In conclusion, the coupling pattern between hemodynamics and CSF is reduced in AD and it correlates with MMSE score.In-vivo microscopical studies indicate that brain cerebrospinal fluid (CSF) transport driven by blood vessel pulsations is reduced in the early stages of Alzheimer’s disease (AD). We hypothesized that the coupling pattern between cerebrovascular pulsations and CSF is altered in AD, and this can be measured using multi-wavelength functional near-infrared spectroscopy (fNIRS). To study this, we quantified simultaneously cerebral hemo- and CSF hydrodynamics in early AD patients and age-matched healthy controls. Physiological pulsations were analysed in the vasomotor very low frequency (VLF 0.008–0.1 Hz), respiratory (Resp. 0.1–0.6 Hz), and cardiac (Card. 0.6–5 Hz) bands. A sliding time window cross-correlation approach was used to estimate the temporal stability of the cerebrovascular-CSF coupling. We investigated how the lag time series variation of the coupling differs between AD patients and control. The couplings involving deoxyhemoglobin (HbR) and CSF water, along with their first derivative, in the cardiac band demonstrated significant difference between AD patients and controls. Furthermore, the lag time series variation of HbR-CSF in the cardiac band provided a significant relationship, p-value = 0.04 and r Since AD is a progressive NDD2, particularly having an early diagnosis is of highly importance. ADI also wrote that MRI or computer tomography (CT) is highly recommended in early evaluation of dementia, while positron emission tomography (PET) and single photon emission computed tomography (SPECT) are used for more complex cases. The main challenges of using these methods are radiation safety, requirements of educated personal to use the imaging technology and high invest and maintenance costs, limiting their wider usage. Nevertheless, functional neuroimaging offers a great potential for early diagnostics of NDDs as it may possibly detect deviation in neuronal activities before neuronal damage3.Early diagnostics of the neurodegenerative diseases (NDDs), such as Alzheimer’s Disease (AD), are of high need. According to Alzheimer’s Disease International (ADI), 75% people with dementia are not diagnosed and it may reach up to 90% in low- and middle-income countries4. Furthermore, impaired neurovascular coupling is thought to contribute to tissue injury, loss of function, and cellular degeneration over time5. Interestingly, dysfunction of the neurovascular unit (NVU) controlling the coupling, has been linked with various neurodegenerative diseases (NDDs) including Alzheimer disease (AD)6. Impaired cerebrovascular reactivity, CBF reductions, CBF dysregulation and blood brain barrier (BBB) disfunction are now recognized to be early events in the AD pathophysiological cascade7.Functional near infrared spectroscopy (fNIRS) is commonly used for measuring human cerebral hemodynamics, e.g., to decipher the coupling between neural activity and cerebral blood flow (CBF) controlled by the neurovascular unit (NVU). The fNIRS technology development in recent years has made it possible to realise it also as a small, wearable brain monitoring device, making it an interesting potential alternative as a first-line brain diagnostic tool. Numerous studies show that NVU dysfunction leads to altered neurovascular coupling can be disrupted due to diseases and aging8, where function of BBB, cardiovascular and cerebrospinal fluid (CSF) conduits inside the BBB driven by physiological pulsations is of high interest. In particular, the mechanism between CSF and cerebrovascular function and their coupling have awaken increased interest9. For instance, a very recent study by Nedergaard’s group shows that dynamic changes in vascular diameter drive perivascular glymphatic CSF inflow and this can occur in the absence of neural activation10. In 2019, Fultz et al., showed a strong correlation between CSF and negative of the first derivative of blood oxygen dependent level (BOLD) in a human sleep study where BOLD and CSF flow were computed by fMRI11. Further, Han et al. found out that the degree of correlation between BOLD and CSF was altered in AD patients compared to controls9. Abnormalities in cardiovascular impulse propagation with increased speed and ever reversal flow and increased BOLD signal amplitude variability have been detected in AD with fast fMRI13 in areas indicating increased BBB permeability14. Also, transcranial Doppler ultrasound (TCD) study showed that cerebral blood flow variability was reduced in AD despite increased blood pressure variability15. These findings indicate an impairment in the cerebral blood flow in rigid, narrowed and non-responsive blood vessels and suggest reduced clearance of CSF waste and metabolites that predisposes to neurodegeneration (NDD) and cognitive decline.These parameters are also linked with the glymphatic system16, and Arai et al. monitored the hemodynamic response from frontal, left and right parietal, and occipital areas when employing verbal fluency task17. They found that controls had higher activation index than AD patients based on the amplitude changes in the oxyhemoglobin (HbO) waveform during the task. Fladby et al. showed dysfunction in temporal lobe based on olfactory response, a response due to odorous substances detection18. As a complement to studies using fMRI, Zeller et al. utilised fNIRS to measure both HbO and deoxyhemoglobin (HbR) from midline parietal (Pz) area from AD patients and controls while doing modified Benton Line Orientation Task3, and found fNIRS as a promising tool to separate AD patients from controls. Furthermore, it seems that cerebral hemodynamics in AD is different compared to healthy controls also in resting state, as it was shown by Ferdinando et al.19.In addition to fMRI, fNIRS shows potential for detecting hemodynamic abnormalities in patients with AD performing controlled tasks. For instance, Li et al. found differences in hemodynamic response in frontal lobe when doing digit verbal span task20. Furthermore, our fNIRS method showed that vascular fluctuations of cerebral blood and brain water concentrations have high anticorrelation22. One particular advantage of the fNIRS technique is its ability to simultaneously measure the cerebral hydrodynamics of HbO, HbR, total hemoglobin (HbT) and CSF with high temporal resolution enabling possibility to effectively study the temporal dynamics and stability of the hydrodynamic couplings. In this present study, we explored the temporal stability of the coupling pattern between the hemodynamic vs. CSF in AD patients and age-matched controls. We found that the hydrodynamic coupling between the brain blood and CSF signal was altered in the cardiac band.Myllylä et al. introduced fNIRS based method to measure non-invasively in human brain water variations in subarachnoid space reflecting CSF dynamics and validated it in fMRIIn this study we analysed temporal stability of the coupling pattern between cortical brain blood signals vs. CSF water signals over time in different time windows and overlaps. Based on the CSF water vs. blood  cross-correlation analysis, see supplementary Tables 2 = 0.16). Figure We also conducted a regression analysis of mini-mental state examination (MMSE) score against these variations, see Fig. We studied hemodynamic–CSF coupling pattern in AD patients and age-matched controls using novel multiwavelength fNIRS method. We applied a sliding window cross-correlation analysis to quantify the coupling pattern between cortical brain hemodynamics and CSF signals. We found that especially in the cardiac band, the controls had higher time domain lag variation in the following pair of HbR-CSF, dHbR-CSF, HbR-dCSF, and dHbR-dCSF compared to AD patients, see Tables 9. Their finding was in concordance with our finding, that the coupling pattern variation involving the venous HbR signal provided good separation between AD patients and controls. The whole brain BOLD and CSF measured from IV ventricle may be more sensitive to T2 CSF flow changes11, while our CSF measure is more sensitive to CSF pulsations detected locally mainly from the subarachnoid space of the NIRS measurement volume. It might explain why we achieved good separation from the cardiac band (0.6–5 Hz), while Han et al. got the results within 0.01–0.1 Hz band. In our case, the sliding window approach is not as sensitive as Han et al. method for slow VLF vasomotor waves. However, the faster hydrodynamic coupling changes in the cardiac frequencies do also occur in between venous to CSF signals. Furthermore, these variations reflect significant relationships with MMSE , see also Fig. 12, as well as well as with the fast 800 Hz sampling frequency. They both seemed providing good reflection for cognitive capability as well. However, larger number of subjects must be studied to confirm this. Future work includes data collection from more subjects and study the correlation with the mild, moderate, and severe state of AD.We found that especially in the very fast with minimal overlapping window the coupling of the venous HbR blood signal and CSF signal seems to be altered into monotonous signal over time in AD. In