Sara Violante

Lodewijk Ijlst

Henk Van Lenthe

Isabel Tavares De Almeida

Ronald J Wanders

Fátima V Ventura
email: fatima.ventura@ff.ul.pt

Carnitine palmitoyltransferase 2: New insights on the substrate specificity and implications for acylcarnitine profiling

Keywords: mFAO -mitochondrial fatty acid β-oxidation, CPT1 -carnitine palmitoyltransferase Acylcarnitines, Carnitine palmitoyltransferase 2 (CPT2), Long-chain acyl-CoA metabolites, Fatty acid oxidation (FAO), Mitochondrial Trifunctional Protein

Introduction

Mitochondrial fatty acid β-oxidation (mFAO) is the most important source of energy, especially for high-energy demanding tissues such as the heart and skeletal muscle [START_REF] Eaton | Mammalian mitochondrial beta-oxidation[END_REF]. In order to be metabolized in the mitochondria, long-chain fatty acids must first undergo During fasting or when the energy demand is increased, fatty acid oxidation is crucial for cellular energy homeostasis. Pathologies involving one or several defects of the mitochondrial fatty acid β-oxidation system, especially those concerning long-chain fatty acids, are complex and clinically heterogeneous. Affected patients usually present hypoketotic hypoglycaemia with hepatic, cardiac and muscular symptoms [START_REF] Roe | Mitochondrial Fatty Acid Oxidation Disorders[END_REF][START_REF] Wanders | Disorders of mitochondrial fatty acyl-CoA beta-oxidation[END_REF]. In as their correspondent carnitine esters [START_REF] Ventura | Quantitative acylcarnitine profiling in fibroblasts using [U-13C] palmitic acid: an improved tool for the diagnosis of fatty acid oxidation defects[END_REF][START_REF] Sim | Strategies for the diagnosis of mitochondrial fatty acid beta-oxidation disorders[END_REF].

It is hypothesized that the acylcarnitines are formed by carnitine palmitoyltransferase 2, although formal proof is lacking for most of the acyl-CoAs. This would be followed by the export of these acylcarnitines from the mitochondria and the cell, processes not yet definitively clarified. Some work has been done pointing towards reversibility of the physiologic mechanism of the carnitine shuttle [START_REF] Ventura | Carnitine palmitoyltransferase II specificity towards beta-oxidation intermediates-evidence for a reverse carnitine cycle in mitochondria[END_REF][START_REF] Schaefer | Characterisation of carnitine palmitoyltransferases in patients with a carnitine palmitoyltransferase deficiency: implications for diagnosis and therapy[END_REF]. However the lack of a comprehensive study on this subject and specifically on human CPT2 prompted us to investigate the complete substrate specificity of this enzyme. The data described in this paper provides new insights into the specific role of CPT2 in the export of toxic acyl-CoAs from the mitochondria into the cytosol and subsequently into the extracellular space.

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Materials and Methods

Strains of Saccharomyces cerevisiae and growth media

The ∆cat2 (cat2::KAN) and ∆fox2 (fox2::KAN) deletion mutants of S. cerevisiae strain BY4742 (Matα; his3∆1; leu2∆0; lys2∆0; ura3∆0) were used. The strain was cultured in rich glucose medium, YPD (glucose 20 g/L, peptone 20 g/L and yeast extract 10 g/L) and minimal glucose medium, YNBD (glucose 3 g/L or 20 g/L and yeast nitrogen base without aminoacids 6.7 g/L). For plates, agar 20 g/L was added. Galactose medium contained yeast extract 1 g/L, yeast nitrogen base without aminoacids 6.7 g/L and galactose 200 g/L. Aminoacids were added (2 mg/mL) as required. Yeast nitrogen base, yeast extract, peptone and agar were obtained from Difco Laboratories Inc. (Detroit, MI).

Glucose and galactose were obtained from Sigma-Aldrich (St. Louis, MO, USA).

Plasmids and Cell Culture conditions

The plasmid used (pYES2-CPT2) was a generous gift from Dr. F. Taroni, Milano, Italy [START_REF] Taroni | Molecular characterization of inherited carnitine palmitoyltransferase II deficiency[END_REF]. Confirmation of the sequence was achieved by direct sequencing which showed a correct insertion into the pYES2 vector of the complete open reading frame of the human CPT2, including the region coding for the corresponding mitochondrial targeting signal. Transformation of the ∆cat2 and ∆fox2 mutants with the pYES2-CPT2 plasmid was performed using the lithium acetate method, as described elsewhere [START_REF] Becker | High-efficiency transformation of yeast by electroporation[END_REF].

Transformed cells were harvested by centrifugation, spread on 3 g/L YNBD plates containing aminoacids as required and cultured for two days at 28°C.

Growth conditions and preparation of yeast homogenates

Cells were grown on minimal 20 g/L glucose medium for at least 24 h at 225 rpm and 28°C in a gyro shaker and then shifted by centrifugation to galactose medium. Cells from overnight cultures grown on galactose medium were harvested and treated with zymolyase as described elsewhere [START_REF] Yaffe | Analysis of mitochondrial function and assembly[END_REF]. The resulting protoplasts were homogenized by sonication (3 times, 10 s at 8W) on ice and suspended in PBS with Complete mini tablets containing a cocktail of protease inhibitors (Roche; Basel Switzerland). Protein concentration of the yeast homogenates was determined using the bicinchoninic acid

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assay (BCA, Sigma-Aldrich, St. Louis, MO, USA) [START_REF] Smith | Measurement of protein using bicinchoninic acid[END_REF] and human serum albumin as a reference substance. Spray Ionization Tandem Mass Spectrometry (ESI-MS/MS). Negative controls were performed as described above, using yeast homogenates transformed with the empty vector.

Inhibition studies upon carnitine palmitoyltransferase 2 activity

The effect of trans-2-C16:1-CoA on CPT2 activity was determined by measuring its activity in the presence of different concentrations of this compound (0 -20 µM) and using C16-CoA as substrate (0 -40 µM). Activity was measured as described above with

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7 some modifications. After 5 min incubation at 37ºC the reaction was terminated by adding 750 µL acetonitrile containing 100 pmol d3C3-, 100 pmol d3C8-and 50 pmol d3C16-carnitine internal standards. In order to gain 10 times more sensitivity, the samples were analysed on UPLC-MS/MS without derivatization.

Results

Determination of carnitine palmitoyltransferase 2 activity using different acyl-CoA esters

In order to determine the substrate specificity of human CPT2, we transformed the ∆cat2 S. cerevisiae mutant (BY4742 cat2::KAN) with a plasmid expressing human CPT2 (see section 2.2). This mutant has no carnitine acetyltransferase activity (cat2, converting acetyl-CoA into acetylcarnitine). Immunoblot analysis after subcellular fractionation on Nycodenz gradient showed that the protein is localized in the mitochondria, although not fully processed to maturity (results not shown). More important, the heterologously expressed human CPT2 is in its active form and thus used for subsequent kinetic measurements. The results depicted in Fig. 1A show that CPT2 is well expressed and is active towards medium (C8-C12) and long-chain (C14-C18) acyl-CoA esters. Virtually no activity was found with short-and very long-chain acyl-CoA esters (Fig. 1A). Furthermore, CPT2 showed activity towards the cis-5 and cis-9 unsaturated acyl-CoAs tested (Fig. 1B) and the mitochondrial β-oxidation intermediates 3-OH and 3-ketopalmitoyl-CoA (Fig. 1C). Surprisingly, different trans-2-enoyl-CoA, which are intermediates of mFAO, including trans-2-C16:1-CoA and trans-2-C12:1-CoA, were found to be poor substrates for CPT2. We observed that trans-2-C16:1 and trans-2-C12:1-CoA intermediates show 1.5% and 1% activity respectively when compared with the straight-chain acyl-CoAs intermediates of the same chain-length (C16-CoA and C12-CoA). In the same manner, only a small percentage of activity (approximately 1.5%) was found when comparing with other enoyl substrates (cis-5 and cis-9-C14:1-CoA) (Fig. 1B).

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Breakdown of different aminoacids including the branched-chain amino acids also involves CoA esters. When tested, none of these acyl-CoAs were found to be a substrate for CPT2 (see Table 1). Intermediates from peroxisomal fatty acid β-oxidation, namely 4,8-dimethylnonanoyl-CoA (DMN-CoA), 2,6-dimethylheptanoyl-CoA (DMH-CoA) and pristanoyl-CoA, were also investigated as potential substrates for CPT2. Interestingly, CPT2 was found to be active with DMN-CoA which is in line with data in literature [START_REF] Verhoeven | Studies on the oxidation of phytanic acid and pristanic acid in human fibroblasts by acylcarnitine analysis[END_REF],

whereas virtually no activity was found with the substrates pristanoyl-CoA or DMH-CoA (Fig. 1D).

Determination of the enzymatic kinetic parameters of CPT2

In order to resolve whether trans-2-enoyl-CoAs may act as inhibitors of CPT2 activity, we determined the kinetic parameters of CPT2 (K m and V max ) using C16-CoA and trans-2-C16:1-CoA as substrates. Employing the Lineweaver-Burk linearization of the Michaelis-Menten equation, we found that although trans-2-C16:1-CoA and C16-

CoA have approximately the same affinity for CPT2 (K m(C16-CoA) = 7.1 µM and K m(trans-2- C16:1-CoA) = 8.1 µM), the catalytic efficiency (here expressed as the ratio V max /K m ) for C16-CoA is approximately 20-fold higher than for trans-2-C16:1-CoA (Table 2). These results indicate that trans-2-enoyl-CoAs might act as inhibitors of CPT2.

