Knowledge of the prevalence of HPV infection among adolescent and early adult girls is essential to determining the best age for the introduction of HPV vaccine, monitoring vaccine efficacy, and giving insight into determinants of persistent high-risk HPV infection, a necessary cause of cervical cancer. Yet, there have been limited studies of HPV infection among adolescent and early adult girls in low-and-middle-income countries.10-DEIA/LiPA25 and analyzed data using Stata 14®.In this cross-sectional study, we randomly selected 205 girls, aged 9–20 years, from 10 schools in central Nigeria. We obtained informed consent and assent, collected data, and trained participants to self-collect vaginal samples using swab stick. We genotyped HPV using SPFThe mean (SD) age of the girls was 14.9 (2.3) years. We found HPV in 13.2% of vaginal swabs. The earliest age at which anyHPV and hrHPV infections were detected was 10 and 12 years respectively. The prevalence of any HPV peaked at 16 and 17 years, hrHPV at 16 years, lrHPV at 17 and 18 years and multiple hrHPV 18 years of age. The prevalence of hrHPV infection was 1.5% among the 9–12 years age group, 2.9% among 13–16 years and 3.4% among 17–20 years old. The commonest hrHPV types detected were 52 (3.9%), 18 (1.5%) and 51 (2.4%). The most common lrHPV types was 6 (2.9%).The prevalence of HPV infection in these urbanized young girls in Nigeria is high and commences after 9 years of age. HPV vaccination in this population should start at 9 years of age or younger to prevent the establishment of persistent HPV infection.The online version contains supplementary material available at 10.1186/s12879-022-07215-7. The prevalence of HPV was 13.2% among adolescent girls in Jos, North-Central Nigeria.The earliest age at vaginal HPV infection was 11 years of age.The most predominant hrHPV type in vaginal swab was HPV 52.These findings point to the need for introduction of HPV vaccine starting at 9 years of age among girls in Nigeria to give maximum protection.The online version contains supplementary material available at 10.1186/s12879-022-07215-7. Human papillomavirus (HPV) infection is the commonest oncogenic virus infection in the world. It is associated with cancers of the cervix, anus, vagina, oropharynx, vulva, oral cavity, penis and larynx , 2. In 2HPV vaccination has been shown to be efficacious for prevention of infection and associated diseases  The vaccSubstantial progress has been made in reducing HPV prevalence in developed countries like the USA  and AustWhile there has been several studies of the prevalence of HPV infection among HIV positive and negative women in Nigeria , 19, datWe randomly selected 15 out of 300 registered high schools in Jos, North Central Nigeria for this study and received permission to conduct the study in 10 (66.7%) of the schools. Between May and November 2016, we contacted 700 female students and their parents, of whom 232 girls and their parents agreed to participate in the study giving a 33.1% response rate. We obtained consent from the parents of girls younger than 18 years of age and assent from the girls. Older girls gave consent in their self-cognizance.We conducted face-to-face interview with the girls in company of female nurses or researchers using structured questionnaires that covered socioeconomic factors, family characteristics, gynecologic history, sexual hygiene and practices. Questionnaire designs were guided by the research questions and literature review. The questionnaires were pre-tested on 10 individuals to evaluate their performance and corrected as required.Remove the swab from the reclosable pack.Gently insert the cotton swab into the vagina while squatting or standing with legs apart.Gently turn the swab three times and remove.Replace the swab in the plastic pack.Repeat this procedure with the second swab stick.Swabs were inserted for about 3 cm and NC was always present to guide the girlsPrior to sample collection, we trained study participants on how to self-obtain vaginal samples using sterile swab sticks—a small cotton swab on a wooden handle packaged in an individual reclosable plastic pack. After the training, each girl was given 2 swab sticks and instructed to:The first collected vulvo-vaginal swab was immediately stored on ice pack in a cooler until transported to the Plateau State Human Virology Research Centre (PLASVIREC) sample repository for storage at −80 °C, usually within three hours of collection. The second swab was immediately applied on Hydrion pH test paper   the colour change was compared with the provided standards.Collected samples were transported to the African Collaborative Centre for Microbiome and Genomics Research (ACCME), Institute of Human Virology, Abuja, Nigeria for HPV detection and genotyping. To prepare the vulvo-vaginal swab for DNA extraction, 1 millilitre of PBS was added to the dry swabs inside the cryovials and vortexed to dislodge the cells from the cotton wool.10  which simultaneously detects HPV types 2, 3, 4, 5, 6, 7, 8, 11, 13, 14, 16, 18, 20, 26, 27, 28, 30, 31, 32, 33, 34, 35, 37, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 55 (re-classified as a subtype of HPV44), 56, 57, 58, 59, 61, 62, 64 (re-classified as a subtype of HPV34), 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 81, 82, 83, 84, 85, 86, 87, 89, 90, 91, 95, 97, 102, 106, 114 and 115. Positive samples were tested with reverse hybridization line probe assay (LiPA25) which can simultaneously identify 25 HPV types  according to the manufacturer’s  instructions. This is a highly sensitive broad-spectrum PCR-based assay for qualitative detection of HPV and suitable for epidemiological studies.DNA extraction was carried out using Cador® Pathogen 96 QIAcube® HT Kit  on QIAcube HT . The DNA was amplified and HPV detected using SPFThe study protocol was approved by the institutional review board of the Jos University Teaching Hospital (JUTH). Written informed consent was obtained from the parent/guardian of participants who were less than 18 year of age and assent obtained from the participants themselves. For participants who were greater than or equal 18 years of age, they provided written informed consent prior to enrolment.p-value < 0.05 as statistically significant.We entered data into REDCap electronic database and analyzed using Stata version 14 . High-risk HPV types were defined as types 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 88, 73 and 82, including the probable hrHPV types and low-risk types were defined as types 6, 11, 40, 42, 43, 44, 54, 61, 68/73, 70, 72, 74, 81 and CP6108 . Univarip-value 0.67). Most of the excluded participants  were from public schools while the remaining ten participants (37.0%) were from private schools. About half of the remaining participants, , were recruited from public schools while 49.3% (101/205) were from private schools . There were no significant differences between girls who were unable to collect their samples and were excluded from the study and those who remained in the study.We excluded 27 participants (11.6%) because they were unable to collect vulvo-vaginal swab leaving 205 participants. The mean age (SD) of the excluded girls was 14.7 (1.97) years while that of the remaining study participants was 14.9 (2.34) years (p = 0.03). Only 9.3% (19/186) of the girls reported positive history of sexual intercourse and this was significantly different (p = 0.01) comparing HPV positive (22.2%) and HPV negative (7.3%) girls. The mean (SD) age at first sexual intercourse for girls who reported positive sexual history was 13.3 (3.58) years, and this was of marginal statistical significance comparing HPV positive (12.0(3.46)) and HPV negative (16.2(1.72) girls (p = 0.07). Most of the girls  who reported sexual intercourse first experienced it at 15 years of age or less and 15.8% (3/19) experienced it before the age of 10 years. Among HPV positive girls, most  of those who have ever had sex had it after 15 years of age compared to only 23.1% (3/13) of HPV negative girls (p = 0.04). Most of the girls with sexual experience  reported one or two lifetime sexual partners while two girls (10.5%) reported more than two sexual partners. All the sexually experienced HPV negative girls had one or two sexual partners while 66.7% (4/6) of the HPV positive girls had one or two sexual partners and the rest (33.3%) have had three or four sexual partners (p = 0.09). Of the girls who had positive sexual history, 11 (57.9%) reported that the sex was forced and without their consent but the prevalence of this not different comparing HPV positive and HPV negative girls (p = 1.00). Among those girls with positive sexual history who reported the age of their sexual partners, most  were older than 21 years of age, as were the sexual partners of all the HPV positive girls compared to only 33.3% (1/3) of the HPV negative girls (p = 0.11).Some 13.2% (27/205) of the girls had any HPV infection. There was no difference in type of school attended and socio-economic status of HPV positive compared with HPV negative girls. Most  of the girls in this study had attained menarche and there was significant difference in menarcheal status comparing HPV positive (88.9%) and HPV negative (68.0%) girls , history of masturbation, use of toys for masturbation, use of condoms, douching, and pH of the vagina comparing HPV positive and HPV negative girls. The girls in this study discussed sex more with others than with their mothers or fathers. Among the others they discussed sex with, the most common were friends, aunts, and sisters. They had low level of knowledge about HPV infection or HPV vaccination Table .Table 1CThe prevalence of any HPV infection in this study was 13.2% (27/205), while that of hrHPV was 59.3% (16/27), lrHPV was 55.6% (15/27), mixed high and low risk HPV was 14.8% (4/27) and multiple hrHPV was 22.2% (6/27). The age-specific and age group prevalence of any HPV among study participants are shown in Fig. 10LiPA25 in this study population. We detected nine hrHPV types and the commonest hrHPV types were 52 (3.9%), 18 (1.5%) and 51 (2.4%. The other hrHPV types detected in this study were 58 (1.0%), 66 (1.0%), 45 (0.5%), and 31 (0.5%). We did not detect any HPV type 16 in this study sample. The most common lrHPV type we identified was type 6 (2.9%) followed by type 44 (1.0%). Other lrHPV types detected were types 68/73 (0.5%), and 74 (0.5). We did not detect lrHPV type 11 among this cohort of adolescent girls. The distribution of these HPV types in relation to the 27 positive participants is shown in Fig. The prevalence of unknown HPV types found in this study population was 2.4%, suggesting substantial prevalence of HPV types beyond those detected using SPFThe age at first detection of HPV types 18, 45, 66, 6, and 74 was 16 years, while HPV types 51, 52, and 58 was detected at 12 years. HPV types 31, 44 and 68/74 were first detected at 19, 15 and 17 years respectively.p-value = 0.04) was significantly associated with risk of anyHPV. There was no statistically significant associations between low or high risk HPV and any other variable in multivariable analyses : 3.11 (1.04–9.34), es Table .Table 2APrevious studies in Nigeria have reported the prevalence of HPV infection in the general population of women and also among HIV positive women , 19. Up The overall prevalence of HPV found in vaginal swab in this population of adolescent females irrespective of the sample type was 13.2%. Globally, the overall prevalence of HPV infection varies according to geographical regions. Our finding is lower than the 24.5% finding among adolescent girls age 14–19 years in the USA before the introduction of HPV vaccination , 66.7% aWe found significant associations between any HPV infection, menarcheal status and history of ever having sex. Association with age at first sex was marginally significant with most HPV positive girls having first had sex at more than 15 years of age compared with HPV negative girls, most of whom had their first sexual experience at 15 years of age or less. Age is an important determinant of HPV vaccination. The WHO recommends vaccine administration as early as age 9 years targeting girls before the onset of sexual activity. In this study, the earliest age at which anyHPV infections was detected was 11 years. HPV Infection peaked at age 16 years and declined with increasing age. This corroborates with previous findings that adolescent girls have higher exposure to HPV infection  and coulThe high HPV infection prevalence of 13.2% recorded in this study is of public health concern and underscores the importance of introduction of HPV vaccines among adolescents in Nigeria for the prevention of HPV infection and associated malignancies. Although more than 90% of all infection with any HPV resolve without treatment within 2 years approximately 10% of infection fail to resolve, resulting in persistent infection with the virus . PersistHPV vaccination is best administered to HPV naïve individuals and before the onset of sexual activities . The earThe major limitation of the study is the small sample size which reduced the power of the analyses, particularly for exploration of associations between group and type-specific HPV infections and their risk factors. Furthermore, this study was carried out among school attending adolescent and early adult girls. The findings may not be wholly generalizable to girls outside of school settings where other factors are likely to play a role. An important avenue for future research may be to consider including girls who are out of school to give a more generalizable outcome.To the best of our knowledge, this is the first HPV study to be conducted in Nigeria focused on young girls who are the primary target group for HPV vaccine. Knowledge about HPV infection prevalence among adolescent girls had provided an important gateway into HPV vaccine introduction in and vaccine success evaluation. Our findings indicate that HPV vaccination starting at 9 year of age would adequately cover the at-risk population in Nigeria. The study lays the foundation for future monitoring of vaccine efficacy and give insight into the determinants of persistent high-risk HPV infection (hrHPV) among the study population.Additional file 1. HPV among adolescent and young adult grils_dataset.


