detected by the applied molecular tests. Microbial colonization of extracorporeal circuits was also demonstrated by Moore et al.,18 showing biofilm contamination in 54.2% of continuous VV hemofiltration circuits by electron microscopic investigation of tubing samples. In our study, Gram-positive bacteria and especially staphylococci were the most prevalent pathogens detected on the MO surfaces, which were also reported to be the most frequently occurring etiologic agents of BSI in ECMO patients.2,3,7,19 Data from a retrospective review by O’Neill et al.6 showed Candida sp. infections in 38% of ECMO patients; these pathogens were mainly isolated from urine and blood specimens. Fungal BSIs made up 19% of the total BSIs. The authors attributed the high rate of fungal infection to broad-spectrum antibiotic use in ECMO patients. A retrospective study by Gardner et al.13 reported a fungal infection rate of 12% in a series of 261 pediatric patients on ECMO. These authors concluded that the presence of fungal infection increased mortality and that routine fluconazole prophylaxis decreased the incidence of fungal infections.
In our study, some pathogens detected on MO surfaces by PCR were also found by blood culture or by culture of specimens obtained from the infectious focus, which demonstrate a possible correlation between infection and membrane colonization in these ECMO cases. Patients with positive PCR but negative blood culture with no additional microbiological findings could be caused by prior antimicrobial treatment, resulting in suppressed microbial growth. Several pathogens that were detected by blood culture during the ECMO period were not found on the oxygenator membranes. Given that all these culture results were generated before MO sampling, a PCR-negative result might be due to the absence of pathogens on the MO and thus to the successful antimicrobial treatment of BSI. Apart from this, it is to be assumed that not every BSI is accompanied by MO colonization, and vice versa.
In comparison studies between PCR-based and blood culture–based diagnostics, the inability to recover microorganisms in culture has also attributed to previous antimicrobial therapy.11,20 The molecular test, in contrast, is a cultivation-independent diagnostic method. In this context, it is important to note that the applied molecular test is aimed at achieving the detection of only viable microorganisms due to the elimination of free pathogen DNA during the extraction procedure.17 In addition, some pathogens detected only by PCR are difficult to grow in culture which is attributed to nonoptimized cultivation media for fastidious bacteria and fungi.21 Timely identification of the causative pathogen is important for adequate antimibrobial therapy of these high-risk patients. The molecular test can be completed within 4 hours, and definitive identification can be performed within additional 3 to 4 hours using modern, fast-cycle sequencing systems. Compared with culture results, the initial Gram-staining results are typically obtained within 1 to 3 days, and definitive identification of infectious agents often requires 3 to 5 days.
However, the clinical relevance of the PCR results is unknown, and there are many complications, which place patients undergoing ECMO at an increased risk of death. Microbial colonization seems to increase the risk of sepsis. Some acquired MO infections might be successfully treated by adequate antimicrobial intervention due to positive blood culture results or clinical evaluation. However, the treatment of implant-associated infections is extremely difficult and implants infected by virulent pathogens have to be removed.9 Persistent septic complications during ECMO support should therefore be discussed as an indication for MO exchange, improving antimicrobial treatment and septic patient outcome.
In contrast to our findings, Müller et al.10 detected bacteria in just a single MO in a population of 10 septic patients receiving ECMO using conventional culture techniques. Growth inhibition of pathogens in culture could not be excluded by the authors because all the ECMO patients were on a broad-spectrum antibiotic treatment. In that regard, and contrary to the clinical situation, we often observe blood culture–negative results in septic ECMO patients on antibiotic treatment, as shown in our study for illustration. A retrospective four-center review by Elerian et al.22 concluded that daily surveillance by blood cultures is not useful in the early detection of sepsis in neonatal ECMO while on antibiotic prophylaxis, mainly based on the low positive blood culture rate of 0.9%. Bloodstream infections occurred in 8 of 177 neonates (4.5%). The authors have not taken into account the aspect of potential growth inhibition in culture in the presence of antibiotics. Moreover, many of the clinical signs and symptoms associated with infections are missing in ECMO patients and standard definitions may not apply.3 In the sepsis diagnosis of ECMO patients, the molecular test could represent a useful option to detect, confirm, and monitor infections. A survey of extracorporeal life support organization (ELSO)-participating centers reported that most ELSO centers gave prophylactic antibiotics (74%), and approximately half of the centers (49%) routinely performed surveillance cultures.23
There are several limitations in this pilot study. Colonization of membrane oxygenator was considered in isolation as one complication in ECMO treatment in a small study population (20 patients). Larger clinical investigations with an increased number of ECMO patients and additional clinical parameters are required to evaluate the initial PCR findings and the clinical relevance. To support the indication for MO exchange, a BSI monitoring and subsequent MO analysis by PCR should be performed on ECMO patients to evaluate a correlation between systemic infections and MO colonization. Furthermore, our investigations of oxygenator membranes represent only part of the whole MO, so that false-negative results due to missing local microorganisms or cell numbers below the limit of detection of the molecular system may not be excluded. The high contamination rate in our ECMO population can also induce to discuss about prophylactic antibiotic application to the ECMO setting before ECMO initiation in subsequent studies.
Bacteria and fungi can adhere to artificial membranes of MOs in patients requiring ECMO. The initial results indicate that the applied universal rDNA PCR test is well suited to detect and identify pathogens and mixed infections on membrane surfaces in ECMO patients. With regard to frequently occurring blood culture–negative infections in septic ECMO patients due to antimicrobial treatment and based on the PCR results, the change of MO should be always considered when a patient on ECMO shows clinical or laboratory signs of infections. The rapid detection of pathogens by the molecular test (8 hours from extraction to sequence results) followed by targeted antibiotic therapy and MO replacement could probably decrease the risk of mortality and of ongoing sepsis in ECMO patients.
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extracorporeal membrane oxygenation; membrane oxygenator; PCR; microbiological colonization; sepsis