Introduction. Osteomyelitis is a severe orthopaedic complication which is difficult to diagnose and treat. Previous experimental studies mainly focussed on evaluating osteomyelitis in the presence of an implant or used a sclerosing agent to promote infection onset. In contrast, we focused on the longitudinal assessment of a nonimplant related osteomyelitis. Methods. An intramedullary tibial infection with S. aureus was established in NZW rabbits. Clinical and haematological infection status was evaluated weekly, combined with X-ray radiographs, biweekly injections of calcium binding fluorophores, and postmortem micro-CT. The development of the infection was assessed by micro-PET at consecutive time points using 18F-FDG as an infection tracer. Results. The intramedullary contamination of the rabbit tibia resulted in an osteomyelitis. Haematological parameters confirmed infection in mainly the first postoperative weeks , while micro-PET was able to detect the infection from the first post-operative week onward until the end of the study. Conclusions. This study shows that osteomyelitis in the rabbit can be induced without use of an implant or sclerosing agent. The sequential follow-up indicates that the diagnostic value of each infection parameter is time point dependant. Furthermore, from all parameters used, the diagnostic value of  18F-FDG micro-PET is the most versatile to assess the presence of an orthopaedic infection in this model. During the last decades, much research has been conducted into prevention, diagnostics, and treatment modalities for orthopaedic infections. Most research studies focus on treatment or prevention and not on the diagnosis of bone infection. However, novel imaging modalities are made available in the clinical evaluation of osteomyelitic lesions, that is, combined 18F-FDG PET [The preclinical evaluation of any diagnostic tool requires a stable and consistent experimental preclinical model with a broad collection of relevant read-out parameters to yield reliable data with a precise follow-up. In this way, preclinical osteomyelitis models can be highly informative on the development of the disease and the accompanying diagnosis by novel tools like -FDG PET , 3.To investigate the development of a nonimplant related osteomyelitis over time and to define a stable preclinical model, we aimed to establish an osteomyelitic lesion in the tibiae of rabbits, without the use of a sclerosing agent, since this destructs the local vascularisation of the bone and reduces the local immune capacity , 5.18F-FDG as an infection specific micro-PET tracer on multiple time points during follow-up.To investigate the potential of novel diagnostic approaches, we evaluated the sequential use of By combining the results of this study with our previously reported studies , 7, we aEleven specified pathogen free (SPF) female New Zealand White (NZW) rabbits , with a weight of 3.5–4 kg (approximately 6 months of age) were used in this study. Animals were allowed to acclimatize for 2 weeks before surgery was performed.During the study, each animal served as its own control since preoperative measurements had been performed; these functioned as a baseline measurement. The nonoperated contralateral leg was also used as a control for the uncontaminated condition . Furthermore, preoperative micro-PET scans were supplemented with a historic control group  to reducThe experimental follow-up scheme is displayed in This study was approved by the Maastricht University Animal Ethics Committee .5 CFU S. aureus  in 100 μL saline.Animals were anaesthetized according to our previously published study . Subsequ5 CFU per contamination, based on OD600 measurements . The inoculation dose and preparation were based on previous studies [The inoculation dose was freshly prepared before surgery from an overnight culture and diluted with sterile saline to an average concentration of 3.8 × 10 studies . To confAfter contamination, the defect was sealed with bone wax  and the surrounding tissue was flushed with sterile saline. The patella tendon was apprimated with 4 resorbable sutures and the skin by 6 single intracutaneous inverted sutures ; aluminum-spray  was applied to protect the wound.The animals were monitored during the 6-week follow-up for the use of their hind legs, the appearance of the wound, and on general signs of infection . Body weight and temperature were measured and blood was collected by venipuncture from the jugular vein at the day of surgery and every week thereafter until the end of the experiment. Blood samples were collected weekly and analysed for changes in erythrocyte sedimentation rate (ESR) , leucocyte differentiation , and C-reactive protein levels (CRP) .Standard X-ray radiographs were collected according to our previously described study . All radEx vivo micro-CT imaging of the affected tibiae was performed after 6-week follow-up, directly postmortem. The micro-CT-images were acquired on an X-rad 225  as described previously [eviously .18F-FDG micro-PET was conducted to assess the local metabolic glucose uptake in the contaminated tissue, since infection is associated with an increased metabolic glucose turnover. Imaging data was collected by a preoperative scan and three postoperative scans  of each animal.The entire micro-PET procedure of approximately 2 hours was performed under tiletamine-zolazepam sedation, initiated by a 15 mg/kg intramuscular dose and 3 additional intramuscular injections of 7.5 mg/kg. The time between the first and the subsequent second dose was 20 minutes, all injections following thereafter were given with 45-minute intervals.18F-FDG  were diluted with sterile saline to a volume of 1 mL and were subsequently injected in the ear vein of the rabbit. Residual activity in the syringe was measured on a CRC-25R dose calibrator  to calculate the initially injected 18F-FDG dose. A 1-hour incubation period was taken into account to allow uptake of the 18F-FDG in the area of interest.The rabbits were fixed in a custom made PVC restrainer, which allowed the rabbit to breath freely without allowing movements of the hind legs . Fifty M vastus lateralis to serve as a measurement for the soft tissue 18F-FDG-uptake. The standardized uptake value (SUV) was calculated from the total activity in the selected VOI, corrected for the weight of the animal and the activity of 18F-FDG in the animal at the time of emission scanning. Activity at the time of the scan was corrected for the injected activity and the calibration factor of the micro-PET and dose calibrator.Data were analysed with the ASIPRO VM software package . The imaging data were reconstructed by the OSEM2D protocol with an isotropic voxel size of 0.87 mm to align the tibial intramedullary cavity with the coronal, sagittal, and transversal planes to keep the volumes of interest (VOI) of all scans equal. A cylindrical VOI of 10.4 mm in diameter and 25.1 mm long (12 voxels in diameter and 29 voxels long) was used as a contour around the affected bony tissue. An equally sized VOI was placed in the contralateral leg on the equivalent location, to serve as an internal control. Each leg also contained an equally sized VOI in the S. aureus on tellurite glycine agar plates. S. aureus growth was identified by the presence of black colonies, due to the coagulase positive character of the species. Other, coagulase negative bacterial species  would appear as white colonies. A 5 mm piece of the distal part of the tuberositas tibiae was excised from the tibia with a surgical drill . After weight measurement, it was homogenized in 10 mL sterile saline  at 6000 rpm. The homogenates were cultured on tellurite glycine agar plates. After 24 hours, culture dishes were quantified for the specific bacterial growth.After sacrifice, the tibiae were dissected aseptically. Swabs were taken from the knee joint cavity. To assess soft tissue infection, these swabs were evaluated for the presence ofDuring the experimental follow-up, three different calcium binding fluorophores were administered, by subcutaneous injection, to follow bone apposition and mineralization over time, which are to be detected in histological sections. Injection of Calcein Green  was performed at 2 weeks and Xylenol Orange  at 4 weeks, and Calcein Blue  was injected on the day before sacrifice.μm sections were obtained using a saw microtome . Sections were analysed and digitized by light microscopy . The localization of calcium binding fluorophores in the bony tissue was visualized by fluorescence microscopy  on unstained PMMA sections. Acquired images were merged using Photoshop CS3  to generate overview images.After sacrifice and sampling for bone culture, tibiae were fixated in 4% formaldehyde/PBS for 4 weeks and embedded in polymethylmethacrylate (PMMA) . After polymerization, sections were stained according to Masson-Goldner  and Gram (without a safranin counterstain) and subsequently 50 18F-FDG). Therefore, the statistical analysis of the micro-PET-data was performed with a combination of the Wilcoxon Signed Ranks Test and the Mann-Whitney U Test. The significance level was determined at P ≤ 0.05. Graphical representation of the data was performed in GraphPad Prism 5 , and error bars represent standard error of mean.SPSS 21  was used for the statistical analyses. Each animal served as its own preoperative healthy control. Data were checked for normality using the Shapiro-Wilk test. Differences between time points were determined by a Wilcoxon Signed Ranks Test for nonparametric significance. In case of the micro-PET imaging, we randomly selected 3 animals to serve as a healthy preoperative control. The data of these 3 animals were supplemented by a historic control group of 10 animals , to redu5 CFU S. aureus via a transpatellar tibial plateau administration route. Nine animals completed the 6-week follow-up, which all developed an osteomyelitis in the right proximal tibia after peroperative contamination. One animal did not recuperate from the surgical procedure and another animal was sacrificed 3 weeks after surgery due to complications (humane endpoints were defined as extensive weight loss of >20% and severe soft tissue infection).Eleven rabbits were intramedullary  contaminated with 3.8 × 10P ≤ 0.049). All animals reduced weight significantly compared to preoperative values and did not regain their preoperative bodyweight values during follow-up (P ≤ 0.011).After recovery from the surgical procedure, all animals showed limited function of the operated leg with partial weight bearing. Body temperature and weight  were monitored on a weekly basis. In comparison to the preoperative measurements, a significant increase in body temperature was noted in all but the second postoperative week (P = 0.042).During the six-week follow-up period, the ESR increased during the first weeks after surgery compared to the preoperative rate, with a significant increase only at two weeks after surgery (P ≤ 0.038).CRP levels however showed a significant increase in the first 5 weeks after surgery (P ≤ 0.017). The neutrophilic granulocyte fraction was only significantly different on the second and sixth postoperative week (P ≤ 0.050). The monocyte fraction was significantly increased in all but the second postoperative week (P ≤ 0.033). The basophilic granulocyte fraction was significantly increased from the third postoperative week onward (P ≤ 0.038). The eosinophilic granulocyte fraction was only significantly different at the fifth postoperative week (P = 0.042).The weekly assessment of the leucocyte differentiation  indicateThe bone morphological changes initiated by the peroperative contamination of the tibia were detectable on X-ray radiographs from the 3rd postoperative week onwards , when scP = 0.371; right leg P = 0.112).Quantification of the micro-PET tracer uptake indicated that there was no statistical difference in tracer uptake in the proximal part of the tibia between the experimental preoperative group and the historic control group  (left leP = 0.753).There was no statistical difference in tracer uptake between the left and right leg of the combined preoperative control group (including the historic control group) .The 18F-FDG uptake, indicated by the standardized uptake value (SUV) in the operated and contaminated right leg when compared to the uncontaminated left leg (P ≤ 0.012).All postoperative time points showed an increased  S. aureus in 4 out of 9 cases, while bone tissue homogenate cultures tested positive for S. aureus in all cases  are to be expected, whereas diseased bone remodelling would show calcium deposition in an outward direction in clearly defined layers . This allowed a clear discrimination between the normal left (unoperated and uncontaminated) and the right (operated and infected) tibia of all animals. The calcium binding fluorophores indicated undisputable signs of periosteal elevation and cortical thickening, in the infected tibiae ; these h18F-FDG to detect implant infection by PET-imaging [Osteomyelitis remains to be a major complication after an orthopaedic intervention or after bone trauma , 10. The-imaging , we can Animal models for experimental (implant-related) osteomyelitis, like ours, are being used to evaluate the application resorbable biomaterials and antimicrobial coatings and their antimicrobial properties –14. For Common haematological parameters like ESR, CRP, and leucocyte differentiation are potent parameters for infection detection. However—there are some practical difficulties—the ESR is difficult to determine due to the used capillary detection system in our experimental setup, resulting in large deviations at the moment of readout. This