Introduction: Biomarkers that will reliably predict the onset of Alzheimer’s disease (AD) are urgently needed. Although cerebrospinal fluid (CSF) amyloid beta 1–42 (Aβ42), total tau, and phosphorylated tau can be used to complement the clinical diagnosis of AD, amnestic mild cognitive impairment (aMCI), the prodromal phase of AD, is heterogeneous. Biomarkers should be able to determine which patients with aMCI are at greatest risk of AD. Histological studies and animal models indicate that amyloid beta 1–43 (Aβ43) aggregates early, and may play a role in the pathological process of AD. We have examined levels of CSF Aβ43 in a 2-year longitudinal study of aMCI and early AD.Materials and Methods: Cerebrospinal fluid was collected at baseline, and after one and 2 years from patients with AD (n = 19), and patients with aMCI (n = 42). Of these, 21 progressed to AD during the 2 years of study, whereas 21 did not. Controls (n = 32) were lumbar punctured at baseline only. CSF analyses of Aβ43, Aβ42, and total tau were carried out with ELISA.Results: At baseline, CSF Aβ43, CSF Aβ42 and ratios with total tau could be used to separate controls from all three patient groups. CSF Aβ43, but not Aβ42, could separate patients with aMCI who progressed to AD during the 2 years of follow-up, from those that did not. The CSF total tau/Aβ43 ratio had a slightly but significantly larger area under the receiver operating characteristic curve when compared to the CSF total tau/Aβ42 ratio. CSF Aβ43 levels, but not Aβ42 levels, decreased from baseline to 2 years in the AD group.Discussion and Conclusion: CSF Aβ43 was demonstrated to be significantly reduced in patients already by the time that aMCI or AD was diagnosed, compared to controls, and this change must have occurred during the preclinical period. Since our results suggested that CSF Aβ43 distinguishes between subgroups of patients with aMCI better than CSF Aβ42, it may prove to be a useful additional biomarker for identifying aMCI patients at greatest risk of AD. According to the proposed new diagnostic criteria for Alzheimer’s disease (AD), early AD can be diagnosed if the prodromal phase  is present, and at least one biomarker is positive, though these biomarkers have yet to be validated . BiomarkLevels of amyloid beta 1–42 (Aβ42) in cerebrospinal fluid (CSF) correlate inversely with brain parenchymal Aβ42 load, as visualized by positron emission tomography (PET) . AdditioCompared to Aβ42, amyloid beta 1–43 (Aβ43) has an additional threonine at the C-terminal, and is thought to be more aggregation-prone than Aβ42 . Aβ43 waTo date there are few studies assessing CSF Aβ43 for its potential as a biomarker in early AD. It has been demonstrated that CSF Aβ43 was decreased in MCI and AD patients, as well as being positively correlated to CSF Aβ42 , despiteIn the present study, we examined CSF Aβ43 levels at baseline, and longitudinal levels over a 2-year period, in samples from patients with aMCI that progressed to AD during this period, as opposed to those that did not. Levels in patients with AD from baseline were also studied, and results for healthy control individuals were available at baseline. Comparisons were made with CSF levels of Aβ42, including correlations, and ratios with CSF t-tau.n = 19) were diagnosed according to the NINCDS–ADRDA criteria (n = 42) according to the International Working Group on Mild Cognitive Impairment Criteria  and assigned to this study from 2009 onward. Patients were referred to the clinic by general practitioners, and were diagnosed by a neurologist (SBS). Patients with early AD  genotyping was performed on blood samples from all study participants as described elsewhere  , as welllsewhere . DiagnosCerebrospinal fluid was usually collected early in the morning with patients lying on their side, and the puncture was at the L4/L5 or L5/S1 level. The first 2.5 mL CSF was used for routine clinical investigation. Aliquots of CSF (1 mL) were then collected directly into 2 mL polypropylene cryovials (Corning) immersed in ice-water. Samples were not centrifuged unless contaminated by blood (four samples), and were frozen within 30 min of lumbar puncture, and stored at -80°C until analysis.Cerebrospinal fluid was analyzed using ELISA monoplex kits . Cross-reactivity for Aβ42 in the Aβ43 ELISA was given as <1%. Although this would contribute slightly to measurements for Aβ43, it would be a constant for both control and patient groups. Aβ43 was reported to have 50x less affinity than Aβ42 for the antibodies in the Aβ42 kit.Samples were thawed in ice-water prior to analysis, and kits were run according to the manufacturers’ instructions. No samples underwent more than one freeze-thaw cycle, and all were analyzed undiluted, in duplicate. Both control and patient samples were included on each plate, and samples from patients in the three groups were evenly distributed across the plates. For the Aβ43 assay, an internal control was run on 5 of 6 plates, yielding an inter-assay CV of 14.4%, and an intra-assay CV of 5.1%.p-values <0.05 were considered statistically significant.Statistical analyses were carried out using SPSS version 22 (IBM), Stata version 13.1., and R version 2.13.1. For all analyses, post hoc test for pairwise comparisons if significant p-values were obtained. Longitudinal MMSE-scores were analyzed in a mixed linear model. Biomarker concentrations at baseline, after 1 year and after 2 years, were log-transformed to the natural logarithm (ln) to approximate to a normal distribution before comparison of groups by one-way ANOVA, followed by the LSD post hoc test for pairwise group comparisons. Pearson’s correlation coefficient (r) for correlations between biomarkers was also calculated from log-transformed values.Demographic data and MMSE scores at baseline were assessed using Pearson’s chi-square test for dichotomous variables, or one-way ANOVA for continuous variables, followed by the least-significant difference (LSD) Receiver operating characteristic (ROC) curves were made to assess the diagnostic accuracy for individual biomarkers, or combinations of them, and the area under each ROC curve (AUC) was calculated.Youden’s index [(sensitivity + specificity)-1] was determined to find exploratory cut-offs (not shown) where the sum of sensitivity and specificity was maximized, for biomarkers or ratios of biomarkers. Pairwise comparisons of AUC between Aβ43 and Aβ42, or ratios including these two biomarkers and total tau protein, were made for pairs of diagnostic groups (DeLong method). Longitudinal data have been analyzed using a mixed linear model. To account for repeated measurements, de-identified patient ID was included as a random effect. The combination of group and time was included as a fixed effect. All biomarker concentrations were log-transformed to the natural logarithm (ln) to approximate to a normal distribution before analysis in the mixed linear model.APOE genotype. Age and gender were found not to be confounding factors in the present study, and correction for APOE genotype would have resulted in many small groups, so was not done. There are results indicating that CSF Aβ42 levels reflect amyloid deposition in the brain independent of APOE 𝜀4 status .Table 1. There were no significant differences in gender distribution between the four participant groups , despite a preponderance of females in the control group. Neither the age at inclusion nor the duration of education was significantly different between the four groups. Moreover, no significant difference in the age at onset of symptoms, or duration of symptoms was found between the patient groups.Demographic data are summarized in APOE 𝜀4 allele in the pMCI subgroup, and amongst patients with AD, compared to the control group (both p ≤ 0.002). There was also a higher prevalence of the APOE 𝜀4 allele in the AD group compared to the sMCI subgroup (p = 0.015). The distribution of APOE 𝜀4 alleles was not significantly different between controls and the sMCI subgroup, between patients with AD and the pMCI subgroup, or between sMCI and pMCI subgroups.There was a higher prevalence of the p ≤ 0.010). There were decreases in MMSE-scores from baseline to 2 years in the pMCI and AD groups (both p < 0.001), but not in the sMCI group.At baseline, MMSE-scores varied between all four participant groups . The CSF Aβ43 level in the control group was higher than in any patient group . The control group was also significantly different from all patient groups for the other three analytes or ratios . There were differences between AD and the sMCI subgroup for Aβ43 and the t-tau/Aβ43 and t-tau/Aβ42 ratios , but for Aβ42 there was only a weak trend (p = 0.086). Aβ43 and the t-tau/Aβ43 and t-tau/Aβ42 ratios varied between the sMCI and pMCI subgroups , but no difference for Aβ42 levels was found. No significant differences were found between patients with AD and the pMCI subgroup for any analytes or ratios.Overall, all four analytes or ratios varied between groups at baseline , as were t-tau/Aβ43 and t-tau/Aβ42 . After 1 year, Aβ43 and Aβ42 were still correlated in the sMCI and pMCI groups , but not the AD group. After 2 years, Aβ43 and Aβ42 were correlated in all three patient groups . T-tau/Aβ43 and t-tau/Aβ42 were correlated in all three patient groups both after 1 year and after 2 years .Cerebrospinal fluid Aβ43 and Aβ42 were correlated in all four participant groups at baseline . Both CSF Aβ43 and Aβ42 showed similar specificity in distinguishing control individuals from patients with AD, but the sensitivity was slightly higher for Aβ43. CSF Aβ43 and Aβ42 were also excellent at separating controls from patients in the pMCI subgroup, with AUCs over 0.90. Sensitivity and specificity were all over 90%, except for slightly lower sensitivity of Aβ42. For separating controls from patients in the sMCI subgroup, all AUC values were over 0.7, and specificity was high (87–100%), whereas sensitivity was much lower (53–74%). On the other hand, the sMCI subgroup was well separated from AD patients by the