Ear, nose, and throat involvement are common in eosinophilic granulomatosis with polyangiitis (EGPA). Among otologic manifestation, middle ear effusion (MEE) is less recognized but a problematic condition as it may progress to hearing impairment when left untreated. This study aimed to evaluate the characteristics, risk factors and clinical outcomes of MEE in EGPA patients.This is a case–control study of patients who were diagnosed and treated for EGPA from January 1995 to November 2018. Patients with ear symptoms  were assessed by otologists and were included in the case group (n = 23) if clinically relevant. The other patients without MEE were included in the control group (n = 52). Risk of MEE was calculated using the Cox proportional-hazard model.P = 0.030] , early onset of ear symptom after systemic therapy (< 6 months)  and continuing the maintenance steroid without cessation  were independently associated with a risk of MEE. To control MEE, 16 (69.6%) patients had to increase maintenance steroid dose and 9 (39.1%) patients experienced recurrent MEE whenever maintenance dose was tapered.During median follow-up of 9.9 years, 23 (30.7%) out of 75 patients had MEE. In MEE group, 12 (52.2%) patients had hearing loss; conductive type in 10  and mixed type in two . In multivariable regression analysis, major organ involvement at diagnosis (adjusted hazard ratio [aHR] 65.4; 95% confidence interval [CI], 1.50—2838.39; MEE is a common but frequently neglected condition in EGPA which is often intractable. The maintenance steroid dose should be adequately adjusted to control MEE and to prevent from progressive hearing loss. Novel biologic agents possibly have a role in controlling MEE in EGPA. Eosinophilic granulomatosis with polyangiitis (EGPA), previously known as Churg–Strauss syndrome, is a rare systemic disease characterized by eosinophil-rich granulomatous inflammation often involving the respiratory tract . The mosThe ear, nose, and throat involvements are frequent in EGPA. Rhinitis has been reported in 15–75% of total cases, occurring before, or at the onset of asthma. Chronic sinusitis has been reported in 60–80% of cases, and PNS abnormality is accompanied in 75% –10. As fThe otologic manifestations of EGPA were reported as rare events, based on a small number of individuals . HoweverThis is a case control study. Patients diagnosed and treated for EGPA from January 1995 to November 2018 were retrospectively identified from the prospectively collected EGPA registry data at Samsung Medical Center . EGPA was diagnosed according to ACR criteria . After cPatients with ear symptoms  at regular outpatient visits were referred to otologists and evaluated for MEE. When a thickened, bulging and pale tympanic membrane with a viscous or gelatinous secretion in the middle ear cavity was observed by otomicroscopy, samples from middle ear effusion or middle ear mucosa were collected through the tympanic membrane perforation or by myringotomy. The samples were fixed in formalin, embedded in paraffin, and then stained with hematoxylin and eosin to be observed under a light microscope for histological confirmation of the presence of the accumulation of eosinophils Fig.  18]. Th. Th18]. Patients with MEE underwent myringotomy/ventilation tube (VT) insertion and/or intratympanic steroid injection to control ear symptoms at the discretion of otologists. Although the choice of MEE treatment differed according to each patient’s situation, a myringotomy/VT insertion was performed to control MEE, and then intratympanic steroid injection was performed if there was no improvement. The dose of maintenance oral corticosteroids was increased upon agreement of both allergists and otologists.The main purpose of this study was to identify the factors associated with MEE in EGPA patients. Data on patient demographics and EGPA treatment-related features were complemented by a review of electronic medical records. Age, sex, fulfilled criteria of EGPA, involved major organs  at diagnosis, and treatment details of EGPA were prospectively collected in the EGPA registry. In patients with MEE, the following data were obtained from electronic medical records to identify the factors associated with MEE: onset and type of ear symptom, previous history of MEE before a diagnosis of EGPA, severity and type of hearing loss (if present), and the treatment regimen for MEE. The last follow-up data were retrieved on March 12, 2021.Patients with MEE were periodically evaluated by otomicroscopy for improvement or aggravation of MEE and audiometry for accompanying hearing loss, if present. Hearing levels were evaluated using pure-tone audiometry  in a sound-proof chamber. Hearing loss was defined based on pure-tone average, calculated using thresholds at 0.5, 1, 2, and 4 kHz, according to the National Institute on Deafness and Other Communication Disorders. Functionally normal hearing was defined as a PTA level < 15 dB.U test was used to compare continuous variables. The difference in eosinophil count before and onset of MEE was compared using the paired t-test. Hazard ratios (HRs) and 95% confidence intervals (CI) for the risk of MEE were calculated using the Cox proportional-hazard model. Multivariable analysis for MEE included clinically important variables or statistically significant variables in the univariate analysis: age, sex, previous history of MEE before diagnosis of EGPA, PNS abnormalities at initial diagnosis, blood eosinophil count at initial diagnosis, major organ  involvement (any vs none), maintenance steroid cessation after vasculitis activity was controlled (tapered off vs not tapered), time of ear symptom after initial systemic therapy (< 6 months vs ≥ 6 months) [P -value < 0.05 was considered statistically significant. All analyses were performed using Stata .Data are reported as numbers with percentages in parentheses for categorical variables and as medians with interquartile ranges (IQRs) in parentheses for continuous variables. Categorical variables were compared using Pearson’s chi-square test or Fisher’s exact test, and the Mann–Whitney  months) . All tesP = 0.085). Female patients comprised 52.0% of the study population.From January 1995 to November 2018, 136 subjects were referred in suspicion of EGPA, and 113 patients were diagnosed as EGPA. After excluding patients with no treatment or follow-up data, 75 patients were included as study population , and there were no association with MEE when the blood eosinophil count was categorized by ≥ 5000/μL , which is the currently widely accepted cut-off level for severe hypereosinophilia. Compared with the patients in the no MEE group, the patients in the MEE group had a significantly higher level of inflammatory markers, ESR , and CRP . Compared with the 10 (18.5%) patients with ANCA positivity in the no MEE group, none in the MEE group showed positive for ANCA (P = 0.024). To control the vasculitis activity, 57 patients (76.0%) were treated with intravenous cyclophosphamide therapy and 18 patients (24.0%) received only steroids. Overall, demographic data, clinical manifestations, or treatment regimens were not statistically significantly different between the MEE and no MEE groups.At the time of diagnosis, 73 (97.3%) patients had asthma. Peripheral neuropathy was presented in 56 (74.7%) patients, PNS abnormalities in 56 (74.7%), extravascular eosinophils on biopsy specimen in 50 (66.7%), peripheral eosinophilia (> 10%) in 38 (50.7%). The most common organ involvement was lung in 36 (48.0%) patients, followed by heart in 11 (14.7%), gastrointestinal tract in 9 (12.0%), kidney in 2 (2.7%), and brain in 2 (2.7%). The median blood eosinophil counts at the time of initial diagnosis of EGPA did not differ between the groups   patients, followed by impaired hearing in 12 (52.5%), tinnitus in 7 30.4%), otorrhea in 6 (26.1%), and otalgia in 4 (17.4%). Among the 15 (65.2%) patients who underwent audiometry under suspicion of hearing loss, 12  were confirmed, with both ears affected in 8 . Conductive hearing loss was observed in 10  patients and mixed type of hearing loss in 2  patients. The blood eosinophil count was significantly higher at the onset of MEE, compared to before MEE   and continuing the maintenance steroid without cessation  were independently associated with a risk of MEE, after adjusting for age, sex, previous history of MEE, PNS abnormalities at diagnosis, eosinophils count at diagnosis, ESR/CRP level at diagnosis, major organ involvement at diagnosis, cessation of maintenance steroids, and early onset of early symptom after systemic therapy.Factors associated with MEE in EGPA patients were evaluated Table . In multTo control the ear symptoms, myringotomy/VT insertion was performed in 10 43.5%) patients Table . Intraty.5% patieEGPA is a systematic disease that has a significant effect on ear, nose and throat. Compared with other involvements, otologic manifestations have been less understood . In the . have reported that 39% of EGPA patients had ear symptoms at initial presentation [Ashman et alentation . This isentation –24.In MEE patients, otologic manifestations appeared mostly within median 4 years after diagnosis. Ear symptoms occur in 30% of patients after apparent suppression of vasculitis activity, even when maintenance steroids were completely tapered off. EGPA follows three distinct clinical phases: a prodromal phase characterized by asthma and rhinosinusitis, an eosinophilic phase marked by peripheral eosinophilia and organ involvement, and a vasculitic phase with clinical manifestations due to small-vessel vasculitis , 25. TheMajor organ involvement at diagnosis of EGPA, early onset (< 6 months) of ear symptom after systemic therapy, and continuing the maintenance steroid were independently associated with MEE. Surprisingly, blood eosinophil counts at diagnosis were not associated with occurrence of MEE, although blood eosinophil counts at the onset of ear symptoms was higher than before MEE occurred, when the disease activity of EGPA was considered relatively stable. As the ear discharges were rich in eosinophils, peripheral eosinophilia may have been a trigger for tissue infiltration of eosinophils.In several studies, neuritis of the vestibulocochlear nerve, or vasculitis of the internal auditory artery, was suggested to suddenly progress to hearing loss , 26. HowFor all EGPA patients, once the disease activity of vasculitis was controlled, the maintenance dose of oral corticosteroids was gradually tapered to as low a dose as possible or even tapered off entirely to minimize the long-term side effect of steroids. Nevertheless, patients with MEE had to temporarily increase the maintenance steroid dose. Local administration of steroids is reported to be effective for localized symptoms in MEE . In the Of these 23 MEE patients, 9 had recurrent episodes of MEE and had to increase and then again decrease steroids repeatedly to control the ear symptom. In these patients, a lifelong low-dose maintenance therapy of steroid may be necessary. The potential side effects of long-term steroid use cannot be ignored , 31 . DeThe present study had a few limitations. First, racial differences must be considered. For instance, Asians show less positivity for ANCA. However, the main results agree with the previous publications from worldwide. Second, cell count analysis for eosinophils were not possible in this study as the samples were often too thick and dense, and the exact eosinophil counts could not be assessed. Last, the sample size may have not been large enough to detect other possible associations not reaching statistical significance in the multivariate analysis. However, EGPA itself is a rare disease, and to the best of our knowledge, this is the largest comparative study, with the longest follow-up, to focus on the otologic manifestations of EGPA. In addition, a close collaboration with an otologist was maintained and all cases were evaluated in an interdisciplinary manner.In conclusion, MEE is a common but frequently disregarded condition in EGPA which is often intractable. The maintenance dose of steroid for EGPA should be adequately adjusted to control the ear symptoms and to prevent progressive hearing loss. Novel biologic agents may have role in controlling MEE in EGPA.


