Proteins and bacteria on the skin of amphibians can protect them from diseases. Marking frogs with elastomer tags had no effect on stress hormone production or protein release, but lead to a slightly greater abundance of skin microbes. This marking technique may be a comparatively safe option for amphibians. Batrachochytrium dendrobatidis. Events that cause disruption of symbiotic bacterial communities or deplete peptide stores could increase the susceptibility of individuals to disease and may have implications for amphibians involved in probiotic trials or time course studies that investigate symbiotic bacterial communities. It has previously been shown that passive integrated transponder tagging of frogs causes a rapid (within 24 h) and major proliferation of micro-organisms on the skin. Here, we show that marking of red-eyed tree frogs  with visible elastomer has no effect on adrenal response  or peptide production, although there was evidence of a slightly greater microbial abundance associated with the skin of marked frogs 2 weeks after tagging. The results indicate that visible elastomer may be a preferable marking technique to passive integrated transponder tagging, particularly in the context of probiotic trials or time course studies that investigate symbiotic bacterial communities. More work is required to determine the effects of different marking techniques on physiological responses of amphibians, whether these physiological responses are consistent across host species and whether such ‘non-invasive’ marking methods affect the susceptibility of amphibians to infectious pathogens, such as B. dendrobatidis.Amphibians possess innate immune defences, including antimicrobial peptides and symbiotic bacterial communities, that can protect them from infectious diseases, including chytridiomycosis. On-going research is attempting to use amphibian symbiotic bacteria to develop probiotic treatments that can protect hosts from the causative agent of chytridiomycosis, the fungal pathogen  Batrachochytrium dendrobatidis, from a number of amphibian species  tagging or visible implant elastomer (VIE) dyes, are now more commonly used. The existing literature indicates that these marking techniques have few negative effects on amphibians  marked with VIE or toe-clipped under varying degrees of anaesthesia did not exhibit increased noradrenalineor adrenaline levels in comparison to unmarked salamanders. Handling has been shown to increase circulating glucocorticoid concentrations in a range of amphibian species .There are limited studies that have investigated the effects of marking techniques on steroid  production in amphibians.  species , 2013, aThe Chester Zoo Ethical Committee and the University of Manchester Ethics Committee approved this study prior to starting. This study did not require a UK Home Office licence because VIE tagging is an approved method for the identification of frogs, and data were collected during routine marking of frogs for identification purposes. Frogs were checked daily throughout the study and for 2 weeks after for any signs of adverse reaction to any of the techniques used, none of which was observed.A. callidryas were used in this study; eight  in the control (unmarked) group and eight  in the marked group. All frogs were from the same clutch of F3 generation captive-bred individuals and had not previously been involved in any other studies. Prior to the start of the experiment, frogs were group housed in 60 × 45 × 45 cm glass tanks  with a naturalistic set-up consisting of a LECA base for drainage and a soil layer covered with dried oak leaves, and planted with a peace lily and cuttings of devil's ivy. Two weeks prior to the start of the study, frogs were randomly assigned to 16 individual experimental housing tubs (30 × 18 × 20 cm ExoTerra® plastic faunariums), with frogs from the same group housed tanks spread across treatment groups to allow for prior variation in bacterial communities. Individual housing was required to distinguish faecal samples between individuals and to avoid the potential for bacterial communities to be confounded by group housing during the study. Experimental tanks were lined with damp paper towels and contained a water dish and a cutting of devil's ivy. Paper towels and water dishes were changed twice weekly. All tanks were maintained with a ZooMed Reptisun® 10.0 ultraviolet strip light with reflectors and a Philips daylight bulb with reflectors, both of which were on a 10:14 light:dark cycle. Frogs were fed three or four times weekly with black crickets (Gryllus bimaculatus) gut-loaded on fresh fruit and vegetables and dusted with Nutrobal .A total of 16 adult Faecal glucocorticoid metabolite concentrations’ section below for further details). On day 8, frogs were sampled for bacterial communities and peptide release as described below . This provided baseline data for each individual, which is referred to hereafter as ‘start of study’. On day 14, frogs in the ‘marked’ group were tagged using VIE dye (see ‘Marking techniques’ below for details). On day 15, all frogs were sampled again for bacterial communities and peptide release, and these data are referred to as ‘post-marking’. On day 28, bacterial communities and peptide release were sampled again, and these data are referred to as ‘end of study’.After a 2 week habituation period in the individual housing, faecal samples were collected daily from day 1 until the end of the study (day 28) to measure faecal glucocorticoid metabolite concentrations . This method was chosen because frogs were due to be rehomed at another institution and it was necessary to distinguish these frogs from existing A. callidryas in the collection at the receiving institution without the requirement for individual identification. The elastomer dye was prepared according to the manufacturer's instructions. Gloves were worn throughout marking and changed between frogs to minimize cross-contamination. During marking, the frog was restrained on a flat and stable surface that had previously been sterilized, and the sterile needle containing the dye was inserted smoothly and quickly under the skin of the underside of the tibiofibular . The marking process was standardized and took ∼30 s per individual. In order to determine whether the whole marking process affected physiological traits of frogs, control frogs were not sham injected or manipulated at the post-marking time point. All frogs were monitored for the remainder of the study and for 2 weeks after the end of the study for any signs of adverse reaction, none of which was observed.A. callidryas are nocturnal and so defaecate at night), and any faecal samples were frozen at −20°C for subsequent analyses. Gloves were used to handle samples and changed between to avoid contamination. Glucocorticoid metabolite analyses were conducted at the Chester Zoo's Wildlife Endocrinology Laboratory.Tanks were checked daily within an hour of the lights coming on , and 50 μl per well was loaded onto a 96-well Nunc-Immuno Maxisorp (Thermo-Fisher Scientific) microtiter plate, which was covered with a plate sealer and left overnight at 4°C. Plates were washed five times  and then 50 μl per well of each standard and sample (3.9–1000 pg per well or samples diluted 1:10 in EIA buffer) were loaded in duplicate with 50 μl per well of horseradish peroxidase conjugate diluted in EIA buffer (1:70 000). Following incubation in the dark for 2 h at room temperature, plates were washed five times and incubated with 100 μl per well of substrate , left to develop at room temperature in the dark and measured at 405 nm at optical density 0.8–1.0. The corticosterone EIA was biochemically validated for measuring corticosterone metabolites in A. callidryas faeces through parallelism  and matrix interference assessment . The cross-reactivities for corticosterone antiserum CJM006 have been reported elsewhere .Glucocorticoid metabolites (corticosterone) were analysed using a modified enzyme immunoassay (EIA) as previously described  and stanpost hoc analysis for pairwise comparisons.Final corticosterone metabolite concentrations were calculated per gram of wet faecal mass. Data collected before any handling or marking events days 1–7) were assigned as ‘start of study’, data collected from 1 day post-marking until 5 days post-marking (days 16–19) were assigned as ‘post-marking’, and data collected from day 20 until day 28 were assigned as ‘end of study’. Raw data values for faecal glucocorticoid metabolite concentrations indicated an increase at around 3–5 days post-marking . Frogs were then swabbed on their ventral surface 20 times using sterile swabs . Gloves were worn throughout the swabbing process, which took ∼30 s per frog, and changed between individuals to minimize cross-contamination. Swabbing methods were consistent across individuals, treatment groups and time points. Swabs were placed into 1 m NaCl2 solution and serial dilutions constructed up to a concentration of 10−2. Concentrations of 10−1 and 10−2 were plated out on low-nutrient agar . Bacterial and fungal colony abundances were counted every 2 days until negligible new growth was observed . Bacterial and fungal counts were then multiplied by their respective dilution factors and averaged for each