Drosophila provides a powerful platform to investigate the signaling cascades upstream and downstream of Nf1, and the fly model exhibits similar behavioral phenotypes to mammalian models. In order to understand how loss of Nf1 affects motor behavior in flies, we combined traditional activity monitoring with video analysis of grooming behavior. In nf1 mutants, spontaneous grooming was increased up to 7x. This increase in activity was distinct from previously described dopamine-dependent hyperactivity, as dopamine transporter mutants exhibited slightly decreased grooming. Finally, we found that relative grooming frequencies can be compared in standard activity monitors that measure infrared beam breaks, enabling the use of activity monitors as an automated method to screen for grooming phenotypes. Overall, these data suggest that loss of nf1 produces excessive activity that is manifested as increased grooming, providing a platform to dissect the molecular genetics of neurofibromin signaling across neuronal circuits.Neurofibromatosis I is a common genetic disorder that results in tumor formation, and predisposes individuals to a range of cognitive/behavioral symptoms, including deficits in attention, visuospatial skills, learning, language development, and sleep, and autism spectrum disorder-like traits. The  Multiple human genetic disorders result in cognitive dysfunction. One of the most common inherited genetic disorders is neurofibromatosis, type I (NF-1), a disorder characterized by nerve sheath tumors, and other visible characteristics affecting the skin and eyes. In addition, some form of cognitive dysfunction is present in approximately 80% of individuals with NF-1, making it the most common monogenic disorder that affects cognitive function . Cognitinf1 gene exhibit reduced performance in learning and memory assays , and attention deficit hyperactivity disorder (ADHD) . These phenotypes provide a platform to dissect the alterations in signaling cascades, and ultimately neuronal function, that result from neurodevelopmental disorders.To develop a platform that allows rapid, semi-automated screening for modifiers of neuronal dysfunction in NF-1, we examined m of Nf1 , and devnf1P1 mutation was backcrossed for six generations into the wCS10 genetic background. Dopamine transporter, fumin (fmn), mutant flies were compared to their control strain w1118  maintained at 25°, 60% relative humidity, and kept on a 12:12 light:dark cycle. The Drosophila activity monitors (Trikinetics). The DAM2 (upright) model was used in experiments not requiring video monitoring. Glass tubes were prepared as follows. Each tube was punched through a 1 cm thick piece of room-temperature (hardened) food containing 5% sucrose and 2% agar. This food plug was covered with a black cap to reduce desiccation, and an EPDM rubber O ring was fitted to the outside of the tube to maintain lateral alignment in the monitor. Flies were anesthetized with CO2, and males were collected into DAM tubes, with one genotype loaded per monitor. Each monitor was placed into the incubator perpendicular to, and equidistant from, the white LED light source in the incubator. Activity was recorded with a 1 min sampling interval using the native Trikinetics software, and combined into standard 30 min bins for plotting in some figures as noted. Data from the first  day/night period was excluded from analysis. Data were collected for 5 d. Day and night activity counts were summed for each fly, independent of day. For each fly, data from each time bin was averaged across 5 d, and then these data points were averaged across flies, to plot day and night activity across zeitgeber time. Custom Matlab (Mathworks) scripts were used to plot sleep and activity profiles. Sleep was calculated using a standard 5 min inactivity window  model was used. Four male flies were loaded into glass DAM tubes , and placed into a DAM4 monitor. Beam crossings were recorded in 10 s bins using the native Trikinetics software. The black area below each tube in the monitor was covered with white tape to enhance the contrast between the fly and background. The incubator was illuminated during the 12 hr day cycle with white LEDs (380 lux at the monitor location), with separately controlled red LEDs providing 24 hr illumination for video monitoring (combined red/white: 485 lux). Two monochrome Firefly MV 1394a cameras (Point Gray) were used to collect videos of the flies in the tubes, in 10 min increments, for 24 hr in total. The cameras were mounted in the incubator, 6 cm from the top of the DAM4 monitor. Each camera was fitted with a Fujinon YV2.8 × 2.8SA-2 lens, and focused on the center of the tubes. Videos were collected with a custom Matlab (Mathworks) script, using the Image Acquisition Toolbox, at 7.5 frames per sec with Motion JPEG 2000 compression. Four flies were captured per experiment (two per camera). Videos were observed offline and compared to the activity trace recorded by the monitor. All monitor-recorded events were scored as either grooming , or locomotion  by an observer blind to the genotype.An open field arena was constructed, 2.85 mm in height and 25.4 mm in diameter, consisting of an opaque (white) acrylic lateral boundary covered on the top and bottom with two clear polycarbonate sheets. The apparatus was illuminated from below with white LEDs that were filtered through a sheet of white acrylic; the light intensity was measured at 720 lux in the location of the fly. A 1394a camera (as above) was mounted 5 cm above the arena. A single male fly was loaded into the arena with an aspirator, and recorded at 7.5 frames per second, 640 × 480 with Motion JPEG 2000 compression. Two videos were collected for each fly, one immediately after loading into the chamber (0–5 min), and one after a 15 min acclimation period (15–20 min). Videos of control and experimental genotype flies were alternated to distribute any circadian variation equally across all groups. Manual scoring of videos was carried out by an observer blind to the genotype. Start and stop frames were noted for each grooming event, which was further categorized according to which body part the fly was grooming: front legs, head/eye, abdomen, wings, or hind legs , where the upper bound C = 100, t = time, and r and k are constants. Mean frequency (n = 30–32 per genotype). Fly strains and Matlab scripts are available upon request.Statistical analyses were carried out in Graphpad Prism. Data were considered normally distributed if no significant deviation from normality was detected by the D’Agostino-Pearson omnibus test. Normally distributed data were compared with Student’s requency  was calcThe authors state that all data necessary for confirming the conclusions presented in the article are represented fully within the article.Drosophila, we placed flies into infrared activity monitors that measure the number of times a fly crosses an infrared beam in the center of a glass tube . Each event recorded by the activity monitor was located in the time-matched video frames, and marked as either grooming or locomotion (nf1P1 mutants represent grooming events rather than locomotion. To confirm that this was the case for similar (though less frequent) activity spikes in wild-type controls, we analyzed videos of wCS10 flies in the activity monitors. Similar to nf1P1 mutants, high-amplitude spikes in activity represented mainly grooming events in wCS10 controls, with all events of 10+ beam crossings per 10 s bin representing grooming   mutant, monitors , and in monitors . fmn mut mutants . There w mutants , nor a sol flies . A seconmn flies  exhibitemn flies .nf1 mutants, we analyzed videos of flies in a backlit open field arena in 5 min intervals . This configuration allowed us to quantify behavior in a less spatially confined environment, providing ample opportunity for locomotion and grooming. Similar to a previous report, we noted that the flies exhibited a tendency toward exploration of the boundary region (wCS10 controls groomed 21.4% of the time (482.4 ± 99.2 frames out of 2250) (nf1P1 mutants groomed 49.0% of the time (1102.0 ± 106.0 frames), a significant increase over the control . After a 15 min acclimation period, the nf1 mutants showed no decay in activity, and again groomed significantly more than the wCS10 controls (P < 0.001). The magnitude of this effect was striking, with the nf1 mutants grooming 7× more than controls. Control wCS10 flies exhibited a trend toward less grooming after the acclimation period, similar to a locomotor decay effect observed in previous studies  . The nf1 studies , there w mutants . Overallgrooming . Howevergrooming .nf1 grooming phenotype with fmn mutants by analyzing video of fmn mutants and w1118 controls in the open field arena. In contrast to the nf1 mutants, fmn flies exhibited a modest decrease in grooming , and there was a significant decrease in head grooming in video 1 in File S1 . Analysis of bout duration and frequency showed that genotype significantly affected the bout duration, with nf1P1 flies exhibiting longer bouts, and fmn shorter than controls at both time points . These data suggest that both Nf1 and DAT loss of function increased total activity levels registered in infrared activity monitors, but that the phenotypes were qualitatively and quantitatively distinct when analyzed in terms of grooming vs. locomotion.Finally, we compared the grooming . The decDrosophila. The magnitude of the effect is the largest reported to date, to our knowledge, with a sevenfold increase in grooming in an open field arena after 15 min of acclimation. High-resolution analysis and video tracking of nf1 and fmn (DAT) mutants revealed that changes in activity observed in traditional infrared activity monitors can result from changes in locomotor activity and/or grooming. Nf1 flies exhibiting increased grooming showed a histogram skewed toward higher values, with a concomitant increase in mean frequency. In contrast, fmn mutants that exhibited increased locomotor activity showed elevated baseline activity that was concentrated on low frequency values, with no significant change in the mean frequency. In other words, they crossed the beam at least once in a larger number of bins, as they patrolled back and forth more consistently, but did not exhibit an increase in the mean number of beam crossing per bin. Therefore, IR activity monitors can be used to observe both locomotion and grooming behavior in Drosophila. The contributions of these two components can be estimated by examining activity histograms, single-fly activity traces, and mean frequency of beam-crossing events.The present data demonstrate that loss of Nf1 produces an excessive grooming phenotype in IR activity monitors are commonly used, and we suggest they may be used as a high-throughput screening tool to identify genes or neuronal subsets involved in this complex behavior. This approach complements methods that offer more detail but require more time or specialized resources, such as manual scoring, imaging of dust cleaning (fmn) mutants produced distinct phenotypes in our hands, both in terms of diurnal activity patterns, and grooming. The diurnal activity pattern observed in fmn mutants is consistent with their previously-reported L:D phenotype , rather than a circuit specific to a body part lower in the suppression hierarchy. Under these assumptions, introduction to a new environment triggers an overall increase in grooming drive, stimulating grooming down the suppression hierarchy in a temporally sequential manner. The fly starts grooming from the head and later incorporates grooming of other body parts. It is notable that sensory neurons from Nf1 +/− mice are hyperexcitable (Drosophila model is a tractable system in which to investigate the contributions of both sensory and central circuits to Nf1 behavioral phenotypes.Grooming has been reported to follow the pattern of a suppression hierarchy (Drosophila are an excellent model organism to study signaling/signal transduction, genetic interactions and modifiers, and fundamental cellular physiology. The large Nf1 phenotype provides a potentially powerful platform to dissect the alterations in signaling cascades, and ultimately neuronal function, that result from neurofibromatosis-1.Known signaling functions of Nf1 are highly conserved, and the loss of Nf1 presumably affects neuronal function in fundamentally similar ways across taxa. The most well-characterized biochemical function of Nf1 is negative regulation of Ras signaling via its GAP-related domain . Uncover


