Synthetic sensing materials  are some of the most attractive components of chemical/biosensors because of their long-term stability and low cost of production. However, the strategy for the practical design of these materials toward specific molecular recognition in water is not established yet. For the construction of artificial material-based chemical/biosensors, the bottom-up assembly of these materials is one of the effective methods. This is because the driving forces of molecular recognition on the receptors could be enhanced by the integration of such kinds of materials at the ‘interfaces’, such as the boundary portion between the liquid and solid phases. Additionally, the molecular assembly of such self-assembled monolayers (SAMs) can easily be installed in transducer devices. Thus, we believe that nanosensor platforms that consist of synthetic receptor membranes on the transducer surfaces can be applied to powerful tools for high-throughput analyses of the required targets. In this review, we briefly summarize a comprehensive overview that includes the preparation techniques for molecular assemblies, the characterization methods of the interfaces, and a few examples of receptor assembly-based chemical/biosensing platforms on each transduction mechanism. Molecular recognition systems in organisms contribute to the tuning of biological functions . For insSynthetic receptors  are some of the most suitable platform materials for the development of chemical/biosensors owing to their chemical/physical stability, low cost of production, and their fine-tuning ability to select the required targets . HoweverIn this review, we briefly summarize a research overview including the construction methods of molecular assemblies, the characterization techniques for the interfaces, and the rational design and demonstrative examples of “nanosensors”  utilizing each transduction mechanism.This section introduces representative methods for the installation of molecular assemblies at the interfaces. In general, the sensing ability of single-molecule receptors in a bulk aqueous phase is lower than that of naturally-derived molecular recognition materials because the molecular interactions between the receptors and the analytes are strongly affected by their hydrations. This means that any extractions of analytes from the target medium in the presence of various interferents by using organic solvents are generally required for the achievement of selective detection of analytes in artificial receptor-based sensors in practical applications. In contrast, the interface microenvironments enable the enhancement of the molecular recognition ability of synthetic receptors because the hydration effect can be reduced at the interfaces. This is possible because the interfacial dielectric constant is much lower than in bulk water . FurtherLangmuir–Blodgett (LB) films have been investigated earlier for both fundamental material properties and practical applications in nanotechnology . In thisTo obtain the functionalized substrate, the LB film should be separated onto a solid surface from the liquid phase. Initially, the substrate is immersed perpendicularly into the water containing the LB molecules. Then, the LB film is successfully transferred onto the solid surface from the water by raising the substrate from the solution carefully c. The trSpontaneously formed molecular monolayers at solid surfaces are called self-assembled monolayers (SAMs) . In geneTo confirm the installation of molecular assemblies for each sensor, characterizations of the material-functionalized interfaces at the sensing portion should be performed. Since there are many evaluation criteria of the molecular assemblies at the interface , various analyses for the assemblies from diversified standpoints are required. In this section, standard techniques for the characterization of synthetic receptor membranes in the sensor platforms are described.Contact angle goniometry (CAG) analysis is the most utilized method to confirm the formation of molecular assemblies at the interfaces because it is easy to use and is rapid . ContactγL, γS, and γSL are the interfacial tensions at the liquid surface, the solid surface, and the liquid–solid interface, respectively. Θ is the contact angle, which consists of the three-phase contact line , the kinetic energy of the electron as measured values, the vacuum energy, and the Fermi energy, respectively  or electron spectroscopy for chemical analysis (ESCA) . When thectively a. Herein−1) irradiates the molecules, the incident light is absorbed at a specific wavenumber due to the vibration and/or stretching of the target molecules in accordance with the chemical bonding state in the compounds. Thus, the FT-IR measurement can evaluate the chemical information of the molecules as with the abovementioned elemental analyses. The attenuated total reflection (ATR) method is one of the popular methods to perform the interfacial analysis based on FT-IR. In the ATR method, the chemical information at the interface can be easily obtained by measuring the total reflection beam from the sample  . The elee sample b. While Since the function of molecular assemblies follows their macro and microscopic structures , direct Molecular assemblies formed in an orderly line at the interfaces strongly influence interfacial electrical potentials . This is attributed to the dipole moment of the arranged molecules in the assemblies . TherefoIn contrast, measurement samples on photoemission yield spectroscopy (PYS) are not confined in solid materials because PYS with an open counter can perform under atmospheric conditions . In factMore individual techniques have been applied for the characterization of substrate surfaces. A fluorescent labeling technique is one of the simple tools to observe mesoscopic structures on the substrate surfaces . The intTo develop chemical/biosensing platforms based on synthetic receptor membranes for practical applications such as environmental assessment and diagnoses, the combination of receptor assemblies with platforms is required to retrieve the molecular recognition at the interfaces. Herein, we summarized various types of chemical/biosensors that consist of artificial receptor membranes suitable for each transduction mechanism . The senTo perform chemical sensing based on synthetic receptor membranes, colorimetric or fluorometric approaches have been widely studied, owing to their simplicity and rapid detection quality . To achi3CO2−, H2PO4−, Cl−) based on a thiourea monolayer was reported ethylenediamine; TPEDA) on the substrate. The fluorescence intensity in the prepared SAMs immobilized with various molecular components increased/decreased upon the addition of each analyte. The versatile fluorescence-quenching phenomenon seen could not only be derived from the intermolecular bonding between the individual fluorophores and analytes but might also be affected by changing the highly-ordered structures of the SAMs induced by the analyte recognition at the sensing portions. The analyte selectivity of the SAMs reflected the molecular recognition ability of each sensing moiety. Importantly, the sensitivity in the SAM-based fluorometric sensors was much higher than that of in the colorimetric sensors  by using the SPR sensor functionalized with a calix[4]crown SAM, which has the specific sensing ability for K+ (+) (detection limit: 1 pM), suggesting that the combination of the SPR sensors and the synthetic receptor membranes can synergistically boost the sensing ability of each other. The protein detection studies based on the SAM-modified SPR sensor were also carried out by Huang et al. ..+) by usg et al. . In thesg et al. . Moreoveg et al. ,62,63. JSurface-enhanced Raman scattering spectroscopy (SERS) methods are also powerful approaches to determine the molecular recognition behavior of receptors at the interfaces with high sensitivity . In SERSMoreover, infrared reflection–absorption spectroscopy (IRAS) methods were employed for minute analysis of the molecular recognition behavior of the sensing membranes at the interfaces . In the Although optical method-based chemical/biosensors exhibit high-sensitive responses to the addition of analytes, a large-sized and complicated equipment  is required for the quantitative determination of the analyte information. To achieve the on-site analyses of target species with high accuracy and quantitativity in the optical sensors, the handheld systems utilizing light-emitting diodes (LEDs)-based light sources combined with photodiodes or digital cameras are useful. However, the improvement of sensitivity in such compact measurement setup is generally difficult due to their low signal-to-noise ratio. Toward that end, electrochemical sensors are some of the more effective tools for the development of chemical analyzers utilizing synthetic receptors. For instance, cyclic voltammetry (CV) methods enable the sensitive detection of analytes. This is because a molecular complex, which consists of target species and sensing membranes on the sensing electrode, can be oxidized or reduced through electrochemical reactions at the specific potential . For exa4dcbpy)2] was made to react with the 4mpy-SAM on an Au electrode. The formed SAM [=Ru(H4dcbpy)2(4mpy)] had important features for the detection of dopamine on the electrode. First, Ru acts as the redox reactive site for enhancing the output current. Second, the dicarboxylic moiety can selectively recognize amine targets  (4dcbpy)2-based SAM, relatively sensitive and selective detection of dopamine was performed by utilizing the electrochemical