individual. Data for bacterial abundance and fungal abundance were analysed separately in RStudio (using the lme4 package) for effects of treatment, time point and the interaction between treatment and time point using a generalized linear mixed model with Poisson distribution, with individual included as a random effect. Tukey's post hoc analysis was conducted for pairwise comparisons.Microbial communities were collected using similar methods to those previously described by Prior to sampling for cutaneous microbes, frogs were rinsed on their dorsal and ventral surfaces by pouring 50 ml of ultrapure water over each surface five times. The final volume of liquid collected was recorded, and peptides were extracted from the water using previously described techniques  according to the manufacturer's instructions, except that bradykinin  was used to establish a standard curve  but not treatment  or the interaction between treatment and time  and between control frogs post-marking and at the end of the study (P = 0.001), with end-of-study faecal glucocorticoid metabolite concentrations being lower at the end of the study than at the other two time points , and post hoc analyses showed that faecal glucocorticoid metabolite concentrations at the end of the study were significantly lower than those at the start of the study (P < 0.001) and post-marking (P < 0.001) across all frogs, but there were no significant differences between faecal glucocorticoid metabolite concentrations at the start of the study and post-marking (P = 0.377).There was a significant effect of time  and the interaction between treatment and time  on bacterial abundance, but not of treatment alone . Post hoc analyses showed overall that there were significant decreases in bacterial abundance over time for both treatment groups, and the bacterial abundance associated with control frogs at the end of the study was significantly lower than that of marked frogs at all time points  on the abundance of fungi associated with the skin of frogs, but no significant effects of treatment  or the interaction between time and treatment . Post hoc analyses showed that marked frogs had a significantly greater fungal abundance at the end of the study than post-marking , treatment  or the interaction between treatment and time  on the release of peptides by frogs .There were no significant effects of time , with frogs maintained individually exhibiting lower faecal glucocorticoid metabolite concentrations than those maintained in groups of two or eight, and those maintained in group housing exhibiting a decline in glucocorticoid metabolite concentrations over the 4 week study period.In this study, we found no evidence that VIE marking of frogs leads to an increase in faecal glucocorticoid metabolite concentrations in comparison to unmanipulated control animals. It has previously been shown that marking ng alone , b, 2013A. certus and A. glyphus, and adrenocorticotrophic hormone challenges in these species indicated a similar lag time in glucocorticoid metabolite deposition in the faeces as for A. callidryas in this study , indicating that these species may primarily deposit glucocorticoid metabolites in their faeces.Faecal samples have been used to measure glucocorticoid metabolite concentrations in a range of taxa . llidryas , fungi wB. dendrobatidis after tagging with VIE and other marking systems. Moreover, culturing methods are known to underestimate microbial diversity greatly  on the skin of marked frogs is unclear. The VIE tags were unlikely to be completely sterile due to the preparation methods, and the greater abundance of microbes may reflect a minor infection from the tags. Alternatively, it is possible that the act of marking caused a disruption in the dynamics of the bacterial communities that may have had delayed effects for the microbial community or elicited a physiological response in the frogs. For example, studies in rats and humans have shown increases in immunoglobulins, lymphocytes and macrophages in response to injection of silicone  than for PIT tagging (18 gauge or 1.27 mm), and the ‘foreign body’ inserted under the skin is much smaller; therefore, frogs undergoing PIT tagging may experience greater adrenal activity than those that are VIE tagged, leading to differences in the responses of the microbial community .The results presented here are in contrast to the results found by A. callidryas. This result is expected given that VIE marking also had no effect of faecal glucocorticoid metabolite concentrations, which have the potential to inhibit the production of new peptides for storage , particularly in the context of probiotic trials or time course studies that investigate symbiotic bacterial communities. Although more work is required to determine differences between host species in response to different marking techniques, as well as the effects of marking on the susceptibility of amphibians to disease, these results together with those of Overall, we have shown that there is no effect of VIE marking on adrenal response  or peptide release of Conservation Physiology onlineSupplementary material is available at .This work was supported by a Biotechnology and Biological Sciences Research Council (BBSRC) PhD studentship to R.E.A.


Quantitation of huntingtin protein in the brain is needed, both as a marker of Huntington disease (HD) progression and for use in clinical gene silencing trials. Measurement of huntingtin in cerebrospinal fluid could be a biomarker of brain huntingtin, but traditional protein quantitation methods have failed to detect huntingtin in cerebrospinal fluid. Using micro-bead based immunoprecipitation and flow cytometry (IP-FCM), we have developed a highly sensitive mutant huntingtin detection assay. The sensitivity of huntingtin IP-FCM enables accurate detection of mutant huntingtin protein in the cerebrospinal fluid of HD patients and model mice, demonstrating that mutant huntingtin levels in cerebrospinal fluid reflect brain levels, increasing with disease stage and decreasing following brain huntingtin suppression. This technique has potential applications as a research tool and as a clinical biomarker. HTT) gene56789101112in vivo?Huntington disease (HD) is a fatal, inherited neurodegenerative disease that is uniquely caused by expansion of a polyglutamine encoding CAG trinucleotide repeat in the huntingtin . IP-FCM is a highly sensitive method of protein detection that can be applied to either single proteins or those found in multiprotein complexes205.5), which suggested that this combination might be able to detect extremely low abundance HTT protein, as might be found in CSF.IP-FCM requires a pair of antibodies, a capture and a probe, that are simultaneously able to bind their protein target in its native state. Thus, we first performed a screen using 12 anti-HTT antibodies previously validated in assays performed in native conditions  or cKO brain lysate, serially diluted in 2- or 5-fold steps, and assayed using HBD4/MW1 or MW1/BKP1. As shown in Next, we performed a dilution experiment with recombinant protein to determine the sensitivity of the assays. A fusion protein consisting of HTT amino acids (aa) 1–171 (with 15 or 65 Q) and aa 1744–2234  was prodHTT IP-FCM was used to measure HTT protein in CSF of control, premanifest HD mutation carriers, early/mid stage HD, and late stage HD individuals . MW1/BKPSupporting this, CSF muHTT IP-FCM signal correlated with multiple clinical measures in premanifest and early/mid HD individuals. A strong correlation between CSF muHTT protein level and universal Huntington’s disease rating scale (UHDRS) motor score was observed in premanifest individuals (p = 0.003) and to a lesser degree in early/mid HD individuals (p = 0.0241), but not in late stage HD individuals (p = 0.4170) . We founTo investigate the effect of brain HTT lowering on CSF HTT levels, Hu97/18 mice were treated with a human HTT specific ASO expected to suppress both wt and muHTT protein . Four diThe response of CSF muHTT protein levels to brain HTT suppression indicates that the brain is a source of CSF muHTT protein. In addition, the lack of muHTT protein signal in CSF of some premanifest HD mutation carriers suggests that muHTT protein may not be a normal component of CSF and that it may be released into CSF in a disease state. To further investigate the source of muHTT protein in the CSF, we used BACHD miceAs the fundamental cause of HD, reducing levels of muHTT protein in the brain has substantial therapeutic potential, and a number of HTT lowering therapies are currently in pre-clinical and clinical development. Clinical trials of HTT lowering therapies will require biomarkers of brain HTT levels in order to validate and quantify target engagement and efficacy and to inform appropriate dosing. The HTT IP-FCM assays developed here represent essential tools for this purpose.Moreover, these assays are valuable research tools that can be used to investigate features of HD and the HTT protein. For instance, the two assays developed here, though both specific for muHTT protein, likely detect different pools of muHTT. With MW1 binding in exon 1 and HDB4 binding at an epitope C-terminal to all known cleavage sites, this assay likely detects full-length muHTT protein. The