Liver stiffness measurements (LSM) have been serially assessed 1, 3, and 5 years after HCV clearance in 655 patients who have been treated with DAAs. p < 0.0001) at the end of the follow-up. Likewise, LSM decreased in cirrhotics from 29.66 ± 14.25 kPa to 22.50 ± 11.16 kPa (p < 0.0001). The proportions of F1, F2, F3, and F4 patients at the baseline were 17.7%, 17.9%, 16.6%, and 47.8%, which became 56.5%, 4.1%, 4.9%, and 34.5%, respectively, with a substantial reversal of cirrhosis in 87 patients (27.7%) at the end of follow-up.  The mean age was 51.44 ± 10 years. 73% of patients were males. 48% were cirrhotics. In noncirrhotics, the mean LSM was significantly decreased from 8.29 ± 2.3 kPa to 4.03 ± 1.0 kPa (F0–F2) usually improves shortly after treatment, while measurements reflecting advanced fibrosis (F3–F4) take a longer time to regress to lower fibrosis stages. There was an overall significant regression of liver stiffness in all patients after sustained HCV eradication. Liver stiffness reflecting mild fibrosis ( Hepatitis C virus (HCV) infection is a major worldwide health problem. Around 70% of persons acutely infected with HCV will develop chronic HCV infection. The global estimate of chronic HCV is about 71 million people (1% of the world population). The progression of hepatic fibrosis with excess deposition of an extracellular matrix is the most serious consequence of chronic hepatitis if left untreated. 15–30% of those with chronic liver injury will ultimately end in liver cirrhosis within 20 years with risk of cirrhosis-related complications including portal hypertension and hepatocellular carcinoma (HCC). The overall HCV related mortality is about 400,000 deaths every year [HCV treatment has been revolutionized since the introduction of direct acting antiviral agents (DAAs) in 2014. DAAs have been associated with high rates of sustained virological response (SVR) exceeding 95% with an excellent safety and tolerability profile .However, the primary goal of HCV therapy is to achieve SVR, and the improvement of liver fibrosis remains the most important prognostic indicator. Therefore, it is necessary to determine how far virological clearance is associated with fibrosis regression .Transient elastography is the most widely validated and approved noninvasive technique used to assess liver fibrosis with adequate accuracy and reproducibility. Although liver biopsy is the gold standard for the evaluation of liver fibrosis, it has been gradually replaced by TE because of its potential complications  and poor tolerability, particularly if serially required –10.In many recent studies –19, liveIn the current study, we aimed to evaluate the liver stiffness serially over 5 years after the successful eradication of HCV among Egyptian patients treated with DAAs.The current study was conducted on 703 patients with chronic HCV who were treated with different interferon-free regimens of DAAs and successfully achieved SVR. Patients were recruited from hepatitis C virology clinic, national liver institute, Egypt. A written informed consent was obtained from each patient before inclusion. The study protocol complied with the ethical principles of Declaration of Helsinki (1975) and has been approved by the Local Institutional Review Board of National Liver Institute, Menoufia University.2, body mass index (BMI) of more than 32, alcohol abuse, HCC, extrahepatic malignancy, immunosuppressive therapy, solid organ transplantation, and poorly controlled diabetes mellitus (HbA1c more than 9%).The following categories of patients have been ruled out: cirrhosis with Child–Pugh score more than 7, HBV, autoimmune hepatitis, chronic kidney disease with eGRF <30 ml/min/1.73 mPotential concurrent etiologies of chronic liver disease were excluded through a panel of investigations including HBs Ag, anti-HBc, and autoimmune profile , anti-smooth muscle antibodies (SMA), and anti-liver kidney microsomal antibodies (anti-LKM) , 21.DAA regimens were given according to the recommendations of the European Association for the Study of the Liver  and the protocol approved by the Egyptian National Committee for the Control of Viral Hepatitis (NCCVH), 2015 , 23: sofSustained virological clearance was considered when HCV RNA becomes undetectable at week 12 after the completion of DAA therapy (SVR-12).Patients have been prospectively followed for changes in liver stiffness using the Fibroscan 502 device  at the time of achieving sustained virological clearance (SVR-12) and then at 1, 3, and 5 years. The standard M probe was basically used. Readings were expressed in kilopascals (kPa), and those with a success rate  more than 60% and interquartile range less than 30% were only considered. Liver stiffness measurement (LSM) represents the median of at least 10 valid measurements.F0-1) was considered when LSM is less than 7.1 kPa, while F2, F3, and F4 (cirrhosis) were considered when LSM is (7.1 − 9.4), (9.5 − 12.4), and (≥12.5) kPa, respectively [While the patient is lying in the dorsal decubitus with the right arm maximally abducted, acquisitions were applied through the intercostal spaces. The tip of the probe was covered with a lubricant gel before being applied to the intercostal skin. The selected liver portion to apply acquisitions had been estimated by the operator to be at least 6 cm thick and free of large vascular structures. The software determined whether each measurement was successful or not. Nonsignificant fibrosis . Graphical illustrations were created using Microsoft Excel 2013. The descriptive statistics for quantitative variables were presented as mean and SD, while qualitative variables were presented as numbers and percent. The comparison of qualitative data was performed using the chi-square test or Fisher's exact test, where appropriate. The continuous variables across time were compared using the paired F3), 117 patients (17.9%) with mild fibrosis (F2), and 116 patients (17.7%) with minimal or insignificant fibrosis (F0-1). 66.4% of the cirrhotic patients were Child–Pugh class A. The rest were Child–Pugh class B (33.6%). The mean LSM was 29.66 ± 14.25 kPa in cirrhotics and 8.29 ± 2.31 kPa in noncirrhotics.Among the initially eligible patients who fulfill the inclusion and exclusion criteria, missed follow-up has been reported in 48 patients. Therefore, the ultimate number of included patients who completed the follow-up and underwent final analysis was 655 patients . The basp < 0.0001). In noncirrhotics, LSM steadily decreased over time where it became 4.03 ± 1.0 kPa (p < 0.0001) at the 5th year with a 51.4% decrease as compared with the baseline value. In the same stream, LSM significantly declined in cirrhotic patients till it became 22.50 ± 11.16 kPa at the 5th year with a 24.1% decrease as compared with the baseline LSM. Although LSM decreased significantly in both noncirrhotic and cirrhotic patients, the rate of decline was greater, faster, and more significant in noncirrhotics than in cirrhotics at the earlier time points of the follow-up (at SVR and 1 year). Beyond the first year, cirrhotic patients attained more prominent trends of decline till the end of the follow-up. The detailed ANOVA statistics and post hoc analysis are shown in In general, there were significant serial decline in LSM in all patients at each time point of the follow-up along the course of the study (p < 0.0001). Unlikely, high significant comparisons in cirrhotic patients were only noted when comparing measurements at relatively remote time points, i.e., LSM at baseline with LSM at 3 and 5 years and LSM at SVR versus LSM at 3 and 5 years.In noncirrhotics, the mean decline in LSM, as compared with the baseline values, was 2.4, 3.4, 4, and 4.3 kPa at SVR, 1, 3, and 5 years, respectively. On the other hand, it was 1.3, 3.8, 5.9, and 7.2 kPa, consecutively, in cirrhotic patients. Detailed comparative statistics between mean difference changes in LSM at each time point and other time points are shown in The changes in the proportions of each fibrosis stage in the studied patients at each time point of the follow-up are shown in .The number of cirrhotic patients at the baseline was 313. This number decreased to 226 at the end of the follow-up. This means that cirrhosis has regressed to lower fibrosis stages in 87 patients (27.7%).F3 patients has regressed from 16.6% at the baseline to 4.9% at the end of the follow-up with transition to lower fibrosis stages in 70.5% of these patients. Regression to lower fibrosis stages was more prominent in F2 patients where 77.1% of these patients have regressed to F0-F1.The ratio of F4, F3, and F2. This could be referred to HCV eradication; the basic underlying etiology of liver fibrosis and lack of any additive liver insult.As being noted, the incremental increase in the ratio of F0-1 over the follow-up period from 17.7% at the baseline to 56.5% came in parallel with regression in the proportions of F0-F1) and mild fibrosis (F2), while trivial insignificant progression  has been reported in only 5 patients (4.6%) with F3 fibrosis at the end of follow-up. Yet, none of these patients has progressed to F4. These patients remained stable with no clinical complications till the end of the study.Noteworthy, we did not report any increase in LSM in patients with minimal (p=0.09) at the end of the follow-up. Of these patients, 6 had clinical and laboratory deterioration, 5 of them died, and 1 received liver transplantation. Two patients developed HCC. The remaining 20 patients were clinically stable till the end of the follow-up without any