CDKL5) cause early-onset epileptic encephalopathy, a neurodevelopmental disorder with similarities to Rett Syndrome. Here we describe the physiological, molecular, and behavioral phenotyping of a Cdkl5 conditional knockout mouse model of CDKL5 disorder. Behavioral analysis of constitutive Cdkl5 knockout mice revealed key features of the human disorder, including limb clasping, hypoactivity, and abnormal eye tracking. Anatomical, physiological, and molecular analysis of the knockout uncovered potential pathological substrates of the disorder, including reduced dendritic arborization of cortical neurons, abnormal electroencephalograph (EEG) responses to convulsant treatment, decreased visual evoked responses (VEPs), and alterations in the Akt/rpS6 signaling pathway. Selective knockout of Cdkl5 in excitatory and inhibitory forebrain neurons allowed us to map the behavioral features of the disorder to separable cell-types. These findings identify physiological and molecular deficits in specific forebrain neuron populations as possible pathological substrates in CDKL5 disorder.Mutations in cyclin-dependent kinase-like 5 ( CDKL5) gene cause early-onset epileptic encephalopathy MECP2 gene Cdkl5 gene expresses two isoforms, with expression segregated to neurons and astrocytes in vitro, suggesting a possible molecular link between CDKL5 disorder and Rett Syndrome Mutations in the X-linked cyclin-dependent kinase-like 5  activity and visual evoked potentials (VEPs). Anatomical analysis was aimed at identifying aberrant morphological features of neurons, including cortical neuron dendritic arborization, reported following the developmental knockdown of Cdkl5 in rat Mecp2 knockout mice Mecp2 knockout mice and thought to be relevant to Rett Syndrome. Finally, we used a conditional knockout approach to map the behavioral features identified to distinct populations of forebrain neurons. Our findings disclose a series of behavioral phenotypes homologous to those described in CDKL5 disorder and demonstrate the underlying neuronal cell-types and brain regions. In addition, our data reveal common deficits in a specific signaling pathway in Cdkl5 and Mecp2 knockout mouse models, suggesting potentially overlapping molecular deficits in CDKL5 disorder and Rett Syndrome.Here we generated and characterized mice carrying a targeted conditional knockout allele of All procedures were approved by The Institutional Animal Care and Use Committee (IACUC) of The European Molecular Biology Laboratory (EMBL) and were conducted according to the Italian Ministry of Health and commensurate with NIH guidelines for the ethical treatment of animals . All surgery was performed under anesthesia with tribromoethanol 250 mg/Kg (avertin). After anesthesia, for fresh brains collections mice were sacrificed by cervical dislocation while for fixed tissues collections mice were perfused transcardially with 4% paraformaldehyde. All efforts were made to minimize suffering.Cdkl5KO/+ females with Cdkl5KO/Y males and Cdkl5KO/X females with +/Y male. Littermate controls were used for all experiments. A portion of the behavioral data derived from knockout mice containing the neomycin selection cassette. No difference in behavior was noted between this allele and the neo-negative allele and the results were combined. For dendritic reconstructions mice with sparse fluorescent labeling of cortical neurons were obtained by crossing Thy1::GFP/Thy1::GFP;+/Y males with +/+;Cdkl5KO/X females and Thy1::GFP/+;Cdkl5KO/Y males with +/+;Cdkl5KO/X females. After weaning, mice were housed three to five per cage on a 12 h light/dark cycle (lights off at 19:00 h) in a temperature-controlled environment (21±2 C) with food and water provided ad libitum. For tissues collections all surgery was performed under anesthesia with tribromoethanol 250 mg/Kg (avertin). All efforts were made to minimize suffering.All mice were handled according to protocols approved by the Italian Ministry of Health and commensurate with NIH guidelines for the ethical treatment of animals. Mice for testing were produced by crossing Cdkl5 (ENSMUSE00000346596) was subcloned into a pDTA targeting plasmid by recombineering-mediated transfer from a 178-kb genomic fragment containing the C57BL/6J mouse Cdkl5 locus . A loxP site was inserted 806 bp upstream of the exon by recombineering-mediated insertion of a loxP-flanked pEM7::kanamycin gene and subsequent Cre recombination. An FRT-flanked pEM7/PGK::neomycin selection cassette was inserted 347 bp downstream of exon 4. The plasmid was linearized with NruI before electroporation into ES cells . G418-resistent clones were identified and screened by long-range PCR. Hybridization with a specific probe for the 5′ and 3′ arms was used to confirm PCR results. Two independent positive ES cell clones were injected into C57BL/6N host embryos using a piezo-drill assisted 8-cell stage injection procedure developed at EMBL. Four out of five offspring  provided germline transmission. Positive offspring were crossed to C57BL/6J congenic FLP-deleter mice Cdkl5 null allele.A 10 kb genomic fragment containing exon 4 of Mice were anesthetized  and perfused transcardially with 4% paraformaldehyde at 2 months of life. Brains were removed from the skull and post-fixed overnight at 4 C. Coronal sections  were cut on a vibratome  in 0.1 M phosphate buffer. For immunofluorescence brain sections were washed in phosphate-buffered saline 0.5% Triton-X and transferred in a 15 mM sodium citrate solution, pH 8.0 for 30 min at 80 C, then washed in phosphate-buffered saline with 0.5% Triton-X and blocked with blocking buffer , incubated with primary antibodies overnight at 4 C , incubated with an appropriate Alexa Fluor secondary antibody (1 h at room temperature), stained with DAPI, and mounted in Moviol . Images were acquired on a confocal microscope . For neuronal reconstruction, brain sections were washed in phosphate-buffered saline, stained with DAPI, and mounted in Moviol . Total dendritic length and Sholl analysis was measured using ImageJ software from confocal images.Mice were anesthetized  and perfused transcardially with 4% paraformaldehyde at 2 months of life. Brains were removed from the skull and post-fixed overnight at 4°C. The right hemisphere was dehydrated through a series of ascending ethanol concentrations, embedded in paraffin, and cut with a microtome (8 µm) and mounted on poly-lysine slides. One of 20 sections from the dentate gyrus was stained with toluidine blue according to the Nissl method. Bright field images  were acquired with a Coolsnap-Pro digital camera  and anatomical measurements carried out with Image Pro Plus software (Media Cybernetics).2O2 for 3 min. Sections were mounted on gelatin-coated glass slides and observed with a light microscope  equipped with a CCD camera . Quantitation of immunolabeling experiments was carried out from optical density (OD) measurements on 10× micrographs using ImageJ by an operator blind to genotype. Measurements of OD in cortex were obtained from 100×50 µm measuring boxes that were randomly placed in each cortical layer. Histograms illustrate the average OD obtained from three repeated measures in 4–5 sections per experimental animal. The mean OD of the corpus callosum was subtracted as background staining.Animals were anesthetized with chloral hydrate and transcardially perfused with ice cold 4% paraformaldehyde in 0.1 M phosphate buffer . After perfusion, the brains were dissected and kept in the same fixative solution overnight at 4 C. After several washes in 0.1 M PB, brains were cryoprotected by immersion in 10%, 20%, and 30% sucrose solutions. One brain hemisphere was cut in 30 µm sections with a cryostat, collected in phosphate buffered saline and processed for free-floating immunohistochemistry as described 3VO4, 0.5 mM PMSF, protease inhibitor mixture , and 50 mM NaF was added to all buffers. Homogenates were mixed for 20 min at 4 C and centrifuged 20 min at 4 C at maximum speed. Supernatant was collected and stored at −80 C. The protein content was determined by bicinchoninic acid assay . For the preparation of hippocampal extracts from P19 mice, tissues were homogenized in RIPA buffer ). Proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes, blocked in 5% milk, TBS, 0.1% Tween, and incubated with rabbit anti-CDKL5 1∶250 (Sigma-Aldrich), mouse anti-Cdkl5 1∶250 (see below), and rabbit anti-MECP2 (1∶1000), anti-AKT (1∶1000), anti-p-AKT (1∶1000), anti-BDNF  and anti-tubulin  overnight at 4 C, incubated with secondary antibodies (1 h at room temperature), developed using ECL detection , and images acquired and quantified using a digital camera .Mice at 2 months of life were decapitated and brains rapidly collected on ice and frozen in liquid nitrogen. One hemisphere was homogenized in lysis buffer  using an automated dounce. A protease/phosphatase inhibitor mix composed of 0.5 mM NaCdkl5 exons using 1× PCR Buffer , 0.5 units of Dream Taq (Promega) and 200 mM each dNTPs . For SYBER green qPCR  exons were amplified using primers against exons 2–3 and 9–10 and control primers against Abl.Brains were collected from mice at 2 months, washed in PBS-DEPC and rapidly frozen in liquid nitrogen. One hemisphere was homogenized using an automated dounce and total RNA extracted  and converted into first-strand cDNA  using oligo-dT according to the manufacturer's protocol. For semi-quantitative PCR, DNA was amplified using primers against Male mice (2–4 months) were anesthetized with ketamine/xylazine supplemented with isofluorane as needed, kept on a heating pad to maintain body temperature at 35±1 C, and immobilized in a stereotaxic frame. An incision above the skull was cut and burr holes drilled into the skull. Four stainless steel screws were used as electrodes placed bilaterally above hippocampus  and frontal cortex . Ground and reference screws were anchored on the posterior and middle portions of the skull, respectively. A wireless Neurologger 2A recording device (400 HZ sampling rate) acquired and stored data in real-time for later downloading Mice were suspended by their tail for 2 min and hind-limb clasping was assessed from video recordings. Clasping was defined as present if it occurred for more than 5 seconds in an animal.Locomotion was measured using activity-monitoring cages similar in size, shape, and material to the home cage . A mouse was placed in the chamber at least 3 h before recording started. Relative activity was monitored continuously for four days and binned into 12 h epochs.Mice were placed in the center of a 50×50 cm open arena equipped with video tracking and infrared rearing detection systems . Cumulative distance travelled was collected in 5 min intervals for 30 min.Mice were placed on a 12 cm diameter platform within a 28 cm visual-tracking drum . The opto-kinetic response (head tracking) was assessed from video recordings. Each mouse was tested using vertical stripes (6 mm and 15 mm width) at two speeds (2 rpm and 4 rpm) and data were averages as genotype effects were similar across conditions.2). VEP amplitude decreases with increasing stimulus spatial frequency; visual acuity was obtained by extrapolation to zero amplitude of the linear regression through the last four to five data points in a curve where VEP amplitude is plotted against log spatial frequency Animals were anesthetized with urethane  and placed in a stereotaxic frame with full viewing of the visual stimulus. Additional doses of urethane were used, if necessary, to keep the anesthesia level stable throughout the experiment. Body temperature was monitored with a rectal probe and maintained at 37.0 C with a heating pad. A hole was drilled bilaterally in the skull, overlying the binocular portion of the primary visual cortex (binocular area Oc1B). After exposure of the brain