Cryptosporidium parvum, Raoultella terrigena, and bacteriophages P22 and MS2 before and after biofilm development to determine average log reduction (ALR) for each organism and the role of the biofilm. There was no significant difference in performance between the two designs, and both designs showed significant levels of removal . Removal levels meet or exceeded Environmental Protection Agency (EPA) standards for microbial purifiers. Exploratory test results suggested that mature biofilm formation contributed 1–2 log10 reductions. Future work is recommended to determine field viability.Two point-of-use drinking water treatment systems designed using a carbon filter and foam material as a possible alternative to traditional biosand systems were evaluated for removal of bacteria, protozoa, and viruses. Two configurations were tested: the foam material was positioned vertically around the carbon filter in the sleeve unit or horizontally in the disk unit. The filtration systems were challenged with  An estimated 884 million people currently rely on unimproved drinking water sources including unprotected wells and springs; small and large water tanks; surface water .2Often in these areas, access to clean water requires long trips to a distant source, usually on foot. This transportation process can increase the risk of contamination.The usefulness of a point-of-use device cannot be measured strictly by the reduction of pathogenic organisms, a complete view of a device must be considered before deployment. Biological, economic, and behavioral factors all play a role in determining devices ultimate effectiveness in the field. A filtration system must be able to maintain adequate levels of microbial reduction for long periods as well as be able to process large volumes of water.6Gravity-fed point-of-use devices, specifically, have been proven effective in mitigating gastrointestinal disease in many of these scenarios.10The biosand design does suffer from some drawbacks. It can be time consuming to deploy, the sand/gravel component makes it heavy and/or costly to transport, or reliant on local materials for the media. Maintenance of the biosand system can also be cumbersome if it requires a complete change of all the internal sand and gravel.Incorporated as part of the design of some point-of-use filters are layers of organic material and benign growth of microorganisms into a structure known as biofilms. Biofilms have been shown to capture and retain a range of microbes from water, particularly protozoaThe objective of this research study was to evaluate the removal of select microorganisms by two novel point-of-use filtration designs. These “biofilter” systems, as they will be referred to in this article, use a foam material to support the growth of biofilms. The biofilter designs are intended as possible alternatives to other gravity-fed point-of-use filters such as the biosand system. They are intended as potential new candidates for deployment among rural populations requiring in-home point-of-use water filtration devices. The biofilter systems have no sand or gravel layers, which could make transport, deployment, and maintenance easier in some respects. The biofilter gravity-fed system uses a multiple barrier approach including water biofiltration and a delivered chlorine disinfection. A multi-barrier approach has been shown to be effective in producing a robust process capable of removing and/or inactivating a variety of microbial pathogens.Two multi-barrier point-of-use drinking water treatment systems using a carbon filter and foam material were compared for removal of bacteria, protozoa, and viruses from water.The two biofilter systems were built by Amway, Ada, MI. They were designed with a foam material configured in a layer to provide mechanical filtration and a platform for biofilm formation, followed by a carbon filter block. In the biofilter sleeve unit , the foaTwo types of water  were used for the evaluation of the biofiltration units per NSF Protocol P231 for testing of microbiological water purifiers . The maiRaoultella terrigena, represented enteric bacteria, and heat-killed Cryptosporidium parvum oocysts served as parasitic protozoan pathogens with influent levels between 9.98 × 104 ± 1.26 × 104 PFU/mL, (N = 24); 4.63 × 106 ± 7.59 × 105 PFU/mL, (N = 22); 7.81 × 108 ± 7.59 × 107 CFU/100 mL (N = 26); and 1.23 × 107 ± 3.24 × 106 oocysts/L (N = 8), respectively  and 0.5 mL log-phase host bacteria Escherichia coli (ATCC# 700891), and then poured into a petri plate containing a bottom layer of trypticase soy agar medium. After solidification, the plates were incubated at 36 ± 1.0°C for 16–24 hours prior to enumeration of plaques. Plaques were counted, and the MS2 concentration was computed as plaque-forming units (PFU) per milliliter (mL). Samples were analyzed over wide range of concentrations in quadruplicate. MS2 was added to the challenge water to a targeted concentration of > 1 × 105 PFU/mL.Bacteriophage MS2 (ATCC 15597-B1) was prepared using a double agar overlay and assayed in accordance with a modified version of Environmental Protection Agency (EPA) Method 1602 without MgClSalmonella typhimurium  was used as a host.Bacteriophage P22 were prepared and assessed with a similar modified EPA Method 1602.R. terrigena (ATCC 33257) was assayed using membrane filtration technique according to American Public Health Association (APHA) standard method 9222 B.R. terrigena concentration was computed as colony-forming units (CFU) per 100 mL of water. Live R. terrigena was added to challenge water to target concentrations of 1 × 106 CFU/100 mL.C. parvum oocysts obtained from Waterborne Inc.  were included at target concentrations of 1 × 106 oocysts/L. For C. parvum analysis, samples were analyzed using EPA method 1623 for Cryptosporidium and Giardia in water by filtration, immunomagnetic separation, and fluorescent-labeled antibodies.Cryptosporidium and anti-Giardia antibodies, separated from the extraneous materials using a magnet, and then oocysts and cysts were removed from the magnetic bead complex and enumerated as described previously.20Heat-inactivated ® S-Pack; Pall Corporation), and the filtered bacteria was grown on HPC media . Samples were incubated for 48 ± 2 hours at 35 ± 2°C, and the colonies were enumerated visually.Biofilm maturation was determined and monitored using heterotrophic plate count (HPC) as outlined in APHA standard method 9215B.The challenge water was seeded with representative organisms for enteric virus, bacteria, and parasite groups. The biofilter systems were tested in two phases: pre-conditioning phase and challenge phase. During the pre-conditioning phase, maintenance water was passed through the biofilter units for 24 days to establish a biofilm on the foam filters. Biofilm maturation was determined and monitored using HPC bacteria enumeration. The biofilm was considered mature when HPC levels plateaued. The challenge phase consisted of a weekly schedule in which 20 L/d of maintenance water was passed through the filter systems for 6 days, and then on the seventh day  challenge water seeded with the representative microorganisms was passed through the filter systems. To evaluate the contribution of the biofilm formation on performance of the biofilter units, an additional challenge was conducted after the filters were cleaned and the biofilm removed.The systems were run a total of 113 days, with 1,696 L through each unit. A total of 11 challenge days were performed. The biofilm required 24 days to develop and mature by running general test water.On challenge days, water samples (1 L) were taken from six biofilter system sites : influenPrior to testing the contribution of biofilm formation on microorganism removal by the biofilter units, the foam filter in the disk unit was replaced with a new, unused disk filter as it was not easy to remove and wash the buildup of the biofilm after use. The foam in the sleeve unit was easily removed, so it was washed in dechlorinated water to remove the biofilm, and reused. To simplify the comparison, the flocculation bucket and chlorination step were removed from the biofilter systems. The challenge was then conducted without preconditioning .R. terrigena, and C. parvum, as described previously.Samples were collected from influent location (INF) prior to adding the challenge water, seeded with the select microorganisms, to the units. Samples were collected from the prechlorination location for the sleeve unit, and the prechlorination and basin sites for the disk unit. To generate a valid comparison between the systems without preconditioning, 2 L samples were taken at 50% and 120% of each units standing water volumes, designated V1 and V2, respectively . The staR. terrigena and between prechlorination and effluent sites for C. parvum..The goals of the statistical analysis were to examine the data for significant differences in reductions between systems, sampling sites, pre- and post-maturation conditions, and microorganism types. Data sets were assessed for normality using the Shapiro–Wilk test. Several data sets did not follow normal distribution, and no mathematical transformation of the data sets was found that produced normal distributions from all of the data sets. Therefore, nonparametric statistical tests were used for analysis. Analysis of variance (ANOVA) on ranks was used to detect statistical differences in ALR between sites in individual units. ANOVA on ranks was also used to compare the ALR between influent and flocculation, influent and prechlorination, flocculation and prechlorination, and prechlorination and chlorination for all organisms to examine potential differences between the sleeve unit and the disk unit. It was also used to compare the ALR between the sleeve unit and the disk unit between chlorination and effluent for MS2, P22, and The observed flow rates in the sleeve unit averaged 371 ± 32.11 mL/min during challenge events and 386 ± 38.95 mL/min during non-challenge events. The disk unit experienced flow rates of 420 ± 37.19 mL/min during challenge events and 443 ± 72.01 mL/min during non-challenge events. During the experiments without biofilm development, the filters were cleaned and flow rates were increased to 820 mL/min and 740 mL/min for the sleeve and disk units, respectively.P ≤ 0.01). However, no statistically significant differences between the sleeve and disk were found when comparing each organism's ALR from the following intervals: flocculation (INF-FLOC), influent to prechlorination (INF-PRECHL), flocculation to prechlorination (FLOC-PRECHL), chlorination (PRECHL-CHL), chlorination to effluent (CHL-EFF) , or prechlorination to effluent (PRECHL-EFF) (C. parvum). The P value for the interval comparisons was ≤ 0.01. There was no significant difference between the overall performance (influent to effluent) of the sleeve unit and the disk unit (P ≤ 0.01). An alpha of 0.05 was used for statistical significance in all tests.In the sleeve unit, P22 virus showed an overall 6.16 ± 0.64 ALR from influent to effluent