Aggregation of α-synuclein is central to the pathophysiology of PD. Biomarkers related to α-synuclein may be informative for PD diagnosis/progression.To analyze α-synuclein in CSF in drug-naïve PD, healthy controls, and prodromal PD in the Parkinson’s Progression Markers Initiative.Over up to 36-month follow-up, CSF total α-synuclein and its association with MDS-UPDRS motor scores, cognitive assessments, and dopamine transporter imaging were assessed.The inception cohort included PD , healthy controls , hyposmics , and idiopathic rapid eye movement sleep behavior disorder . Baseline CSF α-synuclein was lower in manifest and prodromal PD versus healthy controls. Longitudinal α-synuclein decreased significantly in PD at 24 and 36 months, did not change in prodromal PD over 12 months, and trended toward an increase in healthy controls. The decrease in PD was not shown when CSF samples with high hemoglobin concentration were removed from the analysis. CSF α-synuclein changes did not correlate with longitudinal MDS-UPDRS motor scores or dopamine transporter scan.CSF α-synuclein decreases early in the disease, preceding motor PD. CSF α-synuclein does not correlate with progression and therefore does not reflect ongoing dopaminergic neurodegeneration. Decreased CSF α-synuclein may be an indirect index of changes in the balance between α-synuclein secretion, solubility, or aggregation in the brain, reflecting its overall turnover. Additional biomarkers more directly related to α-synuclein pathophysiology and disease progression and other markers to be identified by, for example, proteomics and metabolomics are needed. Intracellular aggregation and intercellular spread of pathological forms of α-synuclein (α-syn) are central to the progressive neurodegeneration of PD.5 and may serve as a marker to assist in diagnosis and prognosticating progression. We recently reported that CSF α-syn levels in de novo PD showed minimal change over 12 months.6 Longitudinal changes in CSF α-syn and other biomarkers have been examined in other PD cohorts for up to 2 years with discrepant findings.8 Subject selection, preanalytical factors, and different assays may have contributed to discrepancies  are decreased in PD and other synucleinopathiescies see . StudiesWe therefore analyzed the levels of total α-syn in longitudinal CSF samples of PD participants and healthy controls (HCs), and in a cohort of prodromal PD. We did not measure subspecies or posttranslationally modified forms of α-syn. We hypothesized that CSF α-syn would change over 36 months with PD progression, that decreased levels would be present in prodromal PD, and that CSF α-syn would correlate with clinical measures or imaging indices of progression.9 newly diagnosed, drug-naïve PD patients (N = 423) and age- and sex-matched HCs (N = 196) were included (http://ppmi-info.org/study-design). Inclusion and exclusion criteria have been published elsewhere.9 Briefly, inclusion criteria for PD participants were the following: (1) aged >30 years; (2) presence of two of the following: bradykinesia, rigidity, and resting tremor or an asymmetric resting tremor or asymmetric bradykinesia; (3) diagnosis made within the last 24 months; (4) PD drug naïvety, and (5) dopamine transporter (DaT) deficit in the putamen on 123-I Ioflupane DaT imaging by central reading.The PPMI (Parkinson’s Progression Markers Initiative) is an ongoing, prospective, longitudinal, observational, international multicenter study that aims to identify biomarkers for the progression of PD. As described,10 was based on the following criteria: (1) 18% of any electromyography (EMG) activity in m. mentalis, 32% of any EMG activity in mentalis and flexor digitorum superficialis ; (2) 27% of any EMG activity in m. mentalis and 32% of any EMG activity in m. mentalis and FDS (in 30-second bins). In 2 cases, a central PSG reading was not available because of technical difficulties with electronic PSG transfer, but these participants had a clinical diagnosis of iRBD by the site investigator, including previous PSG, and also had to show decreased DaT imaging.Between July 2013 and March 2015 participants with isolated (iRBD) rapid eye movement (REM) sleep behavior disorder (RBD) or isolated hyposmia were recruited in PPMI centers for the prodromal part of PPMI. iRBD participants met the following criteria: (1) men or women aged ≥60 years and (2) confirmation of RBD by polysomnography (PSG) with central reading (details below) and/or clinical diagnosis of RBD by the site investigator, including existing PSG. Central PSG interpretationhttp://www.ppmi-info.org/study-design/research-documentsand-sops/.Hyposmic participants were aged ≥60 years with olfaction at or below the 10th percentile by age and sex, as determined by the University of Pennsylvania Smell Identification Test (UPSIT). All iRBD and hyposmic participants also required confirmation from the imaging core at the Institute for Neurodegenerative Disorders that screening DaTscan (or vesicular monoamine transporter type 2/PET scan for sites where DaTscan is not available) was read as eligible. Around 80% of the prodromal participants were selected with a DaT deficit similar to participants with early PD, and 20% were selected with no DaT deficit. Prodromal subjects without DaT deficit were similar in age, sex, and risk profile to those with mild-to-moderate DaT deficit. Exclusion criteria can be found in the study protocol at www.ppmi-info.org).This article is based on α-syn analyses from CSF samples obtained from PD and HCs at baseline and 6-, 12-, 24-, and 36-month visits and for prodromal subjects at baseline and 6- and 12-month visits; overall data were downloaded December 4, 2017 from the PPMI database . Aliquots of 0.5 mL of frozen CSF were used by BioLegend  to measure CSF hemoglobin levels and CSF total α-syn with a sandwich-type immunoassay . In the analyses below, we excluded three CSF values in PD subjects as outliers: all were >5,000 pg/mL , and in all 3 subjects, subsequent longitudinal CSF levels of α-syn were substantially (>50%) lower.CSF was collected using standardized lumbar puncture procedures. Sample handling, shipment, and storage were carried out as described11 In brief, motor assessment used the revised UPDRS published by the International Parkinson and Movement Disorder Society .12 Use of medications for PD was recorded at each visit after baseline assessment and is expressed as levodopa equivalent doses (LEDs)13 and stratified according to LED subtotal from dopamine replacement or dopamine agonists.The clinical assessment battery is described on the PPMI website and has been published previously.14 The REM Sleep Behavior Disorder Screening Questionnaire (RBDSQ) was used to assess subjectively reported symptoms of RBD.15Cognitive testing included the Montreal Cognitive Assessment (MoCA) and psychometric tests of memory , processing speed/attention , executive function/working memory , and visuospatial abilities (Benton Judgment of Line Orientation [BJLO] test).9 Quantitative DaTscan measures in striatal binding ratio (SBR) of caudate, putamen, or striatal uptake were used in our analyses.Dopamine imaging was performed by DaTscan using standardized methods.APOE genotypes, MAPT, and single-nucleotide polymorphisms related to SNCA , measured by the PPMI Genetics Core.16To examine whether selected genetic variants were associated with CSF biomarkers, we used data for SNCA transcripts were analyzed as documented in the Laboratory of Neuroimaging (LONI) database (https://ida.loni.usc.edu/pages/access/studyData.jsp?categoryId=7&subCategoryId=52) by assaying transcript counts in human blood in a high-precision nanoString gene expression assay. PAXgene tubes  were collected by venipuncture according to standardized protocols (http://www.ppmi-info.org/study-design/research-documents-andsops/), incubated at room temperature for 24 hours, frozen, and shipped on dry ice. RNA extraction, followed the PAXgene procedure and quality control, was performed using the RNA Integrity Number package.17 The SNCA probes used target the boundaries of exon 3 and exon 4 (termed E3E4-SNCA), transcripts specifically with a long 3-untranslated region (3UTR) region (termed 3UTR-1 and 3UTR2-SNCA), transcripts that skip exon 5 (termed E4E6-SNCA), or the rate shot SNCA-007 transcript isoform (Enseml ID ENST00000506691) that comprises exons 1–4.t tests or chi-square were used to compare baseline demographic and clinical variables in participants with longitudinal CSF data versus participants who only had baseline CSF data; these comparisons were performed separately in all four groups. Repeated-measures linear mixed models were used to test for changes over time in CSF α-syn levels separately by group. In addition, repeated-measures linear mixed models were used to examine longitudinal relationships between CSF α-syn levels and PD medication use.Statistical analyses were performed using SAS software  on data retrieved from the PPMI data portal at the LONI at the University of Southern California. All tests performed using CSF α-syn were rank-based. P values <0.2 were included in a multivariable model. Finally, a backward selection process was used to remove variables individually until all variables remaining in the model were significant at the 0.1 level.Simple linear models were used to analyze potential baseline predictors of baseline CSF α-syn, separately in PD and HCs. First, the univariate relationship between each predictor and CSF α-syn level was examined. Then, any variables that had univariate associations with a Spearman rank-correlation