Sudan falls in the meningitis belt where most global cases of bacterial meningitis are reported. Highly accurate decision support tools have been developed by international specialized societies to guide the diagnosis and limit unnecessary hospital admissions and prolonged antibiotic use that have been frequently reported from countries around the world. The goals of this study are to critically evaluate the clinical decision of bacterial meningitis in children in Sudan using clinical prediction rules and to identify the current bacterial aetiology.n = 503). Cerebrospinal fluid (CSF) specimens were obtained from all patients while clinical and demographic data were available for only 404. Conventional laboratory investigations were performed. The clinical decision was evaluated by the International Classification of Diseases–Clinical Modification code 320.9 and the Bacterial Meningitis Score. Ethical clearance and permissions were obtained.This cross-sectional hospital-based study was conducted in October to July of 2010 in a major referral pediatric hospital in Khartoum, Sudan. Febrile children age 1 day to 15 years who were provisionally diagnosed as having meningitis on admission were included  with confirmed bacterial aetiology as Proven Bacterial Meningitis. Neisseria meningitidis was identified in 0.7% (3/404) and Streptococcus pneumoniae in another 0.7%. Typical laboratory findings  were seen in 5 (83%). Clinically, patients showed fever, seizures, chills, headache, vomiting, stiff neck and bulging fontanelle. All confirmed cases were less than 5 years old and were admitted in summer. All patients were prescribed with antibiotics; they were all recovered and discharged.Out of 503 provisionally diagnosed bacterial meningitis patients, the final clinical confirmation was assigned to 55.9%. When codes were applied; 5.7% (23/404) with CSF pleocytosis were re-classified as N. meningitidis and S. pneumoniae, but with significant decrease in prevalence. Some cases showed atypical clinical and laboratory findings.Bacterial meningitis is over-diagnosed in hospitals in Khartoum therefore clinical prediction rules must be adopted and applied to guide the clinical decision. The sole bacterial aetiology in this selected group of Sudanese children remain The online version of this article (10.1186/s12887-019-1684-3) contains supplementary material, which is available to authorized users. Alert and Epidemic definitions) ) presented with fever and seizures, other symptoms are shown on . All 357  cases, with available hospital-stay data, did not suffer from complications and were discharged.The vast majority of cases with full records on clinical data . Headache, naturally, was evaluated in 1 year olds and above where most patients  were in age group (1–5 years), 13% (3/23) in age group (6–10 years) and 22% (5/23) in age group (≥11 years). Age groups and seizures showed significant difference (p = 0.046) but no association. Seizures were seen in all 4 patients in age group (1–29 days) and all 6 patients in (6–10 years), in 92.5% (124/134) in age group (1–11 months), 93.3% (194/208) in age group (1–5 years) and 66.7% (6/9) in age group (≥11 years). Highly significant difference was observed between season and stiff neck (p = 0.00) accompanied with significant but weak association (Corre = 0.23 [95% CI: 0.13 to 0.33]). The vast majority of those with stiff neck were admitted in summer  and 3 in winter . Statistical difference between season and bulging fontanelle (among <1.5 years olds) was detected with high significance (p<0.001) and strong correlation (Corre = 0.54 [95% CI=: 0.43 to 0.64]). All cases with bulging fontanelle (age 0 to 18 months [n = 219]) were admitted in summer . No significant differences and associations were found between the remaining demographic and clinical data.Statistically significant difference and association were obtained between age groups and headache (p<0.001) but weak association (Corre = 0.27 [95% CI: 0.19 to 0.35]) were observed between hospital diagnosis and season of admission. On cross-tabulations; amongst patients diagnosed as having BM, 50.3% (100/199) were admitted in summer and 49.8% (99/199) in winter. All patients were treated during hospitalization by first line and/or second line antibiotic therapies  of all admissions during the study period were diagnosed as having a CNS associated condition; 55.9% (199/356) as bacterial (septic) meningitis, 0.6% (2/356) brain abscess and 5.3% (19/356) febrile convulsions. The remaining 38.2% (136/356) were diagnosed as having other infections; data is provided in the Additional file 20/356 ofp<0.001, Corre = + 0.77 [95% CI: 0.72 to 0.81]) were identified between the duration of hospital stay and final hospital diagnosis. Findings of cross-tabulations are summarized on Table Period of hospital stay was evaluated; median 6 days with range of 1 to 38 days. Highly significant and strong positive association (3). Significant WBC count (≥5 cells/mm3) was identified in 5.2% (21/404). Median CSF cell count for values above 5 cells/mm3 was 3000 with range of 50 to 33,400 cells. CSF specimens with high WBC count (>100 cells/mm3) showed neutrophilic predominance; reaching in average 80% and those with low WBC count (≤100 cells/mm3) showed lymphocytic predominance; reaching 60%. Abnormal CSF glucose concentration (< 45 or > 100 mg/dl) was seen in 11.9% (54/453). Abnormal CSF protein concentration (< 14 or > 45 mg/dl) was seen in 47.7% (215/451). Direct Gram stain preparation showed Gram positive diplococci in 0.7% (3/451 [52 missing]) and Gram negative diplococci in another 0.7%. On CSF culture for rapidly growing bacteria, all specimens that were Gram positive on direct preparations yielded S. pneumoniae  and all those that were Gram negative yielded N. meningitidis . A single bacterium was isolated from each patient; there were no polymicrobial infections. Two out of the 3 (66.7%) N. meningitidis isolates were identified as W135 serotype. Further laboratory data is provided in the Additional file All of the 503 patients were subjected to LP, nevertheless, 8.8% (44/503) of the withdrawn CSF specimens were traumatic, therefore, cytological and chemical analyses were not performed. The vast majority  of CSF specimens that were non-traumatic showed no WBCs  suffered also from chills, vomiting, bulging fontanel and altered mental status. She was the only female. They were all infants (1–11 months). The female and 1 of the 2 males (66.7%) were admitted in summer; the third in winter. The female CSF was bloody; the rest were turbid. Turbid specimens showed high CSF WBC count (≥1000 cells/cmm3) and all showed neutrophilic predominance (≥70%). All specimens had low CSF glucose level (< 45 mg/dl) and high CSF proteins level (> 45 mg/dl). All 3 patients had a confirmed hospital diagnosis of bacterial meningitis. They were all treated with penicillin, ampicillin and/or cephalosporins. Further data is provided in the Additional file All 3 patients with positive bacterial culture for N. meningitidis serotype W135 was identified in the isolates from the two infants. All patients were females, they all admitted in summer. Non typical conventional laboratory results were seen; the case that was presented with only 4 symptoms had a clear CSF, normal WBC count (<5 cells/mm3), normal CSF glucose level (45-100 mg/dl), and normal CSF protein level (14-45 mg/dl). The second infant had a traumatic CSF; therefore, no cytological or chemical analyses were performed. The toddler’s CSF WBC count revealed leukocytosis (> 1001 cells/mm3); low CSF glucose level (< 45 mg/dl) and high CSF protein level (> 45 mg/dl). All patients had a confirmed hospital diagnosis of septic meningitis. They were all treated with penicillin or ampicillin and cephalosporins. Further data is provided in the Additional file All the same, all 3 patients presented with fever and seizures. One of the 3 (33.3%); a toddler in the age group 1–5 years), suffered also from headache and vomiting. The remaining 2 (66.7%) infants (1–11 months) presented also with chills, 1 (33.3%) of them with vomiting and the other with stiff neck and bulging fontanel. –5 years,Peripheral Blood Absolute Neutrophil Count ≥ 10,000 Cells/μL) of the BMS was unavailable; therefore, 4 criteria were used in this analysis. When 1 or more prediction rules were fulfilled, patients were considered at High Risk of BM  were identified in the study of Nigrovic et al [In reference to the data from hospital records, BM was assigned for 56% of the study population. This number seems to be exaggerated since neither an ic et al  among 32ic et al  and Amaric et al  identifiic et al  and Oostic et al  identifiic et al ; most prWhen it comes to diagnosing a debilitating and potentially fatal illness in a pediatric population of a country located in the meningitis belt, health care officers in Sudan often tend not to dismiss a poorly suspected case. Fear of misdiagnosis or late treatment result in over diagnosis that could lead to an increased hospitalization costs as evident by the extended periods of hospitalizations Table . Over diS. pneumoniae and N. meningitidis as the predominant pathogens of BM in children of all ages in all regions. In previous studies, pneumococcal disease was usually identified in relatively small number of cases. Dubos [S. pneumoniae meningitis among patients with CSF pleocytosis. Even though in a meningitis outbreak situation, only 6% (n = 149) was reported as S. pneumoniae among 40% with bacterial aetiology [S. pneumoniae as the most common cause of BM in children ranging from 22.5% in Europe and 41.1% in Africa [N. meningitidis in 5% (9/166) and 9% (220/2475) respectively, closely comparable to our findings of 7.5%. On the other hand, studies in Mali reported 44% meningococcal meningitis and 31% pneumococcal meningitis [Pneumococcal and meningococcal conditions were equally identified in 7.5% among those with proven infectious meningitis in this