over 11 challenges. The foam, carbon stage, and biofilm accounted for 4.50 ± 0.42 ALR alone (FLOC-PRECHL). Chlorination (PRECHL-EFF) could not be fully evaluated due to low incoming concentration; it accounted for a measurable 0.16 ± 0.31 ALR. If tested in isolation from the rest of the system with a high concentration of challenge organisms, a higher ALR would be expected between the prechlorination and effluent sampling points. On the basis of the chlorine residual and contact times tested in isolation from the rest of the system with a high concentration of challenge organisms, a higher ALR would be expected between the prechlorination and effluent sampling points.In the disk unit, P22 showed an overall 6.24 ± 0.46 ALR (influent to effluent) over 10 challenges. The carbon filter showed 2.40 ± 0.04 ALR (basin to prechlorination). The contribution of the carbon filter in contrast to the foam filter and biofilm could only be evaluated in the disk system as there was no way to sample these separately in the sleeve unit. Prechlorination to effluent accounted for > 0.01 ± 0.00 ALR in the disk unit see .With biofilm maturation, the sleeve unit exhibited a total removal and inactivation of 4.55 ± 1.26 ALR across the entire system in nine challenges (influent to effluent). The combined foam, carbon filter, and biofilm stage accounted for 2.92 ± 0.51 ALR (flocculation to prechlorination). The chlorination step (prechlorination to effluent) in the sleeve unit inactivated the remaining MS2 virus. From the prechlorination step to the chlorination basin > 0.03 ± 0.10 ALR were observable see .0.54 ± 0.50 ALR (basin to prechlorination). The contribution of the carbon filter in contrast to the foam filter and biofilm could only be evaluated in the disk system as there was no way to sample these separately in the sleeve unit.In the disk unit, MS2 showed an overall 4.55 ± 1.04 ALR (influent to effluent). Of this, 1.78 ± 1.30 ALR was from the flocculation stage (influent to flocculation). The carbon filter showed R. terrigena bacterium showed an overall 8.47 ± 0.55 ALR over 10 challenges (influent to effluent). Foam, carbon filtration, and biofilm (flocculation to prechlorination) accounted for 5.82 ± 1.09 ALR. Chlorination (PRECHL-EFF) showed > 1.72 ± 1.01 ALR.In the sleeve unit, R. terrigena was sampled in 9 to 11 challenges (see N = 9). The foam and biofilm layer alone (FLOC-BSN) accounted for 2.74 ± 1.30 ALR, and the carbon filter (BSN-PRECHL) was responsible for 3.98 ± 1.49 ALR (N = 10). The contribution of the carbon filter in contrast to the foam filter and biofilm could only be evaluated in the disk system as there was no way to sample these separately in the sleeve unit. Chlorination (PRECHL-EFF) process accounted for > 0.73 ± 0.58 log10 reduction in the disk unit (N = 10).In the disk unit, nges see . It showC. parvum was not quantified.This pathogen was sampled in three to four challenges. The sleeve unit had a total of 6.46 ± 0.64 ALR over the entire system (INF-EFF). In the foam and carbon layer (FLOC-PRECHL), 5.51 ± 0.85 ALR was observed. The effect of the chlorination process on C. parvum showed a total of 6.45 ± 0.86 ALR across the whole system (INF-EFF). The foam/biofilm layer by itself (FLOC-BSN) removed 3.71 ± 0.51 ALR of C. parvum and the carbon filter (BSN-PRECHL) removed 1.98 ± 0.89 ALR. The contribution of the carbon filter in contrast to the foam filter and biofilm could only be evaluated in the disk system as there was no way to sample these separately in the sleeve unit. The effect of the chlorination process on C. parvum reduction was not quantified.In the disk unit, The free chlorine measured an average of 2.14 ± 1.76 mg/L in the sleeve unit and 3.33 ± 1.85 mg/L in the disk unit as the water flowed over a chlorine tablet and into the reservoir. Contact time of chlorine within the reservoir was less than 10 minutes during challenges. Final effluent (after dechlorination) measured an average of 0.00 ± 0.00 mg/L in both units.10 reductions recorded during a challenge event. The sleeve unit had two sampling points in this challenge . The disk unit had an additional sample collection point that allowed the removal capacity of the isolated foam disk to be examined. This data represent the systems challenged without biofilm conditioning. The designation PRECHL is used for consistency, but as there were no chlorination steps involved in this challenge, the PRECHL point should be considered the endpoint for the entire system of this experiment.A preliminary investigation into the removals associated with the foam biofilter units without a biofilm was undertaken. The sleeve unit exhibited a total of 3.5 ALR of MS2 overall (INF-PRECHL) averaged over the two volume points. The disk unit showed 0.3 ALR across the foam layer without biofilm (INF-BSN); 3.5 ALR across the entire system through two time points (INF-PRECHL).The sleeve unit exhibited 3 ALR in total (INF-PRECHL) over two volume points. The disk unit showed 3 ALR overall (INF-PRECHL) across two volume points. The foam layer without biofilm (INF-BSN) contributed 0.4 ALR over two volume points. The sleeve unit experienced 4 ALR in total (INF-PRECHL) over the two volume points. The disk unit had 4 ALR overall (INF-PRECHL) over the two volume points. The foam layer of the disk unit had 4.95 ALR (INF-BSN), though this result is influenced by the limit of detection.The sleeve unit showed a 3.5 ALR in total (INF-PRECHL) over the volume points. The disk unit experienced a 4 ALR overall (INF-PRECHL). The foam layer without biofilm contributed 0.2 ALR (INF-BSN).The overall goal of this study was to determine the performance of the two multi-barrier filtration systems that used a foam material in a sleeve and disk format as a filter and to support a biofilm. This assessment focused on performance differences between the two configurations.P ≤ 0.01). There was no significant difference between the overall performance of the sleeve unit and the disk unit (P ≤ 0.01) during any stage of the system . The alpha value used for significance for all tests was 0.05.In both the sleeve and disk units, there was a significant difference between the initial pathogen concentrations at the influent and at the effluent (R. terrigena and P22 than the foam (P ≤ 0.01). The foam/biofilm was better able to reduce MS2 and C. parvum compared with the carbon filter (P ≤ 0.01). It is not clear whether this might be due to attachment to the carbon or biofilms. Abbaszadegan and others examined a system with carbon filters and UV light disinfection. In their analysis, the carbon stage of the system contributed 2 log10 reduction of viruses.10 reduction values of P22 in the disk unit but greater than with the observed reduction of MS2 in the current study.10 removal of C. parvum in accordance with National Sanitation Foundation standards. This is 1 log10 higher than the observed reduction in the biofilter disk unit attributable to the carbon alone in the current study.In both the sleeve unit and the disk unit, the organisms were reduced to non-detectable levels. Within the units, the majority of reduction occurred between flocculation and the pre-chlorine stage (after the carbon filter), showing that the foam and carbon filtration system components were highly effective in pathogen removal. Within the disk unit, the effect of the foam/biofilm layer and the carbon filter were examined separately. The carbon filter demonstrated better capability to reduce 10 reduction value.10 reduction values of 2–7 .® Water Purifier  utilizing flocculation and chlorination demonstrated > 7.3 to > 9.96 log10 reduction for bacteria, ≥ 4.78 to > 6.06 log10 reduction for virus, and 3.55–4.32 for protozoa.22Accurate quantification of the inactivation due to chlorination phase was complicated because of the low starting concentration of organisms in the water after treatment entering the disinfection chamber of the units. The chlorine stage reduction values were calculated to be no more than 1.72 ± 1.01 ALR, but the chlorine stage likely has additional reduction capacity that was not observable with the current study design. During challenges, free chlorine residuals ranged from 0.8 to up to 2.1 mg/L (2.14 ± 1.76 average) in sleeve unit prior to dechlorination and from 0.8 to 3.5 mg/L (3.33 ± 1.85 average) in the disk unit of these biofilter systems. Other point-of-use devices utilizing only chlorine demonstrated a baseline of 3 logThe removal performance of the biofilter was explored without preconditioning the systems. This round of testing was meant to simulate the performance of new systems without a biofilm. This could happen when users neglect instructions and use the devices prior to the development of a mature biofilm. Two volumes were evaluated to examine how increase in water passed through the unit (representing two times the standing volume of the systems) influenced the removal.R. terrigena at V1 in the sleeve unit were similar to post-conditioning reduction values. Thus, the role of the biofilm could not be shown except for the parasites, where preconditioning reduction values were lower for C. parvum by 1.81 ALR.It was anticipated that there would be less removal without preconditioning due to a lack of biofilm development. The preconditioning reduction values of MS2 virus, P22 virus, and 10 higher without a biofilm as compared with the filtration unit with mature biofilm formation. All other microbes showed average reduction values for the challenge organisms at least 2 log10 lower as compared with those seen from conditioned biofilter units. These results suggest that for systems without a biofilm, as a greater volume is pushed through the system there is generally a decrease in the removal of microorganisms, suggesting that the microbes initially attach to the filter and then detach as more water flows through the system. The presence of a biofilm may prevent the organisms from being released. There may be a limit as to how much microbe retention the biofilm can support; the release rate of pathogens, particularly protozoa, may be dependent on many variables.12After V2 sampling in the sleeve unit, average bacteriophage MS2 reduction was approximately 1 logR. terrigena. FLOC to PRECHL pre-conditioning reduction values were at least 1 ALR lower than the post-conditioning reduction values.The data from the disk unit preconditioning data set show a notable decrease in the reduction values from flocculation to basin sampling locations compared with the post-conditioning data set, for all organisms except 10 reduction in the biofilter systems for P22 virus and C. parvum . This could represent a key factor in the differences between the pre- and post-conditioning reduction values. Other studies using sand filtration and biofilms cite change in flow rates as contributing factors to changes in removal.These differences between the pre- and post-conditioning reduction values suggest that the biofilm layer itself may be contributing from 1 to 2 logrvum see . The rolR. terrigena removal improved between V1 and V2 