Neonicotinoids (NN) are selective neurotoxic pesticides that bind to insect but also mammal nicotinic acetycholine receptors (nAChRs). As the most widely used class of insecticides worldwide, they are ubiquitously found in the environment, wildlife, and foods, and thus of special concern for their impacts on the environment and human health. nAChRs are vital to proper brain organization during the prenatal period and play important roles in various motor, emotional, and cognitive functions. Little is known on children’s contamination by NN. In a pilot study we tested the hypothesis that children’s cerebro-spinal fluid (CSF) can be contaminated by NN.NN were analysed in leftover CSF, blood, and urine samples from children treated for leukaemias and lymphomas and undergoing therapeutic lumbar punctions. We monitored all neonicotinoids approved on the global market and some of their most common metabolites by ultra-high performance liquid chromatography-tandem mass spectrometry.N-desmethyl-acetamiprid , the major metabolite of acetamiprid. All but one urine samples were positive for ≥ one NN. A statistically significant linear relationship was found between plasma/urine and CSF N-desmethyl-acetamiprid concentrations.From August to December 2020, 14 children were consecutively included in the study. Median age was 8 years (range 3–18). All CSF and plasma samples were positive for at least one NN. Nine (64%) CSF samples and 13 (93%) plasma samples contained more than one NN. Thirteen (93%) CSF samples had We have developed a reliable analytical method that revealed multiple NN and/or their metabolites in children’s CSF, plasma, and urine. Our data suggest that contamination by multiple NN is not only an environmental hazard for non-target insects such as bees but also potentially for children.The online version contains supplementary material available at 10.1186/s12940-021-00821-z. In 2017, United Nations rapporteurs called for a new global treaty to regulate and phase out the use of hazardous pesticides in farming as chronic exposure to pesticides had been linked to various neurological disorders. Pregnant women, foetuses, and children were also considered as particularly vulnerable [Residential, household or parental exposures to pesticides have been associated with young adult brain tumors, respectively childhood leukemia and paediatric non-central nervous system solid tumors –5.Among pesticides, neonicotinoids (NN), which are selectively neurotoxic and bind to nicotinic acetycholine receptors (nAChRs), are of special concern for their impacts on the environment and human health since they are the most widely used class of insecticides worldwide  and are In mammals, nAChRs are vital to proper brain organization during the prenatal period . In addiN-desmethyl-acetamiprid (Desm-ACT) was detected in 14/57 (24·6%) very low birth weight infants within 48 h of birth [Data on NN distribution and metabolism in humans are scarce . There aof birth . This leof birth . Assessiof birth .The general aims of this pilot study were to develop an analytical protocol to measure NN in children’s CSF and to assess if NN are present in children’s CSF. Our hypothesis was that NN could be found in children’s CSF, thereby representing a potential central nervous system (CNS) exposure.In a prospective paediatric observational study in Switzerland, we conducted a comparative analysis of CSF, blood and urine to develop and test the analytical method and compare the measured concentrations in these three body fluids.conscious sedation or anaesthesia before injecting 8–15 mL of various chemotherapeutic agents; two mL of CSF was reserved for routine chemical, biological, and cytological investigations and 2 mL of the leftover CSF was collected for NN analysis. On the same occasion, blood was drawn for clinical purposes from a subcutaneous implantable device. From the four mL drawn to rinse the device , the first 2 mL were discarded, and the following 2 mL were kept  for NN analysis. The patient also voided freely a 5–10 mL aliquot of urine in a clean tube. CSF and urine were initially kept in 15 mL polypropylene tubes. All samples were centrifuged, to collect plasma from the blood samples and to remove sediments from the CSF and urine samples. The supernatants were stored at − 80 °C.To study children’s CSF in an ethically acceptable way, we included, as a convenience sample, children with oncologic disease whose CSF had to be removed for clinical reasons in large enough volumes to allow aliquots to be collected for research purposes. All children were diagnosed and treated at the Department of paediatrics, Lausanne University Hospital, where they were consecutively included between August and December 2020. After initial diagnostic procedures, all children (0–18 years) presenting acute lymphoblastic (ALL), myeloblastic (AML) leukaemia or non-Hodgkin lymphoma (NHL) and were planned to receive routine intrathecal chemotherapy were approached and included in the study after informed consent was obtained. Intrathecal treatments consisted in removing up to 8 mL of CSF by lumbar tap under 34. All neonicotinoids and related insecticides approved on the global market and their most commonly detected metabolites were targeted: acetamiprid, clothianidin, thiamethoxam, thiacloprid, imidacloprid, dinotefuran, nitempyram, sulfoxaflor, flupyradifurone, Desm-ACT, imidacloprid-olefin (IMI-olefin), desnitro-imidacloprid (IMI-NH), and 6-chloronicotinic acid (6-CAN). The column used for the separation was an Acquity UPLC HSS T3 . Mobiles phases were milli-Q water + 0.05% formic acid + 1 mM ammonium formate (phase A) and acetonitrile + 0.05% formic acid (phase B). The gradient program started at 0% phase B and increased linearly to 37.5% in 7.5 min, followed by a rapid increase to 100% B in 0.5 min, wash at 100% B for 2 min and reequilibration at 0% B for 4 min. The MS source conditions were identical to those presented in Kammoun and colleagues [Aliquots of 0.2 mL of CSF, plasma or urine were placed into 2.0 mL microcentrifuge tubes, to which 580 μL of acetonitrile and 20 μL of a solution of isotopically labelled internal standards (IS) were added. The IS solution contained acetamiprid-d3 , clothianidin-d3 (CDN Isotopes), thiamethoxam-d4 (CDN Isotopes), thiacloprid-d4 (CDN Isotopes), imidacloprid-d4 (CDN Isotopes), dinotefuran-d3 , and nitempyram-13C-d3  at a concentration of 50 ng/mL in acetonitrile. The mixture was swiftly vortexed and ultrasonicated to precipitate proteins. After centrifugation, the supernatant was partially evaporated to a volume of ca. 50–100 μL, diluted with 950 μL of a 2% formic acid solution in water and submitted to a purification procedure using solid-phase extraction (SPE) cartridges. The SPE cartridge was first conditioned with 1 mL of methanol, equilibrated with 1 mL of 2% formic acid, loaded with the sample, washed with 1 mL of 0.1% formic acid and finally eluted with 1 mL of methanol. The fraction was evaporated to dryness and reconstituted in 200 μL of methanol 25% in water, ultrasonicated, centrifuged, and filtered through 13 mm hydrophilic PTFE filters. A 2.5 μL of the resulting extract was injected into an ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) system composed of an Acquity UPLC I-Class and a TQ-XS triple quadrupole . NN were detected and quantified according to Kammoun and colleagueslleagues  except tn = 4 for each matrix). We could not perform validation at multiple concentrations due to too restricted sample availability. Coefficients of variations (%RSD) were below 10% for all analytes and accuracies always ranged between 80 and 110% except for desnitro-imidacloprid in CSF and imidacloprid-olefin in plasma for which lower but still acceptable accuracy values of 74.1 and 67.1%, respectively, were obtained (supplementary Table The performances of the method in terms of precision (expressed as % relative standard deviation (RSD)) and accuracy (expressed in %) were evaluated by spiking a known amount of NN in the different matrices before sample preparation  on a Cobas 8000® modular analyzer .Urinary concentrations can vary widely over the period of elimination of xenobiotics and largely depend on fluid intake. The two most common methods for correction of metabolite concentrations in urine are the measure of specific gravity and that of creatinine . The speStatistical analyses were carried out in R statistical software . To asseAs this pilot study was only meant to test the feasibility of the NN measurements in children’s CSF, the only patient’s characteristics that were obtained were genders and ages and they were not considered as relevant confounding factors from a medical standpoint.The study was approved by our local Institutional Review Board . All teenagers/parents gave their written consent.n = 9) or AML (n = 2) or NHL (n = 3) were consecutively included. M/F ratio was 1.0, median age 8 years (range 3–18). All CSF total protein contents were within institutional normal ranges (150–450 mg/l).Over a 4 months period, 14 children with ALL   alleviates the difficulties related to CSF collection. However, starting from small sample volumes may have an impact on positive detection rates, which are often correlated to the method sensitivity . Therefo3,4 or CNS/non CNS solid tumors2,5. Such exposome outcomes to pesticides hazards have also recently been reported for neurodevelopmental issues [Our findings suggest that ALL, AML or NHL children living in Switzerland are exposed to multiple NN. Our study population was small and highly selected and is thus not representative of a large paediatric population, with or without a haematological cancer. As no control population of healthy children was included, we could not analyse if these multiple exposures to NN were specific to children with leukaemia/lymphoma. As we did not study potential exposures to any dietary pesticides by food or residential proximity to NN treated crops/livestock/pets, we could not analyse if these multiple exposures to NN were associated with increased individual environmental risks. The exposure to multiple NN in all included patients could represent the tip of the iceberg of a larger, but not analysed, exposure to pesticides. Although the Environmental Protection Agency has classified acetamiprid, clothianidin, dinotefuran, imidacloprid, and thiamethoxam as “Not Likely to be Carcinogenic to Humans” , and whil issues .To our knowledge, this is the first study on the presence of NN in humans’ CSF. Surprisingly, we observed remarkable differences in concentrations between acetamiprid and its metabolite Desm-ACT in CSF. We have found no data on absorption and distribution of NN and their metabolites in humans. Acetamiprid and Desm-ACT are structurally very similar and have comparable hydrophobicity , thus they should theoretically be found in the same body compartments. While we cannot totally exclude a Desm-ACT specific blood-to-CSF transfer, the observed differences in concentrations may be better explained by a prolonged half-life of Desm-ACT compared to ACT in the context of low-dose chronic exposure, resulting in the detection of the former only. The normal CSF total protein concentrations in the 14 studied patients argues against a meningeal inflammation and, thus, against an increased blood-to-CSF permeability such as in bacterial meningitis .Our work describes a reliable analytical procedure to measure NN in human CSF. Our data show that children with ALL, AML and NHL living in Switzerland are exposed to multiple NN. They also show that Desm-ACT freely diffuses into children’s CSF and that other NN than Desm-ACT are to be found in childrens’ CSF. No data exist on human blood-to-brain NN transfer, via the BBB or CSF-to-brain trans-neuroependymal route. The latter’s ultrastructure and ontogeny  as well Additional file 1: Supplementary Table 1. UHPLC-MS/MS compound-specific parameters used for the quantification of neonicotinoids. RT, retention time; ESI, electrospray; CV, cone voltage; CE, collision energy.Additional file 2: Supplementary Table 2. Validation parameters for the different analytes in the different matrices. IS; internal standard; LOQ limit of quantification; CSF cerebrospinal fluid.Additional file 3: Supplementary Table 3. Detailed results of neonicotinoids and neonicotinoid metabolites in cerebro-spinal fluid, plasma and urine of 14 children with haematological cancers. CSF, cerebro-spinal fluid; LOD, limit of detection; LOQ, limit of quantification; * Corrected for specific gravity.Additional file 4: Supplementary Figure 1. (CSF occurence). Number of samples with neonicotinoids > lower limit of quantification, per neonicotinoid, in cerebrospinal fluid in 14 children with haematological cancers. NN, neonicotinoid; CSF, cerebro-spinal fluid; LOQ, lower limit of quantification.Additional file 5: Supplementary Figure 2. (plasma occurence). Number of samples with neonicotinoids > lower limit of quantification, per neonicotinoid, in plasma in 14 children with haematological cancers. NN, neonicotinoid; LOQ, lower limit of quantification.Additional file 6: Supplementary Table 4. (models summary). Summary statistics of the regression models, with or without extreme value (cook distance > 1).Additional file 7: Supplementary Table 5. (models coefficients). Coefficient and associated p-values of the regression models, with or without extreme value (Cook distance > 1).