study. A recent meta-analysis  coverings. Dubos  reportedetiology . Never tn Africa . A reducn Africa  and Ghann Africa , where Dn Africa  and Turcn Africa  identifiAll our patients were admitted in the high risky season that is described climatologically as dry compared to about 65% of all cases that were recorded in the Ghanaian study in this season . This peDiagnostic signs for BM in young children are unclear; they do not often exhibit the general symptoms and may only be irritable and look unwell . All casS. pneumoniae and in only one third with N. meningitidis, while another third showed normal CSF picture. Normal CSF cellular count in those with positive CSF culture can be demonstrated, however, rarely [Typical findings on CSF analysis , 38 were, rarely . Garges , rarely  conclude, rarely .H. influenzae in a small proportion of 2% [H. influenzae meningitis in children has become so rare that a case could hardly be seen. The study of Dubos [H. influenzae in only 2.5% (27/1084). Amongst published articles within our reach were those from Mali which stated that the country has suffered high morbidity and mortality of BM and high occurrence of H. influenzae meningitis [H. influenzae in 23% [Hib was not identified in this study probably as the result of the introduction of Hib vaccine to the pediatric population in Sudan which was first started in 1976 and later on in 2001 . Followion of 2% . Furtheron of 2%  affirmedof Dubos  identifiof Dubos  detectedningitis , 43. Hibe in 23%  and a poe in 23% . The obse in 23%  and fairS. pneumoniae and N. meningitidis are the sole pathogens of pediatric BM and H. influenzae is no longer an aetiology. Many cases showed atypical clinical and laboratory findings. Codes and criteria for the diagnosis of BM are not followed and that consequence leads to over-diagnosis and over-prescription of antibiotics. It is therefore of paramount importance that the decision of BM should be guided by internationally validated clinical prediction rules.In conclusion, the study has indicated a significant reduction in the occurrence of BM. Additional file 1:BM in Children in Sudan . It includes 3 tables in the following order. Hospital Diagnosis (Non CNS Conditions): Shows frequencies and percentages of cases diagnosed by the hospital as having conditions affecting systems other than the CNS. Conventional Laboratory Data: Demonstrates detailed laboratory findings for all cases. Findings on Cases with Positive Bacterial Aetiology: Shows detailed findings for cases with confirmed bacterial meningitis. (DOCX 27 kb)Supplementary materials are provided in the file:


Neuroactive steroids seem to be implicated in a variety of neurophysiological and behavioral processes, such as sleep, learning, memory, stress, feeding and aging. Numerous studies have also addressed this implication in various cerebral disorders and diseases. Yet, the correlation and association between steroids in the periphery, e.g. blood, and the central compartments, e.g. cerebrospinal fluid (CSF), have not yet been comprehensively assessed. As the brain is not directly accessible, and the collection of human CSF usually requires invasive procedures, easier accessible compartments, such as blood, have always attracted attention. However, studies in humans are scarce. In the present study we determined estradiol, progesterone and testosterone levels in CSF and serum of 22 males without cerebral disorders or diseases.Samples were taken under conditions corresponding closest to basal conditions with patients expecting only spinal anesthesia and minor surgery. All samples per patient were collected concomitantly. Total estradiol, progesterone and testosterone concentrations were measured by electro-chemiluminescence immunoassay. The strength of correlation was assessed by Spearman’s rank correlation coefficient. Correlation analysis revealed merely weak to very weak correlations for estradiol, progesterone and testosterone respectively between the CSF and serum compartments.Total steroid levels of estradiol, progesterone and testosterone in CSF and serum of males without neurological disorders were determined. Weak to very weak correlations between CSF and serum were found thus suggesting that concentrations in the periphery do not parallel concentrations in the central compartments. Further research is needed to clarify to what extent and under which conditions serum levels of estradiol, progesterone and testosterone may possibly serve as a biomarker reflecting the respective concentrations in the CSF or in the brain. Neuroactive steroids seem to be implicated in a variety of neurophysiological and behavioral processes, such as sleep, learning, memory, stress, anxiety, feeding, aging –3. NumerHowever, a general dilemma of human studies on neuroactive steroids is that the brain is not directly accessible. Even presuming that steroid levels in the CSF may reflect steroid activity in the brain, the dilemma in principle persists as human CSF also is not easy to obtain and usually requires invasive procedures such as lumbar puncture or ventricular drainage. Thus, the issue of whether concentrations of steroids in easier accessible compartments, such as blood, may adequately reflect neuropeptide activity in the CSF remains subjected to studies .The present study determined levels of estradiol, progesterone and testosterone in CSF and blood of males without neurological disorders or diseases. The aim of the study was to evaluate the correlations of these levels between the two compartments.The study was approved by the ethics committee of the medical faculty of the Technische Universität München (Project number 2410/09). CSF and blood samples of 22 male patients  were prospectively analyzed. Written informed consent was provided by all patients. CSF and blood samples were obtained concomitantly during spinal anesthesia for elective minor urologic or orthopedic surgery. All patients were otherwise healthy. None of the patients suffered from neurological or psychiatric disorders. CSF samples were taken before the intrathecal injection of the local anesthetic. All samples were collected into plastic serum tubes and analyzed immediately.Total estradiol, progesterone and testosterone concentrations in CSF and serum were measured on a Roche Mannheim Cobas e 411 immunoassay analyzer using an electro-chemiluminescence immunoassay .Strength of bivariate monotonous correlation of quantitative data was assessed by Spearman’s rank correlation coefficient. An absolute value of the correlation coefficient of 0.00–0.19 indicates a very weak correlation, 0.20–0.39 a weak correlation, 0.40–0.59 a moderate correlation, 0.60–0.79 a strong correlation and a value of 0.80–1.0 a very strong correlation. Exploratory hypothesis testing was performed on two-sided 5% significance levels and R version 3.5.0  was used for all computations.Patient characteristics are listed in Table In humans, the correlation and association between steroids in the periphery, e.g. blood, and the central compartments, e.g. CSF, have not yet been comprehensively assessed. This issue has long been addressed and remains subjected to studies in order to shed light on a complex system of peripheral and central steroid synthesis and steroid penetration across the blood–brain and blood–CSF barrier , 13. ThiAn early study in humans by Backstrom et al. , publishIn view of the numerous studies discussing the alteration of steroid levels in the CSF of patients with neurological disorders , 20, 21,The discussion and careful interpretation of the results of the present study must also take into account general considerations on the concepts on steroid metabolism in central and peripheral compartments. In the periphery, the steroid hormones progesterone, testosterone and estradiol are synthesized from cholesterol in classic steroidogenic tissues, e.g. in the adrenal, gonad and placenta, and may exert their effects in the periphery. Peripheral steroid hormones may cross the blood–brain barrier and act centrally on brain tissues to regulate neuronal functions , 23. It The present study, with respect to the steroids estradiol, progesterone and testosterone, demonstrated merely weak to very weak correlations between serum and cerebrospinal fluid steroid levels. The careful comparison of the results of the present study with the results of the above mentioned studies –14, 19 tThe strength of the present study is the study cohort consisting of males without neurological disorders since, to our knowledge, investigations on the correlations of steroids levels in concomitantly collected samples of CSF and blood in healthy humans are very scarce. A further strength of the study is that samples were taken under conditions corresponding closest to basal conditions with patients expecting only spinal anesthesia and minor surgery. In contrast, in the above mentioned studies patients usually suffered from neurological disorders or diseases; thus, in such studies, a possible independent regulatory capacity of steroid metabolism patterns in central and peripheral compartments may have been affected by disease-related patterns.Still, limitations of this study must be considered. The validity of steroid analysis has long been a matter of debate and the literature reveals the use of various detection methods , e.g. raTotal steroid levels of estradiol, progesterone and testosterone in CSF and serum of males without neurological disorders were determined. Samples were taken under conditions corresponding closest to basal conditions. Merely weak to very weak correlations between CSF and serum were found thus suggesting that concentrations in the periphery do not parallel concentrations in the central compartments. Further research is needed to clarify to what extent and under which conditions serum levels of estradiol, progesterone and testosterone may possibly serve as a biomarker reflecting the respective concentrations in the CSF or in the brain.