The chemistry of ORMOSIL coatings and the production of CeO2 ceramic nanocontainers are described in detail for reproduction by other researchers. The anticorrosion properties of these novel coatings were investigated through frequency response analysis (FRA). As a result, the coatings HDG-ORMOSIL + CeO2 (5-ATDT) were better than the samples of HDG steel, HDG-ORMOSIL, and HDG-ORMOSIL + CeO2 (EMPTY) by a factor of 1033.60, 109.21, and 7.76 in terms of anticorrosion protection, respectively.This paper reports developing an innovative method of anticorrosion protection based on organically modified silica (ORMOSIL) enriched with CeO The term corrosion is accredited to various definitions. According to corrosion science, corrosion relates to the reaction of a solid to its environment. According to corrosion engineering, corrosion relates to the interaction of a metal with its environment. The result is the degradation of the function of the metal due to a change in its properties. The term corrosion refers to the process and not the result of corrosion damage, wear and tear. The importance of corrosion is significant and determined by its consequences on the economy of the countries . A frequIn the recent past, one used chromium (VI) to protect metals. However, due to the carcinogenic consequences of Cr (VI), there has been a great need to develop new protection methods based on polymeric coatings that are friendly to the environment and humans . Just reHot-dip galvanized zinc (HDG) coating is ideal for protection against corrosion of steel bars in concrete structures used in marine environments . The rec2 ceramic nanocontainers filled with 5-amino-1, 3, 4-thiadiazole-2-thiol (5-ATDT)  n-CH2-CH (C6H5)–} which end with a charge. The reactions that take place during polymerization are the following: Styrene monomer was chosen for polymerization because it develops polystyrene roots {Initiation of PolymerizationPropagation of PolymerizationEnd Polymerization2) layer using aqueous solution Ce (acac) 3 and PVP, as shown in The polystyrene nanospheres were coated through the sol–gel method to form a cerium oxide (CeO2 coated polystyrene. The coating of polystyrene nanospheres with Ce (acac) 3 gives nanospheres 180 +/− 10 nm, including gold coating.−1. 2 nanospheres after the first  suggests the formation of crystalline cerianite sintering the coated nanospheres at the 600 °C. The XRD pattern suggests of formation of pure CeO2 nanocontainers. The nanocontainers prepared were then filled with a corrosion inhibitor such as 5-amino-1, 3, 4-thiadiazole-2-thiol (5-ATDT). First, the corrosion inhibitor was dissolved in an acetone solvent to produce a saturated solution. Next, a small number of nanocontainers was placed in a sealed container. The air is removed from the container and, consequently, from the nanocontainers with a vacuum system. Then, the saturated solution with the corrosion inhibitor enters the sealed container, stirring the whole mixture at room temperature for 12 h. Finally, the nanocontainers were filled with an inhibitor, and the loaded nanocontainers were collected via centrifugation, followed by a vacuum drying process for 12 h.2 nanocontainers loaded with 5-ATDT, the % loss of mass between 200 °C and 550 °C is 21, 91%, or 2439 μg. This diagram shows sudden weight losses corresponding to the oxidative degradations of the corrosion inhibitor c. Comparw/w. 2 (EMPTY), and finally, the coating containing filled nanocontainers with corrosion inhibitor was named HDG-ORMOSIL + CeO2 (5-ATDT). SEM was used to determine the thickness of the coatings in the ORMOSIL CeO2 (EMPTY) and ORMOSIL CeO2 (5-ATDT) coatings exposed the two samples to a solution of 0.5 M NaCl at ambient temperature for eight days was about ~500 μm. HDG frames were dipped in the solution containing epoxy resin -ORMOSIL-nanocontainers six times at a 32 cm/min speed. The frames remain in the solution for 1 min. Then, the coated frames undergo aging treatment at a temperature of 70 °C for four days. The following nomenclature was used in the following discussion: the coating without additives (nanocontainers empty or filled) was named HDG-ORMOSIL, and the coating containing empty nanocontainers was named HDG-ORMOSIL + CeOp of the bare HDG steel, ORMOSIL HDG steel, ORMOSIL plus CeO2 (empty) HDG steel, and ORMOSIL plus CeO2 (5-ATDT) HDG steel were 1.62 × 103, 1.54 × 104, 21.62 × 104, and 16.78 × 105 Ohm cm2, respectively [p of ORMOSIL HDG steel, ORMOSIL CeO2 (empty) HDG steel, and ORMOSIL CeO2 (5-ATDT) HDG steel were 7.76, 109.21, and 1033.60 times greater than the bare metal, respectively [2 (5-ATDT) HDG sample due to added inhibitor release from the CeO2 nanocontainers. It is also mentioned that the coating ORMOSIL containing 5-ATDT is a bit better (4.0 × 104 Ohm) than the one that is without 5-ATDT (1.54 × 104 Ohm) but much worse than the one it contains CeO2 (5-ATDT) (16.78 × 104 Ohm). One can explain this value to entrapment of 5-ATDT in ORMOSIL and is not free to act to source the metal from corrosion as much as the 5-ATDT built into the CeO2 nanocontainers. This result proves the usefulness of nanocontainers for their anticorrosion protection. In the diagrams showing the phase with the measurement frequency, observing different behavior between the samples. The spectrum into three areas, 1, 2, and 3, attributed to localized corrosion, iron oxide complexes, and barrier effect [The test involves exposing the coated frames to a solution of 0.5 M NaCl for 30, 60, 90, and 180 h. The EIS assessed the coated samples’ protective capabilities and corrosion mechanism. ectively . One canectively . These op of HDG-ORMOSIL + CeO2 (EMPTY) and HDG-ORMOSIL + CeO2 (5-ATDT) after exposure to a solution of 0.5 M NaCl at ambient temperature for 30, 60, 90, and 180 h.2.HDG steel, among others, is used widely to strengthen concrete in harsh environments ,18,27,282 nanocontainers consist of a pure crystalline phase of CeO2 cerianite. For the first time, the existence of a vacuum device was disclosed and developed in the laboratory for filling the nanocontainers with corrosion inhibitors. The TGA measurements reveal that the occupancy rate in 5-ATDT in nanocontainers reaches a percentage corresponding to 2439 μg. This quantity is released on demand to impinge protection to metals against corrosion in the sample HDG-ORMOSIL + CeO2 (5-ATDT) compared to samples of galvanized steel, HDG-ORMOSIL, and HDG-ORMOSIL + CeO2 (EMPTY). The protection increase is 7.76, 109.21, and 1033.60 times more tremendous in HDG-ORMOSIL + CeO2 (EMPTY), HDG-ORMOSIL, and bare metal. As far as phase angle measurements are concerned 2, partly covered by ZnO, and finally by a zone of β-Zn(OH)2 [From the XRD diagram , the CeOoncerned , the resdroxides ,29. Chlo2 (EMPTY), and ORMOSIL + CeO2 (5-ATDT) form a barrier, which in the last sample is more pronounced. Therefore, in the following, the ORMOSIL + CeO2 (EMPTY) and ORMOSIL + CeO2 (5-ATDT) coatings are emphasized and measured after exhibiting to the salt solution for 30, 60, 90, and 180 h. First, one notices that the ORMOSIL + CeO2 (EMPTY) exhibits a clear improvement in the ORMOSIL coating  concerning the ORMOSIL coatings with empty CeO2 nanocontainers. The 5-ATDT entrapped in the CeO2 nanocontainers released from them as time exposure in the salt solution increased accounts for the other performance. The anticorrosion performance first increases with the time up to 60 h of exposure, and after this time, decreases with increasing time in the salt solution but is still much better than in the coatings with the empty nanocontainers. As the time reaches 60 h, the nanocontainers deplete, leaving behind nanocontainers with fewer inhibitors reducing their performance. This behavior suggests the “self-healing” effect that offers the filled nanocontainers with 5-ATDT [2 nanocontainers [2 (5-ATDT) will also need the employment of Raman and EIS spectroscopies to precisely determine the kind of barrier formed on our HDG substrate. This additional work is out of the scope of the present paper. Now, one can base the interpretation of our observations on the great literature work via a possible 5-ATDT protective coating creation. In our work, the 5-ATDT comes from the release from the charged CeO2 nanocontainers in the ORMOSIL coatings contributing to the self-healing phenomenon observed in our samples  using a corrosive environment NaCl of 0.5 M at ambient temperature. As a result, ORMOSIL coating with CeO2 (5-ATDT) demonstrated improved properties compared to galvanized steel, ORMOSIL, and ORMOSIL + CeO2 (EMPTY) coatings. In addition, the ORMOSIL coating with CeO2 (5-ATDT) possesses a self-healing capability.New technology is presented for the protection of HDG against corrosion used in concrete offered by the filled CeO


Bordetella bronchiseptica. The patient became non-ambulatory 3 days after CSF collection. He was treated with low-dose prednisone for 3.5 months and doxycycline for 1 year. Recheck CSF analysis 1 month after diagnosis showed reduction of inflammation and 3 months after diagnosis revealed only increased protein. The patient improved neurologically over several months and was weakly ambulatory 5 months and fully ambulatory 7 months after diagnosis. Whole genome sequencing of the bacterial isolate and a live modified intranasal vaccine similar to the one the dog had been vaccinated with 7 weeks before diagnosis was similar but not an exact match. Bacterial meningitis should be considered, and culture of CSF is recommended, in cases of neutrophilic pleocytosis of CSF.A 15-month-old male neutered Wirehaired Pointer mixed-breed dog presented with fever and cervical pain. Cerebrospinal fluid (CSF) analysis showed neutrophilic pleocytosis with intracellular bacteria, and culture of CSF grew  Bordetella bronchiseptica in a dog.Bacterial meningoencephalomyelitis (BMEM) has rarely been reported in veterinary medicine . The mosA 15-month-old male neutered Wirehaired Pointer mixed-breed dog weighing 16.4 kg presented to the University of Wisconsin Veterinary Care (UWVC) for a 3.5-week history of cervical pain. The dog had been treated with carprofen [2 mg/kg Per Os (PO) q12h] for acute cervical pain and a fever of 105°F 3.5 weeks before presentation. Two weeks before presentation, the carprofen was discontinued, and he was started on prednisone (0.6 mg/kg PO q12h for 5 days and then 0.6 mg/kg PO q24h), gabapentin (12 mg/kg PO q8h), and tramadol (0.3 mg/kg PO q8h). Diarrhea developed after a few days. Five days before presentation, gabapentin and tramadol were discontinued, and acetaminophen (1.8 mg/kg PO q8h), ondansetron (0.5 mg/kg PO q8h), omeprazole (1.2 mg/kg PO q24h), and pregabalin (3 mg/kg PO q12h) were started. He had been off prednisone for 2 days at the time of the examination. On the day before initial neurologic examination, the dog was admitted to UWVC for worsening cervical pain and was placed on fentanyl [2–5μg/kg/h intravenously (IV)] overnight. In addition, he had been treated for gastroenteritis and a honking cough 5 months before presentation.On physical examination, the dog had a body condition score of 5/9. All findings were within normal limits except for cervical pain and reluctance moving the neck. Neurologic examination was within normal limits except for absent paw placement in both pelvic limbs, a slow stiff gait, and cervical pain on palpation and manipulation of the neck. The