Pavlos Myrianthefs

Sophia L Markantonis

Penelope Evaggelopoulou

Stratos Despotelis

Effimia Evodia

Dimitris Panidis

George Baltopoulos

Monitoring plasma voriconazole levels following intravenous administration in critically ill patients: an observational study

Keywords:

E-mail address: kyroudi@pharm.uoa.gr (S.L. Markantonis).

A c c e p t e d M a n u s c r i p t ABSTRACT Data relating to the pharmacokinetics of voriconazole in critically ill patients are lacking. A prospective observational study was conducted on 18 non-consecutive critically ill patients aged 24-97 years, comprising 12 patients with normal renal function (NRF) [creatinine clearance (CL Cr ) ≥60 mL/min] and 6 patients with moderate renal impairment (MRI) (CL Cr 40-55 mL/min), administered voriconazole intravenously (6 mg/kg loading dose and 3-4 mg/kg twice daily thereafter) in order to determine the suitability of these doses in this patient population. Steady-state blood levels were monitored and liver and renal function were recorded throughout treatment. Large variability in patient plasma levels was observed, ranging from 37% at ≤1 mg/L (minimum inhibitory concentration at which, for most fungal pathogens, 90% of isolates are susceptible) to 19% at >5.5 mg/L. Moreover, maintaining trough concentrations above clinical breakpoints was not consistently achieved because 16/30 (53%) were ≤1 mg/L. In a few MRI patients, average concentrations were found to be significantly different compared with those of NRF patients administered the same dose, however this difference was not noted in pharmacokinetic parameters following dose normalisation. None of the patients experienced deterioration in renal or liver function. Recommended voriconazole doses are inadequate to achieve drug concentrations >1 g/mL over the entire dosing interval in some critically ill patients.

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

Introduction

Fungal infections remain an important and serious infectious complication for critically ill Intensive Care Unit (ICU) patients. These patients are particularly susceptible to fungal infections because of physical barrier disruption, immune system depression, excessive antibiotic use, poor gut motility and the application οf a plethora of medical and surgical interventions including central venous catheter placement and other intravascular devices as well as abdominal surgery. Several antifungal treatment strategies are applied in ICU patients (prophylactic, empirical, pre-emptive and definite treatment), but in the clinical setting the optimal timing, dosage, duration and choice of antifungal agents largely remain a matter of controversy [START_REF] Blot | Is Candida really a threat in the ICU?[END_REF][START_REF] Rüping | Patients at high risk of invasive fungal infections: when and how to treat[END_REF].

Voriconazole, a widely used broad-spectrum triazole antifungal agent, is frequently used in ICU patients with severe fungal infections. It is extensively metabolised hepatically by the cytochrome P450 (CYP) system and <2% of an orally or intravenously administered dose is excreted unchanged in the urine [START_REF]Briefing document for voriconazole (oral and intravenous formulations)[END_REF]. To date, clinical studies have indicated that clearance of voriconazole is not influenced by renal function [START_REF]Briefing document for voriconazole (oral and intravenous formulations)[END_REF]. However, accumulation of the potentially nephrotoxic solubilising excipient sulfobutyl ether--cyclodextrin (SBECD) contained in its intravenous (i.v.) formulation has been described in patients with moderate to severe renal impairment [serum creatinine (Sr Cr ) levels >2.5 mg/dL] due to a linear relationship between clearance of the excipient and creatinine clearance (CL Cr ) [START_REF] Tomaszewski | The pharmacokinetics and safety of sulphobutylether--cyclodextrin (SBECD)[END_REF][START_REF] Mach | Accumulation of the solvent vehicle sulphobutylether beta cyclodextrin sodium in critically ill patients treated with intravenous voriconazole under renal replacement therapy[END_REF]. Furthermore, whilst receiving i.v. voriconazole, reversible elevations in Sr Cr from baseline were documented in clinical trials on patients with impaired renal function [START_REF]Background document for the Antiviral Drug Products Advisory Committee meeting[END_REF][START_REF] Abel | Pharmacokinetics, safety and tolerance of voriconazole in renally impaired subjects: two prospective, multicentre, open-label, parallel-group volunteer studies[END_REF]. Therefore, in patients with reduced renal function (Sr Cr levels >2.5 mg/dL), oral voriconazole is A c c e p t e d M a n u s c r i p t recommended unless assessment of the risk-benefit ratio justifies the use of i.v. voriconazole [START_REF]Briefing document for voriconazole (oral and intravenous formulations)[END_REF].

Voriconazole exhibits ca. 100-fold variability in drug levels in individuals receiving the same dosage [START_REF] Hope | Therapeutic drug monitoring for triazoles[END_REF]. Random concentrations <2.05 mg/L have been associated with suboptimal therapeutic outcome, and high concentrations with elevated liver function test values [START_REF] Tan | Investigation of the potential relationships between plasma voriconazole concentrations and visual adverse events or liver function test abnormalities[END_REF] as well as central nervous system [START_REF] Pascual | Voriconazole therapeutic drug monitoring in patients with invasive mycoses improves efficacy and safety outcomes[END_REF], ocular and other potential dose-related toxicities [START_REF] Boyd | Adverse reactions to voriconazole[END_REF]. High voriconazole levels have been noted in patients with critical illness [START_REF] Hope | Therapeutic drug monitoring for triazoles[END_REF]. Therefore, there is a need to establish what steady-state levels are achieved in the critically ill following i.v. infusion of voriconazole and to interpret their effect on outcome [START_REF] Hope | Therapeutic drug monitoring for triazoles[END_REF], liver function [START_REF] Tan | Investigation of the potential relationships between plasma voriconazole concentrations and visual adverse events or liver function test abnormalities[END_REF], renal function due to SBECD accumulation [START_REF] Tomaszewski | The pharmacokinetics and safety of sulphobutylether--cyclodextrin (SBECD)[END_REF][START_REF] Mach | Accumulation of the solvent vehicle sulphobutylether beta cyclodextrin sodium in critically ill patients treated with intravenous voriconazole under renal replacement therapy[END_REF][START_REF]Background document for the Antiviral Drug Products Advisory Committee meeting[END_REF] and other potential concentration-related toxicities [START_REF] Boyd | Adverse reactions to voriconazole[END_REF] in order to provide appropriate information to facilitate dose selection and dosage adjustment requirements in these patients. Accordingly, the aim of this study was to measure voriconazole plasma levels following i.v. administration to critically ill patients with or without renal dysfunction and to investigate its safety profile in terms of adverse effects on renal function.

Patients and methods

Study design

This was a prospective, open-label, uncontrolled observational study. The study protocol submitted for review stated that only patients receiving voriconazole as medically indicated according to the attending physician would be eligible for participation and that eight blood samples would be collected from each patient to

Study population

Patients were included in the study if they had an indication to receive voriconazole as prophylactic, empirical, pre-emptive or definite treatment.

Inclusion criteria

Inclusion criteria were i.v. administration of voriconazole for 72 h, age >18 years, normal hepatic function, and normal renal function (NRF) (CL Cr ≥ 60 mL/min) up to moderate renal impairment (MRI) (CL Cr 40-55 mL/min).

Exclusion criteria

Exclusion criteria were administration of medications known to be inhibitors or inducers of or to in any way interact with voriconazole and/or a history of hypersensitivity to azoles. Patients were divided into two subgroups according to their renal function status, i.e.

NRF or MRI (see Inclusion criteria, Section 2.3).

Liver and renal function tests were followed up at baseline, Day 5, Day 10 and at the end of therapy to identify possible adverse effects. Liver function tests included serum alanine and aspartate aminotransferases, alkaline phosphatase, -glutamyl transpeptidase, total bilirubin and conjugated bilirubin. To assess the impact of i.v. voriconazole administration on renal and liver function: (i) renal damage due to accumulation of the solvent vehicle SBECD in study patients with MRI was defined as an increase of ≥2 times the initial Sr Cr level, or start of an extrarenal depuration procedure during voriconazole therapy; and (ii) liver damage was defined as an increase of ≥4 times the initial serum concentration of liver enzymes, or ≥2 times in patients with previous impaired liver function, as previously characterised by Alvarez-Lerma et al. [START_REF] Alvarez-Lerma | Voriconazole Study Group in Critically Ill Patients. Impact of intravenous administration of voriconazole in critically ill patients with impaired renal function[END_REF].

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

Drug administration and collection and analysis of plasma samples

All patients were administered a loading dose of 6 mg/kg voriconazole as a 30-min i.v. infusion followed by a maintenance dose of 3-4 mg/kg as a 30-min i.v. infusion every 12 h for a period of at least 72 h. Blood samples (5 mL) were collected from patients through an arterial catheter just before the initiation of infusion, at the end of infusion and at 1.5, 2.5, 4, 6, 9 and 12 h thereafter. All samples were taken when voriconazole blood levels were considered to be at steady state. All collected samples were centrifuged immediately at 3500 rpm for 5 min at ca. 4 C and the acquired plasma was stored at -70 C until analysis.

Voriconazole plasma concentrations were determined using a previously validated high-performance liquid chromatography (HPLC) method [START_REF] Gage | A rapid HPLC assay for voriconazole in human plasma[END_REF]. Calibration curves for voriconazole were linear over the range 200-10 000 ng/mL. The lower limit of quantification was 50 ng/mL. Coefficients of variation (CVs) for intraday and interday precision were 0.5-0.6% and 1.2-7.5%, respectively, at drug concentrations of 200, 600, 4000 and 10 000 ng/mL.

Statistical analysis

Quantitative variables are expressed as mean ± standard deviation (S.D.) for normally distributed variables or as median and range when the distribution departed from normality. Study variables (demographic data, APACHE II , SAPS II and SOFA scores, use of potentially nephrotoxic drugs) in patients with NRF and those with MRI were compared using Student's t-test or Mann-Whitney U-test for continuous A c c e p t e d M a n u s c r i p t variables or using Fisher's exact test for categorical data. All reported P-values are two-sided. A P-value of <0.05 was considered statistically significant. Statistical processing and data analysis were performed with GraphPad Prism 4 software (GraphPad Software Inc., La Jolla, CA).

Pharmacokinetic evaluations

Pharmacokinetic parameters for voriconazole in serum were estimated from the concentration-time data of individual patients by non-compartmental, steady-state analysis using the WinNonlin pharmacokinetic software package (Pharsight Corp., Mountain View, CA). Blood sampling was performed on Day 3.8 ± 0.2. In total, 139 plasma samples were measured (ca. 8 samples from each patient). For two patients (Patients 16 and 17) only 12-h C min values were available, whilst in another patient (Patient 3) the estimated elimination half-life (t 1/2 ) was 68.4h so a comparison could not be made (Table 3)The median (range) minimum plasma concentration (C min ) was 0.79 mg/L (0.08-5.62 mg/L) at time 0 and 0.80 mg/L (0.15-5.95 mg/L) at 12 h, indicating that steady state had been achieved in the 13 patients for which both C min values were determined..

