Dirk Vogelaers

PhD David De Bels

MD Frédéric Forêt

MD Sophie Cran

MD Eric Gilbert

MD Karen Schoonheydt

Prof. Dr Stijn Blot
email: stijn.blot@ugent.be

PhD, for the Anthicus Study

Patterns of Antimicrobial Therapy in Severe Nosocomial Infections: Empiric Choices, Proportion of Appropriate Therapy, and Modification Rates-A Multicenter, Observational Survey in Critically Ill Patients

Keywords: Proportion of Appropriate Therapy, and Modification Rates-A Multicenter, Observational Survey in Critically Ill Patients antibiotic resistance -multidrug resistance -intensive care -infectionappropriate therapy Antibiotic resistance

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A c c e p t e d M a n u s c r i p t Introduction

To avoid nosocomial infection remains a daily challenge for healthcare workers involved in the care of critically ill [1]. The prevalence of infection strongly depends on the presence of particular iatrogenic risk factors such as the use of indwelling devices and extensive surgery, as well as the severity of underlying disease and critical illness. Despite several large scaled efforts to improve prevention of healthcare-associated infection [2][3][4][5][6][7], in general about 20% to 50% of patients hospitalized in intensive care units (ICUs) experience infection, either hospital or ICU-acquired [8,9].

Severe nosocomial infection carries a substantial economic burden due to extensive antimicrobial consumption and, even more importantly, increased length of hospitalization [10][11][12][13]. In addition, severe infection seriously compromises the odds of survival. Attributable mortality rates vary from as low as zero percent to a dramatic 50% dependent of the type of infection, the causative pathogen, patient age, associated co-morbidities, and overall quality of the anti-infective approach [13][14][15][16][17][18][19][20][21]. Among all the modalities that are to be fulfilled in the aim to optimize clinical patient outcomes, early initiation of the proper antimicrobial agent is a cornerstone [1].

Failure to timely administer appropriate antimicrobial therapy results in a dramatic increase in fatality rates [22][23][24][25]. Selecting an empiric regimen which covers the causative pathogen, however, is hampered by the presence of multidrug resistant (MDR) micro-organisms which is the utmost important cause of empiric inappropriate therapy. Since antimicrobial consumption in itself is a major trigger for A c c e p t e d M a n u s c r i p t MDR development, physicians are frequently urged to thoughtfully use 'last-line antibiotics'. In fact, the challenge is to achieve high rates of empiric appropriate therapy in order to optimize patient survival, while avoiding unnecessary use of antibiotics with the intention to minimize microbial selection pressure, and hence, MDR development [1,26].

In order to accomplish this goal, in the past decade two main strategies have been proposed. In the surveillance-assisted strategy, the empiric regimen is selected mainly based on the presence or absence of MDR pathogens in routine surveillance cultures. The strength of this approach is the high negative predictive value of surveillance cultures to predict MDR involvement in subsequent infection. On the condition that cultures are taken at least twice weekly, this strategy is capable to combine high rates of empiric appropriate therapy with reduced consumption of antibiotics in pneumonia and bacteremia [27][28][29][30][31][32][33]. The cost-effectiveness of this approach, however, remains questionable. Another approach is the so-called 'deescalation strategy' which, on the condition of risk factors for MDR involvement, recommends empiric start with a regimen which covers most potential MDR pathogens such as extended-spectrum beta-lactamase producing (ESBL) Enterobacteriaceae, non-fermenting Gram-negative bacteria, and methicillin-resistant Staphylococcus aureus (MRSA) [34][35][36]. Once culture results are available, and if feasible, narrowing the antimicrobial spectrum is advised. This concept, often referred to as 'de-escalation', is widely used and advocated [37][38][39][40]. This strategy has been demonstrated to be successful and safe, and may also reduce antibiotic use and hence limiting the emergence of MDR [36,41].

A c c e p t e d M a n u s c r i p t

The objective of the present study was to prospectively investigate patterns of antimicrobial therapy in critically ill patients with nosocomial infection. More precisely we formulated the following research questions: (i) What is the rate of empiric appropriate or adequate therapy achieved? (ii) Which antibiotic selections allow for the highest rates of empiric appropriate or adequate therapy, either in the presence or absence of risk factors for MDR pathogens? (iii) What is the rate by which empiric therapy (either appropriate/adequate or not) is adapted? Inclusion criteria. Eligible patients were those who provided informed consent, were at least 18 year of age, were hospitalized in the ICU, and experienced severe hospitalacquired infection, either pneumonia, intra-abdominal infection, primary bloodstream infection, or secondary bloodstream infection originating from another source than pneumonia or intra-abdominal infection (e.g. bacteremia secondary to urinary tract infection or sinusitis).

Definitions. Infections were considered hospital-or ICU-acquired when diagnosed at least 48 hours after respectively hospital or ICU admission. Severe bacterial infections were defined following the International Sepsis Forum Consensus Conference on Definitions of Infection in the ICU [42]. Definitions of invasive fungal infections are described elsewhere [43,44]. Pneumonia was considered ventilatorassociated when occurring after at least 48 hours of mechanical ventilation. For the purpose of the study, only the first episode per patient was considered. Pneumonia or intra-abdominal infection with bacteremic breakthrough (i.e. positive blood cultures) were classified according to the primary infection. Bloodstream infections A c c e p t e d M a n u s c r i p t secondary to other sources than pneumonia or intra-abdominal infection were classified as secondary bloodstream infection. Definitions of MDR are described elsewhere [10,32,45]. Candida spp. were considered MDR when resistant to fluconazole.

Following the epidemiological scope of the study aiming at insights into empiric therapy in general, we also wanted to include microbiologically undocumented infections with obvious signs of clinical sepsis. For this purpose we made a difference between appropriate and adequate empiric therapy. The term appropriate therapy was valid for microbiologically documented infectious episodes and was defined as in vitro susceptibility of the causative pathogen and clinical response to the agent administered. The term adequate therapy was used in non-microbiologically documented infections and was defined as favorable clinical response within five days of therapy (resolution of signs of sepsis).

Severity of disease was assessed by means of the acute physiology and chronic health evaluation (APACHE) II score [46]. The following co-morbid conditions were registered: respiratory disease (chronic restrictive, obstructive or pulmonary vascular disease resulting in severe exercise restriction), cardiac disease (New York Heart Association Class IV), diabetes mellitus, hepatic disease (cirrhosis and portal hypertension; episodes of past upper gastro-intestinal bleeding attributed to portal hypertension or prior episodes of hepatic failure or encephalopathy), renal disease (chronic glomerulonephritis, nephropathy, or chronic kidney disease), neurologic A c c e p t e d M a n u s c r i p t disease (impairment of alertness or confusion) malignancy (hematologic cancer or solid tumor), and neutropenia (absolute neutrophil count <1500 cells/mm³).

To assess relationships between empiric antibiotic selection and rates of appropriate or adequate therapy, the empiric antimicrobial schemes were grouped according to the spectrum of pathogens covered. Table 1 describes the classification of empiric antimicrobial schemes. Because of the small numbers of empiric schemes covering MRSA and the relative low occurrence rate of MRSA in the cohort, empiric antibiotic schemes were classified in three major groups: (i) coverage of ESBL-Enterobacteriaceae + non-fermenting Gram-negative bacteria (meropenem [MER]-based schemes), (ii) coverage of non-fermenting Gram-negative bacteria (schemes including an antipseumononal agent), and (iii) schemes without coverage of either ESBL-Enterobacteriaceae nor non-fermenting Gram-negative bacteria (first line agents). In this analysis the added value of vancomycin as empiric therapy in order to cover methicillin resistant Gram-positive pathogens was investigated separately.

Prior antibiotic exposure was defined as the administration of antimicrobial agents within one month preceding the current infectious episode. Prior hospitalization was defined as an hospital admission within the four months preceding the current ICU admission.

Data analyses. Continuous variables are described as median (interquartile range

[IQR]) and discrete variables as n (%). For comparisons between groups the Mann Whitney U test and Fisher Exact test or Chi-square test were used as appropriate.

Independent relationships with empiric appropriate or adequate therapy were 2. Primary infections were pneumonia (n=127, of which 66 ventilator-associated), intraabdominal infection (n=23) bloodstream infection (n=21, of which 9 primary, 1 catheter-related, and 12 secondary to another source than pneumonia or intraabdominal infection). One hundred and fifteen infections were ICU-acquired (66,7%). About 75% of infections were microbiologically documented (Figure 1). This represented 129 microbiologically documented infections in which 165 microorganisms were isolated. Gram-negative bacteria were most common (n=122, 73.9%) with Pseudomonas aeruginosa (n=29), Escherichia coli (n=26), Enterobacter aerogenes (n=21), Klebsiella pneumonia (n=7), and Klebsiella oxytocca (n=7) as predominant pathogens. Among the 42 Gram-positive bacteria, Staphylococcus aureus (n=22), Streptococcus pneumoniae (n=11), and Enterococci (n=5) were most frequently isolated.

One fungal pathogen was isolated. In 64 of the 129 microbiologically documented infections MDR pathogens were involved (49.6%). Figure 1 describes the breakdown of the different types of resistance involved in these infections.

Risk factors for multidrug resistance (MDR).

Risk factors for MDR were assessed in order to evaluate empiric antimicrobial regimens relative to their presence. No specific underlying conditions appeared to predispose for MDR involvement. MDR involvement was more frequent in patients with compared to without prior Hospitalization in another ward prior to ICU admission was also recognized as significantly associated with MDR involvement (59.0% vs. 40.9%, p=0.041; relative risk 2.08, 95% CI: 1.03-4.22). Figure 3 shows a breakdown of microbiologically documented infections according to the presence of these two risk factors for MDR, and subsequent MDR involvement.

