Horizontal gene transfer (HGT), an important evolutionary mechanism observed in prokaryotes, is the transmission of genetic material across phylogenetically distant species. In recent years, the availability of complete genomes has facilitated the comprehensive analysis of HGT and highlighted its emerging role in the adaptation and evolution of eukaryotes. Endophytes represent an ecologically favored association, which highlights its beneficial attributes to the environment, in agriculture and in healthcare. The HGT phenomenon in endophytes, which features an important biological mechanism for their evolutionary adaptation within the host plant and simultaneously confers “novel traits” to the associated microbes, is not yet completely understood. With a focus on the emerging implications of HGT events in the evolution of biological species, the present review discusses the occurrence of HGT in endophytes and its socio-economic importance in the current perspective. To our knowledge, this review is the first report that provides a comprehensive insight into the impact of HGT in the adaptation and evolution of endophytes. Horizontal gene transfer (HGT) refers to the transmission of genetic material across the genomes of biological organisms by processes other than fertilization. HGT is a universal phenomenon observed in bacterial, fungal, and eukaryotic genomes ,2. HowevAlternaria and Fusarium fungi [Bombyx mori) [In the past decade, the availability of eukaryotic genomes through high-throughput sequencing has promoted the research on the occurrence and mechanism of HGT in eukaryotes . Additioum fungi , nematodum fungi ,13, humaum fungi , plants yx mori) , and playx mori) . Extensiyx mori) ; howeveryx mori) ,19. Moreyx mori) ,21, acquyx mori) , and evoyx mori) ,24. Triticum durum) and corn (Zea mays) [Endophytes represent the ecologically favored association between plants and microbes, providing multiple advantages for the plant and the environment. Endophytic associations are gaining momentum socio-economically due to their potential applications in agriculture, industries, and healthcare. The HGT phenomenon in endophytes, which highlights an important biological mechanism for their evolutionary adaptation within the host plant and simultaneously confers “novel traits” to the associated microbes, remains less studied. Studies in endophytic bacteria revealed its role in toluene biodegradation and disease reduction in wheat (ea mays) . Moreoveea mays) . Table 1The phenomenon of HGT differs from the vertical transmission of genetic material from parent to offspring ,34. DistThe discovery of HGT across diverse biological species suggests its strong evolutionary role and highlights its significance in species adaptation and survival. For example, fungi and ciliates acquired the genes for carbohydrate metabolism from a ruminant animal ,41. StudHGT differs from other mechanism of genetic transmission since it does not involve parent–offspring inheritance, and therefore, might occur between distantly related species. Furthermore, the transfer of genetic material is rapid, rendering it an important mechanism for microbial adaptation to new environmental niches ,46. TherAgrobacterium tumefaciens), virus [The HGT mechanisms in prokaryotes, including transformation, conjugation, and transduction, have been well documented; however, HGT in eukaryotes might be more complex, particularly in plants, and might involve vector-mediated or direct pathways . The dir), virus , fungi [), virus , and nem), virus . Studies have shown that adaptation of bacteria to host plant involves diversification and evolutionary processes, while switching from parasitism to mutualism . The bacThe HGT phenomenon, widely regarded as a mechanism favored by evolution, aids the acquisition of novel traits by the associated species. Previous studies have reported the transfer of genetic material between microorganisms ,64 and mThe role of HGT in prokaryotic evolution depends on the amount of genes transferred, their integration in microbial genomes, and the phylogenetic relationship between different species . SuccessE. coli, indicating that HGT events were not limited by any barriers [The occurrence of HGT in prokaryotic genomes challenged the construction of phylogenetic relationships between biological species. The existence of HGT events in microbial genomes make it difficult to establish a correct phylogenetic relationship between microbes; however, it was suggested that the main genes are not transferred and a phylogenetic tree might be constructed using this pattern ,69. Furtbarriers . The HGTbarriers . In the barriers ,72,73.Physcomitrella patens via HGT [HGT has been well-documented and is considered to be a key evolutionary factor in prokaryotic genomes; however, its significance in eukaryotes remains undetermined ,49. The  via HGT . These a via HGT . The identification and annotation of complete genomes in biological organisms have shown the frequent occurrence of HGT events, resulting in a chimeric organism, with multiple DNA from different genomes. Considering the emerging importance of HGT events in the evolutionary process , understStudying gene distribution patterns: Gene transfer between different species results in the acquisition of new genes. Therefore, studying gene distribution patterns with uneven occurrence might lead to the identification of HGT events. However, uneven distribution patterns might also be caused by processes such as sequence divergence or gene loss . AdditioComparison of phylogenetic trees: One possible method to determine the occurrence of HGT event is the analysis of phylogenetic trees of different genes, based on the assumption that HGT events might lead different genes to have different evolutionary trees. However, this method is not accurate since various factors, such as ortholog/paralog misidentification, occurrence of convergence, and incorrect alignment of gene sequences ,86,87, mStudying unusual genome composition: A consistent uniformity is present in genome composition and phenotype and the presence of foreign genes due to HGT events can be detected by identifying genomic regions with unusual composition  . This meSimilarity search between genomes: A common method for the identification of HGT events in genomes is to search for maximum similarity between genes. HGT is very likely between genes of distantly related species. This is a common and fast method, however, its disadvantages include less accuracy and uncertainty regarding the number of best matches to search for, identification of orthologs, and uncertainty with multi-domain proteins ,90. For P. putida W619-TCE, an endophyte of poplar plant for degradation of TCE soil contaminant [T. durum, Z. mays, and B. cepacia [Rhizobium and Xanthomonas, revealed its significance as a mechanism for plant adaptation and survival [Burkholderia seminalis TC3.4.2R3, where it is suggested to be a possible adaptation mechanism. Further studies on the four-gene cluster would provide valuable information about the biosynthetic mechanism of antifungal compounds [The mutualistic association between endophytes and host plants has beneficial consequences for both plant development and microbial adaptation. In the evolution of biological species, HGT events were considered to be a strong, evolutionary mechanism for conferring new traits and assisting adaptation to adverse environments. However, reports on gene transfer between plants and endophytes were limited, suggesting that a more comprehensive research is required for understanding HGT signatures and their possible implications on the associated organisms. Significant studies on HGT in endophytes include the following—genetic manipulation of taminant ; arsenictaminant ; toluenetaminant ; endophy cepacia . These ssurvival . Studiesompounds .Plants are dynamic communities colonized by complex microbial communities that influence plant growth and development. The association of microbial communities with host plant provides multiple advantages to both the microbe and the host plant. These microorganisms spend a part of or an entire life cycle inside the plant and might be present externally (rhizosphere) or internally (endosphere). Studies have suggested that the association of a microbe influences plant growth and development and confers resistance to environmental stresses ,96, whilAmborella trichopoda, through HGT events [Arabidopsis thaliana, Oryza sativa, Populus trichocarpa, and Sorghum bicolor revealed fewer HGT events, suggesting their less frequent occurrence. One remarkable example is the first reported HGT of the β-1,6-glucanase gene from endophytic fungi to grasses [The HGT phenomenon was a key event that conferred novel traits to both prokaryotes and eukaryotes, during evolution ,38,101. T events . HoweverT events ,36,97. W grasses . The stu grasses . In the Endophytic fungi have a long evolutionary history of association with land plants, affecting almost all plant species . Fungal P. vittata were effective arsenic accumulators inside the host plant. The study discussed arsenic resistance and its associated genes in the endophytic bacteria, which are responsible for arsenic accumulation [P. vittata roots and characterized for arsenic tolerance. Results showed that the exchange of arsenite transporter genes arsB/ACR3(2), possibly occurred through HGT between endophytic bacteria [T. durum and Z. mays, induced toluene degradation and promoted plant growth [B. cepacia FX2 containing the gene for catechol 2, 3-dioxygenase (C23O) on a plasmid, effectively degraded toluene in contaminated soil [Recent studies highlighted the emerging importance of gene transfer events as a possible alternative mechanism, to acquire “novel traits” for potential socio-economic applications. Studies have been performed to understand the importance of HGT events between plants and endophytes, particularly the acquisition of “novel traits” by the associated organisms. Some endophytes were found to improve phytoremediation, with HGT as a possible mechanism of gene transfer. A recent study reported that the endophytic bacteria from mulation . In totabacteria . A similt growth . The endted soil . The chated soil . Moreoveted soil ,43. HGT is considered to be a significant process in the evolution of prokaryotes, although it remains less studied as a controversial subject in eukaryotes . SeveralThe occurrence of HGT as a major evolutionary event has been described in various studies ,94. HoweT. durum, Z. mays, and B. cepacia [In recent times, only a few studies have emphasized the advantages of utilizing HGT events in endophytes, and their potential applications in environmental and agricultural research. These studies include arsenic hyperaccumulation in endophytic bacteria , endophy cepacia , and tol cepacia . These sWith the progress in whole genome sequencing, the availability of “big data” has enabled the study of entire genomes of biological organisms. The study of HGT in eukaryotic genomes has become feasible, leading to the discovery of genes linked with HGT in different taxonomic groups of eukaryotes . In rece