matched healthy controls the CSF signal has more variance in its coupling pattern to the regional venous blood signal pulsations. Han et al. performed the coupling analysis using a relatively slow sampling rate of the BOLD data with TR of 2.3 s. They calculated BOLD signal by averaging the whole brain area and CSF from IV ventricle and did not use sliding window approach but rather a whole time series approach more commonly used in fMRI. They found that the overall coupling between the venous derived BOLD signal and CSF was reduced gradually from control to significant memory concern (SMC), mild cognitive impairment (MCI), and lastly AD12, linked to brain tissue atrophy23 that leads to stiffening brain tissue24. Stiffened tissue and blood vessels may fail to both pulsate and drive CSF along perivascular spaces. Furthermore, stiffened arterioles fail to respond to local demands of activation hyperremia that is normally dynamically ongoing as driven by neurovascular coupling15. In other words, the damaged arterioles seem to only convey monotonic flow pulsations that fail respond to rapid neurovascular control cues. The reduced dynamics in the coupling between the HbR and CSF signals may further reflect reduced dynamics in the blood flow drive of CSF.In our data the variation in AD patients were always smaller than that in controls. The AD patients suffer from vascular wall pathologyFrom the three physiological bands involved in the analysis, the cardiac band showed promising results with all window width and overlap parameters combination to separate AD patients from controls, see heatmaps in Fig. To validate this hypothesis, we conducted further experiments on the cardiac band by reducing the signal length up to 25% of the original one to mimic the situation in the respiratory band. It meant the signal of interest only lasted for 1.25 min. The length of the sample reduced drastically and even the lowest number of samples among all window width and overlap parameter was about 30. Interestingly, only HbR-CSF and dHbR-dCSF pair showed their consistencies with the same number of hits. When the same procedure was applied to fit to mimic situation in the VLF band, we got similar results as in the respiratory band. Thus, the length of the sample must be considered carefully. Looking at the results from the VLF band, see Fig. There is a general agreement that CSF flow is driven by physiological signals generated by heart function, breathing, body/muscle motions, and vasomotor activity. Interestingly, the cerebrovascular-CSF coupling, and their pulsations measured in macro scale by the presented NIRS technique seem to reflect these interactions. Aging changes the mechanical properties of brain and vessel tissue stiffness, however, in AD patients these changes are more significant, and seems to affect the cerebrovascular-CSF coupling. Furthermore, our results indicate that the coupling is more drastically altered associated with the cardiac activity.We envisage that fNIRS could be used in near future as a supportive technique to assess changes in fast brain temporal hydrodynamics in AD, while more detailed diagnosis is performed using the golden standard. The fNIRS is portable, easy to use non-invasive measure of that offers a good tool for analysing temporal dynamics due to the fast sampling of brain hydrodynamics. FNIRS can be easily used also outside hospital enabling a possibility to monitor the brain condition and progress of AD more frequently for a large aging population.19.The data collection followed the guidelines established by the Declaration of Helsinki and all participants signed informed consent letter prior to the measurements. All measurements took place in Oulu University Hospital, Oulu, Finland. This study was approved by the regional medical research ethics committee of the Wellbeing services county of North Ostrobothnia. A sample of 8 patients  with AD and 14 age-matched controls  participated in this study, who received detailed explanation before signing the consent letter. Most of the participants also filled the mini-mental state exam (MMSE) prior to the measurements. Based on the MMSE score, 22.8 ± 1.6 and 28.5 ± 1.5 for AD and controls respectively, AD patients were categorized in a mild dementia25.During measurement, all participants were in a supine position in the fast fMRI chamber (10 Hz) and multiwavelength fNIRS probes, 690, 810, 830, and 980 nm, were placed on the left side of the forehead. The light source was in the middle of the forehead, while the light detector was 3 cm away from the source. These optodes were placed about 3 cm above the eyebrow. We measured using both fNIRS and fMRI simultaneously for 5 min, and the participants were instructed to simply lay still comfortably and focus on the visual screen cross in resting state26. MRI compatible optical fibres used in the NIRS device were fabricated by Schott and then customized for the application by Fiberoptics Technology. Having a refractive index of approximately 1.58 and an internal transmittance of 0.999 in the range of 600–1060 nm, the fibres can guide 4 light sources to 1 fibre output. With a diameter of 2.5 mm and a length of 10 mm, the tip of the output has a 90° bend and is attached to the head by a plastic fibre clip. All fibres have the same physical dimensions and measure of 10 m in length to allow combining NIRS measurements with MRI, such that the NIRS device can be placed outside the MRI room with only the optodes and fibres brought inside the room through a waveguide in the wall. This arrangement makes it possible to ensure the MRI compatibility of the NIRS measurement. Our fNIRS device uses wavelengths of 690, 810, 830 and 980 nm produced by high-power light-emitting diodes, each modulated at a specific frequency and then demodulated in the receiver amplifier. The wavelength of 980 nm is selected to ensure high sensitivity for water (CSF) dynamics, since it is strongly absorbed by water20. Sufficient penetration depth to measure brain cortex and subarachnoid space were ensured by using 3 cm source-detector distance, based on light propagation studies in the brain28.The fNIRS device utilizes frequency-coding technique to label different wavelengths, highly sensitive detection and lock-in amplification technique with a very narrow bandwidth filter (1–10 mHz) in the receiver to minimize noise29.Our fNIRS device sampled the measurement at 800 Hz and it was developed for other studies as well, which requires high temporal resolution, e.g., pulse shape analysis. We employed modified Beer-Lambert law (MBLL) to calculate concentration changes of HbO, HbR, and water with the following extinction coefficients: 690 nm: 0.312, 2.1392, 0.0205; 830 nm: 1.0507, 0.7804, 0.1459; 980 nm: 1.2513, 0.4233, 2.1491, and differential path length factor of 5.93. Total hemoglobin (HbT) is the sum of HbO and HbR. The concentration of CSF water was quantified based on the method presented in paper21. We extended this width up to 1.5 times of the starting width with two values in between to get four window width values for each band. For example, the window width parameters in the VLF band were 120, 140, 160, and 180 s. Using a larger window, we should be able to analyse signal more than one period of oscillation. We used 25%, 50%, and 75% overlap when the window was shifted, see Fig. The analysis was conducted in the very low frequency or VLF (0.008–0.1 Hz), respiratory (0.1–0.6 Hz), and cardiac (0.6–5 Hz) bands. We analysed these coupling relationships in the original sampling and downsampling frequencies, 800 Hz and 10 Hz, respectively. Downsampling from 800 to 10 Hz enabled more comprehensive analysis with MREG data in the future. A sliding window with pre-defined width for each band was set. The window width was determined based on the maximum wavelength of the targeted physiological signal source in the selected bands; in the VLF vasomotor band the window was set to 120 s, in the respiratory band for 10 s, and 2 s for the cardiac band11, we were interested to find out whether we could analyse the hemodynamic-CSF coupling with our fNIRS system and whether these couplings were different between AD patients and controls. For this reason, we analysed the following measured signal pairs: HbO-CSF, HbR-CSF, and HbT-CSF. Motivated by the works of Han et al.9 to use include signal amplitude change speed in time-domain by both fMRI BOLD, we also used the first derivative of fNIRS signals. Hence, the following pairs: dHbO-CSF, dHbR-CSF, dHbT-CSF, HbO-dCSF, HbR-dCSF, HbT-dCSF, dHbO-dCSF, dHbR-dCSF, and dHbT-dCSF, where the prefix ‘d’ denotes the first derivative of the corresponding signals, were used.Similar to preceding BOLD studiesThese pairs were subjected to cross-correlation operation within a pre-defined sliding window, see Fig. From the samples of the lag time series in Fig. Based on Kolmogorov–Smirnov method, we found that data distribution from each group was not normal. Moreover, since the number of subjects participated in this study was small, we utilised non-parametric test, i.e., Wilcoxon-test, to evaluate significant difference between AD patients and control at 0.05 significant difference.Supplementary Tables.


