Protein concentrations and activities of ChAT and AChE were measured in CSF of 18 patients with mild AD prior to and after 3 months of treatment with galantamine (n = 12) or placebo (n = 6), followed by nine additional months of galantamine treatment in all patients. A Cholinergic index was defined as the ratio of ChAT to AChE in CSF and was evaluated in relation to the in vivo AChE activity, the nicotinic binding sites and different measures of cognition. Besides an expected inhibition of AChE activity, galantamine treatment was accompanied by a mild increase in CSF ChAT activity. Thereby, the Cholinergic index was significantly increased in the Galantamine group (60% ± 14) after 3 months compared to baseline (p < 0.0023) or (p < 0.0004). This index remained high in the Galantamine group compared to baseline (54% ± 11) at 12 months follow-up, while it showed an increase in the Placebo group when they switched to active galantamine treatment . Furthermore, the in vivo brain AChE activity (assessed by PET) correlated with the CSF Cholinergic index at 12 months . The CSF Cholinergic index also correlated with ADAS-Cog and some other neuropsychological tests at 12 months. This is the first study assessing a CSF Cholinergic index in relation to treatment with a cholinesterase inhibitor. The treatment-specific increase in CSF ChAT activity suggests that cholinesterase-inhibitors may also increase the ACh-biosynthesis capacity in the patients. Additional studies are warranted to evaluate the utility of the CSF Cholinergic index as a biomeasure of therapeutic effect in AD.Alzheimer’s disease (AD) is a progressive disease with early degeneration of the central cholinergic neurons. Currently, three of four AD drugs act by inhibiting the acetylcholine (ACh) degrading enzyme, acetylcholinesterase (AChE). Efficacy of these drugs depends on available amount of ACh, which is biosynthesized by choline acetyltransferase (ChAT). We investigated whether treatment with a cholinesterase-inhibitor, galantamine, alters the relative levels of AChE to ChAT in cerebrospinal fluid (CSF) and whether levels of these CSF biomarkers correlate with  The central cholinergic pathways have prominent roles in learning and memory. The severity of cholinergic deficits in patients with Alzheimer’s disease (AD) correlates with cognitive impairment, which have led to the development of cholinesterase inhibitors (ChEIs). The currently registered ChEIs as AD therapeutics putatively act by inhibiting the degradation of acetylcholine (ACh) in the synaptic cleft by the synaptic acetylcholinesterase (AChE), prolonging ACh’s action on its receptors  by a glycophosphatidylinositol anchoring protein. Measurement of changes in the RBC AChE activity following treatment with ChEIs has also been used to define the inhibition level of AChE in patients treated with ChEIs  following treatment with ChEIs . This is because a remodeling and/or regeneration of the cholinergic synapses will most likely require extra expression of both ChAT and AChE. In this context, recent reports of the presence of a soluble variant of ChAT in both plasma and CSF has provided a unique opportunity . Whole-blood was collected in standard tubes containing EDTA as anticoagulant. Then, blood was also collected after 3 and 12 months during the study. CSF samples, taken at baseline and after 3 and 12 months of treatment, were collected by lumbar puncture in the L3/L4 or L4/L5 interspace at mornings between 9–12 a.m. All CSF samples were kept frozen in small aliquots at –80°C, until the assay. More detailed information have been given previously  were coated overnight at 4°C with 75 μl of monoclonal anti-human ChAT antibody . The wells for choline standards and their blanks were not coated with the capturing antibody. On the following day, the plates were washed with PBS and tapped dry for the next step (as described below in the assay procedure).Undiluted CSF samples (10 μl/well) were applied both unmodified  and as modified denatured samples . The denatured samples served as controls for both the ChAT activity and the endogenous concentration of choline in the samples. The denatured CSF samples were prepared by heating in a thermal cycler, with three cycles of 8 min at 98°C, and 30 s at 60°C.Two series of standards were used on each plate, a choline standard sets for quantifying the choline concentration in the wells, and a ChAT protein standard series for quantifying the amount of ChAT protein in the samples. The choline standards were prepared by two-fold serial dilutions of choline chloride solution in TBS buffer . The highest choline standard point was 50 μM, and 50 μl/well (in triplicates) of each choline standards were applied into the wells of the microtiter plate. As the ChAT protein standard, a calibrated pooled human plasma sample was used. Like the CSF samples, the ChAT protein standards were applied both as native (unmodified) and as denatured samples . The highest ChAT protein standard had a concentration of 1.14 μg/ml, which was prepared by 50 times dilution of the calibrated pooled plasma sample. The denatured standards were prepared as described above for CSF samples.Cocktail-A and -B. The Cocktail-A was prepared in the TBS buffer, and contained 18.75 μM of choline chloride , 0.75 mM of eserine hemisulfate , 1.25U/mL of phosphotransacetylase , 8.75 mM of acetyl phosphate , and 62.5 μM acetyl-coenzyme A . The Cocktail A was incubated for 10–15 min at 38°C prior to the assay procedure. The Cocktail-B was prepared in PBS , and contained 0.93 U/ml of choline oxidase , 1/5,000 Streptavidin horseradish peroxidase (P6782 Sigma-Aldrich), 6.3 mM of phenol , and 3 mM of 4-aminoantipyrine . Both cocktails should be prepared freshly.The ChAT assay requires two master-mix cocktails: Cocktail-B was added to all wells, including the wells containing the choline standards. The reaction was then monitored continuously at 1–2 min intervals at 500 nm for changes in absorbance for as long as was deemed necessary  using a Tecan Infinite M1000 microplate reader. This step quantifies both the ChAT activity and the choline concentration in the samples.The unmodified and denatured CSF samples (10 μl/well), the ChAT protein standards (10 μl/well), or the TBS buffer  were applied to the wells of the anti-ChAT antibody pre-coated microtiter plate. The choline standards (50 μl/well) were applied to un-coated wells on the plate. Then, 40 μl/well of the Cocktail-A was applied to all wells but the choline standards, using a multichannel pipette. The plate was then sealed and incubated for 2 h in a humid chamber at 38.5°C under gentle shaking. The plate was cooled briefly on ice-water bath and centrifuged gently. The sealing tape was removed and 25 μl/well of the 0.05% , and incubated with 100 μl/well of a blocking buffer (TBS-T0.05% containing 5% BSA and 0.01% sodium azide) for 30 min at 38°C in a humid chamber under gentle shaking. The plate was then washed as before, and incubated for 60 min with 50 μl/well of a working dilution of 1:1,700 of the detecting anti-ChAT antibody  at 38°C in a humid chamber under gentle shaking. Then 25 μl/well of a working solution of a secondary swine anti-rabbit polyclonal antibody  was added to the wells and incubated for two more hours at room temperature under gentle shaking. The plate was washed four times with TBS-T0.05% and one time with 1 M diethanolamine buffer . Finally, 75 μl/well of a solution of the alkaline phosphatase substrate . The absorbance was read at 405 nm until the absorbance of plasma standard reached 2–2.5.After this step, the plate was sealed and incubated overnight at 4°C. On the following day, the plate was washed three times for 5 min with TBS-Td − Cn)/ChAT activity =  containing the denatured and native samples, respectively, t is the incubation time with the Cocktail-A (in minutes) at 38°C, and Vs is the 10 μl volume of the samples applied into the wells (in mL). The ChAT protein was determined as ng/mL CSF from the second part of the assay protocol, which is similar to a conventional ELISA assay.ChAT activity (pmol/min/mL samples) was calculated according to the following formula: functionalChAT activity (ChATF). It should be noted that the ChATF activity has the unit of pmol/min/ng of ChAT protein. This integration of ChAT activity and protein is necessary for direct comparison between changes in ChAT activity and inhibition of AChE following galantamine (see below).The ChAT activity can also be expressed in relation to its protein concentration by dividing the ChAT activity (expressed in pmol/min/mL) to the ChAT protein concentration in the samples (ng/mL). For the simplicity, this parameter will be referred to in the following texts as The methodological details for measuring RBC and CSF AChE activities have been described previously , which is an ACh analog, to measure the reduction of cerebral cortical AChE activity in the brain. In the 11C-PMP-PET, the rate constant k3 for hydrolysis of 11C PMP by AChE according to a kinetic model developed in Kuhl et al.  in different brain regions at the follow-up intervals. Thus, the k3 value has a direct relationship with AChE activity. For cortical regions, the AChE activity was calculated as the average of k3 values from the “region of interest” (ROI) in the anterior cingulate, frontal, frontal association, parietal, parieto-temporal, temporal, primary visual, sensory motor and temporal medial lobe cortices of the left and right brain hemispheres.PET was used with the tracer N-. The k2* has an inverse relationship with the number of nicotinic binding sites in the brain.PET with Z-scores of the three cognitive domains were calculated as described before , and after 3 and 12 months of treatment. The details are reported previously  was followed by the Bonferroni–Dunn post hoc analysis that tested the significance of results at each time point compared with baseline or between groups. Correlation analyses were performed using correlation Z-tests. When deemed necessary, the correlations were asserted using the nonparametric Spearman Rank Correlation and were visualized using simple regression plots.Data are expressed as mean values and standard error of the mean (SEM) or otherwise as stated. The effects of treatment at different time intervals compared to baseline were assessed using repeated measures (RM) analysis of variance (ANOVA) for the double-blind phase as well as open-label phase. The comparison between the groups was done using ANOVA analysis. A significant ANOVA result  or placebo (the Placebo group). During the Phase II of the study, the patients in the Galantamine group continued on galantamine for a total of 12 months. At the beginning of the phase II of the study, the Placebo group, however, started on receiving galantamine and continued on this drug for a total of 9 months .The study design and the demographic of the patients are shown schematically in the Galantamine group) was withdrawn as a result of second-degree atrioventricular block, considered possibly related to galantamine. Overall, 17 subjects completed the double blind Phase I of the study which was for 3 months. During the second phase, i.e., the open-label phase II, three more subjects were withdrawn: one from the Placebo-Galantamine group owing to chronic lymphocytic leukemia , and the others from the Galantamine arm of the study: one patient due to cardiac arrhythmia and syncope , and one patient as a result of non-compliance. Of the remaining 14 patients, one could not complete the neuropsychological tests at 12 month due to myocardial infarction . It should be also noted that due to technical issues with PET scan validation and data acquisition, a valid AChE PET scan was available for only 15 patients  at baseline and 3 months but 11 valid AChE PET scans at 12 months. In case of 11C-nicotine PET scan, data were for similar technical issues available for 17 patients  at baseline and 3 months but 14 valid 11C-nicotine PET scans at 12 months. Thus, in principle there were a total of four dropout and 14 subjects completed the 12 months study.Of the initial 18 subjects , during Furthermore and particularly relevant to the CSF analyses, at the time of conducting the CSF ChAT measurements there were not enough CSF left either at the 3 months or the 12 months follow-up for some of the patients. Given that the statistical analyses were done on repeated