P < .001) with a PTA 1‐year post‐HCT of 50 ± 0.7 dB to 23 ± 11 dB 13 years post‐HCT. Bone conduction threshold worsened with a PTA 1 year post‐HCT of 10 ± 7 dB to 18 ± 9 dB 13 years post‐HCT (P = .34). The degree of HL varied from mainly mild‐severe early after HCT to normal‐mild at longer follow‐up. The type of HL consisted of mainly conductive in the first years post‐HCT in contrast to mainly sensorineural at longer follow‐up. MRIs of the cerebellopontine angle did not show abnormalities. HL is still seen in patients with MPS despite HCT and consists of a conductive type early after HCT in contrast to a sensorineural type at longer follow‐up in the majority of cases. Yearly follow‐up of HL is necessary to timely intervene, as hearing is important in the speech and language development of children and their academic achievements.Hearing loss is frequently seen in mucopolysaccharidoses (MPS) patients. Although hematopoietic cell transplantation (HCT) increases overall survival, disease progression is observed in certain tissues. This study describes the course of hearing loss (HL) over time in transplanted MPS patients. Transplanted MPS patients between 2003 and 2018 were included and received yearly audiological evaluation, including auditory brainstem response (ABR) or pure tone audiometry (PTA). Twenty‐eight MPS‐1 and four MPS‐6 patients were analyzed with a median follow‐up of 5 years (range 11 months–16 years). Air conduction threshold improved significantly over time (  Yearly follow‐up of hearing loss in transplanted MPS patients is necessary to enable timely intervention, as hearing is important in the speech and language development of children and their academic achievements.1Hearing loss is a common feature in all subtypes of the mucopolysaccharidoses (MPS).Hearing loss in MPS may be conductive, sensorineural or mixed in origin. The conductive component of hearing loss is the result of seromucinous otitis, ossicular chain deformities or disruption, or arthropathy.Hematopoietic cell transplantation has been used as treatment for MPS‐1, MPS‐2, MPS‐3, MPS‐4, and MPS‐6 with variable success.22.1All MPS‐1 and ‐6 patients treated with HCT in the Wilhelmina Children's Hospital (WKZ) of the University Medical Centre Utrecht (UMCU), The Netherlands between 2003 and 2019, were included in this study. The patients were evaluated by an experienced otorhinolaryngologist and received a hearing assessment on a yearly basis at the Sylvia Toth Center for Multidisciplinary Follow up after Hematopoietic Cell Transplantation. This study is based on retrospectively collected patient data acquired during care‐as‐usual and has been exempted for approval according to the responsible institutional committee Medical Research Involving Human Subjects Act. The MPS subtype, age at transplantation, enzyme activity level after transplantation , otorhinolaryngological and audiological interventions, including the insertion of a ventilation tube in the ear drum and hearing aid use were collected from the medical records.2.2Pure tone audiometry was performed repeatedly to assess hearing levels. In uncooperative patients due to a young age or cognitive impairment, auditory brainstem response (ABR) was performed during natural sleep or sedation. The hearing loss was evaluated according to the threshold in the better ear. The results of pure tone audiometry and ABR were analyzed separately.2.2.1Click‐evoked auditory brainstem response audiometry was performed in a soundproof room during natural sleep . The ABR waveform was elicited by a click‐evoked stimulus of 0.1 ms per click, with a repetition rate of 11.7 clicks/s and alternating polarity. Electrodes were placed on both mastoids with a reference at the vertex and a ground electrode on the forehead and then band pass filtered. Response thresholds were determined by changing the stimulus level with step sizes of 10 dB down and 5 dB up method until no response was found. Experienced clinical specialists analyzed the waveform and defined the auditory brainstem response threshold in dBnHL as the lowest level at which a wave V was repeatedly present. The degree of hearing level in dB HL was agreed to be 10 dB below the estimated threshold in dBnHL as measured by this method.2.2.2An experienced audiology assistant performed the hearing test by air (AC) and bone conduction (BC) for the frequencies 250, 500, 1000, 2000, 4000, and 8000 Hz in a quiet room and masking as appropriate. The hearing loss levels were determined by taking the four‐frequency pure tone average (PTA) of the air conduction threshold (500‐1000‐2000‐4000 Hz).The degree of hearing loss was categorized according to the World Health Organization (WHO) International Classification of Impairments, Disabilities, and Handicaps (WHO‐ICIDH) standard.The criteria of the Genetic Deafness study group2.3MRIs of the cerebellopontine angle were performed in a random selection of MPS‐1 patients during care‐as‐usual brain MRIs. Sagittal spin‐echo T1‐weighted images, and transverse turbo spin‐echo T2‐weighted and diffusion weighted single‐shot images were performed. Furthermore, transverse 3D T1 and T2‐weighted images were performed. The transversal 3D weighted‐image was repeated after administration of gadolinium. All images were performed on a Philips Achieva, Ingenia, or Ingenia Elition 1.5 T or 3.0 T magnetic resonance system.2.4P‐values <.05 were considered significant. The R project  for statistical computing version 3.4.1 with the packages “lme4,” “rms,” and “ggplot2” was used for all analyses and for the creation of figures.Baseline characteristics are reported as frequencies and percentages for categorical variables and median (range) for continuous variables. The course of hearing loss over time after transplantation was analyzed with a linear mixed model. To account for nonlinearity in air conduction and the air‐bone gap, restricted cubic splines with three knots were added to the model. The four‐frequency PTA of the better ear was used as the dependent variable for AC and BC thresholds as well as for the air‐bone gap. Based on previous literature, age at time of transplantation, subtype, and enzyme level after transplantation were considered important covariates and included as fixed effects. A random intercept was included per individual to account for individual variation at baseline and a random slope (for time) for dependency across the repeated measurements within the same individual during follow‐up. To analyze the course of hearing loss for the separate frequencies, data of both ears was included. To account for dependency between both ears a random intercept per ear was included additional to the random intercept per individual. Final coefficients were estimated using restricted maximum likelihood and significance of parameters was assessed with a likelihood ratio test between the model with and without the parameter. The model assumptions including normal distributed residuals, random effects, and homogeneity of variance were confirmed visually. 