Inhibition studies with trans-2-C16:1-CoA

To verify if trans-2-C16:1-CoA may act as an inhibitor of the conversion of acyl-CoA esters into carnitine derivatives catalysed by CPT2, inhibition studies were performed in the ∆fox2 S. cerevisiae mutant (BY4742 fox2::KAN) transformed with the plasmid described in section 2.2 from Materials and Methods. This mutant lacks 2-enoyl-CoA hydratase activity (fox2, converting trans-2-enoyl-CoA esters into L-3hydroxyacyl-CoA esters). The results shown for each concentration of inhibitor reveal a small variation (6%) concerning the V max while an increase of approximately 30% in the K m is observed. This suggests that trans-2-C16:1-CoA acts as a competitive inhibitor of CPT2 with a K i of 18.8 µM (Fig. 2 and Table 3).

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Discussion

The work described in this paper clearly shows that CPT2, when operating in the reverse direction (acyl-CoA + carnitine → acylcarnitine + CoA), accepts, at least in vitro, a range of different medium and long-chain straight-chain acyl-CoAs (Fig. 1A) and converts them into the respective acylcarnitines. These results are in agreement with the data reported by Schaefer et al. [START_REF] Schaefer | Characterisation of carnitine palmitoyltransferases in patients with a carnitine palmitoyltransferase deficiency: implications for diagnosis and therapy[END_REF] in permeabilized human fibroblasts. In this model of study the substrate specificity of a malonyl-CoA insensitive acyltransferase, identified as CPT2, differs to some extent from the results obtained by us using recombinant human CPT2. According to these authors, CPT2 shows higher activity with substrates ranging from C8 to C16-CoA with C12-CoA as the preferred substrate followed by C8-CoA, which was also recognized as the preferred substrate for the recombinant rat liver CPT2 studied by Johnson et al. [START_REF] Johnson | Over-expression and characterization of active recombinant rat liver carnitine palmitoyltransferase II using baculovirus[END_REF]. Our results, however, show that CPT2 has specificity towards longer acyl-CoA esters ranging from C8 to C20-CoA with C10 to C14-CoA as the preferred substrates (Fig. 1A), which compares well previous data on CPT2 in bovine liver mitochondria [START_REF] Ramsay | The soluble carnitine palmitoyltransferase from bovine liver. A comparison with the enzymes from peroxisomes and from the mitochondrial inner membrane[END_REF], chick embryo liver [START_REF] Ishii | The presence of peroxisomal carnitine palmitoyltransferase in chick embryo liver[END_REF] and beef heart [START_REF] Clarke | Isolation and purification of mitochondrial carnitine octanoyltransferase activities from beef heart[END_REF].

Concerning the possible conversion of mFAO intermediates into their corresponding acylcarnitine esters by CPT2 we observed that this enzyme accepts different mFAO intermediates as substrates, including 3-OH and 3-keto acyl-CoA esters, though less efficiently (Fig. 1C). Nevertheless, trans-2-enoyl-intermediates were found to be poor substrates for the enzyme which suggests that CPT2 may not be as active towards trans-2-enoyl-CoAs as shown for the other mFAO intermediates or other unsaturated acyl-CoA esters (Fig. 1B). This data is consistent with our previous work using purified human CPT2 protein [START_REF] Ventura | Carnitine palmitoyltransferase II specificity towards beta-oxidation intermediates-evidence for a reverse carnitine cycle in mitochondria[END_REF]. The trans double bond in the trans-2-enoyl-CoA esters may cause a spatial constraint in the binding of these intermediates to the active site of the enzyme causing the reaction to occur slower than with other substrates. These findings may well explain the fact that trans-2-enoyl-carnitine intermediates have never been reported neither in plasma nor in fibroblasts acylcarnitine profiles from patients with mFAO disorders [START_REF] Ventura | Quantitative acylcarnitine profiling in fibroblasts using [U-13C] palmitic acid: an improved tool for the diagnosis of fatty acid oxidation defects[END_REF]. The catalytic efficiency of CPT2 with the substrates trans-2-enoyl-CoAs is much lower than when handling the substrate palmitoyl-CoA (Table 2) and therefore the reaction is expected to proceed very slowly. Accordingly, enoyl intermediates were found

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to act as competitive inhibitors of CPT2 activity (Table 3; Fig. 3). This may lead to the accumulation of such intermediates within the mitochondrial matrix in specific mFAO deficiencies, which would block further clearance of other toxic acyl-CoA species accumulating in these disorders. Trans-2-enoyl-CoAs are known toxic intermediates [START_REF] Ventura | Differential inhibitory effect of long-chain acyl-CoA esters on succinate and glutamate transport into rat liver mitochondria and its possible implications for long-chain fatty acid oxidation defects[END_REF][START_REF] Ventura | Inhibition of oxidative phosphorylation by palmitoyl-CoA in digitonin permeabilized fibroblasts: implications for long-chain fatty acid beta-oxidation disorders[END_REF][START_REF] Treem | Acute fatty liver of pregnancy, hemolysis, elevated liver enzymes, and low platelets syndrome, and long chain 3hydroxyacyl-coenzyme A dehydrogenase deficiency[END_REF] which are expected to accumulate intramitochondrially in mitochondrial trifunctional protein (MTP) and long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD)

deficiencies. In addition to the symptoms usually observed in all mFAO disorders, MTP and LCHAD deficient patients also present severe characteristic symptoms including neurological and ophthalmological manifestations (peripheral neuropathy and retinopathy) and serious maternal complications during pregnancy [START_REF] Treem | Acute fatty liver of pregnancy, hemolysis, elevated liver enzymes, and low platelets syndrome, and long chain 3hydroxyacyl-coenzyme A dehydrogenase deficiency[END_REF][START_REF] Tyni | Pregnancy complications are frequent in longchain 3-hydroxyacyl-coenzyme A dehydrogenase deficiency[END_REF][START_REF] Tyni | Ophthalmologic findings in long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency caused by the G1528C mutation: a new type of hereditary metabolic chorioretinopathy[END_REF][START_REF] Tyni | Long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency[END_REF]. It is unlikely that such severe abnormalities result from the simple accumulation of long-chain intermediates as they do not emerge in patients suffering from other long-chain mFAO disorders such as a deficiency in very long-chain acyl-CoA dehydrogenase (VLCAD) [START_REF] Andresen | Clear correlation of genotype with disease phenotype in very-longchain acyl-CoA dehydrogenase deficiency[END_REF]. Such clinical facts suggest different pathological mechanisms underlying these disorders. This may in part be explained by the potential inhibition of CPT2 by trans-2enoyl-CoAs which only accumulate in MTP and LCHAD deficiencies.

CPT2 activity was also evaluated with some peroxisomal FAO substrates and intermediates. Only 4,8-dimethylnonanoyl-CoA (DMN-CoA) shows reactivity with the enzyme (Fig. 1D). This is in line with the pristanic acid degradation route, where the first steps are known to take place in the peroxisome [START_REF] Verhoeven | Studies on the oxidation of phytanic acid and pristanic acid in human fibroblasts by acylcarnitine analysis[END_REF]. Peroxisomal degradation of pristanic acid proceeds for three cycles of β-oxidation yielding the intermediate DMN-

CoA, probably converted by carnitine octanoyltransferase into the respective carnitine ester [START_REF] Ferdinandusse | Molecular cloning and expression of human carnitine octanoyltransferase: evidence for its role in the peroxisomal beta-oxidation of branched-chain fatty acids[END_REF] and further exported into the cytoplasm. Further oxidation requires the mitochondrial import of DMN-carnitine and its conversion back into DMN-CoA. One additional cycle of β-oxidation in mitochondria then leads to the formation of 2,6dimethylheptanoyl-CoA (DMH-CoA) [START_REF] Verhoeven | The metabolism of phytanic acid and pristanic acid in man: a review[END_REF]. The metabolic fate of DMH-CoA is not known [START_REF] Verhoeven | The metabolism of phytanic acid and pristanic acid in man: a review[END_REF] but its destiny, according to our results, does not entail its conversion to the corresponding carnitine ester, at least as catalyzed by CPT2. The importance of these data for the in vivo situation remains to be resolved especially since neither DMN nor DMH carnitine derivatives have ever been reported among the acylcarnitine profiles of any Also several acyl-CoA esters formed during branched-chain aminoacid oxidation were tested and were not found to be substrates for CPT2. The presence of the corresponding carnitine intermediates suggests that these may be formed by the action of other transferases, including carnitine acetyltransferase (CRAT). A more detailed study on the substrate specificity of CRAT is lacking in literature, thus requiring further investigation in future work.

Conclusions

In summary although acylcarnitines have been recognized over the last years as important biomarkers for the early detection of mitochondrial [START_REF] Pasquali | Biochemical findings in common inborn errors of metabolism[END_REF][START_REF] Millington | Application of fast atom bombardment with tandem mass spectrometry and liquid chromatography/mass spectrometry to the analysis of acylcarnitines in human urine, blood, and tissue[END_REF][START_REF] Rashed | Diagnosis of inborn errors of metabolism from blood spots by acylcarnitines and amino acids profiling using automated electrospray tandem mass spectrometry[END_REF][START_REF] Chace | Rapid diagnosis of MCAD deficiency: quantitative analysis of octanoylcarnitine and other acylcarnitines in newborn blood spots by tandem mass spectrometry[END_REF][START_REF] Vreken | Quantitative plasma acylcarnitine analysis using electrospray tandem mass spectrometry for the diagnosis of organic acidaemias and fatty acid oxidation defects[END_REF][START_REF] Costa | Quantitative analysis of plasma acylcarnitines using gas chromatography chemical ionization mass fragmentography[END_REF] and peroxisomal [START_REF] Verhoeven | Studies on the oxidation of phytanic acid and pristanic acid in human fibroblasts by acylcarnitine analysis[END_REF] fatty acid oxidation disorders, peroxisomal biogenesis defects [START_REF] Duranti | Urine acylcarnitine analysis by ESI-MS/MS: a new tool for the diagnosis of peroxisomal biogenesis disorders[END_REF] and branchedchain aminoacid oxidation disorders [START_REF] Pasquali | Biochemical findings in common inborn errors of metabolism[END_REF] and acylcarnitine profiling in blood allows a rapid and effective screening of the aforementioned inborn errors of metabolism, the studies presented here clearly demonstrate that the profile of acylcarnitines as observed in plasma may not reflect the profile of acyl-CoA species which accumulate within the mitochondrial matrix under certain conditions, at least not fully. If an intermediate containing a trans-2 double bond accumulates intramitochondrially, the respective carnitine esters might be missed in the diagnosis. Hence, the data gathered in the present paper support the rationale for the relevance of determining mitochondrial acyl-CoA esters profile [START_REF] Minkler | Novel isolation procedure for short-, medium-, and long-chain acyl-coenzyme A esters from tissue[END_REF]. Undoubtedly this approach may be crucial for the elucidation of the diagnosis and further treatment of mFAO disorders as well as for the understanding of the pathogenic mechanisms involved in these inborn errors of metabolism.