sensor. Furthermore, a SAM of 4-mercaptobenzoic acid (MBA) was also utilized for the achievement of sensitive and selective detection of another neurotransmitter (histamine)  is an effective way to monitor nervous activity owing to their high-sensitivity . Howeveropamine) b 75]. B. B4dcbpystamine) b by usinstamine) c 47]. A. A4dcbpyField-effect transistors (FETs) can be employed as the transducers of potentiometric sensors because electrical characteristics in these semiconductor devices (output current and/or voltage) can directly reflect the changes in the electrical potential of the sensing electrodes . More im+) by using a silicon nanowire FET modified with a 15-crown-5-ether monolayer . B. BII-dpaomplexes , the eleated FET d. Based -dpa SAM . This isractions .In general, electrochemical sensors including FETs detect electrical charges (electroactive sites) of analytes, suggesting that the electrical detection of non-charged molecules  by FETs is still a challenging issue. To develop an FET-based sensor for non-charged saccharides, we employed a phenylboronic acid (PBA) SAM as the receptor membrane a 52]. P. P52]. PAs a unique example of electrical signal-based chemical sensing, Sivalingam et al. demonstrated a light modulation system of output signals in metalloporphyrin-coated ZnO nanorod electrodes for the detection of cysteine . In the p-xylene in water was successfully detected by using the QCM sensor modified with a nickel-phthalocyanine (NiII-pc) SAM [II-pc [To study the molecular recognition information of synthetic receptor membranes, more physical parameters can also be utilized like the aforementioned optical or electrical signals. Quartz crystal microbalance (QCM)-type sensors are able to detect the “weight” of the captured analytes on the receptor layer . The osc-pc) SAM . The detM [II-pc . These sM [II-pc .Elastic waves at the interfaces decorated with receptor membranes have also been utilized as output signals for chemical sensing. The sensing mechanism of surface acoustic wave (SAW)-type sensors is as follows: the captured molecules on the surface of the SAW device functionalized with synthetic receptors affect the frequency of the SAW, suggesting that the shifts in the frequency reflect the sensing information of the receptors sensitively . AlthougIn this review, we have provided an overview of the various chemical/biosensing platforms functionalized with synthetic receptor membranes ranging from the basic strategy for materials design to sensing applications. While synthetic receptors are effective materials to construct on-site sensing systems, these material-based sensors have never been popular for practical applications due to their low selectivity for the required targets as compared to biomaterials such as antibodies and enzymes. However, specific detection of the targets in organisms is achieved by integration of the molecular recognition moieties. Encouraged by this effective strategy for molecular recognition in nature, the bottom-up integration of synthetic receptors as the membranes at the interfaces has been employed for enhancing the sensing ability of artificial materials. The integration of molecular recognition moieties at the interfaces was successfully demonstrated by using the typical methods for the preparation of molecular assemblies, such as LB films or SAMs. The installation of the assemblies can be evaluated by various characterization techniques for the interfaces. The target selectivity of the synthetic receptor membranes was much higher than that of the single-molecule receptors because the driving forces for the target detection based on such materials are amplified at the interfacial fields. In addition, the presented molecular assemblies attached to the sensors are not only utilized as receptor functions, but these membranes can be also applied for the prevention of non-specific absorption of unintentional molecules onto the sensors . SignifiThe sensitive detection of macromolecular targets  by using electrical devices is generally difficult, albeit their attractive features. Therefore, there is still room for the further development of biosensing devices by using other transduction mechanisms. However, we already proposed the bottom-up  or top-dFrom the viewpoint of supramolecular analytical chemistry, the fascinating point is that the modification of the sensing ability of the synthetic receptor assemblies can be easily accomplished. Recently, synthetic receptors combined with statistical analysis techniques, which are called chemosensor arrays, have been widely applied to achieve the simultaneous detection of multiple targets . In a ch


Subjects with patch and plaque KS lesions responded more favorably to treatment than those with nodular lesions. Pre-treatment and post-treatment levels of plasma KSHV-DNA, HIV-1 viral load, KSHV-Ab responses, cytokines, and T-cell populations did not predict the KS treatment response. Elevated KSHV-humoral and cytokine responses persisted in participants with KS despite a clinical KS response. While patch and plaque KS lesions were more common among treatment responders, none of the analyzed viral and immunological parameters distinguished responders from non-responders at baseline or after treatment.Kaposi’s sarcoma-associated herpes virus (KSHV) is the etiologic agent for Kaposi’s sarcoma (KS). The prognostic utility of KSHV and HIV-1 (human immunodeficiency virus) viremia as well as immunological parameters in clinical management of participants with KS is unclear. The objective of this study was to investigate viral and immunological parameters as predictors of KS treatment responses in participants with KS from sub-Saharan Africa (SSA). Plasma KSHV-DNA, HIV-1 viral load, total anti-KSHV antibody, KSHV-neutralizing antibody (nAb), cytokine/chemokine levels, and T-cell differentiation subsets were quantified before and after KS treatment in 13 participants with KS and in 13 KSHV-infected asymptomatic control individuals. One-way analysis of variance and the Mann-Whitney t-test were used to assess differences between groups where  Kaposi’s sarcoma (KS), which is a multifocal angio-proliferative sarcoma commonly found on skin and mucosal surfaces, is caused by the Kaposi’s sarcoma-associated herpes virus (KSHV) or human herpes virus-8 (HHV-8) . KSHV in+ T-cell counts and a suppressed HIV-1 plasma viral load , effector [CD197−/CD45RO− (TE)], effector memory [CD197−/CD45RO+ (TEM)], central memory [CD197+/hCD45RO+ (TCM)] CD4+, and CD8+ T-cell populations were quantified by flow cytometry. Their activation (CD38+/Human Leucocyte Antigen/HLA-DR+), senescence (CD57+/CD28−/hCD27−), and proliferation (Ki67+) profiles were also investigated. Compared to controls, there was an increase in proportions of TN (p = 0.005) and a decrease of TEM CD8+ T-cells (p = 0.001) in KS responders  in KS responders when compared to the controls  showed plasma KSHV viral load did not predict survival in T0 or T1-staged participants with KS [+ T-cell count and HIV-1 viremia did not predict the KS treatment response. Similarly, a Zambian study on participants with early KS, to evaluate the longitudinal response to KS treatment with ART alone, found that HIV-1 viremia and CD4 count before and after treatment also did not differentiate KS responders from non-responders . The lack of outcome association with both CD4+ T-cell count and HIV-1 viremia is also supported by recent reports of incident KS in individuals with high CD4+ T-cell counts and suppressed HIV-1 viral load [+ T-cell count and HIV-1 viremia could be due to an indirect impact of HIV on immune function as opposed to simply CD4+ T-cell depletion.Although KSHV viremia has been investigated as a tumor biomarker ,35, a st with KS ,37. Cons with KS . However with KS . Additioral load ,11,12,13+, CD8+ T-cells, NK, and myeloid derived suppressor cell populations have been suggested to be biomarkers for lung cancer and non-Hodgkin’s lymphoma treatment responses [+ T-cell help and cytotoxic activity of CD4+ and CD8+ T-cells play an important role in eliminating infected cells and cancer cells [+ TE and CD8+ TEM cells in participants with KS. However, reduction in CD4+ TE and CD8+ TEM was also evident in responders suggesting possible T-cell qualitative differentials between responders and non-responders. Unfortunately, whether decreases in CD4+ TE and CD8+ TEM, or the noted increase in CD8+ naïve T-cells corresponded to decreased KSHV Ag-specific effector functions could not be determined. Assessment of differentials in functionality of T-cells between treatment responders and non-responders will be important in identifying predictors of the KS treatment response.Changes in immune parameters such as CD4esponses ,39. CD4+er cells . Poor KSCytokine dysregulation has often been associated with KS development ,42. HoweIn acute viral infections, nAb responses often correlate with protection or control. However, for Epstein-Barr virus (EBV) and KSHV, high antibody titers are often associated with disease ,46. Our It is puzzling that none of the immune parameters assessed predicted KS treatment response despite the strong association between KS and immune suppression. Furthermore, it was noted that a reduction in size and number of KS lesions after treatment did not correlate with the magnitude of the immunological responses measured to date. This implies that the response requires modulation of the qualitative or functional aspects of immune control. The lack of significant changes potentially explains the short-lived clinical responses and high recurrence rates reported in other studies ,21. As sA limitation of our study is the small sample size. The stringent recruitment criteria to ensure that only confirmed KS cases are analyzed has limited our sample size and, hence, the depth of our analyses. Thus, it is unlikely that subtle immunological differences between the treatment responders and non-responders can be detected. Additionally, late disease presentation due to the lack of mechanisms for identification of early KS, together with losses during follow-up, have limited our sample size and the depth of our investigations. Larger studies with close patient follow-up are warranted to fully characterize viral and immunological factors associated with the KS treatment response. However, this study highlights the fact that immune dysregulation in participants with KS persists despite a reduction in size and number of lesions in some participants. Additionally, the tested viral and immunological parameters did not predict the KS treatment response in SSA.