Asma Mani

Tao Feng

Albert Gandioso

Robin Vinck

Anna Notaro

Lisa Gourdon

Pierre Burckel

Bruno Saubaméa

Olivier Blacque

Kevin Cariou

Jamel-Eddine - Eddine Belgaied

Hui Chao

Gilles Gasser
email: gilles.gasser@chimeparistech.psl.eu

Dr Franz Heinemann

Structurally Simple Osmium (II) Polypyridyl Complexes as Photosensitizers for Photodynamic Therapy in the Near Infrared

Keywords: Bioinorganic Chemistry, Infrared, Medicinal Inorganic Chemistry, Osmium, Photodynamic Therapy

Five osmium(II) polypyridyl complexes of the general formula [Os(4,7-diphenyl-1,10phenanthroline)2L] 2+ were synthesized as photosensitizers for photodynamic therapy by varying the nature of the ligand L. Thanks to the pronounced π-extended structure of the ligands and the heavy atom effect provided by the osmium center, these complexes exhibit a high absorption in the near-infrared (NIR) region (up to 740 nm), unlike related ruthenium complexes. This led to promising phototoxicity in vitro against cancer cells cultured as 2D cell layers but also in multicellular tumor spheroids upon irradiation at 740 nm. The complex [Os(4,7-diphenyl-1,10-phenanthroline)2(2,2'-bipyridine)] 2+ was found to be the most efficient against various cancer cell lines, with high phototoxicity indexes. Experiments on CT26 tumorbearing BALB/c mice also indicate that the Os(II) complexes could significantly reduce tumor growth under 740 nm laser irradiation. The high phototoxicity in the biological window of this structurally simple complex makes it a promising photosensitizer for cancer treatment.

Introduction

Cancer is a multifactorial global issue, assumed to be the second leading cause of death in the world. According to World Health Organization, it was responsible for 9.6 million deaths in 2018. [START_REF]Cancer[END_REF] Different treatments, including radiotherapy and chemotherapy, can be used to treat this disease, quite often in combination with surgery. [START_REF] Lazic | [END_REF]3] However, these adjuvant treatments usually lead to severe side effects due to their lack of selectivity for diseased tissues. For this reason, many studies were conducted to develop more efficient and selective alternative treatments, as evidenced by the fact that over 60% of all current experimental trials worldwide are focusing on cancer treatment. [3] Photodynamic therapy (PDT) was discovered as a promising alternative/complementary technique to treat several types of cancer as well as infectious and skin diseases. [4] It relies on the simultaneous action of a photosensitizer (PS), light and oxygen. Once administrated to the patient, the PS accumulated in the target tissues is exposed to light at a specific wavelength.

The PS then reaches a relatively unstable excited singlet state, followed by an intersystem crossing from the PS singlet state to its longer-lived triplet state. [4][5][6][7] From the excited triplet state, the chromophore can then decay to the ground state by a vibronic radiation-less relaxation, producing phosphorescence, or interact with the biological environment by two different mechanisms referred to as type I or type II. In a type I mechanism, the exchange of a proton or electron occurs between the triplet excited state of the PS and the surrounding molecular oxygen to generate reactive oxygen species (ROS), such as the superoxide anion radical (O2 •-), the hydroxyl radical and other free radicals. These ROS are powerful oxidant agents capable of causing oxidative damage and hence cellular death. Alternatively, the type II mechanism is favored in oxygenated environments where the PS as a triplet state transfers its energy to ground-state molecular oxygen ( 3 O2) to produce singlet oxygen ( 1 O2), known for its high reactivity leading to the formation of adducts with organic substrates. [6] With its low lifetime (τ < 3 µs), singlet oxygen exhibits a low intracellular diffusion distance estimated by 2-4.10 -6 cm 2 .s -1 leading to a low distance of action (~0.01-0.155 μm). [7], [8] The characteristics of 1 O2 impart PDT with a localized effect, making it a highly selective cancer treatment.

Porphyrin-based compounds were the first developed and the most investigated PSs. However, these first-generation PSs are often insoluble in water, have poor light absorption and induce prolonged skin photosensitivity following treatment. [7] To tackle these problems, in addition to a second generation of organic PSs, metal-based PSs were developed, using different metals such as Ru(II), Pt(II), Pt(IV), Os(II), Re(I) or Ir(III). Among these, Ru(II) remains undoubtedly the most studied metal in PDT, especially Ru(II) polypyridyl complexes, thanks to their tunable photophysical and biological properties. [9][10][11][12][13] Ru(II) polypyridyl complexes have also emerged as promising agents in Photo-Activated ChemoTherapy (PACT) thanks to their photolabile properties. [12,14] Many Ru-based PSs were evaluated in our group and others, especially coordinated with substituted 1,10-phenanthroline and 2,2'-bipyridine ligands, unveiling compounds with promising photodynamic properties. [4,9,[15][16][17][18] Ru(II) based complexes using 4,7-diphenyl-1,10-phenanthroline (DIP) ligand were efficient as PDT agents upon irradiation at 595 nm. [19] To ensure optimal light penetration in the tissues to treat deep-seated or large tumors, the excitation wavelength of the PS should ideally lie in the range of 630-850 nm.

Ru(II) complexes involving a tris-heteroleptic scaffold were used as immunoprotective photosensitizers upon irradiation at 733 nm. [20] In contrast, using two-photon instead of onephoton irradiation enables the excitation of the complexes with less energetic radiations, and thus at higher wavelengths. As such, different polypyridyl complexes based on Ru(II) or Ir(II) as well as bimetallic Ru(II)-Pt(II) complexes were shown to be photoactive in the NIR region, upon two-photon irradiation at up to 830 nm. [21] The promising results obtained with ruthenium led us to focus on other transition metals to further improve their PDT potential. In this endeavor, osmium was shown to be the metal of choice to design new polypyridyl complexes with activation wavelengths in the NIR region. [START_REF] Lazic | [END_REF] Osmium-based complexes have been proposed as DNA-binding agents in diagnostic probes for oncology. Some osmium polypyridyl complexes containing a dppz (dipyridophenazine) ligand have shown a high luminescence response at 750 nm when bound to DNA-quadruplexes. [22] Due to their spectacular luminescent properties, osmium-based complexes were applied to cancer treatment. The complex [(Os(DIP))2pppp] 4+ revealed a high photothermal activity upon irradiation at 808 nm toward human melanoma cells. [23] McFarland et al. have also reported osmium-based PSs with exceptional photophysical properties. These complexes revealed high phototoxicity toward cancer cells upon irradiation at 730 nm. [24] However, this remarkable PDT activity is limited by the sophisticated structure of the oligothiophene-derivatized phenanthroline ligand used to prepare these complexes. In addition, no proof of in vivo efficacy was established for these complexes. [24] Lazic et al. have also developed an osmium-based PS called TLD 1829 containing the auxiliary π-extended 2,2′-biquinoline (biq) ligand. This PS demonstrated high in vivo activity against murine colon cancer after irradiation at 808 nm. [START_REF] Lazic | [END_REF] The selection of complexes bearing ligands with a simpler structure, such as the DIP, is therefore of high interest. We have thus decided to develop new osmium-based PSs, with the potential to be irradiated in the NIR, enabling potentially a higher penetration depth during PDT treatments. In this work, we present the synthesis and characterization of five new osmium (II) polypyridyl complexes 1-5 (Figure 1). These complexes were generated from a common osmium precursor complex bearing two DIP ligands, coordinated to different bipyridine or phenanthroline derivatives: 2,2'-bipyridine (bpy), 4,4'-dimethyl 2,2'-bipyridine (dmbpy), 4,4'diamine-2,2'-bipyridine (dnbpy), 1,10-phenanthroline (phen). The homoleptic complex 5 was obtained by coordination of the precursor with a third DIP ligand. The PDT potential of the newly synthesized complexes was then evaluated in vitro against various cancer cell lines: cervical cancer cells A2780, human and mouse colon cancer cells (HT29 and CT26). The cellular uptake and phototoxicity on 3D multicellular tumor spheroids (MCTS) were also investigated in CT-26 cells. Encouraged by promising in vitro results, we investigated the efficacy of the most promising complex (1) in a CT26 tumor-bearing mouse model. Our findings suggest that, with their simple structure, these new complexes have the potential to become clinically efficient one-photon NIR PDT PSs.

Results and Discussion

Synthesis and characterization

The synthesis of the dichloride osmium complex Os(DIP)2Cl2 has already been reported in previous works, without detailed characterization. [25,26] However, preventing the formation of the byproduct [Os(DIP)3] 2+ [27] while repeating the described procedure appeared challenging.