Can cell-free DNA analysis of cerebrospinal fluid provide additional diagnostic utility beyond cytologic assessment?In this diagnostic study of 43 cerebrospinal fluid samples from 22 patients with leptomeningeal disease confirmed by cytologic assessment who did not have parenchymal tumors abutting their cerebrospinal fluid, tumor-derived cell-free DNA was detected in the cerebrospinal fluid of 40 (93%) samples, whereas 31 (72%) of the samples were positive for malignant neoplasm as determined by cytologic analysis, a significant difference.These findings suggest that cerebrospinal fluid cell-free DNA analysis may be more sensitive than cytologic analysis for diagnosing leptomeningeal disease. Leptomeningeal disease (LMD) is a devastating complication of cancer that is frequently underdiagnosed owing to the low sensitivity of cerebrospinal fluid (CSF) cytologic assessment, the current benchmark diagnostic method. Improving diagnostic sensitivity may lead to improved treatment decisions.To assess whether cell-free DNA (cfDNA) analysis of CSF may be used to diagnose LMD more accurately than cytologic analysis.This diagnostic study conducted in a neuro-oncology clinic at 2 large, tertiary medical centers assessed the use of genomic sequencing of CSF samples obtained from 30 patients with suspected or confirmed LMD from 2015 through 2018 to identify tumor-derived cfDNA. From the same CSF samples, cytologic analyses were conducted, and the results of the 2 tests were compared. This study consisted of 2 patient populations: 22 patients with cytologically confirmed LMD without parenchymal tumors abutting their CSF and 8 patients with parenchymal brain metastases with no evidence of LMD. Patients were considered positive for the presence of LMD if previous CSF cytologic analysis was positive for malignant cells. The analysis was conducted from 2015 to 2018.The primary outcome was the diagnostic accuracy of cfDNA analysis, defined as the number of tests that resulted in correct diagnoses out of the total number of tests assayed. Hypotheses were formed before data collection.P = .02). Of 43 LMD-positive samples, CSF cfDNA analysis was sensitive to LMD in 40 samples , and cytologic analysis was sensitive to LMD in 31 samples , a significant difference (P = .02). For 3 patients with parenchymal brain metastases abutting the CSF and no suspicion of LMD, cytologic findings were negative for LMD in all 3 patients, whereas cfDNA findings were positive in all 3 patients.In total, 30 patients , primarily presenting with metastatic solid malignant neoplasms, participated in this study. For 48 follow-up samples from patients previously diagnosed via cytologic analysis as having LMD with no parenchymal tumor abutting CSF, cfDNA findings were accurate in the assessment of LMD in 45 samples , whereas cytologic analysis was accurate in 36 samples , a significant difference (This diagnostic study found improved sensitivity and accuracy of cfDNA CSF testing vs cytologic assessment for diagnosing LMD with the exception of parenchymal tumors abutting CSF, suggesting improved ability to diagnosis LMD. Consideration of incorporating CSF cfDNA analysis into clinical care is warranted. This diagnostic study compares the sensitivity and accuracy for detecting leptomeningeal disease via genomic sequencing of cell-free DNA with that of cytologic assessment of cerebrospinal fluid obtained from patients with suspected or cytologically confirmed leptomeningeal disease. Leptomeningeal disease is typically diagnosed via identification of malignant cells in cerebrospinal fluid (CSF), most commonly obtained via lumbar puncture or ventriculoperitoneal (VP) shunt access.Leptomeningeal disease (LMD) is a devastating complication of solid and liquid systemic and central nervous system malignant neoplasms. Leptomeningeal disease occurs in 4% to 15% of solid tumors and in 7% to 15% of liquid malignant neoplasms3 Methods have been proposed to limit the rate of false-negative cytologic results. Sampling near the anatomical location of LMD has been shown to improve the sensitivity of cytologic analyses.4 In addition, several studies have shown the use of repeated lumbar punctures to decrease false-negative cytologic diagnoses.6 False-negative rates of cytologic findings are also reduced by increasing the volume of CSF acquired,4 although this approach is often not clinically feasible. Other methods, including neurologic examination and neuroimaging, are similarly known to lack sensitivity in the diagnosis of LMD.7 Given the rapid progression of LMD, there is an important unmet clinical need for a more sensitive diagnostic test.Cerebrospinal fluid cytologic analysis, the currently most reliable diagnostic method, has a sensitivity of only approximately 75% and is known to produce persistently negative results in approximately 10% of patients.9 for LMD are based on positive CSF cytologic findings because MRI has poor specificity.11 Many conditions may result in enhancing leptomeninges on MRI scans, which may lead to an incorrect diagnosis . The direct observation of tumor cells in the CSF is confirmatory of leptomeningeal carcinomatosis. However, CSF cytologic analysis is limited by poor sensitivity.6 In clinical practice, this limitation leads to a delay in diagnosis and treatment and a barrier to clinical trial enrollment.10Magnetic resonance imaging (MRI), a potential alternative to cytologic analysis, lacks specificity, and an incorrect diagnosis of LMD by imaging alone may result in unnecessary treatments and patient harm and may reduce the ability to study new treatments objectively. Current clinical trial designs13 By contrast, the cancer fraction is much higher in CSF cfDNA than in plasma cfDNA owing to the much lower amount of nontumor DNA,14 and detection of alterations in the somatic copy number from CSF cfDNA has been shown.15 In addition, we reasoned that because CSF cfDNA is stable after extraction, it does not require immediate processing. Cytologic examination of CSF is known to be highly sensitive to handling procedures, and samples must be processed quickly after collection.7 By contrast, cfDNA has been shown to produce stable estimates of copy number after storage for 2 weeks at −80 °C following a single centrifugation.16 Thus, we hypothesized that a CSF cfDNA diagnostic test would excel in the detection of LMD. In this retrospective diagnostic study, we sought to establish proof-of-principle LMD diagnosis via genomic sequencing of CSF-derived cfDNA.To address the need for a better diagnostic test for LMD, we propose the use of CSF-derived cell-free DNA  were excluded from analysis because CSF cfDNA was found to not be specific for LMD in this event. This report follows the Standards for Reporting of Diagnostic Accuracy (STARD) reporting guideline for diagnostic studies. Institutional review board approval was previously obtained from the Dana-Farber/Harvard Cancer Center. Written informed consent for the study of tissue specimens, including blood and CSF samples, was obtained from all patients in a manner consistent with the Common Rule requirements and the Declaration of Helsinki.26 No one received compensation or was offered any incentive for participating in this study.Samples of CSF were obtained via lumbar puncture or through routine VP shunt access. The VP shunts used for CSF analysis were assembled as follows: the ventricular catheter was introduced through the skull and then connected to a flushing reservoir with an on/off valve that was in turn connected in-line to a shunt valve that was connected to a distal peritoneal catheter. Any CSF samples obtained from an open surgery were excluded. All patients were reviewed for parenchymal metastasis or recent craniotomy. Patients who had received a diagnosis of suspected or confirmed LMD and had consented to participate in a clinical trial of LMDg for 10 minutes at room temperature. Plasma was separated from blood and centrifuged an additional 10 minutes at 19 000g to remove any remaining impurities before final transfer to storage tubes, which were stored at −80 °C.Whole blood was centrifuged at 1900Cell-free DNA was isolated from blood plasma and CSF using a QIASymphony instrument with the QIASymphony DSP Circulating DNA Kit per the manufacturer’s (Qiagen) protocol. Libraries were prepared using a KAPA HyperPrep kit. A subset of libraries was prepared with unique molecular identifier adapters (Integrated DNA Technologies). Prepared libraries were sequenced on HiSeq X, HiSeq 2500, or HiSeq 4000 instruments to a targeted mean depth of 0.1X. Samples were aligned to hg19 using bwa-mem, version 0.7.7-r441.27 Cancer fraction was inferred using the ichorCNA tool, with a maximum copy number of 5, using chromosomes 1 to 22 with provided references for hg19 GC content, mappability, and centromere locations as well as a panel of reference standards.28 The cfDNA was considered to be diagnostically positive for LMD if ichorCNA inferred a nonzero cancer fraction in the given sample  as the independent factor; concentration and cancer fraction were transformed using log32 A statistical significance threshold of .05 was used for analysis, and all reported tests are 2-sided unless otherwise indicated.Secondarily, the study sought to assess differences in sensitivities, specificities, negative predictive values, positive predictive values, and accuracies between cytologic and cfDNA analyses. Furthermore, the Fisher exact test was used to assess whether sampling location was associated with the outcomes of either cfDNA or cytologic analyses. Statistical analyses were performed with R, version 3.4.0  and the epiR package.The study included a total of 30 patients . The primary histologic finding of patients in this cohort was solid malignant neoplasm, primarily breast cancer (17 patients), with the exception of 1 patient with acute myeloid leukemia. Most patients in this cohort had parenchymal brain metastases (23 with and 7 without). Overall, 22 patients were previously diagnosed as having LMD by using cytologic analyses, whereas 8 patients had no symptomatic, radiographic, or cytologic evidence of LMD. This cohort of 30 patients yielded 51 samples, including 8 LMD-negative samples from patients without radiographic, symptomatic, or cytologic evidence of LMD. All 8 LMD-negative samples were obtained from patients with parenchymal brain metastases. Of these 8 patients, 3 had PTACSF, whereas the remaining 5 had PTs that did not abut the CSF .In 3 LMD-negative samples obtained from patients with PTACSF, CSF cfDNA analysis was positive for the presence of tumor despite the patients lacking radiographic, symptomatic, or cytologic evidence of LMD. In LMD-negative samples from patients with PTs, CSF cfDNA analyses produced results negative for LMD in all 5 samples. Because of these false-positive results, patients with PTACSF were removed from the cohort for further investigation of diagnostic performance. The remaining cohort contained 43 LMD-positive samples and 5 LMD-negative samples.P = .01, McNemar test) (eTable 2 in the P = .02). Of 43 LMD-positive samples, CSF cfDNA analysis was sensitive to LMD in 40 samples , and cytologic analysis was sensitive to LMD in 31 samples ; thus, CSF cfDNA analysis was significantly more sensitive than cytologic analysis (P = .02). In LMD-positive samples, CSF cfDNA and cytologic analysis results disagreed in 11 samples. In 10 of these samples, the cytologic analysis finding was incorrectly negative. In the remaining sample, which was from a patient with acute myeloid leukemia, the only such patient in the cohort, the CSF cfDNA analysis finding was incorrectly negative   A.P < .001). There was no significant difference in inferred cancer fraction between LMD-positive samples and