33.1In total, 190 hearing observations of 32 patients were analyzed. MPS‐1 was the most frequent MPS subtype . Within the phenotypic range of MPS‐1, Hurler syndrome was more frequent  compared to Hurler‐Scheie . Four patients were diagnosed with MPS‐6 (12%). The median age at transplantation for all patients was 1 year (range 2 months–5 years). Nearly all patients had leukocyte enzyme activity levels in the normal range post‐HCT . The median follow‐up after HCT consisted of 5.0 years (range 11 months–16 years).3.23.2.1ABR was performed in 12 patients  aged between 0 and 6 years. In 6 patients (50%) ABR was performed before HCT and all these patients were <2 years. Post‐HCT ABR were performed in patients aged 2 to 6 years with a median follow‐up of 1.5 years (range 1‐3 years). The average hearing threshold determined by ABR pre‐HCT was 43 dBeHL (dB estimated hearing level). The average hearing threshold determined by ABR post‐HCT was 39 dBeHL. None of these patients had normal hearing with a threshold of <20 dB hearing loss in both ears. A conductive or mixed type hearing loss was seen in the majority of the patients .3.2.2P < .001; Figure Pure tone audiometry was performed longitudinally 179 times in 22 patients from the age of 3 years and onwards (maximum age at latest follow‐up was 17 years). One year after HCT, the air conducted mean PTA was 50 dB (95% CI 49‐51 dB), after 3 years this was 34 dB (95% CI 26‐44 dB), after 5 years 30 dB (95% CI 22‐37 dB), after 10 years 24 dB (95% CI 20‐28 dB), and after 13 years 23 dB (95% CI 12‐33 dB) . Three years after HCT, 38% had normal, 31% mild, and 31% moderate hearing loss  with a median age of 4 years (range 1‐14 years). Two patients had the MPS‐6 subtype. Fifteen patients (81%) received these ≥2 times. Of the 32 MPS patients, 12 patients were prescribed hearing amplifications (38%). All were MPS‐1 patients. The majority used bilateral hearing aids (83%), with the exception of 2 patients who received a bone conduction device (13%). The median age at the start of hearing aids use was 4 years (range 2‐15 years) at a median follow‐up of 3 years (range 1‐14 years).3.3MRIs of the cerebellopontine angle were performed in 10 patients  at median follow‐up of 12 years (range 4‐16 years). None showed any abnormalities in the n. cochlearis, n. facialis, or n. vestibularis inferior/superior, respectively. Furthermore, no abnormalities in the anatomy of the cochlea or inner ear were seen.4This longitudinal analysis, spanning more than 15 years of repeated audiological evaluation in MPS‐1 and ‐6 children after HCT at a young age, shows hearing impairment despite HCT. Although an initial improvement is seen, a significant number of patients will likely need hearing aids before reaching adulthood. The initial improvement is seen in air conduction in the first 5 years of life after HCT. Thereafter, air conduction thresholds are more or less stable with a mean hearing loss of 20 to 30 dB. Bone conduction, however, shows a slow deterioration over time which could eventually lead to the need of a hearing aid or even a cochlear implant. This knowledge is important for caregivers counseling these patients.Detailed analysis of hearing after HCT in MPS patients was reported previously in two other studies and the results are in line with our data.Eustachian tube dysfunction, recurrent middle ear infections and thickened middle ear mucosa by GAG deposits have been considered important factors of conductive hearing loss in MPS patients. The improvement of air conduction in the first years after HCT might be due to a combination of the natural improvement in the occurrence of chronic otitis in young children by age and a beneficial effect of HCT especially on the mucosal tissue, both resulting in a reduction of otitis episodes.The bone conducted threshold slowly increases over time especially in the middle range frequencies. This implies that hearing loss actually is progressing despite HCT. As disease progression is seen in transplanted patients especially in connective tissue ,The exact mechanism of SNHL in MPS patients is unknown, however Kariya et al.The MPS are rare diseases, therefore studying them inevitably results in a small number of patients, which is a limitation of this study. Unfortunately, no control data was available, which hampers to determine the specific effect of HCT on hearing. Also, because transplantation is performed in children at a very young age hearing assessments determining the exact hearing level before HCT were missing which hindered comparison pre‐ and post‐intervention. Due to the longitudinal aspect of the study, however, we were able to show the course of hearing loss over time after HCT. Finally, as this was a retrospective study, we only had data collected from care‐as‐usual.Concluding, hearing loss is still seen after long‐term follow‐up in MPS‐1 and ‐6 children despite HCT at an early age. When looking in‐depth with a maximum follow‐up of 16 years, air conduction is initially improving after HCT. Bone conduction however is slowly worsening over time, which could be a sign of disease progression in HCT‐treated patients. To improve the quality of life of these patients, new therapies should focus on how to stop post‐transplant disease progression. Until then, MPS‐1 and ‐6 patients awaiting HCT should be informed that most likely ventilation tubes and/or hearing aids may be necessary at some point. Furthermore, after HCT, yearly and long‐term evaluation with a hearing assessment by tone audiometry and potentially otoacoustic emissions is indicated for early diagnosis of the severity and type of hearing loss and to initiate treatment of hearing loss to ensure the requirements for adequate speech and language development and academic performance in these patients.All authors state they have no competitive  interests in this study.Brigitte T.A. van den Broek: contributed to the design and planning and to the acquisition of the data, performed analyses, interpreted the results and drafted the manuscript including figures, and serves as guarantor for the article; Adriana L. Smit: contributed to the design and planning and to the acquisition of the data, interpret the data, and critically revised the manuscript; Jaap Jan Boelens: contributed to the acquisition of the data, interpret the data, and critically revised the manuscript; Peter M. van Hasselt: contributed to the acquisition of the data, interpret the data, and critically revised the manuscript; all authors were responsible for the medical care of the patients.The data that support the findings of this study are available from the author, PMvH, upon reasonable request.Supplementary Figure S1 Air conduction, bone conduction and air‐bone gap per individual patient over time (blue lines) for the different threshold test frequencies . The black line depicts the average course over time for a patient with normal enzyme activity levels after HCT and mean age at transplantation (1.22 years).Click here for additional data file.


Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) is an immune-mediated peripheral neuropathy that is currently classified into several clinical subtypes, which are presumed to have different pathogenic mechanisms. Recently, studies identified a subgroup of patients with CIDP who were positive for IgG4 autoantibodies against paranodal proteins, such as neurofascin-155 and contactin-1, who respond poorly to first-line therapies for typical CIDP, including intravenous immunoglobulin therapy.This study aims to evaluate the efficacy and safety of intravenous rituximab according to IgG4 autoantibody status in patients with refractory CIDP.The Evaluation of the Efficacy and Safety of Rituximab in Refractory CIDP Patients with IgG4 Autoantibodies in the Exploratory Clinical (RECIPE) trial consists of 2 cohorts: a multicenter, placebo-controlled, randomized study cohort of 15 patients with IgG4 autoantibody-positive CIDP (rituximab:placebo = 2:1) and an open-label trial cohort of 10 patients with antibody-negative CIDP. The primary endpoint is improvement in functional outcome assessed using the adjusted Inflammatory Neuropathy Cause and Treatment Disability Scale score at 26, 38, or 52 weeks after the start of treatment with rituximab in patients with CIDP and anti-paranodal protein antibodies. Secondary outcome measures include grip strength, manual muscle testing sum scores, results of nerve conduction studies, and other functional scales.We plan to enroll 25 cases for the full analysis set. Recruitment is ongoing, with 14 patients enrolled as of January 2020. Enrollment will close in September 2020, and the study is planned to end in December 2021.This randomized controlled trial will determine if rituximab is safe and effective in patients with anti-paranodal antibodies. An open-label study will provide additional data on the effects of rituximab in patients with antibody-negative CIDP. The results of the RECIPE trial are expected to provide evidence for the positioning of rituximab as a pathogenesis-based therapeutic for refractory CIDP.ClinicalTrials.gov NCT03864185, https://clinicaltrials.gov/ct2/show/NCT03864185 ; The Japan Registry of Clinical Trials jRCT2041180037, https://jrct.niph.go.jp/en-latest-detail/jRCT2041180037DERR1-10.2196/17117 Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) is an acquired demyelinating peripheral neuropathy characterized by weakness and sensory disturbance in the extremities that develops over a period of at least 2 months. The current diagnostic criteria for CIDP were published by the European Federation of Neurological Societies/Peripheral Nerve Society (EFNS/PNS) and include clinical and electrophysiological findings with supporting features that strengthen the diagnosis -4. The gAutoantibodies targeting myelin components, inflammatory cytokines, and the complement pathway are assumed to be the humoral factors modifying immune status in CIDP. Although many autoantibodies against various myelin antigens, such as P0, PMP22, Cx32, beta-tubulin, LM1, and sulfatide, have been investigated, none has been implicated in the pathogenesis of CIDP ,12. AutoTherefore, we planned this prospective clinical trial to investigate the efficacy of rituximab in refractory IgG4 autoantibody–positive CIDP by comparing with placebo in IgG4 autoantibody–positive patients and with rituximab in IgG4 autoantibody–negative patients as a reference. This investigator-initiated clinical trial has been named “The Evaluation of Efficacy and Safety of Rituximab  in Refractory Chronic Inflammatory Demyelinating Polyneuropathy (CIDP) Patients with Immunoglobulin G4 (IgG4) Autoantibodies in the Exploratory Clinical Trial” . The design of the trial was developed in consultation with and approved by the Pharmaceuticals and Medical Device Agency (PMDA), which is responsible for reviewing new pharmaceuticals in Japan.Here, we provide the detailed