This paper describes an untargeted NMR metabolomics study to identify potential intracellular donor-dependent and donor-independent metabolic markers of proliferation and osteogenic differentiation of human adipose mesenchymal stem cells (hAMSCs). The hAMSCs of two donors with distinct proliferating/osteogenic characteristics were fully characterized regarding their polar endometabolome during proliferation and osteogenesis. An 18-metabolites signature  was suggested to be potentially descriptive of cell proliferation, independently of the donor. In addition, a set of 11 metabolites was proposed to compose a possible donor-independent signature of osteogenesis, mostly involving changes in taurine, glutathione, methylguanidine, adenosine, inosine, uridine, and creatine/phosphocreatine, choline/phosphocholine and ethanolamine/phosphocholine ratios. The proposed signatures were validated for a third donor, although they require further validation in a larger donor cohort. We believe that this proof of concept paves the way to exploit metabolic markers to monitor  cell proliferation and the osteogenic ability of different donors. Mesenchymal stem cells (MSCs) have become the basis of numerous bioengineering studies due to their ability to differentiate into multiple cell lineages  and secrete bioactive factors essential for tissue repair ,2,3. HowInter-donor variability is perhaps one of the major contributions to MSCs heterogeneity, potentially reflecting differences in donor age , gender To the best of our knowledge, only a few metabolomic studies of MSCs heterogeneity have been carried out ,28,29,30In this work, untargeted NMR metabolomics was carried out in hAMSCs, for the first time to our knowledge, to search for osteogenesis-specific metabolic markers, which may be considered independent of donor origin and of underlying cell proliferation metabolism. To achieve this, the detailed polar endometabolome adaptations of hAMSCs harvested from two randomly chosen donors, found to differ in proliferating and differentiation extensions, were measured at several timepoints throughout 21 days, under osteogenic and basal (control) conditions. The detailed and holistic nature of NMR metabolomics ,36,37 enhAMSCs harvested from two randomly chosen healthy donors (1 and 2) were considered in this study. Adipose tissue from donor 1 was collected through abdominoplasty, under a cooperation agreement between the COMPASS Research Group from CICECO-University of Aveiro and Hospital da Luz, Aveiro, Portugal, upon approval of the hospital Ethics Committee. The patient’s informed consent was obtained under anonymous conditions . Samples were processed within 24 h after surgery, and hAMSCs isolation was carried out as previously described . The hAM6 cells/flask . After reaching nearly 100% confluence under the above-mentioned conditions, the culture medium was exchanged and supplemented with osteogenic differentiation factors, namely, 10 mM β-glycerophosphate , 50 μg/mL L-ascorbic acid , and 10 nM dexamethasone . For both donors 1 and 2, parallel control experiments were carried out . The medium was exchanged twice a week for 21 days. On days 0, 1, 4, 7, 14, and 21, cells were trypsinized  and rinsed twice in phosphate-buffered saline (PBS) solution, to avoid medium contamination. For donor 3, day 4 samples could not be collected. At least 1 × 106 cells per pellet were used for metabolomics. The above experiments were carried out in 48-well plates to assess cellular proliferation and osteodifferentiation. At each timepoint, plated cells were scraped, rinsed twice with PBS, lysed by osmotic/thermal shock and stored at −80 °C. Media samples (>3 mL) were also collected.Prior to osteoinduction, hAMSCs were harvested and seeded at a density of 0.5 × 10escribed ) and coln = 6 samples/timepoint) were centrifuged , and supernatants used for double-stranded DNA (dsDNA) quantification . Dye fluorescence was assessed at emission and excitation wavelengths 528 ± 10 nm and 485 ± 10 nm, respectively . dsDNA concentrations were calculated using a calibration curve in the 0.0−2.0 μg/mL concentration range.Cell lysates were thawed at room temperature (RT), exposed to ultrasounds (10 min) and kept at −20 °C overnight. This was repeated twice to improve DNA extraction. Cell lysates (n = 6 samples per timepoint). The mixture was incubated at 37 °C for approximately 1 h and then 80 μL of 3 M NaOH (in MiliQ water) was added. pNP absorbance at 450 nm was measured  and ALP activity was calculated using a pNP calibration curve (0.0−0.5 mM). For calcium quantification and determination of osteocalcin  was assntiation ) protocontiation ,37. ALP g, 20 min, RT), and polar and lipophilic phases were separated. Polar extracts were dried under vacuum and stored at −80 °C until analysis . Importantly, the above conditions constituted an improved protocol in relation to that used for donor 3 cells, reported earlier [2O  containing 0.1 mM 3-(trimethylsilyl)-propionic-2,2,3,3-d4 acid  for chemical shift referencing. After vortexing, 550 μL were transferred to 5 mm NMR tubes. NMR spectra were recorded on a Bruker Avance III spectrometer operating at 500.13 MHz for 1H (298 K). Standard unidimensional spectra were acquired with the noesypr1d pulse sequence under the following conditions: 7002.801 Hz spectral width, 32 k data points, 2.3 s acquisition time, 4 s relaxation delay (d1) and 512 scans. Each free induction decay was zero-filled to 64 k points, multiplied by a 0.3 Hz exponential line-broadening function and Fourier transformed. Spectra were manually phased and baseline corrected. As described before [1H-1H total correlation (TOCSY) and 1H-13C heteronuclear single quantum correlation (HSQC) spectra, spiking experiments, the literature and spectral databases, such as the Bruker BBIOREFCODE AMIX database, the human metabolome database (HMDB) and Chenomx NMR Suite . Statistical total spectroscopy (STOCSY) [Intracellular metabolites were extracted using a methanol-chloroform-water method described elsewhere . Briefly earlier ,37. Polad before ,37, peak(STOCSY)  was used1H NMR spectra using SIMCA-P 11.5 , upon exclusion of water (5.11−4.69 ppm) and TSP (0.30−0.00 ppm) spectral regions from the matrices. Due to possible contamination, methanol (3.38−3.34 ppm) and ethanol (3.68−3.63 and 1.21−1.17 ppm) regions were also excluded. Spectra were aligned using recursive segment-wise peak alignment [Multivariate analysis was applied to the full resolution lignment , to minilignment . Multivalignment ,37. hAMSCs cultured in basal medium (controls) presented overall similar dsDNA patterns (indicative of cell proliferation) to those observed in osteogenic media until day 14 , except 1H NMR spectra of the polar extracts of hAMSCs from donor 1  +), uridine diphospho-N-acetylglucosamine (UDP-GlcNAc), ethanolamine (Etn), 1-methylnicotinamide (1-MNA) and the unassigned resonance at δ 3.48. We hypothesize that the trajectories followed by these 21 metabolites , with amino acid levels at day 21 approaching those at day 0 (blank rectangles in the right column in δ 3.48 (found to correlate to membrane precursors and adenosine through STOCSY).In order to propose possible osteogenic-specific donor-independent metabolites , selected metabolite trajectories were identified when meeting the following two conditions simultaneously: (i) specificity for osteogenic differentiation , and (ii) apparent donor-independence . We propose that 11 metabolites may serve as candidates for osteogenic-specific and donor-independent markers : (1) taum-inositol feature in both P1 and P2 but lack the symbol †  the 6 robust proliferation markers: alanine, aspartate, proline, tyrosine, ADP and ATP; (ii) the 11 proposed osteogenic-specific metabolites, found in section O1/O2 and in addition to the Cr/PCr, Cho/PCho, Etn/PCho ratios; and (iii) the 5 metabolites found to be osteogenic-specific at least for one of donors 1 and 2: BCAAs, glycine and phenylalanine) were sought for an independently chosen donor 3, in order to attempt some marker validation . This work has compared the detailed metabolic profiling of two randomly chosen donors, during cell proliferation alone and osteogenic differentiation. Donor 1 was found to be more osteogenic  and slightly more proliferating , compared to donor 2. As only two donors were considered, discussion of the reasons for these distinct characteristics is out of scope here, however, the metabolic information found may be correlated to the different donor biochemical parameters.+ levels, increased inosine and decreased 1-MNA), and protein glycosylation (decreased UDP-GlcNAc) [Cell proliferation in donor 1 is accompanied by a well-defined metabolic trajectory, which is not as clear in donor 2. However, amino acids generally tend to accumulate before day 4 in both donors (perhaps in preparation for use for protein synthesis) ,61, is g-GlcNAc) ,62 also -GlcNAc)  to vary Upon osteogenic cues, the most osteogenic donor (donor 1) exhibits a clearer metabolic switch from day 7 to day 14, the overall process being accompanied by generally more persisting changes in several amino acids. This indicated that a higher osteogenic differentiation extension is clearly reflected in amino acid metabolism, at least including their involvement in the biosynthesis of osteogenic protein markers, but also probably as substrates to feed into the TCA cycle. More extensive osteogenesis also leads to lower levels of creatine  and of the cell membrane precursors Cho and Etn. Indeed, the increases in Cr/PCr, Cho/PCho and Etn/PCho ratios identified before as markers of osteogenesis are not as pronounced for the more extensively osteogenic donor. In relation to Cho/PCho and Etn/PCho ratios, it is unclear at this stage if their diminished levels result from differentiation and/or proliferation mechanisms, as the more osteogenic donor 1 also shows slightly enhanced cell proliferation. Furthermore, choline levels are also known to depend on intra/extracellular cross-talk mechanisms , which mIn spite of the above-discussed metabolic differences between the two donors, some osteogenic-specific metabolite changes could be advanced as donor-independent characteristics of osteogenic differentiation. These comprise increased GSH (oxidative protection), increased Cho and Etn and corresponding ratios to PCho , This paper describes, for the first time to our knowledge, an untargeted NMR metabolomics study to measure hAMSCs polar endometabolome as a function of proliferation and osteogenic differentiation, to identify donor-dependent and donor-independent metabolic features of each process, firstly considering two donors. A set of 18 metabolites could be identified as a donor-independent marker of cell proliferation, including particularly robust variations in alanine, aspartate, proline, tyrosine, ATP and ADP, which are not affected by osteogenic differentiation. Regarding osteogenesis, a set of 11 metabolites were proposed as composing an osteogenic-specific donor-independent signature. This signature includes altered levels of taurine, PCr (and Cr/PCr ratios), GSH, methylguanidine, adenosine, inosine, uridine, Cho, Etn, PCho (and Cho/PCho and Etn/PCho ratios) and a still unassigned resonance at 3.48 ppm. In particular, some metabolites previously suggested to relate to osteogenesis were now linked preferably to the underlying cell proliferation process. Although the fact that only two donors were considered in detail is a clear limitation of this work, their distinct proliferation and osteogenic abilities enabled putative correlation between these properties and the metabolic adaptations to be advanced. Interestingly, many of the above signatures could be confirmed for a third donor, although the results reported here necessarily require validation in a larger cohort of donors, then also enabling a more robust correlation between the metabolic profile and (i) biochemical markers of proliferation and osteogenesis and (ii) donor characteristics. Future developments may entail the use of a validated metabolic signature of successful osteogenic differentiation to help monitor and guide the process into a pure lineage outcome. We further propose that such metabolic features may also potentially be used to predict the osteogenic ability of different donors/tissues at an early stage of the process, and even help evaluate in vivo post-implantation MSC performance.