Non‐suppurative encephalitides in a variety of species, including humans and dogs, have been linked to infection by astroviruses and bornaviruses.To determine whether or not ribonucleic acid of astroviruses or bornaviruses was present in the cerebrospinal fluid (CSF) of dogs with clinically diagnosed meningoencephalomyelitis of unknown etiology (MUE).Twenty‐five client‐owned dogs evaluated by CSF analysis at a single university referral hospital.Prospective case‐control study. Cerebrospinal fluid was collected from clinically diagnosed MUE and control cases and evaluated by reverse‐transcriptase polymerase chain reaction for the presence of astrovirus and bornavirus.Neither astrovirus nor bornavirus nucleic acids were identified in CSF collected from 20 clinically diagnosed MUE and 5 control cases.The negative results of this investigation suggest that astrovirus and bornavirus are not commonly detectable in CSF of dogs with MUE. CNScentral nervous systemCSFcerebrospinal fluidESBEEuropean sporadic bovine encephalitisFFPEformalin‐fixed paraffin‐embeddedGMEgranulomatous meningoencephalitisMUEmeningoencephalomyelitis of unknown etiologyNLEnecrotizing leukoencephalitisNMEnecrotizing meningoencephalitisRT‐PCRreverse‐transcriptase polymerase chain reactionTNCCtotal nucleated cell count1Meningoencephalomyelitis of unknown etiology (MUE) is the umbrella term applied to a common clinical diagnosis of noninfectious inflammatory central nervous system (CNS) disease. It most frequently encompasses granulomatous meningoencephalitis (GME), necrotizing meningoencephalitis (NME), and necrotizing leukoencephalitis (NLE), which are distinguished based on histopathologic characteristics. Efforts at identifying common etiologies for MUE have been unsuccessful. Etiologies that have been suggested include genetic, neoplastic, infectious, and autoimmune.Immunohistochemical studies and response by MUE dogs to immunosuppression support an immune‐mediated pathogenesis.Non‐suppurative encephalitides have been linked to RNA viruses in the families Astroviridae and Bornaviridae, in a number of other species. Recently, encephalitis in cattle, mink, swine, sheep, and immunocompromised humans has been associated with astroviruses.Immunosuppression of dogs with MUE is the current standard of care. Despite aggressive treatment, the course of disease is usually fatal. Identification of the underlying etiology of this disease complex is crucial to improve treatment plans and prognosis. The objective of this investigation was to evaluate using PCR‐based methods for the presence of astroviruses and bornaviruses in the CSF of dogs clinically diagnosed with MUE.22.1Toxoplasma gondii, Neospora caninum, and Cryptococcus neoformans. If necropsy results became available, these were reviewed for confirmation of MUE diagnosis.This study was approved by the University of Florida's Institutional Animal Care and Use Committee Study #201609667. As part of this prospective case‐control study, CSF was collected from client‐owned dogs evaluated at the University of Florida Small Animal Hospital Neurology Service for CNS disease from March 2016 through October 2018. All dogs underwent a complete physical and neurological examination by a neurology resident or board‐certified neurologist. Only dog breeds previously documented with GME, NME, or NLE were considered for inclusion. These included Terrier‐breed, Chihuahua, Brussels Griffon, Pug, French Bulldog, Papillon, Shih Tzu, Pekingese, Boston Terrier, Yorkshire Terrier, Cotton de Tulear, West Highland White Terrier, Poodle, Pomeranian, Dachshund, and Maltese or a cross of any of these breeds.Toxoplasma gondii, Neospora caninum, or Cryptococcus neoformans. Samples were also excluded if the dog was diagnosed with an alternate CNS disease associated with a pleocytosis. Such diseases included CNS neoplasia, fibrocartilaginous embolism, and compressive myelopathy associated with intervertebral disc disease.Dogs were excluded from the study if they were identified as a breed other than those listed in the inclusion criteria or had positive serum titers for Dogs were considered as negative controls if they met the inclusion criteria, with the exception of the cerebrospinal fluid (CSF) analysis. Negative control samples were included if the TNCC was less than 5 cells/μL on CSF analysis.A power analysis was done to determine sample size to detect the presence or absence of astroviruses or bornaviruses in dogs with MUE.The CSF samples were obtained only if clinically indicated for each animal via routine cerebellomedullary or lumbar cisternal puncture from anesthetized patients. Unless dog size limited collection volume, an aliquot of 0.2 mL from the collected sample was aseptically placed into a sterile tube for inclusion into the study. The samples were stored at −80°C.2.2Avastrovirus and mamastrovirus, of Astroviridae, Astr4380F (5′GAYTGGRCNCGNTWYGATGGNA‐CIAT‐3′) and reverse primer Astr4811R (5′‐GGYTTNACCCA‐CATNCCAAA‐3′), targeting conserved regions of astrovirus ORF1b (RNA‐dependent RNA polymerase).Orthobornavirus and Carbovirus, via PCR were used as previously described.Parrot bornavirus 4 was used as a positive control  according the manufacturer's instructions. The extracted RNA was stored in duplicates as single‐use aliquots at −80°C until PCR amplification. An internal control was performed on each sample to confirm for the presence of 18S (RNA) , and submitted to Genewiz  for sequencing.33.1Cerebrospinal fluid was collected from 20 MUE cases and 5 control dogs. The MUE cases included 4 terrier breeds, 4 Malteses, 4 Shih Tzus, 3 Chihuahuas, 2 Poodles, and 1 of each of Pomeranian, Brussels Griffon, and Yorkshire Terrier. They included 10 neutered males, 1 intact male, and 9 spayed females that ranged in age from 2 to 13 years (median 6 years). One of each of Chihuahua, Pomeranian, Yorkshire Terrier, Poodle, and Maltese breeds were included as negative controls. Of the negative controls, 2 were neutered males and 3 were spayed females. Neurolocalization of the MUE cases was intracranial in 12, spinal in 6 and multifocal in 2. Cerebrospinal fluid was collected from the atlanto‐occipital cistern in 19 of the MUE cases and in all control cases. In 1 MUE case, CSF was collected from the lumbar cistern. Prior to CSF collection, 2 of the MUE cases received a single dose of glucocorticoid, and another 2 MUE cases were treated with an antibiotic . Cerebrospinal fluid analysis revealed a pleocytosis in all MUE cases; mean TNCC 159.6 . Given these results, we can state with 95% confidence that the prevalence of infection with astrovirus or bornavirus detectable by PCR of CSF is less than 10% in the clinically diagnosed MUE population. Given that MUE is a clinical diagnosis based on magnetic resonance imaging and CSF analysis, obtaining a definitive histopathologic diagnosis of GME, NLE, or NME is often not clinically feasible and biopsy is, in many cases, contraindicated due to the severity of the patient's clinical signs. However, CSF is routinely collected for clinical diagnosis and thus a more available substrate for research in client owned animals.Previous work has focused on the examination of brain tissue in the quest for putative infectious agents. In a study of formalin‐fixed paraffin‐embedded (FFPE) brain tissue, PCR did not identify the presence of specific DNA viruses in 22 dogs diagnosed with NME, NLE, or GME.Astroviruses have generally been associated with clinical signs of gastroenteritis and less commonly respiratory disease.Bornavirus disease is associated with non‐suppurative encephalomyelitis in horses and sheep in endemic areas of central Europe.Several limitations apply to this work. Only a small number of cases were evaluated. The use of 20 clinically diagnosed MUE cases only allows to state with 95% confidence that the prevalence of PCR‐detectable astrovirus in CSF of clinically diagnosed MUE cases is less than 10%.In conclusion, no evidence of astrovirus or bornavirus RNA was identified in the CSF of dogs with MUE. This suggests that neither astrovirus nor bornavirus was present in the CSF but could have been present in the CNS parenchyma of dogs clinically diagnosed with MUE, that the organisms were present in lower quantities or were not detectable by the PCR assays, that the infectious agent may have been cleared by the time of testing, or that they are not involved in the etiology of clinically diagnosed MUE. Further investigation of astroviruses and bornaviruses in brain samples collected via biopsy or at the time of necropsy should be considered.Authors declare no conflict of interest.Authors declare no off‐label use of antimicrobials.Approved by the University of Florida's IACUC; IACUC Study #201609667. Approval by the College of Veterinary Medicine's Veterinary Hospital Research Review Committee (VHRRC) and a client consent form were not necessary as this is a tissue‐only study.Authors declare human ethics approval was not needed for this study.


The aim of this study was to evaluate the clinical outcome of autotransplantation of mature third molars to fresh molar extraction sockets using 3D replicas.Ten patients underwent teeth autotransplantation with or without GBR. We observed the mobility, percussion, radiography examination, the probing depth and the masticatory function of the transplanted teeth during 2 years following up, which were transplanted into fresh molar sockets by using 3D replicas, and GBR when it is necessary.The average extra-oral time of donor tooth had been shortened to 1.65 min when used the 3D replica. Some probing depth of the transplanted tooth were deeper than 3 mm at 4 or 5 weeks temporarily. And one patient felt slight sensitive when chewing with soft food at 4 weeks, then disappeared. The clinical examination of the autotransplantation teeth during 1 year follow-up showed no sign of failure.The tooth autotransplantation using 3D replica with or without GBR is an effective method which can reduce the extra-oral time of the donor teeth and may result in less failure. The tooth autotransplantation is a predictable method to replace a tooth that needs to be extracted due to caries, trauma, or tooth fracture. Since it was first introduced by Fauchard in his book, Le Chirurgien Dentiste, in 1728, the clinical protocol had been developed for hundreds of years . Its briMany previous studies have demonstrated that third molars, premolars, impacted teeth and supernumerary teeth can be a donor tooth in the clinical practice –8. The iResearchers are constantly exploring how to shorten the extraoral time of donor tooth, reduce the damage to the periodontal tissue, and improve the surgeon’s skill. There are many methods attempting to reduce the extraoral time of donor tooth . With thThe bone defect between the prepared socket and the donor tooth is inevitable when transplantation was done in a larger fresh extraction socket. And the bone graft materials were need to fill the bone defect. Yu et al. have autotransplanted canine combined with guided bone regeneration, which show an acceptable result during 7.1 years following up . The tecTherefore, the purpose of this study is to evaluate the clinical outcomes of the transplantation of third molar to fresh first or second molar extraction sockets by using the 3D replica of donor teeth and grafting with autogenous bone to fill the gap between the tooth and the prepared socket when necessary.This was a retrospective observational study of autotransplantation of third molars into fresh first or second molar extraction sockets simultaneously using a 3D replica and grafting with autogenous bone mixed with concentrated growth factors (CGFs) in 10 patients  (ages ranging from 19 to 42 years) between September 2016 and August 2017. All the patients were consecutively collected from Department of Oral and