design of this investigator-initiated clinical trial in refractory CIDP patients with or without IgG4 autoantibodies as an exploratory study in Japan .The primary objectives of the RECIPE trial are to evaluate the efficacy and safety of intravenous rituximab in patients with refractory CIDP according to their IgG4 autoantibody status. This trial consists of a multicenter, placebo-controlled, randomized, double-blind, parallel-group, comparative study in 15 patients with CIDP and IgG4 autoantibodies (CNTN1 or NF155) who will be allocated to a rituximab group (n=10) or a placebo group (n=5) and an open-label study in 10 patients with IgG4 autoantibody–negative CIDP. The outline of this study is shown in The primary endpoint is improvement in the adjusted Inflammatory Neuropathy Cause and Treatment (INCAT) Disability Scale score at 26, 38, or 52 weeks after the start of treatment. Efficacy is based on the effectiveness rate, which is the proportion of patients who achieve the primary endpoint of ≥1-point improvement in the INCAT Disability Scale score compared with baseline. Our specific hypothesis is that the effectiveness rate will exceed a prespecified threshold of 15% in patents with IgG4 autoantibody–positive CIDP in the rituximab-treated group. The data from patients with IgG4 autoantibody–negative CIDP will serve as a reference.The RECIPE trial will investigate the efficacy of rituximab in patients with refractory IgG4 autoantibody–positive and IgG4 autoantibody–negative CIDP. Anti-NF155 and anti-CNTN1 autoantibody status will be confirmed by enzyme-linked immunosorbent assay as described in the literature ,35.Patients who meet the following criteria are eligible to participate:CIDP diagnosed according to the modified EFNS/PNS criteria -4 prior Positive or negative serum IgG4 autoantibody (CNTN1 or NF155) status confirmed prior to enrollmentCIDP refractory to treatment with corticosteroids for 12 weeks and IVIg for 8 weeks prior to enrollment or unable to receive or continue treatment with corticosteroids and IVIgTotal adjusted INCAT Disability Scale scores of 2 to 8 at both preliminary enrollment and enrollment, with no decrease in total score between preliminary enrollment and enrollmentAge ≥12 yearsAble to provide voluntary written consent after receiving adequate information about the study Patients are excluded for any of the following reasons:Any differential diagnosis defined in the modified EFNS/PNS diagnostic criteria -4Initiation or an increased dose of corticosteroids within 12 weeks before enrollmentInitiation or an increased dose of IVIg within 8 weeks before enrollmentPlasmapheresis within 8 weeks before enrollment or disease refractory to 8 weeks of plasma exchange or double-filtration plasmapheresisInitiation or an increased dose of an immunosuppressant  within 12 weeks before enrollmentHistory of hematopoietic stem cell transplantationPrevious treatment with rituximabParticipation in another clinical study within 3 months before enrollment or current participation in another study1c ≥7%)Poorly controlled diabetes , neutropenia (<1000/mm3), or lymphopenia (<500/mm3) at enrollmentLeukopenia Pregnancy or potential for pregnancy, lactation, or unwillingness to use contraception during the study periodDeemed unsuitable for participation by an investigator or sub-investigatorRegarding item 11, patients with positive hepatitis B surface antibody or core antibody can be enrolled when a hepatitis B virus-DNA test is negative (below the limit of detection), and hepatitis B virus-DNA and aspartate/alanine transaminase levels are monitored at fixed intervals.The investigators obtain written informed consent from all patients who are potentially eligible for the study. After confirmation of inclusion criteria 4 to 6 and exclusion criteria 1, 6, 7, and 13 to 16, patients are preliminarily enrolled via an electronic data capture system . At the second screening, the investigators check all the inclusion and exclusion criteria. Patients confirmed to have IgG4 autoantibody–positive CIDP are randomly assigned to the rituximab group or the placebo group at a ratio of 2:1 using the stratified permuted block method according to their adjusted INCAT Disability Scale score (2 to 4 or 5 to 8). Patients with IgG4 autoantibody–negative CIDP are automatically allocated to the rituximab group.2 or placebo is administered intravenously once weekly for 4 weeks. This is the same dosage as used in previous studies involving CIDP patients treated with rituximab [Rituximab 375 mg/mituximab ,27,30,31Hematopoietic stem cell transplantation, plasma exchange therapy (simple plasma exchange or double membrane filtration), and any drug that could affect immune status is prohibited during the trial in view of their potential to affect the efficacy of rituximab. For the same reason, starting or increasing the dose of corticosteroid, IVIg, or immunosuppressant therapy is not permitted for the duration of the study.All participants are followed for 52 weeks after the first dose of investigational medication . All datSecondary endpoints include grip strength, as measured using a vigorimeter (left/right); the Rasch-built Overall Disability Scale; Medical Research Council sum score; median, ulnar, tibial, and peroneal motor nerve conduction studies, including motor nerve conduction velocity and distal and proximal latency, amplitude, and duration of the compound muscle action potential after distal and proximal stimulation; protein levels in the cerebrospinal fluid; B cell (CD19-positive and CD20-positive) counts and T-cell  counts; expression of human anti-chimeric antibodies; and serum rituximab level.The exploratory endpoints include serum antibody titers of IgG4, serum antibody titers of anti-CNTN1 and anti-NF155 IgG subclasses, and serum neurofilament level.The safety endpoints include adverse events and vital signs and laboratory tests.P<0.05 regarded as statistically significant, we estimated that 8 cases would be needed to achieve 80% power. Accordingly, 10 patients with IgG4 autoantibody–positive CIDP will be enrolled for the rituximab group, allowing for a 20% dropout rate.The trial includes patients with CIDP that is refractory to both IVIg and corticosteroids or patients who are unable to receive or continue these therapies. Most eligible patients would be unlikely to achieve an improvement of ≥1 point on the adjusted INCAT Disability Scale even if conventional therapies were continued. An immunosuppressant  is generally considered to be an effective second-line treatment in about 30% of patients with refractory CIDP . HoweverThe PMDA suggested inclusion of a placebo control group for the patients who are IgG4 autoantibody–positive. We agreed to include a placebo group of 5 patients for reference purposes rather than for strict statistical comparison, given the rarity of CIDP and feasibility considerations. The placebo group has been designated as a reference group to confirm that the estimated values do not deviate markedly from the defined thresholds. Patients with IgG4 autoantibody–negative CIDP are included in another reference group to compare their response to rituximab treatment with that of patients with IgG4 autoantibody–positive CIDP. This reference group will consist of 10 patients, which is the same as the number of patients with IgG4 autoantibody–positive CIDP.P values <0.05 will be considered statistically significant.All statistical analyses will be performed on an intention-to-treat basis. The primary analysis will compare the adjusted INCAT Disability Scale scores recorded before treatment (at enrollment) with those recorded at 26, 38, or 52 weeks after the start of treatment. We will calculate the proportion and 95% CI of IgG4 autoantibody–positive patients in the rituximab group who achieve an improvement of ≥1 point at any of evaluation point after 26 weeks, from baseline. The 95% CI will be calculated using the Clopper-Pearson method. Continuous variables will be analyzed using descriptive statistics, and categorical variables will be calculated as the frequency and proportion. All statistical analyses will be performed using SAS version 9.4 . All statistical tests will be two-sided. Monitoring and auditing will include systematic independent examination according to the study protocol, applicable regulatory requirements, and standard operating procedures.The study protocol complies with the Declaration of Helsinki  and the The necessary information for the RECIPE trial has been uploaded to ClinicalTrials.gov  and the Japan Registry of Clinical Trials .The first patient completed registration in April 2019 and received an investigational treatment in May 2019. Recruitment of patients for the RECIPE trial is ongoing at the four participating hospitals. As of January 2020, 14 cases have been enrolled. The targeted accrual is 25 cases for the full analysis set. Enrollment will close in September 2020, and the study is scheduled to end in December 2021.There have been some reports on the epidemiology of IgG4 antibody–positive CIDP ,16,17. TRituximab can be expected to be effective in cases of refractory CIDP -32. SomeThis article has described the design and protocol being used in the RECIPE trial. We outlined the characteristics of patients with IgG4 autoantibody–positive CIDP and discussed some critical considerations for these patients. The RECIPE trial is the first randomized controlled trial of rituximab for IgG4 autoantibody–positive CIDP. It is anticipated that the results will lead to a pathogenesis-oriented therapeutic strategy that can target specific phenotypes of CIDP and confirm the efficacy of rituximab in refractory IgG4 autoantibody–negative CIDP.