issue makes this method of determination less accurate. The determination of the CRP concentration on the other hand is performed by ELISA, which is a very sensitive method, resulting in a more accurate readout. However, both ESR and CRP show the same trend, which is increased levels in the first weeks after surgery, after which the levels slowly decrease.The leucocyte differentiation however shows a specific increase or decrease of a specific fraction of the leucocyte pool. For example, acute infection is generally related to a decrease in the lymphocyte fraction and a related increase in favour of the neutrophil and monocyte fraction. When we combine these haematological findings with the fact that the infection remains active (even when the ESR and CRP are decreasing but the shift in leucocyte differentiation remains present) our data suggests that the infection is stabilizing. This finding was confirmed by our previously performed study on orthopaedic implant infections.X-ray radiographs allow detection of bone infection due to morphological changes like infection-initiated mineralization and osteolysis. Radiography is often used on single time points. The use of multiple imaging moments during follow-up allows monitoring of the bone morphological changes in the affected region. By detecting periosteal elevation, focal loss of cortex integrity, and osteolysis, an osteomyelitic lesion can be detected in time during radiological follow-up and thus allows monitoring of infection progression, whereas the presence of an implant may disturb radiologic detection of infection hallmarks due to implant related scatter of the X-rays; nonetheless, the influence (on radiologic imaging) of the implant will remain equal during follow-up which will not hamper the sequential evaluation of bone mineralization and morphology over time.18F-FDG has been described as a clinical PET tracer for metabolic active processes  [18F-FDG as a micro-PET tracer for the detection of osteomyelitis in relation to other relevant infection parameters, especially since 18F-FDG micro-PET indicates abnormal metabolically active areas in the body [18F-FDG micro-PET allows differentiation between contaminated and uncontaminated bony tissue within one week after surgery. Due to the ability of 18F-FDG micro-PET to distinguish between infected and uninfected tissue, this could be a powerful tool to assess novel infection interventions . However, previously published studies have shown that the presence of an implant or an osteotomy/fracture can hamper early detection by PET due to implant-related scatter of the excited photons on the metallic implant surface or by tracer uptake due to a local sterile inflammation (fracture) [18F-FDG is a very sensitive method to detect increased metabolic activity in the bony tissue; however, the specificity is dedicated not only towards infection but also towards sterile inflammation due to tissue damage  [ctivity) , 21. Togthe body , 22. Furracture) , 22. Thiracture) , 22.Bacterial culture is considered the golden standard for the detection of active bone infections –25. The Calcium binding fluorophores have been used in the past to follow bone remodelling and the mineralization of teeth –30. In c18F-FDG microPET, post-mortem microCT and calcium bindingfluorophores provide optional information on the infection and its related bone mineralization, they are not absolutely necessary to determine if an antimicrobial coating or biomaterial is effective to prevent an osteomyelitis. While a combined follow-up with X-ray radiographs, histology, bacterial culture and haematological analysis will provide sufficient information to determine antimicrobial efficacy. However, when interested in prophylactic approaches, the determination of delayed onset of the actual infection, or infection mediated bone remodelling and osteolysis, 18F-FDG micro-PET, and postmortem micro-CT, and the use of calcium binding fluorophores provide more in depth information about timing, metabolic activity, and bone mineralization.Our collected data provides novel insight into osteomyelitis development and suggestions of parameter usage in both preclinical and clinical perspectives. In the preclinical setting, the body weight and temperature provide general information about the condition of the animal and should be regarded as such. The use of weekly assessment of the CRP levels and leucocyte differentiation, in combination with weekly X-ray radiographs, histology, and bacterial culture, is recommended. Although 18F-FDG PET  could considerably contribute to the early detection of an orthopaedic infection allowing early infection intervention and treatment in the clinical situation.When translated to the clinical situation, however, the situation is different, and CRP and leucocyte differentiation will still be useful and so will X-ray radiographs, but calcium binding fluorophores are not an option. Based on our data, 18F-FDG PET is a potent diagnostic tool for the early detection of orthopaedic infections, which can be of great value when applied in the clinical situation.Our study describes the detection of different bone infection parameters and their correlations in an experimental osteomyelitis animal model (independent of the presence of an implant) and provides information on which parameters would be the most optimal infection parameters to be of use in the preclinical and potentially the clinical setting. Furthermore, our study showed that


Changes in lens may reflect the status of systemic health of human beings but the supporting evidences are not well summarized yet. We aimed to determine the relationship of age-related cataract, cataract surgery and long-term mortality by pooling the results of published population-based studies.We searched PubMed and Embase from their inception till March, 2014 for population-based studies reporting the associations of any subtypes of age-related cataract, cataract surgery with all-cause mortality. We pooled the effect estimates (hazards ratios [HRs]) under a random effects model.P<0.001; I2 = 64.2%). In the meta-analysis of 9 study findings, adults with nuclear cataract were at higher risks of mortality . In the meta-analysis of 8 study findings, cortical cataract was associated with higher risks of mortality . In the meta-analysis of 6 study findings, PSC cataract was associated with higher risks of mortality . The association between cataract surgery and mortality was marginally non-significant by pooling 8 study findings .Totally, we identified 10 unique population-based studies including 39,659 individuals at baseline reporting the associations of any subtypes of cataract with all-cause mortality from 6 countries. The presence of any cataract including cataract surgery was significantly associated with a higher risk of death (pooled