t-tau/Aβ43-ratio, with a slightly higher sensitivity than the corresponding t-tau/Aβ42-ratio. Aβ43 alone also separated these two groups , while Aβ42 alone was not significant. The pMCI and AD patient groups were not separated significantly by any of the biomarkers or ratios thereof. However, the sMCI and pMCI subgroups were separated by ratios t-tau/Aβ43 and t-tau/Aβ42 . T-tau/Aβ43 had higher specificity (90%), whereas t-tau/Aβ42 had higher sensitivity (85%). When comparing AUC between sMCI and pMCI subgroups, the t-tau/Aβ43-ratio gave a larger AUC than the t-tau/Aβ42-ratio (p = 0.040), but for all other comparisons of Aβ43 and Aβ42, or comparisons of ratios t-tau/Aβ43 and t-tau/Aβ42 between two and two diagnostic groups, there were no significant differences in AUC.Receiver operating characteristic plots (not shown) were created for baseline levels of biomarkers or ratios thereof, and statistics were derived for pairwise comparisons of groups , but not for CSF Aβ42. For CSF Aβ43, the t-tau/Aβ43 and t-tau/Aβ42 ratios, there were differences between AD and the sMCI subgroup , and the sMCI and pMCI subgroups .After 1 year, group levels varied for CSF Aβ43 and the t-tau/Aβ43 and t-tau/Aβ42 ratios , but not for CSF Aβ42. For CSF Aβ43, the t-tau/Aβ43, and t-tau/Aβ42 ratios, there were differences between AD and the sMCI subgroup , and the sMCI and pMCI subgroups . No significant differences were found between patients with AD and the pMCI subgroup for any analytes or ratios at either one or 2 years.After 2 years, there were variations in group levels for CSF Aβ43 and the t-tau/Aβ43 and t-tau/Aβ42 ratios  and AD groups (p = 0.005), but no significant changes were observed from year one to year two after inclusion in these two groups. In the AD group, CSF Aβ43 levels were decreased from baseline to 2 years after inclusion (p = 0.003), and levels were stable over 2 years in the sMCI group. For CSF Aβ42, levels were stable over 2 years in all three patient groups.CSF Aβ43 showed a decrease from baseline to 1 year after inclusion in both pMCI (p = 0.002). For sMCI, the increase was significant from year one to year two (p = 0.004), but not from baseline to 1 year after inclusion. For pMCI, the increase was significant from baseline to year one (p = 0.010), but not from year one to year two.For the t-tau/Aβ43-ratio, levels were stable over 2 years in the AD group. In the sMCI and pMCI groups, the ratio increased from baseline to 2 years (both p ≤ 0.003). For sMCI, there was an increase from year one to year two (p = 0.015), but not from baseline to 1 year after inclusion. For pMCI, there was an increase from baseline to year one (p = 0.018), but not from year one to year two.For the t-tau/Aβ42-ratio, levels were stable over 2 years in the AD group. In the sMCI and pMCI subgroups, the ratio increased from baseline to 2 years thereafter .To the best of our knowledge, this is the first longitudinal study of CSF Aβ43 in connection with aMCI and AD. A significant reduction in the CSF Aβ43 level over the 2 years following baseline was observed in the AD group, whereas no significant longitudinal changes in CSF Aβ43 levels were observed in the sMCI and pMCI groups. The longitudinal changes during the 2 years of study were substantially less in all patient groups compared to the concentration difference between the control group and patient groups at baseline. This is an indication that the reduction of CSF Aβ43 is most pronounced during the preclinical phase of disease, which may take place slowly over many years. It is certainly clear that changes in CSF Aβ43 concentration over the 2 years of our study are slow . Since the standard deviation was relatively small in the AD group, the reduction was significant. However, this was not the case in the pMCI or sMCI subgroups where standard deviations were larger, and indeed there was hardly any change over 2 years in the sMCI subgroup.The sMCI subgroup was probably heterogeneous, and although it contained individuals who have converted to AD since the end of the study (data not shown), it probably also contains individuals with control levels that will never convert to any neurodegenerative disease . OverallDespite the heterogeneity of the sMCI subgroup, baseline mean levels of CSF Aβ43 (but not CSF Aβ42) distinguished significantly between the two aMCI subgroups, even though the groups were small. Contrary to CSF Aβ43, CSF Aβ42 levels were stable from baseline to 2 years in all three patient groups, in agreement with an earlier longitudinal study showing that CSF Aβ42 levels were stable over 4 years in MCI patients , and in CSF Aβ43 and Aβ42 were highly correlated in all four participant groups at baseline. Aβ43 and Aβ42 are suggested to be produced from different routes of enzymic cleavage, where three amino acids are cleaved off the peptides Aβ48 and Aβ49 in two steps to produce Aβ42 and Aβ43, respectively . The cloThis is a small study with groups of limited size. In an earlier study of similar size that compared AD patients with controls, CSF Aβ43 was found to have similar specificity (97%) but much lower sensitivity (52%) and lower AUC (0.77) than in the current study . Such diOur findings suggest that CSF Aβ43, either alone or in a ratio with CSF t-tau, may be a useful additional discriminator for patients with aMCI that will progress to AD within a short period of time.CL planned and performed the laboratory work, analyzed data, and wrote the manuscript. SS performed the clinical examination of all study participants together with GG. AS, IM, and GBe planned and performed the laboratory work. IB and ØS advised on statistical analysis. SS, GBr, and LW contributed to the conceptual design of the study, and supervised the project. All authors contributed to critical revision and finalization of the manuscript.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.


While there is some clinical utility in measuring these markers individually, their usage in routine clinical testing remains challenging, in part due to substantial overlap of concentrations between healthy controls and diseased subjects. In contrast, measurement of different analytes in a single sample from individual patients in parallel appears to considerably improve the accuracy of AD or PD diagnosis. Here, we report the development and initial characterization of a first, electrochemiluminescence-based multiplex immunoassay for the simultaneous quantification of all four proteins (‘tetraplex’) in as little as 50 μl of CSF. In analytical performance experiments, we assessed its sensitivity, spike-recovery rate, parallelism and dilution linearity as well as the intra- and inter-assay variability. Using our in-house calibrators, we recorded a lower limit of detection for α-synuclein, β-amyloid42, DJ-1, and t-tau of 1.95, 1.24, 5.63, and 4.05 pg/ml, respectively. The corresponding, linear concentration range covered >3 orders of magnitude. In diluted CSF samples (up to 1:4), spike-recovery rates ranged from a low of 55% for β-amyloid42 to a high of 98% for DJ-1. Hillslopes ranged from 1.03 to 1.30, and inter-assay variability demonstrated very high reproducibility. Our newly established tetraplex assay represents a significant technical advance for fluid-based biomarker studies in neurodegenerative disorders allowing the simultaneous measurement of four pivotal makers in single CSF specimens. It provides exceptional sensitivity, accuracy and speed.The quantification of four distinct proteins (α-synuclein, β-amyloid Hong et al.  (SULFO-TAG) according to the manufacturer’s instructions, except for ADx201, which was biotinylated and used in combination with 0.5 μg/ml streptavidin-SULFO-TAG (MSD). Ruthenium tagged antibodies are quantified using electrochemiluminescence (ECL) technology. Unspecific binding of AF3995 and ADx201 was quenched by adding 0.1% mouse  and goat immunoglobulin  to the detection antibody solution.42). PBS-T was used as blank control. Concentrations as mentioned on the data sheet were used for the study.Calibration curves were prepared with recombinant proteins dissolved in 1% BSA/0.05% PBS-Tween20 (PBS-T) covering a concentration range from 6.1 to 25.000 pg/ml  or 0.73 to 3000 pg/ml  instead of 1% BSA/PBS-T. Calibrators and samples were applied to the system at 25 μl for 1h in singleplex and 50 μl for 2h in multiplex assays.The antibodies for the multiplex assay plates were spotted in the facilities of MSD according to their standard operation procedures. The following coating conditions were applied: 0.5x MSD coating concentration of AF3995, 1x coating concentration of MJF1, ADx215 and MSD´s AβFor the qualification experiments, randomly selected CSF samples from controls of the “Kassel cohort” were used. Details on sample processing and diagnostic criteria were published recently .42, DJ-1 and t-tau protein, respectively). Therefore, CSF samples were diluted up to 1:4 in all further experiments. First, recovery rates were determined in CSF, prepared by pooling eight individual CSF samples that were then diluted 1:4 with Diluent 35. In this experiment, calibrator proteins were spiked individually at a final concentration of 250, 500, and 1,000 pg/ml for Aβ42 and t-tau protein or 625, 1,250, and 2,500 pg/ml for aSyn and DJ-1. In parallel, all four proteins were also spiked as an entire set at the same concentrations into our pooled CSF specimen. Spiking was performed by adding one volume of individual or combined calibrators to nine volumes of CSF. Endogenous levels of protein were determined by spiking one volume Diluent 35 without calibrators to nine volumes of CSF. The latter is important in order to account for the dilution effect by the addition of the diluent to the CSF. In addition, nine volumes of Diluent 35 were spiked with one volume of the calibrators to verify the exact concentration of each analyte that was spiked into the samples. Finally, we determined spike-recovery rates in individual CSF samples with a procedure identical to that described above, but using only pooled protein