Mycoplasma hyopneumoniae is the major pathogen causing enzootic pneumonia in pigs. M. hyopneumoniae infection can lead to considerable economic losses in the pig-breeding industry. Here, this study established a first-order absorption, one-compartment model to study the relationship between the pharmacokinetics/pharmacodynamics (PK/PD) index of tilmicosin against M. hyopneumoniae in vitro. We simulated different drug concentrations of timicosin in the fluid lining the lung epithelia of pigs. The minimum inhibitory concentration (MIC) of tilmicosin against M. hyopneumoniae with an inoculum of 106 CFU/mL was 1.6 μg/mL using the microdilution method. Static time–kill curves showed that if the drug concentration >1 MIC, the antibacterial effect showed different degrees of inhibition. At 32 MIC, the amount of bacteria decreased by 3.16 log10 CFU/mL, thereby achieving a mycoplasmacidal effect. The M. hyopneumoniae count was reduced from 3.61 to 5.11 log10 CFU/mL upon incubation for 96 h in a dynamic model with a dose of 40–200 mg, thereby achieving mycoplasmacidal activity. The area under the concentration-time curve over 96 h divided by the MIC (AUC0–96 h/MIC) was the best-fit PK/PD parameters for predicting the antibacterial activity of tilmicosin against M. hyopneumoniae (R2 = 0.99), suggesting that tilmicosin had concentration-dependent activity. The estimated value for AUC0–96 h/MIC for 2log10 (CFU/mL) reduction and 3log10 (CFU/mL) reduction from baseline was 70.55 h and 96.72 h. Four M. hyopneumoniae strains (M1–M4) with reduced sensitivity to tilmicosin were isolated from the four dose groups. The susceptibility of these strains to tylosin, erythromycin and lincomycin was also reduced significantly. For sequencing analyses of 23S rRNA, an acquired A2058G transition in region V was found only in resistant M. hyopneumoniae strains . In conclusion, in an in vitro model, the effect of tilmicosin against M. hyopneumoniae was concentration-dependent and had a therapeutic effect. These results will help to design the optimal dosing regimen for tilmicosin in M. hyopneumoniae infection, and minimize the emergence of resistant bacteria. Mycoplasma hyopneumoniae is the primary pathogen of mycoplasmal pneumonia in pigs. M. hyopneumoniae is widespread in various regions, and can cause huge economic losses to the pig industry [M. hyopneumoniae can transmit vertically [industry . Infecteindustry . Once flrtically ,4.M. hyopneumoniae [Mycoplasma species [M. hyopneumoniae infections. It is combined with the 50S large subunit of bacterial ribosomes to exert an antibacterial effect, rather than acting on the cell wall. However, the unreasonable use and abuse of macrolides has led to the emergence of drug resistance [Antibiotic treatment is one of the main methods for controlling eumoniae . Tilmico species . It has  species ,8. The usistance ,10. Mostsistance . Resistasistance ,13.M. hyopneumoniae, the pharmacokinetic/pharmacodynamic (PK/PD) profiles of tilmicosin against M. hyopneumoniae are very limited. Also, establishing an infection model of M. hyopneumoniae in vivo is challenging. Therefore, it is a feasible to establish an in vitro dynamic model to evaluate the effect of tilmicosin against M. hyopneumoniae. In vitro PK/PD models have been used widely to optimize dose regimens, monitor antimicrobial activity and prevent the emergence of resistant bacteria [M. hyopneumoniae.Due to the difficulty of culturing and counting bacteria . Such mobacteria . Moreovebacteria ,17,18. TM. hyopneumoniae; in this way, we could investigate the mechanism of resistance. This model can be used as a reference to optimize the dosing regimen for tilmicosin against M. hyopneumoniae.This study wished to apply the one-compartment infection model in vitro to determine the PK/PD indices of tilmicosin against M. hyopneumoniae (ATCC 25934) was obtained from the Chinese Veterinary Microorganism Culture Collection Center  and stored at −80 °C. Tilmicosin (75.8%), tylosin (82.6%), erythromycin (85.0%), tiamulin (99.0%), doxycycline (85.8%) and enrofloxacin (99.0%) were kindly supplied by Guangdong Dahuanong Animal Health Products . Amikacin (99.0%) and lincomycin (84.6%) were purchased from Guangdong Puboxing Animal Health Products  and stored at −80 °C before use.A standard strain of A fresh stock solution (1280 mg/L) of each antibacterial agent was prepared for each experiment. Broth medium base was purchased from Qingdao Hope Biological Technology . The reduced form of nicotinamide adenine dinucleotide was obtained from Beijing Newprobe Biotechnology . L-Cysteine was purchased from Beijing Solarbio Science and Technology .M. hyopneumoniae was determined by a modified version of the MIC assay, as described by Tanner and Wu [5, 106 and 107 CFU/mL (100 μL) were added to an equal volume of drug-containing medium in each well in a 96-well plate. The tilmicosin concentration in the 96-well plate was 0.05–12.8 μg/mL. A growth control (lacking antibiotic), sterility control (sterile broth at pH 7.7) and endpoint control (blank medium at pH 6.5) were included. Plates were incubated at 37 °C in a humidified atmosphere of 5% CO2 until the growth group and endpoint control were the same color. The MIC was defined as the minimal concentration of antibacterial agent that resulted in no color change. All experiments were carried out in triplicate.The MIC of tilmicosin against r and Wu . Briefly5, 106 and 107 CFU/mL were also applied to the drug plates. A blank-growth control group was also used and comprised cells spread on plates lacking the drug. Plates were incubated for ≥7 days. The lowest concentration without M. hyopneumoniae growth was determined as the MIC. All experiments were carried out in triplicate.According to the method described by Hannan et al. , plates M. hyopneumoniae were added to a bottle in turn and then mixed. The tilmicosin concentration in the culture system was in a certain range . A growth control (not exposed to the drug) and a sterility control (medium at pH 7.7 without the drug or M. hyopneumoniae) were indispensable. Penicillin bottles were cultured for 60 h in the environments described above. Aliquots (100 μL) of the culture were taken from each bottle at 0, 1, 3, 6, 9, 12, 24, 36, 48 and 60 h to detect the M. hyopneumoniae population. After 7 days, the results were read using an inverted microscope .Four milliliters of blank medium, 0.5 mL of 10-times the final drug concentration, and 0.5 mL of logarithmic This study used a previously described in vitro dynamic model . This exBriefly, the model system consists of three parts: the first is an absorption chamber containing a drug medium as a site of administration; the second is a central chamber which comprises 300 mL of sterile medium ) and a 10-mL dialysis tube ); the third part is a reserve room for fresh media. At the same time, the waste liquid is collected in the waste liquid chamber.M. hyopneumoniae was studied. The hollow fiber systems have cartridges containing hundreds of hollow fibers which separate the bacteria in the peripheral compartment from the central compartment. The large number of hollow fibers provide a large surface area for drugs to readily diffuse between the central reservoir and the peripheral compartment.In this experiment, by referring to the pharmacokinetic data obtained from the in vivo pharmacokinetic experiments of tilmicosin, a fitting analysis was performed to obtain the required pharmacokinetic parameters and set the flow rate of the pump. Then, the pump model was used to simulate the absorption and elimination process of tilmicosin. Based on this, the relationship between the pharmacokinetic and pharmacodynamic effects of different doses of tilmicosin against M. hyopneumoniae and PK characteristics of tilmicosin in pigs. The parameter values of absorption half-life, elimination half-life and flow rate of peristaltic pumps were 12.17 h, 17.16 h and 0.29 mL/min, respectively. The dosage regimen for this experiment was based on the clinically recommended dosage of tilmicosin and several doses above and below it. The antibacterial effect of tilmicosin was simulated and observed at, and above or below, the recommended dose against M. hyopneumoniae. Eight dose groups  were designed for the in vitro dynamic model. Increasing the speed of the magnetic stirrer as the drug was injected into the absorption chamber achieved a rapid balance between the inside and outside of the dialysis membrane simultaneously.The model parameters were determined by the colonization site of M. hyopneumoniae.Samples (2 mL) were collected from the EC 1, 3, 6, 9, 12, 24, 36, 48, 72 and 96 h after administration, and then stored at −20 °C until analyses. Samples (100 μL) were taken from the IC before dosing, as well as 6, 12, 24, 36, 48, 72 and 96 h after administration. The collected samples were used to detect the number of, and