To clarify the risk factors for subsidence of titanium mesh cage (TMC) following single-level anterior cervical corpectomy and fusion (ACCF) to reduce subsidence.n = 31) and non-subsidence groups (n = 42). Medical records and radiological parameters such as age, sex, operation level, segmental angle (SA), cervical sagittal angle (CSA), height of anterior (HAE) and posterior endplate (HPE), ratio of anterior (RAE) and posterior endplate (RPE), the alignment of TMC, the global cervical Hounsfield Units (HU) were analyzed. Clinical results were evaluated using the Japanese Orthopedic Association (JOA) scoring system and the Visual Analog Scale (VAS).The present retrospective cohort study included 73 consecutive patients who underwent single-level ACCF. Patients were divided into subsidence . In multivariate logistic regression analysis, RAE > 1.18 , alignment of TMC > 3°  and the global cervical HU value< 333  were independently associated with subsidence. Linear regression analysis revealed that RAE is significantly positive related to the extent of subsidence .Subsidence occurred in 31 of 73 (42.5%) patients. Comparison between the groups showed significant differences in the value of RAE, the alignment of TMC and the global cervical HU value (Our findings suggest that the value of RAE more than 1.18, alignment of TMC and poor bone mineral density are the risk factors for subsidence. TMC subsidence does not negatively affect the clinical outcomes after operation. Avoiding over expansion of intervertebral height, optimizing placing of TMC and initiation of anti-osteoporosis treatments 6 months prior to surgery might help surgeons to reduce subsidence after ACCF. Anterior cervical corpectomy and fusion (ACCF) has been a highly effective treatment for various pathologies including posterior osteophytes of vertebral, ossified posterior longitudinal ligament (OPLL), prolapse of free nucleus pulposus, and tumors. It has been particularly shown to improve the long-term outcomes of patients as it sufficiently removes spinal cord compressions , 2. AfteThe present retrospective cohort study included 73 consecutive patients who underwent single-level anterior cervical corpectomy and fusion with the use of TMC  between March 2012 and October 2015. To achieve stability, anterior plate  was also employed in all the patients. Written and informed consent has been obtained from each patient. Corpectomies were performed for various pathologies such as posterior osteophytes of vertebral, OPLL and prolapse of free nucleus pulposus. There were 58 patients with cervical spondylotic myelopathy and 15 patients with OPLL. The enrolled patients included 37 males and 36 females and the mean age at surgery was 58.18 years (range 30–77). The level of corpectomy and fusion was as follows: C3 in 1 case, C4 in 14 cases, C5 in 29 cases and C6 in 29 cases. The exclusion criteria were: trauma, infection, tumor, posterior fusion, revision surgery and inability to measure due to the overlap of shoulders. Fifteen patients had one comorbid illness whereas 10 patients had more than one comorbidity. Hypertension was diagnosed in 16 patients, diabetes mellitus in 13 patients, and coronary disease in 6 patients.All operations were performed by an experienced spine surgeon (H. T.). Under general anesthesia, the patient was maintained in the supine position with neck slightly extended. We approached the cervical spine through a standard channel technique in all patients. Once the vertebral levels were carefully identified by intraoperative radiography, the intervertebral space was expanded with a distractor system. Following necessary discectomies, the vertebral bodies were removed and autologous bone chips obtained from corpectomy were used as bone graft material. Then remove the posterior longitudinal ligament and osteophytes; and carefully prepare the endplate in each case to minimize subsidence. After complete decompression, an appropriate length of TMC was selected and filled with autologous bone fragments taken from the excised vertebra, with end caps employed in some cases. The TMC was then inserted into the corpectomy defect and the location of the cage was confirmed using intraoperative fluoroscopy. Finally, an appropriately sized anterior cervical locking plate system was applied in all patients for further stabilization. The patients were instructed to wear Philadelphia collars for 6 weeks after surgery.Anteroposterior, lateral radiographs taken with the patient in a standing position were obtained before operation, immediately after operation. In addition, anteroposterior, lateral, flexion–extension lateral radiographs were taken at 3, 6, 12, 18, 24 months postoperatively and then annually thereafter. All the patients were observed for at least 24 months after surgery. Radiological assessment included segmental angle (SA), cervical sagittal angle (CSA), height of anterior (HAB) and posterior border (HPB), height of anterior (HAE) and posterior endplate (HPE), ratio of anterior (RAE) and posterior endplate (RPE), the alignment of TMC, the extent of subsidence (ES) and the global cervical Hounsfield Units (HU) value. The SA was defined as the angle between the borders of endplates above and below the affected segment  scoring system for functional neurological status and the Visual Analog Scale (VAS) for arm and neck pain before operation, immediately after operation and at the final follow-up. All surgery-related complications including wound infection, dysphonia, dysphagia and implant-related complications were recorded immediately after operation, 3, 6, 12, 18, 24 months postoperatively and then annually thereafter.2 test and Fisher’s exact test. Factors with p < 0.05 were selected into the multivariate logistic model to identify the risk factors for subsidence of TMC. The best cut-offs of parameters were established using the receiver operating characteristics (ROC) curve. A linear regression analysis was performed to evaluate the relationship between the RAE and the extent of subsidence. The p value < 0.05 was considered statistically significant.Statistical evaluation was performed using SPSS software. Results were presented as the mean ± standard deviation. Variables between the subsidence and non-subsidence group were compared using the Student’s t-test, Mann-Whitney U test, χp = 0.001), indicating that the loss of anterior intervertebral height was more significant than that of posterior intervertebral height.No patients were lost to follow-up in this study. All the patients were diagnosed and divided into subsidence group  and non-subsidence group . In the subsidence group, the preoperative HAB and HPB were 54.56 ± 4.81 and 53.81 ± 4.93 mm. Immediately after operation, the HAB and HPB increased to 58.70 ± 4.70 and 56.24 ± 4.60 mm, respectively. At the last follow-up, these values were 54.13 ± 4.42 and 52.92 ± 4.57 mm. A significant difference was observed between the loss of anterior and posterior intervertebral height in the subsidence group(p < 0.001) larger than that of patients in the non-subsidence group (1.12 ± 0.08). The alignment of TMC and the global cervical HU value were also found significantly associated with the risk of subsidence . The fusion rate in the subsidence and non-subsidence group was 27 patients (87.1%) and 39 patients (92.9%) 6 months after the surgery, respectively (p = 0.448). Bone fusion was achieved in all patients at the last follow-up.Comparison between the subsidence and non-subsidence group Table  showed tP < 0.001). However, there were no significant differences of the VAS and JOA scores between the subsidence and non-subsidence groups immediately after operation and at the final follow-up . No patients showed wound infection, dysphonia after surgery. Dysphagia occurred in 3 patients (4.1%), and spontaneously resolved within 3 months postoperatively. Three patients underwent revision surgery for reconstruction failures , 0.739 (p = 0.001), 0.795 (p < 0.001) and the best thresholds for RAE, alignment of TMC, global cervical HU value were 1.18 , 3  and 333 . In multivariate logistic regression analysis, RAE > 1.18 , alignment of TMC > 3°  and the global cervical HU value< 333  were independently associated with subsidence .Moreover, the value of RAE had a significant effect on the extent of subsidence. As evidenced by the simple linear regression Fig. , the subAnterior cervical decompression and fusion is one of the efficient methods of treating degenerative cervical diseases with satisfactory results. When the compression is extended to the vertebral body levels, anterior cervical corpectomy and fusion (ACCF) offers sufficient operating space and decompression extents . HoweverSome studies have shown that many factors may affect subsidence. It is generally accepted that the risk of subsidence may increase in elderly people and in women because of the osteoporosis associated with the ageing process . HoweverIn the present study, the risk of subsidence might increase in patients with over expansion of intervertebral space during operation, which was represented by the value of RAE. In a biomechanical study, increased higher distractive force on the endplate was recorded with lager graft. Furthermore, the distractive force required for graft insertion and subsequent graft compressive force were linearly related . MoreoveThe correlation between subsidence and clinical outcomes remains controversial. Some previous studies , 7, 24 rThere are a series of limitations that need to be addressed regarding this study. First, the main limitation lies in the retrospective nature. Second, it was a single-center study and the number of the cases was limited resulting to being underpowered to find more independent risk factors. Finally, the minimum follow-up of this study was 24 months and therefore the present findings cannot be interpreted as long-term results. Due to these limitations, prospective studies with large samples and long follow-up period are needed to further examine the results in our study.Our findings suggest that the value of RAE more than 1.18, alignment of TMC and poor bone mineral density are the risk factors for subsidence. TMC subsidence does not negatively affect the clinical outcomes after operation. Avoiding over expansion of intervertebral height, optimizing placing of TMC and initiation of anti-osteoporosis treatments 6 months prior to surgery might help surgeons to reduce subsidence after ACCF.Additional file 1. Figure S1. The contact area between the TMC and endplate would be reduced when the cage was placed obliquely into the intervertebral space.