+ and six were HIV-1−. Consenting subjects with complete clinical and treatment information were recruited. Only participants with KS who were newly diagnosed, histologically confirmed, and KS biopsy KSHV-DNA PCR positive at baseline were included. This is to avoid the approximately 35% misdiagnosed cases resulting from only clinical and/or histopathology diagnosed cases in SSA [The cohort was comprised of 26 study subjects who were ≥18 years of age and of both genders from Ocean Road Cancer Institute (ORCI), Dar es Salaam including 13 participants with KS and 13 KSHV-infected asymptomatic controls. Seven of the controls were HIV-1s in SSA ,49. PeriAccording to ORCI treatment guidelines, all participants with EpKS, who were not on ART, began ART immediately after diagnosis. Depending on the extent of KS disease, patients are then treated with combined chemotherapy consisting of six cycles of adriamycin, bleomycin, and vinblastine/vincristine (ABV) or radiotherapy (XRT). In this study, the KS treatment response was determined 3 months after completion of ABV chemotherapy or XRT. Some KS treatment responders maintained their remission status up to 12 months after completion of treatment. A responder was defined under the AIDS Clinical Trial Group (ACTG) clinical response criteria for a complete and partial response while non-responders included stable and progressive disease . For staHIV-1 infection status and quantification of plasma HIV-1 viral load before and after treatment were performed as previously described . Extract50).The rKSHV.219 was generated and tittered on HEK293T cells as previously described ,51. LevePlasma cytokines and chemokines were quantified with Becton-Dickinson Cytometric-Bead-Array (CBA) FlexSet kits, according to the manufacturer’s protocol. The standard sensitivity array (picograms/mL) included interleukin-4 (IL-4), chemokine CxCL10, interleukin-5 (IL-5), and transforming growth factor-β (TGF-β). The enhanced sensitivity array (femtograms/mL) included interferon-ɣ (IFN-ɣ), interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-12p70 (IL-12p70), interleukin-17A (IL-17A), interleukin-10 (IL-10), and tumor necrosis factor- α (TNF-α). Raw data were collected on an Accuri C6 Plus cytometer  and analyzed with FlowJo version-10 .To immunophenotype cell populations, PBMC were thawed, washed, split into two panels, and stained for 10 min in the dark at room temperature with antibodies against the following human surface markers: CD3 Percp.Cy5.5 , CD4, CD8-FITC, CD45RO-APC, CD197-AlexaFluor-700, CD38-BV510, HLA-DR-PE.Cy7, CD28-BV711, CD27 PE-Dazzle-594, CD57-APC, and CD107a BV421. The cells were fixed using Perm/fix  for 20 min at 4 °C in the dark, and then stained for intracellular Ki67  or IFN-γ . Raw data were quantified by FACSAria  and analyzed with FlowJo version-10.+/HIV-1+) similar to EpKS patients in order to control for viral co-infection and investigate the effect of cancer (KS) in the background of HIV-1 co-infection on T-cell phenotypes while the other group of controls was  similar to EnKS patients in order to control for KSHV infection and investigate the effect of cancer (KS) in the absence of HIV-1 co-infection on T-cell phenotypes. Similar analyses on cytokine and antibody responses have been reported previously [p-values < 0.05 were considered significant.Viral nucleic acid detection in plasma, antibody, and cytokine responses as well as T-cell immunophenotypes were stratified into high and low levels before and after KS treatment. Correlation with the treatment response was analyzed. One-way analysis of variance with Tukey’s multiple comparison test and non-parametric Mann-Whitney t-test were employed to determine differences between groups. One control group was (KSHVeviously . The effIn summary, there was a trend toward favorable treatment outcomes in patch and plaque stages of KS. Our study tested KSHV and HIV-1 viremia, CD4 T-cell counts, KSHV-humoral responses, various cytokine responses, and the magnitude of T-cell population subsets as predictors of the KS treatment response but did not find significant associations. An important finding of this study is that, despite favorable clinical responses in some participants with KS, immunological dysregulation observed at baseline, persisted in these patients in remission, and this likely contributes to the high KS recurrence rates.


In this issue of the Journal, Zganjar et al.  report tKS, a group of chromosomal disorders characterized by the presence of 1 or more extra X chromosomes is typically associated with hyalinization of the seminiferous tubules, azoospermia, and infertility . The molEven though all cells in the KS embryo carry a 47, XXY chromosomal complement, a small fraction of germ cells in the prepubertal testis are diploid 46, XY), likely due to a random loss of the extra X chromosome during 1 of many spermatogonial mitotic divisions , 6, 7. T, XY, likMen with KS have been historically considered sterile; however, the combination of TESE and intracytoplasmic sperm injection (ICSI), has enabled some men with KS to achieve fertility . A meta-Damani et al.  demonstrThe clinical value the KS adolescent derives from testosterone treatment and the need for such therapy is based upon the notion that testosterone levels, which are typically normal or low normal in late puberty , decreasTesticular sperm extraction is not without some downside; it is a surgical procedure that requires anesthesia, imposes some financial and emotional burden, and maybe associated with a decrease in circulating testosterone levels that may persist for 12 or more months after the procedure. Furthermore, it is often difficult for an adolescent boy to make an informed decision about future fertility which may appear abstract at that age. The adult with KS and his partner may be in a substantially better position to take into account the intensity of their desire to have a child, the success rates of TESE and ICSI, and the financial and emotional burdens of a surgical procedure on the patient and that of the treatment cycle to harvest oocyte from his partner.Thus, there is no compelling reason that TESE has to be performed during adolescence , 4 and n


Gliosarcoma is a variant of glioblastoma with equally poor prognosis and characterized by mixed glial and mesenchymal pathology. Metastasis is not uncommon but the involvement of the spinal cord is rare, and comprehensive genetic characterization of spinal gliosarcoma is lacking. We describe a patient initially diagnosed with a low-grade brain glioma via biopsy, followed by adjuvant radiation and temozolomide treatment. Nearly 2 years after diagnosis, she developed neurological deficits from an intradural, extramedullary tumor anterior to the spinal cord at T4, which was resected and diagnosed as gliosarcoma. Whole-exome sequencing (WES) of this tumor revealed a hypermutated phenotype, characterized by somatic mutations in key DNA mismatch repair (MMR) pathway genes, an abundance of C>T transitions within the identified somatic single nucleotide variations, and microsatellite stability, together consistent with temozolomide-mediated hypermutagenesis. This is the first report of a hypermutator phenotype in gliosarcoma, which may represent a novel genomic mechanism of progression from lower grade glioma. Similar to glioblastoma, patients diagnosed with gliosarcoma exhibit poor prognosis and overall survival2. Metastasis is relatively common within the central nervous system (CNS), as well as systemic visceral sites1. Gliosarcomas involving the spinal cord are rare, making up approximately 1% of all malignant gliomas of the spine3 and may represent metastasis from intracranial tumors or less commonly, de novo formation5. To date, comprehensive genomic characterization of gliosarcomas remains vastly under-reported, compared to gliobastomas.Gliosarcomas represent a variant of glioblastoma, characterized by mixed histopathological features of glial and mesenchymal originsIn this study, we report a patient with a spinal cord gliosarcoma with prior history of a low-grade brain glioma treated with adjuvant chemoradiation. Whole-exome sequencing (WES) of the resected spinal tumor revealed the first case of a hypermutator phenotype to be described in gliosarcoma.TP53 was positive. Further molecular testing was negative for the EGFR variant III, IDH1 mutation, and MGMT promoter methylation. She was subsequently referred to our institution for further adjuvant treatment, where she underwent adjuvant radiation therapy and temozolomide treatment, as well as use of tumor-treating fields (TTF) with the Optune device . A brain MRI obtained several months after