susceptibility to, R2 > 0.99) was defined by six calibration standards of tilmicosin, with a final concentration ranging from 5 ng/mL to 500 ng/mL. The limit of detection (LoD) and limit of quantification (LoQ) in the medium were 0.5 ng/mL and 1 ng/mL, respectively.The tilmicosin concentration in the medium was analyzed using a high-performance liquid chromatography unit  and a triple quadrupole mass spectrometer  equipped with an electrospray ionization source. The analytical method used was as described by Huang et al. . The stamax/MIC), the area under the concentration–time curve over 96 h divided by the MIC (AUC0–96 h/MIC) and the cumulative time at which the concentration exceeds the MIC (%T > MIC).Three important PK/PD indices were calculated by integrating PK parameters and MIC values in vitro: the peak concentration by MIC . We chose the inhibitory sigmoid Emax model to analyze data:E is the anti- M. hyopneumoniae effect, Emax is the change in the amount of M. hyopneumoniae in the control group at a 96-h interval, E0 is the largest anti- M. hyopneumoniae effect, determined as log10CFU/mL reduction at the same interval, eC represents the PK/PD indices , N is the Hill coefficient that describes the steepness of the PK/PD indices–effect curve, and EC50 is the corresponding PK/PD value when the anti- M. hyopneumoniae effect reaches 50% of the maximum antibacterial effect. R2 was calculated for each assay.The correlation between the PK/PD indices and antimicrobial activity against This study used the method described by Huang et al.  with sliM. hyopneumoniae with inoculums of 105, 106, and 107 CFU/mL was 0.8, 1.6 and 1.6 μg/mL using the microdilution method, and 3.2, 6.4 and 6.4 μg/mL using the agar dilution method, respectively.The MIC of tilmicosin against M. hyopneumoniae are shown in 10 CFU/mL. The number of bacteria in the blank-growth control group increased by 1.82 log10 CFU/mL. When the drug concentration was >1 MIC, the antibacterial effect showed different degrees of inhibition. The bacteria at 2, 4, 8 and 16 MIC decreased by 0.742, 0.858, 2.03 and 2.65 log10 CFU/mL, respectively. At 32 MIC, the amount of bacteria decreased by 3.16 log10 CFU/mL, thereby achieving a mycoplasmacidal effect. In summary, the antibacterial effect of tilmicosin against M. hyopneumoniae was more obvious with an increase in the drug concentration.The in vitro static bactericidal curves of different concentrations of tilmicosin against M. hyopneumoniae took a long time to occur. With an increase in drug dose, tilmicosin showed increased activity against M. hyopneumoniae. The M. hyopneumoniae count was reduced from 3.61 to 5.11 log10 CFU/mL upon incubation for 96 h in a dynamic model with a dose of 40–200 mg, and achieved mycoplasmacidal activity.The bactericidal curve of tilmicosin from the in vitro dynamic model at different clinically recommended doses is shown in R2 > 0.99). The relative errors of elimination half-life and absorption half-life were −5.6 and 9.28%, respectively, both of which were within the normal range of ±15%. The LoD and LoQ for the developed method were 0.5 ng/mL and 1 ng/mL, respectively.The time–concentration curves for the in vitro dynamic model are shown in Emax model, the correlation coefficient between AUC0−96 h/MIC, %T > MIC, Cmax/MIC and the antibacterial effect was 0.99, 0.91 and 0.99, respectively. Cmax/MIC and AUC0–96 h/MIC were the best-fit PK/PD parameters for predicting the antibacterial activity of tilmicosin against M. hyopneumoniae, which suggested that tilmicosin had concentration-dependent activity. The estimated values for Cmax/MIC and AUC0–96 h/MIC for 2log10 (CFU/mL) reduction and 3log10 (CFU/mL) reduction from baseline were 1.44 and 1.91, and 70.55 h and 96.72 h, respectively, during a 96-h treatment period with tilmicosin. The obtained parameters of E0, Emax and EC50, and the Hill coefficient, are listed in The relationships between antibacterial effect and the PK/PD parameters of this dynamic model are shown in M. hyopneumoniae strains  with reduced sensitivity to tilmicosin were isolated from the four dose groups . The MIC of these strains to tilmicosin ranged from 25.6 μg/mL to 1638.4 μg/mL. Moreover, the MIC of strains M3 and M4 was significantly higher than that of the standard strains. M. hyopneumoniae strains , and a resistance mutation was not found in L4 or L22.Four M. hyopneumoniae infection is a major, worldwide problem in the pig industry. If M. hyopneumoniae is mixed with other pathogenic microorganisms, porcine respiratory disease complex can occur [Mycoplasmal pneumonia caused by an occur ,22.M. hyopneumoniae presents technical difficulties in culturing and agar-plate enumeration, both in the culture environment and in the addition of nutrients [M. hyopneumoniae grows slowly, and is inhibited competitively by other bacteria, so the isolation of M. hyopneumoniae strains is difficult [M. hyopneumoniae are scarce.The rational use of antibiotics is the main measure to prevent and treat mycoplasmal pneumonia. However, utrients . M. hyopifficult . Hence, Pseudomonas aeruginosa using an in vitro dynamic model. They showed that polymyxin B had rapid and concentration-dependent bactericidal activity against P. aeruginosa, and that the effect was diminished if the amount of bacteria was increased. Also, study of the effect of doxycycline against Mycoplasma gallisepticum in an in vitro model led to the determination of the optimal PK/PD parameters to prevent drug resistance [The in vitro dynamic model has been used widely to study PK/PD interactions. Tam et al.  investigsistance .M. hyopneumoniae which simulated the PK of tilmicosin in bronchoalveolar lavage fluid after a single oral dose. The advantage of this model is that when it is difficult to establish an animal-infection model, the interaction between the drug and bacteria can be elucidated, and the change in drug sensitivity of the pathogen can be observed to study the drug-resistance mechanism.Here, this study reported on an in vitro PK/PD model of M. hyopneumoniae field isolates by a broth microdilution method. They showed that the MIC range of tilmicosin against M. hyopneumoniae was 0.25–16 μg/mL. Felde and colleagues reported that the MIC of tilmicosin against M. hyopneumoniae was 0.25–64 μg/mL [Mycoplasma species is 103–105 CFU/mL [5–107 CFU/mL, and the inoculum used in the in vitro dynamic model experiment was high (107 CFU/mL).Vicca et al.  determin64 μg/mL . Compare5 CFU/mL . HoweverMycoplasma species or MIC determination [−6 to 10−8) [There were three main reasons for choosing a high inoculum. First, turbidity has little effect on the growth of mination . Second,to 10−8) . Therefo10 CFU/mL. It is worth noting that the number of bacteria was slightly reduced in the blank-growth control group and low-dose group (10 mg). There are two reasons for this result. First, the nutritional conditions required for M. hyopneumoniae growth were compromised slightly due to the long period of the dynamic model test. Second, in the low-dose group, there was a postantibiotic sub-MIC effect (PA-SME), which may have slightly reduced the amount of bacteria.The time–kill curves in Emax model-fitting confirmed that the effect of tilmicosin on M. hyopneumoniae was concentration-dependent, and the parameters AUC96h/MIC and Cmax/MIC showed a strong correlation with antibacterial effects (R2 = 0.99). Most studies have shown that the %T > MIC parameter of macrolides is significantly associated with antimicrobial activity [24 h/MIC parameter [96h/MIC and Cmax/MIC in this experiment are the lack of fitting analysis data and the relatively close trend. Finally, it was decided that AUC96h/MIC should be used as the optimal parameter.The results of activity ,33. Howearameter . Therefoarameter . A numbearameter ,37,38,39M. hyopneumoniae using drug-containing agar plates. Four strains were obtained with significantly reduced sensitivity to tilmicosin. Studies on the mechanism of resistance of Mycoplasma species to macrolides are limited to mutations in drug-target molecules and the efflux of antibacterial-active substances. It was found that A2058G mutation in domain V of the 23S rRNA gene (M3 and M4) was associated with resistance. No resistance-related differences were found in the ribosomal proteins L4 and L22. Studies have also shown that macrolide-resistant M. hyopneumoniae strains isolated from animals have mutations at positions 2057, 2058, 2059 or 2064 in the 23S rRNA gene [The study screened resistant strains of RNA gene .M. hyopneumoniae was tested, and testing clinical isolates is necessary to confirm our findings.This study had two main limitations. First, all experiments were undertaken in vitro. Although the effects of the drug on bacteria were studied carefully and thoroughly, the effects of the immune system of an animal on microorganisms were not considered. Second, only one standard strain of M. hyopneumoniae. In the in vitro dynamic model, tilmicosin produced a maximal anti- M. hyopneumoniae effect of a 5.11 log10 (CFU/mL) reduction. The antibacterial effect of tilmicosin was concentration-dependent, and the best-fit PK/PD parameters were the AUC0–96 h/MIC (R2 = 0.99). The estimated values for AUC0–96 h/MIC for 2log10 (CFU/mL) reduction and 3log10 (CFU/mL) reduction from baseline were 70.55 h and 96.72 h. The A2058G mutation in region V of the 23S rRNA gene was found in the M3 and M4 strains. These results provide a reliable reference for animal experiments in vivo, and may help in the design of more rational treatments for M. hyopneumoniae infection.This was the first study on the PK/PD relationship of tilmicosin against