measured analyses only the subjects that had data at baseline, 3 months and/or 12 months could be compared.the Galantamine group at the 3-months follow-up . No significant changes in the RBC AChE activity was observed in the Placebo group at 3 months. At the 12-month follow-up, however, the RBC AChE inhibition in the Placebo-Galantamine group was similar to the inhibition level in the Galantamine group .The descriptive results regarding the changes in the RBC and CSF AChE activities are reported elsewhere .This estimated CSF AChE inhibition is shown in 3 months . In the F) in CSF.Since the CSF AChE activity was normalized to the AChE protein levels, it was necessary to also integrate the overall ChAT activity (in pmol/min/mL CSF) with its protein in CSF (in ng/mL), to estimate changes in the level of functional ChAT activity . Noteworthy, repeated-measure ANOVA includes only four subjects in the Placebo group who had 12-months data. These analyses indicated that the Galantamine group had 47% higher levels of ChAT than the Placebo group  to that of AChE (in nmol/min/μg) in CSF (ChATF/AChEF activities). Thus, an increase in this ratio means a net increase in ACh levels.Cholinergic signaling is most likely regulated through a balance between synthesis, release and degradation of ACh. We hence defined a At 3 months, the Cholinergic index differed significantly between the Galantamine and the Placebo groups . The Galantamine group showed also about 60% significant increase when compared to baseline , indicating that in this group the improved cholinergic index was maintained for up to 12 months of galantamine treatment. Most importantly, at the 12 months follow-up, a similar increase in this index was observed in the Placebo-Galantamine group. This increase was significant compared to both the baseline  by PET.At 3 months, the in vivo AChE activity (k3) in the whole brain correlated with the CSF ChATF activity , and the CSF Cholinergic index , as assessed in all patients as one group.At 12 months, significant correlations were observed between the in vivo AChE activity (k3) and the ChATF activity as well as the Cholinergic index in CSF, which are summarized in At baseline, the CSF AChE activity correlated positively with a number of nicotinic binding sites in the pons  and the left and right primary visual cortex , parietal cortex , cingulate gyrus , and the whole brain  and right parieto-temporal cortex . We did not use the CSF Cholinergic index in these analyses since it is composed as a ratio between functional ChAT and AChE activities in CSF.The functional ChAT activity in CSF at baseline also correlated positively with the number of nicotinic binding sites in left thalamus  and AChE-R variants .F activity or the Cholinergic index and the nicotinic binding sites in the brain in the Galantamine or the Placebogroup.No significant correlations were observed between ChATAt 12 months, the correlation analyses indicated an inverse association between the RBC AChE inhibition and the relative changes in the nicotinic binding sites in the brain regions ( regions . Similar regions  as well  regions .the Placebo group) was again restored following the longer-term of 9–12 months of galantamine treatment  following treatment with ChEIs, like galantamine. This Cholinergic index showed a clear increase as response to ChEI-treatment.Here, we described for the first time the effect of galantamine treatment on CSF levels of two important enzymes that maintain the steady state of ACh in the brain, in particular, the core-cholinergic enzyme responsible for biosynthesis of ACh, i.e., ChAT. The first 3 months of the study had a double-blinded placebo-controlled protocol, which allowed us to specifically address the biodynamic relationship of AChE and ChAT enzymes in response to galantamine treatment. We showed that specific inhibition of the CSF AChE activity by galantamine treatment was accompanied by a mild increase of the activity of ChATF activity, the Cholinergic index and the AChE activity to the in vivo AChE inhibition and nicotinic binding sites in several brain regions assessed by PET in AD patients prior to and after treatment with galantamine.In this study, we further assessed the changes in these CSF parameters with the clinical and paraclinical measures in the patients. This way we showed presence of a dynamic pattern of interdependency of the CSF ChATin vivo AChE inhibition in the majority of the regions of the brain, in particular the cortical regions in these patients assessed by PET  was instead accompanied by an increase in the in vivo nicotinic binding sites in the majority of the brain regions. Intuitively, these findings appear unexpected and paradoxical with regards to the pattern of the observations at the baseline. This is because a high AChE inhibition implies less ACh degradation and thereby a prolonged action of ACh on its receptors within the synaptic cleft  may be able to be manifested as improvement in the CSF cholinergic index, or simply how this index may differ among patients at different stage of the disease. Here, it is crucial to refer to the rational for treatment with ChEIs. These drugs by definition require a certain amount of ACh production (by ChAT) and release into the synapses. In other words, these drugs will not work if the production and release of ACh into synapses is heavily affected or is absent. Given that the cholinergic neurons degenerate early and progressively in the course of AD, it is expected that each remaining cholinergic neuron will with time have fewer synaptic interfaces at its projection sites in the brain that fulfill this criteria as the disease progresses. Thereby, it is plausible that not all of the degenerating synapses of a cholinergic neuron can be stimulated by treatment with ChEIs. This might explain the limited and moderate clinical efficacy observed following treatment with ChEIs. Given that even at mild stage of AD, a substantial number of cholinergic synapses may have already degenerated beyond the rescue point, it is not very surprising that the observed increase in soluble ChAT activity was quite mild in the current study following galantamine treatment. This, in turn, suggests that a higher degree of increase in soluble ChAT should be expected if treatment initiated at milder stage of the disease or even at asymptomatic stages.This study has several limitations. It was primarily designed as a PET investigation, thus only included a small group of mild AD patients. These features make it necessary to be cautious when interpreting the data, since the small sample size may result in a lack of statistical power, although this might make the significant results even more interesting. Also, relatively large numbers of statistical analyses were performed on the PET measurement in relation to the CSF cholinergic biomarkers and cognitive function, which were not corrected for multiple comparisons. This may render some of the results liable to be obtained by chance. Therefore, we weighted the overall patterns of the results rather than isolated findings. The number of available data at the 12 months follow-up was particularly small because either no CSF sample or PET scan was available for a few of the patients.F activity, the RBC or CSF AChE activity (or inhibition) and the in vivo AChE activity and nicotinic binding sites in the brain, measured by PET. The pattern of the findings indicates a time-dependent biodynamic remodeling and/or reorganization, indicative of establishment of a new bio-equilibrium in the function of the cholinergic neuronal interfaces following ChEI-treatment vs. placebo as well as the treatment duration. Altogether, these observations suggest that a neuronal and/or synaptic remodeling occurred in response to stimulation with a ChEI’s treatment, which in turn provide a mechanistic basis for the transient stabilization of cognition and delayed progression of the disease observed in AD patients following therapy with ChEIs (Giacobini, In summary, we report for the first time that a cholinesterase inhibitor increases the CSF level of the ACh synthesizing enzyme, choline acetyltransferase. We also report here distinct time and treatment-dependent associations between the CSF ChATThe datasets generated for this study are available on request to the corresponding author.The studies involving human participants were reviewed and approved by The Ethics Committee of Huddinge University Hospital, Sweden. The patients/participants provided their written informed consent to participate in this study.in vivo AChE and nicotine binding analyses. OA performed the neuropsychological assessments on the patients. All authors read and approved this final draft of the manuscript.TD-S and AKarami conceived and designed the CSF experiments; set up and performed the CSF AChE and ChAT assays and performed statistical analysis of the data. AKarami, TD-S and ME wrote the first draft and did the initial revisions. AN conceived and designed the original galantamine-PET study. AKadir and AN did the The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


To compare the sensitivity of identification of penile plaques in the erect and flaccid penises by ultrasound in patients with Peyronie’s disease (PD).A total of 75 PD patients were screened by palpation and ultrasonography for penile lesions in both flaccid and erect penises induced by prostaglandin E1 (PG-1) injection.P < 0.01) or palpation (P < 0.0005) The type of penile lesions identified by ultrasonography included tunical thickening, calcifications, septal fibrosis, and intracavernosal fibrosis. The ratios of these lesions confirmed by ultrasound were 52.6, 33.6, 6.0, and 7.8%, respectively, in the flaccid penises, and 55.8, 28.3, 8.7, and 7.2%, respectively, in the erect penises.A total of 138 lesions were identified by ultrasound in the erect penises induced by injection of PG-1. However, only 74.6% of the lesions (103) were detectable by the palpation of the flaccid penises, and 84.1% (116) by ultrasound of the flaccid penises. The ultrasound confirmed 99 of the palpated lesions in the flaccid penises. The detection rate of lesions in drug-induced erect penises by ultrasound was significantly higher than those in the flaccid penises by the ultrasound (Drug-induced erection can be used in suspicious PD patients when penile lesion is not identified by palpation or ultrasound in the flaccid penis. Peyronie’s disease (PD) is a common penile disorder with a prevalence between 3.2–8.9% . PD is cPeyronie’s disease can be initially diagnosed based on patient’s history and palpation of the penis , and is Ultrasound is typically conducted on the flaccid penis. However, ultrasound on flaccid penis fails to reveal the lesions which can be detected by palpation in some PD patients, and pharmaceutical induced erection is required . ErectioAll patients with PD were recruited from the Andrology Center of our hospital between the period of March, 2015 to May, 2018. A patient was included in the study if a palpable nodule, focal hardening, or bending of the axis of the penis during erection was found in the flaccid or erect penis. Exclusion criteria were congenital penile curvature and previous PD surgery. A total of 75 patients with median age of 45 years, ranging from 29 to 70 years were eventually included in this study. All participants provided written informed content as well. The study was approved by the Ethics Committee of our institute.A veteran urologist examined the penis on all the PD patients. A palpable penile plaque identified in the flaccid state was further confirmed by stretching the penis with one hand while gently compressing the penile shaft between the fingers and thumb of the other hand. The number, size, and location of the plaques were recorded, and the symptoms of pain were thoroughly evaluated.A Doppler ultrasound was used to visualize penile plaques following palpation. The GE Logq E9 or Philips IU22 units with a 5.0–15.0 MHZ linear-array transducer was used.Two ultrasound protocols were adopted to compare their sensitivities in diagnosing PD. In the first protocol, the penis was checked at the flaccid state. In the second protocol, 10 mg of prostaglandin E1 (PG-1) was injected into the corpus cavernosum to induce erection. A second dose of PG-1 was administered if the penis was not fully erect. All 6 patients obtained