  to its transport into the mitochondrial matrix. The import of activated long-chain fatty acids (long-chain acyl-CoAs) into the mitochondrial matrix is handled by the carnitine shuttle. This system operates by the combined action of carnitine palmitoyltransferase 1 (CPT1, EC 2.3.1.21), carnitine/acylcarnitine translocase (CACT, SLC25A20) and carnitine palmitoyltransferase 2 (CPT2, EC 2.3.1.21) and requires the presence of L-carnitine. Long-chain acyl-CoA esters are first converted into the corresponding carnitine esters by CPT1, followed by transport of the resulting acylcarnitines across the mitochondrial membrane by CACT, located in the inner mitochondrial membrane, in exchange with free carnitine. The final step of this cycle, catalyzed by CPT2, reconverts the acylcarnitines back into the respective acyl-CoA esters that can then undergo β-oxidation [2-4].

  addition to the symptoms usually observed in mFAO disorders, those affecting longchain mFAO such as deficiencies at the level of very long-chain acyl-CoA dehydrogenase (VLCAD), long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) and mitochondrial trifunctional protein (MTP) tend to be more severe and present unusual features. Such complications may be caused by the accumulation of toxic intermediates as a consequence of the impaired β-oxidation of long-chain fatty acids[START_REF] Wanders | Disorders of mitochondrial fatty acyl-CoA beta-oxidation[END_REF][START_REF] Boer | Mitochondrial trifunctional protein deficiency: a severe fatty acid oxidation disorder with cardiac and neurologic involvement[END_REF]. An impairment in mFAO, as in genetic FAO deficiencies, gives rise to the intracellular accumulation of acyl-CoAs and its β-oxidation intermediates, which may be further metabolized by alternative oxidative pathways, such as ω and ω-1 oxidation, leading to the production of dicarboxylic acids and/or elimination as acylglycines and/or acylcarnitines with secondary L-carnitine depletion. Acylcarnitines are currently used in neonatal screening programs as biomarkers for the diagnosis of mFAO disorders.Nevertheless, the aetiology of these acylcarnitines is still not completely elucidated. It is usually considered that the observed acylcarnitine profiles reflect the A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT 4 intramitochondrially accumulating acyl-CoAs which are exported out of the mitochondria

2. 4 .

 4 Acyl-CoA esters preparation Trans-2-dodecenoyl-CoA (C12:1-CoA) and trans-2-hexadecenoyl-CoA (C16:1-CoA), were enzymatically synthesized from the corresponding saturated CoA esters using acyl-CoA oxidase. Cis-5-tetradecenoyl-CoA (C14:1-CoA) was synthesized as described by Rasmussen and co-workers [16]. Pristanoyl-CoA, 4,8-dimethylnonanoyl-CoA and 2,6dimethylheptanoyl-CoA were synthesized by Prof. Dr. G. Dacremont, Belgium. All other CoA esters were obtained from Sigma-Aldrich (St. Louis, MO, USA).2.5. Determination of carnitine palmitoyltransferase 2 activity using different acyl-CoA estersCarnitine palmitoyltransferase 2 activity was determined using the method described by van Vlies and co-workers[START_REF] Van Vlies | An improved enzyme assay for carnitine palmitoyl transferase I in fibroblasts using tandem mass spectrometry[END_REF]. The standard mixture contained 150 mM potassium chloride, 25 mM Tris-HCl pH 7.4, 2 mM EDTA, 10 mM potassium phosphate buffer pH 7.4, 1 mg/mL bovine serum albumin (BSA) essentially fatty acid free, 500 µM Lcarnitine and 25 µM of each acyl-CoA ester to a final volume of 150 µL. The reaction was initiated by the addition of 20 µL of sample (S. cerevisiae homogenate) and was allowed to proceed at 37ºC. After 10 min incubation, the reaction was terminated by adding 750 µL acetonitrile containing 50 pmol d3C3-, 50 pmol d3C8-and 25 pmol d3C16-carnitine internal standards. After derivatization of the produced acylcarnitines with 1-propanol/acetylchloride 4/1 (v/v), these intermediates were quantified by Electro

  block should lead to the concomitant accumulation of mitochondrially metabolized peroxisomal FAO intermediates.

Table 1

 1 Evaluation of CPT2 activity with acyl-CoAs formed upon branched-chain aminoacids oxidation. Activity is expressed as percentage of control (C16-CoA was used as a control substrate)

	CoA-ester	CPT2 activity (% of control)
	C16-CoA	100
	isovaleryl-CoA	<0.1
	isobutyryl-CoA	<0.1
	acetoacetyl-CoA	<0.1
	DL-3-hydroxybutyryl-CoA	<0.1
	3-hydroxyisobutyryl-CoA	<0.1
	2-methylacetoacetyl-CoA	<0.1
	methylcrotonyl-CoA	<0.1
	glutaryl-CoA	<0.1
	3-hydroxy-3-methylglutaryl-CoA	<0.1
	2-methylbutyryl-CoA	<0.1
	2-methyl-3-hydroxybutyryl-CoA	<0.1

Table 3

 3 Kinetic parameters K m and V max of CPT2 for C16:0-CoA as substrate with different trans-2-C16:1-CoA concentrations. The K i value for CPT2 with trans-2-C16:1-CoA was 18.8 µM. The reactions were measured at 37ºC for 5 min in a standard incubation medium described in Materials and Methods with C16:0-CoA from 0 to 40 µM. The produced acylcarnitines were quantified by UPLC-MS. Values were calculated from non-linear regression plots using the Berkley Madonna ™ software.

			Trans-2-C16:1-CoA
		0 µM	5 µM	10 µM	20 µM
	K m app (µM)	7.1	11.6	13.5	15.0
	V max app (pmol/min.mg)	1156	1175	1120	1004

Acknowledgments

The authors gratefully acknowledge the help of Prof. Dr. Graça Soveral in the kinetic data analysis and Dr. Carlo van Roermund in the cell culture experiments. This work was financially supported by Fundação para a Ciência e Tecnologia (FCT), Lisboa, Portugal, by a grant awarded to Sara Violante (SFRH/BD/38074/2007).

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Purpose: To evaluate the outcome of surgery and radiotherapy in the treatment of sphenoorbital meningioma (SOM).

Method: A retrospective study of 66 consecutive cases treated with surgery for SOM with a minimum follow-up of 4 years. Clinical and radiological information were compared before and after the following surgical approaches: frontotemporal craniotomy, frontotemporal craniotomy combined with orbitozygomatic resection, and extended lateral orbitotomy alone.

Results:

The median age at presentation was 46 (range, 26-68) years and the median follow-up after surgery was 102 (48-288) months. In total, 48 (73%) patients showed preoperative visual deterioration, with visual field defects. All patients had proptosis at presentation (mean±SD = 6.4±3.0 mm). Surgery for patients with SOM arrested visual deterioration in 61% and improved vision in 30% of cases. Furthermore, a substantial reduction of proptosis was achieved in 85% of patients. The proptosis in our group was reduced by 2.6±2.6 mm. There was no correlation between surgical approach and proptosis reduction (P = 0.125). The recurrence rate was 17%. Only one of 15 patients who underwent radiotherapy showed signs of recurrence.

2 Conclusions: The surgical aims in the treatment of SOM should be the restoration of visual acuity and reduction of proptosis, rather than complete tumour removal. The surgical approach can be tailored to individual cases. We recommend radiotherapy in cases of subtotally removed SOM.