Gender minority individuals are understudied in clinical trials. Realizing the potential of HIV prevention options requires understanding product tolerability across diverse groups vulnerable to HIV acquisition. Within HVTN 704/HPTN 085, a phase 2b trial of the broadly neutralizing antibody (bnAb) VRC01 for HIV prevention, analyses of associations between gender identity and solicited adverse events (solAE) after infusions of VRC01 or saline placebo have not been reported.HVTN 704/HPTN 085 enrolled men who have sex with men and transgender (TG) participants from Brazil, Peru, Switzerland and the US and randomized them 1:1:1 to receive an infusion every 8 weeks  of VRC01 30 mg/kg, VRC01 10 mg/kg or placebo. solAE were recorded for 3 days after each infusion. Gender was defined by self-report and sex assigned at birth. Odds ratios of the association of gender  and frequency and severity of solAE were adjusted for age, race and ethnicity.Tab. 1). Overall, 867 (37.7%) CM and 107 (42.5%) gender minority participants reported a solAE (Tab. 2). Gender minority participants were more likely to report solAE than CM overall  and among placebo recipients  (Tab. 3). SolAE severity (≥Grade 2) did not significantly differ overall . Grade 2 solAE were reported after 1% and 2% of infusions among CM and gender minority participants, respectively. Grade 3 and 4 events were rare in both groups (< 0.1%). Infusion completion did not differ.Of 2,552 participants, 162 identified as TG women (6.3%), 75 as other identity (2.9%) and 15 as TG men (0.6%). Gender minority participants identified as Black/African American (8.7%), other (59.9%) and White (21.8%); 68.3% identified as Hispanic/Latino (HVTN 704/HPTN 085 is the first large-scale HIV bnAb preventive trial allowing assessment of associations between gender identity and solAE. Gender minority participants had more frequent solAE, but importantly, infusion completion did not differ and severe solAEs were rare. HIV prevention and bnAb trials must engage and include gender minority individuals to evaluate the tolerability of novel agents.Srilatha Edupuganti, MD MPH FIDSA, Sanofi: Grant/Research Support


Applying strictly-determined detection limits, 12 cytokines were found in serum and two in CSF. Higher serum MCP-1 levels (p = 0.007) were present in FES versus controls, which correlated with serum IgG . Finally, IL-18 levels correlated with body weight in FES . This study demonstrates potential limitations in the sensitivity of multiplex cytokine assays for CSF studies in mental disorders and suggests that some published studies in this area should be re-evaluated.We determined cytokine levels in paired serum/CSF samples from first-episode schizophrenia (FES) participants (The online version contains supplementary material available at 10.1007/s00406-023-01569-y. Increasing evidence suggests that neuroinflammation contributes to the pathogenesis of psychiatric disorders, including schizophrenia –6. As maSince multiplex immunoassays can show low sensitivity and poor correlation with corresponding singleplex methods , 15, it Although the companies who developed these assays may claim dynamic ranges with low LODs, none are capable of accurately measuring concentrations that give readings in the range of blank samples. Threshold levels of analytes must be present to produce signals that can be distinguished above this noise , 22. We The study was performed according to German laws, the Declaration of Helsinki, and local institutional review board guidelines. Participants gave written informed consent. CSF and sera were obtained from 20 FES in-patients diagnosed according to ICD10 and AWMF-S3 guidelines  for whomn = 21) had headache (n = 3), pseudotumor cerebri (n = 3) or initially unclear neurological symptoms (n = 15) with no history of psychiatric disorders and underwent lumbar puncture to rule out subarachnoid hemorrhage, infectious or autoimmune central nervous system disease. They were matched for age, gender and body mass index. Routine CSF parameters were within the normal range and showed no significant group differences.Controls , CSF and sera were centrifuged and the supernatants stored at − 80 °C.Concentrations of 13 cytokines  were determined in triplicate using the LEGENDplex™ Human Inflammation Panel 1  according to manufacturer instructions. This is a fluorescence–coded microsphere-based multiplex immuoassay for the detection and quantitation of analytes by flow cytometry. Sera were diluted twofold and CSF tested undiluted. Standard curves of each cytokine were analysed in duplicate on two separate occasions. The LOB and LOD were determined for each assay according to CLSI guidelines , 22, usiFor statistical analysis, we used only those cytokines for which at least 2 of the 3 replicates and > 50% of samples gave readings above the LOD.U tests. MCP-1 serum levels (p < 0.001) were significantly higher in smokers. Therefore, diagnosis-dependent differences in MCP-1 were calculated by ART  with the covariate smoking. Due to the exploratory nature of the study, group statistics were not corrected for multiple comparisons.Data were analyzed using R (v4.0.5). Chi-square tests were performed to calculate group differences regarding gender and smoking. Corrected PANSS scores were derived by subtraction of minimum from raw scores . Most dap-values  to identify correlations of cytokines with routine blood/CSF, demographic and clinical parameters. Cliff’s delta (δ) was used to assess effect sizes   compared to controls (n = 19) . This was confirmed by ART with the covariate smoking .Neither of the two measurable CSF cytokines showed significant differences in concentrations between the groups Table . The useR = − 750; p < 0.001; q = 0.013) compared to controls  , albumin quotient  or IgG index .Of the 36 parameters determined (Supplementary Table ST2), there were no correlations with serum-MCP-1 apart from a significant negative correlation with serum IgG in FES , IL-18 was significantly correlated with body weight  in FES but not controls  .This study was limited as we only detected 2 out of 13 cytokines in CSF samples, obviating comparisons with cytokine readings in serum samples. Also, the small sample size of this exploratory investigation necessitates validation in larger cohorts. Regarding the biological findings, we did not assess the potential links of MCP-1 and IL-18 with metabolism-related disturbances as no measures of insulin resistance or visceral fat accumulation were available. Finally, although we controlled for the potential effects of antipsychotic medication, disease duration and smoking, we could not account for other influences like nutrition and sleep, and the use of headache patients as controls might bias the results as inflammatory causes may play a role in different forms of headache.To our knowledge, this is the first multiplex cytokine immunoassay study of paired serum/CSF samples from FES patients and controls using criteria based on strictly determined LODs. The main advantage of using the CLSI guideline is a reduction of false positives as this approach takes into account the variance of both the blank and lowest concentration samplesSupplementary file1 (DOCX 15 KB)Supplementary file2 (XLSX 51 KB)Supplementary file3 (DOCX 12 KB)Supplementary file4 (DOCX 265 KB)Below is the link to the electronic supplementary material.


Fever without a focus is a common reason for medical evaluations, hospitalizations, and the antimicrobial treatment of infants younger than 90 days. The presence of cerebrospinal fluid (CSF) pleocytosis could be challenge for clinicians who treat febrile young infants with urinary tract infection (UTI). We evaluated the factors associated with sterile CSF pleocytosis and the clinical outcomes of the patients.3.A retrospective review of patients aged 29–90 days with febrile UTIs who underwent a non-traumatic lumbar puncture (LP) at Pusan National University Hospital from January 2010 to December 2020 was conducted. CSF pleocytosis was defined as white blood cell (WBC) counts ≥9/mmr = 0.234; P = 0.003). Thirty-three patients had CSF pleocytosis . The time from fever onset to the hospital visit, peripheral blood platelet counts, and CRP level at admission were statistically significant in patients with sterile CSF pleocytosis compared to those without CSF pleocytosis. In the multiple logistic regression, only CRP was independently associated with sterile CSF pleocytosis . The proportion of fever defervescence by hospital day 2 was 87.9% in patients with CSF pleocytosis and 89.4% in those without CSF pleocytosis (P = 0.759). There was no statistical difference in the fever defervescence curves between the two patient groups (P = 0.567). No patients had neurological manifestations or complications.A total of 156 patients with UTI were eligible for this study. Four (2.6%) had concomitant bacteremia. However, no patients had culture-proven bacterial meningitis. In correlation analysis, although weak strength, CSF WBC counts were positively correlated with C-reactive protein (CRP) level (Spearman Coexisting sterile CSF pleocytosis among febrile infants with UTIs suggest a systemic inflammatory response. However, the clinical outcomes between the two groups were similar. A selective LP should be considered in young infants with evidence of UTI, and inappropriate antibiotic therapy for sterile CSF pleocytosis should be avoided. SeriousDespite the low prevalence of coexisting bacterial meningitis, the majority of febrile young infants undergo lumbar puncture (LP) to obtain cerebrospinal fluid (CSF) samples regardless of the urinalysis results. The need for LP among young infants with a presumptive UTI has been questioned for decades , 21, 22.The aims of this study were to investigate the prevalence of bacterial meningitis and sterile CSF pleocytosis among febrile infants 29–90 days of age with UTIs. In addition, we evaluated the factors associated with sterile CSF pleocytosis in those patients