in the disk unit. Removal performance for P22 virus showed no change in the disk unit, but removal performance for C. parvum decreased. These varying results will require many more replicates and further experiments for investigating types of biofilms and the mechanisms involved in improving filtration.In the sleeve unit, the removal performance improved for MS2 virus between V1 and V2 but decreased for all other challenge organisms. In the disk unit, removal performance for MS2 was more variable. MS2 virus and A comparison of the sleeve and disk biofilter systems to other point-of-use filtration systems demonstrate removal and inactivation of microbial loads similar to, if not exceeding, those of other water filtration devices. In a review of point-of-use water purification devices, several different methods for removal and/or inactivation of microbial loads were compared: porous ceramic filtration (PCF), biosand filtration (BSF), polyethylene bottles with solar UV and heat disinfection (SODIS), free-chlorine treatment, and coagulation and chlorination treatment.6Cryptosporidium that requires an intact physical barrier for removal, as it is resistant to inactivation by chlorine. The biofilm is disrupted when the foam layer of the biofilters are cleaned, which may produce variability in filtration performance.Apart from performance measures, practical factors such as ease of use must also be taken into account when considering the implementation of biofilter. In comparison to other gravity-fed point-of-use devices such as sand filters, the biofilter designs offer more portability. The major difference between the sleeve unit and the disk unit was the position of the foam filter. In practice, the foam disk configuration was more difficult to use. The foam disk was quite fragile and required greater care to not puncture the foam during the cleaning procedure. This fragility could lead to eventual damage resulting in the reduction in the ability of the system to remove pathogens, particularly The biofilter designs are multi-barrier systems and there are advantages and disadvantages to these designs. The foam system is light weight and has a small footprint compared with biosand filters. Initial setup of the biofilters was considerably less labor intensive and more straightforward. The biofilter materials also lend themselves a uniformity in construction that may be advantageous. The biofilter cartridge is vulnerable to mishandling when being installed. It must be screwed into a pipe located at the bottom of the basin, requiring a certain amount of torque that could easily be over-applied, or the cartridge could be incorrectly positioned.10 reduction power over the entire system, though additional and more testing of this scenario may reveal a more definitive answer to this question.Another possible weakness of the biofilter system is the need for additional substances to maintain full functionality, specifically the flocculation and chlorine steps. A constant supply of flocculating agents  must be supplied to users of the biofilters to maximize settling efficiency. Overall system performance could become compromised in the event when the flocculating agents run out. A similar problem exists with the chlorine tablets used in the holding reservoir. In our evaluations, the chlorine tablets required replacement after 93 days of use, constituting 13–15 L water filtered each day. In the event when flocculation and chlorination substances are depleted and those substances become unavailable to users, the pathogen reduction power of the system may become compromised. According to the results of the post-flocculation and prechlorination removals in 10 reduction for protozoan cysts, 4 log10 for viruses, and 6 log10 reduction for bacteria.In conclusion, these units meet the standards set forth by the U.S. EPA for microbial purifiers, 3 logAdditional study should be conducted evaluating long-term use of the biofilter in a laboratory setting. This study should include long duration of use as well as large volumes and frequency of use to test the filter design under more strenuous conditions. Evaluation of filter performance before and after cleaning procedures should be performed more thoroughly to reinforce or refute the preliminary tests performed in this research study exploring the role of the biofilm in the biofilter system.Additional work should evaluate the use of the filter in a field environment, monitoring important factors such as compliance, disease prevalence, and durability in a real-world setting. A performance test before and after the puncture of the foam disk or sleeve would provide for an understanding of how resilient the filter system would be to damage in the field. Evaluation of performance without the flocculation or chlorination steps would reveal a baseline standard of performance in the event of a disrupted supply chain for renewable materials.


Engystomops pustulosus), to disparate predators approaching from different spatial planes: a terrestrial predator (snake) and an aerial predator (bat). Túngara frogs showed consistently distinct escape responses when attacked by terrestrial versus aerial predators. The frogs fled away from the snake models (Median: 131°). In stark contrast, the frogs moved toward the bat models (Median: 27°); effectively undercutting the bat’s flight path. Our results reveal that prey escape trajectories reflect the specificity of their predators’ attacks. This study emphasizes the flexibility of strategies performed by prey to outcompete predators with diverse modes of attack.The escape response of an organism is generally its last line of defense against a predator. Because the effectiveness of an escape varies with the approach behaviour of the predator, it should be advantageous for prey to alter their escape trajectories depending on the mode of predator attack. To test this hypothesis we examined the escape responses of a single prey species, the ground-dwelling túngara frog ( Prey use a diverse range of anti-predatory strategies ; foremosTyto alba), for instance, move swiftly and quietly towards spiny mice (Acomys cahirinus). Spiny mice do not have the straight-line speed to outrun a swooping barn owl [The importance of selecting the ‘best’ escape trajectory is no more apparent than when prey cannot outpace their attacker , 15. Barbarn owl . Insteadbarn owl ,16. Preybarn owl . Prey exApodemous mystacinus), for example, run when exposed to most predators [Acomys cahirinus [Parus caeruleus [Engystomops pustulosus (Shreve 1941), formerly Physalaemus pustulosus), to attacks by predators with distinct hunting behaviours .It is well recognised that some prey vary their responses to different predators using behaviours ranging from freezing to fleeing ,14. Woodredators ,19 but eredators . Howeveraeruleus ). In conaeruleus  and refeTrachops cirrhosus, attack túngara frogs from the air using the advertisement calls of male frogs to detect and localise them [Leptodeira annulata) [Philander opossum) [Leptodactylus savagei) [Rhinella marina, formerly Bufo marinus) [Potamocarcinus richmondi; [Sericopelma rubronitens) [Túngara frogs are small palatable anurans that are abundant and widespread in Middle America. Male túngara frogs aggregate in temporary ponds to advertise for females, but their calls make them vulnerable to being preyed on by a variety of predators. Fringe-lipped bats, ise them ,23. Túngnnulata) , opossumopossum) ; South Asavagei) , cane tomarinus)  and invechmondi;  and spidonitens) . The brein-situ, we stimulated continued calling by broadcasting a synthetic male túngara frog call played in a continuous loop. Calls were synthesised using software developed by J. Schwartz . The playback stimulus consisted of bouts of the synthetic call and periods of silence such as those found in natural calling bouts of túngara frogs [-1 using a Sony Nightshot DCR-SR45 camcorder with Sony HVL-IRM infrared lights, and later digitised them using ImageJ v1.4u [We conducted this study in the field, around the facilities of the Smithsonian Tropical Research Institute in Gamboa, Panama  from July to August 2011. To simulate the natural conditions under which signalling frogs are attacked, we tested túngara frogs only when calling. We found calling males in shallow puddles within drainage lines in Gamboa between 1900–2300hrs. Leaving the frogs ra frogs . We usedad libitum. Frogs were fed every 2–3 days with live termites. When the testing of all individuals was completed, we released all frogs at their respective capture sites.To prevent retesting, after each trial we captured and temporarily removed the tested individuals from the population. We housed captive frogs in the laboratory in a individual large plastic container (1.5x0.5x0.5m) lined with leaf litter and given water We used models of naturally occurring predators to examine the escape trajectories of túngara frogs in response to aerial and terrestrial predators. A major aerial predator of túngara frogs is the fringed-lipped bat, which is attracted to the frogs’ advertisement calls . We creaLeptodeira annulata (45.5–61 cm), another known predator of calling túngara frogs [We simulated a ground predator using a rubber snake equivalent in size and shape to a cat-eye snake ra frogs . We placBoth model predators were only presented to actively calling male frogs. Given that frogs attacked by bat models remained in the immediate vicinity and would often resume calling after a silent interval, we tested each frog with the bat model twice. When presented with the snake model, however, frogs moved farther away than when presented with the bat model. The frogs exposed to the snake model were often difficult to locate once they fled and did not resume calling. For this reason, frogs presented with the snake model could only be tested once.We transformed digitised x-y coordinates from the video into polar coordinates using an arctan transformation. We calculated the extent to which the frog rotated in response to the model as the difference between the directions the frog faced before and after exposure to the stimulus. Frog flight paths were expressed relative to the model’s approach trajectory. A 0° escape corresponds to the frog moving directly toward the model while 180° represents the frog fleeing directly away from it. A negative or positive prefix refers to the frog fleeing to the left or right of the approaching axis of the predator.To determine if the angle the frog faced influenced the direction the frog fled we conducted a correlation test for circular data following . To examTo compare the circular distributions between predator models, we employed the Watson’s two-sample test of homogeneity, which is appropriate for non-normal data and, is robust for small sample sizes . Frog esAll animal handling and experimental procedures were reviewed and approved by the Smithsonian Tropical Research Institute IACUC #2011-0616-2014-11. Permits were granted by the Autoridad Nacional del Ambiente .ind = 10, ntrials = 17, 59.3±3.6 cm; call