HR: 1.43, 95% CI, 1.21, 2.02; All subtypes of age-related cataract were associated with an increased mortality with nuclear cataract having the strongest association among the 3 cataract subtypes. However, cataract surgery was not significantly related to mortality. These findings indicated that changes in lens may serve as markers for ageing and systemic health in general population. Visual impairment (VI) is a global health burden, associated with increased mortality in different ethnic groups To address this gap, we conducted a systematic review and meta-analysis to examine the association between different subtypes of age-related cataract, cataract surgery and long term mortality from available population-based studies.The study was approved by the Ethics Committee, Medical College of Soochow University and followed the tenets of the Declaration of Helsinki.The method for performing this meta-analysis is similar with our previously work on overweight/obesity and cataract We included studies if they were population-based, reported any subtypes of age-related cataract or cataract surgery as an independent variable and all-cause mortality as the outcome measure. The Age-Related Eye Disease Study For each study in the analyses, information on first author, publication year, study name, study location, ethnic group, sample size at baseline, age range of the study participants at baseline, follow-up periods, definitions of age-related cataract and mortality, summary estimates and corresponding 95% CI, and confounding factors adjusted for were extracted and recorded in a form designed previously.The study quality were assessed using the tool described by Sanderson et al 2 Statistic The meta-analysis was performed using Stata version 12.0 . We meta-analyzed the fully-adjusted study-specific risk estimates under random effects model, accounting for both within and between study variability. All-cause mortality was treated as the outcome measure while nuclear, cortical, PSC cataract or cataract surgery were analyzed as the independent variable. Statistical heterogeneity among studies was evaluated using IThe literature search yielded 4288 unique titles from the two electronic databases. In total, 91 articles were retrieved for full-text review, 12 of which met all the predefined inclusion criteria. However, two studies reported 2 results at different follow-up periods Characteristics of the 10 studies included in the meta-analysis are summarized in All studies graded cataract based on standardized protocols such as the Wisconsin grading system, the Wilmer grading system or the Lens Opacity Classification System P<0.001; I2 = 64.2%). In the meta-analysis of 9 study findings, adults with nuclear cataract were at higher risks of mortality   (2 = 67.3%) (2 = 76.6%) Figure 2P>0.05) and Begg's test  in all analyses.There was no evidence of publication bias as indicated by a non-significant Egger test  and increased mortality reported in previous studies. In addition, we found that the association of nuclear cataract with increased mortality was strongest among all of the three cataract subtypes. Nuclear sclerosis of the lens may be a marker for changes in structural proteins of the whole body, mainly resulting from oxidation that cause diminished function Compared with nuclear cataract, we found a weaker but significant association between cortical cataract and less survival. In the Blue Mountains Eye Study, the association between cortical cataract and increased mortality was no longer significant in persons without diabetes, indicating that the observed association may be confounded by the presence of diabetes The association of PSC cataract and mortality was also positively significant in this meta-analysis although there were fewer studies reporting this association compared with other subtypes. At current stage, we were unable to provide a reasonable biological explanation for this observed association. Animal studies have demonstrated that injection of peroxidative substances into the vitreous resulted in the formation of PSC cataract We found a marginally non-significant association between cataract surgery and mortality, which may be a balanced effect of aged-related cataract and good knowledge of health. Adults who have undergone cataract surgery certainly had cataract previously, which may increase the risk of long term mortality. On the other hand, they tend to have better knowledge of health and healthier lifestyle compared with those who did not. For example, in the Beaver Dam Eye Study, adults with cataract surgery were more likely to take vitamin supplements Our meta-analysis may have some important clinical implications. Previous studies have shown that age-related cataract is associated with systemic comorbidities There are several strengths of the meta-analysis. The qualities of the included studies were relatively high with population-based samples achieving reasonable follow-up rates and the use of standardized grading systems for age-related cataract. Limitations of this meta-analysis should also be acknowledged. First, the potential biases in the original studies, methodological issues and different strategies for adjusting for confounders could affect the results from this meta-analysis. The longitudinal association of age-related cataract and mortality may have been confounded by other unadjusted factors or selection bias. Second, different studies used different cataract grading system, sampling strategies, follow-up period, and ways of mortality measures, which would affect the estimates of mortality. These differences could have also contributed to the observed high heterogeneity across studies. Thirdly, most included studies only reported the association between cataract and all-cause mortality and therefore we were unable to summarize the association between cataract and the cause of mortality. Fourthly, the number of the contributing prospective studies was small and therefore, detailed subgroups analysis may not be feasible. Finally, although there was no evidence of publication bias as indicated by a non-significant Egger test and Begg's test, publication bias could still be of concern because studies that report statistically significant results are more likely to get published than studies that report non-significant results, and this could have distorted the findings of our meta-analyses.In conclusion, this systematic review and meta-analysis confirmed a longitudinal associations of all age-related cataract subtypes but not cataract surgery with increased mortality. The association was strongest for nuclear cataract. Clinically, ophthalmologists and clinicians should be aware that lens imaging may be a useful tool for the assessment of ageing of the whole body.Checklist S1PRISMA checklist.(DOC)Click here for additional data file.