Intranasal ketamine has recently gained interest in human medicine, not only for its sedative, anaesthetic or analgesic properties, but also in the management of treatment resistant depression, where it has been shown to be an effective, fast acting alternative treatment. Since several similarities are reported between human psychiatric disorders and canine anxiety disorders, intranasal ketamine could serve as an alternative treatment for anxiety disordered dogs. However, to the authors knowledge, intranasal administration of ketamine and its pharmacokinetics have never been described in dogs. Therefore, this study aimed to examine the pharmacokinetics, absolute bioavailability and tolerability of intranasal ketamine administration compared with intravenous administration. Seven healthy, adult laboratory Beagle dogs were included in this randomized crossover study. The dogs received 2 mg/kg body weight ketamine intravenously (IV) or intranasally (IN), with a two-week wash-out period. Prior to ketamine administration, dogs were sedated intramuscularly with dexmedetomidine. Venous blood samples were collected at fixed times until 480 min post-administration and ketamine plasma concentrations were determined by liquid chromatography-tandem mass spectrometry. Cardiovascular parameters and sedation scores were recorded at the same time points. Non-compartmental pharmacokinetic analysis revealed a rapid (Tmax = 0.25 ± 0.14 h) and complete IN bioavailability (F = 147.65 ± 49.97%). Elimination half-life was similar between both administration routes . Heart rate and sedation scores were significantly higher at 5 and 10 min following IV administration compared to IN administration, but not at the later time-points. Generally ketamine is administered intravenously, intramuscularly or subcutaneously. Recently, intranasal administration of ketamine has gained attention in human medicine. Intranasal drug administration delivers drugs directly to the central nervous system, bypassing the blood brain barrier and is associated with a fast onset of action . In addition, intranasal drug delivery avoids painful parenteral administration. Intranasal ketamine has been successfully used for sedation and premedication of pediatric patients [–] and in pain management of both children and adults [–]. Another application lies in psychiatry, where intranasal ketamine has been shown to be safe and effective for the treatment of major depressive disorders [–]. Moreover, recently, the US Food and Drug Administration approved S-ketamine nasal spray as an new therapy for treatment resistant depression. Since anxiety disorders in dogs show several similarities with human mood disorders [–], intranasal ketamine could also be a valuable alternative treatment for certain canine behavioural disorders. Brain imaging studies have reported similar abnormalities in regional cerebral blood flow of certain brain regions in dogs with pathological anxiety and in humans suffering from depression and anxiety disorders [–]. Furthermore, altered perfusion of these brain regions following intravenous subanesthetic ketamine administration has been demonstrated both in humans and dogs [–]. Additionally, functional imaging studies have demonstrated altered function of the serotonergic system in several cortical brain regions, both in humans and dogs suffering from mood and anxiety disorders [–]. Since the management of behavioural disorders in dogs is challenging and treatment outcome is often unsatisfactory, there is a need for faster and more effective treatment strategies [–]. Therefore, intranasal ketamine could be a promising adjunctive treatment in the management of canine anxiety disorders, complementary to standard behavioural therapy and pharmacotherapy. The intranasal route could also offer opportunities when ketamine is used for its anesthetic, sedative or analgesic properties in small animals. For example, when intravenous catheter placement is difficult due to medical or behavioural issues or for the ambulatory treatment of chronic pain. In cats, it has been demonstrated that intranasal administration of ketamine-midazolam is effective to induce sedation . Moreover, compared with intramuscular administration of the same ketamine-midazolam combination, there were no differences in the measured parameters associated with sedation except for time to sternal recumbency, which was more rapid in the intranasal group. In rabbits, intranasal ketamine and S-ketamine was successfully used in combination with medetomidine to induce anesthesia . Induction was more rapid compared to rabbits receiving medetomidine-ketamine intramuscularly or subcutaneously . As rabbits are easily stressed, rapid onset of induction is desirable in this species. Intranasal ketamine has also been used in combination with dexmedetomidine and morphine to provide deep sedation sufficient for routine clinical examinations in rabbits . However, to the authors knowledge, intranasal ketamine has never been used in dogs and its pharmacokinetics have never been described. Therefore, the primary objective of the present study was to determine and compare plasma concentrations of ketamine after intravenous and intranasal administration of 2 mg/kg body weight (BW) ketamine in healthy dogs. Secondly, the tolerability of a single intranasal ketamine administration was assessed.Ketamine is a dissociative anesthetic commonly used in veterinary medicine, mainly for induction and maintenance of anesthesia, but also for pain management in the peri- and postoperative period 2 and twice a day, they were allowed to run and play outside in an enclosed play area, enriched with climbing platforms, hiding places and small bushes. In addition, the dogs were regularly walked by students of the Faculty of Veterinary Medicine. Food was withheld for at least 12 hours before the start of the experiments, but water was provided ad libitum.Seven neutered adult laboratory Beagles  were included in a randomized crossover study. The animals were classified as healthy based on general clinical examination. Experiments were approved by the local Ethical Committee of the Faculty of Veterinary Medicine and of Bioscience Engineering, Ghent University (EC 2018_03) and all manipulations were performed according to good animal practice. Welfare of the animals was respected at each time and great care was taken to avoid stress and anxiety. No animals were sacrificed. The dogs were provided by the Small Animal Department of the Faculty of Veterinary Medicine and were purchased from Marshall BioResources . The dogs were socially-housed in small groups (2 to 8 dogs), according to the European and Belgian legislation and received environmental enrichment . The bedding material in the inner part of the housing facility consisted of wood shavings. The dogs had permanent access to an outside area of 15 mwww.randomizer.org). Two routes of administration of ketamine were examined: the intravenous (IV) and the intranasal route (IN). Following a two-week wash-out period, each dog underwent the same protocol but receiving ketamine through a different administration route. Prior to each ketamine administration, the dogs were sedated intramuscular with dexmedetomidine . Following the placement of an IV 22G over-the-needle catheter  in one of the cephalic veins, the dogs were allowed to relax in a quiet room. The first blood sample (T0) was taken 20 minutes after dexmedetomidine injection and was followed by the administration of ketamine.The dogs were randomly allocated to a two-period crossover design by the principal investigator, using an online randomization program  was used for IV injection. IV ketamine was administered at a dose of 2 mg/kg BW through the cephalic catheter, which was flushed with a standardized volume of saline prior to and following injection. For the nasal spray, an aqueous solution containing 2 mg/kg BW racemic ketamine  dissolved in 0.9% NaCl was administered to the mucous membranes of the nose using a mucosal atomization device . The total volume was fixed at 0.5 mL and was divided over the two nostrils. The MAD converted the aqueous solution into a fine mist creating a film coating the nasal mucosa. During the nasal administration, dogs were held in sternal recumbency with the head and neck gently dorsoflexed and were kept in this position for approximately 1 min after nasal delivery. Blood samples (each 2 mL) were collected from the vena jugularis before ketamine administration (T0) and at 2, 5, 10, 20, 30, 60, 120, 240, 360 and 480 min after IV ketamine administration. Following IN administration of ketamine, blood samples were taken at 5, 10, 20, 30, 45, 60, 120, 240, 360 and 480 min after dosing. Blood samples were immediately transferred into tubes containing lithium heparin and separated by centrifugation within 2 h at 3,500 rpm for 10 min. The plasma was harvested and stored at -80°C until analysis.A commercially available racemic ketamine preparation .  to 15 (maximum sedation)  .Together with the scoring of sedation, the following physiological variables were monitored: heart rate, obtained by auscultation, respiratory rate, obtained by direct observation of thoracic excursions and systolic blood pressure, using an ultrasonic Doppler flow detector and an inflatable cuff.. The chromatographic system consisted of a Waters Alliance 2690 separation module and autosampler of the same type . Chromatographic separation was achieved on an Acquity UPLC® BEH C18 column (particle diameter: 1.7 μm) (Waters). The mobile phases were (A) 0.1% formic acid in UPLC water and (B) 0.1% formic acid in methanol. The following gradient elution program was run: 0–1.0 min , 1.0–2.0 min (linear gradient to 5% A), 2.0–5.5 min , 5.5–6.0 (linear gradient to 95% A), 6.0–10.0 min . Flow rate was set at 300 μL/min.Sample preparation was done as described by Devreese et al. (2015) m/z 238.4 > 125.1/179.2 and for the internal standard (d3-ketamine) m/z 241.4 > 125.1/179.2. The method was validated according to a protocol previously described by . The limit of quantification (LOQ) was 20 ng/mL and the linear range was 20–10,000 ng/mL.The LC column effluent was interfaced to a Waters Quattro Premier triple quadrupole mass spectrometer equipped with a heated electrospray ionization (h-ESI) probe operating in the positive ionization mode . Acquisition was performed in the selected reaction monitoring (SRM) mode. The two most intense product ions of the precursor ion were monitored in the SRM mode for quantification and identification, respectively. For ketamine 0-4h area under the plasma concentration-time curve from 0 to 4 hours post-administration; AUC0-∞ area under the plasma concentration-time curve from 0 to infinity; Cmax maximal plasma concentration (IN); C0 plasma concentration at time zero (IV); Tmax time to maximal plasma concentration (IN); Vd volume of distribution; Cl total body clearance; T1/2el terminal elimination half-life; kel elimination rate constant. Vd and Cl values after IN administration were not corrected for IN bioavailability (F). The absolute IN F, expressed as percentage, was calculated according to the following formula:Plasma concentration-time profiles were modelled by non-compartmental analysis (NCA) using Phoenix 8.4 . Plasma concentrations measured at 360 and 480 min after ketamine administration fell below the LOQ and were therefore not taken into account for pharmacokinetic analysis. The following major pharmacokinetic parameters were calculated: AUCA Student t-test was used to compare both administration routes (IV and IN) for each pharmacokinetic parameter. The level of significance was set at 0.05. For the evaluation of heart rate, respiratory rate, systolic blood pressure and sedation scores, linear mixed models were fitted onto the data set using dog as a random effect and time, treatment and their interaction as fixed effects as the data could be assumed to be normally distributed according to the Shapiro-Wilks test. Next, the two administration routes were compared at seven different time points  and tested at the Bonferroni adjusted significance level of 0.05/7 = 0.0071. Measurements at 360 and 480 min were not included in the analysis since plasma concentrations at these time points were also not taken into account for pharmacokinetic analysis.Mean ketamine plasma concentration versus time curves for each administration route are displayed in p <0.001) and 10 min (p <0.001) after