features of liver decompensation.Among cirrhotic patients, 28 (8.9%) had a nonsignificant increase in LSM  . Of those patients, 5 successfully managed with locoregional therapy, and 3 passed away due to aggressive and infiltrative tumor associated with rapid clinical deterioration.The overall mortality has been reported in 11 patients (1.65%): 5 due to liver deterioration and failure, 3 due to aggressive HCC, and 3 due to non-liver-related causes (2 cardiopulmonary diseases and 1 cerebrovascular stroke).HCV is a major leading cause of liver cirrhosis and its related complications. The annual rates of liver decompensation, transplantation, and HCC attributed to HCV are 6.37%, 4.58%, and 3.36%, respectively .Egypt has the highest HCV burden in the world –28. SincDespite this big achievement in the primary goal of treatment, which is viral clearance, we have no confirmed data about the secondary goal, which is minimizing or preventing complications. Doubtless, this secondary goal is closely reflected by the regression of liver fibrosis. Unfortunately, we have no definitive data about long-term changes in liver fibrosis, particularly in patients with advanced fibrosis and cirrhosis, after successful eradication of HCV. This was the basic motivation to perform the current study.p < 0.001). Furthermore, posthoc analysis revealed a significant decline when comparing LSM at any given time point with other time points of the follow-up except when comparing LSM at the 4th and 5th years (p=0.44). The mean overall decline in LSM in those patients at the end of the follow-up was 4.26 kPa, representing about 51% of the baseline value (4.03 ± 1.0 versus 8.29 ± 2.31). Notably, 78.6% (3.35 kPa) of this decline has been achieved in the first year posttreatment (4.94 ± 1.89 versus 8.29 ± 2.31). This means that liver fibrosis, whenever frank cirrhosis is not reached, could be resolved shortly after successful treatment.In the view of the available literature, we will discuss the principal findings of the present study. Globally, there was a significant decrease in LSM in noncirrhotics . However, the posthoc analysis was statistically nonsignificant when comparing LSM at each two successive time points , while it was significant when comparing LSM at nonsuccessive and remote time points . This means that changes in LSM, in those patients, take longer time to attain a statistically significant value. Therefore, a long-term follow-up of liver stiffness, when intended, should be optimized to be at longer intervals of at least 3 years. However, this should not defer regular periodic HCC surveillance as these patients are still at a risk of developing HCC.In addition, there was an overall significant decline in LSM among cirrhotic patients  has been achieved in the first year, 27.6% (2.1 kPa) in the 3rd year, and 17.1% (1.3 kPa) in the 5th year. It is worthy to note that the overall decline in LSM over 5 years was 24% of the baseline value. This indicates that the extracellular matrix (ECM) in those patients is more compact with excess interlacing fibers, which need longer time to be degraded. The actual time required for complete degradation is not yet established. However, undoubtedly the higher the baseline LSM, the longer the time required for the degradation of ECM.Furthermore, with that reported decline in LSM of 7.16 kPa, we could expect the reversal of cirrhosis in patients with baseline LSM of less than 20 kPa within 5 years after successful treatment.p < 0.0001) over a follow-up of 96 weeks [p < 0.001) 48 weeks after treatment. However, this study was conducted on HCV/HIV coinfected patients, and 52% of them have undergone treatment. Additionally, the sample size was so limited (31 patients). Another cohort by Bachofner et al. [p < 0.001) within 18 months after DAA treatment representing 34.2% decline in the baseline LSM; however, this study was limited by being retrospective.Data from many studies with a relatively longer follow-up came in agreement with our findings; first, a German study by Pietsch et al., LSM has significantly decreased from 13.1 to 7.9 kPa , F2, and F3 at the baseline were 17.7%, 17.9%, and 16.6%, respectively. These ratios became 56.5%, 4.1%, and 4.9% at the 5th year, respectively. The achieved regression in liver stiffness has been reflected as increased ratios of lower fibrosis stages at the expense of ratios of higher fibrosis stages.Changes in the ratios representing different stages of fibrosis along the follow-up were notable and favorable. LSM concordant to F4 patients at baseline were 313 (47.8%) and became 226 (34.5%) at the 5th year, which means that cirrhosis has been regressed to lower stages of fibrosis in 87 patients (27.8%). In a cohort conducted on 304 patients treated with DAAs, there was about 20% decrease in the proportion of F4 patients (p < 0.0001) at 24 weeks after treatment [F4 patients.In the same stream, the number of reatment . Our resF4 patients over a follow-up of 2 years. These findings are close to what we found at 3 years, where cirrhosis has been reversed in 234 (25.2%) patients. In addition, they reported progression from F3 to F4 in 11.4% of patients. This is inconsistent with our results where we reported a trivial progression of LSM in only 4.6% of F3 patients without transition to F4. These differences may be referred to variability in the sample size (F3 patients were 631 in the Shiha study versus 109 in the current study). In addition, the reference range of LSM for each fibrosis stage was quite different in both studies (F3 was 10.2 − 16.3 kPa in Shiha cohort versus 9.5 − 12.4 kPa in the current study while F4 was >16.3 kPa versus >12.4 kPa in our study). In addition, Pietsch et al. reported a higher rate (17%) of fibrosis progression over a follow-up of 96 weeks. However, this study was limited by the relatively small sample size (143 patients). In addition, the increase in LSM was nonlinear with an initial decrease at week 24 followed by an increase between weeks 24 and 96, and the authors could not provide a convincing explanation for this [An Egyptian study conducted by Shiha et al.  showed tfor this .p=0.09), we could not find an explanation for this, particularly, since we had excluded all major possible concomitant causes that could affect the liver, basically HBV, autoimmune hepatitis, hepatotoxic drugs, and alcohol abuse. In addition, patients with a raised suspicion of nonalcoholic fatty liver disease (NAFLD) were also excluded, including obese patients and with poorly controlled diabetes. Surprisingly, BMI has decreased at the end of the study when compared with the baseline value (27.9 ± 1.5 kg/m2 versus 28.3 ± 1.4 kg/m2). Despite this, liver histology remains a mandatory requirement to exclude NAFLD, in view of a recent term of “lean NAFLD,” which indicates the possibility of NAFLD to occur in patients with normal BMI.In the current study, we would like to clarify that we have reported an unexpected increase in liver stiffness in 28 patients among sustained responders. This increase was in cirrhotics only. Although this increase was statistically insignificant  [p < 0.001) [The reported rate of HCC development after the treatment of HCV is quite variable among different studies as some of these reports came from retrospective single-center studies and due to variability in the sample size and duration of the follow-up periods. In addition, some of these studies included cirrhotic patients only, while others included both cirrhotic and noncirrhotic patients. Furthermore, some studies were carried out on responders only and others included all patients with or without SVR. –41. In tp=0.008) . In addi< 0.001) . Among 2< 0.001) . AnotherAll these reports were consistent with our results. In our study, the rate of HCC development was 1.2% of all patients and 2.6% of cirrhotic patients. This rate is quite lower than that reported in some of the previous studies. This difference could be attributed to the heterogeneity of inclusion criteria in our cohort and these studies where we selectively included sustained responders. In addition, cirrhotic patients with Child–Pugh score >7 (late class B and class C), who are more prone to develop HCC, were not included in our study. This could share in part the reported lower rate of de novo HCC among our patients.Indeed, liver fibrosis is an important risk factor for HCC development, where patients with established cirrhosis are at a higher risk than those in lower fibrosis stages , 43. TheOne of the basic limitations of the present study was the inability to include patients with advanced and decompensated cirrhosis, whereas these patients were ineligible to receive the available DAAs at the time of performing the study. Another limitation is unfeasibility to perform paired histological assessment to verify and confirm that the improvement in LSM reflects a true reversal of fibrosis and regaining of normal lobular parenchymal architecture and does not represent a mere reflection of ameliorated liver inflammation.Finally, we can conclude that HCV elimination after treatment with DAAs is generally associated with a significant regression of hepatic fibrosis. Early and mild fibrosis usually improves and could substantially vanish at earlier time; however, advanced fibrosis and cirrhosis take longer time periods to achieve significant improvement. In addition, we have to emphasize that successful viral clearance in cirrhotic patients does not preclude the risk for developing HCC and does not obviate the need for continued HCC surveillance.