completion of chemoradiation and TTF showed radiographic response with stable small foci of enhancement and drastic reduction in perilesional edema  of the brain, which revealed a predominantly non-enhancing right parietal lesion with patchy enhancing foci extending to the splenium of the corpus callosum Fig. . Furtherema Fig. . ClinicaMGMT promoter methylation but was otherwise negative for IDH1 mutation and ATRX loss.Six months after completion of chemotherapy, she presented with increasing gait instability of two weeks duration and worsening thoracic back pain, localized between her shoulder blades. MRI of her thoracic and lumbar spine was obtained, demonstrating a homogenously enhancing dural-based, extramedullary lesion anterior to the spinal cord at T4 with moderate cord compression, causing edema extending from T2 to T7 Fig. . She wasThe patient was subsequently discharged to rehabilitation and exhibited some improvement in lower extremity strength, albeit remained non-ambulatory and required significant assistance with activities of daily living. Further treatment with adjuvant chemoradiation was considered but given the unfavorable prognosis from new gliosarcoma diagnosis and poor performance status, she elected to undergo hospice care without further imaging or treatment and subsequently expired one month after surgery.6. We detected 2372 somatic SNVs with a 96% C>T transition ratio  pathway, predicted to be deleterious based on scores from the following computational prediction tools: SIFT7, Polyphen2 HDIV Prediction8, Polyphen2 HVAR Prediction8, FATHMM9, MetaSVM10, and MutationTaster11. These included deleterious missense (p.P656L) and stop-gain (p.W105X) mutations in MSH6 and a stop-gain mutation in MSH3 (p.R727X). Additionally, there was a heterozygous germline variant in MSH2 (p.H46Q) of unknown significance. Somatic CNV analysis revealed amplifications of chromosomes 7, 20, and X, deletions on chromosomes 10, 13, 17, and focal CNV events on chromosomes 1 and 5. 27% of the genome was altered by CNV and/or loss-of-heterozygosity events.WES was performed on the gliosarcoma specimen, together with matching normal blood, to identify somatic single nucleotide variations (SNVs), insertions/deletions (INDEL), and copy number variations (CNV). The protocol for sequencing and analysis was in accordance with our previously described methods12, we performed further computational analysis with MSIsensor, an algorithm that predicts the microsatellite status of tumors from WES data13. The calculated MSI-score was 0.15, indicative of microsatellite stability per the tool guidelines, which uses a cut-off score of 3.5 for MSI.Given the relationship between microsatellite instability (MSI) and MMR deficiency in systemic cancers14. As such, most cases of gliosarcomas involving the spinal cord have been secondary to metastasis from intracranial origin19. In contrast, primary gliosarcomas of the spinal cord are less common22 and are vastly outnumbered by glioblastoma when analyzing primary malignant gliomas of the spinal cord3. Interestingly, these tumors may present as intramedullary23, as well as intradural, extramedullary lesions22, regardless of primary or metastatic etiology. Similar to their intracranial counterparts, spinal cord gliosarcomas demonstrate avid and typically homogenous enhancement, which in cases of intradural, extramedullary location, may be mistaken for a meningioma, as demonstrated in our patient. A prior history of a primary brain tumor must raise suspicion for an alternative diagnosis and highlights the need for surgical intervention to aid in definitive tissue diagnosis.Gliosarcomas have a high predilection for metastasis to both visceral sites as well as the CNS, via hematogenous and leptomeningeal spreadIDH-wildtype astrocytomas with grade II histology may exhibit survival outcomes akin to IDH-wildtype glioblastoma25. Based on recommendations from the Consortium to Inform Molecular and Practical Approaches to CNS Tumor Taxonomy (cIMPACT-NOW)26, molecular testing to assess for EGFR amplification, chromosome 7 gain/chromosome 10 loss, and TERT promoter mutations to diagnose a diffuse astrocytic glioma, IDH-wildtype, with molecular features of glioblastoma, WHO grade IV could have been performed to predict increased biological aggression and perhaps tailor clinical management28. The existing literature of metastatic gliosarcomas to the spinal cord have all been from primary intracranial gliosarcoma, so the notion that our patient’s gliosarcoma represented metastasis, despite the difference in WHO grade from her initial tumor, would be unique. There have been few reports of progression of low-grade gliomas into intracranial gliosarcomas, in instances with and without adjuvant chemoradiation31. These studies confirmed metastatic origin through single nucleotide polymorphism (SNP) arrays and fluorescence in-situ hybridization (FISH) for chromosomal analysis, but to date, comprehensive sequencing to delineate the genetic changes that may underlie either malignant progression into gliosarcomatous pathology or de novo spinal cord gliosarcoma formation have not been described.It remains unclear whether the gliosarcoma in our patient represented metastasis from her intracranial astrocytoma or de novo formation of a new primary tumor. This may have been addressed with additional WES data or targeted molecular testing from her original brain tumor, but unfortunately, inadequate tissue was available for further genomic studies. Additionally, further molecular testing of her brain tumor would have been helpful, as 34 and particularly after therapy-driven changes in tumor evolution at the time of recurrence. In particular, treatment with temozolomide may induce mutations in the MMR pathway, leading to therapeutic resistance and acquisition of the hypermutated phenotype36. Strikingly, we found a hypermutated phenotype in our patient’s specimen, including mutations in MMR genes, MSH3 and MSH6, which previously have not been associated with gliosarcoma pathology. Likewise, the abundance of C>T transitions detected within the somatic SNVs of our patient’s tumor was consistent with a MMR deficient hypermutated phenotype. We further explored the possibility of MSI, particularly given the strong correlation between MSI and MMR deficiency in systemic cancers that may serve as biomarkers for response to immunotherapies40. Utilizing the MSIsensor computational tool, we did not detect MSI despite the hypermutator phenotype of our patient’s tumor, a result consistent with prior studies failing to reveal an association between MSI and temozolomide-mediated hypermutagenesis in glioblastoma41. Taken together, these findings are consistent with a hypermutated phenotype secondary to temozolomide therapy in our patient’s gliosarcoma. Unfortunately, the WES data were not available in a timely enough manner to guide further treatment for our patient, such as the possibility of immunotherapy. Considering the clinical timeline of temozolomide treatment for her initial intracranial glioma but prior to spinal gliosarcoma diagnosis, this result also supports the notion of gliosarcomatous transformation from her lower grade glioma, rather than de novo tumor formation. We acknowledge that the inability to perform further genetic testing of her brain tumor is a key limitation of this study. Lastly, there are few reports of comprehensive genome sequencing of gliosarcomas, which have described key somatic mutations in known oncogenes including TP53, PTEN, RB1, and NF1 as well as amplification events of EGFR, CDK4/6, PDGFRA, MDM2, AKT1, and MET44. However, this case represents the first report of hypermutagenesis within gliosarcoma and raises the possibility that acquisition of this genotype may be a mechanism of gliosarcomatous transformation from lower grade tumors.In recent years, the prevalence of hypermutagenesis has been described across a variety of cancers, including gliomasMSH2 variant. Sa et al identified a subset of treatment-naïve gliomas with de novo hypermutagenesis that had not been previously exposed to chemoradiation at the time of diagnosis45. Notably, these patients demonstrated germline mutations of MMR associated genes, including MSH2, MSH3, MSH6, and MLH3 and relevant family histories of various cancers, together suggestive of diagnosis of Lynch syndrome. Likewise, gliosarcoma has been described in Turcot syndrome46, a condition characterized by familial predisposition to colorectal and brain cancers, secondary to germline mutations of the MMR genes47. In contrast, our patient’s germline variant in MSH2 has been previously characterized as a benign variant, reported in individuals with colorectal, prostate, and ovarian cancers but not meeting clinical criteria for Lynch or Turcot syndrome or showing strong evidence for causality49. Furthermore, the lack of a second hit for MSH2 supports the notion of our patient’s variant as benign. As such, it remains unlikely that our patient’s germline MSH2 variant contributed to de novo tumor formation and/or acquisition of a hypermutator phenotype.We considered whether our patient’s gliosarcoma arose de novo with a predisposition from her detected germline In summary, we describe the first case of a hypermutator phenotype associated with a gliosarcoma, characterized by somatic mutations in known MMR genes and consistent with temozolomide-mediated hypermutagenesis. Based on patterns of the described literature, it is more likely that our patient’s spinal cord gliosarcoma represents malignant progression and metastasis from her previous low-grade brain astrocytoma. As gliosarcoma