LMD-negative samples with PTACSF. The inferred cancer fraction in LMD-negative samples was significantly higher for patients with PTACSF than for patients with PTs (P < .001). Patient type  was associated overall with cancer fraction (P < .001) . The median cancer fraction inferred in LMD-negative samples with PTACSF was 66.7% . The median cancer fraction inferred in LMD-negative samples with PTs was 0%. The inferred cancer fraction in LMD-positive samples was significantly higher than in LMD-negative samples with PTs ( < .001) C.P = .16) , and in LMD-negative samples with PTACSF, it was 0.0725 ng/µL . The median concentration of CSF cfDNA in LMD-negative samples with PTs was 0.0234 ng/µL . The concentration of CSF cfDNA was not significantly different among patients with LMD, PTACSF, or PTs; thus, patient type was not associated overall with concentration (P = .16) D.For patient DFCI-022, 5 samples were analyzed for CSF cfDNA. Four of 5 samples were positive for LMD, but 1 was negative. The 4 positive samples had similar estimates of cancer fraction  in CSF cfDNA was 2.16 . The median log2 (peak ratio) in plasma cfDNA was 3.51 . The difference in the peak ratios between plasma cfDNA and CSF cfDNA was statistically significant   with a definitive diagnosis of LMD from breast cancer assessed by MRI and clinical symptoms had multiple CSF samples with cytologic findings negative for LMD . She ini10 The tumor-derived cfDNA fraction increased following initiation of pembrolizumab treatment  and cytologic (P = .14) analyses, the probability of a positive result was not significantly associated with the sampling method (Fisher exact tests).Contingency tables were constructed for CSF cfDNA and cytologic analyses to determine whether the probability of a positive result was different for shunts vs lumbar punctures , which likely represented large amounts of noncancer-derived cfDNA as a result of the surgery.22 This evidence suggests that a CSF cfDNA diagnostic test for LMD should not be used for patients with PTACSF. For such patients, traditional methods, including cytology and MRI, may be preferred. Furthermore, the associations among tumor proximity to CSF, PT volume, and the false-positive rate of CSF cfDNA analysis identification of LMD warrant further investigation. Other challenges to consider in the potential use of CSF cfDNA for detecting LMD include neurosurgical procedures and intracranial radiation, both of which may alter the diagnostic results of CSF cfDNA by introducing additional normal or tumor-derived DNA into the CSF than is otherwise present.35This study found that, in this cohort of patients, the specificities of CSF cfDNA analysis and CSF cytologic analysis were the same for assessing the presence of LMD with the exception of patients with PTACSF. The detection of tumor-derived cfDNA from PTACSF—and also the lack of detection of tumor-derived cfDNA from PT—agrees with previous reports in glioma.28 The detection of somatic copy number alterations by low-pass genomic sequencing is robust to different primary histologic approaches; however, the sensitivity is lower in malignant neoplasms with near-diploid genomes, such as acute myeloid leukemia.36 Deeper sequencing using the detection of somatic nucleotide variations as a method for detecting LMD would likely remediate this insufficiency. Deeper sequencing of CSF cfDNA could improve overall diagnostic performance in general by providing additional power to resolve aneuploidy as well as by enabling analysis based on the presence of somatic variants.Future studies will investigate clinical and tumor-specific attributes that may be associated with sensitivity of CSF cfDNA, such as the fraction of cancer-derived cfDNA present, depth of sequencing, concentration of CSF cfDNA, and the percentage of the genome that is aneuploid.3This study shows the potential for the use of CSF-derived cfDNA analysis in the diagnosis of LMD, and we urge follow-up studies to investigate the use of a Clinical Laboratory Improvement Amendments–certified cfDNA diagnostic assay for incorporation into diagnostic guidelines. Furthermore, we recommend prospective clinical trials in LMD to include CSF cfDNA analysis concurrent with cytologic assessment for clarification of the utility of CSF cfDNA analysis as a diagnostic test and marker of treatment response. To our knowledge, this is the first study to directly compare the diagnostic performance of cytologic and cfDNA analyses using samples collected during the same lumbar puncture or VP shunt draw, which enabled direct comparison of their performance. As a result, we showed improved diagnostic accuracy and sensitivity of the CSF-derived cfDNA analysis for the diagnosis of LMD compared with CSF cytologic assessment, which is the current benchmark diagnostic method. Furthermore, we showed the ability of CSF cfDNA analysis to diagnose LMD among patients whose cytologic findings had been persistently negative, a known phenomenon in CSF cytologic analysis.We showed that next-generation sequencing of CSF cfDNA has the potential to yield not only improved diagnostic performance but also metrics, concentration, and cancer fraction that may be informative of response to treatment or disease progression. Improved diagnosis of LMD has the potential to lead to improved treatment decisions and patient outcomes and suggests that consideration of incorporating CSF cfDNA analysis into LMD diagnostic workflows is warranted.


Background: Recent emerging evidence has highlighted the potential critical role of cerebrospinal fluid (CSF) in cerebral waste clearance and immunomodulation. It is already very well-established that the central nervous system (CNS) is completely submerged in CSF on a macro-level; but to what extent is this true on a micro-level? Specifically, within the peri-neural and peri-vascular spaces within the CNS parenchyma. Therefore, the objective of this study was to use magnetic resonance imaging (MRI) to simultaneously map the presence of CSF within all peri-neural  and peri-vascular spaces in vivo in humans. Four MRI protocols each with five participants were used to image the CSF in the brain and spinal cord. Our findings indicated that all CNS neuro- and vascular-communication channels are surrounded with CSF. In other words, all peri-neural spaces surrounding the cranial and spinal nerves as well as all peri-vascular spaces surrounding MRI-visible vasculature were filled with CSF. These findings suggest that anatomically, substance exchange between the brain parenchyma and outside tissues including lymphatic ones can only occur through CSF pathways and/or vascular pathways, warranting further investigation into its implications in cerebral waste clearance and immunity. The critical involvement of cerebrospinal fluid (CSF) in metabolic cerebral waste clearance (CWC) has gained a lot of momentum lately. Our traditional understanding of CSF circulation is that it is produced by the choroid plexus and circulates through the ventricles and subarachnoid spaces, exiting directly into the dural venous sinuses via arachnoid granulations/villi –5. RecenIn light of the importance of CSF physiology, researchers have focused their attention on investigating CWC CSF outflow pathways, out of the central nervous system (CNS). The traditional outflow pathway via the arachnoid granulations/villi is becoming increasingly unpopular due to the lack of physiological evidence to support it , 21. Thiin vivo imaging methods. Therefore, the objective of this study was to use magnetic resonance imaging (MRI) to simultaneously map the presence of CSF within all peri-neural  and peri-vascular spaces in vivo in humans.Although our understanding of CSF physiology remains relatively rudimentary and is still very much under investigation, one concept has become increasingly apparent: all CNS neuro- and vascular-communication channels are surrounded with CSF. It is already very well-established that the CNS is completely submerged in CSF on a macro-level; but now we know that this is true even on a micro-level, specifically within the peri-neural and peri-vascular spaces within the CNS parenchyma. Most studies have asynchronously investigated these spaces and these investigations have been mainly conducted in animals and human cadavers. Moreover, although CSF-filled peri-vascular spaces in several brain regions have been illustrated in several studies, to our knowledge, there is no systematic study focusing on whether all cerebral vasculature (with the exception of capillaries) are surrounded with CSF, and this can only be revealed using To prove the concept, we designed four types of experiments each with five healthy participants . Each protocol was tailored to a specific area of investigation and anatomical structures, such as cranial nerves and their corresponding perineural spaces, cerebral vasculature and their corresponding perivascular spaces, and different segments of the spinal cord. The study was approved by local Institutional Review Board. Written consent was obtained prior to each MRI experiment. Various sequences were used to image CSF in the brain and spinal cord at 3T .Sampling Perfection with Application optimized Contrasts using different flip angle Evolution” (T2W-SPACE) sequence to image the CSF and cranial nerves with hyperintense CSF signal and hypointense signal for cranial nerves and blood vessels , as well as the diameter ratio of the cranial nerve diameter over the total diameter , for all 12 cranial nerves in healthy humans (The first experiment employed a 3D T2-weighted (T2W) “ vessels . This sey humans , 2.STrategically Acquired Gradient Echo” (STAGE) imaging to simultaneously obtain bright-blood and dark-blood images  and bright-blood (T1W) which were used to visualize the blood vessels and perivascular spaces in the order of 100 μm. In addition, to acquire a pure CSF image with suppressed blood signals, we used a 3D turbo-spin echo (TSE) sequence with TR/TE = 2,000/345 ms and an isotropic voxel size of 0.5 mm. With such a long echo time, the images were heavily T2-weighted such that all tissues other than CSF (very long T2) were suppressed. Given the long TR/TE and high-resolution 3D sampling scheme, the sequence took half an hour covering the central half of the brain in the sagittal plane.For the second experiment, in order to image CSF in peri-vascular spaces and to demonstrate that all MRI-visible cerebral blood vessels were surrounded by CSF, we used high-resolution “d images –41. The * weighted spoiled gradient echo sequence  was used to acquire images with hyperintense CSF and blood vessels, and hypointense nerves. This sequence is one of the clinical sequences for cervical protocol. Imaging parameters were: TR = 587 ms, TE = 17 ms, flip angle = 30°, in-plane resolution = 0.5 × 0.5 mm2, slice thickness = 3 mm, scanning time = 3 min.The third experiment was performed on the cervical spinal cord at the C4/C5 level. A 2D T22, slice thickness = 4 mm, scanning time = 2 min. This sequence, with sub-millimeter in-plane resolution, provided high nerve-to-CSF contrast. The 12 pairs of cranial nerves and spinal nerves were labeled by experienced neuroradiologist on images from the first, third and fourth experiments.The fourth and final experiment was for imaging the lumbar spinal nerves at L3/L4 level. A regular T2W-TSE sequence was used with the following imaging parameters: TR = 5,490 ms, TE = 87 ms, in-plane resolution = 0.6 × 0.6 mmAll MRI data were first visually evaluated to label nerves and vessels on representative images. Representative images of the first experiment are shown in The CNS neuro-communication channels consist of 12 pairs of cranial nerves and 31 pairs of spinal nerves. Images from the first experiment were