Accordingly, there is an urgent need to find out reliable specific biomarkers for PD. GPR37 is an orphan G protein-coupled receptor that toxically accumulates in autosomal recessive juvenile parkinsonism. Here, we investigated whether GPR37 is upregulated in sporadic PD, and thus a suitable potential biomarker for PD.GPR37 protein density and mRNA expression in postmortem substantia nigra (SN) from PD patients were analysed by immunoblot and RT-qPCR, respectively. The presence of peptides from the N-terminus-cleaved domain of GPR37 (i.e. ecto-GPR37) in human cerebrospinal fluid (CSF) was determined by liquid chromatography-mass spectrometric analysis. An engineered in-house nanoluciferase-based immunoassay was used to quantify ecto-GPR37 in CSF samples from neurological control (NC) subjects, PD patients and Alzheimer’s disease (AD) patients.GPR37 protein density and mRNA expression were significantly augmented in sporadic PD. Increased amounts of ecto-GPR37 peptides in the CSF samples from PD patients were identified by mass spectrometry and quantified by the in-house ELISA method. However, the CSF total α-synuclein level in PD patients did not differ from that in NC subjects. Similarly, the cortical GPR37 mRNA expression and CSF ecto-GPR37 levels in AD patients were also unaltered.GPR37 expression is increased in SN of sporadic PD patients. The ecto-GPR37 peptides are significantly increased in the CSF of PD patients, but not in AD patients. These results open perspectives and encourage further clinical studies to confirm the validity and utility of ecto-GPR37 as a potential PD biomarker.The online version contains supplementary material available at 10.1186/s40035-021-00232-7. Parkinson’s disease (PD) is the second most common type of neurodegenerative disorder , with haPARK2 gene involved in ubiquitination and proteasome-mediated degradation/clearance of misfolded proteins /μl in ELISA buffer) for 1 h at 22 °C in the dark. Finally, the plates were washed (3 × 1 min) with ELISA buffer and 90 μl of ELISA buffer was added to each well before incorporating 10 μl of ELISA buffer solution containing 10 μM coelenterazine 400a . After 1-min incubation, the end-point luminescence was determined using a CLARIOstar Optima plate-reader  and the output luminescence was reported as the integral RLU. In addition, a standard curve was generated using known concentrations of either mouse or human ecto-GPR37SNAP in each experiment.CSF ecto-GPR37 was measured with the home-made competitive ELISA method. In brief, streptavidine-coated 96-well white plates (ThermoFisher Scientific) were blocked with serum-free DMEM medium (100 μl/well) for 1 h at 22 °C. After washing (3 × 1 min) with ELISA buffer , the wells were coated with 100 μl of anti-rabbit IgG-biotin antibody  in the ELISA buffer for 2 h at 22 °C. Subsequently, the plates were washed with the ELISA buffer (3 × 1 min) and incubated with rabbit anti-GPR37 (mouse) or rabbit anti-GPR37 (human) antibody (1 μg/ml in ELISA buffer) for 2 h at 22 °C. Then, 3 μl of either mouse or human CSF was added to each well containing 47 μl of ELISA buffer and incubated overnight at 4 °C in a humid chamber. Next day, the plates were washed  with ELISA buffer and incubated with 50 μl of purified ecto-GPR37The α-synuclein concentration was measured using the LEGEND MAX™ Human α-Synuclein ELISA Kit  according to the manufacturer’s instructions. Briefly, a microtiter plate pre-coated with the capture antibody was washed 4 times, added with 20 μl of CSF at 1:10 dilution in reagent buffer, and incubated overnight at 4 °C with shaking. Next day, the plate was washed 4 times, followed by addition of the biotinylated detection antibody to each well and incubation for 2 h at room temperature (RT). The unbound detection antibody was removed by 4 washes. The streptavidin-HRP conjugate was added to each well, incubated for 1 h at RT and then the plate was washed 4 times to remove the remaining conjugate. Finally, chemiluminescent substrate was added to each well and signals were read using a Tecan Spark 10 M plate reader .t test or one-way analysis of variance (ANOVA) with Dunnett’s post-hoc test using GraphPad Prism 6.01 . P < 0.05 was considered as statistically significant. The distribution of CSF ecto-GPR37 did not follow Gaussian distribution , so the data were analysed with non-parametric Mann-Whitney U-tests. The diagnostic accuracy by CSF ecto-GPR37 levels was assessed using the receiver operating characteristic (ROC) curve analysis. The correlation coefficients (rho) were calculated using the Spearman two-tailed correlation test.Data are presented as mean ± standard error of mean (SEM) and were analysed by Student’s https://www.cellsignal.com/products/primary-antibodies/gpr37-d4c8h-rabbit-mab/64407). These results indicate that in human native tissues (e.g. SN), GPR37 may be N-terminally processed, thus suggesting the existence of a heterogenous pattern of GPR37 species.Metalloprotease-mediated GPR37 N-terminus cleavage leads to ectodomain shedding in a heterologous expression system . This poP = 0.0057) increased at Braak PD stage 6  when all the GPR37 forms (i.e. precursor plus N-terminus cleaved forms) were considered  3.1-fold increase in GPR37 mRNA expression in SN of Braak PD stage 5/6 subjects as compared to the NC subjects . Six unique peptides corresponding to the N-terminal domain of the human GPR37 in pooled CSF samples from NC and PD patients were detected  in the CSF. To confirm the identity of ecto-GPR37 in CSF, we immunoprecipitated ecto-GPR37 using the anti-GPR37-N antibody and the isolated peptides were analysed by liquid chromatography coupled to mass spectrometry . Subsequently, we determined the ecto-GPR37 density in mouse and human CSF. First, CSF from wild-type and GPR37−/− mice was assayed with the specific mouse GPR37-NL-based ELISA assay. While significant amount of ecto-GPR37 was detected in the CSF from GPR37+/+ mice, which was determined to be 286.8 ± 77.3 pg/ml, ecto-GPR37 was undetectable in all CSF volumes from the GPR37−/− mice assayed . First, we engineered specific mouse or human GPR37 constructs containing HA and NL tags at the N-terminus  of NC and PD subjects using the specific human GPR37 NL-based ELISA assay. While the NCs had an average ecto-GPR37 content of 185 ± 17 pg/ml in the CSF, the PD patients had 282 ± 18 pg/ml ecto-GPR37 in the CSF. Thus, ecto-GPR37 was significantly (P = 0.019) increased in the CSF of PD patients  and PD (n = 41) subjects was assayed. Again, significantly higher levels of CSF ecto-GPR37 were found in the PD subjects (334 ± 28 pg/ml) when compared to the NCs (159 ± 21 pg/ml) (P = 0.001)  from NCs (n = 45), with the area under the curve (AUC) of 0.808 (0.713–0.900) (P < 0.0001)     by RT-qPCR. There were no significant changes in GPR37 mRNA expression in either FC or EC among NC, AD3/4 and AD5/6 subjects  was calculated using the Spearman two-tailed correlation test. Supplementary Figure S2. Correlations of CSF ecto-GPR37 levels with disease duration (a), Unified Parkinson’s Disease Scale  total (b) and part 3 (c), Hoehn & Yahr (H&Y) scale (n = 40) (d), Montreal Cognitive Assessment scores  evaluating the cognitive impairment (e), Hospital Anxiety and Depression scale  (f) and L-dopa equivalent doses  (g). The correlation coefficients (r) was calculated using the Spearman two-tailed correlation test.