Introduction: Fibroadenomas (FAs) and phyllodes tumors (PTs) are less prevalent but allied to have malignant transformation in many instances. It is a challenge to diagnose the phyllodes by conventional trucut biopsy technique.Objective: To evaluate the histological characteristics of tumors labeled as fibroepithelial lesions of breast tissues on trucut biopsy and compare with a diagnosis on excision biopsy.Methods and materials: It was a descriptive cross-sectional study that was carried out in Shaukat Khanum Memorial Hospital and Research Centre within six years from January 2015 to January 2021. In trucut samples, stromal cellularity, stromal cell nuclear atypia, mitotic count, stromal overgrowth, the enhancement of stromal cellularity adjacent to epithelium were scrutinized. In each category, the activity was seen as absent, mild, moderate, or severe. Mitotic activity was graded as 0-1, 0-5, 5-10, >10.Results: A total of 125 patients were registered for the study. The mean age of patients in our study was 33.86 ± 9.95 years. The mean size was 41.02 ± 27.38 mm with a mean lump duration of 7.52 ± 5.34 months. In the FA group, the trucut sampling report showed the stromal cellularity as mild in 62 (69.7%) and stromal cell nuclear atypia as absent in 68 (76.4%) cases. But in the phyllodes tumor group, the stromal cellularity was severe in 10 (27.8%) patients and stromal cell nuclear atypia was severe in five (13.9%). The ultimate outcome showed that 89 (71.2%) patients had FA and 36 (28.8%) had PT at excision.Conclusion: Assessment of tumor size, stromal cellularity, mitoses, and enhancement of stromal cellularity adjacent to epithelium are useful markers for diagnosing the PT in trucut needle biopsy. In women, breast cancer is the most common of all tumors . Breast PTs are uncommon fibroepithelial tumors that are cataloged as benign, borderline, and malignant histologically. Regardless of the fact that only around 10% of these tumors are malignant, even benign tumors may increase in size and relapse. Because of their proclivity for local recurrence, patients are generally treated by surgical excision with negative surgical margins ,5.An FA is a painless, firm, mobile breast lump that frequently affects women between 20 and 35 years. Bilateral FAs and multiple FAs are frequently reported in many cases. Screening mammography could detect the nonpalpable FAs and sclerosis with “popcorn” calcifications. Ultrasound assists in detecting and portraying FAs precisely ,7.In many institutions, Excision biopsy is preferred over trucut biopsy for phyllodes . A signiDespite the use of numerous diagnostic markers, the differential diagnosis of fibroepithelial tissues with trucut biopsy is sometimes challenging and imprecise. And it happens particularly due to the heterogeneity of the stromal component and enhanced cellularity of tumors . So, patRegarding the management of patients, it is either observation and monitoring of the size of the tumor over a period of time or excision at a given moment. So, it is crucial to determine the reliable markers on trucut biopsy, that could help in understanding the benign or malignant nature of an FA. The aim of our study was to evaluate the clinical and histological characteristics of tumors that were labeled as fibroepithelial lesions after trucut tissue biopsy and comparing with diagnosis based on excision biopsy.It was a descriptive cross-sectional study, carried out in Shaukat Khanum Memorial Hospital and Research Centre from January 2015 to January 2021. We included all the patients who underwent surgical excision of breast lesions after trucut biopsy result of a fibroepithelial tumor after IRB approval of Shaukat Khanum memorial cancer hospital and research center (SKMCH &RC) IRB # EX-30-12-19-02. Patients with no excisional biopsy were excluded from the study.Histopathological evaluation was performed by two pathologists of the hospital in the department of histopathology. On trucut biopsy samples, we executed the activities of the stromal cellularity, stromal cell nuclear atypia, mitotic count, stromal overgrowth, the enhancement of stromal cellularity adjacent to the epithelium in all samples. In each category, these (above mentioned) activities were seen as absent, mild, moderate, or severe. Mitotic activity was graded as 0-1, 5-10, >10. While in the enhancement of stromal cellularity adjacent to the epithelium, we recognized the absence, not applicable, or the presence of activity. “Not applicable” applied to cases in which the trucut core was not sufficient to differentiate the epithelium and stromal activity. In excision samples, FA and phyllodes were diagnosed based on specific markers. We strained to identify the parameters in trucut biopsy, which were corresponding to the diagnosis based on the excisional biopsy.Each category was graded based on peculiar histopathological features. For stromal cellularity, most cellular area of the slide was assessed. Approximately twice the cellularity of that of normal perilobular stroma, with none or rare stromal nuclei, was labeled as mild and within close contiguity with many stromal cells, nuclei were labeled as Marked stromal cellularity. The intermediate degree was between mild and marked with cellularity more than twice that of normal perilobular stroma and scattered stromal nuclei.2. Stromal overgrowth was defined as the presence of stroma without epithelium in any low-power field to high power field (×40). The relative proportion of stroma to the epithelium was estimated semi-quantitatively as the overall relative percentage of stroma compared with epithelium present in at least three representative low-power fields [Stromal cell nuclear atypia was graded as 1 (mild), 2 (moderate), or 3 (marked). Stromal cells with grade 1 nuclei have small, uniform nuclei, with evenly dispersed chromatin. Grade 2 stromal nuclei were moderately increased in size, with occasional nucleoli and grade 3 nuclei much variable in nuclear size and shape, with irregular nuclear Membranes. Stromal cell mitotic figures were counted in the most mitotically active areas and expressed as per mmr fields .Mean with standard deviation were calculated for continuous variables like age of the patient, size of the tumor, duration of clinical presentation. Categorical data like a side of the tumor, location of the tumor, specific surgical procedure, ultrasonographic characteristics, and histopathologic results from trucut biopsy and excisional biopsy were analyzed with frequency and percentages. The outcome was confirmed as FA or PT on excisional biopsy. The Chi-square test was also used to check the significant difference at p-value (p < 0.05). Statistical analysis was performed with SPSS 23 version .We enrolled a total of 125 patients. The mean age of patients was 33.86 ± 9.95 years. Fifty patients (40.0%) had lump in left breast, while 75 (60.0%) on right side. The mean size was 41.02 ± 27.38 mm with a mean lump duration of 7.52 ± 5.34 months. The mammographic findings showed a circumscribed hypoechoic lobulated mass in 57.2%, heterogeneously hypoechoic mass with lobulated margin in 30.8%, and irregular hypoechoic mass in 12.0%  cases, while more than 30 mm in 45 (51%). While regarding phyllodes, only five (14%) were less than 30 mm, and 31 (86%) were found to have an ultrasonographic size of more than 30 mm Table .Regarding the procedure types, 84 (67.2%) patients underwent a lumpectomy, mastectomy in five (4.0%), nipple-sparing central segmentectomy in one (0.8%), nipple-sparing segmentectomy in one (0.8%), segmentectomy was performed in four (3.2%), wide local excision was performed in 26 (20.8%), and wire localized lumpectomy was performed in four (3.2%) patients.The outcome showed that 89 (71.2%) patients had FA at excision and 36 (28.8%) patients had PTs. The final histology also revealed the different types of FA and PT on excisional biopsy.We found that in trucut specimens of patients who were finally diagnosed with FA, the stromal cellularity was mild in 62 (69.7%), moderate in 27 (30.3%), and no severe stromal cellularity. While, in PT patients, the stromal cellularity was mild in nine (25.0%), moderate in 17 (47.2%), and severe in 10 (27.8%) patients.Regarding the stromal cell nuclear atypia on trucut specimens, it was absent in 68 (76.4%) and mild in 21 (23.6%), but moderate or severe atypia was absent. Similarly, in PTs, the stromal cell nuclear atypia was absent in 30 (30.6%), mild in 15 (41.7%), moderate in five (13.9%), and severe in five (13.9%) patients.Mitotic count was 0-1 in 89 (100%) patients of FA. While in PT, the mitotic count was found 0-1 in 18 (50.0%) patients, 1-5 in six (16.7%), 5-10 in five 13.9%), >10 in seven (19.4%) patients , mild in 21 (23.6%), and moderate in 17 (19.1%). But, in PT the stromal overgrowth was absent in one (2.8%), mild in nine (25.0%), moderate in 19 (52.8%), and severe in seven (19.4%) patients.In the FA group, the enhancement of stromal cellularity adjacent to the epithelium was absent in 59 (66.3%) and present in 30 (33.7%). While in PT, it was absent in 20 (55.6%), not applicable to two (5.6%), and present in 14 (38.9%) patients  patients had PT at excisional biopsy who were diagnosed as FA on trucut samples. Al-Arnawoot et al. conducted a study with 134 cellular fibroepithelial lesions (CFEL). In their study, they found that Eighty-nine 89 (66%) were FA and 44 (34%) were phyllodes  .Differentiating PT from FA in the preoperative context is a matter of great importance in clinical practice since the conservative management of patients can be worrisome. In a study by Reis et al., they found that fibroepithelial tumors diagnosed after the trucut biopsy, but having a larger size of more than 30 mm on ultrasound, have a greater probability of being pre-malignant. So, fibroepithelial lesions larger than 30 cm are more likely to be PT and are coherent with the existing medical literature . SignifiIn our study, the upgrade rate from fibroepithelial lesions to PTs is 48.3%. These included mainly the tumors with a size of more than 30 mm, increased stromal cellularity, and fibroepithelial lesions with spindle cell proliferation. Moreover, our study also highlights that PTs signify severe stromal cellularity 10 (27.8%)  and severe stromal overgrowth 07 (19.4%)  on trucut biopsy specimens as compared to FAs who had mild to moderates activities of each primarily. These features are necessary to be considered for diagnosing the phyllodes as a marker of surgical excision. Zhou et al. had described the sensitivity of core needle biopsy (CNB) at diagnosing benign, borderline, and malignant PTs as 4.9% (2/41), 4.2% (3/71), and 25.0% (4/16), respectively, that showed poor accuracy as a guide for surgical decisions if considered without these histological features . Likewise, the enhancement of stromal cellularity adjacent to epithelium may reveal a better emblem to assign a patient either to be observed or plan for excision of the tumor . In a study by Jacobs et al., stromal cellularity enhancement was observed 33% in the FA group and 63% in the phyllodes group [As in our study, the findings showed that stromal cellularity, atypia, and mitotic count were different in the FA and the phyllodes. Yasir et al. conducted a study and enrolled 64 patients. Twenty-seven (42.2%) of 64 were diagnosed as PT (24 as benign patients and three borderlines) and 37 (57.8%) as cellular FA on excision biopsy. All features except for increased stromal cellularity were statistically significant in that study (Figure For better-quality management of patients, it is imperative to differentiate between the FA and phyllodes. These tumors have variable courses and outcomes as also found in some other studies ,19. LastThere are few confines of our study. It was a retrospective analysis. Trucut biopsy was performed by different personals, rendering a variable chance of histology result on trucut biopsy. Although our results are statistically significant, we need large data to validate our conclusions. Further, there is a need for comparative diagnostic modalities for dual verification of impressions like magnetic resonance imaging. So, in this reverence, we are arranging a multi-institutional prospective study over a large period of time to find the precise fallouts that could be executed over a large scale for better influence.Assessment of tumor size, stromal cellularity, mitoses, and enhancement of stromal cellularity adjacent to the epithelium in trucut needle biopsy of breast tumors are useful markers for judging the odds of being an FA or PT. The tumors with a size of more than 30 mm, with increased stromal cellularity, and fibroepithelial lesions with spindle cell proliferation are appropriate indicators for excision.A combination of indicators is better than a single marker. We suggest that patients with tumor size < 30 mm, mild stromal cellularity, stomal overgrowth, low mitotic activity, and mild stromal cellularity adjacent to epithelium on trucut biopsy specimens may be observed and followed over time.