satisfactory erection after the second injection of PG-1. Then, patients were examined in a supine position in a warm and quiet private room. The flaccid and erect penises were longitudinally and transversely checked from the sulcus coronarius to the base. The location, size, number, and morphological characteristics of plaques were recorded. The plaques were further classified into 4 types according to their locations and calcification status; tunical thickening (tunica thickness is greater than 2 mm), septal fibrosis, intracavernous fibrosis, and penile calcifications , 2012.P-value <0.05 was defined as statistically significant.All data were statistically analyzed with SPSS 12.0 software . The number and types of plaques were analyzed with the Wilcoxon test. A Patients had various symptoms or noticed penile abnormalities for a duration of 4 to 42 months . Pain duA total of 138 penis plaques were identified by ultrasound in 75 PD patients after PG-1 induced erection. Approximately 52% of the plaques were less than 1.5 cm in length, 37% ranged from 1.5 to 3.0 cm, and 11% were larger than 3.0 cm. The thickness of the plaques varied from 0.2 to 1.6 cm.Ultrasound was able to distinguish penile plaques with different characteristics. Approximately 28.3% of the plaques were calcified, in which 15.9% were fibrosis, and 55.8% were tunical thickening . Among tCompared with the number of penis plaques (138) found in the erect state, palpation in the flaccid state alone was able to identify 74.6% of them (103/138) in 71 out of 75 patients . The othUltrasound detected 84.1% (116/138) of the plaques in 71 out of 75 patients when the penis was examined in the flaccid state. The frequencies of tunical thickening, calcifications , septal P < 0.05) in comparison with palpation. Application of ultrasound on drug-induced erection revealed that 28 patients had solitary lesion, 34 had 2 separate lesions, 10 had 3 lesions, and 3 had 4 lesions, which was significantly different from those with palpation (P < 0.0005) or ultrasound in the flaccid penis (P < 0.01).Palpation of the flaccid penis alone found one single plaque in each of 45 patients, 2 isolated plaques in 20 patients, and 3 plaques in the other 6 patients . UltrasoUltrasound detected 99 of the 103 palpable lesions (96%) in flaccid penis. However, 17 additional lesions were found by ultrasound imaging. In addition, 8 out of the 17 additional lesions were located at the intracavernosum, 6 at the septum, and 3 at the ventral tunica albuginea of corpora cavernosa behind the corpus spongiosum. Therefore, a total of 116 plaques were identified in the flaccid penis with ultrasound.P < 0.01).All 120 lesions detected with palpation or sonography in the flaccid penis were identified in the erect penis by ultrasound . The plaThe present study, to the best of knowledge, for the first time investigated the sensitivity of ultrasound in identifying penile plaques in Chinese PD patients. More penile plaques were identified when ultrasound was performed on a prostaglandin-induced erect penis compared with a flaccid one. The findings provide evidence that drug-induced erection modality can be used in patients with suspicious PD history and palpation but negative ultrasound findings in the flaccid penis.PD may be diagnosed by palpation of penile plaques . HoweverIn the present study, the ultrasound was able to detect 96 and 100% of palpated penile plaques in the flaccid and erection penis, respectively. Our results are similar to some studies , howeverThe present study demonstrated that the ultrasound performed better in identifying penile lesions when the penis was in the erect state. Dorsal palpable penile nodules in 4 PD patients were not detected by the ultrasound in the flaccid penis; however, they were well characterized by ultrasound in the erect penis. Furthermore, 18 new lesions were discovered by the ultrasound in the erect penis. The tunica albuginea in the flaccid penis is thick, which can attenuate signal from the lesions within the corpora cavernosa. However, the tunica albuginea is stretched and becomes thinner due to the expansion of the corpora cavernosa in the erect penis. A penile lesion, which is usually not stretchable, can then be visualized by ultrasound due to the increased contrast enhancement between the lesion and peripheral normal tissue. Therefore, ultrasound in an erect penis can increase the chance of identifying a lesion which is not well contrast with the surrounding tissues, especially in the tunica albuginea and septum. However, the ultrasound was able to identify calcified lesions efficiently regardless of the penis state, which may be due to the distinctive shadow generated by the calcified lesions.The present study had some limitations. Given the size of our patient population, and not all types of PD plaques were represented. In addition, ultrasound images were provided by one sonographer in both the flaccid and erect penis, which may cause bias in the data collection. Therefore, a second independent sonographer should be included in the future study.The ultrasound screening of penile plaques in the erect penis was more sensitive than in the flaccid state for Chinese PD patients. Ultrasound of the erect penis can improve identification of lesions which are not obvious, especially lesions in the tunica albuginea and septum. Our results also suggest that ultrasound in erect penis should be considered in all suspicious PD patients in the clinical routine.All datasets generated for this study are included in the manuscript and/or the supplementary files.JL, YL, and DZ conceived of the study, participated in its design and drafted the manuscript. JL performed the examination of ultrasonography. XL and XS helped in data acquisition, analysis and interpretation. XL and SS helped in literature review and statistical analysis.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Bifidobacterium accounted for 29.9% and 18.1% of OTUs in the stools of donors #1 and #2, respectively. Lactobacillus accounted for 16.3% and 2.9% of the stools of donors #1 and #2, respectively. Furthermore, through longitudinal monitoring of the patient, we identified 6 OTUs that were particularly sensitive to the different FMT complements. Together, we present a case report suggesting that the overall diversity of the intestinal microbiota may not be the only important element in the selection of an effective FMT donor.Almost 90% of patients undergoing hematopoietic stem cell transplantation (HSCT) experience diarrheal episodes, which represent a severe, often life-threatening complication for these patients. Although fecal microbiota transplantation (FMT) represents an alternative treatment option for infection-related diarrhea, the application of FMT in HSCT patients is greatly restricted for safety reasons. Furthermore, the therapeutic outcomes of FMT as a diarrhea treatment are somewhat related to the choice of the FMT donor. Here, we comprehensively profiled the dynamic changes in the intestinal microbiota after FMT from two donors with different feeding patterns and the same severely diarrheal recipient undergoing HSCT via a 45-day clinical observation. Importantly, no adverse events attributed to FMT were observed. The stool volume and frequency of the patient were reduced when we used feces from donor #1 (mixed feeding), but these changes were not observed after FMT from donor #2 (exclusive breastfeeding). Interestingly, no obvious differences in overall diversity (Shannon) or richness (Chao1) between the two donors were observed. Additionally,  Almost 90% of patients undergoing hematopoietic stem cell transplantation (HSCT) –4 experiClostridium difficile infection that cannot be eliminated with antibiotics alone [The gut microbiome plays important roles in regulating diarrhea-related functions, including as a protective barrier to incoming pathogens by colonization competition  and in tcs alone .Given that FMT carries a potential risk of infection , 21, the2, days −5 to −4); GVHD prophylaxis consisted of antithymocyte globulin (ATG), cyclosporin A (CSA), mycophenolate mofetil (MMF), and short-term methotrexate (sMTX). ATG  was given at a dose of 10 mg/kg from days −5 to −2. CsA  was administered starting on day −10, and the trough concentration was adjusted to 150–300 ng/ml. MMF was administered orally starting on day −10  and was withdrawn on day +45 for haploidentical donor (HID) HSCT. sMTX was administered intravenously at 15 mg/m2 on day +1 and 10 mg/m2 on days +3, +6, and +11. Neutrophil and platelet engraftment occurred on days +15 and +23, respectively.Patient #A14, a 56-year-old male subject, was diagnosed with acute myeloid leukemia (AML-M4) in October 2016. A decision was made for haploidentical HSCT with his son as the donor on 28 March 2017. He received grafts from 5/10 HLA-matched peripheral blood stem cells (PBSCs) and bone marrow (BM) stem cells. The conditioning regimen was modified BUCY . Because of insufficient evidence of GVHD, neither enteroscopy (sigmoidoscopy and colonoscopy) nor glucocorticoid therapy was performed for this patient. Virological tests showed that cytomegalovirus (CMV) DNA and Epstein–Barr virus (EBV) DNA were not present in the blood. Antiviral therapy was given acyclovir (0.4 Po Bid) to prevent herpes virus infection. Although the patient's temperature returned to normal and abdominal pain relieved after anti-infection treatment, the diarrhea did not alleviate. Stool screening showed no 1st) was performed on day 2 after cessation of diarrhea treatment  for another 7 consecutive days. Afterward, we similarly suspended FMT and continually observed the dynamic changes in the intestinal microbiota for one week. A standardized protocol for donor screening and FMT administration developed by infectious disease and BM transplant specialists was approved by the hospital quality and safety officer. This study was approved by the hospital ethics committee. Informed consent was obtained from both the parents of the stool donors and the FMT recipient. The study was conducted in accordance with the Declaration of Helsinki.The first stage of FMT . The primers were used as follows: 338F, 5′-ACTCCTACGGGAGGCAGCAG-3' and 806R, 5′-GGACTACHVGGGTWTCTAAT-3'. The PCR cycling conditions were as follows: 95°C for 5 min; 28 cycles of 45 s at 95°C, 50 s at 55°C, 45 s at 72°C, and a final extension at 72°C for 10 min. Purified amplicons were pooled in equimolar amounts and paired-end sequenced (2 × 300) on an Illumina MiSeq platform according to the standard protocols. The sequence data were compiled and filtered for quality. The unique sequence set was classified into operational taxonomic units (OTUs) under a threshold of 97% identity using UCLUST. Chimeric sequences were identified and removed using USEARCH (version 8.0.1623). The taxonomy of each 16S rRNA gene sequence was analyzed by UCLUST against the Silva 119 16S rRNA database using a confidence threshold of 90%. Taxonomic assignments of each OTU were made by similarity using the GLSEARCH program. Alpha diversity values were calculated using Faith's phylogenetic diversity [DNA was extracted from thawed stool samples using the E.Z.N.A. Stool DNA kit  according to the manufacturer's instructions. The quality of the DNA was evaluated by 2% agarose gel electrophoresis and spectrophotometry. The V3-V4 region of the 16S rRNA gene was amplified using FastPfu DNA Polymerase , and bar plots were created in R. The nonparametric Kruskal–Wallis test was performed using GraphPad Prism 4.0 software . 