Length of ICU stay prior to the development of infection, however, was negatively associated with the risk of MDR involvement. Patients who experienced infections without MDR pathogens developed this infection after a median of 3 days (IQR: 2-7 days), while infections caused by MDR pathogens occurred after a median of 2 days (IQR: 0-4.25 days), (p=0.002).

Rates of empiric appropriate or adequate therapy and subsequent adaptation. The presence of MDR pathogens in infections was associated with a significant lower rate of empiric appropriate therapy: 87.7% vs. 35.9% (p<0.001). Rates of empiric appropriate and adequate therapy are illustrated in Figure 2. In 80 of the 129 microbiologically documented infections empiric therapy was appropriate (62.0%). In nonmicrobiologically documented infections, empiric therapy was judged adequate in 29 of 42 infections (69.0%). In 106 patients (62.0%) empiric therapy was adapted: in 60 cases following initial inappropriate or inadequate therapy, and in 46 patients in order to fine tune empiric therapy.

A c c e p t e d M a n u s c r i p t 13 Empiric antimicrobial selection and rates of appropriate or adequate therapy. In 12 infections the causative pathogen was methicillin-resistant (10 MRSA, 2 methicillin-resistant Staphylococcus epidermidis). Vancomycin was 14 times administered as an empiric agent, but in only three cases in which a methicillin-resistant pathogen proved to be involved. As such the rate of inappropriate therapy in case of methicillin-resistance involvement was 75%. Due to the relative low prevalence of methicillin-resistance as well as the low added value of initiating a glycopeptide, the association (or not) of such an agent did not alter the study results in this particular cohort. Therefore, we analyzed rates of empiric appropriate or adequate therapy irrespective of the coverage of MRSA. This is illustrated by Figure 4a. Coverage of ESBL-Enterobacteriaceae (by MER-based empiric schemes) allowed for significantly higher rates of appropriate or adequate therapy. Because the advantage of an antibiotic agent which covers ESBL-Enterobacteriaceae is obvious in settings with a high prevalence of MDR, we hypothesized that the benefit of using a MER-based empiric scheme would fade away when only patients without risk factors for MDR were taken into account. However, in this analysis empiric schemes covering ESBL-Enterobacteriaceae remained superior (Figure 4b).

When MER-based empiric schemes were compared to other empiric regimens, a difference of approximately 30% in empiric appropriate or adequate therapy was observed when all patients were taken into account (89.2% vs. 56.0%; p<0.001), and this distinction remained when only patients without risk factors for MDR were considered (83.6% vs. 53.8%; p=0.040). A c c e p t e d M a n u s c r i p t

Discussion

In this prospective, observational study, which aims to reflect real life daily practice, rates of empiric appropriate or adequate therapy were only 63.7%. About half of the infections were caused by MDR micro-organisms, mostly ESBL-Enterobacteriaceae and P. aeruginosa. As such, only the inclusion of meropenem in the empiric scheme, allowed for acceptable rates of appropriate therapy (approximately 90%).

Several reasons for the low rate of appropriate or adequate therapy can be proposed.

The overall prevalence of MDR in this particular cohort is high (~50%) and the rate of appropriate or adequate therapy in infections caused by MDR pathogens was very poor (35.9%). MDR is well recognized as a major determinant of inappropriate therapy [47][48][49][50][51][52]. However, to a certain extent the involvement of MDR can be predicted on basis of some typical risk factors of which the most essential are prior antibiotic exposure and a length of ICU stay (or mechanical ventilation in case of pneumonia) of >7 days [32,53]. Other risk factors such as particular underlying diseases (e.g. chronic obstructive pulmonary disease) and recent surgery/hospitalization can also be taken into account but generally are not included in the principal risk factors. Strangely enough, in this cohort of ICU patients with nosocomial infection some of the classic patterns failed to predict the involvement of MDR. For instance, prior hospitalization (<4 months of the current hospital admission) was not associated with a higher likelihood of MDR, while length of ICU stay was even inversely related to MDR involvement. On the other hand, prior antibiotic exposure and hospitalization in a general ward prior to ICU admission A c c e p t e d M a n u s c r i p t appeared to have a significant relationship with MDR involvement. Importantly, however, although these relationships were statistically significant, MDR pathogens were isolated in about 40% of patients in the absence of these risk factors. As a consequence, the predictive value of these risk factors was low. As such, the generally accepted concepts of risk perception for MDR involvement failed in this particular cohort. We have no plausible explanation for this observation. Given the relationship between prior hospitalization at a general ward and MDR, the relative high number of infections already present at time of ICU admission, and the short length of stay in the ICU prior to infection, it appears that there exists a serious problem of MDR in general wards in Belgian hospitals, thereby contributing to the low rate of empiric appropriate therapy in ICUs. Indeed, already a decade ago a higher incidence of Enterobacter aerogenes with an increasing resistance pattern has been noticed in Belgian hospitals [54,55].

Following the high rate of ESBL-Enterobacteriaceae and P. aeruginosa, an MER-based empiric scheme allowed for the highest rates of appropriate/adequate therapy. Due to the failure of classic risk factors to predict MDR involvement performance rates of empiric schemes did not substantially alter when only patients without risk factors were considered (Figure 3). Hence, based on the present study an approach including coverage of both ESBL-Enterobacteriaceae and P. aeruginosa seems highly warranted. Without firm supportive knowledge of local ecology in ICU and non-ICU settings indicating low MDR levels, an empiric strategy which does not cover ESBL-Enterobacteriaceae and P. aeruginosa is potentially dangerous, and as such supports the de-escalation strategy. The present findings however, with the failure of MDR A c c e p t e d M a n u s c r i p t prediction based on general characteristics in particular, provide perspectives for the surveillance assisted approach in which individual colonization status is a major element in steering empiric therapy.

In 62% of patients empiric therapy was adapted (n=106). In 60 cases adaptation followed initial inappropriate or inadequate therapy, whereas in 46 patients empiric therapy could be fine-tuned (true de-escalation). Noteworthy, when only microbiologically documented infections are considered, 4 out of 129 patients received inappropriate therapy even after the availability of culture results (Figure 1).

In MER-based empiric schemes (n=37) therapy was continued in 23 and adapted in 14 patients (in 4 cases because of initial inappropriate therapy and in 10 cases in order to narrow down the spectrum). As this study supports the approach of empiric coverage of ESBL-Enterobacteriaceae and P. aeruginosa, we stress the importance of deescalation whenever possible. Baran et al. found that previous exposition to carbapenems was 44% in patients with infections caused by imipenem-resistant Acinetobacter baumannii, whereas only 12% in patients infected by imipenemsusceptible A. baumannii isolates [56]. Yet, in multivariate analysis, previous antibiotic exposure and not carbapenem exposure in particular appeared to be an independent risk factor for imipenem-resistance. The link between exposure and specific resistance development is obvious and should not be disregarded, but this suggests that the mission to reduce microbial selection pressure is valid for all antimicrobial agents and not only for carbapenems. Further, based on the

A c c e p t e d M a n u s c r i p t

Surveillance of Antimicrobial Use and Antimicrobial Resistance in German ICUs (SARI project), Meyer E et al. identified carbapenem use as an independent predictor for a higher Stenotrophomonas maltophilia incidence [57]. Although this data do not disclose a causal relationship, it is highly suggestive for the specific selection pressure of this pathogen. Therefore, excessive use of carbapenems is to be avoided by a strict de-escalation strategy and by avoidance of unnecessary long therapies.

The most important advantage of this study is its non-interventional design, reflecting real daily practice, thereby disclosing the true pain points in empiric antibiotic therapy in critically ill patients. Due to the multicenter approach the data might be valid for many tertiary care centers in Belgium and abroad.

This study has limitations. First, no outcome data are available as the primary aim of the study was to describe antibiotic prescription patterns and how they perform in terms of appropriate therapy. Yet, the relationship between initial empiric failure to cover the causative pathogen and adverse outcomes has been demonstrated repeatedly and is generally accepted as an important quality indicator [58,59].

Secondly, the cohort does not represent a consecutive series of ICU patients with nosocomial infections. Probably this might have lead to the selection bias towards more severe infections, with a higher likelihood of MDR involvement. Yet, for what concerns risk factors for MDR, including less severe infections would probably not have changed the observations regarding the relationship between MDR infection and risk factors.

A c c e p t e d M a n u s c r i p t

In conclusion, in this prospective study reflecting real life practice in ICU patients with nosocomial infections, the rate of appropriate or adequate empiric therapy was 63.7%. This study demonstrated that classic risk factors for MDR such as prior antibiotic exposure and length of ICU stay, may be insufficient to predict MDR involvement. As such, empiric first-line use of MER allowed for significantly higher rates of appropriate or adequate therapy, irrespective of presence of these risk factors, and may be recommended in settings with a high prevalence of MDR pathogens. In addition, these data illustrate the necessity for strict infection prevention and control. involved in the care of critically ill [1]. Despite several large scaled efforts to improve prevention of healthcare-associated infection [2][3][4][5][6][7], in general about 20% to 50% of patients hospitalized in intensive care units (ICUs) experience infection, either hospital or ICU-acquired [8,9].

Declarations

MDR, multidrug resistance

Severe nosocomial infection carries a substantial economic burden due to antimicrobial consumption and increased length of hospitalization [10][11][12][13]. In addition, severe infection seriously compromises survival. Attributable mortality varies from zero, to as much as 50% depending on a variety of factors [13][14][15][16][17][18][19][20][21]. Early initiation of the appropriate antimicrobial agents is a cornerstone in optimising clinical patient outcomes, [1]. Failure of timely administration of appropriate antimicrobial therapy results in a dramatic increase in mortality [22][23][24][25]. Selecting an empiric regimen which covers causative pathogens, however, is hampered by the presence of multidrug resistant (MDR) micro-organisms which is the utmost important cause of empiric inappropriate therapy. Since antimicrobial consumption itself is a major trigger for MDR development, physicians are frequently urged to use "last-resort antibiotics" thoughtfully. The challenge is to achieve high rates of empiric appropriate therapy, while avoiding unnecessary use of antibiotics, and hence, MDR development [1,26].