The aim of the study was to determine the between-trial and between-day reliability of the Glazer protocol and our multi-activity surface electromyography (sEMG) measurement protocol for pelvic floor muscle (PFM) evaluation. The bioelectrical activity of PFM was collected using an endovaginal electrode in 30 young, Caucasian, nulliparous women . The between-trial and between-day reliability of the original Glazer protocol and the new multi-activity sEMG protocol were assessed during the following phases: pre-baseline rest, phasic (flick) contractions, tonic contractions, endurance contraction, and post-baseline rest. The Glazer protocol was characterized by poor and moderate measurement reliability. The time-domain parameters for the rise and fall of the signal amplitude and median frequency showed poor between-trial and between-day reliability. The mean and peak amplitudes indicated mainly good between-trial and moderate between-days reliability. Our protocol showed moderate to excellent reliability of both time-domain and quantitative parameters of muscle recruitment. In our protocol, the frequency-domain parameters describing muscle fatigue demonstrated much higher reliability than in the case of the Glazer protocol. The most important information obtained in this study was the significant improvement of diagnostic validity in PFM bioelectrical activity evaluation. The higher reliability of our sEMG protocol compared to original Glazer protocol allowed us to suggest that protocol modifications and changes in sEMG signal processing methods were effective in the improvement of PFM assessment quality. The new parameters calculated from the sEMG signal proposed in our sEMG protocol allowed us to obtain additional clinically important information about PFM dysfunctions regarding specific deficits of muscle contraction such as decrease in muscle strength; endurance or coordination related to, e.g., stress urinary incontinence; or pelvic floor muscle imbalance after childbirth. The pelvic floor muscles (PFM) have dual function—providing trunk stability and continence ,2,3. It Some authors have assessed the reliability of PFM bioelectrical activity, but the vast majority took only selected types of muscle contraction into account, such as short maximal contraction or endurance contraction lasting a several dozen seconds ,14,18,19r = 0.86, p < 0.001) of sEMG amplitude only between two measurements. Therefore, due to the weak statistical methods employed, these findings regarding reliability should be considered with caution.In the literature, the reliability of PFM sEMG measurements are equivocal. In some studies, good reliability has been reported regarding the sEMG signal evaluated both qualitatively and quantitatively ,14,18,19Most often in subjects suffering from incontinence, we can observe complex changes in PFM neuromuscular control, strength, endurance, and coordination. Therefore, for proper diagnosis, a complex evaluative approach is required ,10,15,21When sEMG is used clinically as well as in research, the reliability of this measurement is fundamental for interpretation of the obtained results. Despite the common use of sEMG in PFM function evaluation, the reliability of this measurement has not been fully established. Therefore, there is a need to verify the reliability of PFM sEMG evaluation including different types, strength, and duration of muscle contractions, and also to reduce the sEMG signal variability by appropriate signal processing.Because there is a lack of studies in which the reliability of PFM sEMG assessment would be comprehensively evaluated, in this study, the aforementioned issue is undertaken for the first time. The purpose of this study is to determine the between-trial and between-day reliability of the multi-activity sEMG measurement protocol for PFM clinical evaluation, which includes all phases described by Glazer et al. . MoreoveIn this study, 30 young, Caucasian, nulliparous women  without pelvic floor muscle ailments were evaluated. They were recreationally active and did not engage in regular physical training. They did not have any symptoms of urinary incontinence and did not experience any spinal pain in the 6-month period prior to enrolment in the study. They were informed in detail about the research protocol and gave their written informed consent to participate in the study. All procedures were performed in accordance with the 1964 Helsinki declaration and its later amendments. Approval of the Ethical Committee of Rzeszów University (4 January 2015) was obtained for this study.Bioelectrical activity of the PFM was collected using the Life-Care two-sided endovaginal electrode . The signal was registered with 16-bit accuracy at a sampling rate of 1500 Hz using the Noraxon G2 TeleMyo 2400 unit  ,25.PFM activity was recorded in supine position with a pillow underneath the participant’s head. The hips and knees were gently flexed, supported by a pillow under the knees, and the lumbar spine was in a neutral position. The women were asked to empty their bladder prior to electrode application. After electrode application, to better familiarize the subjects with testing procedures, we got them to perform a short trial of phasic, tonic, and endurance contractions. After 10 min of rest in supine position, the measurement was performed. During all measurements, participants were verbally instructed to perform the PFM contraction without the use of the abdominal, gluteal, or hip adductor muscles.Visit 1—For the between-trial reliability, 2 measurements were performed with 15 min of rest between them. The reliability was calculated between the first and second measurement.Visit 2—For the between-day reliability, 1 measurement was performed 2–3 days later, during the second visit in the laboratory. The reliability was calculated between the first measurement from Visit 1 and the measurement from Visit 2.One 60-s rest (pre-baseline).Five phasic (flick) contractions (2-s contraction with a 2-s rest in-between).Five phasic (flick) contractions (2-s contraction with a 10-s rest in-between).Five 10-s tonic contractions, with a 10-s rest in-between.One 60-s endurance contraction.One 60-s rest (post-baseline).The between-trial and between-day reliability of the original Glazer protocol and the new multi-activity sEMG protocol were performed during a single measurement. The measurement of PFM sEMG activity included all phases required by both protocols:One 60-s rest (pre-baseline)—the women were instructed to feel the PFM in rest.Five phasic (flick) contractions—the women were instructed to contract the PFM as quickly as possible (2-s contraction with a 2-s rest in-between).Five 10-s tonic contractions, with a 10-s rest in-between—the women were instructed to contract the PFM as strongly as possible, hold the contraction for 10 s, and then fully relax the PFM after contraction remaining relaxed for 10 s.One 60-s endurance contraction—the women were instructed to contract the PFM at such a level as to hold it for 60 s.One 60-s rest (post-baseline)—the women were instructed to feel the PFM in rest.The Glazer protocol consisted of 5 activities :One 60-sAverage mean amplitude (μV)Mean amplitude variability (%)One 60-s rest (pre-baseline):Average peak amplitude (μV)—the result was the mean value from 5 contractions.Time before peak (s)—the result was the mean value from 5 contractions.Time after peak (s)—the result was the mean value from 5 contractions.Five 2-s phasic (flick) contractions with 2-s rest in-between:Average mean amplitude (μV)—the result was the mean value from 5 contractions.Average peak amplitude (μV)—the result was the mean value from 5 contractions.Time before peak (s)—the result was the mean value from 5 contractions.Time after peak (s)—the result was the mean value from 5 contractions.Five 10-s tonic contractions, with a 10-s rest in-between:Median frequency (Hz)Average mean amplitude (μV)Mean amplitude variability (%)One 60-s endurance contraction:Average mean amplitude (μV)Mean amplitude variability (%)One 60-s rest (post-baseline):The following sEMG signal parameters were calculated for the Glazer protocol :One 60-sOne 60-s rest —the women were instructed to feel the PFM in rest.Five 2-s phasic (flick) contractions, with a 10-s rest in-between—the women were instructed to contract the PFM as quickly as possible, and then quickly and fully relax the PFM immediately after contraction.Five 10-s tonic contractions, with a 10-s rest in-between—the women were