RNA sequencing analysis demonstrated that G-CSF enhanced the immunosuppressive function of MDSCs by upregulating gamma-glutamyltransferase (GGT) 1. Moreover, in the EL4 lymphoma-bearing neutropenic mouse model, administration of recombinant G-CSF increased the number of MDSCs and attenuated the anti-cancer effect of chemotherapy. We showed that the combination of GGsTop, a GGT inhibitor, could prevent G-CSF-induced tumor growth, without affecting the promotion of myelopoiesis by G-CSF. These results suggest that targeting GGT1 can mitigate G-CSF-induced enhanced immunosuppressive functions of MDSCs and can eliminate the tumor-promoting effect of G-CSF. Furthermore, GGsTop could be an attractive combination agent during G-CSF treatment for FN in patients with cancer.Myeloid-derived suppressor cells (MDSCs) are major immunosuppressive cells that accumulate in tumor-bearing hosts. Since MDSCs suppress anti-tumor immunity and promote tumor progression, they are promising targets for cancer immunotherapy. Granulocyte colony-stimulating factor (G-CSF) is an agent used for the treatment of chemotherapy-induced febrile neutropenia (FN) in patients with cancer. However, several reports have revealed that G-CSF plays crucial immune-related adverse roles in tumor progression through MDSCs. In this study, we showed that MDSCs differentiated in the presence of G-CSF  MDSCs play important roles in the suppression of anti-tumor immunity, resulting in the exacerbation of cancer . Phenotyhenotype . M-MDSCshenotype . HoweverFebrile neutropenia (FN) is a deadly complication associated with cancer chemotherapy, usually indicating infection and sepsis, and commonly occurs following the initial cycles of myelosuppressive therapy . PreventFemale C57BL/6J mice were purchased from Japan SLC . All mice were bred and maintained under specific pathogen-free conditions and used for experiments at 6–8 weeks of age. All experimental procedures in this study were performed in accordance with the institutional guidelines for animal experiments at the Osaka University, Japan.The EL4 cell lines were purchased from the American Type Culture Collection (ATCC) and maintained in RPMI-1640 medium  supplemented with 10% fetal bovine serum  and 1% Antibiotic-Antimycotic Mixed Stock Solution (100 ×) . Cells were cultured according to the ATCC guidelines and used within 1 month of thawing from an early passage  lots.in vitro differentiation of bone marrow (BM) cells into MDSCs was performed as described previously  supplemented with 2% FBS (2% FBS/PBS). The cell suspension was first blocked with TruStain fcX (anti-mouse CD16/32) antibody (BioLegend) for 5 min, and then stained with the following antibodies (Abs) for 15 min at 4°C: APC anti-mouse CD11b, Pacific Blue anti-mouse Gr-1, PE anti-mouse F4/80, FITC anti-mouse CD11c, APC-Cy7 anti-mouse Ly-6C, FITC anti-mouse Ly-6G, Pacific Blue anti-mouse CD4, and FITC anti-mouse CD8α (BioLegend). Next, the cells were washed and resuspended in 2% FBS/PBS. Shortly before performing measurements, a 7-amino actinomycin D viability staining solution (BioLegend) was added to each sample to stain dead cells. Flow cytometry analysis was performed on a BD FACSCanto II flow cytometer (BD Biosciences), and results were analyzed using the FlowJo software .+ T cells or CD8+ T cells were isolated from the spleens of C57BL/6J mice using the MojoSort magnetic cell separation system as previously described (+ T cells or CD8+ T cells were incubated with in vitro differentiated MDSCs at different ratios in a 96-well plate cultured with anti-mouse CD3ɛ Ab/anti-mouse CD28 Ab (BioLegend). After 3 days of incubation at 37°C in 5% CO2, the proliferation of CD4+ and CD8+ T cells determined by the eFluor 670 fluorescence intensity was analyzed using flow cytometry.CD4escribed  and then+Ly-6Cint) and M-MDSCs (Ly-6G−Ly-6Chi) were purified from in vitro-differentiated MDSCs cultured with or without the addition of G-CSF by fluorescence-activated cell sorting . Total RNA was extracted from the in vitro differentiated MDSCs or sorted MDSC subsets and used to synthesize cDNA using the QuantiTect reverse transcription kit  following the manufacturer’s instructions. qRT-PCR was performed using SYBR Premix Ex Taq  on a CFX96 touch real-time PCR detection system (Bio-Rad). The specific primer sequences are listed in Gapdh) was used as a reference gene, and relative expressions of other genes were calculated using the 2−ΔΔCt method.PMN-MDSCs  according to the manufacturer’s protocol. RNA libraries were prepared for sequencing using a TruSeq stranded mRNA sample prep kit  according to the manufacturer’s instructions. Whole transcriptome sequencing was performed on RNA samples using the Illumina HiSeq 2500 platform in a 75 bp single-end mode. Volcano plot representation and pathway enrichment analysis of the differentially expressed genes were performed using BioJupies tools with adjusted hange >2 . Raw dat6 cells) were homogenized in 200 µL of GGT assay buffer and centrifuged at 13,000 × g for 10 min following which the supernatant was collected. After reacting with the GGT substrate at 37°C for 40 min, fluorescence (365/460 nm) was measured using a microplate reader SpectraMax iD5 (Molecular Devices). GGT activity was calculated according to the protocol of the GGT Activity Colorimetric Assay Kit .MDSCs  according to the manufacturer’s protocol. Briefly, a glutamate standard or MDSC culture medium was added to a 96-well microplate; the working solution was then added to each well. After incubating the microplate at 37°C for 30 min, the absorbance was measured at 450 nm using SpectraMax iD5 (Molecular Devices). The glutamate concentration in each sample was calculated using a standard curve.The 5 MDSCs were treated with 2′, 7′dichlorofluorescin diacetate (DCFH-DA) working solution and incubated for 30 min. Subsequently, the MDSCs were washed twice with Hanks’ Balanced Salt Solution, and ROS production was measured using a BD FACSCanto II flow cytometer (BD Biosciences).Intracellular ROS was assayed using a ROS Assay Kit-Highly Sensitive DCFH-DA (Dojindo) according to the manufacturer’s protocol. Briefly, 2 × 105 cells/mouse) were injected subcutaneously into the lower right flank of C57BL/6J mice. Seven days after inoculation with EL4 cells, mice were intraperitoneally administered a single dose of CPA  to create a neutropenic mouse model as described previously (3) = 0.5 × (Length × Width2).EL4 lymphoma cells  using GraphPad Prism 7.0 (GraphPad Software). Statistical significance was set at p < 0.05.Significant differences were assessed using Student’s in vitro MDSCs). Freshly isolated BM cells were cultured for 4 days in a medium supplemented with or without G-CSF and GM-CSF to examine whether G-CSF affects the differentiation of in vitro MDSCs. G-CSF did not affect the percentage of MDSCs (CD11b+Gr-1+), dendritic cells , or macrophages (Gr-1-F4/80+) differentiated from BM cells, as shown in +Ly-6G−Ly-6Chi phenotype increased in the presence of G-CSF, whereas PMN-MDSCs with the CD11b+Ly-6G+Ly-6Cint phenotype decreased  in more than four cancers, including Nos2, proprotein convertase subtilisin/kexin type 9 (Pcsk9), PRKC apoptosis WT1 regulator (Pawr), and gamma-glutamyltransferase 1 (Ggt1), were identified as candidate factors involved in the immunosuppressive ability of MDSCs using the GEPIA web server (Ggt1 gene.Next, among the top 20 upregulated genes by G-CSF, genes associated with poor prognosis (Logrank b server  (Supplemb server . GGT1 isb server , which wb server . Furtherb server . Thus, tGgt1 by G-CSF in MDSCs was verified by qRT-PCR (Ggt1 mRNA expression was more upregulated in M-MDSCs than in PMN-MDSCs upon G-CSF stimulation, suggesting that M-MDSCs can play an important role in increasing the G-CSF-promoted immunosuppressive effects (in vitro MDSCs by G-CSF. Consistent with Ggt1 expression, the immunosuppressive activity of in vitro MDSCs was significantly increased by G-CSF, while the combination of GGsTop and G-CSF attenuated the immunosuppressive activity to the same level as that in the control group (in vitro MDSCs. Furthermore, we examined the effect of GGsTop alone or the G-CSF/GGsTop combination on Arg1, Nos2, and ROS expression. The G-CSF/GGsTop combination group downregulated the expression of Arg1 and Nos2. Unexpectedly, GGsTop alone upregulated their expression (Arg1 and Nos2 upregulation by GGsTop alone did not increase the immunosuppressive ability of MDSCs (Arg1 and Nos2 did not play a decisive role in increasing the MDSC immunosuppressive ability caused by G-CSF. In addition, GGsTop did not abrogate the expression of ROS caused by G-CSF, suggesting that ROS is not a key factor either (The upregulation of the mRNA of  qRT-PCR . Besides effects . To eluc effects . There w effects , suggest effects . We furtol group . Additiopression . As Arg1of MDSCs , suggestr either . These rThe key role of GGT1 is to hydrolyze glutathione. We speculated that metabolites of glutathione, such as glutamate and cysteinyl glycine, obtained by the action of GGT1, enhance the immunosuppressive function of MDSCs. Elevated glutamate concentrations are commonly observed in tumor patients, and studies indicate that glutamate