surface, the dura was removed. A glass micropipette  was inserted perpendicularly to the stereotaxis plane into the cortex contralateral to the measured eye. In most experiments, microelectrodes were inserted 3.1–3.3 mm lateral to the intersection between sagittal- and lambdoid-sutures and advanced 100 µm within the cortex. Electrical signals were amplified , bandpass filtered (0.3–100 Hz), and averaged (at least 50 blocks of 2 events each) in synchrony with the stimulus contrast reversal. Transient VEPs in response to abrupt contrast reversal (1 Hz) were evaluated by measuring the peak-to-baseline amplitude and peak latency of the major component. VEPs in response to a blank stimulus were also frequently recorded to estimate of noise. Visual stimuli were horizontal sinusoidal gratings of different spatial frequency and contrast generated by a VSG2/2 card  and presented on a computer display  fitting test, and then paired comparisons. For immunohistochemistry, data were statistically analyzed by Student's t-test and one or two-way ANOVA using Prism software (GraphPad). For the open field, data were analyzed in 5 min increments using a two-way ANOVA with repeated measuresData were analyzed using Student's t-test  as well as one-or two-way ANOVA  using Prism software . Cdkl5 was produced by germline deletion of exon 4 of a Cdkl5 conditional knockout allele produced by standard gene targeting in embryonic stem cells . Western blot analysis of whole brain extracts  raised against the C-terminus of mouse Cdkl5  with co-localization in the nucleus with the nuclear speckle marker SC35, as previously reported in cultured cells A constitutive knockout allele of Cdkl5 knockout mice showed normal viability, body weight, and absolute as well as relative brain weight . A general behavioral screen Cdkl5 knockout mice while no, or very low levels of clasping were seen in wild-type littermates  suggesting that the deficit did not reflect a reduced capacity for locomotion. Next, we measured head tracking responses to a continuously moving visual stimulus in the visual drum test Cdkl5 knockout mice showed a significant decrease in the number of head tracks performed in the test compared to wild-type littermates while homozygous female knockouts showed a trend for reduced head tracking  recordings from implanted surface electrodes in freely behaving animals did not reveal spontaneous epileptiform activity in hemizygous male Cdkl5 knockout mice  suggesting that the relative seizure resistence of the founder background was not masking an epileptic phenotype in the knockout. These data reveal that while Cdkl5 knockout mice do not exhibit spontaneous seizures or increased seizure susceptibility, they do show abnormal EEG response to pro-convulsant treatment.Although early onset seizures are a prominent feature of CDKL5 disorder, no evidence for spontaneous seizures emerged during videotaped observations of adult Mecp2 knockout mice CDKL5 mutations. However, siRNA-mediated knockdown of Cdkl5 causes a reduction in dendritic arborization both in vitro and in vivoCdkl5 knockout mice to mice carrying the Thy1::GFP transgene ) pyramidal neurons. Total length of apical dendritic arbors was significantly reduced in homozygous female and hemizygous male Cdkl5 knockout cortical and hippocampal pyramidal neurons compared to wild-type littermates, while dendritic arbor length of cortical neurons in heterozygous female Cdkl5 knockout mice showed an intermediate mean distribution ( and ). Notably, dendritic arbor length in heterozygous female mice showed a bimodal distribution  consistent with a cell autonomous function of the X-linked Cdkl5 gene in cells in which either one or the other X chromosome has been inactivated. Reduced dendritic arborization was associated with a significant reduction in cortical thickness in both homozygous female and hemizygous male Cdkl5 knockout mice when compared to wild-type littermates with intermediate levels seen in heterozygous female knockouts .Dendritic arborization is significantly reduced in cortical pyramidal neurons from both Rett Syndrome subjects Mecp2 knockout mice might be similarly affected in Cdkl5 knockouts. Western blots on whole brain extracts revealed no change in Mecp2 protein levels in Cdkl5 knockout mice . Levels of BDNF immunoreactivity, reported to be reduced in Mecp2 knockout brain Cdkl5 knockout brain . However, decreased levels of phosphorylated Akt were observed in extracts of hippocampus from Cdkl5 knockouts when compared to wild-type littermates . For deletion in forebrain GABAergic neurons  we crossed the Cdkl5 conditional knockout allele with the Dlx5/6::Cre transgene Cdkl5 conditional knockout allele with the Emx1::Cre transgene Dlx5/6 and Emx1 conditional knockout mice appeared outwardly normal at birth and showed normal body weight and viability when compared to littermate controls . A general behavioral screen Emx1-conditional, but not Dlx5/6-conditional Cdkl5 knockout mice when compared to control littermates  for decreased head tracking compared to control littermates  allele of Cdkl5 knockout mice recapitulate several core features of CDKL5 disorder and serve as a useful animal model of the disorder. In particular, Cdkl5 knockout mice showed hind-limb clasping, hypoactivity, defective head tracking, and abnormal EEG responses to convulsants (–S3). We interpret these findings as an indication that Cdkl5 impacts on the development of epilepsy in humans in a manner significantly different from mice. However, our findings of an altered profile of EEG burst activity following kainic acid treatment of Cdkl5 knockout and wild-type mice ( and S3), may nevertheless be useful to identify epileptic mechanisms impacted by Cdkl5 in the human condition.Constitutive ulsants –3, featCdkl5 knockout mice partially overlaps with that of Mecp2 knockouts. Both mutants showed hind-limb clasping, hypoactivity, and reduced dendritic branching of cortical neurons. In addition, cortical neurons in both knockouts showed reduced phosphorylation of rpS6, a ribosomal regulatory subunit and modulator of protein translation Cdkl5 knockout mice ( and ) suggesting that this pathways may not play a major role in common features of CDKL5 disorder and Rett Syndrome.The phenotype of The non-overlapping intracellular localization of Cdkl5 and Mecp2 proteins further argues that they act via different signaling mechanisms to affect common targets. While Mecp2 is nuclear and associated with heterochromatin Cdkl5 in glutamatergic cortical neurons (with Emx1::Cre) and GABAergic forebrain neurons (with Dlx5/6::Cre) revealed a double dissociation of behavioral phenotypes , but constrasts with the marked hypolocomotion we see in a familiar environment  Genomic organization of the Cdkl5 locus showing critical exon 4 (ENSMUSE00000346596), the targeting construct, successfully targeted Cdkl5 locus (genotyping primers indicated by arrows), FRT-deleted conditional Cdkl5 knockout allele, and Cre-deleted constitutive Cdkl5 knockout allele used in the present study. (B) Confirmation of homologous recombinants by long-range PCR at 5′end (top panel) and 3′end (bottom panel). PCR products of positive clones (lane 1 and 2) showed specific signals when hybridized with 5′ and 3′ radioactive-labelled DNA probes. No signal was detected in PCR-negative samples, consistent with them being non-homologous or unmodified (lane 3 and 4). (C) mRNA expression of Cdkl5 exons as estimated by Affymetrix microarray hybridization in wild-type and Cdkl5 knockout brain confirmed absence of the deleted exon 4 in knockout mice, but normal expression of remaining exons consistent with an escape from nonsense-mediated decay. (D) Semi-quantitative PCR on total brain RNA from female wild-type and Cdkl5 mutant mice with primers spanning exons confirmed an absence of exon 4, but normal levels of exons 2, 3, and 5 in mutant mice. (E) Quantitative real-time PCR on total brain RNA from female wild-type and Cdkl5 knockout mice revealed normal levels of expression of upstream (exon 2–3) and downstream (exon 9–10) Cdkl5 exons. (F) Anti-Cdkl5 and SC35 immunofluorescence analysis of S1 cortex brain sections from adult male knockout (KO) mice. .(TIF)Click here for additional data file.Figure S2Normal body and brain weight and novelty-induced locomotion in Cdkl5 knockout mice. (A) No difference was observed in body weight of female and male wild-type and Cdkl5 mutant mice (6 weeks old). (B) No difference was detected in total distance travelled in a novel open arena by adult female and male wild-type and Cdkl5 mutant mice . (C) Representative images of dissected brains from female wild-type, heterozygous, and homozygous Cdkl5 knockout mice. (D) No difference in relative brain to body weight was detected between genotypes . (E) Normal viability was observed of female and male wild-type and Cdkl5 mutant mice (at 6 months of life) .(TIF)Click here for additional data file.Figure S3Seizure response in Cdkl5 knockout mice backcrossed to DBA genetic background. Representative electroencephalogram (EEG) traces recorded from surface electrodes placed over the somatosensory cortex in freely moving male (A) wild-type (WT) and (B) Cdkl5 knockout (KO) mice following 3 generations backcrossing to the DBA2/J strain. Power spectra of EEG recordings showed decreased power in Cdkl5 knockouts when compared to wild-type littermates at low frequencies both under (C) baseline conditions and (D) in mice injected with kainic acid .(TIF)Click here for additional data file.Figure S4Reduced thickness of cortical and hippocampal layers in Cdkl5 knockout mice. (A) Representative image showing region of S1 cortex used for quantification of cortical thickness in wild-type and Cdkl5 mutant mice. (B) Representative image showing features used for quantification of hippocampal layer thickness in wild-type and Cdkl5 mutant mice. (C–F) A significant decrease in thickness was observed in hippocampal CA1 stratum oriens (but not stratum laconosum or radiatum) and the lower and upper blades of dentate gyrus molecular layer in female and male Cdkl5 mutant mice compared to wild-type littermates  .  Representative images of reconstructed neurons from adult male wild-type  and Cdkl5 knockout  mice. (I) Total dendrite length was significantly reduced in male Cdkl5 knockout mice .(TIF)Click here for additional data file.Figure S5Reduced dendrite complexity in Cdkl5 knockout mice. Classical Sholl analysis with radii increasing in 20 µm increments. Sholl analysis measures apical dendrite length within each sphere plotted against radius from soma. Significant decreases in the numbers of intersections between the dendrites and the Sholl circles from the neuronal somata occurred only between 100 µm and 130 µm in cortical neurons (A) and between 80 µm and 120 µm and 140 µm and 160 µm in hyppocampal dendrite (B) in mutant Cdkl5 male mice ((TIF)Click here for additional data file.Figure S6No change observed in Mecp2 and BDNF levels in Cdkl5 knockout mice. Quantification of western blot data failed to detect a change in immunostaining against (A) Mecp2, and (B) BDNF protein in mutant Cdkl5 mice compared with wild-type controls  .(TIF)Click here for additional data file.Figure S7Reduced p-rpS6 protein in Cdkl5 knockout mice. Representative micrographs showing the immunohistochemistry for total rpS6 (A) in the S1 cortex of both female and male wild-type and Cdkl5 mutant mice. Quantitation of immunoreactivity signals revealed no change in total rpS6 protein (B) in both male and female mutants, a significant decrease of phospho-rpS6 (240/244) in (C) female and (D) male mutants, and a decrease of phospho-rpS6(235/236) that was only significant in layer V of (E) female mutants, while it shows only a trend in (F) male KOs .(TIF)Click here for additional data file.Figure S8Normal body and brain weight (A) No difference was observed in body weight of heterozygous female and hemizygous male (A) Dlx5/6-  and (B) Emx1  conditional Cdkl5 knockout mice at 8 weeks of life. Normal viability was observed at 6 months of life of heterozygous female and hemizygous male (C) Dlx5/6-  and (D) Emx1  conditional Cdkl5 knockout.(TIF)Click here for additional data file.