Results

In

No correlation was found between mean concentrations measured and voriconazole

doses administered in the 18 study patients (Table 3). Significant intersubject variation in plasma concentrations was observed at each sampling time for those patients administered 200 mg (n = 4) (CV 80-120%) and 240 mg (n = 4) (CV 96-145%). An increase in dose from 200 mg (4 patients) to 300 mg (2 patients) to 400 mg (2 patients) did not achieve a 2.3-2.5-fold increase in mean steady-state plasma concentration, as previously reported [START_REF]Briefing document for voriconazole (oral and intravenous formulations)[END_REF], with mean concentrations of 2.8 ± 2.4 mg/L, A c c e p t e d M a n u s c r i p t 2.9 ± 2.0 mg/L and 2.7 ± 2.3mg/L, respectively. Corresponding mean maximum plasma concentration (C max ) values of voriconazole were 5.2, 6.2 and 6.9 mg/L, respectively.

In the subgroups of patients with and without renal function impairment, the mean (S.D.) values for CL Cr throughout voriconazole administration are shown in Table 2.

The number of patients in both groups administered potentially nephrotoxic drugs concomitantly with voriconazole were: colistin (5) or gentamicin [START_REF] Blot | Is Candida really a threat in the ICU?[END_REF] or hydrocortisone (1) alone; colistin with vancomycin (3) or hydrocortisone (2) or both (2); vancomycin with hydrocortisone (2); and gentamicin with vancomycin (1). There were no statistically significant differences between patients with normal renal function and renal insufficiency at the start of voriconazole treatment with regard to demographic data, underlying illnesses or concomitant administration of nephrotoxic drugs, with the exception of age (P = 0.0267). Average steady-state concentrations in the MRI compared with the NRF patients were significantly different following administration of the 320 mg dose (P < 0.05) and 240 mg dose (P < 0.001), but were not significant for the 200 mg and 300 mg doses. Steady-state pharmacokinetic parameters for voriconazole, estimated from the concentration-time data for all 18 critically ill patients with or without renal impairment, are presented in Table 3. Following dose normalisation based on the mean administered voriconazole dose of 272 mg, median (range) area under the curve (AUC) and apparent volume of distribution and clearance at steady state (Vd app,ss and CL app,ss , respectively) values for NRF patients were 12.9 mg h/L (2.4-100.6 mg h/L), 246.6 L (36.7-951.9 L) and 20.6 L/h (3.0-113.2 L/h) and for MRI patients were 44.4 mg h/L (5.1-93.6 mg h/L), 198.1 L (28.8-498.2 L) and 5.7 L/h (4-59.5 L/h), respectively (not statistically significant) (Table 3).

A c c e p t e d

M a n u s c r i p t

Discussion

In the present study, we found that the recommended voriconazole dose of 3-4 mg/kg twice daily was inadequate to achieve and maintain drug concentrations >1 g/mL over the entire dosing interval (trough level) in some of our critically ill patients.

Moreover, in accordance with our defined safety criteria, i.v. voriconazole treatment was not associated with either renal or liver damage in NRF and MRI patients.

To our knowledge, this is the first study to describe the pharmacokinetics of voriconazole in critically ill patients with or without renal insufficiency. Voriconazole exhibits non-linear pharmacokinetics due to saturation of its metabolism and as a result small changes in the i.v. dose may result in disproportionate changes in plasma concentrations [START_REF] Trifilio | Monitoring plasma voriconazole levels may be necessary to avoid subtherapeutic levels in hematopoietic stem cell transplant recipients[END_REF]. In addition, terminal half-life estimations are dependent on the dose and are not indicative of accumulation or elimination of the drug. In the study patients, mean plasma concentrations did not increase with increasing dose of the drug from 200 mg to 400 mg. No correlation was found between administered voriconazole dose and achieved plasma concentration. Standard doses of voriconazole produced highly variable drug levels in plasma from <0.1 mg/L to 11.4 mg/L (Table 3). Multiple factors have been found to be associated with this large variability: non-linear saturable pharmacokinetics; drug-drug interactions; liver disease; patient age (in particular with respect to children); and genetic polymorphism of the cytochrome CYP2C19 [START_REF] Johnson | Voriconazole: a new triazole antifungal agent[END_REF][START_REF] Ashley | Pharmacology of systemic antifungal agents[END_REF][START_REF] Hyland | Identification of the cytochrome P450 enzymes involved in the N-oxidation of voriconazole[END_REF][START_REF] Walsh | Pharmacokinetics and safety of intravenous voriconazole in children after single-or multiple-dose administration[END_REF]. Some recent clinical observations have suggested that underdosing and overdosing of voriconazole may influence the efficacy and safety of therapy [START_REF] Boyd | Adverse reactions to voriconazole[END_REF][START_REF] Smith | Voriconazole therapeutic drug monitoring[END_REF][START_REF] Denning | Efficacy and safety of voriconazole in the treatment of acute invasive aspergillosis[END_REF][START_REF] Imhof | Neurological adverse events to voriconazole: evidence for therapeutic drug monitoring[END_REF][START_REF] Trifilio | Voriconazole therapeutic drug monitoring in allogeneic hematopoietic stem cell transplant recipients[END_REF].

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

A few studies have attempted to evaluate the relationship between voriconazole exposure and treatment efficacy. In vitro pharmacodynamic studies have shown the maximum fungicidal activity of voriconazole is around two to three times the minimum inhibitory concentration (MIC) [START_REF] Purkins | Pharmacokinetics and safety of voriconazole following intravenous-to oral-dose escalation regimens[END_REF][START_REF] Andes | In vivo pharmacokinetics and pharmacodynamics of a new triazole, voriconazole, in a murine candidiasis model[END_REF]. Based on a review of laboratory and clinical data, the Antifungal Susceptibility Subcommittee of the Clinical and Laboratory Standards Institute have proposed interpretive MIC breakpoints for voriconazole and Candida spp. as follows: susceptible ≤1 mg/L; susceptible dose-dependent, 2 mg/L; and resistant ≥4 mg/L [START_REF] Pfaller | Correlation of MIC with outcome for Candida species tested against voriconazole: analysis and proposal for interpretive breakpoints[END_REF]. These recommendations suggest that voriconazole concentrations should be maintained >1 mg/L over the entire dosing interval (trough level). In addition, voriconazole trough blood levels >5.5 mg/L have been reported to be possibly associated with toxicity [START_REF] Pascual | Voriconazole therapeutic drug monitoring in patients with invasive mycoses improves efficacy and safety outcomes[END_REF]. Large variability in voriconazole levels was observed in our patients, ranging from 37% at ≤1 mg/L (the MIC at which, for most fungal pathogens, 90% of isolates are susceptible) to 19% at >5.5 mg/L. In fact, in 7 of the 18 study patients 80% of the voriconazole levels measured were ≤1 mg/L. Sixteen (53%) of the thirty measured trough concentrations were ≤1 mg/L and only 6% were >5.5 mg/L.

The very limited literature pertinent to the administration of voriconazole in critically ill patients did not allow direct comparisons to be made between our estimated pharmacokinetic parameters and those of previous studies. In an earlier study in eight critically ill patients administered voriconazole 200 mg or 250 mg twice daily, mean ± S.D. plasma concentrations of voriconazole for trough and peak were, respectively, 4.6 ± 2.8 mg/L and 6.4 ± 4.3 mg/L [26] compared with 1.65 ± 2.02 mg/L and 5.2 ± 3.9 mg/L in our study patients administered 200-250 mg doses. In the present study, the mean estimated Vd app,ss value of 290.9 L or 4.5 L/kg following A c c e p t e d M a n u s c r i p t dose normalisation (mean administered dose 272 mg) was similar to that reported previously, i.e. 2-4.6 L/kg [START_REF] Theuretzbacher | Pharmacokinetic/pharmacodynamic profile of voriconazole[END_REF]. In contrast, our t 1/2 estimations ranged from 5.8 h to 21.8 h compared with a reported t 1/2 of ca. 6 h [START_REF] Theuretzbacher | Pharmacokinetic/pharmacodynamic profile of voriconazole[END_REF]. However, our estimations were longer than the duration of sampling in three of the NRF patients (see Table 3) and therefore must be regarded as only rough estimations.

The lack of influence of renal function on the pharmacokinetics of voriconazole has been reported [START_REF]Briefing document for voriconazole (oral and intravenous formulations)[END_REF][START_REF] Abel | Pharmacokinetics, safety and tolerance of voriconazole in renally impaired subjects: two prospective, multicentre, open-label, parallel-group volunteer studies[END_REF]. The mean voriconazole concentrations in our MRI patients compared with NRF patients were found to be significantly different following administration of 320 mg and 240 mg doses, but this finding was based on a very limited number of patients, i.e. two NRF patients versus one MRI patient and three NRF patients versus one MRI patient, respectively, so the results cannot be considered definitive. Furthermore, pharmacokinetic parameters following dose normalisation did not differ significantly between the study patients with and without renal impairment.

Conclusions

The principal finding of this observational study was that i.v. administration of voriconazole in 18 critically ill patients was associated with a 37% prevalence of possibly inadequate plasma levels (i.e. ≤1 mg/L). No clear reason can be offered to explain these low concentrations besides the possibility that certain patients may have been homozygous extensive metabolisers, particularly since our patients were not receiving concomitant medications that might have induced voriconazole metabolism.

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

Similar to other voriconazole studies, a wide range of concentrations was observed.

A greater than proportional increase in exposure (AUC) was not observed with increasing drug dose. Higher voriconazole plasma concentrations were observed in a few patients with MRI compared with NRF, but despite probable SBECD accumulation, deterioration of renal function was not noted in these patients. Also, none of the patients experienced deterioration in liver function during voriconazole administration.

This study had several limitations. The small sample size, unknown patient genotypes and range of voriconazole doses administered limited the value of the results. However, the findings indicate that administration of voriconazole in critically ill patients needs to be studied further, as additional data are required to facilitate the therapeutic drug monitoring of this antifungal agent in this patient population.

A

  c c e p t e d M a n u s c r i p t University of Athens, Panepistimiopolis, Athens 157.71, Greece. Tel.: +30 210 727 4676; fax: +30 210 727 4027.

A

  c c e p t e d M a n u s c r i p t monitor steady-state drug levels and to determine voriconazole pharmacokinetic parameters. The Scientific and Ethics Committees of the 'KAT' Hospital (Athens, Greece) and the Athens University School of Nursing deliberated over the requirements of the protocol in light of the frequent blood sampling for routine laboratory tests and cultures typically needed for optimal patient care in ICUs and the intended naturalistic nature of the study and decided to approve the study protocol waiving informed consent from each patient.