used to visualize all 12 pairs of the cranial nerves , 2. As iThe spinal nerves were visualized on the images from the third and fourth experiments . SimilarThe high-resolution (in the order of 100 μm) sT2W . Once agin vivo in humans; as opposed to previous studies that have asynchronously investigated this, mainly in animals and human cadavers. Our findings indicate that all brain parenchyma and spinal cord communication channels, both neuro- and vascular-communication channels, are encased in CSF. The diameter ratios, nerve diameter over total diameter , ranged from 0.6–0.8. It logically follows that CSF must play a critical role in substance exchange between the brain parenchyma/spinal cord and its communication channels as well as its surrounding environment/tissue—a role that has been historically underestimated and understudied. Our findings are consistent with previous literature that have asynchronously investigated CSF-filled peri-neural spaces, mostly in the context of CWC lymphatic-associated CSF outflow pathways, as well as peri-vascular spaces , in the peri-neural spaces. Therefore, CSF must play a critical role in regulating the communication between the cranial nerves and their surroundings. One such type of communication regulation is with the lymphatic system. In fact, the interaction of CSF with the lymphatic system is only beginning to be understood. There is a growing body of evidence to suggest that CSF tracer eventually makes it into the deep cervical lymph nodes , 36, 37.Our findings also drive home the point that all MRI-visible vasculature is surrounded with CSF-filled peri-vascular spaces. Therefore, CSF must play a critical role in regulating the communication between the blood and the brain parenchyma, as well as CWC at large. In 1985, Rennels et al. introduced the idea of a fluid circulation system throughout the CNS via peri-vascular spaces . But it Starting 6 years ago, gadolinium-based contrast agent (GCA) residual deposition in the brain parenchyma with chronic administration has been documented , 62. GCAin vivo in humans. Third, our study utilized MRI which is minimally invasive and therefore caused minimal disruption to the biological system. In the past, most MRI studies of the peri-neural and peri-vascular spaces have been mainly conducted in animals , all MRI-visible vasculature and spinal nerves. Second, our study was performed  animals , 68.On the other hand, the major limitation of our study is that we did not investigate the underlying mechanisms of how CSF made it into or out of these peri-vascular and peri-neural spaces. Although this has been a topic-of-interest as of late, and several strides have been made to address this topic, the latest of which has been the establishment of the glymphatic pathway, the CSF efflux mechanisms remain unclear. One current question of interest is: “What is the underlying pathway/mechanism by which CSF drains into the meningeal lymphatics, and how is this pathway/mechanism related to CSF-filled peri-vascular and peri-neural spaces?” Another major limitation is that we did not investigate if and how CSF communicates with the CNS neuro- and vascular-communication channels themselves , nor a quantitative morphometric measurement of the arterial and venous blood vessels and their corresponding peri-vascular spaces. There is growing evidence to suggest that CSF in peri-vascular spaces might be involved in the direct transport of cerebral waste into the parenchymal vasculature, perhaps through transcellular mechanisms , 69, 70.In conclusion, our findings indicate that all CNS neuro- and vascular-communication channels are surrounded with CSF. In other words, all peri-neural spaces surrounding the cranial and spinal nerves as well as all peri-vascular spaces surrounding MRI-visible vasculature were filled with CSF. These findings highlight the extent to which CSF infiltrates the parenchyma and its importance, therefore, warranting further investigation into its potential implications.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Wayne State University Institutional Review Board. The patients/participants provided their written informed consent to participate in this study.LF participated in conceptualization, data acquisition, data analysis, manuscript writing, and manuscript editing. YC participated in conceptualization, investigation, data acquisition, data analysis, manuscript writing, and manuscript editing. SX participated in data acquisition, data analysis, and manuscript editing. EH participated in methodology, data analysis, and manuscript editing. JH participated in conceptualization, methodology, data analysis, and manuscript editing. QJ participated in conceptualization, methodology, data analysis, and manuscript editing. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Metabolite reprogramming in bacterial meningitis was greatest among phosphatidylcholines, and concentrations of all 54 phosphatidylcholines were significantly (p = 1.2 × 10−25–1.5 × 10−4) higher than in controls. Indeed, all biomarkers for bacterial meningitis vs. viral meningitis/encephalitis with an AUC ≥ 0.86  were phosphatidylcholines. Four of the five most accurate (AUC ≥ 0.9) phosphatidylcholine biomarkers had higher sensitivity and negative predictive values than CSF lactate or cell count. Concentrations of the 10 most accurate phosphatidylcholine biomarkers were lower in meningitis due to opportunistic pathogens than in meningitis due to typical meningitis pathogens, and they correlated most strongly with parameters reflecting blood–CSF barrier dysfunction and CSF lactate (r = 0.73–0.82), less so with CSF cell count, and not with blood CRP. In contrast to the elevated phosphatidylcholine concentrations in CSF, serum concentrations remained relatively unchanged. Taken together, these results suggest that increased free CSF phosphatidylcholines are sensitive biomarkers for bacterial meningitis and do not merely reflect inflammation but are associated with local disease and a shift in CNS metabolism. The identification of CSF biomarkers for bacterial meningitis can potentially improve diagnosis and understanding of pathogenesis, and the differentiation from viral CNS infections is of particular clinical importance. Considering that substantial changes in CSF metabolites in CNS infections have recently been demonstrated, we compared concentrations of 188 metabolites in CSF samples from patients with bacterial meningitis ( Despite all advances in diagnosis and treatment, bacterial meningitis continues to feature high morbidity and mortality. Untreated, it can be fatal in 50% of cases, and even when diagnosed early and treated adequately, 8–15% of the patients die, typically within 48 h of symptom onset. Furthermore, 10–20% of the survivors develop permanent sequelae including hearing loss and cognitive dysfunction . This isConsidering the high risk of neuronal damage and long-term sequelae in bacterial meningitis, there are efforts to expand the spectrum of treatments with anti-inflammatory and neuroprotective therapies ,12. TherPhosphatidylcholines are common constituents of cell membranes and also occur in free form in body fluids including blood and CSF. They are important substrates of phospholipases and other enzymes that can release fatty acid moieties such as arachidonic acid and linoleic acid, which can be catabolized further to yield a variety of lipid modulators of inflammation. In the brain, they play additional important roles in intraneuronal signal transduction and in regulating local levels of the neurotransmitter acetylcholine . InhibitWe have previously assembled a cohort of 221 patients with bacterial, viral, autoimmune, and noninflammatory neuropsychiatric disorders and measured CSF concentrations of 188 small molecules using a targeted metabolomic approach based on mass spectrometry. By analyzing subsets of the data, we identified highly accurate biomarkers for CNS involvement in varicella-zoster virus (VZV) reactivation , for theN = 132) from patients with bacterial meningitis , viral meningitis or encephalitis (n = 34), consisting of herpes simplex virus encephalitis , varicella zoster virus meningoencephalitis , enterovirus meningitis , and noninflamed controls (n = 66) comprising normal pressure hydrocephalus (n = 35), Bell’s palsy (n = 11), and Gilles de la Tourette syndrome (n = 20). Serum samples, which were obtained at the time of lumbar puncture, were available from a subset of participants . Diagnostic criteria (case definitions) are summarized in This analysis is based on part of a cohort of CSF samples from 221 patients, which were collected between 2005 and 2013. Other aspects of this cohort have been described in ,18,19,20The following parameters were determined after lumbar puncture: CSF cell count, CSF protein concentration, CSF lactate concentration, Q-albumin ratio , and IgG index . Blood–CSF Barrier (BCB) dysfunction was scored from 0 (no dysfunction) to 3 (severe dysfunction), using age-corrected Q-albumin as described in . Periphehttps://biocrates.com/mxp-quant-500-kit, accessed on 15 December 2020). Details about internal standards and quality controls (QC) are given in the manufacturer’s application note [CSF metabolite concentrations were measured on a triple-quadrupole mass spectrometer  with an electrospray-ionization ion source coupled to a high-performance liquid chromatography system  and the AbsoluteIDQ™ p180 kit and MetIDQ™ software , as described in detail in . Serum cion note . The QuaSince our previous studies have shown that CSF metabolite biomarkers are usually more highly concentrated in the more inflamed diagnostic group, we only included analytes that were detected above limit of detection  in at least 80% of the bacterial meningitis samples, whereas concentrations in the comparator groups were not part of inclusion criteria. One analyte (PC.ae.C42.4) had to be removed from the analysis due to erratic values likely resulting from a technical error. Based on this screen, we included 100 analytes in the subsequent analyses, comprising 54 phosphatidylcholines (PCs), 5 lysophosphatidylcholines (lysoPCs), 12 sphingomyelins (SMs), 23 amino acids (AAs) and amino acid metabolites (AAMs), 5 acylcarnitines (ACs), and the sum of hexoses . All conAs the data were non-normally distributed, the Spearman method was used to assess correlations and the Mann–Whitney U and Kruskall–Wallis tests were used to compare differences in median values between groups and across groups, respectively. The Chi-squared test was used to assess differences in categorical parameters. For principal component analysis (PCA), data were log-transformed and analyzed using R package “FactoMineR”  and the Clinical and sociodemographic data revealed the differences expected from the natural history of the disease entities, including pronounced leukocytosis and elevated blood CRP in peripheral blood, and elevated CSF cell count, lactate levels , and disA principle component analysis revealed a clear separation of bacterial meningitis from the control samples and a somewhat less pronounced separation from the viral CNS infections A,B. Of nAnalysis of mean Euclidian distances confirmed that CSF metabolite reprogramming in bacterial meningitis vs. controls or viral CNS infections was by far greatest in phosphatidylcholines and least in lysophosphatidylcholines and acylcarnitines C. Thus, A hierarchical clustering analysis showed a clade (node marked 1) with broad upregulation of glycerophospho- and sphingolipids in bacterial meningitis but, to a lesser extent, also some viral samples  . Two sman = 79) biomarkers, defined as having an AUC ≥ 0.8, a lower CI not crossing below the chance line of 0.5, and an asymptotic p value of <0.05. Of note, even