and compared the clinical outcomes according to the presence of sterile CSF pleocytosis.2.2.1.3 of CSF specimen) were excluded from this study.A retrospective, cross-sectional study was conducted at a tertiary-care university hospital, Pusan National University Hospital, the Republic of Korea. We investigated febrile infants aged 29–90 days who simultaneously underwent urinalysis with urine culture, blood culture, and LP to evaluate fever without a focus between January 2010 and December 2020. Of these, infants diagnosed with UTI were eventually included in this study. The presence of fever was defined as axillary or tympanic temperature ≥38°C in the hospital or at home within 24 h. Infants with unstable vital signs or specific symptoms and signs other than fever at the hospital visit, those receiving antibiotics in the preceding 48 h before cultures acquisition, and those with traumatic LP  per high powered field, or positive leukocyte esterase, or nitrite] . Bacterie values . Concomi2.2.We collected data on patient demographics, past medical history, onset time of fever, and general appearance of the patients   at the i2.3.U test. Fever defervescence curves according to hospital day between the two groups were compared by the log-rank test. There were no missing data.We used descriptive statistics, including medians, interquartile ranges (IQRs), counts, and proportions with 95% confidence intervals (95% CIs). We divided the patients into two groups according to the presence of sterile CSF pleocytosis, and compared the clinical variables. Categorical variables were compared by Fisher's exact test. Continuous variables were compared by the Mann–Whitney P-values of <0.05 were considered statistically significant. Data were analyzed using Prism 9.3.1 .The correlation between continuous variables and CSF WBC counts was performed by Spearman's correlation analysis. Multiple logistic regression analysis was performed to assess the independent factors associated with sterile CSF pleocytosis, and adjusted odds ratios (aORs) and 95% CIs were calculated. We chose potential predictors using statistically significant variables in univariable analysis and included inflammatory markers, regardless of statistical significance. We determined the cutoff values that maximized the area under the receiver operating characteristic curve. We tested for multicollinearity with variance inflation factors and performed the Hosmer-Lemeshow test for goodness-of-fit. Two-sided 3.3.1.Escherichia coli . Concomitant bacteremia was observed in four infants : 4.2% (3/72) of infants aged 29–60 days; 1.2% (1/84) of infants aged 61–90 days. However, no patients had culture-proven bacterial meningitis. Enterovirus PCR or multiplex PCR for microbial detection in CSF specimens was performed in 135 patients (86.5%). Enterovirus positivity was observed in one patient whose CSF WBC count was 1/mm3.During the study period, 751 infants aged 29–90 days had fever without a focus. Of these, 568 patients received urinalysis with urine culture, blood culture, and LP at the same time. A total of 156 patients with febrile UTIs were finally eligible for this study . The cha3.2.3 . There was no difference in CSF WBC counts between infants aged 29–60 days and 61–90 days  (P = 0.435). For patients with CSF pleocytosis, the median CSF protein value was higher than in those without CSF pleocytosis  . Sterileocytosis . Among p3.3.. 8 h; P = 0.037) and higher platelet counts  and CRP values . There were no significant differences in general appearances at the initial visit, the degree of pyuria, peripheral blood WBC counts, and ANCs. In patients with CSF pleocytosis, the proportion of grade 3 or higher hydronephrosis was higher but not statistically significant .A comparison of the clinical characteristics at admission according to the presence of sterile CSF pleocytosis was described in 3, ANC of 6,710/mm3, platelet count of 415,500/mm3, and CRP of 3.425 mg/dl (P = 0.019).The time from fever onset to the hospital visit, peripheral blood WBC count, ANC, platelet count, and CRP vale were determined to be potential predictors associated with the presence of CSF pleocytosis. The cutoff values for the laboratory values were as follows: peripheral blood WBC count of 15,080/mm25 mg/dl . In the 25 mg/dl , only CR3.4.P = 0.759). There was no statistical difference in defervescence curves between the two patient groups (P = 0.567). The median duration of hospitalization was 7 days  in all patients, and there was no difference between the two groups. Among all study patients, no patients had neurological manifestations during hospitalization or complications after hospital discharge.There was no significant difference in defervescence after hospitalization according to the presence of CSF pleocytosis. Fever subsided within 24 h in the majority of the patients. The proportion of patients with fever defervescence by hospital day 2 was 87.9% in those with CSF pleocytosis and 89.4% in those without CSF pleocytosis . CRP was the only independent factor associated with CSF pleocytosis. Clinical outcomes were similar regardless of CSF pleocytosis, and no patients had neurological complications.. 1.1% ], but there was no difference in meningitis risk between the two groups (0.4% vs. 0.6%). No cases of bacterial meningitis (0/697) were found in infants 29–60 days old with positive urinalysis results, whereas 0.2% of those with negative urinalysis had meningitis . These studies suggest that having a UTI does not increase the probability of bacterial meningitis, and LP is not essentially needed in the evaluation of fever in young infants with a presumptive UTI beyond the neonatal period. The American Academy of Pediatrics (AAP) guidelines recommend performing LP on well-appearing febrile infants 29–60 days old with positive urinalysis results, if any inflammatory markers level are abnormal  (3) were similar between well-appearing term infants and the other infants  among 3,868 infants aged 29–90 days with evidence of UTI . In anot3) but made no mention of traumatic LP. The prevalence of CSF pleocytosis was 29.1%  in infants aged 29–60 days  was 15.9%   .. 42.1%; P = 0.02)  . However = 0.02) . Thus, eA plausible hypothesis for sterile CSF pleocytosis is that systemic inflammation and cytokine release resulting from bacterial UTI might cause meningeal inflammation and lead to CSF pleocytosis , 13, 20.Our study had several limitations. First, it was a retrospective single-center study, which introduces selection bias and concerns about incomplete data. Second, data on viral tests than enterovirus on CSF specimens were insufficient. Third, although based on univariable analysis, the potential predictors associated with CSF pleocytosis were focused on inflammatory markers. However, we applied criteria and definitions based on clinically accepted consensuses. While some previously published studies included patients with traumatic LP or those with negative urine culture results, our study included only patients with culture-proven UTIs and non-traumatic LPs. Furthermore, we evaluated the independent predictors associated with sterile CSF pleocytosis.In conclusion, the overall risk of UTI and concomitant bacterial meningitis was very low in febrile infants aged 29–90 days. Sterile CSF pleocytosis was frequently observed in those with UTIs. Although coexisting sterile CSF pleocytosis with UTI may suggest a systemic inflammatory response, it did not seem to worsen clinical outcomes in healthy infants. The performance of LP in young infants with evidence of UTIs should be decided prudently, and inappropriate antibiotic therapy for sterile CSF pleocytosis should be avoided.


To identify beliefs and knowledge about tummy time (TT) practice and its repercussions on motor development.  Longitudinal study carried out with parents/caregivers of infants older than 30 days of life. Two assessments were performed. A structured interview was conducted, while the babies were between one and six months old to identify beliefs, knowledge about TT, and the motor milestone achievement expected for the age. At six to 12 months, the risk of motor development delay was tracked using the survey of well-being of young infant questionnaire (SWYC). p = 0.01). Only three infants from the control group were at risk of delayed motor development (p = 0.10).  41 families responded to the SWYC questionnaire . 31.70% reported that it was not important to put the infants in a prone position while awake, and 70.70% said they are afraid their babies would become breathless when positioned in a prone position. 85.70% of infants from the TT group showed typical development, while 55% of the control group showed atypical development for their age ( Most of the families feel insecure about proning their babies and fear breathlessness when positioned. Acquisition of motor milestones prevailed in the TT group, suggesting an association between TT practice and motor milestone achievement. Motor milestone achievement in the first years of life reflects the maturation and development of the central nervous system and is affected by intrinsic  and extrA way to prevent developmental delay in newborns is providing infants with adequate stimuli with tummy time (TT), encouraged and supervised by an adult . TT or aIn this sense, TT practice is included in most guidelines on infant health since it correlates with positive long-term outcomes  –10. Its Because studies have demonstrated a positive association between TT and the infant's development, , 15–17 iThis longitudinal study was conducted at an education and research center located in Northeastern Brazil. All parents/caregivers received verbal and written explanations regarding study objectives and methodology and signed an informed consent form. This study was approved by the research ethics committee  and performed according to the Declaration of Helsinki.A convenience sample of parents/caregivers was recruited between October and December 2019, in a childcare ambulatory. Parents/caregivers of full-term infants aged > 30 days were included. Those who did not complete the survey of well-being of young children (SWYC) questionnaire were excluded. Although there are several instruments available for identifying signs of risk for developmental delays, the SWYC is particularly advantageous due to its ease of use as a first-line screening tool and its ability to assess children following age-specific domains .