BKP1/MW1 assay, however, employs two antibodies binding in exon 1 of HTT, indicating that this assay likely detects both full-length and N-terminal fragments of muHTT, which are known to be pathogenic and enriched in HD striatum28In this study, we have used HTT IP-FCM to determine that muHTT protein increases in the CSF of HD mutation carriers with disease stage. CAG tract length correlates with CSF muHTT level in HD mutation carriers, as do the clinical measures disease burden and age of onset, both of which are influenced by CAG tract length.We also identified correlations between CSF muHTT IP-FCM signal and multiple motor and cognitive clinical measures that are independent of CAG tract length, indicating a significant contribution of disease to CSF muHTT protein level. Additionally, in some premanifest HD mutation carriers, no detectable muHTT protein was observed in CSF. Interestingly, we found that premanifest individuals that lacked muHTT protein in CSF were also those that did not score on the UHDRS motor test, while those that did have muHTT in CSF also had sub-clinical motor deficits. This reflects the contribution of disease to CSF muHTT protein level irrespective of CAG tract length. For example, subjects 31 and 41 both have 45 CAG and were collected at 39 years of age, yielding identical disease burdens and predicted onsets. However, subject 31 has a low UHDRS motor score of 2 and a low CSF HTT signal (MFI 63), while subject 41 has a high sub-clinical UHDRS motor score of 8 and a high CSF HTT signal (MFI 332). Taken together, these results suggest that muHTT protein may not be a normal component of CSF or is only present in minute amounts, but is released to the CSF in a disease state, possibly as a result of injured or dying cells. This hypothesis is supported by our finding that QA injection induces a transient increase in CSF muHTT protein levels. Overall, we find that CSF muHTT IP-FCM signal varies with multiple factors including CAG tract length, disease burden, disease stage, motor and cognitive scores, and other unknown factors. However, for longitudinal studies within the same patients, the contribution of CAG tract length and other non-disease related factors to CSF muHTT IP-FCM signal would be the same in baseline and subsequent samples, allowing for accurate relative quantitation. Thus, HTT IP-FCM in CSF could provide a biomarker of HD onset.Additionally, using HTT IP-FCM, we have found that brain HTT suppression results in reduction of muHTT protein in CSF of HD model mice, and that CSF muHTT levels are correlated with brain muHTT levels in ASO treated animals. Moreover, we find that knocking out muHTT selectively in the nervous system of BACHD mice results in a dramatic reduction of CSF muHTT protein. These results indicate that the brain is a major contributor, if not the source, of CSF muHTT protein, and that muHTT IP-FCM in CSF could provide a critically needed pharmacodynamic and target engagement biomarker for upcoming HTT lowering clinical trials.These findings also have far-reaching implications for a wide range of CNS indications, including idiopathic diseases for which IP-FCM CSF biomarkers could be identified as predictors of disease onset in at risk individuals or as biomarkers in therapeutic trials.BKP1, HD46, and HD650 antibodies236/L, range 0–171; median leukocyte count 1.0 × 106/L, range 0–17).All human samples were collected with an approved protocol and in accordance with the guidelines of the institutional review board of the University of British Columbia and the full informed consent of the subjects. Cerebrospinal fluid from HD gene positive and control individuals was collected at the University of British Columbia’s Centre for Huntington Disease. Thirty subjects with the HD gene mutation and 7 control subjects, age-matched and lacking the HD mutation, were recruited see . CSF wasHTT gene  was delivered by intracerebroventricular (ICV) injection as inHu97/18Brain HTT protein was quantified by allelic separation immunoblotting as in2PO4 1.25, MgCl2 1, NaHCO3 26, CaCl2 2 Dextrose 25). A dilution series was made and IP-FCM was performed (see below).For the calibration curves, a recombinant Htt fusion protein was generated that contained the antibody binding sites for MW1, BKP1  and HDB4 (aa1844–2131) . Recombi4 beads in 5 ul NP40 buffer were mixed with either 25 ul of recombinant protein in ACSF, or 50 ul of brain lysate, or 50 ul of human CSF, or 10 ul of mouse CSF and left to immunoprecipitate HTT overnight at 4 °C with rotation to prevent beads settling out of suspension. Beads were then washed in IP-FCM buffer , 0.01% Sodium Azide) and incubated with biotinylated probe antibodies for two hours, followed by another wash in IP-FCM buffer, incubation with 1:200 Streptavidin-PE (BD Biosciences) for one hour, a final wash, and measurement on an Accuri flow cytometer (BD Biosciences). Bead doublets were gated out based on forward scatter area vs. forward scatter height plots, and a singlet bead gate was defined based on forward scatter height vs. side scatter height. All samples were run in duplicate , and the average of the median fluorescence intensity in the FL2 channel in the singlet bead gate indicated the abundance of HTT in the sample.The IP-FCM technique has been previously described20212 and p values indicated on graphs. P values of less than 0.05 were considered statistically significant. N’s represent individual mice or human samples. All IP-FCM samples, except for mouse CSF samples where sufficient volume was not available, were run in duplicate and averaged to obtain the single value for each sample.Differences between sample means were analyzed by two-tailed Student’s T-tests, and one or two-way ANOVAs were used for multiple comparisons, indicated in the text. Correlations were analyzed by linear regression with rHow to cite this article: Southwell, A. L. et al. Ultrasensitive measurement of huntingtin protein in cerebrospinal fluid demonstrates increase with Huntington disease stage and decrease following brain huntingtin suppression. Sci. Rep.5, 12166; doi: 10.1038/srep12166 (2015).


R0). Decreasing R0 while augmenting cross-sectional collection area with multiple donors significantly increases terminal exciton delivery efficiency within dendrimers compared with the first linear constructs. Förster modelling confirms that best results are obtained when there are multiple interacting FRET pathways rather than independent channels by which excitons travel from initial donor(s) to final acceptor.DNA demonstrates a remarkable capacity for creating designer nanostructures and devices. A growing number of these structures utilize Förster resonance energy transfer (FRET) as part of the device's functionality, readout or characterization, and, as device sophistication increases so do the concomitant FRET requirements. Here we create multi-dye FRET cascades and assess how well DNA can marshal organic dyes into nanoantennae that focus excitonic energy. We evaluate 36 increasingly complex designs including linear, bifurcated, Holliday junction, 8-arm star and dendrimers involving up to five different dyes engaging in four-consecutive FRET steps, while systematically varying fluorophore spacing by Förster distance ( DNA is a useful molecule with which to construct nanomaterials with controllable functionalities. Here, the authors fabricate photonic wires by appending dye molecules at set positions along DNA structures, and show how FRET performance can be tuned by modifying dye separation. Structural DNA technology can create nanoassemblies having almost any conceivable multi-dimensional shape ranging from nanoscale world maps to gears and nanoflasks123426781068141718192224252678293122133The most advanced use of DNA-organized fluorophores and FRET has been photonic wires, in which dyes are arranged with the goal of producing directed or sequential energy transfer35391720R0). Optimizing dye placement by decreasing their spacing while increasing effective collection area with multiple initial donors in different geometries provides for >500-fold increase in terminal exciton delivery efficiency within the dendrimer structures in direct comparison to the first linear four-dye construct  placed at 1.5 × R0. Detailed Förster modelling reveals that crucial to the enhancement is the number of FRET pathways, where the best results are observed when there are multiple interacting rather than independent channels by which excitons travel from initial donor(s) to final acceptor. These studies also reveal certain non-idealities that appear to be explainable by formation inefficiency, inadequate fluorophore performance and a lack of control over dye orientation. Our results suggest possible design criteria by which increasingly sophisticated FRET-based DNA devices may be achievable.Here, we utilize the power of DNA architecture to move beyond linear photonic wires to achieve more sophisticated DNA-arranged networks involving as many as 85 organic dye molecules engaged in programmable FRET cascades. We evaluate 36 antenna designs of increasing complexity by assembling >550 different DNA constructs. These include linear, bifurcated, Holliday junction, 8-arm star and 2:1, 3:1 or 4:1 branching dendrimer structures with either