ketamine administration, with a higher heart rate following IV administration. At the same time points, significant differences in sedation scores were also observed (p <0.001) with higher sedation scores following IV administration compared to IN administration. No significant differences between the two administration routes were found for the respiratory rate and systolic blood pressure.Concerning the heart rate, significant differences between the two administration routes were found at two time points: 5 , mean Cl after IV administration of ketamine was 3492 ± 1038 mL/h/kg, while volume of distribution at steady state (Vss) was 4060.3 ± 2405.7 mL/kg. Romagnoli et al. (2017) reported separate Cl values for S- (4309.2 ± 768 mL/h/kg) and R-ketamine (4048.8 ± 640.8 mL/h/kg) following IV racemic ketamine administration, with a volume of distribution for the central compartment of 750 ± 370 mL/kg. The study of Sandbaumhüter et al. consisted of two groups of dogs receiving ketamine, with one group anesthetized with sevoflurane and one group sedated with medetomidine. In the sevoflurane group, Cl was 3341.4 ± 664.2 mL/h/kg for R-ketamine and 3490.2 ± 642 mL/h/kg for S-ketamine with Vss of 1630 ± 1170 mL/kg and 1620 ± 1230 mL/kg respectively. In the medetomidine group Cl was 2844.6 ± 474 mL/h/kg for R-ketamine and 2745.6 ± 996.6 mL/h/kg for S-ketamine, with Vss of 3370 ± 660 mL/kg and 3130 ± 660 mL/kg respectively. Elimination half-life was comparable with that reported in the study of Pypendop and Ilkiw (2005) (1.57 ± 0.61 h), but was longer than documented by Romagnoli et al. (2017) (0.26 ± 0.12 h for S-ketamine and 0.26 ± 0.09 h for R-ketamine) and Sandbaumhüter et al. (2016) . The longer half-life of ketamine in the medetomidine group compared with the sevoflurane group in the study of Sandbaumhütter et al. (2017) indicates a slower elimination in the latter group. The lower clearance and subsequent longer half-life in the current study compared to the literature is in agreement with this and could be explained by the fact that the dogs were sedated with dexmedetomidine. Dexmedetomidine and medetomidine are strong inhibitors of the N-demethylation of ketamine to norketamine by competition for the binding site on cytochrome P450 enzymes . Since the formation of norketamine is a major pathway in the biotransformation and hence elimination of ketamine, inhibition of norketamine formation can explain the slower elimination of ketamine when combined with dexmedetomidine. Moreover, since ketamine is a drug with a high hepatic extraction ratio in the dog , another explanation for the observed lower clearance and longer half-life could be the decreased cardiac output and reduced hepatic blood flow associated with dexmedetomidine administration . In agreement with this, dexmedetomidine has also been shown to alter thiopental pharmacokinetics, most probably by decreasing cardiac output and regional blood flow .Total body clearance (Cl) of ketamine was lower compared to other veterinary studies examining the pharmacokinetics of ketamine in dogs , investigating the analgesic effects of a low-dose constant rate infusion of ketamine, showed that ketamine’s antinociceptive effects were evident when plasma concentrations ranged between 220–370 ng/mL. Another study investigating antinociceptive effects of different ketamine infusion regimes found that the serum concentration of ketamine to produce mechanical antinociceptive effects is above 200 ng/mL . In the present study, mean Cmax after IN administration was 1694.48 ng/mL and mean plasma concentrations stayed above 200 ng/mL until 2 h after ketamine administration, indicating antinociceptive effects in this time frame. This implies that a single IN administration of 2 mg/kg BW ketamine has relatively long lasting analgesic effects in dogs. IN ketamine could therefore serve as a valuable alternative when IV catheter placement is difficult. However, these findings should be interpreted with caution, since this study was not designed to assess the analgesic or sedative potential of IN ketamine. Further studies evaluating these clinical effects of IN administered ketamine at different doses are necessary.Few data are available on ketamine’s effective analgesic serum concentrations. A study of Bergadano et al. (2009) , which is in agreement with our observations. Following ketamine administration, heart rate increases in both groups, due to ketamine’s sympathomimetic effects . However, the stimulatory effects of ketamine on the cardiovascular system were attenuated by the dexmedetomidine comedication. Heart rate and sedation scores were higher following IV administration compared to IN administration the first 10 min after ketamine administration. Following IV ketamine administration, heart rate increased to 86 ± 22 bpm, while after intranasal administration, only a small increase in heart rate was observed. These differences in heart rates and sedation scores can be explained by the fact that, during the first 10 min following ketamine administration, plasma concentrations were higher following IV administration compared to IN administration.In both groups heart rate was low before ketamine administration, due to the sedation with dexmedetomidine (47 ± 5 beats per min (bpm) in the IV group and 43 ± 5 bpm in the IN group). It is known that dexmedetomidine causes bradycardia, with normal expected heart rates ranging between 45 and 60 bpm . In another study, examining nasal administration of diazepam in dogs, several dogs developed salivation and reverse sneezing following intranasal administration . Also in cats, a sneezing and snorting reaction following intranasal administration of a ketamine-midazolam combination has been reported . However, in the current study, coughing was also observed in 3 out of 7 dogs and head shaking was present in one dog. These reactions may be due to the bitter taste of ketamine, which could have caused irritation and an unpleasant sensation in the dogs. In human medicine, patients receiving intranasal ketamine frequently complain about a bad taste in the mouth, due to drug runoff into the pharynx .The seven dogs completed the study without clinical significant or serious adverse events. The IN administration was easy to perform and generally well tolerated by the sedated dogs, with short duration mild adverse effects associated with the route of administration. The most frequently observed adverse event in this study was a short sneezing and snorting reaction, which was present in 4 out of 7 dogs. A brief episode of sneezing during or after intranasal administration was also reported in dogs receiving intranasal midazolam . On the other hand, ketamine may reduce the bradycardia and hypotension associated with dexmedetomidine administration . However, the use of co-medication can have an influence on drug disposition and pharmacokinetics.A limitation of this study is the fact that the dogs were sedated with dexmedetomidine prior to the ketamine administration. Dexmedetomidine was administered to avoid behavioural and cardiovascular adverse effects due to the ketamine administration. Dexmedetomidine-ketamine is a frequently used combination in veterinary medicine as the two drugs are complementary to each other and side effects of both drugs are minimized when used together. Dexmedetomidine may attenuate or prevent tachycardia, hypertension, delirium, excitement and hallucinations caused by ketamine IN administration of 2 mg/kg BW ketamine to healthy dogs, sedated with dexmedetomidine, was well tolerated by all of the dogs. Rapid systemic absorption and complete bioavailability of IN ketamine were demonstrated. These findings encourage the use of IN ketamine in veterinary medicine, not only as anesthetic, sedative or analgesic but potentially also in the treatment of dogs with anxiety disorders. Further studies are necessary to assess the clinical effects of IN ketamine in anxiety-disordered dogs.S1 Table. The degree of sedation was assessed with a modified numeric rating scale ranging from 0 (no sedation) to 15 (maximum sedation), adapted from Gurney et al. (2009).(DOCX)Click here for additional data file.S2 TableT: min; HR: beats/min; RR: breaths/min; SAP: mm Hg; NA: not available. The degree of sedation was assessed with a modified numeric rating scale ranging from 0 (no sedation) to 15 (maximum sedation), adapted from Gurney et al. (2009).(DOCX)Click here for additional data file.S3 TableT: min; HR: beats/min; RR: breaths/min; SAP: mm Hg; NA: not available. The degree of sedation was assessed with a modified numeric rating scale ranging from 0 (no sedation) to 15 (maximum sedation), adapted from Gurney et al. (2009).(DOCX)Click here for additional data file.


M. hominis clinical strains associated with various pathological conditions of the urogenital tract has not been explored hitherto. Here we analyzed the genetic diversity and phylogenetic relationships among 59 M. hominis Tunisian clinical isolates, categorized as gynecological infections- or infertility-associated pathotypes. For this purpose, we developed an expanded multilocus sequence typing (eMLST) scheme, combining the previously reported multilocus sequence typing (MLST) loci  with a new selected set of putative virulence genes , referred herein to as multi-virulence-locus sequence typing (MVLST) loci. In doing so, M. hominis population was segregated into two distinct genetic lineages, which were differentially associated with each pathotype. Such a clear dichotomy was supported by several phylogenetic and population genetic analysis tools. Recombination was found to take place, but not sufficient enough to break down the overall clonal population structure of M. hominis, most likely as a result of purifying selection, which accommodated the most fit clones. In sum, and owing to the eMLST scheme described herein, we provide insightful data on the phylogenetics of M. hominis, arguing for the existence of genetically differentiable urogenital pathotypes.To our knowledge, the phylodistribution of  Mycoplasma hominis, which belongs to the Mycoplasmataceae family, in the Mollicutes class, was the first mycoplasma species isolated from humans in 19371. It resides, as a commensal, in the lower urogenital tract of healthy persons. Under certain circumstances, M. hominis can cause a variety of genital infections such as bacterial vaginosis, pelvic inflammatory disease, and cervicitis2. This microorganism seems to be associated with pregnancy complications and neonatal diseases3. In addition, several studies reported the pathogenic role of M. hominis in infertility5. More interestingly, this species has been linked to a wide range of extragenital infections , especially in immunocompromised patients8.M. hominis, several molecular typing systems have been developed. These include Pulse-Field Gel Electrophoresis (PFGE), Restriction Fragment Length Polymorphism (RFLP) analysis, Amplified Fragment Length Polymorphism (AFLP), and Random Amplified Polymorphic DNA (RADP). All these methods have revealed a high degree of both genetic and antigenic heterogeneity among M. hominis strains12. Although informative, these approaches proved to be quite difficult to reproduce and standardize between laboratories13. Recently, a molecular typing method based on Multiple-Locus Variable-Number Tandem Repeat Analysis (MLVA) has been developed14. This method proved discriminatory enough to be used for large epidemiological studies, and could also be useful to address transmission dynamics of M. hominis at the individual level. However, there was no obvious association between the MLVA type and the isolate’s characteristics14. Multilocus Sequence Typing (MLST) assay, targeting housekeeping genes, was found faster and easier to perform, and it exhibited a higher discriminatory power than did other subtyping methods15. It was first proposed in 1998 for the characterization of isolates of the human pathogen Neisseria meningitidis16, and has subsequently been applied to the epidemiologic studies of several bacterial species including mycoplasmas, such as Mycoplasma agalactiae, Mycoplasma bovis, Mycoplasma pneumoniae, and Mycoplasma synoviae19. MLST results are unambiguous, highly reproducible, and portable; and hence, they can be easily comparable between laboratories via worldwide web databases, including MLST (http://www.mlst.net/) and PubMLST (http://pubmlst.org/). An MLST approach was