FOXC1 variants, 13 PITX2 variants, and two gross deletions spanning FOXC1 were detected in 35 out of 55 (63.6%) patients. 12 FOXC1 variants, 9 PITX2 variants, and two gross deletions were novel. There was a wide range of variability and severity in ocular and systemic manifestations displayed in our patients. Patients with FOXC1 variants were diagnosed at a younger age and had a lower prevalence of systemic manifestations than patients harboring PITX2 variants and those without variants. To our best knowledge, this is the largest study of Chinese patients with ARS to date. Our findings expand the genetic spectrum of ARS and reveal genotype-phenotype correlations in Chinese patients with ARS. Genetic and clinical heterogeneity were present in our patients. Awareness of the extensive characterization may aid in the clinical management and genetic counseling of patients with this rare disease.Axenfeld–Rieger Syndrome (ARS) is a rare disease with a wide spectrum of ocular and systemic manifestations. The genetic spectrum of Chinese patients with ARS and genotype-phenotype correlations have yet to be described. To explore the molecular and clinical features in Chinese patients, fifty-five patients clinically diagnosed with ARS from independent families were recruited. Complete ophthalmic examinations and next generation sequencing of anterior segment dysgenesis associated genes were performed in all patients, and segregation in available relatives was verified using Sanger sequencing. 18  Axenfeld–Rieger Syndrome  is an autosomal dominant developmental disorder that has both clinical and genetic heterogeneity, and primarily affects the anterior segment structure of the eye . The preFOXC1 and PITX2 are two major causative genes of ARS. They are estimated to explain disease pathogenesis in approximately 40–63% of patients with ARS (FOXC1 is a member of the large forkhead box (FOX) transcription factor family, whereas PITX2 is a member of bicoid-like homeodomain transcription factor family. FOXC1 and PITX2 are both transcription factors that are coexpressed in the periocular mesenchyme and play key roles in the regulation of embryonic development  measurement , and ultrasound biomicroscopy . Perimetry was performed in cooperative children and adults using the Octopus 101  or Humphrey Visual Field Analyzer 750 .Whole blood samples of all patients and available relatives were collected for genomic DNA extraction by Gentra PureGene blood kits  according to the manufacturer’s instructions. Genetic testing was performed in 55 patients using next generation sequencing between October 2016 and June 2020. A panel of 289 genes associated with ASD disorders was sequenced by the Illumina Miseq platform  with the 2 × 300 bp paired-end read module. The average depth was 100x, and 90% of the target region was covered above 40x. Low quality bases (< Q20) were removed using SolexaQA .Whole genome sequencing was performed in two patients. In brief, a total of 0.2 μg DNA per sample was fragmented using sonication to a size of 350 bp. The DNA fragments then underwent end-repairing and A-tailing, and ligation was performed with the full-length adapters for Illumina sequencing, followed by polymerase chain reaction amplification and purification. After library quality assessment, clustering of the index-coded samples was performed on the cBot Cluster Generation System using the Illumina PE Cluster Kit (Illumina), and DNA libraries were sequenced on the Illumina platform and 150 bp paired-end reads were generated.http://www.hgmd.cf.ac.uk/ac/index.php), Clinvar (https://www.ncbi.nlm.nih.gov/clinvar/), and 1,000 Genomes Project . The copy number variants were analyzed by calculating the sequencing depth of each region covered by probes. The ExomeDepth Package (FOXC1) and NM_153427.2 (PITX2) were used for mutation nomenclature. Novel variants were classified into five categories according to the American College of Medical Genetics and Genomics (ACMG) guidelines: pathogenic, likely pathogenic, variant of uncertain significance (VUS), likely benign, and benign  . The det Package  was alsod benign . Conservd benign . Polymord benign , Sortingd benign , and Prod benign  were appp-value of 0.05.Statistical analyses were performed using SPSS version 20.0 . Age is presented as the median (range). Patients were classified into three groups according to their genotypes. Differences in age with non-normal distributions were assessed using the Kruskal-Wallis test with Dunn’s post-hoc test. Differences between categorical variables among the three groups were assessed using chi-square test or Fisher’s exact test with Bonferroni correction for post-hoc multiple comparisons. The statistical significance was set at Fifty-five Chinese patients with ARS from independent families were enrolled in our study. These included 22 females (40.0%) and 33 males (60.0%). The median age at diagnosis was 14.0 (0.1–64.0) years. 25 patients (45.5%) had family histories of ARS or glaucoma. 53 patients (96.4%) were diagnosed with glaucoma and 36 patients (65.5%) had systemic features.FOXC1, 13 variants in PITX2 and two gross deletions of 6p25 , with 19 patients carrying the FOXC1 variants, 14 patients carrying the PITX2 variants, and two patients carrying gross deletions of 6p25. No variants were found in the remaining 20 patients. Among the 35 patients in whom variants were detected, six patients carried de novo variants, 11 patients carried heterozygous variants inherited from their parents, and data of parents of 18 patients were unavailable.In total, 33 variants were identified in 35 patients, including 18 variants in  of 6p25 , 2. AmonFOXC1 mutations and two gross deletions of 6p25 spanning FOXC1 were detected in 21 patients  (FOXC1 variants (15/18) were located in the forkhead domain, which is the DNA interaction function domain of FOXC1  who presented with both ocular and systemic disorders with very early onset ages. To further determine the exact range of gross deletions in these two patients, whole-genome sequencing was used. By checking the depth of coverage across the whole genome, a large deletion (Chr 6:1478641-2694750) spanning FOXC1, GMDS, C6orf195, and MYLK4 was detected in patient No. 20, and a large deletion (Chr 6:382441-2333070) spanning IRF4, EXOC2, HUS1B, FOXQ1, FOXF2, FOXC1, and GMDS was detected in patient No. 21  . Most ofof FOXC1 . Six of escribed . Other 1 Provean , locatedt No. 21 .PITX2 variants in 14 patients   , includi, 14/55) . Most of, 14/55) . Nine ofFOXC1 variants, n = 19), PITX2 group , and negative group . The median age at diagnosis was 5.0 (0.1–40.0) years in the FOXC1 group, 24.0 (0.8–64.0) years in the PITX2 group, and 15.0 (0.3–46.0) years in the negative group. The diagnosis age of the FOXC1 group was significantly lesser than that of the PITX2 group (p = 0.006) and the negative group (p = 0.048). However, there was no significant difference in the diagnosis age between PITX2 group and the negative group (p > 0.99). The ratio of patients that were diagnosed before the age of 1 year was statistically significantly different among the three groups (p = 0.007). It was 47.4% (9/19) in the FOXC1 group, but only 7.1% (1/14) in the PITX2 group and 10.0% (2/20) in the negative group.Except for the two patients carrying gross deletions, other patients were divided into three groups: FOXC1 group  received medication only, 84.9% (45/53) received both medication and surgical treatments, and 60.4% (32/53) underwent at least two surgeries for glaucoma. Three patients carrying FOXC1 variants  underwent penetrating keratoplasties because of corneal decompensation.As the most frequent consequence of ARS, glaucoma was found in 53 patients (96.4%). Apart from one patient (No. 3) with p.Y81_S82insY in FOXC1 variants or gross deletions spanning FOXC1 had congenital heart diseases. Other systemic manifestations in the patients included hearing loss, growth disorder, depressive disorder, and kidney stone. The prevalence of systemic manifestations was statistically significantly lower in the FOXC1 group  than in the PITX2 group  and in the negative group ; however, there was no significant difference between the PITX2 group and the negative group (p = 0.13).Furthermore, 36 (65.5%) patients presented with systemic anomalies. The most common systemic manifestations detected in our patients were dental and craniofacial abnormalities. Tooth agenesis presented as hypodontia and microdontia . The craIn this study, we investigated 55 Chinese patients with ARS from independent families, and analyzed their genotypes, phenotypes, and genotype-phenotype correlations.FOXC1 and PITX2 were the two major causative genes of ARS, which has genetic heterogeneity. To date, the variant rates of FOXC1 and PITX2 in individuals with ARS have been reported in a limited number of studies. In an ASD cohort mainly from Belgium or Netherlands, FOXC1 and PITX2 disruptions were recorded in 24% (19/80) and 16% (13/80) of patients, respectively (FOXC1 and PITX2 variants were found to account for 63% of ARS probands (24/38) in a multi-racial study, with 8% (3/38) harboring FOXC1 variants and 55% (21/38) harboring PITX2 variants (PITX2 variants were detected in 55% (11/20) of patients, while no FOXC1 variants were detected  of patients with ARS; FOXC1 variants were detected in 34.5% (19/55) of probands, gross deletions of 6p25 were detected in 3.6% (2/55) of probands and PITX2 variants were detected in 25.5% (14/55) of probands. There were no pathogenic variants found in the other 20 patients. The pathogenic genes of these patients still require exploration. By increasing the number of studied patients, we have greatly expanded the genetic spectrum of Chinese patients with ARS.ARS is a rare disease with autosomal dominant inheritance. All of the patients in our study who carried variants are heterozygous. ectively . Whereasvariants . In anotdetected . The varFOXC1 variants were detected in 19 patients. Most of our detected variants were located in the forkhead domain, which is a conserved 110 amino acid sequence . Similarly, Souzeau et al. reported that the age of glaucoma diagnosis was significantly lower in FOXC1 carriers than in the PITX2 carriers  and in the negative group . Furthermore, FOXC1 was more likely to be associated with congenital heart disease, while PITX2 was always associated with dental anomalies and/or umbilical anomalies, and this is supported by previous studies (FOXC1 or PITX2 variants had a similar age of diagnosis, and similar prevalence and spectrum of systemic features compared to those harboring PITX2 variants. This suggests that the undiscovered pathogenic genes of ARS might have a similar function to the PITX2 gene.Furthermore, the genotype-phenotype analysis in our study revealed that the carriers . Almost terature . Thus, l studies . Our stuFOXC1 variants, PITX2 variants or gross deletions spanning FOXC1, out of which 23 were novel. The genotype and phenotype spectrums of Chinese patients with ARS have been greatly expanded by our study. Because of the early age of onset, high risk for glaucoma, and combined systematic disorders, genetic testing is recommended for patients with ARS, in order to make an early and precise diagnosis.To our best knowledge, this study enrolled the largest number of Chinese patients with ARS to date. In total, 55 Chinese patients with ARS from independent families were studied both clinically and genetically. We found that 63.6% of patients carried 33