coefficients between changes in CSF α-syn levels and changes in clinical progression variables and changes in DaTscan measures were reported, and also for SNCA transcript information and CSF α-syn levels. Kruskal-Wallis H tests were used to test for associations between CSF α-syn levels and genetic variables.P = 0.039), SDMT (P < 0.001), LNS (P = 0.031), and in BJLO (P = 0.002). Prodromal hyposmic participants with baseline data had worse cognitive performance  and lower mean caudate SBR values (P = 0.011) on DaTscan. iRBD participants with milder iRBD by RBDSQ (<6) were more likely to drop out after baseline assessment .The study enrolled 423 PD, 196 HC, 39 iRBD, and 26 hyposmic participants. From these participants, 376 PD, 173 HC, 32 prodromal iRBD, and 16 prodromal hyposmic participants had complete data, including CSF; their demographic and clinical data at baseline visits are shown in P < 0.0001). Changes in CSF α-syn in PD, controls, and both prodromal cohorts over time are shown in P = 0.032), whereas levels did not change in the control group .CSF α-syn levels were significantly lower in PD compared to HC across all visits .Among prodromal groups, the hyposmic participants showed the lowest mean CSF α-syn levels, whereas iRBD participants had intermediate levels between HCs and PD. In both prodromal groups, CSF α-syn remained relatively stable over the study interval from baseline to 6 and 12 months (P = 0.007), and height  were significant predictors of baseline CSF α-syn in PD participants (data not shown). Changes in CSF α-syn were not related to changes in MDS-UPDRS III, MoCA, and DaTscan values (P > 0.05) in PD, and had a relationship to MoCA changes in HC over 36 months .In multivariate regression analysis, older age , but it lost significance when we excluded samples with hemoglobin <200 ng/mL  and REM sleep behavior symptoms (RBDSQ >6) and found no significant correlation between clinical progression and CSF α-syn in these subgroups (data not shown). In PPMI PD subjects, we previously observed a greater decrease in CSF α-syn levels over 12 months in subjects who took dopamine medications, with a weak relationship with LED.APOE e4, MAPT, and polymorphisms in the SNCA gene (SNCA rs356181 and rs3910105) were not associated with baseline or longitudinal change of CSF α-syn in PD and HCs (P > 0.05). Another recent study found that a polygenic hazard score also showed no association with CSF α-syn levels.18SNCA transcripts were not associated with baseline and longitudinal α-syn in PD or in HC (data not shown).Genetic variants in P < 0.001), with a systematic shift toward lower values of α-syn in the 2016 analyses relative to those of 2013 (data not shown). The shift may be attributed to preanalytical factors in CSF sample handling  or analytical/assay factors, which can occur with the manual performance of multiple 96-well ELISA plates. Ongoing studies of α-syn measurement, including mass spectrometry, will address some of these issues.19A subset of PD participants and HCs had CSF α-syn measured in 2013 and again  in 2016. Levels from both analyses were strongly correlated  trial, in analyses that used different assays and inclusion criteria.20 One study that reported an increase over time only included samples from participants with diagnostic likelihood of clinical PD estimated at 90% to 100%, whereas another study also excluded subjects with other neurological disorders identified during follow-up.22 In contrast, the study reporting a decrease included the entire DATATOP cohort without selection.Longitudinal changes in CSF α-syn and other biomarkers in PD have been examined in other cohorts for up to 2 years: two studies show increasing CSF levels over time,6 To assess how early in the disease course CSF α-syn may decrease, we also evaluated levels in prodromal participants with hyposmia and iRBD, both of which carry high risk for PD or related disorders.24 Overall, CSF α-syn decreased longitudinally in PD and increased slightly (nonsignificant) in HCs over 36 months. The magnitude of change was small and was no longer significant when around 50% of CSF samples with hemoglobin contamination were removed. Because of the high abundance of α-syn in blood, blood contamination during lumbar puncture impacts CSF α-syn. At baseline, the PD-HC differences in CSF α-syn remained significant even when samples with high hemoglobin concentrations (>200 ng/mL) were included. Therefore, the loss of longitudinal significance with exclusion for high hemoglobin concentrations could be explained by the lower numbers of PD subjects at baseline (n = 187 vs. n = 376), but could also indicate no longitudinal change of CSF α-syn in PD.The strengths of the PPMI include multicenter recruitment, clinical, biosample, and imaging standardization, high rates of follow-up, and inclusion of prodromal patients at risk for PD. We have now extended the interval of follow-up for CSF biomarkers in PD and HCs in the PPMI to 36 months compared to our previous analyses.25 with some variability; one earlier study that included iRBD had only a small number of controls.26 Despite marked overlap of individual values with HC, mean levels in PPMI PD subjects were significantly lower than HC across all visits. The reason for the decrease remains unclear. One explanation is that CSF α-syn is decreased because of intracellular aggregation of α-syn in the brain. A recent review27 summarizes the different pathways involved in the degradation of intracellular α-syn that includes chaperone-mediated autophagy, endosomal, and proteasomal degradation as well as macroautophagy. Extracellular α-syn may represent other clearance pathways and is subject to proteolysis by extracellular proteases, such as neurosin, that has been detected in CSF28 and that inversely correlated with α-syn accumulation in brains with dementia with Lewy bodies (DLB).29The 10% to 15% decrease in CSF α-syn in PD versus controls is a consistent finding across cohorts30 iRBD is a highly specific prodromal condition with a high conversion rate (>80%) to an α-syn aggregation disorder after 16 years.31 PD subjects with RBD may have a more aggressive form of PD with cognitive decline.32 iRBD may progress to more aggressive α-syn aggregation disorders  and—rarely—to MSA. Greater neurodegeneration may contribute to the finding of higher levels in the iRBD than the PD and hyposmia groups.Our findings that subjects with likely prodromal PD  already have decreased CSF α-syn is consistent with significant pathology being present during these prodromal stages, analogous to the decrease in CSF amyloid beta 42 in Alzheimer’s disease.It is unclear whether the decrease of CSF α-syn develops even earlier during prodromal PD or may represent a trait that is a risk factor for PD. In the PPMI, CSF α-syn levels in HC subjects are followed longitudinally to see whether those with lower levels may develop PD later.SNCA transcripts supports a state, rather than a trait, marker. How α-syn gets into CSF is incompletely understood, although a recent study indicates that neuronal activity, particularly at excitatory synapses, is a major contributor to its release.33 In contrast to PD, levels of α-syn are markedly increased in CSF in Creutzfeldt-Jakob disease, where rapid and progressive neuronal death occurs.34The lack of a relationship between CSF α-syn and genetic risk factors or 35 To date, based on thorough neurological judgement at each visit, there are three misdiagnoses among the PD subjects analyzed here: 2 were diagnosed with MSA (1 with autopsy confirmation) and 1 with corticobasal degeneration. Further clinical follow-up, and the approved brain donation program in the PPMI as well as future biomarker approaches, for example, the ratio of α-syn/tau protein,36 neurofilament light chain,37 or others could help to distinguish PD from atypical PD syndromes.The reasons for CSF α-syn variability remain to be determined. One explanation could be partly attributed to misdiagnoses that are not excluded in this cohort and that are reported to be a problem among de novo PD subjects.The decrease of CSF α-syn in PD over 36 months did not correlate with progression of motor and nonmotor symptoms, or with a decrease of dopamine transporter signal, both robust indices of PD progression. Therefore, the events that result in decreased CSF α-syn do not appear to directly drive PD progression. We confirmed the earlier association of symptomatic medication with greater decline in CSF α-syn in PD, for unclear reasons. We could not identify other predictors of changes in CSF besides age and possibly BMI.38 Further progress in diagnostic and progression biomarkers will benefit from analysis of abnormal forms of α-syn42 and of novel biomarkers identified through methods such as proteomics and metabolomics.Although we observed a significant decrease of CSF α-syn in PD over 36 months’ follow-up, and levels tended to be stably decreased within patients, CSF α-syn, as measured with the assay used, will not serve as a diagnostic marker for PD and is unlikely to be a sole outcome measure for clinical trials or progression. Substantial overlap between PD and HC groups may result from biological or genetic variability, (co)medication, comorbidities, or other factors. Furthermore, subtypes of PD may reflect different pathophysiological factors, with clinical heterogeneity. Other CSF biomarkers are currently being analyzed in the PPMI, including total and phosphorylated tau protein, and β-amyloid 1–42, reflecting different pathological contributions to cognitive and motor progression.