Introduction

Sphenoorbital meningiomas (SOM) originate from the dura of the sphenoid wing, involve the orbit and cause visual deterioration and proptosis. [START_REF] Shrivastava | Sphenoorbital meningiomas: Surgical limitations and lessons learned in their long-term management[END_REF][START_REF] Ringel | Microsurgical technique and results of a series of 63 spheno-orbital meningiomas[END_REF][START_REF] Carrizo | Current surgical treatment for sphenoorbital meningiomas[END_REF][START_REF] Sandalcioglu | Spheno-orbital meningiomas: interdisciplinary surgical approach, resectability and long-term results[END_REF][START_REF] Scarone | Long-term results with exophthalmos in a surgical series of 30 sphenoorbital meningiomas. Clinical article[END_REF][START_REF] Honeybul | Sphenoid wing meningioma en plaque: a clinical review[END_REF][START_REF] Roser | Sphenoid wing meningiomas with osseous involvement[END_REF][START_REF] Jesús | Surgical management of meningioma en plaque of the sphenoid ridge[END_REF][START_REF] Schick | Management of meningiomas en plaque of the sphenoid wing[END_REF][START_REF] Mourits | Orbital meningiomas, the Utrecht experience[END_REF][START_REF] Pompili | Hyperostosing meningiomas of the sphenoid ridge-clinical features, surgical therapy, and long-term observations: review of 49 cases[END_REF][START_REF] Heufelder | Reconstructive and ophthalmologic outcomes following resection of spheno-orbital meningiomas[END_REF] Given their extensive dural and orbital involvement, adequate resection of SOM is difficult, leading to a recurrence rate of up to 50%. [START_REF] Abbott | Pterional meningioma en plaque; report of a case of thirty-six years' duration[END_REF][START_REF] Adegbite | The recurrence of intracranial meningiomas after surgical treatment[END_REF][START_REF] Cophignon | Limits to radical treatment of sphenoorbital meningiomas[END_REF] The main aim of surgery is to restore the deteriorated vision and treat proptosis. [START_REF] Shrivastava | Sphenoorbital meningiomas: Surgical limitations and lessons learned in their long-term management[END_REF][START_REF] Ringel | Microsurgical technique and results of a series of 63 spheno-orbital meningiomas[END_REF][START_REF] Carrizo | Current surgical treatment for sphenoorbital meningiomas[END_REF][START_REF] Sandalcioglu | Spheno-orbital meningiomas: interdisciplinary surgical approach, resectability and long-term results[END_REF][START_REF] Scarone | Long-term results with exophthalmos in a surgical series of 30 sphenoorbital meningiomas. Clinical article[END_REF][START_REF] Honeybul | Sphenoid wing meningioma en plaque: a clinical review[END_REF][START_REF] Roser | Sphenoid wing meningiomas with osseous involvement[END_REF][START_REF] Jesús | Surgical management of meningioma en plaque of the sphenoid ridge[END_REF][START_REF] Schick | Management of meningiomas en plaque of the sphenoid wing[END_REF][START_REF] Mourits | Orbital meningiomas, the Utrecht experience[END_REF][START_REF] Pompili | Hyperostosing meningiomas of the sphenoid ridge-clinical features, surgical therapy, and long-term observations: review of 49 cases[END_REF][START_REF] Heufelder | Reconstructive and ophthalmologic outcomes following resection of spheno-orbital meningiomas[END_REF] Adequate treatment strategies and surgical approaches remain controversial. Some authors have reported that longstanding tumour-related proptosis cannot be treated surgically and that it should therefore not be attempted. [START_REF] Castellano | Pterional meningiomas en plaque[END_REF] Others advocate early, aggressive surgical therapy as the initial treatment. [START_REF] Bikmaz | Management of bone-invasive, hyperostotic sphenoid wing meningiomas[END_REF] Postoperative radiotherapy is recommended for subtotally removed or recurrent SOMs, [START_REF] Maroon | Recurrent sphenoorbital meningioma[END_REF] although little information is available regarding the efficacy and side effects of this treatment.

This study was performed to evaluate different surgical approaches used in two centres over a period of two decades. We studied long-term surgical outcomes and the effects and complications associated with postoperative radiotherapy in SOM.

Patients and Methods

We examined the records of 90 patients treated between 1980 and 2007 at the Orbital Centre of the Academic Medical Centre, University of Amsterdam (n = 61), and at the University Medical Centre in Utrecht (n = 29) with a diagnosis of SOM.

Sixty-six patients in this group underwent surgery at the Orbital Centre of the Academic Medical Centre, University of Amsterdam (n = 40), and the University Medical Centre in Utrecht (n = 26). Fifty-one patients with optic neuropathy combined with disfiguring proptosis and 15 with disfiguring proptosis were indicated for surgery. Twenty-three patients underwent no surgery due to stable visual acuity and the absence of optic neuropathy. Despite visual deterioration in the amblyopic eye, one patient declined surgical intervention. Only patients who underwent surgery were included in this study.

The clinical history, surgical approach, and outcome were obtained retrospectively from a review of patient charts and imaging. Our analysis considered neurological symptoms and their duration, ophthalmological examinations, consisting of testing the patient's visual acuity (Snellen notation) and pupil responses, funduscopy, tonometry, and Goldmann or Humphrey perimetry for visual field defects. Ocular motility was also evaluated.

Diplopia was considered present when the patient experienced double vision (in any gaze direction) that disappeared with one eye closed. The degree of proptosis was determined using a Hertel exophthalmometer. In Amsterdam, an Oculus Hertel (Oculus Optikgeräte GmbH, Wetzlar, Germany) was used and in Utrecht, a Zeiss Hertel (Carl Zeiss, Jena, Germany) was used.

All patients underwent preoperative computed tomography (CT) or magnetic resonance imaging (MRI; n = 51) with contrast. Imaging was scheduled immediately after surgery, at 3 and 12 months postoperatively, and annually thereafter for 5 years, and then at longer intervals, depending on the presence or absence of residual or recurrent tumours.

In patients who underwent radiotherapy, tear production was assessed using the Schirmer test and corneal dryness was classified as no corneal change, corneal stippling, limited to the inferior periphery, more extended stippling, ulceration and clouding postradiation cataract. When necessary, fluorescein angiography was performed to evaluate any postradiation retinopathy. After radiotherapy, all patients underwent detailed endocrinological testing.

Surgical procedures

Frontotemporal (pterional) craniotomy was performed as described. [START_REF] Yasargil | Microneurosurgery[END_REF] The greater wing of the sphenoid ridge was removed to the lateral limit of the lesser wing. When hyperostotic, the anterior clinoid was removed extradurally and the superior orbital fissure (SOF) was unroofed. The optic canal was unroofed, either intra-or extradurally, and the inferior optic strut was removed. When intracranial or intraorbital soft tissue was present, it was resected.

Orbitozygomatic craniotomy (OZ) [START_REF] Al-Mefty | Zygomatic approach to skull-base lesions[END_REF] is an extension of the frontotemporal approach. The superior and lateral orbital rims were mobilised with additional removal of part of the lateral wall along the zygoma and orbital roof, which provides access to the floor of the anterior and middle cranial fossae. This was performed in either one or two pieces, with the superior and lateral rim.

Extended lateral orbitotomy was performed through either a coronal or lid crease incision. In this approach, the classical lateral orbitotomy bone incision described by Berke was enlarged from the deep sphenoid wing up to the SOF. [START_REF] Rootman | Orbital Surgery[END_REF][START_REF] Kennerdell | Surgical approaches to orbital tumors[END_REF]

Reconstruction

Periorbital reconstruction was not performed. The superior and lateral orbital rim bone was reattached. The roof and lateral wall of the orbit were routinely reconstructed with a split cranial bone graft and secured to the orbital rim by titanium miniplates. In two cases, this was performed using titanium mesh. Dural defects were reconstructed with autologous galea grafts, which included convexity and basal defects. If there was a large cavity between the orbital and dural reconstruction and the cranioplasty, an autologous fat graft, harvested from the abdomen, was used to fill this and prevent a postoperative pseudomeningocoele. The temporal muscle was resuspended and sutured meticulously.

Radiotherapy

Postoperatively, 15 patients underwent radiotherapy of 54 Gy in 1.8-Gy fractions. The inclusion criteria for radiotherapy were large rest tumour and involvement of the cavernous sinus, while the exclusion criteria were the presence of diabetes mellitus and hypertension.

Statistical Analyses

Visual acuity and pre-and postoperative proptosis are described as means±SD.

Differences between the three different surgical approaches were compared using the Kruskal-Wallis test. Proptosis reduction was compared between the three approaches with correction for preoperative proptosis (nonparametric ANOVA). All analyses were carried out using the SPSS software (ver. 16 for Windows; SPSS, Chicago, IL, USA). In all analyses, P < 0.05 was deemed to indicate statistical significance.

Results

The median age at presentation was 46 (range, 26-68) years. The study population consisted of 61 (92%) females and five (8%) males. The left and right orbits were involved in 35 and 31 patients, respectively. The median follow-up after surgery was 102 months (48-288 months) and the median period between presentation and surgery was 24 months (1-119 months). In total, 51 (77%) patients had preoperative progressive visual deterioration with visual field defects. Three patients had no light perception (NLP) preoperatively. All patients had proptosis at presentation, with a mean of 5.89±2.89 mm (mean±SD). At presentation, seven (11%) patients had diplopia and 19 (29%) had headaches or retrobulbar pain.

Surgery

The surgical approach was frontotemporal craniotomy in 45 patients and this approach was combined with orbitozygomatic resection in 10 patients. Forty-six patients in this group had optic neuropathy due to compression of the optic nerve, five had disfiguring proptosis with hyperostosis of the optic canal and four had proptosis combined with retrobulbar pain. As all of these 55 patients had disfiguring proptosis, this was also addressed at the time of surgery.

An extended lateral orbitotomy was performed in 11 patients. Patients in this group had only disfiguring proptosis and two had severe proptosis with stretching of the optic nerve.

Craniotomies

Fifty-five patients underwent a large unilateral frontotemporal craniotomy, followed by removal of additional tumour-infiltrated bone via an extradural approach in all cases.

Ten procedures also included OZ resection. The orbital roof was resected in 49 cases and the lateral orbital wall was removed in 35. The optic canal and SOF were opened and decompressed in 51 and 29 cases, respectively. After opening the orbit, periorbital resection or stripping was necessary in 32 procedures. Extensive en plaque dural involvement was found in 45 cases; resection of the convexity-dura was necessary in 39 cases.

Reconstruction was performed in 9 of 10 patients treated via the frontotemporal approach in combination with OZ. The reconstruction used calvarial bone in seven patients and titanium mesh in two; both the lateral and orbital roof were reconstructed in all nine patients (Fig. 1).

Orbitotomy

In 11 patients who underwent lateral orbitotomy, the lateral wall of the orbit until the SOF was removed; in five cases, the orbital roof was also partially removed. In six procedures, periorbital resection was also performed due to tumour infiltration. In five cases in which there was infiltration of the ocular muscles, only the exophytic tumour was removed. None of these patients underwent reconstruction. All lateral orbitotomies were performed by orbital surgeons (Fig. 1).

Radiotherapy

Fifteen patients underwent secondary radiotherapy after surgical excision. All 15 had significant residual tumours, which also involved the cavernous sinus in four cases. Two patients underwent radiotherapy after debulking of a recurrent lesion. Only one of these patients showed further recurrence after follow-up for 38 months. In four patients, the tumour volume was reduced to less than 10%. In all four, there was significant soft tissue involvement.