cessation and fleeing: Nind = 21, ntrials = 38, 39.5±1.8 cm). The height at which the bat model flew above the frog did not, however, influence the escape angle of fleeing individuals  = 0.15). In response to the snake model, all individuals responded by fleeing. The time from predator detection to fleeing, however, was significantly longer in frogs exposed to the snake model than in response to the bat model .Individuals threatened by the approaching predator model would sequentially perform the following defensive behaviours: (1) cease calling keeping their body still inflated , (2) cease calling and rapidly deflate both their body and vocal sac, (3) jump to safety or dive under the water surface. This observed graded defensive response is consistent with those reported in previous experiments using predator models with túngara frogs . The froU2 = 0.0591, P > 0.1; U2 = 0.027, P > 0.1; U2 = 0.065, P > 0.1). In contrast, the escape response angles by frogs to both the bat and snake models were directional .The angles at which the model approached the frogs were random and homogenous, with no directionality for either the bat . Additionally, the angles of frog rotation for both predator models were not significantly different to each other . The frogs actively rotated their bodies until reaching a direction to escape. The angles of rotation for both the bat and snake models did not vary from random . Instead, the frogs fled at angles ranging between 10–60° relative to the bat model’s angle of approach , which concurs with findings from previous studies that have used snake models  or obserPsammodromus algirus), which are seemingly ‘hard-wired’ to escape in the direction they are facing when attacked even though this strategy at times puts them in the path of the predator ([Meriones unguiculatus) respond to a non-ecologically relevant stimulus that approaches from above by fleeing in the direction they are facing resulting in some escape trajectories in the direction of the approaching object [Studies to date have shown that prey escape trajectories are characteristically constrained to the hemisphere opposite to the threat (90–180°); that is, fleeing in a direction opposite or perpendicular to the predator’s approach (reviewed in ). Escaperedator (. Similarg object . TúngaraParus major) presented with a raptor model at a range of different aerial angles of approach modulate their take-off angle. Great tits take off at shallower angles in response to predators with steep attack trajectories (45°), while they use higher angles in response to the same predator approaching with shallow attack trajectories (15°) [Flexibility in prey fleeing response, such as the behaviour described in this study, is widespread across prey species. For instance, male great tits (es (15°) . Our resTyto alba) avoid capture more often than mice dragged away from them [While movement toward an approaching predator is risky, it is possible that it is the most effective method of predator avoidance when prey cannot outpace their attacker. It is common that prey adjust the flight initiation distances to outpace the predators they confront ,49. Fleerom them . Althougrom them . FurtherIn túngara frogs, several lines of evidence suggest that preferentially moving towards an aerial threat likely increases the rate of successful escape. First, the resultant fleeing velocity of the frog escaping towards the predator is higher than the resultant velocity the frog could achieve by moving away from it given the combined velocity of the approaching predator and the prey moving in the same direction. Second, the frog effectively increases its distance from the bat without having to cover as much ground (i.e. combined distance the frog jumps and the distance the bat overshoots the capture site before turning). Together, increased relative fleeing velocity and increased fleeing distance provide the bat with less time to access and respond to the prey than if the prey moves away or perpendicular to the predator. Once the frog moves toward the bat and the bat passes over the frog, the frog stops moving and remains silent. Fringe-lipped bats rely heavily on prey-emitted acoustic cues to localise their prey , and likCraugastor spp), for instance, presented with metallic objects moving towards them from various elevations, jumped away or had random escape trajectories relative to the angle at which the object approached [Rana pipens), tested in response to black squares moved in a pendulum fashion towards the frog, consistently responded by turning away from the object [It is well documented that frogs typically flee away from attacking terrestrial predators ,39,57,58e object ,62. How Lithobates catesbeianus) respond differently to a simulated object moving towards the frog from different areas in their visual field. When the stimulus was presented in the frontal visual field, the frog jumped forward. In contrast, the individuals turned and jumped towards the stimulus when it was presented in the caudal visual field [In this study, túngara frogs occupied human-constructed drainage lines. Although these calling sites are typical for this species often found in urban environments, it is unclear whether túngara frogs show similar escape responses in more natural settings. As many other animals, frogs have ‘instinctual’ spatial awareness to avoid known physical barriers when escaping . Unexpecal field . These rTo our knowledge, ours is the first study to investigate the escape trajectories of a single prey species confronted with both terrestrial and aerial simulated predators in the prey’s home environment. Our study demonstrates that túngara frogs use different escape trajectories in response to attacks by bats and snakes. In nature, prey confront multiple predators that use different attack tactics . Predato


In a fully MHC mismatched mouse kidney allograft model, we describe the synthesis of an immunotoxin, consisting of the F(ab′)2 fragment of a monoclonal antibody against the donor MHC class II molecule I‐Ak conjugated with the plant‐derived ribosomal inactivating protein gelonin. This anti–I‐Ak gelonin immunotoxin depletes I‐Ak expressing cells specifically in vitro and in vivo. When given to recipients of kidney allografts, it resulted in indefinite graft survival with normal graft function, presence of Foxp3+ cells within donor grafts, diminished donor‐specific antibody formation, and delayed rejection of subsequent donor‐type skin grafts. Strategies aimed at the donor arm of the immune system using agents such as immunotoxins may be a useful adjuvant to existing recipient‐orientated immunosuppression.Rejection of donor organs depends on the trafficking of donor passenger leukocytes to the secondary lymphoid organs of the recipient to elicit an immune response via the direct antigen presentation pathway. Therefore, the depletion of passenger leukocytes may be clinically applicable as a strategy to improve graft survival. Because major histocompatibility complex (MHC) class II MHC class II–specific immunotoxin allows long‐term murine kidney allograft survival without apparent protective immune effects. BUNblood urea nitrogenDCdendritic cellELISpotenzyme‐linked immunospotEYFPenhanced yellow fluorescent proteinIFNγinterferon‐γMHCmajor histocompatibility complexTLOtertiary lymphoid organThe key initiating event during the rejection of transplanted organs is the interaction between donor passenger leukocytes and recipient T cells. This was first demonstrated by a series of experiments where donor grafts were depleted of passenger leukocytes by being “parked“ in primary recipients under cover of immunosuppression before being retransplanted While modern antirejection treatments have concentrated on modifying the immune system of the recipient, relatively less work has been done on modifying that of the donor, with very few strategies that have been developed for the clinic. A pilot study perfusing human donor kidneys with anti‐CD45 antibodies to deplete donor passenger leukocytes before transplantation resulted in a reduced incidence of rejection in vitro before transplantation Irradiation of rat livers or hearts to remove donor passenger leukocytes before transplantation can prolong graft survival Studies using antidonor antibodies, including anti–MHC class II antibodies, have yielded mixed results. In a rat model, antidonor MHC class II antibody prolonged the survival of heart allografts Gelonium multiforum, which causes cell death by inhibiting protein synthesis.Strategies such as parking or irradiation of donor organs are difficult or impossible to translate to the clinic. We aim to use an antidonor MHC class II antibody to deliver a toxin to donor MHC class II–expressing cells, to delete this population in a clinically applicable manner. The use of an immunotoxin, more commonly used in cancer therapy, allows targeted cell death without the need for complement activation or antibody‐dependent cell‐mediated cytotoxicity associated with the use of antibodies, and the inflammation that this may cause, which may promote alloreactivity. We have used the plant toxin gelonin, a ribosome inactivating protein found in the plant An MHC class II immunotoxin has previously been used in a pancreas allograft model k gelonin immunotoxin, abbreviated as I‐Ak‐gelonin) to deplete the MHC class II+ donor passenger leukocyte population in a fully mismatched mouse model of kidney transplantation, to investigate the effect of this treatment on graft survival.In this study, we used an antibody against a single donor MHC class II molecule conjugated to gelonin (anti–I‐Ab/k), FVB (H‐2q), C57BL/6 (H‐2b), CBA.Ca (H‐2k), and BALB/c (H‐2d) mice were purchased from Harlan Limited . C57BL/6 mice expressing the enhanced yellow fluorescent protein (EYFP) transgene on all cells  and BL/6 EYFP × CBA F1 mice were bred in‐house. All mice were kept under specific pathogen‐free conditions.All animals were between the age of 8 and 12 weeks and used in accordance with the Animals (Scientific Procedures) Act of 1986. Female C57BL/6 × CBA F1  was digested into F(ab′)2 and Fc fragments using an F(ab′)2 preparation kit  according to the manufacturer's instructions. The F(ab′)2 fragments were conjugated to gelonin  as previously described 2 fragments were modified with 3‐(2‐pyridyldithio) propionic acid N‐hydroxysuccinimide ester  and incubated with a 5‐fold molar excess of gelonin, modified with 2‐iminothiolane (Sigma), for 20 hours before the reaction was terminated with 2 mmol/L iodoacetamide (Sigma). Unconjugated gelonin and F(ab′)2 fragments were removed via passage through a HiTrap rProtein A FF column  and a HiTrap Blue HP column (SLS), respectively.A mouse anti‐mouse MHC class II I‐A6 cells/mL in a 48‐well plate, with either unconjugated antibody, unconjugated F(ab′)2, or I‐Ak‐gelonin, all at a concentration of 400 nmol/L. After 72 hours in culture at 37°C, 5% CO2, cells were removed for flow cytometry. Cells were stained with anti–I‐Ek‐PE , which recognizes the other MHC class II molecule on target