Maxillofacial Surgery, Affiliated Stomatological Hospital of Fujian Medical University. All patients were informed about the surgical treatment procedure. The study design was performed in accordance with the Helsinki Declaration (revised in 2008).First or second molar need to be extracted.Third molar with mature root need to be extracted.Recipient site without local acute inflammatory.The rest bone height of the recipient site is enough for the donor tooth .Systemic diseases such as diabetes mellitus and hypertension, which is not suit for oral surgery, were absent.The patients included in this study reach the following criteria:All patients underwent a low-dose CBCT imaging using the NewTom GiANO  with voxel size 0.150 mm, tube voltage of 90 kV, current of 7.00 mA, and exposure time of 9 s. All patients received a cone-beam computed tomography (CBCT) examination to analysis the compromised tooth and the donor tooth (the stage of the root development and the shape of the root), then the mesio-distal, bucco-lingual dimension, height of the donor tooth and the recipient site were measured to evaluate the adaptability of the donor tooth to the fresh extraction socket as it showed in Fig. After comparing the root of the donor tooth with the extracted tooth, we can know that whether the extraction socket need to be prepared or GBR it needed to filled the gap between the donor tooth and the extraction socket. For patients with horizontal alveolar bone or buccal bone loss at the recipient site, GBR would also need performed. And if GBR is needed, the concentrated growth factors (CGFs) would be prepared before surgery. Whole blood drew from the patient was centrifuged using a tabletop centrifuge  and it was divided into four layers as described by Bozkurt et al. The CGF layer, which was the second growth factor and stem cell layer of the four layers, was separated using sterile scissor.The CBCT data was imported into Materialize Proplan software, this allowed the segmentation of the donor tooth as it show in Fig. All the surgical proceduces were performed by the same surgeon, who had more than 20 years of experience in oral surgery. Block anesthesia of the inferior alveolar nerve was performed when the donor teeth and the recipient site were in the mandibular; local anesthesia was performed when the donor teeth or the recipient site were in the maxillary. Local anesthesia was achieved with articaine chlorhydrate 4% and adrenaline 1:100000.A crevicular incision was made from second premolar to third molar, and the vertical releasing incision in distal side was made if necessary. The compromised molar was extracted by minimally invasive maneuver, using high-speed fissure bur (SINOL) and a dental elevator or forceps (Stoma). The preparation of recipient site was done by piezosurgery according the root shape of the 3D replica of the donor teeth, which was sterilized by ethylene oxide before surgery. For the patients who need performed GBR, which was derived from the CBCT analysis before surgery, the bone fragment was collected during the preparation process. The impacted tooth was also extracted by minimally invasive technique, using a dental elevator or forceps (Stoma). We put the donor teeth into the recipient socket and checked whether it achieve an optimal fit. If there was bone defect around the donor tooth or the fresh extraction socket was larger than the root of the donor tooth, we grafted the autogenous bone to fill the gaps or the bone defects. Then the bone graft area was covered by CGF membrane which was done before surgery. Finally, the flap was repositioned and sutured. The transplanted teeth were stabilized with splints attached to the adjacent teeth which were carried out with a multi-layer fiber-glass band. The brief surgical procedure of the tooth autotransplantation was showed in Fig. After surgery, all the patients received mouth rinsing for 1 week. After 1 week, the sutures were removed and the wound was cleaned by normal saline. The preparation of the root canal was performed 2 weeks after surgery and the filling of the root canal was done 5 weeks after surgery. The multi-layer fiber-glass band was removed 5 weeks after surgery.Follow-up recalls were scheduled for1, 2, 4 weeks and for 3, 6,12, 24 months. At each time of the follow-up the mobility and percussion were checked, while the probing depth of the mesial-buccal, buccal, distal-buccal, mesial lingual, lingual, and distal-lingual of the transplantated teeth and the masticatory function were checked 1, 3, 6, 12 and 24 months after surgery. The radiography examination was taken before surgery and immediate, 1, 3, 12, 24 months after surgery. We defined the normal masticatory function as patients’ ability to chew normal food without pain or discomfort. The primary success criteria of the transplanted tooth were followed according to the described by Tsukiboshi . In termRetrospective, we evaluated 10 patients  who underwent transplantation of their third molar to their fresh first or second molar extraction socket, using a 3D replica. Transplantation of the mature impacted tooth to compromised molar which was in the same region occurred in six cases, the other were in different region. The basic information of the patients is recorded Table The CBCT measurements and the Materialize Proplan software allowed the surgeon to evaluate which mature impacted tooth is best donor tooth to the recipient extraction socket beforehand according to the mesio-distal and bucco-lingual dimension and height of the donor tooth and the recipient site and the number and shape of the root, and whether the GBR is needed. The site of the donor tooth, recipient site, reason for extraction, and whether guided bone regeneration (GBR) was performed are also showed in Table The 3D replica model was used as a guide for preparing the fresh extraction molar socket. When the 3D replica was best fit for the prepared socket, we started to extract the donor tooth. The extraoral time of each case were showed in Table All the patients met the criteria of the success as we enumerated the points previously, and no periodontal pocket, mobility, inflammation and absorption of the root were found. No mobility was found in any cases during the follow-up period and only one patient felt slight pain from percussion of the transplanted tooth at 4 weeks. In addition, only one patient felt slight sensitivity when chewing soft food at 4 weeks. In terms of probing depth, three patients’ probing depth was deeper than 3 mm, and one patient’s probing depth was deeper than 4 mm at 4 weeks, all the probing sites were distal-buccal/lingual. Meanwhile, the probing depth at the distal-buccal/lingual site was deeper than 3 mm in one patient at 3-months follow-up whose probing depth was deeper than 4 mm at 4 weeks. The probing depths in other transplanted teeth were normal at all follow-ups. The specific data about the related clinical symptoms appear in Table In our retrospective study, the tooth autotransplantation, using 3D replica with or without GBR, was an efficient method with a 100% success rate during two-years follow-up, according the success criteria previously mentioned. As EzEldeen reported that the CBCT-guided tooth autotransplantation could be adopted as an alternative for the conventional approach with the help of 3D analysis . VerweijIn the present study, we used a 3D model of donor tooth as a replica to prepared the recipient socket in order to preserve the periodontal ligament of the donor tooth. Firstly, the 3D replica of donor tooth can replace the real one to determine whether the recipient socket is ideally suited for the donor tooth which process would damage the periodontal ligament seriously. Second, the use of the 3D replica of donor tooth shorten the extraoral time to 0–4 min in our surgery. Meanwhile the use of minimally invasive technique can reduce the damage of the periodontal ligament during the extraction of the donor tooth. Andreasen et al. reported that the normal periodontal healing would proceed if the extraoral time of the donor tooth was less than 18 min . The extIn our cases, there are two cases cost 3.5 and 4 min due to the error range of the 3D replica, that is the inaccuracy of the model. The accuracy of the 3D replica model is important to the process of the surgery. The accuracy of the 3D replica model also effected the fitness of the donor teeth to the recipient socket. Many factors may affect the accuracy of the replica model, such as the data from the CBCT, the material shrinkage during the building or postcuring and the minimal thickness of the layers . So far The use of 3D replica model of donor tooth can not only reduce the damage to the periodontal ligament but also increase the ease of the surgery of the tooth autotransplantation and decrease the requirement for the experience of the surgeon. Verweij et al. demonstrated that the surgery time of the autotransplantation when using replica model can be shorten to less than 30 min even if the surgery was done by a less experienced surgeon . ShahbazMany other factors affect the success of the autotransplantation tooth. Yoshino et al. analyzed the influence of age on the tooth autotransplantation and found that the younger the patient is, the higher success rate of the tooth autotransplantation, the success rate was lower in the 55–69 years old group . Sugai eThe third molars extracted for autotransplantation in all cases in the present study were mature teeth with developed roots, so the revascularization of the pulp is not likely to happen after transplantation and needed root canal therapy , 34. SomThe success rate of the autotransplantation, using 3D replica, is high, but the long-term survival rate still need to be observed, and the precise of the tooth autotransplantation need not only a 3D replica as a guide but also a preparation guide of the recipient site and a guide for occlusion, all of which still need more research.The tooth autotransplantation using 3D replica with or without GBR is an effective method which can reduce the extra-oral time of the donor teeth and may result in less failure. And the new 3D replica of donor tooth can make the surgery of tooth autotransplantation much easier and faster.