A c c e p t e d M a n u s c r i p t 2 . 5 .

 25 Patient data collected and recorded For each eligible patient, their age, sex, weight, height, CL Cr , urea level, hepatic function tests, disease severity at the time of admission to the ICU based on the Acute Physiology and Chronic Health Evaluation (APACHE) II score, Simplified Acute Physiology Score II (SAPS II), Sequential Organ Failure Assessment (SOFA) scores and Glasgow scale, voriconazole dose and concomitant administration of potentially nephrotoxic drugs such as aminoglycosides, colistin, vancomycin and teicoplanin were recorded.

  total, 18 ICU patients aged 24-97 years required antifungal therapy and participated in the study between March 2006 and February 2008. Six of these patients were characterised as having MRI and twelve as having NRF at the beginning of voriconazole treatment. The demographic characteristics of the patients are presented in

A

  c c e p t e d M a n u s c r i p t Fourteen patients received empirical antibiotic treatment, two patients received preemptive treatment and two were treated for microbiologically documented candidaemia. Voriconazole was administered on Day 14.8 ± 3.3 of ICU hospitalisation for a mean duration of 11.0 ± 3.6 days. Mean length of ICU stay was 46.7 ± 22.5 days. Other co-administered antimicrobial agents were linezolid (4 patients), meropenem (14 patients), vancomycin (8 patients) and colistin (12 patients). All drugs were administered as clinically indicated by the attending physician.

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

 1

Table 1 .

 1 All patients required intubation and mechanical ventilation for the reasons listed in Table1. Laboratory values regarding white blood cell counts and liver and renal function tests during voriconazole administration are shown in

Table 2 .

 2 During the course of treatment with voriconazole, no clinically significant renal or liver function deterioration/damage (as defined in Section 2.5) occurred in any of the patients with or without renal impairment.

Table 2

 2 Evolution of laboratory parameters ± 128.7 267.6 ± 388.6 179.0 ± 155.6 332.8 ± 553.8 EOT, end of therapy; CL Cr , creatinine clearance; WBC, white blood cell; AST, aspartate aminotransferase; ALT, alanine aminotransferase; ALP, alkaline phosphatase; TBIL, total bilirubin; GGTP, -glutamyl transpeptidase.

		Baseline	Day 5	Day 10	EOT
	All patients
	CL Cr (mL/min)	112.2 ± 67.3 98.8 ± 57.8	109.4 ± 53.2 104.6 ± 52.6
	WBC count (cells/L) 11221 ± 4838 10589 ± 4698 9107 ± 4368 9307 ± 4176
	AST (U/L)	53.4 ± 54.5	45.4 ± 68.2	46.7 ± 22.7	80.1 ± 103.2
	ALT (U/L)	81.0 ± 112.5 46.8 ± 37.2	57.91 ± 41.9 69.3 ± 81.5
	ALP (U/L)	99.6 ± 58.9	103.4 ± 49.1 125.1 ± 44.8 132.3 ± 81.6
	TBIL (mg/dL)	0.6 ± 0.4	0.9 ± 1.4	0.6 ± 0.7	1.7 ± 2.7
	GGTP (U/L)	146.5 ± 193.3 171.9 ± 225.7 164.0 ± 122.1 248.8 ± 355.9
	Patients with normal CL Cr (>60 mL/min)
	CL Cr (mL/min)	140.1 ± 57.5 122.5 ± 53.7 129.4 ± 44.2 129.5 ± 44.2
	WBC count (cells/L) 10314 ± 5252 9721 ± 3860 8004 ± 2197 7626 ± 3208
	AST (U/L)	61.5 ± 61.7	30.5 ± 14.8	46.7 ± 25.2	72.3 ± 85.9
	ALT (U/L)	95.71 ± 128.0 49.7 ± 44.5	54.7 ± 46.1	84.6 ± 104.8
	ALP (U/L)	110.5 ± 66.0 101.4 ± 45.9 128.1 ± 49.2 134.9 ± 90.9
	TBIL (mg/dL)	0.5 ± 0.2	0.5 ± 0.4	0.6 ± 0.5	0.6 ± 0.4
	GGTP (U/L)	166.1 ± 214.3 128.5 ± 96.69 160.7± 124.6 196.3 ± 183.7
	Patients with impaired CL Cr (40-55 mL/min)
	CL Cr (mL/min)	39.6 ± 14.9	12498 ± 6237 56.0 ± 37.9	54.8 ± 25.2

Table 3

 3 Steady-state pharmacokinetic parameters of voriconazole, estimated from the concentration-time data by non-compartmental analysis, following intravenous administration in critically ill patients (N = 18) with either normal renal function or moderate renal impairment

	Patient	Dose	C ave (S.D.)	C min 0 h	C min 12 h	C max	t 1/2	After dose normalisation (with a mean
	No.	(mg/12 h)	(mg/L)	(mg/L)	(mg/L)	(mg/L)	(h)	administered dose of 272 mg)
								CL app,ss (L/h)	Vd app,ss (L)	AUC 0-12 h (mg h/L)
	1	240	0.78 (0.88)	0.17	0.15	2.7	7.0	38.6	386.6	8.0
	2	200	2.94 (1.10)	1.99	1.82	5.3	9.5	8.2	111.8	45.5
	3 a	320	4.55 (1.10)	3.12	4.04	7.0	68.4 5.1	498.2	45.8
	4	250	7.73 (1.51)	5.62	5.95	10.4	8.6	3.0	36.7	100.6
	5	400	4.26 (2.56)	2.33	2.66	9.7	21.8 5.9	186.9	31.1
	6 a	245	0.50 (0.65)	N/A	N/A	1.9	3.8	59.5	329.4	5.1
	7 a	200	1.51 (1.95)	0.08	0.18	5.5	13	21.7	407.4	17.0
	8 a	200	6.03 (2.00)	4.01	3.6	8.9	9.3	4.0	53.2	93.6
	9	320	1.78 (1.30)	0.55	0.72	4.7	6.3	14.3	130.1	16.2
	10	240	0.23 (0.24)	N/A	N/A	0.7	5.8	113.2	951. 9	2.4
	11	200	0.60 (0.25)	0.32	0.40	1.1	14.3 39.9	822.5	9.3
	12 a	300	4.33 (2.42)	2.09	2.10	9.6	8.1	5.7	66.8	42.9
	13	280	5.71 (1.98)	3.61	3.90	9.6	15.0 4.2	89.8	63.5
	14	240	1.09 (1.23)	0.33	0.27	3.8	8.6	28.0	346.4	11.0
	15 a	240	4.69 (3.71)	0.78	0.8	11.4	3.7	5.6	28.8	55.5

Funding

This research was funded entirely by the University of Athens.

Competing interests

None declared.

Ethical approval

Scientific and Ethics Committees of 'KAT' Hospital and the Athens University School of Nursing (Athens, Greece).


Matthew E Falagas

Evridiki K Vouloumanou

Evagelia Baskouta

Petros I Rafailidis

Kostantinos Polyzos

Jordi Rello

Treatment options for 2009 H1N1 influenza: evaluation of the published evidence

Keywords:

E-mail address: m.falagas@aibs.gr (M.E. Falagas).

A c c e p t e d M a n u s c r i p t ABSTRACT We evaluated the evidence regarding the effectiveness of various treatment strategies used for 2009 H1N1 influenza by reviewing available relevant studies. In total, 22 studies (15 cohort studies involving >10 patients, 5 cohort studies with ≤10 patients and 2 case reports) were included. A total of 3020 patients [1068 (35.4%) critically ill, 1722 (57.0%) hospitalised and 230 (7.6%) outpatients, including 909 (30.1%) children] were involved. Notably, 487 (16.1%) were obese [body mass index (BMI) >30)], 362 (12.0%) had asthma or chronic obstructive pulmonary disease and 255 (8.4%) were pregnant. Antiviral treatment was administered to 1622 patients (53.7%), of whom 661 (40.8%) received oseltamivir monotherapy. Corticosteroids were administered in 323 (31.8%) of 1016 patients for whom relevant data were available. Similarly, 633 (85.0%) of 745 patients received antibiotics. Comparative data from the largest included study (involving 1088 patients) indicated that administration of antivirals within 2 days from symptom onset was significantly associated with reduced mortality (P < 0.001). In summary, the scarcity of comparative available data hampered the establishment of any firm conclusions regarding the benefit that various treatment strategies may confer to patients with 2009 H1N1 influenza. Studies with a comparative design as well as randomised studies are needed to clarify further this issue of major importance.

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

Introduction

Influenza is an infection with significant morbidity and mortality that always attracts the interest of the health authorities, the media and the public [START_REF] Falagas | Reaction to the threat of influenza pandemic: the mass media and the public[END_REF][START_REF] Peppas | An analysis of patient house calls in the area of Attica, Greece[END_REF].

During the previous century, humanity has engaged in encounters with three influenza pandemics that have been associated with considerable mortality.

Notably, the Spanish pandemic of 1918 caused more deaths than the First World War. Consequently, national health authorities, as well as the World Health Organization (WHO), were always alert in order to identify a potential influenza pandemic early.

We are currently experiencing a new influenza pandemic, caused by the 2009 H1N1 influenza virus. From the beginning of the pandemic up to 1 November 2009, more than 199 countries had reported confirmed 2009 H1N1 cases, including over 6000 associated deaths [START_REF]Pandemic (H1N1)[END_REF]. Preliminary evidence from experience with this new pandemic has already appeared in the literature [START_REF] Anzic Influenza | Critical care services and 2009 H1N1 influenza in Australia and New Zealand[END_REF][START_REF] Dawood | Emergence of a novel swine-origin influenza A (H1N1) virus in humans[END_REF][START_REF] Hackett | Clinical characteristics of paediatric H1N1 admissions in Birmingham, UK[END_REF][START_REF] Perez-Padilla | INER Working Group on Influenza. Pneumonia and respiratory failure from swine-origin influenza A (H1N1) in Mexico[END_REF].

According to this evidence, 2009 H1N1 influenza appears to occur more frequently in young adults and children, whereas fatal cases refer mainly to older individuals [START_REF] Louie | California Pandemic (H1N1) Working Group. Factors associated with death or hospitalization due to pandemic 2009 influenza A(H1N1) infection in California[END_REF]. Neuraminidase inhibitors (NAIs), in particular oseltamivir, have been used for the treatment of patients with 2009 H1N1 influenza.

However, cases with oseltamivir-resistant pandemic strains have also been reported [START_REF] Centers For | Oseltamivir-resistant 2009 pandemic influenza A (H1N1) virus infection in two summer campers receiving prophylaxis-North Carolina, 2009[END_REF][START_REF] Centers For | Oseltamivir-resistant novel influenza A (H1N1) virus infection in two immunosuppressed patients-Seattle, Washington, 2009[END_REF]. In addition, use of corticosteroids has been reported in some recent studies involving critically ill patients with 2009 H1N1 influenza.

