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Eur J Cardiothorac Surg 2005;27:434-440
© 2005 Elsevier Science NL


Aortic root abscess and secondary infective mitral valve disease: results of surgical endocarditis treatment

Henryk Siniawski*, Onnen Grauhan, Michael Hofmann, Miralem Pasic, Yuguo Weng, Charles Yankah, Hans Lehmkuhl, Roland Hetzer

Department of Cardiothoracic and Vascular Surgery, Deutsches Herzzentrum Berlin, Augustenburger Platz 1, 13353 Berlin, Germany

Received 17 September 2004; received in revised form 16 November 2004; accepted 1 December 2004.

* Corresponding author. Tel: +49 30 4593 2012; fax: +49 30 4593 1003. (E-mail: siniawski{at}dhzb.de).


    Abstract
 Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusions
 References
 
Objective: Mortality in active infective endocarditis (AIE) is substantial and reinfection can strongly influence outcome. Assessment of factors influencing mortality is important. We studied 108 (33%) patients suffering from root abscess out of a total of 327 AIE patients admitted to the Deutsches Herzzentrum Berlin for surgical treatment between 1996 and 2003. Among them were 53 (25.5% of all patients) who were diagnosed as having secondary infective mitral valve disease (SMVD). Mean age was 53±14.2 years; there were 37 men and 16 women. Methods: Risk factors were assessed on the basis of clinical, hemodynamic, echocardiographic and surgical information. Mean follow-up was 325±251 days with complete echocardiographic examination performed in patients with double valve surgery. The differences between groups were analyzed using Student's t-test. Multivariate analysis for the whole group suffering from abscess was performed to identify independent factors influencing mortality. Results: In 19 (35.8%) patients suffering from SMVD mitral valve reconstruction was undertaken and mitral valve replacement was performed in 34 (64.2%) patients. There were 27 patients treated with a Shelhigh prosthesis: 18 with double valve replacement (both Shelhigh) and nine with an aortic Shelhigh prosthesis and concomitant mitral valve reconstruction. Homografts were used in 17 patients, with mitral reconstruction in 10 and a stented mitral prosthesis in seven. In nine cases two stented valve prostheses were used. The calculated mean Doppler gradient for homografts and Shelhigh in aortic position was 12 (±5.7) and 15 (±4.6), respectively (NS). The following predisposing factors for mortality were assessed: severe damage of aortic annulus (OR 4.65, CI 1.22–17.1, P=0.0159); septic shock (OR 3.44, CI 0.85–13.9, P=0.07) and poor ejection fraction (<40%), and dilated LV. Conclusions: Excessive mortality reaching 29% was noted in patients suffering from AIE with aortic ring abscess and SMVD requiring double valve surgery. Double valve surgery with semi-stented Dacron-free valve prostheses is associated with a low rate of reinfection and good function of the implants. The most potent independent risk factors for death were septic shock and severe aortic root destruction.

Key Words: Double valve endocarditis • Destructive endocarditis • Secondary mitral valve endocarditis • Jet lesion • Superstentless valve substitute • Aortic root abscess


    1. Introduction
 Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusions
 References
 
Active infective endocarditis is a serious disease, particularly when the infection spreads below the primary infected valve. The treatment of choice is surgery, which is why this form of endocarditis is often called ‘surgical disease’ [1]. The most common primary site of infection is the aortic root. The destructive process can progressively and very rapidly lead to total root destruction. The destruction can also spread into the mitral valve area causing mitral valve dysfunction [2]. In some cases double valve surgery is necessary to save the life of the patient. Mortality in simple endocarditis is relatively high and especially when aortic root destruction takes place and sepsis is present high mortality is to be expected. It is common opinion that the mortality rate in patients with destructive endocarditis is unacceptably high and until today new technological developments have not helped to lower it. One important factor influencing postoperative mortality is the reinfection rate which per se has a grave prognosis. It is believed that the sensitivity of newly implanted valves to reinfection is associated with the fabric material used in producing valve prostheses. The patients suffering from abscess and treated with a homograft in aortic position have a significantly lower reinfection rate than those receiving a classical prosthesis [3]. In recent years our attention has been focused on valves with special antiphlogistic preparation as prostheses which could be used in patients with high risk of reinfection. Our newly published results in patients suffering from aortic root abscess are promising. The preliminary data of this contribution are focussed on the importance of valve selection in extended and double valve endocarditis [4]