∗ Power software, version 3.1.9.4), considering the risk of cranial asymmetry in infants with (25.8 ± 21.4) and without (48.7 ± 28.9) reduced time in prone (6). With Cohen's d effect size of .90, a power of .80, and α error of .05, the minimal number of infants was estimated as 38 infants .Sample size was calculated , sociodemographic data regarding parents/caregivers (education and occupation of parents), pregnancy, and infant data , and investigation about other infant products/services as smart steps/walkers or sessions of multiprofessional team .Motor development was assessed between one and six months by motor milestone achievement expected for the age of six to 12 months old using the SWYC.TT practice was investigated using a structured questionnaire composed of four domains. Domain #1 is about where and who cared for the infant. Domain #2 assessed the approach of parents and infants related to TT practice. In this domain, we investigated frequency and time dedicated to prone positioning (zero to 30 minutes or more), starting age, place where it is usually performed , stimuli , infant reaction , perception , and parental feeling and confidence regarding TT practice and factors influencing decision-making. For the latter, we presented concepts to parents/caregivers and asked if they agreed or not with the statements. Infants whose parents reported setting aside time to perform prone positioning daily were allocated to the TT group, and the remaining infants were allocated to the control group. Domain #3 evaluated the knowledge of parents/caregivers regarding TT using statements classified as “true” or “false,” while Domain #4 identified where the knowledge was acquired. The interview lasted about 10 minutes and was conducted in a private environment.To track the motor milestone achievement expected for the age, a set of figures representing possible motor skills was presented to parents/caregivers and asked if the infant could perform them. It was considered a “typical infant” when motor milestones expected for his/her age were achieved, or “atypical infant” when he/she did not achieve the motor milestones expected for the age. The key milestone expected for infants aged between one and three months was to lift the head in the prone position, whereas rolling alone was considered for infants aged between four and six months .The risk of motor development delay was tracked using the SWYC questionnaire between the age of six to 12 months. The moments of the evaluations were carried out according to the three stages of motor development determined by each corresponding SWYC form . For this, a telephone interview was conducted with parents/caregivers responsible for the infant's care. SWYC is a free, fast, and easy online tool to monitor infants' development aged between one and 65 months. It was recently validated for the Brazilian population and presents three major domains: motor development, family's behavior, and risk factors  , 25.For this study, only the “developmental milestones” domain was applied, which investigates the infant's behavior using 10 questions according to age group.  The studp value of <0.05 was considered significant for all statistical analyses.GraphPad Prism software version 6  was used for statistical analyses. Data normality was verified using the Kolmogorov-Smirnov test. The Mann–Whitney test was used to compare data between groups, while the Chi-square test investigated associations between TT and both, the motor milestones and risk of developmental delay. Data are shown as median, 25-75% interquartile range, and 95% confidence interval (95% CI). A Fifty-nine parents/caregivers were interviewed about TT and 41 responded to the SWYC questionnaire at the age of six to 12 months. Of the 41 infants included, 21 were allocated in the TT group, five (12.20%) had been hospitalized, three (7.30%) were on mechanical ventilation during the neonatal period, 34 (82.90%) attended monthly medical consultations, and six (14.60%) were followed up weekly by a professional from the multiprofessional team . No infants used smart steps or walkers. Most parents are afraid of positioning the baby in prone (70.70%) and 31.70% reported that positioning infants in prone while awake is not that important. Factors related to decision-making on TT practice are shown in In the TT group, twelve infants (57.10%) remained in prone positioning for one to five minutes daily, eight (38.10%) infants remained in six to eleven minutes in the position, and only one (4.80%) remained in prone for more than thirty minutes. Almost 52.40% of the studied families feel confident positioning the baby in prone. In the control group, eleven infants did not achieve motor milestones at six months of age, and three of them were at risk of motor development delay as assessed by SWYC at 12 months of age. Associations between TT and motor milestones are presented in Data suggest that infants aged between one and six months practicing prone positioning daily are more likely to achieve motor skills expected for their age, such as antigravity control of cervical muscles and rolling. However, infants who did not practice prone positioning daily were not at risk of developmental delay at the age of six to 12 months.Corroborating partially with these results, in Taiwan, Kuo et al. (2008) followed 288 infants and found that rolling and crawling acquisitions at the age of four to 24 months occurred significantly early in the group that practiced TT . Kuo et Although different studies corroborate that daily practice of TT can contribute to motor milestone achievement , 26, 27 Kuo et al. (2008) demonstrated that 20 minutes was reasonable for TT practice and suggested time does not influence the motor milestone achievement . Many paWe believe that TT offers the infant a range of possibilities regardless of daily practice since it increases the visual field . BesidesOther studies reinforce that adaptation of information to local language and culture contributes to adherence, while lack of access to information, especially in poorer and underprivileged families, contributes to delay the dissemination of such practice –34. RicaIn order to determine strategies to support parent's implementation of TT, occupational therapists have provided a new model to plan educational programs . The plaFrom the perspective of motor development, only three infants from the control group presented SWYC scores indicating risk. Despite this, the absence of prone positioning practice was not associated with the risk of developmental delay, probably because families from the control group may have experienced prone positioning during the follow-up interval. Thus, the lack of strict follow-up may have impaired data interpretation, and it characterizes a limitation of this study. Previous studies performed in developed countries showed that low adherence is reversed after adopting and disseminating policies encouraging TT practice , 33, 35.Data suggests that infants aged between one and six months practicing prone positioning daily are more likely to achieve motor skills expected for the age, such as antigravity control of cervical muscles and rolling. Considering the demographic, socioeconomic, and behavioral particularities of the Brazilian context, the benefits pointed out in this study may motivate new, larger, and further studies, contributing to public policies and disseminating and implementing the TT practice.


Detection of α‐synuclein aggregates by seed amplification is a promising Parkinson disease biomarker assay. Understanding intraindividual relationships of α‐synuclein measures could inform optimal biomarker development. The objectives were to test accuracy of α‐synuclein seed amplification assay in central  and peripheral (submandibular gland) sources, compare to total α‐synuclein measures, and investigate within‐subject relationships.n = 59) and compared to healthy controls (n = 21). Motor and non‐motor measures and dopamine transporter scans were obtained. Four measures of α‐synuclein were compared: seed amplification assay in cerebrospinal fluid and formalin‐fixed paraffin‐embedded submandibular gland, total α‐synuclein quantified in biofluids using enzyme‐linked immunoassay, and aggregated α‐synuclein in submandibular gland detected by immunohistochemistry. Accuracy of seed amplification assay for Parkinson disease diagnosis was examined and within‐subject α‐synuclein measures were compared.The Systemic Synuclein Sampling Study aimed to characterize α‐synuclein in multiple tissues and biofluids within Parkinson disease subjects  Parkinson disease participants were positive for both cerebrospinal fluid and submandibular gland seed amplification assay. Comparing accuracy for Parkinson disease diagnosis of different α‐synuclein measures, cerebrospinal fluid seed amplification assay was the highest (Youden Index = 83.1%). 98.3% of all Parkinson disease cases had ≥1 measure of α‐synuclein positive.α‐synuclein seed amplification assay  had higher sensitivity and specificity compared to total α‐synuclein measures, and within‐subject relationships of central and peripheral α‐synuclein measures emerged. SCNA show a gene dosage effect.