In order to achieve this goal, two main strategies have been proposed. In the surveillance-assisted strategy, the empiric regimen is selected mainly based on the A c c e p t e d M a n u s c r i p t presence or absence of MDR pathogens in routine surveillance cultures. The strength of this approach is the high negative predictive value of surveillance cultures to predict MDR involvement in subsequent infection. If cultures are taken at least twice weekly, this strategy is capable to combine high rates of empiric appropriate therapy with reduced consumption of antibiotics in pneumonia and bacteremia [27][28][29][30][31][32][33]. The cost-effectiveness of this approach, however, remains unclear. Another approach is the "de-escalation strategy" which, if there are risk factors for MDR, recommends an empiric start with a regimen which covers most potential MDR pathogens such as extended-spectrum beta-lactamase producing (ESBL) Enterobacteriaceae, nonfermenting Gram-negative bacteria, and meticillin-resistant Staphylococcus aureus (MRSA) [34][35][36]. Once culture results are available, and if feasible, narrowing the antimicrobial spectrum is advised. This concept, often referred to as "de-escalation", is widely used and advocated [37][38][39][40]. This strategy has been shown to be successful and safe, and may also reduce antibiotic use and hence limit the emergence of MDR [36,41].

The To assess relationships between empiric antibiotic selection and rates of appropriate and adequate therapy, the empiric antimicrobial schemes were grouped according to the spectrum of pathogens covered. Table 1 gives Prior antibiotic exposure was defined as the administration of antimicrobial agents within one month preceding the current infectious episode. Prior hospitalization was defined as an hospital admission within the four months preceding the current ICU admission. glycopeptide, the association of such an agent did not alter the study results in this particular cohort. Therefore, we analyzed rates of empiric appropriate or adequate therapy irrespective of the coverage of MRSA (Figure 4a). Coverage of of ESBLproducing Enterobacteriaceae (by MER-based empiric schemes) allowed for significantly higher rates of appropriate or adequate therapy. We hypothesized that the benefit of using a MER-based empiric scheme would disappear when only patients without risk factors for MDR were taken into account. However, in this analysis empiric schemes covering of ESBL-producing Enterobacteriaceae remained superior (Figure 4b).

Data analyses. For comparisons between groups of continuous variables the

When [47][48][49][50][51][52]. The involvement of MDR can be partly predicted by some typical risk factors such as prior antibiotic exposure and length of ICU stay (or mechanical ventilation in case of pneumonia) of >7 days [32,53]. Other risk factors such as underlying diseases and recent surgery/hospitalization can also be taken into account. However, in our cohort of ICU patients with nosocomial infection some of the recognized risk factors failed to predict the involvement of MDR. Also, MDR pathogens were isolated in about 40% of patients in the absence of these risk factors. As a consequence, the predictive value of these risk factors was low. As such, the generally accepted concepts of risk for MDR involvement were not supported by this particular cohort.

It appears that there exists a serious problem of MDR in general wards in Belgian hospitals, thereby contributing to the low rate of appropriate empiric therapy.

A c c e p t e d M a n u s c r i p t was 44% in patients with infections caused by imipenem-resistant Acinetobacter baumannii, but only 12% in patients infected by imipenem-susceptible strains [56].

However multivariate analysis showed it was previous antibiotic exposure, and not carbapenem exposure specifically, that was an independent risk factor for imipenemresistance. The link between exposure and specific resistance development is

intuitive, but such findings suggest the aim of reducing microbial selection pressure is valid for all antimicrobial agents. Further, based on the Surveillance of Antimicrobial Use and Antimicrobial Resistance in German ICUs (SARI project),

Meyer E et al. identified carbapenem use as an independent predictor for increased incidence of Stenotrophomonas maltophilia [57].

This study has its limitations. Firstly, no outcome data are available since the primary aim of the study was to describe antibiotic prescription patterns and how they perform in terms of appropriate therapy. However, the relationship between initial empiric failure to cover the causative pathogen and adverse outcomes was demonstrated repeatedly, and is generally accepted as an important quality indicator [58,59]. Secondly, the cohort does not represent a consecutive series of ICU patients with nosocomial infections This may have led to selection bias for more severe infections, with a higher likelihood of MDR involvement, although there is no reason to believe this would have changed the relationship between MDR infection and risk factors.

In conclusion, in this prospective study, which reflects real life practice in ICU patients with nosocomial infections, the rate of appropriate or adequate empiric A c c e p t e d M a n u s c r i p

MDR, multidrug resistance

Introduction

Avoiding nosocomial infection remains a daily challenge for healthcare workers involved in care of the critically ill [1]. The prevalence of infection strongly depends on the presence of particular iatrogenic risk factors, such as the use of indwelling devices and extensive surgery, as well as the severity of underlying disease and critical illness. Despite several large-scale efforts to improve the prevention of healthcare-associated infection [2][3][4][5][6][7], in general ca. 20-50% of patients hospitalised in the Intensive Care Unit (ICU) experience infection, either hospital-or ICU-acquired [8,9].

Severe nosocomial infection carries a substantial economic burden owing to extensive antimicrobial consumption and, even more importantly, increased length of hospitalisation [10][11][12][13]. In addition, severe infection seriously compromises the likelihood of survival. Attributable mortality rates vary from as low as 0% to a dramatic 50% depending of the type of infection, the causative pathogen, patient age, associated co-morbidities and overall quality of the anti-infective approach [13][14][15][16][17][18][19][20][21]. Among all the processes that should be fulfilled with the aim of optimising clinical patient outcome, early initiation of the proper antimicrobial agent is a cornerstone [1].

Failure to administer appropriate antimicrobial therapy in a timely manner results in a dramatic increase in the fatality rate [22][23][24][25]. However, selecting an empirical regimen that covers the causative pathogen is hampered by the presence of multidrug-resistant (MDR) microorganisms, which is the most important cause of inappropriate empirical therapy. Since antimicrobial consumption in itself is a major trigger for the development of multidrug resistance, physicians are frequently urged A c c e p t e d M a n u s c r i p t 6 to use 'last-line antibiotics' carefully. In fact, the challenge is to achieve high rates of appropriate empirical therapy in order to optimise patient survival whilst avoiding unnecessary use of antibiotics with the intention to minimise microbial selection pressure and hence development of multidrug resistance [1,26].

To accomplish this goal, in the past decade two main strategies have been proposed.

In the surveillance-assisted strategy, the empirical regimen is selected mainly based on the presence or absence of MDR pathogens in routine surveillance cultures. The strength of this approach is the high negative predictive value of surveillance cultures in predicting the involvement of MDR microorganisms in subsequent infection. On the condition that cultures are taken at least twice weekly, this strategy is able to combine high rates of appropriate empirical therapy with reduced consumption of antibiotics in pneumonia and bacteraemia [27][28][29][30][31][32][33]. However, the cost effectiveness of this approach remains questionable. Another approach is the so-called 'deescalation strategy' that, on the condition of risk factors for MDR involvement, recommends initiation of empirical therapy with a regimen that covers most potential MDR pathogens such as extended-spectrum -lactamase (ESBL)-producing Enterobacteriaceae, non-fermenting Gram-negative bacteria and meticillin-resistant

Staphylococcus aureus (MRSA) [34][35][36]. Once culture results are available, and if feasible, narrowing the antimicrobial spectrum is advised. This concept, often referred to as 'de-escalation', is widely used and advocated [37][38][39][40]. This strategy has been demonstrated to be successful and safe and may also reduce antibiotic use and hence limit the emergence of multidrug resistance [36,41].

A c c e p t e d M a n u s c r i p t

Methods

Study design

A prospective, multicentre observational study was performed between February 2006 and June 2007. In total, 24 Belgian ICUs participated. Informed consent was requested from all patients. In all centres, antimicrobial prescribing was done or supervised by the attending senior intensive care physician.

Inclusion criteria

Eligible patients were those who provided informed consent, were at least 18 year of age, hospitalised in the ICU and experienced severe hospital-acquired infection, either pneumonia, intra-abdominal infection, primary bloodstream infection or secondary bloodstream infection originating from a source other than pneumonia or intra-abdominal infection (e.g. bacteraemia secondary to urinary tract infection or sinusitis).

A c c e p t e d M a n u s c r i p t 8

Definitions

Infections were considered hospital-or ICU-acquired when they were diagnosed >48 h after hospital or ICU admission, respectively. Severe bacterial infections were defined following the International Sepsis Forum Consensus Conference on Definitions of Infection in the ICU [42]. Definitions of invasive fungal infections are described elsewhere [43,44]. Pneumonia was considered ventilator-associated when occurring after >48 h of mechanical ventilation. For the purpose of the study, only the first episode per patient was considered. Pneumonia or intra-abdominal infections with bacteraemic breakthrough (i.e. positive blood cultures) were classified according to the primary infection. Bloodstream infection secondary to sources other than pneumonia or intra-abdominal infection were classified as secondary bloodstream infection.

Definitions of multidrug resistance are described elsewhere [10,32,45]. Candida spp.

were considered MDR when resistant to fluconazole.