Outcome

Visual acuity

Forty (61%) patients showed either stabilisation of their visual acuity or an improvement of less than two lines on the Snellen chart. Three eyes from this group maintained a visual acuity of NLP (no light perception). Fifteen had a visual acuity of more than 0.8 preoperatively, which remained stable postoperatively. Twenty (30%) had improved visual acuity. Eight improved by more than four lines on the Snellen chart and 12 others by more than two lines.

There was no significant difference (P = 0.195) in postoperative vision between the different approaches. All except two patients with visual deterioration underwent decompression of the optic canal through a craniotomy. The two exceptions had proptosis of more than 10 mm with stretching of the optic nerve and underwent only extended lateral orbitotomy with total removal of the lateral orbital wall.

Proptosis

The mean proptosis, measured with a Hertel exophthalmometer, in this group was 6.4±3.0 mm preoperatively and 3.8±2.3 mm postoperatively. The mean reduction in proptosis was 2.6±2.6 mm. Seventeen orbits showed a proptosis reduction of more than 5 mm, while ten showed no change in proptosis compared with preoperative values.

One patient who underwent no reconstruction of the orbit developed enophthalmos of 2 mm. Fifty patients had a residual proptosis exceeding 2 mm. Patients who underwent lateral orbitotomy and combined craniotomy and OZ had the greatest mean preoperative proptosis of 7.9±3.4 and 7.7±2.9 mm, respectively. The mean proptosis with frontotemporal craniotomy only was 5.9±2.9 mm. However, there was no significant (P = 0.125) difference in the postoperative reduction in proptosis among the approaches (Fig. 2).

Diplopia

New diplopia was observed in 40 patients; it was transient in 32 cases. Two patients showed no eye movement in the first weeks after surgery; in both patients, the lateral wall was removed completely. Within 6 months, eye movement recovered completely, with no diplopia. Eight patients (12%) still showed diplopia; three of these underwent strabismus surgery with satisfactory results. In six patients after a frontotemporal craniotomy, the cause of the diplopia was third nerve palsy; thus, no strabismus surgery was performed. Ptosis surgery was performed in one patient with partial third nerve palsy and no diplopia. Nine patients with diplopia in the primary position preoperatively still had diplopia postoperatively and underwent surgical correction.

Pain

Of 19 patients with retrobulbar pain after surgery, 16 had no further pain. All of these patients underwent craniotomies.

Complications

Surgery

Five patients developed subgaleal cerebrospinal fluid (CSF) accumulation, which regressed spontaneously in four cases; one patient required surgical revision of the dural leak and placement of a lumboperitoneal shunt. After craniotomies, permanent third nerve palsies were seen in six cases, and fourth and sixth nerve palsies occurred in one patient each. Permanent trigeminal and facial nerve palsies were present in six and three patients, respectively.

In two patients who underwent OZ, the lateral excursion of the mandible was reduced by approximately 50%.

Radiotherapy

Three patients developed severe punctate keratopathy necessitating the daily use of lubricants. One patient also developed radiation retinopathy, but had stable visual acuity of 0.5 after laser treatment. During follow-up, one patient developed pituitary function impairment. There was no death due to treatment or SOM in this study population.

Recurrence

Eleven (17%) patients showed recurrence (regrowth). The median time between surgery and recurrence was 46 months (10-108 months). Macroscopic radical excision was achieved in only six patients. Of 61 patients with rest tumour, 15 underwent radiotherapy. Despite the larger size of the rest tumour in this group, only one of these 15 patients (7%) showed recurrence.

The signs of recurrence in four patients were a combination of visual deterioration and increased proptosis, while four patients showed only a mild increase in proptosis (< 3 mm). Four patients with visual deterioration underwent second craniotomy-two had improved visual acuity and two other patients with NLP underwent two other debulking surgeries and the affected eye was ultimately enucleated because of corneal ulceration caused by proptosis.

Discussion

Symptom-oriented surgery for SOM is directed primarily at optic nerve decompression when there is decreased visual acuity. In our series, the visual acuity improved in 30% of cases and remained the same in 61% of cases. The cause of optic neuropathy was addressed and treated in all of the patients with optic neuropathy. This was probably related to the degree and period of optic nerve compression. In our series, the proptosis was reduced by a mean of 2.6 mm from preoperative values, ranging from 2 to 14 mm. Seventeen (26%) patients showed a marked reduction in proptosis by more than 5 mm and up to 11 mm. Excluding one case of enophthalmos, residual proptosis was present in 50 (76%) of the remaining patients. Nevertheless, 85% showed an improvement of more than 2 mm. All patients in our series underwent periodical proptosis measurements with a Hertel exophthalmometer. There have been only a few previous attempts to quantify postoperative proptosis in SOM. [START_REF] Ringel | Microsurgical technique and results of a series of 63 spheno-orbital meningiomas[END_REF][START_REF] Scarone | Long-term results with exophthalmos in a surgical series of 30 sphenoorbital meningiomas. Clinical article[END_REF][START_REF] Heufelder | Reconstructive and ophthalmologic outcomes following resection of spheno-orbital meningiomas[END_REF] Ringel et al. reported improvement of proptosis in 43 of 56 patients (77%) with no change in proptosis in the remaining 13 patients (23%). [START_REF] Ringel | Microsurgical technique and results of a series of 63 spheno-orbital meningiomas[END_REF] A recent study quantified the proptosis by MRI and reported significant residual proptosis in 53% of cases. [START_REF] Scarone | Long-term results with exophthalmos in a surgical series of 30 sphenoorbital meningiomas. Clinical article[END_REF] We believe that the cause of this significant residual proptosis was long-standing congestion and fibrosis, resulting in less mobile orbital contents. Despite this residual proptosis in the surgical treatment of SOM, the proptosis improves in most cases. Surgery also seemed to be effective at relieving pain associated with SOM; 16 of the 19 patients with reported pain showed no sign of pain postoperatively. We believe that pain is an underreported symptom in these series.

Our recurrence rate of 17% was consistent with other recently published series with long-term follow-up. [START_REF] Shrivastava | Sphenoorbital meningiomas: Surgical limitations and lessons learned in their long-term management[END_REF][START_REF] Ringel | Microsurgical technique and results of a series of 63 spheno-orbital meningiomas[END_REF][START_REF] Carrizo | Current surgical treatment for sphenoorbital meningiomas[END_REF][START_REF] Sandalcioglu | Spheno-orbital meningiomas: interdisciplinary surgical approach, resectability and long-term results[END_REF][START_REF] Scarone | Long-term results with exophthalmos in a surgical series of 30 sphenoorbital meningiomas. Clinical article[END_REF][START_REF] Honeybul | Sphenoid wing meningioma en plaque: a clinical review[END_REF][START_REF] Roser | Sphenoid wing meningiomas with osseous involvement[END_REF][START_REF] Jesús | Surgical management of meningioma en plaque of the sphenoid ridge[END_REF][START_REF] Schick | Management of meningiomas en plaque of the sphenoid wing[END_REF][START_REF] Mourits | Orbital meningiomas, the Utrecht experience[END_REF][START_REF] Pompili | Hyperostosing meningiomas of the sphenoid ridge-clinical features, surgical therapy, and long-term observations: review of 49 cases[END_REF][START_REF] Heufelder | Reconstructive and ophthalmologic outcomes following resection of spheno-orbital meningiomas[END_REF] These recurrence rates are much lower than those in earlier studies, which reported recurrence rates of 30-50%. [START_REF] Abbott | Pterional meningioma en plaque; report of a case of thirty-six years' duration[END_REF][START_REF] Adegbite | The recurrence of intracranial meningiomas after surgical treatment[END_REF] Maroon et al. listed the following reasons for the high recurrence rate in SOM: failure to diagnose the tumour early because the symptoms were confused with those of other clinical entities, such as fibrous dysplasia and Graves' disease; inadequate resection, due to the involvement of important neurovascular structures; and the surgeon's concern of iatrogenic death and serious complications in radical resections. [START_REF] Maroon | Recurrent sphenoorbital meningioma[END_REF] New imaging techniques appear to have improved the early diagnosis and effective surgical resection. To resect SOM, different surgical approaches are used, including transzygomatic, pterional, frontotemporal, and combined transcranialtransmalar and cranioorbital approaches. All of these approaches allow sufficient access to the orbit and middle fossa base for bony and soft-tissue tumour resection and decompression of the SOF and optic canal. [START_REF] Yasargil | Microneurosurgery[END_REF][START_REF] Al-Mefty | Zygomatic approach to skull-base lesions[END_REF][START_REF]Supraorbital-pterional approach to skull base lesions[END_REF][START_REF] Hassler | Extradural and intradural microsurgical approaches to lesions of the optic canal and the superior orbital fissure[END_REF] Extended lateral orbitotomy or total lateral orbitotomy has been used routinely for severe cases of Graves' orbitopathy and tumours in the orbital apex. This approach is effective in patients with SOM in which the main symptom is disfiguring proptosis, without involving the risks of a craniotomy. [START_REF] Rootman | Orbital Surgery[END_REF][START_REF] Kennerdell | Surgical approaches to orbital tumors[END_REF][START_REF] Kim | Total lateral orbitotomy[END_REF][START_REF] Mariniello | Spheno-orbital meningiomas: surgical approaches and outcome according to the intraorbital tumor extent[END_REF] Recently, Lund reported 12 patients with SOM who underwent endoscopic endonasal medial orbital wall decompression and decompression of the optic canal in eight patients with opticopathy and visual deterioration. [START_REF] Lund | Endoscopic transnasal orbital decompression for visual failure due to sphenoid wing meningioma[END_REF] In these patients, the opticopathy improved and the visual acuity improved by 1-4 lines on the Snellen chart.

Depending on the extent of the orbital wall and roof resection, most authors recommend firm reconstruction of the orbital walls to avoid pulsating enophthalmos and diplopia.