Corynebacterium glutamicum cell mass (CGCM) as a protein supplement and a source of cell wall fragments supporting the growth and intestinal health of nursery pigs. Thirty-two pigs (21 d of age) were allotted to four treatments (n = 8) based on the randomized block design with sex and initial body weight (BW) as blocks. The main effect was the dietary supplementation of lysed CGCM  replacing blood plasma and fed in two phases . Feed intake and BW were measured at the end of each phase. Pigs were euthanized on day 21 to collect jejunal tissue and mucosa to evaluate intestinal health. Ileal digesta were collected to measure the apparent ileal digestibility of nutrients in diets. Data were analyzed using Proc Mixed and Reg of SAS. Increasing daily intake of CGCM increased  ADG of pigs. Increasing CGCM supplementation affected  the relative abundance of Lactobacillaceae (minimum: 26.4% at 1.2% CGCM), Helicobacteraceae (maximum: 29.3% at 1.2% CGCM), and Campylobacteraceae (maximum: 9.0% at 1.0% CGCM). Increasing CGCM supplementation affected  the concentrations of immunoglobulin G (maximum: 4.94 µg/mg of protein at 1.0% CGCM) and protein carbonyl , whereas linearly decreased (P < 0.05) malondialdehyde (MDA) in the proximal jejunal mucosa. Increasing CGCM supplemention affected  intestinal enterocyte proliferation rate (maximum: 13.3% at 1.0% CGCM), whereas it did not affect intestinal morphology and the nutrient digestibility. In conclusion, supplementing 1.0% to 1.2%, reducing blood plasma supplementation by 0.7% to 0.9%, respectively, increased potential pathogenic microbiota associated in the jejunal mucosa resulting in increased immune response, enterocyte proliferation, and PC concentration. However, supplementing diets with 2.1% CGCM, replacing 1.5% blood plasma, improved growth performance, and reduced MDA without affecting nutrient digestibility, intestinal morphology, and microbiota in the jejunal mucosa. In this study, based on the polynomial contrast, supplementing 1.0% to 1.2% CGCM suppressed the benefits from blood plasma, whereas supplementing 2.1% CGCM showed functional benefits of CGCM with similar effects from blood plasma supplementation.The objective was to determine the nutritional and functional values of lysed  Soybean meal has been predominantly used as a protein supplement in pig production. However, the antinutritional factors such as allergenic proteins, trypsin inhibitors, and flatulence producing oligosaccharides limit its use in nursery pig diets . PreviouMethylococcus capsulatus , processed vegetable protein, and insect protein . The SCPpsulatus  and Coryutamicum  can overCorynebacterium glutamicum is a Gram-positive bacteria, commonly used to produce AA, and generally recognized as safe (Corynebacterium glutamicum cell mass (CGCM) is removed by filtration and then disposed of or used as fertilizers  at 900 bars for 4 s at 30 °C with a maximum allowance of 70 °C in the holding tube. After the holding time, the CGCM in deionized water flowed to a serpentine tube immersed into chilled water for rapid cooling. The lysis process was completed in four cycles in a continuous system . The lysed CGCM was dried using a freeze dryer . The lysing process was based on the study by Lysine broth including intact CGCM was obtained from CJ Bio . The lysine broth containing 15% to 20% dry matter (DM) was centrifuged at 3,100 × Images of CGCM before and after the lysis were taken using a variable pressure scanning electron microscope  at the 5 kV efficient voltage and 10 mm working distance to obtain the percentage of lysed cells. Each image of CGCM from before and after the lysis  was evenn = 8) based on the randomized complete block design with initial BW (light and heavy) and sex (gilts and barrows) as blocking criteria. All pigs were housed in individual pens and had ad libitum access to water and the assigned experimental diets for 21 d based on two phases: phase 1 (days 0 to 10) and phase 2 (days 11 to 21). Dietary treatments consisted of a basal diet with lysed CGCM at four levels  of CGCM replacing blood plasma, based on Lys content . Therefore, up to 1.5% blood plasma was replaced by 2.1% CGCM. All experimental diets were formulated to meet or exceed the nutrient requirements based on the study by Thirty-two nursery pigs (16 barrows and 16 gilts) at 21 d of age with initial body weight (BW) of 7.2 ± 0.6 kg were used for the experiment. Pigs were allotted to four dietary treatments  very hard and dry stool, (2) firm stool, (3) normal stool, (4) loose stool, and (5) watery stool with no shape following After 21 d of feeding, all pigs were euthanized by exsanguination after the penetration of a captive bolt to the head to collect samples. Sections of proximal  and distal  jejunum were collected and rinsed with 0.9% saline solution to collect mucosa and tissue. The jejunal mucosa was collected into 2 mL microcentrifuge tubes and immediately frozen in liquid nitrogen, and then stored at −80 °C for determination of immune status, oxidative stress markers, and microbiota. Proximal and distal jejunum tissues (5 cm) were collected into 50 mL polypropylene tubes with 40 mL of 10% buffered formaldehyde solution for histological evaluation. Ileal digesta was collected into 150 mL containers placed on ice and then stored at −20 °C for analysis of apparent ileal digestibility (AID).Distal jejunal mucosa samples were used to extract DNA using QIAamp Fast DNA Stool Mini Kit  following the description from g at 4 °C as described by Jejunal mucosa samples (1 g) from microcentrifuge tubes were taken and added with 2 mL of PBS solution into 5 mL polypropylene tubes. Mucosa samples were homogenized using a tissue homogenizer  for 30 s on ice and transferred to a new 2 mL microcentrifuge tube for centrifugation for 15 min at 14,000 × The concentrations of total protein, tumor necrosis factor alpha (TNFα), interleukin-8 (IL-8), malondialdehyde (MDA), protein carbonyl (PC), IgA, and IgG were analyzed by the colorimetric method and the absorbance was measured on a plate reader  and the Gen5 Data Analysis Software (BioTek Instruments).The concentration of total protein was analyzed by using Pierce BCA Protein Assay Kit  following procedures of The concentration of TNFα was analyzed in proximal and distal mucosa using the Porcine TNFα Immunoassay Kit  as described by Two sections of proximal and distal jejunum were cut and placed into cassettes and sent to North Carolina State University College of Veterinary Medicine Histopathology Lab  for immunohistochemistry staining with Ki-67 assay. One slide represented one pig. Pictures were taken using a microscope  and Infinity Analyze and Capture software .http://imagejs.org). The percentage of Ki-67-positive cells was used as an indicator of the enterocyte proliferation rate in the crypt (Pictures were taken at magnification 40× to measure villus height (VH) and width (VW), crypt depth (CD), and villus height to crypt depth ratio (VH:CD). Lengths of 15 well-shaped villi and corresponding crypts were measured in each slide. Pictures of 15 well-shaped crypts were taken from each slide at magnification 100× and cropped to calculate the percentage of the Ki-67-positive cells to the number of epithelial cells in a crypt by operating the ImageJS . Phase 2 diets and freeze dried ileal digesta were ground to fine powder form. The concentration of titanium dioxide in the diets and digesta was measured by following Apparent ileal digestibility of DM, GE, EE, CP, and AA was calculated as previously described by 2 in a diet/TiO2 in digesta) × (nutrient in digesta/nutrient in a diet)]AID% = 100 × {1 – [(TiOP < 0.05), the procedure RSREG was used to predict the critical value and the stationary point. The linear and quadratic effects of daily intake (g/d) of CGCM were tested by REG procedures. The analysis of fecal score data was performed by using Kruskal–Wallis test with Dwass, Steel, Critchlow-Fligner method option for pairwise two-sided multiple comparisons following P < 0.05 and 0.05 ≤ P < 0.10 were considered as a tendency.All data except for fecal score were analyzed based on a randomized complete block design using Mixed procedure by SAS 9.3 . Initial BW and sex were considered as blocks. Dietary treatments were defined as fixed effects and blocks were random effects. The LSMEANS statement was used to calculate mean values for all treatments. Linear and quadratic effects of increasing CGCM supplementation were tested by orthogonal polynomial contrasts using the CONTRAST statement. When a quadratic effect was significant (P < 0.05) the proportion of lysed Corynebacterium glutamicum cells from 4.3% in intact CGCM to 58%  fecal score than pigs fed 0% CGCM on day 19 of the experiment, whereas no differences were observed among other treatments (Pigs fed 0.7% CGCM had lower (eatments .P < 0.05) on the alpha diversity of jejunal mucosa-associated microbiota estimated with Shannon index at family (maximum 2.8 at 1.0% CGCM), genus (maximum 2.7 at 1.0% CGCM), and species (maximum 3.8 at 1.0% CGCM) levels (P < 0.05) on the alpha diversity of jejunal mucosa-associated microbiota estimated with Simpson index at family (maximum 0.8 at 1.1% CGCM), genus (maximum 0.7 at 1.1% CGCM), and species (maximum 0.9 at 1.1% CGCM) levels.Increasing CGCM supplementation had quadratic effects  levels . IncreasP = 0.094) at the family level with the maximum 0.75 at 5.4 g/d CGCM intake . Increasing the daily CGCM intake tended to have the quadratic effects on the alpha diversity of jejunal mucosa-associated microbiota estimated with Shannon (P = 0.091) and Simpson (P = 0.079) indexes at the genus level with the maximum at 2.71 and 0.74, when the daily CGCM intakes were 4.7  and 5.4 g/d , respectively. Increasing the daily CGCM intake had the quadratic effects (P < 0.05) on the alpha diversity of jejunal mucosa-associated microbiota estimated with Shannon and Simpson indexes at the species level with the maximum at 3.88 and 0.88, when the daily CGCM intake was 5.0 g/d , increasing the daily CGCM intake tended to have quadratic effects on the alpha diversity of jejunal mucosa-associated microbiota estimated with Simpson index  on the minimum relative abundances of Firmicutes (minimum 37.8% at 1.1% CGCM) and Proteobacteria (maximum 44.0% at 1.1% CGCM) and tended to have a quadratic effect (P = 0.058) on the relative abundance of Actinobacteria (P = 0.055) on the relative abundance of Firmicutes with minimum 37.5% at 5.4 g/d CGCM intake (P < 0.05) on the relative abundance of Proteobacteria with maximum abundance at 46.5%, when the daily CGCM intake was 5.6 g/d  on the relative abundance of Lactobacillaceae (minimum 26.4% at 1.2% CGCM), Helicobacteraceae (maximum 29.3% at 1.2% CGCM), and Campylobacteraceae (maximum 9.0% at 1.0% CGCM) (P = 0.060) on the relative abundance of Propionibacteriaceae, whereas linearly reduced (P < 0.05) the relative abundance of Bifidobacteriaceae. When the data were analyzed based on the daily CGCM intake (g/d), increasing the daily CGCM intake tended to have quadratic effects on the relative abundance of Helicobacteraceae (P = 0.053) and Campylobacteraceae (P = 0.098) with minimum abundance at 31.3% and 9.0% when the daily CGCM intake was 5.8 g/d  and 5.1 g/d , respectively. Increasing the daily CGCM intake tended to linearly reduce (P = 0.067) the relative abundance of Bifidobacteriaceae .At the family level, increasing CGCM supplementation had quadratic effects (0% CGCM) . IncreasLactobacillus (P = 0.056) and Propionibacterium (P = 0.073) and had quadratic effects (P < 0.05) on the relative abundance of Helicobacter (31.9%) and Campylobacter (9.4%) at 1.2% and 1.0% CGCM, respectively (P = 0.068) the relative abundance of Megasphaera and linearly reduced (P < 0.05) relative abundance of Bifidobacterium. When the data were analyzed based on the daily CGCM intake (g/d), increasing the daily CGCM intake had a quadratic effect (P < 0.05) on the relative abundance of Helicobacter with 33.9% at 5.7 g/d CGCM intake . Increasing the daily CGCM intake tended to linearly reduce (P = 0.064) the relative abundance of Bifidobacterium .At the genus level, increasing CGCM supplementation tended to have quadratic effects on the relative abundance of ectively . IncreasP = 0.023) the relative abundance of Prevotella sp. (P = 0.096) and tended to have a quadratic effect (P = 0.090) on the relative abundance of Lactobacillus kitasatonis and tended to linearly reduce (P = 0.053) the relative abundance of Corynebacterium glutamicum. Increasing CGCM supplementation had a quadratic effect (P < 0.05) on the relative abundance of Pelomonas puraquae (maximum 1.1% at 1.0% CGCM). When the data were analyzed based on the daily CGCM intake (g/d), increasing the daily CGCM intake tended to linearly reduce (P = 0.077) the relative abundance of Prevotella sp. (P < 0.05) on the relative abundances of Pelomonas puraquae with minimum abundance at 1.2%, when the daily CGCM intake was 4.8 g/d  on the concentration of IgA and had quadratic effects (P < 0.05) on the concentrations of IgG (maximum 4.94 µg/mg of protein at 1.0% CGCM) and PC (maximum 6.12 nmol/mg of protein at 1.1% CGCM). Whereas increasing CGCM supplementation linearly reduced (P < 0.05) the concentration of MDA. When the data were analyzed based on daily CGCM intake (g/d), increasing the daily intake of CGCM tended to have a quadratic effect (P = 0.080) on the concentration of PC with maximum 6.19 nmol/mg of protein at 4.9 g/d CGCM intake  