also developed for M. hominis, and was mainly used to evaluate the frequency of recombination events rather than to investigate the evolutionary relationships between strains20. More recently, Jironkin et al., based on the genome sequence of 18 M. hominis isolates, has identified three sets of seven genes that could be used as minimum multilocus sequence typing schemas21.To better understand the epidemiology and the mode of spread of M. hominis isolates according to their involvement in particular pathological conditions. Towards this purpose, we sought here to develop an expanded multilocus sequence typing (eMLST) scheme, which included both housekeeping genes and a set of putative virulence loci, and to assess its ability to distinguish between M. hominis clinical strains associated with gynecological infections or infertility.However, as far as could be ascertained, none of the existing typing approaches has shown the potential to reliably categorize gyrB) to 36 (uvrA), resulting in an allelic variation of 3 (gyrB) to 11 (uvrA and tuf) (Table gyrB) to 0.840 (gap) and the ratio dN/dS ranged from 0 (for gyrB and ftsY) to 0.0798 (for gap). The Tajima’s D values ranged from −0.267 (tuf) to −0.737 (gap) and showed no statistical significance, a finding that might be interpreted as the result of purifying selection (Table The number of polymorphic sites varied from 2 (vaa) to 18 (lmp1). The H value ranged from 0.7 (vaa) to 0.907 (lmp1), and the ratio dN/dS varied from 0.0843 (for p60) to 0.2741 (for lmp3), which suggests that the virulence genes evolved under purifying selection. The Tajima’s D statistic varied from −0.019 (vaa) to −0.632 (lmp1).The data reporting the allelic variations are summarized in Table M. hominis isolates evaluated by MLST were assigned to 21 STs , thus justifying their combination in an eMLST scheme. The SID of the MVLST gene subset was very close to that of eMLST, suggesting its possible use at first intention, inasmuch as the highest overall congruence, as assessed by the adjusted Rand index (AR), was found between eMLST and MVLST (97.4%)  of eMLST was 0.956, which is an ideal cutoff value for a molecular typing methodM. hominis isolates, three phylogenetic trees based on the concatenated gene sequences from the MLST, MVLST, and eMLST data, were obtained using the Neighbor-joining (NJ) method. For the three schemes, two major lineages, A and B, were distinguished, yet not supported by the bootstrap analysis. The dendrogram generated from MLST data showed that the majority of STs were grouped into lineage A (15 STs), and it also revealed that infertility-associated STs did not constitute a distinct lineage from the gynecological infections-associated ones  further confirmed the high level of genetic differentiation between the two lineages.In order to gain deeper insights into the phylodistribution of ges Fig. . Lineageges Fig. . These rges Fig. . The FSTM. hominis clinical strains associated with gynecological infections or infertility, in Tunisia, are genetically differentiable.Taken together, the above-stated data suggested that uvrA, gap, and p60 were network-like with parallelogram structures indicative of recombination, which was statistically confirmed by the phi-test (P < 0.05). By contrast, no recombination could be detected for tuf, lmp1, lmp3, ftsY, p120’, and vaa, whose phi-test P values were >0.05. For gyrB, there were too few informative characters to be used in the phi-test. However, strong evidence for recombination  was observed within and across lineages when the concatenated sequences of eSTs of the entire population were used , indicating that recombination occurred much less frequently than mutation. By contrast, recombination seems to be a major driving evolutionary force in lineage B, since it was found to occur almost 8 times more likely than mutation.A split decomposition analysis, searching evidence for recombination among the ten genes, revealed different structures in the split graphs  of 0.3103 (P < 0.001) for the whole M. hominis population indicated a tendency of linkage disequilibrium between the alleles, a finding minimizing the role of recombination . However, even in case of lineage B, which was found to undergo a high recombination rate, an IAS value of 0.7171 (P < 0.001) indicated persistence of clonally evolving eSTs over time . This finding is in line with previous studies, which revealed the higher incidence of M. hominis29. Despite the relatively small number of isolates included in this study (59 strains), 21 STs, 29 VTs, and 30 eST were distinguished by MLST, MVLST, and eMLST, respectively. Such a finding lends further support to the heterogeneous nature of M. hominis, as reported previously21. Hence, it seems clearly that increased genetic diversity of M. hominis was mainly brought about by virulence genes, as indicated by their average of haplotype diversity of 0.83. This might be the result of their frequent exposure to environmental changes. By contrast, and as expected, the average of haplotype diversity of 0.61 found in M. hominis housekeeping genes was comparable to other mycoplasmas species, such as Mycoplasma bovis (average of 0.64)17 and Mycoplasma hyorhinis (average of 0.651)30. Previous studies have confirmed that virulence gene-based typing schemes (MVLST) afford higher resolution than did MLST, making them the best surrogate approaches to carry out reliable epidemiological investigations32. However, neither MVLST nor MLST data could be unambiguously correlated with the clinical status of the studied strain collections.The ten gene loci targeted in our eMLST scheme were all readily amplified by PCR, making them good candidates for multilocus sequence typing of M. hominis strains and their clear categorization into two distinct lineages, with each corresponding to a specific pathotype. Indeed, all infertility-associated strains fell into one lineage (lineage A), while all gynecological infections-associated strains belonged to another separate lineage (lineage B). Such a clear distinction between the two pathotypes could not be established either by MLST or MVLST gene sets-derived data. This finding was consistent with the conclusions drawn from previous studies, showing that the combination of housekeping and virulence genes provides a more convenient tool for studying the epidemiology of pathogenic bacteria. For example, the combination of MLST and MVLST analyses of Vibrio cholerae was able to differentiate toxigenic from non-toxigenic strains31. Moreover, a correlation between ureaplasma subgroup 2 and genitourinary tract disease outcomes has been revealed by an eMLST scheme33.Here, owing to the development of the eMLST scheme, we have been able to unambiguously elucidate the genetic relatedness of 59 M. hominis pathotypes into two distinct genetic lineages may help to identify pathogenicity determinants which might locate to lineage-specific genetic changes. However, the eMLST scheme developed herein should be tested on larger, clinically and geographically diverse, strain collections to confirm its promising value.The segregation of M. hominis population, recombination could play a role in generating diversity, as previously suggested20. This was particularly noticeable within lineage B , since recombination was found to occur more frequently than mutation, a finding in sharp contrast with the mutation-driven evolution of lineage A (infertitlity-associated pathotype). However, both lineages displayed ISA values indicative of significant linkage disequilibrium between their respective eSTs suggesting a clonal population structure. The observed recombination within lineage B is irreconcilable with a strictly clonal population structure. Theoretically, a clonal population structure under a high rate of recombination can be observed due to non-random and limited sampling, as in this case. Furthermore, estimation of linkage disequilibrium using a single representative from each of the 5 eSTs indicated slightly lower but still significant non-zero ISA values, excluding the hypothesis of an epidemic population structure34. The clonal population structure of M. hominis is also supported by the observation that the same genotypes are repeatedly isolated over the years. For example, eST3, which is the most frequent genotype, was represented by isolates from temporally separated periods (2004–2016), suggesting that this type may be more fit than other potential variants. A recent study has also highlighted the existence of Ureaplasma urealyticum specific epidemic clonal lineages that are more likely to be transmitted between infertile couples and to be associated with clinical manifestations35.Our data indicate that despite an overall clonal evolution of M. hominis could be explained by purifying selection which retrieved deleterious amino acid changes in order to preserve the functionally optimal gene sequence. Indeed, both housekeeping and virulence genes were found to be evolving under purifying selection as suggested by dN/dS ratios and the negative values of Tajima’s D statistic. This finding is not without precedent, since similar results have been reported for Listeria monocytogenes36 and Staphylococcus lugdunensis32. Hence, aside from preserving the most fit pathotype clones, purifying selection might also contribute to maintain gene sequences that define a given pathotype.Yet, the fact that recombination did not disrupt the phylogeny of M. hominis, occurrence of recombination, evolution of M. hominis under purifying selection, and the ability of eMLST to differentiate distinct genetic lineages) was in sharp concordance with previous reports33.The main weakness of this study consists in the limited size of the sample set and the lack of geographical and clinical diversity. However, despite these limitations, the majority of our results  were recovered from infertile patients and 10 strains (17%) from patients suffering from gynecological infections (Supplementary Table M. hominis reference strain PG21 (ATCC 23114) was included in this study.A total of 59 etal.37. Briefly, cells from 2 ml of logarithmic-phase culture of each M. hominis isolate were harvested by centrifugation at 24,000 × g for 20 min. DNA was isolated by treatment with 200 μl of digestion buffer  and proteinase K (100 μg/ml). Lysates were incubated for 1 h at 60 °C, then for 10 min at 100 °C, and finally stored at −20 °C.For PCR, samples were prepared as described by kong gyrB, tuf, ftsY, uvrA, gap) was previously published and used as an MLST scheme20. Here we first screened 8 known M. hominis putative virulence genes, p120, p120’, lmp1, lmp3, vaa, p60, p75, and p80, in order to be included in our eMLST scheme, as a multi-virulence-locus sequence typing (MVLST) gene subset. To be included in the MVLST gene subset, a gene must be (i) amplified in all strains, (ii) amplified and sequenced using a single primer pair, and (iii) endowed with a higher discriminatory power. Only five virulence genes  met the above inclusion criteria. Hence, our eMLST scheme consisted of a 10-loci combination .An eMLST scheme consisting in the combination of housekeeping and putative virulence gene loci was developed. The set of housekeeping genes , 0.4 µM of each primer , 200 µM dNTPs , 1.25 U of Taq DNA polymerase , and 10 µl of treated sample. An Applied Biosystems Thermal Cycler 2720  was set up with a first cycle of denaturation for 5 min at 94 °C, followed by 35 cycles of denaturation at 94 °C for 0.45 min, annealing at 58 °C for 0.45 min, elongation at 72 °C for 1 min, and a final extension step of 7 min at 72 °C. The PCR products were electrophoresed on 1.5% agarose gel and stained with ethidium bromide  to visualize the DNA bands, whose sizes were determined using a 100-bp DNA ladder . PCR products were then purified with both Exonuclease I  and Shrimp Alkaline Phosphatase  as per manufacturer’s instructions. Sequencing reactions were performed using a BigDye Terminator v3.1 cycle sequencing kit , according to the manufacturer’s recommendations, and were run in an ABI 3130 genetic analyzer . Both strands from each PCR amplicon were sequenced twice.The primer sequences of each selected locus are displayed in Supplementary Table 38. For each locus, distinct allele sequences were assigned an arbitrary allele number for identification. Each isolate was characterized by a pattern of numbers defining its allelic profile: sequence type (ST) for MLST  and virulence type (VT) for MVLST  gene sets. An expanded sequence type (eST) was assigned for the 10 eMLST genes of all tested strains.The nucleotide sequences obtained were aligned with reference sequences downloaded from GenBank by using BioEdit Sequence Alignment Editor version 7.2.539. To investigate whether positive or negative selection had occurred at the protein level, the average non-synonymous/synonymous substitution rate ratio (dN/dS) was calculated. dN/dS < 1 indicates that the relative genes were mainly affected by purifying selection during the population evolution, dN/dS = 1 indicates neutral selection, and dN/dS > 1 indicates positive selection. The DnaSP software was also used to calculate Tajima’s D statistic in order to test the neutrality of the observed DNA polymorphisms40.Information for each locus including the number of polymorphic sites and the haplotype diversity (H) were calculated using DnaSP genetic software version 5.1http://www.comparingpartitions.info/). The discriminatory ability was evaluated using the Simpson’s index of diversity, SID, with 95% confidence intervals, as described by Hunter and Gaston41. It refers to the probability that two strains sampled randomly in the collection belong to two different types. An index greater than 0.90 is considered desirable if the typing results are to be interpreted with confidence41. Concordance between eMLST and other typing schemes (MLST or MVLST) was calculated using the Adjusted Rand index (AR)42 and the Adjusted Wallace coefficient (AW)43. AR value close to 0 indicates a lack of congruence between the typing schemes, while value close to 1 indicates a high level of congruence. AW value indicates the probability that two strains classified as the same type by one typing scheme will also be classified as the same one when using the other typing scheme.The discriminatory power, the degree of congruence among different typing schemes, and the statistical analysis were determined via an online tool (44 (http://eburst.mlst.net/). The software determined clonal complexes (CC) from the data, suggesting a consensus ancestral type. To analyse the 59 M. hominis strains, we applied a DLV (Double Locus Variable) criteria for all schemes. This means that the maximum difference allowed within the same CC was that of two loci.To determine closely related genotypes within highly similar clusters, the data was analysed using the BURST version 3 algorithm implemented in the eBURST web server45 method with a Kimura’s two-parameter distance model and 1,000 bootstrap replications using MEGA version 6.0646. STRUCTURE software version 2.3.448 with linkage model was used to identify the ancestral populations for each eST. The K-value that generated the highest posterior probability was used as the probable number of ancestral populations. Ten individual runs per value of K (chosen between 2 and 10) were performed using 200,000 burn-in iterations and 300,000 Markov Chain Monte Carlo (MCMC) iterations. The STRUCTURE Harvester49, which implements the Evanno method, was used to identify the most probable groups (K) that best fit the data. The K-value that generated the highest median posterior probability was used as the probable number of ancestral populations. We estimated the fixation index (FST) to mesure the degree of population differenciation using 100,000 genotypic permutations in Arlequin 3.550. In general, FST values range from 0 (no differentiation) to 1 (complete differentiation). Basically, an FST value of 0.05–0.15 indicates low differentiation, while FST > 0.30 indicates high differentiation51.Three phylogenetic trees from the concatenated sequences of MLST (2361 pb), MVLST (1728 pb), and eMLST (4089 bp) gene sets, and phylogenetic trees based on the allele sequences of each gene, were constructed by the Neighbor-Joining (NJ)The relationships between the eST and the clinical manifestations of all isolates were determined using a Minimum Spanning Tree (MST) analysis performed with BioNumerics software .52. The pairwise homoplasy index (phi) test53 implemented in SplitTree4 for recombination was performed, and the P-value < 0.05 indicated recombination existed. LDhat programme implemented in Recombination Detection Program (RDP) version 4.8354 was used to calculate the per site recombination/mutation ratio (ρ/θ)55 based on the concatenated sequences of ten loci with 100,000 MCMC iterations. Linkage disequilibrium from allelic data was evaluated by calculating the standardized index of association (ISA) using LIAN version 3.757 (http://guanine.evolbio.mpg.de/cgi-bin/lian/lian.cgi.pl/query). IAS value significantly different from 0 indicates that a population is clonal (linkage disequilibrium), while IAS equal to zero indicates a recombining population structure (linkage equilibrium). IAS was tested by using Monte Carlo methods with 1,000 iterations on allelic profile.The split network of eSTs and individual loci were generated by using neighbor-net method using SplitTree4Nucleotide sequences of the ten eMLST allelic variants were submitted to GenBank, and their accession numbers are provided in Supplementary Table Supplementary Information


Clostridium difficile infection (CDI) is one of the most common causes of nosocomial infectious diarrhea in children during anticancer therapy or undergoing hematopoietic stem cell transplantation (HSCT) in Europe. Immunosuppression in these patients is a risk factor for CDI. Malignant diseases, age, acute graft-versus-host disease (aGVHD), HLA mismatch, or use of total body irradiation may play an important role in CDI course. The aim of this study was to evaluate the incidence, course, and outcome of CDI in children treated for malignancy or undergoing HSCT. Between 2012 and 2015, a total number of 1846 patients were treated for malignancy in Polish pediatric oncological centers (PHO group) and 342 underwent transplantation (HSCT group). In PHO group, episodes of CDI occurred in 210 patients (14%). The incidence of CDI was higher in patients with hematological malignancies in comparison to that with solid tumors. Patients with acute myeloblastic leukemia had shorter time to episode of CDI than those with acute lymphoblastic leukemia. Patients over 5 years and treated for acute leukemia had more severe clinical course of disease in PHO group. In HSCT group, CDI occurred in 29 (8%) patients. The incidence of CDI was higher in patients transplanted for acute leukemia. The recurrence rate was 14.7% in PHO and 20.7% in HSCT patients. CDI incidence was highest in patients with hematological malignancies. Most of patients experienced mild CDI. Age < 5 years and diagnosis other than acute leukemia were the positive prognostic factors influencing clinical CDI course. Clostridium difficile (CD) was described for the first time in 1935. It was found in stool specimens from healthy neonates and classified as a commensal [Clostridium difficile infection (CDI) has become one of the most common causes of nosocomial infectious diarrhea in Europe and in the USA, resulting in a high morbidity and mortality among hospitalized patients. Due to common use of broad-spectrum antibiotics, aging of the population, and an increasing number of severe diseases requiring healthcare interventions, problem of CDI is growing [. The rate of CDI has doubled over the last 10 years [C. difficile strains usually produce two toxins: cytotoxin B and enterotoxin—toxin A. Sometimes binary toxin can be present, which is connected with severe CDI course. The main symptoms of infection are diarrhea, vomiting, abdominal pain, and fever [ommensal , 3. Nowa growing –8. The rnd fever –13. Somend fever , 15.CD colitis is a serious complication in immunosuppressed patients which can even end up in septicemia and a fatal outcome –19. ThisBetween 1 January 2012 and 31 December 2015, a total number of 1846 pediatric patients were treated for malignancy and 342 HSCTs were performed in 15 Polish oncological centers. Children were divided into two groups: patients treated with standard chemotherapy and/or radiotherapy for hematological malignancies or solid tumors (PHO group) and transplant recipients (HSCT group). Informed consent was obtained from parents of participants of the study.Patients in PHO and HSCT groups were divided into three subgroups according to primary diagnosis: group C—acute lymphoblastic leukemia (ALL), acute myeloblastic leukemia (AML), Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NHL), and myelodysplastic syndromes (MDS); group L—solid tumors (ST); and group B—other .In PHO group, prophylactic trimethoprim/sulfamethoxazole was administered in all patients starting from the day of diagnosis of malignancy. This prophylaxis was suspended during treatment with methotrexate in ALL patients and during high doses of cytarabine in AML patients. Patients with acute leukemia received mold-active prophylaxis with itraconazole or posaconazole. In the remaining low-risk cohorts, oral fluconazole prophylaxis was used.All HSCT patients were followed starting from day of transplantation up to at least 6 months post transplantation. HSCT patients were hospitalized in single-bed rooms. Antimicrobial prophylaxis consisted of oral ciprofloxacin and colistin from the beginning of the conditioning regimen. Oral or intravenous acyclovir and oral posaconazole were started at the onset of allogenic conditioning regimen, while fluconazole was used in autologous transplantation. Prophylactic trimethoprim/sulfamethoxazole was given to all patients before and after HSCT until at least 1 month after the end of immunosuppression. Antifungal prophylaxis with posaconazole was continued until the end of immunosuppressive therapy. First-line intravenous antibiotic empiric therapy usually included a broad-spectrum beta-lactam and aminoglycoside.C. difficile , without evidence of another infectious cause  [C. difficile, rotavirus, adenovirus, and multi-resistant bacteria, as a part of routine monitoring. Ribotyping and endoscopic examination was not routinely performed; gastroscopy and/or colonoscopy was done in case of suspicion of gut GVHD.CDI was diagnosed in patients having diarrhea (more than three semiliquid or liquid stools within 24 h) and a positive stool test result for the presence of toxigenic oridium) , 14. StoTime to CDI diagnosis in PHO group was defined as a time from the day of beginning of oncological therapy to CDI episode, while in HSCT patients from the day of transplant to CDI episode.CDI recurrence was defined as a new episode of clinically and microbiologically documented CDI separated by complete resolution of clinical symptoms of primary infection with at least 4 weeks interval.Severe CDI course was defined by CDI first-line treatment failure which meant lack of improvement in stool consistency after 3 days, the need to add the second drug, new signs of severe colitis, the need of admission to an intensive care unit, and/or death related to CDI , 18.In case of clinical symptoms and positive A or B toxin or binary toxin of CD before high-dose chemotherapy, treatment with oral metronidazole (3 × 10 mg/kg) was used for 10–14 days. In case of busulfan-containing regimens, oral vancomycin (4 × 10 mg/kg) was usually administered. In case of severe clinical course of CDI, intravenous metronidazole, vancomycin, teicoplanin , or oral rifaximin (3 × 200 mg) were administered, usually in combination therapy.U test for continuous variables. Hazard ratio (HR) and 95% confidence intervals (95%CI) are shown. The cumulative incidences of CDI were assessed using competing risk analysis and Gray’s test. A multivariate logistic regression using the stepwise model selection method was used to evaluate potential risk factors that might influence donor outcome variables. The following risk factors were analyzed in PHO group: age, primary diagnosis, time from diagnosis to CDI, and duration of treatment. The following risk factors were analyzed in HSCT group: age, primary diagnosis, time from diagnosis to CDI, duration of treatment, type of transplant , HLA match, intensity of conditioning (myeloablative vs others), the use of TBI, source of graft , and GVHD. P < 0.05 was regarded as significant.To compare differences between groups, the chi-square test or Fisher exact test was used for categorical variables and the Mann-Whitney n = 1846) included 386 children with ALL, 74 with AML, 114 with NHL, 108 with HL, and 1105 with ST, and 59 were diagnosed with other