cells so that binding of I‐Ak‐gelonin to the target cells would not hinder the detection of MHC class II+ cells and a live/dead cell stain , used according to the manufacturer's instructions. Cells were acquired using a FACScalibur flow cytometer  and analyzed using Cellquest software version V3.3 (BD).Single‐cell suspensions were obtained from spleens harvested from CBA and BALB/c mice. Red blood cells were lysed and the splenocytes plated at 2 × 10k‐gelonin in killing I‐EK+ cells in vivo was tested. Single‐cell suspensions were obtained from spleens harvested from BL/6 EYFP × CBA F1 mice. Red blood cells were lysed, and the splenocytes were injected intravenously into recipient mice (one spleen per recipient). I‐Ak‐gelonin was then injected intravenously into these mice. Spleens of recipient mice were harvested 20 hours later, and flow cytometry was performed as described here earlier, using only anti–I‐Ek‐PE antibody.The effectiveness of the I‐Ak‐gelonin was given intravenously on the day of transplant. The recipient left native kidney was removed at the time of transplantation. The remaining right native kidney was removed 1 week after transplantation, leaving the donor graft life sustaining. Before and at regular intervals after the second native nephrectomy, blood samples were obtained and measurements of blood urea nitrogen (BUN) were made to monitor graft function, using Infinity Urea  according to the manufacturer's instructions. Heart transplantation was performed as described previously Mouse renal transplantation was performed as described previously Periodic acid–Schiff staining was performed on 2‐μm‐thick paraffin sections as previously described Immunohistochemistry was performed on 5‐μm‐thick frozen sections as previously described Serum from recipient mice were used for the detection of donor‐specific alloantibodies as previously described 5 responder cells.Enzyme‐linked immunospot (ELISpot) assays were performed as previously described Unpaired two‐tailed Student's t‐tests were used for all results except survival, which was tested by the log rank‐sum test. Values are expressed as mean ± standard error of mean.k expressing) with I‐Ak‐gelonin for 72 hours resulted in a doubling of cell death in the MHC class II+ population, demonstrating its effectiveness in vitro k (inherited from the CBA parent) that is recognized by the I‐Ak‐gelonin immunotoxin. Splenocyte injection was used instead of kidney transplantation here because it results in a higher number of cells that can be tracked, giving a more accurate dose titration. Splenocytes were injected into recipients with or without I‐Ak‐gelonin treatment given intravenously, and 1 day later, their spleens were harvested for flow cytometry. This experiment was repeated several times with different doses of I‐Ak‐gelonin. One representative experiment is shown in Figures k. After injection of 40 or 100 μg of I‐Ak‐gelonin, this population was reduced to 0.04%. The donor‐derived nontarget population (EYFP+ but negative for I‐Ek) was not affected by I‐Ak‐gelonin injection, at 0.03% and 0.04% with or without immunotoxin injection, respectively. The dose of 40 μg I‐Ak‐gelonin was chosen for use in the kidney transplant study.BL/6 EYFP × CBA F1 mice were used as splenocyte donors as they express both the EYFP protein, which allows tracking of donor cells in recipient mice, and the MHC class II molecule I‐A+ cells on graft survival, target antigen I‐Ak–expressing C57BL/6 × CBA F1 kidneys were transplanted into fully MHC mismatched FVB recipients that do not express the target antigen I‐Ak. In naive recipients, 40% of grafts were rejected acutely, with 40% undergoing chronic rejection and the remaining 20% surviving long term  showed mononuclear cell infiltration, including tubulitis, and tubular necrosis Figure A. Of thee Figure C. Mononue Figure D. In thee Figure E. Foxp3 s Figure F, suggesk‐gelonin was also tested in a heart transplantation model, using the same donor–recipient strain combination. Treatment of recipients with I‐Ak‐gelonin led to a slight prolongation of heart graft survival from a median survival time of 17.5 to 31.5 days (n = 4).I‐Ak‐gelonin was administered to FVB recipients of C57BL/6 hearts, which do not express I‐Ak and whose passenger leukocytes should therefore not be depleted by the immunotoxin. Treatment of recipients with I‐Ak‐gelonin had no effect on graft survival .To ensure that the effect seen is donor specific, I‐Ak‐gelonin were given challenge skin grafts at around 100 days posttransplantation. The original donor strain, C57BL/6 × CBA F1, the two parental strains C57BL/6 and CBA , as well as third‐party BALB/c mice were used as donors. C57BL/6 × CBA F1 donor skin showed prolonged survival .Sections of the transplanted heart were stained for Foxp3 expression. The number of Foxp3k‐gelonin–reated splenocytes . This was probably due to the indirect pathway, because by the time point chosen (day 10 posttransplantation) we would expect most donor passenger leukocytes to have been killed. The addition of donor cells as stimulators resulted in an increase in IFNγ production through the direct pathway in both control and I‐Ak‐gelonin–treated groups , while no or very little increase was seen when third‐party cells were used as stimulators   production in response to either alloantigen presented via the indirect pathway only (using lysed donor splenocytes as stimulator cells) or alloantigen presented by both the direct and indirect pathways (using intact donor splenocytes as stimulator cells). Because DCs were not removed from the recipient splenocyte preparation, an IFNγ response was seen even without the addition of donor cells as stimulators but with no differences between control and I‐A+ cells), but antibodies cause cell death through complement activation or antibody‐dependent cell‐mediated cytotoxicity, both of which cause inflammation. When used to target donor cells, this has the potential to exacerbate the alloimmune response. Immunotoxins combine the specificity of antibodies with killing by toxin delivery, which may avoid these potential problems.Given the importance of donor passenger leukocytes in the alloimmune response, it makes sense to develop strategies to deplete them to improve graft survival, such as using monoclonal antibodies or immunotoxins. Antibodies have the advantage of allowing specific depletion of the target cell populations and are already being routinely used in the clinic to deplete or modify recipient cells 2 component.Gelonin, the ribosome‐inactivating protein used here, is unable to enter cells in its unconjugated form. Instead, it relies on antibody internalization for delivery into the cytoplasm and therefore is highly specific. To avoid complement activation and antibody‐dependent cell‐mediated cytotoxicity, the antibody used here was digested to remove the Fk‐gelonin on graft survival due to the range of outcomes seen, allowing the immunotoxin's effect on both acute and chronic rejection and on long‐term survival to be studied. The use of F1 donors also enabled us to use either parental strain as challenge graft donors.The C57BL/6 × CBA F1 to FVB kidney transplant model was chosen to study the effect of I‐ALong‐term graft survival was seen in treated kidney recipients, while heart recipients showed a modest prolongation of survival. This may be because heart grafts are more immunogenic than kidney grafts. As the immunotoxin would not have depleted donor passenger leukocytes immediately, the initial exposure of the immune system of the recipient may have resulted in the rejection of the heart grafts, albeit at a delayed tempo. Kidney grafts however, are more “tolerogenic,” and the reduced immune response resulting from the immunotoxin treatment may have been sufficient to induce indefinite graft survival. Long‐term acceptors of kidney grafts demonstrated prolonged survival of donor‐type skin grafts, but their survival was not indefinite. This is not surprising because skin is far more immunogenic than kidney grafts, and it is not unusual to observe split tolerance. In addition, skin‐specific antigens may also play a role here.Tolerance was clearly not induced in the heart transplant model. If translated to the clinic, immunotoxin treatment would not be a means to induce indefinite graft survival but may be a useful adjuvant to prevent rejection. Because it is donor specific, the risk of opportunistic infections and malignancies should not be increased.In the kidney transplant model, we did observe indefinite graft survival. It has been shown that donor passenger leukocytes are essential for tolerance induction The indefinite survival induced in kidney transplant recipients suggests that the immunotoxin itself is unlikely to have any detrimental effects on the graft and can protect it from the rejection process. Mouse endothelial cells can upregulate MHC class II following transplantation k‐gelonin depletes donor passenger leukocytes, it dampens the direct pathway of allorecognition. Due to this interference with the direct pathway of allorecognition, it might have been expected that the I‐Ak‐gelonin would have an effect on acute rejection, which is thought to primarily be caused by the direct pathway, but no effect on chronic rejection, which is thought to be mediated mainly by the indirect pathway. However, I‐Ak‐gelonin administration prevented both acute and chronic rejection and instead induced long‐term survival of the kidney grafts and extended the survival of challenge skin grafts. This may be due to the fact that the direct pathway of allorecognition is the first to appear and is the bigger response in terms of the percentage of T cells that can recognize foreign MHC class II directly [estimated at between 1% and 7% of T cells k‐gelonin. The presence of Foxp3+ cells [which have previously been shown to be important in spontaneous acceptance of kidney allografts k‐gelonin is in agreement with this. However, there was very little difference between the number of Foxp3+ cells seen in heart grafts 10 days after transplantation in treatment and control groups. This may be because heart grafts are more immunogenic, and therefore any beneficial effect of the immunotoxin is not as obvious.Because the I‐Ak‐gelonin on the direct and indirect pathways separately, ELISpot assays were performed in which either whole donor splenocytes were used, meaning antigen could be presented by both direct and indirect pathways, or lysed donor splenocytes were used, meaning antigen could be presented only by the indirect pathway k‐gelonin–treated and control groups. This may be due to the time point chosen (10 days posttransplantation), which may be too late to detect any effect of the I‐Ak‐gelonin treatment. Also, the ELISpots were carried out on heart transplant recipients, which were less sensitive to I‐Ak‐gelonin treatment.In an attempt to determine the effect of I‐Ak‐gelonin did reduce the level of circulating donor‐specific antibodies in kidney recipients, presumably because of a reduction in the number of alloantigens available. However, the presence of circulating donor‐specific antibodies, albeit at lower levels, in I‐Ak‐gelonin–treated recipients of kidney allografts suggests an active immune interaction with the graft, as does the presence of TLOs within these grafts. In a long‐term surviving DBA/2 to C57BL/6 model of kidney transplantation, the presence of donor‐specific antibodies does not affect graft function (unpublished information) and TLO formation is indirectly proportional to BUN k‐gelonin–treated recipients.Treatment with I‐AOur data suggest that depletion of donor passenger leukocytes, resulting in indefinite kidney graft survival and good graft function, can be achieved with a single treatment of an antidonor MHC class II immunotoxin administered to the recipient after transplantation. Current antirejection treatment is almost exclusively directed against the immune response of the recipient. The approach described here, directed specifically against cells of donor origin, opens a new, simple, and safe therapeutic avenue that can be used in conjunction with conventional therapy.American Journal of Transplantation.The authors of this manuscript have no conflicts of interest to disclose as described by the