two-, four- or five-dye types, engaged in one-, three- or four-consecutive FRET steps, respectively, where inter-fluorophore spacings are systematically varied in increments of dye-pair Förster distances (R0 constructs the dyes have enough separation so that we did not observe adverse fluorophore–fluorophore interactions such as heterodimer formation (data not shown). See In choosing the dyes, the possibility of them being photophysically perturbed/quenched by DNA is well-known although this phenomena still lacks a predictive understanding4242444546474830353739de novo structures and adaptations from previous reports; see R0 , characterizing the range of FRET interactions in the point-dipole approximationWith downstream utility in mind, our design approach was kept simple: all structures should self-assemble by one-pot hybridization without requiring enzymatic ligation, and to avoid potential degradation in yield, structures would be directly assessed without post-assembly purification. The three different structural systems studied here  are basen→Cy5  at nominal values of 0.75 × , 0.85 × , 1.0 × , 1.25 × and 1.5 × R0 with predicted FRET efficiencies ranging from ~80% to 5%, respectively. The second system . Given that these assemblies were designed to sample a linearly increasing range of either initial discrete Cy3n donor or three-dye [Cy3→Cy3.5→Cy5]n photonic wire donors, the latter 1.0 × and 1.5 × R0 spacings were included to specifically evaluate if multiple donors at longer spacings could augment energy transfer by increasing the probability that FRET occurs.The two-dye single-FRET step system, designated Cy3n→Cy5 , consistd system  involvedn→Cy3.5]n→Cy5]n→Cy5.5 with n=2,3,4 . Due to the structural complexity , only dye spacings of 0.5 × R0 were studied. One five-dye dendrimer design (2→Cy3]2→Cy3.5]2→Cy5/AF647]2→Cy5.5 configuration with either Cy5/Alexa Fluor 647 (AF647) in the fourth penultimate position. In addition to the two-, four- and five-dye photonic wire/dendrimer constructs with their full complement of dyes, we analyzed all partially functionalized forms, with dye display evolving from initial donor to fully decorated structure. Structures with one- or two-dyes missing were also examined to estimate longer range FRET contributions. Unlabelled DNA was always substituted for labelled DNA to retain the same underlying structure. Although depicted in a flat two-dimensional (2D) perspective  or in a three-step FRET cascade across a ~180 nm portion of the spectrum to a terminal Cy5.5 (λmax em. 694 nm). Alternatively, AF488 can be excited at 460–490 nm and transfer energy across ~250 nm in a four-step cascade providing for Cy5.5 sensitization. Relevant dye photophysical parameters allowed calculation of spectral overlap integrals (J) and R0 values for each donor–acceptor pair . All samples were prepared in 2.5 × PBS without Mg2+ to avoid dye quenching issues at high millimolar ionic concentrations521→Cy5 system as dye spacing was varied in comparison to Cy3 alone ; definitions in Methods, E estimates efficiency of exciton delivery. TEF utilizes a single upstream excitation and monitors output from the final acceptor with the 1.5 × R0 linear constructs typically used as reference. Since the latter has the fewest initial donors and largest dye separations, it manifests weakest sensitization and so TEF will give large values. Its merits are that it thus amplifies improvements while also providing perspective on final output performance across all assemblies in a given system as donor number(s), dye spacing and geometries vary. AE looks at relative efficiency increases between a higher energy excitation/sensitization of multiple donors and direct excitation of a given acceptor, providing a measure of efficiency within the same structure. Plotting TEF as a function of donor number for each donor–acceptor spacing in the Cy3n→Cy5 system shows that acceptor output can be magnified >60 times by decreasing inter-fluorophore separation to 0.75 × R0 and increasing donor number to four , antenna effect (AE) and exciton end-to-end efficiency  to 27 (33) to 64 (43) (E at 23% (The dendrimeric systems have larger numbers of chromophores arranged with higher packing densities. Designs are based on Luo’s structures 64 (43) , and ini 64 (43) . NormaliE at 23% . We estiE at 23% , and par2→Cy3]2→Cy3.5]2→Cy5]2→Cy5.5 construct with 0.5 × R0 dye spacing. Spectra collected as they evolve to incorporate all five dyes , suggesting that the problem is associated with DNA assembly rather than dye performance (2→Cy3.5]2→Cy5]2→Cy5.5 four-dye 2:1 dendrimer structure with the Cy3.5 ester dye (flexible tether) replaced by one inserted into the DNA as a localized phosphoramidite .We extended the 2:1 dendrimer design by adding an initial AF488 donor to create a [[AF488ive dyes  show sigive dyes . Despiteectively . Modelliectively  and 28. formance . Lastly,ramidite . Here E ectively . The incE and TEF for the photonic wire and dendrimer systems (R0 construct as a reference (left axis). These data also show that TEF can still be enhanced by ~12 × even in the linear 1.5 × R0 constructs despite three intervening FRET steps if donor wire number and geometry are optimized. This enhancement approaches 40 × at 0.5 × R0 dye spacing. If the sensitized emission of Cy5.5 from the linear 0.5 × R0 construct is used as a reference (right axis), a nearly 12-fold enhancement is observed in the 2:1 dendrimer along with a 40-fold enhancement in the 3:1 dendrimer, in a manner that exceeds a simple ratiometric relationship between initial donor(s) and terminal acceptor. This is a striking example of how DNA can pattern molecular dyes to markedly increase fluorescent output. Normalized emission spectra for the 0.5 × R0 2:1 dendrimer (Cy3.5 phosphoramidite) and the eight-arm photonic wire star structures were directly compared  with branching ratios of 2:1, 3:1 and 4:1 n, reflecting basic antenna properties. However, increases are not continuous as issues plague the more complex structures. Net exciton delivery efficiency E over three or four FRET steps is substantially improved compared with that in our previous work39E of ~20% across four-FRET steps combined with AE of 3.9 and TEF of 550 , would not have been provided with use of only purified structures regardless of the assembly method. Following a first pass of construct formation, we did attempt to improve FRET E and formation efficiency through optimization of hybridization. Several different hybridization permutations were attempted with select linear and dendrimer structures, with improvements only seen for two of the dendrimers. Increasing to 12 h hybridization resulted in a doubling of the 3:1 and 4:1 dendrimer E values although FPLC analysis revealed assembly was only ≤10% more efficient (data not shown), suggesting improvement in placement of key internal FRET dye components . Data values of the latter are only used here for brevity. No improvements to the 2:1 dendrimer assembly or E were seen. Systematic-parametric testing of hybridization protocols could certainly help improve formation efficiency for a desired structure, however, this was beyond the current scope due to the variety of different systems involved.Our experimental approach did not include DNA assembly purification but instead looked at ensemble properties of unpurified product. Our structures are relatively simple in the number of oligos per construct, which both simplifies experiments and complicates analysis. This was necessitated by the difficulties of separating these fully formed products from partial structures of similar size and charge. In contrast, when using an origami assembly-based approach, separation from excess staple strands is easier due to the dramatic difference in size of the final assembly, but the cost associated with hundreds of different oligos for each construct and the need for excess dye-labelled staples made this approach impractical (cost prohibitive). Far denser dye-networks are probably achievable on origami. It should be noted, however, that insight into one of our more interesting and potentially important findings, that multiple redundant FRET pathways compensate for assembly deficiencies in the dendrimer architecture  to a more rigid version by using a double phosphoramidite insertion . This improved E and AE by ~60% and 65%, respectively  is relatively small, primarily because of the 1/6 power dependence over separation, which in the best case of parallel dipoles results in R0 being only ~35% larger than when randomly oriented. To examine the impact of having oriented dipoles on the anywhere-to-end efficiencies of FRET networks more closely, we compared simulations with oriented dipoles to results obtained for the dynamically averaged random dipoles (κ2=2/3) limit; the latter were used for all other simulations (κ2=2/3) is compared with that obtained when optimally oriented parallel to the DNA axis of each arm. Since these arms are allowed to bend out of the plane, the optimal dipole orientation will be maintained only within a given arm and not between arms. The main plot R0. For example, in the two-dye system , despite poor assembly . There is only a small gain in ideal E when going from a 3:1 to 4:1 dendrimer (E (16–19%) comparable to the four-dye dendrimers even with an extra FRET step. Finally, controlling dipole orientation remains challenging. For a single-FRET step, a random static distribution of dipoles can limit energy transfer efficiency because more ways to achieve relatively poor dipole–dipole orientations exist than favourable orientations. This penalty accrues with the length of the FRET cascade  with the exception of the Cy3.5 and internal labelled Cy5.5-functionalized strands, which were purchased from Operon Biotechnologies, Inc. . 