Dyscolus and Dercylus.Páramo is a tropical alpine ecosystem present in the northern Andes. Its patchy distribution imposes limits and barriers to specialist inhabitants. We aim to assess the effects of this habitat distribution on divergence across two independently flightless ground beetle lineages, in the genera One nuclear and one mitochondrial gene from 110 individuals from 10 sites across the two lineages were sequenced and analyzed using a combination of phylogenetics, population genetic analyses, and niche modeling methods.Dyscolus alpinus, where one dominant haplotype is found in four out of the six populations analyzed for both molecular markers. However, complete population isolation was revealed in species of the genus Dercylus, where high levels of differentiation exist at species and population level for both genes. Maximum entropy models of species in the Dercylus lineage show overlapping distributions. Still, species distributions appear to be restricted to small areas across the Andes.The two lineages show different degrees of population subdivision. Low levels of gene flow were found in Dyscolus alpinus appears to be a better disperser than species in the genus Dercylus.Even though both beetle lineages are flightless, the dispersal ability of each beetle lineage appears to influence the genetic diversity across fragmented páramo populations, where  The distribution of a species is determined by environmental conditions, ecological interactions of species, and dispersal dynamics. In particular, the dispersal ability of species plays an important role in determining species ranges and biogeographical patterns . The evoFlightlessness has been recorded in insect faunas in stable habitats such as oceanic islands and caves , as wellFlightlessness in carabid beetles has been the focus of several studies, some of which support the idea that wing loss promoted genetic isolation across populations . HoweverThe higher elevations of the northern Andes host a unique plant community known as páramo. Limited to elevations above 3,000 m, páramo is patchily distributed among the many higher peaks and volcanos in the range. Páramo contains a high number of endemic species, which are mostly are restricted to small elevational ranges , is a micropterous species present in the northern and central portion of the Ecuadorian Andes, between 2,750 and 4,200 m, in páramo, subpáramo, and montane forest . The second beetle lineage assessed comprises multiple species in the genus DercylusDercylus is a new world genus with 35 species (LinodercylusDercylus orbiculatusDercylus praepilatusDercylus cordicollis (In this study we focused on two flightless ground beetle (Coleoptera: Carabidae) lineages from páramo. Most of the ground beetles present in páramo are micropterous (76% of species), with only few macropterous and brachypterous species reported . To exame forest . MembersDyscolus. Of 320  species , includi species . All Ecudicollis , and twoThe aim of this study is to analyze the effects of montane isolation in these two flightless beetle lineages from páramo, and to ask how their phylogenetic structure and population connectivity have been affected by potential geographical barriers, their dispersal abilities, and their ages of divergence. We analyzed two molecular markers, cytochrome c oxidase subunit I (COI) and carbamoyl phosphate synthetase 2 (CAD), alone and combined, to determine phylogenetic relationships among individuals for each lineage, population genetic diversity and structure, and approximate ages of each clade with the use of molecular clocks. In addition to the analyses of the molecular data, geographic information was used to generate models of species distribution as a way of assessing habitat continuity.The study area encompassed the Ecuadorian Andes, where all the sampling took place above 3,100 m in the páramo ecosystem. The selection of sites was based on previous records of occurrence of the target taxa, and potential geographical barriers. Some of these potential barriers include: dry valleys, rivers, faults, and the east/west split of the Andes mountain range . In partDyscolus alpinus was found in six out of the eight sites sampled (Dercylus specimens were collected in five sites (Dercylus cordicollis (SIMT265) from Cotacachi, this sample was included for the analysis of Dercylus cordicollis populations. Outgroup taxa were chosen based on availability of fresh material and taxa considered closely related carabid lineage within the Harpalinae. Outgroup taxon sampling included 12 taxa: two were collected in the Ecuadorian páramo; six taxa were collected in the southeastern United States; and four additional samples were retrieved from GenBank  using GeneJet’s Genomic DNA Purification Kit  following the manufacturer’s instructions. A fragment of the mitochondrial cytochrome c oxidase subunit I (COI) gene was amplified using the primers TL2–N–3014  and aligned in MAFFT v.7  togetherDyscolus alpinus; (2) Dercylus orbiculatus; and (3) Dercylus species together  in order to contrast inter– and intrapopulation variability. Parameters estimated included: estimated haplotype diversity (h), nucleotide diversity (π), number of polymorphic sites (S), number of migrants per generation (Nm), and Tajima’s D neutrality test statistic (D). ɸST values were calculated to assess the population structure, while an analysis of the molecular variance (AMOVA) was performed to test the influence of geographical barriers, including the rivers, dry valleys discussed above, and the division between populations of eastern and western cordilleras. A Mantel test of correlation between genetic and geographic distances was performed using 1,000 randomizations . Lastly, Structure 2.3.4. (K), using no admixture for the mtDNA and admixture for the nuclear gene. A total of 10 iterations were run per potential K, and the presence of one to eight populations were tested for both beetle lineages.The levels of genetic diversity were calculated using Arlequin 3.5.2.2  and DnaSe 2.3.4.  was usedPhylogenetic analyses were performed to determine relationships among individuals for each lineage and among lineages. To explore marker-specific effects, mitochondrial and nuclear data sets were initially analyzed separately, but combined data were used to generate the most robust hypotheses for each beetle lineage using MrBayes 3.2  and RAxMDyscolus alpinus, one point of fossil calibration was used to designate minimum node ages, using a log-normal distribution and enforced monophyly. This fossil represents the same tribe as Dyscolus (Platynini): a fossil of Limodromus from the late Eocene (37.2–33.9 Mya), of Baltic origin  from the Middle Eocene (44.1 Mya), of Baltic origin  from the Eocene (37.2–33.9 Mya) of Teller County, Colorado (http://tree.bio.ed.ac.uk/software/tracer/) to ensure effective samples sizes. Trees were visualized in TreeAnnotator 2.0.02 (http://beast.bio.ed.ac.uk), using maximum clade credibility after a 10% burn–in.To understand the timing and rate of diversification for each ground beetle lineage, divergence times were estimated using an uncorrelated relaxed clock in BEAST 2.0  using thc origin . The divc origin , and PteColorado . In addiDercylus was modeled to determine if species distribution overlapped, using present bioclimatic variables at 2.5’ spatial resolution ), BIO5 = Max Temperature of Warmest Month, BIO6 = Min Temperature of Coldest Month, BIO12 = Annual Precipitation, BIO13 = Precipitation of Wettest Month and BIO14 = Precipitation of Driest Month. The analyses ran for 10 replicates of 500 iterations and using a 10% training presence, using the default settings for the convergence threshold (10−5). For each species, the output from the average run was reclassified using ArcMap 10.4.1  with a 10% threshold. Model performance was evaluated using the area under the curve (AUC) calculated by MAXENT, where values between 0.7 and 0.9 indicated a good discrimination . Haplotype 1 is the most widespread across populations, found in four of six sites (h) is shown in h = 0.71). The overall nucleotide diversity of COI was low (π = 0.00–0.01). Structure analyses tended to overestimate the number of populations; results from multiple simulations show that the number of populations using this data set is one (K = 1). Results from the neutrality test for the overall values , as well as on most populations are not significant  between genetic (ɸST) and geographical distances.The portion of the COI gene analyzed for ix sites . Unique ix sites . Haplotynificant . Cayambenificant . AdditioDyscolus alpinus spanned 744 bp, with 64 parsimony informative sites. These represented five haplotypes (GenBank accessions MK440260–MK440264), with no heterozygote individuals observed. Haplotype 1 is found in four of the six populations. Unique haplotypes were recorded from Atillo (H4) and Cayambe (H5). Haplotype networks for both genes show that most haplotypes are present at multiple sites (h) and nucleotide diversity was low for CAD data set (h = 0.91). Structure analyses of the nuclear data set showed three populations (K = 3). Similar to the results from the mitochondrial data, most neutrality tests using CAD  were not significant, with Cayambe and Pichincha as exceptions. Again, however, these apparent exceptions exhibit few haplotypes, inconsistent with a population expansion scenario. Results from the Mantel test show there was no correlation between genetic and geographic distances .The CAD data set for le sites . The ovedata set . Among aDyscolus alpinus shows that most of the variation occurs within populations , which suggest moderate levels of population subdivision among populations. When ɸST values were compared on a population–by–population basis, some genetic differentiation was found among populations : ɸST = –0.10. The CAD data set shows lower genetic differentiation among all populations of Dyscolus alpinus . The overall number of migrants between Dyscolus populations (Nm = 0.31) indicates that there is population subdivision across sites. Higher levels of genetic connectivity were observed between Mojanda and Cayambe (Nm = 1.79), results that are also supported by the low ɸST values between these two sites . No haplotypes were shared among sites  among Dercylus species and populations. Structure analyses of Dercylus orbiculatus individuals all resolved as one population, even though the two sites do not share haplotypes. These analyses were also performed for the entire clade, and one population was found (K = 1). Neutrality test values were not significant , with the exception of the population of Dercylus orbiculatus from Culebrillas, for which significantly positive D and Fs were found  between genetic and geographical