In an effort to improve the accuracy of an AD diagnosis, it is important to be able to distinguish between AD and other types of dementia (non-AD). The concentration ratio of Aβ42 to Aβ40 (Aβ42/40 Ratio) has been suggested to be superior to the concentration of Aβ42 alone when identifying patients with AD. This article reviews the available evidence on the use of the CSF Aβ42/40 ratio in the diagnosis of AD. Based on the body of evidence presented herein, it is the conclusion of the current working group that the CSF Aβ42/40 ratio, rather than the absolute value of CSF Aβ42, should be used when analysing CSF AD biomarkers to improve the percentage of appropriately diagnosed patients.The cerebrospinal fluid (CSF) biochemical markers (biomarkers) Amyloidβ 42 (Aβ The clinical manifestation of AD is generally preceded by a relatively symptom-free preclinical phase . After tiagnosis , 7. Impoiagnosis –10.Depending on their age, approximately 30–50% of patients with MCI will develop ADD within 5 years . Therefo40, has been reported to be unaltered in AD, the concentration ratio of Aβ42 to Aβ40 (Aβ42/40 ratio) has been suggested to be superior to the concentration of Aβ42 alone in discriminating patients with AD [42/40 ratio in AD diagnosis.In an effort to improve the accuracy of an AD diagnosis, it is important to be able to distinguish between AD and other types of dementia (non-AD) that are not characterized by amyloid pathology. Although the concentration of another amyloid peptide species, Aβ with AD , 13. To 42/40 ratio in the (i) differential diagnosis of AD dementia vs non-AD dementias and (ii) prediction of subsequent development of AD dementia in cases with MCI, and to discuss its value in comparison to other CSF biomarkers and compared to other diagnostic modalities, such as Aβ positron emission tomography (Aβ-PET). In addition, the effects of non-AD pathologies and pre-analytical handling on the various CSF biomarkers were also discussed. In order to achieve these goals, a working group was brought together to critically evaluate the evidence for use of the CSF Aβ42/40 ratio in the diagnosis of AD and a consensus paper was drafted reviewing the advantages and disadvantages surrounding the use of the CSF Aβ42/40 ratio.Thus, the aim of the current article was to review the available evidence on the use of the CSF Aβ42/40 ratio to detect Aβ pathology, which is an approach to normalize the Aβ42 CSF concentration for the total Aβ CSF concentration . In contrast, this paper does not address approaches to interpret the overall pattern of the AD CSF biomarkers that combine results derived from the two distinct AD pathologies (amyloidosis and neurodegeneration) to form any kind of ‘ratios’.This review addresses the advantages and disadvantages of the Aβ42/40’ and ‘Aβ40’ and excluding those, whose primary scope was not the role of the Aβ42/40 ratio in AD diagnostics. The results of the discussions and the evidence gathered during the meetings are presented in this paper.A group of experts in the field of AD biomarkers were brought together, and several meetings of this working group were conducted. During the meetings, discussions took place based both on evidence gathered from scientific publications and the experience of the group members, as experts in the field. Studies were selected to be included in this paper based on an independent review by at least two  co-authors of the report, with MEDLINE database as the primary source of the studies. Searches were conducted using keywords, such as ‘Aβ42/40 ratio to the existing panel of biomarkers could improve the accuracy of the differential diagnosis of AD from other dementia disorders. Here we provide details of 16 studies that have compared the diagnostic accuracy of CSF biomarkers to diagnose ADD versus non-ADDs. These studies demonstrate the usefulness of the CSF Aβ42/40 ratio for the diagnosis of AD in patients with dementia. Studies with relevant data are also summarized in Table Due to similar clinical manifestations and overlapping brain pathologies, differentiation of AD from other neurodegenerative disorders may prove difficult even with the aid of biomarkers. For example, the symptoms and biomarker patterns observed in patients with dementia with Lewy bodies (DLB) or subcortical vascular dementia (VaD) sometimes closely resemble those of AD, which makes differential diagnosis difficult and decreases the diagnostic accuracy of the core CSF AD biomarkers, especially in the early stages of the disease. Therefore, evidence was gathered on whether adding the CSF Aβp <  0.001), but Aβ40 levels did not show any significant differences between the groups. The reduction of Aβ42 levels in AD also resulted in a significant increase in the Aβ40/42 ratio  as an improved marker. The authors therefore concluded that the Aβ ratio is another important marker for AD.In a study of patients with ADD, normal controls, patients with non-ADD and patients with other neurological diseases, Shoji et al.  found th42, Aβ40 and total tau (T-tau) in order to compare their accuracy in discriminating patients with ADD, non-ADD and control subjects. The results showed that concentrations of Aβ42 were decreased (p <  0.001) and of T-tau were increased (p <  0.001) in ADD patients, while Aβ40 concentrations did not differ significantly among the groups. For all groups when the Aβ42/40 ratio was used, more patients were classified correctly, compared to when the Aβ42 concentration alone was used . The improvement of the diagnostic accuracy reported in this study was not significant, probably due to the small numbers of subjects and a clear ceiling effect .Lewczuk et al.  measured40/42 and Aβ38/42 ratios were significantly altered in AD. They also found that the Aβ40/38 ratio was the only one that differentiated clearly control subjects from FTD subjects, while not being significant between AD and FTD. In the ROC curves, they found that for FTD versus AD diagnosis, the best AUCs for amyloid biomarkers were the Aβ38/42 ratio and the Innogenetics Aβ/Tau index (IATI) (AUCs = 0.87). However, the Aβ40/42 ratio or Aβ42 alone had very close and statistically undifferentiated AUC values. The authors concluded that the Aβ38/42, Aβ40/42 and the IATI ratios were also better than individual biomarkers to identify AD therefore justifying their clinical relevance.Gabelle et al.  evaluate42 concentrations might not only result from AD pathology but may also be related to total Aβ peptide concentrations. In such cases, healthy individuals with relatively low total Aβ might be misdiagnosed as having ‘pathologically low’ Aβ42 concentrations, and vice versa, AD subjects with high total Aβ might be misinterpreted as normal Aβ42 carriers. It was therefore concluded that the analysis of CSF Aβ42 alone (i.e. without Aβ40) might lead to misinterpretation of the neurochemical dementia diagnostics outcome in subjects with constitutively high or low concentrations of total Aβ peptides. Consequently, the authors conclude that the CSF Aβ42/40 ratio can possibly improve the reliability of the neurochemical dementia diagnosis.In another study carried out by Wiltfang et al. , the aut42/40 ratio to the existing panel of biomarkers, Aβ42, Aβ40 and tau phosphorylated at threonine 181 (P-tau181) was compared to the panel without the addition of the ratio. The results showed that the CSF Aβ42/40 ratio was significantly decreased in AD patients (0.043 ± 0.021) compared to non-AD patients  and controls . Following receiver operating characteristic (ROC) analysis, the optimal cut-offs discriminating the groups were defined as the values maximising the sum of the sensitivity and the specificity. Although the difference between the areas under the ROC curves (AUC) for Aβ42 and Aβ42/40 turned out to be insignificant, the diagnostic accuracy of the decision tree that contained Aβ42, Aβ40, P-tau181 and the Aβ42/40 ratio was significantly better than the diagnostic accuracy of the decision tree without Aβ40 and the Aβ42/40 ratio . The authors concluded that there was no difference in CSF Aβ40 levels found between AD and non-AD patients, but that adding CSF Aβ40 and the CSF Aβ42/40 ratio to a biomarker-based decision tree, might have an added value for discriminating AD from non-AD patients in cases with intermediate CSF P-tau181 values.A study by Slaets et al.  compared42/40 ratio could be used for differentiating AD from DLB and Parkinson’s Disease Dementia (PDD). The primary finding of this study was that the CSF Aβ42/40 ratio increased discrimination of AD from PDD and DLB compared with either of the two Aβ biomarkers individually. Of note, in this study, Aβ40 was significantly higher in AD. Furthermore, the authors concluded that use of the Aβ42/40 ratio could improve the differentiation of AD from PDD and DLB.Nutu et al.  evaluate40), along with the core CSF markers T-tau, P-tau181 and Aβ42, in the discrimination between two large dementia groups of FTD (n = 107), AD (n = 107) and non-demented subjects (n = 33) was evaluated. The authors found that their data ‘taken together’ indicated that ‘although CSF Aβ40 has no added value in the distinction between AD and FTD patients, it might be useful in the discrimination between AD and FTD patients from non-demented controls, and therefore could be considered in patients diagnostic work-up’.In a study by Baldeiras et al. , the add42/40 ratio would reduce the frequency of indeterminate CSF profiles. They found that, on the basis of local optimum cut-offs for Aβ42 and P-tau181, 22% of patients had indeterminate CSF profiles. The systematic use of the Aβ42/40 ratio instead of Aβ42 levels alone decreased the number of indeterminate profiles , but it failed to improve the classification of subjects . In contrast, use of the Aβ42/40 ratio instead of Aβ42 levels alone in patients with a discrepancy between P-tau181 and Aβ42 led to a reduction by half of the number of indeterminate profiles  and was also in agreement with clinician diagnosis . The authors therefore concluded that in patients with a discrepancy between CSF P-tau181 and CSF Aβ42, the assessment of the Aβ42/40 ratio led to a reliable biological conclusion in over 50% of cases that agreed with a clinician’s diagnosis.In a prospective study of subjects with cognitive disorders at three French memory centres , Dumurgier et al.  assessed42/40 ratio could be used to improve the accuracy of diagnostically relevant conclusions in patients with ambiguous CSF Aβ42 or tau results. They found that one third of the biomarker profiles of patients with atypical dementia were ambiguous. The addition of the CSF Aβ42/40 ratio increased the proportion of interpretable profiles from 69 to 87%, without changing the conclusion when the usual biomarkers (Aβ42 and P-tau181) were concordant. The authors therefore concluded that their results support the use of the Aβ42/40 ratio in addition to the usual CSF AD biomarkers for patients with ambiguous profiles. They added that this method could be specifically directed to this population (i.e. patients with ambiguous results) in order to improve the level of certainty for clinical routine practice.Sauvee et al.  investig42/40 ratio and CSF Aβ42 alone. Analysis of Aβ42 gave a sensitivity and specificity of 69.3% and 88.9%, respectively, whereas the Aβ42/40 ratio showed significantly improved performance with sensitivity and specificity of 93.3% and 100%, respectively. Thus, the authors concluded that the CSF Aβ42/40 ratio concentration shows significantly better diagnostic performance compared to the CSF Aβ42 concentration alone. It should be noted, however, that this study must not be interpreted as providing absolute values of the diagnostic accuracies, but their relative comparison.Lewczuk et al.  also com42/40 ratio under the assumption that Aβ40 closely represents the total cerebral Aβ load. They found that the Aβ42/40 ratio improves differentiation of AD patients from VaD, DLB and non-ADD patients when compared to Aβ42 alone. Furthermore, they found that the Aβ42/40 ratio is a more easily interpretable alternative to the combination of Aβ42, P-tau181 and T-tau when differentiating AD from either frontotemporal dementia (FTD) or other non-ADDs. Since they found different Aβ40 concentrations in the various dementia groups, the authors also added that it can be debated if the Aβ42/40 ratio is a good representation of the Aβ42 fraction of the total Aβ load and thus eliminates inter-individual differences in total Aβ concentrations.In another study including various CSF biomarkers, Spies et al.  investig42/40 ratio improved the diagnostic performance of Aβ42 in most differential diagnostic situations. Struyfs et al. [42/40 ratio was the best biomarker to distinguish between AD-MCI and FTD.A study of patients with AAD, non-ADDs , MCI due to AD and non-demented controls found that the CSF Aβs et al.  also fou42/40 ratio, as well as the CSF Aβ42/38 ratio, was ‘superior biomarkers of AD pathology compared with Aβ42 alone’. Using three commercially available CSF biomarker immunoassays, this study found that the CSF Aβ42/40 and Aβ42/38 ratios improved differentiation of AD from non-ADs, especially when separating AD from DLB/PDD and VaD.Similarly, Janelidze et al.  also fou42/40 and Aβ42/38 ratios instead of Aβ42. They suggest that it might be that subcortical pathologies not specific to AD, such as WMLs and alpha-synuclein pathology, cause reduced levels of