After several purification attempts by silica gel chromatography, the precursor complex was successfully isolated from byproducts by precipitation from acetone in an ethanol/acetone mixture (50:1) (v/v). All the ligands were commercially available except dnbpy, which was synthesized as previously reported. [28] The final compounds were obtained by heating the precursor complex at 90 °C with the corresponding ligand in degassed ethylene glycol. Detailed procedures and characterizations are provided in the supporting information. The structure of all complexes was confirmed by 1 H and 13 C NMR spectroscopy (Figure S1-S12) and highresolution mass spectrometry and their purity was evaluated by elemental analysis and HPLC (Figure S13). Complexes 4 and 5 were successfully crystallized by slow diffusion of diethyl ether in acetone or acetonitrile (CH3CN), respectively. Single crystal X-ray diffraction studies were carried out. [29] Crystal data, structure refinement parameters and molecular structures are provided in the supporting information as well as in Figure 2 and

Photophysical properties

The UV-visible spectrum of the different polypyridyl complexes was recorded to investigate their electronic behavior in the desired phototherapeutic window (i.e. 600-850 nm). [30] We note that the baseline for the precursor is not ideal potentially due to the formation of nanoparticles.

The absorption spectrum of complexes 1-5 shows a panchromatic absorption from 240 to 840 nm, and finally a weaker broadband covering the region 650-750 nm. [25][26][27][28] This latter can be explained by the spin-forbidden MLCT transitions due to the direct singlet-triplet transition of the PS. [START_REF] Lazic | [END_REF]31] These transitions are explained by the strong spin-orbit coupling of osmium, often encountered in heavy atoms. [32] A similar result recently reported on [Os(phen)3] 2+ and [Os(DIP)3] 2+ confirms our assumptions. [24], [27] Unlike their ruthenium counterparts, these complexes have therefore the potential to be phototoxic upon irradiation at wavelengths above 595 nm. [19] Figure 4. UV/vis/NIR spectra of complexes 1-5 and their precursor complex in CH3CN.

Singlet oxygen ( 1 O2) is the main toxic species for a PS working through the type II mechanism. [33,34] Therefore, the production of singlet oxygen upon irradiation of the PSs was quantitatively evaluated in CH3CN and Phosphate Buffer Sulfate (PBS) using two methods. [35,36] As an indirect method, the decrease in absorbance of p-nitrosodimethylaniline in the presence of the photosensitizers and imidazole as 1 O2 scavenger was monitored as a function of the irradiation time (450 nm). The results obtained by this indirect method were confirmed by a direct method based on the quantification of the characteristic luminescence produced by the relaxation of 1 O2. [35,36] The singlet oxygen quantum yields were then calculated using

[Ru(bpy)3]Cl2 and phenalenone as references in PBS and CH3CN, respectively.

Both methods showed that all complexes but 3 efficiently sensitize oxygen with quantum yields of 35-50% in aerated CH3CN and 2.6-6.2% in aerated PBS, Table 1. The lower yields obtained with complex 3 may be explained by the presence of a diamine group, which is able to quench the singlet oxygen, as reported previously. [37], [38] The quenching mechanism is due to a charge transfer between the ground state of a nitrogen-containing compound and singlet oxygen. [38] The quantum yield in aqueous media appeared lower than in CH3CN. The low singlet oxygen production in aqueous media can be related to the strong quenching properties of water. [START_REF] Belousova | Laser Optics[END_REF] Unfortunately, the singlet oxygen production yield could not be determined in deuterated water D2O using the direct method as the luminescence signal was under the detection limit. Despite low 1 O2 production yields in polar aqueous media, our compounds could still prove to be efficient PSs since cells include apolar environments. Indeed, it was previously demonstrated that, upon interaction with hydrophobic components of the cell such as DNA, PSs that otherwise produce a low level of 1 O2 in water can efficiently sensitize molecular oxygen. [40] (Photo-)toxicity

To evaluate the (photo-)cytotoxic effect of the synthesized complexes, a screening was performed on cervical cancer cells (A2780) and non-cancerous retinal pigment epithelial (RPE-1) cells. For this purpose, cells were incubated with 0.1, 1, or 10 µM of compounds 1-5 in the dark for 4 hours. After washing, they were then either kept in the dark or irradiated for 1 h at 620 nm (spectral half-width: 32 nm, 60 min, 1.88 mW cm -2 , 6.7 J cm -2 ), 645 nm (spectral halfwidth: 32 nm, 60 min, 2.50 mW cm -2 , 9.0 J cm -2 ), 670 nm (spectral half-width: 32 nm, 60 min, 3.75 mW cm -2 , 13.5 J cm -2 ), 740 nm (spectral half-width: 32 nm, 60 min, 3.50 mW cm -2 , 12.6 J cm -2 ). The cell viability was determined 2 days later using a fluorometric assay. For comparative purposes, the clinically approved PDT photosensitizer Protoporphyrin IX (PpIX)

was included in this study.

This first screening showed that compounds 1-5 have no cytotoxic at up to >10 μM in the dark in A2780 and RPE-1 cells (Figure S20). In contrast, all compounds induced high phototoxicity upon light exposure at all tested wavelengths in the two cell lines (Figure S21).

While complexes 1, 3 and 4 led to a potent reduction of cell viability at concentrations as low as 1 µM, complexes 2 and 5 only revealed their phototoxic effect at 10 µM. Of note, no significant selectivity towards cancer cells was observed in this preliminary assay. Importantly, we could demonstrate that complexes 1-4 were able to have a phototoxic effect up to 740 nm.

In light of these promising results, the phototoxicity of the complexes was evaluated in more detail. Because of their structural similarity with 1 (for 2) and their relatively poor phototoxicity (for 5), respectively both complexes 2 and 5 were excluded from further investigations. The concentration of complexes 1, 3, and 4 needed to kill 50% of the cells (IC50) was determined following 4 hours of incubation and 1 hour of irradiation at 740 nm or 4 hours of incubation in the dark in A2780 and RPE-1 cells, Table 2. All complexes appeared less toxic than PpIX in the dark (IC50 = 3 ± 2 μM), with 1 and 3 exhibiting the lowest cytotoxicity (IC50 = 58 ± 9 μM and 62 ± 10 μM, respectively). With the exception of PpIX, the IC50 of every compound significantly decreased upon light irradiation at 740 nm, confirming their potential as PDT PSs.

Of note, 1 displayed the highest phototoxicity index (PI = 118), defined as the ratio between dark toxicity and phototoxicity, against A2780 cells. Similar results were obtained on healthy RPE-1 cells, confirming the absence of selectivity towards cancer cells. Since complex 1, as a PF6 salt, was revealed to be the most efficient PS at 740 nm, and in view of future in vivo experiments in a colon tumor mouse model, an additional phototoxicity evaluation was performed on human and mouse colon adenocarcinoma (HT29 and CT26) cells.

For this purpose, the PF6 -counter ion was exchanged by chloride ions Cl -, using an Amberlite IRA-410, to ensure better solubility in biological fluids, mainly later for in vivo tests. The results presented in Table 2 show that complex 1 displays similar and high phototoxicity in the two cell lines (IC50 = 0.33 ± 0.03 μM and 0.34 ± 0.06 μM for HT29 and CT26, respectively). Of note, the same phototoxicity was observed with the structurally similar ruthenium complex [Ru(DIP)2dmbpy] 2+ in CT26 cells upon irradiation at 540 nm using the same low light dose (14.2 J.cm -2 ) (IC50= 0.34 ± 0.005 μM). [19] In contrast, the phototoxicity of the osmium complex [Os(DIP)2(dpp)] 2+ , where dpp = 2,3-bis(2-pyridyl)pyrazine, was previously tested in the rat glioma cells F98, following irradiation at 625 nm and showed a significantly lower anticancer activity (IC50 = 86.1± 8.5 μM) than that our complex 1. [41] The dinuclear osmium complexes [(Os(DIP))2pppp] 4+ , described previously, reported a high photothermal activity toward human melanoma cells, using higher complex concentration (≥10 μM) upon irradiation at 808 nm. [23] Furthermore, complex 1 with the Cl -counterion was found to be cytotoxic after irradiation with near-infrared light in the micromolar range in hypoxic conditions (IC50, 740 nm = 4.75 ± 0.30 μM, IC50, dark = 46.44 ± 1.0 μM, PI = 10) (Figure S23-25). Importantly, we could demonstrate that complex 1 exhibits a phototoxic effect up to 740 nm, even under hypoxia conditions, an important requirement for a PDT agent.

These impressive results of complex 1 against the three tested cell lines A2780, CT-26, and HT29 at 740 nm, a NIR wavelength, prompted us to submit it to additional experiments before undergoing in vivo studies. These impressive results of complex 1 against the three tested cell lines A2780, CT-26, and HT29 at 740 nm, a NIR wavelength, prompted us to submit it to additional experiments before undergoing in vivo studies.

Photostability

An important parameter to assess is the (photo-)stability of a PS. For this reason, the stability of 1-5 was evaluated. The stability was first determined in the dark by observing changes in the complexes' UV-Vis spectrum over 48 hours of incubation in different media. This experiment suggests that all complexes are stable in CH3CN, PBS, and DMSO (Figure S15-S17). Of note, complexes were found to be poorly soluble in PBS, requiring supplementation with 1% DMSO.

Since PDT requires light application, the photocytotoxicity activity of photosensitizer candidates is strongly influenced by their photostability upon irradiation. For this reason, we performed photo-stability studies of complexes 1-5 at 37°C in biologically relevant media (i.e. PBS and DMEM medium (Gibco) supplemented with 10% FBS) (Figure S18 andS19) upon irradiation at 740 nm for 1 h. Importantly, no significant changes in the absorption spectra of complexes 1-5 were observed in PBS and DMEM after 1 h of irradiation.