distances.In the case of ng sites , and oveng sites . Eight hre found . Lastly,Dercylus species was 669 bp with 24 parsimony informative sites (GenBank accessions MK440236–MK440251). A total of 13 alleles were identified across the Dercylus lineage and the overall nucleotide diversity was low (π = 0.00–0.02). Four alleles were recovered from Dercylus orbiculatus populations, and three alleles from Dercylus cordicollis, with one shared with Dercylus from Cotacachi , whereas results from the analyses of the whole clade show five distinct populations (K = 5), corresponding to each species. Neutrality tests supported significantly positive Fs for Dercylus cordicollis from Pichincha  between genetic (ɸST) and geographical distances.The portion of the CAD gene amplified for achi H5; . Four aldicollis . Structuichincha . ResultsDercylus orbiculatus, occurring across the southern Ecuadorian Andes . A high level of genetic differentiation was also observed when ɸST values were compared between populations and species in COI data set .The AMOVA showed that most of the genetic variation was found among groups when testing the effect of geographical barriers . The higan Andes . The higdata set . In thisST (ɸST = 0.93) shows high levels of population differentiation. When each population was compared against each other, we found moderate to complete population subdivision between some sites , as well as between Pichincha and Cotacachi (ɸST = 0.00), Pichincha and Salinas (ɸST = 0.00), Salinas and Atillo (ɸST = 0.03), and Salinas and Cotacachi (ɸST = 0.00). The number of migrants per generation was calculated for two populations of Dercylus orbiculatus (Cajas and Culebrillas), which revealed low gene flow (Nm = 0.39).In the case of the nuclear coding gene CAD, the overall value of ɸDyscolus alpinus yields similar resolution  as sister to the Dyscolus alpinus clade . The timing of splitting–events for the Dyscolus alpinus clade , and between Dercylus cordicollis and Dercylus sp. from Cotacachi , Dercylus from Cotacachi  and Dercylus sp. from Atillo  during the Miocene (16.4–6.04 Mya). Dercylus cordicollis, on the other hand, originated in the Pliocene , and Dercylus praepilatus  in the Pleistocene. Only these last two diversifications were contemporary with the diversification of most páramo plants , shows a low haplotypic diversity across sampled populations. In Dercylus (entirely micropterous), however, high levels of differentiation were found at the species and population levels. The reasons for these differences are not entirely clear.The effect of dispersal ability on population genetic structure and diversification represents a rich focus of study . InsectsDyscolus alpinus, a dominant haplotype for each marker is found in four out of the six populations analyzed, with one site (Mojanda) exhibiting unusually high diversity. The diversity at this site could be associated with multiple colonization events due to dynamic habitat shifts from volcanic activity dated prior to the Last Glacial Maximum  to other sites. Some population pairs on opposite sides of the cordillera also present high population structure , but this is not the case for most sites, so simple E–W and N–S groupings show minimal explanatory power. High ɸST values found among sites on the same side of the cordillera could have resulted from habitat discontinuity reinforced by the presence of major geographical barriers such as major rivers and dry valleys , whereas the higher population structure in the COI data set probably results from both higher mutation rates and shorter coalescence times of mitochondrial DNA  species, high levels of differentiation are reported at the species and population levels for both genes. The mitochondrial marker showed no shared haplotypes among sites and species, whereas CAD showed three shared haplotypes, generally shared by proximate populations . Species in the genus Dercylus show smaller distributions , sharing no haplotypes in either marker. It is, however, intriguing that the three CAD haplotypes at Culebrillas appear to be derived from the single common one at Cajas. This may be the basis for Structure only recovering one population (K = 1) with COI and CAD sequences, suggesting that in the past these populations had higher connectivity levels. Based on AMOVA results, the Chimbo and Chanchan river valleys, which overlap with the Pallatanga fault  also seem to have contributed toward the genetic heterogeneity seen in Dercylus in the eastern cordillera, considering the high ɸST values (Dercylus cordicollis (Pichincha) and Dercylus sp. (from Cotacachi). Divergence time estimates show that the species in the Dercylus clade originated during the Miocene, Pliocene and Pleistocene  and Dercylus praepilatus .For the genus ibutions . Howeverga fault , appear T values  and the T values . Lastly,Dercylus largely support species distinctions, the status of Dercylus orbiculatus (sampled from both Cajas and Culebrillas) is less clear. It is resolved as a paraphyletic grade by COI, as well as in the combined data tree, but monophyletic by CAD, with strong support. While this difference may be a result of the difference in rate of nucleotide change between genes , perhaps facilitated by Quaternary glaciations, when páramo moved to lower elevation areas such as the inter–Andean valleys . How, then, Dyscolus alpinus has achieved its broad distribution, while Dercylus species have largely remained confined to restricted areas, remains an open question.When results from these two flightless lineages were compared to the patterns observed in n páramo , a commo valleys . Still, Understanding the history of movements and diversification of organisms in alpine tropical ecosystems has considerable importance, given the effects that climate warming is having on high elevation faunas . Beetle Pelmatellus columbianus exhibit broad elevational ranges  and sustain higher levels of gene flow among populations (Dyscolus alpinus, to high levels of differentiation among species and populations within the Dercylus lineage. Although species in the genus Dercylus show similar predicted distributional ranges, less genetic cohesiveness was found among populations, possibly as a result of division of the ecosystem by abrupt topographic features. Species with smaller elevation ranges are probably more susceptible to climate change, and elevational shifts toward higher elevations have been already been reported among ground beetle species from páramo (The effects of mountain isolation vary among páramo species. The genetic diversity found in both ground beetle lineages analyzed to date appears to be influenced by the range size and dispersal capability for each beetle lineage. While macropterous species such as ulations , flightlm páramo . The pat10.7717/peerj.7226/supp-1Supplemental Information 1Outgroups used in this study as also listed in this table. Accession numbers are recorded under each gene column.Click here for additional data file.10.7717/peerj.7226/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.7226/supp-3Supplemental Information 3Click here for additional data file.


Different guidelines are adopted in clinics and countries to assess pure tone hearing sensitivity in children with otitis media with effusion (OME). Some guidelines specify a broad range of audiometric frequencies that must be tested and from which average thresholds determined, while others leave test frequencies unspecified. For guidelines that suggest specific frequencies there are various pure tone frequencies and frequency ranges given. The present study investigated whether (1) a full range of audiometric frequencies is required to evaluate hearing loss caused by OME in children, or if neighboring frequencies provide essentially the same threshold information, and (2) if different combinations of test frequency pure tone averaging calculations may affect decision criteria for surgical treatment. In a retrospective cohort study, right and left ear air conduction pure tone threshold data were obtained, from 125 Hz to 8 kHz, for 96 children with OME aged 4 to 12 years. Paired t-tests, correlation tests  and absolute differences were used to examine the relationships among pure tone audiometric (PTA) frequencies for all ears with hearing loss. 168 ears were found to have OME-related hearing loss. Only the 125 Hz—250 Hz comparison showed no statistically significant difference between neighboring thresholds. However, only the 4 kHz and 8 kHz comparison showed a clinically significant mean difference of ≥ 10 dB. When viewing individual differences, comparison between 250 Hz and 500 Hz, 125 Hz and 500 Hz, and 4 kHz and 8 kHz, showed a large number of ears with clinically significant differences between test frequencies. Comparisons among low frequency 3 PTA average , high frequency 3 PTA average , and 4 frequency PTA average  showed no statistically significant differences, with very strong correlations for all comparisons. In addition, for all the combinations of PTA averages, no clinically significant differences were found for the various comparisons or among individual results. Clinically, testing hearing sensitivity in the 125 Hz to 8 kHz range is worthwhile in evaluating hearing sensitivity in children with OME due to large individual variability across audiometric frequencies. However, frequencies tested for criterion averages for surgical treatments of children with OME may be restricted to 3 frequency PTA averages, either an average of 500 Hz, 1 kHz, 2 kHz or an average of 1 kHz, 2 kHz, 4 kHz, as no clinically significant differences were found using these or a 4 frequency averaging technique. For research purposes, 250 Hz can proxy for hearing thresholds at 125 Hz; and the low frequency 3 PTA average, high frequency 3 PTA average and 4 frequency PTA average may be used interchangeably, as no statistically significant differences were found among these measures. Otitis media is a term for a spectrum of middle ear disease, with acute otitis media and otitis media with effusion being a very common illnesses among paediatric patients –3. In AuAcute otitis media (AOM) is an infection of the middle ear caused by bacteria or viruses with a rapid onset ,9. SymptThe presence of fluid in the middle ear results in increasing stiffness and mass of the middle ear system, which affects the transmission of sound from the outer ear to the inner ear, resulting in conductive hearing loss . HearingStudies indicate that approximately 28% of newly diagnosed OME cases recover after 3 months without treatment and 75% of OME as a sequela of AOM also recovered spontaneously by 12 weeks . AccordiIf OME persists for 3 months or more (classified as chronic OME) only around 30% of the cases may