With improved access to antiretroviral therapy (ART), HIV infection is becoming a chronic illness. Preliminary data suggest that HIV-infected children have a higher risk of disabilities, including hearing impairment, although data are sparse. This study aimed to estimate the prevalence and types of hearing loss in HIV-infected children in Lilongwe, Malawi.This was a cross-sectional survey of 380 HIV-infected children aged 4–14 years attending ART clinic in Lilongwe between December 2013-March 2014. Data was collected through pediatric quality of life and sociodemographic questionnaires, electronic medical record review, and detailed audiologic testing. Hearing loss was defined as >20 decibels hearing level (dBHL) in either ear. Predictors of hearing loss were explored by regression analysis generating age- and sex-adjusted odds ratios. Children with significant hearing loss were fitted with hearing aids.Of 380 patients, 24% had hearing loss: 82% conductive, 14% sensorineural, and 4% mixed. Twenty-one patients (23% of those with hearing loss) were referred for hearing aid fitting. There was a higher prevalence of hearing loss in children with history of frequent ear infections  and ear drainage . Hearing loss was linked to history of WHO Stage 3  or Stage 4  and history of malnutrition , but not to duration of ART or CD4. Only 40% of caregivers accurately perceived their child’s hearing loss. Children with hearing impairment were less likely to attend school and had poorer emotional (p = 0.02) and school functioning (p = 0.04).There is an urgent need for improved screening tools, identification and treatment of hearing problems in HIV-infected children, as hearing loss was common in this group and affected school functioning and quality of life. Clear strategies were identified for prevention and treatment, since most hearing loss was conductive in nature, likely due to frequent ear infections, and many children with hearing loss qualified for hearing aids. Screening strategies need to be developed and tested since caregivers were not reliable at identifying hearing loss, and often mis-identified children with normal hearing as having hearing loss. Children with frequent ear infections, ear drainage, TB, severe HIV disease, or low BMI should receive more frequent ear assessments and hearing evaluations. HIV currently affects an estimated 35 million people worldwide. Over the past decade, quality HIV care and access to ART has become much more widely available throughout Africa. The expansion in access to ART globally has reduced HIV-related morbidity and mortality ,2 and coOne million of these HIV- infected patients live in Malawi, and 150,000 are children less than 15 years of age . Malawi To date, there are few studies looking at hearing loss in HIV-infected children, and many have been small in size. All have found a high prevalence of hearing loss in HIV-infected children, ranging between 6.4–84.8% , which iA recent study at the Baylor College of Medicine Clinical Centre of Excellence (BCOE) in Lilongwe, Malawi reported higher prevalence of a range of disabilities in children living with HIV when compared to their HIV-uninfected siblings . OverallThis study was reviewed and approved by both the Malawi National Health Sciences Research Committee (NHSRC Protocol #1200) and the Baylor College of Medicine Institutional Review Board (BCM Protocol H-33667). Full written informed consent was obtained from adult caregivers, and assent was obtained from children 10 years and older unless they did not have adequate cognitive ability to understand the study due to disability. Children who did not have cognitive ability for assent were included in the study, as long as their caregiver provided informed consent and a study clinician documented inability of the child to provide assent due to cognitive delay. Participation in the study was entirely voluntary and confidential, and participants were free to withdraw at any time without any consequences.This was a cross-sectional study conducted from December 2013 to March 2014.Participants were HIV-infected children 4 to 14 years of age attending the Baylor College of Medicine Children’s Clinical Center of Excellence (BCOE) in Lilongwe, Malawi. The BCOE in Lilongwe serves as a tertiary referral center for pediatric HIV care and treatment, and has approximately 3500 patients actively enrolled. This represents the largest cohort of pediatric HIV-infected children in Malawi. While data are not available for all children participating in the study, the vast majority of children attending care at BCOE are perinatally-infected. Participants were selected from a convenience sample of HIV-infected children aged 4 to 14 years who were attending clinic at the BCOE and who were accompanied by their primary caregiver. Caregiver report of hearing loss was not a criterion for enrollment. After informed consent was completed, a research assistant completed two questionnaires with the caregiver and/or child. Of the 380 participants, 93 children completed questionnaires. The children were then transported to African Bible College (ABC) Hearing Clinic and Training Centre, where trained audiology staff performed extensive audiologic testing. ABC is a 5-minute drive from the BCOE. On occasions when large numbers of older children were enrolled, a portable testing van containing two soundproof booths was parked at BCOE and testing was done onsite. Fifty-six children were tested using the audiotrailer. Younger children and children unable to be assessed using age-appropriate subjective methods required the larger booths at ABC for play audiometry, visual reinforcement audiometry (VRA), and other objective diagnostic assessment equipment. Both the audiotrailer and the permanent audiobooths at ABC were custom built in South Africa by the company Audiometry Specialists, using the same materials for the paneling. The audiotrailer was constructed to meet industrial hearing conservation program standards for mobile testing units. The ambient noise levels of the audiotrailer are assessed annually by an independent accredited calibrator from Johannesburg, using South African Bureau of Standards (SABS) 0182/ 1998 maximum allowable noise measurements in dB of sound pressure level (dBSPL). All relevant octave and interoctave frequency measures from ambient noise from 125 Hz to 8KHz were under the maximum allowable limits. The diagnostic audiometers in the audiotrailer were calibrated by the same technician who calibrates the audiometers in the hearing clinic booths and measures the ambient noise levels of the audiotrailer. Testing location was not included in statistical analysis.th percentile as per the Ministry of Health guidelines at the time of the study [Sociodemographic information was collected on age, gender, caregiver education, caregiver HIV status, household size, household location, family income, source of water, asset ownership, and food security. Information was also collected on school attendance and performance, history of medical illnesses, and signs of hearing impairment. If a caregiver reported that they believed their child had hearing impairment, an additional set of questions was asked concerning the nature of the disability . The following HIV-related data were extracted from the electronic medical records at BCOE on the date of the audiology testing: timing and duration of ART, CD4+ cell count, WHO status at enrollment, current WHO status, history of TB, nutritional status, history of streptomycin exposure, documented history of hearing or ear problems. All children on ART were on combination antiretroviral therapy (cART) with three antiretroviral medications. Regimens were demarcated as non-nucleoside reverse transcriptase inhibitor (NNRTI) or protease inhibitor (PI) based regimens. History of malnutrition was determined by documented weight for height less than the 85he study .™), which has been translated into Chichewa and validated in Malawi [™ within the appropriate age category if they had assented and were cognitively able to understand the questionnaire.Caregivers completed the Caregiver version of the Pediatric Quality of Life Inventory , and audiometry. The AccuScreen Pro was used for TEOAE testing and uses a non-linear click sequence stimulus with click rate of approximately 60 Hz and sampling rate of 16 kHz. The pass/refer criteria were evaluated according to AccuScreen binomial statistics. The criterion level used by the AccuScreen Pro signal detection algorithm for the presence of a time-locked signal is 99.7%. All audiometric assessment included in the study was age-appropriate and performed under headphones. Pure-tone audiometry using a button or hand-raise response was the preferred method. However, for those children aged 4–6 years, a play task was used to elicit the child’s response. If any cognitive impairment impacted pure-tone or play testing, this was noted by the tester as an issue in preventing a reliable assessment of hearing, and an alternative method was conducted. Eight children were unable to complete pure-tone or play audiometry due to developmental delay; three of these children successfully completed testing using VRA  under headphones, and four children successfully completed VRA under freefield. One child required auditory steady state testing under sedation due to severe neurocognitive impairment, with stimulus presented through insert earphones. Audiometry testing was performed using an Amplivox 240 Portable Diagnostic Audiometer at the following frequencies: 500, 1000, 2000, 3000, 4000, 8000 Hz. Bone conduction frequencies were tested at 500 Hz, 1000 Hz, 2000 Hz and 4000 Hz. Pure tone average was used at four frequencies  to determine the average hearing threshold level. Hearing loss was defined as greater than 20 decibels hearing level (dBHL) in either ear. Categories of hearing loss were defined as follows: normal (up to 20), mild (21–40), moderate (41–65), severe (66–90), and profound (≥91). The type of hearing loss was determined based on the relationship of the four air and bone conduction thresholds. If the bone conduction level was within 10dBHL of the air conduction level, the hearing loss was considered to be sensorineural. If the bone conduction level was within the normal range of hearing (≤20dBHL), then the hearing loss was conductive. If the bone conduction level was outside the normal range, but still better than the air-conduction threshold by more than 10dBHL, then the hearing loss was considered to be mixed. Also, if the hearing loss was clearly conductive at some frequencies and sensorineural at other frequencies, it was considered mixed, based on the definition “a mixed hearing loss is the combination of a sensorineural loss and a conductive loss in the same ear” .Children who qualified were fitted with hearing aids. Qualification for hearing aids was determined by an audiologist and was based upon a combination of clinical and social criteria. A bone conduction hearing aid was considered for children with a chronic and recent history of discharging ears who had a mild to severe air conduction audiometric threshold average (30–85 dBHL) in the better ear, and a normal or mild bone conduction audiometric threshold average (≤35 dBHL). An air conduction hearing aid (behind the ear type), was considered for children where their ear did not have ear discharge issues or other contraindications, but had a mild to profound degree of hearing loss (average air conduction of ≥30 to ≤105 dBHL) which was sensorineural, conductive, or mixed in nature. Hearing aid fitting was then discussed with the child and guardian to ensure there was a recognized functional hearing loss requiring rehabilitation and that the device would be appropriately used and cared for.