P <0.001); there was no significant difference in the rate of sputum conversion between the GAT-containing regimens and the standard regimen . There were no significant differences in the incidence of death from any cause, including TB, nor were there serious adverse events between the MOX- or GAT-containing regimens and the standard regimen. In conclusion, MOX or GAT might not have the ability to shorten treatment duration in the initial therapy for tuberculosis despite the non-inferiority or even slightly better efficacy in the early phase of treatment compared with the standard regimen. Furthermore, it is safe to include MOX or GAT in initial TB treatment.Moxifloxacin (MOX) and gatifloxacin (GAT) have exhibited promising mycobactericidal activity, and a number of clinical trials have been conducted in recent decades to compare the treatment efficacy of MOX-containing and/or GAT-containing regimens with the standard regimen. The aim of this meta-analysis for clinical trials of MOX- or GAT-containing regimens was to evaluate their treatment efficacy and safety in initial therapy for drug-sensitive tuberculosis (TB). Databases were searched for randomized controlled trials, and nine studies with 6980 patients were included. We found that fluoroquinolone substitution for isoniazid or ethambutol in short-course regimens might result in more frequent unfavorable treatment outcomes compared with the standard regimen—in particular, an increased incidence of relapse. In a per-protocol analysis, MOX-containing regimens had slightly higher rates of sputum culture conversion at two months than the standard regimen (RR 1.08, 95% CI 1.04–1.11,  Mycobacterium tuberculosis, and it remains a major global health problem. The World Health Organization estimated approximately 9.0 million new TB cases and 1.5 million TB-related deaths in 2013.1 The current recommended therapy for new cases of drug-susceptible TB is a standard 6-month regimen with four first-line drugs: isoniazid, rifampicin, ethambutol and pyrazinamide. The treatment success rate can reach up to 86% among newly diagnosed TB cases.1 However, the treatment duration is too long to achieve excellent compliance, which contributes to relapse and drug resistance. Therefore, new drugs or new drug combinations are required to simplify the regimen or to shorten the course of treatment with a non-inferior therapeutic effect.4Tuberculosis (TB) is an infectious disease caused by in vitro9 and the promising early sterilizing effect in animals12 led to the suggestion that fluoroquinolones (FQs) might reduce the duration of treatment in new TB cases. The fourth-generation FQs gatifloxacin (GAT) and moxifloxacin (MOX) have shown promising mycobactericidal activity,16 and a number of clinical trials have been conducted in recent decades to compare the treatment efficacy of MOX-containing and/or GAT-containing regimens with the standard regimen. In these regimens, MOX or GAT was used as the substitution for or as an addition to the established standard regimen. Previous reviews have indicated that insufficient evidence was available to assess the efficacy and safety of MOX or GAT in the first-line regimen for primary pulmonary TB and that much larger trials were required.18 A recent meta-analysis concerning the efficacy and safety of MOX plus first-line therapy failed to include relevant newly published randomized clinical trials (RCTs) and did not evaluate the relapse rates of these regimens.19 Given the inferiority of four-month FQ-containing regimens compared with the six-month standard regimen for drug-susceptible TB in recent large-scale trials,22 we speculate whether these FQ-containing regimens have sufficient anti-TB efficacy in vivo. Because the evidence obtained via RCTs seemed contradictory and insufficient to address this issue, we summarized the clinical trials of MOX- or GAT-containing regimens and conducted a meta-analysis to evaluate their treatment efficacy and safety in drug-susceptible TB patients.Both the anti-mycobacterial activity 23 compliant literature search strategy was performed. The electronic databases MEDLINE (1966 to November 2014) and EMBASE (1974 to November 2014) were searched by two reviewers (R. and L.). We attempted to identify all relevant trials regardless of language. All of the titles and abstracts generated by this search strategy were independently reviewed. After the initial screening process, the full texts of the eligible articles were reviewed against the predefined inclusion criteria. In addition, we scrutinized the reference lists of each eligible paper for any omitted studies.The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA)Studies were eligible for inclusion if they were RCTs that evaluated the MOX- or GAT-containing regimen for drug-sensitive pulmonary tuberculosis. Studies were excluded if they were not RCTs or if they lacked desired outcomes. Ethical approval was waived for this meta-analysis because the data we extracted were open-access from individual studies that had obtained ethics approval.Two researchers (R. and L.) independently extracted data from studies using a pre-defined data extraction form and resolved any discrepancies in the extracted data through discussion. The basic characteristics of the studies were recorded, including author; year of publication; country; number of participants in each treatment group; mean age and gender of participants; HIV prevalence; detailed treatment regimens of each arm; and duration of follow-up.The rate of unfavorable outcomes was set as the primary outcome. The unfavorable outcomes were treatment failure, relapse, and death or withdrawal from the trial at the end of follow-up. The rate of sputum culture conversion at the end of the intensive phase (2-month) was set as the secondary efficacy outcome. Considering the possible impact of HIV status, we had planned to assess the primary and secondary outcomes in subgroups according to HIV status, but approximately half of the studies that included HIV-infected patients did not report the relevant data, and for those that reported data according to HIV status, the outcomes of HIV-positive patients were similar to those of HIV-negative patients. We therefore did not perform a subgroup analysis. The safety outcomes were the rates of death from any cause, TB-related death and serious adverse events. We extracted the number of treatment failures, relapses, deaths, sputum culture conversions and serious adverse events, as well as the number of participants in each treatment group. We presented dichotomous data and combined them using risk ratios (RR) with 95% confidence intervals (CIs).24 We determined whether the selected studies were appropriate for inclusion in the meta-analysis by considering six domains: sequence generation, allocation concealment, blinding , incomplete outcome data, selective outcome reporting and other sources of bias (2 test and inconsistency (I2) statistic.25 The fixed-effects model was used to calculate the RR for I2 <50%, and the random-effects model was used for I2 ≥ 50%. P values <0.05 suggested statistical significance.The risk of bias for each study was assessed using the Cochrane Collaboration's tool for assessing the risk of bias. of bias . Data an31  .22Three studies examined the efficacy of short-course MOX-containing regimens, and all of the unfavorable outcome rates for these 4-month regimens were higher than those of the standard regimen.ue 0.42) . In cont21 and one GAT-containing study22 compared the unfavorable outcome rate between HIV-positive patients and HIV-negative patients, and another MOX-containing study26 set the two-month sputum conversion rate as the end point. We therefore did not conduct this subgroup analysis. However, all seven of these studies reported no difference in effect based on HIV status.We aimed to conduct subgroup analysis according to HIV status to measure the possible impact of HIV co-infection. Among the seven studies that enrolled patients who were co-infected with HIV, only four reported