demonstrate bilateral spontaneous resolution after 12 months . TherefoAs shown in In summary, the aims of the present study were: (1) to investigate the correlation between 125 Hz and 250 Hz, 250 Hz and 500 Hz, 125 Hz and 500 Hz (a near-neighboring low frequency pair), and between 4 kHz and 8 kHz when assessing hearing levels in children with otitis media with effusion, to understand how much of the variation in one frequency is related to an adjacent frequency; (2) to evaluate the degree of relationship among hearing threshold average calculations, namely in the overall pure tone average (125 Hz– 8kHz), low frequency 3 PTA average , high frequency 3 PTA average  and 4 frequency PTA average  in children with OME-related hearing loss, to determine the degree of relationship among PTA averages; and (3) to determine whether any differences among relationships noted in (1) and (2) were clinically significant, with a clinically significant difference defined as a difference between compared thresholds of ≥10 dB.Children aged from 4 to 12 years, who were diagnosed with otitis media with effusion, were assessed during December 2014 to August 2015. All participants were recruited from the Department of Otorhinolaryngology—Head and Neck Surgery, Shenzhen Children’s Hospital, China. Children attended the clinic due to parental concern regarding hearing loss or for follow-up appointments after acute otitis media or OME. Parents provided informed written consent approving their child’s participation in the research program. Children with unilateral/bilateral mixed hearing loss, with ventilation tubes in situ, and those who were unable to provide a full range of pure tone audiometry frequency thresholds were excluded from the present study. A sample size of n ≥ 133 ears was targeted, to achieve a power of 80% and a significance level of 1% for detecting an effect size of 0.3 between pairs. The present study extends previously published work on audiometric profiles with the same cohort  and usedA portable otoscope  was used to examine for clinical signs of OME. A 226 Hz tympanometer  at 85 dB SPL with sweep rate of 50 daPa/s, calibrated to ANSI S3.39–1987 (R 2007) standards  was usedAll participants were identified with otitis media with effusion through otoscopic examination, tympanometry and ipsilateral acoustic reflex assessment by an ENT specialist. Children with diagnosed otitis media with effusion were further tested for their hearing sensitivity with pure tone audiometry. Clinical otoscopic signs of OME included retracted tympanic membrane, foreshortened handle of malleus, absence of cone of light, discoloration of tympanic membrane, and observable bubbles and fluid levels . Jerger’Children with diagnosed OME had their hearing sensitivity tested with pure tone audiometry. Air conduction pure tone thresholds at 125 Hz, 250 Hz, 500 Hz, 1 kHz, 2 kHz, 4 kHz, and 8 kHz were tested. Bone conduction thresholds (250 Hz to 4 kHz) were obtained only if the air conduction hearing thresholds were worse than 20 dB HL. Children with bone conduction thresholds of ≥ 25 dB HL at 500 Hz, 1 kHz or 4 kHz were considered as having a sensorineural hearing loss component to the hearing loss and excluded from the study. 2 kHz bone conduction thresholds were not included as a consistent 2 kHz bone conduction dip is often shown in the pure tone audiogram of children with otitis media with effusion .Pure tone frequency comparisons and pure tone average comparisons were analyzed at both group and individual levels. The pure tone frequency comparisons that were investigated were 125 Hz and 250 Hz; 250 Hz and 500 Hz, 125 Hz and 500 Hz, and 4 kHz and 8 kHz. The pure tone average comparisons examined were the overall PTA average (125 Hz– 8 kHz) with the low frequency 3 PTA average ; the overall PTA average with the high frequency 3 PTA average ; the overall PTA average with the 4 frequency PTA average ; the 4 frequency PTA average with the low frequency 3 PTA average; the 4 frequency PTA average with the high frequency 3 PTA average; and the low frequency 3 PTA average with the high frequency 3 PTA average. Group level analysis took the average of all the OME ears in the data set and individual level analysis examined the various comparisons in each participant ear. The comparisons were selected (a) in view of their saliency to typical OME audiometric configurations , hence tFor group level comparisons, paired t-tests were performed to investigate statistically significant differences. An alpha level of p < 0.01 was used to determine statistical significance. Clinically significant differences were also determined for the mean differences. In usual clinical practice, threshold measurements are determined in 5 dB steps . When thMore than one estimation of agreement is recommended as no one measure is definitive . TherefoAt the individual analysis level, Bland-Altman analysis was performed to indicate absolute threshold differences among individuals in each comparison . The perNinety-six children  were diagnosed with otitis media with effusion (OME). Seventy-two children had bilateral OME and 24 had unilateral OME. A total of 168 ears were diagnosed with otitis media with effusion.A paired t-test for bilateral OME ears and an independent t-test for unilateral OME ears was performed to check for statistically and clinically significant differences between right and left ears in each frequency with a 99% confidence interval. A rigorous confidence interval was chosen in view of the large sample sizes and as only clinically minor differences between ears were observed in the data set. Only 250 Hz in the bilateral OME condition showed a statistically significant difference between right and left ears  and no frequencies showed clinically significant differences between the ears, i.e., all had < 10 dB HL differences. No frequencies in the unilateral OME comparison showed clinically or statistically significant differences. Therefore, right and left ears were combined for the following analyses. The Shapiro-Wilk test in addition to means, standard deviations, skewness and kurtosis values were used to check for the normality of all threshold data sets. All threshold frequencies and PTA averages were considered to be normally distributed and parametric tests were therefore used in all analyses.Averaging across all ears, thresholds of 28 dB HL at 125 Hz, 29 dB HL at 250 Hz, 27 dB HL at 500 Hz, 27 dB HL at 4 kHz and 39 dB HL at 8 kHz were found. Paired t-tests were performed between 125 Hz and 250 Hz; 250 Hz and 500 Hz; 125 Hz and 500 Hz; and 4 kHz and 8 kHz. The mean differences between the frequency comparisons are shown in Correlation tests consistently showed a strong positive correlation in all conditions. The coefficients of Pearson’s r, Cronbach’s alpha and intraclass correlation for the different frequency conditions are shown in Absolute differences for the thresholds of the frequencies of interest among all the ears with OME were calculated with Bland-Altman analyses. The percentage of ears outside of the limit of agreement (LOA) range for clinically significant differences of ≥ 10 dB and the percentage of ears outside of the statistical 95% LOA range for the frequencies of interest are shown in The various PTA averages for all ears were determined. These were found to be 29 dB HL in the overall PTA average, 27 dB HL in the low frequency 3 PTA average, 27 dB HL in the high frequency 3 PTA average, and 27 dB HL in the 4 frequency PTA average. Mean differences between averages and paired t-tests results for all PTA averaging combinations, including overall PTA average (125 Hz– 8 kHz), low frequency 3 PTA average , high frequency 3 PTA average , and 4 frequency PTA average  are shown in Correlations among the combinations of PTA averages were analyzed using Pearson’s r, Cronbach’s alpha and intraclass correlation procedures. All correlation coefficients were > 0.9, indicating very strong positive correlations for all examined combinations of averages .Absolute differences in each ear were derived using Bland-Altman analyses. The percentage of ears outside of the LOA range for clinically significant differences of ≥ 10 dB and the percentage of ears outside of the statistical 95% LOA range of the PTA average comparisons are shown in Frequency correlations between 125 Hz and 250 Hz, 250 Hz and 500 Hz, 125 Hz and 500 Hz, and 4 kHz and 8 kHz, were investigated to examine whether a full range of frequencies, 125 Hz to 8 kHz, is necessary to evaluate hearing loss in children with otitis media with effusion (OME). As shown in For the comparison between 125 Hz and 250 Hz, less than 10% of the OME ears showed absolute differences beyond the LOA range with the clinically significant criterion of ≥ 10 dB. Twenty percent and 28% of the OME ears were outside of the LOA range for clinically significant differences of ≥ 10 dB for the comparison between 250 Hz and 500 Hz, and 125 Hz and 500 Hz respectively. This suggests hearing sensitivity at 125 Hz, 250 Hz, and 500 Hz in children with OME differs considerably within individuals and that, clinically, any hearing threshold at 125 Hz, 250 Hz and 500 Hz cannot be replaced by another of the three thresholds. When comparing 4 kHz and 8 kHz at a clinical significant criterion of ≥ 10 dB, 63%—more than half the OME ears—were outside of the LOA range. This indicated that many OME ears showed large differences across neighboring frequencies. Despite high statistical correlation between 4 kHz and 8 kHz, a clinically significant difference was shown in the group level analysis, and intrasubject differences were up to 50 dB between 4 kHz and 8 kHz. Overall, this analysis suggests that at both the group and individual levels, 8 kHz cannot be replaced by 4 kHz when testing hearing sensitivity in individual children with OME.Results revealed that hearing sensitivity at 4 kHz and 8 kHz showed the largest difference when compared with other frequency comparisons. Due to dysfunction of the Eustachian tube in OME children, stiffness in the middle ear increases. As stiffness affects the transmission of low frequency sound the most, OME at an early stage shows low frequency hearing loss. However, in later stages of OME, fluid accumulates in the middle ear, increasing the mass in the middle ear. Since high frequency sound transmission is mostly affected by mass, high frequency hearing loss then occurs .A full range of PTA frequencies (125 Hz to 8 kHz), as shown from the present study, is necessary to precisely evaluate hearing sensitivity in children with OME in clinical settings. On the other hand, for research purposes, as the comparison between 125 Hz and 250 Hz showed no statistically significant differences, high correlations between the thresholds