Furthermore, we evaluated relationship between sex hormone-binding globulin (SHBG), gonadal status and clinical and virologic parameters.The prevalence of low testosterone and symptoms of hypogonadism in HIV-infected men is still debated. We aimed to estimate the prevalence and type of hypogonadism in HIV-infected males complaining about sexual symptoms, and to evaluate the role of calculated free testosterone (cFT) We retrospectively evaluated 169 HIV-infected men with sexual symptoms, with TT available. Among them, we selected 94 patients with TT, SHBG, cFT, and luteinizing hormone (LH) available, and classified hypogonadism into overt (low TT and/or low cFT) and compensated . Comparison was performed by non-parametric Kruskal–Wallis test and Spearman’s correlation was calculated to verify the possible associations.Overt and compensated hypogonadism were found in 20.2% and 13.8% of patients, respectively. With reliance on TT alone, only 10.6% of patients would have met diagnosis. SHBG values were elevated in one third of patients, and higher in men with compensated hypogonadism. Significant positive correlation was found between SHBG and HIV infection duration, TT and LH.Only a complete hormonal profile can properly diagnose and classify hypogonadism in HIV-infected men complaining about sexual symptoms. TT alone reliance may lead to half of diagnoses missing, while lack of gonadotropin prevents the identification of compensated hypogonadism. This largely comes from high SHBG, which seems to play a central role in the pathogenesis of hypogonadism in this population. Since the early stages of HIV epidemic, hypogonadism has been recognized as a known frequent associated condition. Hypogonadism is a clinical syndrome caused by failure of the testis to produce physiological amounts of testosterone and/or a normal number of spermatozoa, depending on alteration at different levels of the hypothalamic–pituitary–testicular axis . ImportaAlthough the association between HIV and hypogonadism is well known , the exaDespite many authors have already investigated the prevalence of hypogonadism in HIV-infected men, some possible limitations must be considered. First of all, clinical symptoms of hypogonadism were rarely considered as diagnostic criteria, and most of the papers focused only on testosterone values to make diagnosis . MoreoveTherefore, we aimed to estimate the prevalence of true hypogonadism  in HIV-infected males complaining about sexual symptoms, using different and gradually more specific biochemical criteria, to classify hypogonadism according to gonadotropin levels and evaluate the rate of misdiagnosis resulting from the use of TT alone versus a complete hormonal profile, including cFT and LH measurement. Furthermore, we aimed to evaluate possible relationship between gonadal status and some clinical and virological parameters.An observational retrospective study was performed. We assessed for inclusion 189 HIV-infected males, sent for symptoms of hypogonadism from HIV-specialist to the outpatient clinic of Endocrinology over a period from 2012 to 2019.Inclusion criteria were: age > 18 years, serologically documented HIV infection in stable condition under cART and presence of self-referred sexual symptoms , the availability of HIV infection parameters , and blood samples for hormonal parameters carried out at central hospital laboratory. Patients treated with androgens or drugs with potential detrimental role on gonadal function were excluded, as well as those with hormonal assays not analyzed in central hospital laboratory.We performed three-step inclusion criteria, as shown in Fig. Cutoff value for lower limit of TT was set at 3.46 ng/ml; normal range for LH was established between 1.5 and 9.4 mUI/ml; cFT values were considered low if ≤ 65 pg/ml , 17, wheFigure http://www.issam.ch/freetesto.htm), that combine TT, SHBG and albumin. CD4 count was assessed by flow cytometry. Interassay coefficient of variance for TT, LH and SHBG was < 5%.All blood samples were obtained between 8.00 and 10.00 a.m., after a 12-h overnight fast, and were analyzed at central hospital laboratory . TT and LH were determined using CMIA, SHBG using CLIA (chemiluminescence immunoassay). cFT was calculated with Vermeulen equation  and informed consent was obtained from all participants.H test, followed by post-hoc Dunn’s test when a significant difference was found. The Spearman’s correlation was calculated to verify the possible association with TT and cFT of the variables used. P values ≤ 0.05 were considered significant.GraphPad Prism version 5.1  was used for statistical analysis. Since the variables were not normally distributed , comparison among medians of the quantitative variables were performed by non-parametric Kruskal–Wallis An overview of the clinical information of the 169 patients, grouped according to TT levels , TT, SHBG and cFT , and complete hormonal profile, including TT, SHBG, cFT and LH  is summarized in Table When considering Group A, TT levels below 3.46 ng/ml were found in 14/169 (8.3%) patients. When also SHBG and cFT were assessed , overt hypogonadism was found in 22/118 (18.6%) patients. In detail, of these 22 patients, only 10 would have met diagnosis based on TT values. Therefore, the addition of cFT in Group B allowed the identification of 12 further patients, who otherwise would have been considered eugonadal, with a neat increase in diagnosis rate of 2.2 times.We then focused our study only on patients with complete hormonal profile available, including TT, SHBG, cFT and LH . We found overt hypogonadism in 19/94 (20.2%) patients. The assessment of LH values allowed us to classify hypogonadism for these patients, and we found that 5/19 (26.3%) showed primary hypogonadism and 14/19 (73.7%) had secondary and/or normogonadotropic hypogonadism. Moreover, 13/94 (13.8%) patients, despite having TT and cFT within normal range, showed clearly increased LH values, fitting the definition of compensated hypogonadism, and bringing the total number of patients suffering from any gonadal axis alteration to 32/94 (34.0%).If we had used TT alone as diagnostic criterium in these patients, as we did in Group A, only 10 out of 94 (10.6%) would have met hypogonadism diagnosis, a prevalence close to what we actually found in Group A. The addition of cFT has, therefore, allowed us to diagnose overt hypogonadism in 19 patients instead of 10, with an increase of 1.9 times; otherwise, these 9 patients with low cFT but normal TT would have been considered eugonadal or with compensated form, if considering TT alone or TT and LH.Subsequently, a comparison among groups according to gonadal status was performed for Group C (94 patients), as shown in Table Once again, we found, as expected, significant differences between groups for TT, cFT and gonadotropins (P < 0.001). Interestingly, SHBG values were higher in men with compensated hypogonadism with respect to the other two groups, even if this difference did not reach statistical significance. Remarkably, mean SHBG value was close to upper limit (63 nmol/l), and 34 patients (36.2%) showed values over 70 nmol/l. Among them, 18 (52.9%) were eugonadal.No significant differences were found between groups for age, BMI, and CD4 + cell count. Conversely, we found a statistically significant difference in HIV infection duration (P < 0.05), longer in patients with compensated hypogonadism than in patients with overt hypogonadism or eugonadism. cART duration was found close to significance, again longer in patients with compensated hypogonadism than in other two classes.Figure Figure We found overt hypogonadism, considered as the association of low TT and/or cFT levels with clinical symptoms of androgen deficit, in 20.2% of patients, in substantial agreement with previously reported data , 18–21. http://www.issam.ch/freetesto.htm, that combine TT, SHBG and albumin [When considering biochemical definition of hypogonadism, it is essential to clarify the role of free testosterone. Basically, only 2–4% of serum testosterone circulates as free and biologically active, whereas about two thirds are tightly bound to SHBG and one third is weakly bound to albumin. This implies that in conditions with a known increase of SHBG, such as HIV infection , liver d albumin . Further albumin .. [In our study, the use of TT alone would have resulted in a potential loss of almost 50% of diagnoses. Monroe et al.  expresse. .. [As expected, SHBG values were generally high in our study, in accordance with literature for HIV population . Higher . . They su. . However.  showed t. . Therefo. .vs those with ED [A strength of the present study is that all patients were symptomatic for hypogonadism (by inclusion criteria), which is hardly reported in literature to our knowledge, since only few authors considered clinical presentation of hypogonadism , 21, 25. with ED  found th with ED .The present study has some limitations. Firstly, it was a retrospective cross-sectional study on a relatively low number of patients, and not all patients had a complete hormonal profile. Moreover, hormonal measurements were carried out a single time and were not repeated. This is a portrait of real-life clinical practice, with a wide temporal range, and visits performed by different endocrinologists. Moreover, guidelines for hypogonadism diagnosis evolved in last years. Therefore, the lack of SHBG and/or LH measurement in some of the participants in the full sample (Group A) is due to different clinical practices and temporal changes over time, and was unlikely due to patient characteristics and does not represent a selection bias. Furthermore, due to its retrospective nature, oestradiol assay, useful to better investigate gonadal function, was not available for many patients and a control group is lacking.In conclusion, using a complete hormonal profile, including SHBG and cFT, we found a prevalence of 20.2% of true overt hypogonadism in HIV-infected males complaining about sexual symptoms. Furthermore, gonadotropin measurement allowed us to better classify hypogonadism, and to identify compensated forms, thus increasing the prevalence of overall hypothalamic–pituitary–gonadal axis alteration to 34.0%. This finding is essential, since only a complete hormonal profile can properly recognize and classify hypogonadism in these patients. In fact, TT alone failed to diagnose half of the cases, and lack of gonadotropins prevented the identification of patients with compensated hypogonadism, which accounted for nearly 14% of the total. Although longitudinal data would better clarify some aspects on the onset and progression of hypogonadism, our data highlight some interesting pathophysiological data underlying hypogonadism in this population, especially on the role of SHBG.