all CSF Aβ species, including Aβ42. A second explanation for the improved diagnostic accuracy of the Aβ42/40 and Aβ42/38 ratios could be that differences in the overall production and clearance of Aβ probably contribute to inter-individual variability in total CSF Aβ levels. This is supported by the present finding that in CSF Aβ42 correlates with Aβ38 and Aβ40 even in healthy controls. Consequently, when detecting Aβ42 brain pathology with CSF Aβ42, using ratios to Aβ40 or Aβ38 might correct for inter-individual differences in total Aβ levels.The authors point to several potential explanations for the improved accuracy when using the CSF Aβ42/40 ratio was found to aid diagnosis. The study by Bousiges et al. [42/40 ratio remained unchanged in DLB patients between the prodromal and demented stages, contrary to what was observed in AD. The Aβ42/40 ratio therefore makes it possible to distinguish between the two pathologies ‘at a time when the differential diagnosis is difficult’.In another study, which evaluated the differential diagnosis of DLB and AD, once again, the CSF Aβs et al.  found th42/40 ratio was added to a previously described PLM scale (Paris-Lille-Montpellier study), which combines Aβ42, T-tau and P-tau181 biomarkers, in order to evaluate an optimized PLMR scale . Nine hundred and four participants (342 AD and 562 non-AD) were studied, in two chronologically different cohorts (400 Mtp-1 and 504 Mtp-2). For AD patients, the mean CSF Aβ42 and CSF Aβ42/40 ratio was 553 ± 216 pg/mL and 0.069 ± 0.022 pg/mL in Mtp-1 and 702 ± 335 pg/mL and 0.045 ± 0.020 pg/mL in Mtp-2. The distribution of AD and non-AD differed between the PLM and the PLMR scales (p < 0.0001). The percentage AD well-classified (class 3) increased with PLMR from 38 to 83% in Mpt-1 and from 33 to 53% in Mpt-2. A sharp reduction of the discordant profiles going from 34 to 16.3% and from 37.5 to 19.8%, for Mtp-1 and Mtp-2 respectively, was also observed. The authors concluded that the integration of the Aβ42/40 ratio in the PLMR scale resulted in an easy-to-use tool which reduced the discrepancies in biologically doubtful cases and increased the confidence in the diagnosis.Finally, in a study by Lehmann et al. , the Aβ442 alone and that become correctly classified with the Aβ42/40 ratio. Assuming normal distribution of Aβ40 across the population of interest [40  leads to misdiagnoses of ca. 5–10% of cases. This is further confirmed by Baldeiras et al. [40 concentrations corrected diagnosis in AD patients with non-pathological CSF Aβ42 levels in 76.2% of cases using the CSF Aβ42/40 ratio.In order to try to assess what is the overall impact on diagnosis, an estimation was made of what the actual percentage of patients that are misdiagnosed by Aβinterest , a very s et al. , who fous et al.  report t42/40 ratio for the diagnosis of AD in patients with dementia. The CSF Aβ42/40 ratio is also better than CSF Aβ42 alone at distinguishing AD dementia from non-AD dementias, not only from controls. The evidence therefore strongly suggests that the CSF Aβ42/40 ratio, rather than CSF Aβ42 alone, should be used in the clinical work-up of AD, as a way to improve the diagnostic accuracy for distinguishing ADD from other dementia disorders.In summary, the accumulation of evidence clearly points to the usefulness of the CSF Aβ42/40 ratio in accurately predicting progression to ADD. Studies with relevant data are also summarized in Table With disease-modifying therapies on the horizon, there is a need to be able to predict the risk of developing ADD before the onset of dementia, i.e. during the MCI and subjective cognitive decline (SCD) stages. The increased percentage of MCI subjects with pathologic CSF who convert to ADD, compared to those having normal CSF, is considered a very strong argument in favour of the use of CSF biomarkers as predictors of MCI to ADD conversion. Here we provide details of six studies that have compared the diagnostic accuracy of CSF biomarkers when predicting the development of ADD in patients with MCI. All of the studies show the added value of the CSF Aβ42/40 ratio in addition to Aβ42) correlated better with the ratio of pre-dementia subjects having progressed to ADD than the score based on three biomarkers [In a study validating the previously introduced Erlangen Score interpretation algorithm , Lewczukomarkers .40 and Aβ42 in CSF were measured in a cohort of patients with MCI (n = 137) in relation to the final diagnosis after 4–6 years of follow-up. The Aβ42 concentration at baseline and the Aβ42/40 ratio were significantly decreased in MCI patients who developed ADD compared to cognitively stable MCI patients and MCI patients who developed other forms of dementia (p < 0.001). The baseline levels of Aβ40 were similar in all MCI groups but correlated with change in Mini-Mental State Examination scores in converters to ADD. The Aβ42/40 ratio was superior to Aβ42 concentration with regard to identifying incipient AD in MCI (p < 0.05). The authors concluded that the data provides support for the view that amyloid precursor protein metabolism is disturbed in early sporadic AD and points to the usefulness of the Aβ42/40 ratio as a predictive biomarker for AD.In a study by Hansson et al. , baselin42/40 and Aβ42/38 ratios were higher compared to that of Aβ42 alone (p < 0.01) when using the electrochemiluminescence technology of the Meso Scale Discovery (MSD) platform to quantify amyloid in MCI patients. However, Aβ42 quantified with a bead-based multiplexing (xMAP) technology performed as well as the MSD Aβ42/40 ratio.A study by Hertze et al.  investig40, Aβ42, T-tau and P-tau181 in patients with AD, stable MCI (MCI-MCI) and MCI evolving into ADD (MCI-AD) in order to evaluate the power of each biomarker and/or their combination in predicting AD progression. Although they found that inclusion of the Aβ42/40 ratio instead of Aβ42 alone did not improve the prediction power of the model in the multivariate analysis, when univariate statistics were employed, they found that the Aβ42/40 ratio had an increased sensitivity with respect to Aβ42.A 4-year follow-up study carried out by Parnetti et al.  measured42 by the Aβ42/40 ratio resulted in a significant increase in the proportion of interpretable biological profiles  of MCI patients, due to a reduction by half in the number of suspected non-Alzheimer pathophysiology cases and an increase in the proportion of the high-AD-likelihood subgroup. In their study, the risk of progression to ADD was highest in the ‘high-likelihood’ group and increased when the Aβ42/40 ratio, instead of Aβ42, combined with T-tau and P-tau181 was used for biomarker-based categorization.In a recently published paper, Baldeiras et al.  showed t42, Aβ42/40, T-tau and P-tau181) were numerically superior over the performance of a single biomarker index in predicting MCI subjects who progressed to AD. In this dataset, however, the Aβ42/Aβ40 ratio was not consistently superior to Aβ42 alone for predicting AD dementia in MCI patients.Frölich et al.  investig42/40 ratio in accurately predicting progression to ADD.Together these reports provide evidence that clearly demonstrates the added value of the CSF Aβ38 concentration as the reference isomer for normalization of the Aβ42 concentration (in the form of Aβ42/38 ratio). However, it needs to be stressed that neither the results of these studies, nor the physiological rationale behind using Aβ38 instead of Aβ40 is convincing enough to replace Aβ40.In the last years, studies have also been published applying the CSF Aβ42 and Aβ-PET have both been found to correlate highly with brain biopsy findings [42 and increased retention of amyloid tracers in the brain on PET are considered the earliest biomarkers of AD, although some evidence suggests that the alterations measurable in the CSF occur earlier [42 as an early diagnostic marker is the possibility to detect regional Aβ depositions that might occur before the global neocortical signal becomes pathologic. On the other hand, analysis of CSF offers a quantitative result of the net effect of the biomarkers. Running costs of CSF analysis are 10- to 15-fold lower than those for Aβ-PET . CSF analysis is also more accessible to patients, it does not require the patient and caregiver to travel to a distant centre equipped with PET facilities and it enables simultaneous analysis of dozens of biomarkers in one sample volume, including biomarkers of neurodegeneration, neuroinflammation and others [CSF Aβfindings , 36. Dec earlier . A potend others –39. In cFinally, although lumbar puncture is frequently regarded as an invasive procedure, serious complications are extremely rare, with the most common, headache, only being observed in 2% of the elderly population and being easily treated with simple analgesia . On the 42 and Aβ-PET status was observed. In fact, they found that 10–20% of healthy individuals and MCI patients showed a mismatch in CSF Aβ42 and Aβ-PET status. In their study, they found that the CSF Aβ42/40 and Aβ42/38 ratios better predict abnormal cortical amyloid deposition  compared with Aβ42. The ratios increased the classification performance both for patients who were falsely classified as positive (by low CSF Aβ42) and for patients who were falsely classified as negative (by high CSF Aβ42).In Cohort 1 of a study carried out by Janelidze et al. , which i42/40 and Aβ42/38 ratios instead of Aβ42, was that subcortical pathologies not specific to AD could cause reduced levels of all CSF Aβ species, including Aβ42, but not the ratios. For example, in patients with MCI, low CSF Aβ42, Aβ40 and Aβ38 were all linked to subcortical injury, including increased white matter lesions (WML) and enlarged lateral ventricles. The mechanisms underlying these associations are likely related to dysregulation in β amyloid precursor protein pathways with a general decline in the production of Aβ.One possible explanation proposed by the authors for the improved concordance between Aβ-PET and CSF Aβ, when using the Aβ42/40 ratio corresponded better than Aβ42 with PET results, with a larger proportion of concordant cases  and a larger area under the ROC curve  associated with the ratio. For both CSF biomarkers, the percentage of CSF-abnormal/PET-normal cases was larger than that of CSF-normal/PET abnormal cases. The authors concluded that the CSF Aβ42/40 ratio is superior to Aβ42 alone as a marker of amyloid-positivity by PET, which may be explained by the fact that the ratio compensates for general between-individual variations in CSF total Aβ concentrations. Furthermore, they speculated, that the fact that there was a higher proportion of subjects with pathologic CSF (as reflected by Aβ42 or Aβ42/40) and normal PET compared to those with pathologic PET and normal CSF might suggest that CSF reflects amyloidosis earlier than PET does. Similar conclusions based on earlier findings in CSF compared to PET were also made by Mattsson et al. and Palmqvist et al. [A study by Lewczuk et al.  reportedt et al. .42 levels measured using five different immunoassays [Innotest, Modified Innotest , fully automated Lumipulse, Euroimmun and MSD assays) and visual read of Aβ-PET images in 262 patients with MCI or SCD. They found that although the accuracy to correlate with visual 18F-flutemetamol PET was decreased when using newer Aβ42 assays, this limitation is overcome by using the CSF Aβ42/40 ratio. They also found that the CSF Aβ42/40 ratio from the newer assays showed improved accuracy for detection of cortical Aβ fibrils as measured by PET. Moreover, the sensitivities and specificities of these newer assays were less influenced by moderate changes in the cut-offs when the CSF Aβ42/40 ratio was used, a finding that is important when samples will be analysed consecutively over time.Janelidze et al.  studied 42, Leuzy et al. [42/40 