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

Taking all the above factors into consideration, we sought to collect and evaluate the currently available published evidence in order to contribute to further understanding of treatment aspects of 2009 H1N1 influenza.

Data sources

Studies to be included in this review were identified from searches performed in PubMed and Scopus databases, both last assessed in November 2009. The search term applied to both databases was '(swine flu OR H1N1 infection OR novel influenza) AND (treatment OR therapy)'. Bibliographies of relevant articles were also hand-searched.

Study selection criteria

Two reviewers (EB and KP) independently performed the literature search, assessed the retrieved studies for eligibility for inclusion, and extracted the data.

To be considered eligible for inclusion in the review, an article should have provided data regarding strategies used to treat patients of any age and from any clinical setting with 2009 H1N1 influenza infection worldwide. In addition, only articles for which a full-text version could be obtained were included in the review.

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

Data extraction

Data extracted from each of the evaluated articles included: study design; characteristics of the evaluated patients; country to which each specific study referred; number of included patients with co-morbidity and/or any risk factors considered to be associated with severe complications from seasonal influenza; characteristics of the antiviral treatment administered (type, dosage, duration, time of initiation); other treatment administered (if any); and any complications from the 2009 H1N1 infection or any adverse event reported. In addition, information regarding the outcome of the 2009 H1N1 infection, in particular mortality, were also extracted. The abovementioned extracted data are presented in Table 1.

With regard to the patient populations enrolled, the studies included in this review were classified as follows: studies involving exclusively or a majority of patients with 2009 H1N1 influenza admitted to the Intensive Care Unit (ICU); studies involving exclusively or a majority of patients hospitalised due to 2009 H1N1 influenza; and studies involving exclusively or a majority of outpatients with 2009 H1N1 influenza. If a study involving mixed populations provided specific data for these subpopulations, the study was classified in more than one of the abovementioned categories.

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

Comparisons

In an attempt to evaluate the impact of antiviral treatment as well as the impact of early administration of antiviral treatment (within 2 days from symptom onset) on mortality, percentages of those who received antiviral treatment and who died versus those who did not receive antiviral treatment were compared for the subgroup of studies providing relevant data. All comparisons were performed using OpenEpi software [START_REF] Dean | Open source epidemiologic statistics for public health, version 2.2.1[END_REF].

Study characteristics

A total of 22 individual articles (15 cohort studies involving >10 patients [START_REF] Anzic Influenza | Critical care services and 2009 H1N1 influenza in Australia and New Zealand[END_REF][START_REF] Dawood | Emergence of a novel swine-origin influenza A (H1N1) virus in humans[END_REF][START_REF] Hackett | Clinical characteristics of paediatric H1N1 admissions in Birmingham, UK[END_REF][START_REF] Perez-Padilla | INER Working Group on Influenza. Pneumonia and respiratory failure from swine-origin influenza A (H1N1) in Mexico[END_REF][START_REF] Louie | California Pandemic (H1N1) Working Group. Factors associated with death or hospitalization due to pandemic 2009 influenza A(H1N1) infection in California[END_REF][START_REF] Kumar | Canadian Critical Care Trials Group H1N1 Collaborative. Critically ill patients with 2009 influenza A(H1N1) infection in Canada[END_REF][START_REF] Domínguez-Cherit | Critically ill patients with 2009 influenza A(H1N1) in Mexico[END_REF][START_REF]Surveillance for pediatric deaths associated with 2009 pandemic influenza A (H1N1) virus infection-United States, April-August 2009[END_REF][START_REF] Rello | H1N1 SEMICYUC Working Group. Intensive care adult patients with severe A c c e p t e d M a n u s c r i p t respiratory failure caused by influenza A (H1N1)v in Spain[END_REF][START_REF] Raffo | Influenza A(H1N1) epidemic in Argentina. Experience in a National General Hospital (Hospital Nacional Alejandro Posadas)[END_REF][START_REF] Jain | Hospitalized patients with 2009 H1N1 influenza in the United States, April-June 2009[END_REF][START_REF] Jamieson | Novel Influenza A (H1N1) Pregnancy Working Group. H1N1 2009 influenza virus infection during pregnancy in the USA[END_REF][START_REF] Komiya | Clinical features of cases of influenza A (H1N1)v in Osaka prefecture, Japan, May 2009[END_REF][START_REF] Koliou | Epidemiological and clinical characteristics of influenza A(H1N1)v infection in children: the first 45 cases in Cyprus, June-August 2009[END_REF][START_REF] Cdc | Bacterial coinfections in lung tissue specimens from fatal cases of 2009 pandemic influenza A (H1N1)-United States, May-August 2009[END_REF], 5 cohort studies involving up to 10 patients [START_REF]Intensive-care patients with severe novel influenza A (H1N1) virus infection-Michigan, June 2009[END_REF][START_REF] Liang | New influenza A (H1N1) 2009 in Singapore: the first ten adult imported cases[END_REF][START_REF] Centers For | Neurologic complications associated with novel influenza A (H1N1) virus infection in children-Dallas, Texas, May 2009[END_REF][START_REF]Novel influenza A (H1N1) virus infections in three pregnant women-United States, April-May 2009[END_REF][START_REF] Bin | National Influenza A Pandemic (H1N1) 2009 Clinical Investigation Group. Clinical and epidemiologic characteristics of 3 early cases of influenza A pandemic (H1N1) 2009 virus infection, People's Republic of China[END_REF] and 2 case reports [START_REF] Wiebe | Atypical clinical presentation of H1N1 influenza in a dialysis patient[END_REF][START_REF] Cheng | Fatal coinfection with swine origin influenza virus A/H1N1 and community-acquired methicillin-resistant Staphylococcus aureus[END_REF]) were regarded as eligible for inclusion in the review. Nine of these studies referred to the USA [START_REF] Dawood | Emergence of a novel swine-origin influenza A (H1N1) virus in humans[END_REF][START_REF] Louie | California Pandemic (H1N1) Working Group. Factors associated with death or hospitalization due to pandemic 2009 influenza A(H1N1) infection in California[END_REF][START_REF]Surveillance for pediatric deaths associated with 2009 pandemic influenza A (H1N1) virus infection-United States, April-August 2009[END_REF][START_REF] Jain | Hospitalized patients with 2009 H1N1 influenza in the United States, April-June 2009[END_REF][START_REF] Jamieson | Novel Influenza A (H1N1) Pregnancy Working Group. H1N1 2009 influenza virus infection during pregnancy in the USA[END_REF][START_REF] Cdc | Bacterial coinfections in lung tissue specimens from fatal cases of 2009 pandemic influenza A (H1N1)-United States, May-August 2009[END_REF][START_REF]Intensive-care patients with severe novel influenza A (H1N1) virus infection-Michigan, June 2009[END_REF][START_REF] Centers For | Neurologic complications associated with novel influenza A (H1N1) virus infection in children-Dallas, Texas, May 2009[END_REF][START_REF]Novel influenza A (H1N1) virus infections in three pregnant women-United States, April-May 2009[END_REF], two to Mexico [START_REF] Perez-Padilla | INER Working Group on Influenza. Pneumonia and respiratory failure from swine-origin influenza A (H1N1) in Mexico[END_REF][START_REF] Domínguez-Cherit | Critically ill patients with 2009 influenza A(H1N1) in Mexico[END_REF], two to Canada [START_REF] Kumar | Canadian Critical Care Trials Group H1N1 Collaborative. Critically ill patients with 2009 influenza A(H1N1) infection in Canada[END_REF][START_REF] Wiebe | Atypical clinical presentation of H1N1 influenza in a dialysis patient[END_REF], three to European countries (UK [START_REF] Hackett | Clinical characteristics of paediatric H1N1 admissions in Birmingham, UK[END_REF], Spain [START_REF] Rello | H1N1 SEMICYUC Working Group. Intensive care adult patients with severe A c c e p t e d M a n u s c r i p t respiratory failure caused by influenza A (H1N1)v in Spain[END_REF] and Cyprus [START_REF] Koliou | Epidemiological and clinical characteristics of influenza A(H1N1)v infection in children: the first 45 cases in Cyprus, June-August 2009[END_REF]), two to China [START_REF] Bin | National Influenza A Pandemic (H1N1) 2009 Clinical Investigation Group. Clinical and epidemiologic characteristics of 3 early cases of influenza A pandemic (H1N1) 2009 virus infection, People's Republic of China[END_REF][START_REF] Cheng | Fatal coinfection with swine origin influenza virus A/H1N1 and community-acquired methicillin-resistant Staphylococcus aureus[END_REF] and the remaining to Argentina [START_REF] Raffo | Influenza A(H1N1) epidemic in Argentina. Experience in a National General Hospital (Hospital Nacional Alejandro Posadas)[END_REF], Australia and New Zealand [START_REF] Anzic Influenza | Critical care services and 2009 H1N1 influenza in Australia and New Zealand[END_REF], Japan [START_REF] Komiya | Clinical features of cases of influenza A (H1N1)v in Osaka prefecture, Japan, May 2009[END_REF] and Singapore [START_REF] Liang | New influenza A (H1N1) 2009 in Singapore: the first ten adult imported cases[END_REF]. In addition, 7 of the 20 included cohort studies and case series were prospective [START_REF] Anzic Influenza | Critical care services and 2009 H1N1 influenza in Australia and New Zealand[END_REF][START_REF] Kumar | Canadian Critical Care Trials Group H1N1 Collaborative. Critically ill patients with 2009 influenza A(H1N1) infection in Canada[END_REF][START_REF] Raffo | Influenza A(H1N1) epidemic in Argentina. Experience in a National General Hospital (Hospital Nacional Alejandro Posadas)[END_REF][START_REF] Jain | Hospitalized patients with 2009 H1N1 influenza in the United States, April-June 2009[END_REF][START_REF] Komiya | Clinical features of cases of influenza A (H1N1)v in Osaka prefecture, Japan, May 2009[END_REF][START_REF] Liang | New influenza A (H1N1) 2009 in Singapore: the first ten adult imported cases[END_REF][START_REF] Bin | National Influenza A Pandemic (H1N1) 2009 Clinical Investigation Group. Clinical and epidemiologic characteristics of 3 early cases of influenza A pandemic (H1N1) 2009 virus infection, People's Republic of China[END_REF], whereas the remaining 13 were retrospective [START_REF] Dawood | Emergence of a novel swine-origin influenza A (H1N1) virus in humans[END_REF][START_REF] Hackett | Clinical characteristics of paediatric H1N1 admissions in Birmingham, UK[END_REF][START_REF] Perez-Padilla | INER Working Group on Influenza. Pneumonia and respiratory failure from swine-origin influenza A (H1N1) in Mexico[END_REF][START_REF] Louie | California Pandemic (H1N1) Working Group. Factors associated with death or hospitalization due to pandemic 2009 influenza A(H1N1) infection in California[END_REF][START_REF] Domínguez-Cherit | Critically ill patients with 2009 influenza A(H1N1) in Mexico[END_REF][START_REF]Surveillance for pediatric deaths associated with 2009 pandemic influenza A (H1N1) virus infection-United States, April-August 2009[END_REF][START_REF] Rello | H1N1 SEMICYUC Working Group. Intensive care adult patients with severe A c c e p t e d M a n u s c r i p t respiratory failure caused by influenza A (H1N1)v in Spain[END_REF][START_REF] Jamieson | Novel Influenza A (H1N1) Pregnancy Working Group. H1N1 2009 influenza virus infection during pregnancy in the USA[END_REF][START_REF] Koliou | Epidemiological and clinical characteristics of influenza A(H1N1)v infection in children: the first 45 cases in Cyprus, June-August 2009[END_REF][START_REF] Cdc | Bacterial coinfections in lung tissue specimens from fatal cases of 2009 pandemic influenza A (H1N1)-United States, May-August 2009[END_REF][START_REF]Intensive-care patients with severe novel influenza A (H1N1) virus infection-Michigan, June 2009[END_REF][START_REF] Centers For | Neurologic complications associated with novel influenza A (H1N1) virus infection in children-Dallas, Texas, May 2009[END_REF][START_REF]Novel influenza A (H1N1) virus infections in three pregnant women-United States, April-May 2009[END_REF]. Regarding the 15 cohort studies involving >10 patients, 5 involved mainly ICU patients [START_REF] Anzic Influenza | Critical care services and 2009 H1N1 influenza in Australia and New Zealand[END_REF][START_REF] Kumar | Canadian Critical Care Trials Group H1N1 Collaborative. Critically ill patients with 2009 influenza A(H1N1) infection in Canada[END_REF][START_REF] Domínguez-Cherit | Critically ill patients with 2009 influenza A(H1N1) in Mexico[END_REF][START_REF]Surveillance for pediatric deaths associated with 2009 pandemic influenza A (H1N1) virus infection-United States, April-August 2009[END_REF][START_REF] Rello | H1N1 SEMICYUC Working Group. Intensive care adult patients with severe A c c e p t e d M a n u s c r i p t respiratory failure caused by influenza A (H1N1)v in Spain[END_REF], 6 involved mainly hospitalised patients [START_REF] Dawood | Emergence of a novel swine-origin influenza A (H1N1) virus in humans[END_REF][START_REF] Hackett | Clinical characteristics of paediatric H1N1 admissions in Birmingham, UK[END_REF][START_REF] Perez-Padilla | INER Working Group on Influenza. Pneumonia and respiratory failure from swine-origin influenza A (H1N1) in Mexico[END_REF][START_REF] Louie | California Pandemic (H1N1) Working Group. Factors associated with death or hospitalization due to pandemic 2009 influenza A(H1N1) infection in California[END_REF][START_REF] Jain | Hospitalized patients with 2009 H1N1 influenza in the United States, April-June 2009[END_REF][START_REF] Jamieson | Novel Influenza A (H1N1) Pregnancy Working Group. H1N1 2009 influenza virus infection during pregnancy in the USA[END_REF]] and 3 involved mainly outpatients [START_REF] Komiya | Clinical features of cases of influenza A (H1N1)v in Osaka prefecture, Japan, May 2009[END_REF][START_REF] Koliou | Epidemiological and clinical characteristics of influenza A(H1N1)v infection in children: the first 45 cases in Cyprus, June-August 2009[END_REF][START_REF] Cdc | Bacterial coinfections in lung tissue specimens from fatal cases of 2009 pandemic influenza A (H1N1)-United States, May-August 2009[END_REF]. The remaining 1 of these 15 cohort studies involved a mixed population of ICU and hospitalised patients A c c e p t e d M a n u s c r i p t [START_REF] Raffo | Influenza A(H1N1) epidemic in Argentina. Experience in a National General Hospital (Hospital Nacional Alejandro Posadas)[END_REF]. Specific data were provided for these two subpopulations, thus this study was classified into two categories (data are presented in Table 1).