could regulate the immunosuppressive activity of MDSCs on the proliferation of T cells . TherefoGiven that G-CSF enhances the immunosuppressive function and proliferation of MDSCs, it is possible that G-CSF causes side effects through MDSCs when used to prevent FN. We then tested whether G-CSF promoted MDSCs and tumor progression using the EL4 lymphoma-bearing neutropenic mouse model. In the EL4 tumor-bearing mouse model, administration of the anti-tumor drug CPA reduced white blood cells, including neutrophils, monocytes, and lymphocytes to 50% below the pretreatment value measured using an XT-2000i hematology analyzer, leading to neutropenia . CPA strTo date, G-CSF administration is commonly used in the management of cancer patients and dramatically reduces the risks associated with chemotherapy-induced FN . Howeverin vitro MDSCs and RNA-seq analysis and identified that GGT1 played a pivotal role in enhancing the immunosuppressive function of MDSCs by G-CSF. GGT1 is a member of the GGT family, which also includes other proteins, such as GGT2, GGT3P, GGT4P, GGT5, GGT6, GGT7, and GGT8P. Among these, GGT1 and GGT5 are the only two enzymes with catalytic activity (+ T cell proliferation, similar to G-CSF (+ T cells may express lower glutamate receptor levels, resulting in the relative resistance of CD8+ T cells to the increased suppressive function of MDSCs cultured with G-CSF. These results indicate that the mechanism by which G-CSF enhances the immunosuppressive ability of MDSCs may involve GGT-induced increased glutamate levels.To eliminate the side effects of G-CSF, it is necessary to elucidate the direct mechanism underlying the action of G-CSF on MDSCs. We utilized activity , and theactivity . Therefoactivity . Our resactivity . Recent activity . Previouto G-CSF . The expto G-CSF ; thus, Cin vivo CPA-induced neutropenic tumor model, indicating that GGsTop can eliminate the effect of G-CSF in promoting tumor growth. Inhibition of GGT1 by GGsTop did not affect the viability and proliferation of MDSCs in vitro, as assessed by cell number quantification. The numbers of white blood cells, neutrophils, monocytes, and lymphocytes were similar after 6 days of treatment with GGsTop compared to PBS control in vivo. Accordingly, the inhibition of GGT1 by another inhibitor, acivicin, also showed no difference in microglia, macrophages, and whole blood cell numbers, but altered the glutathione metabolism of those cells (GGsTop is now commercially available and its beneficial properties, such as being non-toxic, highly selective, and potent irreversible inhibitor of GGT activity, have been reported under a variety of experimental conditions . For exase cells . These rIn conclusion, our study showed that G-CSF directly enhanced the proliferation and immunosuppressive function of MDSCs and identified GGT1 as a novel factor involved in the enhanced immunosuppressive function of MDSCs induced by G-CSF. Furthermore, our results demonstrated that tumor progression in the chemotherapy-induced neutropenic mouse model was promoted by G-CSF, and the specific GGT inhibitor, GGsTop, prevented G-CSF-induced EL4 lymphoma progression. Although our findings suggest that GGsTop exerts its effects by targeting GGT1 on MDSCs, GGT1 is also expressed in a variety of cells other than MDSCs, and the possibility that GGsTop functions through other cellular mechanisms cannot be excluded, thus suggesting the requirement for further studies. Our study also still lacks clinical human experimental data to prove the safety and efficacy of GGsTop, the existing results suggest that GGsTop would be effective in future clinical applications. These findings should contribute to the development of safer and more effective treatment strategies for FN in patients with cancer.


Tacrolimus is characterized by high pharmacokinetic variability in combination with a narrow therapeutic range. However, influence of donor CYP3A5 genotype and graft-to-recipient body weight ratio (GRWR) on tacrolimus' pharmacokinetics after pediatric living donor liver transplantation (LDLT) remains unclear. n = 40), SS/EX (n = 38), LS/NEX (n = 48), and LS/EX (n = 48). Pharmacokinetics of tacrolimus and clinical outcomes were analyzed.  A total of 174 LDLT recipients (<6 y) were grouped according to donor CYP3A5 genotypes (nonexpressor (NEX) or expressor (EX)) and GRWR  or ≥3.0% ): SS/NEX  and GRWR  were independent impact factors for C/D ratios 3, 7, 14, and 30 days after transplantation. Donor CYP3A5-EX genotype was associated with significantly increasing risks of infectious complications and significantly lower Cylex ATP values. However, no significant difference was observed in acute rejections among 4 groups.  The relationships between the concentration-dose ratio and donor CYP3A5 genotypes and graft size were examined 3, 7, 14, and 30 days after the transplantation. Tacrolimus C0 levels varied greatly among groups, although recipients started with the same initial dosage. LS/EX recipients had significantly lower C0 levels in comparison with those of other groups. The use of CYP3A5-EX-grafts and a greater GRWR both resulted in significantly higher TAC dose requirements and lower C/D ratios. However, the significance of GRWR no longer exists 3 months after transplantation. The multivariate generalized linear mixed model analysis showed that donor CYP3A5 genotypes ( Monitoring of C0 levels alone is not reliable to guide tacrolimus administration. Donor CYP3A5 and GRWR both significantly affect tacrolimus pharmacokinetics after pediatric LDLT. The use of Cylex ATP tests would be helpful to avoid overimmunosuppression. Grafts from living donors have currently become the first choice for pediatric liver transplantation (LT) in Asian countries because of the severe shortage of deceased donor organs in children. In experienced transplant centers, living donor liver transplantation (LDLT) in children has satisfactory short-term and long-term outcomes with the 5-year graft survival rate reaching more than 85% –4. HowevTacrolimus (TAC) is the most common calcineurin inhibitor used for the prevention and treatment of rejection in patients undergoing solid organ transplantation. High TAC exposure leads to increased risks of infections, renal injury, or other toxic effects, while a low dosage of TAC may cause acute or chronic rejections, and even graft loss or death in severe cases. TAC has a narrow therapeutic range and a high interindividual variability in terms of pharmacokinetics –7. Espec∗3/∗3) of donor CYP3A5 (n = 40); the SS/EX (expressor) group had a GRWR < 3.0% and presence (∗1/∗1 or ∗1/∗3) of donor CYP3A5 (n = 38); the LS (large-size)/NEX group had a GRWR ≥ 3.0% and absence of donor CYP3A5 (n = 48); and the SS/EX group had a GRWR ≥ 3.0% and presence of donor CYP3A5 (n = 48). The baseline characteristics and TAC's pharmacokinetic parameters were analyzed and compared between the 4 groups. This study was approved by the institutional review board of Renji Hospital, and written informed consents were obtained from all parents of recipients.Children who underwent LDLT before the age of 6 years between June 2009 and December 2014 were included in further patient screening. Exclusion criteria are shown as below: (1) retransplantation (0 case); (2) combined organ transplantation (0 case); (3) diagnosis with malignant tumor (0 case); (4) graft loss or death within 1 month after LT (9 cases); (5) use of cyclosporine-based regimen as the initial immunosuppression regimen (1 case); (6) data missing (17 cases). Finally, 174 children and their corresponding donors were included in our study and categorized into 4 groups by GRWR and donor CYP3A5 genotype: the SS /NEX (nonexpressor) group had a GRWR < 3.0% and absence  was used as the initial immunosuppressive regimen. TAC was started on day 1 after LT with an initial dosage at 0.1-0.15 mg/kg/day, and the target trough level (termed C0 level) was 8-12 ng/ml during the first month after LT, 7-10 ng/ml from the 2nd to 6th month, 5-8 ng/ml from the 7th to 12th month, and around 5 ng/ml thereafter. If TAC could not reach the target C0 level despite a twice increase of dosage, patients would use a triple-drug regimen with mycophenolate mofetil added or switch to a cyclosporine-based regimen. The use of methylprednisolone was started at 10 mg/kg during LT and at 4 mg/kg on day 1 after LT. The dose of methylprednisolone was gradually tapered by 4 to 8 mg per day and maintained with prednisone 2.5 to 5 mg daily taken orally, which was withdrawn within 3 months after LT [One milliliter of ethylenediamine tetraacetic acid-treated venous whole blood was collected 12 hours after previous TAC administration. Blood TAC C0 level was tested using the microparticulate enzyme immunoassay . TAC's weight-adjusted doses and C0 levels were recorded after each blood test, and C/D ratios were calculated using the C0 levels (ng/ml) divided by the weight-adjusted dose (mg/kg/d), which was used as an alternative method for estimation of TAC clearance , 13, 14.A > G  was tested for the 174 children and their corresponding donors. We extracted the genomic DNA from leukocytes using a QIAamp Blood Kit . The PCR-based sequencing process and primer information were