paw placement deficits were attributed to the fentanyl treatment. On the basis of the signalment, history, and presentation, the top differential diagnosis was steroid responsive meningitis arteritis, although a meningoencephalomyelitis of unknown origin or an infectious cause could not be ruled out.A chemistry panel was performed and was unremarkable. Complete blood count showed a leukocytosis with a mature neutrophilia .The dog was routinely anesthetized for cerebrospinal fluid (CSF) collection. The CSF from the cerebellomedullary cistern was milky white and cloudy in appearance and showed an increase in total protein content , mild blood contamination  and a neutrophilic pleocytosis . The lumbar CSF was blood tinged and hazy and had an increase in protein content , mild blood contamination , and a neutrophilic pleocytosis  . The CSFThe following day, the dog appeared more ataxic. He was mildly obtunded and ambulatory with a mild paraparesis and moderate proprioceptive ataxia. Paw placement test was absent in the pelvic limbs and normal in the thoracic limbs. The change was initially attributed to the treatment with fentanyl constant rate infusion (CRI) overnight and the anesthesia.Bordetella bronchiseptica. On further questioning the owner, the dog had received a modified live Bordetella vaccine intranasally 3.5 weeks before onset of clinical signs. The azathioprine was discontinued.On further review of the CSF slides, there were a few intracellular basophilic short bacterial rods in the neutrophils . CultureTM probiotic capsule (1 capsule PO q24), and continued ondansetron and acetaminophen at previous doses. At the time of discharge, he was mildly obtunded and ambulatory paraparetic with absent paw placement in both pelvic limbs. He became non-ambulatory paraparetic 3 days after the spinal fluid collection.The dog was discharged the following day with prednisone (0.45 mg/kg PO q24h), doxycycline (4.5 mg/kg PO q12h), ProviableOne month after diagnosis, the dog was able to stand on his own but remained non-ambulatory. He was fecally incontinent and able to urinate on his own, but manual expression of the urinary bladder by the owner was required for complete emptying. The patient had regular visits to a physical therapist for rehabilitation and had a home exercise program. On neurologic examination he was bright, alert, and responsive (BAR), with normal cranial nerve examination. He was non-ambulatory paraparetic with good voluntary motor function. Paw placement was absent in both pelvic limbs and normal in the thoracic limbs. Spinal reflexes were normal, except for crossed extensor reflex in the pelvic limbs. There was no pain noted on palpation of the spine including the neck, and there was full range of motion of the neck.Repeat CSF analysis was done to evaluate response to treatment. The CSF  showed an increased total protein (53 mg/dl), a red blood cell count of 2/μl, and a lymphocytic pleocytosis . Lumbar collection of CSF showed an increased total protein (324 mg/dl), a red blood cell count of 3/μl, and a lymphocytic pleocytosis . No organisms were seen. The interpretation was chronic inflammation or ongoing antigen stimulation.TM were continued at the previous doses.The dose of prednisone was increased (0.45 mg/kg PO q12h), and the doxycycline and ProviableThree months after diagnosis, the dog was BAR, a ventral strabismus oculus dexter (OD) was present, and he was non-ambulatory tetraparetic with the pelvic limbs appearing weaker than the thoracic limbs. Paw placement was absent in both pelvic limbs and the right thoracic limb. The patella reflexes were increased and there was a crossed extensor reflex bilaterally in the pelvic limbs. There was no pain on vertebral column palpation. Gamma-glutamyl transferase was mildly elevated , and alkaline phosphatase was elevated . White blood cell count was within normal limits (WNL), and there was a mild non-regenerative anemia . Given the clinical progression and change in neurologic status, a magnetic resonance imaging (MRI) to evaluate for structural lesions of the cervical and thoracic spine and brain were performed  . The imaThe lumbar CSF performed at this time contained an elevated protein with mild blood contamination and a normal cell count . Cisternal CSF showed a mildly elevated protein with a normal cell count . Culture of CSF showed no growth.TM were continued.The dose of prednisone was reduced . The doxycycline and ProviableOver the next 4 weeks, the dog continued to improve in attitude—being more playful and alert at home. On recheck 4 weeks later (5 months after diagnosis), he was able to take 10 steps with a tetraparesis worse in the pelvic limbs and generalized proprioceptive ataxia. The remainder of the examination was unchanged.Seven months after diagnosis, the dog was ambulatory with a spastic paraparesis and a generalized proprioceptive ataxia. The fecal incontinence and inability to fully void resolved 11 months after diagnosis, and he was able to walk for longer distances—still with a paraparesis and generalized proprioceptive ataxia. The crossed extensor reflex in the pelvic limbs was still present. One year after diagnosis, the dog was ambulatory with a spastic paraparesis and a mild to moderate proprioceptive ataxia in the pelvic limbs. The doxycycline was discontinued.Bordetella bronchiseptica cultured from the CSF showed that the isolates were 50,525 single nucleotid polymorphism (SNP) apart, suggesting that the isolates were very close but not completely identical.Whole genome sequencing of a Bordetella vaccine similar to what the dog received in March of 2020 and the Bordetella bronchiseptica meningoencephalomyelitis in a dog. Bordetella bronchiseptica is one of the causative agents of canine infectious tracheobronchitis and is a gram-negative, aerobic coccobacillus. Transmission occurs after direct contact with infected dogs or contaminated material from aerosolized microdroplets. After the 6 days of incubation period, the bacteria attach to and replicate on the cilia of respiratory epithelium. Toxin produced by the bacteria impair phagocytic function and induce ciliostasis. The bacteria can invade host cells and thus avoid immunologic defense mechanisms and establish a persistent infection. The bacteria persist in the epithelium for 3 months before it is cleared by local antibody production. The organism has been isolated from the upper respiratory tracts from clinically healthy dogs. Dogs recently vaccinated may still develop clinical signs. Adverse reactions reported after vaccination include a cough or nasal discharge 2–5 days after inoculation , via the olfactory pathways or trigeminal pathways, after overcoming the nasal mucosal barrier (Our patient received an intranasal modified live Bordetella vaccine 3.5 weeks before onset of cervical pain, which was 7 weeks before the diagnosis of  barrier . The genThe choice of antibiotic was based on susceptibility of the isolate and that doxycycline has good penetration into the central nervous system. Treatment with prednisone is controversial in infectious disease but has been recommended at low doses for the short-term in infectious meningoencephalomyelitis to reduce the inflammatory response as the organism dies from the treatment . The risThe worsening of neurologic signs and the findings on MRI may have been due to damage to the spinal cord parenchyma from the infection, although as stated above, a residual bacterial infection could not fully be ruled out.Bordetella species that infects humans is Bordetella pertussis, which is the causative agent for whooping cough. Bordetella bronchiseptica is infrequently reported as cause for airway infection in immunocompromised people, and only four reports of meningitis have been reported in people. It was diagnosed in one patient secondary to a traumatic CSF leak after a fall, in one patient who underwent surgery after having been kicked by a horse, and in two patients after brain surgery. All patients had household pets or were in close contact with dogs (The more common ith dogs –20.The mortality rate in people with bacterial meningitis is 5–40%, and 30% of survivors have permanent neurologic deficits . A revieBordetella bronchiseptica in a dog. BMEM should be ruled out in cases presenting with a fever and neutrophilic pleocytosis on CSF. Culture of CSF in case of neutrophilic pleocytosis is highly recommended, even if the yield for positive culture is low. Recovery from BMEM may be slow with residual neurologic deficits.In conclusion, we report the first case of BMEM caused by info@cosmosid.com.The SNP was done at a third party, CosmosID laboratory. Questions can be directed to Written informed consent was obtained from the owners of the dog for the publication of this case report.HR, DD, and SC all handled the case in the clinic and helped writing the manuscript. 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The as made catalyst, with a unique distorted IrO2 structure, is discovered to significantly boost the performance for pH‐universal oxygen evolution reaction (OER). Based on experimental results and theoretical calculations, the distorted IrO2 active sites with flexible redox states in IrO2/V2O5 server as electrophilic centers balance the adsorption of oxo‐intermediates and effectively facilitate the process of O—O coupling, eventually propelling the fast turnover of water oxidation. As a result, IrO2/V2O5 demonstrates not only ultralow overpotentials at 10 mA cm−2  for OER, but also high‐performance overall water electrolysis over a broad pH range, with a potential of mere 1.50 V (pH = 0), 1.65 V (pH = 7) or 1.49 V (pH = 14) at 10 mA cm−2. In addition, SOSI can simultaneously secure the distorted active sites and thus remarkably improving the catalytic stability, making it a promising strategy to develop high‐performance catalytic systems.Constructing strong oxide‐support interaction (SOSI) is compelling for modulating the atomic configurations and electronic structures of supported catalysts. Herein, ultrafine iridium oxide nanoclusters (≈1 nm) are anchored on vanadium oxide support , the distorted IrO2 active sites with flexible redox states in IrO2/V2O5 server as electrophilic centers to balance the adsorption of oxo‐intermediates and effectively propel the fast turnover of water oxidation, eventually achieving high‐performance water splitting in a wide pH range.Here, highly dispersed IrO Electrocatalysts are the central part of the hydrogen and oxygen evolution reactions (HER and OER). Significant success has been realized in the field of HER and overpotential at 10 mA cm−2 can be lower than 20 mV. To date, the large‐scale industrial water electrolysis is hindered by the huge energy consumption derived from the large overpotential of OER. Basically, producing one oxygen molecule involves the sluggish kinetics of four electrons transfer and the formation of O—O bond. Consequently, exploring advanced OER electrocatalysts is vital for improving the energy conversion efficiency of water electrolyzers and has been drawn considerable attention in recent years. In principle, the binding energies of multiple intermediates  in OER are correlated with each other. Typically, the adsorption energy difference between *OOH and *OH is about 3.2 ± 0.2 eV because of scaling relation of the two intermediates without regarding of catalyst type. According to the volcano plot of ηOER versus (ΔGO* − ΔGOH*), the catalyst with oxygen binding that is neither too strong nor too weak shows optimal activity. To date, several approaches have been reported for modulating oxygen intermediate adsorption energy, including substituting with foreign elements, heterostructure modulating, strain engineering, nanoscopic confinement and so on. So far, the overpotentials values of state‐of‐the‐art OER catalysts at 10 mA cm−2 are smaller than 200 mV, however, most of them can only be applied in a certain pH or specific working scenarios. In order to meet the complicate working conditions, it is highly urgent to develop a high‐performance catalyst that is universally compatible to optimize the reliability during operation.Electricity‐driven water splitting to produce hydrogen  would be appropriate building blocks. Though IrO2 is an excellent benchmark catalyst for OER in acidic media, the binding between IrO2 and oxygen‐related intermediates is still strong, limiting its OER performance. A large space exists for the property enhancement of IrO2 by modifying geometric and electronic structures, which can lead to optimized interaction between the catalyst and oxygen intermediates. Recently, owing to their excellent interactions with molecules or ions, outstanding catalytic activities, and/or strong electron‐electron correlations, nanostructured vanadium oxides‐based composite materials have been extensively studied for energy conversion  in the recent years. Benefit from the multiple valence states, V2O5 exhibits redox and Lewis acid/base properties, which makes strong interaction with foreign active materials. On the one hand, strong interaction is beneficial to regulate the intrinsic activity of active species, and on the other hand, it also plays a role in stabilizing the substrate. Additionally, vanadium oxide can facilitate the hydrolysis kinetics and provide hydrogen protons during electrochemical process, which has been considered important in surface catalytic reactions. Inspired by the aforementioned discussions, we envisioned whether a rational modification of IrO2 on VOx support could integrally construct a supported catalyst with strong oxide‐support interaction, and improve catalytic activity and durability.In the past decades, constructing strong metal/metal oxide‐support interaction (SMSI/SOSI) has attracted great attentions due to its intriguing capabilities for designing advanced catalysts with unique properties.31 On 2/V2O5 catalysts in which vanadium containing MOF MIL‐88B (V) was used as a host. After impregnating with Ir precursor and then annealing under air atmosphere, IrO2 nanoclusters (≈1 nm), with unique distorted and deformed lattice fringes induced by SOSI, are uniformly embedded in the porous network of V2O5. Contrast experiments and theoretical calculations indicated that the SOSI between IrO2 and V2O5, i.e., the SOSI induced lattice fringes’ distortion exposed more catalytic active sites and enhanced the redox states of electrophilic IrO2, can significantly promote the OER performance of IrO2 over a wide pH range  as well as durability.Herein, a simple method was developed to prepare highly dispersed IrO22.12/V2O5 hybrid oxides is illustrated in Scheme  in which vanadium chloride and p‐phthalic acid were used as raw materials. And then Ir(III) ions can be readily hosted within MIL‐88B (V) by means of impregnation. An apparent color changes from dark green to brown can be observed over the parent MIL‐88B (V) and Ir(III)/MIL‐88B (V), which indicates the successful incorporation of the Ir(III) ions into MIL‐88B (V). The power X‐ray diffractions (XRD) patterns of both MIL‐88B (V) and Ir(III)/MIL‐88B (V)  to form Ir‐V mixed material in the pores of MIL‐88B (V) but was adsorbed on the pore surface. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images of MIL‐88B (V) and Ir‐MIL‐88B (V) exhibit similar shuttle‐like morphology /MIL‐88B (V) under air atmosphere. As a comparison, MIL‐88B (V) was treated in the same condition to produce yellow powder V2O5. As shown in Figure 2/V2O5 inherits the original shuttle‐like morphology of MIL‐88B (V) with rough surface and porous structure. However, SEM and TEM images  as shown in Figure 2 (JCPDS No. 86–0330) and orthorhombic V2O5 (JCPDS No. 09–0387) phase in IrO2/V2O5. Compared to IrO2 analog, however, the (110) characteristic reflection in the IrO2/V2O5 sample broadens and shifts to a lower degree at 2θ = 27.6°, indicating an lattice expansion feature of IrO2 induced by the known effects of strain on both the position and width of diffraction peaks. Moreover, high‐resolution transmission electron microscope (HRTEM) image and corresponding line‐scan profile  with mean size of ≈1 nm are uniformly embedded on porous skeleton of V2O5. Scanning transmission electron microscopy (STEM) and corresponding elemental mapping images  with similar particle size distribution and Ir loading (19.1 wt%) of IrO2 (Table2/HPC sample possesses regular lattice fringes (interplanar distances of 0.223 nm) without obvious distortion. Furthermore, IrO2 was loaded on the synthesized V2O5 by wet impregnation and postcalcination as comparison. Although, it is not as obvious as the IrO2/V2O5 sample, HRTEM images and XRD pattern indeed showed the formation of distorted IrO2 NCs , X‐ray photoelectron spectroscopy (XPS), and density functional theory (DFT) calculations were carried out. Figure 2‐TPR profiles of IrO2/V2O5, IrO2/HPC and V2O5. The reduction profile of IrO2/HPC is characterized by a single and sharp peak at 197 °C, assigned to the reduction of iridium oxide species, which is similar to reduction temperature of commercial IrO2 (210 °C)  of V reduced to low oxidation state. With regard to IrO2/V2O5 hybrid oxide, the H2‐TPR profile shows the typically two reduction peaks. Obvious shift and broaden of iridium oxide reduction peak to a higher temperature region (310 °C) is clearly observed, indicating that the iridium oxide species supported on V2O5 are more difficult to be reduced. What's more, the shoulder peak around 348 °C is detected, which may be attributed to iridium oxide species intimately interacted with the V2O5 support at the interface. Meanwhile, the reduction temperature of V2O5 species in IrO2/V2O5 moves to lower temperature region (440–550 °C) comparison to pure V2O5. Based on the results of H2‐TPR measurement, we convinced that the strong interaction between IrO2 species and V2O5 support is successfully constructed.To further figure out the effect of SOSI on the properties of catalysts, H species,46 whi2 and IrO2/HPC . As excepted, apart from typical V5+ signal, the intensity of V4+ peak significantly enhances in IrO2/V2O5 hybrid oxide (AV4+:AV5+ = 0.3). These results point to the highly strong electron coupling effect between IrO2 NCs and V2O5 support with the charge transfer from the IrO2 to V2O5. DFT calculations were additionally performed for analyzing the charge transfer and internal interactions between IrO2 NCs and V2O5 and C supports, respectively. As shown in Figure 2/V2O5. According to Bader charge analysis, a partial charge transfer of 1.03 e− from Ir to the V2O5 was found, while only charge transfer of 0.08 e− is discovered in IrO2/C system, which is in good agreement with the XPS analysis. The projected density of states (pDOS) curves of IrO2/V2O5 , along with commercial rutile IrO2 , commercial IrO2 , and V2O5 support. Notably, the acidic OER catalytic activity is also superior to most of the recently reported landmark catalysts  in neutral‐pH media, which opens the door to the realization of efficient and biocompatible systems for energy storage and conversion, as well as for direct seawater splitting. The comparison of OER activity with state‐of‐art catalysts also indicates that IrO2/V2O5 ranks among the top neutral OER catalysts  was first performed to investigate the interface charge transfer process of the electrode. The semicircular diameter of IrO2/V2O5 in Nyquist plots is much smaller than that of IrO2/HPC, commercial IrO2 and V2O5 in all‐pH range, as revealed in Figure 2 and V2O5 support. The high density of active sites plays an important role in the improvement of catalytic activity. In order to quantitatively analyze the number of active sites, underpotential deposition of copper (Cu‐UPD) was then conducted to measure the active site density. As is seen in Figure 2 (4.80 × 10−8 mol) and IrO2/HPC (4.94 × 10−8 mol), an increase in the number of active sites can be observed for IrO2/V2O5 (5.51 × 10−8 mol). As mentioned above, the small NCs (≈1 nm) consists of unique structure distortion, which endows the IrO2/V2O5 catalyst with a high exposure of IrO2 active sites as well as a high accessibility for substrate molecules. The turnover frequencies (TOFs) based on the number of active sites of the catalyst was calculated to evaluate the intrinsic activity of a catalyst. As illustrated in Figure 2/V2O5 even reach 0.27, 0.19, and 0.05 O2 s−1, which are about 3.0, 6.3, and 2.5 folds of those of IrO2/HPC , and far surpassing commercial IrO2 in acidic, alkaline and neutral media, respectively. As one of precious metal, the mass activity of Ir‐based electrocatalysts is of great importance for industrial application. Mass activity of IrO2/V2O5 is 287, 207, and 56 A gIr−1, which is about 3.4, 7.7, and 2.7 folds of those of IrO2/HPC , and 1–2 orders of magnitude larger than those of commercial IrO2 in acidic, alkaline and neutral media at the overpotential of 300 mV  protocol on Au electrode . The static chronopotentiometry test (at 10 mA cm−2) is conventional way to evaluate the stability, as plotted in Figure 2/V2O5 catalyst represents a relatively stable horizontal line in different electrolyte (even in the acidic media) during 20 operating hours, while IrO2/HPC and commercial IrO2 exhibit a progressive activity decay within a few hours, demonstrating the outstanding activity retention of IrO2/V2O5, which is competitive to the state‐of‐art Ir‐based OER catalysts reported in literatures  is available on the surface. In sharp contrast, as shown in Figure 2 NCs loaded on HPC after stability test are obviously agglomerated. Notably, static protocol is not sufficient to evaluate the stability of Ir‐based OER catalysts. Therefore, the transient ADT protocol endows the catalyst to distinctly higher currents (double layer charging and fast redox reactions) than the static protocol at same potential, which is benefit to save time to investigate the durability. As seen in the Figure RHE and shifted to lower potential, which represents the typical transition of IrIII+ to IrIV+, agreeing with above results of Ir4f spectra. The strong change in redox activity during ADTs is reflected by the increase in Qanodic . The same process is performed on IrO2/HPC in Figure 2SO4 are determined by ICP‐MS measurement. To show this more clearly, when the amount of dissolved Ir is normalized to time and to the geometric surface area, as shown in the Figure 2/V2O5 (0.21 ug h−1 or 33.0 ug cm−2) is much smaller than those of IrO2/HPC (0.33 ug h−1 or 51.3 ug cm−2). Meanwhile, we observed that the V/Ir mass ratio of IrO2/V2O5 decreases from initial 2.1 to 1.7 due to the faster V dissolution rate (0.59 ug h−1 or 93.0 ug cm−2). Furthermore, as displayed in Figure jgeo, jspec, and jmass) remaining percentage of IrO2/V2O5 at 1.53 VRHE are 2 times higher than those of IrO2/HPC. These results collectively confirm that the SOSI effect