and PC (P = 0.051).In the distal jejunal mucosa, increasing CGCM supplementation did not affect the concentration of TNFα, IgA, IgG, and MDA in nursery pigs . IncreasP = 0.090) VH:CD in the proximal jejunal mucosa and had a quadratic effect (P < 0.05) on the maximum intestinal enterocyte proliferation rate (13.3%) at 1.0% CGCM in the distal jejunal mucosa.Increasing CGCM supplementation did not affect VH, VW, and CD in proximal and distal jejunum . WhereasIncreasing CGCM supplementation did not affect the AID of DM, GE, EE, CP, and AA in nursery pig diets .This study demonstrated that CGCM can be supplemented up to 2.1%, replacing 1.5% blood plasma, in diets without affecting the growth performance and intestinal health of nursery pigs. Additionally, increasing daily CGCM intake increased the ADG of pigs. The results observed in this study are in accordance with previous studies showing that SCP can be used as alternative protein supplements to replace plant or animal protein supplements improving growth performance in broilers  and pigsHelicobacteraceae, which belongs to Proteobacteria, is commonly associated to unhealthy pigs (Lactobacillaceae in Firmicutes is predominant in healthy pigs (Previous studies have demonstrated that diet composition greatly affected the intestinal microbiota . Interesthy pigs , whereasthy pigs .Lactobacillaceae, which can increase the production of anti-inflammatory cytokines and, consequently, reduce proinflammatory cytokines (Corynebacterium glutamicum, contains TA and Slp that can affect the intestinal microbiota by immunomodulatory effects (Lactobacillus attaches to receptors on the intestinal epithelial cells and, consequently, reduces the availability of receptors for the adherence of potential pathogens (Helicobacteraceae. However, supplementing 2.1% CGCM, replacing 1.5% blood plasma, in diets may have provided sufficient TA and Slp for their functional roles reducing the relative abundance of Proteobacteria and increasing Firmicutes in the jejunal mucosa.Blood plasma not only is highly digestible but also provides around 20% of IgG that plays an essential role in the immune response by affecting the host immune system and intestinal microbiota , 2018. Iytokines . The cel effects  and by c effects . Previouathogens . In thisThe alpha diversity of intestinal microbiota can also be affected by the diet composition . The carHelicobacteraceae and Campylobacteraceae, can affect the immune system and oxidative stress status in the jejunum of nursery pigs (Lactobacillaceae can be associated with the synthesis of anti-inflammatory cytokines (The modulation of intestinal microbiota is generally associated with changes in the immune response (Duarte and ery pigs . In respery pigs . Conversytokines .Corynebacterium glutamicum, contains CWGs that have immunomodulatory functions, reducing excessive immune reactions (Previous study showed that pigs fed blood plasma in diets improved intestinal health by preventing from the production of proinflammatory cytokines, such as TNFα, IL-8, and interferon-γ, resulting in apoptosis of infected cells and the production of additional proinflammatory cytokines and chemokines . Therefoeactions . The CWGeactions . Accordieactions . In the The activation of the immune system can increase the production of reactive oxygen species leading to an increase in oxidative stress . MalondiThe oxidative stress can cause damage in the enterocytes by oxidizing lipids, proteins, and DNA resulting in cellular apoptosis, consequently, affecting the intestinal morphology . IncreasIn conclusion, based on the polynomial model, supplementing CGCM at 1.0% to 1.2%, reducing the blood plasma supplementation by 0.7% to 0.9%, respectively, increased potential pathogenic microbiota resulting in increased immune response, enterocyte proliferation, and PC concentration. However, supplementing CGCM at 2.1%, reducing blood plasma supplementation by 1.5%, improved growth performance, and reduced MDA concentration without affecting nutrient digestibility, intestinal morphology, and microbiota in the jejunal mucosa. Collectively, supplementing 1.0% to 1.2% CGCM suppressed the benefits from blood plasma, whereas supplementing 2.1% CGCM showed functional benefits of CGCM with similar effects from blood plasma supplementation.


This study aimed to evaluate whether an algorithm  with skin neoplasms suspected of malignancy by either physicians or patients were recruited in two tertiary care centers located in South Korea. An artificial intelligence (AI) group  was diagnosed via routine examination with photographic review and assistance by the algorithm, whereas the control group  was diagnosed only via routine examination with a photographic review. The accuracy of the nondermatologists before and after the interventions was compared.P = .008). The number of differential diagnoses of the participants increased from 1.9 ± 0.5 to 2.2 ± 0.6 after the assistance (P < .001). In the control group, the difference in the Top-1 accuracy between before and after reviewing photographs was not significant , and the number of differential diagnoses did not significantly increase .Among the AI group, the accuracy of the first impression  after the assistance of AI was higher than that before the assistance (46.5%, In real-world settings, AI augmented the diagnostic accuracy of trainee doctors. The limitation of this study is that the algorithm was tested only for Asians recruited from a single region. Additional international randomized controlled trials involving various ethnicities are required. For specific quantifiable problems, artificial intelligence (AI) has demonstrated performance comparable with that of specialists in the medical field . In partIn dermatology, AI could analyze dermoscopic and clinical images as accurately as dermatologists in reader tests –8. Howevhttps://modelderm.com) to diagnose 178 skin diseases and predict the chance of malignancy in previous studies [https://b2019.modelderm.com) [We have developed a skin disease classifier  .A few algorithms have been tested in a prospective real-world setting where the expertise of the user affects the accuracy , and thehttp://modelderm.com) was described in previous studies [https://github.com/nvidia/caffe; version 0.17.2, CUDA 10.0, cuDNN 7.6.2), we trained our CNN models using a transfer learning method using ImageNet pretrained models. Histogram normalization was performed as a preprocessing step before training the models. The output values of SE-Net [The training history of our algorithm  using the following formula: Malignancy score =  output + SCC in situ output + keratoacanthoma output + malignant melanoma output) × 100 + (actinic keratosis output + ulcer output) × 20.The algorithm reports an overall risk of malignancy as “Low”, “Medium,” or “High”. The algorithm reports the risk of malignancy as “Low” when the malignancy score is below 10, “Medium” when the score is between 10 and 20, and “High” when the score is over 20.https://b2019.modelderm.com) developed in our previous study [After obtaining approval from the institutional review board of Asan Medical Center (2018–1130), a prospective study was performed at two tertiary care centers in Korea  between February 1, 2020, and November 7, 2020. The algorithm  who had skin neoplasms suspected of malignancy by either patient or physician were recruited. Exclusion criteria were patient refusal, broken blindness, the wrong version of the algorithm, non-real-time analysis, and exposure of the biopsy results in the referral note . If firshttp://b2019.modelderm.com. After referring to the algorithm’s three diagnoses and the malignancy score, they were given an opportunity to modify their initial diagnoses. In the control group, trainees just reviewed the photographs once again then provided the after-diagnoses.A total of 10 attending physicians (11.4 ± 8.8 years’ experience after board certification), 11 dermatology trainees, and 7 intern doctors participated in this study . AttendiP value of < .05 was considered statistically significant.Top accuracy was calculated as an evaluating metric. Top-(n) accuracy is the accuracy of the Top-(n) diagnoses. If any one of the Top-(n) diagnoses is correct, it counts as “correct.” Only an exact diagnosis was recorded as correct. For evaluating the sensitivity and specificity of malignancy prediction, the physicians’ diagnoses were transformed into either malignant or benign. Top accuracies were compared using two-tailed paired Wilcoxon signed-rank tests (R version 3.5.3), and a P = .008/ < .001/ < .001)  .The Top-1/Top-2/Top-3 accuracies of the attending dermatologists were 61.8%/ 69.4%/ 71.5%, respectively, and those of the standalone algorithm were 53.5%/ 66.0%/ 70.8%, respectively. In 42.4% (61/144) cases, the Top-1 diagnosis of the algorithm was coherent with that of the trainees, and in 50.0% (72/144) cases, the Top-1 of the algorithm was coherent with that of the attending physicians. The Top-1 of the trainees was coherent with that of the attending physicians in 52.8% (76/144) cases.The trainees revised 28.5% (41/144) of their Top-1 diagnosis after reviewing three diagnoses of the algorithm. A total of 70% (29/41) of their revised answers were correct, whereas 29% (12/41) of their revised answers were incorrect.P = .77/ = .06). The sensitivity/specificity of the attending dermatologists was 82.6%/ 91.7% and that of the patients were 56.5%/ 42.6%. The sensitivity/specificity derived from the Top-1 diagnosis of the algorithm was 52.2%/ 93.4%. The sensitivity/specificity at the threshold of the risk “Medium” using the malignancy score was 95.7%/ 60.3% and that at the threshold of the risk “High” was 82.6%/ 70.2% .The differences of the Top-1/Top-2/Top-3 accuracies between before and after reviewing photographs were not significant . The sensitivity/specificity of the attending dermatologists was 79.3%/ 90.2% and that of the patients was 48.1%/ 44.5%.For determining malignancy, the sensitivity/specificity derived from the Top-1 diagnosis was 65.5%/ 81.3% before reviewing and 65.5%/ 86.6% after reviewing (P = .57).The number of differential diagnoses by the trainees had not changed significantly . Summarized key results were described in The differences of the Top-1/Top-2/Top-3 accuracies between the AI group and the Control were not significant  with similar sensitivity and specificity of dermatologists .Reflecting these points on the diagnostic excellence of AI, the concept of augmented intelligence has recently emerged. Augmented intelligence is a term that focuses on the assistive role of AI, emphasizing that augmented intelligence is designed to enhance human intelligence and the clinician-patient relationship rather than substitute it . The AmeIn this study, although the Top-1 accuracy of the standalone algorithm (53.5%) was comparable with that of the trainees (46.5%), the Top-1 accuracy of the augmented trainees (58.3%) was significantly higher. This augmentation could be owing to different strategies between humans and CNNs , 24. TheThe augmentation may be achieved when the accuracy of the algorithm is higher or at least comparable with that of the user. In the study using dermoscopic images, the physicians with the least experience were the most frequently augmented . For neoP = .76). Our algorithm was developed with numerous benign crops to cope with the false-positive problem in detecting skin cancer using unprocessed images [The sensitivity derived from the Top-1 prediction of the algorithm was low (52.2%), as noted previously . Consequd images  and a muConsidering that our study population was limited to Asians, our results cannot be generalized in other circumstances. In completely different settings , the stWe could not demonstrate the superiority of the AI Group over the Control Group in the manner of the randomized controlled trial. There was not a power and sample size calculation before initiating the study. Patients were randomly recruited but were not recruited consecutively. In addition, the two groups were not truly comparable. As shownIn the real-world setting, the standalone performance of the algorithm was comparable with that of the trainees, although the performance of the algorithm was reported to be comparable with dermatologists in the artificial setting . NeverthS1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S1 File(XLSX)Click here for additional data file.S2 File(PDF)Click here for additional data file.