1st), in which stool samples were obtained from donor #1, although the frequency of liquid stools was increased at the beginning of FMT1st, the number of bowel movements began to decline significantly from the third sample collection point (3FMT1st) onward. Meanwhile, the patient stool mass was dramatically decreased from 1260 g (1FMT1st) to 470 g (3FMT1st). During the 7-day follow-up after cessation of the first FMT, the shape of the feces was obviously improved , we returned to stool samples from donor #1 to conduct the third FMT (FMT3rd). Similarly, during the 7-day FMT period, we found significant improvements in both the shape and frequency of the patient's stool . However, the dominant genera were different among these samples. In samples with low diversity (inverse Simpson index < 2), signs of diarrhea were found in 42.8% (3/7) of samples. Enterobacter was the dominant genus in 71.4% (5/7) of the samples.To assess the dynamic changes in the gut microbiome during FMT, we sequenced DNA from stool samples collected at the baseline (B point) and the indicated time points  with 16SStreptococcus, which accounted for 94.3% of the sampled OTUs, while Bifidobacterium and Enterococcus accounted for only 2% and 1.2%, respectively. For the donors, we observed that Bifidobacterium (29.8%) and Faecalibacterium (28.8%) were the dominant genera in donors #1 and #2, respectively. Other highly represented genera in both donors included Lactobacillus , Veillonella , Streptococcus , Haemophilus , and Escherichia-Shigella . Notably, similar compositions were observed in the samples from donor #1 in both FMT1st and FMT2nd procedures (Streptococcus (72.2%) and Bifidobacterium (18.6%). During the second FMT from donor #2, the microbiota mainly comprised Enterobacter (76.5%), Lactobacillus (7.8%), and Veillonella (7.0%). When the patient received the final FMT from the second donor #1 sample, Lactobacillus (23.8%), Escherichia-Shigella (19.9%), and Enterobacter (23.3%) were the core OTUs of the microbiome. Meanwhile, we identified 6 OTUs that were more sensitive to changes during FMT  among the 114 recognized OTUs  segregated from the other samples  from the gastrointestinal tract . A pilotnfection . This ste groups . Severale groups . Importae groups . This raC. difficile-associated diarrhea [C. difficile infection was not found in this HSCT patient, the diversity and fitness levels of the fecal microbiome were extremely low at baseline compared with those of a healthy person. Thus, we tried to use feces from healthy donors to rebuild the intestinal microecology of this severe diarrheal patient undergoing HSCT. As we assumed, the diversity and richness of the patient's microbiome were significantly improved after FMT, regardless of the donor that we used. In accordance with previous reports [Bifidobacteria, Streptococci, and Lactobacillus spp. Increasing evidence has now demonstrated that some microbial species  can restore the disrupted gut epithelium that causes the diarrhea commonly experienced by HSCT patients through upregulation or phosphorylation of tight junction proteins [The community of microorganisms within the human gut is critical to health and functions with a level of complexity comparable to that of an organ system . Therefodiarrhea . Althoug reports , 41, we proteins –44. DespFaecalibacterium as the dominant taxa in the stool of the exclusively breastfed donor #2  [Lactobacilli, three species of Bifidobacteria, and Saccharomyces boulardii) can prevent irinotecan-related diarrhea, and Lactobacillus GG can reduce the frequency of severe diarrhea caused by 5FU-based chemotherapy [Bifidobacterium accounted for 29.9% of the OTUs in the stool of donor #1 and 18.1% in the stool of donor #2  than that of donor #2 (0.6%). Furthermore, through longitudinal monitoring of the patient, we outlined 6 OTUs that were more sensitive to changes caused by FMT  among the 114 recognized OTUs  might have been one of the possible factors, reducing the incidence of diarrhea when we used stool from donor #1.Antibiotic-associated diarrhea can be reduced to 8% in patients with the administration of llus GG) . Animal otherapy . In our donor #2 ; meanwhiectively . Interes Figures . AccordiLactobacillus, Bifidobacteria, and Saccharomyces boulardii, might be the key factor in choosing the right FMT donor when clinicians treat noninfectious diarrhea. Because this was a small FMT substitution, we are expanding this work by building a larger cohort and using a machine learning method to predict the effects of FMT on diarrhea treatment in leukemia patients by analyzing the intestinal microbiota of FMT donors and recipients.Overall, we report the confirmation of the safety of FMT for treating an HSCT patient with diarrhea. FMT offers promise as a potential treatment option for noninfectious diarrhea after HSCT. Importantly, the therapeutic outcomes of diarrhea were somewhat related to the choice of FMT donors. Here, we found that 1-year-old infants can serve as safe FMT donors for treating diarrhea in HSCT patients. Furthermore, the overall diversity of the intestinal microbiota may not be the only important element in the selection of an effective FMT donor. The richness of probiotics, including


To determine diagnostic accuracy of Cerebro Spinal Fluid (CSF) Adenosine De-Aminase (ADA) in detecting Tuberculous Meningitis (TBM) keeping CSF Polymerase Chain Reaction (PCR) for Mycobacterium Deoxy Ribonucleic Acid (DNA) as gold standard.This cross sectional validation study was conducted at Department of General Medicine of PNS Shifa Naval Hospital Karachi, Pakistan from Oct 2015 to Mar 2017 for a total duration of one and a half year. One hundred and thirty six patients were included. The diagnosis of TBM was based clinically on symptoms like fever, headache, altered mental state and signs of meningeal irritation with CSF findings of increased proteins, low glucose and lymphocytic pleocytosis. Lumbar puncture was done and approximately 4ml of CSF sample was withdrawn for analysis. Diagnosis of TBM was confirmed by doing CSF PCR test for mycobacterium tuberculosis DNA.Total 136 patients were enrolled in this study. Mean age in our study was 47.09±12.80 years, whereas frequency and percentages of male and female patients was 102 (75%) and 34 (25%) respectively. The diagnostic accuracy, sensitivity, specificity, positive predictive value and negative predictive value of CSF ADA level in detecting TBM was 71.32%, 84.21%, 95.45%, 98.97% and 53.85% respectively.The study concludes that diagnostic accuracy of CSF ADA in detecting TBM is high which is proposed as an investigation to differentiate it from other causes of meningitis in places where PCR test is not available. Meningitis is a medical, neurological and sometimes neurosurgical emergency that requires a multi-disciplinary approach. Most common types are bacterial, viral and Tuberculous Meningitis (TBM). TBM is more prevalent in the developing countries, especially in socioecomically low status countries.3Jarvis et al in a study conducted in Cape Town, South Africa concluded that 28% cases of the total 4961 suspected cases were due to TBM.5The diagnosis of TBM cannot be made on the basis of clinical findings alone because almost all types of meningitis present with more or less the same findings. Diagnosis requires observation of tubercle bacilli in the Ziehl-Neelsen staining of the Cerebro Spinal Fluid (CSF) but it lacks sensitivity. Similarly CSF culture is also diagnostic but the results are often too late, takes 4-6weeks.Adenosine deaminase (ADA) is an enzyme which is involved in purine metabolism in which it is responsible for conversation of adenosine and deoxyadenosine to inosine and deoxyinosine respectively resulting in release of ammonia. In TBM tubercle bacilli results in cell mediated immune response which causes release of ADA by T cells but it is also released in other conditions which involve activation of cell mediated immunity.International studies have been performed to evaluate the diagnostic and predictive value of CSF ADA in TBM patients. Agarwal et al in a hospital based study showed that ADA estimation in CSF with a cut-off value of 10U/L is simple, cost effective and less time consuming method for making a diagnosis of TBM, especially when there is a difficulty in differentiating whether it is due to tuberculous etiology or non tuberculous etiology.This cross-sectional study was conducted at PNS Shifa Naval Hospital, Karachi from October 2015 to March 2017 over a period of one and a half year. After approval from the hospital’s ethical and research committee, informed written consent was obtained from the patient or his next of kin in case patient lacked decision making capacity, after explaining the purpose and benefits of study. All indoor patients meeting the inclusion criteria were included in the study aged between 12 to 85 years. The diagnosis of TBM was based on symptoms like fever, headache and altered mental state and signs like neck stiffness and positive kerning’s sign with CSF routine examination (R/E) findings of increased proteins, low glucose and lymphocytic pleocytosis. Diagnosis of TBM was confirmed by doing CSF PCR test for Mycobacterium TB DNA. Patients who were not willing for initial diagnostic tests were excluded.All patients were subjected to detailed history and examination. Infarct, hemorrhage and Space occupying lesion (SOL) brain was ruled out on CT scan brain. The fundus was examined to rule out papilloedema. Within one hour of admission, Lumber puncture was done and approximately 4ml of CSF sample was withdrawn under strict aseptic technique and was sent to specified laboratory of the hospital. CSF sample about 2ml was used for cell count, biochemistry and smears for gram staining and staining for AFB and 1ml for ADA levels estimation under supervision of pathologist. ADA activity in CSF was estimated according to the method of Galanti and Giusti colorimetric method and was expressed as U/L. Remaining sample of CSF was used for PCR for MTB DNA for confirmation. PCR machine used was smart cycler which determines real time pcr which involves DNA extraction and amplification. PCR was used as gold standard because culture results takes 2-3 weeks to come and determination of AFB in CSF lacks sensitivity.Frequencies and percentages were calculated for qualitative variables like gender, true positive and true negative. For quantitative variables like age, mean and standard deviation was used. Effect modifiers like age and gender were controlled by stratification. All results were presented as tables and graphs. Post stratification chi-square test was applied. p value ≤ 0.05 was considered statistically significant.A total 136 patients were enrolled in the study. Mean age of study population was 47.09+12.80 years with ranges from 20-85 years. Out of 136, 102 (75%) were males, and 34 (25%) were females with a M:F ratio of 3:1 but patients were randomly selected who visited hospital during the study time frame. A 2x2 table was constructed for comparison, given in TBM is a common disease in socioeconomically low status countries. In India every year about 0.5 million patients die because of tuberculosis, 8.3% of which is occurring in children only. Emergence of resistance to multiple drugs in tuberculosis and occurrence of Acquired Immuno-Deficiency Syndrome (AIDS) along with TB has further worsen the disease outcome. Among extrapulmonary TB, TBM is the most serious form with incidence of 7 to 12% of all cases of TB in developing countries. If there is any delay in diagnosing of the disease or starting of effective treatment than it can result in permanent neurological damage in up to 25% of cases resulting in poor prognosis. The methods available for the diagnosis of TBM were checked but all were found to be of low sensitivity and specificity.1Many methods are available for the diagnosis of TB. Light microscopy is used for the detection of Acid Fast Bacilli (AFB) in a smear throughout the world and the rate of detection is 30-40%.ADA is basically an enzyme with very high levels in T-lymphocytes where its role is in the differentiation of lymphoid cells. During lymphocytes antigenic responses ADA levels increases. Defects in cell mediated and humoral immunity is associated with low levels of ADA making the patient prone to many opportunistic infections.11Rana et alSaini et al.CSF ADA average cost is Rs. One thousand and CSF PCR cost is around Rs. Five thousand. CSF ADA test is readily available while PCR test is not available everywhere.The study concludes that diagnostic accuracy of CSF ADA in detecting TBM is high (71.32%) which proposed it as an investigation to differentiate it from other causes of meningitis in places where PCR test is not available. As in Pakistan, PCR for Mycobacterium Tuberculosis DNA in CSF is not available everywhere and therefore CSF ADA can be readily available parameter in this regard. This test is cheap and results can be available early as compared to CSF PCR test so it can be used for detecting TBM.AH conceived, designed the study, did data collection.ZAA did editing and finally approved manuscript.SHR did statistical analysis, manuscript writing.SA did editing, manuscript writing.