The aim of this study was to assess the risk factors influencing mortality of patients suffering from infective aortic valve endocarditis complicated by infection extension into the aortic periannular area and additionally to examine the results of surgery in patients in whom infection spread from the aortic to the mitral valve necessitating double valve surgery. This part of the study focussed on the postoperative mortality and reinfection rates in patients treated with the new valve implants in aortic and mitral position compared with homografts and classical implants and those treated by an additional mitral valve reconstruction procedure. The large number of patients included in the study made it possible to assess risk factors responsible for operative mortality using multivariate analysis which we believe can prospectively assess the chance of survival of individual patients in dependence on defined factors.


    2. Methods
 Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusions
 References
 
2.1. Patients
Between December 1996 and December 2003 a total of 327 patients suffering from active infective aortic valve endocarditis were admitted to the Deutsches Herzzentrum Berlin for surgical treatment (Table 1). The patients came from all over Germany. There were 208 (63.6%) patients suffering from active aortic valve endocarditis including 108 (90 men and 18 women) in whom aortic root abscess was diagnosed. The mean age of patients suffering from root abscess was 53.3 (±14.15) years. There were 36 (34%) patients with previous aortic prosthesis implantation, thus suffering from prosthetic endocarditis, and 72 (66%) patients with native aortic valve endocarditis. Among them were 53 patients (25.5% of all patients) who were diagnosed as having secondary infective mitral valve disease (SMVD) requiring double valve surgery (DVS). The mean age was 53±14.2 years; there were 37 men and 16 women. Most of the patients scheduled for DVS suffered from root abscess and only in six patients was secondary infection of the mitral valve without abscess found (jet lesion). This group was analyzed to assess the mortality and reinfection rate according to the valve substitutes used.


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Table 1. Preoperative diagnosis according to complete echocardiographic investigation
 
The 108 consecutive surgical patients suffering from aortic ring abscess (including SMVD patients) formed the group which was extensively studied for assessment of independent factors influencing mortality and reinfection rate (Table 2).


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Table 2. Characteristics of group of patients suffering from abscess (n=108) and endocarditic double valve disease (n=53)
 
Clinical information was obtained by interviewing the patients and/or family members as well as the doctors responsible for their treatment and by studying medical documents and medical results.

Infective endocarditis was diagnosed as ‘active’ when the patients required surgery before completion of the standard course of antibiotic treatment. Emergency surgery was included and defined as surgery under life-threatening conditions.

2.2. Echocardiography
Preoperative and postoperative echocardiographic examination (including transesophageal echocardiography) was performed using the commercially available ALOKA 5.5 echo machine equipped with a 3.5 multifrequent ultrasonic transthoracic probe (ranging between 2.5 and 5.0MHz) with second harmonic imaging modality and a rotary transesophageal probe (multiplane) with multifrequent modality (3.5–7.0MHz).

The time of investigation was as follows: preoperative investigation (on the day of operation, which in urgent cases was the day of admission), intraoperatively if a reconstruction procedure was undertaken, and on the day of discharge or if hemodynamic instability developed. Follow-up investigation was carried out routinely 6 months after operation and at annual intervals thereafter.

Active infective endocarditis was diagnosed if the patient had positive blood cultures (prospective diagnosis) or valve cultures (postoperative diagnosis) or signs of ongoing sepsis (epinephrine use with echocardiographic signs of endocarditis) or echocardiographically detected development of abscess, or if the patient suffered from recurrent embolic events with echocardiographically demonstrated presence of vegetation.

2.3. Definitions of abscess
Abscess was established when an echo-dense or echo-lucent area visible in TEE was seen as a structure anatomically localized in the annulus.

2.3.1. Localized abscess
A localized abscess was one that is not larger than two aortic cusps and was defined ultrasonically.

2.3.2. Aortoventricular dehiscence
Aortoventricular dehiscence is defined as a separation (discontinuity) between the aorta and the left ventricle of more than half of its circumference. Dehiscence in the ultrasonic image usually (but not always) means an echo-lucent area recognized as a separation of structures.