remains a rare entity within primary malignant CNS tumors, even more so within primary spinal cord tumors, further comprehensive genomic characterization is needed to understand the genetic factors that can lead to malignant transformation from lower grade gliomas, as well as de novo formation of gliosarcomas.This study was approved by Yale University’s Human Investigations Committee and Human Research Protection Program. Written informed consent was obtained from the parents of the patient. Specimens from resected tumors were evaluated microscopically by a board-certified neuropathologist.50. Microsatellite status was determined with MSIsensor, a publically available computational algorithm designed to predict microsatellite stability based on WES data13.Genomic DNA from the resected tumor specimen and blood were isolated, and exome capture was performed with IDT xGen Exome Research Panel v1 with the additional spike-in regions of ~620 kb of RefGene coding regions. Sequencing of the captured regions was performed at Yale Center for Genome Analysis (YCGA) using Illumina NovaSeq6000 with 2 × 100 bp reads. Mean coverage of 152.4x and 230.6x was achieved for normal and tumor, respectively. Pre-processing of the raw data, including alignment, PCR duplicate identification, re-alignment around INDELs, and base quality score recalibration was performed “GATK Best Practices” guidelines (GATK v4.1.8.1). Somatic variant calling for SNV/INDELs together with CNV analysis and annotation was performed as previously described in reports from our institutionFurther information on research design is available in the REPORTING SUMMARY


Oleh Hornykiewicz , by demonstrating the loss of dopamine neurons in Parkinson’s disease, introducing the effort to treat the disorder with L-DOPA, and other innovative research, improved the lives of countless individuals and transformed neurology and medical science. Here we celebrate the life and great achievements of an outstanding scientist. Oleh Hornykiewicz did not have an easy start in life. He was born in November 1926 in Lviv , an area with many nationalities contested over the centuries by Ukraine, Prussia, Lithuania, Sweden, Austria-Hungary, Cossacks, Poland, and Russia. In the beginning of 1940, his family of Ukrainian / Austro-Hungarian origin, moved to Vienna, where he, as a 13 year old immigrant, had to learn German for his schooling. The years in Austria under the Nazi regime and later in post-war years were far from easy. In 1945, after a fierce battle, Vienna was occupied by Allied and Soviet armies and brought to terrible conditions with robberies, revenges, rapes, poverty, and food shortages mainly caused by Soviet army. These experiences produced in the young Oleh a strong character that helped him later to deal with disputes, fights, and frustrations throughout life. At the same time those early difficulties gave him the capacity to fully enjoy the important rewards  Blaschko’s department in Oxford. Blaschko’s laboratory was then the center for research in catecholamines, particularly due to their characterizations of monoamine oxidase and DOPA decarboxylase.1 He also tested L-DOPA and found the same effects.In Blaschko’s group, he devoted a couple of years to the study of the cardiovascular effects of dopamine. He showed that the effects were due directly to dopamine and not to a metabolite of dopamine.2 Weil-Malherbe and Bone,3 and Carlsson et al.4 he had read in Oxford, he became a staunch supporter of dopamine’s role as a neurotransmitter. He was particularly impressed by Montagu’s finding that the same compound whose effects he had been studying on blood pressure, was found in the central nervous system, which he described as “bordering, for me, on the sensational”.Hornykiewicz credited Blaschko’s advice to continue to focus on dopamine in his research for his subsequent successful career. Returning to Vienna following reports on dopamine in the brain by Montagu,6 and the reversal by exogenous L-DOPA administration of reserpine’s induced akinesia consequent to brain catecholamines depletion.7Oleh, who was very fair with giving credit to colleagues, was further impressed by the demonstration of dopamine’s high concentration in basal ganglia of healthy human brain8 Hornykiewicz was also the first to suggest that the loss of these neurons in the midbrain was responsible for the loss of dopamine in the striatum, and in that way was fundamental to introducing the nigrostriatal pathway.9While the loss of the substantia nigra neurons that were pigmented due to the presence of neuromelanin was observed in 1919 by the Russian neuropathologist, Konstantin Tretiakoff, the neurochemical properties of these neurons were unknown. After establishing his laboratory at the University of Vienna, Oleh Hornykiewicz and his young postdoc, Herbert Ehringer, set out to measure brain dopamine and norepinephrine in normal adult human post-mortem brains and in patients who had died of Parkinson disease and postencephalitic parkinsonism. Comparing the concentrations of dopamine and norepinephrine in multiple brain regions, they found a selective dopamine depletion in the caudate nucleus and putamen in patients with both forms of parkinsonism.10 and in his words in an interview with Barbara Sommer stated:The obvious next step from that discovery was to attempt to treat Parkinson’s patients by administering dopamine’s precursor, levodopa that could reach the brain crossing the blood brain barrier. It took over a year to convince his clinical partner, the neurologist Walther Birkmayer, to attempt intravenous injection of levodopa in patients with Parkinson disease, mostly because Birkmayer was believing that a loss of hypothalamic serotonin, rather than dopamine, was responsible for the disorder. Eventually, they administered small doses of levodopa intravenously to five parkinsonian patients, resulting in the miraculous “awakening” immortalized in the movie now visible at several scientific sites. He succeeded in the beginning of July 1961“I still remember, of course. I was present at that time in the Lainz hospital and watched the results. It was a spectacular moment to see the patients who could not walk, could not get up from bed, could not stand up when seated, start walking. They all performed these activities like normal. Speech became better. The associated movements, the face expressions; they started laughing and then actually crying with joy. These were patients who could not be helped by any doctor. And then levodopa produced the effects. It was really very spectacular.”11, millions of parkinsonian patients now enjoy decades of acceptable life in spite of their progressive neurodegenerative condition.Thanks to that historical event, along with subsequent clinical investigations including the administration of high oral doses of levodopa introduced by Cotzias et al.In 1967 Hornykiewicz was offered a professorship at the University of Toronto, Clarke Institute of Psychiatry, where he led the Psychopharmacology Section until he returned to Vienna as Professor of Biochemical Pharmacology in 1976, retaining his scientific commitment in Toronto until the early 1990s. Back in Vienna he was instrumental in the creation of the new Centre for Brain Research at the University.Oleh Hornykiewicz was a gentleman and a family man. With his wife, Christine, they had Maria, Nikolai, Stephan, and Joseph and together enjoyed an intense family life until Christine’s passing away in 2017. Hornykiewicz was an “independent thinker” like most ingenious people. Obviously, he was open minded and listened to all scientific opinions, but at the end he drew his personal conclusions and road maps for the experimental paths that led him to great achievements. Although exposed to different mentors whom he respected highly, he was not a follower of anyone. Hornykiewicz’s work was novel and centered on human brain disease. Sometimes having one prevailing mentor induces people to remain intellectually pupils for their entire life. When meeting somebody for the first time, within the blink of an eye, he immediately understood who you were, your standing, and your potential. Even though he was a highly respected and recognized scientist, he always remained very open to listening and discussing opinions with anybody, without arrogance . He demonstrated that the post-mortem biochemical analysis of nervous tissue provides reliable and relevant clues to the pathogenesis “in vita” of diseases, giving birth to the field of “pathobiochemistry” that has led us to the contemporary understanding of many neuropsychiatric disorders.He provided an example of how it is important for scientists to have original, independent ideas rather than follow the fads of the moment, and how once those ideas are corroborated by solid experimental evidence, to be brave enough to carry them forward. That means we have to be ready to fight for our ideas, but this is obviously not a life for all humans. His driving force was scientific curiosity and not a wish for power and career, unlike what is commonly sought by many. Even in the final days of his life, talking with him you could feel his excitement and intellectual curiosity to understand even the minutiae of science and life Fig. . He suggThe methods he used were quite simple but straightforward and accurately carried out. In life, Oleh showed how undergoing difficult situations in youth may help people to grow strong and ready to face and solve problems in the rest of their life. He also provided an example of how a stable, loving family could provide support during a challenging scientific career.