instructed to contract the PFM as strongly as possible, hold the contraction for 10 s, and then fully relax the PFM after contraction remaining relaxed for 10 s.One 60-s endurance contraction—the women were instructed to contract the PFM at such a level as to hold it for 60 s.One 60-s rest (post-baseline)—the women were instructed to feel the PFM in resting position.Our multi-activity sEMG protocol consisted of 5 activities:Average mean amplitude (μV)—the value was calculated separately for each of the 3 intervals: I-5s, II-5s, III-50s.Mean amplitude variability (%)—the value was calculated separately for each of the 3 intervals: I-5s, II-5s, III-50s.One 60-s rest (pre-baseline):Average peak amplitude from contraction phase (μV)—the result was the mean value from 5 contractions.Onset to offset time (s) (contraction duration)—the result was the mean value from 5 contractions.Average mean amplitude from rest in-between phase (μV)—the result was the mean value from 5 rests.Onset to peak time (s) (time of amplitude increase)—the result was the mean value from 5 contractions.Peak to offset time (s) (time of amplitude decrease)—the result was the mean value from 5 contractions.Five 2-s phasic (flick) contractions with 10-s rest in-between:Average mean amplitude (μV)—the result was the mean value from 5 contractions.Mean amplitude variability (%)—the result was the mean value from 5 contractions.Median frequency (Hz)—the result was the mean value from 5 contractions.Mean frequency (Hz)—the result was the mean value from 5 contractions.Five 10-s tonic contractions, with a 10-s rest in-between:Median frequency (Hz)—the result was the mean value from 6 intervals lasting 10 s each.Mean frequency (Hz)—the result was the mean value from 6 intervals lasting 10 s each.Average mean amplitude (μV)—the result was the mean value from 6 intervals lasting 10 s each.Mean amplitude variability (%)—the result was the mean value from 6 intervals lasting 10 s each.One 60-s endurance contraction:Average mean amplitude (μV)Mean amplitude variability (%)One 60-s rest (post-baseline):The following sEMG signal parameters were calculated for our sEMG protocol:The sEMG data were filtered using the built-in hardware first-order high-pass filter set to 10 Hz ± 10% cut-off. The raw sEMG data were visually checked for artefacts. The root mean square (RMS) value was determined over a 200-msec window ,25. Thenr) for both between-trial and between-day comparisons. The level of statistical significance was set at (p < 0.05).Statistical analysis was carried out using the STATISTICA 12.0 software. To assess the normality of variables distribution, we performed the Shapiro–Wilk test. The between-trial and between-day reliability of the sEMG variables were determined using intraclass correlation coefficients (ICC). We used the ICC  model according to Shrout and Fleiss . The intr) between the first and second measurements were noted (r = 0.52–0.91)  .r) compared to their between-trial values. time before peak, time after peak, and the mean amplitude variability during the pre-baseline rest and during endurance contraction phases demonstrated poor reliability (ICC ranged from 0.08 to 0.44 and r ranged from 0.08 to 0.39)  . The rel.37–0.76 .All phases of our protocol demonstrated higher reliability compared to the Glazer protocol. Muscle timing parameters such as onset to offset time, onset to peak time, and peak to offset time showed good and excellent reliability (ICC = 0.85–0.91). The average mean and peak amplitude during rest, flick, tonic, and endurance contractions presented good and excellent reliability (ICC = 0.80–0.95) . In our The between-day reliability of our protocol was slightly lower compared to between-trial values. Muscle timing parameters such as onset to offset time, onset to peak time, and peak to offset time showed moderate and good reliability (ICC = 0.66–0.90). The average mean and peak amplitude during rest, flick, tonic, and endurance contractions presented moderate and good reliability (ICC = 0.62–0.80) . The relThe most important observation of this study is that the Glazer protocol commonly used to assess PFM bioelectrical activity is characterized by poor and moderate measurement reliability. The time-domain parameters for the rise and fall of the signal amplitude proposed by Glazer showed poor reliability, which means that timing analysis of the sEMG signal in the original Glazer protocol did not provide information on the temporal characteristics of PFM recruitment. This problem concerns both the between-trial and between-day reliability. Moreover, sEMG signal analysis of muscle fatigue assessment in a 60-s isometric contraction was inconclusive. The poor reliability of the median frequency did not provide useful information regarding fatigue of the tested muscles. Other parameters characterizing the PFM strength  indicated mainly good between-trial and moderate between-day reliability. Thus, verification of the measurement reliability of all phases of the original Glazer protocol and all parameters proposed by the authors of this test indicated its moderate diagnostic value.In this work, the modification of the test performance itself in relation to the Glazer protocol was not large. The greatest changes were made for the registered sEMG signal processing. The main element of our protocol compared to Glazer’s proposal was determination of many additional sEMG signal parameters, which, according to the sEMG signal analysis methodology, allow for assessment of muscle recruitment timing, fatigue, or endurance. In individual phases of the protocol, many additional sub-phases were added, which enabled the calculation of signal parameters not only for the entire phase, but also took signal variability into account during its duration by separate analysis of relevant sub-phases. This approach allowed to largely eliminate errors resulting from incorrect determination of phase boundaries in the protocol and from natural sEMG signal variability.The results obtained in this way showed moderate to excellent reliability of both time-domain and quantitative parameters of muscle recruitment. The time-domain parameters proposed in our protocol characterizing the muscle recruitment during flick and tonic contractions allowed for reliable assessment of the PFM condition. Moreover, the method of quantitative analysis of the signal amplitude, indicating the amount of muscle recruitment, allowed us to obtain reliable information. In our protocol, the frequency-domain parameters describing muscle fatigue demonstrated much higher reliability than in the Glazer protocol. The sEMG signal processing for each of the six 10-s parts separately and then the averaging the result for them significantly improved the reliability.Moreover, the diagnostic value of the original Glazer protocol was verified in this study for the first time. The measurement reliability of all phases in the Glazer protocol has not yet been assessed, even by the test author himself. Glazer et al.  reportedIn this study, for most parameters of the Glazer protocol compared to our protocol, lower reliability coefficients were obtained. Moreover, our protocol demonstrated only slight deterioration of between-day ICC values compared to between-trial ICC. In the Glazer protocol, the between-day ICCs were much lower than the between-trial ICCs. This may have been due to the fact that in the Glazer protocol, the natural variability of the sEMG signal was additionally augmented by the variability resulting from improper signal processing. Within our protocol, a decrease in between-day ICC was also observed, however, only for a few parameters, and this decrease was much smaller than in the Glazer protocol. Perhaps such a result was obtained thanks to the more complex sEMG analysis, taking many specific aspects of signal processing not used in the Glazer protocol into account.r = 0.20–0.58 (bipolar) and r = 0.37–0.82 (unipolar), respectively, and normalization of the sEMG amplitude did not significantly improve the obtained values [Auchincloss et al.  analyzedr = 0.76–0.97, and the authors did not provide a coefficient of variation for the signal amplitude [Many works to date in which the reliability of intravaginal PFM bioelectrical activity has been evaluated have had some methodological errors. Thorp et al.  examinedmplitude .r = 0.86, p < 0.001). However, the correlation coefficient did not reflect the real measurement reliability because it did not take such factors as