detailed data. Two MOX-containing studiesThe rate of sputum conversion was reported in each study included in our analysis and was used as the primary efficacy point in several studies. In this meta-analysis, the pooled rates of sputum culture conversion at two months of treatment were calculated as a secondary outcome. Because a considerable number of randomized patients failed to meet the inclusion criteria, were lost to follow-up, or withdrew, a per-protocol analysis was conducted.P value <0.00001) .31 We detected moderate heterogeneity, with I2 = 41%. In addition, solid culture medium seemed to produce more negative culture results than liquid culture. In the per-protocol analysis of data from solid culture results, we found that MOX-containing regimens resulted in a much higher sputum culture conversion rate at two months [1934 (90.0%) of 2148 patients] than did the standard regimen [800 (78.1%) of 1024 patients] with statistical significance   of 2580 patients] than did the standard regimen [1011 (84.3%) of 1306 patients] with statistical significance  . A simil0.00001) . HoweverP value = 0.13)  of 897 patients] was slightly higher than that in the control groups [680 (82.4%) of 825 patients], but no significant difference was found  . However2I value was very low. As with MOX, there was no significant difference in the number of serious adverse events between GAT-containing regimens and the standard regimen  corresponded to only slight differences in the conversion rates (86.6% vs. 84.3%). Of the phase two trials included in our meta-analysis, most investigators used 2-month sputum culture conversions as their primary efficacy endpoint, which is a surrogate marker for the final treatment outcome. Two-month culture status has been demonstrated in earlier studies to be a good marker for the efficacy of TB treatment regimen, but the observed correlation among populations is not strong enough to reliably predict treatment outcomes such as relapse. Based on the detailed data provided in 21 whereas a higher relapse rate was reported when the regimens were shortened to 4 months in the three phase three clinical trials.22 The higher relapse rate indicated that MOX or GAT substitution might not permit a reduction in treatment duration. This result could be influenced by the shorter therapeutic period. In future studies, more robust surrogate markers of treatment efficacy are needed to select suitable regimens for shortening tuberculosis treatment that can be further assessed in phase three studies. Interestingly, Wallis and his colleagues developed a meta-regression model in 2013 to predict relapse risk using treatment duration and the 2-month sputum culture positive rate as predictors, and the analysis using this model of published2-month data for MOX-containing regimens indicated that it would result in relapse rates similar to those of standard therapy only if administered for over five months.33 They further assessed and refined the model in 2015 using more recent RCTs. This finding may provide information regarding treatment duration for future TB trials. Regretfully, a number of RCTs comparing non-shortened FQ-containing regimens with the standard regimen lacked data describing relapses and deaths due to an insufficient duration of follow-up.29 Thus, we could not determine whether non-shortened FQ-containing regimens resulted in better outcomes than the standard regimen. One study indicated that supplementing the controlled regimen with MOX could significantly hasten culture conversion.31 However, this study was an interim analysis, and research regarding the rate of treatment success and relapse is in progress. Whether adding MOX or GAT to the standard regimen successfully shortens the initial treatment regimen remains unknown, and more studies are needed. Furthermore, the contradictory outcomes of animal studies and human trials indicate that current available mouse models do not fully recapitulate human TB infection; caution should be exercised when interpreting these studies.MOX, as is the case with isoniazid, has greater early bactericidal activity than other standard drugs. Previous studies have indicated that MOX greatly accelerated the speed of culture conversion and produced a stable cure when combined with rifampin and pyrazinamide in a mouse model.34 and MOX was shown to prolong the QTc interval.35 Our meta-analysis revealed that there were no significant differences between the groups regarding the percentage of serious adverse events that were considered relevant to the studied medications. However, studies of the use of GAT to treat TB were insufficient compared to those for MOX. In the safety analysis, there were no significant differences in the incidence of death from any causes, TB-related deaths between FQ-containing regimens and the standard regimen. Adverse events were recorded in each study, especially for certain symptoms: GAT had been reported to cause incidents of hypoglycemia and hyperglycemia,M. tuberculosis isolates, thereby hampering the treatment of multi-drug resistant TB (MDR-TB) in more difficult situations. FQs currently play a very important role in the treatment of MDR-TB36 and are strongly recommended by the WHO guidelines for the programmatic management of drug-resistant tuberculosis.37 They are significantly associated with cure—an effect that is more pronounced for later-generation FQs, such as levofloxacin, MOX and GAT.37 However, several studies have reported a high prevalence of FQ-resistant TB in China,38 India39 and the Philippines.40 In Migliori's meta-analysis, TB patients were found to have a three-fold higher risk of acquiring FQ-resistant TB when prescribed FQs before TB diagnosis compared to non-FQ-exposed patients .41 In another meta-analysis, previous exposure to FQs was determined to be a risk factor for FQ-resistant TB; it was found that 20.8% of patients exposed to FQs for more than 10 days, 60 days prior to a TB diagnosis had FQ-resistant TB compared to only 1.6% of those taking an FQ for less than 10 days.42 Thus, if we use MOX for initial therapy, it is possible that FQ-resistant MDR-TB strains will flourish, leading to the development of extensively drug-resistant tuberculosis.Furthermore, the use of FQs for initial tuberculosis therapy raises concerns regarding whether the application of FQs in drug-sensitive TB will increase the rate of FQ-resistant This meta-analysis has several limitations. First, variation in treatment duration and the follow-up period among the studies resulted in the inability to calculate a pooled estimate of the rate of unfavorable outcomes. Studies that use FQ as a component in the first-line non-shortened regimen with an adequate follow-up duration are needed to fully evaluate the efficacy of FQs. Second, although culture conversion is the most widely supported surrogate endpoint, the discrepancy between sputum culture conversion rate at two months and treatment outcome indicated that the relapse rate after the completion of therapy could be a precise indicator of effectiveness. However, among the nine included studies, only four studies reported the number of relapses. Third, only a handful of studies using GAT to treat TB were identified, and the duration of treatment and follow-up varied, which contributed to the moderate heterogeneity when we calculated the pooled estimates. Thus, the calculation of the rates of death and adverse events between GAT-containing regimens and the standard regimen may be crude, and the precise efficacy and safety of GAT in initial therapy of TB requires additional study. Fourth, although pooling the results using different culture methods may result in greater heterogeneity, the results should not impact the main finding.In conclusion, MOX or GAT might not be able to shorten treatment duration in the initial therapy for TB, despite their equivalent or even slightly better efficacy in early phase of treatment compared with the standard regimen. Nevertheless, it is safe to include MOX or GAT in initial TB treatment.