New discoveries based on genetic and epigenetic evidence have significantly expanded the understanding of diffuse gliomas. Molecular biomarkers detected in diffuse gliomas are not only potential targets for radiotherapy, chemotherapy, and immunotherapy, but are also able to guide surgical treatment. Previous studies have suggested that the optimal extent of resection of diffuse gliomas varies according to the expression of specific molecular biomarkers. However, the specific guiding role of these biomarkers in the resection of diffuse gliomas has not been systemically analyzed. This review summarizes several critical molecular biomarkers of tumorigenesis and progression in diffuse gliomas and discusses different strategies of tumor resection in the context of varying genetic expression. With ongoing study and advances in technology, molecular biomarkers will play a more important role in glioma resection and maximize the survival benefit from surgery for diffuse gliomas. Histologically, diffuse gliomas originate from aberrant neural progenitor stem cells of the central nervous system and composed of either astrocytes or oligodendrocytes. Although the morphology-based classification system from the World Health Organization (WHO) in 2007 is highly significant for the characterization of diffuse gliomas, it is unable to explain the evident differences in prognosis of patients with the same histopathology , 2. MoleThe goal of traditional treatment strategies for diffuse gliomas was to achieve maximum resection in the context of protecting neurological and functional integrity, and further adjuvant radiotherapy and/or chemotherapy is applied according to the extent of resection (EOR) and histopathology . The degMultiple retrospective studies and large meta-analyses have demonstrated that, regardless of low- or high-grade, primary or recurrent, greater EOR can significantly prolong both progression-free survival (PFS) and overall survival (OS) of diffuse gliomas compared with partial resection or biopsy –9. HowevIsocitrate dehydrogenase (IDH) is an enzyme participating in the tricarboxylic acid cycle. IDH 1 and IDH 2 are two subtypes of IDH and responsible for converting isocitrate to alpha-ketoglutarate in the cytoplasm and mitochondria, thereby influencing the metabolic process. Studies have confirmed that, although IDH mutation occurs in the early stages of glioma formation and may affect DNA demethylation and lead to tumorigenesis, the exact mechanism remains unclear . IDH mutIn patients with diffuse astrocytoma, the effect of surgical resection on prognosis is different according to IDH expression and pathological grade. A previous study involving 228 adults with supra-tentorial low-grade gliomas (WHO grade 2) found that postoperative tumor volume exerted a negative effect on OS. The impact was more evident in astrocytoma with IDH mutations, in which any tumor residual will significantly reduce OS. Therefore, maximal resection in the primary operation is crucial, and a “second-look” operation may be necessary to ensure total resection for IDH-mutant tumors, if it can be performed safely . Other sRegarding anaplastic diffuse astrocytoma, a study analyzed the relationship between EOR and prognosis and revealed that GTR can significantly prolong OS of patients with IDH-mutant anaplastic diffuse astrocytoma but not wild-type . SimilarOn reviewing the existing literature, we conclude that radical surgical treatment is beneficial for patients with diffuse astrocytoma. Even if the tumor is located in a functional area, it is worthwhile to realize total resection at the cost of few partially unessential function because of the brain plasticity. Relatively conservative surgical treatment is recommended only for patients with IDH wild-type malignant diffuse astrocytomas Fig. .Fig. 1GFUBP1 and CIC genes, which promote tumorigenesis and cell proliferation [1p/19q chromosome codeletion results from a balanced translocation of the short arm of chromosome 1 and the centromere region of chromosome 19 and leads to the subsequent deletion of other chromosome arms . It commferation , 31. In feration , 33.GTR is not always recommended for diffuse gliomas with IDH mutation and 1p/19q codeletion, especially for those located in the eloquent area. A previous study demonstrated that, in low-grade diffuse gliomas with IDH mutation and 1p/19q codeletion, GTR would not significantly prolong OS . CompareO6-methylguanine-DNA methyltransferase (MGMT) is a DNA damage repair protein that removes guanine alkylation and prevents apoptosis , 36. TheMGMT promoter methylation helps to improve EOR. In MGMT promoter methylated GBMs, intraoperative ultrasound, intraoperative MRI, and 5-aminolevulinic acid (5-ALA) fluorescence staining can more accurately determine tumor boundaries, reduce the false-negative rate, and, ultimately, improve EOR . Sharma Epidermal growth factor receptor (EGFR) is a tyrosine kinase, which is commonly activated or overexpressed in malignant astrocytoma, with the most common form of recombinant amplification being the EGFR variant type III (EGFRvIII) . EGFRvIIIn the past few years, 5-ALA has been established as an effective intraoperative tool to increase EOR in high-grade gliomas . Fontanap53 is one of the most well-known tumor suppressor proteins to date, involving virtually all cancers, including gliomas. TP53 gene mutation involves multiple regulatory factors and is believed to promote malignant progression of low-grade glioma . ATRX isCD133  is a membrane-bound glycoprotein that may be involved in cell differentiation and epithelial to mesenchymal transition and is a marker of human neural stem cells . A previIn addition to the molecular markers mentioned above, there are many other molecular variants associated with diffuse gliomas. Further study is necessary to explore the relationship between molecular biomarkers and tumor resection to improve therapeutic effects and patient prognosis.Based on the evidence mentioned above, molecular biology is, in fact, able to guide tumor resection to improve survival outcomes of patients with diffuse gliomas. Hence, preoperative prediction and intraoperative detection of molecular biomarkers are critical for glioma surgery. Several techniques have been applied to acquire both histological and molecular characteristics intraoperatively, including rapid frozen section, mass spectrometry, and real-time polymerase chain reaction, among others , 56. ExcMRI yields a significant amount of information about tumors. Recently, radiomics has emerged with the development of image feature extraction and machine learning techniques. Radiomics can automatically extract high-throughput and high-dimensional image features from medical imaging data aimed at obtaining more objective, quantitative, and invisible tumor features. Subsequently, these features could be combined with the results of molecular detection methods to establish prediction models to achieve accurate prediction before surgery , 59. BasIn summary, both preoperative and intraoperative techniques, including radiomics, real-time polymerase chain reaction, gap-enhanced Raman tags, molecular nanoprobes, and other fluorescence staining techniques based on molecular biomarkers, can provide valuable evidence of molecular biomarker expression and tumor margins, which could further affect the design and implementation of surgical strategies to treat diffuse gliomas. The development and application of these new techniques could establish a foundation for guiding surgical strategies with molecular biomarkers and further the progress of individualized medicine.The main limitation of the present literature review was that all studies were retrospectively designed. This was inevitable because no randomized controlled clinical trials have been conducted. Additionally, the number of studies that focused on the correlation between molecular biomarkers and EOR is limited, especially when taking functional area into consideration. Thus, the level of evidence for EOR recommendations based on molecular biomarkers is relatively low.The current study revealed that molecular biomarkers can inform the design of surgical strategies for diffuse gliomas. In the era of precision medicine, individualized rather than “one-size-fits-all” operative strategies developed by comprehensive consideration of functional status and molecular biological characteristics can maximize the survival benefit to patients, which would represent a major trend in the surgical treatment of diffuse gliomas in the future.