performed as described previously [The single nucleotide polymorphism of CYP3A5 at position 6986eviously . The forCD4 + T-lymphocyte functional assay  was performed for assessment of cellular immunity functions according to our previous study . In brieP values <0.05 indicated statistically significant. All measurement data were tested for normality using the Shapiro-Wilk method. The independent-samples t-test analysis was used for variables with normal distribution, while the nonparametric statistical analysis (Mann–Whitney test) was applied to abnormal-distribution variables. Categorical data were analyzed using χ2 test. The generalized linear mixed model (GLMM) was used to analyze each factor that might have influenced TAC C/D ratios, and any variables identified as statistically significant in the univariate GLMM analysis were included in the multivariate GLMM analysis to obtain independent factors affecting TAC C/D ratios. Logistic analysis was used to study the correlations between CYP3A5 genotype and infection, rejection status, respectively, then the relationship between CYP3A5 genotype and infection, rejection status, respectively. The rejection-free survival rate was estimated using Kaplan-Meier method , and differences between groups were compared using the log-rank test.SPSS 19.0 software  was used for statistical analysis. ∗1 and 244 (70.1%) for ∗3, and those in recipients were 85 (24.4%) for ∗1 and 263 (75.6%) for ∗3, respectively. Detail frequencies of CYP3A5 genotypes in donors and recipients are shown in TAC dosages were adjusted according to target C0 levels at different time point after LT. However, we found that most C0 levels were outside the target ranges, and proportions of C0 levels within the target ranges were only 28.9%, 42.0%, 22.4%, 35.0%, 42.4%, 32.3%, and 30.4% at 3 days, 7 days, 14 days, 1 month, 3 months, 6 months, and 12 months after LT, respectively. Moreover, C0 levels varied greatly among SS/NEX, SS/EX, LS/NEX, and LS/EX groups, even though recipients started with the same initial dosage. Figures Compared with NEX-graft recipients, EX-graft recipients had a significant higher TAC dose requirement as well as a significant lower C/D ratio within 12 months after LT . FurtherF = 11.876, P = 0.01) and GRWR  were independent impact factors for C/D ratios 3, 7, 14, and 30 days after LT , recipient CYP3A5 , and the ABO compatibility  were independent impact factors for C/D ratios 3, 6, and 12 months after LT , including recipient age , recipient gender, recipient weight , recipient height (<70 cm or ≥70 cm), recipients' ABO blood group, ABO compatibility , PELD score , primary diagnosis (cholestatic liver disease or other), previous Kasai procedure (with or without), graft weight (<250 g or ≥250 g), GRWR (<3.0% or ≥3.0%), recipients' CYP3A5 genotype (EX or NEX), donor CYP3A5 genotype (EX or NEX), donor age (<40 years or ≥40 years), donor gender, donor body mass index (BMI) , donor ABO blood group, and donor-to-recipient relationship . We found that 8 variables had significant impacts, including recipient age, recipient weight, recipient height, PELD score, GRWR, recipient CYP3A5, donor CYP3A5, and donor ABO blood group. Finally, the multivariate GLMM analysis showed that donor CYP3A5 (after LT . Similarafter LT .P = 0.010) and SS/EX groups , but the LS/NEX group merely showed a slight trend in the increasing risk of sepsis  (P = 0.0035), the LS/NEX group , and the LS/EX group  all presented significantly lower Cylex ATP levels compared with the SS/NEX group . Meanwhile, we found that the LS/EX group had the highest proportion of children with low immune functions , due to an increasing use of TAC doses to obtain satisfactory C0 levels (P = 0.032) were likely to have bacterial infection after LT  children experienced one or more episodes of infections within 3 months after LT, in which incidences of bacterial infection, fungal infection, Epstein-Barr virus infection, cytomegalovirus infection, and other infections were 35.1%, 5.2%, 16.1%, 16.1%, and 2.3%, respectively. Posttransplant infections were classified according to 2012 International Guidelines for Management of Severe Sepsis and Septic Shock . In comp= 0.147) . Namely,0 levels . What isafter LT . The resP = 0.6). However, CYP3A5-expressed patients prone to have shorter rejection-free survival time (P = 0.3).Rejection-free survival rates in SS/NEX, SS/EX, LS/NEX, and LS/EX groups were 67.0%, 52.6%, 66.1%, and 59.8% at 1 year, 57.4%, 49.3%, 55.9%, and 44.9% at 3 years, and 55.0%, 44.7%, 54.1%, and 43.8% at 5 years, respectively. No significant difference was observed in rejection-free survival rate between these groups , P = 0.6val time , P = 0.3TAC's therapeutic use is complicated by its pharmacokinetic variability. The variable pharmacokinetic property of immunosuppressants may have a significant adverse effect on long-term outcomes after solid organ transplantation, secondary to an increased risk of acute and chronic rejections and drug toxicity. Thus, periodical monitoring of blood TAC C0 levels is recommended to judge whether TAC has reached the therapeutic window and detects possible rejections or overimmunosuppression. TAC's pharmacokinetic in transplantation is affected by various factors such as the patient age, ethnic groups, genetic factor, transplant organ type, coadministration with food or medication, gastrointestinal functions, and small intestinal p-glycoprotein –24. In p∗1 allele (the wild type) have a high amount of CYP3A5 enzymes in the liver and small intestine. Ethnicity-dependent pharmacokinetic differences were reported in expression of CYP3A5, with African people (60%) characterized by more active CYP3A5 enzymes than white people (33%) [∗1 and ∗3 alleles in Chinese population [∗1 in Chinese people was approximately 24-30%, and those who carried the ∗1 allele consisted of at least 40% of all cases. Based on our previous results, CYP3A5 genotypes in both donors and recipients had consistent influences on pharmacokinetic profile of TAC with contributions of 32.35% and 6.0% to the attributable variance in the C/D ratio, respectively [TAC is metabolized by CYP3A enzymes into demethylated and hydroxylated metabolites, which catalyses metabolic reactions with extensive effects on the biological activities of drugs, pretoxic chemicals, and endogenous substances in human liver and small intestine . CYP3A5 le (33%) , 26. Ourpulation , and datectively . It is uectively , 28, whiInterestingly, weight-adjusted doses of TAC were often much higher in infantile recipients than in older children regardless of CYP3A5 genotypes, indicating a decrease of TAC clearance corresponding to children's growth. Small children usually require approximately twice as much as weight-adjusted doses of TAC in adults to achieve the same C0 levels. However, younger children often tend to have a higher GRWR in LDLT using left lateral segment. The relationship between recipient age and GRWR probably plays an important role in pharmacokinetic variability of TAC over age, and this may explain why recipient age or weight are not independent impact factors on the TAC C/D ratio. In this study, results were much more explicit and reliable using multivariate GLMM analyses with C/D ratios in multiple levels (different time points after LT), which minimized the possible interlevel selection bias in single-level regression model. The recipient CYP3A5 genotype determines the amount of CYP3A5 protein in the small intestine, where TAC is absorbed. TAC is highly lipophilic and easily spreads into intestinal epithelial cells after oral administration. CYP3A5 enzymes in the small intestine reduce the oral bioavailability of TAC –31. ConsOur data show children with an EX-graft suffered from higher risks of infections after LT than those with a NEX-graft even if they have lower C0 levels of TAC, suggesting that the TAC C0 level is not a reliable index to truly reflect patients' situation of immunosuppression. However, this is probably related to the increasing doses of TAC to reach the target C0 levels in this group of patients. Alak and Moy  revealedThere are several underlying limitations in our study. First, data in this study were retrospectively reviewed from the pediatric LT database, and the sample size was subject to the nature of a single-center study. Second, disproportionate percentages of children who switched from TAC to cyclosporine across the 4 groups might cause selection bias in multivariate GLMM analyses. Third, amounts of TAC metabolites in recipients and CYP3A5 enzyme activities in donor livers were not measured in this study, and how TAC metabolites affect immune functions after pediatric LDLT still needs to be further clarified in a prospective study.In conclusion, donor CYP3A5-EX-type and a greater GRWR are both associated with significantly higher TAC dose requirements and lower C/D ratios after pediatric LDLT. However, significance of GRWR vanished after 3 months posttransplant. High doses of TAC in recipients with EX-grafts would result in increasing risks of infectious complications. Thus, monitoring of immune responses using Cylex ATP levels would be helpful to establish individualized immunosuppressant regimens.