between V2O5 and IrO2 can efficiently reduce the detrimental effects of agglomeration of particles and degradation of intrinsic or mass activity of Ir active sites.Stability is an important criterion to evaluate the performance of catalysts. The stability of the IrOnalysis).57 The2.44O8 cluster supported on V2O5 (001) substrate along with periodically interconnected rutile IrO2 (110) as comparison. The elementary electrochemical steps for proposed the 4e− mechanism of OER on Ir4O8/V2O5 and IrO2 under acid and alkaline media are presented in Figure  The Ir atoms in both models serve as the active sites in the OER process. The computed energy landscapes of the OER steps at U = 0 and 1.23 V (acid OER) or 0.408 V  for Ir4O8/V2O5 and IrO2 are shown in Figure 4O8/V2O5 and IrO2 under the equilibrium potential, respectively, indicating that Ir4O8/V2O5 is more referable for OER. As demonstrated in Figure 2 , the obvious reduced oxygen adsorption energy (ΔEO* = 2.02 eV) and enlarged Ir—O* bond length (L = 1.82 Å) on Ir4O8/V2O5 may result in the relatively higher OER activity. This is consistent with the results by Nørskov and coworkers that catalyst can bind O too strongly and the overall reaction is limited by the O—O coupling process, and appropriately weakening the strength of M—O* is beneficial to reduce the barrier for the formation of M—OOH*.DFT calculations were performed to unveil the mechanism of the OER from the view of atomic and electronic structures. Based on the amorphous and distorted iridium oxide structure observed in HRTEM, the OER computational model adopt Irn Figure 59 The2O5 and IrO2 weakens the binding of IrO2 with oxygen. To figure out the surface electronic features of catalyst is conducive to better understanding the discrepancy of oxygen adsorption energy, therefore, the d‐band centers of the Ir atoms of Ir4O8/V2O5 and IrO2 were calculated . It is well known that the lower the center of d‐band relates, the higher the antibonding states, leading to weak adsorption  were calculated to represent the bonding and antibonding filling states of Ir‐O*. As illustrated in Figure 4O8/V2O5 (−2.67 eV) and IrO2 (−2.84 eV) by calculating the energy of COHP integral up to the Fermi level, where the more positive ICOHP value on Ir4O8/V2O5 indicates the weaker bonding. In rutile IrO2, O—O bond formation may be hindered by its “rigid” periodically linked structure, while distorted structures of Ir4O8/V2O5 enables the iridium oxide species more “flexible” during the oxidation‐reduction cycles to accelerate the hydroxyl oxidation and the subsequent O—O bond formation, which is supported by the comparison of the cyclic voltammetry (CV) curves of IrO2/V2O5 and IrO2  and commercial IrO2 . Meanwhile, the IrO2/V2O5 shows lowest Tafel slopes and charge transfer resistance among the catalysts, manifesting its fastest reaction kinetics. Impressively, in all three conditions, IrO2/V2O5 is demonstrated as a very robust electrocatalyst with negligible degradation of activity after 20 h of continuous operation. Hence, the IrO2/V2O5 is expected to act as a “universally compatible” electrocatalyst that simultaneously shows excellent HER and OER performances over the entire pH range. In order to demonstrate the practical applications, we loaded the IrO2/V2O5 electrocatalysts on carbon fiber paper (CP) as anode and cathode to construct a two‐electrode water splitting cell in 0.5 M H2SO4, 1 m PBS and 1 m KOH solutions . In the experiments of all‐pH overall water splitting, the potential required for two‐electrode IrO2/V2O5@CP (±) electrolyzer is only 1.49, 1.65, and 1.50 V to reach a current density of 10 mA cm−2 in alkaline, neutral, and acidic media, exceeding the commercial (−)Pt/C@CP||IrO2@CP(+) electrolyzer  system exhibits excellent stability in all‐pH condition by chronopotentiometry tests Pt/C@CP||IrO2@CP(+) electrolyzer deactivates rapidly within a few hours. We detected the generated H2 and O2 from IrO2/V2O5@CP (±) electrolyzer by on‐line gas chromatography  is also demonstrated to further promote the application. This work provides a road map in accelerating OER kinetics in different media to boost the efficiency of water electrolysis and shed light on the extension of SOSI to a variety of catalyst systems for enhancing their activity and stability.In summary, we have developed a simple method to synthesize highly effective IrO43▪xH2O, 298.58), vanadium(III) chloride , and p‐phthalic acid  were purchased from Aladdin Inc. The commercial IrO2 was purchased from Sigma‐Aldrich.Iridium(III) chloride hydrate (IrCl3) and 332 mg of terephthalic acid (C8H6O4) were mixed in a 50 mL beaker. Then, a solution containing 2 mL of hydrochloric acid (1 м HCl) and 10 mL of ethyl alcohol (C2H5OH) was added under vigorous stirring at room temperature. After 30 min, the resulting green suspension was further dispersed with ultrasonic treatment for 15 min. Finally, the mixture was transferred to a 50 mL Teflon‐lined stainless‐steel autoclave and heated in an electrical oven at 120 °C for 48 h. The prepared MIL‐88B (V) was placed at 50 °C under vacuum for 12 h for future use.In a typical procedure, 314 mg of vanadium (III) chloride  was slowly added into the IrCl3 solution under stirring. To ensure the uniform distribution of IrCl3, the resulting mixture was further kept stirring at room temperature for 12 h to allow the compete loading of IrCl3 into MIL‐88B (V) pores. After impregnation, the product was recovered by centrifugation, and washed four times with ethanol to remove IrCl3 remained on the outer surface of MIL‐88B (V) material. Finally, the resulting Ir(III)/MIL‐88B (V) was dried at 50 °C under vacuum for 10 h. Ir(III)/MIL‐88B (V) powder was placed in muffle furnace and heated to T  at a heating rate of 5 °C min−1 and held for 4 h. After cooling down to room temperatures, the resulting black products were collected, and denoted as IrO2/V2O5‐T. Notably, the typical IrO2/V2O5 material was obtained at 400 °C unless otherwise specified. For comparison, V2O5 was obtained by annealing MIL‐88B (V) powder at 400 °C.Typically, 50 mg IrCl2O5 were impregnated by 6.2 mL of IrCl3▪xH2O aqueous solution (10 mg mL−1 based on precursor). Then water was evaporated at 60 °C and the powders were dried at 120 °C for 3 h, followed by calcination in muffle furnace at 400 °C for another 4 h.Typically, in order to ensure the consistent loading of Ir, 0.2 g hierarchically porous carbon (HPC) or synthesized Vα, λ = 1.5418 Å, operating at 40 kV/40 mA). The morphologies were characterized by transmission electron microscope  and scanning electron microscope . The high‐resolution transmission electron microscope (HRTEM) were carried out by an FEI Tecnai G2 F20S‐Twin HRTEM working at 200 kV. The aberration‐corrected high‐angle annular darkfield scanning transmission electron microscope (AC HAADF‐STEM) images and the corresponding atomic‐resolution energy‐dispersive X‐ray spectroscopy element mappings were recorded by a Titan 80–300 scanning/transmission electron microscope operated at 300 kV. N2 adsorption–desorption measurements were carried out at 77 K using a Quantachrome SI‐MP Instrument. The surface area of the samples was estimated by method of Brunauer–Emmett–Teller (BET). Elemental analysis of Ir and V in the samples was detected by a Thermo IRIS Intrepid II inductively coupled plasma‐optical emission spectrometry (ICP‐OES). X‐ray photoemission spectroscopy were obtained on an ESCALAB MARK II spherical analyzer with a monochromatized Al Kα source (1486.6 eV) with a base pressure in the lower 2 × 10−7 mbar range. The binding energy (BE) was calibrated by setting the C 1s BE to 284.8 eV with respect to the Fermi level. High‐resolution spectra were acquired with an analyzer pass energy of 40 eV. The XPS spectra were fit after subtraction of a Shirley background with the available XPSPEAK 4.1 software. In all fits, the peak separation and the peak area ratios between the Ir 4f7/2 and the Ir 4f5/2 components were constrained to 3 eV and 4:3, respectively. The detailed description about the fitting models, lineshape definition, peak position, restrained parameters summarized in the Tables Powder X‐ray diffraction patterns (PXRD) of samples were recorded using a Bruker D8 Advance diffractometer (Cu K2‐TPR measurement using hydrogen (H2) as a reductant. Briefly, 100 mg of catalyst sample with grain sizes of 60–80 mesh were loaded in a quartz reactor and pretreated with a ultrapure Ar gas (with flow rate of 30 mL min−1) at 200 °C for 2 h to remove the surface‐adsorbed species. The pretreated sample was then cooled to room temperature, following which the sample was heated to 800 °C under the mixed gas flow (10 vol% H2 in Ar with flow rate of 30 mL min−1) at a heating rate of 2 °C min−1. H2 consumption was measured using an online thermal conductivity detector (TCD).The mobility and redox properties of catalyst samples were evaluated via H2/V2O5, V2O5, commercial IrO2) together with 1 mg acetylene black were dispersed in 480 µL ethanol solution with 20 µL Nafion solution (5 wt%) by sonication to form a homogeneous ink. Then, the same Ir content with 0.1 mg cm−2 was loaded onto the surface of a glassy carbon electrode  to serve as the working electrode for the electrochemical tests. A saturated calomel electrode (SCE) and carbon rod were used as the reference electrode and the counter electrode, respectively. The reference electrode was also calibrated using the Pt RDE as working electrode in highly pure H2‐saturated electrolyte. All potentials were referenced to a reversible hydrogen electrode (RHE). For OER test, linear sweep voltammetry (LSV) was carried out in 1.0 м KOH, 1.0 м PBS, and 0.5 м H2SO4 at a scan rate of 1 mV s−1 after purging with O2 (99.999%) for at least 30 min and the HER test was also performed in accordance with above similar operations, except that O2 is replaced with H2 before the test. All the polarization curves are the steady ones after several scans with 95% iR compensation. The current density was calculated against the geometric area (0.196 cm2) of the electrode to obtain the specific activity. Electrochemical impedance spectroscopy (EIS) measurements were performed from 100 kHz to 0.01 Hz with an amplitude of 5 mV at an overpotential of 100 mV for HER and 300 mV for OER. The stability measurements were performed by static chronopotentiometry test in 1.0 м KOH, 1.0 м PBS, and 0.5 м H2SO4 and transient accelerated degradation test (ADT) protocol in 0.5 м H2SO4 on Au electronode . Chronopotentiometry was measured at 10 mA cm−2 to represent typical static long‐term stability tests. ADT tests were conducted as follows. Typically, square‐wave voltammetry consisted of 10 000 cycles were conducted between a lower potential of 0.05 VRHE and an higher potential of 1.5 VRHE. Each cycle was maintained for 4 s. Every 2500 cycles were interrupted for LSV and CV scans . The leaking extent Ir and V from the electrode to electrolyte was determined by ICP‐MS (NexION 2000).All the electrochemical measurements were carried out with a three‐electrode setup using a CHI 760E electrochemical workstation . 