Corynebacterium striatum isolated from blood cultures. A total of 64 isolates from 51 patients identified as C. striatum by matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and 16S rRNA gene sequencing were assessed. More than two blood cultures were positive in 25 (48.1%) patients. Diabetes, solid tumor, and a history of previous exposure to antibiotics were more common in patients with multiple positive blood cultures. Charlson comorbidity scores were also higher, and more isolates were recovered after 48 hours of hospital stay in patients with multiple positive blood cultures. Strains recovered from patients with multiple positive blood cultures produced significantly more biofilm. Based on multilocus sequence typing (MLST), sequence type (ST) 20 (31.3%) was the most dominant, followed by ST2 (20.3%) and ST23 (10.9%). There was no relationship between the number of positive blood culture sets and sequence typing. In multivariate analyses, Carlson comorbidity score  and biofilm formation were associated with multiple positive blood cultures . This study provides evidence that the biofilm phenotype could contribute to determining the clinical significance of C. striatum in patients with severe underlying conditions. The predominance of certain STs suggests the relatedness of C. striatum infection and the nosocomial environment.This study aimed to identify clinical or microbiological factors affecting the clinical relevance of  Corynebacterium striatum is one of the most frequently isolated species of coryneform bacteria in clinical microbiology laboratories . Bi. BiC. stAntibiotic susceptibility testing was performed using E-test  strips on Mueller-Hinton agar plates supplemented with 5% sheep blood  . The breC. striatum bloodstream isolates. Patients were divided into two groups according to the number of positive blood cultures . The source of bacteremia, the appropriateness of antibiotic therapies, and the treatment outcomes were also evaluated in patients with multiple positive blood cultures. The Institutional Review Boards of the two participating hospitals  approved this study. A waiver of consent was granted given the retrospective nature of the project.Medical records were reviewed for all patients with 8 colony forming unit(CFU)/mL bacterial suspension in brain-heart infusion (BHI) broth were added to each well of the 96-well plate and incubated at 37°C for 48 h. Next, each well was washed twice with 200 μL phosphate-buffered saline . Biofilms were fixed with 200 μL of 99% methanol and stained with 2% crystal violet. The dye was eluted with 160 μL of 95% ethanol and the optical density (OD) at 595 nm (OD595) was measured using an Epoch Microplate Spectrophotometer . The negative controls contained only BHI. Subtractions of the mean OD595 of triplicates from the negative control were used for analysis.Biofilm formation in non-duplicate isolates was evaluated on the negatively charged polystyrene surfaces of flat-bottomed 96-well microtiter plates . A totalgyrA, and partial rpoB genes were also amplified and sequenced for all C. striatum bloodstream isolates [http://www.ebi.ac.uk/ena, HE586270 to HE586300). For each gene, previously reported allele numbers were used when exact matches were found in the European Molecular Biology Laboratory (EMBL) databases; otherwise, new numbers were arbitrarily assigned [C. striatum isolates and HE586270 for 16S rRNA, HE586276 for ITS1, HE586288 for gyrA and HE586295 for partial rpoB as references, respectively.In addition to the 16S rRNA gene, the internal transcribed spacer 1 (ITS1), isolates . Primersisolates . Obtaineassigned . The num2 tests were used to compare categorical variables. Continuous variables were analyzed using the Mann-Whitney U tests or paired t-tests. One-way ANOVA or the Kruskal-Wallis test were used to compare the means of three or more continuous variables. Multivariate analysis to evaluate the factors associated with multiple positive blood cultures was performed in the enter mode with selected variables because of the limited sample size [P-values < 0.05 were considered statistically significant. SPSS v.18.0  and GraphPad Prism v.5.03  were used for data analyses.Fisher’s exact tests or Pearson χple size . All tesC. striatum and were subjected to 16S rRNA sequencing; 64 (recovered from 51 patients) had ≥ 99% similarity to one of the following C. striatum strains: AY008302, JF342700, JX974430, or KJ934789. A discrepancy between MALDI-TOF MS and 16S rRNA sequencing was found in a single strain showing 99% similarity to C. simulans (AF537604), which is closely related to C. striatum based on a previous phylogenetic analysis study [Of the 300 gram-positive bacilli isolated from blood samples, 138 (46.0%) were identified to the species-level by MALDI-TOF MS. Of these, 65 (21.7%) were identified as is study .C. striatum isolates were obtained from 51 patients. More than two blood cultures were positive in 25 (48.1%) patients, while the other 26 (51.9%) patients had one positive blood culture. The prevalence of diabetes  and solid tumors  was higher among patients with multiple positive blood tests compared to patients with a single positive blood culture. The Charlson comorbidity scores  were also higher in patients with multiple positive blood cultures. Isolates recovered after 48 h of hospital stay  or previous exposure to antibiotics  were more common among patients with multiple positive blood cultures than in those with a single positive blood culture , cefotaxime (MIC90 > 32), clindamycin (MIC90 > 256), erythromycin (MIC90 > 256), and levofloxacin (MIC90 > 32). There was no difference in antibiotic susceptibility between groups with multiple and single positive cultures .P = 0.03) and biofilm formation  were independently associated with multiple positive blood cultures , and biofilm production–were included in the analysis. Charlson comorbidity score , consistent with other studies [C. striatum, this species warrants greater attention among gram-positive bacilli [C. striatum blood cultures. Therefore, this study was performed to identify a predictive factor for clinical relevance using 64 C. striatum bloodstream isolates recovered from 51 patients between 2006 and 2016. To the best of our knowledge, this is the first study to investigate the microbiological and clinical characteristics, as well as the genetic relatedness, of bloodstream isolates in a longitudinal manner.Since MALDI-TOF MS was introduced to our clinical lab in October 2014, detailed identification of gram-positive bacilli has been performed. Over two years, 87,241 blood cultures were submitted and 713 gram-positive bacilli were recovered. Among these strains, C. striatum infections were related to underlying medical conditions including exposure to broad spectrum antibiotics, immunocompromised hosts, invasive medical procedures, and implanted medical devices [C. striatum as related to invasive infection.The number of positive blood cultures is one proven methodology that can help differentiate true infection from contamination . Thus, w devices –7. HowevC. striatum blood isolates, we tested antibiotic susceptibility, genotyping using MLST, and biofilm production ability. To the best of our knowledge, no single marker robust enough to be of clinical value has been identified. Staphylococcus epidermidis, which is similar to C. striatum, is a common inhabitant of the skin and mucous membranes and also causes opportunistic infection related to medical devices. S. epidermidis isolates causing catheter related bloodstream infections have been shown to be multidrug resistant, icaA positive (which involves biofilm formation), and belong to specific PFGE or MLST types, compared to those from colonized healthcare workers [gyrA, and rpoB genes possess adequate variability for discriminating between the MLST types of C. striatum strains [et al. also assessed the 16S rRNA gene and observed variability via phylogenetic analyses [C. striatum isolates were recovered after 48 hours of hospital stay in the current study. As there is no standard MLST of C. striatum and only a limited number of studies have addressed this issue, it remains somewhat unclear which strains are commonly circulating in Korea or other areas. However, as ST2 was also the most frequently observed ST in previous studies, it is possible that ST2 is the most common strain circulating in hospital settings [C. striatum isolates in the single positive blood culture group originated from healthcare workers or patients. In the present study, C. striatum biofilm production was higher in strains isolated from the multiple positive blood culture group compared to the non-blood culture controls or isolates from patients with single positive blood cultures. Even within the same ST, a higher level of biofilm production was observed in strains from multiple positive culture patients compared to the single positive culture group. The difference in biofilm formation was not associated with ST, consistent with a previous study by Qin et al. showing no relationship between PFGE type and level of biofilm formation [C. striatum strains may have evolved the ability to form robust biofilms under specific circumstances. This hypothesis might be supported by previous studies demonstrating that the increased level of biofilm formation was associated with the infection status and not strain clonality in Staphylococcus aureus and Enterococcal disease [et al. showed that biofilm production in C. striatum was related to PFGE type [In the present study, in order to investigate whether distinct microbiological characteristics are related to the clinical relevance of  workers . A previ strains . Howeveranalyses . Based osettings , 30. Fursettings . These formation . It is p disease , 37. In FGE type . ConsideC. striatum strains are multidrug resistant. As earlier studies have described broad sensitivity to various antibiotics [C. striatum strains were isolated. An emerging resistance to daptomycin has been reported in several studies [et al. reported that the multidrug resistance phenotype of C. striatum is associated with specific PFGE types [C. striatum isolates were multidrug resistant, which is consistent with our finding [Most isolates tested in this study were resistant to penicillin, cefotaxime, clindamycin, erythromycin, and levofloxacin. This is in agreement with recent studies that have shown that clinical ibiotics , 38, whiibiotics , it is a studies , 39, 40;GE types . However finding , 22.C. striatum blood isolates were collected during the study period. Thus, there could have been selection bias, despite random sample collection. Second, as patients were divided based on the number of positive blood cultures, there is a possibility that contamination could have caused some patients to have multiple positive tests. However, we regarded this classification as relatively objective based on previous findings demonstrating the importance of the number of positive blood cultures for interpreting the clinical significance of C. striatum [C. striatum isolates. Thus, our results may help physicians understand the clinical relevance of C. striatum and aid in managing infections more appropriately.This study had several limitations. First, it was a retrospective study and not all striatum , 9, 10. C. striatum bacteremia. C. striatum isolates recovered from patients with multiple positive blood cultures had a significantly higher level of biofilm production, suggesting that biofilms are an important virulence factor. The predominance of specific STs during specific timeframes suggests the relatedness of C. striatum infections and the nosocomial environment. Clinicians should be aware of the possibility of invasive C. striatum infections in patients with severe underlying diseases and biofilm related infections. Timely, appropriate treatments and the implementation of strict infection control measures to prevent the spread of C. striatum are warranted.Patients with severe underlying diseases were more likely to develop clinically significant S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.