Background: Right-sided living donor kidneys have longer renal arteries and shorter veins that make vascular anastomosis more challenging. We sought to determine whether recipients of right-sided living donor kidneys have worse outcomes than left-sided kidney recipients.Methods: An observational analysis of the Australia and New Zealand Dialysis and Transplant Registry (ANZDATA) was undertaken. We used adjusted logistic regression to determine the association between side and delayed graft function (DGF) and time-stratified adjusted cox regression models for graft and patient survivals.Results: Between 2004 and 2018, 4,050 living donor kidney transplants were conducted with 696 (17.2%) using right kidneys. With reference to left kidneys, the adjusted OR (95% CI) for DGF was 2.01 (1.31–3.09) for recipients with right kidneys. Within 30 days, 46 allografts (1.4%) were lost, with major causes of overall graft loss being technical, primary non-function and death. Recipients of right donor kidneys experienced a greater risk of early graft loss , but not beyond 30 days .Conclusion: Technical challenge is the most common cause of early graft loss. The risk of early graft loss among recipients who received right kidneys is doubled compared to those who received left living donor kidneys.  The effect of the side of the living donor kidney on graft and recipient outcomes remains a subject of debate. Transplant surgeons prefer left-sided living donor kidneys because the longer renal vein facilitates implantation of the donor kidney to the deeply situated recipient right iliac vein –8. CompaThe increased technical difficulty of implanting a right donor kidney may predict the greater risk of thrombosis, delayed graft function (DGF) and graft loss for recipients of right compared to left kidneys , 12–14. Ethics approval was granted by the Western Sydney Local Health District Human Research Ethics Committee ((6063) 2019/ETH09846) and the ANZDATA executive. This manuscript was prepared following the Strengthening the Reporting of Observational studies in Epidemiology (STROBE) guidelines .An observational analysis of the Australia and New Zealand Dialysis and Transplant (ANZDATA) registry was undertaken from January 2004 to the end of 2018. Paediatric recipients  and non-primary grafts  were excluded from the analysis, as these populations are expected to have different characteristics and outcome profiles that may be inadequately captured by measured characteristics. Cases with missing data on the key exposure, kidney side, were not included  .The key exposure of interest was the side, left or right, of the living donor kidney. Donor baseline characteristics included for analysis were age, sex, ethnicity, smoking, family history of diabetes mellitus, diabetes mellitus, hypertension, and body mass index (BMI).Recipient baseline characteristics included age, sex, ethnicity, primary kidney disease, smoking, diabetes mellitus, body mass index, cytomegalovirus (CMV) and Epstein-Barr virus (EBV) status, history of chronic lung disease, cardiovascular disease, hepatitis or cancer, and time on dialysis before transplantation. The primary kidney disease of the recipient was classified into glomerulonephritis, polycystic kidney disease, reflux nephropathy, vascular, diabetes mellitus and other. Cardiovascular disease was defined as a history of any one of coronary artery disease, peripheral vascular disease or cerebrovascular disease. Kidney donor surgery and implantation characteristics included the procedure date, donor and recipient relationship, human leukocyte (HLA) mismatches, total ischemia time, number of renal arteries and veins anastomosed, number of ureters, operation type and approach. The relationship between the donor and recipient was classified as related or unrelated. Total ischaemia time was the sum of warm and cold ischemia times, from donor renal artery interruption to the release of the renal artery in the recipient. The information collected by ANZDATA on DGF changed in 2017, from recording grafts requiring dialysis within 72 h to grafts requiring dialysis within 7 days after transplantation. Our analysis therefore defined DGF as recipients who required dialysis within 7 days of transplantation.The patient relevant outcomes included in these analyses were overall graft loss, death censored graft loss, all-cause death and DGF. We also sought to compare the cause of early graft loss in the first 30 days after transplantation, between left and right living donor kidneys. Overall graft loss was defined as transplant nephrectomy, recommencing long term dialysis, re-transplantation or death from any cause. Time to graft loss was the period from the date of transplantation until the date of graft failure or death, with cases censored for loss to follow-up and the end of the study period. For death-censored graft loss, recipients were censored at the time of death, loss to follow-up or the end of the study period, whichever one came first. Patient survival was defined as the time from transplantation until patient death, censored for loss to follow up and the end of the study period.p-values less than 0.05 were considered statistically significant.Categorical variables are presented as counts with percentages and compared using Pearson chi-square tests. Non-normally distributed continuous variables are presented as medians with interquartile ranges and compared using Mann-Whitney U tests. A Pearson’s product-moment correlation was performed to determine the relationship between the total number of transplants performed at each centre and the percentage of right kidneys transplanted. The 17 centres that performed transplants in 2018 were included to capture the centres that are well established and currently active. p-value <0.25 on univariate analysis were included in the initial model, as well as kidney side. We then used a step-wise backward elimination process until the variables with p < 0.05 remained in the final model. To examine the effect of the change in definition of DGF in 2017, a two-level categorical variable representing the different periods was constructed from the year of transplant  and added to the final multivariable model. The binomial logistic regression analysis was then fitted using a random effect model to account for clustering of DGF within centres.Adjusted binomial logistic modelling was used to determine the association between donor kidney side and DGF. Variables with a Time to event outcomes  were analysed using the Kaplan-Meier method and differences in survival curves were compared using the log-rank test.p-value of less than 0.25 on univariable analysis. The least significant variables were then removed from the base model using a step-wise backward elimination process until only variables with p < 0.05 remained in the final parsimonious model. The linearity of continuous variables was assessed by dividing into categories and examining the trend.Adjusted cox regression modelling was used to assess the association between the side of the kidney and allograft outcomes. For each outcome, the initial multivariable model included variables with a The proportional hazards assumptions of the Cox models considering the whole study period (from 2004 to 2018) were tested and the Schoenfeld residuals were plotted for each variable. There was no deviation from the assumption with the key exposure (side of kidney) for overall graft loss, death-censored graft loss and overall mortality. The models were then fitted with the predetermined division at 30 days. Thirty days was selected to elucidate the differences between early and late recipient outcomes and as a clinically relevant timepoint used in studies investigating early kidney graft loss . For eacTo assess the robustness of our results, the Cox proportional hazard regression analyses were also fitted using a random effect model (frailty model) to account for clustering of graft loss and mortality risk within centres. Additionally, a three-level categorical variable for transplant year was constructed  and added to the final Cox models to assess for era effects, this term was removed from the model if it was not significant.Analyses were performed using IBM SPSS v25  and RStudio .There were 4,985 living donor transplants between 2004 and 2018. After excluding paediatric recipients, non-primary grafts, and donor kidneys with missing data on kidney side, the recipient cohort of 4,050 living donor transplants included 3,354 (82.8%) left kidneys and 696 (17.2%) right kidneys . The basThere were no differences between left and right kidney donors . Recipiep < 0.01) (p = 0.02). During the time period of the study, 10 of the 27 transplant centres closed or merged with others. Between 2004 and 2018, the proportion the transplanted kidneys each year that were right sided was stable .There was variation in the proportion of right kidneys transplanted between the 27 transplant centres in this study (Pearson chi-square  < 0.01) . There wp = 0.001). Right kidneys were associated with an increased risk of DGF (adjusted odds ratio (OR) (95% CI) 2.01 [1.31–3.09]), adjusting for total ischemia time, time on dialysis before transplant, number of HLA mismatches, donor hypertension and primary kidney disease (DGF was reported in 3.0% of transplants. Recipients of right kidneys were more likely to experience DGF with 86 recipients (2.6%) of left kidneys compared to 34 (4.9%) of right kidneys affected  cases . During p-value = 0.005)  . Overall= 0.005) .p-value = 0.005)  . The ove= 0.005) .Patient survival was not significantly different between left and right kidney recipients at 30 days, 1 year or 5 years. Thirty days after transplantation, the survival of recipients of left and right kidneys was 99.7% (95% CI 99.5–99.9%) and 99.5% (95% CI 98.7–99.9%) respectively . Overallp = 0.03 (p = 0.03) and the recipient having type 1 diabetes mellitus   . After 3(p = 0.8 . In the p = 0.04) (p = 0.04)  . Grafts  = 0.04) . After 3 = 0.04) . In the p = 0.5, and after the first 30 days aHR 1.22 [95% CI 0.95–1.57], p = 0.1)  . In the This large registry-based study demonstrates that adult recipients of primary right living donor kidneys have a two-fold increased risk of DGF, and graft loss and death-censored graft loss within the first 30 days of transplantation. Primary non-function accounted for 9% of left kidneys and 36% of right kidneys that were lost in the first 30 days after transplantation. Patient survival and graft survival beyond 30 days were not associated with living donor kidney side.The association between living donor kidney side and recipient outcomes has mostly been studied previously in small, single-centre studies with disparate results, complicating the debate as to whether more right-sided nephrectomies should be undertaken. An OPTN registry-based retrospective analysis from 2000 to 2009 reached similar conclusions to our study, with lower effect sizes; right living donor kidneys experienced a 1.4 (95% CI 1.2–1.5) increased risk of DGF and a 1.1 (95% CI 0.85–1.5) increased risk of graft loss . In contOur multi-centre and registry-based study had sufficient power to examine differences in DGF, patient survival and graft survival. Furthermore, the findings of our study corroborated prior work that compared outcomes between transplanted left and right deceased donor kidneys . The infHowever, the demonstrated higher incidence of DGF and PNF-related graft loss in right living donor kidneys in the first 30 days after transplantion supports increased surgical challenges associated with transplanting right living donor kidneys. In the first 30 days, 64.3% of right kidneys that were lost were lost due to either primary non-function or technical causes compared to 59.4% of left kidneys that were lost. Expanding data collection to include important factors such as vascular anastomosis times, and intra-operative and post-operative complications could help determine if there are increased surgical challenges with right living donor kidney transplantation. For example, although anastomosis times were not captured by the ANZDATA registry, right deceased donor kidneys have been shown to have longer anastomosis times .Our study has a number of limitations. Indication bias remains a possibility. We were unable to account for inter-centre decision-making variations that might influence outcomes such as indications for right donor nephrectomies in preference to left. This clinical decision evaluates the risk of surgery on either side and aims to maximise the