Cellular uptake and localization studies

The cellular uptake of our osmium complexes 1-5 was then investigated in CT-26 cells by determining the amount of Os inside the cells using inductively coupled plasma mass spectrometry (ICP-MS) after 4 h of incubation at 10 μM, (Figure 5). The Os complex [Os(DIP)3] (PF6)2 ( 5) was found to have the highest uptake, almost three times larger compared to the rest of the complexes. This is probably due to the higher lipophilicity of the DIP ligand compared to bpy and phen. The uptake of 1 and 2 is also 2-fold higher in comparison to complexes 3 and 4. The lower uptake of complexes 3 and 4 might be due to the lower solubility of these complexes. The incorporation of NH2 into the bpy (complex 3) decreases the internalization of the complex. Unfortunately, we could not establish a relationship between the accumulation and their photo-toxicity values because all the complexes described herein present similar photo-toxicity activity.

To get further insight into the internalization mechanism of complex 1, its subcellular localization was determined by confocal microscopy using the intrinsic luminescence of our Os(II) complex (λex = 448 nm, λem = 645-730 nm). In CT26 cells incubated with 5 µM complex 1 for 4 hours, the luminescence appeared as both a diffuse and a punctate signal suggesting the accumulation of the complex in both the cytosol and some vesicular compartment (Figure 5).

However, the complex did not accumulate in the nucleus and mitochondria as shown by the absence of colocalization with Hoechst 33342 and MitoTracker Green respectively.

Phototoxicity on 3D multicellular tumor spheroids (MCTS)

Complex 1 was found to be the most promising candidate among the series of osmium complexes investigated in a 2D cell model. Due to its remarkably high and promising photocytotoxicity, we explored its activity in a multicellular tumor spheroids (MCTS) model.

In 3D spheroids, MCTS simulates the conditions in clinically treated tumors, including hypoxia environment, and extracellular matrix deposition. Additionally, the growth pattern, metabolism, and gene expression mimic the complexity of the initial stages of solid tumors. These features allow for a reasonable estimation of in vivo antitumor activity, qualifying MCTS as a more reliable model than monolayer cell cultures for advanced cancer research. [42,43] An experiment was therefore performed to evaluate the time-dependent effect on the growth of MCTS treated with complex 1. CT26 MCTSs (ca. 550 μm in diameter) were treated with increasing concentrations of 1 (0,1, 1, 10, 30, and 100 μM). After 36 h of incubation, a luminescence signal is observed in the spheroids (Figure S22). The medium was then exchanged with fresh medium, and cells were kept in the dark or irradiated for 1 h at 740 nm.

Following this treatment, half of the medium in the wells was exchanged every two days, and pictures of the spheroids were taken (Figure 6). Importantly, CT26 MCTSs treated with the highest concentrations of 30 μM and 100 μM of complex 1 had a reduced diameter. In contrast, no effect was seen in MCTSs treated with the complex and kept in the dark (100 μM) in comparison to untreated MCTS. In addition, complex 1 was tested via a luminescent cell viability assay in CT-26 MCTS (single graphs are available in Figure S23). Complex 1 displayed high cytotoxicity toward CT-26 MCTS with IC50 ≈ 31± 6 μM. This result is comparable to the IC50 obtained with [Ru(DIP)2 dmbpy] 2+ toward HeLa MCTS. [19] Overall, the outstanding activities shown by complex 1 in the monolayer cell model were confirmed in an MCTS model. This is of high interest since the center of spheroids is considered hypoxic (i.e., with a low concentration of oxygen) and it could have been anticipated that our complex would not be efficient. These findings are a powerful motivation for further investigation of complex 1 as a novel potential photosensitizer agent in photodynamic therapy.

Dynamic Light Scattering (DLS)

We previously observed that Ru(II) complexes incorporating two DIP ligands could form aggregates in isotonic aqueous solutions. [44] While they form large aggregates in PBS, we recently showed that they can form smaller nanoparticles in the presence of plasmatic proteins.

As it is the case with positively charged gold nanoparticles, we postulated that the presence of salt decreases the repulsive electrostatic interaction between the Ru(II) complexes, resulting in their aggregation. [45] This aggregation was prevented in culture medium by the coating effect provided by plasmatic proteins, leading to the formation of sub-micron nanoparticles. [46] As this aggregation behavior could affect the biodistribution of the complexes in vivo, we performed a Dynamic Light Scattering (DLS) analysis to investigate the aggregation behaviour of complex 1 with the Cl -counterion in 10% FBS in PBS. The DLS analysis of 10% FBS in PBS without the complex revealed the existence of nanometric objects with a mean diameter of 8.87 nm, which could correspond to serum albumin. [47] In the presence of complex 1 with Cl counterion, a subtle shift in the mean diameter of the objects from 8.87 to 12.68 could be observed (Figure S14). This small shift could be explained by the binding of the complex to albumin, as observed previously with other drugs. [48] In 100% PBS, complex 1 with the Cl -counterion tends to form larger particles that sediment, which can be observed with the naked eye and is confirmed by the high polydispersity index obtained by DLS. Therefore, in contrast to our previously described Ru(II) complexes, for which nanoparticles of up to 350 nm were observed in the presence of plasmatic proteins, complex 1 appears to be soluble in this medium, probably thanks to its binding to plasmatic proteins.

In vivo Studies

Encouraged by the promising results obtained in vitro with complex 1 in 2D and 3D models, its PDT efficacy was further investigated in a CT26 tumor-bearing BALB/c mouse model. One hour following intratumoral injection, the mice were either kept in the dark or exposed to onephoton irradiation using a 740 nm laser (50 mW, 12.6 J. cm -2 , 300 s). The tumor volume and body weight of each mouse was measured and recorded every two days for two weeks.

According to the tumor growth inhibition curve (Figure 7A), the tumors treated with complex 1 and light were found to be nearly eradicated within a single procedure. On day 14, the normalized tumor volume in the complex 1 + Light group was 4.67, 4.56 and 4.36-fold smaller than the control group, the group treated with light only and the group treated with complex 1 without irradiation, respectively (Figure 7A, C andD). Importantly, the animals treated with complex 1 behaved normally, without signs of pain, stress or discomfort and did not lose weight, suggestive of the high biocompatibility of the compound (Figure 7B). After the treatment, all major organs (i.e., heart, liver, spleen, lung, kidney, brain, intestine) as well as the tumor tissues were histologically examined by the hematoxylin-eosin stain. While no pathological alterations or injuries were observed for all organs (Figure S24), significant damages including karyopyknosis and widespread areas of apoptotic nuclei were noticed in the tumor tissue (Figure 7E). Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) stain was employed to analyze tumor tissues. The green fluorescence signals, indicative of DNA strand breaks during apoptosis, for the treatment with Complex 1 + Light were observed, indicative of the strong therapeutic effect. As discussed in the introduction, the Os-based complex TLD 1829 showed high survival in murine population bearing colon cancer as well, after irradiation at 808 nm (600 J.cm -2 , 4 h). [START_REF] Lazic | [END_REF] TLD 1829 and complex 1 showed comparable in vivo activity with the advantage of lower light exposure of complex 1 (740 nm, 12.6 J.cm -2 , 1h) compared to TLD1829 (808 nm, 600 J.cm -2 , 4 h). Overall, this study demonstrated the important potential of complex 1 for photodynamic therapy in the biological window.

Conclusion

In summary, we were able to prepare and characterize structurally simple Os(II) polypyridyl complexes bearing two bathophenanthroline ligands. These complexes showed excellent photophysical properties, including high 1 O2 production quantum yields. Importantly, they displayed a panchromatic absorption which enables the irradiation of the PS at wavelengths up to 740 nm. This wavelength is much higher in comparison to the maximum wavelength at which the ruthenium analog [Ru(DIP)2dmbpy] 2+ can be excited. Cell experiments on all complexes in non-cancerous retinal pigment epithelium (RPE-1) and cervical cancer cells A2780 showed no cytotoxicity in the dark and intense toxicity following light irradiation. Importantly, complex 1, with its simple structure, was found to have a promising PI value at 740 nm with low dark toxicity and an IC50 in the nanomolar range following irradiation. It also proved to be extremely stable and highly phototoxic against human and mouse colon cancer cells (HT29 and CT26).

The high 1 O2 production quantum yield and absorption properties of complex 1 endow it with excellent PDT efficacy in vivo. Such simple Os(II) polypyridyl complexes may indeed become a promising antitumor therapeutic agent for future clinical applications. Overall, the results presented in this article confirm that osmium polypyridyl complexes might be the next generation of highly efficient photosensitizers for PDT.

Table of Contents

Osmium-based polypyridyl complexes have been developed as photosensitizers for one-photon photodynamic therapy in the near-infrared.

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Figure 1 .

 1 Figure 1. General synthesis route for the osmium complexes 1-5.

Figure 3 .