Patient characteristics

A total of 3020 patients with confirmed or probable 2009 H1N1 influenza infection were evaluated, including 909 (30.1%) children aged <18 years. From the total of 3020 reported patients, 1068 (35.4%) were ICU patients, 1722 (57.0%) were hospitalised patients in general wards and 230 (7.6%) were outpatients. In addition, 255 (8.4%) of the 3020 patients were pregnant, 487 (16.1%) were obese [body mass index (BMI) >30)] and 136 (4.5%) were morbidly obese (BMI > 40). Furthermore, 362 (12.0%) of the 3020 patients had asthma or chronic obstructive pulmonary disease (COPD). Detailed data regarding the proportion of included patients with specific co-morbidities are presented in Table 1.

Antiviral treatment characteristics

A total of 1622 (53.7%) of the 3020 reported patients received antiviral treatment. Specifically, 661 (40.8%) of these 1622 patients received only oseltamivir, 95 (5.9%) received only zanamivir (including 2 patients who switched to oseltamivir), 8 (0.5%) received only amantadine and 1 (0.06%) received only rimantadine. In addition, 25 (1.5%) of the 1622 patients received a combination of oseltamivir and amantadine, 18 (1.1%) received a combination Data regarding the administered dosages of antiviral treatment were scarcely reported. Specifically, a dosage of 150 mg oseltamivir twice daily was administered in the ICU patients included in two studies [START_REF] Rello | H1N1 SEMICYUC Working Group. Intensive care adult patients with severe A c c e p t e d M a n u s c r i p t respiratory failure caused by influenza A (H1N1)v in Spain[END_REF][START_REF]Intensive-care patients with severe novel influenza A (H1N1) virus infection-Michigan, June 2009[END_REF], whereas in another study regular oseltamivir doses were administered in the included paediatric ICU patients [START_REF] Raffo | Influenza A(H1N1) epidemic in Argentina. Experience in a National General Hospital (Hospital Nacional Alejandro Posadas)[END_REF]. An oseltamivir dosage of 75 mg twice daily was administered in the hospitalised patients involved in two studies [START_REF] Perez-Padilla | INER Working Group on Influenza. Pneumonia and respiratory failure from swine-origin influenza A (H1N1) in Mexico[END_REF][START_REF] Raffo | Influenza A(H1N1) epidemic in Argentina. Experience in a National General Hospital (Hospital Nacional Alejandro Posadas)[END_REF] as well as in outpatients involved in another study [START_REF] Bin | National Influenza A Pandemic (H1N1) 2009 Clinical Investigation Group. Clinical and epidemiologic characteristics of 3 early cases of influenza A pandemic (H1N1) 2009 virus infection, People's Republic of China[END_REF]. Data regarding the duration of antiviral treatment were also scarcely reported. Specifically, oseltamivir dosage schedules beyond the standard 5-day course were administered in ICU patients reported from two studies [START_REF] Rello | H1N1 SEMICYUC Working Group. Intensive care adult patients with severe A c c e p t e d M a n u s c r i p t respiratory failure caused by influenza A (H1N1)v in Spain[END_REF][START_REF]Intensive-care patients with severe novel influenza A (H1N1) virus infection-Michigan, June 2009[END_REF], whereas a 5-day oseltamivir course was administered in paediatric outpatients according to another study [START_REF] Koliou | Epidemiological and clinical characteristics of influenza A(H1N1)v infection in children: the first 45 cases in Cyprus, June-August 2009[END_REF].

Regarding the time of initiation of antiviral treatment, oseltamivir was administered within 2 days from symptom onset in 529 (35.3%) of 1498 patients for whom relevant data were available. Detailed data regarding the duration as well as the time of initiation of antiviral treatment are presented in Table 1.

Other treatment characteristics

Corticosteroids were administered in 323 (31.8%) [232 (71.8%) were ICU patients and 91 (28.2%) were hospitalised patients] of the 1016 patients for whom relevant data were available. Of the 745 patients with available relevant data, 633 (85.0%) received antibiotics. Among 548 patients with available A c c e p t e d M a n u s c r i p t relevant data, 205 (37.4%) received vasopressor drugs. Recombinant activated protein C was administered in two ICU patients. Specific data regarding the type of treatment other than antivirals administered to patients included in the evaluated studies appear in Table 1.

Complications

Viral pneumonia or acute respiratory distress syndrome (ARDS) occurred in 1055 (55.9%) of a total of 1886 patients for whom relevant data were available.

In particular, the majority of these 1886 patients were included in three studies [START_REF] Anzic Influenza | Critical care services and 2009 H1N1 influenza in Australia and New Zealand[END_REF][START_REF] Louie | California Pandemic (H1N1) Working Group. Factors associated with death or hospitalization due to pandemic 2009 influenza A(H1N1) infection in California[END_REF][START_REF] Jain | Hospitalized patients with 2009 H1N1 influenza in the United States, April-June 2009[END_REF]. Percentages of patients with viral pneumonia or ARDS ranged between 34.6% and 65.6% among the three studies. In another study providing relevant data for 32 ICU patients, the reported percentage of patients with viral pneumonitis was 90.6% [START_REF] Rello | H1N1 SEMICYUC Working Group. Intensive care adult patients with severe A c c e p t e d M a n u s c r i p t respiratory failure caused by influenza A (H1N1)v in Spain[END_REF]. Bacterial pneumonia complicated H1N1 infection in 290 (12.8%) of 2263 evaluated patients. Detailed data regarding the complications observed in the evaluated patients are presented in Table 1.

Mortality

Of the 3020 reported patients, 386 (12.8%) died. Specifically, 216 deaths (20.2%) occurred in a total of 1068 ICU patients, 156 deaths (9.1%) occurred in a total of 1722 hospitalised patients and 14 deaths (6.1%) occurred in a total of 230 outpatients. In addition, 60 children (6.7%) died from a total of 901 with available mortality data. Specifically, in the largest of the included studies, 110 (14.8%) of the 744 included adults died, whereas 8 (2.3%) of the 344 included A c c e p t e d M a n u s c r i p t children died [START_REF] Louie | California Pandemic (H1N1) Working Group. Factors associated with death or hospitalization due to pandemic 2009 influenza A(H1N1) infection in California[END_REF]. The scarcity of data regarding the proportions of patients with co-morbidities among those who died precluded any further evaluations.