though the overall differences appeared to be smaller, 57 biomarkers were identified for the clinically relevant differentiation between bacterial meningitis and viral CNS infections (p = 1.2 × 10−25–1.5 × 10−4) elevated in bacterial meningitis vs. controls, and 53/54 (p = 3.2 × 10−10–3.5 × 10−3) in bacterial meningitis vs. viral CNS infections. Indeed, they constituted 51 of the 79 (65%) total biomarker candidates for bacterial meningitis vs. controls, and 46/57 (80%) for the distinction from viral CNS infections , closely followed by Q-IgG, protein, BCB dysfunction, all of which reflect dysfunction of the BCB, whereas increased protein can also be a sign of cell death. The correlation with CSF lactate was similar, whereas correlation with CSF cell count was only moderate, and those with blood leukocyte count and CRP were weak. In terms of metabolite subgroups, phosphatidylcholines and sphingomyelins exhibited the strongest correlations, again correlating most strongly with parameters reflecting BCB dysfunction and lactate, much less with cell count and only minimally with blood parameters. Of note, the only significant (p ≤ 0.05) negative correlations were those of hexoses (in CSF mostly represented by glucose) with lactate and cell count. Taken together, these results strongly suggest that the observed changes in overall metabolite populations and in the phosphatidylcholine biomarkers (i) reflect local CNS disease more than systemic manifestations and (ii) are most closely associated with disruption of the BCB, cell death, and a disease-associated shift in central carbon metabolism to glycolysis. We then tested to what extent the observed changes in bacterial meningitis in the various CSF metabolite groups correlated with parameters of local (measured in CSF) and systemic (measured in blood) inflammation . The strp = 0.009–0.048), whereas lactate (p = 0.08) and leukocyte count (p = 0.55) did not achieve this degree of significance  and infection with opportunistic pathogens, which are more likely to be pathogenic in immunocompromised or multimorbid individuals. Dividing the bacterial meningitis samples into two groups, typical and opportunistic meningitis pathogens . We performed a targeted metabolomic analysis of CSF samples from patients with bacterial meningitis, viral CNS infections and noninflammatory controls, and identified free phosphatidylcholines as the molecular class with the highest biomarker potential, in particular to increase sensitivity and negative predictive value for a diagnosis of bacterial meningitis, and to distinguish between meningitis due to typical vs. opportunistic bacterial pathogens. We previously found that increased phosphatidylcholines were among the most accurate CSF biomarkers to detect CNS involvement in VZV reactivation  and enteFrom the clinical point of view, it is notable that CSF lactate turned out to be the metabolite biomarker with the overall greatest accuracy, thus further validating lactate as a routine CSF biomarker to differentiate between bacterial and aseptic meningitis . Even thOur study is unique in that we were able to compare concentrations of the phosphatidylcholine biomarkers in paired CSF and serum samples. In peripheral blood, concentrations of many phosphatidylcholines decrease in systemic inflammatory disorders. This may be due to a combination of alterations in membrane signaling, catabolism by free phospholipases and other enzymes in blood, and removal in the reticuloendothelial system  lists 376 endogenous CSF metabolites, and a broader screening would likely detect additional and perhaps more accurate biomarkers. On the other hand, we used prospectively collected specimens that were processed according to unified protocols and had a definitive identification of bacterial/viral pathogens.Even though our study features a sample size typical of a single-center cohort, the conclusions are limited as we could not perform an external validation. This is particularly true for the comparison “typical” vs. “opportunistic” pathogens. In addition, the exact molecular identity of some of the investigated phosphatidylcholines is uncertain, as the designated molecular species may actually correspond to up to six isobars or isomers with known chemical structures . In addiOur results underscore the value of CSF lipid profiling for the discovery of biomarkers for CNS infections and show that raised phosphatidylcholine concentrations in CSF are not the result of systemic inflammation but reflect local disease activity, likely by measuring release from perturbed cell membranes. Measuring free phosphatidylcholines in CSF may provide added value to diagnosis and risk stratification of patients with a clinical suspicion of meningitis, specifically by providing high sensitivity and NPV, distinguishing between patients infected with classic vs. atypical meningitis pathogens, and identifying those at greatest risk of neuronal damage and clinical complications. Further research should focus on identifying the exact molecular species that define the discriminatory ability of the identified phosphatidylcholine biomarkers, validating their diagnostic performance in larger cohorts, assessing their prognostic value in longitudinal study designs, and ultimately developing simpler assays that can be used as rapid diagnostics at the time of lumbar puncture.


P < 0.001). The correlation between MRI-TT and pDOI was good . MRI-TT were significantly greater than pDOI in all axial and coronal T2WI and CE-T1WI (P < 0.001). In multivariate Cox proportional hazards analysis, MRI-TT measured on axial CE-T1WI yielded a significant prognostic value for OS . MRI-TT demonstrated excellent intra- and inter-rater agreements as well as high correlation with pDOI. MRI-TT may serve as a prognostic predictor in patients with tongue SCC.This study aimed to assess the prognostic value of MRI-measured tumor thickness (MRI-TT) in patients with tongue squamous cell carcinoma (SCC). This single-center retrospective cohort study included 133 pathologically confirmed tongue SCC patients between January 2009 and October 2019. MRI measurements of tongue SCC were based on axial and coronal T2-weighted (T2WI) and contrast-enhanced T1-weighted (CE-T1WI) images. Two radiologists independently measured MRI-TT. Intraclass correlation coefficients (ICC) were calculated for inter-rater agreements. Spearman’s rank correlation between MRI-TT and pathologic depth of invasion (pDOI) was assessed. Cox proportional hazards analyses on recurrence-free (RFS) and overall survival (OS) were performed for MRI-TT and pDOI. Kaplan–Meier survival curves were plotted with log-rank tests. The intra- and inter-rater agreements of MRI-TT were excellent (ICC: 0.829–0.897, all  The prognostic significance of pathologic depth of invasion (pDOI) has been well-established8, such that pDOI was introduced in the recent 8th Edition of the American Joint Committee on Cancer (AJCC) and the Union for International Cancer Control (UICC), as a crucial part of T-staging in oral SCC11.Oral cavity squamous cell carcinoma (SCC) is the 8th most common cancer globally12. While the 8th Edition of the UICC/AJCC criteria proposed three classifications for the pDOI 11, there is no consensus regarding the reliable cutoff value of MRI-TT for prognostication as of yet.Prior to surgical treatment, preoperative MRI allows measurement of the tongue SCC thickness, which is often used for evaluating clinical T-stage. However, pDOI and MRI-measured tumor thickness (MRI-TT) are intrinsically different in that MRI-TT does not account for pathologic findings, such as epithelial involvement16. Several studies have found that MRI-TT is an independent prognostic factor for predicting occult nodal metastasis22 and overall survival (OS)23 in oral cavity SCC. However, prognostic value of MRI-TT for recurrence-free survival (RFS)  has not yet been investigated. Furthermore, defining the optimal MRI-TT measurement method deserves more research attention.pDOI is the gold standard reference that can only be determined postoperatively. A number of recent studies have investigated preoperative MRI for risk assessment in patients with tongue SCCAddressing the prognostic value of MRI-TT, with respect to the gold standard pDOI, would aid in preoperative risk-stratification of patients. Therefore, this study aimed to determine the intra- and inter-rater agreement on various methods of measuring MRI-TT and to assess their performances for predicting RFS and OS in patients with tongue SCC.A total of 133 patients were retrospectively selected. The patient selection process is depicted in Fig. P < 0.001) (Table P < 0.001) (Table P < 0.001) and were higher among the different methods of MRI-TT .The intra- and inter-rater agreements for all the MRI measurements were excellent . CE-T1WI axial thickness of greater than 18.3 mm was associated with a greater risk of recurrence . pDOI grade was significantly associated with greater risks of recurrences . Coronal measurements of MRI-TT predicted recurrences, but without statistical significances . Among the clinical variables, age was significantly associated with RFS . In multivariate analysis including age, axial T2WI, and pDOI grade, age remained as a significant variable . The Kaplan–Meier curves of MRI-TT and pDOI demonstrated similar results . pDOI grade was significantly associated with worse prognosis . Coronal measurements of MRI-TT demonstrated significances in predicting OS . Among the clinical variables, age and T-stage of T1/T2 were significantly associated with OS . The Kaplan–Meier curves of MRI-TT and pDOI demonstrated similar results .The hazards ratios and 95% confidence intervals for OS are summarized in Table The current study demonstrated excellent intra- and inter-rater reliabilities of MRI-TT, which also showed high correlation with pDOI. In multivariate analysis, axial CE-T1WI of > 23 mm demonstrated statistical significance with respect to OS while pDOI grade did not. The results of this study suggest the potential use of MRI-TT in preoperative risk-stratification of tongue SCC patients.15 reported the prognostic MRI depths of tongue SCC that enabled the prediction of overall and disease-specific survival as 11 mm, which is similar to our cutoff value of 11.7 mm determined on axial T2WI. Their image analysis used pathologic specimen as reference template for measurement, allowing MRI measurement much closer to the pDOI. Our findings based solely on MRI measurements might provide a more practical prerequisite for risk-stratification of patients preoperatively.A previous study by Mao et al.24 who showed a mean difference of 3.2 ± 4.9 mm on T2WI and 3.0 ± 4.4 mm on CE-T1WI. However, their study did not specify whether the measurements were based on either axial or coronal MRI. The current study demonstrated that axial measurements on both T2WI and CE-T1WI have smaller mean differences compared to the coronal measurements. This finding could further shed light on choosing the optimal reconstruction method of MRI in determining MRI-TT.Our reported mean difference between MRI-TT and pDOI of 2.7–4.1 mm was similar to a study by Yesuratnam et al.25, whereas the current study included a large sample size of patients with various T and N stages. This strengthens our findings to be more applicable in a wider range of disease severity for patients with tongue SCC. Another strength of this study includes the image analysis based on multi-parametric (T2 and CE-T1WI) and multi-reconstructed MRI , providing four different measurement methods. A similar study by Murakami et al.13 also employed three different methods of measurement on MRI; however, in addition to the larger sample size of our cohort, the survival analysis of this study