diseases. HSCT group included 342 transplants . Children were transplanted due to ALL (n = 86), AML (n = 51), NHL/HL (n = 23), severe aplastic anemia (SAA) and other bone marrow failure syndromes (n = 53), ST (n = 62), PID (n = 36), or other diseases (n = 31).PHO group (P < 0.0001)  , as well as in patients with malignant diseases vs non-malignant ones . Majority (24/29) of infected patients underwent myeloablative conditioning. In allogeneic setting, 21/24 patients underwent matched unrelated donor (MUD-HSCT), 2/24 had matched sibling donor (MSD-HSCT), and one patient was given a haploidentical transplantation. Bone marrow and peripheral blood were the transplant source in 7/24 and 17/24 allo-HSCT patients with CDI, respectively.In HSCT group, episodes of CDI were diagnosed in 29 (8%) patients, including 24 (8.9%) in allo-HSCT recipients and 5 (6.7%) in children after auto-HSCT Fig. c. TwentyP < 0.001).The main symptoms in PHO group were diarrhea and fever. The median time to develop CDI in all groups was 6.3 months from the diagnosis of the malignancy and beginning of anticancer therapy. CDI occurred earlier in group C than in group L patients . Median time of CDI treatment was 12 days (range 7–203 days). Anti-CDI treatment was successful in first-line therapy with metronidazole , vancomycin or teicoplanin , and rifaximin . The success rate in second-line treatment was 100% for vancomycin (13/13), rifaximin (8/8), and metronidazole (1/1). Nine patients were not given any medications, and data were not available in nine patients. Several patients were given intravenous immunoglobulins. Probiotics were used rarely due to neutropenia, and no fecal microbiota transplantation (FMT) was performed among PHO patients during the period of the study.The median time to the CDI was shorter in AML patients than in ALL patients  after HSCT. CDI occurred in 82.8% of cases during the first month after transplantation. The median time of treatment of CDI among HSCT recipients was 12 days (range 7–98 days), and it was the same as in PHO group. Treatment was successful in first-line therapy with metronidazole , vancomycin (1/2), and rifaximin (2/3). The second-line treatment was successful in 92.3% cases after the use of vancomycin (6/7) and rifaximin (5/5) or metronidazole (1/1). In HSCT group, probiotics were used rarely and FMT was not performed during the period of the study. In case of hipogammaglobulinemia (< 5 g/L), the majority of patients received immunoglobulin supplementation.P < 0.0001). The efficacy of metronidazole in first-line treatment of CDI was higher in PHO vs that in HSCT .The results of first-line treatment of CDI were better in PHO vs those in HSCT patients . One case of death with concomitant CDI was reported, in 17-year-old boy with SAA  with no signs of aGVHD. The primary cause of death in these cases was a generalized inflammatory response syndrome with multi-organ failure.Clinically and microbiologically documented CDI recurrences were observed in 31/210 (14.8%) PHO and in 6/29 (20.7%) HSCT patients . Patients with diagnosis of acute leukemia had clinically more severe disease . There was no significant impact of sex, time to CDI, and duration of treatment. Among children who underwent HSCT, no factor contributed to severity of disease and outcome, although there was a trend towards more severe clinical course of CDI in patients treated for acute leukemia or older than 5 years  may facilitate with transmission of C. difficile and play an important role in this population.Another important risk factor is age. Among pediatric patients, the risk of CDI varies by age, as older children are regarded to have a lower rate of CDI –30. NeonAccording to our observation, severe clinical course of CDI occurred more often in older group: in PHO group, we observed that younger patients (< 5 years) had milder course of disease. Similar trend was found among HSCT recipients.We did not find HLA matching status, intensity of conditioning, use of TBI, or GVHD to be a significant risk factor for a longer period of treatment or more severe CDI course, as some authors reported , 21, 23.Salmonella, E. coli, norovirus, and colitis ulcerosa and in patients suffering from inflammatory diseases or receiving anti-inflammatory  or antiviral drugs [The main manifestations of CDI infection are diarrhea and abdominal pain which can also be found in patients with other infections such as adenovirus, norovirus, al drugs , 32. Chaal drugs .Treatment algorithm for CDI in hematological patients depends on the severity of infection. Metronidazole remains still the “gold standard” in treatment of CDI. Due to the intolerance or in case of recurrent infection , some authors recommend to use vancomycin. For patients with complicated CDI with ileus or toxic colitis and/or significant abdominal distention, vancomycin delivered orally (500 mg four times per day) and/or per rectum (500 mg in a volume of 500 mL four times a day) plus intravenous metronidazole (500 mg three times a day) is the treatment of choice. Supportive care including intravenous fluid resuscitation and electrolyte replacement should be delivered to all patients with severe CDI. In the absence of significant abdominal distention or ileus, oral or enteral feeding should be continued , 34. In Metronidazole was the first choice in our study population. In severe cases, we used vancomycin in monotherapy, or together with metronidazole in combination therapy. In some cases, we administered also rifaximin or teicoplanin with very good efficacy. The clinical presentation of CDI in pediatric patients in most cases presented as mild-to-moderate diarrhea, abdominal pain, and vomiting. Severe complications of CDI, such as fulminant CDI or pseudomembranous colitis, are not common among children –18. NeitCDI occurred in 14% PHO and 8.5% HSCT patients, including 6.7% patients after autologous HSCT. The highest incidence occurred in patients with hematological malignancies both after transplantation and during conventional chemotherapy. Children with AML have shorter time to CDI than patients with ALL; however, most of patients experienced relatively mild clinical course with adequate response to antibiotic therapy. We confirm metronidazole to be the first choice option in CDI treatment in PHO patients. Almost all children responded well to the treatment with limited adverse effects, observed only in a few patients. CDI rarely contributed to death among children during conventional oncological treatment or in transplant recipients. In non-HSCT children, younger age (< 5 years) and diagnosis other than acute leukemia were the prognostic factors for less severe clinical CDI course.


From 34 patients, a total of 354 afatinib plasma concentration values were available for the population PK analysis. Significant covariates in the population PK model included aspartate aminotransferase and creatinine clearance on CL/F, and age and body mass index on V/F. Results of simulation based on final PK model indicated that hepatic impairment had a significant effect on afatinib levels in plasma after multiple dosing. Afatinib trough plasma concentrations on day 8 were higher in patients with adverse events of grade 3 or higher. The population PK analysis showed that hepatic impairment affected afatinib PK parameters and contributed to the high inter-patient variability and high plasma concentrations of afatinib following multiple treatments.To investigate the exposure–safety relationships of afatinib in Japanese population, we performed population pharmacokinetics (PK) analysis of afatinib in Japanese advanced non-small cell lung cancer patients harboring epidermal growth factor receptor mutation. Plasma samples were collected at 0.5–1, 2–3, 8–12, and 24 h after oral afatinib (40 mg) administration on day 1 and day 8. Plasma afatinib concentrations were determined using high-performance liquid chromatography. Data was analyzed following the population approach and using the software Phoenix EGFR mutation-positive non-small-cell lung cancer (NSCLC)1. Afatinib is a second generation, irreversible ErbB family blocker, which shows inhibitory activity against EGFR, human EGFR 2 (HER2) and 4 (HER4), with IC50 values of 0.5, 14, and 1 nM, respectively4. Afatinib has also been reported to perform significantly better than standard chemotherapy in improving the response rate (RR) and progression-free survival (PFS) of patients with EGFR mutation-positive advanced NSCLC in a first-line setting6. A LUX-lung 7 study showed that the PFS, time-to-treatment failure, and RR with afatinib were superior to those with gefitinib in patients with treatment-naive lung adenocarcinoma having EGFR mutation. However, owing to its phase-IIb nature, this study did not have the power to draw any definite conclusions, and there was no significant difference between the two drugs in terms of the overall survival (OS)8. Recently, the superiority of afatinib to erlotinib was demonstrated in patients with squamous cell lung cancer who had progressed on platinum-based chemotherapy9. The most common adverse events (AEs) for afatinib include rash or acne, diarrhea, and stomatitis whereas hepatic impairment occurs less frequently9. Although these AEs are typically manageable, dose reduction and treatment discontinuation are frequently required. However, comprehensive therapeutic effects are difficult if the drug is not used continuously at its full dose.Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) such as gefitinib, erlotinib, afatinib, and osimertinib are the standard first-line therapies for patients with 4. Afatinib plasma level shows high inter-patient variability5. Therefore, monitoring blood afatinib concentration is important for its dose adjustment and treatment continuation.A pharmacokinetics (PK) analysis showed that plasma concentrations of afatinib peaked at 3–4 h after administration and declined with a half-life of 37 h at steady state10. Freiwald et al. studied the population PK of afatinib based on the data from 927 cancer patients including 764 NSCLC patients in 7 Phase II or III studies. Age, smoking history, alcohol consumption, and presence of liver metastases did not show significant impact on the afatinib exposure11. However, best of our knowledge, we have only identified one report describing population PK model of afatinib which lacks information on the exposure-safety relationship. The present population PK analysis aimed to investigate the exposure-safety relationships of afatinib in Japanese NSCLC patients with EGFR mutation.The population PK approach is often used for PK analysis in a clinical study, as a substitute for or in addition to the standard PK approaches2. A relatively large inter-individual variability was observed in aspartate aminotransferase (AST) (13–65 IU/L), alanine aminotransferase (ALT) (5–77 IU/L), and creatinine clearance (Ccr) (42.3–131.8 mL/min) levels. Among the patients, four had a AST/ALT of higher than limit normal and ten had a Ccr of less than limit normal .A total of 354 afatinib plasma concentration values from 34 patients were used for the population PK analysis. The demographic and baseline characteristics of the participants are described in Table Matrix diagram was performed by the IBM SPSS Statistics 23 software  and the results have been described in Fig. 2; AST 25.3 IU/L; Ccr 79.9 mL/min) who received 40 mg afatinib once daily, the typical CL/F was 20.0 L/h and V/F was 795.8 L. The CV% for each population mean parameter (fixed effects) estimate was ≤43.5%. Inter-individual variability (random effects) was estimated for all parameters  in the one-compartment model. Relatively large inter-individual variability was observed in ka (ωka = 92.9%) and CL/F (ωCL/F = 76.6%). The CV% for each inter-individual variability parameter and the residual variability  was ≤30.3% and 7.2%, respectively. Each median value of the population PK parameter estimates obtained using the bootstrap procedure was similar to that obtained from the original data set, indicating that the final model adequately estimated the model parameters. The 97.5th confidence intervals obtained from bootstrap analysis include the zero. This result was expected because the number of patients (n = 34) was small and large sample size is needed to more precisely estimate inter-individual variability parameters.Fig. The significant covariates in the population PK model included AST and Ccr on