Fungal burden in the cerebrospinal fluid is an important determinant of mortality in cryptococcal meningitis, but its use in aiding clinical decision making is hampered by the time involved to perform quantitative cultures. Here, we demonstrate the potential of flow cytometry as a novel and rapid technique to address this issue. Cerebro culture . This raCryptococcus counts per milliliter of CSF. Statistical analyses were performed using Stata version 12 (StataCorp).A prospective cohort study was conducted in Cape Town, South Africa. All participants provided written informed consent; surrogate consent was obtained from the next of kin for patients with impaired consciousness. Ethical approval was obtained from the University of Cape Town  and Liverpool School of Tropical Medicine  research ethics committees. HIV-infected persons age ≥18 years with a first episode of cryptococcal meningitis (diagnosed by antigen test or culture) were enrolled within 48 h of diagnosis, and lumbar puncture performed to measure the cerebrospinal fluid (CSF) opening pressure. CSF fungal burden was assessed with quantitative culture  and cryp10 CFU/ml of CSF , as measured by quantitative culture, and 4.53 log10Cryptococcus yeasts per ml of CSF , as measured by flow cytometry. The median CrAg LFA titer was 1:8,000 .Sixty HIV-infected patients with cryptococcal meningitis were enrolled, with a median CD4 count of 34 cells/μl of CSF. CSF samples were available for 58 participants, 36 of whom had not received any amphotericin B prior to enrollment. The median CSF volume collected for flow cytometry was 7 ml . The median fungal burden was 4.74 logr = 0.91; P < 0.0001) (P < 0.0001). Linear regression showed that the quantitative culture result could accurately be predicted from flow cytometry counting . The agreement between flow cytometry counting and quantitative culture was also assessed using a Bland-Altman plot (10 CFU/ml of CSF and only 6.9% (4/58) of values outside the 95% limits of agreement. These outlying values were found mainly among participants with low fungal burdens (<500 CFU/ml of CSF), whereas flow cytometry counting produced values that were approximately 1- to 2-log10 CFU/ml of CSF higher. Similarly strong correlation and agreement between the two measurement techniques were also noted when analysis was restricted to the 36 participants who had not received any antifungal therapy prior to CSF sampling  of the values outside the limits of agreement   and CrAgman plot . This sh< 0.0001 ; the meagreement .in vitro [These findings suggest that flow cytometry has the ability to provide a rapid and accurate measurement of fungal burden in persons with HIV-associated cryptococcal meningitis. If combined with a cryptococcal viability stain (as previously demonstrated in vitro ), flow cDue to the well-recognized toxicity of amphotericin B , there i− cell found in the CSF. Given that all participants had laboratory-confirmed cryptococcal meningitis, a lysis buffer was used to ensure that no erythrocytes remained in the CSF, and host leukocytes were excluded using the panleukocyte marker CD45; we feel this is a reasonable assumption and that our results are valid. However, to be a fully robust clinical assay, an anticryptococcal stain would ideally be incorporated into the panel and the assay validated on CSF samples from patients who do not have CM. This would have the additional benefit of improving the accuracy of flow cytometric counting at low fungal burdens, where debris or miscellaneous cells may have interfered with the counting process. In addition, we examined only the use of flow cytometric counting to assess fungal burden at baseline and did not assess changes on antifungal therapy. Future work should aim to incorporate a cryptococcal viability marker to address this issue, as previously assessed in vitro (There were a number of limitations to this study. No cryptococcus-specific stain was used to identify cryptococci; instead, they were assumed to be any CD45


For more information about the data, please refer to the related original article Knowledge about the normal human cerebrospinal fluid (CSF) proteome serves as a baseline reference for CSF biomarker discovery and provides insight into CSF physiology. In this study, high-pH reverse-phase liquid chromatography (hp-RPLC) was first integrated with a TripleTOF 5600 mass spectrometer to comprehensively profile the normal CSF proteome. A total of 49,836 unique peptides and 3256 non-redundant proteins were identified. To obtain high-confidence results, 2513 proteins with at least 2 unique peptides were further selected as bona fide CSF proteins. Nearly 30% of the identified CSF proteins have not been previously reported in the normal CSF proteome. More than 25% of the CSF proteins were components of CNS cell microenvironments, and network analyses indicated their roles in the pathogenesis of neurological diseases. The top canonical pathway in which the CSF proteins participated was axon guidance signaling. More than one-third of the CSF proteins (788 proteins) were related to neurological diseases, and these proteins constitute potential CSF biomarker candidates. The mapping results can be freely downloaded at  Value of the data•This study identified the largest high-confidence dataset of the human CSF proteome.•Some CSF proteins׳ abundances are quantified by the iBAQ method.•High proportion of the CSF proteins is microenvironment components of CNS cells.•High proportion of the CSF proteins participate in neurocyte connectivity.•A large part of CSF proteins are biomarker candidates of neurological diseases.11.11.2CSF pooled from 14 patients (7 women and 7 men) who received spinal anesthesia before non-neurological operations was subjected to a global proteomic analysis. The pooled sample was first depleted of 14 high-abundance proteins with an immunoaffinity column. The flow-through proteins, bound proteins, and original proteins (extracted from the CSF samples that were not subjected to immunoaffinity depletion) were collected separately, digested according to the filter-aided sample preparation method 1.31.3.1A TripleTOF 5600 mass spectrometer from AB Sciex  and an ACQUITY UPLC system from Waters  were used.1.3.2Deionized water from a MilliQ RG ultrapure water system  was used at all times. HPLC-grade acetonitrile and formic acid, ammonium bicarbonate, iodoacetamide, dithiothreitol, sequencing-grade modified trypsin, and protease-inhibitor PMSF (phenylmethanesulfonyl fluoride) were purchased from Sigma-Aldrich .1.3.3g to remove cellular components and subsequently aliquoted and stored at −80 °C for further analysis. A total of 14 samples from 14 individuals  were selected and subjected to quantitation by the Bradford method CSF samples were collected by lumbar puncture from patients who received spinal anesthesia before non-neurological operations at Beijing Tiantan Hospital. These patients were checked by an independent medical doctor to rule out neurological diseases and recent medication use. Following collection, a subsample of each CSF sample was sent to a clinical laboratory for routine CSF diagnostics. The remaining sample was immediately centrifuged for 10 min at 2500×1.3.41-acid glycoprotein, α2-macroglobulin, apolipoprotein A-I, apolipoprotein A-II, fibrinogen, complement C3, and transthyretin) using a 4.6×50 mm Human 14 affinity LC column  with a Waters HPLC system . The separations were performed according to the manufacturer׳s instructions regarding column usage and loading capacity. The flow-through protein sample and bound protein sample were collected separately. The two samples and the non-depleted CSF sample  were subjected to the sample handling and analysis procedures described below.The pooled CSF sample, which contained approximately 1 mg of CSF protein, was depleted of 14 high-abundance proteins  overnight at 37 °C. After digestion, the three peptide mixtures derived from the flow-through, the bound sample and the non-depleted CSF sample were desalted on a Waters Oasis C18 solid-phase extraction column and lyophilized for HPLC separation.A filter-aided sample preparation method 1.3.62O, pH=10). The elution gradient was 5–30% buffer B2  for 60 min. The eluted peptides were collected as one fraction per minute, and the 60 fractions collected were re-suspended in 0.1% formic acid and pooled into 30 fractions. A total of 90 fractions produced from the three peptide mixtures were analyzed by LC-MS/MS.The three lyophilized peptide mixtures were fractionated with a high-pH RPLC column from Waters . Each peptide mixture was loaded onto the column in buffer A2 . The elution gradient was 5–30% buffer B1  for 40 min. A TripleTOF 5600 mass spectrometer was used to analyze the fractions. The MS data were acquired using the high-sensitivity mode with the following parameters: 30 data-dependent MS/MS scans per full scan, full scans acquired at a resolution of 40,000 and MS/MS scans at a resolution of 20,000, rolling collision energy, charge state screening (including precursors with charge states of +2–+4), dynamic exclusion (exclusion duration 15 s), MS/MS scan range of 100–1800 m/z, and a scan time of 100 ms.1.3.8www.uniprot.org) using Mascot software, version 2.3.02 . Trypsin cleavage specificity was set with a maximum number of allowed missed cleavages of two. Carbamidomethylation (C) was set as a fixed modification. The searches were performed using a peptide and product ion tolerance of 0.05 Da. The resulting dataset was further filtered using the decoy database method in Scaffold (v 4.3.2).The MS/MS spectra were searched against the Swiss-Prot human database from the UniProt website (1.3.9(1)The protein intensities were first computed by Progenesis LC-MS  as the sum of all identified peptide intensities .(2)The protein intensities were then divided by the number of theoretically observable peptides .(3)The resulting intensities were iBAQ values, which are shown as “Absolute iBAQ intensities” in column K of (4)The relative iBAQ intensities (in column L of (5)The relative iBAQ intensities were applied to estimate the relative protein abundances .(6)The protein abundances (or concentrations) in CSF were finally calculated by multiplying the relative iBAQ intensities by 0.3 (the protein concentration of the pooled CSF sample).Protein abundances were estimated using the iBAQ algorithm 1.3.10http://www.ingenuity.com) and the PANTHER classification system (http://www.pantherdb.org/genes/batchIdSearch.jsp) All identified CSF proteins were subjected to network and functional analyses using ingenuity pathway analysis (IPA) version 9.0 (