Mycobaterium bovis is the main cause of infection in water buffalo  and bovines and is of great concern for human health and for buffalo producers in Italy. The bTB eradication programme is based on slaughterhouse surveillance and intradermal skin tests. Other in vivo diagnostic methods such as the interferon-gamma (IFN-γ) assay have been developed and are widely used in cattle to accelerate the elimination of bTB positive animals. The present study is the first to assess the use and performance of IFN-γ assays, which is used as an ancillary test for bTB diagnosis in water buffalo, and presents the results of a field-evaluation of the assay from 2012 to 2019 during the buffalo bTB eradication programme in Italy. The study involved 489 buffaloes with a positive result to the single intradermal tuberculin test (SITT). The IFN-γ assays and single intradermal comparative tuberculin test were used as confirmation tests. Then, a total of 458 buffaloes, reared on officially tuberculosis-free (OTF) herds, that were confirmed bTB-free for at least the last 6 years were subjected to IFN-γ testing. Furthermore, to evaluate the IFN-γ test in an OTF herd with Paratuberculosis (PTB) infection, 103 buffaloes were subjected to SITT and IFN-γ test simultaneously. Four interpretative criteria were used, and the IFN-γ test showed high levels of accuracy, with sensitivity levels between 75.3% (CI 95% 71.2–79.0%) and 98.4% (CI 95% 96.7–99.4%) and specificity levels between 94.3% (CI 95% 91.2–96.50%) and 98.5% (CI 95% 96.9–99.4%), depending on the criterion used. Finally, in the OTF herd with PTB infection, in buffalo, the IFN-γ test displayed high specificity values according to all 4 interpretative criteria, with specificity levels between 96.7% (CI 95% 88.4–99.5%) and 100% (CI 95% 96.2–100%), while SITT specificity proved unsatisfactory, with a level of 45.3% (CI 95% 35.0–55.7%). Our results showed that the IFN-γ test in the buffalo species could reach high Sensitivity and Specificity values, and that the level of Sensitivity and Specificity could be chosen based on the interpretative criterion and the antigens used depending on the health status of the herd and the epidemiological context of the territory. The IFN-γ test and the use of different interpretative criteria proved to be useful to implement bTB diagnostic strategies in buffalo herds, with the possibility of a flexible use of the assay.Bovine tuberculosis (bTB) is a worldwide zoonosis that affects many species of domestic and wild animals.  Mycobacterium tuberculosis and Mycobacterium bovis; therefore, one of the objectives of the WHO is to improve the surveillance and reporting of bovine TB (bTB) in livestock and wildlife and to augment the capacity of the animal health sector to reduce the prevalence of bTB.Tuberculosis (TB) is a zoonosis of global importance, causing major economic losses and trade restrictions. In the year 2018, 10 million people contracted TB worldwide, of whom 1.5 million died  was recorded in Egypt in 1986  territory. Improve the diagnosis of bTB is a real challenge because this disease is still causing serious economic and genetic losses as a result of the slaughter of infected animals and the depreciation of milk, which is subject to obligatory heat treatment on M. bovis infected farms, in a territory whose economy is closely linked to buffalo dairy products.The prevalence and incidence rates of bTB in the Campania buffalo population were 7.30 and 3.50%, respectively, in 2018 and 13.80 and 8.9% in 2019. It should be noted that about 6,500 animals were slaughtered in 2018 and 4,600 in 2019 as a result of “test and cull” strategy adopted during bTB outbreaks. The cost to the Campania region for compensating buffalo farmers exceeded €20 million in the 2019 (data from Campania region report). It would therefore be extremely advantageous to have an accurate diagnostic strategy that could rapidly reveal bTB outbreaks during the early stage of Syncerus caffer or in B. bubalis , recognized by the World Organization of Animal Health and the European Commission as the screening test, used in swamp buffalo , 9 and i bubalis .M. terrae, M. nonchromogenicum, M. vaccae, Mycobacterium avium subsp. avium, and M. avium subsp. paratuberculosis (MAP), might reduce its Sp , such as e its Sp , 12. More its Sp .M. bovis and those infected with M. avium complex or environmental mycobacteria  is used primarily as an ancillary test for positive or inconclusive reactors in the SITT . Therefore, these criteria should be used in high bTB prevalence context either in herds or territories.The same performance was observed in the data-set where the inconclusive results had been removed, and the assessment of the accuracy was performed in 718 animals .In fact, the second and third criteria introduce the possibility of animals without an outcome, the second criterion gives 14.5% (137/947) of IN results and the third criterion 19.4% (184/947) ND results. In general, these animals are more difficult to define. When evaluating all the criteria these “difficult animals” were removed and the criterion 2 and 3 had shown better values of Se (98.6% CI 95% 97.0–99.5%) in contrast to the criterion 1 (97.7% CI 95% 95.8–98.9%) and criterion 4 (79.5% CI 95% 75.4–83.2%).However, with the second criterion, among 137 IN animals, only 11 were expected true positive and the other 126 were expected true negative. For the criterion 3, which is based on the relationship between two couples of PPDB and PPDA, among the 184 ND animals, 54 were expected true positive and 130 were expected true negative, because this criterion is the most conservative one. In fact, this criterion provides different steps and controls to define the correct sample and result; in terms of accuracy, it therefore achieves better performance , but leaves more subjects without an outcome. Similar to other reports , 32 a maIn addition, our study also suggests that using the ESAT6/CFP10 cocktail (fourth criterion), in addition to PPDs, minimizes the possibility of obtaining a false-positive result. It could therefore be a useful tool for diagnosing bTB in herds or territories in which the prevalence of bTB is low.The four criteria also showed high levels of precision as the reproducibility and repeatability values were very satisfactory, and the tests were carried out in accredited public laboratories that have been performing the IFN-γ test for several years.in vivo and in vitro bTB assays is the cross-reactivity with responses induced by exposure to NTM, including MAP, we wanted to assess the Sp of the 4 criteria of the IFN-γ test in a particular but frequent situation a buffalo herd negative to bTB (OTF) but PTB infected for several years. In buffalo, as in cattle, infection/exposure to NTM can interfere with bTB diagnosis, because the composition of PPDs includes several antigens that can cross-react with environmental mycobacteria and this may lead to false positive reactions  lower than the values reported in cattle in the literature between 75.5 and 99.0% .M. bovis-negative (The lack of concordance between the Sp of the IFN-γ test (96.7% CI 95% 88.5% 89.6–100% CI95% 96.20–100%) and the Sp of SITT (45.3% CI 95% 35.0–55.7%) in the OTF herds with MAP infection was due to the use of the avian PPDs in the IFN-γ test, that were able to correctly identify MAP-infected animals and therefore classify them as negative , 69, 94.In summary, our data indicate that, in buffalo, the IFN-γ assay is an excellent test and shows good accuracy which ranged from 96.42% (CI95% 95.14-97.70%) to 98.00% (CI95% 97.01-98.99%) for the three best criteria.Since in cattle, the diagnostic Se of bTB positive animals improves when SITT is used in combination with the IFN-γ test , 82, we In the present study, the Se of the IFN-γ test in buffalo, which ranged from 94.70% CI95% 92.30-96.50%) to 98.04% (CI95% 96.70-99.40%) for the best three criteria, was comparable to that indicated in cattle, with a Se estimated median of 87.6% with a range between 73 and 100% , 30.-96.50% tWith regard to Sp, our values in the buffalo ranged between 97.20% (CI95% 95.20-98.50%) to 98.50% (CI95% 96.90-99.40%) for the best three criteria, and we have achieved the best performance reported in cattle, with a Sp median value of 96.6% (range: 85.0–99.6%) , 30.B. bubalis displayed higher Se and Sp levels than those reported in S. caffer by Michael et al. , explain why the usefulness of SITT in the diagnosis of bTB in buffaloes is still debated  due to the highest number of inconclusive results (140/489) and 41 false-negative results  and among the 41 false-negative results, identified a mean of 36 bTB positive buffaloes.M. bovis. Furthermore, its different interpretative criteria and antigens can be adopted according to the objective to be pursued and the epidemiological context (The IFN-γ test has been proved as an objective method, as it utilizes a standard procedure and is not affected by the subjectivity of the operator, in contrast to SITT, which could be influenced by several factors that can interfere with Sp and Se , 30. Mor context . Our res context .Finally, countries that gained bTB eradication in cattle, including Australia, focused their attention on the herd rather than on the individual animal; these countries had considered SITT as primary screening test for bTB in herds because of its low accuracy , while aIn summary, our study provides new data on the Se and Sp of the IFN-γ test comparing four interpretative criteria for bTB diagnosis in water buffalo under field conditions.Our results showed that the IFN-γ test in the buffalo species could reach high Se and Sp values, and that the level of Se and Sp could be chosen according to the interpretative criterion and the antigens used, depending on the health status of the herd and the epidemiological context of the territory.M. bovis compared to SITT.In addition, the 4 interpretive criteria, in OTF herds with PTB, proved to be particularly useful in drastically reducing false positivity reaction for Based on our results, in order to improve bTB diagnostic Se in buffalo herds, IFN-γ assays could be used in parallel with the SITT to identify the largest number of infected buffaloes in bTB outbreaks. Meanwhile, in order to improve bTB diagnostic Sp, IFN-γ assays could be used in series with SITT to limit false positive results in buffalo herds that are officially bTB-free.Starting from the reported experience in cattle and the data of our study, in territories where bTB is still present, such as the Campania region, the use of the IFN-γ assay can support successfully the bTB eradication programme in buffalo.