2.5 × PBS  was diluted from a 10 × DNAse/RNAase-free stock purchased from Sigma-Aldrich . DNAse/RNAase-free water was also obtained from Sigma-Aldrich. Black Corning 96 and 384 well nonbinding surface microtitre plates were obtained from Sigma-Aldrich.2 was excluded due to the potentially deleterious effects high concentrations of such ions can have on dye fluorescence2 has been previously validatedStock solutions of DNA were diluted into 2.5 × PBS at 20 μM working concentration. Individual samples were assembled stepwise from component DNA (20 μM) in 0.5 ml PCR or 1.5 ml Eppendorf tubes to a final concentration of 1 μM in ~110 μl of 2.5 × PBS. This concentration range was empirically tested to match the linear portion of fluorescent response in later instrumental data collection, see For the 2 h PCR-based annealing conditions used for the dendrimer structures, solutions were annealed on an Eppendorf Mastercycler Thermal cycler. The standard annealing conditions were 95 °C for 4 min with a 1 °C ramp down per minute until 4 °C was reached. For the 12 h annealing condition, the same thermal cycler was used, but the program was modified such that it was held at 95 °C for 5 min and ramped down to 60 °C at a rate of 1 °C for every 4 min. The temperature was held at 60° for 2 h. The temperature was ramped down again to 50° at a rate of 1 °C per 4 min and held at 50 °C for 2 h. A final ramp at 1 °C every 4 min was done until a final temperature of 4 °C was reached. The hold temperatures of 60° and 50° were chosen as they are just below the average melt temperature of the medium- and short-length strands, respectively. Replicate structures were assembled for the full constructs and all control permutations thereof with one or more dyes missing to estimate other FRET pathways.Each structure to be tested was typically independently assembled at least in triplicate, which usually entailed performing experiments over several days. Assembled structures were aliquoted into microtitre well plates and fluorescence was collected on a Tecan Infinite M1000 Dual Monochromator Multifunction Microtiter Plate Reader  equipped with a xenon flash lamp using 515 nm excitation for Cy3 and 465 nm for AF488. 400 Hz flash frequency was used with a 40 μs integration time. Emission spectra were collected from 530 to 850 nm in 1 nm increments and exported into an Excel spreadsheet for later data processing and analysis. The Tecan performs an automatic, calibrated adjustment on collected data for nonlinear detector response in the near-infra red, that is, internally corrected. spFRET analysis is described in J(λ), for FRET conjugates. The Förster distance (R0), corresponding to donor–acceptor separation resulting in 50% energy transfer efficiency was also calculated for each donor–acceptor pair using the expressionDonor emission, acceptor absorption and molar extinction coefficients of each dye were used to calculate the spectral overlap integral, QD is the fluorescence quantum yield (QY) of the donor, and κ2 is the dipole orientation factor that we often, but not always, take to be 2/3 as is appropriate for the quasi-random dipole orientations characteristic of these constructsWhere, χ2-values. Satisfactory fits were confirmed by residual plots, which consisted of the fit data subtracted from the raw data, and were reduced to baseline noise with respect to the experimental data sets. The decomposed components of the best-fit curves were numerically integrated to quantify the PL contribution from individual fluorophores within a given construct. These data were then used to determine several parameters including donor PL loss, acceptor sensitization, E and TEF. The general approach closely follows the analysis of FRET-based DNA photonic wires previously reported39Each construct was processed by collecting spectra from each configuration along with the direct excitation components of each dye; the latter  were then subtracted from the composite. Finally, corrected spectra were decomposed into component parts corresponding to the quenched initial dye and sensitized remaining dyes. See 3940E, of each construct was calculated using the expression3739The end-to-end energy transfer efficiency, ΦAD is the integrated PL area of the terminal acceptor in the presence of donor, ΦA is the integrated PL area of the terminal acceptor in the absence of donor, ΦD is the integrated PL area of the donor in the absence of acceptor, and QA and QD are the QY of the terminal acceptor and donor, respectively. E provides a means to assess the terminal acceptor re-emission following sensitization from an upstream fluorophore, while also accounting for the QY of the initial donor and terminal acceptor. The values of ΦD and ΦA were determined by numerical integration of PL area fits from molar equivalent samples containing only the donor or acceptor of interest, respectively. In multiFRET step constructs ΦAD in where E accounts for energy that has entered the system through the initial donor as well as from direct excitation of intermediary fluorophores serving as donors. For example, in full Cy3–Cy3.5–Cy5–Cy5.5 constructs the majority of energy is introduced to the system via direct excitation of Cy3. However, some energy is introduced through direct excitation of Cy3.5 and to a lesser extent Cy5. Therefore, the magnitude of E reflects the amount of terminal acceptor sensitization arising from energy that has entered the system through any upstream fluorophore, of which the Cy3 emission provides the significant majority.In constructs possessing multiple donors, ED, and acceptor re-emission efficiency, EA, for each donor–acceptor pair within a particular construct. Direct excitation spectra fits for each fluorophore  were subtracted from fit emission data sets to minimize contributions to the composite PL intensity arising from direct excitation of fluorophores subsequent to the initial donor. Following the scaling/subtraction method outlined above, the resulting spectra were decomposed to determine the contributions from the primary donor emission and the sensitized acceptor contribution in a particular configuration. These data were numerically integrated and used to calculate ED and EA for each donor–acceptor conjugate according toA similar analysis was employed to quantify the average FRET donor efficiency, andFDA is the integrated PL area of the donor in the presence of acceptor, FD is the integrated PL area of the donor in the absence of acceptor, and FAD is the integrated PL area of the acceptor in the presence of donor. Equation 4 is simplified here and must also take into account the quantum yields of the donor and acceptor as in Equation 2. In constructs with multiple fluorophores, the efficiency for each donor–acceptor pair is analyzed as an independent step, regardless of whether the donor was directly excited and/or sensitized.where The antennae effect (AE) was measured for all systems and is defined here asandICy5,515 nm, ICy5,635 nm and ICy5.5,515 nm, ICy5.5,685 nm are the fluorescence intensities (decomposed area under the curve) of the terminal Cy5 or Cy5.5 following excitation of the initial Cy3 donor at 515 nm and direct excitation at 635/685 nm, respectively. AE are given as amplification factors.where R0) was then set to unity and all other data scaled up by this value. The result is a series of normalized data points reporting the terminal acceptor PL intensity accounting for the various linear and dendrimeric constructs. TEF is applied regardless of donor number, configuration or donor–acceptor separation. TEF was determined for the two-dye and four-dye constructs by:TEF was introduced to allow for a comparison of the PL intensity of a terminal acceptor (Cy5 or Cy5.5) across all FRET constructs regardless of geometry  or donor number. First, PL data for a given construct were normalized by the intensity of the Cy3-DNA conjugate since molar equivalence was maintained across all data sets. This accounted for any instrument variation during data collection. Next, a scaling factor was introduced to account for presumptive number of active Cy3 dyes within a particular construct: 1 for linear, 2 for the bifurcated, 4 for the Holliday junction, 8 for the eight-arm star and 2:1 dendrimer, 27 for the 3:1 dendrimer and 64 for the 4:1 dendrimer. The PL intensity of terminal acceptor, determined from decomposition of composite spectra, was subjected to this normalization and scaling procedure and tabulated. Finally, the terminal acceptor PL intensity with the lowest value . The quantities in Φm bywhere on dye m . The matQm is the quantum yield of dye m, ρk).Supplementary Figures 1-53, Supplementary Tables 1-67, Supplementary Notes 1-5, Supplementary Discussion, Supplementary Methods and Supplementary References.