calibrators. Addition of proteins was done immediately prior to analysis without additional freezing of samples.Initial experiments to determine the recovery rates of spiked recombinant proteins in undiluted CSF samples indicated matrix interference for each analyte, resulting in low relative values  and three individual CSF samples for analysis. Values were re-calculated taking into account the dilution factor as described .Raw data were analyzed using Workbench 3.0 software (MSD).Our study was carried out in accordance with the Declaration of Helsinki and with the informed written consent of all patients or, in the case of cognitive impairment, their next of kin. Our study was approved by the ethics committees of the University of Göttingen and the Landesärztekammer Hessen (Germany).42, t-tau and aSyn were performed according to instructions and protocols provided by the supplier .Quantification of AβResults with CVs <20% for replicate determinations of analyte concentrations were considered reliable according to published guidelines –25. GrouWe first optimized an established, sandwich-type aSyn ELISA ,6 and deThe general characteristics of tetraplex assay performance are summarized in 42). The calibrators were prepared in serial four-fold dilutions ranging from 25,000–0 pg/ml  or 3,000–0 pg/ml (AβThe lower limits of detection (LLOD), as determined by Workbench 3.0 software, varied between 1.24 pg/ml and 5.63 pg/ml and therefore confirmed the high sensitivity of each component of the assay . For LLOPotential interactions in our assay format with other proteins present in the biological fluid specimen were analyzed in two different ways. First, calibration curves of pooled and individual proteins were compared. Calibration curves were compared within the same assay run. As shown in 42), respectively  and 3,000 pg/ml  are presented in 42 concentration, normalized protein concentrations differed by more than 20% from the reference concentration. At higher dilutions normalized concentrations were comparable to the reference values until, in the case of t-tau protein, the concentration approached the detection limit and therefore protein quantification became more inconsistent.Dilution linearity was analyzed using serial two-fold dilutions of three individual CSF samples spiked with an approximately 100-fold endogenous concentration of aSyn, DJ-1, and t-tau protein as well as a 25-fold endogenous concentration of Aβ42, one out of four samples measured at 1:4 dilution deviated by slightly more than 20% from the reference value measured at 1:8 dilution . These results point to matrix effects interfering with the protein quantification at higher CSF concentration.Parallelism was analyzed using serial two-fold dilutions of CSF, and using undiluted CSF as the highest concentration. Results normalized for concentrations in CSF diluted at 1:8 are presented in 42, 105.7 ± 3.0% for DJ-1, and 119.5 ± 9.7% for t-tau protein , 2 cases for DJ-1 (range: 23.3–30.5%) and 3 cases for t-tau protein (range: 21.9–30.0%). For diluted CSF samples, CVs above 20% were found in ten cases for aSyn (range: 20.4–28.3%), 16 cases for Aβ42 (range: 22.5–46.3%), one case for DJ-1 (CV: 28.7%) and 27 cases for t-tau protein (range: 20.5–141.1%).Intra-assay reproducibility was determined by measuring CSF samples in duplicate. In total, 36 undiluted CSF samples were measured and 192 were measured at 1:4 dilution. Most samples showed CVs below 20%. For undiluted CSF samples, CV values above 20% were found in 3 cases for aSyn (range: 23.9–35.4%), 5 cases for Aβ42 (23.2 and 27.5%). T-tau protein quantification yielded CVs above 20% in 20 cases. With the exception of four CSF samples that exhibited relatively high t-tau protein concentrations, the other 16 CSF samples had t-tau protein concentrations at or near the lower limit of detection (i.e. below 20 pg/ml). Quantification of aSyn and DJ-1 concentrations always gave CVs below 20%.Inter-assay variability was determined by measuring 40 CSF samples at 1:4 dilution in two independent experiments. Two CSF samples showed CVs above 20% for quantification of Aβ42, t-tau and aSyn. As expected, the results revealed a high correlation between them for all three proteins tested   .2 for aSyn: 0.504, for DJ-1: 0.791, for Tau: 0.726)  without evidence of typical PD and eight patients with idiopathic PD. Twenty five μl of CSF were measured undiluted both in our singleplex assays for aSyn, DJ-1 and t-tau protein and by tetraplex assay . Overall: 0.726) .42 and t-tau protein in CSF on an ECL-platform; furthermore, its performance was substantiated according to the most stringent criteria, such as those published in internationally recommended guidelines for new assay validation [In this study we describe the first generation of a tetraplex immunoassay that simultaneously quantifies aSyn, DJ-1, Aβlidation –25. WithSingleplex immunoassays are suitable to measure individual protein concentrations without the problem of potential interference of antibodies. However, multiple assays need to be performed to quantify several proteins. Furthermore traditional ELISAs often require sample incubation over night.The major advantage of a multiplex assay format is that it reduces time, cost and processing time. The whole assay may be performed within less than four hours. In addition this format may help to increases diagnostic accuracy by measuring four analytes in a single sample without the need to run multiple assay plates, which can introduce variability due to multiple handling steps of the same stock specimen. Therefore it is desirable to derive considerably more information from minimal sample volumes . A disadThe ECL technology is widely used in different research areas ,28. Most42, in order to improve diagnostic accuracy by identifying PD pathology or by excluding AD pathology.Our assay development efforts were aimed at multiplexing four proteins associated with Parkinson´s or Alzheimer´s disease pathology, i.e. aSyn, DJ-1, t-tau protein and Aβ42. These data show that in the case of Aβ42 a ten-fold increase in protein concentration corresponds to a 13-fold increase in signal intensity. In other words, a 13-fold increase in signal intensity is required to measure a ten-fold increase in protein concentration. For the other proteins the correlation was much better. Mean Hillslopes ranged from 1.03 to 1.15. In addition, LLODs for all proteins were far lower than the lowest concentrations measured in our clinical samples (1.24–5.63 pg/ml).The general performance of our established multiplex assay as indicated by the Hillslopes and LLODs showed high sensitivity for each of the four proteins of interest. Hillslopes define the correlation of increases in concentration and signal intensity. A sensitive assay should have very close correlation between these two metrics. An ideal Hillslope of 1 demonstrates that a two-fold or ten-fold increase in concentration corresponds to a two-fold or ten-fold increase in signal intensity, respectively. In our experiments we observed mean Hillslopes of 1.30 for AβThe calibration curves were found to be highly reproducible. The mean signal intensities obtained at all concentrations performed in duplicate in seven experiments gave CV values of less than 20% in most cases, indicating that quantification over the entire concentration range is reliable –25.Both in undiluted and in diluted CSF we found that most of the duplicate determinations had CVs below 20%, except for t-tau protein. Signal readings for t-tau protein in most cases was detected at the lower end of the calibration curve, where slight changes in signal reading have a strong impact on concentration assignment. This is very evident when diluted samples were analyzed. In this case the proportion of t-tau protein measurements with high CVs increased and therefore became less reliable. Quantification of the other proteins was comparable in two experiments. These results clearly demonstrate that finding a single dilution factor that permits the measurement of multiple proteins in a given sample may be difficult, as has been reported .All proteins of interest in this study tend to form either homo- or hetero-oligomers or to promote aggregation of other proteins –33. We t42. These results clearly show that the combinations of antibodies used in this study are suitable for quantification of the proteins in a multiplex format. We observed that using individual CSF samples led to variable recovery rates. This may be attributed to matrix effects which can be reduced by further diluting the CSF. Further reduction of CSF concentration will also reduce t-tau protein concentrations to levels below the limit of detection, at least in our cohort. The analytical sensitivity of the t-tau assay can be improved by combining it with other monoclonal antibodies or modifying the method of labelling the detector antibody. Nevertheless, this multiplex assay format may be suitable for analysing t-tau protein concentrations in other neurodegenerative diseases that are accompanied by significant cell destruction (for example Alzheimer’s disease and Creutzfeldt-Jakob disease).Recovery rates determined in several experiments showed that these were higher than 50% for all proteins and nearly 100% for t-tau protein and DJ-1. They were slightly lower for aSyn and AβOur multiplex assay does not necessarily preclude problems that arise with immunoassay performance in general, such as lab-to-lab variability, lot-to-lot variability, lack of certified reference material, etc. ,35. Howe42. We speculated that similar observations will be made when diluted samples from PD patients are compared to neurological controls. Currently we are not able to definitely state whether the effect of diluting CSF will lead to a more comparable concentration assignment between the two different assay formats. The answer would require that in the case of DJ1 sample dilution must have a very strong effect in the singleplex assay in order get comparable results to the multiplex assay while for