Antiviral treatment and mortality

Three studies provided specific data in order to be included in the analysis regarding the impact of antiviral treatment on the outcome of 2009 H1N1 infection (death versus survival) [START_REF] Louie | California Pandemic (H1N1) Working Group. Factors associated with death or hospitalization due to pandemic 2009 influenza A(H1N1) infection in California[END_REF][START_REF] Raffo | Influenza A(H1N1) epidemic in Argentina. Experience in a National General Hospital (Hospital Nacional Alejandro Posadas)[END_REF][START_REF] Jain | Hospitalized patients with 2009 H1N1 influenza in the United States, April-June 2009[END_REF]. One of these three studies was the largest study included in the review, involving a total of 1088 patients [START_REF] Louie | California Pandemic (H1N1) Working Group. Factors associated with death or hospitalization due to pandemic 2009 influenza A(H1N1) infection in California[END_REF]. The differences observed for these three studies were non-significant, with the exception of the comparison regarding the subgroup of patients older than 18 years who were included in the study of Louie et al. [START_REF] Louie | California Pandemic (H1N1) Working Group. Factors associated with death or hospitalization due to pandemic 2009 influenza A(H1N1) infection in California[END_REF]. Detailed data are presented in Table 2. In addition, another study, which did not provided adequate data in order to be included in the above comparisons, reported that survivors were most likely to have received treatment with NAIs compared with those who died (odds ratio = 7.4, 95% confidence interval 1.8-31.0; P = 0.006) (Table 1) [START_REF] Domínguez-Cherit | Critically ill patients with 2009 influenza A(H1N1) in Mexico[END_REF].

Early administration of antiviral treatment and mortality

Adequate data for the comparison regarding the impact of early administration of antiviral treatment (within 2 days of symptom onset) were provided from one study [START_REF] Louie | California Pandemic (H1N1) Working Group. Factors associated with death or hospitalization due to pandemic 2009 influenza A(H1N1) infection in California[END_REF]. Statistically significant differences were observed in the comparisons regarding the subgroup of patients who were older than 18 years as well as in patients of all ages combined. Specific data are presented in Table 2. In A c c e p t e d M a n u s c r i p t addition, in the multivariate analysis of another study [START_REF] Jain | Hospitalized patients with 2009 H1N1 influenza in the United States, April-June 2009[END_REF] the administration of antiviral treatment within 2 days of symptom onset was significantly associated with a favourable outcome of the infection.

Discussion

According to the findings of this review, approximately one-half of patients with 2009 H1N1 influenza infection included in the reviewed studies received antiviral treatment (mainly oseltamivir). Notably, the majority of these patients were hospitalised and ICU patients. However, the scarcity of comparative data provided from the studies included in the review as well as the lack of available relevant studies with a randomised design precluded us from assessing the effectiveness of the various treatment regimens, as well as their combinations, in reducing morbidity and mortality from 2009 H1N1 influenza. However, the impact of early administration of antiviral treatment (within 2 days of the onset of influenza symptom) in reducing mortality from 2009 H1N1 infection appears to be much more clear according to the findings of this review.

Novel H1N1 influenza began in Mexico in April 2009 and quickly spread worldwide, confirming its pandemic nature. Up to 8 November 2009, more than 206 countries and overseas territories or communities have reported laboratoryconfirmed cases of 2009 H1N1 influenza, whereas the reported deaths associated with this infection, up to this writing, have risen up to 6250 [START_REF]Pandemic (H1N1)[END_REF]. On the other hand, since testing and reporting of 2009 H1N1 influenza cases is not obligatory, these data may be underestimations of the real respective numbers.

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

Various probabilistic models may account for a one hundred-fold higher incidence of the disease for every laboratory-confirmed case [START_REF] Reed | Estimates of the prevalence of pandemic (H1N1) 2009, United States[END_REF]. Preliminary data also suggest that this new infection occurs more frequently in children and young adults [START_REF] Kelly | Interim analysis of pandemic influenza (H1N1) 2009 in Australia: surveillance trends, age of infection and effectiveness of seasonal vaccination[END_REF]. In this review, children younger than 18 years represented a considerable percentage (30%) of the included patients.

NAIs, especially oseltamivir that has the advantage of being available in an oral formulation, have been used for the treatment of seasonal influenza. NAIs have been proven effective in reducing seasonal influenza symptoms, yet their effectiveness in reducing influenza complications and mortality still remains unclear [START_REF] Burch | Prescription of anti-influenza drugs for healthy adults: a systematic review and meta-analysis[END_REF][START_REF] Jefferson | Neuraminidase inhibitors for preventing and treating influenza in healthy adults: systematic review and meta-analysis[END_REF]. Based on this experience from seasonal influenza, NAIs have been used to treat patients with 2009 H1N1 influenza. Specifically, in this review more than one-half of the evaluated patients (53.7%) received antiviral treatment. Notably, the majority of these patients received oseltamivir monotherapy, whereas lower percentages received monotherapy with other antivirals or combinations of antivirals.

The dosage schedules of oseltamivir treatment varied considerably among the included studies. Specifically, maximum dosages of 150 mg twice daily and for a duration longer than the conventional 5-day regimen were administered to a considerable number of ICU patients involved in the evaluated studies.

Corticosteroids were also administered in a considerable proportion (31.8%) of the evaluated patients for whom relevant data were available. Most of these patients were ICU patients with co-morbidity. Owing to the lack of comparative have occurred in a total of over 503 536 confirmed influenza cases [START_REF]Pandemic (H1N1)[END_REF]. An explanation for the discrepancy between the mortality rate of 2009 H1N1 infection observed in this review and the one reported from the WHO is the fact that a considerable proportion of the 2009 H1N1 influenza cases presented in the studies included in this review had co-morbidity. Specifically, 16.1% of the evaluated patients were obese and 12.0% had asthma or COPD. Moreover, 8.4% of the evaluated patients were pregnant. Pregnancy has already been associated with higher morbidity from seasonal influenza [START_REF] Dodds | Impact of influenza exposure on rates of hospital admissions and physician visits because of respiratory illness among pregnant women[END_REF]. Current reports also suggest that pregnant women have an increased risk for complications A c c e p t e d M a n u s c r i p t from 2009 H1N1 influenza [START_REF] Jamieson | Novel Influenza A (H1N1) Pregnancy Working Group. H1N1 2009 influenza virus infection during pregnancy in the USA[END_REF]. In addition, selection bias may be a reason for the relatively high mortality and morbidity observed in this review, since 92% of the reported patients were hospitalised and ICU patients. However, if hospitalisation is warranted for 2009 H1N1 influenza, physicians have to be alert to treat the disease and its complications. In view of this finding despite the lack of definitive data for 2009 H1N1 influenza, treatment with antivirals for those hospitalised is probably warranted. To await the results of randomised controlled trials (RCTs) as the pandemic goes on may not be practical. Even ethical issues may arise, i.e. who should get the medication. Publication bias may also have led to a relatively high reported mortality in this review.

Our attempt to evaluate the effectiveness of antiviral treatment in reducing mortality from 2009 H1N1 influenza was hampered by the lack of studies with a randomised design as well as by the scarcity of comparative data. Of note, no RCT focusing on the treatment of patients with 2009 pandemic influenza was available up to this writing. On the other hand, despite the fact that randomisation procedures could eventually manage to eliminate any known or unknown confounding factors regarding the effectiveness of different treatment strategies used for 2009 pandemic influenza, one should bear in mind that during the critical times of a pandemic such as the influenza pandemic that we are currently witnessing, there might be insufficient time to conduct RCTs.

Moreover, ethical issues might also be raised in regard to the conduction of such trials. In this regard, collection and evaluation of relevant observational data from different clinical settings may aid considerably in the understanding This review has specific limitations that should be taken into consideration in the interpretation of its findings. First, considerable heterogeneity was observed among the patient populations presented in the included studies. Specifically, patients with differences in the severity of the 2009 H1N1 influenza infection, such as ICU patients, hospitalised patients and outpatients, were evaluated. Moreover, mixed populations, including otherwise healthy patients and patients with co-morbidity or risk factors considered to predispose to serious influenza infection such as pregnancy or obesity, were involved in the included studies. In this regard, the generalisability of the findings to influenza treatment in general practice may be limited. In addition, all of the studies included in this review were observational studies describing the strategies used to treat 2009 H1N1 infection of involved patients, whereas no relevant RCTs were identified through the literature searches. In this regard, many factors could have possibly confounded the findings of the individual studies included. Moreover, a considerable number of studies included in the review were retrospective studies. Consequently, the bias of retrospective data collection should also be considered in the interpretation of the findings of this review. The e Overall hospitalisation rate and/or fatality/100 000 for all age groups: 2.8, ranging from 11.9 in infants <1 year to 1.5 in those ≥70 years. In infants <1 year, the highest hospitalisation rates/100 000 were in infants 1 month old (35.8) and 2 months old (21.1); between 3 months and 12 months the hospitalisation rates ranged from 4.2 to 12.6/100 000.

f Obesity, BMI 30-39.9 in adults ≥18 years or BMI percentile 95-100 in children 2-18 years old; and morbid obesity, BMI ≥ 40 in adults only; the BMI was not calculated in pregnant women.

g Commonly used antibiotics were ceftriaxone (94 patients), azithromycin (84 patients), vancomycin (56 patients) and levofloxacin (47 patients).

h Including asthma, chronic lung disease, developmental delay, neuromuscular disease, immunodeficiency, prematurity, recurrent chest infections, and metabolic and endocrine disease.

i Arterial hypertension (3 patients), non-type 1 diabetes mellitus (3 patients, 1 of whom also had hypertension), asthma (2 patients) and obstructive sleep apnoea (1 patient).

  author. Tel.: +30 694 61 10 000; fax: +30 210 68 39 605.

A

  c c e p t e d M a n u s c r i p t of oseltamivir and rimantadine and 1 (0.06%) received a combination of oseltamivir and zanamivir.

  A c c e p t e d M a n u s c r i p t data provided from the non-randomised studies included in this review, evaluation of the potential benefit that corticosteroids might confer on the outcome of the infection was hampered. Of note, a large number of evaluated patients for whom relevant data were available received antibiotics. On the other hand, bacterial pneumonia complicated 2009 H1N1 influenza infection in 12.8% of the 2263 evaluated patients. This leads to the explanation that a significant number of patients received antibiotics on an empirical basis before a definitive diagnosis of 2009 H1N1 influenza was established. In contrast, high percentages (up to 90.6%) of patients were reported to have viral pneumonitis caused from the 2009 H1N1 influenza virus or ARDS in the studies evaluated in this review.According to our findings, the mortality rate of 2009 H1N1 influenza is 12.8%.Most of the patients who died were ICU and hospitalised patients. Official data from the WHO suggest that as of 8 November 2009 at least 6260 deaths (1.2%)

A

  c c e p t e d M a n u s c r i p t and clarification of the field of 2009 pandemic influenza treatment. However, the favourable impact that early administration of antiviral treatment (within 2 days of symptom onset) has on mortality became more obvious from the comparisons performed in this review. The need for early administration of antiviral treatment was also borne out by individual studies included in the review[START_REF] Rello | H1N1 SEMICYUC Working Group. Intensive care adult patients with severe A c c e p t e d M a n u s c r i p t respiratory failure caused by influenza A (H1N1)v in Spain[END_REF].

