Idiopathic normal pressure hydrocephalus (iNPH), the most common type of adult‐onset hydrocephalus, is a potentially reversible neuropsychiatric entity characterized by dilated ventricles, cognitive deficit, gait apraxia, and urinary incontinence. Despite its relatively typical imaging features and clinical symptoms, the pathogenesis and pathophysiology of iNPH remain unclear. In this review, we summarize current pathogenetic conceptions of iNPH and its pathophysiological features that lead to neurological deficits. The common consensus is that ventriculomegaly resulting from cerebrospinal fluid (CSF) dynamics could initiate a vicious cycle of neurological damages in iNPH. Pathophysiological factors including hypoperfusion, glymphatic impairment, disturbance of metabolism, astrogliosis, neuroinflammation, and blood‐brain barrier disruption jointly cause white matter and gray matter lesions, and eventually lead to various iNPH symptoms. Also, we review the current treatment options and discuss the prospective treatment strategies for iNPH. CSF diversion with ventriculoperitoneal or lumboperitonealshunts remains as the standard therapy, while its complications prompt attempts to refine shunt insertion and develop new therapeutic procedures. Recent progress on advanced biomaterials and improved understanding of pathogenesis offers new avenues to treat iNPH. In this review, we summarized the pathogenesis of idiopathic normal pressure hydrocephalus (iNPH) and its pathophysiological features that lead to neurological deficits. We also reviewed the current treatment strategies for iNPH and discussed prospective future therapies. It is characterized by dementia, gait disturbance, and urinary incontinence (known as Hakim's triad).2We searched Medline/PubMed database for the relevant literature using the using the Medical Subject Headings (MeSH) terms “Hydrocephalus, Normal Pressure,” and following keywords “pathogenesis,” “pathophysiology”. The titles and abstracts were reviewed. And 78 articles pertaining to the INPH were included. Non‐English language articles; gray literature publications; editorials and commentaries were excluded. Additional references were obtained from bibliography of selected articles.3In the physiological condition, CSF flow has both bulk and pulsatile components. Driven by the continuous production of CSF, the CSF bulk flow moves from lateral ventricles to the third ventricle through the interventricular foramen. It continues to flow across the aqueduct of Sylvius down to the fourth ventricle. Further, through the foramen of Magendie and foramen of Luschka, CSF flows to the subarachnoid space where it is reabsorbed into blood circulation.Abnormal CSF dynamics has been a major research focus in the etiology of iNPH.3.1It has long been observed on MRI images of iNPH patients that the aqueduct flow voids might be an indicator of hyperdynamic CSF flow through the aqueduct.3.2To fully understand the role of abnormal CSF dynamics in the pathogenesis of iNPH, the concept of abnormal CSF drainage should not be overlooked. It has long been acknowledged that in the normal condition, the CSF in the subarachnoid space is reabsorbed through the arachnoid granulations and taken up into the superior sagittal sinus due to the pressure gradientforce.Studies about abnormal CSF drainage in iNPH mostly focus on the venous‐related route. Bateman et al4Ventricular enlargement induced by abnormal CSF dynamics increases mechanical stress on the parenchyma and blood vessels, causing hypoperfusion and consequent hypoxia in iNPH.Multiple imaging modalities including MRI, computed tomography, single photon emission computed tomography, or positron emission tomography (PET) indicate both global and regional CBF reduction in the PVWM, gray matter, and basal ganglia in iNPH patients.Hypoperfusion could further leads to a series of pathophysiological changes of brain tissue including alterations of metabolism, gliosis, neuroinflammation, and blood‐brain barrier impairments.4.1It is generally acknowledged that hypoperfusion and hypoxia could disturb the normal cellular homeostasis, especially the oxygen‐related energy metabolism.Some metabolites that have been analyzed in iNPH could serve as valuable biomarkers. In iNPH patients, the value of N‐acetylaspartate (NAA)/creatine was correlated with the MMSE score, and concentrations of NAA and total N‐acetylaspartate (tNA) were significantly declined in thalamus compared to healthy controls.4.2Astrogliosis is evident in iNPH patients. GFAP  is commonly used as a marker for reactive astrocytes.Neuroinflammatory responses, which are characterized by the release of inflammatory mediators, such as cytokines and chemokines,4.3Blood‐brain barrier (BBB) refers to the barrier between the blood and the brain parenchyma, forming mainly by tight junction‐sealed brain endothelial cells.5The recently discovered glymphatic system facilitates the bulk flow of CSF into the brain along the para‐arterial space, the interstitial space and eventually into the para‐venous space. This pathway is mediated by Aquaporin‐4 (AQP4) channels at astrocytic perivascular endfeet.Impaired glymphatic system is observed in iNPH individuals. Using contrast‐enhanced MRI, delayed removal of the intrathecal CSF tracer gadobutrol was observed in iNPH.A direct consequence of the glymphatic system disturbance is the reduced clearance of neurotoxic substances, such as beta amyloid (Aβ) and hyperphosphorylated tau (HP tau).6Hypoperfusion, glymphatic impairment, and other aforementioned pathophysiological changes in iNPH culminate in brain lesion and functional deficits. White matter lesions are prominent in iNPH.White matter lesions in iNPH could be characterized by hyperintensities on T2‐Fluid‐attenuated inversion recovery (FLAIR) MRI imaging, especially the periventricular white matter hyperintensities (PVH).Pathologically, damages on both myelin and myelin‐sheathed axons have been detected in iNPH. Neurofilament light chain (NFL) is a protein that functions to maintain axonal architecture. Myelin basic protein (MBP) is a vital structural protein of myelin.7It is known that the axon of an unhealthy neuron may progressively dies back over weeks, beginning distally and spreading proximally toward the cell body.The structural and functional organizations in gray matter have been analyzed in iNPH. In a structural network study, larger global network modularity and network decentralization of frontal, temporal, posterior cingulate and insula cortices were observed in iNPH patients compared to controls.8Cerebrospinal fluid diversion with ventriculoperitoneal or lumboperitoneal shunts is currently the first‐line and standard therapy for iNPH.The reversal of abnormal CSF dynamics could be the principal mechanism underlying the therapeutic effect of CSF diversion. Drainage of excessive CSF restores normal CSF pulsatility and directly compensates for the insufficient CSF absorption in iNPH patients.There are currently no FDA‐approved pharmacological treatments for iNPH. Clinical trials suggest that acetazolamide, a carbonic anhydrase inhibitor, can reduce periventricular white matter hyperintensities and improve symptoms in iNPH.Recent years have witnessed a better understanding of iNPH pathogenesis and pathophysiology accompanied by bioengineering advances in medical devices. Novel treatment strategies are on the horizon. Shunt obstruction and infection remain critical concerns in iNPH patients with shunt surgery.Heterogeneous clinical outcomes after surgical treatments clearly indicate that it is not enough merely to drain the excessive CSF. This procedure does not address all pathophysiological problems underlying iNPH.9Here, we provide a comprehensive review on the pathogenesis of iNPH and various mechanisms leading to neurological deficits. Instead of being totally idiopathic, etiologic factors and pathophysiological changes might constitute a vicious, deregulating loop that eventually result in clinical symptoms of iNPH Figure . We alsoThe authors declare no conflict of interest.


Background: Cerebrospinal (CSF) fluid leaks with associated pneumocephalus (PNC) represent a condition bearing serious risks for the patient, with little data available in the literature. Reported success rates of endoscopic skull base repair are lower when PNC is associated than in the case of simple CSF leaks. The present study represents an analysis of our experience with endoscopic management of this condition. Methods: Records of patients with pneumocephalus and associated CSF leaks, who underwent endoscopic skull base repair, were reviewed. Demographics, history, etiology of PNC, size of defect, surgical approach, reconstruction technique and complications were evaluated. Results: Twenty patients with CSF leaks and PNC underwent endonasal repair by the senior author between 2005 and 2019. Defect size was larger than 15 mm in all cases. All patients presented either worsening of PNC under conservative treatment or tension PNC. First-attempt closure of the defect was successful in all patients (100%), with resolution of the pneumocephalus. One patient developed a synechia in the proximity of the frontal ostium, as a postoperative complication. The mean follow-up was 39 months (range: 15–94 months). Conclusion: The present study represents a proposed argument for earlier endoscopic endonasal treatment in patients presenting CSF leaks and pneumocephalus. Simple pneumocephalus is a more frequent entity, usually with a self-limited evolution contained by conservative measures [Pneumocephalus (PNC) is caused by entrapment of air inside the cranial cavity, usually indicating a breach in the craniodural barrier. Most common etiologies include trauma, iatrogenic , intracranial gas-producing microorganisms, congenital defects, neoplasms, CSF shunt-related complications or even spontaneous defects ,2,3. SimAs a result of the multifactorial etiology and diversified therapeutic approaches, there is no consensus regarding treatment of pneumocephalus other than recommendations based on observations from centers with a high incidence of this pathology. Endoscopic skull base repair has become the treatment of choice for CSF leaks, with success rates over 90%; however, when PNC is associated, success rates appear to be lower. The present case series exemplifies our experience regarding endoscopic endonasal skull base repair (ESBR) in patients with persistent pneumocephalus and CSF leak, following