Maroon et al. reported 200 cases of orbital wall and roof resection without reconstruction, in which no cases showed permanent pulsating enophthalmos. [START_REF] Maroon | Recurrent sphenoorbital meningioma[END_REF] DeMonte et al. concluded that partial or complete orbital roof resection, isolated or combined with lateral or medial orbital wall defects, did not require routine reconstruction when the periorbita was not resected. [START_REF] Demonte | Ophthalmological outcome after orbital entry during anterior and anterolateral skull base surgery[END_REF] We advocate reconstruction of the orbital roof and the lateral orbital rim. We consider reconstruction of the lateral orbital wall to be unnecessary in most cases. Studies of orbital decompression in Graves' orbitopathy have shown that lateral wall decompression can reduce the proptosis by up to 2 mm. [START_REF] Goldberg | The lacrimal keyhole, orbital door jamb and basin of the inferior orbital fissure. Three areas of deep bone in the lateral orbit[END_REF][START_REF] Baldeschi | The removal of the deep lateral wall in orbital decompression: its contribution to exophthalmos reduction and influence on consecutive diplopia[END_REF] As significant residual proptosis was present with all surgical approaches in SOM in these series, no further reconstruction of the lateral orbital wall may contribute to further proptosis reduction, while reconstruction of the orbit roof is sufficient to avoid pulsating enophthalmos. Techniques have improved since 1952, when Castellano et al. reported a high surgical mortality rate (20%) associated with SOM and concluded that SOM should be used only as a last resort. [START_REF] Castellano | Pterional meningiomas en plaque[END_REF] Although the mortality rate has decreased, some recent studies still reported mortality rates of 3-6%. [START_REF] Ringel | Microsurgical technique and results of a series of 63 spheno-orbital meningiomas[END_REF][START_REF] Carrizo | Current surgical treatment for sphenoorbital meningiomas[END_REF][START_REF] Pompili | Hyperostosing meningiomas of the sphenoid ridge-clinical features, surgical therapy, and long-term observations: review of 49 cases[END_REF][START_REF] Mariniello | Spheno-orbital meningiomas: surgical approaches and outcome according to the intraorbital tumor extent[END_REF] Other complications, such as vision loss and transient (6-84%) or permanent (7-30%) cranial nerve deficits, are frequent. [START_REF] Shrivastava | Sphenoorbital meningiomas: Surgical limitations and lessons learned in their long-term management[END_REF][START_REF] Ringel | Microsurgical technique and results of a series of 63 spheno-orbital meningiomas[END_REF][START_REF] Carrizo | Current surgical treatment for sphenoorbital meningiomas[END_REF][START_REF] Sandalcioglu | Spheno-orbital meningiomas: interdisciplinary surgical approach, resectability and long-term results[END_REF][START_REF] Scarone | Long-term results with exophthalmos in a surgical series of 30 sphenoorbital meningiomas. Clinical article[END_REF][START_REF] Honeybul | Sphenoid wing meningioma en plaque: a clinical review[END_REF][START_REF] Roser | Sphenoid wing meningiomas with osseous involvement[END_REF][START_REF] Jesús | Surgical management of meningioma en plaque of the sphenoid ridge[END_REF][START_REF] Schick | Management of meningiomas en plaque of the sphenoid wing[END_REF][START_REF] Mourits | Orbital meningiomas, the Utrecht experience[END_REF][START_REF] Pompili | Hyperostosing meningiomas of the sphenoid ridge-clinical features, surgical therapy, and long-term observations: review of 49 cases[END_REF][START_REF] Heufelder | Reconstructive and ophthalmologic outcomes following resection of spheno-orbital meningiomas[END_REF] Although life-threatening complications were not seen in this series, postoperative visual deterioration occurred in 6 of 66 cases (9%), oculomotor nerve palsy in 5 of 66 cases (7%) patients, and facial nerve palsy in 3 of 66 cases (5%).

Radiotherapy is often used to treat SOM. Peele et al. delivered a total dose of 45 Gy to subtotally or recurrent SOMs and none of the 42 patients in the radiation treatment group experienced recurrence during an observation period of 4.2 years, whereas 21 of the 44 patients in the non-radiation treatment group later presented with recurrent tumour growth. [START_REF] Peele | The role of postoperative irradiation in the management of sphenoid wing meningiomas. A preliminary report[END_REF] Another study indicated a decrease in size of skull base meningiomas in 51% of patients, stabilisation in 47% of cases and increased tumour size in 2% of cases. [START_REF] Goldsmith | Postoperative irradiation for subtotally resected meningiomas. A retrospective analysis of 140 patients treated from 1967 to 1990[END_REF] In our series, postoperative radiotherapy in subtotally removed SOM resulted in a reduction in tumour size and stabilisation in 27% and 67% of cases, respectively.

In conclusion, the aim of surgery in the treatment of SOM should be restoration of visual acuity and reduction of proptosis, rather than complete bony tumour removal.

The surgical approach can be tailored to individual cases. Decompression of the optic canal can be achieved either through a combined frontotemporal approach and OZ or a frontotemporal approach alone. However, the frontotemporal approach combined with OZ in these series was not associated with better visual or proptosis outcome.

When the major symptom is proptosis without optic canal stenosis, extended lateral orbitotomy alone can be preformed to avoid the complications of a craniotomy.

As the majority of SOMs are resected subtotally, postoperative radiotherapy may provide better control of tumour growth. As retrospective studies, by their nature, have potential bias, these case series should be followed in future by prospective studies.

Table 1 . 22 Figure - 2 1 .

 12221 Fig. 1 A-B Preoperative and postoperative T1-weighted MR fat suppression sequences obtained post-Gd administration, showing a sphenoorbital meningioma before and after frontotemporal craniotomy combined with orbitozygomatic resection. C-D Preoperative and postoperative CT scan axial views showing a sphenoorbital meningioma before and after extended lateral orbitotomy.


James Cohen Stuart
email: j.cohenstuart@umcutrecht.nl

Maurine A Leverstein-Van Hall

Nashwan Al Naiemi

Alex Van Belkum

Marc Bonten

James Cohen

Marjolein Kluytmans

Jan Kluytmans

Greet Vos Christina Vandenbroucke-Grauls

Andreas Voss

Guideline for phenotypic screening and confirmation of carbapenemases in Enterobacteriaceae

Keywords:

Detection of carbapenemases in Enterobacteriaceae includes a screening step followed by a genotypic and optional phenotypic confirmatory step. For all Enterobacteriaceae, the meropenem screening breakpoint to detect carbapenemases is set at ≥0.5 mg/L or a zone diameter of ≤23 mm (10 g disk loading). For Escherichia coli, Klebsiella spp., Salmonella spp., Enterobacter spp. and Citrobacter spp., the imipenem screening breakpoint is set at ≥2 mg/L or a zone diameter ≤21 mm. Ertapenem is not advised as an indicator carbapenem as it has a lower specificity compared with imipenem and meropenem. On the first isolate from a patient with a positive carbapenemase screen test, a polymerase chain reaction (PCR)-based test should be performed to detect carbapenemase genes. However, if genotypic confirmation is not immediately available, phenotypic confirmation tests should be performed to avoid delayed reporting of carbapenemase-producers to the clinic. Recommended phenotypic confirmation tests are the modified Hodge test as well as carbapenemase inhibition tests with boronic acid for Ambler class A carbapenemases and with ethylene diamine tetra-acetic acid (EDTA) or dipicolinic acid for metallo-carbapenemases.

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

Introduction

The rapid emergence and dissemination of Enterobacteriaceae that are resistant to carbapenems such as imipenem and meropenem poses a considerable threat to clinical patient care and public health. Carbapenemase-producing strains are characterised by their resistance to virtually all -lactam antibiotics, including the cephalosporins and carbapenems, as well as to fluoroquinolones, aminoglycosides and co-trimoxazole. Invasive infections with these strains are associated with high rates of morbidity and mortality [START_REF] Bratu | Rapid spread of carbapenem-resistant Klebsiella pneumoniae in New York City: a new threat to our antibiotic armamentarium[END_REF][START_REF] Souli | An outbreak of infection due to -lactamase Klebsiella pneumoniae carbapenemase 2-producing K. pneumoniae in a Greek University Hospital: molecular characterization, epidemiology, and outcomes[END_REF].

The carbapenemases fall into three classes according to their amino acid sequence: Ambler class A (serine carbapenemases); class B (metallo-carbapenemases); and class D (OXA carbapenemases). Within these classes, further divisions are made (see Table 1) and new variants are frequently encountered [START_REF] Queenan | Carbapenemases: the versatile -lactamases[END_REF]. The rapid emergence and spread of carbapenemase-producing strains is mainly caused by epidemics of bacteria bearing plasmid-mediated KPC (class A), VIM-1 and NDM (class B) and OXA-48 (class D) enzymes. Carbapenem minimum inhibitory concentrations (MICs) observed in carbapenemase-producing microorganisms can exhibit considerable variation depending on the type and expression of carbapenemase enzyme, the bacterial species and the presence of other resistance mechanisms such as cephalosporinases [extended-spectrum -lactamase (ESBL) and AmpC], reduced permeability and/or efflux pumps [START_REF] Falcone | Infections with VIM-1 metallo--lactamase-producing Enterobacter cloacae and their correlation with clinical outcome[END_REF][START_REF] Pasteran | Sensitive screening tests for suspected class A carbapenemase production in species of Enterobacteriaceae[END_REF][START_REF] Tenover | Carbapenem resistance in Klebsiella pneumoniae not detected by automated susceptibility testing[END_REF]. Increased carbapenem MICs in Enterobacteriaceae may also result from high expression of AmpC or CTX-M ESBLs in combination with porin alterations [START_REF] Pasteran | Sensitive screening tests for suspected class A carbapenemase production in species of Enterobacteriaceae[END_REF][START_REF] Woodford | Ertapenem resistance among Klebsiella and Enterobacter submitted in the UK to a reference laboratory[END_REF].