Following the epidemiological scope of the study aiming at insights into empirical therapy in general, we also wanted to include microbiologically undocumented infections with obvious signs of clinical sepsis. For this purpose, a difference was made between appropriate and adequate empirical therapy. The term appropriate therapy was valid for microbiologically documented infectious episodes and was defined as in vitro susceptibility of the causative pathogen and clinical response to the agent administered. The term adequate therapy was used in nonmicrobiologically documented infections and was defined as favourable clinical response within 5 days of therapy (resolution of signs of sepsis). To assess relationships between empirical antibiotic selection and rates of appropriate or adequate therapy, the empirical antimicrobial schemes were grouped according to the spectrum of pathogens covered.

Results

Demographics

During the study period, 198 patients were included. Owing to incomplete patient files, 27 patients were excluded, resulting in a final database containing 171 patients.

Patient characteristics are described in Table 2. Primary infections were pneumonia and bloodstream infection (n = 21, of which 9 were primary, 1 was catheter-related and 11 secondary to a source other than pneumonia or intra-abdominal infection).

One hundred and fifteen infections (67.3%) were ICU-acquired. Approximately 75% of infections were microbiologically documented (Fig. 1). This represented 129 microbiologically documented infections in which 165 microorganisms were isolated.

Risk factors for multidrug resistance

Risk factors for multidrug resistance were assessed in order to evaluate empirical antimicrobial regimens relative to their presence.

Rates of empirical appropriate or adequate therapy and subsequent adaptation

The presence of MDR pathogens in infections was associated with a significant lower rate of empirical appropriate therapy (87.7% vs. 35.9%; P < 0.001). Rates of empirical appropriate and adequate therapy are illustrated in Fig. 3. Empirical therapy was appropriate in 80 (62.0%) of the 129 microbiologically documented infections. In the 42 non-microbiologically documented infections, empirical therapy was judged adequate in 29 (69.0%). Empirical therapy was adapted in 106 patients (62.0%), in 60 cases following initial inappropriate or inadequate therapy and in 46 patients to fine-tune empirical therapy.

Empirical antimicrobial selection and rates of appropriate or adequate therapy

In 12 infections the causative pathogen was meticillin-resistant (10 MRSA and 2 meticillin-resistant Staphylococcus epidermidis). Vancomycin was administered 14 times as an empirical agent, but in only 3 cases in which a meticillin-resistant pathogen proved to be involved. As such, the rate of inappropriate therapy in the A c c e p t e d M a n u s c r i p t 13 case of meticillin resistance involvement was 75%. Owing to the relative low prevalence of meticillin resistance as well as the low added value of initiating a glycopeptide, the association (or not) of such an agent did not alter the study results in this particular cohort. Therefore, rates of empirical appropriate or adequate therapy were analysed irrespective of the coverage of MRSA. This is illustrated in Fig. 4a.

Coverage of ESBL-producing Enterobacteriaceae (by meropenem-based empirical schemes) allowed for significantly higher rates of appropriate or adequate therapy.

Because the advantage of an antibiotic agent that covers ESBL-producing

Enterobacteriaceae is obvious in settings with a high prevalence of multidrug resistance, it was hypothesised that the benefit of using a meropenem-based empirical scheme would fade away when only patients without risk factors for MDR were taken into account. However, in this analysis empirical schemes covering ESBL-producing Enterobacteriaceae remained superior (Fig. 4b).

When as a major determinant of inappropriate therapy [47][48][49][50][51][52]. However, to a certain extent the involvement of multidrug resistance can be predicted on the basis of some typical risk factors, of which the most essential are prior antibiotic exposure and length of ICU stay (or mechanical ventilation in the case of pneumonia) of >7 days [32,53].

Other risk factors, such as a particular underlying diseases (e.g. chronic obstructive pulmonary disease) and recent surgery/hospitalisation, can also be taken into account but generally are not included in the principal risk factors. Strangely enough, in this cohort of ICU patients with nosocomial infection some of the classic patterns failed to predict the involvement of multidrug resistance. For instance, prior hospitalisation (within 4 months of the current hospital admission) was not associated with a higher likelihood of multidrug resistance, whilst length of ICU stay was even A c c e p t e d M a n u s c r i p t 15 inversely related to MDR involvement. On the other hand, prior antibiotic exposure and hospitalisation in a general ward prior to ICU admission appeared to have a significant relationship with MDR involvement. Importantly, however, although these relationships were statistically significant, MDR pathogens were isolated in ca. 40% of patients in the absence of these risk factors. As a consequence, the predictive value of these risk factors was low. As such, the generally accepted concepts of risk perception for MDR involvement failed in this particular cohort. We have no plausible explanation for this observation. Given the relationship between prior hospitalisation in a general ward and multidrug resistance, the relatively high number of infections already present at the time of ICU admission and the short length of stay in the ICU prior to infection, it appears that there exists a serious problem of multidrug resistance in general wards in Belgian hospitals, thereby contributing to the low rate of empirical appropriate therapy in ICUs. Indeed, already a decade ago a higher incidence of E. aerogenes with an increasing resistance pattern had been noticed in Belgian hospitals [54,55].

Following the high prevalence rate of ESBL-producing Enterobacteriaceae and P.

aeruginosa, a meropenem-based empirical scheme allowed for the highest rates of appropriate/adequate therapy. Owing to the failure of classic risk factors to predict MDR involvement (Fig. 2), performance rates of empirical schemes did not substantially alter when only patients without risk factors were considered (Fig. 4b).

Hence, based on the present study, an approach including coverage both of ESBLproducing Enterobacteriaceae and P. aeruginosa seems highly warranted. Without firm supportive knowledge of local ecology in ICU and non-ICU settings indicating low MDR levels, an empirical strategy that does not cover ESBL-producing Resistance in German ICUs (SARI project), Meyer et al. [57] identified carbapenem use as an independent predictor for a higher Stenotrophomonas maltophilia incidence. Although these data do not disclose a causal relationship, they are highly suggestive for the specific selection pressure of this pathogen. Therefore, excessive use of carbapenems is to be avoided by a strict de-escalation strategy and by avoidance of unnecessary long therapies.

The most important advantage of this study is its non-interventional design, reflecting real daily practice, thereby disclosing the true problem points in empirical antibiotic therapy in critically ill patients. Owing to the multicentre approach, the data might be valid for many tertiary care centres in Belgium and abroad.

However, the study has limitations. First, no outcome data are available as the primary aim of the study was to describe antibiotic prescription patterns and how they perform in terms of appropriate therapy. However, the relationship between failure of initial empirical therapy to cover the causative pathogen and adverse outcomes has been demonstrated repeatedly and is generally accepted as an important quality indicator [58,59]. Second, the cohort does not represent a consecutive series of ICU patients with nosocomial infections. It is possible that this might have led to a selection bias towards more severe infections, with a higher likelihood of MDR involvement.

In conclusion, in this prospective study reflecting real-life practice in ICU patients with nosocomial infections, the rate of appropriate or adequate empirical therapy was 63.7%. This study demonstrated that classic risk factors for multidrug resistance such a Data are n (%) unless otherwise stated.

A c c e p t e d M a n u s c r i p t 7 Methods

 7 Study design. A prospective multicenter observational study was performed betweenFebruary 2006 and June 2007. In total 24 Belgian ICUs participated. In all centers the study was approved by the local ethics committee and informed consent was requested. In all centers antimicrobial prescribing was done or supervised by the attending senior intensivist.

A

  c c e p t e d M a n u s c r i p t 10 assessed by means of a logistic regression analysis. Variables taken into account in the logistic regression analysis either showed a moderate relationship in univariate analysis or a logic relationship with the dependent variable. Variables considered were: age, APACHE II score, underlying diseases, hospitalization in another ward prior to ICU admission, prior antibiotic exposure, and empiric antibiotic schemes.Results of the regression analysis are reported as odds ratios (OR) and 95% confidence intervals (CI).

A

  c c e p t e d M a n u s c r i p t Results Demographics. During the study period 198 patients were included. Due to incomplete patient files 27 patients were excluded, resulting in a final database containing 171 patients. Patient characteristics are described in Table

A

  c c e p t e d M a n u s c r i p t antibiotic exposure (74.3% vs. 41.3%, p=0.001; relative risk 4.26, 95% CI: 1.80-10.09).

A c c e p t e d M a n u s c r i p t 14

 14 scheme (OR 18.1, 95% CI 4.8 to 68.1; p<0.001) and the presence of MDR pathogens

Funding:

  This study was sponsored by an unrestricted grant by AstraZeneca, Belgium. Dr. Blot is supported by a grant from the European Society of Intensive Care Medicine and iMDsoft Patient Safety Research Award 2008.Competing Interests: No conflicts of interest to declareEthical Approval: For this non-interventional, observational study, ethical approval was given by the local ethics committees of the participating centres (n=24).

A c c e p t e d M a n u s c r i p t 20 *

 20 The ANTHICUS Study Investigators are: O. Abid (RHMS, Ath, Belgium), S. Cran (CHIREC, Brussels, Belgium), D. De Bels (CHU Brugmann, Brussels, Belgium), F. De Leener (CHR St. joseph-Warquignies, Boussu, Belgium), L. Finianos (Clinique St. Joseph, Liège, Belgium), F. Forêt (CHR St. Joseph-Warquignies, Mons, Belgium), M. Genard (CIU Ambroise Paré, Mons, Belgium), E. Gilbert (CHR du Tournaisis, Tournai, Belgium), E. Khodadadi (IRIS Sud-Bracops, Brussels, Belgium), S. Machayekhi (CH Hornu Frameries, Hornu), D. Mircev (IRIS Sud-Etterbeek-Ixelles, Brussels, Belgium), D. Neuberg (CH de L'Ardenne, Libramont, Belgium), J.-Y. Piette (CH Bois de l'Abbaye, Seraign, Belgium), P. Serpe (CH ND Bruyères, Chenee, Belgium), F. Beernaert (H. Hart Ziekenhuis, Eeklo, Belgium), K. De Decker (Antwerp University Hospital, Antwerp, Belgium), K. De Ridder (AZ Maria Middelares, Sint-Niklaas, Belgium), C. Declercq (St. Josef Ziekenhuis, Izegem, Belgium), I. Demeyer (OLV Aalst, Aalst, Belgium), B. Nonneman (ASZ, Aalst, Belgium), K. Schoonheydt (ZNA, Antwerp, Belgium, W. Swinnen (AZ St. Blasius, Dendermonde, Belgium), L. Wosteyn (ASZ, Roeselaere, Belgium).