failure of conservative measures.2 therapy for 24 to 48 h, bed rest in Trendelenburg position, prophylactic antimicrobial therapy and lumbar drains and were advised to refrain from Valsalva or similar actions. Three-dimensional volume rendering (VR) of CT scans was obtained with the AW Volumeshare 5 software  by using the preset “Air Structure” Approval of this study was granted by the ethics committee research council (number 197/25.05.2020). The current retrospective review includes medical records of patients with pneumocephalus who had undergone endoscopic endonasal repair, by the senior author, between 2005 and 2020. Records of all patients with CSF leaks undergoing endonasal repair were analyzed, and only patients with associated PNC were included in the study. All included patients were hospitalized in the neurosurgical department and were treated according to the hospital’s CSF leak treatment protocol. After obtaining the diagnosis of pneumocephalus on high resolution CT scans, patients with simple pneumocephalus were treated conservatively by 100% OPreoperative assessment of the defect was performed on a thin sliced CT scan and MRI. After identification of the defect, the mucosa and displaced bone fragments were removed, and the defect was measured with a curette. During surgery, in cases with defects difficult to identify, after complete spheno–ethmoidectomy, topical 10% fluorescein was applied on the skull base for guidance . Reconstruction was performed using only autologous materials. A multilayer technique was employed: an inlay mucosa or fascia lata, septal cartilage to reinforce the first layer and fascia lata graft as overlay. Surgicel and gelfoam were used to secure the repair, and a fat graft was applied as a buttress for defects located in the lateral sphenoid recess and frontal sinus posterior wall. Perioperative prophylactic antibiotherapy was administered intravenously for 48 h and continued orally until removal of the nasal packing. The main cause for PNC was a persistent dural breach following cranialization  with a mean age of 44.5 (range: 21–67) years were diagnosed with pneumocephalus and had undergone transnasal endoscopic repair by a single surgeon S.A.; . The defA successful first-attempt repair by means of transnasal endoscopic surgery was accomplished in all patients (100%), with closure of CSF leak and resolution of pneumocephalus. Regarding postoperative complications, one patient developed a synechia of the frontal sinus neo-ostium, requiring revision surgery. One patient presented tumor recurrence and was treated in the neurosurgical department.Most frequently, pneumocephalus is a complication of trauma or a recent neurosurgical procedure ,4. ThereBefore the emergence of modern imaging techniques, the incidence of PNC was reported to be under 1% in patients with craniofacial trauma, the diagnosis being made on the basis of a plain X-ray ,12. HoweRegarding risk factors for development of PNC, abnormal body mass index (BMI) is linked to higher incidence of postoperative CSF leak and meningitis . AllenswPlacement of lumbar drains is controversial, some authors stating that PNC can be worsened by lumbar drains as a consequence of intracranial negative pressure ,18,19,20The occurrence of postoperative pneumocephalus indicates a breach between the cranial cavity and the external environment, the mechanism being mutual with CSF leak formation. The difference between simple CSF leak and that associated with pneumocephalus is that in the case of the latter, PNC transfers contaminated air inside the sterile intracranial area. Although a higher incidence of intracranial infections has been observed in patients with postoperative CSF leaks, little is mentioned in the literature about the implications of pneumocephalus ,16. Guo Craniodural defects are considered to have an inadequate healing due to the incapacity of the dura to regenerate, the defect being covered by a single layer of mucosa . EnjamelThere is no consensus regarding pneumocephalus treatment, possibly due to the variate etiologies; however, certain conservative approaches are generally accepted, namely bed rest, oxygen therapy, lumbar drainage, airtight nasal packing and maintaining the patient in the Trendelenburg position. In case medical measures are insufficient, surgical temporizing options for decompression include needle aspiration, burr holes, ventriculostomy and craniotomy ,4,21. A Although success rates of ESBR in CSF leak closure are over 90% ,28,29, aRegarding defect size, Del Gaudio observed the inefficiency of conservative measures in defects larger than 15 mm, an observation supported by the present study, taking into consideration that all defects were larger than 15 mm and all patients failed conservative measures . A partiEven though there is no consensus regarding treatment of pneumocephalus, first-line conservative measures are usually adopted. However, CSF leaks with associated PNC are a particular entity due to greater risks for the patient to develop complications. This study has shown high success rates of ESBR in CSF leak closure with resolution of pneumocephalus, contradicting another similar study and providing an argument for early endoscopic skull base reconstruction, even if the defect is not very large, not to mention the advantage of avoiding the long-term risk of ascending meningitis. We consider these arguments to recommend early endoscopic intervention in patients with pneumocephalus accompanying CSF leaks, or at least to present them as an option for these patients.


Conventional genetic analyzers require surgically obtained tumor tissues to confirm the molecular diagnosis of diffuse glioma. Recent technical breakthroughs have enabled increased utilization of cell-free tumor DNA (ctDNA) in body fluids as a reliable resource for molecular diagnosis in various cancers. Here, we tested the application of a chip-based digital PCR system for the less invasive diagnosis  of diffuse glioma using the cerebrospinal fluid (CSF).IDH1 R132H, TERT promoter (C228T and C250T), and H3F3A (K27M) on the QuantStudio® 3D Digital PCR System. These results were compared with their corresponding tumor DNA samples.CSF samples from 34 patients with diffuse glioma were collected from the surgical field during craniotomy. Preoperative lumbar CSF collection was also performed in 11 patients. Extracted ctDNA was used to analyze diagnostic point mutations in We detected either of the diagnostic mutations in tumor DNA samples from 28 of 34 patients. Among them, we achieved precise molecular diagnoses using intracranial CSF in 20 (71%). Univariate analyses revealed that the World Health Organization (WHO) grade (p = 0.0034), radiographic enhancement (p = 0.0006), and Mib1 index (p = 0.01) were significant predictors of precise CSF-based molecular diagnosis. We precisely diagnosed WHO grade III or IV diffuse gliomas using lumbar CSF obtained from 6 (87%) of 7 patients with tumors harboring any mutation.We established a novel, non-invasive molecular diagnostic method using a chip-based digital PCR system targeting ctDNA derived from CSF with high sensitivity and specificity, especially for high-grade gliomas.The online version of this article (10.1007/s11060-020-03682-7) contains supplementary material, which is available to authorized users. H3 K27M mutation ], diagnosis of which was confirmed by molecular findings [Molecular diagnosis of diffuse gliomas is conventionally performed using surgically obtained tumor tissues; however, the 2016 central nervous system (CNS) World Health Organization (WHO) classification defined a novel tumor [diffuse midline glioma, IDH1 R132H (IDH1), H3F3A K27M (H3 K27M), and TERT promoter (pTERT) mutations. Additionally, we evaluated the feasibility of this assay using CSF samples from patients with diffuse glioma.Recent progress in genetic analysis technologies such as digital PCR systems allows precise detection of genetic alterations from small samples , 3 and hIntracranial CSF samples were collected from adult and adolescent patients between July 2017 and November 2019 during surgical resection of intracranial tumors under a preoperative diagnosis of diffuse glioma. We collected 3–10 mL of CSF from the operative field immediately after opening the dura under general anesthesia to prevent contamination with DNA released from the resected tumor during surgical manipulation. We also preoperatively collected 1–6 mL of CSF by lumbar puncture from enrolled adult and adolescent patients who underwent tumor resection of the intracranial tumor under preoperative diagnosis of diffuse glioma without highly elevated intracranial pressure between March 2019 and February 2020. We excluded non-glioma patients after confirmation of pathological diagnosis.g for 10 min within 1 h of collection to remove residual cells and stored them at − 80 °C as described with slight modification [We centrifuged CSF samples at 5000 fication . DNA wasCSF samples were obtained and analyzed in accordance with the Declaration of Helsinki, with the approval of the Ethics Committee of our institute, and with written consent from patients.