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The SITT and SICTT and collection of blood samples on which the IFN-tests were carried out was conducted as part of The National and Regional Buffalo TB Eradication Programme, in compliance with the EU trade  Council AM, ED, MP, MB, PM, and NV carried out the conceptualization of the study and performed the supervision. AM, PM, AD, IA, LS, and AC developed the methodology. NV and LC dealt with the software. AM, AD, NV, IA, FG, LS, and PM carried out the validation. NV, LC, AM, PM, MB, and MP performed the formal analysis. AM, AD, IA, LS, AC, and FG performed the investigation. AM, ED, and PM addressed the resources. NV, LC, AM, and LS performed the data care. AM, NV, PM, IA, MB, and MP prepared the original drafts for writing. AM, NV, PM, MB, MP, and ED carried out the writing—review and editing. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Antimicrobial resistance (AMR) should be tackled through a One Health approach, as stated in the World Health Organization Global Action Plan on AMR. We describe the landscape of AMR surveillance in the European Union/European Economic Area (EU/EEA) and underline a gap regarding veterinary medicine. Current AMR surveillance efforts are of limited help to veterinary practitioners and policymakers seeking to improve antimicrobial stewardship in animal health. We propose to establish the European Antimicrobial Resistance Surveillance network in Veterinary medicine (EARS-Vet) to report on the AMR situation, follow AMR trends and detect emerging AMR in selected bacterial pathogens of animals. This information could be useful to advise policymakers, explore efficacy of interventions, support antimicrobial stewardship initiatives, (re-)evaluate marketing authorisations of antimicrobials, generate epidemiological cut-off values, assess risk of zoonotic AMR transmission and evaluate the burden of AMR in animal health. EARS-Vet could be integrated with other AMR monitoring systems in the animal and medical sectors to ensure a One Health approach. Herein, we present a strategy to establish EARS-Vet as a network of national surveillance systems and highlight challenges of data harmonisation and bias. Strong political commitment at national and EU/EEA levels is required for the success of EARS-Vet. Salmonella and Campylobacter infections  when relevant, for emerging diseases or antimicrobial-resistant pathogens” [In order to support prudent veterinary use of antimicrobial therapy, and to implement a strong One Health strategy for AMR surveillance and antimicrobial consumption (AMC) control in Europe, building a European Antimicrobial Resistance Surveillance network in Veterinary medicine (EARS-Vet) should be considered. Such a network could be integrated with EARS-Net, FWD-Net, the EFSA monitoring for AMR in zoonotic and indicator bacteria and the European Surveillance of Veterinary Antimicrobial Consumption (ESVAC). This proposal, which we elaborate in the following sections, results from a collective agreement within the EU Joint Action on Antimicrobial Resistance and Healthcare-Associated Infections (EU-JAMRAI). It is in line with Regulation (EU) 2016/429 on transmissible animal diseases , which states that thogens” , and RegEARS-Vet would aim to describe the AMR situation, follow AMR trends and detect emerging AMR in bacterial pathogens of animals in Europe. The data generated could be used for (i) advising policy, (ii) monitoring interventions, (iii) evaluating marketing authorisations of antimicrobials, (iv) supporting antimicrobial stewardship initiatives, (v) generating epidemiological cut-off values, (vi) supporting risk assessment and (vii) estimating antimicrobial resistance burden.Data generated through EARS-Vet could serve policymakers in devising more targeted and efficient interventions based on a better understanding of the AMR situation and its evolution, as well as of the links between AMC and AMR in animals and humans. At present, these links are investigated in the Joint Inter-Agency Antimicrobial Consumption and Resistance Analysis (JIACRA) reports , using AEARS-Vet data would also allow for monitoring of the impact of European efforts to tackle AMR in the animal sector, such as the EU One Health Action Plan against AMR and the Animal Health Law . They coMoreover, EARS-Vet data would support antimicrobial stewardship initiatives, especially the development of veterinary antimicrobial treatment guidelines, which need accurate resistance data of animal pathogens. However, because of differences between countries in terms of animal populations, production systems, bacterial diseases, available authorised antimicrobials and resistance levels, such recommendations would need to be tailored to each country and only general indications could be defined at the European level. In addition, interpretative criteria for AST, i.e. epidemiological cut-off values and clinical breakpoints, are currently missing for many common combinations of animal species, bacterial species and infection sites. This challenges the interpretation of AST results in the veterinary field and from an epidemiological perspective. EARS-Vet could facilitate the defining of missing interpretative criteria by sharing its distributions of AST results to breakpoint-setting organisations such as the Veterinary Committee on Antimicrobial Susceptibility Testing (VetCAST).EARS-Vet would support assessment of the risk of AMR transmission from animals to humans via non-food-borne routes, e.g. by direct contact with companion or food-producing animals. This would complement the risk assessments performed by EFSA for the food-borne pathway.Finally, EARS-Vet could provide information to aid in assessing the burden of AMR in animal health, e.g. attributable deaths caused by infections with antibiotic-resistant bacteria. Such an estimation could be similar to the burden assessments performed in the human sector , which aEARS-Vet would provide the necessary coordination to collectively define common microbiological and epidemiological standards for the surveillance of AMR in animal bacterial pathogens in Europe, as well as a strategy to reach effective harmonisation. More broadly, EARS-Vet would represent an opportunity to build a European scientific community and knowledge hub to support the establishment, improvement and harmonisation of respective national surveillance systems, the interpretation of surveillance outputs and their translation into interventions. This EARS-Vet community could also prove useful in urgent contexts; for example, when an emerging resistance mechanism is discovered and its spread across Europe needs to be quickly evaluated, as experienced in 2015 upon the discovery of plasmid-mediated colistin resistance .One of the first steps in designing EARS-Vet would be the definition of a surveillance framework, including its: (i) surveillance scope , age categories, bacterial species, specimens and antimicrobials it covers), (ii) AST standards, (iii) metadata to be collected (including minimum and desirable variables), (iv) data governance, (v) frequency of data reporting, (vi) general data management system and (vii) required procedures , as well as (viii) how data would be analysed and communicated.This framework should ensure that EARS-Vet can meet the aforementioned surveillance objectives and facilitate broad country participation. It should also address two major challenges for EARS-Vet: the current lack of method harmonisation and possible sampling biases.To reach harmonisation, we suggest that standards are defined in an inclusive and bottom-up approach, i.e. according to what is considered relevant and feasible within countries. In the beginning, we envisage a transition period where different AST standards would be accepted, as originally done by EARS-Net over two decades, before accepting only those methods complying with the European Committee on Antimicrobial Susceptibility Testing (EUCAST).The possible sampling biases are linked to the fact that national surveillance systems usually collect AST results routinely produced in veterinary diagnostic laboratories. As culture of veterinary diagnostic specimens is often not performed until treatment failure, AMR levels tend to be overestimated compared with first-time infections . Such biSeveral successful initiatives, such as EARS-Net and ESVAC, have proven that a European surveillance network can be developed gradually, starting with a group of countries collaborating despite initial lack of harmonisation. Long-term success of EARS-Vet would depend on political commitment at national and European levels, accompanied by sustainable funding. In particular, the European Commission and the EU/EEA countries could play a role in supporting the development of national surveillance systems for AMR in diseased animals and fostering the participation of national agencies in the EARS-Vet initiative.From a regulatory perspective, the Animal Health Law (applying from April 2021) presents the opportunity to implement AMR surveillance in bacterial pathogens of animals in the EU for the first time. Thus, developing EARS-Vet is timely and could set the basis for a future regulated surveillance network.Antimicrobial resistance in animal bacterial pathogens represents a gap in the European One Health strategy on AMR surveillance. Here, we provide a perspective on the multiple anticipated benefits of establishing EARS-Vet and show how such a system could complement the existing European monitoring systems for AMR and AMC, in humans and animals coordinated by ECDC, EFSA and EMA. We have devised a pragmatic strategy to build EARS-Vet, which results from a collective agreement of EU-JAMRAI partners involved in the veterinary and medical sectors. It addresses the two main obstacles that EARS-Vet would face: the current lack of harmonisation in terms of methods and possibly biased surveillance data. In the coming years, more and more countries are expected to build national surveillance systems for AMR in diseased animals. In the absence of a European-level framework, this could lead to a further lack of harmonisation. The Animal Health Law, applying from April 2021, presents the opportunity for possible regulation of AMR surveillance in diseased animals in the EU/EEA, which would coincide well with the establishment of EARS-Vet.