™ were reverse-scored and linearly transformed to a 0–100 scale  with higher scores indicating better quality of life. Scale scores were computed as the sum of the items divided by the number of items answered. If more than half the items were missing data, then the scale score was not calculated. Age-sex-adjusted logistic regression analyses were then completed to compare those with hearing loss versus no hearing loss.Age- and sex-adjusted odds ratios were generated through logistic regression to assess the relationship between the presence of hearing loss and HIV and sociodemographic characteristics. For all analyses, the case definition was hearing loss greater than 20 dBHL in the worse ear. The PedsQLSensitivity of TEOAE as a screener for hearing loss was calculated by measuring the proportion of people with hearing loss (as defined by audiometry) who did not pass the TEOAE test. Specificity of TEOAE as a screener was calculated by measuring the proportion of people without hearing loss (as defined by audiometry) who passed the TEOAE test.Of the 380 children aged 4 to 14 years recruited and tested, 90 (24%) patients had hearing loss in either ear . Overall, 36 (9%) had an average hearing threshold of >20 dBHL in the better ear. Of the children with hearing loss in either ear, 82% of the hearing loss was conductive, 14% was sensorineural, and 4% was mixed. Degrees of hearing loss by type are included in There is weak evidence that the sensorineural hearing loss is associated with more profound levels of hearing loss, but it is difficult to make a clear statement given the small numbers. Among children with hearing loss, 21 (23%) were referred for hearing aid fitting, 63% were not recommended for hearing aid, and 14% were recommended to be reviewed at a later date. Most children were fitted with bilateral hearing aids; children fitted with unilateral hearing aids had either a very mild high frequency hearing loss in the non-fitted ear or an unaidable profound hearing loss in the non-fitted ear.Most of the ears with conductive hearing loss had abnormal physical findings. For the 26 ears with moderate or severe conductive hearing loss, 62% had perforated eardrums, 8% had an acute otitis media with intact eardrum, 12% had a fungal infection, 38% had discharge, and 20% had more than one abnormal finding. A higher percentage of patients with hearing loss had discharge in the external ear canal and perforated eardrum at the time of testing than those patients without hearing loss. Other otoscopy findings such as ear canal inflammation, foreign body, or dullness of eardrum were not related to hearing loss. All patients with otitis media, otitis externa, fungal infections, effusions, or other medical issues were treated and re-evaluated at a later date. Patients with discharge and perforation were also evaluated at a later date after the physical exam findings had resolved for repeat testing. Presence of wax was defined if it was obstructing or occluding the ear canal. The eardrum was more likely to be abnormal in those patients with hearing loss. Only 16% of patient’s ears with hearing loss had normal tympanometry. The majority with abnormal tympanometry had no observable middle ear mobility for compliance with normal canal volume (Bmid), indicating a middle ear effusion. In comparison, 79% of ears in patients without hearing loss had type A tympanometry, indicating normal mobility and pressure. A higher percentage of ears in patients with hearing loss did not pass the transient evoked otoacoustic emissions test (TEOAE) when compared with ears without hearing loss. If OAE was used as the screening test for hearing loss, this would have 79% sensitivity and 95% specificity.Mean age of all participants was 8.6 ± 3.2 years). Ages were similar in those with hearing loss (mean age 8.5 ± 3.3 years) and those without hearing loss (mean age = 8.6 years ± 3.2 years). There was no difference in age or gender comparing those with and without hearing loss. Children with hearing loss tended to be more likely to report experiencing other disabilities. Orphan status and current caregiver were not related to hearing loss. Children from families with higher socioeconomic assets were less likely to have hearing loss than those from lower socioeconomic status. Family income, location , food security, and parental levels of education were not related to hearing loss.Only 40% of caregivers of children with hearing loss accurately perceived the hearing loss, though hearing impairment was related to caregiver perceived hearing loss. Of those with hearing loss perceived by the caregiver, 67% had previously discussed the concern with a clinician. Those with hearing loss were more likely to have had a hearing or ear problem noted in the electronic medical record (EMR) and were more likely to have previously sought treatment. Positive answers to caregiver screening questions were related to hearing loss, including needing to repeat things to the child, the child having difficulty following instructions, and the child not speaking clearly. Family history of hearing loss was not related to presence of hearing loss.Among the children with perceived hearing loss, caregivers attributed this to unknown causes (60%), illness (30%) and rarely to HIV (10%). Caregivers noted hearing loss at an average of 6.0 years (SD = 3.0 years). Among the children with hearing loss by audiometric testing, only 7 (19%) had received a previous hearing test, and 3 of these children had already been fitted with hearing aids prior to the study. Services sought for hearing loss included hospital (25%), specialist clinic (22%), private doctor (6%) and other services (3%); the remaining children had not sought any services.There was a higher prevalence of hearing loss in children with history of frequent ear infections and ear drainage. History of TB treatment tended to be more common among children with hearing loss; only 4 children with hearing loss had been exposed to the ototoxic medication streptomycin as part of their TB treatment. Place of birth, history of overnight admissions, recent outpatient treatment for an illness, history of meningitis, and current body mass index (BMI) were not related to hearing loss.Almost all children with hearing loss (98%) were on ART, and average duration was 4.6 years. There was no significant difference between the age of entry into care, the age of ART initiation, or duration of ART in children with and without hearing loss. Children with hearing loss were more likely to have been WHO Stage 3 or Stage 4 at enrollment in clinic, rather than Stage 1. Current WHO Stage, and current or nadir CD4 <200 were not related to hearing impairment. For those with any degree of malnutrition recorded in the EMR, there was a higher association with hearing loss. Various ART regimens were not significantly different between children with and without hearing loss.Among children above 5 years, those with hearing loss tended to be less likely to be attending school. However, if the child was attending school, hearing loss did not seem to affect whether they were in the correct class at school for their age or their school performance. For those children not attending school, lack of money was the main factor (35%), while lack of transport (23%) and child too sick (19%) were other major factors; the remaining did not identify the reason. Children with hearing loss reported poorer emotional and school functioning, but not poorer overall quality of life, physical health or social functioning.The overall prevalence of hearing loss in any ear among children with HIV in this study was 24%, and was largely conductive in nature. Overall, 5.5% of all children tested required a hearing aid, almost one in four of those with hearing loss. Few of the children with hearing loss had previously undergone audiometry or had been referred for relevant services. Factors significantly associated with hearing loss included WHO Stage 3 or 4 at enrollment, perceived hearing loss by caregiver, documentation of hearing or ear problems in the medical record, history of frequent ear infections or ear drainage, and positive screens for reported signs of hearing impairment. Children with HIV and hearing loss were more likely to report other disabilities, less likely to attend school, and had poorer emotional and school functioning.Most previous studies have reported the prevalence of hearing loss among children living with HIV to be 20–30% –13,16, aWhen reviewing clinical factors associated with hearing loss in HIV-infected children, we found that hearing loss was more likely in those with more severe illness at enrollment (WHO Stage 3 or 4), similar to other studies where hearing loss was more common in children with history of AIDS-defining illnesses . Our finPredominant types of hearing loss in HIV-infected children have varied across studies, with several studies reporting the majority with conductive hearing loss ,16, as iThis study shows a large unmet need for ear examination and hearing services among children living with HIV in Malawi. This finding is concerning, especially given that the vast majority of children were already benefiting from ART and therefore in regular contact with health care professionals. Caregivers were unreliable at perceiving hearing loss: only 40% of caregivers of children with hearing loss accurately perceived the hearing loss, while 11% of children with no hearing loss were reported by their caregiver to have difficulties hearing. One possible explanation may be that the definition of hearing loss used in this study (>20 dB HL in either ear), resulting in a lack of recognition of hearing loss in one ear. Possible explanations for over-reporting of hearing loss in children with normal audiologic exams may be due to the fluctuating nature of conductive hearing loss, where previous conductive hearing loss has resolved. Additionally, there is likely to be high prevalence of cognitive and developmental delay ,25 and cIn our patients, the high prevalence of conductive hearing loss and high frequency of discharging ears and abnormal tympanic membranes suggest recurrent or chronic otitis as a likely cause of hearing loss. This finding implies that preventive services could focus on the early identification and treatment of ear infections in these children, and different models of developing this intervention should be considered. Furthermore, many of the children with hearing loss could benefit from hearing aids, indicating another point for intervention. Studies are needed to develop screening strategies for hearing loss among children with HIV, as well as prevention and treatment services.Hearing loss related to HIV was demonstrated to impact school attendance and aspects of quality of life, which has not been described previously, and further contributes towards the disadvantages experienced by children with HIV. These impacts may be ameliorated through the provision of hearing aids. Other interventions may also be needed to ensure that children with hearing loss can participate fully in school, such as training of teachers or linking the children with hearing loss to community-based rehabilitation programmes.Strengths of this study include a large sample size, with comprehensive assessments of the ears and hearing completed for every child. A broad range of data was collected through the electronic medical record, patient and caregiver interviews, and formal audiologic assessment. Limitations of this study include lack of an HIV-negative control group. However, data on children in Africa show that the expected prevalence of hearing loss is far lower than that identified in this study . AnotherThere is an urgent need for improved screening tools, identification and treatment of hearing problems in HIV-infected children, as hearing loss was common in this group and affected school functioning and quality of life. Clear strategies were identified for prevention and treatment, since most hearing loss was conductive in nature, likely due to frequent ear infections, and many children with hearing loss qualified for hearing aids. Screening strategies need to be developed and tested, since caregivers were not reliable at identifying hearing loss, and often mis-identified children with normal hearing as having hearing loss. Children with frequent ear infections, ear drainage, TB, severe HIV disease, or low BMI should receive more frequent ear assessments and hearing evaluations.