ratio.In another study analysing the agreement between data on cerebral amyloidosis, derived using Pittsburgh compound B (PiB) PET and immunosorbent assay of CSF Aβy et al.  examined42, P-tau181/Aβ42 and Aβ42/40) best discriminated PiB-positive from PiB-negative individuals.A study by Schindler et al.  studied 42/40 ratio can better predict abnormal cortical amyloid deposition  compared with Aβ42. This then leads to fewer patients being diagnosed as false positive (low CSF Aβ42) or false negative (high CSF Aβ42).These reports show that both Aβ-PET and CSF biomarkers can identify early AD with high accuracy. However, several studies strongly suggest that Aβ alterations in the CSF occur earlier , 43. SimIn clinical practice, CSF biomarker analyses are often carried out in patients with atypical or mixed presentation of dementia. This makes the diagnosis complex and highlights the importance of being able to discriminate AD from other neurodegenerative processes such as FTD, WMLs, VaD, DLB, PDD and cerebral amyloid angiopathy .38, Aβ40 and Aβ42 were inversely correlated with chronic WML volume , but not with acute WML or infarct volumes.Selnes et al.  studied 42/40 ratio and Aβ-PET were not. These results indicate that the presence of WMLs affects the levels of CSF Aβ38, Aβ40 and Aβ42, but not the CSF Aβ42/40 ratio or Aβ-PET.Similarly, van Westen et al.  studied 40 levels were decreased in FTD suggesting that it could represent a diagnostic biomarker in this pathology.Finally, Gabelle et al.  observed42, as well as Aβ38 and Aβ40, might be reduced in CSF due to non-AD related pathologies, but usually, the Aβ42/40 ratio, as well as other Aβ ratios, including Aβ42, are not affected. These studies therefore point to the usefulness of the CSF Aβ42/40 ratio in improving the accuracy of the differential diagnosis in patients with ambiguous biological profiles or with other conditions that might affect the concentrations of the Aβ peptides.It can therefore be concluded that Aβ42 peptide in CSF [Various papers have pointed out pre-analytical and analytical variability between laboratories for the concentration of the Aβe in CSF –54. Tubee in CSF , 56. Thee in CSF .42 and Aβ40 onto the sampling tube surface, regardless of the type of plastic, however with significant differences across different type of plastics [42/40 ratio would provide a complete interpretation of CSF amyloid biomarker results, integrating the impact of plastic tube type.The type of sampling and storage tubes used is an important source of variability because of the tendency of Aβ peptides to adsorb on plastic surfaces , 54, 56.plastics . They al181, Aβ42, Aβ40) in CSF samples in collection tubes made of different materials. The results suggest that the CSF Aβ42/40 ratio and CSF P-tau181 are much less prone to methodologic error introduced by interactions of the biomarkers’ molecules with the test tube surfaces compared with Aβ40 and Aβ42 concentrations, similarly to the concentrations of T-tau. The authors concluded that the CSF Aβ42/40 ratio is a more reliable biomarker than pure Aβ concentrations, as the Aβ42/40 ratio is less altered by interaction with the surface of the collection tubes.Lewczuk et al.  measured42 and Aβ40 concentrations was investigated. CSF from 35 patients was collected in different polypropylene (PP) tubes and stored at − 80 °C. Samples were also subjected to three successive freeze-thaw cycles. The results showed that CSF Aβ42 and Aβ40 values were significantly different depending on the type of collection tube and the number of freeze/thaw cycles. Although the calculation of the CSF Aβ42/40 ratio eliminated the effect of PP tube-dependent variation, this was not the case for freeze-thaw cycle-associated variation. The authors concluded that the use of Aβ42/40 ratio rather than Aβ42 alone could contribute toward pre-analytical standardization, thus allowing for the general use of CSF AD biomarkers in routine practice.In a similar study by Gervaise-Henry et al. , the imp42 concentrations, incubation times, pipettes, vortex intensities and other CSF proteins. They found that every sample transfer from one tube to another resulted in 5% loss of Aβ42 concentration, which reached as high as 10% when small volumes were used. This decrease in concentration was, however, similar for Aβ40, resulting in stable Aβ42/40 ratios over multiple tube transfers. Correspondingly, they conclude that use of the Aβ42/40 ratio overcomes the effect of adsorption-derived Aβ concentration loss and can therefore contribute to increased diagnostic accuracy.Willemse et al.  tested s42/40 ratio mitigates many of the effects of additional freeze/thaw cycles, tube type and CSF volumes for PP storage tubes. In fact, they concluded that the CSF Aβ42/40 ratio is clearly a more robust biomarker than Aβ42 toward (pre-) analytical interfering factors. They also observed that the rate of adsorption to PP recipients is higher for Aβ42 than for the other amyloid isoforms, such as Aβ40, and therefore using the Aβ42/40 ratio does not completely eliminate the effects of binding of Aβ to the tube walls of PP tubes. However, they found that ‘low binding recipients’ are able to reduce the binding of Aβ species to the tube walls. They concluded that integration of the Aβ42/40 ratio and ‘low-binding tubes’ into guidance criteria may speed up worldwide standardization of CSF biomarker analysis.Furthermore, Vanderstichele et al.  determin42/40 ratio would improve the interpretation of CSF amyloid biomarker results, by reducing the impact of these factors on the final outcome. The use of the CSF Aβ42/40 ratio could therefore contribute toward pre-analytical standardization, allowing for the use of CSF AD biomarkers in routine clinical practice.In summary, evidence from studies on the effect of pre-analytical handling on biomarkers of AD suggest that use of the CSF Aβ42/40 ratio is economical and not interpretational in nature. Considering the laboratory costs of the AD biomarkers, the inclusion of Aβ40 increases the costs by ca. €40–50, which is negligible when considering the total costs of the diagnostic work-up and treatment of patients with suspected AD assessed at specialized memory clinics. Furthermore, the amount of CSF sample needed to perform this additional test is also negligible . In addition, interpretation of the results when four biomarkers (instead of three) are used is not more complex, when a solid interpretational algorithm is validated and consequently used.The main disadvantage of the use of the CSF Aβ42/40 ratio compared to CSF Aβ42 alone. In addition, it also appears to be clear that including the CSF Aβ42/40 ratio in the clinical workup of MCI patients improves the accuracy of predicting progression to AD.There is a growing body of evidence that suggests the better diagnostic performance of the CSF Aβ42/40 ratio can also better predict abnormal cortical amyloid deposition  compared with CSF Aβ42. This then leads to fewer patients being diagnosed as false positive (low CSF Aβ42) or false negative (high CSF Aβ42). In addition, there is evidence to suggest that Aβ alterations in CSF occur earlier than they are detectable in Aβ-PET.It has also been shown that both Aβ-PET and CSF biomarkers can identify early amyloid pathology with high accuracy. The CSF Aβ42/40 ratio to the usual panel of CSF AD biomarkers for patients with ambiguous biological profiles also increases the number of interpretable results.Addition of the CSF Aβ42/40 ratio rather than CSF Aβ42 alone contributes toward pre-analytical standardization, removing the effects of (pre-)analytical interfering factors, such as tube type, freeze/thaw cycles and CSF volumes.It has also been found that the use of the CSF Aβ42/40 ratio should always be analysed, irrespective of the results of other AD biomarkers, and without paying attention to whether Aβ42 is normal or pathologic. This is driven by the fact that the Aβ42/40 ratio can equally change the CSF interpretation from ‘normal to pathologic’ as from ‘pathologic to normal’. Analysing the Aβ42/40 ratio only in cases with abnormal Aβ42  would neglect the former scenario. On the other hand, performing Aβ42/40 ratio analysis only in cases with normal Aβ42  would neglect the latter case.The Aβ42/40 ratio and to participate in an internal quality control programme to ensure longitudinal stability in the measurements of Aβ42 and Aβ40, just as it is mandatory to establish reference values and control quality of all other biomarkers.It should be mandatory for every laboratory to establish their own specific reference values (cut-offs) for the Aβ42/40 ratio must be carefully validated. It is beyond the scope of this paper to make suggestions on statistical methods to calculate reference values and/or their transfer from other laboratories or analytical platforms.Following on from (2), it is possible to combine laboratory results obtained on different platforms and from different vendors. The point is only that the reference value for the AβThe working group therefore makes the following recommendations:42/40 ratio, rather than the absolute value of CSF Aβ42, should be used when analysing CSF AD biomarkers to improve the percentage of appropriately diagnosed patients. It is also suggested that the CSF Aβ42/40 ratio could therefore be used as a proxy for amyloid status in AD clinical trials and eventually in clinical care settings.Based on the body of evidence collected here it is the conclusion of the current working group that the CSF Aβ


The purpose of the study is to describe oncologists’ perceptions and opinions about patient eligibility, guidelines, and barriers for use of granulocyte colony-stimulating factor (G-CSF), overall and stratified by their affiliation with the Oncology Care Model (OCM). In May 2018, we invited and recruited practicing US oncologists from a national database for an online survey. Level of agreement was identified using a seven-point scale, ranging from strongly disagree to strongly agree. Of 200 participating oncologists, 70 were OCM-affiliated. Overall, 65% of oncologists agreed or strongly agreed that all patients at high risk of febrile neutropenia (FN) should receive prophylactic G-CSF, and half agreed or strongly agreed that benefits of G-CSF outweigh the potential adverse effects. The most common barriers to G-CSF use for patients at high risk of FN included patient refusal (37.1% of OCM-affiliated oncologists vs. 21.5% of non-OCM-affiliated oncologists), not on protocol/not supported by guidelines (32.9% vs. 23.1%), lack of reimbursement to practice (30.0% vs. 15.4%), and concerns about insurance coverage (22.9% vs. 26.9%). More OCM-affiliated oncologists reported that their practices offer and strongly encourage adherence to a specific protocol for G-CSF use (49.2%) versus non-OCM oncologists (31.3%). Despite recommendations from national guidelines and strong evidence from randomized, controlled clinical trials, only two thirds of oncologists agree or strongly agree that all patients at high risk of FN should receive primary G-CSF prophylaxis. Decisions about G-CSF prophylaxis may be affected by factors other than risk of FN, such as patient choice, practice protocols/guidelines, lack of reimbursement, and insurance coverage.The online version of this article (10.1007/s13187-019-01638-8) contains supplementary material, which is available to authorized users. Febrile neutropenia (FN) following myelosuppressive chemotherapy is associated with significant morbidity, mortality, dose delays, and dose reductions –3. The rIn the USA, the real-world use of G-CSF among patients at high risk of FN does not reflect the recommendations of national guidelines. G-CSF prophylaxis is observed in 17 to 74% of patients receiving chemotherapy regimens associated with high risk of FN, suggesting underutilization of