 3 For complex 4, the Os atom is coordinated to two bathophenanthroline ligands and one substituted phenanthroline ligand through the N atoms in a distorted octahedral geometry. For complex 5, the Os is coordinated to three bathophenanthroline ligands through the N atoms in a distorted octahedral geometry.

Figure 2 .

 2 Figure 2. X-ray molecular structure of complex 4, with hydrogen atoms omitted.

Figure 3 .

 3 Figure 3. X-ray molecular structure of complex 5, with hydrogen atoms omitted.

Figure 4 .

 4 All five complexes show a similar profile, suggesting that the accessible electronic transitions, and the ground and excited states between the compounds are qualitatively similar. Their spectrum is however different from that of their precursor Os(DIP)2Cl2. Complexes 1-5 exhibit three major absorption bands, as is usually observed for related complexes. They show a sharp and intense peak at 280 nm, which can be assigned to the IL ππ* transitions of DIP, two broad peaks (with maxima at ca. 450 and 500 nm, respectively) attributed to the Metal to Ligand Charge Transfer (MLCT) Os(dπ)-ligand(π*),

Figure 5 :

 5 Figure 5: (Left) Cellular uptake of compounds 1-5 by ICP-MS (Right) Cellular uptake of complex 1 by confocal microscopy. A. Mitochondria-specific dye MitoTracker Green (green, exc: 488 nm, em: 513-550 nm) B. Complex 1 (red, exc: 448 nm, em: 645-730 nm) C. Overlay plus nucleus-specific dye Hoechst 33342 (blue, exc: 405 nm, em: 409-448 nm) D. Higher magnification of the boxed area in C.

Figure 6 :

 6 Figure 6: (Left) Changes in the growth kinetics of MCTSs treated with complex 1 at different concentrations (0,1, 1, 10, 30, and 100 μM). Images were collected on days 0, 2, 4, 7, 9, and 12. (Right) MCTS diameter was measured at different time points. Average of three independent measurements.

Figure 7 :

 7 Figure 7 : (A) Average tumor growth curves after the respective treatment (n = 5) Dose: 5.0 mg/kg; Irradiation (740 nm; 50 mW, 12.6 J. cm -2 , 300 s). (B) Time-dependent change in body weight after various treatments on mice. (C) Representative pictures of the tumor after the respective treatments. (D) Individual tumor growth curves after the respective treatments. (E) Haematoxylin-eosin (H&E) stain (scale bar: 200 μm) and TUNEL stain (scale bar: 100 μm) of the tumor tissues after the respective treatments. ***P < 0.001. Average of five independent measurements.

  Wang et al. have reported new dinuclear polypyridyl osmium complexes for

	photothermal	therapy	using	a	bridging	ligand	pppp

(

[1,10]

phenanthrolino[5'',6'':5',6']pyrazino[2',3':5,6]pyrazino [2,3-f][1,10]phenanthroline).

Table 1 .

 1

	Compound	CH3CN	CH3CN	D2O	PBS

1-5 singlet oxygen quantum yields (Φ( 1 O2)) in CH3CN and aqueous solutions determined by direct and indirect methods upon excitation at 450 nm. n.d.=not determinable

Direct method Indirect method Direct method Indirect method

	1	35%	39.7%	n.d	6.2%
	2	37%	41.5%	n.d	4.3%
	3	9%	20.7%	n.d	1.7%
	4	40%	39.9%	n.d	5.9%
	5	50%	43.4%	n.d	2.6%

Table 2 .

 2 IC50 values in normoxia and hypoxia conditions, in the dark and upon irradiation at 740 nm (spectral halfwidth: 32 nm, 60 min, 3.50 mW cm -2 , 12.6 J cm -2 ) for 1, 3, and 4 compared to PpIX on non-cancerous retinal pigment epithelium (RPE-1), cervical cancer (A2780) cells, human colon cancer (HT29) cells and mouse colon adenocarcinoma (CT-26) cells. Average of three independent measurements. n.d.=not determinable.

	Normoxia		Dark	740 nm (1 h, 12.6 J/cm²) PI
	conditions
	PpIX	A2780	3 ± 2 μM	2.2 ± 0.2 μM	2
		RPE-1	4 ± 2 μM	6 ± 2 μM	n.d
	1	A2780	58 ± 9 μM	0.49 ± 0.09 μM	118
		RPE-1	47 ± 2 μM	0.54 ± 0.03 μM	87
	3	A2780	62 ± 10 μM	1.3 ± 0.3 μM	48
		RPE-1	76 ± 9 μM	2.9 ± 0.6 μM	27
	4	A2780	19 ± 2 μM	0.51 ± 0.07 μM	37
		RPE-1	26.7 ± 0.5 μM	0.77 ± 0.06 μM	35
	1*	HT29	14 ± 1 μM	0.33 ± 0.03 μM	42
		CT26	13.1 ± 0.3 μM	0.34 ± 0.06 μM	39
	Hypoxia		Dark	740 nm (1 h, 12.6 J/cm²) PI
	conditions
	PpIX	CT-26	> 100 µM	35.50 ± 0.6 µM	> 2.8
	1*	CT-26	46.44 ± 1.0 µM	4.75 ± 0.3 µM	10
	*the counter ion was exchanged from PF6 -to chloride Cl -

Acknowledgments

This work was financially supported by an ERC Consolidator Grant Photo-MedMet to G. G. (GA 681679), has received support under the program ''Investissements d'Avenir'' launched by the French Government and implemented by the ANR with the reference ANR-10-IDEX-0001-02 PSL (G. G.) and by a Qlife pré-maturation funding (G.G. and R.V.). A.G. thanks the ARC Foundation for cancer research for a postdoctoral Research Fellowship. Part of the ICP-MS measurements was supported by IPGP multidisciplinary program PARI, and by Paris-IdF region SESAME Grant no. 12015908. The authors thank Mathilde Chaboud and Dr. Philippe Goldner for their help in the determination of singlet oxygen quantum yield as well as Dr. Gregory Lefevre for lending the DLS apparatus. This work was also supported by the National Natural Science Foundation of China (No. 22120102002), and the Science and Technology Innovation Program of Hunan Province of China (No. 2021RC5028). L. G. acknowledges the ENS-PSL for her PhD fellowship.

Conflicts of interest

The authors declare no competing financial interests.

SUPPORTING INFORMATION

1 H and 13 C NMR spectra (Fig. S1-S12). HPLC traces (Fig. S13). Size distribution by volume (Fig S14). Absorption spectra for stability evaluation in the dark (Fig. S15-S17). Absorption spectra for photobleaching evaluation (Fig. S18-S19). Viability tests (Fig. S20-S21).

Fluorescence microscopy of CT-26 spheroids treated with complex 1 (Fig. S22). Fluorometric cell viability assay in normoxia conditions in CT26 MCTS after irradiation at 740 nm (Fig. S23). Fluorometric cell viability assay in hypoxia conditions in CT26 MCTS after irradiation at 740 nm (Fig. S24-S25). Hypoxia chamber (Fig. S26). Histological examination of all major organs by a hematoxylin-eosin stain (Fig. S27).


François Caméléna

Gauthier Péan De Ponfilly

Hélène Pailhoriès

Lucas Bonzon

Alexandre Alanio

Thibaut Poncin

Matthieu Lafaurie

François Dépret

Emmanuel Cambau

Sylvain Godreuil

Rachel Chenouard

Alban Le Monnier

Hervé Jacquier

Florence Claude Doucet-Populaire

Multicenter Evaluation of the FilmArray Blood Culture Identification 2 Panel for Pathogen Detection in Bloodstream Infections