Streptococcus pneumoniae from dried mock CSF spots was 14 genomic equivalents (GE)/μL (interquartile range 5.5 GE/μL) or 230 (IQR 65) colony forming units/mL. A prospective clinical evaluation for S. pneumoniae, S. suis and Neisseria meningitidis was performed. Culture and PCR performed on fresh liquid CSF from patients admitted with a clinical diagnosis of meningitis (n = 73) were compared with results derived from dried CSF spots. Four of five fresh PCR-positive CSF samples also tested PCR positive from dried CSF spots, with one patient under the limit of detection. In a retrospective study of S. pneumoniae samples (n = 20), the median  CSF S. pneumoniae bacterial load was 1.1 × 104 GE/μL . Utilizing the optimized methodology, we estimate an extrapolated sensitivity of 90%, based on the range of CSF genome counts found in Laos. Dried CSF filter paper spots could potentially help us to better understand the epidemiology of bacterial meningitis in resource-poor settings and guide empirical treatments and vaccination policies.We investigated whether dried cerebrospinal fluid (CSF) conserved on filter paper can be used as a substrate for accurate PCR diagnosis of important causes of bacterial meningitis in the Lao PDR. Using mock CSF, we investigated and optimized filter paper varieties, paper punch sizes, elution volumes and quantities of DNA template to achieve sensitive and reliable detection of bacterial DNA from filter paper specimens. FTA Elute Micro Card™  was the most sensitive, consistent and practical variety of filter paper. Following optimization, the lower limit of detection for  Streptococcus pneumoniae, Streptococcus suis, Neisseria meningitidis and Haemophilus influenzae account for the majority of cases of community-acquired bacterial meningitis in Laos (LOMWRU unpublished) H. influenzae type b, S. pneumoniae  and N. meningitidis  have had a profound impact on the incidence of bacterial meningitis cases in many regions Bacterial meningitis is a life-threatening disease that carries significant mortality and considerable risk of severe neurological complications. In developing countries, such as the Lao People's Democratic Republic (Laos), the combined mortality rates of all causes of bacterial meningitis are likely to be >20% In Laos, conventional bacteriological and molecular microbiological facilities for the diagnosis of central nervous system disease are confined to the capital city of Vientiane, and antibiotic treatment is often initiated prior to lumbar puncture Reliable procedures are necessary to preserve, transport and test CSF samples, frequently requiring a challenging and costly cold chain to be in place. Effective disease surveillance is thus severely hampered in resource-limited settings, such as those currently present in rural Laos, where procedures for appropriate laboratory diagnosis are suboptimal. Innovative, simple and inexpensive rapid diagnostic tests for detecting pathogen antigens in CSF hold promise et al. S. pneumoniae and H. influenzae type b in CSF-impregnated filter paper strips after 8 months storage at room temperature in sealed plastic bags.Blood dried onto filter paper to diagnose infectious diseases dates back to as early as 1939 S. pneumoniae, S. suis and N. meningitidis. We investigated different types of filter paper and optimized the processing of dried CSF spots to obtain the maximum possible yield of nucleic acid. A prospective pilot study was conducted utilizing these optimized methods to compare PCR results on DNA extracted from dried CSF spots with routine liquid CSF and conventional bacterial culture.Until laboratory capacity is developed in rural Laos, filter paper could serve as a sample preservation method that can easily be delivered to a central laboratory for CSF PCR and therefore allow better understanding of the geographical epidemiology of bacterial meningitis. We investigated whether dried CSF spots would serve as a sensitive and reliable method for defining three of the major causative agents of bacterial meningitis in Laos: Three varieties of filter paper were selected for performance evaluation: the Whatman Grade 903 , the Flinders Technology Associates (FTA) Micro Card™  (referred to as FTA hereafter) and the FTA Elute Micro Card™ (referred to as FTA Elute hereafter) . Whatman 903 requires elution of the organism from the paper followed by lysis and DNA extraction. FTA and FTA Elute papers contain a proprietary mix of reagents that lyse cell walls but stabilize and bind nucleic acids. FTA paper requires a washing protocol, with the paper disc itself being used as a DNA template in the PCR.S. pneumoniae (NCTC 12977) or N. meningitidis (NCTC 10025) grown overnight on 5% goat blood agar. This was incubated for 4 h in 5–7% CO2 at 37°C and then diluted with sterile TSB to achieve a standardized initial concentration  using the Nanodrop 2000 . Ten-fold serial dilutions were prepared and aliquots spotted onto filter paper  and onto blood agar (100 μL) to determine the bacterial load (colony forming units/mL) for each dilution using a micropipette.A mock CSF was prepared by spiking tryptic soy broth (TSB) with a few discrete colonies of National Collection of Type Culture (NCTC) strains of either S. pneumoniae and to demonstrate inter-species reproducibility final methods were repeated with N. meningitidis.Optimization studies were performed using c. 3 μL and 20 μL of CSF, respectively. Biopsy punches were sprayed with 70% ethanol after each punch, dried with tissue paper and then punched into a stack of sterile filter paper a further five times to eliminate cross-contamination Discs (3 or 8 mm) were punched out of the centre of a filter paper circle using skin biopsy punches  and transferred to 1.5-mL microcentrifuge tubes. Three and 8-mm punched discs would contain The FTA Elute manufacturer's instructions were followed with the following modifications: 1 mL of sterile water was used to wash the disc by pulse-vortexing three times for a total of 5 s. The DNA was eluted with sterile water after 22.5 min at 95°C, the mid-point of the manufacturer-recommended time period. FTA was processed following the manufacturer's instructions, except for the use of a slightly larger disc (3 mm in diameter rather than the recommended 2 mm). Whatman Grade 903 discs were processed following the QIAamp DNA Mini kit Dried Blood Spot Protocol  with the following modifications: elution was carried out in 180 μL of buffer ATL with proteinase K incubated at 56°C overnight.n = 25) was initially processed using the QIAamp DNA Mini kit Blood or Body Fluids Protocol (Qiagen) after centrifuging 200 μL of CSF at 16 000 g and re-suspending the pellet in buffer ATL. Subsequently (n = 48), routine extraction was performed on 200 μL unspun CSF with the EZ1 Virus Mini Kit v2.0 for automated, simultaneous purification of viral DNA and RNA as well as bacterial DNA (Qiagen).Liquid CSF (2) was compared with the standard size of 3 mm (7.1 mm2) (c. seven-fold increase in disc surface area). Finally, three different elution volumes were also investigated: 40, 80 and 145 μL. These experiments assumed an even distribution of pathogen DNA across the paper after application.To optimize DNA yield from filter paper, a series of experiments were performed in triplicate. Three different quantities of DNA eluate were used in the PCR: 3, 5 and 10 μL. A larger punch size of 8 mm (50.2 mmn = 73), Vientiane, between July 2011 and March 2012 with a clinical diagnosis of meningitis, who underwent lumbar puncture, had fresh CSF spotted onto FTA Elute. Four circles were spotted with CSF and allowed to dry for 4 h before being stored in an air-conditioned laboratory at c. 20°C in sealed plastic bags containing silica granules to reduce humidity. PCR on dried CSF spots was subsequently compared with PCR performed on liquid CSF and routine culture.Patients admitted to Mahosot Hospital .Patients' CSF samples were cultured on both 5% goat blood and chocolate agar (and MacConkey agar in those <1 year old) and incubated at 37°C in 5–7% COS. pneumoniae, N. meningitidis and S. suis  and comprised at least two no-template controls as well as duplicates of serial dilutions of positive controls, which were quantitated using the Quant-iT PicoGreen assay , corresponding to c. 103, 102, 101 and 100 genomic equivalents (GE)/μL.Three separate real-time quantitative PCRs were performed for is Table . Assays S. pneumoniae PCR-positive CSF samples, using external standards in a quantitative real-time PCR assay.Previously characterized frozen CSF samples collected from patients with bacterial meningitis admitted to Mahosot Hospital between 2005 and 2012 were identified retrospectively. These samples served to quantify the DNA loads in Statistical analysis was performed with Stata  and the figure created using GraphPad software .Lumbar punctures were performed as part of a routine diagnostic service if verbal (2003–2006) or written (2006–2011) consent was given by patients or their parents/guardian. Ethical clearance was granted by the Ethical Review Committee of the Faculty of Medical Sciences, National University of Laos, and the Oxford University Tropical Ethics Research Committee, Oxford, UK.7 cfu/mL to 1 cfu/mL. When applied to quantitative real-time PCR assay with external controls, the DNA templates eluted from FTA Elute consistently led to lower cycle thresholds than DNA eluted from Whatman 903 for the same mock CSF dilutions  , increasing the sensitivity of detection 2) to 8 mm (50.2 mm2) reduced the Ct value by a median of 2.7 (IQR 0.55), equivalent to increasing the sensitivity of detection c. ten-fold , equivalent to a median mock CSF concentration of 230 cfu/mL (IQR 65). Studies to optimize the detection of S. pneumoniae from filter paper were reproducible using N. meningitidis (data not shown).In addition, increasing the punch size of FTA Elute from 3 mm 7.1 mm mm2 to 8c. 20°C in sealed plastic bags containing silica granules for up to 8 months before being processed. Of these, 5/73 (6.8%) tested positive by quantitative real-time PCR on DNA extracted from liquid CSF (four S. pneumoniae and one S. suis) and 4/73 (5%) tested positive by quantitative real-time PCR on DNA from dried CSF spots (three S. pneumoniae and one S. suis). The positive samples were identical, resulting in a sensitivity of 80% (95% CI 29–97%) for PCR using FTA Elute. One sample was not detected by FTA Elute due to a low bacterial load of <10 GE/μL. Of the five positive samples, two were culture positive (both S. pneumoniae). No false-positives occurred, resulting in a specificity of 100% (95% CI 95–100%).FTA Elute performed consistently with a high level of sensitivity throughout the optimization process and was therefore selected for further evaluation. A total of 73 dried CSF spots were prospectively collected from patients admitted with a clinical diagnosis of bacterial meningitis over a 9-month period. Dried CSF spots were stored at S. pneumoniae patients collected over a 7-year period were