α‐synuclein (αSyn) is the pathologic hallmark of Parkinson's disease (PD) and mutations in The Systemic Synuclein Sampling Study (S4) aimed to address these knowledge gapsS4 was a cross‐sectional observational study that enrolled participants from October 2015 to August 2017 at six sites in the United States and Canada. Study methodology have been previously reported.Institutional review board approval was obtained at each site and written informed consent was obtained from each participant.As previously described,n = 59 PD, n = 21 HC) have been previously published.Sample characteristics of the 80 participants of the S4 cohort who contributed at least two fluid and tissue specimens  calculated as the average of putamen and caudate SBR on right and left.Blood, saliva, CSF, and SMG biopsy were collected and processed as described in previous reports.Fmax, value at which a plateau of ThT fluorescence is reached), T50 (time needed to reach 50% of maximum aggregation), and area under the curve (AUC). For each subject, a mean value across the three triplicates was calculated for each measure. Unlike total αSyn measures, CSF SAA is considered robust to hemoglobin (HGB) contamination; after confirming this in our dataset, we included in our analysis all CSF samples for which SAA results were available.CSF SAA was performed as previously described.Fmax). To normalize the signals between samples, %Fmax relative fluorescence units (RFU) was determined by dividing the highest mean RFU for each sample in a plate by the highest or maximal RFU for the positive control on a given plate.SMG SAA was performed as previously describedTotal αSyn in CSF, blood, and saliva were measured using a commercially available ELISA.As for SMG αSyn IHC, as previously described,In S4, 59 individuals with PD and 21 HC were enrolled and contributed at least two biofluids and two tissue specimens. Only those individuals who had available either CSF or SMG SAA results were included in analyses pertaining to those respective assays. Of the 59 with PD, 54 had a CSF sample available, 41 had an SMG sample available, and 38 had both CSF and SMG sample available. All 21 HC had CSF, and 14 had SMG.t tests were used to compare continuous variables among PD versus HC for normally distributed variables, with Wilcoxon rank‐sum tests used where nonparametric testing was appropriate. Chi‐squared test or Fisher's exact test were applied as appropriate to compare categorical variables between groups.Two‐sample Fmax and T50 for CSF, %Fmax and TT for SMG). Optimal cutoffs were defined by the point on the ROC curve that yielded the highest Youden index (J = sensitivity + specificity−1). ROC curves were based on univariate logistic regression models and estimates of the area under the ROC curve (AUC) were also computed.Sensitivity and specificity of the SAA measures in CSF and SMG as PD biomarkers (PD vs. HC) were examined using cutoff values derived from receiver operating characteristic (ROC) curves, conducted for each of the individual SAA measures , as well as measures of PD disease severity were examined within the PD group using spearman rank‐order partial correlations, controlling for age.All statistical analyses were performed using SAS version 9.4 .Sample characteristics of those with either CSF or SMG SAA results available are shown in Table Fmax cutoff of ≥1375 maximized accuracy of classification of subjects as PD versus HC. 50/54 PD were positive and 19/21 HC were negative for CSF SAA, for a sensitivity and specificity of 92.6% and 90.5%, respectively (Youden index = 83.1%). Among the PD group with negative CSF SAA, two were in the moderate PD group and two in the advanced PD group. The sensitivity of SAA was 100%, 88.9%, and 90% in the early, moderate, and advanced groups, respectively; specificity was 90.5% in all three subgroups of disease severity.Receiver operating characteristic analyses indicated that a CSF Fmax was 3309.5 RFU in the PD group and 23 in the healthy control group. The Fmax values for the four PD cases that were negative for CSF SAA were 16, 20, 146, and 481.1 RFU. Thus, one of the four cases enrolled in the PD group had an Fmax value that could indicate a diagnosis multiple system atrophy (MSA).Median Receiver operating characteristic analyses indicated that an SMG TT cutoff of ≤26.9 h maximized accuracy of classification of subjects as PD versus HC. At that cutoff, 30/41 PD were positive and 11/14 HC were negative for SMG SAA, yielding a sensitivity and specificity of 73.2% and 78.6%, respectively (Youden Index = 51.8%). Among the 11 PD participants negative for SMG SAA, five were in the early PD group and five were in the advanced PD group.Positivity of CSF SAA across subgroups of PD severity did not differ Table . In addiFmax and total αSyn in both CSF  and plasma , indicating that higher Fmax correlated with lower total CSF and plasma αSyn.The relationship between SAA in CSF and SMG and total αSyn (ELISA) measures across all biofluids is shown in Table n = 80) are included in the following analyses which contrast the different measures of αSyn in the overall cohort. The prevalence of positivity for each of four measures of αSyn is depicted in Figure Fmax value was 481 RFU , had a positive SMG SAA.All subjects enrolled in S4 who contributed at least two tissues and two fluids . Notably, three samples were positive for CSF total αSyn but not for CSF SAA. In only one of the PD cases with both assays available were results negative on both tests. Among the HC, one was positive for both CSF SAA and total αSyn.In considering CSF αSyn measures, 58/59 PD subjects enrolled in S4 had at least one measure positive. Among the In contrasting SMG SAA to IHC among the PD group, of the 41 with both results available, 20 (48.8%) were positive for both. Among the 30 subjects positive for SMG SAA, SMG IHC was positive in 20 (66.7%). Among 11 participants negative for SAA, IHC was positive in four participants. Among the 17 PD participants negative for IHC, SAA was positive in 10 participants. Notably, three HC were positive for SMG SAA, but were negative for SMG IHC, whereas the one SMG IHC positive HC was negative for SMG SAA.In the PD cohort, 12/59 (20.34%) were positive for all four αSyn measures compared here. Importantly, only one PD subject was negative across all available measures (though missing data for this subject limit interpretation of this finding). Of 35 participants who had either SMG measure positive, all also had a CSF measure positive. Of those with CSF positive by either measure who also had both SMG measures available, 12/26 (46.2%) had positive SMG by either measure.Two controls were positive in both CSF and SMG: one was IHC positive in SMG and had positive CSF SAA, whereas the other had SMG SAA positive and CSF total αSyn positive.Comparing total αSyn measures in CSF and blood in relation to SAA positivity in CSF or SMG in the PD group revealed no significant differences Table .In this study, αSyn SAA, which has high specificity for disease‐associated αSyn, yielded high accuracy as a PD diagnostic biomarker in CSF, a central source, and moderate accuracy in SMG, a peripheral tissue proximal to the CNS. These findings are superior to the performance of previously examined measures,Biomarkers are critically needed to facilitate diagnosis of PD, especially early on in the disease course where diagnostic accuracy may be low, particularly among older indivdiuals.Fmax and total αSyn. While the relationship is conceptually intuitive, any significant relationship between the two measures would be expected to be of small effect size, given the aforementioned considerations related to measures of total αSyn by antibodies, whereby only a small percentage of the overall measure likely reflects pathologic forms of αSyn.et alet al,Fmax and/or T50 was not found. Given that the S4 protocol and the study of Kang et alIn line with the above, in examining the relationship between CSF αSyn measured by SAA and ELISA, we found a moderate but significant relationship between CSF Fmax value that was significantly higher than normal controls but lower than in PD. This pattern has been described in individuals with MSAOur data indicate that the two approaches to measuring αSyn in CSF (SAA and ELISA) provide complementary information. Even with the high sensitivity of CSF SAA, there were three PD subjects negative for CSF SAA but positive for total αSyn by ELISA. One of these subjects had an et alet alInvestigating the distribution of αSyn in peripheral tissue informs proposed models of spread of αSyn during the pathophysiological cascade that are alternately posited to occur first in the CNS or first in the PNS, the latter in the gastrointestinal tract and elsewhere. Chahine Fmax had optimal accuracy, in SMG, the TT metric was optimal and Fmax less sensitive. This may have been due to heat, xylene and EtOH used to remove paraffin, and/or formalin‐induced cross‐linking of αSyn aggregate seeds, the dilute nature of the processed FFPE samples, variability in the total amount of target tissue harvested from each slide, or other factors yet to be investigated.As for the relationship of central (CSF) and peripheral (SMG) measures of αSyn, not surprisingly, there were not any instances of PD subjects having positive SMG but not positive CSF. While it has been hypothesized that PD pathology may begin outside the CNS,A lack of relationship between αSyn SAA measures and clinical measures of PD disease severity is consistent with other studies,S4 study limitations include the small sample size in PD subgroups, limiting comparisons. In addition, the cohort lacks racial and ethnic diversity, limiting generalizability. Nevertheless, S4 remains unique and valuable with the various intraindividual tissue and fluid collection in subjects with different PD stages and HC.In conclusion, the S4 study, by comparing αSyn measured by SAA and ELISA in CSF and SAA and IHC in SMG, illustrates the higher sensitivity and specificity of SAA in these matrices. Our results support the value of other αSyn measures to inform further development of αSyn as a PD biomarker and indicate that multimodal and multifocal assessment of αSyn biomarkers is informative.The authors declare no conflicts of interest related to this work.