Split skin grafting (SSG) is the cornerstone in the treatment of deep burns and large skin defects. Frequently used donor sites are the thigh, abdomen and buttocks. The scalp is less common while considered as a reliable donor site. Advantages are a large surface area, rapid wound healing, cosmetically favourable results and multiple harvests from the same donor site. Complications include scab formation, chronic folliculitis and alopecia but have been recorded sporadically in previous studies. This article evaluates the complication rate of the scalp donor site in the treatment of deep burns in the Beverwijk Burn Centre.A retrospective study was performed of all patients who received a skin graft from the scalp at the Beverwijk Burn Centre between January 2004 and December 2012. Data were collected from medical files of included patients, including gender, age, type of burn  and total body surface area (TBSA) burned at the time of first surgery. Postoperative variables were healing time of the donor site and incidence of complications. During follow-up, the incidence of late complications was reviewed.A total number of 105 grafts were analysed in 93 patients: 58 males (62 %) and 35 females (38 %), with a median age of 2 years and 3 months old. Of the patients, 30 (32 %) had flame burns and 57 (61 %) had scald burns. Eighty-seven percent of patients had a TBSA burned of 5 % or less. All donor sites healed within 14 days. No alopecia or scar hypertrophy developed at the donor sites. Two patients (2.2 %) developed folliculitis; one patient (1.1 %) showed scab formation.The scalp as a donor site in our Burn Centre shows a comparable short-term complication rate to the previous literature, with quick healing and no long-term complications. Therefore, we propose the consideration of the scalp as a primary donor site, especially in young children, where the scalp offers a larger donor site area than the buttocks or thighs. Split-thickness skin grafting (SSG) is the cornerstone in the treatment of deep partial-thickness burns with a large risk of scar formation, full-thickness burns and large skin defects. The common donor sites are the thigh, abdomen and buttocks. In the treatment of deep partial-thickness burns, skin grafting is usually carried out 10 to 14 days post-burn in the Beverwijk Burn Centre.The scalp as a donor site is less common, while it is considered a reliable donor site since its first use in 1964 –4, cosmeChang et al.  define tThis article evaluates the complications of the scalp as a primary donor site in the treatment of burns in the Beverwijk Burn Centre.This retrospective study was conducted in the Red Cross Hospital in Beverwijk, The Netherlands. All patients who received a split-thickness skin graft from the scalp as a donor site between January 2004 and December 2012 were included. All patients were treated per in-hospital protocol, as described below.th to 12th day post burn, the surgery and donor site are discussed with the patient or parents. Especially in children, the scalp is the first choice of donor site. When patients or parents strictly object after explanation and refuse to give their informed consent, a different donor site is chosen.If the wound is judged to be indicated for surgery, usually around the 8In adults, the scalp is the primary choice of donor site in burns above the clavicles because of the good colour match.Contraindications for the use of the scalp are conditions in which impaired wound healing of the scalp is expected, for instance, atrophy of the skin of the scalp, burns of the scalp or pre-existent skin conditions of the scalp such as folliculitis, dermal mycosis or eczema.®Air Dermatome . Haemostasis is achieved by a temporary alginate dressing soaked in adrenaline solution: 10 mg adrenaline in 1 L NaCl 0.9 %  and total body surface area (TBSA) involved at the time of the first surgery. Split-thickness skin grafting was indicated in deep partial- to full-thickness burns. According to our protocol, these are defined as burn wounds unlikely to have healed within 21 days post-burn, assessed on the 10Postoperative variables were the time of healing of the donor site and the incidence of donor site complications, like folliculitis and scab formation. During the follow-up period in the outpatient clinic, the incidence of late donor site complications like alopecia and the development of scar hypertrophy was registered.A total number of 105 grafts in 93 patients was analysed , no medical-ethical approval is necessary according to the WMO :http://www.ccmo.nl/en/file-research).“Retrospective research/research with patient files does not fall under the scope of the WMO as the research subject is not physically involved in the research.” as quoted from the website of the Central Committee of Research involving human subjects. (N/A.


Macrodontia  is a rare shape anomaly that has been used to describe dental gigantism. Mandibular second premolars show an elevated variability of crown morphology, as are its eruptive potential and final position in the dental arch. To date, only eight cases of isolated macrodontia of second premolars have been reported in the literature. This case report presents clinical and radiographic findings of unusual and rare case of isolated unilateral molarization of left mandibular second premolar.How to cite this article: Mangla N, Khinda VIS, Kallar S, Brar GS. Molarization of Mandibular Second Premolar. Int J Clin Pediatr Dent 2014;7(2):137-139. Canoglu4 et al reported an overall prevalence of macrodont permanent teeth to be 0.03 to 1.9% with a higher frequency in males. In most of the cases, macrodontia in mandibular second premolars has been reported in children.78 All the reported unilateral cases of macrodontia involved the right mandibular second premolar.3Macrodontia , is a rare shape anomaly that has been used to describe dental gigantism.45 Isolated form of macrodontia has rarely been reported.49 To date, only eight cases of isolated macrodontia of second premolars have been reported in the literature; five of which have shown bilateral occurrence.14 No case report of unilateral erupted macrodont mandibular second premolar has been reported in the literature.Macrodontia is usually associated with systemic disturbances or syndromes, such as insulin-resistant diabetes, otodental syndrome, facial hemihyperplasia, KBG syndrome, Ekman-Westborg-Julin syndrome and 47 XYY syndrome.This case report presents clinical and radiographic findings of unusual and rare case of isolated unilateral molarization of left mandibular second premolar.A 14-year-old male patient reported to our clinic for routine dental check-up. There was no relevant family and medical history elicited. Extraoral examination revealed no abnormalities. On intraoral examination, dental caries was present involving the buccal pits of permanent mandibular right and left first and second molars. The patient had extrinsic stains and calculus with respect to mandibular anterior region . He presThe mandibular left second premolar had an abnormal ovoid molariform crown with an irregular crescent-shaped crater-like fissure with the convex aspect toward the lingual side, in occlusion (fully erupted) which resulted in severe crowding in mandibular anterior region . ClinicaAustralopithecus robustus, in which the premolars are shaped like molars, with large occlusal surfaces and one, two or three roots.3 The etiology of dental anomalies remains largely unclear but some anomalies in tooth structure, shape and size results by many factors from disorders during the morphodifferentiation stage of development. Identification of specific patterns of associated dental problems could be related with certain genetic and environmental factors contributing to different dental anomaly subphenotypes.7The molar-like morphology of the premolars consists of a reduction of the single vestibular cusp, the shoulders of which appear as small extra cusps. The resulting appearance is the same as that of a mandibular first molar, with three vestibular cusps and three, two, one or no lingual cusps. In studies of dental anthropology and hominid evolution, descriptions are found, such as that of 1011 True macrodontia of a single tooth should not be confused with fusion of teeth, in which early in odontogenesis, the union of two or more teeth results in a single large tooth.10Macrodontia can be classified as true generalized where teeth are larger than normal and are associated with pituitary gigantism, relative generalized where there is presence of normal or slightly larger than normal teeth in small jaws and macrodontia involving single tooth.7 This type of macrodontia is more frequently found in incisors and canines,5812 and has been rarely reported to involve premolars and molars. All the reported unilateral cases of macrodont mandibular second premolars demonstrate the involvement of the right tooth but, in the present case, the left tooth was involved.8According to the classification of macrodontia, this case corresponds to an isolated macrodontia. It is uncommon to see localized macrodontia alone, because generally it is associated with a syndrome.13 Thus, the appearance of severe crowding was a predictable consequence of the increased size of the mandibular second premolar. Under normal conditions, the mesiodistal size of the premolar is less than that of its deciduous predecessors, particularly in the case of mandibular second premolars.31415 Problem of plaque accumulation is found in such cases because of surface notching as in this reported case.16The mesiodistal (11 mm) and buccolingual diameter (11.5 mm) of mandibular second premolar was greater than its normal size of 7 and 8 mm respectively.8The large crown size causes problems with eruption and disrupts the dentition. There are consequent inherent difficulties for the extraction of these teeth. Once erupted, their anatomy predisposes them to caries.The dental anomaly of unilateral macrodontia of mandibular second premolar appears to be extremely rare. Dental professionals should acquire deeper knowledge about this anomaly and carry out careful treatment planning to avoid unexpected problems during dental treatment procedures generated by the ignorance of morphology.