CL/F, as well as age and BMI on V/F. This result of covariate analysis indicated that hepatic and renal impairment had a significant effect on the PK parameters of afatinib. No other evaluated covariate was found to significantly affect the PK parameters of afatinib.Figure 2; Ccr 79.9 ml/min). High steady-state afatinib exposure was expected in patients with hepatic impairment. The 95% prediction interval profile for the typical patient indicated that hepatic impairment is a factor inducing large inter-individual variation in plasma concentration of afatinib. These simulated profiles also suggested that no other significant covariate  notably affected the plasma concentration of afatinib following multiple administration   and higher ka value (0.60 1/h) relative to the results of the study by Freiwald et al. . The effects of Ccr and AST on CL/F and the effect of BMI and age on V/F were significant covariates in Japanese NSCLC patients. Although Ccr was identified as the covariate of CL/F in the previous report11, our results also identified hepatic function marker as a covariate. Contrary to this finding, Freiwald et al. did not observe any significant effect of hepatic impairment on afatinib exposure; however, they determined that a priori dose adjustment based on sex, body weight, renal impairment, levels of performance score, alkaline phosphatase, lactate dehydrogenase, and total protein was not necessary. The different results are possibly due to the limited number of patients with hepatic impairment in their study . Simulations using the final population PK model showed that hepatic impairment had a significant impact on the plasma concentrations of afatinib at steady state, but renal impairment, BMI, and age had no effect. This finding is consistent with a previous report describing that moderate-to-severe renal impairment has a minor effect on the PK of afatinib12. In contrast, Schnell et al. reported that mild to moderate hepatic impairment had no clinically relevant effect on the PK of a single dose of afatinib13. These results suggest that hepatic impairment may affect afatinib levels in plasma at steady state after repeated treatment, and AST levels measured during the pre-dosing period may be used to adjust the afatinib dose. Nonetheless, additional population analyses with a larger sample size of patients with hepatic impairment are required.We developed a population PK model based on plasma afatinib concentration data from Japanese NSCLC patients harboring EGFR mutation. Only one population PK model of afatinib has been reported so far and Freiwald et al. reported a phase III study of afatinib or platinum-based doublet chemotherapy in NSCLC patients with EGFR mutation5. In their study, dose reduction to less than 40 mg/day was required for 52% of the participants, with 19% needing more than one dose reduction. Furthermore, high inter-patient variability was observed in the plasma concentrations of afatinib. Dose modification according to patient tolerability reduced excessive afatinib exposure. The inter-patient variability of pre-dose plasma concentration of afatinib in the 40 mg dose group decreased from 85.0% (day 1 of cycle two) to 66.5% (day 1 of cycle three). LUX-Lung 4 was a Japanese single-arm phase II trial of afatinib in patients previously treated with erlotinib and/or gefitinib. Patients received 50 mg/day afatinib orally in this study; 69.4% of patients required dose reduction to 40 mg/day and 35.5% of patients required further dose reduction to 30 mg/day14.Sequist post-hoc analyses of LUX-Lung 3 and 6 reported the effect of afatinib dose reduction on AEs, PK, and PFS15. In these studies, afatinib trough plasma concentrations on day 22 were higher in patients whose dose was subsequently reduced to 30 mg than in those who remained on the 40 mg dose . This result demonstrated that tolerability-guided dose adjustment allows to reduce treatment-related AEs without affecting treatment efficacy. Our study evaluated the PK of afatinib and investigated its relationship with AEs. The trough plasma concentration of afatinib on day 8 was significantly higher in the patients who required dose reduction or interruption than in those who did not (p = 0.0272). Dose reduction and discontinuation of afatinib appeared to be necessary in many cases, owing to toxicities. This dose discontinuation could lead to impairment of efficacy.Furthermore, et al. conducted a phase II study of first line low dose afatinib in advanced NSCLC patients with EGFR mutations16. Afatinib was orally administered at the starting dose of 20 mg/day. If tolerated, this dose was increased in 10 mg increments up to 50 mg/day. The objective RR was 81.8% and 30.4% reported grade 3 or more AEs. The authors reported that low dose of afatinib at initial treatment with dose modification could be considered a better strategy than the treatment with standard dose. The dose–response relationship of cytotoxic anticancer drugs is important. An appropriate treatment efficacy cannot be expected from insufficient dose regimen.Yokoyama 17. In contrast, Imai et al. reported that gefitinib efficacy in patients with EGFR-mutated advanced NSCLC did not differ according to BSA, body weight, or BMI. However, OS was higher in patients with both exon 19 deletion and low BSA than in patients without these characteristics18. These results suggested that BSA- and body weight-based dose adjustments could affect the treatment with EGFR TKIs. Unlike that of parenteral injection, the dose of oral tablets is difficult to adjust. However, the same dose for all patients seems to be related to differences not only in efficacy but also in toxicity. If the high plasma concentrations of afatinib are responsible for the toxicities, therapeutic drug monitoring (TDM) of plasma afatinib concentrations may help prevent toxicities associated to over-exposure.The standard dose of EGFR TKIs is determined irrespective of body size. A retrospective study reported that the median PFS of patients with higher body surface area (BSA) was worse than that of those with lower BSA19. The solubility of afatinib is high throughout the physiological pH range of 1–7.5, and therefore, no interactions with acid-reducing drugs are expected20. Gefitinib and erlotinib are metabolized primarily by cytochrome P450 (CYP)3A4, CYP3A5, and CYP1A121. Therefore, concurrent use of these agents with CYP3A4 inhibitors, such as macrolide antibiotics, azoles, protease inhibitors, or grapefruit juice, may result in increased drug levels and toxicity. However, afatinib is not metabolized by CYP enzymes22. Afatinib is a substrate of P-glycoprotein and an inhibitor of P-glycoprotein and Breast Cancer Resistance Protein22. Therefore, concurrent therapy with P-glycoprotein inducers may decrease afatinib exposure. Recently, Hayashi et al. reported that genetic polymorphisms related to afatinib had influence on the pharmacokinetics in the early phase and were correlated with the severity of diarrhea23. Second, we did not investigate the effect of meals. According to the instruction in the package label, food should be avoided when administering afatinib22. In particular, a high-fat meal decreases afatinib exposure. In this study, afatinib was administered on an empty stomach, in accordance with the package label instructions. Third, few patients with severe hepatic and renal impairment, severe comorbidities, and poor overall condition, were a part of this study. The results of the current population PK analysis show hepatic function marker AST was identified as a covariate of CL/F, but ALT was not, which may be caused by limited sample size. Therefore, our results may not apply to these patients who have serious organ impairments. The fourth, there are several studies24 in which plasma afatinib concentrations were determined by using LS-MS or LC-MS/MS. In this study, we determined plasma afatinib concentrations with the HPLC system, since HPLC system is generally used at clinical sites due to ease of use and cost effectiveness. Though the sensitivity in determination is lower than the LS-MS or LC-MS/MS, plasma concentration of afatinib in patients is adequately measurable by HPLC system. Finally, our PPK model was not confirmed in a different cohort. Although we evaluated our final PPK model using a visual predictive check and nonparametric bootstrap analysis, a validation study is necessary to confirm the predictability of the final model in a validation cohort.There are some caveats in the present study. First, we did not investigate the potential of drug–drug interactions and genetic polymorphisms of drug metabolite. Antacid treatments decrease the serum concentrations of first-generation EGFR TKIs. Administration of acid-reducing agents and first-generation EGFR TKIs to healthy subjects decreases the area under the plasma drug concentration-time curve and the peak plasma concentration of first-generation EGFR TKIsThe PK parameters of afatinib showed high inter-patient variability. The population PK analysis showed that hepatic impairment affected the PK parameters of afatinib. The trough plasma concentrations of afatinib on day 8 were higher in patients who required dose reduction or interruption due to dose-limiting toxicities than in those who did not. TDM is needed in cancer therapy with TKIs to reduce overexposure-related AEs.The population PK analysis was performed using plasma afatinib samples obtained from 34 patients with NSCLC, who started afatinib therapy at the National Hospital Organization Kinki-Chuo Chest Medical Center and the Osaka Habikino Medical Center between August 2014 and May 2016. All patients met the following criteria: cytologically or histologically confirmed as EGFR mutation-positive NSCLC, with no history of treatment with afatinib in previous chemotherapy. Patients who had undergone gefitinib or erlotinib treatments in the past were included. Afatinib was orally administered at a dose of 40 mg/day once daily until disease progression or intolerable toxicity. Physicians were able to increase the afatinib dose up to 50 mg/day when they judged it was appropriate to increase the dose.The protocol of this study was reviewed and approved by the institutional review board (IRB) at National Hospital Organization Kinki-Chuo Chest Medical Center, Osaka Habikino Medical Center, and Setsunan University. The methods were conducted in accordance with the ethical principles listed in the Declaration of Helsinki. Written informed consent was obtained from all participants prior to enrollment. This study was registered with the University Hospital Medical Information Network (UMIN) Clinical Trials Registry Identifier, UMIN000014181.g (4 °C) for 10 min. The plasma samples obtained were stored at −80 °C until analysis. Plasma concentration of afatinib was determined using a high-performance liquid chromatography system . Further details of the assay have been described in our previous report25.Blood samples were collected at 0.5–1, 2–3, 4–6, 8–12, and 24 h after oral administration of afatinib on day 1, and at 0, 0.5–1, 2–3, 4–6, 8–12, and 24 h after the dose on day 8. All blood samples were collected into potassium ethylenediaminetetraacetic acid-containing tubes and centrifuged immediately at 1800 × ® NLMETM Version 7.0 . Non-compartment model analysis was initially performed to compare the PK parameters with those in previous studies26. The area under the plasma concentration–time curve from 0 h to 24 h after dosing (AUC0–24h) was calculated using the linear trapezoidal rule. The elimination half-life (t1/2) was determined by the terminal portion of the plasma concentration–time curve. To determine the population PK parameters and estimate their variability, the first-order conditional estimation with extended least squares (FOCE-ELS) method was used. Different PK models  were selected based on Akaike’s Information Criteria (AIC), goodness-of-fit plots, including observed (OBS) vs. individual predicted concentrations (IPRED), conditional weighted residuals (CWRES) vs. independent variable (IVAR), and CWRES vs. population predicted concentration (PRED), as well as coefficient of variation (CV) in parameter estimates. A drop of 2 in the AIC value was adapted as the cut-off criterion for PK model improvement. Different absorption models including lag time model were also selected because absorption of TKIs is usually complex. The models were compared based on these criteria, and