A

  c c e p t e d M a n u s c r i p t abovementioned factors precluded the establishment of firm conclusions regarding the effectiveness of various treatment strategies used against 2009 H1N1 influenza.In conclusion, our findings suggest that treatment with NAIs, and specifically administration of NAIs within 2 days of symptom onset, may be associated with a favourable outcome of 2009 H1N1 influenza infection. However, no firm conclusions could be established based only on observational studies. In this regard, comparative studies or randomised studies are needed to clarify further the benefit that various treatment strategies might confer to patients with 2009 H1N1 infection and particularly to specific subpopulations such as critically ill patients as well as patients with risk factors predisposing for influenza complications such as pregnancy. Nevertheless, the severity of the disease in patients in need of hospitalisation probably mandates the use of available specific antivirals.

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

  Factors independently associated with in-hospital mortality (OR, 95% CI; P-value): (a) requirement for invasive ventilation at ICU admission (5.51, 3.05-9.94; P < 0.001); (b) older age (OR/year of age: 1.02, 1.01-1.04; P = 0.002); and (c) any co-existing condition (2.56, 1.52-4.30; P < 0.001). b This observation occurred after excluding patients dying early (within 72 h of illness onset), who may have had less opportunity to be exposed to NAIs. c Invasive bacterial co-infection was defined as laboratory detection of a bacterial pathogen in a specimen from normally sterile site or a postmortem lung biopsy. d -Lactam + fluoroquinolones (n = 20; 62.5%), -lactam + macrolides (n = 6; 18.8%) and -lactam + linezolid (n = 5; 15.6%); 1 patient (3.1%) received levofloxacin as monotherapy.

Table 1

 1 Main characteristics of the studies included in the review

	Edited Table 1
	Reference Study design	Characteristics	Country	Patients with co-	Antiviral	Time of	Other treatment	Complications	Outcomes other	Mortality
			of patients		morbidity/risk factors	treatment:	initiation of			than mortality
			evaluated			Type	antiviral
						Dosage	treatment
						Duration
	Cohort studies involving >10 patients
	Studies involving (mainly) ICU patients
	[4]	Prospective	722 patients	Australia	Pregnant, 66/722	NR	NR	Steroids, 91/494 (18.4%);	Viral	Discharged,	In-hospital
			with confirmed	and New	(9.1%); obese adults			renal replacement,	pneumonitis/ARDS,	505/722	mortality,
			infection	Zealand	(BMI > 35), 172/601			27/506 (5.3%);	336/689 (48.8%);	(69.9%)	103/722
			(median age		(28.6%); diabetes,			vasopressor drugs,	secondary bacterial		(14.3%) a
			40 years)		112/700 (16.0%);			176/498 (35.3%)	pneumonia,
					asthma/COPD,				140/689 (20.3%);
					231/707 (32.7%);				exacerbation of
					chronic heart failure,				airflow limitation,
					74/703 (10.5%); co-				95/689 (13.8%);
					existing conditions,				mechanical
					192/687 (27.9%)				ventilation, 456/706
									(64.6%)

  Intensive Care Unit; BMI, body mass index; COPD, chronic obstructive pulmonary disease; NR, not reported; ARDS, acute respiratory distress syndrome; OR, odds ratio; CI, confidence interval; NAI, neuraminidase inhibitor; NA, not applicable; APACHE, Acute Physiology and Chronic Health Evaluation; SOFA, Sequential Organ Failure Assessment Score; IQR, interquartile range; i.v., intravenous; RSV, respiratory syncytial virus; HIV, human immunodeficiency virus; ILI, influenza-like illness; MSF, multiorgan system failure; VAP, ventilator-associated pneumonia; SICU, Surgical Intensive Care Unit; CAP MRSA, community-acquired pneumonia due to meticillin-resistant Staphylococcus aureus.

	[24] [25] Case reports	Retrospective 3 Prospective 3 patients with pregnant women among 13 with available data (median age 26 years) confirmed infection	USA China	Asthma, 2/3 (67%) None	Oseltamivir, 2/3 (66.7%) NR NR Oseltamivir, 3/3 (100%) 75 mg twice a n u s c r i p t Patient who died, 10 days from onset; patient who survived, 1 day from onset Antibiotics, 2/3 (66.7%); non-steroidal anti-inflammatory medication, 1/3 (33.3%); acetaminophen, 1/3 (33.3%); anti-nausea drugs, 1/3 (33.3%) NR NR daily 5-day course M	ARDS, 1/3 (33.3%) None	Cure, 2/3 (66.7%)-pregnancy proceeding normally; mechanical ventilation, 1/3 (33.3%) Cure, 3/3 (100%) 0/3 (0%) 1/3 (33.3%)
	[26]	NA	32-year-old man	Manitoba,	Renal disease		Oseltamivir	NR	Antibiotics	Mental status Haemodialysis, ICU,	Cure with no Survived	0/4 (0%) NA
	[27] ICU,	NA	presented in end-stage renal disease with fluid overload 42-year-old man previously healthy co-infected with nH1N1 and CAP MRSA A c c e p t e d Canada Hong Kong, China None	75 mg daily 5 days None	NA	, each 1/4 Mechanical ventilation, (25%)] haemodialysis, vancomycin, clindamycin, piperacillin/tazobactam, clarithromycin	changes, 4/4 (100%); seizures, 2/4 (50%); encephalopathy, 2/4 (50%); pneumonia, 1/4 (25%) mechanical ventilation, nosocomial pneumonia (Escherichia coli) Mixed bacterial and viral pneumonia; respiratory, circulatory and acute renal failure	neurological sequelae, 4/4 (100%); intensive care, 1/4 (25%) NA	Death

Funding

None.

Competing interests

None declared.

Ethical approval

Not required.


Matthew E Falagas

Sofia Maraki

Drosos E Karageorgopoulos

Antonia C Kastoris

Anastasios Kapaskelis

George Samonis

Antimicrobial susceptibility of Gram-positive non-urinary isolates to fosfomycin

Keywords:

E-mail address: m.falagas@aibs.gr (M.E. Falagas).

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

ABSTRACT

We aimed to evaluate the antimicrobial activity of fosfomycin against Gram-positive non-urinary isolates collected at the microbiological laboratory of the University Hospital of Heraklion, Crete, Greece, in 2008. Susceptibility testing was performed by the disk diffusion method for a total of 1846 isolates; 1275 isolates (69.1%) were susceptible to fosfomycin. Specifically, 416/419 Staphylococcus aureus (99.3%)

[including 129/130 meticillin-resistant S. aureus (MRSA) isolates] and 745/961 coagulase-negative staphylococci (77.5%) were susceptible to fosfomycin. Among 42 Streptococcus pneumoniae, 64 Streptococcus pyogenes and 93 other streptococcal isolates, 61.9%, 40.6% and 48.4%, respectively, were susceptible to fosfomycin. Fosfomycin was inactive against the 166 enterococcal isolates tested. This old antibiotic may deserve consideration for further studies and use in clinical practice, especially for S. aureus (including MRSA) infections.

Introduction

Antibacterial agents traditionally used for the treatment of infections caused by Gram-positive cocci are becoming increasingly ineffective [START_REF] Levy | Antibacterial resistance worldwide: causes, challenges and responses[END_REF]. The spread of meticillin-resistant staphylococci and penicillin-non-susceptible Streptococcus pneumoniae, in the community or the hospital, and of vancomycin-resistant enterococci, mainly in the hospital, is of great public health importance [START_REF] Arias | Antibiotic-resistant bugs in the 21st century-a clinical super-challenge[END_REF].

Although new antibacterial agents with activity against these pathogens have been developed, each has a specific place in therapy [START_REF] Karageorgopoulos | New antibiotics: optimal use in current clinical practice[END_REF]. Thus, expansion of the current therapeutic options against resistant Gram-positive cocci is an important goal.

Novel therapeutic options may sometimes be identified among older, almost neglected antibiotics. Fosfomycin is an agent that might merit re-evaluation for use against contemporary resistant pathogens [START_REF] Falagas | Fosfomycin: use beyond urinary tract and gastrointestinal infections[END_REF]. It has a unique mechanism of action and a wide spectrum of antimicrobial activity that encompasses both Gram-positive and Gram-negative aerobic bacteria [START_REF] Reeves | Fosfomycin trometamol[END_REF]. In this study, we sought to evaluate retrospectively the in vitro antimicrobial activity of fosfomycin against recent Grampositive non-urinary isolates.

Methods

This study included all Gram-positive clinical isolates originating from sites other than the urinary tract collected over a 1-year period (January-December 2008) at A c c e p t e d M a n u s c r i p t 5 the microbiological laboratory of the 700-bed University Hospital of Heraklion (Heraklion, Crete, Greece). All isolates for which susceptibility testing to fosfomycin had been performed were included. No specific criteria were set throughout the study period for the selection of isolates to be tested for fosfomycin susceptibility.

Only the first isolate of the same species for each patient was included.

Routine laboratory methods were used for specimen processing and culture.

Bacterial species identification was done by standard biochemical methods, the API system or the Vitek 2 automated system (bioMérieux, Marcy l'Etoile, France) [START_REF] Thomson | Specimen collection, transport and processing: bacteriology[END_REF]. Antimicrobial susceptibility testing for all antibiotics was performed by the disk diffusion method following the recommendations of the Clinical and Laboratory Standards Institute (CLSI) [START_REF]Performance standards for antimicrobial susceptibility testing[END_REF]. Specifically, disks used for determination of susceptibility to fosfomycin contained 200 g of fosfomycin plus 25 g of glucose-6-phospate. To interpret susceptibility to fosfomycin, values corresponding to the most relevant CLSI breakpoints were used as provisional breakpoints, i.e. those for Enterococcus faecalis urinary tract isolates.

Results

A total of 1846 Gram-positive, non-urinary, first-patient isolates for which susceptibility to fosfomycin had been tested during the study period was evaluated, representing 87.2% of all the Gram-positive non-urinary isolates examined. The Table 1 shows the origin of the patients who provided the culture specimens from which the isolates grew as well as the type of specimens.

Overall, 1275 (69.1%) of the 1846 studied isolates were found to be susceptible to fosfomycin. Among the antimicrobial agents evaluated, only linezolid, vancomycin, teicoplanin and rifampicin were more active than fosfomycin for all isolates studied, with rates of susceptibility to the above agents of 98.5%, 98.2%, 97.1% and 81.7%, respectively. Tetracycline (68.7% susceptible), trimethoprim/sulfamethoxazole (57.4%), amoxicillin/clavulanic acid (56.3%), gentamicin (52.9%), imipenem (45.0%), clindamycin (44.7%) and ciprofloxacin (44.4%) followed fosfomycin in terms of highest susceptibility rates.