Objective. The aim of this study is to analyze the high-frequency hearing levels in patients with rheumatoid arthritis and to determine the relationship between hearing loss, disease duration, and immunological parameters. Materials and Methods. A descriptive cross-sectional study including fifty-three patients with rheumatoid arthritis was performed. The control group consisted of 71 age- and sex-matched patients from the study population . Both a pure tone audiometry and an extended-high-frequency audiometry were performed. Results. Extended-high-frequency audiometry diagnosed sensorineural hearing loss in 69.8% of the patients which exceeded the results obtained with pure tone audiometry (43% of the patients). This study found significant correlations in patients with sensorineural hearing loss related to age, sex, and serum anti-cardiolipin (aCL) antibody levels. Conclusion. Sensorineural hearing loss must be considered within the clinical context of rheumatoid arthritis. Our results demonstrated that an extended-high-frequency audiometry is a useful audiological test that must be performed within the diagnostic and follow-up testing of patients with rheumatoid arthritis, providing further insight into a disease-modifying treatment or a hearing loss preventive treatment. Autoimmune hearing loss was reported for the first time by McCabe in 1979.  A descriRheumatoid arthritis (RA) is a connective tissue disease that has a disseminated erosive arthropathy associated with systemic manifestations. This immune-mediated disease may entail mainly SNHL –11, whicThe pathogenesis of SNHL in RA still remains unclear, although it is potentially related to vasculitis , neuritiThis study aimed to assess the real prevalence of SNHL in RA using advanced audiometric analysis based on extended-high-frequency audiometry (HFA), thus considering its effectiveness and clinical utility for inclusion in the routine diagnostic hearing tests. Furthermore, we analyzed the immunological parameters to obtain an indicator of the inner ear impairment in these patients.A cross-sectional descriptive study was carried out with comparative cases and controls matched for age and sex.Inclusion Criteria. Fifty-three patients with RA (106 ears), both sexes, aged between 20 and 60, were diagnosed and treated in the office of the Rheumatology and Nephrology Departments of “Severo Ochoa” Hospital . The diagnostic criteria and their respective scoring used for a definitive RA diagnosis were as follows: (A) joint involvement: 1 large joint = 0; 2–10 large joints = 1; 1–3 small joints (with or without involvement of large joints) = 2; 4–10 small joints (with or without involvement of large joints) = 3; >10 joints  = 5; (B) serology (at least 1 test result is needed for classification): Negative Rheumatoid Factor (RF) and negative anti-citrullinated protein antibody (ACPA) = 0; low-positive RF or low-positive ACPA = 2; high-positive RF or high-positive ACPA = 3; (C) acute phase reactants (at least 1 test result is needed for classification): normal C-reactive protein (CRP) and normal Erythrocyte Sedimentation Rate (ESR) = 0; abnormal CRP or abnormal ESR = 1; (D) duration of symptoms: <6 weeks = 0; >6 weeks = 1. The classification criteria from the American College of Rheumatology (ACR) for RA  consider that a score of > or = 6/10 is needed for classification of a patient as having a definitive RA. inclusion criteria: patients who suffered from rheumatoid arthritis, both sexes, aged between 20 and 60.Both a medical history and an ENT examination were performed, using the followingExclusion Criteria. Exclusion criteria are patients who suffered from another immune-mediated disease, inner ear disease, traumatic brain injury, Menière's disease, metabolic diseases, and cardiovascular disease, exposure to noise, and the use of ototoxic medication different from that used in the treatment of RA.A control group of 71 patients (142 ears) was consecutively recruited in “Area 9” of Madrid, from January 2010 to February 2011.The control group was composed of patients with a normal state of health, free from all signs or symptoms of ear disease and from obstructing wax in the ear canal, and with no history of exposure to noise, potentially ototoxic drugs, or familial hearing loss that attended our ENT department for head and neck or rhinologic disorders.If the control population presented hearing thresholds in the limits of the normal range, according to the range of age and sex, assessed by pure tone audiometry (PTA) using frequencies of 125 to 8000 Hz, consequently an extended-high-frequency audiometry (HFA) of 8000 to 18000 Hz was also performed to determine the normal hearing parameters and use those parameters as references for the study.A number of variables have been studied, such as demographic variables (age and sex), hearing variables (hearing thresholds with PTA and HFA), and variables related to the rheumatologic disease . Disease activity is measured as active or nonactive.The first otological test included an initial otoscopy and a hearing screening with PTA and HFA.All the subjects suitable for inclusion were evaluated based on a questionnaire (Annex A in ISO 389-9: 2009) according to a written protocol based on ISO 389-9 recommendations on the determination of reference hearing threshold levels.Audiometric tests were conducted by experienced audiometric technicians. For each test frequency, the signal was manually increased by steps of 5 dB until the test person responded, after which the signal was decreased by 10 dB and increased by 5 dB until response. The intensity to which the listener responded three out of five times was recorded as threshold (masking was used). The pure tone hearing thresholds (125–8000 Hz) were measured with a manual audiometer  and equipped with TDH-39 supra-aural earphones . Bone and air conduction audiometry were performed.HFA thresholds  were determined using a Madsen clinical audiometer  with a Sennheiser HDA 200 closed circumaural earphone . All testing equipment for audiometry was calibrated according to ISO 389-5  and the manufacturer's recommendations. The threshold was defined as the lowest decibel hearing level at which responses occurred in at least 50% of a series of ascending trials  , 27.The limit for the hearing loss to be considered was established when one or more frequencies have higher threshold than the frequency previously identified as normal in the control population .An immunological analysis of all the parameters, including immunological RA activity (CRP and ESR), subgroup markers of RA (RF and antinuclear antibodies), acute phase reactants (APR), serum cryoglobulins, and anti-cardiolipin (aCL) and antithyroid antibodies, was performed.All the procedures used in this study and practiced on each patient were in agreement and regulated with approval from the “Severo Ochoa” Hospital (Madrid) Institutional Review Board and ethical code identified by the MINUTE: 11/09, 25/11/2009, IEC (Independent Ethics Committee) Internal code: 431-A (65/09), and the Declaration of Helsinki (1983). Informed consent was obtained from each participant. The same person performed all interviews. The subjects received no monetary compensation. Informed consent was obtained from all subjects, after being informed of details about the purpose of the study, according to the recommendations from the Ethics Committee in our centre.Statistical Package for the Social Sciences) for Windows. This study used quantitative and qualitative descriptive statistics, Chi-square test, Student's t-test, and Mann-Whitney U test. Logistic regression analysis was achieved by adjusting the estimate for some risk factors, including independent variables such as risk factors related to the patient, subjective hearing or rheumatic disease, with a dependent variable represented by the presence of sensorineural hearing loss. The estimate was established with 95% confidence intervals of the hearing loss prevalence in the PTA (threshold > 30 dB HL) and in the HFA (according to the hearing threshold of the control population for each frequency), adjusted for age  and sex. A p value of <0.05 was considered significant.Statistical analyses were performed using SPSS 15 (The average age of the patients with RA was 50.5 years (interquartile range: 38–59 years), and the patients included 14 males and 39 females with a 26.4%/73.6% male/female ratio. The control population included 71 subjects (142 ears) belonging to Madrid Area 9; the patients were consecutively enrolled, with an average age of 38 years .p = 0.001) and 8000 Hz (p = 0.001) frequencies between patients with RA and the control population.In this study, 43% of the study population with PTA had hearing loss (46 ears). The type of hearing loss was sensorineural, with high-frequency loss predominant. High frequencies were affected in 56.6%, medium frequencies in 17%, and low frequencies in 13.2% of the population. There was a significant difference in hearing thresholds (arithmetic mean) at 6000 Hz (p = 0.038), 6000 (p = 0.004), and 8000 Hz (p = 0.009) frequencies; there were lower auditory hearing thresholds in females compared to males. We observed that in patients with RA there was a statistically significant relationship between the hearing loss diagnosed by PTA and sex (OR = 3.46 (1.29–9.27)) and the age at which it was diagnosed (OR = 1.09 (1.04–1.15)).SNHL was bilateral and symmetric in 74% of the cases. There were no cases of conductive or mixed hearing loss. Our results showed that patients with RA had greater high-frequency loss than the control population (according to age range) . Regardip < 0.0001)  . These r 0.0001) .p = 0.009) and the 10000 Hz (p = 0.026) threshold frequencies, between male and female patients with RA, which demonstrated a greater hearing loss in males than in females.Regarding sex, we obtained a significant difference between the means of the 8000 Hz ).The diagnosis of SNHL in RA reached 69.8% with HFA and 43.4% with PTA. The chance of diagnosing hearing loss with HFA is 33.6 times higher compared to PTA OR = 29 (4.8–1184.43).RA may cause SNHL , similarNevertheless, nowadays the treatment used in RA includes immune-modulating drugs that could control the progression of the SNHL in some patients. Thereby an early diagnosis of a possible subclinical SNHL by means of HFA  in order to allow a prior treatment (transtympanic or systemic corticosteroids) is of paramount importance.(1) PTA in RA. The prevalence of SNHL with PTA in our study (43.4%) is within the same range as described in previous literature, which has been estimated between 25.2 to 60% [2 to 60% , 12, 13.2 to 60% , 31, 32.2 to 60% , who cla2 to 60% , who repA comparison between the study and control groups using PTA revealed a significant hearing loss in patients with RA at frequencies of 6000 and 8000 Hz. Our results differ from other authors, who did not find significant differences between the two populations , 13.Moreover, 73.9% of our patients with RA had bilateral, symmetric SNHL, which was a slightly lower result compared to the results obtained by Özcan et al.: 84.6% with bilateral SNHL , 21. In In our study, we observed a significant relationship between SNHL and sex (OR > 1) with males demonstrating a higher risk of hearing loss and significant differences in high frequencies at approximately 4000, 6000, and 8000 Hz. These findings were consistent with those observed by Dikici et al. . This heThe persistent findings of high-frequency thresholds in female subjects might be explained due to the fact that the female ear canal has smaller average length and volume. Nevertheless, there could be other explanations for the gender difference, such as differences in exposure to workplace or extracurricular activity noise or different hearing levels as a result of a different phylogenetic origin. Moreover, the aging process may be different in males and females. There are many health factors that influence hearing status  which may also influence the differences observed between males and females .