5 mg catalysts (IrOn) could require based on the generated charges during the UPD copper (Cu) stripping , that is, n = QCu / 2F.The underpotential deposition of copper (Cu‐UPD) on Pt, Ru, and Ir was proven to be an ideal method for quantifying the number of active sites and the electrochemically active surface area (ECSA). Hence, the number of active sites (−1) was calculated by the equation: TOF = I/(znF). F (C mol−1) is the Faraday constant, I is the current (A) at corresponding overpotential and the factor z means the number of electrons required to form one hydrogen molecule or oxygen molecule .The turnover frequency  as the carrier. Then, the homogeneous catalyst ink was daubed uniformly in 1 cm2 area of CFP . The polarization curves were measured at a scan rate of 5 mV s−1 in 0.5 м H2SO4, 1.0 м KOH, and 1 м PBS solution. The stability measurements were conducted by long‐term chronoamperometry. The amount of H2 and O2 generated from water splitting was detected by online gas chromatograph (KE CHUANG GC2002) equipped with a Thermal Conductivity Detector (TCD).The full electrolyzer configuration was assembled using two identical IrOσ = 0.05 eV were used. A vacuum of 15 Å was adopted along z‐axis. And (5 × 5 × 1) and (2 × 2 × 1) Monkhoest‐Pack k‐point mesh were used for all samples during electronic structure calculation and the structure optimization, respectively. During structure optimization, the geometry optimization was performed when the convergence criterion on forces became smaller than 0.02 eV Å−1 and the energy difference was <10−4 eV. The crystal orbital Hamiltonian population (COHP) calculation was performed on LOBSTER code.All DFT calculations were performed with Vienna ab‐Initio Simulation Package (VASP) in the form of Perdew–Burke–Ernzerhof (PBE) function to account for exchange‐correlation potential with the on‐site Coulomb Repulsion U term. The projector augmented wave pseudopotentials method was used for describing electron–ion interactions. In this work, the value of U was 3.0 for V and 0 for Ir. All the calculations were performed using the Cambrideg Serial Total Energy Package. The cutoff energy of 400 eV and Gaussian electron smearing method with 2 catalyst was constructed by cleaving the bulk structure with the stable (110) surface. The 4 × 4 supercells were employed. The atoms in the bottom two layers were kept frozen while the remaining were allowed to relax during the slab calculations. To model the IrO2/V2O5 catalyst, the Ir4O8 clusters supported on 4 × 4 supercell of V2O5 (001) were built. The atoms in the bottom two layers of V2O5 (001) were kept frozen while the remaining were allowed to relax during the slab calculations. The value of d‐band center was obtained by using Ed=∫−∞∞Nd(ε)εdε/∫−∞∞Nd(ε)εdε, where ε is the energy referring to E‐Fermi and Nd (ε) is DOS projected onto d‐states or different orbits of d‐states. The charge accumulation and depletion were obtained as ρdifference = ρIrOx‐support − (ρIrOx + ρsupport). All DFT calculations performed in this work are about the origin of the activity of three catalysts for OER. As introduced by Nørskov and coworkers, the acidic OER mechanism was described by a four‐electron transfer process with the following steps, where the symbol “*” represents the active siteThe slab model of commercial IrOThe OER mechanism under alkaline and neutral condition can be described by the following four stepsEads) of species X is calculated by Eads = E(X/slab) – E(X) – E(slab). The thermodynamic free energies were obtained as G = EDFT + EZPE – TS, where EDFT is the DFT ground state, EZPE is zero‐point energy, and S is the entropy. Free energies were used for all absorbed species , and free energy correction values for adsorbates are provided in Table 2 is derived as G(O2) = 2G(H2O) − 2G(H2) + 4.92 (eV). The free energy at an applied overpotential U is calculated as GU = G − neU, where n is the number of (H+ + e−) pairs involved and e is the transferred electron. More precisely, the catalytic performance was estimated by the magnitude of the potential‐determining step for the OER, GOER. This was the specific reaction step in the four‐step mechanism with the largest ΔGThe adsorption energy (The theoretical overpotential, which is independent of pH, at standard conditions is then given by following equationThe authors declare no conflict of interest.Supporting InformationClick here for additional data file.Supplemental Movie 1Click here for additional data file.


Background: Spontaneous intracranial hypotension (SIH) is a rare syndrome characterized by heterogeneity of presentation and prognosis, which can occasionally result in serious complications, such as the formation of subdural hematomas (SDHs). This case series aims to emphasize that SIH remains a diagnostic and therapeutic challenge; it can present with a broad clinical spectrum of symptoms, can lead to SDH and, if conservative treatment fails, an epidural blood patch (EBP) is a viable treatment option. Although the exact etiology of SIH is not known, it is believed to be due to cerebrospinal fluid (CSF) leak or a low CSF pressure. Case Series: Three patients  with ages ranging between 38 and 53 years old who presented with complaints of not only an orthostatic headache, but also a variety of symptoms of SIH, including the formation of two SDHs in one of them, were included in this series. These patients did not respond to conservative management and, subsequently, given the clinical and radiological evidence of SIH, were referred to the Anesthesiology Department for an EBP. Diagnostic workup was facilitated by imaging modalities, including magnetic resonance imaging (MRI) of the brain and spinal cord, prior to the EBP. All three patients were subjected to an EBP with an 18-gauge epidural needle. A total of between 30 and 43 mL of autologous blood was collected from the patients and was injected into the epidural space under strict aseptic conditions. Two lumbar  EBPs and one thoracic (T11–T12) EBP were performed on the three patients, respectively. All patients reported complete resolution of symptoms following the EBPs, while MRI improved substantially. Conclusions: This report describes three cases of SIH with CSF leak originating from the cervical, the thoracic and the lumbar level. The EBP restored CSF pressure and relieved the patients’ persistent symptoms. MRI helps in revealing indirect signs of a low volume of CSF, though it may not be possible to locate the actual site of the leak. In conclusion, EBP is a well-accepted and beneficial treatment modality for SIH when conventional measures fail.  Spontaneous intracranial hypotension (SIH) is a rare syndrome; however, it is highly disabling and characterized by heterogeneity of presentation and prognosis. The hallmark of symptoms is an acute orthostatic headache, but a broad spectrum of clinical manifestations, such as neck pain or stiffness, nausea, tinnitus or photophobia, can also be present. Despite its infrequent occurrence, the increased accessibility to and development of neurological imaging examination, such as a spine magnetic resonance imaging (MRI) and computed tomographic myelography, identify more and more recognized cases . AlthougA 53-year-old male presented to the Neurology Outpatient Department with complaints of episodes of dizziness over the last month, with no signs of vertigo but accompanied by hearing disturbance. He could also sense the onset of dizziness at an earlier stage during each episode along with a feeling of instability accompanied by headache, which was worse while sitting up, but was relieved on lying down. There was no loss of consciousness, no history of craniospinal trauma or recent central neuraxial blockade. On clinical examination, no neurological deficits were elicited and no papilledema was detected. On further neurological examination, the level of consciousness, papillary light reflex and the muscle force of limbs were intact, but mild dysarthria and muscular weakness of the left lower extremity were revealed.1-weighted (T1W) sequence in the transverse plane after intravenous contrast medium and a flair sequence in the transverse plane) showed an excess of pachymeningeal enhancement while two subdural hematomas were detected on the flair sequence: one left frontal (5.5 cm × 6 mm) and a second right temporal–parietal (5.3 cm × 3 mm) (2-weighted (T2W) sequence in the sagittal plane) also showed evidence of two CSF leaks by revealing one fluid collection at the anterior cervical level and another one at the L2–L3 lumbar level. The T1W sequence in the sagittal plane after intravenous contrast medium also demonstrated homogenous pachymeningeal enhancement at the cervical spinal level (MRI study (T × 3 mm) a. Spinalal level b. Imagin2O. Given the clinical and radiological evidence of SIH, the patient was referred to the Anesthesiology Department for an EBP. With the patient resting in a left supine position, an 18-gauge Tuohy needle was inserted into the middle epidural compartment at the lumbar level L2–L3. Blood was collected from the patient’s left basilic vein. A total of 35 mL of autologous blood was injected into the epidural space via an extension pipe under strict aseptic conditions. The patient was advised to stay supine for 24 h and reported immediate relief of symptoms afterwards with a simultaneous reduction of the dizziness. One month later at the follow-up, the patient remained asymptomatic and significant radiological improvement in the thickness of the subdural hematomas was also demonstrated compared to the previous examination; the left frontal one decreased to 5.5 cm × 3 mm and the right temporal–parietal one decreased to 3.5 cm × 4 mm. Their imaging was accompanied by elimination of pachymeningeal enhancement around the cerebral hemispheres on the flair sequence  and the mandibular (V3) branches of the trigeminal nerve at the left side of the face and posterior neck pain and stiffness. Radiologic findings of magnetic resonance of the brain showed significant enhancement of the meninges of brain hemispheres and cerebellum , decreased sensation of the maxillary , although most patients have CSF opening pressures within the normal range, making SIH a misnomer [2O; however, based on the clinical findings and MRI imaging , their diagnosis was undisputed. In fact, it is considered nowadays that SIH results from hypovolemia due to CSF leakage rather than CSF hypotension, and CSF pressure can lie within the normal range [The diagnosis of SIH is based mainly on the orthostatic headache according to the third edition of the International Classification of Headache Disorders (ICHD-3) . The patmisnomer . The firal range ,22. In oal range ,23. FurtImaging modalities which are readily available, non-invasive and easy to perform such as brain computerized tomography (CT) and brain MRI with contrast enhancement can reveal signs of intracranial hypotension, while the exact site of a CSF leak can be identified with spinal neuroimaging, such as spine MRI and MRI myelography. Invasive imaging studies, such as myelography, CT myelography and radioisotopic cisternography, belong to the past due to their risks, invasiveness and the advances in cross-sectional novel imaging techniques . ClassicAs mentioned before, the persistent leakage of CSF causes the characteristic headache of SIH, which may not respond to conservative treatment; this consists of complete bed rest with the head of the bed flat, fluid hydration, application of an abdominal binder, caffeine or analgesic medications. These conservative regimens aim at counteracting CSF loss and cerebral vasodilation. For example, fluid hydration is encouraged with the aim to increase CSF production; however, results