α-Synuclein the effect should be higher in the multiplex assay. Further experiments are needed to address this issue.We compared the quantification results of our multiplex assay with those of singleplex assays in a small cross-sectional study. In these experiments we found that the general trend was comparable between both assay formats, in that the CSF samples from the PD patients had lower concentrations of aSyn, DJ-1 and t-tau protein than those from the patients with normal-pressure hydrocephalus used as clinical controls. These results are consistent with other studies investigating CSF levels of these proteins in PD patients and healthy controls ,36. InteWe found that for defined calibrator concentrations much higher ECL signals were measured on multiplex than on singleplex plates. This was also seen in the samples, although not to the same extent. ECL signals for t-tau protein calibrators and samples were comparably increased on multiplex plates (~3.5-fold). With aSyn, the ECL signals for calibrators and samples on multiplex plates increased by factors of four and two, respectively, while the calibrator readings for DJ-1 increased ~20-fold and sample readings >70-fold on multiplex plates. Therefore DJ-1 concentrations were assigned much higher levels on multiplex plates and aSyn concentrations to lower levels as compared to singleplex plates.We demonstrate the generation and careful characterization of a tetraplex assay for the simultaneous quantification of PD- and AD-associated proteins in as little as 50 μl of CSF volume. The assay is easy to perform, requires less hands-on time as compared to conventional ELISAs and saves precious clinical material. Further experiments are required to validate and apply the assay further, such as to demonstrate its usefulness in routine laboratory practice processing larger numbers of samples than shown here. Major advantages of the tetraplex assay are (1) its superior sensitivity, (2) the broad dynamic range, (3) its high degree of reproducibility; and importantly, (4) the one-step readout of multiple analytes in the same specimen. We are aware that our assay also needs validation by independent groups . Due toIn summary, our tetraplex platform now permits a more objective, faster and more informative interrogation of small volumes of CSF from patients with incurable disorders of the brain. We envision that technological advances such as this one will serve patient stratification, improve the design of clinical trials, allow for disease monitoring, and thus, increase the chance for an urgently needed, positive outcome in future intervention studies.S1 Fig(DOC)Click here for additional data file.S2 Fig(DOC)Click here for additional data file.S1 TableIndicated are mean signal readings and standard deviations. The relatively high standard deviation of Tau protein standard curves may be explained by usage of two different kit lots. This table refers to (DOC)Click here for additional data file.S2 TableRaw data for standard curves performed on individual standards and pooled standards (in duplicate) in a single experiment are indicated. Signal readings for both standard curve formats are highly comparable. This table refers to (DOC)Click here for additional data file.S3 TableIndicated are row positions of a 96 well plate, spot with identity of capture antibodies and the respective results. Positions where specific results were expected are highlighted in grey. In most assay wells no unspecific interactions were observed. Only in a few cases unspecific interactions were found (indicated in red in S3B Table). This table refers to (DOC)Click here for additional data file.S4 TableIndicated are protein concentrations from CSF samples spiked with very high protein concentrations (left section). Results were then adjusted for the fourth dilution (middle section) and normalized for this dilution step (right section). This table refers to (DOC)Click here for additional data file.S5 TableIndicated are protein concentrations from serially diluted CSF samples (left section). Results were then adjusted for dilution factors (middle section) and normalized for the fourth dilution step (right section). This table refers to (DOC)Click here for additional data file.S6 TableIndicated are protein concentrations in spike solutions used in the experiment as well as protein concentrations measured in CSF samples spiked at different concentrations (left section). Endogenous protein concentrations were subtracted and recovery rates were calculated based on proteins concentrations in spike solutions and spiked CSF samples. This table refers to (DOC)Click here for additional data file.S7 TableThis table refers to (DOC)Click here for additional data file.


Early differential diagnosis between acute bacterial and viral meningitis is problematic. We aimed to investigate whether the detection of lipocalin 2, a protein of the acute innate immunity response, may be used as a marker for acute bacterial meningitis.Transgenic mice expressing the human transferrin were infected by intraperitoneal route and were imaged. Cerebrospinal fluid (CSF) was sampled up to 48hours post- infection to measure lipocalin 2. We also tested a collection of 90 and 44 human CSF with confirmed acute bacterial or acute viral meningitis respectively.p < 0.0001) in patients with confirmed acute bacterial meningitis  than in patients with acute viral meningitis  with a sensitivity of 81%, a specificity of 93%, a positive predictive value of 96% and a negative predictive value of 71% in diagnosing acute bacterial meningitis.Lipocalin 2 was detected after 5 h in CSF during experimental infection in mice. Lipocalin 2 levels were significantly higher (Increased levels of lipocalin 2 in cerebrospinal fluid may discriminate between acute bacterial and viral meningitis in patients with clinical syndrome of meningitis. Haemophilus influenzae type b (Hib), Streptococcus pneumoniae (Sp), and Neisseria meningitidis (Nm) are the most frequent agents of ABM. Other agents are also incriminated in infants such as Streptococcus agalactiae and Escherichia coli K1. ABM is a medical emergency and requires immediate management that relies mainly on appropriate and prompt antibiotic treatment. However, the major differential diagnosis of ABM is acute viral meningitis (AVM) that does not require antibiotics and are usually of better prognosis. Laboratory confirmation requires lumber puncture and analysis of the cerebrospinal fluid (CSF). Etiologic diagnosis of ABM is performed by culture and non-culture methods [Acute bacterial meningitis (ABM) is a major cause of morbidity and mortality worldwide. The Wold Health Organization (WHO) estimates that there are around 1 million of cases per year worldwide with 135–200,000 fatal cases[While 71% of patients with ABM may have a leukocyte count of ≥1000 cells/μl in CSF samples, 15% of patients with AVM may display similar counts in CSF and up to 10% of ABM cases may show leukocyte counts of <100 cells/μl,4. Cut-oNeutrophil Gelatinase Associated Lipocalin (NGAL)[The lipocalin 2 (LCN2) is a small protein of 22 kDa involved in iron homeostasis that allows an alternative method to transferrin to deliver iron to the cytoplasm. LCN2 wan (NGAL). LCN2 isn (NGAL),13. We hn (NGAL). Severaln (NGAL). We aimeOne hundred thirty four cerebrospinal fluids (CSF) addressed to the National Reference Center for Meningococci (NRCM) for molecular diagnosis of bacterial meningitis were tested. Available epidemiological data , clinical and biological data, C-reactive protein (CRP) in blood, CSF levels of protein and glucose were analyzed. Confirmed ABM was defined as more than 100 leukocytes in CSF and the detection of bacteria . PCR-based diagnosis was performed as previously described. EnterovN. meningitidis strain LNP24198 is a clinical isolate of serogroup C, serotype 2a and serosubtype P1.7,1 (PorA VR1 = 7-1 and VR2 = 1) that belongs to the clonal complex ST-11[Photorhabdus luminescence luxCDABE operon was purchased from Xenogen Corp., Alameda, CA and was modified by insertion of a Neisseria specific promoter sequence. A 600 bp promoter sequence of the meningococcal porB gene (PporB) was PCR amplified using primers PorB3 (5′-GGTGCTGAAGCACCAAGTGA -3′) and PorB4 (5′- GGCAATCAGGGATTTTTTCA-3′) and subcloned into a BamHI site upstream of the luxCDABE operon to express the luxCDABE operon under the control of the PporB meningococcal promoter N. meningitidis. The generated plasmid was named pDG33. The fragment encompassing the luxCDABE cassette and the porB promoter was extracted by digesting pDG33 with KpnI and SacI restriction enzymes and inserted into BamHI site of the plasmid pTE-KM[aph3’ resistance cassette. The resulting vector was named pDG34 in which, the PporB-luxCDABE-aph3’ was flanked by the meningococcal pilE gene and 120 bp downstream pilE gene to facilitate the recombination upon transformation to obtain the LNP24198lux strain.ex ST-11. The plad pTE-KM, upstreaWe have previously described the use of transgenic mice expressing the human transferrin infected by intraperitoneal route (ip) as an experimental model of meningococcal infection. Mice weEach mouse was infected with standardized inocula of the bioluminescent strain LNP 24198lux. Bacterial infection images were acquired using an IVIS® 100 system  according to instructions from the manufacturer and as previously described. AnalysiThis study was carried out in strict accordance with the European Union Directive 2010/63/EU (and its revision 86/609/EEC) on the protection of animals used for scientific purposes. Our laboratory has the administrative authorization for animal experimentation (Permit Number 75–1554) and the protocol was approved by the Institut Pasteur Review Board that is part of in the Regional Committee of Ethics of Animal Experiments of Paris region (Permit Number: 99–174). CSF samples were initially received for diagnosis that is part of the primary management of suspected meningococcal meningitis. The patients were informed on the secondary use of CSF