Burkholderia pseudomallei is the causative agent of melioidosis, which is increasingly being reported worldwide. Mortality rates as high as 40% have been reported based on clinical patient outcomes in the endemic areas of Australia and Thailand. Novel therapies are needed to reduce treatment duration and adverse effects and improve treatment outcomes. Epetraborole, a novel antibiotic, targets leucyl-tRNA synthetase (LeuRS), an essential enzyme that catalyzes the attachment of leucine to transfer RNA. Epetraborole was evaluated for in vitro activity and efficacy in a murine model to assess clinical relevance against Burkholderia pseudomallei infections for possible treatment of melioidosis. Epetraborole was tested against 13 clinically derived and three reference B. pseudomallei strains that have a broad spectrum of susceptibilities to the standard-of-care (SoC) drugs for melioidosis, which showed that epetraborole exhibited minimal inhibitory concentrations of 0.25–4 μg/mL. Ex vivo studies using THP-1 macrophages confirmed the potency of epetraborole and demonstrated synergy between epetraborole and ceftazidime. In the acute pulmonary murine infection model of melioidosis, epetraborole demonstrated equivalent efficacy when delivered orally or subcutaneously, which compared well with the standard-of-care drug ceftazidime. In addition, adding epetraborole to ceftazidime significantly improved antimicrobial activity in this animal model. This work warrants further exploration of epetraborole as a candidate for treating melioidosis and substantiates LeuRS as a clinically relevant drug target in B. pseudomallei. Burkholderia pseudomallei, commonly found in Southeast Asia, Australia, Central and South America, Mexico, and recently in the Southern United States. The current clinical treatment of melioidosis involving multiple drugs is extensive, and a portion of infections will persist or relapse after the completion of treatment. Resistance to the majority of antibiotics limits the ability to treat and manage the disease effectively. However, Burkholderia pseudomallei is an under-recognized emerging pathogen with a general lack of resources and interest to drive research for new treatments. Epetraborole was initially identified with in vitro and in vivo activity against various medically important Gram-negative bacterial infections and was advanced to treat complicated urinary tract infections. Accordingly, we have assessed the use of epetraborole to treat melioidosis in a mouse model when administered alone or in combination with the current clinically used drug ceftazidime.Our study suggests the repurposing of epetraborole for the treatment of the often rapidly progressing and fatal disease melioidosis is warranted. Melioidosis is caused by the bacterial pathogen  Burkholderia pseudomallei, the causative agent of the tropical disease melioidosis, is an infectious bacterium that causes acute fatal disease in humans, which was first described in 1912 in Rangoon by Whitmore and Khrisnaswami [B. pseudomallei is an under-recognized emerging pathogen in Central and South America, Mexico, and more recently the Southern United States [B. pseudomallei is endemic [B. pseudomallei suggests the disease is endemic in many countries throughout the Americas [d States . Until r endemic . Indeed,Americas . The widAmericas .in vivo resistance mechanisms and natural resistance due to efflux pumps render most drugs ineffective against Burkholderia infections and limit the ability to treat and manage the disease [Treatment of melioidosis is lengthy compared to other Gram-negative bacterial infections, with a minimum of 2 weeks of IV therapy of ceftazidime or meropenem followed by 3–6 months of oral therapy with trimethoprim-sulfamethoxazole . Althoug disease ,10,18–21in vitro and in vivo activity against Gram-negative bacteria. Epetraborole was advanced to a phase 2 clinical trial for complicated urinary tract infections. These studies demonstrated that epetraborole has broad-spectrum potential and LeuRS is a clinically relevant drug target. LeuRS is an essential enzyme for protein synthesis that attaches leucine to tRNALeu. In addition to this aminoacylation active site, LeuRS has an editing site that hydrolyzes incorrect amino acids from its cognate tRNA [Leu in the editing active site via its boron atom binding to the cis-diols of the terminal adenosine of tRNALeu [Leu from being leucylated by the aminoacylation site. Thus, the inhibition of Leu-tRNALeu synthesis ultimately leads to a block in protein synthesis. In addition to epetraborole’s novel mechanism of action, it has physiochemical properties appropriate for a melioidosis drug candidate. Epetraborole has a small molecule weight (MW 237), high polarity with a LogD7.4 of -0.23, very low plasma protein binding, and excellent cell penetration, including human alveolar macrophages [B. pseudomallei.The clinical drug candidate, epetraborole, is a boron-containing inhibitor of the bacterial leucyl-tRNA synthetase (LeuRS). Epetraborole was initially identified with ate tRNA . Epetrab tRNALeu , which prophages ; these ain vitro activity and in vivo efficacy of epetraborole against B. pseudomallei as monotherapy and in combination with the standard of care (SoC) drug ceftazidime. Epetraborole demonstrated potency against a panel of laboratory reference and clinically derived B. pseudomallei strains and efficacy in in vitro, ex vivo, and an acute animal model of melioidosis. The assessment of epetraborole against clinically derived strains and in efficacy models allowed us to directly assess the potential use of epetraborole to treat clinically relevant infections administered alone or in combination with ceftazidime. These data demonstrate that further exploration of epetraborole in the therapy of melioidosis is warranted, which further supports LeuRS as a clinically relevant drug target.Accordingly, we assessed the All studies performed at Colorado State University were conducted in a BSL3 facility dedicated to bacterial pathogen work under the approvals and management of the Biosafety Official. Studies were approved by the Institutional Biosafety Committee and the Institutional Animal Care and Use Committee and performed under approvals PARF 17-095B and IACUC protocol 3796.B. pseudomallei strains were grown to an OD600 of ~0.6 and were frozen in 10% (v/v) glycerol at -80°C as standard bacterial stocks for these studies. For each evaluation, bacteria were prepared fresh by growth from the standard Luria-Bertani (LB) Agar stocks at 37°C for 48–72 hrs. Bacteria recovered from the LB plates were used to inoculate 10 mL LB Broth. Broth cultures were then incubated for 18 hrs at 37°C, passed 1:100, and incubated for an additional 6 hrs at 37°C. Bacteria were then diluted into 1x106 colony forming units (CFU)/mL concentration in cation-adjusted Mueller-Hinton (caMH) Broth , and 50 μL was added to each well for each drug plate for a final inoculum of 5x105 CFU/mL. For MIC determination, the concentration range tested for epetraborole and ceftazidime was 0.03–64 μg/mL in caMH broth. MIC plates were incubated at 37°C for 18 hrs, at which time MIC was determined per CLSI guidelines [P. aeruginosa ATCC 27853 was 2 and 4 μg/mL, respectively, which is within their QC ranges.idelines . The MIC5 activated THP-1 cells per well in a 24-well tissue culture plate were cultured in a complete medium that consisted of Roswell Park Memorial Institute (RPMI) (Invitrogen) and 10% (v/v) fetal bovine serum . THP-1 cells were incubated at 37°C with 5% CO2 for 24 hours (hrs). Bacteria were added to THP-1 cells at a multiplicity of infection of 10 CFU per cell in a 0.5 mL media. The plates were centrifuged at 2,400 X g for 2 min and placed at 37°C/5% CO2 to incubate for 2 hrs. The supernatant was removed, and each well was washed twice with 2 mL phosphate-buffered saline . Epetraborole HCl (AN2 Therapeutics) and ceftazidime (Sigma-Aldrich) were added to each well at 8, 4, 2, 1, 0.5, and 0.25 μg/mL in complete media in triplicate. Media only were added to three wells to serve as a negative drug control. Plates were incubated for 24 hrs, and cells were observed for signs of infection. The cells were washed thrice with 2 mL PBS and lysed with 1 mL sterile 0.05% SDS/water. Each well was thoroughly mixed/scraped and incubated at room temperature for 7 minutes to ensure complete cell lysis. Lysates were then serially diluted at 1:10, and inoculum plated from the neat, 10−1, 10−2, 10−3, and 10−4 dilutions onto LB agar plates. Plates were incubated for 48 hrs at 37°C. Colonies from each plate were counted, and Log10 CFU/mL lysate was calculated.Infected THP-1 macrophages  were used to assess the intracellular effectiveness of these compounds. 2.5x10Epetraborole pharmacokinetics were assessed in female BALB/c mice by Quintara Discovery Inc. . Epetraborole formulation and bioanalysis were performed as described by Hernandez et al. . EighteeB. pseudomallei mouse model of disease for efficacy evaluation used 7–9 week-old BALB/c female mice , which were challenged by intranasal infection with 5,000 CFU/mouse B. pseudomallei 1026b. Animals were anesthetized with a mixture of 100 mg/kg ketamine and 10 mg/kg xylazine delivered intraperitoneally before receiving the inoculum in a 20 μL volume in alternating nostrils. Ceftazidime was formulated in PBS (pH 7.4) to be delivered subcutaneously (SC) at 200, 125, and 25 mg/kg twice a day (BID). Epetraborole was formulated for injection in water (pH 5) to be given SC at 300, 100, and 30 mg/kg or orally (PO) at 30, 15, and 5 mg/kg BID. Dosing for mice assessed with combinatorial drug therapy was 200/30, 125/15, and 25/5 mg/kg ceftazidime and epetraborole. Drugs were delivered 2 hrs post-infection, and repeated doses were given every 24 hrs. The number of viable bacteria in the lung and spleen was determined at 60 hrs post-exposure by plating serial 10-fold dilutions of homogenates onto LB agar and incubating for 48 hrs at 37°C. Bacterial burden was assessed using a one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison tests with significance determined by a P-value < 0.05.Acute B. pseudomallei. Animals were anesthetized with a mixture of 100 mg/kg ketamine and 10 mg/kg xylazine delivered intraperitoneally before receiving the inoculum in a 20 μL volume in alternating nostrils. Ceftazidime was