an important preventive measure –16. TherThe timing of prophylactic pegfilgrastim, the most commonly used G-CSF, is also an issue. National guidelines recommend that pegfilgrastim should be administered 1 to 4 days or 1 to 3 days after the last dose of chemotherapy and that pegfilgrastim administration on the same day as the last dose of chemotherapy is sometimes justified , 12. HowData are also lacking regarding the influence of oncologists’ practice setting. The use of G-CSF may differ in academic centers versus community clinics or in practices participating versus not participating in the Oncology Care Model (OCM). The OCM is a Medicare performance-based payment model that aims to provide higher-quality, more highly coordinated, more patient-centered oncology care at the same or lower cost , 21.The objective of this survey was to describe the perceptions and opinions of US-based oncologists about G-CSF use in patients receiving myelosuppressive chemotherapy. Specifically, we sought to explore oncologists’ perspectives about G-CSF prophylaxis for FN; barriers to G-CSF use across various treatment settings; patient-level risk factors for FN; and differences in G-CSF use, specifically pegfilgrastim, stratified by affiliation with the OCM.This was a national cross-sectional survey of US oncologists conducted by an independent third party, Adelphi Research. Prior to initiation of the study, the protocol was approved by a centralized, US-based independent review board, Quorum Review (QR#33224/1). After approval, oncologists who currently prescribe G-CSFs were invited and recruited from a national database of 13,800 oncologists hosted by M3 Global Research. Oncologists from Vermont and Maine were excluded because of state prohibitions on compensation for surveys, and oncologists from Massachusetts were excluded due to state requirements for double blinding.The study targeted recruitment of approximately 200 US oncologists. Targets for specific subgroups of interest included approximately 75 oncologists affiliated with an academic cancer center and approximately 50 oncologists affiliated with the OCM. Oncologists who consented completed a screening section prior to the actual survey so that their eligibility could be assessed according to the following criteria: (1) licensed and board-certified in the USA in oncology and/or hematology for at least 3 years, (2) treated at least 40 unique oncology patients with chemotherapy in the past 6 months, (3) treated at least six oncology patients with a G-CSF in the past 6 months, (4) self or family member not affiliated with a marketing, pharmaceutical, or biotechnology company, and (5) proportion of professional time devoted to patient care ≥ 40% if affiliated with an academic center or ≥ 60% if affiliated with a community center.The survey questionnaire was developed using standard survey methodology . Before The finalized version of the questionnaire consisted of 58 questions: 12 screening questions, 4 demographic questions, 6 questions on G-CSF use overall and by type, 5 questions about predictors of G-CSF use, 23 questions about types of G-CSF selected, and 8 questions about future G-CSF prescribing. The survey collected information on primary affiliation , patient-level risk factors, criteria considered to determine a patient’s FN risk level, drivers of and barriers to G-CSF use, and perceptions about G-CSFs. The survey took 30 min to complete . The level of agreement was identified in the questionnaire using a seven-point scale, where responses from 1 to 7 indicated strongly disagree, disagree, somewhat disagree, neither agree nor disagree, somewhat agree, agree, and strongly agree, respectively. To minimize missing data, the electronic questionnaire was designed in such a way that a question had to be completed before the subsequent question was made available to the participant.https://www.qresearchsoftware.com).Continuous variables were summarized as mean, standard deviation, median, and interquartile range, and categorical variables were summarized as number and percentage with binomial 95% confidence interval (CI). Outliers were identified as values beyond 3 standard deviations and excluded from the analyses. The seven-point scale for level of agreement was categorized into three groups: strongly disagree or disagree; somewhat disagree, neither agree nor disagree, or somewhat agree; and agree or strongly agree. To understand the differences between groups, survey data was further stratified by OCM and academic center affiliation. Analyses were conducted using Q Research Software  were hematologist-oncologists and 86 (43.0%) were medical oncologists.n = 130; 65.0%) and community clinics . Detailed characteristics of the participating oncologists stratified by institutional affiliation are presented in Table Overall, oncologists reported practicing for an average of 13 years, and each participating oncologist provided G-CSF support for an average of approximately 80 patients in the past 6 months. The most common practice settings reported by oncologists were group practices (36.0%) and major academic centers (25.0%). The majority of the 200 oncologists were primarily affiliated with non-OCM practices (In response to the question regarding what guidelines they follow for G-CSF use (Q.B1a), most oncologists reported awareness of the NCCN guidelines (70.5%), followed by ASCO guidelines (38%), institution-specific or practice-specific guidelines (17%), American Society of Hematology guidelines (12%), no guidelines (11%), Centers for Disease Control and Prevention guidelines (6%), and other (2%).Almost two thirds of oncologists agreed or strongly agreed with the statement that patients at high risk of FN should receive primary G-CSF prophylaxis. According to practice setting, the figures were 57.3% for academic center, 69.6% for community practice, 60.0% for OCM-affiliated, and 67.7% for non-OCM-affiliated. More OCM oncologists reported that their practices strongly encourage adherence to a specific treatment protocol for G-CSF use  compared with non-OCM oncologists . Additionally, oncologists affiliated with practices where specific treatment protocols are strongly encouraged were more likely to say they use G-CSF for primary prophylaxis for patients in the adjuvant chemotherapy setting  compared with oncologists affiliated with practices that somewhat encourage specific treatment protocols .Participants were asked to select the recommended timing for administering pegfilgrastim prefilled syringe (PFS) from four predefined timeframes. The majority of the oncologists in both the OCM 72.5%) and non-OCM (75.0%) subgroups indicated that pegfilgrastim PFS should be administered between 24 and 48 h following completion of chemotherapy administration. Almost a quarter of oncologists in both the OCM (23.5%) and non-OCM (22.8%) subgroups said that the recommended timing of pegfilgrastim PFS is within 24 h of the last day of chemotherapy administration  to 100 (most important). Based on the mean ratings, the factors considered most important in determining whether a patient is at high risk of developing FN were chemotherapy regimen, preexisting neutropenia or bone marrow involvement with tumor, and prior experience of neutropenia . Additionally, half of oncologists (50.0%) agreed or strongly agreed that the benefits of G-CSF therapy outweigh the potential adverse effects, with no difference observed according to OCM affiliation , not being on protocols set by the practice or guidelines (26.5%), and concerns about insurance coverage (25.5%). OCM oncologists perceived barriers to prescribing G-CSF as prophylaxis differently than non-OCM oncologists did, with more OCM oncologists than non-OCM oncologists reporting lack of reimbursement to the practice/organization  and patient refusal  as barriers , 67.0% of oncologists reported that the use was increased somewhat or significantly. Subsequent questions, which allowed oncologists to select multiple reasons for increased use of OBI, showed that oncologists most commonly attributed the increase to convenient dosing (66.7%) and patients’ need for an active lifestyle (45.5%). Non-OCM oncologists reported convenient dosing as an influential factor more frequently than OCM oncologists .The majority of the oncologists in this survey reported adhering to at least one national guideline for G-CSF use –12, but Furthermore, almost a quarter of the oncologists in this survey said that the recommended timing of pegfilgrastim PFS administration is within 24 h of the last dose of chemotherapy administration. This misinterpretation of recommended timing could be one of the reasons for almost 12% of chemotherapy cycles in the real-world setting being given with same-day pegfilgrastim administration .The reasons for same-day pegfilgrastim administrations are multifactorial and have been explored in a survey of oncologists who prescribed at least one same-day dose of pegfilgrastim in the past 6 months . PreviouCompared with non-OCM oncologists, OCM oncologists were more likely to have their practices offer or strongly encourage adherence to a specific protocol for G-CSF use. The OCM sites may be more likely to adhere to a specific protocol for G-CSF use because the practice performance is graded not only on outcomes such as all-cause hospitalization and emergency room visits but also on medication use . AnotherCompared with academic oncologists, community oncologists rated hepatic and renal dysfunction and recent surgery as more important determinants of high risk of FN. The perception of risk factors for FN may be different for community oncologists compared with academic oncologists, and this may explain the slightly higher use of G-CSF for primary prophylaxis reported by community oncologists in this study. This finding is similar to previous survey studies of ASCO oncologists, which detected greater use of prophylactic G-CSF by oncologists practicing in a fee-for-service setting , 27.We restricted the study to US oncologists who met the eligibility criteria, volunteered to participate in an online survey, and had used G-CSFs in their practice. Thus, the results may not be generalizable to non-US oncologists, to US oncologists who were ineligible to participate, or to oncologists who do not use G-CSFs yet treat patients who are otherwise eligible for prophylactic FN treatment. The selection of oncologists using physician panel is not truly random, and those volunteering to participate may not be representative of source population of oncologists. The survey collected information on oncologists’ perceptions that were based on recall; therefore, the results may not reflect routine practice. Substantial variations in patterns of G-CSF use have been noted between oncologists belonging to the same community practice ; howeverDespite G-CSF approval since 1991 and the availability of evidence-based guidelines, FN prophylaxis in appropriate patients remains uneven. Our survey shows that not all oncologists follow national guidelines. Noncompliance with prophylactic G-CSF use in appropriate patients can increase FN risk, leading to increased morbidity and potentially increased health care costs. Further education of oncologists on the downstream consequences of not adhering to national guidelines could help reduce the underutilization and inappropriate timing of G-CSF, consequently reducing the risk of FN. Pegfilgrastim OBI is now increasingly used for next-day pegfilgrastim injection, reducing the travel burden and likely reducing the risk of noncompliance as a result of the travel burden. Barriers to G-CSF, including concerns about lack of reimbursement and patient refusal, differ by OCM affiliation and need to be explored further and overcome to improve coverage for G-CSF prophylaxis in appropriate patients.ESM 1(DOCX 230 kb)