The aim of this study was to investigate the effects of Fadrozole hydrochloride and recombinant human insulin-like growth factor I (rhIGF-I) on female-to-male sex reversal, hatching traits, and body weight of broiler chickens. On the third day of incubation, fertile eggs were randomly assigned to five experimental groups comprising (i) Fadrozole (0.1 mg/egg), (ii) rhIGF-I (100 ng/egg), (iii) Fadrozole (0.1 mg/egg) + rhIGF-I (100 ng/egg), (iv) vehicle injection (10 mM acetic acid and 0.1% BSA), and (v) non-injected eggs. Eggs in the rhIGF-I-injected groups showed the mode of hatching time at the 480th hour of incubation, 12 hours earlier compared to the other groups, with no statistically significant difference in mortality and hatchability. On Day 1 and 42 of production, 90% of genetically female chicks were masculinized using Fadrozole treatment, while 100% female-to-male phenotypic sex reversal was observed in the Fadrozole+rhIGF-I group. Fadrozole equalized the body weight of both genders, although rhIGF-I was effective on the body weight of male chicks only. Interestingly, combined rhIGF-I and Fadrozole could increase the body weight in both sexes compared to the individual injections (P<0.05). These findings revealed that (i) IGF-I-treated chicken embryos were shown to be an effective option for overcoming the very long chicken deprivation period, (ii) the simultaneous treatment with Fadrozole and IGF-I could maximize the female-to-male sex reversal chance, (iii) the increase in the body weight of masculinized chickens via Fadrozole could be equal to their genetically male counterparts, and (iv) the IGF-I effectiveness, specifically along with the application of aromatase inhibitors in female chicks, indicates that estrogen synthesis could be a stumbling block for the IGF-I action mechanism in female embryos. Sexual differentiation in the avian system is directed by the presence or absence of the W chromosome, similar to the Y chromosome in mammals The importance of aromatase is underscored by numerous studies showing that aromatase inhibitors, such as Fadrozole, induce female-to-male sex reversal in ZW females when administered before or during sexual differentiation in ovo administration of IGF-I on embryonic and postnatal growth and development, particularly skeletal muscles, is well documented in broiler chicks and quails Insulin-like growth factors, IGF-1 and IGF-2, play essential roles in mammalian and avian growth and development as a mediator of growth hormone, either as endocrine or autocrine-paracrine effectors in ovo administration of Fadrozole and recombinant human (rh) IGF-I on female-to-male sex reversal and body weight of broiler chicks. Hatching characteristics were also investigated to detect possible detrimental treatment effects.Male broiler chicks have a faster growth rate and better feed efficiency than females and are thus of particular economic interest for farmers. Even though acceptable sex reversal rate has been achieved through aromatase inhibitors, female-to-male sex-reversed chicks do not gain much weight compared to the genetically male chickens in ovo injection was carried out over a large distance from the embryo location to minimize the physical damages done to the embryos. Eggs which did not hatch until Day 22 of incubation were cracked by the staff, and the dead embryos were used for genotyping. No mortality was observed during the production period in all treatment groups. In each measurement session, each chick was handled only for few minutes to avoid unnecessary stress and improve chicken refinements. By the end of Day 42 of production, chicks from all groups were promptly and humanely killed by a panel of experts and used for morphological evaluations. It should be borne in mind that the researchers of the current study had recruited two enthusiastic, well-trained, and skilled staff to thoroughly monitor the chicken’s behavior and physical appearance, as well as feed and water intakes throughout the production period. All the experimental procedures were assessed and approved by the Institutional Animal Care and Ethics Committee in Islamic Azad University, Khorasgan Branch, Isfahan, Iran, and all national and institutional guidelines were appropriately followed.On Day 4 of incubation, an A total number of 550 fertilized commercial eggs coming from the Ross broiler (308) chickens were collected and incubated under standard conditions . On the third day of incubation, 85 hours following the eggs being placed in the incubator, all the set eggs were candled and the air sacks were located. Fertile eggs (65±1.5 g) were randomly divided into five groups with four replicates of 24 eggs; namely i) a Fadrozole hydrochloride-injected  group, ii) a recombinant human (rh) IGF-I injected  group, iii) Fadrozole hydrochloride (0.1 mg per egg) + rhIGF-I (100 ng per egg) injected group, iv) a vehicle injected sham control group containing 10 mM acetic acid  and 0.1% Bovine serum albumin , and v) a non-injected control group. Prior to the injections, the shell surface was wiped using 70% ethanol and a small tiny hole was drilled on the surface of the air sack. Injections were made into the albumen right under the air sack using 23-gauge needles cut to 13 mm, then the holes were sealed with liquid paraffin and the eggs were replaced in the incubator in order to be hatched.ad libitum throughout the experiment.To determine the period after which the incubated eggs hatched (“hatching time”), the hatched chicks from each group was recorded every 3 hours from 456 to 510 hours of incubation. To follow the standards On the day of hatch, the phenotypic sex of randomly selected hatched chicks was evaluated through vent sexing by expert staffs. In summary, following squeezing the vent by fingers and expelling the chick dropping, the vent was opened, and the presence of a bump was considered as the male indicator. Afterwards, these chicks were slaughtered and their liver samples were collected and used to determine their sexual ZZ or ZW genotype. Next, all birds reared for 6 weeks, and the presence or absence of the testis was considered to determine the chicken phenotypic sex following slaughter on Day 42 of production.Genomic DNA was extracted from the liver samples collected on the hatching day or Day 42 after hatch, and then the genetic sex was identified by PCR from the CHD1 gene On Day 1, 2.5 ml of blood was collected by puncturing the heart from four randomly selected birds per replicate (16 chicks per treatment). Blood samples were immediately aliquoted into non-anticoagulant tubes and allowed to clot for 2 hours at 37°C. Then the serum samples were isolated by centrifugation at 2000×g for 10 minutes and stored at −20°C for later analysis. Serum samples were thawed and glucose, total protein, and triglyceride