Homogeneity of thermal tasters and implications for mechanisms and classificationThermal taste is a phenomenon whereby some individuals, known as thermal tasters (TT) experience taste sensations when their tongue is warmed or cooled. It was first reported in 2000 by Cruz and Green  TT rate the intensity of taste stimuli higher than TnT •Researchers interested in the thermal taste status phenomenon will have access to a dataset consisting of a large sample of TTs and their responses both to thermal taste elicitation and to aqueous orosensory stimuli. Together, these results can inform future TTS screening protocols, recruitment targets and classification methods.•TRPM5, a heat-activated cation channel expressed in taste receptor cells, may be involved in the thermally-elicited sweetness experienced by some thermal tasters ,10. Howe•TT rate the intensity of alcoholic beverages and the emotions they elicit higher than TnT 1The primary aim of this manuscript is to visually display the relationship between orosensory responsiveness and TT subgroups. Each figure shows the mean orosensory responsiveness and corresponding standard error to aqueous solutions of sweet, salty, sour, bitter, umami, metallic & astringent stimuli of a TT subgroup compared to TT who are not a part of the subgroup. Readers are referred to the “TT Subgroup Naming Conventions” for full details of the inclusion criteria for each subgroup.The raw data associated with each of the figures can be found in the accompanying file “Thermal Taster Subgroups and Orosensory Responsiveness Dataset.xlxs”. The dataset includes information on each participants cohort (Column A), a unique identifier for each participant  and z-scores of mean orosensory responsiveness for six common orosensations (Columns C-I). The remaining columns provided the TT subgroup status of each participant using a binary (Yes/No) system of coding. “Yes” is used to indicate that a participant is part of the TT subgroup listed in the heading, while “No” indicates that they are not. For example, the participant with ID code 14 is a both a Sweet TT and a Sour TT but is not a Salty TT, a Bitter TT, an Umami TT or a Metallic TT. Readers should consult the “TT Subgroup Naming Conventions” for full details of the inclusion criteria of each TT subgroup.(1)One-factor (n = 254): Patterns in the types of TT responses to thermal elicitation may provide insights into potential TT subgroups. This tab includes the data for TT subgroups based on the orosensation experienced when the tongue was heated or cooled (see (2)Two-factor (n = 254): Each TT belongs to a minimum of three single-factor TT subgroup, suggesting that subgroups based on two factors (e.g. the location and temperature regime) may also be of interest. To this end, Thibodeau et al (3)Spicy (n = 265): Includes an expanded dataset with 25 participants who can be classified as Spicy TT if it is included in the list of valid thermally-elicited sensations ((4)Unmatched (n = 286): Includes an expanded dataset with 32 participants (IDs 266–297) who can be classified as Unmatched TT if participants are not required to report the same thermally-elicited sensations in corresponding trials (see (5)Sample Sizes: As this study is retrospective in nature, not all cohorts were exposed to each of the orosensory stimuli. For convenience, this tab summarizes the sample sizes for each of the TT subgroups.The data is divided across five tabs as follows:Please note: As z-scores are calculated using sample means and standard deviations, small differences in the z-scores of some participants exist (1–254) as the number of participants included in the calculations varied. Each participant is assigned the same unique identifier if they are included in multiple tabs of the spreadsheet, for comparison purposes.2The primary aim of this manuscript was to create a large data set of TT responses. To this end, data from the TTS screening procedures of 12 recruitments drives (‘cohorts’) was combined. 975 participants were recruited from Brock University and the surrounding community, of which 905 completed the study in full. Failure to appropriately use the scales during training led to the exclusion of an additional 124 participants. The final data set includes only the responses for the 297 participants who could be classified as thermal tasters. A description of the experimental design, materials and method is provided next and readers are referred to Thibodeau et al 2.1The thermal taste status of all participants was determined based on the protocol of Bajec and Pickering After providing informed consent and basic demographic information, participants were training on the appropriate use of two intensity scales, the generalized Visual Analogue Scale (gVAS) and the generalized Labeled Magnitude Scale (gLMS). After, a verbal description of the scale from the researcher, participants were asked to rate the maximum intensity of a series of remembered sensations on each of the scales Using a sip-and-spit protocol, participants rinsed with aqueous solutions eliciting common orosensations primarily to aid with the later identification of thermally-elicited sensations (see Table 2 from 2 computer-controlled Peltier device with a thermocouple feedback attached to a toothbrush-sized water-circulated heat sink (thermode). Two different cycles were used: a warming cycle and a cooling cycle. Warming cycles started at 35°C, then cooled to 15°C before final re-warming to 40°C and holding for 1 s. Participants were only asked to rate the maximum intensity of sensations during the warming phase of the cycle. Cooling cycles started at 35°C, with subsequent cooling to 5°C and holding for 10 s. Participants were asked to report any sensations regardless of when they occurred during the cooling cycle.Thermal stimulation was performed using a 64 mmThree locations on the edge of the tongue were tested for each participant: the very tip of tongue along the midline, 1 cm to the left from the midline and 1 cm to the right from the midline. A total of 12 runs were performed for each participant in two blocks. Each block consisted of three warming cycles (one per location) followed by three cooling cycles (one per location). A minimum 3 min break was taken between blocks. All participants rated any sensations  elicited using a paper ballot with individual gLMS scales for each. For the most recent cohorts , the paper ballot was modified by adding gLMS scales for umami and metallic.TTS classification was determined using the methods of Bajec et al 2.2(1)Participants who experienced sweetness above ‘weak’ on the gLMS during both replicates for at least one temperature regime and location combination are referred to as sweet TT. Similarly, participants that report a different orosensation are defined as salty TT, sour TT, bitter TT, umami TT or metallic TT based on the orosensation reported.(2)Participants who reported the same orosensations  above ‘weak’ during both warming replicates for at least one location are referred to as warm TT. Similarly, participants who experience thermally-induced orosensations during cooling are cool TT.(3)Participants who experienced the same orosensations  above ‘weak’ during both warming and/or cooling replicates at the tongue tip are tip TT. Similarly, participants who experience thermally-induced orosensations on the left or right side of the tongue are defined as left TT or right TT.(4)At minimum, each participant belongs to three subgroups; one from each of (1–(3) above. However, TT may belong to more subgroups if they experience multiple thermally-elicited orosensations above ‘weak’ or if the sensation(s) is experience at more than one location or during both temperature regimes. Membership of more than one group is designated by an “&” or “/“. The use of each symbol is demonstrated below using the example of a participant who is both a sweet TT and warm TT.(a)The “&” symbol indicates that the participant is a member of both groups simultaneously. Thus, a sweet&warm TT reports thermally-induced sweetness during both warming replicates at a minimum of one location.(b)A sweet/warm TT could be either a sweet&warm TT or a TT that does not report thermally-induced sweetness during both warming replicates at a minimum of one location. E.g. they could report saltiness during warming and sweetness during cooling.(c)Please note: It is not possible to be a warm&cold TT as the warming and cooling regimes were tested during separate trials. Similarly, it it not possible to be a left&right TT, left&tip TT or right&tip TT. However, as multiple orosensations can be elicited in a single trial, it is possible to use the “&” symbol for two thermally-elicited orosensations. For example, a TT that experiences both thermally-elicited sweetness and sourness at the same location and during the same temperature regime is a sweet&sour TT.TT were divided into subgroups based on the orosensation(s) reported, the temperature regime(s) and the location of the thermally-elicited orosensation(s). The following conventions were followed in classifying and naming the groups:(5)TT subgroups defined in (1)–(3) are referred to as single-factor subgroups because only one criterion was used to classify participants. TT subgroups from (4) are referred to as two-factor subgroups.2.3The mean orosensory responsiveness z-score  was plotted for each TT subgroup comparing it to all other TT who were not part of that subgroup (e.g. sweet TT vs not sweet TT). Subgroups examined include single-factor subgroups based on the naming conventions 1; see , 2; see and 3; .•Prototypical tastes are broadly divided into two classes based on mechanism. G-protein-coupled receptors (GCPR) are responsible for the perception of sweetness, bitterness and umami while ion channels are responsible for the perception of saltiness and sourness •Three mutually exclusive subgroups based on temperature regime are also recognized. Individuals that experience thermally-elicited tastes during only warming, only cooling or both warming and cooling, are referred to as onlywarm TT, onlycool TT and warmandcool TT, respectively .•Two-factor TT subgroups, as outlined in naming convention (4), allow for more precision when defining TT subgroups. This may be important in understanding the association between TTS screening responses and the orosensory advantage of TT. For example, 77 participants are sweet TT but only 64 experience thermally-elicited sweetness during warming. Therefore, by excluding the 13 TT who experience thermally-elicited sweetness during cooling, noise in the dataset may be reduced. As 54 TT subgroups can be established, based on naming convention (4a) alone, a more targeted approach was necessary when selecting two-factor TT subgroups to examine. Two-factor TT subgroups were selected based on the findings of Thibodeau et al Additional TT subgroups were all developed and plotted based on the following criteria:•Consistent with some studies ,13,15,16•TT are required to report the same taste sensation across replicate trials in most but not all ,13,15 stWhile the naming conventions provide a clear definition of a TT, other studies have further expanded their definition of TT. This is possible as the mechanism(s) underlying thermal taste are not well elucidated. The orosensory responsiveness of two additional TT subgroups was investigated by re-classifying participants as follows:Informed consent was obtained from all individual participants and all procedures were cleared by the Brock Research and Ethics Board.The authors declare that they have no known competing financial interests or personal relationships which have, or could be perceived to have, influenced the work reported in this article.


Pythium insidiosum. The disease has been increasingly diagnosed worldwide. P. insidiosum inhabits freshwater and presents in two forms: mycelium and zoospore. Clinical manifestations of pythiosis include an infection of the artery, eye, skin, or gastrointestinal tract. The management of pythiosis is problematic due to the lack of effective treatment. Many patients die from an uncontrolled infection. The drug susceptibility testing provides clinically-useful information that could lead to proper drug selection against P. insidiosum. Currently, no standard CLSI protocol for the drug susceptibility of P. insidiosum is available. This review aims at describing methods and antimicrobial agents for susceptibility testing against P. insidiosum. Several in-house in vitro susceptibility methods  have been established for P. insidiosum. Either mycelium or zoospore can be an inoculum. Rabbit is the commonly-used model of pythiosis for in vivo drug susceptibility testing. Based on the susceptibility results , several antibacterial and antifungal drugs, alone or combination, exhibited an in vitro or in vivo effect against P. insidiosum. Some distinct compounds, antiseptic agents, essential oils, and plant extracts, also show anti-P. insidiosum activities. Successfully medical treatment, guided by the drug susceptibility data, has been reported in some pythiosis patients. Future studies should emphasize finding a novel and effective anti-P. insidiosum drug, standardizing in vitro susceptibility method and correlating drug susceptibility data and clinical outcome of pythiosis patients for a better interpretation of the susceptibility results.Pythiosis is a life-threatening infectious disease of humans and animals caused by the oomycete microorganism  Pythium insidiosum; Susceptibility; Antimicrobial agentImmunology; Microbiology; Pythiosis;  Krajaejun]).This work was supported by Faculty of Graduate Studies, The authors declare no conflict of interest.No additional information is available for this paper.