Figure 1 -Figure 2 -Figure 3 -

 123 Figure 1 -Review of 171 nosocomial infections distributed for microbiological

Figure 4 -

 4 Figure 4 -Rates of empiric appropriate or adequate therapy according to three major groups of empiric antibiotic regimens (coverage of methicillin-resistant pathogens not considered).

Figure 4a ,

 4a Figure 4a, all patients considered (n=170; in one patient no empiric therapy was initiated) Figure 4b, only patients considered without risk factors for multidrug resistance involvement (hospitalization at a general ward prior to ICU admission and prior antibiotic exposure), (n=78). Group 1: coverage of ESBL-Enterobacteriaceae + P. aeruginosa (meropenem-based schemes) Group 2: coverage of P. aeruginosa (schemes containing an antipseudomonal agent)

Figure 2

 2 Figure 2 Click here to download high resolution image

Figure 3

 3 Figure 3 Click here to download high resolution image

Methods

  Study design. A prospective multicenter observational study was performed betweenFebruary 2006 and June 2007. 24 Belgian ICUs participated. In all centres the study was approved by the local ethics committee and informed consent was requested. In all centers antimicrobial prescribing was by, or supervised by, the attending senior intensivist.Inclusion criteria. Eligible patients were those who gave informed consent, were at least 18 years of age, were hospitalized in the ICU, and experienced severe hospitalacquired infection; either pneumonia, intra-abdominal infection, primary or secondary bloodstream infection originating from a source other than pneumonia or intra-abdominal infection (e.g. bacteremia secondary to urinary tract infection or sinusitis).Definitions. Infections were considered hospital-or ICU-acquired when diagnosed at least 48 hours after hospital or ICU admission respectively. Severe bacterial infections were defined according to the International Sepsis Forum Consensus Conference on Definitions of Infection in the ICU[42]. Definitions of invasive fungal infections are described elsewhere[43, 44]. Pneumonia was considered ventilatorassociated when occurring after at least 48 hours of mechanical ventilation. For the purpose of the study, only the first episode per patient was considered. Pneumonia or intra-abdominal infection with bacteraemic breakthrough (i.e. positive blood cultures) were classified according to the primary infection. Bloodstream infections A c c e p t e d M a n u s c r i p t secondary to other sources than pneumonia or intra-abdominal infection were classified as secondary bloodstream infection. Definitions of MDR are described elsewhere[10, 32, 45]. Candida spp. were considered MDR when resistant to fluconazole.Following the epidemiological scope of the study, aiming at insights into empiric therapy in general, we also wanted to include microbiologically unconfirmed infections with obvious signs of clinical sepsis. For this purpose we made a difference between appropriate and adequate empiric therapy. The term appropriate therapy was used for microbiologically documented infectious episodes and was defined as in vitro susceptibility of the causative pathogen with clinical response to the agent administered. The term adequate therapy was used in non-microbiologically confirmed infections, and was defined as favourable clinical response within five days of treatment (resolution of signs of sepsis).Severity of disease was assessed by means of the acute physiology and chronic health evaluation (APACHE) II score[46]. The following co-morbid conditions were recorded: respiratory disease (chronic restrictive, obstructive or pulmonary vascular disease resulting in severe exercise restriction), cardiac disease (New York Heart Association Class IV), diabetes mellitus, hepatic disease (cirrhosis and portal hypertension; episodes of past upper gastro-intestinal bleeding attributed to portal hypertension or prior episodes of hepatic failure or encephalopathy), renal disease (chronic glomerulonephritis, nephropathy, or chronic kidney disease), neurologic A c c e p t e d M a n u s c r i p t disease (impairment of alertness or confusion) malignancy (haematologic or solid tumor), and neutropenia (absolute neutrophil count <1500 cells/mm³).

  schemes were classified in three major groups: (i) coverage of ESBL-producing

A c c e p t e d M a n u s c r i p t 13 in

 13 case of meticillin-resistance involvement was thus 75%. Due to the relative low prevalence of meticillin-resistance and the low added value of initiating a

A c c e p t e d M a n u

  s c r i p t therapy was 63.7%. This study demonstrated that classic risk factors for MDR such as prior antibiotic exposure and length of ICU stay, may be insufficient to predict MDR involvement. As such, empiric first-line use of MER allowed for significantly higher rates of appropriate or adequate therapy, irrespective of presence of these risk factors, and may be recommended in settings with a high prevalence of MDR pathogens. In addition, these data illustrate the necessity for strict infection prevention and control.

Figure 1 -Figure 3 -Figure 2 -

 132 Figure 1 -Review of 171 nosocomial infections distributed for microbiological

Figure 4 -

 4 Figure 4 -Rates of empiric appropriate or adequate therapy according to three major groups of empiric antibiotic regimens (coverage of meticillin-resistant pathogens not considered).

Figure 4a ,A c c e p t e d M a n u s c r i p t 25 Group 2 :

 4a252 Figure 4a, all patients considered (n=170; in one patient no empiric therapy was initiated) Figure 4b, only patients considered without risk factors for multidrug resistance involvement (hospitalization at a general ward prior to ICU admission and prior antibiotic exposure), (n=78). Group 1: coverage of of ESBL-producing and Pseudomonas aeruginosa (meropenem-based schemes)

A c c e p t e d M a n u s c r i p t

  onset of infection (days) 3.8 (0 -6) Data are described as n (%) or median (interquartile range)

  Figure 1 Click here to download high resolution image

Figure 4 Click here to download high resolution image A c c e p t e d M a n u s c r i p t 4 inappropriate

 44 Figure 4 Click here to download high resolution image

A c c e p t e d M a n u s c r i p t 9 Severity

 9 of disease was assessed by means of the Acute Physiology and Chronic Health Evaluation (APACHE) II score[46]. The following co-morbid conditions were registered: respiratory disease (chronic restrictive, obstructive or pulmonary vascular disease resulting in severe exercise restriction); cardiac disease (New York Heart Association Class IV); diabetes mellitus; hepatic disease (cirrhosis and portal hypertension, episodes of past upper gastrointestinal bleeding attributed to portal hypertension or prior episodes of hepatic failure or encephalopathy); renal disease (chronic glomerulonephritis, nephropathy or chronic kidney disease); neurological disease (impairment of alertness or confusion); malignancy (haematological cancer or solid tumour); and neutropenia (absolute neutrophil count <1500 cells/mm 3 ).

A c c e p t e d M a n u s c r i p t 11 (

 11 n = 127, of which 66 were ventilator-associated), intra-abdominal infection (n = 23)

  Gram-negative bacteria were most common (n = 122; 73.9%), with Pseudomonas aeruginosa (n = 29), Escherichia coli (n = 26), Enterobacter aerogenes (n = 21), Klebsiella pneumonia (n = 7) and Klebsiella oxytoca (n = 7) being the predominant pathogens. Among the 42 Gram-positive bacteria, S. aureus (n = 22), Streptococcus pneumoniae (n = 11) and enterococci (n = 5) were most frequently isolated. One fungal pathogen was isolated. MDR pathogens were involved in 64 (49.6%) of the 129 microbiologically documented infections. Fig. 1 shows the breakdown of the different types of resistance involved in these infections.

A c c e p t e d M a n u s c r i p t 12 microbiologically

 12 documented infections according to the presence of these two risk factors for multidrug resistance and subsequent MDR involvement.Length of ICU stay prior to the development of infection was negatively associated with the risk of MDR involvement. Patients who experienced infections without MDR pathogens developed this infection after a median of 3 days (IQR 2-7 days), whilst infections caused by MDR pathogens occurred after a median of 2 days (IQR 0-4.25 days) (P = 0.002).

Fig. 1 .

 1 Fig. 1. Review of 171 nosocomial infections by microbiological documentation and

Fig. 2 .

 2 Fig. 2. Breakdown of microbiologically documented infections according to the

Fig. 3 .

 3 Fig. 3. Rates of empirical appropriate or adequate therapy in Intensive Care Unit

Fig. 4 .

 4 Fig. 4. Rates of empirical appropriate or adequate therapy according to three major

A c c e p t e d M a n u s c r i p t 1 1

 11 Table Empirical antibiotic regimens clustered in six major groups according to the

Table 1 -Empiric antibiotic regimens clustered in six major groups according to the pathogens covered.