formulated in PBS (pH 7.4) to be delivered subcutaneously (SC) at 250 mg/kg for every 4hrs, and epetraborole was formulated for injection in water (pH 5) to be delivered intraperitoneally (IP) at 200 and 100 mg/kg and dosed 12 hours after infection. Combination therapy was assessed by dosing mice with 200 and 100 mg/kg epetraborole IP BID plus the ceftazidime regimen described above. All study drugs were administered 12 hours post-infection for delayed dosing efficacy evaluation. The number of viable bacteria in the lung and spleen was determined at 60 hrs post-exposure by plating serial 10-fold dilutions of homogenates onto LB agar and incubating for 48 hrs at 37°C. Bacterial burden was assessed using a one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison tests with significance determined by a P-value < 0.05.7–9 week-old BALB/c female mice were challenged by intranasal infection with 5,000 CFU/mouse B. pseudomallei laboratory reference strains 1026b and K96243, the clinical representative susceptible strain DD503, and the drug efflux deficient B. pseudomallei strain Bp400 to determine the baseline inhibitory activity of epetraborole and assess as an efflux pump substrate. These tests revealed that epetraborole has a MIC range of 0.25–1 μg/mL across these strains, with DD503 and Bp400 being the most susceptible and the laboratory strains 1026b and K96243 being the least sensitive (Table 1). The in vitro activity of epetraborole was superior to the SoC drug ceftazidime, which has a MIC range of 2–4 μg/mL against these strains. The Bp400 strain, with the BepAB-OprB and AmrAB-OprA efflux pumps deleted, was only 4-fold more susceptible than its parental wild-type strain 1026b [B. pseudomallei strains representative of the drug susceptibility spectrum associated with clinical infections [Table 1). These studies confirm that epetraborole has potency across clinically representative strains comparable to the standard-of-care drugs currently used in the clinical treatment [Epetraborole was screened for anti-burkholderia activity using a standard broth microdilution method . Epetrabfections . Againstreatment .B. pseudomallei 1026b in a Burkholderia ex vivo model. Growth inhibition was determined over a 0.25 to 8 μg/mL concentration series of epetraborole. Epetraborole inhibited intracellular growth of B. pseudomallei in a dose-dependent fashion, demonstrating a ~ 1.5 log10 reduction in CFUs at 4 μg/mL and ~ 2.5 log10 reduction in CFUs at 8 μg/mL . Similar dose-dependent decreases were observed for the control drug, ceftazidime, which demonstrated ~ 2 log10 reduction in CFUs at 4 μg/mL . Epetraborole was also evaluated as a co-therapeutic with ceftazidime to determine epetraborole’s use as a new agent in a combination therapy regimen. A dose of 4 μg/mL of ceftazidime was added to an epetraborole concentration series of 0.25–8 μg/mL, which showed that co-treatment of 1 μg/mL epetraborole with 4 μg/mL of ceftazidime showed a ~ 3.5 log10 reduction in CFUs. This reduction was more significant than the reduction observed for 4 μg/mL of ceftazidime alone . This demonstrates that the 1:4 combination of epetraborole and ceftazidime has an additive effect and can significantly reduce CFUs at lower concentrations than can be achieved with monotherapy. These studies also substantiate that epetraborole can gain access to the bacteria in macrophages.Epetraborole was evaluated against B. pseudomallei animal infection model to determine efficacy. Initially, we chose to dose mice subcutaneously with 30, 100, or 300 mg/kg epetraborole BID, which equates to AUC0-24 of approximately 18.6, 62, 186 mg.h/L, assuming linearity from a single 30 mg/kg SC dose in a satellite PK group that yielded an AUC0-24 of 9.3 mg.h/L (Table 2). In comparison, in humans in 2 studies where epetraborole was dosed at 1500 mg q12h, it achieved exposures with an AUC0-24 of 146 mg.h/L [10 or greater CFU/mL reduction (P<0.05) in the lung compared to the untreated control group . This CFU reduction was more significant than observed in mice treated with comparative doses of 25, 125, or 200 mg/kg ceftazidime BID, which resulted in a decrease of 0.5 to 4 Log10 CFU/mL in the lungs (P<0.05 for 200 mg/kg group). Dissemination of B. pseudomallei to the spleen was not detected in the epetraborole-treated groups, contrasting the observed dissemination in the lower ceftazidime-treated groups . To determine epetraborole efficacy via the oral route, epetraborole was also delivered orally at 30 and 15 mg/kg doses of BID, which equates to an AUC0-24 of approximately 4.9 and 9.7 mg h/L assuming linearity from a single 30 mg/kg PO dose in a satellite PK group that yielded an AUC0-24 of 4.9 mg. h/L. These low oral doses of epetraborole reduced bacterial burden in the lungs by 1.8–3.8 Log10 CFU/mL (p<0.05) with the 30 mg/kg dose of epetraborole resulting in a ~ 3.5 Log10 CFU/mL reduction (P<0.05) in the lung compared to the untreated control group . Oral treatment with 15 mg/kg of epetraborole resulted in a ~ 1.5 Log10 CFU/mL reduction (P<0.05) in the lung. Dissemination to the spleen was not observed in the animals treated orally with 30 mg/kg of epetraborole .Epetraborole was tested in the acute in vivo. Epetraborole and ceftazidime were co-delivered at 30 mg/kg-200 mg/kg, 15 mg/kg-125 mg/kg, or 5 mg/kg-25 mg/kg combinations. These doses of oral epetraborole were chosen not to saturate the efficacy window in this model, thus allowing us to see at least additive efficacy from the combination of epetraborole and ceftazidime. The co-delivery of epetraborole orally at 30 and 15 mg/kg doses of BID and ceftazidime at 125 mg/kg or 200 mg/kg (SC), respectively, resulted in a ~ 5 Log10 CFU/mL reduction in bacterial burden in the lung compared to untreated control . Epetraborole was also effective against disseminated disease in the spleen when delivered orally at 15 or 30 mg/kg as a single agent or combined with 125 or 200 mg/kg ceftazidime (SC) .We further assessed the combination of epetraborole with ceftazidime . Delayed dosing with epetraborole 100 mg/kg and 200 mg/kg IP BID significantly reduced the bacterial burden in the lung by ~ 3.4 and ~ 4.8 Log10 CFU/mL and in the spleen by ~ 3.8 and 4.8 Log10 CFU/mL, respectively . Combining the ceftazidime dose regimen with 100 mg/kg epetraborole treatment enhanced the activity compared to 100 mg/kg epetraborole alone. Together, these studies demonstrate that epetraborole is efficacious against B. pseudomallei when delivered by different routes and offers an additive activity when combined with the SoC drug ceftazidime, even when treatment is initiated after infection.To further validate the potential improvement in clinical outcomes of epetraborole efficacy against the clinically derived strain MSHR435, which has elevated ceftazidime MIC values of 8 μg/mL, was investigated in a delayed dosing model where mice were dosed 12 hrs post-infection (B. pseudomallei is more geographically widespread than previously thought [st, 2023, melioidosis has become a nationally notifiable urgent condition in the United States by the CDC [B. pseudomallei strains and in animals of melioidosis to determine its potential as a clinical candidate that can be used alone or as a co-therapeutic to treat B. pseudomallei infections.Given the high unmet medical need for melioidosis, one of the goals in drug discovery is to identify candidates that can be used alone or in combination to treat this severe, rapidly progressing, potentially life-threatening disease. Once thought to only be endemic in Australia and Asia, recently, melioidosis has been isolated in the Americas at an increasing frequency, indicating that  thought , and, wi the CDC . The lim the CDC ,10. A hiB. pseudomallei strains with differing susceptibility to SoC drugs. An important metric of drug performance is the demonstrated comparable potency of epetraborole against B. pseudomallei strains with a spectrum of drug susceptibilities. Multiple encoded efflux systems have been well documented to impact the natural resistance rate of B. pseudomallei strains because even slight changes in more than one efflux activity would increase the required inhibitory concentration of the drug. Notably, the in vitro activity of epetraborole is minimally impacted by drug efflux that is well known to result in widespread drug resistance in B. pseudomallei. To assess the efficacy potential of epetraborole, the intracellular activity was evaluated using an ex vivo efficacy model routinely used in drug discovery efforts. Epetraborole was highly effective in significantly reducing the bacterial numbers in a dose-dependent manner by several log10 CFU. The activity in this in vitro assay indicates that epetraborole is very effective in killing intracellular bacteria in macrophages. The observed activity in an ex vivo model substantiates the ability of epetraborole to permeate the mammalian cell membrane and maintain activity in the host environment.We have successfully used standardized strain panels to discover two new investigational drug candidates ,29. SimiB. pseudomallei strains, including clinical strains with various susceptibilities to current SoC drugs, and has efficacy in an ex vivo model and a standard lethal mouse models of melioidosis. Epetraborole reduced the bacterial load in the lungs in a dose-dependent fashion. Notably, epetraborole demonstrated efficacy when administrated by different routes at equivalent doses. Consistent with the significant reduction in bacterial load in the lungs, the bacterial load in the spleen was typically at the level of detection or below. Given that 25% of patients on IV SoC therapy die within 1 month, with even a greater amount in 1 year, any improvement in SoC is desperately needed [B. pseudomallei in the clinical setting. Even when epetraborole administration was delayed, it demonstrated efficacy against a clinically derived difficult-to-treat B. pseudomallei strain. These observations are particularly significant given the various intrinsic resistance and drug efflux mechanisms of B. pseudomallei. The potency of epetraborole against clinically derived strains and in vivo efficacy demonstrates the potential for epetraborole to improve clinical outcomes in combination with ceftazidime.This study demonstrated that epetraborole possesses antibacterial activity against a panel of y needed ,31. Ther