are rather temporary and disappointing . On the As for the procedure itself, written informed consent should be obtained from the patient, as well as documentation about the discussion on the risks, benefits and availability of alternative treatment. Strict aseptic conditions should be adhered to, including for the procedure of blood withdrawal. The procedure should not be performed in case of systemic or in situ infection or if there is overt coagulopathy. There is no consensus on the optimal volume of blood required. It has been shown that a larger amount of injected blood predicts a better response to the first epidural blood patch ,35. TherAs already mentioned, all our patients reported complete resolution of their symptoms both during the first hours after the EBP and also one month later at the follow-up without any untoward events. Factors associated with better outcome after the performance of the EBP include older age, absence of tinnitus, intense pachymeningeal enhancement and subdural collections and a larger volume of blood (>20 mL) used. Apparently, sex and body mass index bear no correlation to the success of the EBP . An EBP Apart from EBP, which is considered the gold-standard of therapy, other alternative pain interventions have been attempted, though with limited evidence as yet. Greater occipital nerve block is one of these and seems to act by interrupting the pain transmission to the trigeminus nucleus caudalis  and by reducing central sensitization . AnotherTo sum up, the target of the therapy in cases of SIH is either to increase the CSF pressure or to seal the CSF leak. Taking into consideration the inefficacy of conservative treatment, the debilitating symptoms of SIH and the potential for life-threatening complications, early EBPs are warranted. The literature suggests that the success rate of the first attempt of an EBP ranges between 80 and 90%. In case of persistent symptoms, one might consider repeating the EBP up to two or three times with an interval time of 5 days between the repeated EBPs to limit the risk of spinal cord compression due to excessive EBP volume. However, in case of patients refractory to treatment with EBP headache, the physician should be alerted to review the differential diagnosis .In conclusion, this report is a reminder that SIH may be an under-diagnosed or misdiagnosed syndrome with a variety of imaging features as well as a great diversity of signs and symptoms at presentation not limited to the classic orthostatic headache. Therefore, the diagnosis should not be excluded based on the presence of non-orthostatic headache, normal neuroimaging studies or normal CSF opening pressure. The procedure of an EBP secures the diagnosis and remains the main treatment modality in cases of severe SIH syndrome refractory to conservative management. The majority of reports agree that the EBP should be attempted early in patients failing conservative treatment even if the exact site of the leak cannot be localized. The current case series describes not only the persistent symptoms of the patients but also reports the formation of a SDH, which is a severe complication of SIH. Although the exact etiology of SIH is not known, it is believed to be due to a CSF leak which produces the clinical spectrum. Brain MRI and spine MRI with contrast should be offered as first line investigations in patients with clinical suspicion of SIH and help in demonstrating indirect signs of a low volume of CSF, though it may not be possible to locate the actual site of the leak. In our series, EBPs were performed in order to restore CSF volume; they relieved the patients’ symptoms, suggesting that EBP is a well-accepted and beneficial treatment modality for SIH when conventional measures fail.


The next generation sequencing (NGS) based non-invasive prenatal test (NIPT) has outplayed the traditional serum biochemical tests (SBT) in screen of fetal aneuploidies with a high sensitivity and specificity. However, it has not been widely used as a primary screen tool due to its high cost and the cheaper SBT is still the choice for primary screen even with well-known shortages in sensitivity and specificity. Here, we report a multiplex droplet digital PCR NIPT (dPCR-NIPT) assay that can detect trisomies 21, 18 and 13  in a single tube reaction with a better sensitivity and specificity than the SBT and a much cheaper price than the NGS-NIPT.In this study, the dPCR-NIPT assay’s non-clinical characteristics were evaluated to verify the cell free fetal DNA (cffDNA) fraction enrichment efficiencies, the target cell free DNA (cfDNA) concentration enrichment, the analytical sensitivity, and the sample quality control on the minimum concentration of cfDNA required for the assay. We validated the clinical performance for this assay by blindly testing 283 clinical maternal plasma samples, including 36 trisomic positive samples, from high risk pregnancies to access its sensitivity and specificity. The cost effectiveness of using the dPCR-NIPT assay as the primary screen tool was also analyzed and compared to that of the existing contingent strategy (CS) using the SBT as the primary screen tool and the strategy of NGS-NIPT as the first-tier screen tool in a simulating situation.For the non-clinical characteristics, the sample processing reagents could enrich the cffDNA fraction by around 2 folds, and the analytical sensitivity showed that the assay was able to detect trisomies at a cffDNA fraction as low as 5% and the extracted cfDNA concentration as low as 0.2 ng/μL. By testing the 283 clinical samples, the dPCR-NIPT assay demonstrated a detection sensitivity of 100% and a specificity of 95.12%. Compared to the existing CS and the NGS-NIPT as the first-tier screen strategy, dPCR-NIPT assay used as a primary screen tool followed by the NGS-NIPT rescreen is the most economical approach to screen pregnant women for fetal aneuploidies without sacrificing the positive detection rate.This is the first report on a dPCR-NIPT assay, consisting of all the necessary reagents from sample processing to multiplex dPCR amplification, can detect T21, T18 and T13 in a single tube reaction. The study results reveal that this assay has a sensitivity and specificity superior to the SBT and a cost much lower than the NGS-NIPT. Thus, from both the test performance and the economic benefit points of views, using the dPCR-NIPT assay to replace the SBT as a primary screen tool followed by the NGS-NIPT rescreen would be a better approach than the existing CS for detection of fetal aneuploidies in maternal plasma.The online version contains supplementary material available at 10.1186/s12967-022-03455-y. Trisomy 21 , trisomy 18 , and trisomy 13  are the most common autosomal aneuploidies that cause mental retardation and serious congenital defects of newborns –6, and tSince the discovery of cell free fetal DNA (cffDNA) in maternal plasma in 1997, 21, DNAHere we describe the performances of a multiplex dPCR-NIPT technique for simultaneous detections of fetal T21, T18 or T13 in a single tube reaction on a droplet dPCR platform, as well as its preliminary clinical performance in testing real clinical samples in a clinical environment. By the best of our knowledge at the time this manuscript being prepared, this is the first report to demonstrate that a full dPCR-NIPT technique, consisting of sample processing, cffDNA fraction enriching, the target cell free DNA (cfDNA) concentration enrichments, and dPCR amplification reagents, can simultaneously detect fetal T21, T18 or T13 in maternal plasma samples in a single reaction. The justifications of replacing the SBT with this dPCR-NIPT assay for the upgraded CS as mentioned above are also discussed.To evaluate how this dPCR-NIPT assay would perform in a real clinical setting, we performed the entire assay process by testing true clinical samples in the First Affiliated Hospital of Zhengzhou University. A total of 453 high risk pregnant women with singleton pregnancy were recruited for this study and their high risk statuses were identified by the hospital’s routine first and/or second trimester screens  for fetal aneuploidies. Some of pregnancies in this population directly selected or prescribed by their doctors for the NGS-NIPT screen, and, except a few, all the high risk pregnancies identified by the SBT and/or ultrasound were further rescreened by the NGS-NIPT method to confirm the high risk results and filter out the false positives. The NGS-NIPT high risk pregnancies were finally diagnosed by the invasive CVS/AC procedures. A separate aliquot (ca. 2.5 mL) of each maternal plasma samples was kept a side at -20ºC for this dPCR-NIPT test. Of the 453 samples, 170  were randomly picked and used as training set for onsite training and assay critical procedure validations, as well as for establishing euploid baseline cut-off value for this assay. The clinical characteristics for the training set samples are shown in Additional file The median or average cffDNA fraction in maternal plasma is generally considered around 10% in the first trimester and would dramatically increase to about 20% in the third trimester –47, whilhttps://www.ncbi.nlm.nih.gov). These candidate sequences were analyzed to ensure no primer-dimmers and hairpin-structures exist with an online tool provided by iGeneTech (https://mfeprimer3.igenetech.com). The primers and probes passed above selection steps were sent to ThermoFisher Scientific (https://www.thermofisher.cn) for syntheses and the detection probes of Chr21, Chr18, and Chr13 were labeled with FAM, VIC, and Cy5 fluorophores, respectively, to differentiate the amplification signals. After a series of evaluation experiments, 10 sets primers and probes for each of the three chromosomes  were selected for this dPCR-NIPT assay and the sequence information are presented in Additional file To increase the assay sensitivity, multiplex dPCR was applied to quantitate Chromosomes 21, Chromosome 18, and Chromosome 13 . Candidate primers and probes were chosen for each of the three chromosomes by searching the National Center for Biotechnology Information’s (NCBI) database with its online tools of Nucleotide-BLAST and Primer-BLAST provided on its website , separately, for the sample using Eq. 21/18, R21/13, and R18/13  for the sample using Eq. n is the total number of effective droplets in a dPCR reaction, d is the number of positive droplets, and V is the volume of a single droplet (μL).ChrT/ChrR is the CCN ration between the chromosome being tested (ChrT) and the reference chromosome (ChrR) from the same sample in the same dPCR assay. When R21/18  is calculated. ChrT represents Chr21  and ChrR represents Chr18 , respectively.ChrT/ChrR is the Z score of the RChrT/ChrR of a sample being tested, BL Mean RChrT/ChrR is the mean RChrT/ChrR of a euploid pregnant population that serves as a baseline (BL) RChrT/ChrR for a cut-off value determination, and SD is the standard deviation of BL Mean RChrT/ChrR. When Z21/18  is calculated, the Sample RChrT/ChrR is the R21/18  of a sample being tested and the BL Mean RChrT/ChrR is the mean R21/18  of a euploid pregnant population.Since it is generally accepted in principle that there is no co-existent of two aneuploidies in a singleton pregnancy regarding T21, T18 and T13, thus, when one of these three chromosomes is investigated for fetal aneuploidy, the other two would serve as euploidy references