were found, and less than 10% of the OME ears were outside the statistical 95% LOA range for the 125 Hz and 250 Hz comparison, researchers can assume that 125 Hz thresholds would be statistically equivalent to 250 Hz thresholds at the group level.The comparison between the overall PTA average with the other PTA averages  found statistically significant differences. However, comparison between low frequency 3 PTA average, high frequency 3 PTA average and 4 frequency PTA average did not show statistically significant differences. Furthermore, for all PTA average combinations no clinically significant differences were found in the various comparisons in the group level analysis.Under the group level, by averaging across all OME ears there were statistically significant differences in the comparisons of the different PTA averages. However, effect sizes for the differences were relatively small and no clinically significant differences were shown. The comparison between the 4 frequency PTA average and the 3 frequency PTA averages (low frequency 3 PTA average and high frequency 3 PTA average), and between the low frequency 3 PTA average and the high frequency 3 PTA average showed no statistically or clinically significant differences. All combinations of PTA averages showed very strong positive correlations. At the individual level of analysis, less than 10% of the OME ears in all PTA average comparisons were outside of the LOA range of a clinically significant difference of ≥ 10 dB. This indicated that only a very small number of OME ears showed substantial individual differences for different PTA average comparisons.The above results indicate that using different combinations of test frequencies in calculating PTA average, as used in different clinical guidelines, is not likely to affect the criteria for medical/surgical treatment of children with OME. However, when evaluating the need for surgery in children with OME, the frequencies tested may be restricted to 3 frequency PTA averages, either an average of 500 Hz, 1 kHz, 2 kHz or an average of 1 kHz, 2 kHz, 4 kHz, if a reduced audiological assessment time is desired.In addition, there were high correlations and no statistically significant differences between the 4 frequency PTA average , low frequency 3 PTA average  and high frequency 3 PTA average  comparisons, and less than 10% of the OME ears were outside the 95% statistical LOA range for those PTA average comparisons in an individual level analysis. Hence, for both research and clinical purposes these averages can be validly equated. It should be noted that there are no other known studies to compare the current results to and hence replication studies that focus on similar audiometric data would be of value.In the present study, all participants were recruited from the same hospital in Shenzhen and the children were all Chinese, and aged 4 to 12 years old. The peak incidence periods for OME may occur at 2 and 5 years of age . Thus, tThe present study, to the best of our knowledge, is the first to investigate the relationships between PTA thresholds, and the relationships between different combinations of PTA frequencies used to guide surgical intervention and clinical decision-making in children with OME.Results show that clinically, individuals may exhibit large variability in their hearing thresholds, especially in the high frequencies , with clinically significant differences found. Therefore, testing hearing sensitivity from 125 Hz to 8 kHz is necessary to fully evaluate hearing sensitivity in individual children with OME in clinical settings. For research purposes, 250 Hz can adequately represent hearing thresholds at 125 Hz.When evaluating the need for surgery on children with OME, hearing thresholds to be tested can be minimized to any standard 3 frequency PTA average, either an average of 500 Hz, 1 kHz, 2 kHz or an average of 1 kHz, 2 kHz, 4 kHz. In addition, as the low frequency 3 PTA average , high frequency 3 PTA average  and 4 frequency PTA average  did not show statistical significant differences and less than 10% of OME ears were outside of the statistical 95% limit of agreement range, for research purposes these PTA average comparisons may be used interchangeably.S1 FileSTROBE_checklist_v4_combined_PlosMedicine Chow et al. MS.doc.(DOC)Click here for additional data file.


COQ2. COQ2 gene mutations not only cause primary coenzyme Q10 deficiency but also cause SRNS without extrarenal manifestations. The concept of COQ2 nephropathy has been proposed for a long time. Mutations in the COQ2 gene have rarely been reported. Worldwide, only 5 cases involving 4 families have been reported.Treatment of steroid-resistant nephrotic syndrome (SRNS) remains a challenge for paediatricians. SRNS accounts for 10~20% of childhood cases of nephrotic syndrome (NS). Individuals with SRNS overwhelmingly progress to chronic kidney disease (CKD) and end-stage kidney disease (ESRD). Genetic research is of great significance for diagnosis and treatment. More than 39 recessive or dominant genes have been found to cause human SRNS, including COQ2 defect with no additional systemic symptoms. The patient was identified as a homozygote for the c.832 T > C (p. Cys278Arg) missense mutation and a single base homozygous mutation in ARSB gene in c.1213 + 1G > A. The father and mother were heterozygous mutation carriers in both COQ2 and ARSB, and her healthy sister was only a heterozygous mutation carrier in COQ2. In this case, hormone therapy was ineffective, and progressive deterioration of renal function occurred within 1 week after onset, leading to acute renal failure and eventual death.We present the case of a 6-month-old girl with steroid-resistant glomerulopathy due to a COQ2 and ARSB dual mutant. Kidney diseases caused by COQ2 gene mutations can manifest as SRNS, with poor prognosis. The C. 832 T > c (p.csc 278arg) is a new mutation site. Genetic assessment for children with steroid-resistant nephrotic syndrome, especially in infancy, is very important. Families with a clear family history should receive genetic counselling and prenatal examinations, and children without a family phenotype should also receive genetic screening as early as possible.We reported a consanguinity married family which had  COQ2 gene can lead to coenzyme Q10 (COQ10) deficiency, a type of mitochondrial encephalomyopathy and kidney disease, an inherited mitochondrial disease that mainly affects the kidneys. The severity of kidney disease varies and is not necessarily associated with neurological symptoms. Steroid-resistant nephrotic syndrome (SRNS) accounts for 10~20% of childhood cases of nephrotic syndrome (NS) [COQ2 mutations. Patients with recessively inherited mutations manifest with SRNS in childhood [COQ2 was detected [ARSB gene. To date, 340 mutations have been described, most of which are single base homozygous mutations.Mutations in the hildhood . Overalldetected . At presCOQ2 gene has a homozygous mutation for c.832 T > C (p.cys278arg), and both her parents and sister were heterozygous  mutation. Both parents were carriers of a heterozygous mutation in the ARSB gene, which her sister did not have , hypoalbuminemia , hypercholesterolemia , and lactic acidosis (4.32 mmol/L). The child’s parents are cousins and sisters, both of whom are Han, and they are in good health. Their first child was a girl who was diagnosed with mucopolysaccharides at the age of 6 and died at 11 years old. The second child was a 12-year-old healthy girl. The third girl was diagnosed with nephrotic syndrome at 8 months old; treatment was halted, and the child died. The fourth child was aborted at 5 months. The fifth is the child in this report. The mother had no pregnancy complications and was not on any special medication. The child was treated with methylprednisolone. There was no remission trend in proteinuria, showing hormone resistance. The patient rapidly developed acute renal failure within 1 week and underwent peritoneal dialysis. Renal pathological and gene examinations were performed over time. Light microscopy images showed 48 glomeruli, 4/48 as immature glomeruli, 17/48 of glomerular mesangial cells and matrix hyperplasia with different levels of insert appearance , 15/48 of the glomerular balloon expansion, the expansion of multiple focal proximal convoluted tubules, sliced renal tubular epithelial lesions and interstitial oedema, and the small arteries did not show obvious pathological changes. Immunohistochemical testing of 4 glomeruli results include IgG(−), IgA(−), IgM(−), C3(−), C1q(−), and Fn(−)  is located at 4q21.22-q21.23 and has 17 exons. As early as 1975 and 1990, scholars observed that mutations in the gene encoding mitochondrial coenzyme Q10 synthase could lead to a decrease in COQ10 (MIM607426) content and affect the electron transport of the respiratory chain. In 1992, Matthew N. Ashby, an American scholar, showed that COQ2 could encode polypentene hydroxybenzoate transferase, which is a very important enzyme in the synthesis of COQ10. It can clear oxygen free radicals and reduce oxidative stress reactions. COQ10 deficiency is an autosomal recessive genetic disease that is related to four main clinical phenotypes, including prominent nephropathy and encephalopathy, which are typical infant multi-system diseases. Scholars reported a 33-month-old boy with infant brain myopathy, kidney disease and COQ10 deficiency, and a homozygous missense mutation was found in COQ2 genes after genetic testing. For the first time, COQ2 gene coding mutations can lead to primary COQ10 deficiency. Recent studies have found that mitochondrial nephropathy can occur independently without the manifestations of mitochondrial encephalomyopathy, and hormone therapy and immunosuppressive therapy are ineffective. In 2007, scholars reported two cases of patients with early onset of glomerular lesions, and both cases have found COQ2 gene mutations; the study puts forward the concept of “COQ2 kidney disease”. It was described as a new genetic mitochondrial disease with primary kidney involvement and is characterized by variable renal lesions and the proliferation of mitochondria with an aberration in glomerular cells, and the clinical manifestations may be polymorphic. Additionally, neuromuscular symptoms may complicate the course of disease. The most severe forms are devastating multi-system disorders, including glomerular dysfunction and lactic acidaemia [COQ2 gene, can cause ultrastructural abnormalities of the hiatus membrane channel, hole membrane channel collapse, mitochondrial malformation increase, elevated levels of autophagy and mitosis in renal cells, increased reactive oxygen species, and increased susceptibility to oxidative stress [COQ2 mutation. There