Discussion

The main finding of this study, which evaluated recent Gram-positive non-urinary isolates collected at a university hospital in Greece, is that fosfomycin exhibits high in vitro antimicrobial activity against S. aureus, including meticillin-resistant S. aureus (MRSA). Fosfomycin also shows activity against a substantial proportion of coagulase-negative staphylococci. Fosfomycin may be active against some S. pneumoniae isolates (particularly those without a MDR phenotype) as well as other streptococcal isolates, including Streptococcus pyogenes. However, it appears to be inactive against enterococcal isolates.

Previous studies have also shown high in vitro susceptibility of MRSA to fosfomycin. A recent systematic review on this issue identified 22 relevant studies, the majority of which showed susceptibility of MRSA isolates to fosfomycin of A c c e p t e d M a n u s c r i p t 8 >90% [START_REF] Falagas | Fosfomycin for the treatment of infections caused by Gram-positive cocci with advanced antimicrobial drug resistance: a review of microbiological, animal and clinical studies[END_REF]. Other studies have shown that fosfomycin can be active against penicillin-non-susceptible S. pneumoniae [START_REF] Falagas | Fosfomycin for the treatment of infections caused by Gram-positive cocci with advanced antimicrobial drug resistance: a review of microbiological, animal and clinical studies[END_REF]. It should be mentioned, however, that the majority of the aforementioned studies referred to isolates collected in relatively distant periods in the past. It is also noteworthy that fosfomycin can modify the expression of altered penicillin-binding proteins in MRSA and penicillin-nonsusceptible S. pneumoniae, resulting in synergy with -lactams [9,10].

An important issue for the interpretation of data on antimicrobial susceptibility to fosfomycin refers to the choice of appropriate susceptibility breakpoints. The relevant breakpoints that have been issued by major pertinent organisations vary considerably. Moreover, the CLSI breakpoints used in this study refer to urinary isolates of E. faecalis. Recently, the European Committee on Antimicrobial Susceptibility Testing (EUCAST) issued fosfomycin minimum inhibitory concentration (MIC) breakpoints for staphylococci that are not site-specific. The latter breakpoints are one dilution stricter than the CLSI MIC breakpoints for E. faecalis that correspond to the disk zone diameter breakpoints used in our study.

Fosfomycin has bactericidal antimicrobial activity through a unique mechanism of action that involves inhibition of MurA, an enzyme that catalyses the first committed step in bacterial cell wall synthesis [START_REF] Falagas | Fosfomycin: use beyond urinary tract and gastrointestinal infections[END_REF]. This may partly explain the low levels of cross-resistance that have been noted between fosfomycin and other antibacterial agents [START_REF] Reeves | Fosfomycin trometamol[END_REF]. However, mutational resistance to fosfomycin can arise rather rapidly in A c c e p t e d M a n u s c r i p t 9 vitro after exposure to this agent [START_REF] Ellington | Mutators among CTX-M -lactamase-producing Escherichia coli and risk for the emergence of fosfomycin resistance[END_REF]. Moreover, resistance determinants encoding for fosfomycin resistance have been identified on plasmids [START_REF] Nakaminami | Characterization of the pTZ2162 encoding multidrug efflux gene qacB from Staphylococcus aureus[END_REF]. In Greece, fosfomycin was off the market during the period of our study. Whether resistant strains will predominate if fosfomycin is used at large for the treatment of MRSA infections is thus a matter of concern.

Fosfomycin has been mainly used as single-dose oral therapy for acute uncomplicated cystitis, in the form of fosfomycin tromethamine [START_REF] Reeves | Fosfomycin trometamol[END_REF]. The disodium salt of fosfomycin is also available in certain countries for intravenous (i.v.) administration. Although fosfomycin has not been formally evaluated for the treatment of infections other than those involving the urinary tract, cumulative experience from the use of i.v. fosfomycin for various types of systemic infections appears to be favourable [START_REF] Falagas | Fosfomycin: use beyond urinary tract and gastrointestinal infections[END_REF]. The pharmacokinetic or pharmacodynamic properties of this drug do not limit its role to the treatment of urinary tract infections only [START_REF] Roussos | Clinical significance of the pharmacokinetic and pharmacodynamic characteristics of fosfomycin for the treatment of patients with systemic infections[END_REF].

Apart from urine, fosfomycin appears to achieve clinically relevant concentrations in tissues that MRSA infections can involve, such as skin and subcutaneous tissue, bone, lungs and serum. However, clinical evidence regarding the use of fosfomycin for the treatment of MRSA infections is scarce and consists only of a few reports that have generally showed effectiveness of fosfomycin therapy, commonly in combination with other active agents, in cases of serious staphylococcal infections [START_REF] Falagas | Fosfomycin for the treatment of infections caused by Gram-positive cocci with advanced antimicrobial drug resistance: a review of microbiological, animal and clinical studies[END_REF].

A c c e p t e d M a n u s c r i p t 10 A particular value of fosfomycin in the treatment of MRSA infections lies in the fact that it can be administered orally. Thus, it could prove to be a therapeutic option for patients with hospital-acquired MRSA who need to complete a course of treatment at home. Additionally, it could prove a useful option for the treatment of MRSA infections acquired in the community, as resistance of community-acquired MRSA to commonly used oral antibiotics is increasing [START_REF] Diep | Emergence of multidrug-resistant, community-associated, methicillin-resistant Staphylococcus aureus clone USA300 in men who have sex with men[END_REF]. Furthermore, some of the agents that are active against community-acquired MRSA may be the cause of worrisome toxicity in children, where a substantial proportion of these infections occur, whilst fosfomycin has generally proved safe when used in this population [15].

In conclusion, this study indicates that fosfomycin can have substantial antimicrobial activity against staphylococcal isolates, especially against S. aureus regardless of the presence of meticillin resistance. We suggest further microbiological and clinical evaluation of fosfomycin in this regard, particularly as the armamentarium of orally available anti-MRSA drugs is diminishing.

*

  Corresponding author. Tel.: +30 694 611 0000; fax: +30 210 683 9605.

  1846 study isolates comprised 1380 Staphylococcus spp. (74.8%), 199 A c c e p t e d M a n u s c r i p t 6 Streptococcus spp. (10.8%), 166 Enterococcus spp. (9.0%) and 101 other Grampositive isolates (5.5%) (Corynebacterium spp., Micrococcus spp., Aerococcus spp., Lactococcus spp., Gemella spp., Listeria spp. and Brevibacterium spp.).

  A c c e p t e d M a n u s c r i p t 7 isolates. The subset of 23 multidrug-resistant (MDR) S. pneumoniae isolates (defined as resistant to at least two of the following agents: penicillin, cefuroxime, erythromycin or trimethoprim/sulfamethoxazole) had lower susceptibility to fosfomycin. None of the 166 enterococcal isolates studied was found to be susceptible to fosfomycin. These comprised 115 E. faecalis and 51 Enterococcus faecium isolates; 7 (6.1%) and 19 (37.3%), respectively, were vancomycinresistant. Finally, 5.5% of the 73 Corynebacterium spp. isolates studied were susceptible to fosfomycin.

  Careddu P, Borzani M, Scotti L, Varotto F, Garlaschi L, Fontana P. Treatment of lower urinary tract infections in children: single dose fosfomycin trometamol versus pipemidic acid. Chemioterapia 1987;6:290-4.

Table 2

 2 presents the susceptibility rates to fosfomycin and other selected antibacterial agents tested for the staphylococcal and streptococcal isolates studied. Specifically, 84.1% of the 1380 staphylococcal isolates studied were susceptible to fosfomycin, including 99.3% of the 419 Staphylococcus aureus isolates. The presence of meticillin resistance in S. aureus isolates did not affect susceptibility to fosfomycin. Additionally, 48.7% of all the 199 streptococcal isolates studied were susceptible to fosfomycin, including 61.9% of the 42 S. pneumoniae

Table 1

 1 Main characteristics of the 1846 Gram-positive non-urinary isolates studied

	Characteristic	n (%)
	Origin of patient
	Surgical wards	441 (23.9)
	Medical wards	409 (22.2)
	Intensive Care Unit	386 (20.9)
	Dialysis unit	46 (2.5)
	Adult outpatient units	216 (11.7)
	Paediatric units	294 (15.9)
	Inpatients	283 (96.3)
	Outpatients	11 (3.7)
	Other hospital units	54 (2.9)
	Culture specimen
	Blood	723 (39.2)
	Purulent collections or surgical specimens 552 (29.9)
	Upper respiratory tract	166 (9.0)
	Lower respiratory tract	129 (7.0)
	Normally sterile fluids	126 (6.8)
	Obstetric and gynaecological specimens	51 (2.8)
	Removed catheter tips	39 (2.1)
	Other clinical sites	60 (3.3)

Table 2

 2 Susceptibility to fosfomycin and other selected antibacterial agents of the staphylococcal and streptococcal isolates testedDrugSusceptible isolates [n/N (%)]

		Staphylococcus	MRSA a	CoNS b	Streptococcus	MDR S.	Streptococcus	Other
		aureus			pneumoniae	pneumoniae c	agalactiae	streptococci d
	AMC	ND	ND	ND	18/18 (100)	1/1 (100)	24/24 (100)	55/56 (98.2)
	Ampicillin	40/314 (12.7)	ND	18/374	18/19 (94.7)	ND	24/24 (100)	51/52 (98.1)
				(4.8)
	SAM	198/314 (63.1)	ND	90/374	ND	ND	ND	ND
				(24.1)
	Cefoxitin	24/88 (27.3)	ND	ND	ND	ND	ND	ND
	Cefuroxime	198/314 (63.1)	ND	68/351	25/42 (59.5)	6/23 (26.1)	ND	ND
				(19.4)
	Ceftriaxone	ND	ND	ND	41/42 (97.6)	22/23 (95.7)	ND	ND
	Clindamycin 330/419 (78.8)	93/130	313/961	31/42 (73.8)	12/23 (52.2)	19/25 (76.0)	56/68 (82.4)
			(71.5)	(32.6)
	Ciprofloxacin 287/331 (86.7)	47/66	285/567	ND	ND	ND	ND
			(71.2)	(50.3)
	Erythromycin 304/419 (72.6)	85/130	243/961	22/42 (52.4)	3/23 (13.0)	19/25 (76.0)	41/67 (61.2)
			(65.4)	(25.3)

a The 130 MRSA isolates constitute a subset of the

S. aureus isolates. b Includes Staphylococcus epidermidis, Staphylococcus saprophyticus and Staphylococcus haemolyticus. c The 23 MDR S. pneumoniae isolates constitute a subset of the 42 S. pneumoniae isolates. d Other streptococci include (i) -haemolytic streptococci other than S. pneumoniae and (ii) -haemolytic streptococci other than S. agalactiae or S. pyogenes.

Funding

None.

Competing interests

None declared.