yielded significant prognostic values derived from various measurement methods, potentially providing the optimal method for prognostication.Several similar studies included patients with limited T stages26. One possible explanation is due to shrinkage of specimens after formalin fixation. In a study by Chen et al.27, head and neck cancer specimens’ length, width and depth shrank on average by 4.4% to 6.2% after formalin fixation. However, Umstattd et al.28 found that shrinkage of mucosal margins of oral cavity squamous cell carcinoma specimens occurred mostly between pre-excision and post-excision—prior to formalin fixation, thus suggesting that the shrinkage might be due to intrinsic tissue properties. With the presented data, it is difficult to determine whether the shrinkage occurred immediately after excision or due to formalin-fixation. However, understanding the inherent discrepancy between MRI-TT and pDOI would allow more informed clinical decision making. One interesting side finding was that larger tongue SCC tended to demonstrate weaker correlation between MRI-TT and pDOI, as evidenced in both Bland–Altman and correlation matrix plots. This finding suggests that larger tongue SCC would require more cautious interpretation.While MRI is the imaging modality of choice for assessing tongue SCC, the discrepancy between MRI-TT and pDOI, such as overestimated values of MRI-TT, has been reported in previous literatureOf importance, MRI-TT seemed to show more prognostic value for predicting OS than RFS. In univariate Cox proportional hazards analysis, all four different methods of MRI-TT were found to be significantly associated with OS, whereas one method—axial T2WI—was significantly associated with RFS. In multivariate analysis, only MRI-TT over 23 mm on axial CE-T1WI was found to be independently associated with OS. These weak associations in multivariate results suggest that MRI-TT should be interpreted as a complimentary measure along with other factors.There are several limitations of the study that must be addressed. First, in addition to the retrospective study design, 20 patients had positive surgical margins in their pathologic specimens. However, other than that, the patients were included according to the predefined strict selection criteria, which would have minimized the possibility of selection bias. Second, the standard protocol in our institution employed MRI slice thickness of 4 mm, which could have missed very thin SCC that might have been present. Third, the number of recurrences was small (n = 25) to obtain optimal accuracy of regression coefficients. Studies with a larger number of cases of recurrence and deaths are warranted for precise multivariate survival modelling. Finally, the potential impact of pre-MRI biopsy was not accounted for; the biopsy might have increased the extent of tumor enhancement, possibly yielding overestimated measurements.In conclusion, MRI-TT demonstrated excellent intra- and inter-rater agreements and high correlation with pDOI. MRI-TT above certain threshold thicknesses showed prognostic value in patients with tongue SCC.This single-center retrospective cohort study was approved by the Institutional Review Board of Seoul St. Mary’s Hospital, Seoul, Republic of Korea (KC20RISI0216). Written consent for medical research was waived by the Institutional Review Board of Seoul St. Mary’s Hospital due to the retrospective nature of study. All methods were performed in accordance with the ethical standards of our institutional research committee and with the 1964 Helskinki declaration and its later amendments.The records of consecutive patients diagnosed with tongue SCC at our institution from January 2009 to October 2019 were retrospectively reviewed. The inclusion criteria of the patients were: (1) pathologically diagnosed with tongue SCC and underwent surgery; (2) has available pretreatment MRI with sequences, including axial and coronal T2-weighted images (T2WI) and contrast-enhanced T1-weighted images (CE-T1WI); and (3) does not have distant metastasis at presentation. The exclusion criteria were as follows: (1) prior treatment history; (2) severe artifacts affecting appropriate image analysis; (3) tongue SCC not visible on the MRI scans; and (4) no pDOI report.11. All patients were treated with definitive primary tumor resection and neck dissection. Location of unilateral neck dissection was determined based on the location of primary cancer (i.e. left or right); bilateral modified radical neck dissection was performed for patients with metastatic cervical lymph node at presentation. Indications for adjuvant radiotherapy or chemotherapy were variable but were primarily based on the following findings: positive or close margins found on the resection, advanced T classification, lymphovascular or perineural invasion, multiple nodal metastasis, or extracapsular spread. The tissue samples from resected specimens were formalin-fixed for at least 7 h and embedded in paraffin. Representative tissue areas were stained with hematoxylin and eosin. The pDOI was measured on paraffin-embedded sections by an experienced head and neck pathologist with 20 years of experience. The pDOI was measured as the perpendicular distance from the mucosal surface of tumor to the deepest point of invasion in any plane.The following clinical factors were reviewed from the electronic medical records of our institution: age at diagnosis, sex, type of surgery , history of adjuvant chemotherapy and radiation therapy, and pathologic reports. All patients were assigned a pathological TNM stage according to the 8th Edition of the AJCC/UICC staging system11. RFS was defined as the interval between the date of pathological confirmation of SCC and the date of pathologically confirmed recurrence , last clinical follow-up, or death. Similarly, OS was defined as the interval between the date of diagnosis and the date of death or last clinical follow-up. Patients underwent MRI scans at 6-month intervals during the first two years following the initial surgery, after which MRI was scanned annually. The mean clinical follow-up was 3.4 years (range 0.1–10.7 years) and ended on February 18, 2020.The cutoff values of pDOI to stratify the patients into low-risk or high-risk SCC were 5 mm and 10 mm, respectively, as proposed by the 8th Edition of the AJCC/UICC criteria2; slice thickness, 4 mm; (b) coronal T2WI; repetition time/echo time, 4760/81 ms; matrix, 320 × 256; field of view, 220 × 220 mm2; slice thickness, 4 mm; (c) axial CE-T1WI; repetition time/echo time, 610/16 ms; flip angle, 150°; matrix, 320 × 256; field of view, 200 × 200 mm2; slice thickness, 4 mm; and (d) coronal CE-T1WI; repetition time/echo time, 693/14 ms; flip angle, 150°, matrix, 320 × 250; field of view, 220 × 220 mm2; slice thickness, 4 mm.All MRI were acquired from a 3.0 Tesla scanner  with a 12-channel head coil. The CE-T1WI were acquired with gadolinium-based contrast agent . The acquisition parameters were: (a) axial T2WI; repetition time/echo time, 3600/87 ms; flip angle, 150°; matrix, 320 × 256; field of view, 200 × 200 mmThe two raters with 3 and 8 years of experience in head and neck imaging independently analyzed all the images. To minimize recall bias, the first rater repeated the analysis after three months. Both raters were blinded to the patients’ clinical information during image analysis. Two different MR sequences (T2WI and CE-T1WI) with axial and coronal acquisitions were used to measure the tumor thickness. For both axial and coronal scans, the reference line was drawn along the tumor’s lateral mucosal margin at the most representative image where the tumor appeared largest. A line perpendicular to the reference was drawn to the deepest point and was determined as MRI-TT. All measurements were performed using our in-house picture archiving communication system software. Representative images of the MRI-TT measurements are illustrated in Fig. 29 were plotted for visual assessment of agreements between mean MRI-TT and pDOI. The difference between MRI-TT and pDOI was assessed via paired Wilcoxon signed-rank test. Spearman rank correlation coefficients were calculated and visualized via correlation matrix of the different methods of MRI-TT and between MRI-TT and pDOI. Survival analyses on RFS and OS was performed via Cox proportional hazards models. Kaplan–Meier curves were plotted with log-rank tests to compare the survival curves. The cutoff values for pDOI were grouped into grade 1 (< 5 mm), grade 2 (5–10 mm), and grade 3 (> 10 mm) based on the 8th edition of the AJCC/UICC11, while those for MRI-TT were determined by optimal stratification based on log-rank statistics30. All statistical analyses were performed using R statistical software . Statistical significance was set at P < 0.05.The normality of the MRI-TT and pDOI measurements was tested via Shapiro–Wilk test. The continuous variables were reported as the mean and standard deviation or median and interquartile range, as appropriate. For categorical variables, frequencies and proportions were reported. The intra- and inter-rater reliabilities were evaluated via intraclass correlation coefficients (ICC) with two-way random effects models. Bland–Altman plots


This study aimed to test the hypothesis that routine MRI ankle can be used to evaluate dimensions and correlations between dimensions of single and double fascicular variants of the ATFL and the CFL.P < .05 was considered as significant.We reviewed ankle MRIs for 251 patients. Differences between the length, thickness, width, and length of the bony attachments were evaluated twice. p < 0.001). The average values for the ATFL were thickness, 2.2 ± 0.05 mm; length, 21.5 ± 0.5 mm; and width, 7.6 ± 0.6 mm. The average values for the CFL were thickness, 2.1 ± 0.04 mm; length, 27.5 ± 0.5 mm; and width, 5.6 ± 0.3 mm. A negative correlation was found between length and width for the CFL (p < 0.001).For the ATFL, we observed a negative correlation between thickness and width, with a positive correlation between thickness and length (Routine MRI showed that most dimensions of the ATFL and CFL correlate with each other, which should be considered when planning new reconstruction techniques and developing a virtual biomechanical model of the human foot.III The ankle instability and pain after surgical ligament reconstruction is seen in about 20% of patients, which requires further treatment . Due to The MRI assessment of the thickness of the ATFL and the CFL is relevant for radiologists. The thickness may be a simple and objective factor that reflects the grade of ligament injury. The mean thickness of the ATFL reported in a previous anatomical study was 2.2 ± 0.6 mm, and the corresponding value for the CFL was 2.1 ± 0.5 mm . HoweverThis study aimed to investigate the clinical anatomy of the ATFL and the CFL and their mutual relationship. The authors hypothesized that the thickness, length, and width of single and double fascicular variants of the ATFL and the CFL would correlate with each other.This was a retrospective single center study of 251 clinically indicated MRI scans of the ankle. Our study included 143 females and 108 males; the age range was 18–62 years, with a mean of 32.7 years. The right ankle was examined in 132 cases, and the left ankle was examined in 119 cases.The MRI examinations were performed from January 2018 to May 2020 for non-traumatic indications. The imaging was performed on 1.5 Tesla (34 cases) and 3.0 Tesla (217 cases) MRI machine (Siemens and Philips). The inclusion criteria included use of a dedicated ankle coil and the availability of at least the following sequences: proton density (PD) or T2-weighted sequences without fat saturation in the sagittal, axial, and coronal planes to assess