 1

	Coverage

  Dirk Vogelaers 1 , PhD, David De Bels 2 , MD, Frédéric Forêt 3 , MD, Sophie Cran 4 , MD,

	revised MS
	Abstract
	This prospective, observational multicenter (n=24) study investigated relationships
	between antimicrobial choices and rates of empiric appropriate or adequate therapy,
	and subsequent modification of therapy in 171 ICU patients with severe nosocomial
	Eric Gilbert 5 , MD, Karen Schoonheydt 6 , MD, and Stijn Blot 1 , PhD, for the infections. Appropriate antibiotic therapy was defined as in-vitro susceptibility of the
	ANTHICUS Study Investigators* 1 General Internal Medicine & Infectious Diseases, Ghent University Hospital, Ghent, Affiliations: Belgium M a n u s c r i p t causative pathogen and clinical response to the agent administered. In non-microbiologically documented infections, therapy was considered adequate in the case of favorable clinical response <5 days. Patients had pneumonia (n=127; 66 ventilator-associated), intra-abdominal infection (n=23), and bloodstream infection appropriate therapy M a n u s c r i p t
	Word count: 3146 Address of correspondence: Prof. Dr. S. Blot General Internal Medicine & Infectious Diseases Ghent University Hospital De Pintelaan 185 9000 Ghent, Belgium T: +32 9 332 62 16 A c c e p t e d negative bacteria ("meropenem-based"), (ii) non-fermenting Gram-negative bacteria (schemes with an antipseudomonal agent), and (iii) first-line agents not covering A c c e p t e d
	stijn.blot@UGent.be ESBL-Enterobacteriaceae nor non-fermenting Gram-negative bacteria. Meropenem-
	based schemes allowed for significantly higher rates of appropriate/adequate
	therapy (p<0.001). This benefit remained when only patients without risk factors for
	2

2 Intensive Care Dept., Brugmann University Hospital, Brussels, Belgium 3 Dept. of Internal Medicine -Intensive Care Unit, CHR St. Joseph-Warquignies, Boussu, Belgium 4 Intensive Care Dept., CHIREC, Brussels Belgium 5 Intensive Care Dept., CHR du Tournaisis, Tournai, Belgium 6 Intensive Care Dept., Ziekenhuisnetwerk Antwerpen, Antwerp, Belgium (n=21). Predominant pathogens were Pseudomonas aeruginosa (n=29) Escherichia coli (n=26), Staphylococcus aureus (n=22), and Enterobacter aerogenes (n=21). In 49.6% of infections multidrug resistant (MDR) bacteria were involved, mostly extendedspectrum Beta-lactamase producing Enterobacteriaceae and MDR non-fermenting Gram-negative bacteria. Prior antibiotic exposure and hospitalization in a general ward prior to ICU admission were risk factors for MDR. Empiric therapy was appropriate/adequate in 63.7% of cases. Empiric schemes were classified according to coverage of (i) ESBL-producing Enterobacteriaceae and non-fermenting Gram-MDR were considered (p=0.021). In 106 patients (61%) empiric therapy was modified: in 60 cases following initial inappropriate/inadequate therapy, in 46 patients in order to refine empiric therapy. In this study which reflects real-life practice, first-line use of meropenem provided significantly higher rates of the appropriate/adequate therapy, irrespective of presence of risk factors for MDR. Keywords: antibiotic resistance -multidrug resistance -intensive care -infection -Introduction Avoiding nosocomial infection remains a daily challenge for healthcare workers

  objective of the present study was prospectively to investigate patterns of antimicrobial therapy in critically ill patients with nosocomial infection. More precisely we formulated the following research questions: (i) What is the rate of empiric appropriate or adequate therapy achieved? (ii) Which antibiotic selections allow for the highest rates of empiric appropriate or adequate therapy, either in the presence or absence of risk factors for MDR pathogens? (iii) What is the rate by

which empiric therapy (either appropriate/adequate or not) is subsequently modified?

  MDR involvement (59.0% vs. 40.9%, p=0.041; relative risk 2.08, 95% CI: 1.03-4.22).Figure2shows a breakdown of microbiologically documented infections according to the presence of these two risk factors for MDR, and subsequent MDR involvement.

	analysis or a logic relationship with the dependent variable. Variables considered incomplete patient files 27 patients were excluded, resulting in a final database
	were: age, APACHE II score, underlying diseases, hospitalization in another ward containing 171 patients. Patient characteristics are given in Table 2. Primary
	M a n u s c r i p t prior to ICU admission, prior antibiotic exposure, and empiric antibiotic schemes. Results of the regression analysis are reported as odds ratios (OR) and 95% confidence intervals (CI). M a n u s c r i p t infections were pneumonia (n=127, of which 66 ventilator-associated), intra-abdominal infection (n=23) bloodstream infection (n=21, of which 9 primary, 1 catheter-related, and 12 secondary to a source other than pneumonia or intra-abdominal infection). 115 infections were ICU-acquired (66,7%). Approximately 75% Length of ICU stay prior to the development of infection, however, was negatively associated with the risk of MDR involvement. Patients who experienced infections without MDR pathogens developed this infection after a median of 3 days (IQR: 2-7 days), while infections caused by MDR pathogens occurred after a median of 2 days (IQR: 0-4.25 days), (p=0.002). a n u s c r i p t M empiric appropriate therapy: 87.7% vs. 35.9% (p<0.001). Rates of empiric appropriate
	A c c e p t e d A c c e p t e d Risk factors for multidrug resistance (MDR). Risk factors for MDR were assessed in order to evaluate empiric antimicrobial regimens relative to their presence. No and adequate therapy are illustrated in Figure 3. In 80 of the 129 microbiologically documented infections, empiric therapy was appropriate (62.0%). In non-microbiologically documented infections, empiric therapy was judged adequate in 29 of 42 infections (69.0%). In 106 patients (62.0%) empiric therapy was modified: in 60 cases following initial inappropriate or inadequate therapy, and in 46 patients in order to refine empiric therapy. A c c e p t e d specific underlying conditions appeared to predispose for MDR involvement. MDR Empiric antimicrobial selection and rates of appropriate or adequate therapy. In 12 infections
	Mann involvement was more frequent in patients with prior antibiotic exposure (74.3% vs. the causative pathogen was meticillin-resistant (10 MRSA, 2 meticillin-resistant
	Whitney U test and Fisher Exact test or Chi-square test were used as appropriate. 41.3%, p=0.001; relative risk 4.26, 95% CI: 1.80-10.09). Hospitalization in another Staphylococcus epidermidis). Vancomycin administered empirically in 14 cases, but in
	Independent relationships with empiric appropriate or adequate therapy were ward prior to ICU admission was also recognized as significantly associated with

10

assessed by means of a logistic regression analysis. Variables taken into account in the logistic regression analysis either showed a moderate relationship in univariate

Results

Demographics. During the study period 198 patients were included. Due to of infections were microbiologically documented (Figure

1

). This represented 129 microbiologically documented with 165 micro-organisms. Gram-negative bacteria were most common (n=122, 73.9%) with Pseudomonas aeruginosa (n=29), Escherichia coli (n=26), Enterobacter aerogenes (n=21), Klebsiella pneumonia (n=7), and Klebsiella oxytocca (n=7) as predominant pathogens. Among the 42 Gram-positive bacteria, Staphylococcus aureus (n=22), Streptococcus pneumoniae (n=11), and enterococci (n=5) were most frequently isolated. One fungal pathogen was isolated. In 64 of the 129 microbiologically documented infections MDR pathogens were involved (49.6%).

Rates of empiric appropriate or adequate therapy and subsequent modification. The presence of MDR pathogens in infections was associated with a significant lower rate of only was a meticillin-resistant pathogen involved. The rate of inappropriate therapy

Table 1 -Empiric antibiotic regimens clustered in six major groups according to the pathogens covered.

 1

	Coverage

Table 2 -Characteristics of 171 ICU patients with a nosocomial infection.

 2

	Characteristic
	Gender (male)

Table 2

 2

7

  The objective of the present study was prospectively to investigate patterns of antimicrobial therapy in critically ill patients with nosocomial infection. More precisely, the following research questions were formulated. (i) What is the rate of empirical appropriate or adequate therapy achieved? (ii) Which antibiotic selections allow for the highest rates of empirical appropriate or adequate therapy, either in the presence or absence of risk factors for MDR pathogens? And (iii) what is the rate by which empirical therapy (either appropriate/adequate or not) is adapted?

Table 1

 1

	describes the classification

Table 2

 2 Characteristics of 171 Intensive Care Unit (ICU) patients with nosocomial infections

	Characteristic	n (%) a
	Gender (male)	107 (62.6)
	Age (years) [median (IQR)]	67 (55-76)
	APACHE II score [median (IQR)] Recent trauma/surgery Co-morbidities Respiratory disease Cardiac disease Neurological disease Diabetes mellitus Hepatic disease Renal disease Malignancy Neutropenia Corticosteroid therapy	20 (15-24) 39 (22.8) 139 (81.3) 66 (38.6) 59 (34.5) 28 (16.4) 36 (21.1) 11 (6.4) 20 (11.7) a n u s c r i p t 28 (16.4) 1 (0.6) M 20 (11.7)
	Prior hospitalisation Hospitalisation at another ward prior to ICU admission Prior antibiotic use ICU-acquired infection A c c e p t e d ICU stay before onset of infection (days) [median (IQR)] 3.8 (0-6) 51 (29.8) 84 (49.1) 49 (28.7) 115 (67.3)
		1

ESBL, extended-spectrum -lactamase; MRSA, meticillin-resistant Staphylococcus aureus; MDR, multidrug-resistant; MER, meropenem; VAN, vancomycin; AMI, aminoglycoside; OXA, oxacillin; CIP, ciprofloxacin; PIP/TAZ, piperacillin/tazobactam; LEV, levofloxacin; CAZ, ceftazidime; CEP, cefepime; FLU, fluconazole; AMC, amoxicillin/clavulanic acid; MOX, moxifloxacin; CLA, clarithromycin; AZT, aztreonam; MTR, metronidazole; CEF, cefuroxime; CFX, ceftriaxone; TMO, temocillin. IQR, interquartile range; APACHE, Acute Physiology and Chronic Health Evaluation.

Funding: This study was sponsored by an unrestricted grant by AstraZeneca, Belgium. Dr. Blot is supported by a grant from the European Society of Intensive Care Medicine and iMDsoft Patient Safety Research Award 2008. Funding This study was sponsored by an unrestricted grant from AstraZeneca (Belgium). SB is supported by a grant from the European Society of Intensive Care Medicine and iMDsoft Patient Safety Research Award 2008.