® 3D Digital PCR System . The DNA template, QuantStudio® master mix, and the assay containing primer/probe were mixed according to the manufacturer protocol to obtain a dPCR reaction.Genotyping of cfDNA and tumor DNA was performed using the QuantStudioTERT C228T and pTERT C250T analyses, and a custom TaqMan SNP genotyping assay was used for IDH1 and H3 K27M mutation analyses. The primer and probe sequence data for each assay are described in the Supplemental Digital Content . The final 14.5-µL dPCR mixture was loaded onto a QuantStudio® 3D Digital PCR chip version2 and subjected to PCR amplification using the QuantStudio® 3D GeneAmp PCR system 9700 . Thermal cycling was performed according to the manufacturer’s instructions . Following the reaction, data were analyzed using QuantStudio® 3D Analysis Suite (v.3.1).The TaqMan dPCR liquid biopsy assay (Life Technologies) was used for pIDH1, pTERT C228T or C250T, and H3 K27M point mutations by high-resolution melt analysis and subsequent Sanger sequencing, as described previously [We isolated and purified DNA from snap-frozen (− 80 °C) intraoperative tumor samples obtained from all patients whose CSF was analyzed, using QIAmp DNA mini kits (Qiagen). We confirmed the presence of eviously , 14. Loseviously .Tris–acetate-EDTA (pH 8.0) was used as electrophoresis buffer, and a 1.5% agarose gel was used for the electrophoresis of a sample prepared by mixing 5 µL of DNA template with 6X loading buffer . Electrophoresis was performed using a Mupid R-exu system  at 100 V for 30 min, followed by staining with EtBr solution and detection and imaging using a Limited-STAGE-UV system .t test was used to compare the DNA concentrations at different collection sites. Associations between ctDNA positivity and patient characteristics were assessed using non-parametric tests, including the Wilcoxon rank-sum test (continuous variables) or Fisher’s exact test . All statistical tests were two-sided, with an α value of ≤ 0.05 used to determine statistical significance. Statistical analysis was performed using JMP software .Student’s IDH1 and H3 K27M assays, respectively .To verify the reproducibility of PCR products amplified from CSF-derived cfDNA, two pairs of cfDNA and the corresponding tumor-tissue DNA obtained from different patients were subjected to gel electrophoresis Fig. . DiscretFigure IDH-positive and -negative samples is shown in the Supplemental Digital Content .For mutation analysis, we used a pair of VIC- and FAM-labeled TaqMan probes annealing to wild type and mutant alleles, respectively , 19, andAmong 53 patients from whom intracranial CSF was obtained during surgery, 34 had confirmed, pathologically-diagnosed, diffuse glioma. Table IDH1 R132H (n = 16), pTERT C228T (n = 22) and C250T (n = 32), and H3 K27M (n = 32) assays. There were no false-positive findings among cfDNA results when tumor-tissue DNA results were used as controls. These results confirmed the high specificity of these assays. Because 28 (82%) of 34 tumors harbored at least one mutation in tumor-tissue DNA, we evaluated the reliability of dPCR within these results. The genotypes of all glioblastoma (GBM) samples (n = 7) were confirmed by using dPCR, with the H3 K27M mutation confirmed in 4 of 5 patients with DMG, H3 K27M-mut. IDH and pTERT co-mutations, a hallmark of oligodendroglioma [IDH mutation. Univariate analyses revealed that the WHO grade (p = 0.0034), radiographic enhancement (p = 0.0006), recurrence (p = 0.0084), and Mib1 index (p = 0.01) were significant predictive factors for precise CSF-based molecular diagnosis; however, DNA concentration and radiographic dissemination were not predictive (Table Supplementary Table 5 shows a comparison of the genotype profiles between dPCR results with CSF cfDNA and Sanger sequencing with the corresponding tumor-tissue DNA. Samples without mutation(s) in the corresponding tumor were determined as WT in CSF cfDNA for roglioma –22, wereIDH mutation without radiographic enhancement and a recurrent anaplastic oligodendroglioma manifesting radiographic enhancement and dissemination. The results showed that 6 (87%) of 7 WHO grades III and IV gliomas were precisely diagnosed preoperatively, whereas neither of the grade II tumors could be preoperatively diagnosed. Similar to results obtained from intracranial CSF, lumbar CSF samples did not return false-positive results during genotyping. Notably, grade IV tumors were accurately diagnosed preoperatively, even in patients with cytology class I or without radiographic dissemination . Of the 16 patients undergoing pre-surgery lumbar puncture, pathological diagnosis of diffuse glioma was confirmed in 11, which were enrolled for subsequent cfDNA genotyping Table . Nine ofIDH mutation and the 1p19q co-deletion as essential markers for a molecular diagnosis of diffuse glioma, and the H3 K27M mutation as an essential marker of pediatric glioma [TERT mutation is recognized as a novel essential diagnostic marker based on its presence in GBMs and oligodendrogliomas [TERT mutations as the most prevalent molecular markers detected in IDH-WT GBMs and predictive of aggressive bioactivity of diffuse astrocytomas [IDH-WT diffuse or anaplastic astrocytomas with pTERT mutation as WHO grade IV [IDH-mutant oligodendroglial tumors, the status of 1p19q and pTERT are strongly correlated, with these markers interchangeable when combined with IDH status [H3 K27M-mut predominantly occurs in children and adolescents, high-grade thalamic gliomas from young adults frequently harbor H3 F3A-K27M [H3 K27M-mut included three adult-onset cases. Moreover, the frequency of elderly patients with DMG, H3 K27M-mut is notable [H3 K27M-mut that mimicked a hemispheric malignant glioma [H3 status of an adult hemispheric diffuse glioma affecting the midline should be confirmed even in elderly patients. It is conceivable that the majority of diffuse gliomas can be molecularly diagnosed by confirming IDH, H3 K27M, and pTERT status. In the present study, we selected these genes based on this interpretation and also their status as hotspot point mutations, which enable their detection by dPCR via design of a single TaqMan probe per mutation.In this study, we describe the establishment of a novel method for liquid biopsy targeting the ctDNA in CSF using chip-based dPCR to detect glioma-specific diagnostic driver mutations. The 2016 CNS WHO classification and subsequent cIMPACT-NOW update incorporated various glioma-specific genetic alterations in order to integrate molecular diagnoses , 23, 24.ogliomas . Recent ocytomas . cIMPACTgrade IV , 26. In H status . Althoug notable , 29. We t glioma . Taken tAlthough other liquid biopsy methods for glioma diagnosis exist, sensitivity is essential for selection of analytical methods for future clinical applications. The chip-based dPCR method presented here yielded highly sensitive results using lumbar puncture CSF, especially for diagnosis high-grade diffuse gliomas. Pentsova et al.  collecteTERT, IDH, and H3 K27M, in 15 (88%) of 17 patients using CSF collected from lumbar puncture. This indicated that ddPCR showed comparable or higher sensitivity relative to the dPCR assay in the present study; however, Martinez et al. [Currently, commercially available dPCR systems are theoretically divided into droplet digital (dd) PCR and chip-based dPCR. The sensitivity of ddPCR exceeds that of NGS . Martinez et al.  used thez et al. . Given tOne of the major limitations of the present study is that the reliability of our method seemed to depend on the extent of CSF involvement in tumor distribution. Therefore, our method might be most appropriate for diagnosing advanced high-grade glioma involving CSF. However, we should take into consideration that lumbar puncture is not always indicated for patients with advanced high-grade glioma, because of risk of herniation by space-occupying lesions. These issues are associated with another limitation of the present study that most of our samples were obtained by craniotomy, and lumbar CSF was collected from a limited number of our patients. We detected diagnostic mutations in 28 of 34 patients with glioma using intracranial CSF and the analyzed genes in the remaining six gliomas did not harbor any diagnostic mutations even in tumor tissues. This issue is associated with another limitation that gliomas cannot always be differentially diagnosed from non-glial tumors through genotyping of diagnostic mutations. The sensitivity of our method is lower for lumbar than for intracranial CSF is another limitation. The sensitivity of our method needs to be improved, even for lumbar CSF, before less-invasive liquid biopsies could be routinely used for diagnostic purposes.Here, we showed that molecular analysis of diffuse glioma can be performed using chip-based dPCR of ctDNA extracted from patient CSF. Lumber CSF analysis is a less invasive than conventional molecular diagnostic methods. This approach facilitates further development of novel methods of molecular diagnosis and future paradigm shifts in treatment strategies for and clinical management of glioma.Fig 1 (a) Establishment of threshold values for determining VIC- and FAM-positive results in the IDH1 R132H assay. A negative control assay was performed with a final concentration of 1 ng/μLof the sample. The scatter plot shows bimodal VIC-negative with a positive distribution (left). We established the threshold value for determining VIC positivity at a certain distance (indicated as “X”) apart from the mode point of the VIC histogram (center). The threshold value for determining FAM positivity was set at a certain distance (indicated as “Y”) apart from the mode point of the FAM histogram (right). “X” was calculated as the average distance from the peak value of the first distribution and the lowest value of the second distribution. “Y” was determined as the distance from the mode, with the maximum value of the distributions excluding sporadic high-fluorescence spots (arrow heads) within quadruplicate negative control reactions. (b) Validation of threshold values with lower-concentration samples. We performed negative control assays with final concentrations of 0.1 ng/μL (upper) and 0.01 ng/μL (lower) samples. The results indicated no spots in the upper-left area and only sporadic spots in the upper-right area. (c) Example of a sample with a mutation. Based on our threshold values for VIC- and FAM-labeled samples, mutant spots are shown in the upper-left area (blue). Fig 2 Representative result of chip-based digital PCR for the IDH1 R132 mutation based on FAM- and VIC-fluorescence channels. The clustered yellow dots show empty wells. Blue dots indicate FAM-positive wells involving amplification of a mutant allele, and red dots show VIC-positive wells involving amplification of a wild-type allele. The left image shows a cluster of blue dots indicating a mutant genotype. The right image shows a result for a wild-type genotype. Fig 3 Representative 18-year-old female patient with diffuse midline glioma, H3 K27M-mutation (a) A hyper-intensity lesion with slight gadolinium enhancement extending from the brain stem to cerebellum  was biopsied and diagnosed as diffuse midline glioma. (b) Hematoxylin and eosin staining revealed diffuse infiltration of glioma cells with roundish, monotonous, hyperchromatic nuclei, mainly in the subcortical white matter. (c) Immunohistochemistry revealed H3 K27M-positive cells located sporadically among background neural cells. (d) We extracted DNA from surgically obtained tumor tissue and performed digital PCR to reveal the H3 K27M mutation, which was not detected by Sanger sequencing . (e) Digital PCR revealed the mutation in both intracranial and lumbar cerebrospinal fluids .Supplementary material 1 (PDF 3511 kb) Below is the link to the electronic supplementary material.