signal amplitude variation or measurement error into account. Aukee et al. [r = 0.84–0.97, p < 0.05), however, the authors underlined that the qualitative visual analysis of the signal collected from the muscles indicated its very large variability [r = 0.37–0.42. Moreover, for our protocol, the reliability presented lower values than Glazer et al. [r = 0.58.Thompson et al.  observedr et al.  and AukeThe following factor, which may significantly influence the results, is the group homogeneity. In Glazer’s study , the groAnother problem associated with the clinical utility of PFM activity testing is the sequences and type of muscle contractions. There is no doubt that PFM dysfunction is very complex and occurs with disruption of both PFM strength as well as the endurance and coordination of these muscles . TherefoThe limitation of this research is the fact that the study group consisted of young, nulliparous women, aged 20–27 years, without PFM dysfunction. Therefore, due to the better sEMG signal quality measured for non-dysfunctional muscles compared to muscles with dysfunction, the PFM sEMG reliability reported in our study may be higher than in women after childbirth, or in those above the age of 40, in which the bioelectrical activity of PFM may present greater signal variability.The most important information obtained in this study was the significant improvement of diagnostic validity in PFM bioelectrical activity evaluation. The high reliability of our sEMG protocol allowed us to suggest that protocol modifications and changes in sEMG signal processing in our methods were effective in the improvement of PFM assessment quality. The new parameters calculated from the sEMG signal which were proposed in our sEMG protocol allowed us to obtain additional clinically important information about PFM dysfunctions, regarding specific deficits of muscle contraction such as decrease in muscle strength; endurance or coordination related to e.g., stress urinary incontinence; or pelvic floor muscle imbalance after childbirth. Moreover, in this study, the reliability of all phases in the original Glazer protocol was evaluated.There is a lack of studies in which the reliability of PFM sEMG assessment would be comprehensively evaluated. The purpose of this study was to determine the between-trial and between-day reliability of the multi-activity sEMG measurement protocol for PFM clinical evaluation with the broad aspect of muscle contraction and full signal processing standardisation.The higher reliability of our sEMG protocol compared to original Glazer protocol allowed us to suggest that protocol modifications and changes in sEMG signal processing methods were effective in the improvement of PFM assessment quality. The new parameters calculated from the sEMG signal that were proposed in our sEMG protocol allowed us to obtain additional clinically important information about PFM dysfunctions


Of the measured cytokines in the CSF, only IL-10 levels were significantly increased in NS patients compared to NNS patients (p < 0.001). In a subgroup analysis, the CSF levels of IL-10 were significantly elevated in SNS patients compared to ANS and NNS patients . The CSF IL-10 levels had a significant correlation with the markers of microglial activation and neuronal injury, and they also correlated with CSF rapid plasma reagin (RPR) titer, CSF white blood cell (WBC) count, and CSF protein concentration. The areas under the ROC curve (AUC) of CSF IL-10 in the diagnosis of NS and ANS were 0.920 and 0.891, respectively. The corresponding sensitivities/specificities were 86.7%/91.7% and 83.3%/91.7%, respectively. Therefore, the excessive production of IL-10 might facilitate bacterial persistent infection, play an important role in the pathogenesis of NS, and associate with the progression of the disease. CSF IL-10 concentration had a useful value in the diagnosis of NS, especially in ANS.The aim of this study was to examine the cerebrospinal fluid (CSF) concentrations of proinflammatory and anti-inflammatory cytokines in neurosyphilis (NS), analyze the differences between asymptomatic NS (ANS) and symptomatic NS (SNS), and explore the diagnostic value of these cytokines. We enrolled 45 patients with a diagnosis of NS, including 18 patients with ANS and 27 patients with SNS, whose cerebrospinal fluid (CSF) samples were collected before penicillin therapy. Twelve patients with syphilis but non-NS (NNS) were also included. We measured the CSF levels of interleukin- (IL-) 1 Treponema pallidum infection, which can induce chronic inflammation to the cardiovascular system, the skeletal system, the nervous system, and other human organ systems [T. pallidum could invade the CSF early in the course of disease [T. pallidum could be removed from the CSF by itself in most patients, while those who failed to remove T. pallidum were asymptomatic neurosyphilis patients (ANS) and may developed into SNS [Syphilis is a chronic and systemic sexually transmitted disease caused by  systems , 2. Neur systems , 4. CSF  disease . It was into SNS , 7. HoweT. pallidum infection. T. pallidum infection can cause a strong host-specific immune response as well as the secretion of a large number of proinflammatory cytokines to clear the spirochetes [The cell-mediated immune response plays an important role in the natural course of rochetes , 9. Howerochetes . Thus, tβ, IL-6, IL-17A, IL-21, and TNF-α) and anti-inflammatory (IL-4 and IL-10) cytokines in patients with SNS, ANS, and NNS and determined their association with markers of microglia activation and neuronal injury (sTREM2 and NFL), aiming to investigate the roles of these factors in the pathogenesis and progression of NS. We further investigated the value of CSF cytokines in diagnosing neurosyphilis.In this report, we detected proinflammatory /fluorescent treponemal antibody absorption (FTA-ABS), and (iii) elevated CSF WBC count (>5/μL) or elevated CSF protein concentration (>45 mg/dL) in the absence of other known causes of these abnormalities. The exclusion criteria were as follows: treatment with antibiotics within the last 1 month, and other infectious diseases , autoimmune diseases, and neurodegenerative diseases.The diagnostic criteria for neurosyphilis was based on the guidelines of CDC in the USA and Europe and related literatures –14. ConfAll the enrolled patients underwent lumbar puncture to rule out neurosyphilis, because they either had neurological and/or ophthalmic symptoms/signs or had a serofast status without clinical symptoms/signs. CSF samples were sent for routine examination  and syphilis testing . For the purpose of this study, we divided the NS patients into the ANS group and the SNS group according to whether they had clinical symptoms or not.β, IL-4, IL-6, IL-10, IL-17A, IL-21, and TNF-α. The minimum detection limits were as follows: IL-1β—0.15 pg/mL; IL-4—0.51 pg/mL; IL-6—0.047 pg/mL; IL-10—0.31 pg/mL; IL-17A—0.62 pg/mL; IL-21—0.1 pg/mL; and TNF-α—0.39 pg/mL. In addition, the levels of the sTREM2  and NFL  in CSF were determined by ELISA with a minimum detection limit of 10.5 pg/mL and 7.8 pg/mL, respectively.The CSF samples were centrifuged and stored at -80°C until biomarker measurements were executed. The samples were simultaneously assayed using the MILLIPLEX® MAP Human High-Sensitivity Cytokine/Chemokine Panels  for the following cytokines: IL-1U test when the data were nonparametric or independent two-sample t-test when the data were parametric. Gender differences were assessed by the chi-square test. Correlations were assessed via Spearman's rank correlation coefficient. Receiver operating characteristic (ROC) curves were used to evaluate the diagnostic values. The Youden index was calculated to determine the cut-off value. A two-sided p value < 0.05 was considered statistically significant. The RPR titer measured by the double-dilution method was applied to analyze after a log transformation (log2 1/RPR titer).Statistical analysis was performed by SPSS software (IBM SPSS 25.0 version) or Prism (GraphPad software 7.0 version). Between-group comparisons of continuous data were performed with the nonparametric Mann–Whitney μL), and 12 of them had an increased protein CSF concentration (≥45 mg/dL). Eighteen NS individuals had a negative CSF RPR test result (presumptive NS). Fifteen patients from the presumptive NS group had CSF pleocytosis, and 13 patients had elevated CSF protein concentrations. Twenty-seven patients with SNS were enrolled, including those with