(2) HFA in RA. In most of the studies, the hearing loss diagnosis in patients with RA was performed using pure tone audiometry. In few studies a hearing screening was performed using frequencies higher than 8000 Hz [ 8000 Hz , 34, 35. 8000 Hz , 35 and,Furthermore, 69.8% of our patients with RA were diagnosed with SNHL using HFA, which is a higher percentage compared to those obtained using PTA. Some studies showed that there were no references regarding the index of SNHL using HFA, because previous results were global (125–16000 Hz) , 36.Our results do not show a significant correlation between SNHL diagnosed using HFA and age. This finding takes into consideration that normal age-related hearing loss in patients older than 50 from the control population is expected to have higher thresholds than those with RA since hearing loss is generally observed since early stages of the disease . RegardiIn our study there were statistically significant differences in all of the frequencies studied between the hearing threshold in both the control population and patients with RA. The hearing loss is greater starting at 40 years of age with thresholds higher than 60 dB.Öztürk et al. obtained similar results and found significant differences between the control population and the patients with RA, from a frequency 220 to 16000 Hz . In our We found no correlation between SNHL diagnosed using PTA and HFA and the duration of the disease, which was consistent with other studies , 32, 33.p = 0.029) between SNHL diagnosed using HFA and anti-cardiolipin (aCL) positivity (OR < 1) was observed. This finding might be due to the potential for inner ear thrombosis to be developed, which is found to be associated with high levels of aCL (risk factor for SNHL). In our study, 43.3% of the patients with RA had aCL positivity, which may cause confusion since it is usually associated with SLE [The analysis of immunological parameters using specific laboratory tests was performed without obtaining conclusive results , 38, 39.with SLE –42.No correlation between positivity to anti-nuclear antibodies (ANA), rheumatoid factor (RF), Sjögren's syndrome (SS), aCL, ESR, and SNHL has been reported , 12, 14.There was a higher rate of patients diagnosed with SNHL using HFA  compared to studies previously reported. SNHL in RA was bilateral and predominantly symmetric, affecting mainly high frequencies. Males over 40 had a greater risk of suffering from SNHL.There were more cases of subclinical hearing loss diagnosed using HFA compared to PTA. These results lead us to consider HFA as a necessary test to diagnose subclinical hearing loss in patients with RA. An early intervention in this subgroup of patients may cease the development of hearing loss. Thus, we propose that HFA should be performed as a routine screening not only for the study of RA but also for all of the inner ear disorders , 28, 43.


A male infant with a diagnosis of homocystinuria presented with avascularity of the peripheral retina with a ridge on ophthalmic exam, consistent with a FEVR-like manifestation homocystinuria. Upon follow-up and treatment for homocystinuria, the retinal vascularity improved without the need for prophylactic treatment to the peripheral avascular retina.  Homocystinuria is a rare disorder caused by a defect in methionine metabolism due to inadequate activity of the enzyme cystathionine beta synthase (CBS) . This reFamilial exudative vitreoretinopathy (FEVR) is a progressive eye disease that affects the vascularization of the peripheral retina . The chaIn this unique case report, we describe a male infant affected by a FEVR-like presentation of homocystinuria that resolved with the treatment of the homocystinuria.μmol/L, ref: 4–17 μmol/L) on routine labs. He has no other signs of hypercoagulability and was not given supplemental oxygen. He was referred for genetics evaluation and an ophthalmologic exam at the Bascom Palmer Eye Institute was performed to rule out ectopia lentis.A male infant born in Jackson Memorial Hospital at 36 weeks at 2466 grams via cesarean section for premature rupture of membranes was found to have elevated homocysteine levels  showed grossly attenuated vessels and avascularity of the retina bilaterally. A retinal angiogram was performed and confirmed the diagnosis of avascularity of the peripheral retina, in a FEVR-like distribution Figures .Genetic testing showed a heterogeneous unclassified novel missense variant of the cystathionine beta-synthase (CBS) gene, confirming the diagnosis. The patient was then treated with a methionine-free diet, vitamin B6, folic acid, and betaine powder. Upon observation, fundus photographs showed regression of the peripheral ridge, and it was noticed that the retina vessels grew from zone 2 to zone 3 forming normal vascularization of the retina. In the last follow-up at 2 months, his fundus exam showed a flat retina bilaterally with normal vasculature to the ora serrata.Homocystinuria is a condition associated with increased risk of primary thrombosis . High leDysregulation of vascular growth and maturation is most likely responsible for the FEVR-like retinal manifestation of homocystinuria in this patient. While it is unlikely that treatment of homocystinuria could reverse arterial changes so quickly, prompt treatment may reduce oxidative stress and allow for a return to normal development with proliferation of the retinal vessels. This was evident when the patient showed complete vascularization of the peripheral retina on his final examination.Although ectopia lentis remains one of the most common ocular abnormalities associated with homocystinuria, a complete ocular exam on patients with homocystinuria should be performed to rule out or diagnose retinal abnormalities. Angiography, including wide-field angiography, can be considered to evaluate retinal abnormalities with a vascular component.


Episodes of acute adenolymphangitis (ADL) are often the first clinical sign of lymphatic filariasis (LF). They are often accompanied by swelling of the affected limb, inflammation, fever, and general malaise and lead to the progression of lymphedema. Although ADL episodes have been studied for a century or more, questions still remain as to their etiology. We quantified antibody levels to pathogens that potentially contribute to ADL episodes during and after an episode among lymphedema patients in Léogâne, Haiti. We estimated the proportion of ADL episodes hypothesized to be attributed to specific pathogens.We measured antibody levels to specific pathogens during and following an ADL episode among 41 lymphedema patients enrolled in a cohort study in Léogâne, Haiti. We calculated the absolute and relative changes in antibody levels between the ADL and convalescent time points. We calculated the proportion of episodes that demonstrated a two-fold increase in antibody level for several bacterial, fungal, and filarial pathogens.Streptococcus group A, followed by IgG2 responses to a soluble filarial antigen (BpG2), Streptococcal Pyrogenic Exotoxin B, and an antigen for the fungal pathogen Candida. When comparing the median antibody level during the ADL episode to the median antibody level at the convalescent time point, only the antigens for Pseudomonas species  and Streptolysin O  showed a significant result.Our results showed the greatest proportion of two-fold changes in antibody levels for the carbohydrate antigen Streptococcus A as a potential contributing factor to ADL episodes. Our results add to the current evidence and illustrate the importance of determining the causal role of bacterial and fungal pathogens and immunological antifilarial response in ADL episodes.Although our results are limited by the lack of a control group and few antibody responses, they provide some evidence for infection with  Wuchereria bancrofti, Brugia malayi, and Brugia timori [Lymphatic filariasis (LF) is a neglected tropical disease affecting 68 million people throughout the world . The disa timori , which aWhile often characterized by chronic lymphedema and ultimately elephantiasis of the limbs, the first clinical sign of lower limb LF disease is typically an episode of acute adenolymphangitis (ADL). ADL is characterized by a plaque-like area of relatively diffuse cutaneous inflammation with or without ascending lymphangitis or satellite adenitis . ADL epiStreptococcus A and Staphylococcus as the causal agent in ADL episodes [Wolbachia endosymbiont in ADL episodes [Despite extensive research, the etiology of ADL episodes is yet to be fully understood. Although there has been evidence for bacterial pathogens such as episodes –16, otheepisodes ,18. Otheepisodes  or have episodes . Additioepisodes . The rolepisodes ,23. Fungepisodes , which sUnderstanding the etiology and immunology of ADL episodes is important because they contribute to the progression of chronic LF—a major cause of disability worldwide. Our objective was to understand how antibody response to different antigens changes over the course of an ADL episode in order to inform models of ADL episode causality. To do this, we quantified antibody levels to pathogens that potentially contribute to ADL episodes during and after an episode among lymphedema patients in Léogâne, Haiti.Wuchereria bancrofti. Patients were included if they had lymphedema of the leg, agreed to return to the clinic for follow-up evaluations, and did not have an obvious cause of edema such as a tumor. Data were collected on demographics, number and history of ADL episodes, and leg volume. Patients’ lymphedema was classified into stages using the 7-stage system of Dreyer and colleagues [Between June 1995 and December 1997, lymphedema patients were enrolled in a study to test the feasibility and effectiveness of a basic lymphedema management program . Patientlleagues , and stalleagues . Of 302 A subset of 41 patients of the original 175 patients was used for this analysis. In addition to the demographic, ADL, and leg volume information, serum samples were collected from these patients. During an ADL episode, patients were instructed to report to the clinic where a serum sample was taken. They were then instructed to return to the clinic after the ADL episode had ended for collection of a convalescent serum sample. Attempts were made by the investigators to obtain serum samples from all 175 patients enrolled in the original cohort, but not all patients were able to get to the hospital during an ADL episode as they may have been disabled and others did not consent to have their blood drawn. Thirty three of the 41 patients had serum samples taken during and following one ADL episode comprising 33 paired samples. Eight patients had serum samples during and following multiple ADL episodes: 3 of which had serum drawn during and after 3 episodes comprising 9 paired samples and 5 had serum drawn during and after 2 episodes comprising 10 paired samples. Overall there were a total of 52 paired samples and 104 observations. The current analyses were conducted post-hoc from 2013–2014.The study was approved by the Ethics Committee of Ste. Croix Hospital and by the Institutional Review Board of the Centers for Disease Control and Prevention. To be eligible for participation, patients were required to provide written informed consent.Brugia pahangi antigens that were extracted from adult worms as previously described [Brugia and Wuchereria infections. Isotype-specific responses were measured with isotype-specific monoclonal antibodies. An IgG1 response to Brugia (BpG1) is considered to be a measure of filarial exposure [The samples collected from 1995–1998 were stored at -20°C at the CDC Laboratories in the Division of Parasitic Diseases and Malaria until