were no clinical manifestations outside the kidney, and the condition eventually developed into renal failure [OMIM60982 is locatCOQ2 gene usually lead to early onset SRNS. The parents of the four cases were in consanguineous marriages. Two patients developed acute renal failure (ARF), received peritoneal dialysis, and died at 6 months. If COQ10 deficiency has been identified clinically, but pathogenicity variation is unknown, measurement of COQ10 level in skin fibroblasts can be considered to assess whether it is high risk [Of the six cases reported in the current literature , 9 Tabl, 4 casesigh risk . Di GiovARSB gene is related to mucopolysaccharidosis type VI (MIM no. 253200). ARSB is located on chromosome 5q14.1 and has ubiquitous expression in the kidney, but no renal disease has been reported. It clinically presents multiple systemic symptoms and chronic progression, mainly involving the bone and cardiopulmonary system, cornea, skin, liver, spleen, brain and nerve. Severe MPS VI usually begins before the age of 3 years [. Naglazyme  was approved as an enzyme replacement therapy (ERT) for MPS VI in May 2005 and was considered a safe and effective treatment. With the advent of ERT, early application can benefit patients more, and early recognition and accurate diagnosis of this rare lysosomal storage disease are mandatory for a better and successful outcome [The  3 years . Naglazy outcome . SupportThere is no literature on COQ2 combined with ARSB gene mutations. In this case, the parents of the child were carriers of two pathogenic genes, COQ2 and ARSB, and both diseases were autosomal recessive inheritance. The c.832 T > C(p.cys278Arg) of COQ2 is a newly discovered locus. The mutation type was a single base homozygous mutation (Hom), and this gene locus has not been publicly reported. The child developed renal failure and eventually died, suggesting that the poor prognosis was related to the carrying of homozygous mutant genes. The parents of this child had given birth to a girl with a definite diagnosis of MPS, so we can speculate that the ARSB gene carried by their parents is a pathogenic gene, and there is no related literature about the pathogenicity of this locus for the time being. In this case, there is no MPS VI phenotype, so it is not possible to determine whether kidney damage is related to this gene. At present, there is no literature on renal damage and pathological changes of MPS; thus, it is not clear whether the pathological changes of this case are caused by MPS. In addition, the renal pathology of this case did not find the mitochondrial proliferation manifestations reported in other literature on COQ2 gene mutation. However, it was only 10 d from the onset of this case to the renal pathological examination; the renal pathology likely had not changed due to the short period since onset. At the time of renal pathological examination, the child was in the stage of acute renal failure and needed renal replacement therapy. The pathological results showed that the renal tubular epithelial and interstitial oedema was relatively serious, which may be one of the reasons for the rapid deterioration of renal function.COQ2 is a newly discovered locus. We believe that if the onset age is early, homozygous mutation and/or neuropathy are the risk factors for the severe cases resulting from the COQ2 mutation. Consanguineous marriage is the cause of homozygous mutation. Genetic counselling should be carried out for whole family members, either prenatally or postnatally. Families with autosomal recessive diseases should avoid consanguineous marriages and inform their offspring of the risk of illness. Gene detection should be carried out as early as possible for foetal and normal phenotypes.The c.832 T > C(p.cys278Arg) mutation in


The morbidity and socioeconomic effects of onchocerciasis, a parasitic disease that is primarily endemic in sub-Saharan Africa, have motivated large morbidity and transmission control programmes. Annual community-directed ivermectin treatment has substantially reduced prevalence. Elimination requires intensified efforts, including more efficacious treatments. We compared parasitological efficacy and safety of moxidectin and ivermectin.Onchocerca volvulus microfilariae per mg skin who were not co-infected with Loa loa or lymphatic filariasis microfilaraemic. Participants were randomly allocated, stratified by sex and level of infection, to receive a single oral dose of 8 mg moxidectin or 150 μg/kg ivermectin as overencapsulated oral tablets. The primary efficacy outcome was skin microfilariae density 12 months post treatment. We used a mixed-effects model to test the hypothesis that the primary efficacy outcome in the moxidectin group was 50% or less than that in the ivermectin group. The primary efficacy analysis population were all participants who received the study drug and completed 12-month follow-up (modified intention to treat). This study is registered with ClinicalTrials.gov, number NCT00790998.This double-blind, parallel group, superiority trial was done in four sites in Ghana, Liberia, and the Democratic Republic of the Congo. We enrolled participants (aged ≥12 years) with at least 10 Between April 22, 2009, and Jan 23, 2011, we enrolled and allocated 998 participants to moxidectin and 501 participants to ivermectin. 978 received moxidectin and 494 ivermectin, of which 947 and 480 were included in primary efficacy outcome analyses. At 12 months, skin microfilarial density (microfilariae per mg of skin) was lower in the moxidectin group (adjusted geometric mean 0·6 [95% CI 0·3–1·0]) than in the ivermectin group . Mazzotti  reactions occurred in 967 (99%) of 978 moxidectin-treated participants and in 478 (97%) of 494 ivermectin-treated participants, including ocular reactions (moxidectin 113 [12%] participants and ivermectin 47 [10%] participants), laboratory reactions (788 [81%] and 415 [84%]), and clinical reactions (944 [97%] and 446 [90%]). No serious adverse events were considered to be related to treatment.Skin microfilarial loads  are lower after moxidectin treatment than after ivermectin treatment. Moxidectin would therefore be expected to reduce parasite transmission between treatment rounds more than ivermectin could, thus accelerating progress towards elimination.UNICEF/UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases. Onchocerca volvulus, affects some of the world's most disadvantaged communities, 99% of which are in remote, rural areas in sub-Saharan Africa. Infective larvae, transmitted by the bite of Simulium spp, develop into macrofilariae that reside in subcutaneous and deep tissue nodules, and produce millions of microfilariae during their reproductive lifespan (9–11 years). Microfilariae live for 1–2 years primarily in the skin, from where they are taken up by the vectors, thus fuelling transmission. The host inflammatory reactions to dead microfilariae cause the dermatological, lymphatic, and ocular symptoms of onchocerciasis, including blindness due to microfilariae invading the eyes. The morbidity and socioeconomic effects of onchocerciasis have motivated large control programmes based now on community-directed treatment with ivermectin (CDTI) donated by Merck & Co .2Onchocerciasis, which is an infectious disease caused by the helminth fespan 9–1 years. Evidence before this studyOnchocerca volvulus in Ghana (now complete). No adverse events had occurred that would preclude administration in a larger study.Evaluation of moxidectin for onchocerciasis control and elimination started with non-clinical pharmacology studies funded by TDR. At that time, elimination of onchocerciasis through mass administration of ivermectin was considered feasible in the small American foci, but not across Africa, which is where 99% of the at-risk population live. After external review of the non-clinical pharmacology and safety data, clinical evaluation of moxidectin began. The protocol for this study was based on one published and one unpublished healthy volunteer pharmacokinetic study and blinded safety data from the ongoing phase 2 study in people infected with Added value of this studyThis study confirmed the conclusions from the phase 2 study and five studies in healthy volunteers. Skin microfilarial densities were significantly lower after moxidectin than after ivermectin; the percentage of participants with undetectable microfilariae in skin was significantly higher after moxidectin than after ivermectin treatment to 18 months post treatment ; and the safety profiles suggest moxidectin is suitable for mass treatment. Responses to ivermectin classified in areas with long-term ivermectin mass treatment as ‘suboptimal’ occurred in each of the four study areas without previous community-directed ivermectin treatment.Implications of all the available evidenceO volvulus to ivermectin's embryostatic effect in areas of long-term use. The suboptimal responses to ivermectin we observed show that data on changes in the frequency of such responses with duration of community-directed treatment with ivermectin are needed for conclusions about reduced O volvulus susceptibility or emerging resistance to ivermectin. Our data suggest that moxidectin could accelerate progress towards the elimination of onchocerciasis in Africa, including in areas with suboptimal responses to ivermectin.Modelling and field studies done since the start of this study suggest that annual mass administration of ivermectin could eliminate onchocerciasis in many African foci, while other areas need alternative strategies, including more efficacious drugs. Furthermore, there are concerns about diminishing susceptibility of In 11 of 13 foci in South and Central America (population about 0·56 million), 17–25 twice yearly mass administrations of ivermectin  have or are likely to have eliminated onchocerciasis.Loa loa microfilaraemia, and so-called suboptimal response to ivermectin. The WHO African Programme for Onchocerciasis Control (1995–2015) emphasised the need for alternative treatment strategies, including more efficacious drugs.7Questions remain about whether annual CDTI can eliminate onchocerciasis in areas with particularly high barriers to elimination, such as very high endemicity, loiasis co-endemicity that limits CDTI because of severe reactions in people with very high O volvulus, moxidectin reduced and maintained low skin microfilarial density  in more participants, faster, to lower levels, and for much longer than did ivermectin. Our phase 3 study was designed to determine whether, 1 year after one moxidectin dose, SmfD was 50% or less than the SmfD was after one dose of ivermectin , and to collect additional safety data.Moxidectin is a milbemycin macrocyclic lactone that is not registered