Macrophage colony-stimulating factor (M-CSF), matrix metalloproteinase-2 (MMP-2) and its specific tissue inhibitor (TIMP-2) may play an important role in the pathogenesis of cancer disease. We investigated the plasma levels and diagnostic power  of M-CSF, MMP-2, TIMP-2 and tumor markers CA 125 and SCC-Ag in cervical cancer (CC) patients as compared to control group. The study included 89 patients with cervical cancer. The control group consisted of 50 healthy, untreated women. The plasma levels of M-CSF, MMP-2 and TIMP-2 were determined using ELISA, CA 125 and SCC-Ag – by CMIA method. The median levels of M-CSF, TIMP-2, SCC-Ag and CA 125 in the entire group of CC were significantly different than compared to the healthy women group. MMP-2 showed the highest value of sensitivity from all examined parameters . The highest specificity was obtained by M-CSF (86%). The area under the ROC curve (AUC) of M-CSF (0.8051) was the largest of all the tested parameters (even higher than commonly used tumor markers) in the group of cervical cancer. The combination of M-CSF, MMP-2 or TIMP-2 with SCC antigen resulted in an increase AUCs in all cases . The findings of this study suggest the usefulness of all examined parameters in the diagnostics of CC patients. Out of the tested substances, M-CSF also appears to be the best candidate for cancer diagnostics in all stages of the disease, based on ROC analysis. Cancer of the uterine cervix is the leading cause of death among gynecological cancers in developing countries and the fourth principal cause of cancer-related death in women worldwide . High-riReliable methods for the accurate identification of both the presence and severity of cervical intraepithelial neoplasia (CIN) and extent of spread of invasive carcinomas of the cervix (IC) are critical. The primary objective of cervical cancer screening is to detect cervical intraepithelial neoplasia CIN 3) sufficiently early so that it can be treated to prevent the development of cancer. Screening based on cytological testing (commonly known as the smear test or Pap test) is still the most important diagnostic technique for detecting pre-invasive cervical cancer [ sufficie1-matrix metalloproteinase 1 (MT1-MMP) and TIMP-2 [Macrophage colony-stimulating factor (M-CSF) is a hematopoietic growth factor that stimulates the proliferation and differentiation of monocytes to macrophages. In inflammation M-CSF induces macrophages to secrete cytokines and proteases, thereby enhancing the macrophages’ ability to combat microbial infections . Increasd TIMP-2 . Imbaland TIMP-2 . TIMP-2 d TIMP-2 –21.The aim of this study was to determine plasma levels of M-CSF, MMP-2 and TIMP-2 in comparison to CA 125 and SCC-Ag concentrations in patients with cervical cancer in relation to the control group . Additionally, the diagnostic criteria: sensitivity (SE) and specificity (SP) were defined in the study. Furthermore, the study defined the receiver-operating characteristics (ROC) curve for all the tested parameters alone and in combination with tumor markers (CA 125 and SCC-Ag). The data obtained in the present study may be used in evaluating the usefulness  of the tumor marker panel in the diagnostics of cervical cancer patients.Table Venous blood samples were collected from each patient. Blood was collected into heparin sodium tubes, centrifuged 3500 rpm for 20 min to obtain plasma samples, and stored at −85 °C until assayed.The examined parameters  were measured with enzyme-linked immunosorbent assay (ELISA) , according to the manufacturer’s instructions. This assay employs the quantitative sandwich enzyme immunoassay technique. Plasma concentrations of SCC-Ag and CA 125 were measured by chemiluminescent microparticle immunoassay (CMIA) . The intra-assay coefficient of variation (CV%) of CA 125 is reported to be 2.4% at a mean concentration of 43.50 U/mL, SD = 1.1. SCC-Ag is reported to be 4.3% at a mean concentration of 1.97 ng/mL, SD = 0.085. M-CSF is reported to be 3.4% at a mean concentration of 227 pg/mL, SD = 7.7. MMP-2 is reported to be 3.8% at a mean concentration of 11.20 ng/mL, SD = 0.42, TIMP-2 to be 6.0% at a mean concentration of 2.90 ng/mL, SD = 0.173. The inter-assay coefficient of variation (CV%) of CA 125 is reported to be 3.9% at a mean concentration of 43.50 U/ml, SD = 1.7. SCC-Ag is reported to be 5.1% at a mean concentration of 1.97 ng/mL, SD = 0.1. M-CSF to be 3.1% at a mean concentration of 232 pg/ml, SD = 7.3. MMP-2 is reported to be 6.6% at a mean concentration of 11.10 ng/mL, SD = 0.738, TIMP-2 to be 6.7% at a mean concentration of 2.79 ng/mL, SD = 0.188. The value of intra- and inter-assay CVs were calculated by the manufacturers and enclosed in the reagent kits. The assay does not exhibit cross-reactivity or interference with numerous human cytokines and other growth factors. Duplicate samples were assessed for each patient.p < 0.05. Diagnostic sensitivity (SE) and specificity (SP) were calculated. The cut off values were calculated by Youden’s index (as a criterion for selecting the optimum cut-off point) and for each of the tested parameters was as follows: M-CSF – 394.42 pg/mL; MMP-2 – 155.92 ng/mL; TIMP-2 – 82.12 ng/mL; CA 125–13.44 U/mL; SCC-Ag – 0.89 ng/mL. In the analyses of both diagnostic performance  and ROC curve, only healthy subjects were used as a control group. The construction of the ROC curves was performed using the GraphRoc program for Windows  and the areas under the ROC curve (AUC) were calculated.Statistical analysis was performed using STATISTICA 12.0 PL . Preliminary statistical analysis (Chi-square test) revealed that the tested parameters and tumor marker levels did not follow a normal distribution. Consequently, the Mann–Whitney U test was used for statistical analysis between cancer and control group. Additionally, statistical analysis between groups with different stages of CC was performed using the Kruskal–Wallis test, and a multivariate analysis of various data - with the post-hoc Dwass Steele–Crichlow–Flinger test. Statistically significant differences were defined as comparisons resulting in p < 0.05). Additionally, the median value of TIMP-2 (76.00 ng/mL) in the total cervical cancer group was significantly lower compared with the values in healthy subjects (87.25 ng/mL) (p < 0.05).Table p < 0.05 in all cases). Additionally, we observed significantly higher concentrations of CA 125 in all the analyzed groups in all stages of cervical cancer (with the exception of stage I) in comparison with healthy women. Significantly higher concentrations of MMP-2 in stages III and IV were also observed (p < 0.05). Furthermore, significantly lower concentrations of TIMP-2 in stage I were also noticed (p < 0.05).Similarly, we observed statistically significantly higher concentrations of M-CSF in all the analyzed groups in relation to CC stage compared with healthy women (p < 0.05 and II vs. III and IV p < 0.05). Furthermore, we detected significantly higher plasma levels of CA 125 when stages III and IV was compared to stage I (p < 0.05).We also observed significantly higher concentrations of TIMP-2 in stages III and IV of cancer compared with stage I or II (TIMP-2: I vs. III and IV Diagnostic criteria for tumor markers are sensitivity (SE) and specificity (SP) Table . We indiThe diagnostic SP of the tested parameters was the highest for M-CSF (86%) and was higher than that for CA 125 (68%) and SCC-Ag (74%).p < 0.001; p = 0.0257; p < 0.001; p < 0.001; respectively) The combined analysis of AUC for M-CSF, MMP-2, and TIMP-2 with antigen SCC resulted in an increase in the areas in all cases  , similarly to M-CSF (p = 0.0011), TIMP-2 (p = 0.0064) and CA 125 (p = 0.0188)  and it was marginally higher than SCC-Ag (0.7973). Moreover, the AUCs for M-CSF and TIMP-2, similarly to those for CA 125 and SCC-Ag, were statistically significantly larger in comparison to AUC =0.5   and it was marginally higher than CA 125 . Additionally, the AUCs for M-CSF and MMP-2, similarly as for CA 125 and SCC-Ag, were statistically significantly larger in comparison to AUC =0.5 . .The AUC of M-CSF and MMP-2 demonstrated a distinct increase, concomitant with CC stage, identically to CA 125. In stage I of CC the highest AUC of all the tested parameters was found in SCC-Ag (0.8022) and it was the parameter which was statistically significantly larger in comparison to AUC = 0.5  than in healthy subjects 290.82 pg/mL) . Our obs2 pg/mL .In our study, the sensitivity of MMP-2 was the highest out of all the tested parameters (92.05%). Ghosh et al.  also shoIn this study, the SP of M-CSF (86%) was the highest out of all the studied parameters and was higher than that of SCC-Ag (74%) and CA 125 (68%). In the case of M-CSF, the specificity reported in the paper by Ławicki et al.  on cerviThe area under the ROC curve indicates the clinical usefulness of a tumor marker. In this study, the area under the ROC curve of M-CSF was the largest out of all the tested parameters in the total CC group. Additionally, we observed statistically significantly larger AUCs for the tested parameters (with the exception of MMP-2), compared to AUC = 0.5 (borderline of diagnostic usefulness of the test). The combined analysis of AUC for all the tested parameters with the commonly accepted tumor marker (SCC-Ag) resulted in an increase in the areas in all cases. A study by Ławicki et al.  reportedThe findings of this study suggest the usefulness of M-CSF, MMP-2 and TIMP-2 in the diagnostics of CC patients, particularly in combination with CA 125 and SCC-Ag. The area under the ROC curve was highest for the combination of M-CSF and the commonly accepted tumor markers, which indicates potential clinical significance of plasma M-CSF in the diagnosis of CC. Out of the tested substances, M-CSF also appears to be the best candidate for cancer diagnostics in all stages of the disease, based on ROC analysis.


Climate change driven increases in the frequency of extreme heat events (EHE) and extreme precipitation events (EPE) are contributing to both infectious and non-infectious disease burden, particularly in urban city centers. While the share of urban populations continues to grow, a comprehensive assessment of populations impacted by these threats is lacking. Using data from weather stations, climate models, and urban population growth during 1980–2017, here, we show that the concurrent rise in the frequency of EHE, EPE, and urban populations has resulted in over 500% increases in individuals exposed to EHE and EPE in the 150 most populated cities of the world. Since most of the population increases over the next several decades are projected to take place in city centers within low- and middle-income countries, skillful early warnings and community specific response strategies are urgently needed to minimize public health impacts and associated costs to the global economy. Ongoing climate variability and change are increasing the frequency, duration, and intensity of extreme weather events across the globe ,2. RecenIncreases in frequency of EHE and EPE may be higher in urban areas characterized by high population density and worsening air quality ,26,27. TTo address these issues, we characterized the frequency of EHE and EPE between 1980 and 2017 across the 150 most populated metropolitan locations of the world and estimated the trend in exposed populations using meteorological data from monitoring stations as well as climate models. We further analyzed this data to better characterize seasonal, regional, and temporal trends.We acquired information on the 150 most populated metropolitan locations of the world with populations over 3 million from the World Atlas  and downhttps://www.ncdc.noaa.gov). If a metropolitan location had multiple meteorological stations, we used the one closest to the urban core. If no station data were available, we borrowed information from stations that were located within a 50 km radius of the location boundary or set it to “missing” if no stations were available within the 50 km radius. We used 20 years (01/1980–12/1999) of daily meteorological data  as the base period to calculate calendar day and location-specific 95th percentile thresholds to define EHE and EPE. For example, the baseline data for Beijing for January 16th consisted of all daily observations for Beijing from 1 January to 31 January from 1980 to 1999. If a metropolitan area had more than 30% of daily observations missing during the baseline period (1980–1999), we dropped them from the analysis. Based on the distribution of this data, we identified location and calendar day specific 95th percentile threshold values for TMAX and precipitation, referred to as EHE95, and EPE95 thresholds, respectively. Calendar day-specific TMAX and precipitation values for each metropolitan region were compared with their respective EHE95 and EPE95 thresholds and assigned a value of “1” if they exceeded the threshold, and “0” otherwise [95 were identified as extreme heat events, and days where the daily total precipitation exceeded the EPE95 thresholds were identified as extreme precipitation events.We obtained daily maximum temperature (TMAX) and precipitation data for 1980–2017 from the Global Historical Climatology Network (GHCN) through the National Climate Data Center (NCDC) data portal  and fall  definitions followed suit. We calculated seasonal and yearly sums of EHE and EPE for all metropolitan locations. We computed yearly exposed populations for each city based on the yearly population and frequency of EHE and EPE for the respective cities . We alsoOf the 150 largest cities, 84 had valid weather data during the 1980–2017 period that satisfied our inclusion criteria. On average, these 84 cities were missing less than 8% of daily observations . For theAs depicted in Based on regression analysis, we identified the top 10 metropolitan locations with the highest seasonal rate of increases in EHE and EPE frequencies . Six outWhen we considered cities individually, Bangkok had the fastest rate of seasonal increases in EHE during fall , while Kyiv had the fastest rate of EHE increases during summer . Likewise, Abidjan had the fastest rate of EHE increases during winter , while the rates of increase were highest in Kunming during spring . Overall, we observed higher rates of EHE increases during the summer and fall seasons. Cities such as Hyderabad and Bangkok experienced EHE increases across all seasons while the rate of EHE increases for other locations were confined to only one or two seasons A. For EPWe also explored the relationship between EHE/EPE calculated using weather station data vs. ERA5 reanalysis products . OverallWhile the list of adverse human health impacts that are tied to the ongoing climate change continues to grow, the geographic distribution of populations that are exposed to extreme events and therefore, at risk of experiencing adverse health outcomes are lacking at a global scale. Taking into consideration both the rapid rise in urban populations and the disproportionate increases in EHE and EPE in major cities ,31, we sOur data showing increases in EPE across cities in the Western Pacific Region during the 2010s is consistent with Huang et al. , who tieWhen we compared EHE results that were derived using weather station data to those derived using ERA5 reanalysis products, the agreement was considerably better for EHE compared to EPE. This is expected since simulating precipitation is generally much more challenging for models than simulating temperature . AlthougThere are several strengths of our study. This study focused on potential exposures to extreme heat and precipitation events in the 150 major cities of the world over the past four decades. We further analyzed how these exposures varied across WHO regions and latitudes, which provided valuable information for assessing regional disparities in exposure and potential health impacts. Our findings will inform public health preparedness, particularly across low- and middle-income countries where such data have been lacking. Our study has some limitations as well. First, only 84 out of the 150 cities had weather station level data that matched our selection criteria, which limited our analysis. To overcome this, we used ERA5 reanalysis products. However, limited agreement between ERA5 reanalysis products and weather station level extreme event data, particularly with regards to EPE, raises concerns about just relying on the ERA5 based analysis alone. Nevertheless, these findings also underscore the need for sustained monitoring stations to capture the expected continuation of increases in weather extremes in a warming world.Given the linkage between exposure to extreme events and impaired health ,2,3, ourThe confluence of disproportionate increases in the frequency of extreme weather events in city centers, and rapid rises in urban populations has turned major cities across the globe into flashpoints for climate change driven disease burden with a 500% increase in populations exposed to extreme heat and precipitation events over the past four decades. The projected increases in the frequency of extreme heat events and the continued rises in urban populations combined with ever increasing disparities in income, food, and healthcare access in urban areas will only worsen public health outcomes in these city centers in coming decades. Cities across the globe need to prepare for such increases in exposed populations and begin evaluating the efficacy of their adaptation strategies.