Histoplasma capsulatum by culture in peripheral blood and cerebrospinal fluid (CSF) is extremely rare. We describe the case of a three-month-old Costa Rican immunocompetent infant who presented with shock and hemolytic anemia secondary to disseminated histoplasmosis that was confirmed by bone marrow aspirate and positive peripheral blood and CSF cultures.Disseminated histoplasmosis is the most common clinical presentation of histoplasmosis in human immunodeficiency virus (HIV) negative infants from Costa Rica and Latin America. Initial presentation as septic shock and autoimmune hemolytic anemia is uncommon. Even more, detection of  Histoplasma capsulatum is a dimorphic fungus endemic in certain areas of North, Central, and South America, Africa, and Asia. However, cases have been also reported in Europe. Outbreaks have been found in pigeon or chicken breeders in places where bats are common (caves) and in old demolition sites [on sites .H. capsulatum endemic regions. However, in immunocompetent infants, it develops after exposure to a large or prolonged inoculum of the pathogen, resulting in case fatality rates as high as 40% to 50% [Disseminated histoplasmosis usually occurs in immunocompromised patients who reside in % to 50% . It is m% to 50% .H. capsulatum in peripheral blood is infrequent [In disseminated disease, the highest culture yield is from bone marrow aspirates, being positive in more than 75% of cases . Less cofrequent .H. capsulatum from CSF or brain tissue is diagnostic; however, culture is insensitive, and slow growth may result in significant treatment delay [Central nervous system (CNS) involvement occurs in 5% to 10% of individuals with disseminated histoplasmosis ,5. ThereHere we describe an uncommon and severe case of a Costa Rican infant with disseminated histoplasmosis confirmed by both peripheral blood and CSF cultures, an uncommon scenario in pediatric clinical practice.A three-month-old Costa Rican male was admitted to a peripheral community hospital part of the social security system of Costa Rica (CCSS) because of two days of vomiting. On admission, physical examination revealed a febrile male with severe dehydration, mottled skin, and hepatosplenomegaly Figure .He needed initially 40 cc/kg of intravenous crystalloids. He persisted febrile with abdominal distention and developed a tonic seizure treated with one dose of intravenous diazepam. Samples were obtained for laboratory analysis, including peripheral blood cultures and CSF culture. Following a lumbar puncture, the patient had an apnea and decortication postures; therefore, he needed immediately endotracheal intubation, mechanical ventilation, and one dose of mannitol to reduce the intracranial pressure. A brain ultrasound revealed dense and lumpy material inside the lateral ventricles, an abdomen ultrasound confirmed the hepatosplenomegaly, and intra-abdominal free fluid was seen. Intravenous cefotaxime was started as an empiric therapy of septic shock.A complete blood count was compatible with autoimmune hemolytic anemia  and thrombocytopenia , with normal leukocytes count . One dose each of intravenous immunoglobulin (1 g/kg) and methylprednisolone (30 mg/kg) was administered. Coagulation times and other hematological revealed disseminated intravascular coagulation ; therefore, reanimation was continued with fresh frozen plasma, red blood cells, and cryoprecipitates. Of interest, acute inflammatory markers were negative: C-reactive protein (CRP) of 12 mg/L and procalcitonin of 0.5 mg/mL. Further laboratory investigations revealed normal blood urea nitrogen, creatinine, electrolytes, and liver enzymes. CSF analysis revealed normal glucose (79 mg/dL), elevated proteins (845 mg/dL), lymphocytic pleocytosis , and cerebral bleeding (3 000 erythrocytes), and no bacteria or fungal forms seen on gram stain. He developed refractory septic shock; therefore norepinephrine and epinephrine were started and the patient was transferred to the only national tertiary pediatric center of the country.H. capsulatum were seen at direct microscopy, but no hemophagocytosis was observed. Polymerase chain reaction (PCR) for H. capsulatum in bone marrow aspirate was negative, and culture of bone marrow was not requested. Samples from blood and CSF that initially were cultivated in conventional aerobic bacterial cultures were centrifugated and planted onto Sabouraud’s dextrose agar and Mycosel agar at 35-37°C.On admission, the child continued on refractory septic shock, had pancytopenia, and coagulopathy, and was transferred to the pediatric intensive care unit. Under the suspicion of macrophage activation syndrome, a bone marrow aspirate was performed; blastospores of H. capsulatum  and extended for one month. At the end of four weeks, both the peripheral blood and CSF cultures obtained at the referral community hospital were positive for m Figure .The patient recovered completely with no sequelae. Immunologic studies of the child including HIV, immunoglobulins , nitro blue tetrazolium test (NBT test), and flow cytometry were all normal. A large inoculum of bat guano was located at his home in a rural place of Costa Rica.H. capsulatum depends mainly on the host. Infants, presumably because of the immaturity of their cell-mediated immune system, are a special group that develop severe life-threatening disease when exposed to H. capsulatum [In pediatric patients, the clinical forms of histoplasmosis vary according to age group. In infants, the acute progressive disseminated form predominates (80%) . Dissemipsulatum .Hemolytic anemia is not a common presentation of disseminated histoplasmosis. In our review of the literature, there are only three reported cases and none of these are children. In 2012, Chang et al. described a 65-year-old female with hemolytic anemia and a pulmonary nodule with pleural affection, pancytopenia, and elevated liver enzyme. Diagnosis was made by direct examination of sputum and bone marrow culture . ChejaraH. capsulatum in cultures taken from peripheral blood and CSF is uncommon. One of the reasons is that these patients commonly present with severe thrombocytopenia and coagulopathy, and therefore lumbar puncture cannot be performed or is deferred. Samples of tissue or body fluids that are sent to the laboratory for culture are plated onto Sabouraud’s dextrose agar and incubated at 25°C to allow for growth of the mycelial phase of H. capsulatum. The lysis-centrifugation system has been shown to be more sensitive than automated systems for growing H. capsulatum from blood. Cultures usually yield no growth from CSF, and the diagnosis almost always must be made in a different manner [Histoplasma can be isolated from fungal culture of CSF in only one quarter of patients, and visualization of yeast is rare. Only in 38% of patients, Histoplasma is isolated from CSF culture, or yeast resembling H. capsulatum were observed (in brain tissue histopathology or in CSF) [Isolation of t manner . Wheat e in CSF) . In thisH. capsulatum were seen on bone marrow aspirate, we used the leukocytes concentration technique to isolate the H. capsulatum. CSF and blood were centrifugated at 2,500 rpm for 15 minutes. Centrifugation system was used to concentrate the samples and obtain the buffy coat. The buffy coat was then planted onto Sabouraud’s dextrose agar and Mycosel agar at 35-37°C to allow the growth of the H. capsulatum. Four weeks later, growth of the fungus was seen with this technique in both peripheral blood and CSF samples. In CNS histoplasmosis of immunocompetent hosts, inoculum size is the main determinant of clinical manifestations and isolation [In our case, the samples were taken initially from blood and CSF in conventional aerobic bacterial cultures and were negative. Four days later, when the suspicion of disseminated histoplasmosis was high enough because the blastopores of solation .H. capsulatum by CSF culture is difficult, Bloch et al. [Histoplasma antibody enzyme immunoassay (EIA) for the detection of IgG and IgM antibody in the CSF for the diagnosis of CNS histoplasmosis. In this study, they found that CSF H. capsulatum culture was positive in only 19% of cases with diagnosis of CNS histoplasmosis defined as a patient with CNS inflammation  or brain imaging abnormalities and supporting laboratory studies . This study concludes that testing CSF for anti-Histoplasma IgG and IgM antibody complements antigen detection and improves the sensitivity for diagnosis of meningitis [Histoplasma meningitis earlier.As detection of h et al.  performeH. capsulatum in peripheral blood is infrequent [H. capsulatum [H. capsulatum from blood culture have been described in patients with disseminated histoplasmosis, chronic cavitary histoplasmosis, or acute pulmonary histoplasmosis after large inoculum exposure [Furthermore, even in large studies, detection of frequent . Ouelletfrequent . In a sipsulatum . To our exposure ,11.Histoplasma grew in cultures from at least one sample for each patient [Odio et al. reported 40 patients with disseminated histoplasmosis in Costa Rica from 1983 through 1996 at our children’s hospital . All pat patient . This isH. capsulatum isolated from blood and eight from CSF [H. capsulatum isolated in blood and only two had it isolated in CSF [In other literature from Latin-American, López et al. reported 45 patients with histoplasmosis in Colombia, of which only two had from CSF . In Arged in CSF .Progressive disseminated histoplasmosis in children is fatal if untreated. Amphotericin B deoxycholate (1 mg/kg daily) given for four weeks has been used successfully and with minimal toxicity. In contrast to cases in adults, meningitis that accompanies progressive disseminated histoplasmosis of infancy responds to amphotericin B deoxycholate (given for four to six weeks) without a high rate of relapse. Treatment recommended for progressive disseminated histoplasmosis of infancy does not need to be modified in patients found to have CNS involvement . In our Herein, we described the case of a three-month-old boy with disseminated histoplasmosis who on admission presented in septic shock and developed autoimmune hemolytic anemia, a rare manifestation in children with disseminated histoplasmosis. Although disseminated histoplasmosis is a rare cause of septic shock, physicians should be aware of this possibility in endemic areas in the differential diagnosis of infants with fever, malaise, hepatosplenomegaly, and abnormalities in the complete blood count, especially pancytopenia or any other cytopenia. Culture-proven histoplasmosis from peripheral blood and CSF culture is extremely rare, but if there is high suspicious of histoplasmosis, blood and CSF fungal cultures should be obtained. Using the centrifugation technique in the laboratory increases the possibility of isolation and confirmation.