in chromosome copy number (CCN) comparisons. Based on this concept, we designed the dPCR-NIPT assay for detecting fetal aneuploidies by three major steps: (1) measuring the CCN for Chr21, Chr18, and Chr13 by the dPCR-NIPT assay in a single tube reaction, while the CCN for each chromosome is automatically calculated by the instrument’s software based on Eq. http://www.turtle-tech.cn), which consists of three separate instruments: BioDigital-QING Loader™ for generating one layer of water-in-chamber droplets inside a microfluidic chip , respectively. Thus, a pre-amplification (PA) step is required to boost the target cfDNA concentrations for accurate CCN quantification.For target enrichment, 10 μL of isolated cfDNA from each sample was mixed with 5 μL of pre-amplification master mix (PA-MMX)  and amplified with 6 cycles of PCR with the following cycling conditions: 95 °C for 10 min, 6 cycles of 95 °C for 30 s and 60 °C for 30 s. The PA-MMX contains 10 primer pairs each for Chr21, Chr18, and Chr13 for specifically enriching the designated cfDNA targets on the three chromosomes. Ten (10) μL of the enriched target cfDNA was used in dPCR test after a 6.25-fold dilution, which was to ensure the positive droplet rate fallen within the 30–80% range for more actual CCN measurements. We evaluated the efficacy of cfDNA specific target enrichments by testing 16 normal maternal plasma samples without and with PA.21/18, R21/13, and R18/13, as well as Z21/18, Z21/13, and Z18/13 were sequentially calculated as described above.In a 30 μL multiplex reaction, we added 10 μL of enriched and diluted cfDNA targets into 20 μL of dPCR master mix (MMX) containing the same 30 primer pairs as those in PA-MMX, plus the corresponding detection probes for Chr21, Chr18, and Chr13, the resulted 30 μL dPCR reaction mix was tested by using the Biodigital-Qing dPCR™ system according to the manufacturer’s instruction. The dPCR cycling conditions was as follows: 95 °C for 10 min, 45 cycles of 95 °C for 30 s and 60 °C for 30 s. The total CCN (presented as copies/μL) cumulated from the 10 amplification sequences for Chr21, Chr18, or Chr13 was automatically calculated by the analysis software of the dPCR system according to the Poisson distribution, then, RWe accessed the dPCR-NIPT assay’s analytical sensitive regarding the effect of cffDNA fraction in the maternal plasma on the trisomy detections by testing a panel of artificial T21, T18 or T13 samples. Appropriate amounts of 10 synthetic Chr21, Chr18 or Chr13 targets (simulating cffDNA) matching to the 10 amplification sequences on its corresponding chromosome were added to a purified maternal cfDNA (50 copies/μL) to a final “cffDNA” fraction of 0%, 3%, 5%, 10%, or 15%, 10 μL of the artificial trisomy samples was tested by the dPCR-NIPT assay in 8 replicates.The 283 plasma samples of the testing set were tested by the dPCR-NIPT assay following the manufacturer’s instructions, throughout the testing processes the samples’ true aneuploidy statuses were kept unknown to all operators performing the dPCR test until all sample tests completed and conclusions made. Then, the dPCR-NIPT test results were compared to CVS/AS confirmed results for these samples. Whenever possible, the test results of the dPCR-NIPT were also compared to those of the SBT and NGS-NIPT to evaluate the dPCR-NIPT assay’s clinical performances.To assess the cffDNA enrichment efficiency of the SMP Prep Kit, 10 maternal plasma samples of normal pregnancies with male fetuses were processed with this kit and their cfDNA and cffDNA copy numbers were directly measured with dPCR without PA at cfDNA extraction and cffDNA enrichment stages. The results indicated that the SMP Prep Kit was capable to increase the cffDNA fraction by 1.73–2.42 folds , while the mean R21/18 (SD) of the 16 samples without and with PA were 0.9901 (0.0175) and 0.9841 (0.0225), respective, which are statistically no difference (P > 0.4) , which agrees with the reported results[P > 0.8), except the ≥ 26 gestational week group which was statistically different from the other groups (P < 0.05). In general, the cffDNA fraction for more than 99% of maternal plasma samples would be ≥ 4% with a median value around 10% [There are three main challenges the dPCR-NIPT assay need to overcome before this assay can be implemented in clinical sample testing: the relatively low cffDNA fractions in maternal plasma, generally insufficient total cfDNA amount due to limited available sample volume, and the number of droplets provided by a dPCR system. We resolved the first two sample related challenges by creating and incorporating the general cffDNA fraction enrichment and target cfDNA PA technologies to this dPCR-NIPT assay Table . Of causd results. In contound 10% –47, 50, As for the dPCR system, we calculated the minimum number of droplets required for the NIPT purpose under certain conditions according to the mathematic formulas Eqs.  and 5 puThe BioDigital-QING dPCR™ system used in this study could reliably generate about 20,000 droplets with above 95% effectiveness, meaning a minimum of 19,000 effective droplets routinely available for a dPCR assay. It seemed that the third challenge was also resolved, allowing us to proceed the clinical sample tests.2 value of 0.9995  on a routine basis, however, to determine the cfDNA weight concentration (ng/μL) with a fluorometer is convenient. We used Qubit 3 Fluorometer to measure cfDNA concentration (ng/μL) and established a standard curve to convert ng/μL to c/μL  from group to group. The results reveals that the all samples with a ng/μL concentration ≥ 0.2 had a PDR above 30%, thus, we set the benchmark of minimum cfDNA concentration required right at 0.2 ng/μL for plasma samples to be tested by the dPCR-NIPT assay.We evaluated 103 maternal plasma samples of euploid pregnancies to validate the accuracy of the standard curve for concentration unit conversion and the minimum cfDNA concentration needed to reach 30% PDR. The samples were extracted with the Tage SMP Prep Kit, their ng/μL concentrations were measured and converted to c/μL concentration by using the equation of y = 188.91x-4.5745 from Fig. 21/18, Z21/13 and Z18/13 values would be evaluated against the Z-score cut-off value and the aneuploidy status calling algorithm (Table 21/18 (or Z21/13 or Z18/13) ≥ 1.65 is called T21 (or T21 or T18) positive, a sample with a Z21/18 (or Z21/13 or Z18/13) ≤ -1.65 is called T18 (or T13 or T13) positive, and a sample with a Z21/18 (or Z21/13 or Z18/13) < 1.65 and > -1.65 is called trisomic negative or low risk.Before examining the testing set samples, we established the assay strategies and determined the cut-off criteria. The dPCR-NIPT assay uses Chr21, Chr18 and Chr13 both as the targeting chromosomes and as the reference chromosomes Fig. . When a hm Table . With th21/18, R21/13 and R18/13 values, and the corresponding Z21/18, Z21/13 and Z18/13 values for each sample as described above and determined the aneuploidy status for each sample tested according to the calling algorithm tabulated in Table We then test the 283 maternal plasma samples of the testing set, in a blind manner, according to the assay procedures provided by Tage Biotech, briefly, 2.5 mL of plasma sample was processed with the Tage SMP prep kit on Genefaster’s PF32A Automated Nucleic Acid Extraction System with a final elution volume of 40 μL, the purified cfDNA concentration was measured by Qubit 3 Fluorometer , 10 μL was used for PA (6 cycles), and 10 μL of the PA product was used in the dPCR-NIPT assay after a 6.25-fold dilution. We calculated the CCNs of Chr21, Chr18 and Chr13, the ROf the 283 samples, 8 (2.83%) fell into Scenario 11 and went through the re-test processes with the dPCR-NIPT assay according to the calling algorism Table : 7 of thA receiver operating characteristic (ROC) curve and the area under an ROC curve (AUC) are usually used to assess the performance of a diagnostic test , we plugFor a screen test, the first priority is always the safety, i.e., making the positive detection rate as high as the technology allows while keeping the false positive rate at an acceptable level. Our results verified that the Z-score cut-off value of 1.65 and the calling algorithm Table  applied By testing the 283 clinical maternal plasma samples in a real clinical environment, we validated the dPCR-NIPT assay on aspects of testing performances, clinical operationality, and user friendliness. With existing dPCR system available on the market, we overcame the challenges of restricted sample volume available and the limited cffDNA fraction and cfDNA concentration presented in the maternal plasma samples by effectively incorporating cffDNA enrichment, specific cfDNA target amplifications and a multiplexed assay design into the dPCR-NIPT assay. The overall test results demonstrated that the dPCR-NIPT assay with a minimum of 18,000 droplets has a significantly statistical power to distinguish aneuploidies from euploid noises with a 95% confidence and is readily applied to screen pregnancies for fetal aneuploidies in clinical. This assay can be further improved in three areas, using a dPCR system with multiple fluorescent detection channels and minimum of 80,000 PCR partitions, increasing plasma sample volume to 4 mL, and continuously optimizing the testing reagents to increase accuracy and reduce variations. A dPCR system with at least 80,000 PCR partitions can increase the test confidence to 99.9% when estimated by Eq. NGS-NIPT is a very powerful tool for screening fetal aneuploidies with high test accuracy –29, the Considering the dPCR-NIPT assay has a better sensitivity and specificity, if using it to replace the SBT in the existing CS, the upgraded version would be more effective with a higher positive detection rate and significantly less false positive samples to be resolved by the NGS-NIPT test. A major concern about to use the NGS-NIPT as the first-tier fetal aneuploidy screening test is lack of cost effectiveness due to its relatively higher price at an average of $570 per test with a range of $200-$1,100, while the SBT price is averaged at $53 with a range of $45-$77, depending on the countries –35. If tIn this study, we evaluated the dPCR-NIPT assay’s non-clinical characteristics by verifying the cffDNA fraction enrichment efficiency, the PA uniformity for all multiplex cfDNA specific targets, the analytical sensitivity on artificial trisomic samples with low cffDNA fractions, and the sample quality control on minimum cfDNA concentration required. We validate the clinical performance for this assay by testing 283 plasma samples from high risk pregnancies and demonstrated that the sensitivity and specificity of the dPCR-NIPT assay is superior to those of the SBT. With a brief cost effectiveness analysis based current market prices, compared to the existing CS and the NGS-NIPT as the first-tier screen strategy, the upgraded CS, in which the dPCR-NIPT assay is used as a primary screen tool followed by the NGS-NIPT rescreen, is the most economical approach with improved sensitivity and specificity to screen pregnant women for fetal aneuploidies.Additional file 1: Figure S1. The structure of the microfluidic chip. Figure S2. The BioDigital-QING dPCRTM system. Table S1. Training set sample information. Table S2. Sequence information for primers and probes. Table S3. Estimation of number of droplet required for a dPCR reaction.