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

assay in comparison to the results of standard methods used in routine practice at four tertiary care hospitals. KEYWORDS BCID2, BSI, Biofire, bloodstream infection, FilmArray, molecular diagnosis, rapid diagnosis, genotypic identification, resistance gene, sepsis B loodstream infections (BSI) and sepsis are leading causes of morbidity and mortality, and they constitute a major public health concern, with an estimated burden of 1,200,000 episodes annually in Europe [START_REF] Goto | Overall burden of bloodstream infection and nosocomial bloodstream infection in North America and Europe[END_REF]. Timely, effective antimicrobial treatment is crucial, as delays in treatment initiation are associated with a poorer outcome, particularly in patients developing septic shock [START_REF] Seymour | Time to treatment and mortality during mandated emergency care for sepsis[END_REF][START_REF] Evans | Surviving Sepsis Campaign: international guidelines for management of sepsis and septic shock 2021[END_REF]. The increase in antimicrobial resistance has led to a decrease in the efficacy of empirical antibiotic treatment [START_REF] Peralta | Impact of antibiotic resistance and of adequate empirical antibiotic treatment in the prognosis of patients with Escherichia coli bacteraemia[END_REF][START_REF] Tamma | Combination therapy for treatment of infections with gram-negative bacteria[END_REF]. Rapid and accurate diagnosis of the causal pathogens, and also the identification of antibiotic resistance genes, can improve the outcome for patients with BSI by allowing more targeted therapy, which is becoming ever more important with the increase in antimicrobial drug resistance [START_REF] Lamy | ESCMID Study Group for Bloodstream Infections, Endocarditis and Sepsis (ESGBIES)[END_REF][START_REF] Timsit | Bloodstream infections in critically ill patients: an expert statement[END_REF]. Blood culture (BC) in a microbiology laboratory remains the gold standard technique for identifying the pathogens responsible for BSI. This approach includes culture-based methods for pathogen identification and antimicrobial drug susceptibility testing (AST). These methods yield results 24 to 48 h after the obtainment of a positive BC, and this delay may be critical in the management of patients with sepsis [START_REF] Tabak | Blood culture turnaround time in U.S. acute care hospitals and implications for laboratory process optimization[END_REF]. It is possible to shorten the time to BSI pathogen identification by using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS)-based approaches, with identification directly on positive BC or after a short period of subculture on solid medium [START_REF] Faron | Matrix-assisted desorption ionization time of flight mass spectrometry for the use with positive blood cultures: methodology, performance, and optimization[END_REF][START_REF] Dubourg | Emerging methodologies for pathogen identification in bloodstream infections: an update[END_REF]. However, these methods are subject to several limitations, including major time-consuming changes in laboratory workflows. Molecular assays based on multiplex PCR or microarrays have been marketed in recent years for the identification of bacterial and fungal pathogens from positive BC within 1 to 4 h, shortening both turnaround and hands-on times [START_REF] Lamy | ESCMID Study Group for Bloodstream Infections, Endocarditis and Sepsis (ESGBIES)[END_REF][START_REF] Dubourg | Emerging methodologies for pathogen identification in bloodstream infections: an update[END_REF]. The FilmArray Blood Culture Identification 2 panel (BCID2; Biofire; bioMérieux, Marcy l'Etoile, France), an updated version of the original BCID, is a fully automated microbiological diagnostic assay based on nested multiplex PCR analysis that can detect 11 Gram-positive bacteria, 15 Gram-negative bacteria, 7 fungal pathogens, and 10 antimicrobial resistance genes (see below). The aim of this multicenter study was to evaluate the real-life microbiological performance of the BCID2 assay for identifying bacteria and detecting resistance genes, through comparison with the results of standard methods used in routine practice.

RESULTS

Concordance and discrepancy between the BCID2 panel and culture for microorganism identification. In total, 152 positive BC were included in the analysis: 70% (106/ 152) corresponded to BacT/alert FA plus bottles and 30% (46/152) to BacT/alert FN plus bottles. We obtained 51% (78/152) of the positive BC from the Saint-Louis-Lariboisière-Fernand Widal Hospital Group, 19% (29/152) from Paris Saint-Joseph Hospital Group, 16% (24/152) from Angers University Hospital, and 14% (21/152) from Montpellier University Hospital. The 152 positive BC yielded 130 monomicrobial and 22 polymicrobial cultures. The identification results for pathogens targeted by the BCID2 panel are shown in Table 1, and the details of BC giving discrepant BCID2 and standard culture results are provided in Table 2. Organisms detected in polymicrobial BC are presented in Table S1 of the supplemental material. A total of 177 bacteria from 152 positive BC were identified in culture, of which 90% (88/ 98) of the Gram-negative and 89% (70/79) of the Gram-positive bacteria were correctly detected by BCID2. In addition, the BCID2 detected three Staphylococcus epidermidis, one Staphylococcus lugdunensis, one Staphylococcus spp., and one Streptococcus spp. that were not confirmed by standard culture of the positive BC. In addition, five bacterial isolates recovered from five positive BC, including three that were polymicrobial, were missed by the BCID2 assay. These isolates corresponded to one Escherichia coli and two Citrobacter freundii isolates (genera not included in the BCID2 panel but normally detected as Enterobacterales), one Streptococcus anginosus isolate (normally detected as Streptococcus spp.), and one Enterococcus faecium isolate. For fungi, three yeasts that grew in culture and were targeted by the BCID2 panel were correctly identified, and one Candida albicans isolate was missed by the BCID2 assay.

Fifteen bacterial or fungal pathogens in 15 positive BC (10%, 15/152), corresponding to 8% (15/182) of the isolates grown from BC, were not targeted by the BCID2 panel. These organisms are listed in Table 3. They were isolated from monomicrobial cultures from BC with a time to positivity of >35 h. Seven of the 15 isolates not targeted by the BCID2 panel were species likely to be BC contaminants. Most of the remaining pathogens were Gramnegative and/or anaerobic bacteria. One isolate of Fusarium spp. not targeted by BCID2 was also found in one BC.

Detection of resistance markers targeted by the BCID2 panel. The results for the resistance genes detected by the BCID2 panel are presented in Table 4. Three CTX-M type extended-spectrum b-lactamase (ESBL)-producing E. coli, two Enterobacter cloacae isolates, and one Klebsiella pneumoniae isolate (all confirmed by AST) were also accurately detected by BCID2. The discrepant results corresponded to one ESBL-producing E. coli for which no target was detected with BCID2 and one sample for which Serratia marcescens and bla CTX-M were detected with BCID2 but for which wild-type S. marcescens and AmpC-hyperproducing Citrobacter freundii grew in culture. Additionally, two ESBL-producing E. cloacae isolates, for which BCID2 detected only the E. cloacae without the bla CTX-M gene target, were observed, resulting in the failure of the system to predict this phenotype. AST did not reveal an acquired carbapenem resistance phenotype, and no carbapenemase gene was detected with BCID2. A mecA/C signal was detected for 18 of the 152 samples tested with BCID2. All a PPA, positive percent agreement; NPA, negative percent agreement; OPA, overall percent agreement; k, Cohen's kappa; CI, confidence interval.

these signals were associated with S. epidermidis targets (including one with S. epidermidis and S. lugdunensis targets). All these 18 samples yielded positive cultures for Staphylococcus and 15 were concordant with the growth in culture of methicillin-resistant (MR) S. epidermidis. The remaining three samples yielded non-epidermidis staphylococci on culture, which were methicillin susceptible (MS) for two of these samples. The MS phenotype was confirmed by an immunochromatography test performed from colonies (Alere PBP2a SA culture colony test; Abbott, Scarborough, Maine, USA) [START_REF] Canver | Improved performance of a rapid immunochromatographic assay for the detection of PBP2a in non-Staphylococcus aureus staphylococcal species[END_REF].

Turnaround times of BCID2 testing and culture results. The median time to positivity of the BC was 13.9 h (interquartile range [IQR], 10.3 to 19.5 h). The median turnaround time (TAT; i.e., from bottle processing after flagging as positive to the first report of the results) for BCID2 testing was 3.5 h (IQR, 2.4 to 4.8 h). The median TAT for BCID2 testing was significantly shorter than the 25.7 h (IQR, 18.4 to 27.2 h) required for standard culture (P , 0.001) and the 27.4 h (IQR, 25.7 to 47.6 h) required to obtain AST results (P , 0.001). The instrument used for bacterial identification had no impact on the TAT, while AST results obtained with the Vitek2 instrument had a TAT longer than that obtained by disk diffusion (Table S2).

Detection of pathogen and resistance markers in spiked BC bottles. Isolates in spiked BC bottles included K. pneumoniae (n = 4), E. faecium (n = 2), Pseudomonas aeruginosa (n = 1), S. aureus (n = 1), and Acinetobacter baumannii (n = 1) harboring resistance genes, which included bla CTX-M (n = 2), bla NDM (n = 3), bla KPC (n = 1), bla VIM (n = 2), bla IMP (n = 1), bla OXA-48-like (n = 2), vanA (n = 1), vanB (n = 1), and mecA and MREJ (n = 1). The BCID2 panel identified correctly the nine isolates spiked in four BC bottles and the 14 resistance markers. The characteristics of the isolates and the spiked BC bottles are presented in Table S3.

DISCUSSION

One of the major challenges in BSI diagnosis is decreasing the time between sampling and bacterial identification [START_REF] Lamy | ESCMID Study Group for Bloodstream Infections, Endocarditis and Sepsis (ESGBIES)[END_REF][START_REF] Timsit | Bloodstream infections in critically ill patients: an expert statement[END_REF][START_REF] Alhazzani | Surviving Sepsis Campaign: guidelines on the management of critically ill adults with coronavirus disease 2019 (COVID-19)[END_REF]. Indeed, it generally takes at least 1 or 2 days to obtain identification and AST results following the obtainment of a positive BC. By shortening the time taken to obtain results, it should be possible to decrease the risk of patients being treated inappropriately or unnecessarily with broad-spectrum antimicrobial agents.

In this prospective multicenter study, parallel evaluations of standard culture and BCID2 were performed on 152 positive BC. We found (i) excellent concordance between BCID2 and a traditional workflow and (ii) a significant decrease in turnaround time.