retrieved and their bacterial load quantitated. Genome equivalent counts in this population ranged widely from 1 × 100 to 6.1 × 106 DNA GE/μL  , S. suis (n = 5) and H. influenzae (n = 6) samples were available (data not shown).Stored frozen (−80°C) DNA extracts of CSF samples from 20 well-characterized PCR-positive In this study, we aimed to evaluate the use of dried CSF spots for the identification of three of the major causative agents of bacterial meningitis in Laos and to identify the role/potential limitations of using a filter paper-based surveillance approach to investigate the epidemiology of CNS disease aetiologies.Our findings suggest that this can be accomplished using FTA Elute with the optimized methods, yielding an estimated sensitivity of 90% when compared with the ‘reference standard’ real-time diagnostic PCR assay on liquid CSF. Investigation with mock CSF suggested that FTA Elute gave the most consistent and sensitive results compared with Whatman 903. FTA paper discs used as a DNA template in the PCR proved impractical for testing several pathogens from a single dried CSF spot using single assays. We developed our methods using TSB that does not contain all the constituents of human CSF. However, the prospective evaluation of clinical samples confirmed our findings during the optimization process.c. 10 times less than that used for routine PCR on liquid CSF (20 μL vs. 200 μL). Even this may be slightly overestimated due to possible diffusion outside the marked circle. As a result, despite the use of highly sensitive quantitative real-time PCR assays, those CSF samples with very low bacterial loads can fall under the limit of detection. The optimization studies demonstrated that, beyond the optimal cut-off, DNA concentrations in the eluate declined with increasing elution volumes. A small reduction in DNA concentration was accepted for the final methodology to allow sufficient PCR template to be available. Multiplex PCR can simplify the process of detecting multiple pathogens and potentially reduce costs. However, careful optimization must be performed to ensure that sensitivity and specificity are not reduced c. 230 cfu/mL or 14 GE/μL of CSF could be reliably detected by PCR from CSF dried on filter paper. In a study by La Scolea et al. 3 cfu/mL and 56% had counts >105 cfu/mL. Samples collected in Laos over a 7-year period showed a wide range in GE/μL of S. pneumoniae with a median of 1.1 × 104. This is consistent with other studies that report wide ranges and medians between 4.6 × 104 and 5.7 × 105 DNA copies/μL in Latin America, Australia and Malawi Despite using a larger than manufacturer-recommended punch size to increase the lower limit of detection, the volume of CSF used from paper is H. influenzae and S. pneumoniae from filter paper with sensitivities of 70% and 92% and specificities of 99% and 100%, respectively S. pneumoniae was 5 × 100 to 9.2 × 106, with a median of 1.6 × 104, very similar to that seen in Laos . If other central nervous system pathogens were detectable in CSF using the optimized filter paper method, this would further increase the potential of using this methodology as a surveillance tool in areas without laboratory capacity.An additional limitation was that we were only able to prospectively test our methods on a limited number of samples and pathogens. Other studies have shown that We did not investigate the practicalities and suitability of collecting dried CSF spots in smaller regional hospitals and transporting these for analysis. Although the effects of humidity and storage conditions on dried CSF spots were not assessed, DNA was readily detected after 8 months storage in an air-conditioned room. A simple standard operating procedure was provided to laboratory staff for the preparation of dried CSF spots.FTA Elute paper inactivates pathogens within a short period of application onto the paper Wellcome Trust of Great Britain.


Vascularized composite allotransplantation is a continuously evolving area of modern transplant medicine. Recently, vascularized composite allografts (VCAs) have been formally classified as ‘organs’. In this review, key aspects of VCA procurement are discussed, with a special focus on interaction with the procurement of classical solid organs. In addition, options for a matching and allocation system that ensures VCA donor organs are allocated to the best-suited recipients are looked at. Finally, the different steps needed to promote VCA transplantation in society in general and in the medical community in particular are highlighted. Vascularized composite allotransplantation refers to the transfer of a vascularized human body part containing multiple tissue types  as an anatomical and/or structural unit from a human donor to a human recipient. The first reported unilateral hand transplantation was performed in Ecuador in 1963, but the transplant had to be removed within two weeks due to acute rejection . The firDirective 2004/23/EC of the European Parliament and of the Council on setting standards of quality and safety for the donation, procurement, testing, processing, preservation, storage and distribution of human tissues and cells (European Union [EU] tissue and cell directive) [Directive 2010/45/EU on standards of quality and safety of human organs intended for transplantation (EU organ directive) [A similar development regarding the categorization of VCAs took place in Europe. In rective) , organs rective) , 16. In rective) . In thisrective) . The impA major prerequisite for the procurement of a VCA is the consent by the donor or next of kin. There is general understanding that although VCAs will now be classified as organs, currently existing consent to ‘organ donation’ as documented in donor registries or on donor cards does in general not cover consent to VCA donation. As such, the importance of a transparent and explicit consent for VCA donation is highlighted in the document from the US Department of Health and Human Services classifying VCAs as organs. This will include an adaptation of donor forms and the approach to the family, so that it is clear whether the consent covers the procurement and transplantation of VCA and whether certain body parts are excluded from this consent. In the reports on VCA transplantation from European countries, it was made clear that explicit consent was required from the family for VCA donation, even in countries with presumed consent legislation. In fact, the consent process, especially for facial allograft donation, has shown to be a major challenge •. There Intercultural variation regarding the acceptance of facial transplantation has been reported. In addition, the willingness to accept a face transplant was higher (68 %) than the willingness to donate one’s face after death (41 %) in one investigation ••. ReligVascularized composite allotransplantation is typically not a life-saving treatment; therefore, careful donor selection is of upmost importance to prevent the transmission of any unwanted disorders. A detailed work-up of the donor is essential –26. As bThe surgical details of the procurement procedure vary from one type of VCA to another and are beyond the scope of this review. Extensive experimental data have been gathered for the different types of VCA procurement and transplantation and several reports summarizing current clinical practice have been published ••, 41. TGordon developed a classification system for VCA procurement and transplantation that is based on the relative complexity of the procedure. It shows that upper extremity and face transplantation are characterized by a high degree of complexity, substantially more complex than, for example, abdominal wall transplantation and only outmatched by the complexity of combined VCA transplantation ••, 43.An important aspect for face replantation and transplantation is the knowledge of facial angiosomes first described by Houseman et al. in 2000 . This haAlthough rejection in a sentinel skin graft is not necessarily representative of rejection in the main VCA, the use of a sentinel skin allograft to allow skin biopsies without affecting the VCA has been used .Minimizing immunosuppression to reduce side effects related to this therapy  is an important goal and it is a focus of the field to understand the VCA specificities and potential treatment options .Similar to the fact that the question of consent for VCA procurement must not negatively influence solid organ procurement, there is general agreement that procurement of VCA must not hamper the retrieval of other potentially life-saving organs. Well structured time planning is essential. Hand and forearm should be harvested prior to solid organs if possible to prevent impaired hand perfusion and prolongation of ischemia time , 47. ThiFor face transplantation, multiple vessels, nerves, muscles, and osseous structures have to be identified and dissected carefully, so the procurement is much more complex and typically takes about 7–12 hours. In some cases, face procurement has taken as long as 22 hours ••, 50. TIf the donor is unstable or the VCA procurement is a high-risk procedure, the thoraco-abdominal incision could take place at the beginning of the procurement procedure or be triggered by signs of hemodynamic deterioration of the donor.In any phase of face harvesting with an elevated risk of blood loss it could be an option to have the solid organ teams remaining on stand-by in-house, ideally already scrubbed. Such an approach might pose quite a burden on the explant teams for the solid organs, especially if non-local teams are involved.Often the arrival of the solid organ teams in the operating room (OR) and solid organ retrieval are postponed until shortly prior to the planned VCA explantation.If the face is procured first, coordination with the solid organ teams is of upmost importance, due to the fact that the donor might become unstable during the long preparatory phase. In case a ‘face first’ approach is chosen, the following three strategies regarding access to the thoracic and/or abdominal structures can be distinguished depending on donor stability, risks associated with the face procurement, and local preferences ••, 50.IfIndependent of the selected individual strategy, the aim has to be to prevent solid organ loss.Prior to the procurement of the face, typically a tracheostomy is performed because orotracheal intubation might hamper the surgical procedure ••. This The VCA recovery team is responsible for donor reconstruction, which can pose a special challenge in face transplantation. Creating an adequate aesthetic prosthesis that can be attached to the donor after procurement can be quite demanding, especially in case of face procurement . SeveralStep 1Selection: identifying those patients that are at all suitable for a specific organ among all patients on the waiting list.Step 2Ranking: determining the allocation sequence among all suitable recipients.The organ allocation process has to be transparent, objective and