2.4. Aortic valve endocarditis with secondary infective mitral valve disease (SMVD)
Extension of the lesion from the aortic valve or aortic root into the mitral valve area requiring double valve surgery was designated SMVD. There were two distinguishable processes of infection extension in patients suffering from SMVD.

One is extension ‘per continuitatem’ (the local metastatic form), which leads to severe destructive endocarditis extending below the aortic valve and enlargement of the infected area to include the mitral valve. The second is lesion extension by blood stream metastasis (jet lesion).

Typical complications of extended endocarditis such as ventricular septal defect (VSD) and fistulas were diagnosed according to classical criteria.

2.5. Surgery and indications for surgery
Aortic surgery was performed on the basis of classical criteria published elsewhere [4]. An indication for concomitant mitral surgery was based on the presence of mitral valve destruction with or without severe mitral regurgitation. The type of surgery (choice of valve substitute, stentless or classical prosthesis or mitral valve reconstruction) was selected by the surgeon.

Cardioplegic arrest was achieved using antegrade cold crystalloid cardioplegic solution.

2.6. Valve substitutes
Aortic valve surgery was performed and homografts or the Shelhigh No-React® SuperstentlessTM and Stentless Aortic Valve ConduitTM were the valve substitutes used, according to the surgeon's preference. Concomitant mitral valve surgery was performed, giving preference to mitral reconstruction; when this was not possible the Shelhigh mitral valve prosthesis was inserted. In only a minority of patients were other mechanical or biological valve substitutes used in accordance with our policy of protection from reinfection (Table 3).


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Table 3. In-hospital mortality and reinfection in studied group of patients
 
2.7. Medical treatment
Antibiotic medication was directed specifically at the cultured organism if this was known. If the organism was not known, a broad-spectrum antibiotic was used.

2.8. Statistical analysis
The data are expressed as means and standard deviation. The differences between groups were analyzed using Student's t-test. Multivariate analysis for the whole group suffering from abscess was performed to identify independent factors influencing mortality.


    3. Results
 Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusions
 References
 
For the clinical characteristics of the patients see Table 1.

Before being referred to our institution the patients received drug treatment according to blood culture results when the microorganism could be identified. The average time of the treatment was a mean of 15±12 days in the whole abscess group and 17±9 days for patients with double valve surgery (ns). Severe heart failure (pulmonary congestion in X-ray; clinical evidence of left ventricular failure; auscultative pulmonary rales; presence of gallop rhythm and tachycardia) was present in 41 (37.9%) patients in the whole abscess group and in 31 patients (58.5%) suffering from double valve disease, which represents a highly significant statistical difference (P<0.001). Septic shock was more frequent in the double valve surgery group but the figures did not reach statistical significance. There were 14 patients with double valve surgery (26.4%), in whom vasopressors were used to maintain blood pressure at an adequate level, compared to 24 (20.2%) in the whole abscess group.

Severe destruction of the aortic ring defined as AV dehiscence and presence of VSD showed a slightly higher incidence in the DVS group compared to all abscess patients but the difference did not reach statistical significance. In three patients suffering from double valve disease a fistula into the right ventricle was present.

In the group suffering from double valve disease requiring DVS two different paths of infection extension into the mitral valve were distinguished by echocardiography and confirmed on inspection by the surgeon: in 38 (71.7%) extension by local metastasis and in 15 (28.3%) extension by blood stream metastasis (usually jet lesion at the anterior mitral leaflet or chordae). Characteristics of the path of infection spreading from aortic to mitral valve are shown in Table 2.

3.1. Microorganisms
Microorganisms were collected and typified in 91 (84.3%) patients in the whole abscess group and in 46 (86.8%) patients requiring DVS. The percentage of patients with detected staphylococcal infections was similar in both study groups: 46 (50.5%) for abscess patients and 24 (52.2%) for DVS (ns) (Table 1).