Globally, BCID2 had an excellent overall percent agreement (OPA; 100%) relative to conventional culture for the pathogens present in the panel (Table 1). For all pathogens included in the panel, BCID2 had an overall positive percent agreement (PPA) of 97%, with six microorganisms (two C. freundii 1 and2). These pathogens have also been reported to remain undetected by BCID2 in other studies, mostly in polymicrobial cultures [START_REF] Berinson | Usefulness of BioFire FilmArray BCID2 for blood culture Evaluation of the FilmArray BCID2 Panel for BSI Microbiology Spectrum[END_REF][START_REF] Graff | Clinical impact of the expanded BioFire Blood Culture Identification 2 panel in a U.S. children's hospital[END_REF]. In our study, only two of the undetected bacteria were isolated from polymicrobial cultures, in which they were found with S. epidermidis detected by BCID2 (Table 2). Off-panel bacteria were detected by culture in 10% (15/152) of the samples. This suggested that the panel includes the vast majority of bacteria causing BSI. This proportion of off-panel microorganisms was lower than reported in previous studies evaluating the first version of the BCID panel [START_REF] Southern | Implementation and performance of the BioFire FilmArray Blood Culture Identification panel with antimicrobial treatment recommendations for bloodstream infections at a midwestern academic tertiary hospital[END_REF][START_REF] Zheng | Rapid identification of pathogens from pediatric blood cultures by use of the FilmArray Blood Culture Identification panel[END_REF]. Interestingly, these off-panel pathogens included three obligate anaerobic bacteria: Eggerthella lenta, Bacteroides uniformis, which is not detected by the Bacteroides fragilis target, and Clostridium perfringens. As Bacteroides and Clostridium account for most of the obligate anaerobes identified in BSI, adding pan-Bacteroides and pan-Clostridium targets to the BCID2 panel would facilitate the rapid identification of these genera, which can cause severe infections if not picked up sufficiently early [START_REF] Laupland | Population-based epidemiology and microbiology of community-onset bloodstream infections[END_REF][START_REF] Lafaurie | Utility of anaerobic bottles for the diagnosis of bloodstream infections[END_REF][START_REF] Morel | Non-C. difficile clostridioides bacteremia in intensive care patients, France[END_REF]. Furthermore, the shape of pathogen(s) on the Gram stain allows the microbiologist to suspect certain pathogens, in particular, fungi and Campylobacter, which are not detected by BCID2.The overall performance of BCID2 was consistent with that reported in other studies [START_REF] Berinson | Usefulness of BioFire FilmArray BCID2 for blood culture Evaluation of the FilmArray BCID2 Panel for BSI Microbiology Spectrum[END_REF][START_REF] Graff | Clinical impact of the expanded BioFire Blood Culture Identification 2 panel in a U.S. children's hospital[END_REF][START_REF] Cortazzo | Comparing BioFire Fil-mArray BCID2 and BCID panels for direct detection of bacterial pathogens and antimicrobial resistance genes from positive blood cultures[END_REF][START_REF] Holma | Rapid molecular detection of pathogenic microorganisms and antimicrobial resistance markers in blood cultures: evaluation and utility of the next-generation FilmArray Blood Culture Identification 2 panel[END_REF][START_REF] Sparks | Evaluation of the BioFire Blood Culture Identification 2 panel and impact on patient management and antimicrobial stewardship[END_REF]. Holma et al. performed a study on 80 positive clinical BC samples in which the PPA was 99% and the NPA was 100% for all samples [START_REF] Holma | Rapid molecular detection of pathogenic microorganisms and antimicrobial resistance markers in blood cultures: evaluation and utility of the next-generation FilmArray Blood Culture Identification 2 panel[END_REF]. The BCID2 assay can also detect pathogens in polymicrobial BC. In our study, 22 positive BC yielded polymicrobial cultures (22/152, 14%) containing two to four different microorganisms. We obtained completely concordant results between BCID2 and culture in 73% (16/ 22) of the BC containing multiple organisms (Table S1). However, other BCID studies have shown that the presence of multiple organisms favors discordant results between BCID and culture [START_REF] Southern | Implementation and performance of the BioFire FilmArray Blood Culture Identification panel with antimicrobial treatment recommendations for bloodstream infections at a midwestern academic tertiary hospital[END_REF][START_REF] Blaschke | Rapid identification of pathogens from positive blood cultures by multiplex polymerase chain reaction using the FilmArray system[END_REF][START_REF] Salimnia | Evaluation of the FilmArray Blood Culture Identification panel: results of a multicenter controlled trial[END_REF]. In addition, there are only a few alternative fully automated assays to the BCID2 panel that can provide broad-spectrum species identification and detect antimicrobial resistance markers in positive BC. Verigene System bloodstream infection tests (Nanosphere, Inc., Northbrook, IL, USA) and the ePlex (GenMark Diagnostics, Carlsbad, CA, USA) provide two or three separate panels for Gram-positive, Gram-negative, and yeast-related organisms and resistance gene targets. The ePlex has a sensitivity of 93 to 100% and a specificity of 97 to 100%, and the Verigene System has a sensitivity of 50 to 100% and a specificity of 99 to 100%, depending on the organism targeted [START_REF] Ward | Performance evaluation of the Verigene (Nanosphere) and FilmArray (Bio-Fire) molecular assays for identification of causative organisms in bacterial bloodstream infections[END_REF][START_REF] Huang | Evaluation of the ePlex blood culture identification panels for detection of pathogens in bloodstream infections[END_REF]. Good performance has also been reported for the Unyvero Blood Culture molecular assay (Curetis, Holzgerlingen, Germany), with a global sensitivity of 97% and a specificity of 99 to 100% with results in around 5 h [START_REF] Burrack-Lange | Multicenter assessment of the rapid Unyvero blood culture molecular assay[END_REF]. Recently, a test using a magnetic resonance-based technology (T2Candida and T2Bacteria panels; T2 Biosystem, Lexington, MA, USA) was developed that detects microbial cells directly within whole blood, skipping the BC incubation step. This new test showed a higher sensitivity than BC with a shorter TAT (only a few hours between sampling and results), but the T2Bacteria panel is limited to detection of six bacterial species [START_REF] Clancy | T2 magnetic resonance for the diagnosis of bloodstream infections: charting a path forward[END_REF][START_REF] Nguyen | Performance of the T2Bacteria panel for diagnosing bloodstream infections: a diagnostic accuracy study[END_REF].

The BCID2 panel can detect key genetic markers underlying cephalosporin and carbapenem resistance in Gram-negative bacteria, which is of great potential importance for antimicrobial drug stewardship. The detection of the bla CTX-M gene, the most prevalent ESBL-encoding gene worldwide, and major carbapenemase genes (bla KPC , bla NDM , bla OXA-48 , and bla VIM ) by BCID2 may decrease the time to optimal and effective treatment, particularly in institutions with high rates of resistance in Gram-negative bacteria. In our study, the BCID2 panel detected seven bla CTX-M genes, six of which were confirmed by antimicrobial drug susceptibility testing. However, three ESBL-producing isolates were not detected, including one ESBL-producing E. coli isolate for which the E. coli target was not detected and two E. cloacae strains for which the bacterial target was detected by BCID2 without detection of the bla CTX-M gene. These results can be explained either by a lack of detection of the bla CTX-M gene or by the production of a non-CTX-M enzyme. Indeed, the ESBL-encoding genes of E. cloacae are more diverse than those of E. coli or K. pneumoniae. In France, up to 40% of ESBL-encoding genes in E. cloacae isolates are non-CTX-M-encoding genes [START_REF] Robin | Inventory of extended-spectrum-b-lactamase-producing Enterobacteriaceae in France as assessed by a multicenter study[END_REF]. This rate is higher than that in E. coli or K. pneumoniae, in which 95% of ESBL are encoded by a bla CTX-M gene. These findings suggest that molecular tests may have a limited ability to predict ESBL phenotype when an E. cloacae target is detected. Unfortunately, no molecular analysis was performed to identify the ESBL-encoding genes in either of the E. cloacae isolates. Furthermore, no MR S. aureus was detected with either BCID2 or in culture, but given published BCID data, we would expect BCID2 to detect this resistance well [START_REF] Zheng | Rapid identification of pathogens from pediatric blood cultures by use of the FilmArray Blood Culture Identification panel[END_REF][START_REF] Salimnia | Evaluation of the FilmArray Blood Culture Identification panel: results of a multicenter controlled trial[END_REF]. BCID2 correctly identified all resistance markers in spiked BC, indicating that the assay could detect resistance genes in polymicrobial BC.

The median TAT for organism identification by BCID2 was significantly shorter than that for conventional culture methods, with identification results generally available 18.5 h earlier for the BCID2 assay. The difference between the median TAT (3.5 h) of the BCID2 results compared to the duration of the analysis (;1 h) corresponded to the time to perform the BCID2 in parallel to the standard process, which can be impacted by the number of positive blood cultures daily. This result is similar to those of previous studies on the first version of the BCID panel and for other fully automated assays [START_REF] Ward | Performance evaluation of the Verigene (Nanosphere) and FilmArray (Bio-Fire) molecular assays for identification of causative organisms in bacterial bloodstream infections[END_REF][START_REF] Huang | Evaluation of the ePlex blood culture identification panels for detection of pathogens in bloodstream infections[END_REF][START_REF] Fiori | Optimized use of the MALDI BioTyper system and the FilmArray BCID panel for direct identification of microbial pathogens from positive blood cultures[END_REF]. Alternative methods for rapid identification using a bacterial pellet from positive BC or short-term bacterial incubation on a solid medium have been reported, but these require a reorganization of laboratory workflow to be routinely performed [START_REF] Dubourg | Rapid phenotypic methods to improve the diagnosis of bacterial bloodstream infections: meeting the challenge to reduce the time to result[END_REF]. Rapid identification can affect antimicrobial drug prescription, particularly in conjunction with real-time antibiotic stewardship recommendations [START_REF] Banerjee | Randomized trial of rapid multiplex polymerase chain reaction-based blood culture identification and susceptibility testing[END_REF], as demonstrated by the decrease in median time to optimal therapy in both pediatric and adult populations [START_REF] Messacar | Potential clinical impact of the film array meningitis encephalitis panel in children with suspected central nervous system infections[END_REF][START_REF] Verroken | The impact of a rapid molecular identification test on positive blood cultures from critically ill with bacteremia: a pre-post intervention study[END_REF].