Regarding liver transplantations in Japan, with no progress having been made in deceased donor liver transplantations, a living donor liver transplantation performed on a boy with end-stage liver cirrhosis caused by biliary atresia by Nagasue et al. at Shimane University in November 1989 was the first case of its kind. Unlike deceased donor liver transplantations, living donor liver transplantations have two major advantages. First, because organs are donated from healthy adults, it is possible to transplant organs with better viability compared to deceased donor organs, which have been preserved in cold storage for a long time. Second, depending on the recipient’s condition, it is possible to conduct elective surgery at the optimal time. In Japan, the number of annual liver transplantation cases is approximately 400, with the number of annual pediatric liver transplantation cases stable at approximately 120 cases. The patient survival rate of pediatric liver transplantation cases is relatively good at 89.4% over the course of 1 year, 86.8% over 5 years, 84.4% over 10 years, and 80.9% over 20 years.The liver transplantation program was initiated at the National Center for Child Health and Development, Tokyo, Japan, in November 2005, providing liver transplantation treatment to 510 pediatric patients with end-stage liver disease to date. This article outlines the history of liver transplantations in Japan along with the present state of liver transplantations at the National Center for Child Health and Development. Although the results were initially unsatisfactory, they remarkably improved because of advances in patient selection, surgical techniques, organ preservation methods, immunosuppressive therapy, and perioperative management, among others. In the United States, approximately 7,500 deceased donor liver transplantations are conducted annually; therefore, this can be considered an established medical treatment for patients with end-stage liver diseases. Liver transplantations in Europe and the United States are centered on deceased donor liver transplantation by harvesting organs from brain-dead or cardiac arrest donors. In Japan, the Organ Transplantation Law took effect in 1997, at which time deceased donor liver transplantation became legally feasible. In 2004, the World Health Organization called on member states as the Istanbul Declaration “to take measures to protect the vulnerable groups from transplant tourism and the sale of tissues and organs, including attention to the wider problem of international trafficking in human tissues and organs.” All countries require a professional framework to govern organ donation and transplantation activities with transparency, as well as a transparent regulatory system that ensures donor and recipient safety and the enforcement of standards and prohibitions on unethical practices. Each country should strive to ensure that programs to prevent organ failure are implemented and provide organs to meet the transplant needs of its residents from donors within its own population (2). Thereafter, the “Revised Brain Death Bill” in 2010 enabled organ donations from individuals aged 15 years or younger with the consent of their relatives, resulting in a marginally increased tendency for deceased donor organ donation; however, the demand of patients waiting for organ transplantations has not been sufficiently satisfied.Clinical liver transplantation, a medical treatment with a short history, was initiated in 1963 by TE Starzl et al. in the United States (3). Living donor liver transplantation is a medical treatment that takes advantage of the following two features: the liver is an anatomically divisible organ, and the liver is a regenerable organ.With the background that there has been no progression in deceased donor liver transplantations, liver transplantations in Japan have been centered on living donor liver transplantations using a partial liver from healthy relative donors. The living donor liver transplantation performed on a boy with end-stage liver cirrhosis caused by biliary atresia by Nagasueet al. at Shimane University in 1989 was the first case of its kind in Japan Unlike deceased donor liver transplantations, living donor liver transplantations have two major advantages. First, as organs are donated from healthy adults, it is possible to transplant organs with better viability compared to deceased donor organs that have been preserved in cold storage for a long time. It is possible to sufficiently evaluate donors prior to planned transplant surgery, and because the cold storage duration from organ harvesting to transplantation is short because of organ harvesting and transplantation being conducted at the same facility, it is possible to provide a liver with excellent quality blood vessels to be reconstructed and stable conditions for recipients in many cases. Second, depending upon the recipient’ condition, it is possible to conduct elective surgery at the optimal time, even for patients whose condition has suddenly deteriorated or patients who need very urgent treatment. If the consent and safety of a living donor is guaranteed, it is possible to perform liver transplantation as a scheduled surgery with the cooperation of the anesthesiology department, intensive care unit, radiology department, pathology department, nursing department, and administrative department.(4). In Japan, there is a desire to spread the word and enlighten the population with regard to deceased donor liver transplantations, which are commonly conducted in Europe and the United States.In contrast, the disadvantage of living donor liver transplantation is that a liver resection for organ donation is required in healthy living donors despite there being no medical benefits. As donor deaths due to living donor liver transplantation have been reported previously, it is difficult to guarantee the complete safety of living donors because they undergo medically unnecessary liver resection, which is a significantly invasive surgery (5). In the United States, pediatric liver transplantation cases comprised 7.3% of the total transplantation cases, which is negligible compared with that for adults (6). This does not mean that pediatric liver transplantations are being proactively performed in Japan. This is because liver transplantations are not sufficiently indicated in adults who can benefit by liver transplantations. In Japan, the number of annual liver transplantation cases indicates a decreasing tendency from 2005. Donor deaths caused by living donor liver transplantations were also reported in Japan on May 5th, 2003 (7). Adult living donor liver transplantations, which require a larger volume donor liver resection compared with pediatric living donor liver transplantations, are believed to be declining in terms of the number of cases from the viewpoint living donor safety. Nevertheless, the number of deceased donors has doubled since the revised organ transplant law in 2010; the intensification of efforts to reduce living donor morbidity and increase the number of deceased donors have remained important issues in Japan that require attention.In Japan, 8,347 cases of liver transplantation were conducted over a 26-year period from 1989 to the end of 2017 . The num(5). Cholestatic liver diseases represented by biliary atresia account for 73%. Biliary atresia is an unexplained cholestatic liver disease occurring at a frequency of 1/5,000 to 1/19,000 births and is a serious disease leading to cirrhosis from cholestasis. Kasai surgery (portoenterostomy) was developed by Morio Kasai from Tohoku University in the 1950’s, resulting in a dramatic improvement in the short-term survival rate (8). However, the 20-year survival rate of autologous livers (without liver transplantation) following Kasai surgery is reportedly 49%, suggesting that liver transplantation is required to be performed in childhood for approximately half of the patients as of now (9). Metabolic liver disease accounts for 10% of the total indication of liver transplantation. Metabolic liver disease is diagnosed by clinical symptoms, such as low activity after birth and vomiting, as well as blood sampling results, such as hyperammonemia and acidosis. If medical treatment for metabolic failure fails, liver transplantation is occasionally indicated. Fulminant hepatitis is a disease leading to liver transplantation indication because of the deterioration of liver functions triggered by drug/virus infections, etc., accounting for approximately 9%. Moreover, liver malignancies represented by hepatoblastoma are also surgically unresectable cases and can be indicated for liver transplantation (10). The results of pediatric liver transplantation in Japan indicated a 1-year survival rate of 89.4%, a 5-year survival rate of 86.8%, a 10-year survival rate of 84.4%, and a 20-year survival rate of 80.9% in patients, demonstrating relatively better results than those of deceased donor liver transplantations in Europe and the United States. Although it is necessary to take a life-long immunosuppressive drug, pediatric liver transplantation can be considered as an established general medical treatment (4). However, a decrease in survival rate because of chronic rejection and nonadherence is a major problem in long-term follow-up cases. Going forward, proactive participation of pediatricians in transplantation medicine is desired in Japan.The National Center for Child Health and Development, Tokyo, Japan, launched the liver transplantation program in 2005, and the Organ Transplantation Center was established in May 2011. We have performed 510 cases of liver transplantation as of May 31, 2018 . The num(11). In the event of pediatric living donor liver transplantation, the left lateral segment  of the liver of adult living donors is used as the transplanted liver; however, the average weight of the left lateral segment of the liver of living liver transplantation donors at our center is 244.3 ± 44.0 g, which is still too large a graft for infants suffering from liver failure with a body weight of 6 kg or less. Therefore, our center has developed a hyperreduced graft from the left lateral segment in which the graft was further surgically reduced and resected. With the development of this new surgical procedure, the results of transplantation for neonatal/infant metabolic liver diseases and fulminant hepatitis remarkably improved (12) ((13). In some cases of neonatal metabolic liver disease, because the liver is morphologically normal, transplanting normal hepatocytes into the livers of newborns for the purpose of supplementing the enzyme makes it possible to temporarily avoid severe metabolic decompensation. Thus far, we performed hepatocyte transplantations on two newborns with urea cycle disorder who remain well and visit our center on an outpatient basis without neurological complications.Transplanting a whole liver of an adult into the small body of a child is difficult to adapt because it is impossible to maintain sufficient blood flow to the transplanted liver due to compartment syndrome. It has been reported that the optimal transplanted liver weight for children is 2%–4% of their body weight  ved (12) . Liver t(14). The procedure of living donor liver transplantations can also be sufficiently applied to split-liver transplantations. Since the revision of the law in July 2010, our center has registered 120 pediatric patients with end-stage liver disease on the deceased donor liver transplant waiting list and safely performed split-liver transplantations in 20 patients. If the indications are made stricter, even in children, it is possible to expand the donor pool for brain-death liver transplantations by indicating split-liver transplantations.Even in Europe and the United States, where deceased donor liver transplantations are performed as a major organ transplantation procedure, the number of deceased donor organs available for children is very limited. To reduce the number of deaths of pediatric patients with end-stage liver disease waiting for transplantation due to chronic lack