EFZ induced similar neuronal damage at concentrations of 31.6 ng/ml. We investigated the effect of genotype and blood-brain barrier integrity on EFZ metabolite concentrations in cerebrospinal fluid (CSF). We measured CSF drug concentrations in subjects from two separate study populations: 47 subjects with tuberculous meningitis (TBM) coinfection in Vietnam receiving 800 mg EFZ with standard antituberculous treatment and 25 subjects from the PARTITION study in the United Kingdom without central nervous system infection receiving 600 mg EFZ. EFZ and metabolite concentrations in CSF and plasma were measured and compared with estimates of effectiveness and neurotoxicity from available published in vitro and in vivo data. The effect of the CYP2B6 c.516G→T genotype  was examined. The mean CSF concentrations of EFZ and 8OH-EFZ in the TBM group were 60.3 and 39.3 ng/ml, respectively, and those in the no-TBM group were 15.0 and 5.9 ng/ml, respectively. Plasma EFZ and 8OH-EFZ concentrations were similar between the two groups. CSF EFZ concentrations were above the in vitro toxic concentration in 76% of samples  in the TBM group and 13% of samples  in the no-TBM group. CSF 8OH-EFZ concentrations were above the in vitro toxic concentration in 98% of the TBM group and 87% of the no-TBM group; levels were independent of genotype but correlated with the CSF/plasma albumin ratio. Potentially neurotoxic concentrations of 8OH-EFZ are frequently observed in CSF independently of the CYP2B6 genotype, particularly in those with impaired blood-brain barrier integrity.Efavirenz (EFZ) has been associated with neuropsychiatric side effects. Recently, the 8-hydroxy-EFZ (8OH-EFZ) metabolite has been shown to be a potent neurotoxin  CYP2B6 to yield the most abundant metabolite, 8-hydroxy-EFZ (8OH-EFZ). Comparatively minor alternative metabolic pathways are through CYP2A6 (leading to the 7OH-EFZ metabolite) and CYP3A  toxicity, efavirenz (EFZ) is widely deployed within first-line combination HIV treatment regimens worldwide because of its effectiveness, established safety record, and resilience to hepatic enzyme induction by rifampin in patients who require concomitant therapy against tuberculosis (TB) , 2. EFZ 4–nd CYP3A .CYP2B6 metabolism (–CYP2B6 single nucleotide polymorphism c.516G→T (rs3745274), which encodes a Gln172His amino acid substitution. The CYP2B6 c.516G→T GG genotype is associated with a fast EFV metabolizer status, the GT genotype is associated with an intermediate metabolizer status, and the TT genotype is associated with a slow metabolizer status. Preliminary data suggest that in CYP2B6 slow metabolizers, CYP2A6 represents the dominant route of elimination and may be affected by enzyme inhibition through concomitant isoniazid administration  (The results of pathways . In addipathways . Recentlid (CSF) . That stIn this study, we developed sensitive, accurate, and precise assays for measuring EFZ and its metabolites in CSF. We aimed to characterize the disposition of EFZ and its metabolites within CSF in HIV-infected patients with and without tuberculous meningitis (TBM) and to evaluate the impact of pharmacogenetic variability on drug disposition.The CSF pharmacokinetics of EFV were studied in two separate patient populations. Since these cohorts differ in several characteristics, no statistical comparisons between the two groups were undertaken.3 . All patients were of Southeast Asian ethnicity. Ethics approval was obtained from the Oxford Tropical Research Ethics Committee and the Hospital for Tropical Diseases Scientific and Ethical Committee.In Vietnam, HIV-infected patients over 15 years of age with newly diagnosed TBM  were randomized to receive immediate (within 7 days) or deferred (after 2 months) initiation of antiretroviral therapy as previously described , 22. AmoPenetration of Antiretroviral Therapy into the Nervous System) study . Twenty (80%) subjects were of white ethnicity, 3 (12%) were of black ethnicity, and 2 (8%) were of Asian ethnicity. No subject was receiving antituberculous therapy or other enzyme-inducing medication at the time of sampling. The study was approved by the North Wales Research Ethics Committee .In the United Kingdom, paired plasma and CSF samples were obtained at a single time point from 25 subjects without CNS infection from the United Kingdom PARTITION (EFZ concentrations in plasma and CSF samples from subjects receiving EFZ were determined at steady state (>10 days) , and samHelix pomatia in 0.2 M sodium acetate buffer (pH 5) (2O in 1 mM ammonium acetate), and 20 μl of this solution was analyzed directly by LC-MS/MS on a Thermo Access triple-quadrupole mass spectrometer. Hexobarbital was used as the internal standard. Gradient elution was on a reverse-phase C18 column using 1 mM ammonium acetate in water and acetonitrile. Quantification was by selective reaction monitoring in the negative ionization mode. Accuracy and precision were satisfactory, with a mean bias of 4.8% and an intra-assay coefficient of variability of 6.5%.EFZ concentrations in plasma and CSF were measured by a validated tandem liquid chromatography (LC)-mass spectrometry (MS) method as previously described . Freshlyr (pH 5) . The samAlbumin concentrations in CSF and blood (plasma/serum) were determined by radial immunodiffusion (Bindarid). A CSF/blood albumin ratio indicative of a breach in integrity of the blood-brain barrier was taken to be ≥6.8 for subjects less than 45 years old and ≥10.2 for subjects over 45 years old .in vitro were 31.6, 3.3, and 33.2 ng/ml, respectively .Genomic DNA was purified from whole blood using standard phenol-chloroform extraction methods. Allelic discrimination was performed by TaqMan real-time PCR for determination of the 10 drug/metabolite concentrations were compared using Student's t test and one-way analysis of variance (ANOVA). The Pearson r coefficient was used to determine the correlation between continuous variables. The CD4 count and CSF/plasma ratio of the EFZ concentration were nonparametrically distributed and analyzed using the Mann-Whitney U test. Fisher's exact and chi-square tests were used for categorical demographic data. All analyses were performed using SPSS (version 22) software.The geometric mean logPlasma EFZ concentrations correlated with CSF EFZ concentrations in both groups; however, there was no correlation between plasma EFZ and CSF 8OH-EFZ concentrations . The medCYP2B6 c.516G→T . Allele frequencies were 50% GG genotype, 43% GT genotype, and 7% TT genotype in the TBM group and 43% GG genotype, 48% GT genotype, and 9% TT genotype in the no-TBM group (CYP2B6 c.516G→T was in Hardy-Weinburg equilibrium in both groups (P = 0.912 for the TBM group and 0.672 for the no-TBM group). The CYP2B6 c.516G→T genotype related to the concentration of EFZ in CSF and plasma in both groups. This relationship was not present for the concentrations of the 8OH-EFZ metabolite . CSF 8OH-EFZ concentrations tended to be above the estimated in vitro neurotoxic level in both groups regardless of genotype.Plasma EFZ concentrations were similar between the TBM and no-TBM groups and mostly fell within the estimated therapeutic range, regardless of genotype. CSF EFZ concentrations exceeding the estimated CYP2A6 copy number . The CYP2A6 gene deletion occurred in 8 (17%) subjects and was in Hardy-Weinburg equilibrium (P = 0.394). There was no association of the CYP2A6 copy number with the concentration of EFZ or metabolites in plasma or CSF either singly or in combination with the CYP2B6 genotype. A single subject had the CYP2A6 gene deletion in combination with a homozygous CYP2B6 c.516G→T mutation; in this subject, the EFZ concentration was 6,319.5 ng/ml in plasma and 54.7 ng/ml in CSF.Forty-six samples in the TBM group were successfully genotyped for the In the TBM group, the addition of β-glucuronidase did not significantly alter the concentrations of EFZ . In contrast, the concentrations of 8OH-EFZ were much higher following β-glucuronidase addition. The ratio mean free/total concentration of 8OH-EFZ was 0.064 in plasma and 0.075 in CSF. Without β-glucuronidase, free 8OH-EFZ concentrations were low: mean concentrations were 87.3 ng/ml  in plasma and 3.7 ng/ml  in CSF.Mean 7OH-EFZ concentrations in the TBM group with β-glucuronidase were 75.3 ng/ml in plasma and 3.5 ng/ml in CSF; without β-glucuronidase, 7OH-EFZ levels were below the lower limit of quantification. In the no-TBM group, mean 7OH-EFZ concentrations were 236.6 ng/ml in plasma and 1.3 ng/ml in CSF.The CSF/serum or plasma albumin ratio was abnormal in 35 (90%) subjects in the TBM group and 4 (21%) in the no-TBM group. In the TBM group, the CSF/plasma albumin ratio was positively correlated with the CSF 8OH-EFZ concentration . A nonsiCYP2B6 c.516G→T genotype. In contrast, the concentrations of the neurotoxic metabolite 8OH-EFZ were not related to plasma EFZ concentrations or the CYP2B6 c.516G→T genotype but were correlated with the degree of blood-brain barrier breakdown measured by the CSF/plasma albumin ratio. These data confirm the findings of a recent publication from the ENCORE CNS substudy which demonstrated an association of the CYP2B6 c.516G→T genotype with plasma and CSF EFZ concentrations but not with plasma and CSF metabolite 8OH-EFZ concentrations at doses of 400 mg and 600 mg  since plasma exposures were comparable across both study populations. We observed a strong correlation between plasma and CSF EFZ concentrations, and both were associated with the d 600 mg . We demoCYP2B6 and CYP2A6 are present in the CNS, and expression has been shown to be inducible and subject to genetic variation (–The majority of EFZ metabolites in CSF were present as glucuronide conjugates. This is less likely to be due to CSF trapping of plasma glucuronide (the percentage of free compound was not significantly higher in CSF) and suggests that EFZ metabolites may be conjugated within the CNS. A number of UDP-glucuronosyltransferases have been demonstrated to be present in human brain tissue , 31. EFZariation –34. The riation –.This is the first report of EFZ metabolites in the CSF of patients with TBM. CSF concentrations of EFZ and metabolites were higher in those with a loss of blood-brain barrier integrity due to TBM infection, and the concentrations were the highest in TBM patients with the greatest loss of blood-brain barrier integrity, as measured by the CSF/plasma albumin ratio. As EFZ is >99.75% protein bound in blood , 37, higin vitro neurotoxic concentrations in patients with a combination of TBM infection and the CYP2B6 c.516G→T mutation . In contrast, CSF total 8OH-EFZ concentrations exceeded the in vitro neurotoxic concentration in the majority of subjects with and without TBM, regardless of genotype. This has implications for neuronal damage in patients with TBM, which could contribute to the overall neurological sequelae from this disease. Data from the recent ENCORE CNS substudy demonstrated an association of CSF 8OH-EFZ concentrations with symptoms at 1 year (in vitro neurotoxic levels, as this has important clinical implications. One question is whether EFZ should be avoided in those with impaired blood-brain barrier integrity, in particular, those with a neurological infection, such as TBM. However, as discussed above, such studies will be limited by difficulties in separating EFZ neurotoxicity from the effects of neurological infection. Another question is whether CYP2B6 c.516G→T genotyping in clinical practice would lower the incidence of neurocognitive side effects. Our data suggest that avoiding EFZ in those with the GT or TT genotype would not alter CSF 8OH-EFZ concentrations and, hence, may not be an effective strategy.CSF EFZ concentrations consistently exceeded t 1 year . The mai