The principal limitation of this study was the small number of samples positive for some of the pathogens included in the BCID2 panel. In particular, A. baumannii, Haemophilus influenzae, Streptococcus pneumoniae, and Neisseria meningitidis were not detected, and the small number of cases of fungemia (n = 5) in our study made it impossible to draw firm conclusions about the performance of the BCID2 panel for detecting fungal pathogens. Further studies are needed to evaluate the performance of the BCID2 panel for those pathogens. We were also unable to confirm discrepant results due to the absence of conserved BC materials for supplementary tests. Another limitation of this study was the absence of cultured organisms displaying carbapenem or glycopeptide resistance in positive BC from patients. Other studies, with a higher frequency of resistant bacteria, will be required for clinical evaluations of these specific targets. However, simulated BC showed that BCID2 detected resistant markers, including variants of carbapenemase genes. Finally, the clinical impact of the BCID2 panel on antibiotic treatment was not evaluated here. A few studies have suggested that it may be possible to use BCID2 for antibiotic stewardship purposes, but further studies are required to address this point [START_REF] Graff | Clinical impact of the expanded BioFire Blood Culture Identification 2 panel in a U.S. children's hospital[END_REF][START_REF] Holma | Rapid molecular detection of pathogenic microorganisms and antimicrobial resistance markers in blood cultures: evaluation and utility of the next-generation FilmArray Blood Culture Identification 2 panel[END_REF][START_REF] Sparks | Evaluation of the BioFire Blood Culture Identification 2 panel and impact on patient management and antimicrobial stewardship[END_REF][START_REF] Claeys | Day at the races: comparing BioFire FilmArray blood culture ID panels with Verigene blood culture panel in Gram-negative bloodstream infections using DOOR-MAT analysis[END_REF].

MATERIALS AND METHODS

Population and specimen collection. This study was conducted from March to October 2020 at four tertiary care hospitals distributed in three distinct geographic regions of France (Saint-Louis-Lariboisière-Fernand Widal Hospital Group, AP-HP, Paris; Paris Saint-Joseph Hospital Group, Paris; Angers University Hospital, Angers; and Montpellier University Hospital, Montpellier). BC meeting the following criteria were excluded from the study: previous BC obtained within the last 7 days and included in the study, or no AST performed for a bacterial isolate from a positive BC or invalid BCID2 results (n = 3, invalidity rate of 1.9% [3/155]). BC were included on weekdays during the open hours of each laboratory, at the discretion of the clinical microbiologists responsible for the study at each center. Conventional culture and BCID2 were performed in parallel, and the BCID2 result was not communicated to the prescribing physician.

Standard microbiology methods. FA plus and FN plus BC bottles were incubated in a BacT/Alert VIRTUO system (bioMérieux, France) at 35°C. For bottles considered positive, the contents were fixed for microscopy and Gram staining and were then cultured for isolation of the bacteria and fungi responsible for BSI, in accordance with the current French Guidelines for Medical Microbiology (REMIC v6 2018) [START_REF] Emic | référentiel en microbiologie médicale[END_REF]. One drop (50 mL) from each BC bottle was used to inoculate each of two sheep blood agar plates and one chocolate agar plate (bioMérieux). These plates were incubated at 35°C in ambient air, under an anaerobic atmosphere, and under an atmosphere containing 5% CO 2 , respectively. If Gram-negative rods were detected by Gram staining, a drop of the BC was used to inoculate a Drigalski agar plate. If a polymicrobial BSI was identified by Gram staining, then colistin-nalidixic acid sheep blood and Drigalski agar plates were added. If a fungal element was detected by Gram staining, a Sabouraud agar plate was added. Bacteria were identified with the Vitek2 system (bioMérieux, Marcy l'Etoile, France) and/or MALDI-TOF MS (Microflex LT [Bruker Daltonics, Bremen, Germany] or Vitek MS [bioMérieux, Marcy l'Etoile, France]), depending on the hospital laboratory. AST was performed by the disk-diffusion method, or with the automated Vitek2 system (bioMérieux, France), depending on the laboratory procedure used in the hospital concerned and/or the species identified, and the results were interpreted according to EUCAST recommendations. Detection of ESBL production was based on AST results and additional synergy tests if necessary (Mastdiscs D68C; Mast Group, Bootle, United Kingdom).

FilmArray BCID2 panel testing. The BCID2 test is CE marked for in vitro diagnosis and is FDA cleared. BCID2 testing was performed according to the manufacturer's instructions. Briefly, the contents of the BC bottle were homogenized, and 200 mL was withdrawn and added directly to a dilution buffer, which was then injected into the FA-PP cartridge. Nucleic acid extraction, amplification, detection, and analysis were performed automatically within the cartridge, with a total run time of about 1 h. BCID2 tests were run on the FilmArray V2.0 system. Bacteria, fungi, and resistance genes were analyzed qualitatively with classification as "detected" or "not detected." The panel includes [START_REF] Huang | Evaluation of the ePlex blood culture identification panels for detection of pathogens in bloodstream infections[END_REF]

  [Enterobacterales target]), one E. coli ([Enterobacterales and E. coli targets], one S. anginosus [Streptococcus spp. target], one E. faecium [E. faecium target], and one C. albicans [C. albicans target]) detected by culture alone (Tables

TABLE 1

 1 Concordance between the BCID2 panel and culture for each microorganism identification target a

		No. of samples with BCID2/
		culture result of:			% PPA	% NPA	% OPA
	Organism	+/+	+/2	2/+	2/2	(95% CI)	(95% CI)	(95% CI)	k (95% CI)
	Acinetobacter calcoaceticus-baumannii complex	0	0	0	152		100 (98-100)	(98-100)
	Bacteroides fragilis	5	0	0	147	100 (48-100)	100 (98-100)	(98-100)	1
	Enterobacterales	64	0	3	85	96 (87-99)	100 (96-100)	(95-100)	0.96 (0.92-1)
	Enterobacter cloacae complex	13	0	0	139	100 (75-100)	100 (98-100)	(98-100)	1
	Escherichia coli	31	0	1	120	97 (84-99)	100 (97-100)	(97-100)	0.98 (0.94-1)
	Klebsiella aerogenes	3	0	0	149	100 (29-100)	100 (98-100)	(98-100)	1
	Klebsiella oxytoca	0	0	0	152		100 (98-100)	(98-100)
	Klebsiella pneumoniae group	8	0	0	144	100 (63-100)	100 (98-100)	(98-100)	1
	Proteus spp.	3	0	0	149	100 (29-100)	100 (98-100)	(98-100)	1
	Salmonella	1	0	0	151	100 (0-100)	100 (98-100)	(98-100)	1
	Serratia marcescens	5	0	0	147	100 (48-100)	100 (98-100)	(98-100)	1
	Haemophilus influenzae	0	0	0	152		100 (98-100)	(98-100)
	Neisseria meningitidis	0	0	0	152		100 (98-100)	(98-100)
	Pseudomonas aeruginosa	13	0	0	139	100 (75-100)	100 (98-100)	(98-100)	1
	Stenotrophomonas maltophilia	2	0	0	150	100 (16-100)	100 (98-100)	(98-100)	1
	Enterococcus faecalis	5	0	0	147	100 (48-99)	100 (98-100)	(98-100)	1
	Enterococcus faecium	13	0	1	138	93 (66-99)	100 (98-100)	(97-100)	0.96 (0.88-0.98)
	Listeria monocytogenes	1	0	0	151	100 (0-100)	100 (98-100)	(98-100)	1
	Staphylococcus spp.	32	1	0	119	100 (89-100)	99 (95-100)	(97-100)	0.98 (0.94-0.98)
	Staphylococcus aureus	5	0	0	147	100 (48-100)	100 (98-100)	(98-100)	1
	Staphylococcus epidermidis	19	3	0	130	100 (82-100)	98 (94-100)	(94-100)	0.92 (0.82-0.98)
	Staphylococcus lugdunensis	0	1	0	151		99 (96-100)	(97-100)	0
	Streptococcus spp.	14	1	1	136	93 (68-100)	99 (96-100)	(96-100)	0.93 (0.83-1)
	Streptococcus agalactiae	1	0	0	151	100 (0-100)	100 (98-100)	(98-100)	1
	Streptococcus pneumoniae	0	0	0	152	100 (40-100)	100 (98-100)	(98-100)
	Streptococcus pyogenes	4	0	0	148		100 (98-100)	(98-100)	1
	Candida albicans	2	0	1	149	67 (9-99)	100 (98-100)	(97-100)	0.80 (0.41-1)
	Candida auris	0	0	0	152		100 (98-100)	(98-100)
	Candida glabrata	0	0	0	152		100 (98-100)	(98-100)
	Candida krusei	1	0	0	151	100 (0-100)	100 (98-100)	(98-100)	1
	Candida parapsilosis	0	0	0	152		100 (98-100)	(98-100)
	Candida tropicalis	0	0	0	152		100 (98-100)	(98-100)
	Cryptococcus neoformans	0	0	0	152		100 (98-100)	(98-100)