residual renal function of the donor. However, the shared frailty models demonstrated minimal changes to the estimates when accounting for centre-specific random effects. Even though there were multiple confounding factors adjusted for in the analyses, there are likely to be several unmeasured and residual confounders, such as differential kidney function of the living donor kidneys and individual surgeons’ volumes and expertise. The definition of DGF changed from the need for dialysis within 72 h after transplantation to the need for dialysis within 7 days in 2017. We defined DGF as the need for dialysis within 7 days, therefore this may lead to an underestimation of the overall incidence of DGF. Adjusting for the era of the transplantation in the model did not change the risk factor profile. However, only 2 years of data were captured using the revised ANZDATA Registry definition. Additionally, the outcome ascertainment bias is unlikely to be differential between recipients of left and right living donor kidneys, as the proportion of right kidneys transplanted each year was relatively stable. Strengths of this study are the large cohort with few missing values and cases lost to follow up and the minimal risk of selection bias as the study population represents all primary adult recipients of kidney transplants in Australia and New Zealand.The time period of this ANZDATA based study corresponded to the progressive uptake of laparoscopic donor nephrectomy. The driving force behind this was a consumer-driven preference by prospective living kidney donors and their referring nephrologists to avoid open surgery where possible. In the 15 year study period, 10 of 27 centres ceased to provide a living donor kidney transplantation service, likely driven by their inability to provide laparoscopic surgical expertise. The higher percentage of right nephrectomies performed by open or hand assisted laparoscopic surgery and low overall rates of right nephrectomies likely reflected the increasing uptake of laparoscopic nephrectomies and hesitancy to undertake right laparoscopic nephrectomies during this learning phase, particularly in the early part of the study. Importantly, this careful approach resulted in equivalent recipient outcomes for laparoscopic and open donor surgery, as the type of operation was not a risk factor for graft or patient survival. Furthermore, the era of transplantation was not a risk factor for inferior recipient outcomes. Centres with low volumes of transplants were not excluded in the study, as they remained important data points, and despite the variation in transplant centre volume, the shared frailty models demonstrated minimal changes to the estimates when accounting for centre-specific random effects. Equally, the use of right living donor kidneys (17.2%), which is high by international standards, suggests that it would have been uncommon for recipients in Australia and New Zealand to be denied the opportunity to be transplanted with a living donor kidney because their transplant centre had been reluctant to tackle either donation or transplantation of a right-sided donor kidney.Robot-assisted surgery may have an emerging role in living kidney transplantation and the impact of the side of the living donor kidney should be studied in this context. There has been increasing uptake of robot-assisted kidney transplantation with initial studies indicating that it is non-inferior to open kidney transplantation  and feasIn summary, our results indicate that recipients of right living donor kidneys may have an increased risk of DGF and graft loss in the first 30 days after transplantation. The implication is that the technical challenges of transplanting a right living donor kidney are real, but not to the extent that right-sided kidneys should be excluded, particularly in light of the limitations addressed above. The prospective donor of a right kidney and the recipient should be informed but also reassured that the differences between left and right living donor kidneys are relatively small, confined to the early post-operative period and are similar to those seen in recipients of left or right deceased donor kidneys . Neverth


Background: The association between congenital spigelian hernia and ipsilateral ectopic testis has been debated in the literature, and the management of such cases has yet to be standardized. Both pediatric surgeons and sonographers should be aware of this entity to allow for prompt diagnosis and monitoring/repair because congenital spigelian hernias have a high risk of incarceration.Case Report: A 3-month-old male presented with left-sided abdominal wall swelling present since birth with coexisting left-sided undescended testis. Ultrasound confirmed the suspicion of a congenital spigelian hernia with undescended testis but failed to locate the ectopic testis. Diagnostic laparoscopy and open repair of the spigelian hernia were performed when the patient was 9 months of age. Left-sided orchidopexy was also performed as the left testis was located within the spigelian sac. The patient was asymptomatic at 1-year follow-up.Conclusion: The association between congenital spigelian hernia and ipsilateral ectopic testis requires the surgeon and sonographer to pay special attention to the spigelian hernia sac as it may contain the ectopic testis. Orchidopexy and hernia repair in very young children may be delayed while closely monitoring for incarceration to allow for improvement in immunity, an increase in size of the spermatic cord and vasculature, and avoidance of the stress of 2 separate surgeries. The surgical approach can be laparoscopic or open depending on the experience of the surgeon and the complexity of intraoperative findings. The swelling was associated with a transient increase in size during episodes of crying or coughing followed by spontaneous reduction. The infant was born at term by elective caesarean section to a gravida 2 para 1 (1 live birth) mother with gestational diabetes mellitus.Examination revealed a soft, reducible swelling with an expansile impulse when the infant cried that was located in the posterolateral aspect of the left lower abdominal wall and extended anteriorly onto the left iliac fossa. The left hemiscrotum was empty, and the left testis was not palpable. A provisional diagnosis of a congenital spigelian or lumbar hernia with left undescended testis was considered.Ultrasound of the abdomen revealed 2 abdominal wall defects. The first fascial defect was in the anterior abdominal wall in the left iliac fossa and measured 1.6 cm with a sac extending laterally and measuring 2.8 × 1.2 × 3 cm. A second fascial defect was noted posterosuperior to the first defect in the posterior abdominal wall lateral to the kidney that measured 1.3 × 0.6 cm with a sac measuring 2.7 × 1.2 × 3 cm. The left testis was not visualized on ultrasound, while the opposite side had no abnormalities, consistent with the clinical examination and the initial diagnosis. Diagnostic laparoscopy and open repair were planned for when the infant was 9 months of age.When the patient reached the age of 9 months, diagnostic laparoscopy revealed 2 apparent fascial defects. The larger defect, with clearly defined margins and measuring approximately 2.5 × 2 cm, was present in the lower left abdominal wall approximately 1.5 cm lateral to the lateral umbilical ligament. The sac proceeded anteriorly and then superolaterally in the subcutaneous plane in the anterolateral abdominal wall. The left undescended testis was identified in the hernia sac with the vas deferens and the accompanying vasculature entering the sac at the lateral end of the fascial defect . The gubThe second potential fascial defect was noticed posterosuperiorly as a depression in the parietal muscle bed. However, neither clear margins nor a hernia sac was seen with this fascial defect as identified in the ultrasound. These findings led to the confirmation of the diagnosis of congenital spigelian hernia with ectopic testis.The hernia sac from the first fascial defect was isolated and separated from the testis, vas, and vessels. Although 80% of the size of the opposite testis, the consistency and contour of the left testis were normal. In addition, the length of the vasculature was adequate. A herniotomy was performed flush with the abdominal wall. The testis was rerouted medial to the inferior epigastric vessels and tunneled through the subcutaneous tissue into the scrotum. Left-sided orchidopexy was performed in the conventional manner . The mus7 Most commonly, the spigelian hernia sac protrudes through defects in the transversus abdominis aponeurosis and internal oblique muscle to lie beneath the overlying thick, intact external oblique aponeurosis below the level of the arcuate line.7 Fibers of the transversus abdominis aponeurosis and internal oblique muscle run parallel to each other below the umbilicus, allowing for the development of potential weak points through which a hernia could develop.9 In addition, spigelian hernias are commonly visible lateral to the confluence of the arcuate and semilunar lines because the hernial sac grows laterally as it is limited medially by the rectus sheath and muscle.10 Spigelian hernias are classified based on their relation to the inferior epigastric vessels into the classic  and low types .7 According to Sengar et al, the low variety is more commonly associated with congenital ipsilateral cryptorchidism and is commonly misdiagnosed as direct inguinal hernia.2 Early differentiation of spigelian hernias from other hernias is essential, as the risk of incarceration is notably higher with spigelian hernias, and misdiagnosis could delay management.2 However, our patient presented with the classic type in conjunction with an undescended testis.Spigelian fascia is the aponeurotic part of the transversus abdominis muscle between the lateral border of the rectus abdominis muscle and the semilunar line extending from the ninth costal cartilage to the pubic tubercle. The semilunar line marks the transition between muscle and aponeurosis in the internal oblique muscle.2 While up to one-third of all spigelian hernias are associated with other anomalies including myelomeningocele, anorectal malformations, and skeletal abnormalities, up to 80% of spigelian hernias in males are associated with an ectopic testis.11 Up to 87% of the time, the testis is present in the hernia sac.12 Although physical examination is the mainstay of diagnosis, ultrasound is a useful adjunct to evaluate the hernia prior to surgery and to locate the testis.13 However, in our case, the left testis was not palpable by clinical examination and not visualized by ultrasound. Other authors have reported a similar failure of ultrasound to locate the testis on the side of the spigelian hernia.15 Consequently, the sonographer must maintain a high index of suspicion when suspecting a spigelian hernia in a patient with undescended testis and attempt to locate the testis in the hernia sac.14 Even if ultrasound fails to demonstrate the testis, the surgeon should be prepared to locate it in the hernia sac intraoperatively.12 Because of the nature of the spigelian sac to grow in the superolateral direction and the propensity of the neck to lie near the inguinal ring, the sonographer must also keep in mind that a spigelian hernia could be mistaken for other hernias such as an inguinal hernia or a lumbar hernia.Unlike adult spigelian hernias, which are usually attributable to a chronic increase in intra-abdominal pressure or trauma and have a female preponderance, up to 80% of spigelian hernias in children are thought to be nontraumatic in origin with a male preponderance (3-4:1).13 Special attention must be paid to the location and size of the defect, length of the testicular vasculature, and presence of the inguinal canal and gubernaculum. The location of the defect helps classify the hernia as a classic or a low hernia. The size of the defect determines whether herniorrhaphy or hernioplasty is required. Durham and Ricketts placed an SIS mesh (Cook Biotech), while Singal et al utilized a VYPRO (Vicryl and Prolene) mesh to repair the hernia as the defect was too large for a herniorrhaphy.4 Both authors reported good results with hernioplasty and neither reported recurrence in their cases. If the length of the testicular vasculature is sufficient, a 1-stage orchidopexy can be performed. In most cases, including our own, the length of the testicular vessels was sufficient. No cases of spigelian hernia-ectopic testis