3.2. Surgery
All patients suffering from destructive endocarditic double valve disease received aortic and mitral valve surgery (Table 3). A late stage of jet lesion or metastatic destruction with a large anterior aneurysm and extensive destruction of the subvalvular apparatus was present in 43 cases. In such cases primary mitral valve replacement was undertaken. In 19 (35.8%) less severe cases mitral valve reconstruction was undertaken, and in a further four cases mitral valve replacement had to be carried out following attempted mitral valve reconstruction, since intraoperative echocardiography demonstrated inadequate results. Altogether, mitral valve replacement due to severe damage to the valvular and subvalvular apparatus was necessary in 30 (56.6%) patients.

No prosthetic materials or rings were used in reconstruction procedures. The preferred technique was ring plasty with plication of the mitral leaflets or resection if required. The ring plasty was almost always reinforced with pericardium. In two cases a homograft with anterior leaflet was used to close the mitral perforation. In another five cases a perforation was closed with a pericardial patch and simultaneously ring plasty with pericardial reinforcement was performed.

Twelve patients (22.6% of those who underwent double valve surgery) required aortic root replacement for excessive annular or aortic wall involvement. Eight of these patients underwent homograft root replacement, while four patients received a stentless composite valve graft.

Other types of surgery had to be performed in 11 (20.8%) patients: in eight septic VSD closure and in three fistula to RV and RA closure. There were 27 patients treated with a Shelhigh prosthesis: double valve prosthesis in 18 patients and mitral reconstruction in nine patients. Homografts were used in 17 patients, with mitral reconstruction in 10 and a stented mitral prosthesis in seven. In nine cases double stented valve prostheses were used.

3.3. Hospital mortality and morbidity
Altogether in the group suffering from abscess (108) there were 19 (17.6%) early (60 day) deaths after valve surgery. The other two patients suffering from aortic ring destruction died later of left heart failure. The overall mortality rate (early and during follow up) in this group was 19.4% (21 patients).

In the group treated by double valve surgery there were 14 (26.4%) early (60 day) deaths. In the Shelhigh group seven (25.9%) patients died early after double valve replacement (Table 3); none died later. The cause of death was myocardial failure in 12 cases and septic shock resistant to treatment in two cases.

There were reinfections in five (9.4%) patients requiring reoperation: one in the Shelhigh group (double valve replacement) (3.7%), one with an AV homograft (5.8%) and mitral replacement with a stented valve, and three with double valve replacement with stented valves (33%).

No reinfection was noted after homograft and Shelhigh aortic valve replacement with concomitant mitral valve reconstruction.

3.4. Function of implants in patients after double valve surgery: continuous and color Doppler investigation
Late postoperatively (325±251 days) comparative studies for two different implants in aortic position were performed: for homografts and the Shelhigh stentless prosthesis. The calculated instantaneous (maximal Doppler) gradient and mean pressure gradient through aortic implants were respectively for homografts 19 (±10.4) and 12 (±5.7) and for the Shelhigh stentless prosthesis 24 (±8.4) and 15 (±4.6) (ns for both groups) (Table 4). There was no mitral or aortic valve dysfunction. In two patients trivial paravalvular leakage (one in mitral and another in aortic position) and in another pseudoaneurysm of the LV outflow tract without leakage or valvular dysfunction in Doppler studies were diagnosed postoperatively. Two patients were reoperated upon (with pseudoaneurysm and progressively developed leakage in aortic position); after 1 and 4 years the explanted valves did not show any form of degeneration.


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Table 4. Preoperative and postoperative echocardiographic data in 14 patients (late postoperative investigation)
 
3.5. LV function
LV function was assessed postoperatively and compared to preoperative values. A significant improvement in LVEDD post operatively compared to pre operatively was noted in the group of patients studied as the abscess group (108 pts) (59.3±10 vs. 56±10, respectively) (P<0.001), without any improvement in LV function (EF 52±13 preoperatively vs. 51±12 postoperatively, ns).

LV end-diastolic dimension was not significantly smaller postoperatively in the DVS group (59±14 vs. 55±11mm). LV ejection fraction was also not significantly better (54±14 vs. 56±12%). However, patients requiring DVS who had jet extension mitral valve disease showed significant improvement in LVEDD dimension: 59±19 preoperatively vs. 51±8 postoperatively (P=0.05). The LV function improved in this group but the difference in EF did not reach significant values (58±14 preoperatively vs. 55±10 postoperatively).