TABLE 3

 3 Cultured organisms not targeted by the BCID2 panel

	Sample no.	Bottle type	Time to positivity of BC	Culture	Isolate identification
	7	Aerobic	36 h	Monomicrobial	Achromobacter spp.
	13	Aerobic	43 h	Monomicrobial	Capnocytophaga sputigena
	14	Aerobic	75 h	Monomicrobial	Paenibacillus spp.
	32	Aerobic	46 h	Monomicrobial	Sphingomonas paucimobilis
	70	Aerobic	54 h	Monomicrobial	Corynebacterium mucifaciens
	73	Anaerobic	38 h	Monomicrobial	Gram-positive rod nonidentified by MALDI-TOF MS
	76	Anaerobic	119 h	Monomicrobial	Cutibacterium acnes
	82	Anaerobic	53 h	Monomicrobial	Capnocytophaga sputigena
	89	Anaerobic	48 h	Monomicrobial	Clostridium perfringens
	101	Anaerobic	48 h	Monomicrobial	Bacteroides uniformis
	124	Aerobic	74 h	Monomicrobial	Moraxella osloensis
	140	Aerobic	52 h	Monomicrobial	Campylobacter jejuni
	157	Aerobic	52 h	Monomicrobial	Facklamia languida
	159	Anaerobic	41 h	Monomicrobial	Eggerthella lenta
	165	Aerobic	90 h	Monomicrobial	Fusarium spp.

TABLE 2

 2 Discrepancy of pathogen identification between the BCID2 panel and culture a GNR, Gram-negative rod; GPCC, Gram-positive cocci in clusters; GPCP, Gram-positive cocci in pairs or chains. Microorganisms found by only one method are indicated in bold.

	Sample	Bottle	Gram-staining
	no.	type	result	Culture result	BCID2 result
	17	Aerobic	GNR	C. freundii	None
	21	Aerobic	GPCC, GNR	S. epidermidis, C. freundii	Staphylococcus spp., S. epidermidis
	25	Aerobic	GPCC, GPCP	E. faecalis, S. aureus	E. faecalis, Staphylococcus spp., S. epidermidis, S. aureus
	34	Anaerobic	GNR	S. marcescens	Enterobacterales, S. marcescens, Staphylococcus spp.
	58	Aerobic	GPCC, GPCP	E. faecium, S. epidermidis	E. faecium, Staphylococcus spp., S. epidermidis,
					Streptococcus spp.
	75	Aerobic	GNR	E. coli, P. mirabilis, M. morganii, S. anginosus	Enterobacterales, E. coli, Proteus spp.
	77	Aerobic	GPCC, GPCP	E. faecium, S. epidermidis	Staphylococcus spp., S. epidermidis, S. lugdunensis
	100	Anaerobic	GNR	E. coli	None
	108	Aerobic	GPCC, GNR	P. aeruginosa, S. haemolyticus	P. aeruginosa, Staphylococcus spp., S. epidermidis
	144	Anaerobic	GPCC	S. hominis	Staphylococcus spp., S. epidermidis
	166	Aerobic	Yeast	C. albicans	None

a

TABLE 4

 4 Detection results for resistance markers targeted by the BCID2 panel

		Resistance marker detected by BCID2 a
	Phenotype and organism	bla CTX-M	mecA/C	mecA/C and MREJ	No detection	No detection of relevant organism by BCID2 b
	Presence of ESBL phenotype c	6			2	1
	E. coli	3			0	1
	E. cloacae	2			2	0
	K. pneumoniae	1			0	0
	Absence of ESBL phenotype c	1			5 5	2
	Methicillin-resistant phenotype d		16	0	0	6
	S. epidermidis		15		0	0
	Other Staphylococcus e		1 f		0	6
	Methicillin-susceptible phenotype d		2	0	8	0
	S. aureus			0	5	0
	S. epidermidis		0		3	0
	Other Staphylococcus e		2 g		0	0

a Only if a bacterium allowing the report of the resistance gene was detected. No isolate harbored a phenotype compatible with the presence of carbapenemase genes vanA, vanB, or mcr1.

1. b

No detection of a bacterium allowing the report of the resistance gene of interest. c Based on antimicrobial drug susceptibility testing on Enterobacterales isolates. d Based on antimicrobial drug susceptibility testing on staphylococcal isolates. e Species other than S. aureus, S. epidermidis, and S. lugdunensis. f mecA/C and S. epidermidis detected by BCID2 with a positive culture of methicillin-resistant Staphylococcus haemolyticus. g mecA/C and S. epidermidis detected by BCID2 with a positive culture of methicillin-susceptible Staphylococcus hominis.

  bacterial targets (Enterococcus faecalis, Enterococcus faecium, Listeria monocytogenes, Staphylococcus spp., Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus lugdunensis, Streptococcus spp., Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Acinetobacter calcoaceticus-baumannii complex, Bacteroides fragilis, Haemophilus influenzae, Neisseria meningitidis, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Enterobacterales, Enterobacter cloacae complex, Escherichia coli, Klebsiella aerogenes, Klebsiella pneumoniae group, Klebsiella oxytoca, Proteus spp., Salmonella spp., and Serratia marcescens), seven fungal targets (Candida albicans, Candida auris, Candida glabrata, Candida krusei, Candida Evaluation of the FilmArray BCID2 Panel for BSI Microbiology Spectrum January/February 2023 Volume 11 Issue 1 10.1128/spectrum.02547-22 7 Downloaded from https://journals.asm.org/journal/spectrum on 10 April 2023 by 194.57.207.221.

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ACKNOWLEDGMENTS

We thank the staff at the microbiology laboratories participating in the study. This study was supported by bioMérieux (Marcy L'Etoile, France), which provided consumables. The funders had no role in study design, data collection and interpretation, or manuscript preparation.

F.C. received lecture fees from bioMérieux. F.D. received lecture fees from bioMérieux and Sedana and grants from European Society of Intensive Care Medicine, Société Française d'Anesthésie Reanimation, and the French Ministry of Research. A.A. received lecture fees and travel grants from Gilead Sciences and Pfizer and received grants from Agence Nationale de la Recherche. All other authors report no conflicts of interest.

bioMérieux (Marcy L'Etoile, France), which provided consumables. The funders had no role in study design, data collection and interpretation, or manuscript preparation. F.C. received lecture fees from bioMérieux. F.D. received lecture fees from bioMérieux, Sedana and grants from European Society of Intensive Care medicine, Société Française d'Anesthésie Reanimation and from the French ministry of research. A.A. received lecture fees and travel grants from Gilead science and Pfizer. He received grants from Agence Nationale de la Recherche. Other authors report no conflicts of interest.

parapsilosis, Candida tropicalis, Cryptococcus neoformans/gattii), and 10 antimicrobial resistance genes (ESBL bla CTX- M , carbapenemases bla KPC , bla NDM , bla IMP , bla OXA-48-like , and bla VIM , vancomycin resistance vanA/B, methicillin resistance mecA/C and MREJ, and colistin resistance mcr-1).

Spiking of blood culture bottles. The BC bottles were spiked following the EUCAST RAST QC protocol (www.eucast.org). In brief, a bacterial suspension of 100 to 500 CFU was suspended in 5 mL of sterile sheep blood and inoculated in an aerobic blood culture bottle. The bottles were incubated in the Virtuo instrument until the instrument signaled positive growth, and the specimen underwent subsequently analysis using the BCID2 test as described above. The clinical isolates used to spike BC were characterized by whole-genome sequencing.

Statistical analysis. Positive percent agreement (PPA), negative percent agreement (NPA), overall percent agreement (OPA), Cohen's k coefficient, and two-tailed 95% confidence intervals were calculated by comparing the results for conventional culture (reference method) with those for BCID2 exclusively for the bacterial and fungal pathogens present in the BCID2 panel. A Wilcoxon signed-rank test was used to compare the turnaround time (TAT) between the two methods.

Ethics statement. This study was performed in accordance with the Declaration of Helsinki. The samples used in this study were collected during routine patient management without the need for additional sampling, and the BCID2 result was not communicated to the prescribing physician. As such, this study did not require ethics committee approval or informed consent from the patients.

SUPPLEMENTAL MATERIAL

Supplemental material is available online only. SUPPLEMENTAL FILE 1, PDF file, 0.1 MB.