have been reported in which an ipsilateral gubernaculum or inguinal canal was confirmed by the surgeon, but in at least 69% of spigelian hernia-undescended testis cases, the ipsilateral gubernaculum and/or inguinal canal was absent.1 The absence of the gubernaculum and inguinal canal, the adequate length of the testicular vessels, and the location of the defect of the spigelian hernia play an important role in hypothesizing the pathophysiology of this embryologic association.To delineate the anatomy of the surgical site, we recommend a diagnostic laparoscopy before performing the orchidopexy and open hernia repair.16 leading to discussions to establish the most suitable hypothesis for the characterization of this relationship. Initially, the occurrence of spigelian hernia and ipsilateral undescended testis together was thought to be a coincidence, but as multiple case reports were published, enough evidence accrued to challenge this notion. Although no widely approved consensus has been reached among the pediatric surgery research community, multiple hypotheses have been suggested to explain where the embryologic defect occurred and whether the association between spigelian hernia and ectopic testis is a sequence or a syndrome.Since the late 1990s, the association between congenital spigelian hernias and ipsilateral undescended testis has been increasingly highlighted by many authors,1 Raveenthiran criticized this theory, as the defect in the abdominal wall is very small in most cases and unlikely to result in a significant decrease in intra-abdominal pressure.16 Silberstein et al and Al-Salem reported the absence of the inguinal canal in their cases and hypothesized that the testis takes the path of least resistance through the congenital defects in the spigelian fascia.10 This theory was challenged by several, including Raveenthiran, who believed that for unknown reasons, maldescent of the testis would occur, dragging a processus vaginalis during descent and opening a defect in the aponeurotic layers.16 Raveenthiran reiterated that this potential point of herniation would be opened by increased intra-abdominal pressure caused by other comorbidities.16 However, Rushfeldt et al argued that this explanation did not reflect general embryologic knowledge and that the testis could not drag down a processus vaginalis.12 Rushfeldt et al proposed that the failure of the gubernaculum and, hence, the inguinal canal to form would arrest the descent of the testis, leading it to induce the formation of a spigelian hernia as an “emergency exit.”12 Jones and Hutson disagreed with the hypothesis of Rushfeldt et al because if an undescended testis could induce rescue canals, the incidence of spigelian hernia should approach the incidence of undescended testis, which is untrue.1 Rushfeldt et al hypothesized that the “Spigelian-cryptorchidism syndrome” was characterized by a congenital ipsilateral defect in the spigelian fascia, hernia sac with testis, absence of gubernaculum, and absence of inguinal canal, a hypothesis that several authors accepted.14 Jones and Hutson agreed with Raveenthiran and attempted to explain the unknown reason behind his theory via the analysis of various published animal models on testicular descent.1 They suggested that the gubernaculum becomes located slightly superior to the usual site of attachment toward the scrotal sac, which corresponds to the site of the future spigelian hernia. This orientation results in the migration of the testis into the anterior abdominal wall through the spigelian fascia, leading to the manifestation of an ectopic testis. However, as the genitofemoral nerve plays an essential role in maintaining the gubernaculum but is located further caudally, the gubernaculum loses the trophic support of the nerve and degenerates.1 Hence, this theory explains the absence of the gubernaculum and the inguinal canal, the presence of ectopic testis in the spigelian hernia sac, and the adequate length of testicular vasculature. Although the theory is relatively new with support from only animal models, it received the support of Sengar et al, who believe this theory explains their findings of a higher incidence of low spigelian hernias in association with undescended testis.2 We agree with Raveenthiran16 and Jones and Hutson,1 as inherent defects in both the testis and gubernacular orientation appear to explain all the features of the syndrome proposed by Rushfeldt et al.12 Hormonal crosstalk between the androgen-producing cells of the testis, gubernaculum, and the genitofemoral nerve appears to be critical in testicular descent.1 In particular, testosterone appears to influence the genitofemoral nerve to secrete calcitonin gene-related peptide that mediates gubernacular migration during testicular descent.1 The malfunction of this testosterone-mediated process would explain the features of the spigelian hernia-ectopic testis syndrome. However, the presentation of a classic spigelian hernia with ectopic testis in the sac deviates from the Sengar et al hypothesis and suggests that the Jones and Hutson hypothesis may explain both low and classic spigelian hernia-ectopic testis.2Early researchers suggested that abdominal wall defects may impair the generation of sufficient intra-abdominal pressure that mechanically drives the descent of the testis along with the gubernaculum.14 Vascular damage, tension on and compression of the spermatic cord structures, and scrotal infection are possible complications of surgery and may result in testicular atrophy. Some authors propose that spigelian hernia repair surgery should be done immediately to limit incarceration and that orchidopexy should be delayed until the patient is 1 year old to allow for immunity to improve and for the vas deferens and vasculature to enlarge and attain more length and girth.14 Inan et al suggested the conservative approach of waiting until the patient is 1 year old while monitoring for incarceration.14 We operated on our patient at 9 months of age while closely monitoring for incarceration in the interim between diagnosis and repair. We recommend the approach of intervention at an older age while monitoring for incarceration to avoid the increased stress and risk of potential surgery-related complications associated with 2 separate procedures compared to a single surgery.Some authors question the ideal time to operate.17 Deshmukh et al reported successful results with laparoscopic orchidopexy and repair for spigelian hernia-ectopic testis.17 However, more data are required to definitively recommend complete laparoscopic repair vs the conventional open approach. Hence, as a prudent approach, we recommend that this type of case be approached laparoscopically and converted to an open approach if required, depending on the complexity of operative findings and the surgeon's experience.In uncomplicated cases, complete laparoscopic repair may be a viable option.Regardless of the nature of the relationship between spigelian hernia and ipsilateral ectopic testis, the association is an important consideration for pediatric care providers and sonologists to prevent delayed diagnosis and management. In particular, health care providers should expect to find the ectopic testis in the spigelian hernia sac even if the testis is not clinically or radiologically located. When the condition is diagnosed in early infancy, we recommend that surgery be delayed while monitoring for incarceration to allow for improvement in immunity, an increase in size of the spermatic cord and vasculature, and avoidance of the stress of 2 separate surgeries. Although no study has compared open and laparoscopic approaches for repairing spigelian hernia with ectopic testis, we recommend that this decision be made based on the complexity of intraoperative findings and the surgeon's level of experience.


Magnetic resonance imaging (MRI) has been consistently indicated as the imaging modality of choice in children and neonates. MRI is pivotal for the management and prognostication of different neonatal conditions including neonatal encephalopathy, preterm birth, and congenital heart diseases. While feed and wrap technique may be used to perform MRI scans without sedation, this is not always possible due to MRI sensitivity to motion artifacts and long duration of the procedure.Many scans do not require contrast and the intranasal route offers an effective and relatively non‐invasive way to deliver medications. Therefore, the ideal sedation regimen should obviate the need for an intravenous access, but still enable the completion of the MRI without deep sedation and subsequent complications. Dexmedetomidine is a highly selective α2 adrenergic receptor agonist, which induces dose‐dependent sedation, anxiolysis, and sympatholysis with respiratory drive preservation. Although it has been increasingly used in pediatric sedation for diagnostic imaging, little it is known on its safety and efficacy for procedural sedation in newborns.Recently, the Pediatric Sedation Research Consortium reported the data regarding the safety and efficacy of intranasal dexmedetomidine for sedated MRI examinations in children.However, no neonates were included in the study.Bua et al.The primary aim of our study was to assess safety and effectiveness of the combination of intranasal dexmedetomidine and midazolam as sedation for neonatal MRI compared with intranasal dexmedetomidine or intranasal midazolam alone.We prospectively studied 199 neonates scanned at the MRI Research Centers of the University of Campania “Luigi Vanvitelli” and AORN San Giusepe Moscati, Italy, from September 2019 to July 2021. The first one‐hundred‐one infants received a single dose of intranasal dexmedetomidine 3 mcg/kg 20 min before scheduled MRI and 10 min after dexmedetomidine administration, intranasal midazolam (0.2 mg/kg). The following 98 infants received a single dose of intranasal midazolam (0.2 mg/kg) 10 min before the MRI scan. A historical group of 78 infants born in the previous 2 years and who received intranasal dexmedetomidine (3 mcg/kg) as first‐line drug was used as third comparison group. No changes took place in the MRI protocol during the study period. In all the three groups, intranasal midazolam (0.2 mg/kg) was administered as rescue sedation. All the neonates were continuously monitored from the time of sedation to full waking. Intranasal route was achieved by using a mucosal atomization device.In all the infants, we placed peripheral venous access before the scan and adjusted feeding times (last feed 2 h before the scan), minimized noise with double‐layered hearing protections and light with eye shields during all the scans. The degree of sedation before and after drugs administration was assessed by using N‐Pass Score. A single researcher (PM) scored image quality from 1 (poorest) to 5 (best) for T1 and T2 images based on gray‐white matter differentiation, deep nuclei organization, and the absence of motion artifact, on axial images. An overall quality score of 1–5 was given based on both T1 and T2 scans.Adverse effects of sedation were defined as cough, bradycardias (heart rate <100 beats per minute), desaturations (oxygen saturation <90% lasting >10 s),p < .0001). Desaturations were not self‐limiting in six cases  but responded promptly to supplemental oxygen. No other interventions or assistance was required for any participants.Table Our data show that the use of intranasal dexmedetomidine together with midazolam provided an adequate sedation even in scans of long duration, with low risks of significant adverse effects and high‐quality images. Although dexmedetomidine offers favorable sedation profile with minimal respiratory depressant effects, it has slow onset of action when compared to other sedatives. The combination of dexmedetomidine and midazolam increased the efficacy of procedural sedation in neonates and can be considered an option in case of MRI scans of long duration.In conclusion, intranasal dexmedetomidine as an adjunct to intranasal midazolam offers relatively efficient and effective sedation with minimal adverse effects for neonatal MRI scans when compared to intranasal dexmedetomidine or midazolam alone. Intranasal route offers a viable alternative for neonates who do not have an indwelling intravenous access at the time of their scan.None.The study protocol was approved by the University of Campania “Luigi Vanvitelli” ethics committee.Open Access Funding provided by Universita degli Studi della Campania Luigi Vanvitelli within the CRUI‐CARE Agreement.