general paresis (n = 18), meningovascular NS (n = 5), tabes dorsalis (n = 3), and meningeal NS (n = 1). Eighteen patients with CSF abnormalities had no neurological symptoms, while they were diagnosed as having ANS.Forty-five patients with NS and 12 patients with NNS were included in this study. The clinical characteristics are summarized in p = 0.112 and p = 0.086, respectively). NS patients when compared with NNS patients were characterized by significantly higher levels of serum RPR titer, CSF WBC count, and CSF protein concentration . SNS patients had a higher rate of positive CSF RPR than ANS patients (p < 0.001). No significant differences were observed regarding the serum RPR titer, CSF WBC count, and CSF protein concentration between SNS and ANS patients.There was no significant difference in age and gender between the NS group and the NNS group (p < 0.001). Notably, in the NS subgroup analysis, the CSF levels of IL-10 were significantly elevated in SNS patients compared to ANS and NNS patients , and ANS patients had significantly higher IL-10 levels than NNS patients (p < 0.001) (p = 0.103). There was a trend of statistical difference in the CSF levels of IL-6 between the SNS group and the NNS group (p = 0.055). The CSF levels of IL-17A were below the detection limit. The CSF contents of IL-1β, IL-4, IL-21, and TNF-α were comparable between the NS group and the NNS group . The CSF levels of cytokines in patients with SNS, ANS, and NNS are shown in Patients with NS had significantly higher levels of CSF IL-10 when compared with the NNS group (< 0.001) . Comparep < 0.001). In the NS subgroup analysis, the CSF levels of sTREM2 were much higher in the SNS group than in the ANS and NNS groups . The CSF levels of sTREM2 were also significantly higher in the ANS group than in the NNS group (p = 0.009) (The levels of sTREM2 in the CSF of NS patients were significantly higher than those of NNS patients (= 0.009) .p < 0.001). When we analyzed the NS patients separately, the CSF NFL levels in the SNS group were significantly higher than those in the ANS and NNS groups . The CSF NFL levels in ANS patients were also higher than those in NNS patients (p = 0.017)  . The CSFr = 0.534, p < 0.001)    and CSF < 0.001) .r = 0.616, p < 0.001)  (2 (1/serum RPR titer)  (CSF levels of IL-10 were positively correlated with CSF WBC count (< 0.001)  and CSF < 0.001) . We also= 0.013) .p < 0.001)  was 0.920  . The optp < 0.001)  , and thep < 0.001; AUC = 0.826, 95% CI 0.684-0.968, p = 0.001; respectively)   [Microglia are the “inspectors” of CNS, which continuously monitor the CNS environment, responding to any disturbance in neuronal homeostasis. Once harmful stimuli, such as infection, trauma, and toxic stimuli, are identified, microglia are activated rapidly –31. At dd TNF-α) , 34, whid TNF-α) , 36. Micd TNF-α) , 38. Ourβ phagocytosis by microglia [β load in the transgenic mouse brains, via phagocytosis by activated microglia [IL-10 has become a potential therapeutic target for several neurological disorders. The intracranial administration of IL-10 improved the outcome of EAE . Howevericroglia , while ticroglia . TherefoAt present, the diagnosis for NS remains a major clinical challenge. There is no “gold standard” to establish or exclude the diagnosis of NS. The laboratory diagnosis of NS is based on the abnormal results of CSF immunologic tests and on the elevation of CSF WBC count and protein concentrations. A reactive CSF Venereal Disease Research Laboratory (VDRL) test is generally considered specific for NS. However, the CSF VDRL test has low sensitivity , 42. In Some limitations in our study need to be mentioned. First, the sample size was relatively small. The studies should be verified in a large-scale patient population. Second, the results of blood proinflammatory and anti-inflammatory cytokine profiles were lacking because of the absence of serum samples. Third, the dynamic changes of these cytokines after treatment should be included in this study. Further studies are expected to address these issues.The study found that the CSF IL-10 levels in NS patients were significantly increased compared to those in NNS patients, and they peaked in SNS patients. The CSF IL-10 levels had positive correlation with microglial activation and neuronal injury markers and also with CSF RPR titer, CSF WBC count, and CSF protein concentration. These results indicate that the excessive production of CSF IL-10 might facilitate bacterial persistent infection and play an important role in the pathogenesis of NS; it might also be associated with the progression of the disease. In addition, we also found that CSF IL-10 concentration had a useful value in the diagnosis of NS, especially in ANS.


Midazolam is used for sedation before diagnostic and therapeutic medical procedures by several routes including oral, intravenous, intranasal and intramuscular. This is an update of a Cochrane review published in 2016, which aimed to determine the evidence on the effectiveness of midazolam for sedation when administered before a diagnostic or therapeutic procedure in adults and children.We searched CENTRAL, MEDLINE, Embase and two trials registers up to May 2020 together with reference checking to identify additional studies. We imposed no language restrictions. Randomized controlled trials of midazolam in comparison with placebo or other medications used for sedation were included. Two authors independently extracted data and assessed risk of bias for each included study.I2 = 75%; 2 studies; 123 adults). Risk of difficulty performing procedures was lower in the midazolam group . There was no difference in discomfort . Five trials with 336 children were included in the oral midazolam versus chloral hydrate comparison. Midazolam was less likely to result in moderate sedation . This effect estimate is highly uncertain due to concerns about the risk of bias, imprecision and inconsistency. There was no difference in ratings of anxiety . Midazolam increased risk of incomplete procedures . This effect estimate is uncertain due to concerns about the risk of bias. There were four trials with 359 adults and 77 children included in the oral midazolam versus placebo comparison. Midazolam reduced ratings of anxiety . It is unclear if midazolam has an effect on difficulty performing procedures. Meta-analysis was not performed because there was only one incomplete procedure in the midazolam group in one of the trials. Midazolam reduced pain in one study with 99 adults . The effect estimate is uncertain due to concerns about the risk of bias.Eight new trials were included in this update, which resulted in changed conclusions for the intravenous midazolam versus placebo, oral midazolam versus chloral hydrate and oral midazolam versus placebo comparisons. Effect estimates for all outcomes within the intravenous midazolam versus placebo  are uncertain due to concerns about imprecision and risk of bias. Midazolam resulted in a higher level of sedation than placebo (mean difference (MD) 1.05; 95% confidence interval (95% CI) 0.69 to 1.41; 1 study; 100 adults). There was no difference in anxiety (RR 0.43, 95% CI 0.09 to 1.99; The additional evidence arising from inclusion of new studies in this updated review has not produced sufficient high-quality evidence to determine whether midazolam produces more effective sedation than other medications or placebo in any specific population included in this review. For adults, there was low-quality evidence that intravenous midazolam did not reduce the risk of anxiety or discomfort/pain in comparison to placebo, but the sedation level was higher. By combining results from adults and children, there was low-quality evidence of a large reduction in the risk of procedures being difficult to perform with midazolam in comparison to placebo. The effect estimates for this comparison are uncertain because there was concern about risk of bias and imprecision. There is moderate-quality evidence suggesting that oral midazolam produces less-effective sedation than chloral hydrate for completion of procedures for children undergoing non-invasive diagnostic procedures. Ratings of anxiety were not different between oral midazolam and chloral hydrate. The extent to which giving oral midazolam to adults or children decreases anxiety during procedures compared with placebo is uncertain due to concerns about risk of bias and imprecision. There was moderate-quality evidence from one study that oral midazolam reduced the severity of