Mucormycosis is a rare fungal infection that typically occurs in immunosuppressed patients following chemotherapy or hematopoietic stem cell transplantation.An 11-year-old child with newly developed acute lymphoblastic leukemia suffered from the paroxysmal left chest pain, fever, and hemoptysis.We made a histopathologic diagnosis aided by bronchoscopy techniques, which indicated invasive fungal hyphae that are characteristic of mucormycosis.The patient was treated with oral posaconazole and repeated bronchoscopy interventions for 4 months.The patient's clinical signs and symptoms and signs were no longer present. The prior lung lesions were also no longer observable using radiologic methods, and a 3-month follow-up with the patient showed no signs of mucormycosis recurrence. Finally, the patient was cured, when the cancer chemotherapy was stopped. Close follow-up for another 2 years showed no evidence of recurrence.Mucormycosis diagnosis is difficult as clinical and imaging findings vary. This case demonstrates that posaconazole monotherapy combined with bronchoscopy interventions may be a safe and effective treatment option for pediatric pulmonary mucormycosis. Mucormycosis is often classified by the affected organ system, with 6 forms that include rhinocerebral, pulmonary, cutaneous, gastrointestinal, and disseminated mucormycosis, along with uncommon presentations. Pulmonary mucormycosis is the second most common form and recently increases in incidence of mucorales infections have been reported. However, studies of pediatric patients with mucormycosis are limited. In this report, we present a case of pulmonary mucormycosis in an 11-year-old boy diagnosed with acute lymphoblastic leukemia. The purpose of this study is to characterize the presentation, treatment, and follow-up of pulmonary mucormycosis. The patient was successfully treated with oral posaconazole combined with fiberoptic bronchoscopy, which we believe may be effective in other pediatric pulmonary mucomycosis patients as well.Mucormycetes belong to class Zygomycetes, the order Mucorales, subphylum Mucoromycotina. Rhizopus species are the most common organisms isolated in children.29/L) with elevated C-reactive protein . Galactomannan, 1,3-β-D-glucan, repeat blood, and sputum cultures were negative. Chest CT images revealed a pleural effusion with high-density areas in the left lower lobe  based on bone marrow morphology, immunology, cytogenetics, and molecular biology examinations. Remission induction therapy was initiated in accordance with the Chinese Children's Leukemia Group (CCLG) ALL 2008 protocol (CCLG-ALL 2008). Chest computed tomography (CT) findings were normal on admission  and specific pathologic changes were necessary to make the diagnosis of mucormycosis. Diagnostic methods for mucormycosis infections have advanced in recent years however and now include molecular techniques such as conventional PCR, restriction fragment length polymorphism, DNA sequencing, and real-time PCR. However, the patient refused any of these molecular techniques, as considering the costs.Patients with pulmonary mucormycosis typically present with nonspecific clinical findings including fever, cough, chest pain, and hemoptysis. Disease progression often leads to airway obstruction or invasion of adjacent blood vessels, which can cause asphyxia, thrombosis, tissue necrosis, or mass hemoptysis. Radiographic findings are also typically nonspecific for mucormycosis, as pleural effusions, nodules, halo signs, reverse halo signs, and cavities are associated with many types of fungal infections.–12 and prior studies have shown that children treated by antifungal therapy and surgery have a significantly lower case fatality rate. However, according to the ESCMID/ECMM 2019 guidelines, an early complete surgical treatment for mucormycosis, without additional antifungal therapy, is strongly recommended. Both conventional Amphotericin B and AmB lipid complex are also recommended for first-line antifungal monotherapy mucormycosis. However, amphotericin B toxicity can limit its use and manifests as acute infusion-related reactions and dose-related nephrotoxicity. Additionally, when amphotericin B lipid formulations are not available, posaconazole and isavuconazole are strongly suggested as salvage therapy, or in instances of fungal infections that are refractory or intolerant to amphotericin B.,17 Prophylaxis should be performed with posaconazole delayed release tablets during remission induction chemotherapy for acute myeloid leukaemia or myelodysplastic syndrome. In these indications, posoconazole has played significant role than amphotericin B. Posaconazole was a new azole antifungal drug. Most available clinical data of posaconazole were in the form of case reports.,20 The survival rate of patients who received posaconazole therapy  was higher than those receiving other antifungals in renal transplant recipients with mucormycosis. Posaconazole was available in oral and intravenous formulations, highly lipophilic, and distributed extensively in tissues. Posaconazole was well tolerated in children with a low incidence of adverse effects than that in adults. General safety and efficacy data were lacking for many antifungal drugs and children are typically treated with off-label antifungal drugs in the absence of established guidelines. Despite currently being unlicensed for use in pediatric patients, 2 oral formulations of posaconazole including a gastro-resistant tablet and an oral suspension have been successfully used to prevent and treat invasive fungal disease.,23 To treat systemic fungal infections in children, the recommended posaconazole dose in patients weighing less than 34 kg is 18 to 24 mg/kg daily, given in 4 divided doses. For patients aged 13 years or older or those weighing 34 kg or more, the recommended dose is 800 mg daily, given orally in 4 divided doses. Drug concentrations monitoring is recommended.,24 Here in our research, the patient's weight was less than 34 kg; therefore, we selected the posaconazole dose at 20 mg/kg daily. Optimal individualized treatment is also necessary, as disease heterogeneity results in varying disease course and therapeutic response in patients. In our patient, only pulmonary mucormycosis was diagnosed without any other organs affected, such as rhinocerebral, cutaneous, gastrointestinal, and disseminated mucormycosis.When treating patients with mucormycosis infections, multiple approaches should be used to first remove exacerbating factors for fungal infection. Surgical debridement and antifungal therapies are considered the cornerstone for mucormycosis treatment We show here that oral posaconazole plus bronchoscopy intervention techniques can play a vital role in successful pulmonary mucormycosis management, and limits the need for surgical treatment and intravenous amphotericin B. Importantly, this approach was effective for treating the patient and also reduced healthcare-associated costs. Thus, we posit that monotherapy posaconazole with bronchoscopy interventions may be a safe and effective treatment option for pediatric pulmonary mucormycosis patients.In our patient we also used repeated bronchoscopy techniques which had many diagnostic and therapeutic benefits. BAL and lung tissue removal is known to be beneficial in the diagnosis of many pulmonary diseases.This approach may not be applicable in all situations, as genus and species level identification may sometimes be necessary which requires the use of molecular diagnostic techniques. CT angiography may also be necessary to identify the site and degree of pulmonary thrombosis and embolism. Additionally, once mucor invades major vessels, a surgical lobectomy must be performed to prevent massive hemoptysis and reduce patient mortality.The authors gratefully acknowledge the Microbiology Laboratory of Shandong Provincial Hospital for their efforts.Data curation: Yan Liang.Investigation: Yan Liang, Juan Yang.Methodology: Chunyan Guo, Fengqin Liu.Resources: Yan Liang.Supervision: Xing Chen, Jinrong Wang.Writing – original draft: Yan Liang.Writing – review & editing: Xing Chen.