    4. Discussion
 Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusions
 References
 
This paper deals with complex cases of patients suffering from abscess and its complications. Our cohort of patients was seriously ill: 20% of the whole group (108 with abscess) and one quarter of the double valve surgery group suffered from shock, and a large percentage of patients had signs of left heart failure (37.9 and 58.5 for the whole abscess group and for the double valve surgery group, respectively). The average time of treatment was 15±12 days for development of severe destruction of the aortic root and not significantly longer (17±0.9) when destruction of two valves took place. A high incidence of virulent microorganisms (over 50% for both groups) was responsible for this dynamic development; however, the patients were treated intensively and adequately during the preoperative period.

The percentage of prosthetic infection (33%, n=36) did not vary significantly from that reported in the literature [5]. Prosthetic endocarditis is in fact an extension of infection into tissue behind the valve, even when the abscess is not definable by echocardiography. This implies that the development of abscess in prosthetic endocarditis is a matter of time. In our view surgical intervention is obligatory to treat prosthetic endocarditis; however, there some studies demonstrating good results of medical treatment alone when abscess has not excessively damaged the aortic root [6].

4.1. Surgery
The medical policy was not to reject patients for surgery because of any criteria including septic shock. The strategy of operation in patients suffering from active infective aortic valve endocarditis is dependent on how much destruction of paraannular and subannular tissue has taken place. Invasion of infection into periannular structures of the aortic valve can produce technical problems that can only be managed in dependence on the individual situation [7,8,9]. Most of our cases were treated with a homograft or the Shelhigh No-React® SuperstentlessTM and Stentless Aortic Valve ConduitTM as an alternative. Mitral valve reconstruction was performed when possible but, when destruction of the mitral annulus and subvalvular apparatus was present, this was often not feasible. In such cases the Biomitral ShelhighTM valve was used. Only in a few cases were standard mechanical or stented bioprostheses utilized to treat endocarditis patients according to the preference of the surgeon but a high rate of reinfection was found in this group.

In the present study a significant percentage of the patients (42 out of 108 with abscess=38.8%) had to have double valve surgery. The patient's situation can progressively worsen preoperatively as well as postoperatively when double valve disease occurs. If adequate echocardiographic investigation is done at the right time, this form of endocarditis can be prevented from extending to form double valve disease. It is our feeling that surgery should be done earlier, before the mitral valve becomes involved in the process of infection. Further studies are needed to confirm this impression. This critical statement means that double valve surgery, which was associated with an excessive mortality rate of 26.4%, should be avoided.

4.2. LV function
Left ventricular function was assessed by echocardiography and a statistically significant improvement was noted in the abscess group (59.3±10 vs. 56±10, P=0.001) and in patients suffering from double valve disease treated by double surgery, but only in those who had jet lesion metastasis (EDD 59±19 vs. 51±8 P=0.05). Patients with tissue metastatic disease did not show significant improvement after operation. The ejection fraction in general did not improve significantly after operation. In our studies LV enlargement and poor ejection fraction were factors associated with increased mortality.

4.3. Reinfection: the importance of valve selection for surgery
Reinfection causing extremely high mortality inevitably influences surgical results [10]. The avoidance of reinfection in patients suffering from AIE with infection extension into surrounding tissue is of the utmost importance. It should be stated again that preserved homografts treated with antibiotics as well as fresh homografts are widely used for aortic valve surgery in patients suffering from AIE complicated by root abscess of the native valve and prosthesis [3,11]. The long-term results after homograft implantation are satisfactory [12,13].

The regular valve substitutes have to be taken into account and some authors have reported the utility of tissue [14] or mechanical prostheses [15]. In our studies cited earlier mechanical and biological stented prostheses were associated with a high reinfection rate reaching 20% of cases compared to 4% reported in the homograft group. Very similar results are found in the present study for homografts (3%), but a low reinfection rate similar to that of the homograft was found for stentless valves [3,16].

Our experience shows the reinfection rate in AIE with ring abscess to be lower for the homograft or Shelhigh prosthesis compared to that after aortic valve replacement using standard prostheses. The selection of valves for double valve surgery seems to be a corner-stone in efforts to lower early and late reinfection and mortality.