discomfort/pain for adults during a brief diagnostic procedure in comparison with placebo.The online version contains supplementary material available at 10.1186/s13643-021-01617-5. Anxiety at the time of therapeutic or diagnostic medical procedures is a natural response to the unfamiliar environment and experience , 2. AnxiMidazolam is one of the most commonly used medications for inducing anxiolysis or sedation or both, prior to diagnostic and therapeutic procedures , 7. ThisI2 statistic were first observed. There was an insufficient number of studies to perform subgroup analyses based on age, type of procedure and medical specialty or sensitivity analyses for trials rated low versus moderate or high risk of bias. We used the GRADE system to assess the quality of evidence and created summary of findings tables.A full description of the methods was provided in the original review , so we hWe included eight new trials in this updated review. In total, we included 38 trials with 3344 participants, which compared pre-procedure midazolam via the intravenous, oral and intranasal routes of administration, to either a placebo or alternative medication used for sedation Fig. . The incIntravenous midazolam was compared with placebo in six trials with 633 adult participants –15 and oLevel of sedation on a sedation assessment scaleOne study, which used the Ramsay scale to measure level of sedation, reported on this outcome . Scale sNumeric rating scale of anxiety or number of participants rated as anxiousI2 = 75%; 123 adults; 2 studies; low quality). The quality of this evidence was downgraded to low quality due to concerns about risk of bias and imprecision.There was no difference in anxiety (risk ratio (RR) 0.43; 95% CI 0.09 to 1.99; Proportion of incomplete procedures or where there was difficulty performing the proceduresI2 = 45%; 3 studies; 191 adults and 32 children; low quality). The quality of this evidence was downgraded to low quality due to concerns about risk of bias and imprecision.Risk of difficulty performing procedures was lower in the midazolam group . The quality of this evidence was downgraded to low quality due to concerns about risk of bias and imprecision.There was no difference in discomfort between groups , there was no difference between groups in one study  .Sedation reversalOne trial (100 participants) reported that no participants required sedation reversal in either group .Participant or proceduralist satisfactionI2 = 0%; moderate quality). In the Yuno 1996 trial, participant satisfaction, which was measured on a four-point scale with lower scores indicating greater satisfaction, was better in the midazolam group . Proceduralist satisfaction was also greater in the midazolam group in the same study . The effect estimates for this outcome are uncertain due to concerns about the risk of bias.Four trials, all conducted with adult participants, reported on participant or proceduralist satisfaction  , 13–15. Five trials , with 336 participants compared oral midazolam with chloral hydrate for sedation of children [17–21] . We downgraded the evidence from this meta-analysis to very low quality, due to concerns about the risk of bias, inconsistency and imprecision.Two trials reported on the rate of reaching a level of moderate sedation. Derakhshanfar et al.  reportedNumeric rating scale of anxiety or number of participants rated as anxiousI2 = 0%; 2 studies; 68 participants; low quality). We downgraded the evidence for this outcome to low, due to concerns about the risk of bias and imprecision. To aid interpretation, we converted the estimate for the SMD to an MD using the numerical rating scale in D’Agostino et al. [A numerical rating of anxiety was reported in two trials with 88 participants. The outcome was measured using different scales . We downgraded the quality of evidence to moderate, due to concerns about the risk of bias.Four trials (268 participants) reported on this outcome , 19–21. Discomfort/painNo trials reported this outcome.Within this comparison, no trials reported results for anterograde amnesia, quality of recovery, allergic or anaphylactoid reactions, sedation reversal and patient or proceduralist satisfaction.Disinhibition or excitationThere was no difference in disinhibition or excitation between midazolam or chloral hydrate groups in the Derakhshanfar et al.  trial  by Akil et al. . There wFour trials  with 436 participants compared midazolam administered via the oral route with a placebo . The quality of this evidence was downgraded to low quality due to concerns about the risk of bias and imprecision. To aid interpretation, we converted the estimate for the SMD to an MD using the numerical rating scale from Puttapitakpong et al. [Kuganeswaran et al.  reportedg et al. . Scores Proportion of incomplete procedures or where there was difficulty performing the proceduresThere were no incomplete procedures in either the midazolam or placebo groups in three trials –24. One Discomfort/painIn the Kuganeswaran et al.  trial, wI2 = 99%; 2 trials; 359 participants; low quality). However, the results were inconsistent and imprecise. As such, the quality of evidence was rated as low quality.Within this comparison, no trials reported results for disinhibition or excitation, quality of recovery, allergic or anaphylactoid reactions and sedation reversal. For anterograde amnesia , there was no overall difference between midazolam and placebo in meta-analysis of two trials that enrolled adults undergoing upper  or loweTolerance of procedure or participant cooperationTolerance of the procedure was measured using the Frankl behaviour rating scale  in Akil et al. . There wParticipant cooperation was measured using the Houpt behavioural scale  in Akil et al. . ParticiParticipant or proceduralist satisfactionParticipant satisfaction (measured by participants’ perception that they received inadequate sedation for their procedure) in Kuganeswaran et al.  was supeOne trial with 38 participants compared intranasal midazolam with intranasal dexmedetomidine for sedation in children before laceration repair . EighteeP = 0.007). The proportion of participants who were not anxious during positioning for the procedure was also reported. Participants who scored less than 30 using the modified Yale Preoperative Anxiety Scale were considered ‘not anxious’. More participants in the dexmedetomidine group were not anxious during positioning compared to those in the midazolam group (14/20 (70%) dexmedetomidine; 2/18 (11%) midazolam, P = 0.00). The odds of participants not being anxious during positioning were 19 times higher in the dexmedetomidine group compared to the midazolam group . We rated this evidence as moderate quality, due to concerns about imprecision.Participants’ level of anxiety during patient positioning for the procedure was measured in this trial by the modified Yale Preoperative Anxiety Scale . ParticiOne trial, with 145 children undergoing echocardiography, compared intranasal midazolam with ketamine . There wLevel of sedation was measured every 15 min using the RASS, with levels of ‘awake and calm’, ‘drowsy’ or ‘sedated’. More participants were rated as ‘sedated’ in the midazolam group at 15 min  and 30 min . There was no difference in the level of sedation between groups at 45 min  and 60 min . The effect estimates for this outcome are uncertain due to concerns about imprecision and the risk of bias.There was no difference between groups in the number of participants who were insufficiently sedated to be able to perform the procedure . This effect estimate is uncertain due to concerns about imprecision and the risk of bias.We identified one trial that enrolled 120 participants for this comparison . Forty pYamasaki et al.  reportedDespite inclusion of additional studies in this update, in general, it remains unclear if intravenous midazolam is more effective than placebo for procedural sedation. Overall, we judged the quality of the evidence for the primary outcomes to be low quality. Intravenous midazolam did not reduce the risk of anxiety or discomfort/pain. By combining results from adults and children, there was low-quality evidence of a large reduction in the risk of procedures being difficult to perform with midazolam in comparison to placebo. Also, there was low-quality evidence from multiple studies that midazolam improved participant satisfaction in comparison with placebo. Further studies are needed to increase precision and consequently increase confidence in the effect