Hearing loss, a highly prevalent sensory impairment affecting older adults, is a risk factor for cognition decline. However, there were very limited studies on this association in low-resource countries. This study aimed to assess the association between self-reported hearing loss and cognitive decline, and whether engagement in leisure activities moderated this association among older adults in China.Data were obtained from two waves of the nationally representative survey of China Longitudinal Healthy Longevity Survey (CLHLS) between 2011/12–2014. Eight thousand eight hundred forty-four individuals aged 65 years old or above with a dichotomized measure of self-reported hearing status were included. Modified Mini-Mental Examination (MMSE) was used to measure global cognition. Fixed-effects models were used to estimate whether leisure activity engagement moderated the association of self-perceived hearing loss with global cognitive change in the overall sample and sex subsamples.Self-reported hearing loss was associated with cognitive impairment, with an odds ratio of 2.48 . Sex difference in the association of hearing loss and cognitive impairment was not found. Self-reported hearing loss was associated with cognitive decline, with 8% increase in risk compared with those with normal hearing. Frequent engagement in leisure activities moderated the association between hearing loss and cognitive decline for the whole and male samples.Hearing loss was associated with cognitive decline, and leisure activities engagement moderated the association among males rather than females. Dementia is a major global public health concern. It affected approximately 50 million people worldwide . DementiHearing loss, a prevalent chronic condition in older adults, is a key risk factor of cognitive impairment and dementia –12. ApprAlthough the mechanisms underlying the association of hearing loss with cognitive decline and dementia remain unclear, several hypotheses exist . One posOne potential way in modifying the association of hearing loss with cognitive decline and dementia may be through increased participation in activities. Previous studies indicated that older adults with hearing loss were more likely to have smaller social networks . Since tThough much research has documented that hearing loss was associated with cognitive decline and dementia in developed countries, a very limited number of studies have been done in developing countries, and few have focused on the role of engaging in leisurely activity. Maharani et al.  and FranWe obtained the data from the Chinese Longitudinal Healthy Longevity Survey (CLHLS), a dynamic cohort study that collected first-wave data in 1998, along with six follow-up surveys with the replacement of deceased elders in 2000, 2002, 2005, 2008, 2011 and 2014 . The CLHCLHLS included questions on self-reported hearing difficulties only in waves during 2011/12 and 2014. Therefore, the current study adopted two waves from the CLHLS longitudinal data taken during 2011/2012 to 2014. Accordingly, 9765 and 7192 respondents were interviewed in 2011/12 and 2014, respectively. Of the 9765 respondents interviewed in the 2011/12 survey, 6066 were followed up in the 2014 survey, but 820 respondents were lost to follow-up, while 2879 persons died or were already deceased prior to the 2014 follow-up survey. In addition to the 6066 follow-up respondents, 1125 respondents were newly interviewed in 2014 , 39. DetWe restricted the study sample to 14,309 respondents aged ≥65 years who completed the questionnaire on self-reported hearing difficulties and cognitive function from the 2011/12–14 CLHLS sample. Here, 8844 were from the 2011/12 wave, while 5465 were part of the 2014 wave. Individuals were excluded if they were younger than 65 years of age, had died, or failed to respond to the items comprising the outcome variable.Hearing impairment was defined by responses of self-reported hearing difficulty. Participants were asked the following question to check their hearing status (without using hearing aids): “Do you have any difficulty with your hearing?” “YES” was coded as having hearing impairment, while “No” signified not having hearing impairment.Global cognition is measured using the Chinese version of the Mini-Mental State Examination (MMSE). The Chinese version of MMSE is comprised of 23 items and examines four aspects of cognitive function , which was adapted from the original MMSE developed by Folstein et al. . ConsideWe used the frequency of participation in leisure activities to measure the level of engagement in such activities among older adults. Overall, the survey respondents were asked, “Do you now perform the following activities regularly? (please choose one from the frequency on the right)”. Each specific activity was indexed at five levels: 0 = never; 1 = not every month; 2 = not every week; 3 = not every day; and 4 = almost every day. To obtain a general perspective on activity involvement, the total sum of the scores regarding participation frequency in each activity was computed. Details concerning the leisure activities are shown in Additional file Covariates included several sociodemographic characteristics, health behaviors and health conditions that were potentially associated with cognitive function as in previous studies . These vp < 0.005). Likelihood-ratio tests were also performed, which indicated that the fixed-effects model was more appropriate than the mixed-effects model with LR chi2 = − 3871.60 (p = 1.00). Moreover, the cognitive function was initially treated as a binary variable using a cutoff point of 18 . Fixed effect logit models were conducted to estimate the average odds ratios of cognitive decline related to the occurrence of self-perceived hearing loss during the follow-up period. The time-variant variables were adjusted in these models, including age, smoking, alcohol use, self-reported health, hypertension and diabetes. Second, the continuous form of the cognitive function (the summed MMSE scores) was utilized as the dependent variable to estimate the average change in MMSE scores associated with hearing loss during the follow-up period. Hausman tests indicated that fixed effects models were more appropriate in the estimation compared to random effects models . Furthermore, regarding the potential bias caused by the missing data due to participants being deceased or lost to follow-up, the characteristics of the missing data were reported, and a proportional hazard model was done for the mortality sample as well as a regression analysis using an attribution indicator and baseline dataset, as shown in the Descriptive statistics were used for all study variables. Fixed effect models were used to capture the within-individual association between the occurrence of hearing loss and cognitive function decline over time. The fixed effect models are longitudinal models that were applied in the panel data, which confer benefits in treating unobserved confounders and time-invariant variables as a set of fixed parameters to control effects pertaining to both observed and unobserved time-invariant variables as well as the effect of years on the dependent variable . In thisn = 749) to 6% (n = 336) over two waves, while the proportion for hearing loss decreased from 44% (n = 3937) in wave 1 to 39% (n = 2137) in wave 2. The mean scores for all kinds of leisurely activities increased over time from 9.98 to 10.48 over two waves. The average age was 85.08 (SD = 10.98) in wave 1 and 84.74 (SD = 9.99) in wave 2. Details regarding the sample characteristics are shown in Table Table n = 560) of females having cognitive impairment  and 47% (n = 2231) of females having hearing loss  in wave 1. In these two waves, the elderly male cohort were reported to frequently engage in leisurely activities compared to females. Men were observed to have higher average scores in physical outdoor activities, intellectual activities, recreational activities, and social activities, while women participated more in productive activities. Older men attained better education than older women, with 77% (n = 3693) of females being illiterate  in wave 1 and 73%  in wave 2, respectively. Details regarding the characteristics of the elderly sex subgroups are presented in Table Elderly females had a higher MMSE score than males in both waves. The proportion of cognitive impairment and self-reported hearing loss was found to be higher among older females than in males, with 12%  study documented a moderate association between self-reported and audiometric hearing loss and suggested that self-reported hearing loss may indicate hearing disability, the dichotomized measure of self-reported hearing loss does not provide a very accurate and reliable basis to some extent compared with audiometric hearing loss . MoreoveDespite these limitations, strengths of our current are that our results are based on a nationally representative sample, and a longitudinal analysis with fixed-effect model methods to avoid the potentially strong cross-sectional confounding effects and fix problems on time-invariant omitted variables to some extent. If our results are confirmed by a standard audiometric testing protocol and in other independent studies, our findings potentially have important implications in aging health. Our findings show that hearing loss was negatively associated with cognitive decline and of importance to highlight the role of leisure activities engagement in moderating the association. This implies the Chinese policy maker to consider the role of hearing aids and activity participation in cognition or dementia prevention program.Hearing impairment was negatively associated with cognitive function among older adults in China. Leisure activity moderated the impact of hearing loss on cognitive performance among older men rather than women. These findings lend support to the hypothesis that hearing impairment may be a risk factor for cognitive dysfunction in older adults and that hearing aid use or proper leisure activity engagement could possibly reduce this risk. Given the current lack of standardized audiometric assessment and mechanism identification, further studies are needed to estimate whether hearing loss and leisure activity interventions could reduce cognitive decline in older adults.Additional file 1: Table 1. Classification of leisure activities. Table 2. Characteristics at baseline for missing sample (deceased or lost to follow up). Table 3. Cox proportional hazard model for mortality sample. Table 4. Regression for cognitive impairment with an indicator for attrition using sample from 2011/12 wave.