A valve substitute suitable for the treatment of endocarditis is necessary. The most important qualities of such a valve are that it is infection resistant and has acceptable durability. The Shelhigh valve was chosen for this purpose. The proprietary No-React® detoxification process developed by Shelhigh Inc. eliminates residual glutaraldehyde and ensures stable tissue cross-linking [17]. Results on reducing early calcification and tissue deterioration have been encouraging, based on clinical experience [18,19].

Surgical treatment of AIE complicated by paravalvular abscess is reportedly associated with wide-ranging and high mortality (11.4–38%) and high morbidity [20,21,22]. Wide-ranging mortality probably has to do with the varied conditions of the patient groups studied. The mortality of our abscess group patients was high (17.6%) but this was to be expected because of difficult preoperative conditions. To find out the most important independent risk factors for death, multivariate analysis was used (Table 5). Severe damage of the aortic annulus leading to aortoventricular discontinuity was strongly associated with increased mortality (OR 4.65, CI 1.22–17.1, P=0.0159). Septic shock, with an odds ratio of 3.44 (CI 0.85–13.9, P=0.07), was the factor with the strongest influence in terms of increased mortality. Other important factors influencing the risk of death were: poor ejection fraction (<40%) preoperatively and postoperatively (OR 2.5, CI 1.04–1.2, P=0.0027 and OR 2.7, CI 1.02–1.14, P=0.07, respectively). Postoperatively dilated left chamber (>65mm) was assessed to be a risk factor for death (OR 2.0, CI 1.14–1.22, P=0.0007). This means that those patients with severely destroyed annulus and AV discontinuity or with preoperative use of vasopressors, and those suffering from left heart failure and severe regurgitation and with a low ejection fraction were at greater risk of death compared to those who did not demonstrate such unfavorable morphological and hemodynamic signs (Table 5).


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Table 5. Factors influencing mortality: multivariate analysis of 108 patients suffering from ring abscess
 
In multivariate analysis double valve surgery was an independent risk factor for death when metastatic annulus destruction was defined (odds ratio 4.65, CI 1.22–17.1, P=0.02). Blood metastatic destruction (jet lesion) was not found to be a risk factor in multivariate analysis. Our results indicate that surgery in tissue metastatic patients is a matter of urgency. The type of mitral valve surgery did not influence mortality in the cohort of patients studied.

It is interesting that female gender (OR 5.14, CI 1.45–18.2, P=0.007) was an independent factor influencing increased mortality. The explanation was not found in this series. It is to be noted that the virulent microorganisms were not defined as an independent risk factor for death in the group of patients studied.

It is possible practically to predict the mortality on the basis of assessable factors. The differences in mortality between single and double valve surgery speak a clear language. Operating on patients with active infective endocarditis before deterioration of the heart takes place is the rule of thumb to reach satisfactory operative results. Every effort should be made to avoid double valve surgery. Aggressive surgical treatment has to be advocated in view of the fact that an acceptable valve substitute with anti-inflammatory properties is available.


    5. Conclusions
 Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusions
 References
 
Severe destruction of the annular and subvalvular tissue causes higher early mortality.

Excessive mortality in patients suffering from abscess with destruction extension into the mitral valve requiring double valve surgery was noted.

The path of infection extension also influences mortality: it is high in local metastatic extension and in blood stream extension it is significantly lower.

If recognized early, extension of infection into the mitral valve can be treated by mitral valve reconstruction surgery without risk of reinfection.

Double valve surgery with semi-stented Dacron-free valve prostheses treated antiphlogistically (Shelhigh No-React®) is associated with a low rate of reinfection and good function of the implants.

Multivariate analysis revealed the following predisposing factors for mortality:

– female gender
– early phase of abscess (closed cavity without signs of soluble content)
shock (norepinephrine use)
– poor LV function.


    Acknowledgments
 
We would like to thank Anne Gale ELS of the Deutsches Herzzentrum Berlin for editorial assistance.


    Footnotes
 
Presented at the joint 18th Annual Meeting of the European Association for Cardio-thoracic Surgery and the 12th Annual Meeting of the European Society of Thoracic Surgeons, Leipzig, Germany, September 12–15, 2004.


    References
 Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusions
 References
 

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