for human use. Like ivermectin (an avermectin macrocyclic lactone), moxidectin is widely used by veterinarians.Between April, 2009, and May, 2012, we did a randomised, double-blind, single oral dose, ivermectin-controlled study of moxidectin for superiority in onchocerciasis endemic areas in Ghana (Nkwanta district), Liberia (Lofa county), and the Democratic Republic of the Congo (Nord Ituri and Nord Kivu) without loiasis or previous CDTI.We enrolled male and female volunteers aged 12 years or older, weighing 30 kg or more, who had 10 or more microfilariae per mg of skin. Participants with loiasis or lymphatic filariasis with an intensity of infection greater than 100 microfilariae per mL were to be excluded. Full eligibility criteria are shown in the Volunteers gave consent or assent  through signature or thumbprint in the presence of a literate witness. SmfD was measured in the villages or nearby health clinics by study staff. Volunteers who qualified for further screening were brought to one of the research centres  for 3 days of screening. Those who were eligible stayed another 5 days for treatment and initial follow-up, and were brought to the centre for 1–2 days for each follow-up visit.This study was approved by the Ghana Food and Drugs Authority; Ghana Health Service Ethics Review Committee; Liberia Ministry of Health and Social Welfare; Ethics Committee of the Liberia Institute for Biomedical Research; Ministère de la Santé Publique of the Democratic Republic of the Congo; Ethics Committee of the Ecole de la Santé Publique Université de Kinshasa Democratic Republic of the Congo; and the WHO Ethics Review Committee. Study compliance with International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use Good Clinical Practice guidelines was monitored.vs ≥20 microfilariae per mg skin). Wyeth Research  manufactured and provided 2 mg moxidectin tablets, purchased 3 mg ivermectin tablets from Merck & Co, overencapsulated both tablet types, and provided matching placebo. For each participant, the pharmacists prepared a sealed envelope, labelled with participant identifiers, that contained either four moxidectin-containing capsules or two, three, or four ivermectin-containing capsules plus two, one, or zero placebo capsules (as required by participant weight).At each centre, a pharmacist randomised participants using sponsor-provided computer-generated randomisation lists (block size 6) in a 2:1 ratio to 8 mg moxidectin or 150 μg/kg ivermectin, stratified by sex and level of infection  at screening and at months 1, 6, 12, and 18 using a Holth corneoscleral punch. We weighed each snip before incubation for 8 h or more in isotonic saline. We then counted the microfilariae using a microscope and calculated SmfD as the arithmetic mean of microfilariae per mg skin across all snips.An ophthalmologist did ocular examinations  before treatment, on day 3 or 4 after treatment, and at months 1, 6, 12, and 18.Loa loa infection in the sites in the Democratic Republic of the Congo and requirements for Loa loa testing in Liberia where potential participants might have lived in a loiasis endemic area during the preceding civil conflicts are provided in the We did physical examinations and vital sign measurements before treatment and at each follow up visit, and did serum biochemical, haematological, and urine analyses before treatment, on day 5 post treatment, and at months 0·5, 1, 3, and 6. We did an immunochromatographic card test for lymphatic filariasis during screening and tested for microfilaraemia in those who were positive. Since none of the participants were microfilaraemic, post-treatment tests were not conducted. We did one single-sample Kato-Katz test for intestinal helminths before treatment and, in those who were positive, at month 1. Details on testing for The primary efficacy endpoint was SmfD at 12 months. Secondary efficacy measures were SmfD at months 1, 6, and 18, and the number of live microfilariae in anterior chambers at months 1, 6, 12, and 18 in participants with more than 10 live microfilariae in the anterior chambers across both eyes before treatment. Infection with intestinal helminths at month 1 was an exploratory outcome.O volvulus infection and as adverse events after treatment with microfilaricidal drugs because of accelerated microfilariae death .16We assessed safety in terms of incidence of adverse events, including post-treatment changes in vital signs, symptoms, or laboratory values found through examinations, questions to participants, or spontaneous reporting. International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use criteriaOCRC criteria for Mazzotti reactions differ substantially from National Cancer Institute criteria for grading similar events; OCRC criteria generally reflect much less severe symptoms grade for grade. National Cancer Institute criteria include need for medical intervention for grade 2 sometimes, grade 3 frequently, and grade 4 nearly always, whereas most OCRC criteria grade 4 Mazzotti reactions require no intervention. For symptoms not included in OCRC criteria, we used National Cancer Institute criteria (version 2.0) or provided our own method-specific criteria . Adverse18t-test (α=0·05 and 90% power with loge [SmfD + 1] transformation), assuming SmfD post moxidectin is 0·5 SmfD post ivermectin, and 2:1 moxidectin:ivermectin randomisation . Therefore, we used post-ivermectin SmfD at month 12 from two previous studies  to calculate sample sizes to detect a difference of 50% or more in SmfD at month 12 with a two-sided Student's misation . Even ade [SmfD + 1]) SmfD before analysis. In all mixed-effects models, the site was the random effect. The percentage treatment difference was calculated as the difference in adjusted geometric means (GM) as a percentage of post-ivermectin adjusted GM.We log transformed  and, following peer review, a fixed-effects model and a linear mixed-effects model of percentage change from baseline .For the longitudinal analysis of SmfD at months 1, 6, 12, and 18, we used a mixed-effects model for repeated measures with participant as the statistical unit, baseline SmfD, treatment, sex, level of infection, time, treatment × sex, treatment × level of infection, and treatment × time interaction as fixed effects, and time as a repeated effect for the four SmfD measurements. We compared the number of participants with undetectable SmfD by use of a mixed-effects logistic model with treatment, sex, and level of infection as fixed effects. We calculated adjusted odds ratios. We used a mixed-effects model to compare the percentage reduction from baseline of live microfilariae in the anterior chambers of the eyes at month 12 with sex, level of infection, treatment, treatment × sex, and treatment × level of infection as fixed effects.2 test. We compared by system organ classes and preferred terms or by Mazzotti reactions cluster, group, and sign or symptom, maximum grade, and seriousness across all ages and by age group (adolescents [12–17 years] vs adults [≥18 years]). Since the frequency and severity of Mazzotti reactions can depend on pretreatment SmfD,We used the Medical Dictionary for Regulatory Agencies (version 13.1) to code unrelated adverse events and adverse drug reactions. We used a specific dictionary  to code The primary population analysed was the modified intention-to-treat population . Descriptive statistics included only those with applicable data. Analysis of reductions in live microfilariae in anterior chambers included only those with more than 10 live microfilariae in the anterior chambers across both eyes before treatment who had both eyes evaluated at month 12 .ClinicalTrials.gov, number NCT00790998.We used SAS version 9.3  for statistical analyses. A data monitoring committee reviewed safety data during recruitment, including serious adverse events. This study is registered with The UNICEF/UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR) funded this study. TDR staff participated in study design, data management, analysis plan, interpretation, and manuscript writing. The corresponding author had full access to all data and made the final decision to submit for publication.Because TDR was the sole sponsor of this study from July, 2011, resource limitations delayed availability of all results and dissemination at meetings to 2013–14. In 2014, the non-profit organisation Medicines Development for Global Health assumed sponsorship. To ensure publication and regulatory submissions using the same database, publication preparation was delayed until after Medicines Development for Global Health had completed its blinded database review in 2016. The manuscript was finalised without Medicines Development for Global Health involvement.O volvulus are shown in We recruited participants between April 22, 2009, and Jan 23, 2011. Of 4526 people screened, 1472 (33%) were treated . ParticiMonth 12 SmfD was lower in the moxidectin group (adjusted GM 0·6 microfilariae per mg [95% CI 0·3–1·0]) than in the ivermectin group . The difference in SmfD at month 12 was independent of sex , but differed between pretreatment level of infection , month 6 (by 97%), and month 18 (by 76%) after treatment with moxidectin than with ivermectin .Figure 2The proportion of participants with undetectable SmfD was higher in the moxidectin group than in the ivermectin group  of 131; at month 18, this value was 113 (98%) of 115. In the ivermectin group, 62 (84%) of 74 participants did not have detectable microfilariae at month 12; this value was 55 (90%) of 61 at month 18. The percentage reduction in live microfilariae in the anterior chambers from pretreatment to month 12 did not differ between the two treatments p=0·13; .Both drugs were well tolerated. vs seven [1%] of 494). Severe symptomatic postural hypotension is diagnosed when a person, after 5 or more min in the supine position, is unable to stand up and be still for 2 min because of dizziness or weakness linked to a decrease in blood pressure. Such hypotension does not require intervention and resolves quickly after the individual lies down. People in the moxidectin group had severe symptomatic postural hypotension from treatment day to 2 days post treatment, compared with from 1–3 days post treatment in those treated with ivermectin.The profiles of Mazzotti reactions after both treatments were similar when analysed by severity across all pretreatment SmfD  and by pThe most frequent ocular Mazzotti reactions were pruritus , conjunctivitis , and eye pain . Fewer than 1% of participants had eyelid swelling, ocular discomfort, tearing or watery eyes, blurred vision, and photophobia. Ocular Mazzotti reactions exceeding grade 1 occurred in 1% or fewer participants in both treatment groups, the most