ligament structure. The other sequences, such as T1-weighted turbo spin echo (T1-TSE), PD with fat suppression, or short-T1 inversion recovery (STIR), were used to detect pathology. PD-weighted turbo spin echo (TSE): TE (the echo time) 45 ms, TR (the repetition time) 2800–5000 ms. T2-weighted (TSE) TE 60 ms, TR 3000–5000 ms. T1-weighted: TE 11.5 ms, TR 700–750 ms. Voxel 0.45 × 0.53 × 3.0 mm, slide thickness 3 mm, field of view (FOV) 14 cm. No interslice gap was in our protocol. A dedicated ankle coil was used for MRI acquisition.The patient was in supine position and the location of the ankle joint was maintained by using a dedicated coil Fig.  which waDetailed clinical evaluation of the ankle included in the referral letter. Uniform thickness, homogeneous signal, normal adjacent fat tissue, and no marrow edema or bone abnormalities indicate no trauma. MRI evaluation of the examined ligaments was done simultaneously by two experienced observers. Only normal ATFL and CFL were included in the study.The exclusion criteria Fig.  were ankIn total, 251 MRI examinations of the ankle fulfilled the inclusion and exclusion criteria  to evaluate the inter-observer agreement.First, single, and multi-fascicular ligaments were identified. Then, measurements were assessed following the same protocol Fig. . The appOn the sagittal sequence, the length of the fibular attachment of the ATFL was measured Fig. a. The leConsecutive sagittal slides were used to evaluate the CFL length and the width and length of the fibular insertion Fig. a. The leMeasurements were made at a dedicated diagnostic station using radiological software, with measures to 0.1 mm.p-value was less than .05. Repeated measures (test–retest) reliability analyses via interclass correlation coefficients (ICC)  and ICC) and 95% confidence intervals were obtained. ICC values were interpretated according to Fleiss et al. [The Kolmogorov Smirnov test was used to test for normality. A correlation between ligament dimensions and the TCA was performed using Pearson’s correlation coefficient. Differences between sex, side, and groups of single and double fascicular ligament were considered statistically significant if the s et al. , where as et al. . The datn = 193) . No other variations were identified. The average length of the ATFL was 24.5 ± 3.3 mm, and no significant differences were found between single and double fascicular ATFL (Table p < 0.001). No statistically significant differences were found regarding the length of the talar attachment. The average volume of the ATFL was 250.9 mm3, which corresponds to 0.3 mL, with statistically significant differences between groups (p < 0.001), as shown in Table p > .05).The intra-observer agreement was measured by ICC values which ranged from 0.58 to 0.89 for individual measurements Table  while th3) Figs.  and 5 anOur study revealed very weak but significant negative correlations between the length and thickness in all groups Table , Fig. 6.Variable correlation could be seen between the dimensions of the bone attachments and the other diameters. A negative correlation between the length and the thickness of the ATFL and the length of the fibular attachment in single fascicular ATFL was found Table . The lenn = 175)  CFLs was identified. The average length of the CFL was 33.7 ± 4.1 mm. Statistically significant differences (p < 0.001) between the two groups were found (Table p < 0.001) in volume were revealed, with an average value of 419.8 mm3. The average craniocaudal dimension of the calcaneal attachment of the CFL was 7.7 ± 1.3 mm, with significant difference between groups (p < 0.01) (Table p > .05).The inter-rater ICC values ranged from 0.66 to 0.78 for individual measurements Table , while tp > .05).The inter-rater ICC value for the CFL angle was 0.84  Table . The intWe revealed some statistically significant correlations between the dimensions of the CFL Tables  and 8. SThe width of the fibular attachment was correlated with CFL length, thickness, and width Table . Width wp > .05). We noted two cases of os subfibulare, where the inferior fascicle of the ATFL had an insertion on the accessory bone and a superior fascicle on the fibula.No statistically significant differences between sex, side, and groups of single and double fascicular ligaments or dimensions were found . Only one operator performed the measurements. MRI examinations were performed on two different MRI machines.Most dimensions of the ATFL and the CFL correlated with each other. There are significant differences in dimensions between single and double fascicular ligaments. Routine MRI may be used to assess ATFL and CFL correlations of dimensions. The data from this study may be used for the development of a virtual biomechanical model of the human foot.


The changes of plasma retinol binding protein 4 (RBP4) level after anutrition intervention can indicate the metabolic changes associated withthe delivered intervention.We investigated the changes in plasma RBP4 level among 12- to 18-month-oldchildren after a nutrition intervention and measured its association withsubcutaneous adiposity, maternal characteristics, and inflammation.Z score [LAZ] <−1 to −2 and 270 had LAZ score<−2) were collected from the Bangladesh Environmental Enteric Dysfunctionstudy conducted in Dhaka, Bangladesh. Multivariable linear regression andgeneralized estimation equations (GEE) modeling techniques were used tomeasure the association.Data of 520 undernourished children (250 of them had length-for-ageP > .05) with the percentage changes in child’sheight, weight, and subcutaneous adiposity. But maternal height  and milk intake  were negatively and maternal weight waspositively associated  with the changesin RBP4 levels. The GEE models revealed negative association of RBP4 levelswith C-reactive protein  andα-1-acid glycoprotein .At baseline, median RBP4 level was 19.9 mg/L (interquartile range [IQR]:7.96), and at the end of the intervention, it was 20.6 mg/L (IQR: 9.06).Percentage changes in plasma RBP4 level were not significantly associated(Children whose mothers were taller experienced less increase in plasma RBP4level, and children whose mothers had a higher weight experienced moreincrease in the RBP4 level from baseline. We have also found that CRP andAGP levels and intake of whole milk were negatively associated with theplasma RBP4 level. Plasma RBP4 was found to be reduced afterintake of hypocaloric diets.13 Nutrition scientists are directing different dietary interventions toeradicate undernutrition, in all its forms. A randomized controlled trial, conductedin Ecuador, delivered 1 egg per day for a 6-month period.14 The intervention increased length-for-age Z score (LAZ) by0.63 and weight-for-age Z score by 0.61. In the BangladeshEnvironmental Enteric Dysfunction (BEED) study, we supplemented the usual home dietof the mild, moderate, and severely stunted children with 1 boiled egg and 150 mL ofwhole milk for 90 feeding days and 1 sachet of multiple micronutrient powder dailyfor 60 feeding days.15 We found that the supplementation created a change of +0.27  in moderate/severe stunted (LAZ <−2)children and +0.19  in children withstunting (LAZ: −1 to −2).16 Although the outcome of interest of the study was improvement in lineargrowth, the changes in fatty mass and its associated changes in the markers ofmetabolic risk were also an arena to explore. Measuring the changes of plasma RBP4level after the nutrition intervention is a way to predict any metabolic changesassociated with the delivered intervention. Therefore, we investigated the changesin plasma RBP4 level among 12- to 18-month-old undernourished children after theabove-mentioned nutrition intervention and measured its association withanthropometric status, subcutaneous adiposity, socioeconomic status, and maternalcharacteristics.Undernutrition is a global public health concern. Worldwide, a total of 149 millionpreschool children are suffering from stunting.17 Plasma RBP4, which is regarded as a negative acute phase reactant, is alsofrequently found to be reduced in settings of inflammation or infection.12 Hence, the aim of the current analysis also includes measuring theassociation of plasma RBP4 with the markers of systemic and gut inflammation.Undernutrition is closely associated with infection and inflammation. Systemic andgut inflammation is metabolically expensive and can result in adverse growth outcomes.15The BEED study  is a community-basedintervention study which has been conducted in Bauniabadh slum area of Dhaka,Bangladesh. The study recruited undernourished children who were suffering frommild stunting (LAZ score <−1 to −2) or moderate/severe stunting (LAZ score<−2). Data from a total of 520 (250 mild stunting and 270 moderate/severestunting) children, enrolled between July 2016 and June 2019, were used for thisparticular analysis. The enrollment scheme is shown as a flowchart in 18 A higher WAMI index indicates a better socioeconomic status. Morbiditydata were collected daily for the duration of intervention. Diarrhea was definedas passage of ≥3 loose stools in 24 hours, and fever was defined as an axillarytemperature of >99° F (37.2 °C) by a mercury thermometer.Data on household socioeconomic status was collected from the mother/primarycaregiver of the participants. Data collectors used pretested questionnaires anddocumented the information. WAMI index —a socioeconomic statusindex which includes access to improved water and sanitation, 8 selected assets,maternal education, and household income was used as a representative ofsocioeconomic status of the households.19 Children were weighed with very minimum clothing using a Seca digitalscale , and a Seca infantometer (model no.416) wasused to measure the recumbent length to the nearest 1 mm. The LAZ for each childwas determined using the World Health Organization 2006 child growth standards.20 Harpenden skin fold calipers were used to measure the triceps skinfold(TSF) thicknesses. Standard measuring tapes were used to measure the mid upperarm circumference (MUAC). Seca stable stadiometer for mobile height measurement and Tanita step-on typeweighing scales were used to measure the height and weight of the mothers,respectively. Regular quality control sessions were organized to ensure thereliability of the measurement. We regularly measured the Intraclass correlationcoefficient (ICC) values to check the reliability of the measurers and found theICC values above 0.9 in all the cases. Measuring equipments were also calibrateddaily to maintain the validity of the measurements. Intake of egg and milk andrelated compliance data for the nutritional intervention were collected andrecorded daily throughout the intervention period.Anthropometric measurements were conducted following an established and validated protocol.21 The formula counts the upper arm area as a rectangle shaped unrolled fatrim whose length = upper arm circumference (C) and width = TSF thickness (TS)/2.Here, MUAC indicates the whole length of the total arm area or C, TSF thicknessindicates total width of TS and total upper arm area (TUA) = c2/4π. Hence, the upper arm fat area (UFA) estimate = C × (TS/2), and upperarm muscle area estimate = TUA − UFA. The formula was validated using themagnetic resonance imaging technology.Upper arm fatty and fat-free mass areas were estimated following the mathematicalformula proposed by Rolland-Cachera et al.All biological samples were collected following standard operative procedureswhich were prepared and validated for the study. A total of 5 mL venous bloodand 2 g of stool samples were collected from each of the participants before andafter the nutrition intervention. Blood samples were centrifuged at 4000rotations per minute for 10 minutes to separate the