Transdermal fentanyl is widely used to control pain in cancer patients. The high pharmacokinetic variability of fentanyl is assumed to be due to cytochrome P450 3A-mediated (CYP3A) N-dealkylation to norfentanyl in humans. However, recently published clinical studies question the importance of the described metabolic pathway. In this small study in palliative cancer patients under real-life clinical conditions, the influence of CYP3A on fentanyl variability was investigated. In addition to the determination of midazolam plasma concentration to reveal CYP3A activity, plasma concentrations of fentanyl and its metabolite, norfentanyl, were measured in identical blood samples of 20 patients who participated in an ongoing trial and had been on transdermal fentanyl. Fentanyl, norfentanyl, midazolam, and 1′-OH-midazolam were quantified by liquid chromatography/tandem mass spectrometry. Plasma concentrations of fentanyl and norfentanyl exhibited a large variability. Mean estimated total clearance of fentanyl and mean metabolic clearance of midazolam (as a marker of CYP3A activity) were 75.5 and 36.3 L/h. Both clearances showed a weak correlation and hence a minimal influence of CYP3A on fentanyl elimination. Fentanyl is a synthetic full agonist at µ-receptors with high lipophilicity, submitting a sudden transfer of the agent to central nervous structures2. Cytochrome P450 3A (CYP3A) is the most relevant drug metabolizing enzyme in humans and responsible for metabolism of about half of all available pharmaceuticals. Fentanyl is assumed to be almost exclusively metabolized in the liver by CYP3A-mediated piperidine N-dealkylation to norfentanyl in humans4. The known interindividual high variability of CYP3A activity would result in a high variability of fentanyl exposure and in consequence also the efficacy and the side effects. Moreover, drug interactions are commonly caused by pharmaceuticals acting as an inducer or inhibitor of the CYP enzymes5. As drug interactions are often responsible for the escalation of debilitation symptoms, and therefore responsible for the negative influence of the palliative cancer patients’ quality of life, it is of major significance to be aware of a drug’s CYP-dependent metabolism.Transdermal fentanyl is widely used as a basic therapy in the treatment of pain, which is one of the most common and distressing symptoms of cancer patients near the end of life7. The determination of midazolam plasma concentrations as well as plasma concentrations of fentanyl and its metabolite norfentanyl in identical blood samples offers an exceptional opportunity of investigating the interindividual variability.As part of a clinical trial investigating the CYP3A activity in palliative cancer patients, this analysis is dealing with the metabolism of fentanyl in a special population under real-life clinical conditions in order to increase knowledge about the influence of CYP3A. The activity can be assessed in a subject by measuring a few plasma concentrations after oral intake of a microdose of midazolam, acting as a marker substance, in combination with pharmacokinetic calculations8.The study was approved by the responsible ethics committee of the Medical Faculty at Heidelberg University . Written informed consent was obtained from each patient prior to any study-related activity. This clinical trial was conducted according to the World Medical Association Declaration of Helsinki. It started in April 2016 at the palliative care unit of Heidelberg University Hospital and is still ongoing. The detailed study protocol of the trial was published beforeIncluded patients were selected based on the administration of transdermal fentanyl to be evaluated in this study. Transdermal fentanyl was prescribed by the treating physicians due to a regular drug therapy and not modified by the investigators. Measuring plasma concentrations of substances other than midazolam was predefined in the study protocol and approved by the ethics committee. In addition to the determination of midazolam plasma concentration to reveal a four-hour pharmacokinetic profile after oral intake of a 10 µg microdose as part of the procedure of the main trial, plasma concentrations of fentanyl and its metabolite norfentanyl were measured in identical blood samples.Fentanyl and norfentanyl have been measured by liquid chromatography/tandem mass spectrometry (LC-MS/MS) with electrospray source in positive ionization mode following protein precipitation. Briefly, 100 µl of serum was mixed with fentanyl-d5  as the internal standard, and 1000 µl cold acetonitrile was added. Precipitated proteins were removed by centrifugation . The supernatant was reduced to dryness  and reconstituted in 150 µl of the mobile phase. One µl was injected into the QTRAP 6500 LC-MS/MS-system . Fentanyl and norfentanyl were separated on a Zorbax Eclipse C8 column  by gradient elution . Multiple reaction monitoring was used for specific detection of fentanyl m/z 337→188*, 337→105, norfentanyl m/z 233→84*, 233→55, and fentanyl-d5 m/z 342→105. Transitions marked with an asterisk were used for quantification. Calibration was performed by addition of standard solutions to drug-free matrix  prior to extraction; a blank and a zero sample as well as five quality control samples covering the calibration range were included in each run . Bench top stability was assured for both analytes up to 3 days. Linearity of calibration lines was assessed by regression analysis; correlations coefficients were 0.9994 and 0.9989 for fentanyl and norfentanyl, respectively. According to DIN 32645, the lower limit of detection and of quantitation were 0.02 and 0.05 ng/ml. The relative recovery from serum was on average 89% and 96% for fentanyl and norfentanyl, respectively (n = 6). Matrix effects were <25%. Percent mean within-day imprecision and accuracy of <4% and >93% for fentanyl and of <2% and >95% for norfentanyl were obtained .−1 (midazolam) and 0.255 pg mL−1 (1′-OH-midazolam)9.Plasma concentrations of midazolam and 1′-OH-midazolam were quantified using high-performance liquid chromatography with tandem mass spectrometric (LC/MS/MS) detection methods as previously described with lower limits of quantification of 0.093 pg mL2-4) using Prism® 7.0 . Partial metabolic clearance of midazolam (CLmet) was derived using the established equation CLmet = 5668/(AUC2-4/mg midazolam) as a reliable method to assess CYP3A activity11. In the same 4 samples, fentanyl and norfentanyl concentrations were determined. The samples for AUC2-4 of fentanyl were obtained at any times during this 72-hour dosing interval of the fentanyl patch. Assumptions were made for a constant delivery rate of fentanyl from the patch and also a constant resorption from the subcutaneous depot since the patients were hospitalized and in stable conditions. Therefore, total clearance (CL) of fentanyl was estimated using 2.78% of the dose of the fentanyl patch delivered for the whole 72-h dosing period divided by AUC2-4.Area under the plasma concentration-time curve (AUC) of midazolam was calculated between 2 and 4 hours after midazolam dosing . Data are presented as mean ± SD.Of the 41 patients included in the trial 20 patients were treated with transdermal fentanyl under real-life clinical conditions and thus assessed in this analysis. All of these patients suffered from cancer with impaired liver function based on the laboratory test results. According to the us presented previous findings and accessible patient’s charts none of the patients did receive cancer treatments due to their reduced general health condition at least 2 weeks before the day of the study. No special diet or nutritional supplements were applied. Patient characteristics including laboratory test results are described in Table A dose increase due to the indication of the treating physicians was done in 2 patients at the last patch application before the pharmacokinetic assessment day. In 3 patients, administration of transdermal fentanyl was started at least 2 days earlier, and 18 patients were on constant fentanyl dosing for at least 1 dosing interval. As the evaluated subjects had been on individual patch doses ranging from 12.5 to 175 µg/h, all measured plasma concentrations were normalized to a dose of 25 µg/h transdermal fentanyl.Assuming a constant sustained fentanyl transdermal delivery out of the patches under clinical conditions, plasma levels are anticipated to stay nearly constant. This is of importance as the blood samples were individually taken in relation to midazolam administration which translates to the following time points in relation to the fentanyl patch application: In 10, 5, 5 of the investigated patients the series of 4 blood samples for fentanyl analysis was taken at 2.8 (±1.56), 26.3 (±1.35), 50.3 (±1.35) hours after application of the patch, respectively.Mean dose normalized plasma concentrations of fentanyl and norfentanyl of all evaluated blood samples, in relation to the 3 mentioned mean sampling time points from fentanyl administration (period between application of the patch and blood sampling), are presented in Figs Plasma concentrations of fentanyl, norfentanyl and midazolam in relation to midazolam administration are shown in Fig. Pharmacokinetic data of fentanyl as well as midazolam are presented in Table met. Nonlinear regression analysis of the log transformed fentanyl CL and midazolam CLmet . A slope of 0.08 reveals that about 8% of the total fentanyl clearance (fentanyl metabolism) is related to the midazolam metabolic clearance (marker of CYP3A activity).If metabolism of fentanyl to norfentanyl was mainly due to CYP3A, the total clearance of fentanyl should correlate with the metabolic clearance of midazolam to 1′-OH-midazolam, as the latter is known to be exclusively mediated by CYP3A. Hence, data of this evaluation were used to establish the correlation of fentanyl CL and midazolam CLmet Fig.  resultedet al. who identified substantial interindividual variabilities of fentanyl serum concentrations as well as of the corresponding metabolic ratio among 620 cancer patients on transdermal fentanyl. CYP3A gene polymorphisms can influence transdermal fentanyl metabolism to norfentanyl, but only a small proportion of the variability of serum