Corynebacterium striatum is a non-diphtheric actinobacterium that has been reported as being the causative agent of several different infections, affecting both immunocompetent and immunocompromised patients. Recently, C. striatum has been recognized as a nosocomial pathogen that is responsible for severe infection in critical patients, as well as in fragile and immunocompromised subjects. C. striatum has been described as the etiological agent of bacteremia, central line infections, and endocarditis. We report a case of a 91-year-old woman who was hospitalized due to SARS-CoV-2 infection, who developed C. striatum bacteremia and died despite antimicrobial therapy and clinical efforts. Furthermore, we discuss C. striatum diagnosis and treatment based on evidence from the scientific literature.Bacterial infections, especially those in hospital settings, represent a major complication of COVID-19 patients, complicating management and worsening clinical outcomes.  As the SARS-CoV-2 pandemic persists, bacterial coinfections are reported worldwide as being ascribed to significant morbidity and mortality. Secondary bacterial infections could have occurred in more than 50% of fatal COVID-19 patients [Several studies have analyzed bacterial infections in COVID-19 patients among different countries, reporting a prevalence ranging from 12.4 to 50% [Corynebacterium (C.) striatum is a non-diphtheric Gram-positive, facultative anaerobic, catalase negative, asporogenous, non-motile actinobacterium [C. striatum has been recognized as being a nosocomial emerging pathogen that is associated with several diseases, including cases of bacteremia, endocarditis, and pneumonia [C. striatum infections [acterium . Despiteacterium , in the neumonia . Extendefections . C. striatum bacteremia and died despite antimicrobial therapy and clinical efforts. We also review and analyze the literature evidence on C. striatum diagnosis and treatment. In this report, we describe the uncommon case of a 91-year-old woman who was hospitalized due to SARS-CoV-2 infection, who developed A 91-year-old woman was admitted to our ward from a COVID-19 residential care facility because of the onset of fever (T max: 38 °C) associated with urinary symptoms (dysuria). She received a two-dose vaccination against SARS-CoV-2 . Her medical history was positive for hypertension, chronic atrial fibrillation, stroke, chronic kidney disease, and thyroidectomy. She had uterine cancer with peritoneal metastasis. The patient took levothyroxine, pantoprazole, furosemide, enoxaparin, and canrenone. Upon admission, she was in a poor general state with disturbances in consciousness , was feverish (T: 37.5 °C), her blood pressure (BP) was 120/60 mmHg, her pulse rate was 70 bpm, and the oxygen saturation in the room air was 98%. Physical examination revealed bilateral leg pitting oedema. A peripheral venous catheter along with a urinary catheter were placed. An electrocardiogram showed alterations compatible with her medical history.3/mm3), hemoglobin levels of 11.1 g/dL, and a normal platelet count (259 × 103/mm3). Her inflammatory markers were elevated: C-reactive protein (CRP) was 7.15 mg/dL , and erythrocyte sedimentation rate (ESR) was 48 mm/h . Procalcitonin was negative. Creatinine was 1 mg/dL (eGFR with CKD-EPI was 49.2 mL/min). Liver markers, including coagulation tests (INR 1), were normal. D-dimer levels were also elevated (1.827 ng/mL). HBV, HIV, and HCV serology tested negative. Lab tests also revealed hypopotassemia (2.7 mEq/L). A urine examination showed leukocyturia, whereas the urine culture was negative  along with hypotension (BP 80/45 mmHg). Her lab tests showed increasing levels of CRP (12.29 mg/dL) and procalcitonin (7.68 µg/L). 3; FIB: 100 mg/dL). The INR was 1.40 , as well as decreased platelet counts and fibrinogen levels ) for C. striatum from the blood cultures, despite the progressive amelioration of inflammatory markers (CRP levels up to 1.25 mg/dL) and procalcitonin levels (up to 0.71 µg/L), as well as the initiation of noradrenaline infusion, her clinical status continued to deteriorate until exitus occurred. A SARS-CoV2 molecular test repeatedly produced positive results during the patient’s hospital stay.However, the day after the isolation of Mycoplasma pneumoniae, Staphylococcus aureus, Streptococcus pneumoniae, Escherichia coli, and, to a lesser extent, Klebsiella pneumoniae, Haemophilus influenzae, Pseudomonas aeruginosa, Acinetobacter baumannii, and Bordetella pertussis [Corynebacterium striatum is a nosocomial opportunistic pathogen, and a Gram-positive rod member of the normal human skin microbiota [C. striatum is increasingly being considered to be an emerging pathogen, with most infections being referred to as nosocomial and occurring especially in patients with significant comorbidities or in patients with skin integrity alterations, indwelling devices, and prolonged exposure to broad-spectrum antibiotics [As reported in the literature, the bacteria most commonly isolated in patients who are positive for COVID-19 are ertussis ,7. In socrobiota . In the ibiotics ,8.Although it was previously postulated that only endogenous infection pathways were possible, in recent years, epidemiological studies have revealed the possibility of corynebacteria patient-to-patient transmission via healthcare-personnel hands ,10,11. C. striatum is the causative agent of various illnesses, including skin infections such as cellulitis [llulitis , osteomyllulitis , meningillulitis , pneumonllulitis , bacterellulitis ,18,19,20llulitis . Over thllulitis . C. striatum from culture is often considered as being contamination unless repeated cultures reveal the same result [C. striatum after the inclusion of this pathogen in the database [C. striatum identification is 16S RNA gene sequencing or, alternatively, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF), due to its simplicity and cost efficiency [The isolation of e result . In a redatabase , despitedatabase ,24. Howeficiency . C. striatum bacteremia are associated with a fever >38 °C, and about half of the infected patients improved without therapy, or even despite inappropriate antibiotic treatment. The organisms in these cases were assumed to be contaminants, whereas only those patients recovering after an appropriate antibiotic course were considered to be truly infected. Yamamuro et al. [C. striatum, along with C. jeikeium, caused true bacteremia more frequently than other Corynebacterium species, defining true Corynebacterium spp. as bacteremia cases with at least two sets of positive blood cultures from a patient with signs of infection.Ishiwada et al.  reportedo et al.  reportedC. striatum and C. jeikeium were the two major Corynebacterium species causing bloodstream infections.Abe et al.  also repIn addition, Mushtaq et al.  showed tC. striatum, as has been suggested in several studies [C. striatum recovery from patients’ samples. Indeed, the association between biofilm formation and C. striatum nosocomial outbreaks has been highlighted by De Souza et al. [Furthermore, biofilm production is an important virulence factor of  studies ,30,31. PC. striatum that have been reported over the last few years. Moreover, considering the role of biofilms in infective endocarditis (IE) [C. striatum [This evidence could explain the growing number of bloodstream and catheter-related infections caused by tis (IE) ,33, physstriatum ,24,34,35C. striatum, leading the physician to use empirical therapy without the possibility of conducting appropriate antimicrobial stewardship [C. striatum requires particular settings as well as the use of horse-defibrinated blood and β-NAD for both broth microdilution, MIC evaluation, and disk diffusion in agar. The incubation time before obtaining the results could be up to 44 h, making the tests cumbersome and time consuming, especially in a hospital environment [C. striatum samples are often sent to an external laboratory and, in some cases, the antibiotic susceptibility profile may be communicated to the infectious disease specialist after the patient’s discharge [Corynebacterium spp. being resistant to vancomycin in a clinical case dated back to 1991 [C. striatum are not available on the EUCAST site, the correlation between disk diffusion and the MIC values demonstrated that all of the 248 and 258 Corynebacterium spp. isolates analyzed for vancomycin and linezolid susceptibility had an MIC of ≤1 mg/L  [C. striatum , and the EUCAST breakpoint for both the antibiotics indicate a susceptibility for MIC of ≤2 mg/L [Unlike many other infective agents, the automatized systems of identification  do not perform antimicrobial susceptibility testing (AST) for wardship . Moreoveironment . For theischarge  or deathischarge ,37,38,39ischarge ,40,41—wi to 1991 , and ano to 1991 . Further–1 mg/L) . Notably ≤2 mg/L ,37.C. striatum infections have reported on the use of this antibiotic for 1–6 weeks to eradicate the bacterium [Considering the case that we presented, linezolid had been already administered as an empirical therapy, since vancomycin was ruled out due to both its nephrotoxicity and the patient’s impaired renal function. Overall, linezolid was administered for 4 days before the exitus, whilst other case reports of acterium ,40.However, therapeutic failure should be further investigated to reveal whether this is due to the general condition of the patient, the bacterial superinfection, or to the timing of antibiotic administration.C. striatum seems to be susceptible to daptomycin, whether alone or combined with rifampicin [Promising antibiotic alternatives may be represented by tigecycline or daptomycin . Indeed,fampicin ; despitefampicin ,45. In afampicin , macrolifampicin , fluoroqfampicin , aminoglfampicin , and cotfampicin , have beC. striatum, this latter likely entered the patient’s bloodstream through catheters or indwelling devices after hospitalization. The patient’s temporary amelioration after treatment with ceftriaxone may suggest a primary bacterial infection, probably from the urinary tract—recovered after treatment with third-generation cephalosporin—not caused by Overall, although the patient that we presented had no relevant immunosuppression diseases ,49,50,51C. striatum bacteremia. Moreover, due to the association between C. striatum bacteremia and infective endocarditis, the physician should be aware of the need of consider the diagnosis of IE in order to eventually perform echocardiography. Unfortunately, despite correct management of the infection, we could not successfully treat the patient because of her poor general condition and comorbidity.This case highlights the need to consider the patient’s history of exposure to broad-spectrum antibiotics or chronic diseases to maintain high levels of suspicion toward these kinds of infections. Empirical antibiotic therapy with vancomycin or linezolid should be considered when choosing the appropriate antimicrobial regimen, especially with increasing numbers of reported cases of C. striatum as commensal bacteria leads to the necessity for two positive blood cultures to indicate this bacterium as being the cause of bacteriemia. This could be the first reason for the problematic treatment of these blood infections. Moreover, the correct identification of C. striatum could require systems that are not available in every hospital.In a hospital setting and in emergency cases such as the one reported herein, it is very important to rapidly find the cause of infection and the best therapy to manage it, especially for patients with a high risk of multi-organ failure. Unfortunately, the common presence of