a one-compartment model with first-order absorption was adapted as the final basic PK model.Data was analyzed following the population approach and using a non-linear mixed-effect modeling software, namely Phoenix® NLMETM  and backward elimination (p < 0.001) methods) with −2Log-Likelihood values. The covariate candidates included in the current study were age, height, body weight, BMI, and levels of AST, ALT, serum creatinine (Cre), and Ccr. Individual Ccr values were determined using the Cockcroft-Gault equation. Various covariate effects (power or linear) were initially tested, and continuous covariates were modeled using a linear equation and centered around the mean value of the subjects.Inter-individual variability was modelled exponentially. Different error models  were tested to describe the residual variability accounting for the discrepancies between observed and predicted concentrations. After developing the base population PK model, covariate candidates were evaluated for significance. The covariates were added to the population PK model using the stepwise program in Phoenixth, 50th, and 95th percentiles of plasma concentration of afatinib were simulated to obtain data sets (n = 1000) using the final model parameters. A nonparametric bootstrap procedure (n = 1000) was conducted to compare the parameters with the final model parameters estimated from the original data set, and to obtain the confidence intervals for the model parameters.The final population PK model was evaluated by a visual predictive check and nonparametric bootstrap analysis. For the visual predictive check, the 5th, 50th, and 95th simulated percentiles of afatinib plasma concentration, after oral administration of 40 mg afatinib once daily for 8 days, were simulated (n = 1000) using the obtained fixed- and random-effect parameters. The plasma concentration-time profile in individual patients was simulated using the final population PK model and post hoc estimates of individual PK parameters.The effects of obtained covariates on afatinib plasma concentration were evaluated using final population PK model-based simulations. The 5Toxicity was evaluated based on a numeric grading system, in accordance with the National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE), version 4.0. Two-group comparisons of trough plasma concentration on day 8 were performed using the Wilcoxon rank sum test. Patients were divided into two groups; a grade 0–2 AEs group vs a grade 3–4 AEs group, and a dose reduction/interruption group vs a no change/dose escalation group. The results were considered statistically significant for P < 0.05.The current study was conducted in accordance with the ethical standards of the institutional research committee, and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.Supplementary Information


Atrial fibrillation (AF) is the most common atrial arrhythmia. Our aim was to compare the outcomes of atrial fibrillation treatment with original modified minimally invasive MAZE using monopolar radiofrequency ablation (mi-MAZE) and open surgery MAZE using bipolar radiofrequency ablation (os-MAZE).We searched the associated patients’ information on the medical record system of the First Affiliated Hospital, School of Medicine, Zhejiang University. Statistical Package for Social Sciences (SPSS) was used to analyse the data. The primary outcome is the atrial fibrillation ablation rate 3 months, 6 months, 12 months after operation. And secondary outcome is the postoperative quality of life.P < 0.001).The mi-MAZE group included 42 patients and the os-MAZE group had 65 patients. Three months after surgery, we found that 31 patients (77.5%) in the mi-MAZE group were sinus rhythm and 44 (71.0%) recovered sinus rhythm in the os-MAZE group. We followed up these patients on the phone or in person and scored them on the SF-36 scale. The results were found to be 120.2 ± 8.10 vs 110.6 ± 6.58  between the mi-MAZE group and the os-MAZE group. The postoperative quality of life in mi-MAZE group was higher than that in os-MAZE group. Atrial fibrillation (AF) is the most common atrial arrhythmia, with an activation frequency of 350–600 beats/min. The typical characters of AF are absence of clear P waves, presence of f waves, irregularly irregular ventricular rhythm and QRS complex < 0.12 s. Based on the Global Burden of Diease Database, the global morbidity rate of AF is 0.5% . The risThe etiology and pathophysiological mechanisms of atrial fibrillation are still unclear. The current mainstream hypothesis includes multiple wavelet re-entry hypothesis  and focaRadiofrequency ablation (RF) mainly includes monopolar ablation and bipolar ablation. Martin-Suarez et al. compared bipolar radiofrequency ablation maze, monopolar radiofrequency ablation and interventional therapy . They coThe study included a total of 157 AF patients from two inpatient wards in the First Affiliated Hospital, School of Medicine, Zhejiang University. The minimally invasive MAZE group(mi-MAZE group) included 42 patients who underwent modified minimally invasive MAZE with monopolar radiofrequency ablation and mitral valve surgery from January 1, 2014 to May 31, 2018 , the open surgery MAZE group (os-MAZE group) includes 115 patients underwent traditional bipolar radiofrequency ablation and mitral valve surgery from January 1, 2014 to May 31, 2018 . Patients with a history of atrial fibrillation less than 2 years and a left atrial diameter < 60 mm with mitral valve disease were selected. Eventually 107 AF patients  were included. All patients were diagnosed as AF by conventional 12-lead ECG (electrocardiogram) or 24-h Holter ECG. And echocardiography was used to diagnose mitral valve disease. The primary outcome is the atrial fibrillation ablation rate evaluated by ECG 3 months, 6 months, 12 months after operation. And secondary outcome is the postoperative quality of life, which is assessed by the SF-36 scale [The mi-MAZE group: Our hospital used an original surgery technique for the mi-MAZE group—— After general anesthesia, incision was made in the fourth intercostal space of the right chest. After heparinization, peripheral extracorporeal circulation was established through the femoral artery and vein. Blunt dissection and monopolar ablation of the right superior and inferior pulmonary veins were done during extracorporeal circulation. The left upper and lower pulmonary veins are bluntly separated after heart arrest. The Medtronic Cardioblate flushing radiofrequency system which is connected to the monopolar ablation pen was used for ablation. Left atrial ablation path includes: left and right pulmonary vein ring, the line which connects right superior pulmonary vein and left superior pulmonary vein, the line which connects right lower pulmonary vein and left lower pulmonary vein, the line which connects the incision on interatrial groove and mitral annulus, the line which connects left lower pulmonary vein and mitral annulus, and the line which connects left atrial appendage and left superior pulmonary vein. After the ablation is completed, the valve is replaced or repaired and the left atrial appendage is ligated. The epicardial temporary pacing leads are placed routinely.The os-MAZE group: After general anesthesia, traditional sternotomy was made in the midline. After heparinization, extracorporeal circulation was established through ascending aorta and right atrium. Blunt dissection and bipolar ablation of the right superior and inferior pulmonary veins were done during extracorporeal circulation and the Marshall ligament was cut off. The left upper and lower pulmonary veins are bluntly separated after heart arrest. The Medtronic Cardioblate flushing radiofrequency system which is connected to the bipolar and the monopolar ablation pen was used for ablation. Left atrial ablation lines were the same as in mi-MAZE group. The right atrium ablation lines include: the line which connects superior vena cava and inferior vena cava, the line which connects right atrial anterior wall incision, coronary sinus and tricuspid posterolateral annulus, the line which connects right atrial anterior wall incision and atrial septal fossa, the line which connects the tricuspid anterior leaflet and the right atrial appendage, and the line which connects the tricuspid posterior valve annulus and the incision on right atrial anterior wall. After the ablation is completed, the valve is replaced or repaired and the left atrial appendage is ligated. The epicardial temporary pacing leads were placed routinely.The patients’ surgical approaches were determined by the surgeon.All patients were treated with oral anticoagulation for 3 months. If the mitral valve replacement was not performed and the patient was in normal sinus rhythm, the oral anticoagulant was discontinued the fourth month after surgery.Prevention of arrhythmia is one of the routine treatments. Amiodarone is used as the first choice: intravenous bolus injection of 300 mg, followed by continuous infusion of 1200 mg / 24 h until the first day after surgery. If the patients have no first degree AV (atrioventricular) block, oral administration of amiodarone of 200 mg is given three times a day until discharge. A maintenance regime of 200 mg / day is given for 3 to 6 months. In patients with contraindications to amiodarone, metoprolol or propranolol is administered. Diuretics and isosorbide mononitrate are also routinely used during hospitalization.After their discharge, we followed up the patients by telephone, letter, and outpatient visiting. The effectiveness of the radiofrequency ablation was evaluated by the electrocardiogram and echocardiography 3 months, 6 months and 12 months after surgery. The postoperative quality of life was assessed by the SF-36 (short form 36 questionnaire) scale.P value < 0.05 was considered statistically significant for all analyses.Data were analyzed using SPSS  22.0, using Shapiro-Wilk to test normally distribution. Differences between the mi-MAZE and the os-MAZE group were analyzed using Fisher’s exact test, Pearson’s test, continuous correction test for categorical variables and Student t test and Kolmogorov-Smirnov test for continuous variables. A The study has been approved by the Ethics Committee of the First Affiliated Hospital, School of Medicine, Zhejiang University.P = 0.148). The average age of mi-MAZE group was 56.8 ± 11.29 years and that of the os-MAZE group was 59.5 ± 10.04 years. Heart function was assessed using NYHA classification, and the overall difference between two groups was not significant. Of the 42 patients in the mi-MAZE group, 16 (38.1%) had hypertension, 25 (59.5%) had palpitations, 5 (11.9%) had type 2 diabetes mellitus, and 8 (19.0%) had pulmonary hypertension; One of the 65 patients in the os-MAZE group (1.5%) had COPD, 21 (32.3%) had hypertension, 28 (43.1%) had palpitations, and 14 (21.5%) had type 2 diabetes, 22 (33.8%) with pulmonary hypertension; there was no significant difference between the two groups. Preoperative cardiac ultrasonography measured LAD, LVDd, which were not significantly different. However, average LVEF was significantly lower and LVDs was significantly higher in the mi-MAZE group than that in the os-MAZE group  patients in the mi-MAZE group and 65 (100%) patients in the os-MAZE group recovered sinus rhythm when the operation finished. The two groups also had similar ICU stays and hospital stays Table . During P < 0.001, Table P = 0.028, Table After surgery, we followed up 40 patients (2 patients had wrong telephone numbers) in the mi-MAZE group and 62 patients (3 patients withdraw) in the os-MAZE group. It was found that 31 patients (77.5%) in the mi-MAZE group were sinus rhythm 3 months after operation. However, 1 of the 31 patients became atrial flutter 6 months after operation. After treatment in hospital, he eventually recovered  between the minimally invasive MAZE with monopolar radiofrequency ablation group and the open surgery MAZE with bipolar radiofrequency ablation group. The atrial fibrillation ablation rate of the mi-MAZE group achieved 77.5%. For the 3, 6, and 12 month postoperative SF-36 score, the mi-MAZE group was always significantly higher than the os-MAZE group. If a patient had a history of atrial fibrillation less than 2 years and with a left atrial diameter < 60 mm combined with mitral valve diseases, the minimally invasive monopolar radiofrequency ablation modified MAZE operation would be recommended.