of pediatric deceased donor donors, split-liver transplantation, which involves splitting the liver graft from adult deceased donors into the left lateral segment or the left lobe and the right lobe and transplanting each into a child and an adult, respectively, has been developed Our center often receives referred patients with serious high-risk/complications of other organ disorders in the preoperative state, in addition to many cases in which determining the indications for transplantation, management following surgery, or immunosuppressive therapy is difficult. In recent years, the number of patient referrals where it is difficult to manage the perioperative period in transplantations has been increasing. With the aim of further improving treatment results, it is essential to continue clinical research, establish innovative transplantation treatment methods, and develop human resources at the center. In order to spread understanding regarding pediatric organ transplantations, we have opened our doors to other institutions and are accepting many doctor trainees from home and abroad. Particularly in Islamic countries, deceased donor transplantation is difficult for religious reasons, making living donor transplantation the only treatment procedure for patients with end-stage organ failure. The National Center for Child Health and Development proactively accepts doctors/healthcare professionals from Islamic countries and develops medical professionals familiar with transplantation, making it possible to introduce safe living donor liver transplantation to each country. It is already well known as an international pediatric organ transplant institution. To further pursue the safety of pediatric organ transplantation and improve the prognosis thereof, it is important to establish strict indications of transplantation, postoperative management, immunosuppressive therapy, and infection control methods in deceased donor liver transplantation. The National Center for Child Health and Development not only performs clinical organ transplantations but is also involved in joint research, including, with regard to medical hepatocyte transplantation, the development of new immunosuppressive methods, the control of viral infectious diseases following transplantation, clinical application of multivisceral organ transplantation, and regenerative medicine using embryonic stem cells in cooperation with research centers and clinical research and development centers for future children. Moreover, diseases leading to liver transplantation are diverse, many of which are rare diseases that are rarely seen in daily general practices. There are many details to learn from rare diseases, and we are also intensively studying their causes and treatment methods.Currently, pediatric care in Japan has undergone a significant transformation. Because the system that administers anti-infectious vaccinations has improved and a reduction is expected in the number of hospitalizations because of bacterial meningitis, sepsis, dehydration, and respiratory infections, foundation hospitals for pediatric care must appropriately respond to the necessities of highly advanced medical treatments, including the treatment of newborns with complications, maternal therapy, congenital diseases, pediatric cancer, and organ transplantations.Organ transplantation is a form of medical care established with the intention of providing precious organs to others. There are very few organ donations from brain-dead patients in our country, and most transplantations for pediatric patients with end-stage organ failure are primarily performed as living donor transplantations with relatives as donors. In the case of pediatric patients with end-stage organ failure who have small bodies, size mismatches with organs from living donors are occasionally observed, rendering it difficult to perform the transplantation. Moreover, there are organ transplantations in which it is impossible to perform living donor transplantations, such as heart transplantations. Because there are many pediatric patients whose lives cannot be saved under the present circumstances of organ transplantation and are dependent on living donors, going forward, enlightenment and promotion of deceased donor organ transplantation will be considered very important issues in Japan. The National Center for Child Health and Development will humbly accumulate such cases so that the lives of more pediatric patients with end-stage organs can be saved. In addition, the center will work to develop new transplantation procedures, study immunosuppressive methods and clinical applications of regenerative medicine, and develop international medical professionals. We hope that transplantations at our center will help further the development of pediatric care/medical care in Japan.NoneThis work was supported by grants from the Scientific Research Fund of the Ministry of Education and a Research Grant for Immunology, Allergy and Organ Transplant, Rare and Intractable Disease from the Ministry of Health, Labor and Welfare, Japan .M.K.: study design, writing of the paper, S.S., A.F., R.H., A.U., Y.M., H.K., T.I.: study design, critical revision of the article for clinical content.


Canada has been home to comparatively extreme developments in prescription opioid (PO) availability and related harms  post-2000. Following persistent pan-Canadian increases in PO use, select control measures were implemented and PO dispensing levels—while only inconsistently by province—inverted, and began to plateau or decrease post-2012. We examined annual PO dispensing levels in Canada up until 2018, based on representative prescription sample data from community-based retail pharmacies. Annual prescription-based dispensing data were converted into defined daily doses/1000 population/day by province, and mainly categorized into ‘weak’ and ‘strong’ opioids. All provinces indicated decreasing trends in strong PO levels in most recent years, yet with inter-provincial differences of up to one magnitude in 2018; in about half the provinces, dispensing fell to below-2005 levels. British Columbia had the largest decline in strong PO dispensing from its peak rate (− 48.5%) in 2011. Weak opioid dispensing trends remained more inconsistent and bifurcated across Canada. The distinct effects of individual—including many provincially initiated and governed—PO control measures urgently need to be evaluated. In the meantime, recent reductions in general PO availability across Canada appear to have contributed to shortages in opioid supply for existent, sizable  user populations and may have contributed to recent marked increases in illicit opioid use and harms (including rising deaths). In North America, the epidemiological picture of high availability and use of prescription opioids (POs), and related public health consequences, are well-documented –4. Post-North American governments and regulators began to more actively implement measures to address increasing PO-related harms post-2010 . In CanaWhile PO dispensing peaked in the US in about 2010 and subsequently declined by about 25% in response to control measures implemented, it remains globally unsurpassed for its opioid consumption levels –20. In CIn the following and extending previous work, we briefly examine and update on trends and patterns in PO dispensing in Canada for the period up to 2018, with primary focus on recent developments as well as population health implications.Present analyses are based on annual PO retail dispensing data across Canada  from 2005 to 2018. Raw aggregated update data were obtained from the IQVIA’s CompuScript database, which monitors prescription-based retail transactions for medications, and had been used for previous related analyses –31. TotaThe individual provincial summary of total dispensing data were converted to annual defined daily doses per 1000 population per day (DDD/1000/day) values. DDD estimates were obtained from the World Health Organization’s (WHO) Anatomical Therapeutic Chemical (ATC) classification and DDD measurement methodology, population statistics calculated from the Statistics Canada’s CANSIM table 051-0005 –36. BaseDescriptive analyses for the different PO categories included low- and high-ranking values in dispensing and change rates, by province and over-time Fig. . No ethiIn 2018, British Columbia (BC) (5.1 DDD/1000 pop/day) featured the lowest strong PO dispensing rate and Newfoundland and Labrador (NL) had the highest (10.1 DDD/1000 pop/day), approximately double the rate. Most provinces reported their peak rates for strong PO dispensing in 2011 but no later than 2016. All provinces indicated multi-year decreasing trends in strong PO dispensing in recent years and lower dispensing compared to previous peak rates; four out of the ten provinces indicated strong PO dispensing rates in 2018 that were below 2005-levels. Compared with respective peak years, BC had the greatest (− 49.5% from 2011), Quebec the smallest (− 13.8% from 2016) relative reduction in strong PO dispensing within the study period.Correspondingly, BC (2.6 DDD/1000 pop/day) featured the lowest weak PO dispensing rate and Alberta (AB) had the highest (17.1 DDD/1000 pop/day) in 2018, equating to more than a sixfold difference. All provinces had lower dispensing rates for weak POs compared to previous peak rates observed during the study period, and all but one of the provinces (Saskatchewan [SK]) had weak PO dispensing rates that were below 2005 levels. Compared with respective peak years, ON had the greatest (− 43.1% from 2005) and NL the smallest relative reduction (− 11.8% from 2014) in weak PO dispensing within the study period.The PO dispensing data presented is drawn from a stratified, representative sample of about 6,000  retail pharmacies across Canada, including a continuously refreshed sub-sample providing the pharmaceutical dispensing data comprising the large majority of prescriptions at the national level , 38. TheOur data—building on previous pharmaco-epidemiologic analyses—showed that population-level PO dispensing has undergone distinctly oscillating waves and changes in Canada during the period 2005–2018 , 23, 29.Notably, by 2018, some provinces have reduced their strong PO dispensing to below 2005 levels—a time when more generous opioid dispensing was actively promoted towards improved chronic pain care outcomes –46. WhilIt is reasonable to assume that the pronounced recent decreases especially in strong PO dispensing across Canada are an overall consequence of the various opioid control measures—including opioid product delisting, prescription monitoring and revised prescription guidelines—implemented in Canada over the past decade , 51–53. One would expect that the recent declines in population-level opioid dispensing in Canada co-occurred with parallel benefits in opioid-related public health outcomes. Unfortunately, this appears not to be the case, or reductions from excessive prescribing levels occurred too late, evidence for improvements in pain care outcomes is lacking, and opioid-related mortality and morbidity indicator outcomes have substantially risen further in recent years (e.g. 2017/18) –58. ContFollowing persistent increases that gave Canada one of the highest opioid use rates in the world, an assortment of various policy interventions implemented post-2012 to curb general opioid availability and adverse consequences in the population were introduced, and prescription opioid dispensing showed variably decreasing trends in each of ten provinces. Both the magnitude of the decreases and current opioid dispensing levels continue to be rather heterogeneous across the provinces and so raise questions about differential medical use and needs standards, as well as the effectiveness of policy measures for opioids. From a public health perspective, the substantive decreases in medical opioid availability appears to have led to unintended consequences of supply shortages for non-medical opioid use resulting in extensive adverse outcomes  related to increases in illegal opioid  availability and use.