A c c e p t e d M a n u s c r i p t 15 Indeed, a decade ago a higher incidence of Enterobacter aerogenes with an increasing resistance pattern was noticed in Belgian hospitals [54,55].

Following the high rate of of ESBL-producing Enterobacteriaceae and P. aeruginosa, a MER-based empiric scheme gave the highest rates of appropriate/adequate therapy.

Due to the failure of classic risk factors to predict MDR involvement, performance of empiric schemes did not substantially alter when only patients without risk factors were examined (Figure 2). Hence, the present study suggests coverage of both of ESBL-producing Enterobacteriaceae and P. aeruginosa is warranted and supports the de-escalation strategy. The present findings however, with the failure of MDR prediction based on general characteristics in particular, provide perspectives for the surveillance assisted approach in which individual colonization status is a major element in steering empiric therapy.

In 62% (n=106) of patients empiric therapy was modified. In 60 cases modification followed initial inappropriate or inadequate therapy, in the other 46 patients empiric therapy could be refined (true de-escalation). It is noteworthy that when only microbiologically documented infections are considered, four of 129 patients received inappropriate therapy even after culture results were available (Figure 1). In MER-based empiric schemes (n=37) therapy was continued in 23 and modofied in 14 patients (in four cases because of initial inappropriate therapy and in 10 to narrow the spectrum). This supports the approach of empiric antibiotic coverage followed by de-escalation. Likweise Baran et Prior antibiotic exposure and hospitalisation in a general ward prior to ICU admission were risk factors for multidrug resistance. Empirical therapy was appropriate/adequate in 63.7% of cases. Empirical schemes were classified according to coverage of (i) ESBL-producing Enterobacteriaceae + non-fermenting Gram-negative bacteria (meropenem-based), (ii) non-fermenting Gram-negative bacteria (schemes with an antipseudomonal agent) and (iii) first-line agents not covering ESBL-producing Enterobacteriaceae or non-fermenting Gram-negative bacteria. Meropenem-based schemes allowed for significantly higher rates of appropriate/adequate therapy (P < 0.001). This benefit remained when only patients without risk factors for MDR infection were considered (P = 0.021). Empirical therapy was adapted in 106 patients (62%), in 60 cases following initial Enterobacteriaceae and P. aeruginosa is potentially dangerous and as such supports the de-escalation strategy. However, the present findings, with the failure of MDR prediction based on general characteristics in particular, provide perspectives for the surveillance-assisted approach in which individual colonisation status is a major element in steering empirical therapy.

Empirical therapy was adapted in 62% of patients (n = 106). In 60 cases adaptation followed initial inappropriate or inadequate therapy, whereas in 46 patients empirical therapy could be fine tuned (true de-escalation). Of note is that when only microbiologically documented infections are considered, 4 of 129 patients received inappropriate therapy even after the availability of culture results (Fig. 3). In meropenem-based empirical schemes (n = 37), therapy was continued in 23 patients and adapted in 14 patients (in 4 cases because of initial inappropriate therapy and in 10 cases to narrow down the spectrum). As this study supports the approach of empirical coverage of ESBL-producing Enterobacteriaceae and P. aeruginosa, we stress the importance of de-escalation whenever possible. Baran et al. [56] reported that the rate of previous exposure to carbapenems was 44% in patients with infections caused by imipenem-resistant Acinetobacter baumannii whereas it was only 12% in patients infected by imipenem-susceptible A. baumannii isolates. Yet in multivariate analysis, previous antibiotic exposure and not carbapenem exposure in particular appeared to be an independent risk factor for imipenem resistance. The link between exposure and specific resistance development is obvious and should not be disregarded, but this suggests that the mission to reduce microbial selection pressure is valid for all antimicrobial agents and not only for carbapenems. As such, empirical first-line use of meropenem allowed for significantly higher rates of appropriate or adequate therapy, irrespective of the presence of these risk factors, and may be recommended in settings with a high prevalence of MDR pathogens. In addition, these data illustrate the necessity for strict infection prevention and control.

Furthermore, based on the Surveillance of Antimicrobial Use and Antimicrobial

Competing interests

None declared.

Ethical approval

Ethical approval was given by the local ethics committees of the participating centres (n = 24).


M Mar Rodríguez

F Javier Pastor

Carolina Serena

Josep Guarro
email: josep.guarro@urv.cat

Efficacy of voriconazole in a murine model of invasive paecilomycosis

Keywords:

We studied the efficacy of voriconazole (VRC) and amphotericin B (AMB) in an immunosuppressed murine model of disseminated infection by two strains of Paecilomyces lilacinus. Mice were treated with VRC 60 mg/kg/day orally or AMB 3 mg/kg/day intraperitoneally, beginning 1 day after infection and continuing for 9 days. To avoid rapid clearance of VRC, animals receiving VRC and the control group were given grapefruit juice instead of water. VRC significantly prolonged survival with respect to the group treated with AMB and the control group for both strains (P = 0.005 and P < 0.0001, respectively, for strain FMR 5522; and P = 0.0002 and P < 0.0001, respectively, for strain FMR 8252). VRC reduced the fungal load in the spleen, kidneys and liver of infected mice for both strains tested. Survival of mice challenged with strain FMR 8252 treated with AMB did not differ from that of the control group (P = 0.223), being worse than that of the mice treated with VRC (P = 0.0002). AMB was not able to reduce the tissue burden in any organ with respect to the control group for both strains studied.

A c c e p t e d M a n u s c r i p t

Introduction

Paecilomyces lilacinus is an opportunistic filamentous fungus causing severe infections both in immunocompetent and immunocompromised patients [START_REF] Pastor | Clinical manifestations, treatment and outcome of Paecilomyces lilacinus infections[END_REF][START_REF] Carey | Paecilomyces lilacinus vaginitis in an immuno-competent patient[END_REF][START_REF] Castro | Hyalohyphomycosis by Paecilomyces lilacinus in a renal transplant recipient and a review of human Paecilomyces species infections[END_REF][START_REF] Itin | Cutaneous manifestations of Paecilomyces lilacinus infection induced by a contaminated skin lotion in patients who are severely immunosuppressed[END_REF].

The most frequent clinical manifestations are oculomycosis and cutaneous and subcutaneous infections [START_REF] Gutierrez-Rodero | Cutaneous hyalohyphomycosis caused by Paecilomyces lilacinus in an immunocompetent host successfully treated with itraconazole: case report and review[END_REF][START_REF] Orth | Outbreak of invasive mycoses caused by Paecilomyces lilacinus from a contaminated skin lotion[END_REF][START_REF] Minogue | Cure of Paecilomyces endophthalmitis with multiple intravitreal injections of amphotericin B. Case report[END_REF][START_REF] D'mellow | Intralenticular infections[END_REF][START_REF] Hirst | Non-traumatic mycotic keratitis[END_REF][START_REF] Domniz | Successful treatment of Paecilomyces lilacinus endophthalmitis after foreign body trauma to the cornea[END_REF][START_REF] O'sullivan | Outcome of Paecilomyces species ophthalmic infections treated with systemic voriconazole[END_REF][START_REF] Anderson | Fungal keratitis caused by Paecilomyces lilacinus associated with a retained intracorneal hair[END_REF][START_REF] Okhravi | Paecilomyces lilacinus endophthalmitis with secondary keratitis[END_REF][START_REF] Pintor | Endophthalmitis due to Paecilomyces lilacinus after non-surgical penetrating trauma [in Spanish][END_REF][START_REF] Hecker | Cutaneous Paecilomyces lilacinus infection: report of two novel cases[END_REF][START_REF] Gottlieb | Case report. Successful treatment of cutaneous Paecilomyces lilacinus infection with oral itraconazole in an immune competent host[END_REF][START_REF] Hall | Cutaneous hyalohyphomycosis caused by Paecilomyces lilacinus: report of three cases and review of the literature[END_REF][START_REF] Itin | Cutaneous manifestations of Paecilomyces lilacinus infection induced by a contaminated skin lotion in patients who are severely immunosuppressed[END_REF][START_REF] Yuan | Pathogenesis and outcome of Paecilomyces keratitis[END_REF]. Less frequently, however, this fungus can also disseminate, probably due to this ability to sporulate in tissue, producing numerous conidia that spread haematogenously [START_REF] Pastor | Clinical manifestations, treatment and outcome of Paecilomyces lilacinus infections[END_REF][START_REF] Liu | Morphologic criteria for the preliminary identification of Fusarium, Paecilomyces, and Acremonium species by histopathology[END_REF].