samples for research and they gave their consent for this use. The printed form of this informed consent is sent to our laboratory. This procedure is performed according to the French public health code (Art L1211-2).http://imagej.nih.gov/ij/). All density data were corrected for the background by subtraction of the density of the negative control . Results were then expressed as a ratio of the corrected density obtained for each CSF over the corrected density obtained for the positive control . ELISA was performed with lipocalin2/NGAL Human ELISA kit®  according to the manufacturer’s recommendations. The expression of LCN2 gene in mice was performed by reverse-transcriptase-PCR (RT-PCR) analysis as previously described[Thirty micro liters of human CSF, 15 μl of diluted CSF (see above) or 2 μl of blood from infected mice were separated in 14% SDS- polyacrylamide gels and then transferred onto nitrocellulose membrane. Western blotting was performed as previously described using anescribed.Qualitative data were analyzed using the Chi-square test. Statistically significant differences were assumed when p < 0.05. Geometric means as well as lower and upper 95% confidence intervals were calculated using GraphPad InStat® version 3.06 . Specificity, sensitivity, positive predictive value, negative predictive value and likelihood ratios were calculated as previously described.6 CFU/mice or 107 CFU/mice of the strain LNP24198lux respectively and 3 uninfected mice were used as controls. Mice were imaged at 30 min, 2 h, 5 h and 24 h  was the main bacterial agent that was detected in our samples (n = 88) due to the fact that samples were tested for suspected meningococcal meningitis. One sample was positive for S. pneumoniae (Sp) and another was positive for Streptococcus group A. This group corresponded to confirmed bacterial meningitis. Nm positive samples were of several groups; 73% NmB (n = 66), 12% NmC (n = 11), 7% NmW (n = 6), 6% NmY (n = 5). The multi locus sequence typing (MLST), that determines the clonal complex, was performed for 45 (51% of Nm positive samples): 10 belonged to the hyperinvasive clonal complex ST-11 and 35 belonged to other clonal complexes. The second group (group 2) included 44 CSF that corresponded to PCR-confirmed acute enteroviral meningitis. The characteristics of patients and CSF of each of these two groups are presented in Table We next aimed to test whether lipocalin 2 can be detected in CSF from patients with acute bacterial meningitis. We explored a large collection of CSF samples n = 134) that are received at the National Reference Center for Meningococci for non-culture diagnosis by PCR. The samples were classified in two groups: The first group (group 1) included 90 CSF (67%) that showed more than 100 leukocytes and were positive for at least one of the following tests: PCR, culture, smear detection or slide agglutination. 34 that aWe next tested all the 134 CSF samples from the two groups by Western blotting using antibodies against human LCN2 and using an ELISA kit . Western blotting showed the expected band with an apparent molecular mass of 22 kDa that was observed with variable intensities  and/or corticosteroids may modify LCN2 production. The timing of lumbar puncture may also of importance in interpreting CSF findings. Moreover, the long conservation of CSF samples in the tested collection as well as the conditions of this conservation may also be responsible for the low detection of LCN2 in true ABM cases. These considerations may explain the relative low NPV (71%) in our study. Under these conditions, the high likelihood ratio of a positive result (11.6) indicates that a positive test multiplies the pre-test odds by a factor of 11.6. This means that the test is better at ruling in a condition than ruling it out. This would be of interest in case the incidence of acute bacterial meningitis is to be reduced after the introduction of vaccines targeting agents of ABM. The detection of lipocalin 2 in sera from infected mice may allow discrimination when CSF was not collected or lumber puncture was not feasible.One limitation of our study is that the samples were studied retrospectively and that the samples were enriched for ABM  as there were sent to the NRCM for suspicion of meningococcal meningitis. The development of a rapid test (for example a dipstick test) will open the possibility to perform a multisite study with prospective inclusion of patients suffering of clinical syndrome of meningitis. The performance of such a direct and rapid test for LCN2 detection in CSF and blood will be compared to that of other markers of ABM under the conditions of “in routine” management of ABM.Our data clearly indicate that LCN2 levels in CSF are highly increased in CSF in patients with ABM. We suggest that LCN2 detection in CSF from patients with clinical meningitis may help in differential diagnosis between acute bacterial and viral meningitis and may improve decision making for treatment algorithms in meningitis.The authors declare no conflict of interest.TG, AED and MKT participated in the study design and the preparation of the manuscript, TG, AED, DG and MKT participated in the laboratory experimental work and in the interpretation of data. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/14/276/prepub


Encephalitis is parenchymal brain inflammation due to infectious or immune-mediated processes. However, in 15–60% the cause remains unknown. This study aimed to determine if the cytokine/chemokine-mediated host response can distinguish infectious from immune-mediated cases, and whether this may give a clue to aetiology in those of unknown cause.We measured 38 mediators in serum and cerebrospinal fluid (CSF) of patients from the Health Protection Agency Encephalitis Study. Of serum from 78 patients, 38 had infectious, 20 immune-mediated, and 20 unknown aetiology. Of CSF from 37 patients, 20 had infectious, nine immune-mediated and eight unknown aetiology.Heat-map analysis of CSF mediator interactions was different for infectious and immune-mediated cases, and that of the unknown aetiology group was similar to the infectious pattern. Higher myeloperoxidase (MPO) concentrations were found in infectious than immune-mediated cases, in serum and CSF (p = 0.01 and p = 0.006). Serum MPO was also higher in unknown than immune-mediated cases (p = 0.03). Multivariate analysis selected serum MPO; classifying 31 (91%) as infectious (p = 0.008) and 17 (85%) as unknown (p = 0.009) as opposed to immune-mediated. CSF data also selected MPO classifying 11 (85%) as infectious as opposed to immune-mediated (p = 0.036). CSF neutrophils were detected in eight (62%) infective and one (14%) immune-mediated cases (p = 0.004); CSF MPO correlated with neutrophils (p<0.0001).Mediator profiles of infectious aetiology differed from immune-mediated encephalitis; and those of unknown cause were similar to infectious cases, raising the hypothesis of a possible undiagnosed infectious cause. Particularly, neutrophils and MPO merit further investigation. Encephalitis is a neurological emergency, which presents as headache, cognitive and behavioural disturbance, sometimes with focal neurological signs; many cases progress to seizures, coma and death . The mosThere is mounting evidence that the host inflammatory response, driven by cytokines, chemokines and associated mediators, may play a pivotal role in the pathogenesis of encephalitis –14, and Earlier work has demonstrated the importance of considering the significance of any identified mediators in the pathogenesis of disease in the context of leucocyte subsets within the CNS . The earTherefore, we conducted a study to examine the mediator profiles in the cerebrospinal fluid (CSF) and serum of patients with acute encephalitis to address two questions. Firstly, does the mediator profile differ between those of infectious and immune-mediated aetiologies; and secondly might characteristic mediator profiles suggest whether those of unknown aetiology are more likely to be infectious or immune-mediated.We analysed samples available from adult patients in a previous prospective clinical and diagnostic study- the Health Protection Agency  Aetiological Study of Encephalitis in England; this recruited 203 patients from 24 centres which each recruited over 2 years (2005–2008); methods have been described in detail [Patients provided written informed consent, the procedure for which was approved in the HPA study by the North and East Devon Multicentre Research Ethics Committee (Reference: 05/Q2102/22). This sub-study was approved by the HPA Encephalitis Study Steering Group and also by the Pan-Manchester Research and Development Group for the University of ManchesterSerum and CSF samples were collected at recruitment and stored at -80°C. Thirty mediators  were assessed using cytometric bead array . A further panel for eight matrix metalloproteinases was performed using the same technique where additional volumes were available. Mediators were identified from previous literature and chosen to reflect, in part:Leucocyte proliferation and differentiation ;Leucocyte chemotaxis ;Adhesion molecules ;Markers of BBB permeability ;Antiviral and related peptides ;Broadly pro-inflammatory interleukins ; andBroadly anti-inflammatory interleukins .Fluorescence intensity was determined using a Bio-Rad platform . Standards and samples were analysed in duplicate and mean value used. Standard curves were adjusted at the points of fluorescence intensity saturation, generating a sigmoid curve with 6–8 fluorescence points, as described . To avoiFreeze/thaw cycles were minimised, typically to 2–3, in the central laboratory. Nevertheless, to minimise any potential impact of any possible variation in sample storage, concentrations were median-centred and normalised for each patient, using established methodology ,17. TherThe Mann-Whitney U test was used for categorical variables and Kendall’s rank for continuous variables (SPSS 2011). Step-wise discriminant function analysis was used to develop a