estimates.Based on a meta-analysis of four trials , 19–21, There was low-quality evidence that oral midazolam reduced anxiety in comparison with placebo in adults and children. There was low quality evidence of a reduction in discomfort/pain in one of the 4 studies included in this comparison .There was moderate-quality evidence that children who received midazolam for laceration repair had higher ratings of anxiety compared with dexmedetomidine. Additional trials should be conducted in other similar clinical contexts where motion control is required. Such research is needed to confirm these promising initial findings indicating the potential superiority of intranasal dexmedetomidine over midazolam for this indication. Alongside these trials should be a consideration of the cost-effectiveness of dexmedetomidine in comparison to midazolam for paediatric sedation. Another new comparison was intranasal midazolam versus ketamine. One study was identified with 155 children undergoing echocardiography . Low-quaTrial protocols were not sought for clarifications regarding risk of bias assessments because many included trials in this review were published prior to the establishment of clinical trial registries. For this update, we based our selection of primary outcomes on recommendations from the Sedation Consortium on Endpoints and Procedures for Treatment, Education, and Research Recommendations (SCEPTER) about core outcome domains in clinical trials of in procedural sedation . The secThe additional evidence arising from inclusion of new studies in this updated review has not produced sufficient high-quality evidence to determine whether midazolam produces more effective sedation than other medications in any specific population included in this review. Moderate-quality evidence demonstrated that midazolam administered orally to children who require sedation for motion control during diagnostic procedures produced less-effective sedation compared with chloral hydrate in terms of the ability to complete procedures. Patients appear to prefer to be sedated with midazolam when undergoing a procedure than receive no sedation at all. For this reason, sedation with midazolam could be offered if it is clinically appropriate to do so.Additional file 1. Methods: Expanded description of methods used in the original and updated reviewAdditional file 2. Search strategy: Search terms used for the original and updated reviewAdditional file 3. Study characteristics: Sample and intervention characteristics and outcome descriptions as well as risk of bias assessments for each study included in the updated reviewAdditional file 4. Results: Expanded description of results for all comparisons included in the updated reviewAdditional file 5. Data and analyses: All data and results of meta-analyses


Intraventricular vancomycin is an effective treatment for neonatal ventriculitis, as the cerebrospinal fluid (CSF) vancomycin levels reach adequate concentrations to achieve microbiological cure. There is no robust data on intraventricular vancomycin pharmacokinetics in the preterm population. This pilot population pharmacokinetic modelling study examines the pharmacokinetic behaviour of intraventricular vancomycin in the preterm population of < 28 weeks gestation, to inform the feasibility of future prospective studies.The study comprised 8 preterm infants with neonatal ventriculitis . Population pharmacokinetics (non-linear mixed effects modelling) were described with one- and two-compartment models to fit plasma concentrations of vancomycin. A CSF compartment was added to the plasma modelling and mass transfer examined. Three covariates  and CSF protein) were tested on the final model. Area under the curve (AUC) and average CSF concentration (C average) predictions were generated from the final model and compared with time to microbiological cure.A one-compartment model provided the best fit to the data. There was no appreciable transfer between plasma and CSF. None of the covariates provided a significant reduction in the objective function value (OFV). Generally, time to sterilisation with higher CSF AUC (0-24) and C average tends to be shorter, however this should be interpreted with caution as data is erratic.This pilot population pharmacokinetic analysis provides important information to warrant changes in the management of intraventricular vancomycin treatment in the preterm population, such as the current use of VI as a dosing parameter. Further study with a larger data pool is necessary to investigate the influence of VI on CSF vancomycin and ascertain dosing strategies.  This coThe neurodevelopmental impairment burden in neonatal ventriculitis is yet unquantified in the literature, but it is known that having a shunt alone in post haemorrhagic hydrocephalus increases the risk of neurodevelopmental impairment and outcomes of growth at 18-22 months . AlthougAlthough vancomycin has been used widely in the neonatal population, there is still limited knowledge on its pharmacokinetics, regardless of the route of administration . IV vancIn a review of vancomycin use in neonates over three decades by De Hoog et al in 2004, volume of distribution of intravenous vancomycin in steady state in neonates was found to range between 0.38 and 0.97 L/kg and the clearance ranged between 0.04 to 0.09 L/hour/kg . FurtherStaphylococcus aureus and Staphylococcal epidermidis was noted to be maximum at 5-10 mg/L, when the minimum inhibitory concentration (MIC) was 2mg/L, in adult CSF samples .A retrospective clinical case review of ventriculitis in preterm infants of < 28 weeks gestation was undertaken in Southmead Hospital Neonatal Intensive Care Unit (NICU), in Bristol, United Kingdom. The study period spans from 2009 to 2016. Over this 8-year study period, 8 preterm patients that were treated for ventriculitis, were identified. In this patient cohort, ventriculitis was defined as elevated CSF white cell count (WCC) >20/mmPatient medical records both written and electronic were utilised to collect demographical and pharmacokinetic data on IV vancomycin as well as intraventricular vancomycin. For every patient, the following information was collected:CSF WCC, microbiological CSF culture results, CSF protein levels, IV vancomycin and intraventricular vancomycin doses administered, all available CSF vancomycin levels and serum vancomycin levels, dates and times of IV vancomycin doses, intraventricular vancomycin doses, serum and CSF vancomycin levels.The intraventricular vancomycin treatment doses for the preterm infants were individualised, as per the recommendations by the Medical Microbiologists and in discussion with the attending Consultant Neonatologist.Four starting doses were used in the study's preterm cohort of 3mg, 5mg, 10mg and 15mg.The infants had cranial ultrasound (USS) studies 2-3 times per week, using a Philips HD5 USS machine, through an 8.5 MHz sector transducer. Specific measurement of ventricular index (VI), which is defined as the distance between the falx and the lateral wall of the anterior horn in the coronal plane, was undertaken . The VI Intraventricular vancomycin was given when the ventricular access device was accessed for the routine drainage. Intraventricular vancomycin concentration used was 10 mg/mL, in a sterile preparation, and was given as a slow bolus over 2 minutes. The intraventricular vancomycin was given at the end of the CSF drainage. The ventricular access device was flushed with 1ml of sterile NaCl 0.9% following instillation of the intraventricular vancomycin.CSF samples for vancomycin levels were taken when the ventricular access device was accessed for routine CSF drainage, as needed for clinical management of hydrocephalus, for the individual baby. Due to this routine practice, the CSF vancomycin levels were taken at random time points. Repeat intraventricular vancomycin dose was administered if the level dropped under the threshold of 10mg/L. The target CSF vancomycin concentration was at least ten times above the breakpoint of MIC for CoNS. This was chosen as these were the principal infecting organisms in the defined neonatal cohort and the study hospital's MIC breakpoint for CoNS is 1 mg/L  .The CSF vancomycin measurements were undertaken in the local microbiology laboratory, which is a national referral centre for antibiotic assays, through quantitative microsphere system (QMS® Thermo Scientific)-based immunoassay . The CSF^3 and simultaneously achieve sterile CSF.Time to sterilisation