Between electric gloom,angels and demons dressed in plastic bluehave dragged me through the darkness,to the infinite black stone,where the origin and the end of time are lost.There, in the immense dark solitude,and in the imposed silence of word and thought,She, in my breath, gazed at mewith her eyes of fire,frosty and timeless,without any mercy,without any mercy.


The incidence of cryptococcal meningitis (CM) has gradually increased in recent years. Cerebrospinal fluid (CSF) cytology and cell count are very important for CM on etiology diagnosis and assessment of disease status and therapeutic response. However, the clinical significance of CSF white cell count (WCC) in CM patients is not fully understood. Using longitudinal data of CSF WCC and its relationship with clinical outcomes in CM patients, we aimed to elucidate the clinical significance of this test.We retrospectively analyzed the medical records of 150 CM patients admitted to our hospital between January 2008 and December 2018.P<0.05). CM patients with triple therapy during the induction period presented with a better clinical outcome (P<0.05). Baseline CSF WCC had a moderate positive correlation with peripheral CD4+ T lymphocyte count  and CD4+ T lymphocyte percentage . The best cut-off value to predict a poor clinical outcome was 40 cells/μL during baseline CSF WCC. The predictive model incorporating longitudinal data of CSF WCC had better sensitivity, specificity, and accuracy than a model incorporating only baseline CSF WCC data.CM patients with lower baseline CSF WCC, CSF protein concentration or CD4/CD8 ratio, and those with altered mentation or HIV coinfection were more likely to have poor clinical outcome (Our results indicated that baseline CSF WCC and changes in CSF WCC over time could be used to assess the prognosis of CM patients. Cryptococcus spp., is responsible for significant morbidity and mortality and requires prolonged treatment [Cryptococcal meningitis (CM), caused by reatment . It is areatment . Globallreatment . The increatment . Recent reatment , 5.Lumbar puncture (LP) is a common procedure in CM patients. Aggressive management of elevated intracranial pressure via LP is beneficial to CM patients –8. AccurIn this retrospective study, we aimed to evaluate the clinical significance of longitudinal CSF WCC data and elucidate its relationship with clinical outcomes in patients with CM who were admitted to a university hospital in China between 2008 and 2018.Cryptococcus or positive CSF India ink staining. Exclusion criteria were age under 14 years, suspected or confirmed co-infection with other pathogens, and those who had received antifungal treatment before admission to our hospital.This is a retrospective cohort study was conducted on CM patients admitted to West China Hospital, Sichuan University, Chengdu, Sichuan, China between January 2008 and December 2018. The diagnosis of CM was done based on clinical manifestations and positive CSF culture for Laboratory tests such as routine blood tests, blood biochemistry, and peripheral blood T lymphocyte subsets analysis were carried out. The enzyme-linked immunosorbent assay (ELISA) or chemiluminescence immunoassay was used to detect HIV infection, and positive results were confirmed by western blot . A lymphocyte subset analysis was done by flow cytometry. LP was performed before treatment was initiated. The CSF samples were analyzed immediately for cell count, biochemical parameters, India ink staining, acid-fast smears for tuberculosis (TB), TB DNA analysis, and fungal and bacterial cultures. Latex agglutination (LA) test or lateral flow immunoassay (LFA) was used to detect the CSF CrAg titer. Fungal CSF culture was done in Sabouraud dextrose agar plates. After the initiation of antifungal therapy, CSF examination was performed approximately once a week. All examinations included cell count, biochemical test, India ink staining, and fungal culture.Cryptococcal disease management was done following the Infectious Diseases Society of America and our country’s guidelines, and based on our experience , 7, 15. Clinical outcomes of patients with CM were evaluated at 2 and 10 weeks after diagnosis or initiation of antifungal therapy. Based on clinical manifestations and laboratory findings, clinical outcomes were divided into good or poor. Patients were assigned to the poor outcome group if they had died or the disease has progressed or relapsed. Disease progression was defined as aggravation of consciousness disorders, fever, headaches, and other clinical manifestations, and persistently positive CSF cultures or India ink staining. Relapse was defined as a situation in which CSF culture or India ink staining has turned negative and then positive again, and symptoms and signs of infection appear again after they have disappeared. A good outcome was defined as a situation in which CSF culture or India ink staining turned negative after being positive, and clinical symptoms have improved.t-test for normally distributed continuous variables or the Mann-Whitney U test for non-normal data. Categorical variables were compared using the Chi-squared test or Fisher’s exact test. Associations between quantitative variables were assessed by Spearman’s rank correlation coefficient (r) for non-normal data. An r-value greater than 0.8 was considered strong positive correlation; 0.2 < r < 0.5 was considered as weak correlation. CSF WCC receiver operating characteristic (ROC) curve was plotted, and the area under the curve (AUC) was calculated using the MedCalc software, version 11.5.1.0 .The AUC was used to evaluate the prognosis for CM patients. A machine learning algorithm (C 5.0 decision tree) was implemented in the R 3.5.1 program for Windows to identify the relationship between dynamic changes in CSF WCC and clinical outcome, and the predictive value of the dynamic changes in CSF WCC for clinical outcome. We split the data into training (70%) and test (30%) datasets while preserving the proportions of the good and poor outcome categories. The training data set was used to build a C5.0 decision tree model. We then repeated 10 times a random leave-one-out cross-validation (LOOCV) test to identify the optimal parameters. The testing dataset was then used in the trained model to predict the outcome. We randomly chose 50 seeds to run our model 50 times. The final model results and the features’ importance analysis results  are the mean values of these 50 runs. A two-tailed P-value < 0.05 was considered statistically significant.Statistical analysis was carried out using SPSS for Windows, version 22.0 . Continuous variables were assessed for normality using the Shapiro-Wilk test. Groups were compared using the Student’s P<0.05). The most common clinical symptoms were headache  and vomiting . Altered mentation was more common in the poor outcome than the good outcome group (P<0.05). The most frequent underlying diseases were HIV infection  and immune system disease . A higher percentage of patients in the good outcome group  had no underlying diseases or factors compared to the poor outcome group .A total of 150 patients with CM  were admitted to our hospital from January 2008 to December 2018 and were included in this retrospective study. They were categorized into two groups (good and poor outcome groups) according to their clinical outcome. Clinical improvements after 2 and 10 weeks were observed in 93 and 94 CM patients, respectively. Therefore, data assessed after 10 weeks was used for clinical analysis. The demographic and clinical features of these patients are summarized in Table P < 0.05). The positive rate of CSF India ink staining in the poor outcome group  was higher than in the good outcome group . There was no difference in CSF glucose or chloride levels and the positive rates of CSF fungal culture between the two groups. The percentage of serum CD4+ T lymphocyte  and CD4/CD8 ratio  were lower in the poor outcome group than in the good outcome group.The laboratory tests results in patients with CM are shown in Table P < 0.05), as shown in Table A total of 134 CM patients received antifungal treatment after diagnosis. The remaining patients  did not receive antifungal treatment because their disease had rapidly progressed to death before such treatment could be initiated or because they were abandoned by their family members and were discharged voluntarily. Treatments for the other patients were categorized as monotherapy, two-drug combination, or triple therapy. Most patients  were treated by the two-drug combination, but patients receiving the triple therapy had a better clinical outcome (P < 0.001) and in the non-HIV infection group  were higher than in the control group (P < 0.01).The CM patients were divided in two ways; one division was based on their clinical outcomes, and the other based on their HIV-infection status. As can be seen in Fig. r = 0.738, P < 0.001), as shown in Fig. Results of CD4+ T lymphocyte count in the peripheral blood were obtained in 35 cases. The correlations between CSF WCC and CD4+ T lymphocyte count, CD4+ T lymphocyte percentage, CD8+ T lymphocyte percentage, and CD4/CD8 ratio were analyzed. CSF WCC and CD4+ T lymphocyte count were closely correlated (P < 0.001). The most effective cutoff value of CSF WCC for evaluating poor clinical outcomes in CM patients was 40 cells/μL, for which Youden’s index was 0.58.Figure P < 0.05). The counts decreased faster in this group compared to the poor outcome group. Two predictive models (C5.0 decision tree), using the caret package, revealed the relationship between dynamic changes in CSF WCC and the clinical outcome. The covariables used to predict the outcome in both models were sex, age, Charlson comorbidity index, hemoglobin concentration, peripheral WBC count, neutrophil concentration, treatment strategies, CSF protein concentration, CSF chloride concentration, CSF India ink staining, and CSF culture. Model 1 only used CSF WCC at week 0, while model 2 used CSF WCCs at weeks 0 to 10. The sensitivity, specificity, accuracy, and AUC of model 2 were superior to those of model 1. In both models, CSF WCC at week 0 made a great contribution to predicting the clinical outcome . In model 2, the importance of CSF WCCs from week 1 to week 10 were all greater than zero  had the worst clinical outcome. This finding is similar to previous research results . The immlearance .CM patients with lower baseline CSF WCC and protein level had poor clinical outcomes. Several previous studies have also found that CM patients with lower CSF WCC had a lower survival rate , 23, butCryptococcus from the CSF than amphotericin B alone, amphotericin B with fluconazole, or triple therapy [Nowadays, options for treating CM remain limited. All patients without access to treatment had a poor clinical outcome. The best clinical outcome was of patients with triple therapy. Therefore, early diagnosis and treatment are very important to reduce the mortality of CM patients. Amphotericin B, combined with 5-flucytosine, remains the most commonly used induction therapy regimen for CM, but its therapeutic efficacy is unsatisfactory. One earlier research found that in HIV-associated CM patients, a combined treatment of amphotericin B and 5-flucytosine could more rapidly clear  therapy . A recen therapy . Zhao et therapy . Triple Cryptococcus. In such patients, the CNS inhibits the recruitment of immune cells into the CSF [In this study, we observed lower CSF WCC in the poor outcome group and the HIV-infected group. In addition, CSF WCC was positively correlated with peripheral CD4+ T lymphocyte count, CD4+ T lymphocyte percentage, and the CD4/CD8 ratio. A previous study found a positive correlation between CSF and blood CD4+ counts. Lower numbers of CSF T cells and NK cells, and lower expression of the neutrophil activation marker CD66b26 were also observed in CM patients with a high fungal burden . Lower C the CSF . Our stu the CSF . That stThis study has some limitations. It is a retrospective study in a single hospital, and only part of the patients had data on CD4+ T lymphocyte count. However, this is the single-center study with the largest number of cases. Some tests could not be performed in every patient during diagnosis and treatment, which is more consistent with clinical practice. Further multicenter studies involving primary medical care settings, still need to be performed.This study shows that CM patients with lower baseline CSF WCC, lower CD4+ lymphocyte count, and altered mentation as well as older male CM patients (≥ 60 years) were more likely to have a poor clinical outcome. Triple therapy during the induction period might be an alternative treatment approach for CM patients with poor prognosis. Baseline CSF WCC had a moderate positive correlation with peripheral CD4+ T lymphocyte count. Baseline CSF WCC below 40 cells/μL might indicate poor prognosis in CM patients. Baseline CSF WCC, combined with CSF WCC changes over time, could better predict the prognosis of CM. More aggressive treatment and effective management should be given to patients with poor prognosis.