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

Purpose of this guideline

This guideline aims to standardise and improve carbapenemase detection in Enterobacteriaceae for routine diagnostics in clinical microbiology laboratories.

Although this guideline reflects, as much as possible, the current knowledge on carbapenemases, regular updating will remain necessary owing to ongoing technological developments and emergence of new carbapenemase variants.

Adequate detection of carbapenemase-producing microorganisms in the routine diagnostic laboratory is essential for patient care because (i) it is vital for the correct choice of antibiotic therapy and (ii) appropriate hospital hygiene precautions are indicated for patients harbouring carbapenemase-producing microorganisms as these strains are associated with multiresistance and epidemics that result in reduced patient safety and increased costs.

Strategy for carbapenemase detection

The detection strategy includes a screening step followed by a phenotypic and genotypic confirmation step (see Fig. 1).

Screening is based on detection of reduced susceptibility to carbapenems by carbapenemase-producing isolates compared with isolates of the wild-type population (Table 2). For each class of carbapenemases, and for each species and isolate, the MIC may vary from MICs of the wild-type population to >256 mg/L, dependent on the presence of other resistance mechanisms. 1. the breakpoint MIC should be higher than the highest MIC of the wild-type population (http://www.eucast.org), as the specificity of the screening test may otherwise become too low; and 2. the MIC breakpoint should be lower than the lowest carbapenem MICs described in the literature for strains shown to have a carbapenemase gene.

Meropenem

Based on these criteria, the MIC screening breakpoint for meropenem could be set at ≥0.5 mg/L for all Enterobacteriaceae (Table 2), enabling detection of the vast majority of carbapenemase-producers. Only sporadic carbapenemase-producers with meropenem MICs < 0.5 mg/L will not be detected using this breakpoint [START_REF] Falcone | Infections with VIM-1 metallo--lactamase-producing Enterobacter cloacae and their correlation with clinical outcome[END_REF].

The zone diameter screening breakpoint for meropenem has been set at ≤23 mm. The meropenem zone diameter breakpoint is slightly less sensitive than the MIC screening breakpoint of 0.5 mg/L (84% vs. 100%, respectively) [START_REF] Pasteran | Sensitive screening tests for suspected class A carbapenemase production in species of Enterobacteriaceae[END_REF], but it was shown to detect all VIM-and KPC-producing isolates [START_REF] Vading | Comparison of disc diffusion, Etest, and Vitek 2 for detection of carbapenemase producing Klebsiella pneumoniae with EUCAST and CLSI breakpoint systems[END_REF]. Although a meropenem zone diameter screening breakpoint of ≤27 mm was reported to have a sensitivity of 100% for carbapenemase production [START_REF] Pasteran | Sensitive screening tests for suspected class A carbapenemase production in species of Enterobacteriaceae[END_REF], this would result in an unacceptably high level of false-positive isolates based on the published meropenem zone diameter distributions (http://www.eucast.org).

A c c e p t e d

M a n u s c r i p t 7

Imipenem

For imipenem it is not possible to set a breakpoint for all Enterobacteriaceae as some species (Proteus spp., Serratia spp., Providencia spp. and Morganella morganii) have a high imipenem MIC owing to mechanisms other than carbapenemase production (see Table 2 for epidemiologic cut-offs for imipenem MICs for these species). For pragmatic reasons, and based on the available wild-type MIC distributions, we have chosen to make a distinction in this guideline between species for which the MIC for imipenem can be used as a breakpoint and species for which it cannot. For Escherichia coli, Klebsiella spp., Salmonella spp., Enterobacter spp. and Citrobacter spp. the imipenem breakpoint is ≥2.0 mg/L or a zone diameter ≤21 mm.

Although the imipenem MIC screening breakpoint of ≥2.0 mg/L was shown to have a sensitivity of 79% [START_REF] Pasteran | Sensitive screening tests for suspected class A carbapenemase production in species of Enterobacteriaceae[END_REF], the breakpoint was not set lower because the imipenem MIC distribution of the wild-type population is up to 1 mg/L (Table 2) (http://www.eucast.org). It has been shown that the sensitivity of the imipenem zone diameter screening breakpoint ≤21 mm was 100% [START_REF] Pasteran | Sensitive screening tests for suspected class A carbapenemase production in species of Enterobacteriaceae[END_REF][START_REF] Vading | Comparison of disc diffusion, Etest, and Vitek 2 for detection of carbapenemase producing Klebsiella pneumoniae with EUCAST and CLSI breakpoint systems[END_REF].

Ertapenem

Ertapenem is not advised as an indicator carbapenem in this guideline since it has lower specificity than imipenem and meropenem. Ertapenem is less specific because isolates with AmpC/ESBL and decreased permeability have higher MICs for ertapenem than for imipenem or meropenem [START_REF] Woodford | Ertapenem resistance among Klebsiella and Enterobacter submitted in the UK to a reference laboratory[END_REF]. However, an ertapenem screening breakpoint of 0.5 mg/L could be used (Table 2).

Phenotypic confirmation of carbapenemase production is based on detection of a diffusible carbapenemase and in vitro inhibition of carbapenemase activity upon

Screening methods for carbapenemases

Carbapenemase screening should be a standard component of the susceptibility testing on all Enterobacteriaceae isolated in routine diagnostics. This can take place by assessing the carbapenem MICs or by an alert from the expert system (see Fig. 1).

When using automatic systems for susceptibility testing (e.g. Phoenix, Vitek or MicroScan), panels containing meropenem are preferred. The preferred lowest concentration in the panels should be 0.25 mg/L for meropenem and 1 mg/L for imipenem. If ertapenem is used then the preferred lowest concentration should be 0.25 mg/L. The laboratory should be aware that strains with an MIC above the carbapenemase screening breakpoint but below the clinical breakpoint might nevertheless have a carbapenemase gene (see Table 2). Strains with an MIC for meropenem of 0.5, 1 or 2 mg/L or an MIC for imipenem of 2 mg/L are sensitive according European Committee on Antimicrobial Susceptibility Testing (EUCAST) clinical breakpoints but should still be tested for the presence of a carbapenemase gene (Table 1).

Use of a correct inoculum is important both for broth microdilution methodology and the automatic systems since a moderate decrease in the inoculum may lead to inaccurate susceptibility results. It was shown in a study with 29 KPC-positive A c c e p t e d M a n u s c r i p t 9 isolates that if the standard inoculum was decreased from 10 5 to 10 4 colony-forming units/mL, the imipenem MIC was on average 1.6 doubling dilutions lower [START_REF] Bratu | Rapid spread of carbapenem-resistant Klebsiella pneumoniae in New York City: a new threat to our antibiotic armamentarium[END_REF].

To exclude technical errors and to limit the number of strains to be confirmed for carbapenemase production, a carbapenem MIC above the screening breakpoint measured by an automatic system can be confirmed by Etest (bioMérieux, Marcy l'Etoile. France) with meropenem or imipenem (Fig. 1) on Muller-Hinton agar (Iso-Sensitest agar may underestimate carbapenem MICs of metallo-carbapenemaseproducers owing to low zinc concentrations [START_REF] Walsh | Evaluation of a new Etest for detecting metallo--lactamases in routine clinical testing[END_REF]). Determining the MIC of carbapenemase-positive strains by Etest can be complicated because mutant colonies with higher MICs than the dominant population may be found in the inhibition ellipse. These colonies should be included when interpreting the Etest in accordance with the manufacturer's instructions.

Phenotypic confirmation of carbapenemase production

On the first isolate from a patient with a positive carbapenemase screen test, a PCRbased test should be performed to confirm the presence of carbapenemase genes (Fig. 1). However, if genotypic confirmation is not immediately available, phenotypic confirmation tests can be performed in order to avoid delayed reporting of potential carbapenemase-producers to the clinic. Phenotypic confirmation may be performed using one or two methods, the modified Hodge test and the carbapenemase inhibition tests [START_REF] Pasteran | Sensitive screening tests for suspected class A carbapenemase production in species of Enterobacteriaceae[END_REF][START_REF] Miriagou | Acquired carbapenemases in Gram-negative bacterial pathogens: detection and surveillance issues[END_REF][START_REF] Pasteran | Controlling falsepositive results obtained with the Hodge and Masuda assays for detection of class A carbapenemase in species of Enterobacteriaceae by incorporating boronic acid[END_REF]. The modified Hodge test is used for detection of diffusible carbapenemases, and the inhibition tests are used to distinguish between the different classes of carbapenemases (Tables 3 and4). Test characteristics and

Modified Hodge test

The modified Hodge test (Fig. 2) should be performed according to the guidelines of the Clinical and Laboratory Standards Institute [START_REF]Performance standards for antimicrobial susceptibility testing. Nineteenth informational supplement[END_REF]. The modified Hodge test has a high sensitivity (95-100%) [START_REF] Pasteran | Sensitive screening tests for suspected class A carbapenemase production in species of Enterobacteriaceae[END_REF][START_REF] Miriagou | Acquired carbapenemases in Gram-negative bacterial pathogens: detection and surveillance issues[END_REF]. Disadvantages of this test are possible interpretation difficulties and the fact that different classes of carbapenemases cannot be distinguished. Specificity may be low because CTX-M ESBL-or AmpCproducing isolates with reduced or absent porin expression may give false-positive results [START_REF] Pasteran | Sensitive screening tests for suspected class A carbapenemase production in species of Enterobacteriaceae[END_REF][START_REF] Pasteran | Controlling falsepositive results obtained with the Hodge and Masuda assays for detection of class A carbapenemase in species of Enterobacteriaceae by incorporating boronic acid[END_REF]. However, for detection of class A carbapenemases the specificity of the modified Hodge test can be increased by modifying the test as described by Pasteran et al. [START_REF] Pasteran | Controlling falsepositive results obtained with the Hodge and Masuda assays for detection of class A carbapenemase in species of Enterobacteriaceae by incorporating boronic acid[END_REF] (see Fig. 2).