multivariate model, which underwent a leave-one-out cross-validation to protect against type 1 error; the power of each mediator is expressed using Wilk’s Lambda coefficient, from perfect discriminatory power (0.0) to none (1.0). This assisted by generating a model based not just on absolute concentrations of selected mediator(s), but also with a numerical weighting applied to selected mediator(s). This was generated using data from the majority of patients and tested on a random subset in a split-reliability analysis. Significance was defined as p<0.05.A total of 95 patients were identified, 78 of whom had serum samples of sufficient volume available; these comprised 38 with an infectious aetiology , 1 influenza A, 1 measles, 1 HIV, 1 toxoplasmosis); 20 immune-mediated cases , and 20 of unknown aetiology. For 37 patients CSF samples of sufficient volume were available, 19 with an infectious aetiology , 9 immune-mediated , and 8 of unknown aetiology. Additional samples were available to assess the MMP panel for 62 serum and 18 CSF samples.The following mediators were not identified in >80% of the cohort and were therefore removed from further analysis: TNFR1 and 2, eSelectin, CXCL9, IL-17α, and VEGFα.The mediators that tended to be found at higher concentrations in either the CSF or the serum were similar across all the aetiological groups . SignifiUnivariate analysis of mediator concentrations showed many similarities but also several differences between the aetiological groups Tables  and 4. TIn particular, higher concentrations of MPO were identified in both the serum and CSF of patients with infectious than immune-mediated aetiologies (p = 0.01 and p = 0.006 respectively) Figs  and 3. IHierarchical cluster analysis of mediators from all patients identified two broad clusters of mediators, which were used in the heat-maps. The first cluster of serum mediators contained IL-1α, IL-1β and IL-10, in addition to other mediators; in CSF these same mediators were clustered together with the addition of IL-1RA. The second cluster in serum contained several mediators including IFNα, IFNβ, IFNγ and IFNο; these same mediators clustered together in CSF. Heat-maps derived from serum mediator concentrations showed strong positive correlations between mediators in the first cluster and strong negative correlation in between mediators in the second cluster in all aetiological groups and this did not appear to be substantially different between groups . HoweverTo see which specific mediators might be used to distinguish between aetiological groups we used discriminant function analysis for pairwise comparisons of the groups. For discriminant function analysis of serum data this generated a model that used serum MPO alone to distinguish 31 (89%) of 35 samples as infective and five (31%) of 16 as immune-mediated . This technique also generated a model using serum MPO alone to distinguish 17 (85%) of 20 samples of unknown aetiology and six (38%) of 16 immune-mediated samples ; both were confirmed on the leave-one-out cross validation. No serum variables qualified as distinguishing samples of infective versus unknown aetiology. A serum MPO concentration cut-off of >0.8pg/mL correctly identified 27 (77%) infective, 17 (85%) as unknown, and 8 (50%) immune-mediated cases; to assess the usefulness of this cut off value for MPO this was then applied to samples from 7 patients (3 infective and 4 immune-mediated) that had not been included in the initial analysis. In this split-reliability analysis all three cases were identified as infective but none of the four were identified as immune-mediated.Discriminant function analysis of CSF data generated a model which again used MPO alone to correctly classify 11 (85%) of 13 cases as infective and four (67%) of six cases as immune-mediated ; this was confirmed on the leave-one-out cross validation. A CSF MPO concentration cut-off of 1.0pg/mL correctly classified nine (69%) infective cases and four (67%) immune-mediated cases. The proportion of cases correctly classified was the same when applied to the raw data using a cut-off of 1000pg/mL. To assess the usefulness of this cut off value we assessed this in 9 patients (6 infective and 3 immune mediated) not included in the original analysis. In this split-reliability analysis three of six and one of three cases were classified as infective or immune-mediated respectively.As MPO is released during degranulation of neutrophils, we assessed whether these findings may reflect the relative neutrophil concentration between the different aetiological groups. Eight (62%) of the infective group had detectable neutrophils in the CSF, as opposed to only one (14%) of the immune-mediated group (p = 0.004). Moreover, across the whole cohort there was a strong positive correlation between CSF neutrophil count and CSF MPO concentration  . In the This study has shown that the cytokine and associated mediator profiles in UK patients with infectious encephalitis differ from those with immune-mediated aetiologies, particularly in the CSF. For patients with unknown aetiology patterns in CSF most closely resembled infectious cases, suggesting they may have undiagnosed infection. MPO was the mediator of greatest significance, with higher concentrations in patients with infection, and CSF MPO concentration was strongly correlated with CSF neutrophil count.There are an estimated 0.07–12.6 cases of encephalitis per 100,000/year . The mosTherefore, we analysed CSF and serum samples from 95 patients with encephalitis recruited prospectively in the HPA Encephalitis Study . We founDiscriminant analysis of both CSF and serum identified MPO as the mediator that most strongly discriminated cases of an infective or unknown aetiology from immune-mediated cases. CSF concentrations of MPO correlated with CSF neutrophil count, suggesting that CSF neutrophils may be the primary source of MPO identified. A higher percentage of patients had detectable neutrophils in the CSF in the infectious as opposed to immune-mediated group, although this was not as strong an association as the MPO concentration, perhaps suggesting that the MPO in the CSF may also reflect neutrophil extracellular traps ; these aNeutrophils are known to be an important part of the host response early in viral encephalitis, before lymphocytes predominate; in bacterial central nervous system infections the neutrophil counts predominate, and are typically much higher. Interestingly, neutrophils have been identified recently in murine models of HSV encephalitis, and may be important for reducing viral load, albeit whilst also being associated with the production of, potentially neurotoxic, reactive oxygen species –35. HistOur study suggests that MPO in the CSF could be explored as a biomarker for a potential infectious aetiology in patients with encephalitis. Also serum MPO was also associated with an infective aetiology so may represent a potential biomarker and both require validation in prospective studies. For example interesting developments have been made in point-of-care tests for protein biomarkers, such as the lateral flow assays developed for CSF lactate to distinguish bacterial from viral meningitis . In addiIn CSF there were many more differences in the concentrations of mediators between the aetiological groups. Specifically, higher concentrations of IL-8 were identified in both the infectious and unknown group as opposed to immune-mediated cases, which would be consistent with the key finding of the role of neutrophils in our study, given the important role of IL-8 in neutrophil activation –49. In aInterestingly no significant differences were found in the concentration of any of the mediators between the infectious and unknown groups in either the serum or the CSF. Only one other study has assessed profiles between encephalitis aetiologies and identified a higher concentration of IFNγ and TNFR1 in 13 patients with HSV than 15 with ‘non-herpetic limbic encephalitis’ . We did Pooling the patients with different infectious aetiologies together, allowed analysis of factors which were common between them, and which distinguished them from immune-mediated encephalitis. It also allowed a comparison with those whose aetiology was unknown., This analysis makes the assumption that the pathology is similar in infectious cases. Whilst most is known about HSV infection, which is associated with necrosis, neutrophil invasion, haemorrhagic change and secondary autoimmunity in some, fewer histopathological data are available for other aetiologies ,52,53. IAlthough the patients for whom no aetiology could be identified had a mediator profile suggestive of infection, they had all undergone an extensive work up for infectious causes, through the HPA study ,54. TherIn summary, whilst there were many similarities identified, the cytokine and chemokine profiles in the CSF of patients with encephalitis of an infectious aetiology differed from those with an immune-mediated aetiology; those in whom no cause could be identified best reflected those of an infectious aetiology. MPO may represent a potential biomarker to distinguish encephalitis due to infection versus immune-mediated causes, which may in part relate to neutrophils in the CSF.BDM is an NIHR Academic Clinical Lecturer and this work received support as part of an NIHR Doctoral Research Fellowship (NIHR-DRF-2010-03-97). TS received support from the NIHR Health Protection Research Unit in Emerging and Zoonotic Infections at Liverpool. TS was also supported by the MRC. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. This work was supported by the laboratory team at the Vaccine Evaluation Unit, Public Health England, Manchester, UK. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR, the Department of Health or Public Health England. The samples and data from these patients have been used in associated studies arising from the original Health Protection Agency  Aetiological Study of Encephalitis in England.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.