Contrafreeloading (CFL) is the phenomenon when animals work for a resource although an identical resource is available for free. Possible explanations for CFL are that animals seek context for species-specific behaviours or to control their environments. We investigated whether goats show CFL and whether breeding for productivity traits has altered its occurrence. In a manipulation task, we compared two selection lines: 27 Nigerian dwarf goats, not bred for productivity traits, and 30 dairy goats, bred for high milk yield. Over 10 trials, each goat could perform one of three behaviours: not participating in the trial, feeding for free from an open door, or opening a sliding door for a feed of similar value. The results were analysed using an Item Response Tree (IRTree) generalized linear mixed model (GLMM). The fitted probabilities to participate were > 0.87 over all trials in both selection lines. For dwarf goats, the probability of choosing the closed door, and thereby demonstrating CFL, increased from 0.30 in Trial 1 to 0.53 in Trial 10. For dairy goats, this probability was constant at approximately 0.43. Unlike dwarf goats, dairy goats were faster to approach the closed compared to the open door. Overall, our results suggest that both selection lines were similarly interested in CFL. This phenomenon contradicts optimal foraging theory (e.g.4) which suggests that an animal will maximize the net energy gain by choosing the food source providing most energy for the lowest cost5. Although CFL is documented in captive wildlife and domestic pigs, cattle, goats, and chicken9, it has not been reported in animals living in the wild10. One prominent theory to explain the occurrence of CFL is the Information Primacy Model2. It assumes that CFL is driven by the urge to gather information about optimal food sources in a natural environment where food shortages can occur. As a result, if food deprivation increases, optimal foraging strategies will increase and the preference to gather information decreases. The need to explore the environment might, therefore, be an important adaptive mechanism10 and possibly explain why CFL occurs in animals in captivity2 where food is abundant.Contrafreeloading (CFL) describes the phenomenon when animals, given the choice, work for a resource even though an identical resource is simultaneously available for free11 which is not mutually exclusive from the Information Primacy Model. White’s theory postulates the need of animals to control and modify their surroundings and assumes that the successful performance of a task reinforces itself12 by increasing the perceived control over the environment14. Several studies found that mastering a task can induce positive emotions in farm animals19 that often live in barren environments with little stimulation or possibilities to control their surroundings. For example, in a study by De Jonge et al.6, pigs preferred searching for food rewards in straw rather than receiving the identical rewards freely available from a trough. The authors concluded that the display of CFL could be best explained by the rewarding effect of the anticipation of food while foraging. Therefore, if the need to control aspects of the environment and/or the need to perform species-specific behaviours are the motivators behind CFL, providing tasks to satisfy CFL motivation within a farm setting might enhance animal welfare. To effectively enrich housing conditions, all individuals of a species should frequently take part in the CFL task.Another theory to explain CFL is White’s Competence Theory20 assessed the motivation to learn in cattle. They trained 30 heifers to perform an operant response (nose touch) to access a compartment providing a discrimination task. Although they used positive reinforcement (clicker training), the authors had to omit 10 animals mainly due to poor engagement in the task during the training phase. Participation proportions in the discrimination learning task varied between individuals and ranged from 0–100% of the offered sessions. Also, in studies where all of the individuals participated, huge individual differences in the extent of CFL display were found (i.e.23). For example, in a study with starlings, the percentage of choosing to work for food ranged from 0–100% across individuals21, whereas, in a study on pigs, the relative fraction of ‘earned’ food from total food consumed ranged from 0.4–12%24. Similar variations in CFL levels were reported in goats by Langbein et al.25. In this study, 12 dwarf goats were trained to operate an automated learning device that posed a discrimination task to receive drinking water. They found that 10 out of 12 goats chose to direct, on average, one-third of their total daily button presses towards the device instead of pressing the normal drinker where no prior discrimination task had to be solved. Some goats gained more than 80% of their daily water at the learning device, while other goats gained very little water at the device.The type of training used, and the characteristics of the task offered to measure CFL may affect the proportion of individuals participating in the particular CFL experiment. Meagher et al.6. Goats are considered an intermediate ruminant type in the line of browsing—grazing species26. They feed on a mixture of shrubs, trees, and grasses, often switching seasonally27, and browse for the most nutritive fractions in their food29. In contrast to sheep and cattle, which are mainly grazers, the time spent browsing can make up to 73–93% of their feeding time, depending on season27. A suitable CFL task for goats might resemble this natural browsing behaviour, allowing for oral manipulation of the test apparatus. Additionally, a low-effort and easily executable manipulation task is expected to increase the number of individuals frequently choosing to work for the reward2.It has been postulated that tasks that resemble the species’ foraging behaviours or need low effort to receive the reward will promote CFL32, but also foraging34 and exploration behaviour31 in farm animals. Compared to less productive breeds, pigs selected for high feed efficiency showed less behavioural reactivity towards fear-eliciting stimuli and displayed increased latencies when approaching a novel object and an unfamiliar human31. Such alterations in behaviour may reflect in the motivation to show CFL. Schütz and Jensen33 compared White leghorn chicken, selected for high egg productivity, to red jungle fowl, the ancestor of the domestic chicken, and to Swedish bantam, a domestic breed not strongly selected for production traits. They found that White leghorn chicken obtained a lower proportion of their food through CFL than both junglefowl and bantam chicken. Selection for high egg productivity in White leghorn chicken might thus have either directly reduced their motivation for CFL or indirectly decreased traits such as curiosity and risk-taking that are likely to affect the preferences of animals to perform CFL. Whether selection for high milk yield had a similar effect on the motivation to show CFL is yet unknown. To address this question, goats may represent a suitable species as selection lines differ strongly in milk production performance. Goats specifically selected for the dairy industry, such as Saanen goats, can produce up to 2–3 kg of milk per day35. On the other hand, common pet goats such as Nigerian Dwarf goats were not selected for productivity traits and their milk yield is much lower (0.3 kg per day)36.Domestication in general, and the selection for high productivity in particular, were found to have altered not only stress reactivity25, we expected that goats in our experiment would also show CFL to a certain proportion, i.e., push the closed sliding door open to receive the food reward instead of choosing the free reward. If goats are more motivated to work for a reward instead of receiving it for free, we would expect the approach time towards the closed door to be shorter than towards the open door20. Following up on the findings by Schütz and Jensen33, who showed lower motivation for CFL in the high-productivity chicken line, we assessed whether selection for high milk yield had a similar effect on CFL in goats. We compared dairy goats with a pedigree for high milk production to Nigerian dwarf goats that have not been selected for productivity traits. To increase genetic variability in our sample, we used individuals of two dairy breeds and of their crossbred . Using an Item Response Tree (IRTree) generalized linear mixed model (GLMM)37, the experiment was statistically modelled as a sequence of binary decisions between mutually exclusive behaviours . Additionally, a linear mixed model, with approach time as a continuous response, was used to compare approach times between the open and the closed door.We examined whether domestic goats show CFL and repeatedly do so over several trials in a low-effort manipulation task that resembles their natural foraging behaviours. We provided goats with the choice between receiving a desired food item at an open door or opening a sliding door to access an identical food item. As some dwarf goats readily worked for more than 80% of their daily water intake in a previous CFL study36. All dwarf goats were born in January/February 2017. As it was common practice at the institute in Dummerstorf, dwarf goat kids stayed with their dams for 6 weeks before they were separated. They were moved to Ettenhausen in June 2017. To investigate the effect of selection for high productivity on CFL, we compared dwarf goats to dairy goats with a pedigree for high milk production. To increase genetic variability in our sample38, we used two of the most common high-producing dairy breeds in Switzerland and their crossbred, namely Saanen (n = 15), Chamois coloured (n = 12), and Saanen × Chamois , consisting of a deep-bedded straw area of 11 m2 (approximately 2.8 m × 3.9 m) and a 0.5 m elevated feeding place (3.0 m2). The total area of each dairy goat pen was 17.5 m2 (approximately 3.9 m × 4.5 m), consisting of a deep-bedded straw area of 13.2 m2 (approximately 4.5 m × 2.9 m) and a 0.6 m elevated feeding place (4.3 m2). Hay was provided ad libitum behind a feeding fence and replenished twice a day at approximately 8 am and 4 pm. Each pen had one drinker and a mineral supply. Additional structures in the straw-bedded area included a wooden bench  along the wall of the pen and a