plasma. Aliquots of stooland plasma were immediately frozen at −80 °C. Plasma RBP4 was measured using acommercially available enzyme-linked immunosorbent assay (ELISA) kit (R&DSystems Inc). A face mask was used during the laboratory test to protect the kitreagents from contamination as RBP4 is also found in saliva. Plasma samples wereanalyzed for C-reactive protein , α-1-acid glycoprotein, and ferritin (ORGENTEC Diagnostika GmbH) by using commercial ELISAkits. Stool samples were analyzed for neopterin (GenWay), myeloperoxidase(Alpco), and α-1-antitrypsin  using commercially availableELISA according to kit manuals. Concentrations of each of the biomarkers werecalculated against standards provided by the manufacturers.ttest and Mann-Whitney U test were used to detect thedifferences in the continuous variables, while chi-square test was used forcomparing the categorical variables of the mildly stunted and moderate/severelystunted participants. Percentage changes of different anthropometric indices andplasma RBP4 level were calculated by deducting the after intervention valuesfrom the before intervention values, dividing the outcome by the beforeintervention values and multiplying by 100.Data analysis was conducted in R version 3.6.0 using the “dplyr,” “ggquickeda,”“ggplot2,” and “gee” packages. Exploratory data analysis was done to explore thesocioeconomic, anthropometric, and morbidity status of the participants atbaseline. Variables showing normal distribution are presented as mean andstandard deviations (SDs), whereas variables with skewed distribution arepresented as median and corresponding interquartile ranges (IQRs). Categoricalvariables are presented as frequency and percentages. Student Multivariable linear regression analysis was done to measure the association ofpercentage change of plasma RBP4 level from baseline with subcutaneousadiposity, socioeconomic status, and maternal characteristics. After estimatingthe regression model, we also performed regression diagnostics to check for theassumptions of multicollinearity, homoscedasticity, and normality of residualsof the developed model using variance inflation factor (VIF), residuals versusfitted plot (rvf plot), and quantile–quantile (Q-Q) plot, respectively. We havefound that all the VIF values were less than 2, indicating the absence ofmulticollinearity. The rvf plot showed that the heteroscedastic errors were notvividly present. The Q-Q plot indicates that the residuals were normallydistributed. The results of the postestimation analyses reveal that the modelestimated the coefficient accurately. All these results can be found inSupplementary File 1.22 The GEE method models the repeated responses collected from the sameparticipants over time and produces efficient and unbiased regressionparameters. For the reported analysis, the family was “Gaussian” and the linkfunction was “Identity.” Quasi-likelihood information criterion (QIC) was usedto select the correct covariance structure for building the GEE model, and themultivariable model with unstructured covariance matrix produced the smallestQIC value. Hence, we report the results of the multivariable model that wasconstructed using unstructured covariance matrix with robust variance estimates.We determined the strength of association by estimating the regressioncoefficients and their 95% CIs. In all analyses, a probability value <.05 wasdefined as the cutoff for statistical significance.The association between plasma RBP4 level and the markers of inflammation at 2time points (before and after the nutrition intervention) was examinedlongitudinally using population specific generalized estimation equations (GEE)modeling technique.P > .05; On average, everyday a child consumed 55 g of boiled egg and 149 mL of milk. Eachmicronutrient sachet contained 12.5 mg iron, 5 mg zinc, 300 µg vitamin A, 150 µgfolic acid, and 50 mg of vitamin C (Monimix). After taking the intervention,overall, children gained more fat in their upper arm area than the muscle mass(5.90 vs 4.09 mm). Children from the mild stunting group gained more fat (mean ±SD) in the upper arm area (6.05 ± 21.1 mm) than the moderate/severe stuntinggroup (5.79 ± 19.7 mm), whereas children with moderate/severe stunting gainedmore muscle mass (4.42 ± 9.57 mm) than the mildly stunted group (3.98 ± 8.93mm). But the differences between the groups were not statistically significantP > .05; .At baseline, median RBP4 level was 19.9 mg/L (IQR 7.96 mg/L), and at the end ofthe intervention, it was 20.6 mg/L (IQR: 9.06 mg/L). Overall, the childrengained a median of 1.73% (IQR: 45.0 mg/L) increase in plasma RBP4 level frombaseline. At baseline, moderate/severe stunting group had a higher plasma RBP4level (median [IQR]: 21.0 [9.25] mg/L) than the mildly stunted group (median[IQR]: 18.9 [7.59] mg/L). And at the end line, the values were (median [IQR]:22.1 [10.3] mg/L) and (median [IQR]: 19.6 [8.11] mg/L), respectively .P value <.05) association of the outcome variable withmaternal height, maternal weight, and milk intake. Maternal height  andmilk intake  werenegatively associated with the percentage change of plasma RBP4 level, whereasmaternal weight  waspositively associated with it. Variables indicating the changes inanthropometry, socioeconomic status, and morbidity did not show anystatistically significant (P > .05) association with theoutcome variable in the fully adjusted model.P < .05) negative association between CRP , AGP , and RBP4 levels , protein,calcium, vitamin D, and probiotics present in milk and dairy products might beresponsible for exerting the beneficial effects.23 Short chain fatty acids can independently affect lipid, glucose, andcholesterol metabolism in various tissues.29 Moreover, SCFAs and probiotics have the potential to modulate the effect ofdairy-based foods on cardiometabolic function by interacting with the microbialcommunity of the host gut.30The analyses revealed that, in addition to improving the height and weight of thechildren, a nutrition intervention comprising egg and whole milk can result inaccumulation of more subcutaneous fat than muscle mass in the upper arm area. Butthe intervention did not increase the risk of alteration of metabolic profile as thelevels of RBP4 in plasma did not change significantly after the nutritionintervention. Moreover, milk intake was found to be negatively associated with thepercentage change of plasma RBP4 level, indicating a protective change against thedevelopment of cardiometabolic risk. Whole milk is one of the top sources ofsaturated fat. Although different dietary guidelines recommended reducing intake ofsaturated fat for a better cardiometabolic health,31 The “Developmental Origins of Adult Health and Disease” hypothesis statesthat children facing any unfavorable conditions during the early postnatal periodsmight be at a greater risk of developing adulthood diseases.32 However, the vectors that might potentially modify the mechanisticunderpinnings of the phenomenon are yet to be explored and genetic and epigeneticfactors are the potential candidates for that exploration. It has recently beenhypothesized that maternal diet, which is directly related to the maternal obesity,might alter the methylation status of insulin-like growth factors and related genesin infants.33 Although more studies are needed to prove or refute the hypothesis,scientists have shown that maternal overnutrition could program the metabolicphenotypes of the offspring through sperm tRNA-derived small RNAs.34 Overall, this is an interesting area where we have tried to shed the light,and molecular level studies are now required to explore the exact relation.We have found that children, whose mothers were taller, experienced less increase inplasma RBP4 level from baseline than their counterparts. On the other hand,children, whose mother had a higher weight, experienced more increase in the RBP4level. There is a paucity of data for undernourished infants that might echo ourfindings. But it has been shown that among the older children (aged 6-11 years),maternal obesity exerted a 2-fold risk of developing metabolic syndrome.36 Accordingly, the circulating levels of RBP4 show stronger correlation to thequantity of visceral fat than the subcutaneous fat.36 Hence, changes in subcutaneous fat are unlikely to show any strongassociation with the RBP4 level. Multivariable linear regression done in our studyreveals the same as we have found that changes in fat and fat-free mass of upper armarea were not significantly associated with the changes in plasma RBP4 level.In addition to liver, adipose tissue also synthesizes and secretes RBP4, and theexpression is higher in visceral fat compared to subcutaneous fat.37 Cow’s milk and egg yolk are known to be the rich sources of fatty acids.38 Hence, regular intake of whole milk and chicken eggs might play importantroles in combating inflammation as our study showed a declining trend in the levelsof inflammatory biomarkers after the nutritional intervention. The GEE analysesrevealed that plasma RBP4 level had a statistically significant negative associationwith CRP and AGP. Very few studies have explored the role of RBP4 in systemicinflammation, more so in the absence of obesity and related metabolic alterations. Areview paper published on the role of RBP4 in inflammation revealed mixed results.Of 21 papers reviewed, only 2 studies reported negative relations and 4 studiesreported positive association. But the rest of the studies (15) could not find anyrelation between RBP4 and CRP.40 But being a negative acute phase reactant, the negative association ofcirculatory levels of RBP4 with CRP and AGP implies biological plausibility. Theassociation of RBP4 with the rest of the inflammatory markers showed statisticallyinsignificant relationships with negligible effect sizes.From the descriptive analysis, we have found that the levels of RBP4 increased afterthe nutrition intervention, whereas the levels of most of the positive acute phasereactants (those which increase in response to inflammation) decreased. Thesefindings indicate that intake of animal protein based foods might impact theinflammation process by modifying the actions of positive and negative acute phaseproteins. A Mediterranean diet that contains high amount of monounsaturated and ω-3polyunsaturated fatty acid has shown similar anti-inflammatory effects.This is the first study that has compared the before–after changes of plasma RBP4level among the undernourished children and measured its association with the bodycomposition, biomarkers of inflammation, and maternal nutritional status. But wecould not compare those changes with age and sex matched healthy cohort of controlchildren. Moreover, we did not measure visceral adiposity and serum levels ofvitamin A. These are the limitations of our study.In summary, our study reports that plasma RBP4 level, a marker of metabolic risk andinsulin resistance, is positively associated with mother’s weight but negativelywith mother’s height. Intake of whole milk is negatively associated with thepercentage change of plasma RBP4 level after the nutrition intervention. We havealso found that the markers of systemic inflammation were negatively associated withplasma RBP4 level.Click here for additional data file.Supplemental Material, Supplementary_file for Changes in Retinol Binding Protein4 Level in Undernourished Children After a Nutrition Intervention Are PositivelyAssociated With Mother’s Weight but Negatively With Mother’s Height, Intake ofWhole Milk, and Markers of Systemic Inflammation: Results From a Community-BasedIntervention Study by Subhasish Das, Md Amran Gazi, Md Mehedi Hasan, ShahMohammad Fahim, Md Ashraful Alam, Md Shabab Hossain, Mustafa Mahfuz and TahmeedAhmed in Food and Nutrition Bulletin