reliable. Each step in the allocation process should be well documented to allow full accountability towards the transplant community and the general public. The underlying allocation rules shall be based on sound medical judgment and are typically specific for each organ type. They shall seek to achieve the best use of the organs, avoiding wasting of organs and futile transplantation. Allocation of organs typically takes place in two steps:There are a few general selection criteria: the blood group of donor and recipient have to be compatible. While it is desirable to have a good immunological match between donor and recipient, HLA mismatches between donor and recipient are not a contraindication to transplantation , 51. ThiNext to these general considerations, other prerequisites for the selection of suitable recipients have been proposed. Age difference of donor and recipient should be limited. Results of a computer simulation suggested that it is acceptable when the donor is between 2 decades younger and 1 decade older than the recipient . In FranInstead of using surrogate parameters like age and gender for matching, specific phenotypic matching based on size, especially concerning bones, color and texture of the skin, and soft tissue features has been recommended by several authors ••, 57. ONext to skin color and texture, soft tissue features like the cartilaginous nose, the lips, and the eyebrows are also important in order to let the recipient’s face or limbs look similar to the previous native situation after transplantation. When determining acceptable differences between donor and recipient it probably has to be taken into account that conventional dermatologic techniques and/or conventional makeup could be used to mitigate modest differences between donor and recipient •, 28•. OThe respective donor characterization and reporting to the allocation organization have to be based on these criteria and can then be used in matching. This might include the use of standardized color cards and standardized rules and techniques to determine donor size , 58••.Once suitable recipients have been identified, it is necessary to determine the allocation sequence. Different criteria might be used for this.Outcome of transplantation could be one important criterion. HLA matching between donor and recipient could be taken into account in allocation, because better matching might positively influence long-term results of transplantation due to less acute and chronic rejection. In kidney transplantation it has been shown that with better matching there is a reduced need for immunosuppression, thus mitigating the related short- and long-term complications  . TherefoRecipients with a high level of donor-specific antibodies should be avoided to reduce the risk of graft loss both in the early phase after transplantation and later due to chronic rejection with graft vasculopathy .Next to patient and graft survival, the expected functional and cosmetic transplant results which are influenced by the quality of phenotypic matching between donor and recipient could play a role in allocation. Based on the phenotypic characteristics of donor and recipients, the best match in size, (skin) pigmentation, and texture could be identified and given priority •, 32••.Urgent patients, for example patients with arteriovenous malformation with severe bleeding waiting for a face transplant or patients with severe psychological distress, could be given priority.For highly immunized patients or patients waiting for a combined transplant, it is often extremely difficult to find a suitable donor and expected waiting times are very long. As a compensation for this disadvantage these patients could be given priority in allocation if a suitable donor for them is available.Taking the aspect of general fairness into consideration, waiting time could be used for prioritization, especially as VCA transplantation is not a life-saving procedure.Currently, VCAs are typically allocated locally or regionally (first). One argument in favor of this approach is the expected resulting shorter ischemic time. Some experimental studies showed that extended ischemic time increases tissue damage and has a negative impact on long-term structural and functional outcome, in others an effect of ischemic time on VCA function was not shown . As suchA)Categorical or tier-based systemThe different allocation criteria are used in a sequential manner starting with the most important one. All recipients are subdivided into two or more categories based on the first criterion and each of the resulting categories determines a different priority level. If there is only one recipient in the top priority tier, he/she will get the organ offer. If there is more than one recipient in that category, the next allocation criterion will be used to further subdivide the remaining patients. For example, as a first step, recipients could be subdivided into urgent and elective patients. If there is more than one urgent patient, local patients could get priority in this group. If there is more than one urgent local patient, HLA matching or phenotypic matching could further subdivide groups. As a final tie-breaker, waiting time could be used. While this system is simple to understand and to implement, it has the major disadvantage that there is a strict hierarchy of the criteria from the most to the least important one. This problem is avoided in the score-based system.B)Score-based systemHere all identified allocation criteria are used at the same time to determine the allocation sequence. For each criterion, an individual point value is assigned. The total score is calculated based on a mathematical algorithm using all individual values of the different criteria. A very simple score system could be the sum of the values for the different allocation criteria . However, more complex scores could also be used to reflect the interaction of the different allocation factors .C)Combined systemOften a combination of both systems is used. Patients are subdivided into two or more main tiers and within each tier the allocation sequence is based on a score calculated using other criteria. For example, patients could be subdivided into high-urgent patients and patients with normal urgency. In that case, high-urgent patients would always have priority over patients with standard urgency. Within each tier, the allocation sequence would be based on a score using the other allocation factors.Regarding the interaction of the different allocation factors for determining the allocation sequence, different strategies can be distinguished Fig. :Fig. 1ScVCA transplantation is currently very much based on ad hoc arrangements, typically within an OPO or a region thereof. At present, VCA transplantation is more about looking for a donor for an individual patient than about assigning a VCA from a donor to the most suitable recipient. This approach is quite similar to the situation in the early days of solid organ transplantation. Logistical aspects are – especially in this early phase of setting up VCA transplantation – a strong argument in favor of this local allocation system because donor hospitals have to be involved and the coordination with other teams has to be agreed upon . On the VCA transplantation is currently a low volume area with all the related challenges of training donor hospitals, procurement teams, and transplant centers. Together with the aforementioned aspects of procurement and allocation, several prerequisites for stimulating and spreading VCA transplantation in general and setting up individual transplant programs can be derived.There should be a policy for informing the general public about the need and the benefits of VCA transplantation. At present, the possible demand for VCA transplantation is unclear. Approximately 100,000 upper-extremity amputees are living in the US at the moment , but of Educational meetings with donor hospitals and OPOs are necessary to allow full understanding of the complex donor procedure – VCA transplantations must not come as a surprise to the donor hospital. OPOs have to be especially trained and informed regarding how to approach the family and ask for informed consent. A VCA-tailored algorithm should be used when approaching all potential donor families for VCA, especially face transplantation ••. VitalOrgan allocation organizations have to discuss and develop matching criteria and allocation rules ••. BasedSetting up a VCA transplant program is a complex and challenging process for a transplant center. Several prerequisites have to be met to create a successful program. Requirements for the development of a hand transplant program have been described , 64•, 70Clear rules for the VCA procurement team including the interaction and cooperation with the other procurement teams have to be developed. It is highly recommended to establish common guidelines per type of VCA for all procurement teams. This would make procurement outside the local OPO more easy and feasible. In an interim period, it might be an option that a procurement team visiting a donor hospital outside their own OPO is joined in a type of rendezvous system by a VCA procurement expert from the local (VCA) transplant team for organizational support and to guarantee smooth cooperation with the donor center. This might even include support in donor reconstruction.As long as there are only a few VCA transplant centers, recipients might undergo transplantation in a center quite distant from home. Therefore, cooperation with special training of external doctors to allow local long-term follow-up has to be setup .A new VCA transplant program must only start with its first transplants after formal approval by the responsible authorities , 21. ThiVCA transplantation is a still small but promising section of organ transplantation. The success and possible further growth of this new field will depend on several factors: a coordinated team approach involving multiple disciplines in the VCA transplant center, and excellent cooperation with the respective organ procurement organization as well as transparent interaction with the other solid organ procurement teams. This medical approach has to be accompanied by thorough, well planned public information on all aspects of VCA transplantation, because willingness to donate VCA is the most important but also the most critical prerequisite for expanding the donor pool. The more common VCA transplantation becomes, the more allocation will move away from the current primary local usage of donor VCAs to a broader exchange of these grafts. This will allow better matching of donors and recipients, thereby improving functional and cosmetic short- and long-term results of transplantation. Establishing such a structured and transparent allocation system and further improving outcomes of VCA transplantation will foster general acceptance of this type of transplantation, which could have a further positive effect on VCA donation rates.