Control of coupling of dopant atoms in silicon nanostructures is a fundamental challenge for dopant-based applications. However, it is difficult to find systems of only a few dopants that can be directly addressed and, therefore, experimental demonstration has not yet been obtained. In this work, we identify pairs of donor atoms in the nano-channel of a silicon field-effect transistor and demonstrate merging of the donor-induced potential wells at the interface by applying vertical electric field. This system can be described as an interfacial double-donor molecule. Single-electron tunneling current is used to probe the modification of the potential well. When merging occurs at the interface, the gate capacitance of the potential well suddenly increases, leading to an abrupt shift of the tunneling current peak to lower gate voltages. This is due to the decrease of the system’s charging energy, as confirmed by Coulomb blockade simulations. These results represent the first experimental observation of electric-field-assisted formation of an interfacial double-donor molecule, opening a pathway for designing functional devices using multiple coupled dopant atoms. Dopants have played a key role as carrier providers in conventional solid-state electronic devices. In downscaled metal-oxide-semiconductor field-effect transistors (MOSFETs), discrete dopant atoms significantly affect the subthreshold transport characteristics1245678911121314The effect of vertical electric field on the control of the electron wave function within a system formed by the potential well of a single dopant atom and a nearby interface has been recently studied both experimentally and theoretically16171819For this purpose, we fabricate and characterize transistors with ultrathin Si channels doped with phosphorus (P) donors. In one type of transistors, the channel was doped with lower doping concentration, for which pairs of donors can be found with high probability. We find that, under the application of a vertical electric field, potential wells induced by two neighboring donors can be merged at the interface, forming an interfacial double-donor molecule-like system. In another type of devices, the channel was selectively doped with higher doping concentration. For this case, we identify clusters of multiple P-donors strongly interacting with each other due to their physical proximity, without assistance of an interface.2 layer was thermally grown by dry oxidation as gate oxide, on top of which an Al frontgate was formed. The p-type Si substrate  is used as a backgate through a 150-nm-thick buried oxide (BOX) layer. The frontgate and the backgate provide control of the electric field within the Si channel.A set of devices studied in this work are nano-channel silicon-on-insulator (SOI) MOSFETs, as illustrated in 2O3. Doping concentration was estimated to be ND ≈ 1 × 1018 cm−3 from four-point probe measurements on reference samples. For this concentration, the average distance between neighboring P-donors is ~10 nm. Since the Bohr radius for P-donors in Si (rB) is ~2.5 nm, the P-donors can be considered as isolated from each otherThe top silicon layer was uniformly doped with P-donors by thermal diffusion from a spin-coated silica film containing PFz. The present study mainly focuses on the effect of such electric field on the potential well formed at the interface by a pair of neighboring P-donors.ND > 1 × 1019 cm−3). For such high concentration, selective doping design is necessary to preserve tunnel barriers in the channel so that tunneling conduction can still be observed without the formation of a highly-conductive channel, like in the case of junctionless transistors. We have also fabricated another type of SOI-MOSFETs in which the central region of the channel was selectively doped with higher concentration (IDS-VFG) characteristics were measured at low temperature (T = 5.5 K) with backgate voltage (VBG) as a parameter. In order to observe the effect of the electric field on transport through ground-states only, we map the current peaks only in the low-bias (low-VDS) region. At higher biases (higher VDS), more complex behavior is likely to be observed as transport could occur through multiple interactive dopants. Due to the random distribution of dopants, different behaviors are observed for different devices as a function of electric field. In this report, we present two significantly different cases among our observations. IDS-VFG characteristics at VBG = 0 V for devices labeled A and B, respectively. Several isolated current peaks can be observed before the onset of higher current. These current peaks have irregular intensities (IDS) and do not exhibit any periodicity, which excludes the possibility of a single multiple-electron QD as their origin. The stability diagrams (plots of IDS in the VFG-VDS plane) for each device are also shown in VDS, transport occurs by single-electron tunneling via a single QD, i.e., most likely the ground state of a donor. At higher VDS, more complex structures can be observed in the stability diagrams. Based on the quantitative analysis of these diagrams in the low-VDS region, we extracted typical parameters for each QD, such as the lever-arm factor, α, and the lateral position along the channel. The results of this analysis are shown in 78VDS can be explained based on transport through multiple dopants. The dynamical modification of the potential profile in such multiple-dopant system with the variation in bias and gate voltages could change the equivalent circuit from a single, isolated dopant to interactive, multiple dopants with increasing bias voltage (VDS). Such modification of the potential profile in a multiple-dopant system has been recently reported based on the potential mapping of doped Si nano-channels by Kelvin probe force microscope (KPFM)23VDS region to a multiple-dopant system in the high-VDS region can explain the occurrence of the complex features in the stability diagram at higher biases. Detailed explanations of the stability diagrams are presented in the VDS region only.For several lower-concentration devices, source-drain current vs front gate voltage (1–P3) can be observed in VFG’s . Higher-order current peaks appear near the onset of diffusion current for VFG > 1.7 V. Similarly, for device B, two isolated current peaks (labeled as P1 and P2) can be seen in VFG’s , followed by more complex current peaks for VFG > 1.72 V. The isolated current peaks at the lowest VFG’s can be ascribed to single-electron tunneling via P-donors with deeper ground-state energiesVFG can be ascribed to P-donors with shallower ground states. In addition to the main current peaks, faint current traces can be observed, such as seen for device B in 1. This faint trace is ascribed to tunneling transport through another P-donor energetically close to donor P1. The lower intensity of this current trace is most likely due to higher tunnel resistances for this satellite P-donor. These two donors are also likely located close to each other in the channel, forming an intriguing double-donor system that can be directly addressed by electrical measurements. We study the effect of a vertical electric field on the properties of this double-donor system working as a transport-QD for single-electron tunneling.For device A, three isolated peaks  for which the BOX-substrate junction changes from depletion into accumulation modeVFG-VBG plane are dictated mainly by the relative coupling of the transport-QDs to the front gate and to the back gate . For thin Si channel, as in our devices (~5 nm), CFG and CBG are dominated by the thickness of the gate oxide and buried oxide only. Therefore, these current traces appear as almost parallel to each other in the VFG-VBG plane, even though the QDs have different positions in the channel 19FC is relatively small and the transition occurs by a non-adiabatic tunneling process. On the other hand, if the donor is close to the interface, FC is larger and the transition process is adiabatic. For our measurements, the positive range of VBG covers a range of electric fields of 0-~50 mV/nm. Under such electric fields, transport is expected to occur via hybridized donor-interface QDs if the P-donors are close to the interface. This is the case of our devices that have an ultrathin Si channel (~5 nm), as seen in VBG, for which the electron wave function is extended in the system formed by the donor and the interface well.Under the application of this vertical electric field, the electron wave function moves away from the donor site toward the back interface18VBG. However, for device B, some peculiar behavior is observed in VBG (marked by dashed circles). If we follow the first current peak (P1) up to VBG > ~8.0 V, it is possible to observe a new current trace suddenly appearing at lower VFG. At the same time, the upper current trace still appears to continue for a range of VBG. A similar observation can be made for the second current peak (P2) at VBG > ~10.0 V and for the third current peak (not marked) at VBG > ~8.0 V. The estimated electric field at which these current shifts appear is in the range of 20–40 mV/nm. This is the unique phenomenon that we observe in the present study and understanding its origin is the main focus of the following analysis.For device A, the traces of the current peaks seen in VBG, the transport-QD consists of one P-donor’s potential well. However, as VBG is increased, the potential expands at the interface and in the lateral directionVBG. This merged potential well, formed near the interface, will thus become the new transport-QD having a suddenly increased gate capacitance. The system can be treated from this point on as an interfacial double-donor molecule. This model will be treated in more details in the Discussion section by simulations of potential landscapes and Coulomb blockade transport. The VFG and the increase of gate capacitance.The observation of a sudden shift of the current can be most likely ascribed to a sudden increase in the gate capacitance of the transport-QD. At lower In order to evaluate the possibility of this occurrence, we first estimate the probability of formation of merged donor-induced wells with increasing vertical electric field. For that purpose, we consider that the radius of lateral confinement for the donor-induced well changes with the electric field as calculated in Ref. N is the total number of donors in the channel, r is the radius of the lateral confinement of the donor-induced well and V is the volume of the channel. The total number of donor atoms in the channel is ~14, as calculated from channel dimensions (70 × 40 × 5 nmND = 1 × 1018 cm−3). After calculating the above probability, we can evaluate the number of donor-wells distributed as isolated and as merged. For instance, assuming that all donor atoms are situated at distance 3.8 nm from the Si/BOX interface, this evaluation leads to the results shown in rB from each other. We calculate the probability of finding systems containing 2, 3, and 4 merged P-donor wells assuming a Poisson distribution of the donors in the lateral planewhere A similar probability calculation was also carried out for the case of P-donors situated at 2.7 nm from the Si/BOX interface. The results are presented in Based on this probability calculation, it can be concluded that P-donors located closer to the front interface can readily merge their potential wells at the back interface as electric field is increased. On the other hand, if the donors are located closer to the back interface, there is a significantly lower probability to induce merging of their potential wells. This interpretation may be correlated with a specific P-donor arrangement in device B, which may be more favorable for the formation of the interfacial double-donor molecule system.VFG-VBG diagram within the positive-VBG region, focusing only on the two lowest-VFG prominent current peaks. As a way to emphasize the fine features, a second-order derivative of the current as a function of VFG is also presented in VFG in a range of positive VBG. The regions in which these current shifts occur are marked by dashed rectangles.IDS in the VFG-VBG plane. IDS-VFG characteristics (VBG = 0 V) are also shown as the side panel of IDS as a function of VFG, as illustrated in 31More complex interactions are expected for devices with channels selectively-doped with higher concentration. An example for one such device is shown in VFG as VBG is increased. As suggested earlier, this shift could be related to the merging of neighboring P-donor wells, a phenomenon which can occur with relatively high probability in the lower-concentration devices. Within this model, the electron wave function is first