fentanyl and norfentanyl concentrations can be explained by these polymorphisms. An unexplained large interindividual variability of fentanyl serum concentrations with potential clinical consequences and metabolism of fentanyl to norfentanyl was observed and is still awaiting further research to determine the underlying mechanisms12.This study investigated the variability of exposure of transdermal fentanyl in palliative cancer patients under real-life clinical conditions. Evaluated patients exhibited a large variability in fentanyl plasma concentrations, which is of importance in regards to effectiveness and safety of the transdermal formulation. These findings are in line with a work by Barratt 13. This hypothesis is powered by findings of other groups, that revealed inconsistencies in respect to the metabolism of fentanyl. Concomitant administration of transdermal fentanyl and aprepitant, another CYP3A inhibitor that is extensively metabolized by the enzyme, showed no significant influence on the AUC of fentanyl as it was expected before14. However, norfentanyl was not quantified in that study. Furthermore, the total clearance of intravenous fentanyl was inhibited just weakly though a strong inhibition of the metabolic clearance of fentanyl to norfentanyl was observed to be affected by the antimycotic and strong CYP3A inhibitor ketoconazole in healthy volunteers15. In addition, we re-analysed data from that study in which fentanyl was given intravenously alone and during CYP3A inhibition by ketoconazole, and midazolam was co-administered orally as CYP3A probe drug. This new correlation analysis of the clearances of fentanyl and midazolam under baseline and inhibition conditions results in a weak relationship  by voriconazole and fluconazole (both CYP3A inhibitors) in healthy volunteers. However, AUC ratios between norfentanyl and fentanyl were reduced more than the corresponding clearances, which led to the suggestion that other pathways may be involved in the metabolism of fentanylAlthough the use of a limited sampling strategy to estimate midazolam metabolic clearance has been validated, this method has not been proven for fentanyl, which could be a limitation of our study. Furthermore, a constant fentanyl delivery from the patch during the 72-hour dosing interval into the subcutaneous depot was assumed, however, a constant resorption from the depot into the systemic circulation cannot be assured. Regarding the real-life clinical setting of the trial, an intravenous fentanyl application with secured constant systemic drug application was not realizable in the investigated palliative care patients. However, the correlation of the CYP3A dependent activities of the two substances in identical blood samples illustrates a valuable approach to understanding the variability of transdermal fentanyl effect under clinical conditions.15. According to our findings, this pathway is only responsible for about 8% of the total metabolism of the drug, and hence is not the major factor for the high variability of fentanyl pharmacokinetics. Based on this current observation, no major drug interactions are to be expected even when strong CYP3A inhibitors are concomitantly used.The present study did not confirm the widely accepted assumption that the main route of fentanyl elimination is via CYP3A-mediated N-dealkylation to norfentanyl. The results obtained in this special population, under real-life clinical conditions, supports the recent data obtained in healthy volunteers


Kidney transplantation is the treatment of choice in end-stage renal disease due to Alport syndrome (AS). However, the chances of finding an adequate living-related donor in AS are much worse compared to non-heritable conditions. Successful cases of related living-donor transplantation mostly refer to X-linked AS but are rarely reported in genetically confirmed autosomal AS.COL4A3. While decision-making was based on extensive clinical donor evaluation prior to transplantation, we analyzed the underlying genetic background in retrospect and associated these findings with the phenotype in all available family members. While biallelic COL4A3 variants caused autosomal recessive AS (ARAS) in the son (recipient), heterozygous family members, including the father (donor), showed minimal renal involvement and high-frequency sensorineural hearing impairment later in life indicating mild autosomal dominant Alport syndrome (ADAS). The recipient’s successful participation in the European and World Transplant Games is a testament to the positive outcome of transplantation.We describe the outcome of an exceptional AB0-incompatible kidney donation from father to son in a family with altered In summary, living-related donor transplantation may be successful in autosomal AS, provided that thorough clinical and genetic evaluation of potential donors is performed. However, unrelated kidney transplantation should be given priority upon unpredictable genetic risk. Individual genetic variant interpretation is an important component of personalized donor assessment and will help to better predict genetic risk in the future. COL4A3–5) [Alport syndrome (AS) is a hereditary condition with considerable genotypic and phenotypic variability, caused by variants in genes encoding collagen type IV chains 3, 4, and 5 (OL4A3–5) . Renal mOL4A3–5) . Kidney OL4A3–5) –4. HowevCOL4A5) were traditionally thought to account for the vast majority of AS (XLAS), autosomal inheritance, by means of biallelic (ARAS) or monoallelic (ADAS) COL4A3 or COL4A4 alterations, was recently found to be much more frequent than previously estimated. This shift is mainly due to advent of next-generation sequencing (NGS) techniques in routine diagnostics, resulting in an increased identification of autosomal AS. In XLAS, risk of ESRD ranges from 25% in heterozygous females to 100% in hemizygous males, while in ARAS, ESRD risk is thought to be 100% [While X-linked forms  and 50 ml/min/1.73 m2 (II1) 10 days after LRKTx , with ESRD at age 24 was evaluated for LRKTx. The patient presented with microhematuria and proteinuria since age three. For progressive proteinuria, a renal biopsy was performed 10 years later demonstrating irregular thinning, thickening, and GBM-lamellation upon electron microscopy, allowing for histological diagnosis of AS  participating in the World Transplant Games (WTG) and the European Transplant and Dialysis Sports Championship (ETDSC). Taken together, he won two gold medals, two silver medals, and a bronze medal in five different disciplines Fig. .2 with modest microalbuminuria, the recipient (II1) exhibits a stable baseline eGFR of 48 ml/min/1.73 m2, however with persistent proteinuria of 0.5–1 g/d, due to biopsy-proven chronic transplant glomerulopathy.Seven years after LRKTx, both donor and recipient show moderate renal function at CKD-stage 3a Fig. . While tCOL4A3, COL4A4, and COL4A5 in the index patient and his family and identified compound-heterozygous variants in COL4A3 (NM_000091.4) at c.1909G > A (p.Gly637Arg) and c.4421 T > C (p.Leu1474Pro) in the index patient. Segregation analysis revealed paternal transmission of c.1909G > A and maternal inheritance of c.4421 T > C  [likely pathogenic. On the contrary, the maternal variant c.4421 T > C (p.Leu1474Pro) has been previously reported in AS with compound-heterozygosity [variant of uncertain significance (VUS). Unlike the index brother, both parents showed sensorineural hearing impairment for high frequencies at the age of 62 and 65, respectively. None of the family members had any findings of ocular pathology. To further evaluate for mild renal GBM-irregularities, we retroactively reevaluated pre-donation ultrastructural findings and performed immune histochemical staining for COL4A5 in the donor biopsy specimen from the father. While upon reevaluation, electron microscopy showed mildest GBM-thinning, COL4A5-staining revealed a so-called mosaic pattern with partial COL4A5 deficits in the father’s kidney supporting the histopathological diagnosis of non-progressive TBMN compatible with light ADAS   classifizygosity . Due to Written informed consent was obtained from the index patient and all tested family members.COL4A variants, further impacting glomerular filtration barrier integrity. This applies in particularly to kidney donor evaluation within a family with autosomal AS. Recently, the term autosomal dominant AS has been heavily debated and the current consensus guidelines suggest restricting the term to exceptional cases [COL4A3 variants and the severe clinical features we assume the index patient (recipient) to have ARAS, while his father (donor) harboring a monoallelic COL4A3 variant shows minor clinical signs compatible with light ADAS.With introduction of genetic diagnostics, the complexity of genetic variant interpretation in AS is increasingly recognized as a major challenge to counseling affected patients and their families. The main challenge is to assess a patient’s outcome from variable penetrance, potential digenicity, and possible disease modification by other non-al cases , 13. As COL4A3 encodes the type IV collagen alpha 3 chain. As a triple helix together with two other alpha chains it forms collagen type IV. The primary structure is built by the central triple helix collagenous domain, adjacent to a 7S-domain at the amino-terminus and a non-collagenous (NC1) domain at the carboxy-terminus  is located in the NC1 domain  mostly due to its location in the important collagenous-domain and its absence from the general population  was classified as , and II2In summary, LRKTx remains an exceptional procedure in Alport syndrome but adequate outcomes can be achieved by thorough clinical and genetic donor evaluation. However, seeking alternatives such as living-unrelated kidney donor transplant and/or deceased donor transplant should be given priority in cases of unpredictable genetic risks within the family. In the future, validated AS-prediction scores, taking genic and allelic information into account, will be needed to optimize family counselling, in particular on the question of LRKTx. Genetic variant interpretation is a fundamental part of individual risk assessment and helps to predict renal survival in both donors and recipients.