frequent being eye pruritus (1%).O volvulusMoxidectin was well tolerated without substantial adverse drug reactions, confirming data from the previous study in people infected with The significantly lower SmfD  from month 1 to month 18 after moxidectin than after ivermectin—with 86% treatment difference at month 12—confirms our efficacy-related study hypothesis and is consistent with previous data.Loa loa microfilaraemia that puts them at risk of serious adverse events from microfilaricidal drugs.Moxidectin would be particularly useful to accelerate the elimination of onchocerciasis in situations where there are operational barriers to mass drug administration , where precontrol endemicity was so high that infection prevalence is still high despite long-term CDTI, and where transmission seasons are long or have two peaks.O volvulus susceptibility to ivermectin.Persistent onchocerciasis prevalence and suboptimal responses to ivermectin's embryostatic effect after long-term CDTI in Ghana and Cameroon have raised concerns about decreasing O volvulus susceptibility to ivermectin over time.In each of the four CDTI-naive study areas, some participants met the criteria for suboptimal response to ivermectin's embryostatic effect or for suboptimal response to ivermectin's microfilaricidal effect.The biological mechanism(s) through which moxidectin leads to lower long-term SmfD than does ivermectin are unknown.This study and the phase 2 studyOur study did not include a paediatric population. A pharmacokinetic study to determine a safe dose in children is required.For countries to consider the use of moxidectin for the control of onchocerciasis, moxidectin needs to be registered and manufactured. In 2014, WHO licensed all moxidectin-related data at its disposal to Medicines Development for Global Health. The US Food and Drug Administration assigned the new drug application submitted by Medicines Development for Global Health to priority review. Medicines Development for Global Health is preparing a paediatric pharmacokinetic study and a large multi-dose comparative study29


The general chemical composition of the untreated and treated bark including determination of extractives, suberin, lignin and monosaccharides was also studied. Composition of the residual solid fraction and relative proportions of different components, as a function of the processing conditions, could be established. Organosolv treatment produces a suberin-rich solid fraction, while during hydrothermal and steam explosion treatment cleavage of polysaccharide bonds occurs. This work will provide a deeper fundamental knowledge of the bark chemical composition, thus increasing the utilization efficiency of birch outer bark and may create possibilities to up-scale the fractionation processes.A comparative study among different pretreatment methods used for the fractionation of the birch outer bark components, including steam explosion, hydrothermal and organosolv treatments based on the use of ethanol/water media, is reported. The residual solid fractions have been characterized by ATR-FTIR,  Efficient bioconversion of lignocellulosic materials to ethanol and value-added biochemicals represents a challenging proposition from both energy and environmental viewpoints. Apart from polysaccharides, multiple biomass feedstocks, including cork and outer bark residues, are rich in natural waxes, such as triterpenes, and insoluble polyesters (suberin) that serve as structural components of the outer barriers of plants. In order to improve the utilization of these feedstocks, an in depth delineation of the extraction and biodegradation of these components is needed. Moreover, degradation products, some of which are found almost exclusively in the suberized plant cell walls, may be of considerable value as sources of oleochemicals ,2,3.Betula pendula) is widely distributed in the northern hemisphere, particularly in boreal climates, and is of great commercial significance as it constitutes the dominant hardwood tree species used for pulp production in Northern European countries. The total production of market pulp in Sweden amounts to approximately 3.8 million tons annually, according to The Swedish Forest Industries Fact and Figures, leading to the production of considerable amounts of birch bark as a residual product from log debarking, usually burned for energy production. According to the biorefinery concept, using biomass directly as solid fuel is a waste of valuable material. Instead, the natural and renewable resources should be used to prepare value-added products such as chemicals and materials [Silver birch  layers with an elongated polygonal shape a,b. AfteAnother remarkable feature is the presence of globular structures associated to lignin condensation that were observed on the surface of samples treated with 90:10 and 50:50 ratios of EtOH/water a,b. Whenw/w) and extractives (40% w/w), with a low content of polysaccharides (4.5% w/w) and lignin (9% w/w). These values are similar to those reported in the literature for birch outer bark [Quercus suber L. [w/w) when compared to those from cork (19% w/w). Another possible reason could be that the presence of suberin in the cell walls can limit the diffusion of the liquid mixture [Eucalyptus bark [The compositional analysis of birch outer bark revealed a material mainly composed of the polyester suberin . Release of acetic acid was more extensive when steam explosion and organosolv pretreatment are applied, following the release of hemicellulosic sugars detected in the liquid fraction.The ethanol/water ratio was shown to affect significantly disruption of lignin/hemicellulose ester bonds.The Klason lignin content of the residual solid fractions increased after pretreatment, although a decomposition and removal of lignin aromatic structures can be assumed from FTIR and NMR data. Previous studies report that water-soluble phenolic compounds that are present in bark can condense with lignin during pretreatment and appear as acid-insoluble lignin in the subsequent compositional analysis ,37,39. D−1 were used for monitoring ester bonds and thus suberin presence and structure, while bands at 1513, 1468, 855 and 819 cm−1 were used for lignin aromatic vibrations. Apart from the samples after hydrothermal and treatment with alkali catalyst, there are no detectable changes in the ATR-FTIR spectra indicating suberin removal, while lignin removal is profound in all OS samples. The clear presence of signals from ester groups at 1740 cm−1, associated with the low intensity of signals from acidic groups at 1715 cm−1, indicates the polymeric nature of residual suberin in most of the samples. The latter band is associated with hydrogen bonded carbonyl groups in either esters or acids and reflects the formation of monomers released during the polymerization of suberin molecule. The presence of suberin can be also reflected by the strong intensity of the 1161 cm−1 band from C–O–C asymmetric stretching of high esters. However, one important observation is the reduced intensity of the 1265 cm−1 band from C–O–C symmetric stretching in all pretreated samples, compared to the 1161 cm−1 band, corroborating the idea that cleavage of ester bonds within the polymeric structure of suberin  occured [−1 relative to that at 1161 cm−1 suggests that lignin has been more largely removed in comparison to polysaccharides [Since ATR-FTIR spectra can provide useful information for identifying the presence of certain functional groups or chemical bonds in a molecule or an interaction system, it was applied here to survey the changes in chemical structures before and after extraction by various methods. Bands at 2935–2850, 1740, 1265, 1161, 723 cm occured , while tcharides .2 groups close to the glycerol anchorage points compared to the middle- and end-chain –CH2. Upon 80:20 OS treatment, the opposite effect is observed; 30 ppm carbons resonances from polymethylenic domains are decreased, suggesting that higher concentration of ethanol in the pretreatment liquor leads to middle-and end-chain bonds cleavage. The reduction of 30 ppm carbons in NMR spectra of OS/EtOH samples goes along with the increase of ethanol concentration in the liquid mixture. 13C ssNMR molecular dynamics studies in other suberinic materials, such as cork and potato periderm, have also revealed the presence of two different populations of aliphatic CH2 (methylene) groups, complementing the idea that suberin rigid moieties correspond to the hydrocarbon chains in the more orderly organized parts of the suberin polyester, close to the the glycerol anchorage points, as well as those with more freedom of motion to the hydrocarbon chains in less constrained regions [The NMR spectra of OS/NaOH residual solid show that both main aliphatic bands generally decrease, but the decrease at 33 ppm is more significant than that at 30 ppm, which confirms the preferential removal of these methylenes compared to those at 30 ppm. This is attributed to the presence of alkaline catalyst that cleaves the ester bonds closer to linkage groups, thus removing shorter aliphatic chains, while it may also account for the removal of a higher number of –CH regions ,40. The Scanning electron microscopy was used in order to appreciate the morphological changes after pretreatment. The destruction of the cell layers and phellem configuration in the sample treated with alkaline catalyst is probably related to lignin removal. It is speculated that lignin is mainly located in the middle lamellae, the membrane delimiting neighboring cells; studies has shown that in wood cells at least 50% is concentrated in this compartment ,42. As aBetula pendula was obtained as residual by-product of commercial debarking at Smurfit Kappa pulp mills  with 4.5% of moisture and was ground in a knife mill  using an output sieve of 1 mm × 1 mm. After grounding, dry birch bark samples were soaked by mixing in distilled water. The outer bark fraction, which floated to the top of the water surface, was collected and dried [The analytical protocol applied in this work is illustrated in nd dried ,47. FinaA summary of the processing conditions of each pretreatment is given in o) = Log (t exp(T − Tref)/14.7, where Tref = 100 °C [o) − pH.The treatments were evaluated with the severity correlation, which describes the severity of the pretreatment as a function of treatment time (min) and temperature (°C): Log and using an extinction coefficient of 110 L·g−1·cm−1.Klason and acid-soluble lignin, and carbohydrates contents were determined on the extracted and desuberinized materials after acidolysis. Sulphuric acid  was added to 0.3 g of the material sample, and the mixture was placed in a water bath at 30 °C for1 h after which the sample was diluted