In ophthalmic and skin infections caused by this fungus, several drugs have been used, generally topically, but the optimal treatment for P. lilacinus infection

has not yet been established. Amphotericin B (AMB) is probably the most commonly used drug for treating this infection, with most of the results being negative or unknown [START_REF] Orth | Outbreak of invasive mycoses caused by Paecilomyces lilacinus from a contaminated skin lotion[END_REF][START_REF] D'mellow | Intralenticular infections[END_REF][START_REF] Domniz | Successful treatment of Paecilomyces lilacinus endophthalmitis after foreign body trauma to the cornea[END_REF][START_REF] Hecker | Cutaneous Paecilomyces lilacinus infection: report of two novel cases[END_REF][START_REF] Clark | Paecilomyces lilacinus infection in a heart transplant recipient and successful treatment with terbinafine[END_REF][START_REF] Jade | Paecilomyces lilacinus cellulitis in an immunocompromised patient[END_REF][START_REF] Murciano | Paecilomyces lilacinus infection in an immunocompromised patient[END_REF][START_REF] Legeais | Severe keratomycosis. Diagnosis and treatment [in French][END_REF][START_REF] Scott | Exogenous endophthalmitis caused by amphotericin B-resistant Paecilomyces lilacinus: treatment options and visual outcomes[END_REF][START_REF] Martin | Voriconazole treatment of disseminated Paecilomyces infection in a patient with acquired immunodeficiency syndrome[END_REF], in agreement with the poor in vitro response of P. lilacinus to this antifungal drug [START_REF] Pastor | Clinical manifestations, treatment and outcome of Paecilomyces lilacinus infections[END_REF][START_REF] Aguilar | Antifungal susceptibilities of Paecilomyces species[END_REF][START_REF] Espinel-Ingroff | In vitro fungicidal activities of voriconazole, itraconazole, and amphotericin B against opportunistic moniliaceous and dematiaceous fungi[END_REF]. There is little data on the activity of the newer triazoles either in vitro [START_REF] Espinel-Ingroff | In vitro fungicidal activities of voriconazole, itraconazole, and amphotericin B against opportunistic moniliaceous and dematiaceous fungi[END_REF][START_REF] Espinel-Ingroff | Optimal testing conditions for determining MICs and minimum fungicidal concentrations of new and established antifungal agents for uncommon molds: NCCLS collaborative study[END_REF] or in animal models [START_REF] Pastor | Clinical manifestations, treatment and outcome of Paecilomyces lilacinus infections[END_REF][START_REF] Rodríguez | Posaconazole efficacy in a murine disseminated infection caused by Paecilomyces lilacinus[END_REF], but the available data are promising. Although there is limited clinical experience in the use of voriconazole (VRC), this drug appears to be effective in the treatment of this fungal infection [START_REF] Pastor | Clinical manifestations, treatment and outcome of Paecilomyces lilacinus infections[END_REF][START_REF] Van Schooneveld | Paecilomyces lilacinus infection in a liver transplant patient: case report and review of the literature[END_REF]. The aim of this study was to evaluate the efficacy of VRC in a disseminated P. lilacinus infection in neutropenic mice.

Materials and methods

Isolates FMR 5522 and FMR 8252, from knee joint fluid and of environmental origin, respectively, were used in this study. Isolates were identified following A c c e p t e d M a n u s c r i p t 4 morphological criteria [START_REF] De Hoog | Atlas of clinical fungi[END_REF] and by sequencing several genes. Their in vitro susceptibility to AMB and VRC was tested using the broth microdilution method following Clinical and Laboratory Standards Institute guidelines for filamentous fungi [START_REF]Reference method for broth dilution antifungal susceptibility testing of filamentous fungi: approved standard[END_REF]. Isolates were stored at -80 C and prior to testing they were subcultured on potato dextrose agar (PDA) at 30 C. On the day of infection, cultures on PDA were suspended in sterile saline and filtered through sterile gauze to remove clumps of spores or hyphae. The resulting suspensions were adjusted to the desired inoculum based on haemocytometer counts and by serial plating on PDA to confirm viability.

Male OF1 mice (Charles River, Criffa S.A., Barcelona, Spain) with a mean weight of 30 g were used. Animals were housed in standard boxes with corncob bedding and free access to food and water. Two markers of efficacy were used, namely prolongation of survival and reduction in tissue burden. The former was evaluated through a lethal infection attained by using severe immunosuppression, and the latter through a sublethal infection attained by using moderate immunosuppression. Both regimens had been selected in a previous study [START_REF] Rodríguez | Posaconazole efficacy in a murine disseminated infection caused by Paecilomyces lilacinus[END_REF].

For the survival study, mice were challenged with 1.2  10 8 colony-forming units (CFU)/mouse for strain FMR 5522 or 0.6  10 8 CFU/mouse for strain FMR 8252 [START_REF] Rodríguez | Posaconazole efficacy in a murine disseminated infection caused by Paecilomyces lilacinus[END_REF]. Severe immunosuppression was reached with a dose of cyclophosphamide prior to the infection to achieve moderate immunosuppression. For this study, animals were challenged with 1.2  10 7 CFU/mouse of strain FMR 5522 or 0.6 10 7 CFU/mouse of strain FMR 8252 [START_REF] Rodríguez | Posaconazole efficacy in a murine disseminated infection caused by Paecilomyces lilacinus[END_REF]. Ten mice were used for survival studies and ten for tissue burden studies, the latter group being identified before the study started. AMB (Fungizona ® ; Squibb Industria Farmacéutica S.A., Barcelona, Spain) was administered i.p. at a dose of 3 mg/kg body weight/dose once daily [START_REF] D'mellow | Intralenticular infections[END_REF]. VRC (Vfend ® ; Pfizer Inc., Madrid, Spain) was administered orally at a dose of 60 mg/kg body weight/dose once daily. From 3 days prior to infection, mice receiving VRC and the control group were given grapefruit juice (Hero España, Murcia, Spain) instead of water to block VRC metabolism and to increase its serum concentration in mice to suitable levels for performing treatment studies [START_REF] Sugar | Efficacy of voriconazole in treatment of murine pulmonary blastomycosis[END_REF][START_REF] Graybill | Improving the mouse model for studying the efficacy of voriconazole[END_REF]. The selected dose of VRC has been shown previously to deliver adequate plasma levels in mice when co-administered with grapefruit juice [START_REF] Rodríguez | Effects of double and triple combinations of antifungal drugs in a murine model of disseminated infection by Scedosporium prolificans[END_REF].

Preliminary experiments with strain FMR 5522 (data not shown) demonstrated that VRC 60 mg/kg/day significantly prolonged the survival of mice in comparison with VRC 40 mg/kg and the control group; survival of mice treated with VRC 40 mg/kg was not different from that of the untreated control group. Therefore, only the higher dose was tested in this study. All mice received ceftazidime (5 mg/day subcutaneously) from Days 1 to 10 after infection to prevent bacterial infection. All treatments began 24 h after challenge and the therapies lasted for 10 days. Control animals received no treatment. Survival of mice was evaluated daily for 20 days. For tissue burden studies, mice were sacrificed on Day 11 post infection. Livers, spleens and kidneys were removed

Results

Minimal inhibitory concentrations were 32 g/mL for AMB and 0.5 g/mL for VRC for both strains. Fig. 1 shows the results of the survival studies. VRC 60 mg/kg/day significantly prolonged survival with respect to the group treated with AMB and the control group for both strains. Fig. 2 shows that with both strains the fungal load was at least two log units higher in the spleen and liver than in the kidneys. For strain FMR 8252, VRC 60 mg/kg/day was able to reduce significantly the fungal load with respect to the control group and the group receiving AMB in all three organs (Fig. 2d-f). For strain FMR 5522, VRC 60 mg/kg/day was able to reduce significantly the fungal load with respect to the control group and the group receiving AMB in the spleen and kidneys only (Fig. 2a-c).

Discussion

In this study, we investigated the effect of VRC against a P. lilacinus infection using the same murine model where previously posaconazole showed efficacy A c c e p t e d M a n u s c r i p t 7 [START_REF] Rodríguez | Posaconazole efficacy in a murine disseminated infection caused by Paecilomyces lilacinus[END_REF]. The low virulence of P. lilacinus previously described in an experimental murine model of disseminated infection was also corroborated [START_REF] Pujol | Experimental pathogenicity of three opportunistic Paecilomyces species in a murine model[END_REF]. In the survival study, severe immunosuppression was used to provoke an acute infection, with all of the control animals dying within 10 days post infection. Some data exist on the efficacy of triazoles in human paecilomycosis, especially VRC [START_REF] Martin | Voriconazole treatment of disseminated Paecilomyces infection in a patient with acquired immunodeficiency syndrome[END_REF][START_REF] Van Schooneveld | Paecilomyces lilacinus infection in a liver transplant patient: case report and review of the literature[END_REF][START_REF] Anderson | Fungal keratitis caused by Paecilomyces lilacinus associated with a retained intracorneal hair[END_REF][START_REF] Garbino | Successful treatment of Paecilomyces lilacinus endophthalmitis with voriconazole[END_REF]. Our results agree with these favourable data, since VRC was able to reduce the fungal burden in the three organs studied for one strain and in two organs for the other strain. Our results suggest that VRC may have a clinical role in the treatment of disseminated paecilomycosis.

  200 mg/kg body weight administered intraperitoneally (i.p.) on the day of infection and two doses of 5-fluorouracil, one of 150 mg/kg body weight intravenously (i.v) on the day of infection and another one of 75 mg/kg on Day 5 post infection. For the tissue burden study, mice received cyclophosphamide 200 mg/kg body weight administered i.p. plus 5-fluorouracil 150 mg/kg body weight administered i.v. 1 day A c c e p t e d M a n u s c r i p t 5

  homogenised in 1 mL of sterile saline. Serial 10-fold dilutions of the homogenates were plated on PDA and incubated for 24-72 h at 30 C. Mean survival time was estimated by the Kaplan-Meier method and compared among groups using the log-rank test. Colony counts in tissue burden studies were analysed using the Mann-Whitney U-test. Calculations were made using SPSS 15.0 (SPSS Inc., Chicago, IL) and GraphPad 4.0 for Windows (GraphPad Software Inc., La Jolla, CA).

13 Fig. 1 .

 131 Fig. 1. Cumulative mortality of severely immunosuppressed mice infected with (a) 1.2  10 8 colony-forming units (CFU)/mouse of Paecilomyces lilacinus FMR

Fig. 2 .

 2 Fig. 2. Effect of antifungal treatment with voriconazole 60 mg/kg/day or

Funding

This work was supported by a grant from Fondo de Investigaciones Sanitarias from the Ministerio de Sanidad y Consumo, Spain (PI 050031).

Competing interests

None declared.

Ethical approval

All animal care procedures were supervised and approved by the Universitat Rovira i Virgili Animal Welfare Committee (Reus, Spain).

Days Postinfection

Survival (%)

Edited Figure 1