round wooden table  in the centre of the pen. The goats were between 15 and 18 months old at the start of the study .In Ettenhausen, dwarf and dairy goats were initially housed in one group pen each. At the age of 7–8 months, all goats were moved to pens of 10 goats each: three groups of dairy goats and three groups of dwarf goats. The total area of each dwarf goat pen was 14 m39.All procedures involving animal handling and treatment were approved by the Swiss Cantonal Veterinary Office Thurgau  and were performed in accordance with all relevant Swiss legislative and regulatory requirements and the ASAB/ABS guidelines for the use of animals in researchThe goats were individually tested in a test arena (4.5 m × 2 m). The test apparatus was installed as part of a wall on the shorter side of the test arena and consisted of two identical wooden sliding doors. A metal grid fence (1.35 m high × 1.27 m long) was installed between the two sliding doors to prevent goats from switching sides and feeding from both openings right after each other . Participation and the choice of door type were unambiguous.Goats were individually led into the test arena by a second experimenter (E2) and released near the centre of the room approximately one meter away from the start of the fence  model of the GLMM family to analyse the multivariate behavioural response in our experiment. Such IRTree models allow representing a multivariate behavioural response as a tree of sequential binary responses, enable incorporating hierarchical sampling, and can account for correlated responses as well as repeated testing of the same individuals37. They are well suited for analysing categorical data in behavioural studies, as shown for example for escalating courtship behaviours, antipredator behaviours, and social interactions42.All statistical analyses were performed in R v4.0.241  and an individual slope for the trial number for the two selection lines [Node:SelectionLine:I]. The trial number was included as Trial − 1 to render the intercept to correspond to Trial 1 instead of the non-meaningful Trial 0. As a random effect, we included a random intercept for observation (1|Obs) to ensure that the sequential binary responses corresponding to a single observation shared the same variance and were not treated as independent observations. Furthermore, we specified for each node a random intercept for the location of the closed door, nested within individual, itself nested within pen . This was done to account for potential side bias, repeated testing of the same individual, and potential effects of pen affiliation. Despite the randomization of the location of open and closed door, a side bias was evident; the choice for the closed door was apparently more likely when the closed door was on the left versus the right side .glht function from the R package multcomp44. Both the p-values for fixed effect estimates (glmer function) and for the contrasts (glht function) were obtained using Wald tests. Fitted probabilities and bootstrap confidence bands that were only conditioned on the fixed effects were obtained using the predict.MerMod function  in conjunction with the bootMer function (lme4) for parametric bootstrapping .To investigate the difference in behaviour between the selection lines, we tested selection line contrasts for the fixed effects using the lmer function (lme4 package) and the following model formula:To compare approach times towards the open versus the closed door, we also analysed the approach time as a continuous response in a linear mixed model using the glht function (multcomp package). The R code used to perform the analyses described in this section is provided as Supplementary Material  and only one individual chose in all trials to not participate  and reached a probability of 0.53 in Trial 10  towards the closed door ranged from 0.56 to 0.70 in dwarf goats and from 0.33 to 0.42 in dairy goats Fig. , a diffeIn dwarf goats, the probability for a long approach time towards the open door decreased with increasing trial number (p = 0.01)—from 0.63 in Trial 1 to 0.20 in Trial 10  to 1.8 s  and 2.3  to 2.0 , respectively. Thus, dairy goats approached the closed door faster than the open door (p = 0.002).In line with our hypothesis, goats chose to participate in the experiment and were willing to work for a reward in the presence of an identical, free reward, and thus chose to perform CFL. Contrary to our expectations, the selection line was not related to the overall probability of working for food but rather to changes in goats’ responses with increasing trial numbers. Our results suggest that both selection lines were motivated to work for food, while this motivation was suppressed in early trials in dwarf goats, presumably due to higher stress reactivity.The high proportion of goats that frequently chose to participate in the experiment suggests that we used a suitable experimental setting with a highly desired reward and enough previous habituation to the test arena and the sliding door in order to make participation rewarding for goats. To avoid spatial learning, we randomized the location of the open and the closed door in all trials. However, we found indications that the closed door was more likely to be chosen when located on the left side. As the goats’ pen mates where located on the right side, this bias seems not socially induced. In our statistical models we considered the potential side bias as a random effect nested in individual, thus allowing an individual side bias for each goat.34 and cattle7 which found that animals selected for high productivity were choosing more energy conserving strategies and thus were less likely to show CFL than animals not selected for production traits. In our experiment, the probability in dwarf goats to choose the closed door increased with increasing trial number, while it remained approximately constant for dairy goats . Increasing habituation and positive reinforcement from opening the door might then have increased motivation to choose CFL in subsequent trials. This is in line with the notion that stress reactivity has been reduced in animals selected for high productivity46, which would suggest reduced stress reactivity in dairy goats compared to dwarf goats.In accordance with our expectations, all but four goats exhibited CFL in at least 1 of 10 trials. Overall, the probabilities to choose the closed door were not different between the dwarf and dairy goats. This is in contrast to what we expected and to previous studies on different selection lines of chicken48 found indications for higher fearfulness of dam-reared goats in goat-human encounters as compared to human-reared goats—dam-reared goats exhibited greater behavioural responsiveness in novel situations, as well as longer latencies to approach an unfamiliar human.However, not only a genetic disposition but also differences in rearing may have caused different stress responses between the selection lines in our experiment. Whereas the dairy goats were artificially raised without their mothers, the dwarf goats stayed with their dams for 6 weeks. Previous studies20. Our results only support this hypothesis in dairy goats, which approached the closed door faster. In contrast to dairy goats and not in line with our hypothesis, dwarf goats showed similar approach times towards both doors in the first few trials and tended to approach the open door faster in later trials (Fig. 49, regardless of whether it is genetically based or developed during the ontogenesis.Regarding the effect of door type on approach times, we hypothesised that, if goats are motivated to work for a reward instead of receiving it for free, they would approach the closed door faster than the open doorals Fig. . An expl12. Positive emotions as a result of mastering a task and being in control of the situation have been reported in cattle, pigs, and goats19. This is also in line with White’s Competence theory11, which postulates that animals are motivated to manipulate and control their environment to attain competence. Hence, it is possible that goats were choosing CFL due to positive feedback from executing the manipulation task.Over all trials, the probability of choosing CFL was slightly below 50%, raising the question whether our results could partially be explained by goats randomly choosing the open or closed doors. However, we found the probability of choosing CFL in dwarf goats to increase over trials and the approach time to be affected by door type. This indicates that goats deliberately chose the door type rather than choosing at random. An explanation for the occurrence of CFL over several trials might be attributed to intrinsic rewarding properties of the performance of the task itself 19 and that animals value effort50 such that the incorporation of such challenges into a farm setting can have welfare benefits for the animals51. For a successful implementation of CFL tasks in the husbandry system, it would be necessary to evaluate if the motivation to display CFL is stable in various conditions and also persists over a longer period of time. The high inter- and intra-individual variations in the extent of CFL, which are in accordance with other studies on CFL in dwarf goats25 and in other species52, require further research regarding the motivational background to show CFL in animals.It has been suggested that animals need appropriate cognitive challenges to express control over their environmentOverall, high CFL proportions in both selection lines, increasing interest in approaching the closed door in dwarf goats, and shorter approach times towards the closed door compared to the open door in dairy goats indicate that both dairy and dwarf goats were motivated to work for a resource in the presence of the same resource for free. The two selection lines of goats differed in the changes of the probabilities to choose CFL with increasing trial number and regarding the comparison of approach times towards the open versus towards to closed door. These results might reflect differences in stress reactivity towards the CFL task, potentially related to selection for productivity or differences during ontogeny. Our findings suggest that goats seem to be motivated to solve a CFL task, stressing the need for the provision of cognitive challenges to improve the welfare of farm animals.Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.Supplementary Video 1.