testing was completed in 2000. We measured the antibody level for a variety of filarial, bacterial, and fungal pathogens hypothesized to be associated with ADL episodes. To test for filarial antibodies we used escribed . These aexposure , whereasexposure  and is hexposure ,30. We aCandida and Trichophyton were measured using crude extracts from culture organisms. We measured IgG antibody to the bacterial pathogen Streptococcus A with several different antigens: the virulence factors Streptococcal Pyrogenic Exotoxin A (SPEA) and Streptococcal Pyrogenic Exotoxin B (SPEB), the hemolytic exotoxin Streptolysin O (SLO), and the carbohydrate antigen Streptococcus group A (StrepA). In addition to the streptococcal bacterial pathogens, we measured IgG responses to Pseudomonas using an exotoxin antigen and Staphylococcus using Staphylococcus enterotoxin B (SEB).IgG antibodies to the fungal pathogens Wuchereria bancrofti antigen was used to determine which lymphedema patients were currently infected with the parasite. Assays to detect antibody response to adult filarial antigen were performed using biotinylated monoclonated antibodies  specific for IgG1, IgG2, IgG3, and IgG4. Assays for antibody response to Streptococcus group A , Streptolysin O, Pseudomonas exotoxin , Staphylococcus enterotoxin B , Candida, and Trichophyton were done using biotinylated monoclonal antibodies for total IgG. All antibodies were standardized against a high-titered serum sample included in each assay and arbitrarily assigned an antibody level of 10,000 units per ml. Antibody levels were then transformed into arbitrary units (AU). Plasma levels for all antigens were assayed in triplicate at a final dilution of 1:50. Acute and convalescent samples from a given patient were run on the same plate.The Og4C3 ELISA assay to detectIn exploring potential filarial, bacterial, or fungal pathogens associated with ADL episodes, we conceptualized them using sufficient component cause models (SCCM) . We assuStreptococcus A along with unknown factors U1 is sufficient to cause an episode of ADL. Model 2 states that at minimum, a reaction to filarial worm infection in combination with unknown factors U2 must be present for ADL episode to occur. Model 3 states that at minimum, the presence of a fungal infection such as Trichyophtyon in combination with unknown factors U3 is sufficient to cause an episode of ADL. Model 4 involves a biological interaction of infection with both a bacterial and fungal pathogen. The fungal pathogen causes a lesion on the skin of the affected limb, which then provides an entry point for bacteria and the subsequent response to infection in the form of an ADL episode. The unknown factors stated in these SCCMs may comprise a multitude of environmental, genetic, and immunologic factors. The examples of sufficient component causes displayed in If infection by a particular pathogen immediately prior to an ADL episode contributes to the etiology of the episode, we expect the antibody levels to increase from the ADL to the convalescent time point ,16,36. WFor each antigen, we calculated an absolute change and percent change from the ADL to convalescent time point:Since the distribution of antibody change in this population was not normal, we reported median values for the absolute antibody values, absolute change, and percent change along with the standard deviation and inter-quartile range (IQR). We conducted a Wilcoxon signed rank test to detect a difference between ADL and convalescent median antibody levels.For each antigen we calculated the proportion of antibody response as the number of cases showing an antibody change at the two-fold level divided by the total number of ADL episodes in the sample. The denominator for the proportion is 52 ADL episodes.The majority of the lymphedema patients were female (83%) with a median age of 34 years . Most paCandida and Trichophyton). All responses represent a two-fold positive change of antibody level from the ADL to convalescent time point. Of the 52 paired serum samples, 5 (9.6%) showed an antibody response to the Strep A antigen, four (7.7%) showed a response to the BpG2 antigen, three (5.8%) showed a response to the SPEB antigen, and two (3.9%) showed a response to the Candida antigen. Only one (1.9%) paired sample demonstrated an antibody response to the BpG1, SEB, or SLO antigens. Our samples showed no response at the two-fold level to the BpG3, BpG4, Pseudomonas, and SPEA antigens.Candida had the highest proportions of response respectively.Among the bacterial antigens, Strep A had the highest proportion of two-fold response. Among the filarial and fungal antigens, BpG2 and Pseudomonas  and SLO  showed a significant result. The median difference between paired ADL and convalescent time points ranged from -39.6 for BpG1 to 0.8 for SEB. The median % change between paired ADL and convalescent time points of greatest magnitude was seen for the SLO antigen (-9.6%). The median % change of smallest magnitude was for Trichophyton (-0.1%). SPEA had the greatest positive percent change (2.7%). Although the median % changes are minimal, the IQRs for each antigen are wide, indicating a great amount of variation.Candida. Amongst the bacterial antigens, the Strep A antigen for Streptococcus A infection had the highest proportion of two-fold responsesTo better understand factors associated with ADL episodes among lymphedema patients in Léogâne, Haiti, we quantified antibody levels to specific pathogens during and following an ADL episode. We conceptualized our hypotheses and will discuss our results in the context of sufficient component causal models (SCCM). We calculated the proportion of two-fold antibody responses for different pathogens hypothesized to be associated with ADL episodes. We assumed that those pathogens showing a two-fold change from an ADL to convalescent time point are considered a component cause of ADL episodes. Although a majority of the paired samples did not show a two-fold increase in anti-body levels for the antigens tested, our results showed the greatest proportion of two-fold changes in antibody levels for the Strep A antigen, followed by BpG2, SPEB, and Streptococcus A in combination with one or many unknown factors (U1) could be sufficient to cause an episode of ADL. These findings are consistent with several other studies which found a high proportion of patients undergoing ADL to be infected with group A Streptococcus, leading to the conclusion that group A Streptococcal infection at least in part is associated with episodes of adenolymphangitis [Streptococcus A [Under our assumptions, these results support SCCM 1  indicatihangitis ,13,16. Ococcus A , while t2 must be present for an ADL episode to occur. Although we saw responses for a change in filarial antibodies, the frequency of the filarial responses was less than the frequency of responses to Streptococcus A antigens, providing less evidence for the SCCM 2. Interestingly, the BpG2 filarial antigens had the highest proportion of change among the filarial antigens. Since IgG2 responses to filarial antigens are associated with carbohydrate epitopes, it is possible that these responses reflect cross reactivity stimulated by exposure to non-filarial antigens or even bacterial antigens. Responses to BpG4, indicating adult worm infection [SCCM 2 states that at minimum, filarial worm infection in combination with a set of unknown factors Unfection , would bnfection ,38 as senfection , but thenfection .Candida antigen. This evidence would support SCCM 3  and four-fold (300%) levels, and found that few changed by this magnitude. We also explored the proportion of response at lower levels, but are not confident those levels of change are reproducible. The lack of antibody titer change from an ADL to convalescent time point at the higher levels may be due to several factors. The study population in Léogâne, Haiti is regularly exposed to the pathogens of interest and may already have elevated antibody titers. Therefore, a smaller rise in antibody levels from the ADL episode to the convalescent time point may be indicative of infection in this population.There are several limitations in the design of this study. First, the sample size of 41 lymphedema patients is relatively small, limiting the external validity of our findings. Second, the 41 ADL patients who gave blood during and following an ADL episode may not have been representative of the original cohort of 175 lymphedema patients. Although there were no differences in the gender and age distribution, the distribution of lymphedema stage, duration of lymphedema and rate of ADL episodes reported in the year prior to study enrollment, the 41 ADL patients had a statistically significant higher rate of ADL episodes during the entire study period (1995–1998): 1.42 per person year among patients who provided serum vs. 0.75 per person year among original cohort patients  . The mora priori, we may have missed some component causes of ADL, which may contribute greatly to the ADL episodes in this study population. Because most of the variance in the antibody levels was due to subject and not due to time, our conclusions about which pathogens contributed the most to ADL episodes in this population based on arbitrary responses of change may not be as strong as findings from other studies in which a control population (no ADL episode) was present.Third, all antibody values are based on arbitrary units and not on a calibrated reference scale. Therefore, we can only explore absolute and relative differences in antibody levels between the ADL and convalescent samples and assume certain levels of change indicate infection. Furthermore, it is possible that blood was not drawn from individuals during the most florid period of their ADL episode. Patients were required to provide their own transportation to and from the clinic. Those who did willingly make the trip to the clinic to give blood may have done so after the most severe symptoms of their ADL episode had passed. If this is the case, the antibody levels measured during the ADL episode are probably an overestimate of the levels during the most florid time (assuming antibody levels increase over the period of interest). Thus, the differences in antibody levels from ADL to convalescent time point were probably an underestimate. Also, because we chose which pathogens to test Lastly, we only collected information from 1–3 ADL episodes among each individual in this sample population. The lymphedema patients in the cohort have been exposed to LF for many years and may have experienced many ADL episodes throughout their lives. We assume that the pathogen(s) with the greatest contribution to ADL episodes at this time point will be typical of all ADL episodes in this population.Streptococcus A as a potential contributing factor of ADL episodes. However, such changes were only observed for a small proportion of ADL episodes and the contribution of Streptococcus A to ADL episodes in this cohort may be minimal compared to other unmeasured factors. The overall lack of response of the paired samples may suggest that none of the pathogens tested in this study are a major contributing factor to ADL episodes. Nonetheless, these findings are important within the context of previous research suggesting a relationship between ADL episodes and Streptococcous A and within the more recent focus on LF morbidity control in endemic areas. This work adds to the current evidence and illustrates the importance of determining the causal role of bacterial and fungal pathogens and immunological antifilarial response in ADL episodes.Through a descriptive analysis of the proportion of antibody response at a two-fold level of change, this study provides further, yet limited evidence for infection with S1 TableN = 104 samples.(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.