gradually shifted from the main transport-donor (P1) toward the interface and then it abruptly expands due to merging with a neighboring P-donor (P′1) well. This satellite donor is expected to be energetically and spatially close to the main transport-donor, but it gives rise to a lower-intensity current possibly because of higher tunnel resistances.The main experimental observations for device B with lower doping concentration are schematically summarized in y direction at 10 nm. The y-z cross-sectional potential landscapes for different electric fields are presented as insets in z = 0) ; (ii) at low vertical electric field (Flow); (iii) at higher vertical electric field (Fhigh). For zero-field condition [(i)], the two donor-wells are significantly separated from each other. For a low vertical electric field [(ii)], the potentials of both donors expand towards the interface and slightly in the lateral directionThe system can be illustrated by the electronic potential landscapes induced by the Coulomb potentials of such two P-donors. We consider a case of two donors situated at 3.8 nm and 4.5 nm away from the back interface and separated from each other in the VFG can be excluded. For instance, a current shift may be intuitively ascribed to trapping of a charge carrier into another P-donor or into an interface trap state. However, since the charge carriers are electrons, trapping of such a negative charge should shift the channel potential to higher values and VFG would shift to more positive values. This is inconsistent with the polarity of the experimentally observed shift (towards more negative VFG). An extended argument is introduced in Other possible reasons for the appearance of the new current trace at lower VFG when the donor-wells merge with each other, we study tunneling transport based on the Coulomb blockade theoryVFG.In order to theoretically explain the shift of the current peak to lower 1 works as main conduction path and is coupled to source and drain by tunnel junctions with capacitances CS1 and CD1, respectively. The satellite QD P′1 is coupled to source and drain via tunnel junctions with capacitances C′S1 and C′D1, respectively, but with higher tunnel resistances. The two QDs are coupled to the gates (frontgate and backgate) by non-tunnel capacitors with capacitances  and , respectively.For clarifying this point, we simulate the system of two donor-wells using a circuit of two parallel QDs, as schematically illustrated in VBG-dependent gate capacitance of the QDs. With this phenomenological model, simulation was carried out by using the successive circuits shown in IDS is plotted in the VFG–VBG plane in VBG. VBG is increased. This occurs for VBG beyond the flatband voltage, as the electron wave function expands at the back interface. Finally, VFG. This shift, ascribed to the sudden reduction of the system’s charging energy, is consistent with our experimental observation.The simulation parameters are estimated based on the device geometry and the proposed model. In addition, since the donor-well gradually expands at the interface, its gate capacitance is also expected to gradually increase with increasing electric field. This behavior is also incorporated in the present simulation as a VFG. Indeed, in VBG larger than a critical value. These two current peaks correspond to the double-electron occupancy of the merged QD (PM). This can explain the two current traces experimentally observed and illustrated in VBG, as marked by a dotted rectangle. The higher-VFG trace appears practically in the continuation of the original single-donor-well trace. This suggests that the potential of the interfacial QD containing one electron is comparable with the potential of the original single-donor well. Based on these results shown in this section, it can be concluded that this Coulomb blockade simulation approach provides qualitative support for the interpretation of our experimental findings. Additional simulation data shown in the Finally, it is expected that two electrons can be accommodated in such a double-donor QD, since each P-donor can accommodate only one electron, except at very low temperatures69VFG due to a reduction in the charging energy of the merged interface well. This result can provide a pathway to control the electron wave function in systems of multiple P-donors connected via an interface, essential for a wide range of dopant-based applications.In conclusion, we demonstrated experimentally that two donors in an ultrathin SOI-FET channel can merge their potential wells at an interface by applying vertical electric field. By monitoring the single-electron tunneling current, we observed a transition of the QD from a single-donor well to an interfacial double-donor molecule-like structure. This transition is observed as a sudden shift of the current peak to lower p-type Si substrate (doped with boron (B), with concentration ~1 × 1016 cm−3) is used as a backgate.Samples were fabricated from silicon-on-insulator (SOI) wafers, with a 150-nm-thick buried oxide (BOX) layer. All processes have been carried out in a clean room environment, using CMOS-compatible techniques. Initial SOI thickness was 55 nm, but it was thinned down by sacrificial oxidations and usual oxidation processes to a final thickness of ~5 nm (as measured by transmission electron microscope (TEM) images). Channel patterning was done using an electron-beam lithography (EBL) technique, with the channel width set as the main parameter  for uniformly doped low concentration devices. High quality gate oxide is formed by thermal oxidation process with an average thickness of ~10 nm for all devices. However, for the selectively-doped devices, gate oxide was formed by wet thermal oxidation  in order to minimize lateral thermal diffusion of dopants after selective doping. Standard Al metallization technique is adopted to make the gate, source, and drain contacts. The I-V measurements, carried out mainly at low temperatures (~5.5 K). In all measurements, the source electrode was grounded and a small bias was applied to the drain electrode . The frontgate voltage (VFG) and backgate voltage (VBG) were swept as variable parameters.Electrical characteristics were measured with Agilent 4156C and Agilent B1500A precision semiconductor parameter analyzers using a variable-temperature prober station. The samples were placed in a high-vacuum chamber for the IDS-VFG characteristics are calculated numerically using Monte Carlo simulationVBG. According to the orthodox theory of Coulomb blockade, forward and reverse tunneling rates of electrons across a junction are given by:The theoretical e is an elemental charge and ΔE± is the change in total charging energy due to forward and reverse tunneling events. The temperature T is set to be 5.0 K for the present study, consistent with our experimental conditions. By considering a suitable range of tunneling parameters based on the experimental situation, IDS-VFG characteristics are simulated in the experimental range of positive VBG.where How to cite this article: Samanta, A. et al. Electric-field-assisted formation of an interfacial double-donor molecule in silicon nano-transistors. Sci. Rep.5, 17377; doi: 10.1038/srep17377 (2015).


Background. The group A meningococcal conjugate vaccine, PsA-TT, uses tetanus toxoid (TT) as a carrier protein (PsA-TT). TT as a carrier protein in other conjugate vaccines is known to be immunogenic and generates a robust anti-TT response.Methods. Clinical studies in Africa assessed whether PsA-TT generated tetanus serologic responses when tested in African populations (toddlers to adults). Second, the high acceptance of PsA-TT mass immunization campaigns in the 1- to 29-year age group meant that a sizeable fraction of women of reproductive age received PsA-TT. Incidence data for neonatal tetanus were reviewed for countries with and without PsA-TT campaigns to check whether this had any impact on the incidence.Results. PsA-TT generated robust tetanus serologic responses in 1- to 29-year-olds, similar to those expected after a booster dose of TT. Neonatal cases of tetanus fell by 25% in countries that completed PsA-TT campaigns in 1- to 29-year-olds.Conclusions. Although these data are not yet definitive, they are consistent with the hypothesis that improved community immunity to tetanus as a result of the PsA-TT campaigns may be having an impact on the incidence of neonatal tetanus in sub-Saharan Africa.Clinical Trials Registration. ISRCTN17662153 (PsA-TT 001); ISRTCN78147026 (PsA-TT 002); ISRCTN87739946 (PsA-TT 003); ISRCTN46335400 (PsA-TT 003a); ISRCTN82484612 (PsA-TT 004); CTRI/2009/091/000368 (PsA-TT 005); PACTR ATMR2010030001913177 (PsA-TT 006); and PACTR201110000328305 (PsA-TT 007). Maternal and neonatal tetanus is still a significant, although vaccine-preventable, cause of morbidity and mortality in many developing countries. When tetanus develops, case-fatality rates are high and treatment is often limited by paucity of resources. The Maternal and Neonatal Tetanus Elimination Initiative, relaunched by the World Health Organization (WHO), United Nations Children's Fund, and United Nations Population Fund in 1999 following the initial launch in 1989, has made substantial progress in eliminating maternal and neonatal tetanus ) and Indian (96.2% 327/340]) children (27/340) cThe 3 consecutively produced lots of PsA-TT vaccine, when administered as a single dose in children 5–10 years of age, gave consistent anti-TT IgG GMCs 4 weeks following vaccination of 24.1 , 20.3 , and 25.3  IU/mL. All subjects who were assayed for anti-TT IgG had antibody concentrations ≥0.1 IU/mL after vaccination.P < .0001 for both a full dose of PsA-TT vs EPI only and a half-dose of PsA-TT vs EPI only). By 15 to 18 months of age, the anti-TT IgG GMCs remained significantly higher in children who received PsA-TT than those who had only received the EPI schedule  .In healthy 9- to 12-month-old Malian children, a single immunization with either a full or half dose of PsA-TT elicited a marked boost in anti-TT IgG GMCs. Four weeks following the immunization given to 9- to 12-month-olds, anti-TT IgG GMC was significantly higher in children who received PsA-TT than those who only received the EPI (measles and yellow fever) vaccines  Figure . Four weTable Average annual cases of neonatal tetanus in countries that had mounted regional PsA-TT campaigns Table  were uncFor the 5 countries  that had not yet mounted PsA-TT campaigns Table , the aveIn all age groups and in all countries studied, PsA-TT significantly boosted anti-TT IgG levels. The magnitude of the response was not significantly different compared with a full dose of TT vaccine in healthy Indian adults aged 18–35 years and a Hib-TT vaccine in healthy African toddlers aged 12–23 months. Given the low percentage (48.3%) of 11- to 17-year-old Africans, recruited from Mali, Senegal, and the Gambia, with anti-TT IgG concentrations ≥0.1 IU/mL, an extra booster dose of TT is of huge benefit. as many of these subjects will go on to be future mothers. One striking difference in baseline anti-TT IgG concentrations was demonstrated in the 2- to 10-year age group where Indian children had significantly higher levels than African children. One explanation for this difference lies in the fact that the Indian Universal Immunization Programme recommends at least 7 doses of TT: 3 doses of DTP in infancy, 2 booster doses at 16–24 months, 5–6 years of age, and at 10 and 16 years of age, as opposed to the 3-dose EPI schedule in Africa , 15.The neonatal tetanus surveillance data from meningitis belt countries are interesting but incomplete. In countries that have mounted national campaigns, there has been a decrease in reported cases of 25%. This has not been noted in countries that have not yet mounted campaigns or in countries that had partial campaigns. Neonatal tetanus is an underreported disease, and caution needs to be taken in overinterpreting surveillance data. Nonetheless, the information from countries that have completed PsA-TT campaigns is encouraging and is consistent with the tetanus serologic data from the PsA-TT clinical trials in Africa. Increasing immunity to tetanus in women of reproductive age doubtlessly happened at a result of the introduction of PsA-TT in 1- to 29-year-olds, but the extent to which this has translated to decreases in the incidence of neonatal tetanus needs to be further quantified.Supplementary materials are available at Clinical Infectious Diseases online . Supplementary materials consist of data provided by the author that are published to benefit the reader. The posted materials are not copyedited. The contents of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author.