When symptomatic spondylolysis fail to respond to nonoperative treatment, surgical management may be required. A number of techniques have been described for repair by intrasegmental fixation with good results; however, there are still some problems. We reported a repair technique with temporary intersegmental pedicle screw fixation and autogenous iliac crest graft. The aim of present study is to assess the clinical outcomes of L5 symptomatic spondylolysis with this technique.A retrospective analysis of 128 patients with L5 spondylolysis treated with this method was performed. According to CT scan, the spondylolysis were classified into 3 categories: line, intermediate, and sclerosis type. The diagnostic block test of L5 bilateral pars defect was done in all patients preoperatively. The sagittal and axial CT images were used to determine the bone union. The healing time, complications, number of spina bifida occulta, Japanese Orthopedic Association (JOA) score, and VAS for back pain were recorded. After fixation removal, the rate of ROM preservation at L5S1 was calculated.There were 97 patients (194 pars) followed with mean follow-up of 23 months . The union rate of pars was 82.0% at 12 months and 94.3% at 24 months postoperatively. Low back pain VAS significantly (P < 0.05) improved from preoperative mean value of 7.2 to 1.3 at the final follow-up postoperatively (P < 0.05). JOA score increased significantly postoperatively (P < 0.05) with average improvement rate of 79.3%. The rates of L5S1 ROM preservation were 79.8% and 64.0% after fixation removal at 1 and 2 years postoperatively. There were 3 patients of delayed incision healing without other complications.Although sacrificing L5S1 segment motion temporarily, more stability was obtained with intersegmental fixation. This technique is reliable for spondylolysis repair which has satisfactory symptom relief, high healing rate, low incidence of complications, and preserve a large part of ROM for fixed segment. Lumbar spondylolysis is a bony defect in the pars interarticularis. The current theory believes fatigue or stress fracture of pars with developmental factors is the most widely accepted reason for the occurrence of isthmic defect . PreviouSurgical methods for lumbar spondylolysis could be roughly divided into two categories, fusion and repair. Meaningless sacrifice of fairly normal disc and segmental motion make stricter indication for fusion –7. The cWe describe a direct repair technique with temporary intersegmental pedicle screw fixation and autogenous iliac crest graft. The purpose of this retrospective case series study is to evaluate the clinical outcomes of L5 symptomatic bilateral lumbar spondylolysis with this method after pars block test.Between October 2013 and October 2016, 128 consecutive L5 spondylolysis patients were included in this retrospective study. All of them met the following inclusive criteria: (1) age range (18–36 years); (2) LBP VAS ≥ 5.0 (without radiculopathy); (3) lack of response to conservative treatment for 6 months, including medication and brace; (4) bilateral spondylolysis and no more than I grade spondylolisthesis; and (5) more than 50% pain relief by bilateral pars gap block test  multi-segmental spondylolysis (≥ 2 levels) and (2) Pfirrmann III-V grade disc degeneration , 12 for The patient was placed in the prone position under general anesthesia. A midline incision was performed, the bilateral muscles were stripped off the spinous process, lamina, and the base of the transverse process carefully protecting facet joint capsule intact. The defects were exposed. The entry point of the L5 pedicle screw should be selected as cephalic as possible to provide more bone graft space for the defect. After L5S1 pedicle screws implantation, the fibrous scar tissue was removed completely. The pars defect, corresponding lamina, and transverse process base were decorticated to bleeding with burr. The iliac crest cylindrical bone mass was taken with a circular saw within posterior superior iliac spine leaving the inner and outer plate intact in the same incision. Iliac bone was implanted in the gap and impacted bilaterally. The residual bone fragments were placed from the transverse process to the lamina covering the defect. The appropriate rods were contoured and connected with pedicle screws. When tightening the screw, the defects and bone graft were compressed. The negative drainage was placed and the incision was closed. The lumbar brace was used for 3 months after surgery. When bilateral pars defects union, pedicle screw should be removed through the midline incision and intermuscular approach.All patients should take anteroposterior, lateral, dynamic, double oblique radiography, 3D CT scan, and lumbar MRI preoperatively. The diagnosis was established according to the CT results. Based on the axial CT scan , the claThe surgical data of operating time, blood loss, and complications were noted. The LBP and functional outcomes assessment were carried out using the Japanese Orthopedic Association (JOA) and VAS with regular follow-up plan. The radiographic examination and functional evaluation were followed to 3 months after removal of internal fixation. The ROM of L5S1 was measured before repair and after fixation removal.The time of healing was recorded. The union rate was calculated at different follow-up time according to different reference. All quantitative data were described as mean ± standard deviation. The Student t-test and ANOVA method were undertaken. The difference was statistically significant with P < 0.05.Thirty-one of the patients were lost to follow-up. Night-seven patients data were collected completely with the mean follow-up of 23 months . The average operation time was 121 ± 31 min  with a mean blood loss of 100 ± 21 ml . On the basis of mentioned CT classification, there were 36, 126, and 32 pars in the line, intermediate, and sclerosis type, respectively.At 6 months after operation, there were 41 pars healed (pars union rate 21.1%), and 17 patients healed bilaterally (patient union rate 17.5%). Ninety-four pars healed (48.5%), 34 patients bilaterally (35%) at 9 months; 159 pars healed (82.0%), 72 patients bilaterally (74.2%) at 12 months; and 183 pars healed (94.3%), 90 patients bilaterally (92.8%) at 24 months . The internal fixation of 60 patients and 15 patients with bilateral union were removed at 12 months and 24 months postoperatively. The dynamic lumbar lateral radiograph was taken at 3 months after removal with the L5S1 sagittal ROM of 7.1° ± 3.7° (2.0–14.5°) for 12 months and 5.7° ± 2.7° (2.0–14.0°) for 24 months, respectively. The rate of motion preservation is 79.8% and 64.0% after fixation removal at 1 and 2 years postoperatively.Previous studies had found that the most common lumbar spondylosis involved L5 level , 15. It Although lumbar spondylolysis accounts for around 6% of the general population , most ofIt is no accident that the most controversial point of this technique is temporary fixation of L5S1 motion segment with intersegment pedicle screws. That usually means a degree of segmental motion loss even though temporary stabilization. However, what are the advantages of intersegment fixation? Although there were no biomechanical studies to compare the intersegmental pedicle screw with main intrasegmental stabilization technologies, logically, as the three-column spinal fixation, the biomechanical performances of pedicle screws should be better in control of intersegmental extension and rotation stress . A very There are some limitations to this study. First, because of the retrospective nature of this study, the strength of evidence was not very high in evaluating outcomes of pain and function. Second, part of patients was lost to follow-up which may result in exclusive bias. Third, there were no control group, and the study might have been subjected to intervention biases. Although the size of the sample was big, it was still a single-center study.In conclusion, although the L5S1 motion segment was fixed temporarily with intersegmental pedicle screw, the segment obtained more stability to decrease hyperextension and rotation stress of L5 pars and improve the union rate, especially for patients of CT classification III, grade I spondylolisthesis, mild disc degeneration, and spina bifida occulta. The diagnostic par block test should be recommended in the repair of spondylolysis which is an available link between symptoms and pars defect. The gratifying result was that a large part of segmental motion was preserved. This technique is reliable for lumbar spondylolysis repair which has satisfactory symptom relief, high healing rate, and low incidence of complications.