Widespread implementation of Alzheimer’s disease biomarkers in routine clinical practice requires the establishment of standard operating procedures for pre-analytical handling of cerebrospinal fluid (CSF).Here, CSF collection and storage protocols were optimized for measurements of β-amyloid (Aβ). We investigated the effects of (1) storage temperature, (2) storage time, (3) centrifugation, (4) sample mixing, (5) blood contamination, and (6) collection gradient on CSF levels of Aβ. For each study participant, we used fresh CSF directly collected into a protein low binding (LoB) tube that was analyzed within hours after lumbar puncture (LP) as standard of truth. Aβ42 and Aβ40 were measured in de-identified CSF samples using EUROIMMUN and Mesoscale discovery assays.CSF Aβ42 and Aβ40 were stable for at least 72 h at room temperature (RT), 1 week at 4 °C, and 2 weeks at − 20 °C and − 80 °C. Centrifugation of non-blood-contaminated CSF or mixing of samples before the analysis did not affect Aβ levels. Addition of 0.1–10% blood to CSF that was stored at RT without centrifugation led to a dose- and time-dependent decrease in Aβ42 and Aβ40, while Aβ42/Aβ40 did not change. The effects of blood contamination were mitigated by centrifugation and/or storage at 4 °C or − 20 °C. Aβ levels did not differ between the first to fourth 5-ml portions of CSF.CSF can be stored for up to 72 h at RT, 1 week at 4 °C, or at least 2 weeks at either − 20 °C or − 80 °C before Aβ measurements. Centrifugation of fresh non-blood-contaminated CSF after LP, or mixing before analysis, is not required. In case of visible blood contamination, centrifugation and storage at 4 °C or − 20 °C is recommended. After discarding the first 2 ml, any portion of up to 20 ml of CSF is suitable for Aβ analysis. These findings will be important for the development of a clinical routine protocol for pre-analytical handling of CSF.The online version of this article (10.1186/s13195-019-0517-9) contains supplementary material, which is available to authorized users. Cerebrospinal fluid (CSF) Alzheimer’s disease (AD) biomarkers, β-amyloid 42 (Aβ42) and tau, have been incorporated into the clinical research criteria for AD diagnosis proposed by the US National Institute on Aging–Alzheimer’s Association (NIA-AA)  and InteThe aim of the present study was to study the effects of different pre-analytical handling procedures using fresh CSF analyzed within hours after LP as the standard of truth, paving the way for a unified pre-analytical protocol for clinical routine use. We chose to focus on CSF Aβ measurements, because earlier findings suggest that amyloid peptides, Aβ42 and Aβ40, are particularly sensitive to pre-analytical handling whereas tau is less affected –15. We iDe-identified CSF samples from a total of 59 patients were used in this study. Patients were undergoing LP as a part of the Swedish BioFINDER Study or due to clinical suspicion of normal pressure hydrocephalus at the Memory Clinic, Skåne University Hospital, Sweden. All patients gave their written informed consent allowing their CSF samples to be used for research. Previous studies have shown that compared to polypropylene, LoB material significantly reduces Aβ42 loss due to adsorption to plastic , 16. TheCSF was collected from 12 patients . From all 12 participants, all 10 CSF samples were analyzed within hours (h) after LP . From all 10 participants, all 4 CSF samples were analyzed at baseline . From all 6 participants, all 4 CSF samples were analyzed at baseline . The other 6 tubes  were not centrifuged. From all 4 participants, all 12 CSF tubes were analyzed on the day of collection. After analysis, CSF and CSF-blood samples (centrifuged and non-centrifuged) were stored at either RT or 4°; one tube of neat CSF, CSF-0.1%-blood, and CSF-10%-blood were used per each four conditions: (i) no centrifugation and storage at RT, (ii) no centrifugation and storage at 4 °C, (iii) centrifugation and storage at RT, and (iv) centrifugation and storage at 4 °C . The other 4 tubes  were not centrifuged. From all 4 participants, all 8 CSF tubes were analyzed on the day of collection. After analysis, CSF and CSF-blood samples (centrifuged and non-centrifuged) were stored at RT , and 1 ml was aliquoted into 2-ml LoB tubes  followed by storage at − 80 °C. CSF Aβ42 and Aβ40 were measured in P1–P4 portions using EI and MSD kits.CSF was collected from 10 patients. For each patient, four 5-ml portions (P1-P4) of CSF were collected into 5-ml LoB tubes  after discarding the first 2 ml. CSF samples were centrifuged after LP  and according to the manufacturer’s recommendations. All samples within the same pre-analytical protocol were analyzed using the same batch of the kits. The time delay between LP and the start of the baseline sample analysis was 2–4 h. In MSD runs, samples were analyzed in singlicate because MSD V-plex kit has consistently shown low intra-plate coefficient of variance  in our previous analyses. For EI kit, all samples were analyzed in duplicate. EI and MSD assays were run in parallel. Each plate included 2 quality control (QC) samples  that were analyzed in duplicate.R2 values were computed using the method described by Nakagawa and Schielzeth [MSD , and 11% for Aβ40MSD  assays. Aβ concentrations in the same QC samples analyzed on different plates varied on average by approximately 5% and 10% for EI and MSD assays, respectively. Consequently, for changes in the biomarker levels within these ranges, it was not possible to determine whether they were caused by inter-plate variability or pre-analytical factors. Therefore, only changes exceeding 5% for EI assays and 10% for MSD assays were considered to be due to pre-analytical sample handling and tested in statistical analysis.SPSS version 22  and R version 3.4.3 (RStudio)  were usehielzeth . To furtEI, Aβ40EI, Aβ42/Aβ40EI, Aβ42MSD, Aβ40MSD, and Aβ42/Aβ40MSD were 747 pg/ml (353–1892 pg/ml), 8343 pg/ml (3541–12933 pg/ml), 0.13 (0.05–0.23), 511 pg/ml (198–1554 pg/ml), 6249 pg/ml (2813–12094 pg/ml), and 0.11 (0.04–0.18), respectively  values for Aβ42g/ml 353–892 pg/mlEI , there was a significant time × temperature interaction , and therefore, we performed simple main affects analysis for changes in the biomarker levels that exceeded 5%. In samples kept at 4 °C, Aβ42EI levels were within 95–105% range of baseline at all time points  and 10% after 2 weeks (p < 0.001). Aβ40EI levels were within 95–100% range of baseline at both temperatures and all time points  but no significant time × temperature interaction . Aβ42/Aβ40EI decreased by 8% and 7% when samples were stored for 2 weeks at RT and 4 °C, respectively  and after storage at either RT or 4 °C for 24 h, 48 h, 72 h, 1 week, and 2 weeks  compared to mixed sample (Aβ42 CV = 2.7%).We also assessed the effects of centrifugation after LP and/or blood contamination on Aβ levels in CSF stored at different temperatures for 24 h, 72 h, 1 week, and 2 weeks after collection.First, we investigated the effect of centrifugation on Aβ levels in neat (without added blood) CSF. We did not observe differences in the biomarker levels between neat centrifuged and non-centrifuged CSF samples stored for up to 2 weeks at RT, 4 °C, or − 20 °C  and Aβ40 , there were significant time × treatment group interactions , and therefore, we performed simple main affects analysis for changes in the biomarker levels exceeding 5%. Again, we found that blood contamination led to a dose- and time-dependent decrease (by 14–98%) in Aβ42 and Aβ40  was still visible by the unaided eye, CSF-0.02%-blood (1000 E/μl) and CSF-0.01%-blood (500 E/μl) samples were visually indistinguishable from neat CSF  CSF samples analyzed within hours after LP as the standard of truth. Our findings indicate that CSF could be stored for at least 72 h at RT, 1 week at 4 °C, and 2 weeks at − 20 °C and − 80 °C without significant changes in the levels of Aβ42 or Aβ40. Centrifugation was not needed if the CSF samples were not contaminated with blood. Further, we found that there is no need to mix the CSF samples prior to Aβ measurements, if the samples had not been frozen. In CSF samples that were not centrifuged after LP and stored at RT, blood contamination led to dose- and time-dependent reductions in Aβ42 and Aβ40 levels whereas Aβ42/Aβ40 was stable. The effects of blood contamination on Aβ42 and Aβ40 were partially to fully mitigated by centrifugation and/or storage at lower temperatures (4 °C and − 20 °C). Finally, we did not observe differences in Aβ42 and Aβ40 levels between the first and second to fourth 5-ml portions of 20 ml CSF collected during LP. Although we used LoB tubes from Sarstedt in the present study, our unpublished data indicates no difference in Aβ levels between CSF samples stored in either Sartstedt or Eppendorf LoB tubes. In principle, LoB tubes available from other vendors could also be suitable for CSF storage before Aβ analysis. Still, their performance should be assessed in comparison to Sartstedt or Eppendorf LoB tubes before incorporation in pre-analytical protocols.For practical reasons, it is more suitable to transport CSF to clinical chemistry laboratories for testing at RT or at 4 °C. However, at these temperatures, Aβ42 release from amyloid-binding proteins, adsorption to tube material, or proteolytic degradation may all potentially cause changes in the levels of this peptide , particuFreezing and thawing procedures introduce concentration gradient in biological samples, and consequently, thorough mixing is needed to make samples homogeneous  AccordinCentrifugation of CSF after LP is usually advised to remove white blood cells and, in case of blood contamination, red blood cells that may alter the proteome profile –28. Yet,3–5 × 105 E/μl to CSF samples caused a 5–98% decline in Aβ42 and Aβ40 concentrations which was more pronounced and occurred earlier with increasing degree of blood contamination. At this level, blood contamination was visible by the unaided eye  any portion of up to 20 ml of CSF (after discarding the first 2 ml) could be used for Aβ analysis, (2) CSF should be collected directly into a LoB tube, (3) centrifugation of CSF after LP is not necessary if erythrocyte count is ≤ 1000/μl or no visible blood contamination, (4) fresh CSF samples could be stored at RT for 72 h or at 4 °C for 1 week until analyses, (5) mixing of fresh CSF samples before Aβ measurement is not required, and (6) CSF samples with visible blood contamination should be centrifuged after LP and stored at 4 °C. An alternative approach is to store the CSF collected directly into a LoB tube at − 20 °C or − 80 °C until analyses. The frozen samples should be handled similar to the fresh samples (see above), except that mixing of the thawed samples should be done just before analyses. Although the findings of the present study were very similar for EI and MSD assays, they should be verified for other available Aβ assays/platforms. A final unified pre-analytical protocol must be decided in consensus by the main stakeholders in the field. This effort is currently led by the Alzheimer’s AssociationAdditional file 1:Figure S1. Flowchart of the CSF storage protocol. Figure S2. Flowchart of the CSF mixing protocol. Figure S3. Flowchart of the blood contamination  and centrifugation protocol—EI assay. Figure S4. CSF-blood samples. Figure S5. Samples and analysis in blood contamination and centrifugation protocol—MDS assay. Figure S6. Flowchart of blood contamination at low levels  and the centrifugation protocol—EI assay. Figure S7. Frequency plots of CSF biomarkers. Figure S8. Effects of centrifugation when not adding blood. (DOCX 8700 kb)