Inhibition tests of carbapenemase activity (synergy tests)

The confirmation step is based on in vitro inhibition of carbapenemase activity by addition of an inhibitor specific for a class of carbapenemases (resulting in a reduction in the MIC for the carbapenem). This phenomenon is called synergy between the carbapenem and the inhibitor.

For detection of class A carbapenemases, the inhibitor used is boronic acid (3aminophenylboronic acid). For detection of class B metallo-carbapenemases, ethylene diamine tetra-acetic acid (EDTA) or dipicolinic acid can be used as an inhibitor.

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

It is recommended that these tests be carried out using either combination disks or an Etest strip that contains both a carbapenem and an inhibitor. Table 3 shows the details of the combination tests as recommended by a group of experts from EUCAST and the ESCMID Study Group for Antibiotic Resistance Surveillance (ESGARS) as well as how these should be interpreted [START_REF] Miriagou | Acquired carbapenemases in Gram-negative bacterial pathogens: detection and surveillance issues[END_REF]. Double-disk synergy tests (disk approximation methods) are not recommended since the sensitivity depends on the optimal distance between the disks, which cannot be predicted.

Quality control

The following strains are recommended for quality control: Klebsiella pneumoniae ATCC BAA-1705 (KPC-positive); E. coli ATCC 25922 (carbapenemase-negative); and K. pneumoniae ATCC BAA 1706 (resistant to carbapenems by mechanisms other than carbapenemase, modified Hodge test-negative).

Genotypic confirmation

Genotypic confirmation comprises PCR detection and sequencing of carbapenemase genes. The high diversity of genes with ever-increasing numbers of new variants implies that isolates with a negative genotypic result in the local laboratory setting should be sent to a reference laboratory for further genotypic confirmation.

In The Netherlands, the reference centre is a collaboration between RIVM (Centre for Infectious Disease Control) and the Medical Microbiology Laboratory of University Medical Centre Utrecht. The carbapenemase genes named in

Report to the clinic

Strains confirmed to be carbapenemase-positive should be reported to the clinic as susceptible or resistant according to the clinical breakpoints of EUCAST (http://www.eucast.org). Strains shown to have a carbapenemase gene and an MIC for meropenem of 0.5, 1 or 2 mg/L or an MIC for imipenem of 2 mg/L are therefore reported as susceptible. The clinical significance of the presence of a carbapenemase gene in an isolate with a carbapenemase MIC below the clinical EUCAST breakpoint is currently unknown. The published data on this issue are controversial since a few case reports described carbapenem therapy failure in patients infected with KPC-positive K. pneumoniae with carbapenem MICs in the susceptible range [START_REF] Lomaestro | The spread of Klebsiella pneumoniae carbapenemase-producing K. pneumoniae to upstate New York[END_REF][START_REF] Mathers | Fatal cross infection by carbapenem-resistant Klebsiella in two liver transplant recipients[END_REF]. In contrast, two studies from Greece in patients with bloodstream infections observed no difference in mortality between VIM-negative K. pneumoniae infections and infections with VIM-positive K. pneumoniae having an imipenem MIC ≤ 4 mg/L. Mortality during carbapenem treatment was increased in those patients with VIM-positive K. pneumoniae infections and an imipenem MIC > 4 mg/L [START_REF] Daikos | VIM-1-producing Klebsiella pneumoniae bloodstream infections: analysis of 28 cases[END_REF][START_REF] Daikos | Prospective observational study of the impact of VIM-1 metallo--lactamase on the outcome of patients with Klebsiella pneumoniae bloodstream infections[END_REF]. A comment should therefore be added to the results to indicate that the susceptibility of the microorganism to carbapenems is not clear and that treatment of any infection should be determined in consultation with a microbiologist-physician or an infectious diseases consultant.

Funding

None. b For E. coli, Klebsiella spp. and Enterobacter spp. the zone diameter screening breakpoint for imipenem has been set at ≤21 mm with a disk content of 10 g.

c No zone diameter screening breakpoint has been set for ertapenem owing to the low specificity, although a breakpoint of ≤27 mm (disk content 10 g) was reported to have a sensitivity of 97% and a specificity of 90.5% for KPC-producing K. pneumoniae and E.

coli [START_REF] Lolans | Direct ertapenem disk screening method for identification of KPC-producing Klebsiella pneumoniae and Escherichia coli in surveillance swab specimens[END_REF].

A c c e p t e d M a n u s c r i p t 3 ABSTRACT

 3 Adequate detection of carbapenemase-producing Enterobacteriaceae is crucial for infection control measures and appropriate choice of antimicrobial therapy. This guideline aims to improve the detection of carbapenemase-producing Enterobacteriaceae in the routine setting of clinical microbiology laboratories.

A c c e p t e d M a n u s c r i p t 6 Setting

 6 of the screening breakpoints should therefore be guided by the following principles:

  inhibitor. The genotypic confirmation consists of polymerase chain reaction (PCR) detection and sequencing of carbapenemase genes.

A

  c c e p t e d M a n u s c r i p t 10 validation reports of the inhibition tests for the routine setting, only some of which are commercially available, are limited[START_REF] Miriagou | Acquired carbapenemases in Gram-negative bacterial pathogens: detection and surveillance issues[END_REF].

A

  c c e p t e d M a n u s c r i p t 12 be detected by PCR and sequencing at the University Medical Center Utrecht using in-house PCRs as well as recently published PCRs [14].

Fig. 1 .

 1 Fig. 1. Detection scheme for carbapenemases in Enterobacteriaceae. a The zone

Fig. 2 .

 2 Fig. 2. Protocol for the modified Hodge test. (1) A 0.5 McFarland suspension of

A c c e p t e d M a n u s c r i p t 18 a 1 1

 1811 carbapenem plus 3-aminophenylboronic acid to inhibit AmpC and class A carbapenemases[START_REF] Pasteran | Controlling falsepositive results obtained with the Hodge and Masuda assays for detection of class A carbapenemase in species of Enterobacteriaceae by incorporating boronic acid[END_REF].A c c e p t e d M a n u s c r i p tTable Carbapenemase genes Ambler Class A 9 families (KPC, SME, NMC-A, IMI, PER, GES, SFO, SFC, IBC) Ambler Class B 6 families (VIM, GIM, SIM, NDM, IMP, SPM) Ambler Class D 2 families (OXA, PSE)A c c e p t e d M a n u s c r i p t 1

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

 2 The zone diameter screening breakpoint for meropenem has been set at ≤23 mm with a disk content of 10 g.

A c c e p t e d M a n u s c r i p t 1 3

 13 Table Phenotypic carbapenemase confirmation tests for Class A and B /www.abbiodisk.com McF, McFarland; MHA, Mueller-Hinton agar; EDTA, ethylene diamine tetra-acetic acid; DPA, dipicolinic acid; MBL, metallo-lactamase; N/A, not applicable; MIC, minimum inhibitory concentration.a The brand of Muller-Hinton agar may influence the test characteristics of the metallo-carbapenemase inhibition tests with EDTA [10].b To avoid false-negative results in MBL-producing isolates with imipenem MICs ≤ 4 mg/L, and according to the instructions of the manufacturer, the result of the MBL Etest should be interpreted as non-determinable if the Etest result is imipenem <4 mg/L and imipenem + EDTA <1 mg/L.

1 :

 1 Modified Hodge test 2: APB-meropenem combination disk 3: Meropenem-DPA combination disk 4: Imipenem-EDTA combination disk/Etest Positive Elevated carbapenem MIC confirmed with meropenem or imipenem Etest? No Carbapenemase screening: Escherichia coli, Klebsiella spp., Enterobacter spp., Salmonella spp. and Citrobacter spp.: Expert system positive OR meropenem MIC  0.5 mg/L OR imipenem MIC  2.0 mg/s c r i p t Example of positive modified Hodge test (photo Neil Woodford, Health Protection Agency, UK) meropenem ertapenem imipenem Growth of E. coli ATCC 25922 Inhibition of E. coli ATCC 25922 Increased growth of E. coli ATCC 25922 (disturbed edge of inhibition zone) by inactivation of the carbapenem from the disk by diffusion of the carbapenemase originating from the suspected isolate in the streak Test isolate (suspected carbapenemase production)

Table 1

 1

can currently

Table 2

 2 Carbapenem screening breakpoints, clinical breakpoints and epidemiological cut-off values (mg/L)

	Species	Meropenem		Imipenem		Ertapenem
		Escherichia coli,	Citrobacter spp.,	E.	Klebsiella spp.,	Serratia spp., P.	E. coli, Klebsiella spp.,
		Klebsiella spp.,	Serratia spp.,	coli	Enterobacter spp.,	mirabilis, M.	Enterobacter spp.,
		Enterobacter spp.,	Proteus mirabilis,		Salmonella spp.,	morganii,	Salmonella spp.,
		Salmonella spp.	Morganella		Citrobacter spp.	Providencia	Serratia spp., P.
			morganii,			spp.	mirabilis, M. morganii,
			Providencia spp.				Providencia spp.
	Carbapenemase	≥0.5 a	≥0.5 a	≥2 b ≥2 b	N/A	≥0.5 c
	screening
	breakpoint
	Epidemiological	S ≤0.125	S ≤0.25	S	S ≤1	S ≤4 (Serratia S	S ≤0.064
	cut-off wild-type			≤0.		≤2)
				5
	EUCAST	S ≤2	S ≤2	S	S ≤2	S ≤2	S ≤0.5
	breakpoint			≤2
	CLSI breakpoint	S ≤1	S ≤1	S	S ≤1	S ≤1	S ≤1
				≤1
	N/A, not applicable; S< susceptible; EUCAST, European Committee on Antimicrobial Susceptibility Testing; CLSI, Clinical and
	Laboratory Standards Institute.

Competing interests

None declared.

Ethical approval

Not required.