Aim: Accumulating evidence suggests that neural inflammation plays an important role in psychiatric disorders. We aimed to identify inflammatory cytokines involved in the pathophysiology of such disorders by quantifying them in cerebrospinal fluid (CSF) samples from a large sample of patients with major psychiatric disorders and healthy controls.Methods: The subjects included 94 patients with schizophrenia, 68 with bipolar disorder, 104 with major depressive disorder, and 118 healthy controls, matched for age, sex, and ethnicity (Japanese). Lumbar puncture was performed to collect these CSF samples. A multiplex immunoassay was then performed to measure CSF cytokine levels using magnetic on-bead antibody conjugation for 19 inflammatory cytokines.Results: CSF interferon-β level was significantly higher in total psychiatric patients than in healthy controls (corrected p = 0.000029). In diagnostic group comparisons, CSF interferon-β level was significantly higher in patients with schizophrenia, or bipolar disorder (corrected p = 0.000047 or 0.0034) than in healthy controls.Conclusion: We present novel evidence that CSF IFN-β level showed prominent statistical differences between psychiatric groups and healthy controls. This suggests IFN-β as the most important player among the 19 cytokines tested here in the inflammation-related pathophysiology of major psychiatric disorders. Inflammation has been suggested to play a key role in the pathogenesis and pathophysiology of psychiatric disorders , includiCerebrospinal fluid (CSF) is the optimal biomaterial to examine the molecular status of the central nervous system (CNS) . PreviouMultiplex immunoassays (Bio-Plex and V-plex®) have been validated for the simultaneous measurement of multiple cytokine levels using blood samples from patients with MDD and healthy control subjects . The beaAlthough prior studies have conducted multiplex immunoassays to measure CSF cytokine levels in psychiatric disorders , the higThis study involved 94 patients with schizophrenia , 68 with BD , 104 with MDD , and 118 healthy controls  who were matched for age, sex, and ethnicity (Japanese). The BD group included 22 patients with BD I and 46 with BD II. A total of 384 samples were not based on any power analyses since we have not had data on pre-obtained effect size. All participants were recruited at the National Center of Neurology (NCNP), via advertisements at the NCNP Hospital, on our website, and in local free magazines. Participants were screened for psychiatric disorders by qualified psychiatrists using the Japanese version of the Mini International Neuropsychiatric Interview . ConsensThe Positive and Negative Syndrome Scale (PANSS) was used to evaluate symptoms in patients with schizophrenia , and theg for 10 min) at 4°C and the supernatant was dispended in 0.5-ml aliquots and stored at −80°C in a deep freezer. After a single melting and re-freeze of the sample for the preparation of the 96-well plates, multiplex immunoassays were performed.Lumbar puncture was performed in the left lateral decubitus or a sitting position. Each participant received local anesthesia by lidocaine hydrochloride injection before the puncture. CSF was withdrawn from the L3–L4 or L4–L5 interspace using an atraumatic pencil-point needle . The CSF was collected in a low protein absorption tube  and immediately transferred on ice. The CSF was centrifuged  using magnetic on-bead antibody conjugation for 37 inflammatory cytokines  according to the manufacturer's instructions. CSF samples were diluted to 1:2, and 2 standard samples (S9 and S10) were newly created to extend the assay working range on the basis of the results of a verification assay. The assay was performed on the same day using 384 single CSF samples to secure a large number after confirming that the intra-run and inter-run coefficients of variance for the 37 proteins accounted for less than 10% of the variance in the verification assay . A VIAFLO 96/384 system  was used to apply samples simultaneously into the 96-well plates. To adjust the inter-assay variations between the 96-well plates, 13 randomly selected CSF diluted to 1:2, 2 standards (S4 and S10), and one blank were used as margin samples to fit measures of four plates to those of 1 standard plate that included 8 standard dilution (S4–10) and blank samples. Based on the measures of the margin samples, regression equations were calculated for the 37 proteins using 2-dimensional scatter diagrams between the standard and the other four plates for the inter-plate adjustment. Among the 37 assayed proteins, the measurements of 19 cytokines satisfied the following three criteria and were thus deemed reliable: 1) within the assay working range, 2) coefficients accounting for less than 15% median inter-run variance, and 3) strong Pearson’s correlation coefficients (r > 0.70) in the regression equations of the inter-plate adjustment. The assay data for the 18 remaining cytokines , IL-20, IL-22, IL-27(p28), IL-28A/IFN-λ1, IL-32, IL-34, IL-35, LIGHT/tumor necrosis factor superfamily (TNFSF) 14, matrix metalloproteinases-1, matrix metalloproteinases-2, osteopontin, pentraxin-3, and tumor necrosis factor-related weak inducer of apoptosis/TNFSF12) did not meet some of the criteria and were thus excluded from the following statistical analyses. CSF cytokine levels are represented as pg/ml.p < 0.05/3 = 0.016), Mann–Whitney U tests, Kruskal-Wallis tests, and correlational analyses on measured 19 CSF cytokine levels (p < 0.05/19 = 0.0026). Hence, corrected p-values were calculated 19 x nominal p-values for group comparisons and correlational analyses on the CSF cytokine levels. All statistical tests were 2-tailed and p < 0.05 was considered significant. Statistical analyses were performed using the Statistical Package for the Social Sciences version 25.0 and 27.0 .Categorical and continuous variables were compared between the four diagnostic groups using chi-square tests and analysis of variance (ANOVA), respectively. CSF cytokine levels were compared between patient (both three diagnoses combined and each diagnosis) and control groups and between drug free and non-drug free groups using Mann–Whitney U tests, while effect sizes are shown using r. Kruskal-Wallis tests were used when comparing CSF cytokine levels across the four diagnostic groups. Correlation between CSF cytokine levels and clinical variables were assessed using Pearson’s correlation coefficients, while CSF cytokine levels were compared using unpaired (Student’s or Welch’s) -t tests between sexes. Correlations between CSF cytokine levels and symptom scores or CSF total protein level were assessed using Pearson’s partial correlation coefficient controlled for age, sex, and drug use (only for patients). The correlation matrix for CSF cytokine levels was also assessed with Pearson’s partial correlation coefficient controlled for age, sex, and drug use (only for patients). Bonferroni corrections for multiple testing were applied for 3 ANOVAs for comparison of clinical variables  between each diagnostic and control groups (p = 0.004 and 0.006). There were 11, 5, and 26 drug-free patients with schizophrenia, BD, and MDD, respectively. Correlations between CSF cytokine levels and clinical variables in patients with schizophrenia, BD, or MDD and healthy controls are shown in p < 0.05). Age was significantly and positively correlated with CSF soluble CD163 and soluble TNF-receptor one levels, while age of onset was significantly and positively correlated with CSF soluble CD163 level in patients with MDD (corrected p < 0.05).The clinical characteristics of the participants are shown in n = 266) and the control group are shown in p = 0.000029, p = 0.000047 or 0.0034, respectively, p < 0.05).Comparisons of CSF cytokine levels between the patient group , while no significant correlations were found in patients with BD or MDD. Correlations between CSF cytokine and total protein levels are shown in p < 0.05). Correlation matrices for CSF cytokine levels are shown in p < 0.05).Correlations between CSF cytokine levels and symptom scores are shown in n = 19) and the patient  and controls (n = 118) sample size. Among the 19 reliably measured cytokines, CSF IFN-β level was higher in psychiatric patients than in healthy controls. Even when comparing across all four diagnostic groups, we found that CSF IFN-β level was higher in patients with schizophrenia and in those with BD, in this order, than in healthy controls. Notably, CSF IFN-α2 and IFN-λ1 levels were not significantly altered. These results suggest that CSF IFN-β could be a useful biomarker for psychiatric disorders.To our knowledge, this is the largest multiplex immunoassay study in terms of both the number of cytokines measured , CSF IFN-β level was tended to be higher in patients with MDD than in healthy controls. In line with this finding, CSF IL-6  level was increased in a geriatric depression group compared with a non-depression group in an earlier V-plex® assay  had taken some form of psychotropic medication, although the effects on CSF cytokines were confirmed to be small in statistical comparisons. Second, 19 (51.4%) among the 37 cytokines could only be measured using a Bio-Plex assay kit. Similarly, prior studies have reported that a variety of targeted cytokines cannot be measured with V-plex®  or multiIn conclusion, CSF IFN-β level showed most prominent increases in psychiatric groups compared with healthy controls. Our data suggest that IFN-β could be a useful biomarker in the inflammation-related pathophysiology of major psychiatric disorders.