to a concentration of 3% H−1 as the mobile phase. Organic acids and other by-products in pretreated materials were determined using an Aminex HPX-87H chromatography column with a Cation-H Bio-Rad micro-guard column at 65 °C (Bio-Rad Laboratories), with a mobile phase of 5 mM sulphuric acid at a flow rate of 0.6 mL·min−1.The polysaccharides were determined after acidolysis of the extractive-free material, as described above. The combined filtrates were further subjected to complete digestion by incubating 4.5 mL of each sample with 0.5 mL HCl 4 M at 70 °C for 15 min followed by neutralization with 0.5 mL NaOH·12N. The monomeric sugars produced were quantified with isocratic ion-exchange chromatography using an Aminex HPX-87P column with a De-Ashing Bio-Rad micro-guard column at 85 °C  using Millipore water at a flow rate of 0.5 mL·minv/v). After 3 min, at room temperature, 2 mL of aqueous Na2CO3 (7.5% m/v)was added, then the mixture was vortexed and further incubated in a thermostat at 45 °C for 15 min and the absorbance of the resulting blue colored mixtures was recorded at 765 nm against a blank containing only water.The total phenolic content in the hydrolysate (liquid phase) of the pretreated materials was determined using Folin–Ciocalteu method. Gallic acid (GA) was used as standard and the total phenolic content was expressed as mg GA equivalent/mL. An aliquot (50 μL) of each extract, or of the gallic acid standard, was mixed with 2 mL of Folin–Ciocalteu reagent .ATR-FTIR spectra of extractive—free untreated and pretreated solid fractions were collected on a Bruker IFS 66v/S FTIR spectrometer  using a single reflection ATR cell . Data were recorded at room temperature, in the range of 4000–400 cm13C-NMR analyses were recorded with a Bruker Ascend Aeon WB 400 spectrometer. Data extractive—free untreated and pretreated solid fractions were obtained at 25 °C using standard Bruker pulse programs. 13C-CP/MAS NMR spectra were recorded at 9.48 T using 12 kHz spinning rate and MAS with proton 90° pulses 2.5 µs. Chemical shifts are given in ppm from glycine. The NMR spectra were processed and analyzed with Topspin software .Samples from extractive—free untreated and pretreated materials were dried prior to use and coated with a thin layer of tungsten using a sputter coater. Electron micrographs were recorded using the extreme high-resolution (XHR) FEI Magellan™ 400 system  operated at 3.0 kV. Micrographs are regarded as representative.Betula pendula silver birch barks after different pretreatment methods. The solid fractions obtained present different characteristics depending on the specific process conditions. During hydrothermal and steam explosion pretreatment, cleavage of the hemicellulosic bonds led to the release of hemicellulosic sugars in the liquid fraction, while suberin was partially depolymerized. Organosolv treatment with ethanol/water solvent was proved to be an efficient method for the fractionation of bark components, resulting in removal of significant extractives, cleavage of lignin/hemicellulose bonds, removal of lignin and retaining of suberin components. This work contributes to a deeper fundamental knowledge of the bark chemical composition and the development of pretreatment strategies for a selective component enrichment to facilitate downstream fractionation processes and bark valorization.The present work studies the chemical composition of the European hardwood


The brainstem plays a significant role in migraine pathogenesis, but a relationship between volume alterations of brainstem subregions and migraine aura characteristics has not been sufficiently investigated. The aim of this study is to compare the volume of the brainstem, and its subregions, between patients with a migraine with aura (MwA) and healthy controls (HC), and also to correlate characteristics of MwA and the volume of the brainstem subregions.Forty-two MwA and 42 HCs, balanced by sex and age, were selected for this study. Total brainstem volume changes as well as volume changes in the pons, medulla, midbrain and the superior cerebellar peduncles were investigated in MwA relative to HCs. In addition, the relationships between brainstem subregions and aura characteristics  were explored in MwA.3 vs. 25,179.32 ± 2019.1 mm3; p = .008), as well as the midbrain and pons . Total brainstem volume, as well as volumes of brainstem subregions, were not significantly correlated to the MwA characteristics.MwA patients had a larger brainstem volume relative to HCs (25,941.35 ± 2559.2 mmThe results of this study reveal that a migraine with aura is associated with a larger volume of the brainstem with a particular involvement of the midbrain and pons. •Migraineurs with aura had larger volume of pons and midbrain relative to controls.•Medulla did not significantly differ in volume size between migraineurs and controls.•Volume of brainstem is not linked to disease duration and intensity of headache pain. Strong The majority of advanced neuroimaging migraine studies investigated cortical changes in migraine patients. There have been only a few studies that analyzed brainstem volume together with characteristics of migraine attacks. Smaller volumes of brainstem subregions were detected in migraine patients with allodynia and lower heat pain thresholds . Also, gThe aim of this study is to compare the volume of the brainstem and its subregions, between patients with MwA and healthy controls (HCs), as well as to correlate characteristics of MwA and the volume of brainstem subregions.22.1This study included 42 MwA patients and the same number of HCs balanced by sex and age. The research protocol of this study was approved by the Institutional Review Boards at the Neurology Clinic and the Special Hospital for Prevention and Treatment of Cerebrovascular Diseases (SHPTCD). Written informed consent was obtained from all subjects prior to study participation. Patients with typical MwA were recruited through the Headache Center at the Neurology Clinic and diagnosed using the International Classification of Headache Disorders criteria (ICHD-3) . MwA pat2.2o, 24 slices with 0.47x1x5 mm2 voxels, slice thickness = 5 mm, acquisition matrix 512 × 512) and a three-dimensional T1 weighted fast spoiled gradient-echo (T1-3D-FSPGR) series . T2W images were only used to exclude the presence of brain lesions.MR examinations of participants were performed using a 1.5 T MR scanner with an eight-channel head coil . The imaging protocol consisted of a T2 weighted spin echo (T2W) in the axial plane (Echo time (TE) = 105.8 ms, repetition time (TR) = 5700 ms, flip angle (FA) = 90The images were transferred to the PC workstation and converted to the NifTI-1 (Neuroimaging Informatics Technology Initiative) format using the dcm2nii software. Only T1W FSPGR volumetric images were included in the analysis.http://freesurfer.net) (3 using FLIRT (FMRIB's Linear Image Registration Tool). Each subject's brain segmentation result was carefully reviewed for errors by a trained analyst (MD). Moreover, the total intracranial volume was computed for each subject using the high-resolution T1W images with the fully automated tool “Structural Image Evaluation, using Normalization, of Atrophy” (SIENAX from FSL 5.0 package) ) was done using the FreeSurfer 6.0 version, a freely available automated image analysis software tool with well-established accuracy and reliability for labeling structural anatomy for post-processing neuroimaging data (fer.net) . The toofer.net) . Voxels package) .2.3t-test or Chi-squared test, as appropriate. Statistical significance levels were set at p < .05.Subject demographics and migraine with aura characteristics were reported using descriptive statistics and compared groups using the Independent samples The volumes of the whole brainstem, as well as brainstem subregions, were exported to the R statistical program. General linear model (GLM) analysis was used to investigate differences in volume of the brainstem and their subregions between the MwA and HCs, controlling for the effect of age, sex and total intracranial volume to avoid spurious results. Although MwA and HCs were balanced by age and sex, these values were used as co-variables because of suggested atypical age-related cortical thinning in MwA relative to HCs . The res3The study included 32 females and 10 males with a migraine with typical aura balanced with the same number of HCs. The main demographic and clinical characteristics of participants included in the study are shown in MwA had significantly larger total brainstem volume and the volumes of the pons and midbrain relative to HCs . The medThere were no significant correlations between volumes of the brainstem, as well its subregions, and the duration of the aura, the frequency of the aura, occurrence of somatosensory and dysphasic auras, duration of a headache, intensity of headache pain, and duration of disease. A negative correlation between the occurrence of dysphasia and volume of SCP  did not survive the Bonferroni-Holm correction.4The results of this study reveal that MwA is associated with volume changes of brainstem regions. In particular, MwA patients have larger volumes of the midbrain and pons relative to HCs. In MwA subjects, these grey matter volume changes were not influenced by the duration or frequency of the aura, occurrence of somatosensory and dysphasic symptoms, duration or intensity of headache pain, and duration of disease.Over the past two decades, a number of brain morphometric studies have explored grey matter changes associated with a migraine with aura focusing mainly on the cerebral cortex . These slocus ceruleus, nuclei in the pons, which can be a mechanism through which migraine triggers cause a migraine  T1 sequence with non-isotropic voxels.In conclusion, the brainstem volumetric investigation of 42 migraine patients with typical aura, done with the help of a special designed routine in the software package (FreeSurfer 6.0 version) for investigations of brainstem subregions, revealed a larger volume of the brainstem, more accurately of the pons and midbrain, in MwA patients relative to HCs. Future investigations exploring pons and midbrain resting and evoked activity may provide new evidence supporting the hypothesis that those structures can play a role in migraine aura pathophysiology.Ministry of Education and Science, Republic of Serbia [project no. III 41005], and JZT received research grant support from the Ministry of Education and Science, Republic of Serbia [project no. 175022].The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: IP and MD received research grant support from the The authors declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: IP, MD and JTZ have no conflict of interest.