was defined as length of time taken for CSF WCC to fall to < 20/mmrd IV dose, and before the 3rd subsequent IV doses then after  were evaluated using the nonlinear mixed-effects modelling software NONMEM version 7.3 . The first order conditional estimation method with the interaction option (FOCE-I) was used to estimate PK parameters and variability. R (version 3.0.2) and PsN (version 5.18.2) tools were used for pre- and postprocessing.i is the model parameter for the ith patient, pTV is the typical value of the parameter in the population, η is the departure from the typical value for an individual (IIV) and π is the departure of the parameter from one occasion to another (IOV).The residual variability was initially described using a combined proportional and additive error model.The PK base model was a one-compartment model with first order elimination. The one-compartment PK parameters were clearance (CL) and apparent volume of distribution (V). The inter-individual variability (IIV) and inter-occasion variability (IOV) were assumed to follow a log-normal distribution :(1)pi=pTCovariate testing was performed on both plasma and CSF PK parameters. Covariates were selected for testing based on their biological plausibility of affecting response. Covariates were deemed to be significant according to the likelihood ratio test (-2 × log-likelihood), if a drop in the objective function value (OFV) is 3.84 or higher with one parameter and 5.99 or higher with 2 parameters (p < 0.05).a priori scaled for size and age  was tested using first-order rate constants. We tested 2 covariates of VI and CSF protein, on CSF clearance and volume and CSF clearance respectively.n=1000). Parameter uncertainty was obtained from the standard error of estimate through the covariance step.For model evaluation, decrease in objective function value (OFV), plots of observed versus population predicted concentrations (DV-PRED), observed versus individual predicted concentrations (DV-IPRED), conditional weighted residuals versus time after dose (CWRES-TAD) were utilized. The final population model was evaluated through visual predictive check (VPC) (Area under the curve (AUC) predictions were generated for CSF vancomycin from the final model. We explored the relationships between AUC 0-24 and average CSF concentration /t; t = time) and time to sterilisation of CSF.2.3The protocol for the study was approved by National Health Service (NHS) England Health Research Authority (HRA). The committee did not require parental informed consent due to retrospective design and use of anonymised data collected by clinical staff who would normally have access to these records as part of their clinical work.3Pharmacokinetic data from 8 preterm infants were available for NLME modelling. The median gestation age was 25.3 weeks (range 23.9 - 27.7) and the median postnatal age was 8.7 weeks (range 3.9-23.1). 41 plasma samples (from 6 out of 8 patients who received concomitant IV vancomycin) and 74 CSF samples were available for the pharmacokinetic modelling. One plasma sample was below lower limit of quantification and 1 plasma sample was >100mg/L, most likely attributed to sampling or analytical error. These 2 samples were excluded from the analysis. One patient received two courses of intraventricular vancomycin treatment. We have included only the first episode of treatment (without concomitant IV vancomycin) within our pharmacokinetic analysis for this patient, therefore a further 2 plasma and 7 CSF samples were excluded from the analysis. Patient demographics have been provided in The structural model that provided the best fit to the data was a one-compartment model. A two-compartment model did not provide a significant drop in the OFV. When CSF data was added, the initial assumption was of no transfer between the CSF and plasma. The addition of a transfer rate constant either in both directions and just from plasma to CSF did not significantly improve the model fit. Hence, our pharmacokinetic model did not demonstrate an appreciable transfer between plasma and CSF.We tested the covariates of serum creatinine, VI and CSF protein as described in 0-24 and C average suggest a shorter time to sterilisation with higher AUC (0-24) and C average, as represented in The final model was used to generate CSF AUC predictions. The observed trends between AUC4A vancomycin population pharmacokinetic model was developed in preterm neonates receiving intraventricular and intravenous administration. Neonatal ventriculitis is a rare but significant complication in the described cohort of preterm babies, meaning that systematic study of large groups of patients is challenging. Robust pharmacokinetic data is vital to improve understanding of vancomycin administered by the intraventricular route to inform prescribing and treatment practice. In the preterm population, evidence is lacking on the currently used parameter of VI for intraventricular vancomycin dosing and the effects of important covariates on CSF vancomycin.S.aureus and S.epidermidis was noted to be maximum at 5-10 mg/L, when the MIC was 2mg/L, in adult CSF samples  and clearance (VI/CSF protein) were examined. The intraventricular vancomycin dosing in the patient cohort was guided by the ventricular size, through measurement of VI. This parameter is widely used in the neonatal population, however it is a surrogate marker for ventricular volume and not likely to accurately delineate volume of distribution. Currently, there is insufficient evidence on the use of ventricular size to determine the dose of intraventricular vancomycin, especially in the neonatal population, and the use of this parameter is based on local unit experience . In the The CSF protein was examined as a covariate in this study as 50-55% of vancomycin is protein bound . As the This pharmacokinetic study of intraventricular vancomycin had a few limitations. Firstly, as the pharmacokinetic data was pooled from only 8 preterm patients, the results represent an association. We were also not able to perform intraventricular vancomycin dosing simulation, as doing so would not generate conclusive data due to the small patient cohort. Larger dataset is also needed to further explore and confirm the observed trend of shorter time to sterilisation with higher AUC (0-24) and C average. Furthermore, we did not examine the effects of CSF drainage on the CSF vancomycin level, as in our patient cohort the CSF drainage was fairly constant at 5-10ml/kg/day. There is some evidence to suggest that CSF drainage is associated with CSF vancomycin clearance . This ce5This pilot population pharmacokinetics study has demonstrated that a one-compartment model best described the pooled IV vancomycin and intraventricular vancomycin data from 8 preterm infants of < 28 weeks gestation. There was no appreciable transfer between plasma and CSF, hence intraventricular vancomycin is an effective route for treating ventriculitis. Covariates of VI and CSF protein did not demonstrate any influence on CSF vancomycin, and there was a trend of shorter time to sterilisation with higher AUC (0-24) and C average. Further study with larger data pool is necessary to investigate the influence of VI on CSF vancomycin in particular as the current dosing parameter and ascertain dosing strategies.None.10.13039/501100000395Royal College of Physicians (RCP) through Thomas Watts Eden Paediatric Fellowship (received by first author JMP), The Showering Fund from Southmead Hospital (grant number SF113-received by author MA) and United Kingdom Medical Research Council (MRC) Fellowships (grants P014534 and M008665-received by authors FK and JFS).This work was supported by the The data that support the findings of this study are available from the corresponding author upon reasonable request.Jaya Madhura Parasuraman: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Data curation, Writing - original draft, Writing - review & editing, Visualization, Project administration, Funding acquisition. Frank Kloprogge: Methodology, Software, Validation, Formal analysis, Investigation, Writing - review & editing, Visualization. Joseph Frank Standing: Conceptualization, Methodology, Software, Validation, Formal analysis, Investigation, Resources, Writing - review & editing, Visualization, Supervision. Mahableshwar Albur: Validation, Writing - review & editing. Axel Heep: Conceptualization, Methodology, Resources, Writing - review & editing, Supervision, Project administration, Funding acquisition.