The decision to accept organs from donors with acute kidney injury (AKI) can result in a clinical dilemma in the context of conflicting reports from published literature.The need for This observational study included all deceased donor kidney transplants performed in Australia and New Zealand between 1997 and 2017. The association of donor-AKI, defined according to KDIGO criteria, with all-cause graft failure was evaluated by multivariable Cox regression. Secondary outcomes included death-censored graft failure, death, delayed graft function (DGF) and acute rejection.The study included 10,101 recipients of kidneys from 5,774 deceased donors, of whom 1182 (12%) recipients received kidneys from 662 (11%) donors with AKI. There were 3,259 (32%) all-cause graft failures, which included 1,509 deaths with functioning graft. After adjustment for donor, recipient and transplant characteristics, donor AKI was not associated with all-cause graft failure , death-censored graft failure , death  or graft failure when death was evaluated as a competing event . Donor AKI was not associated with acute rejection but was associated with DGF .Donor AKI stage was not associated with any kidney transplant outcome, except DGF. Use of kidneys with AKI for transplantation appears to be justified. Kidney transplantation is an important treatment for end-stage kidney disease (ESKD) since individuals receiving kidney transplants generally have better survival and quality of life compared with those who remain on dialysis –3. GivenOne such consideration is deceased donors with acute kidney injury (AKI). In a recent study from the United States, AKI defined by Acute Kidney Injury Network criteria occurred in up to 24% of all deceased donors and could potentially contribute significantly to the pool of kidneys offered for transplantation . HoweverTherefore, this study aimed to investigate the associations between donor AKI and recipient transplant outcomes using the Australia and New Zealand Dialysis and Transplant (ANZDATA) Registry and Organ Donation (ANZOD) Registry.Research using ANZDATA and ANZOD Registry data was approved by the Princess Alexandra Hospital Human Research Ethics Committee (LNR/2019/QMS/51797) and the ANZDATA executive (02/04/2019). This manuscript was prepared in accordance with the Strengthening The Reporting of Observational studies in Epidemiology (STROBE) guidelines .This retrospective, observational cohort study included all deceased donors (adult or pediatric) and their kidney transplant recipients (adult or pediatric) in Australia and New Zealand from 1 January 1997 to 31 December 2017. Recipients of multi-organ or repeat kidney transplants during this study period were excluded. Donors with missing admission or terminal serum creatinine (SCr) data were not included as their AKI status could not be confirmed.2 in patients under the age of 18 years [Donor AKI was defined according to the Kidney Disease Improving Global Outcomes (KDIGO) criteria (excluding urine output criteria due to incomplete data on patients) as follows: an increase in SCr level by ≥0.3mg/dL (26.5μmol/L) within 48 h or an increase in a SCr level to ≥1.5 times from time of donor admission to time of organ procurement . The sta18 years .human leukocyte antigen mismatches, panel reactive antibody, body mass index at time of transplant, total ischemia time, induction immunosuppression regimen, transplant era and transplant country.The deceased donor demographic variables extracted were age, race, gender, smoking status, height, weight, hypertension, diabetes mellitus, cause of death, expanded-criteria donor (ECD) , standarThe primary outcome of this study was all-cause graft failure . Secondary outcomes included death-censored graft failure, all-cause mortality (excluding death occurring beyond 30 days of graft failure), acute rejection within 6 months after transplantation , and delayed graft function . Data were censored as of 31st December 2017.Recipient, donor and transplant characteristics are expressed as number (percentage) for categorical variables, mean ± standard deviation for normally distributed continuous variables, and median (interquartile range) for continuous variables that were not normally distributed. Comparisons of characteristics between kidney transplants recipients who received a donor kidney with AKI and those who received a donor kidney without AKI were performed by Chi-square or Fisher’s exact test, as appropriate, for categorical variables. Student’s t-test was performed for normally distributed continuous variables, and Mann-Whitney test or Kruskal-Wallis test for continuous variables that were not normally distributed. Time to event outcomes  by donor AKI status were displayed using Kaplan-Meier survival curves and compared with log-rank tests. Three multivariable Cox proportional hazards regression models using robust standard errors were subsequently constructed using variables with p<0.2 on univariable analyses in addition to variables deemed to be clinically important (donor weight and donor ethnicity) . VariablDuring the study period, 10,710 patients with ESKD received kidney transplants from 5,974 deceased donors. 609 kidney transplant recipients and 200 donors were excluded from further analysis due to missing data or because they received a repeat kidney transplant during the analysis period . TherefoOf 10,101 recipients, 1,182 (12%) transplant recipients received kidneys from 662 donors with AKI; 693 recipients (7%) with stage 1, 264 (3%) with stage 2, and 225 (2%) with stage 3. Of 662 donors with AKI, 91 donors donated both kidneys to 91 individual recipients, 51 only donated one kidney while the remaining 520 donors donated both kidneys to 1040 separate recipients. Recipients of kidneys from donors with AKI were more likely to have diabetes mellitus, be transplanted in Australia , and be on hemodialysis prior to transplantation . Post-trThe total follow-up period of the cohort was 64,682 patient-years with a median follow-up period of 5.2 years (interquartile range 2.0–9.6 years). There were 3,259 (32%) all-cause graft failures, which included 1,509 deaths with a functioning graft. Respective 1-year graft survival rates were 93% (95% CI 92–93%) for donors without AKI vs 91% (95% CI 89–93%) from donors with AKI, 5-year graft survival rates were 81% (95% CI 80–82%) for donors without AKI vs 78% (95% CI 75–80%) from donors with AKI, whilst 10-year graft survival rates were 62% (95% CI 60–63%) vs 58% (95% CI 54–62%). Compared with recipients of kidneys without donor AKI, recipients of kidneys with donor AKI experienced a higher unadjusted hazard of all-cause graft failure  transplant recipients died during the study period including 1509 deaths with functioning graft and 99 deaths within 30 days of graft failure. The most common cause of death was cardiovascular disease (31%), followed by malignancy (27%), sepsis/ infection (20%) and other/unknown (23%). There were no differences in causes of death between the two groups. Respective 5-year patient survival rates were 91% (95% CI 90- f91%) for donors without AKI vs. 89% for donors with AKI (95% CI 87–91%), whilst 10-year patient survival rates were 79% (95% CI 78–81%) vs 76% (95% CI 72–79%). Compared with recipients of kidneys without donor AKI, recipients of kidneys with donor AKI experienced a higher unadjusted hazard of mortality  recipients experienced DGF during the follow-up time. Of these, 523 (44%) were from 1,182 donors with AKI and 2275 (26%) were from 8,919 donors without AKI. The fully adjusted OR for DGF among recipients with AKI donor kidneys was 2.27  recipients experienced acute rejection within 6 months after transplantation. Of these, 243 (21%) were from 1,182 donors with AKI and 1,773 (20%) were from 8,919 donors without AKI. The fully adjusted OR for acute rejection among recipients with AKI donor kidneys was 1.06 , but not with acute rejection .This large Australia and New Zealand study of 10,101 kidney transplant recipients followed over a 21-year period demonstrated that donor AKI was not associated with an increased risk of all-cause graft failure after adjustment for donor, recipient and transplant characteristics. Similar results were observed for the secondary outcomes including death-censored graft failure, graft failure with death as a competing event and all-cause mortality. Donor AKI was not associated with acute rejection but was associated with an increased risk of DGF. Donor AKI stage was not associated with any kidney transplant outcome, except DGF. During the study period, the proportion of donors with AKI remained comparable, but a greater proportion of kidneys from donors with stage 3 AKI were transplanted in the most recent era (2010–2017), with comparable post-transplant outcomes.graft survival or death-censored graft failure in 2,430 kidney transplant recipients between 2010 and 2013. This US study pointed out that the transplant community should consider measures to increase utilization of kidneys from deceased donors with AKI, especially in the context of organ shortage. Kayler et al. [The main finding of no significant association between donor AKI and all-cause graft failure is in keeping with that of a recent US Organ Procurement and Transplantation Network (OPTN) registry study , that alr et al.  analyzedr et al.  reportedr et al.  analyzedgraft survival rates compared with those who received kidneys from donors without AKI [In contrast, a previously published UK Transplant Registry of 11,219 deceased donor kidney transplant recipients between 2003 and 2013 reported that recipients of kidneys from donors with AKI were at increased risk of graft failure after adjustment for confounding factors, and experienced slightly inferior 1-, 3-, and 5-year hout AKI . Althoughout AKI . Howeverhout AKI  analyzedThe current study also reported that all-cause mortality was comparable among recipients who received kidneys from donors with AKI or without AKI after full adjustment, and was not associated with the severity of AKI stage. Several small studies also reported a similar relationship with donor AKI and patient survival. Ali et al.  analyzedIn the present study, the incidence of DGF was significantly higher in donors with AKI and the risk increased progressively with increasing AKI stage. This finding is consistent with that of previous studies , 24, 25.Another novel finding of the present study was that graft failure was not consistently associated with the severity of AKI, as defined by KDIGO stage. This may have been due in part to the relatively small numbers of events in recipients of kidneys with stage 2 and 3 AKI. Moreover, donor kidneys with stage 3 AKI were commonly utilized in the more recent era (2010–2017), and this era was associated with a lower risk of graft failure as compared to the earlier era. However, the shorter follow-up duration may have limited the ability to detect a difference in graft outcomes. There may have also been a component of selection bias, since kidneys with more severe AKI were more likely to have procurement biopsies and may have been discarded more often unless there were other favorable characteristics that supported their preferential selection for transplantation. For example, in the present study, donors with stage 3 AKI tended to be younger, were less likely to be ECD, and were more likely to have died from anoxia. Nevertheless, this result still encourages broader utilization of AKI stage 3 kidneys for transplantation.A recent single center report on 1,113 deceased donor kidney transplants followed for 10 years from Mayo clinic investigators  showed nIn native kidneys, AKI is often associated with increased risks of chronic kidney disease (CKD) progression and ESKD, which increases in a graded manner with the severity of AKI , 30. PotThe strengths of this study lie in its large size and extended duration of follow up. It included all patients who received a deceased donor primary kidney-only transplant in Australia and New Zealand between 1997 and 2017, making the study population representative of a broad range of patient demographics and comorbidities. However, there were some limitations to this study. Firstly, the possibility of selection bias, particularly in donors with more severe AKI, could not be excluded. Even though adjustment was made for an appreciable number of donor, recipient and transplant characteristics, the possibility of residual confounding remained. Secondly, some donors already had significantly elevated SCr concentrations on admission without data on previous kidney function, thereby impairing the ability to diagnose donor AKI based on KDIGO criteria because of delayed and restricted time windows for assessment. Some degree of misclassification was therefore possible. Thirdly, the ANZOD Registry does not record information on duration of decreased urine output, which is one of the important criteria used by KDIGO to define AKI. The registry also does not collect other important information, such as procurement kidney biopsy results, kidney transplant management protocols and concomitant medications. Fourthly, there were relatively small numbers of donors with severe stage 3 AKI, which limited the ability to evaluate the association between donor AKI severity and transplant outcomes. Finally, AKI was solely defined according to the KDIGO AKI classification, which has been endorsed by both the Acute Dialysis Quality Initiative (ADQI) and KDIGO working groups , 35. UseIn conclusion, donor AKI was not associated with a significantly increased risk of all-cause graft failure, mortality, death-censored graft failure and graft failure with death as a competing risk after adjustment for donor, recipient and transplant characteristics. Thus, cautious use of donor kidneys with AKI appears justifiable in a number of kidney transplant circumstances, particularly if associated with immunologic advantage. Further studies are warranted to determine optimal selection and risk stratification of donor kidneys with AKI to help better inform shared decision-making regarding the anticipated benefits and risks of their use in specific recipients.S1 Fig(PNG)Click here for additional data file.S2 Fig(PNG)Click here for additional data file.S3 Fig(PNG)Click here for additional data file.S4 Fig(PNG)Click here for additional data file.S5 Fig(PNG)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.