levels were determined using an auto analyzer following the instructions of the corresponding reagent kit .The collected data of hatchability, sexual mortality, and the rate of sex reversal were analyzed by χ2 test. The data obtained from serum biochemical parameters and productive performance attributes of genetically male and female sexes were subjected to the analysis of variance using the general linear model (GLM) procedure in SAS 9.2 package (each sex in isolation). The Tukey post-hoc test was used to assess any significant differences among treatments in each sex. In addition, an independent t-test was used to perform a mean comparison between genetically male and female chicks in each group. Since the frequency distribution of hatching time can be an important factor for the chicken quality dispersion through the duration of early deprivation period, further descriptive statistics including skewness and kurtosis were measured by Microsoft Office Excel 2007. The P-value set at ≤0.05 was considered as the significant level for mean comparisons. All values are presented as mean ± SEM, unless indicated otherwise.In this study, we evaluated if the administration of Fadrozole and IGF-I, alone or in combination, could affect the hatching traits of broiler chickens. Along with the graphical distribution of hatching time , we measFollowing the hatch, a randomly selected sample of chicks from each group underwent phenotypic and genotypic sexing using vent sexing and PCR-based procedures, respectively . Even thThe treatment effects on serum constituents which were tested on the day of hatching are summarized in The body weight of genetically male and female chicks on Day 1 and 42 of production are presented in In the current study, chicken embryos were treated with recombinant human IGF-I and Fadrozole, as a nonsteroidal anti-aromatase reagent, on Day 3 of incubation. We evaluated the simultaneous and separate effects of these two compounds on female-to-male sex reversal and body weight on the Day 1 and 42 of production as well as the hatching traits. Strong similarity of human and chicken IGF-I protein sequence Sex-reversal was the main agenda of this project. Determination of chicken gender was carried out based on vent phenotype and ZW genotype on the first day of production. Based on phenotype and genotype sexing being in complete agreement, neither sham injection nor rhIGF treatment induced sex-reversal. By contrast, Fadrozole effectively converted females to male chicks with 90% efficiency. The obtained results for Day 1 was consistent with previous reports on aromatase-inhibitors in ovo. The masculine phenotypes were permanently observed in the genetically female chicks through 42 days of production. Application of growth hormones (GH) improved spermatogenesis, serum testosterone, estradiol and serum IGF-I Even though IGF-I did not lead to sex-reversal in this study, 100% of genetically female chicks associated with male characteristics when a combination of IGF-I and Fadrozole was injected We further investigated if the sex-reversal event is associated with the chicken body weight gain. Fadrozole significantly increased the initial body weight of genetically female chicks. Estrogen enhances skeletal muscle catabolism, while testosterone increases skeletal muscle anabolism IGF-I substantially increased male chick body weight at Day 1 and subsequently Day 42 of production. IGF-I plays a crucial role in the growth and development during embryonic period as well as post-hatch period Along with the sex-reversal and weight gain of chicks, we evaluated if the IGF-I and Fadrozole effects can influence the hatching traits, as important factors in the chicken hatchery section. Our results confirm that hatchability and mortality traits were not affected by the treatment groups. However, application of higher doses of Fadrozole led to remarkable decrease in hatchability Results from each 3-hour inspection period of hatchery machine showed that the hatching time was left-skewed and indicated abundance of late hatchers in the control groups. Supplementation of Fadrozole into chicken eggs increased the number of hatched chicks around the mode and afterwards increased the number of late hatchers. The spread of hatching time can be escalated up to 48 hours for late versus early hatchers in ovo treatment of chicken embryos with IGF-I alleviated the newly hatched chick’s early feeding problems in several ways, including shortened and normalized hatching time, increased spread of hatch around the average, and enhanced serum biochemical parameters of the hatchers.Inclusion of recombinant human IGF-I into chicken egg albumen, with or without Fadrozole, noticeably advanced the hatching time. Shortening the hatching window through setting and hatching period was suggested as an important pathway to attenuate possible detrimental effects of the quite long early deprivation period


Regional climate projections are challenging because of large uncertainty particularly stemming from unpredictable, internal variability of the climate system. Here, we examine the internal variability-induced uncertainty in precipitation and surface air temperature (SAT) trends during 2005–2055 over East Asia based on 40 member ensemble projections of the Community Climate System Model Version 3 (CCSM3). The model ensembles are generated from a suite of different atmospheric initial conditions using the same SRES A1B greenhouse gas scenario. We find that projected precipitation trends are subject to considerably larger internal uncertainty and hence have lower confidence, compared to the projected SAT trends in both the boreal winter and summer. Projected SAT trends in winter have relatively higher uncertainty than those in summer. Besides, the lower-level atmospheric circulation has larger uncertainty than that in the mid-level. Based on k-means cluster analysis, we demonstrate that a substantial portion of internally-induced precipitation and SAT trends arises from internal large-scale atmospheric circulation variability. These results highlight the importance of internal climate variability in affecting regional climate projections on multi-decadal timescales. Changes in regional climate are particularly relevant for effective decision-making on how to manage adaptation and mitigation, and how to cope with potential losses and damages at the regional scale in a future warmer climate . HoweverThe uncertainty in past and projected precipitation and surface air temperature (SAT) trends that is attributable to internal variability has been discussed extensively , 12, butThis purpose of this study is to employ large ensemble simulations to examine regional-scale uncertainty in future East Asian climate projections. We first analyze the relative importance of the externally-forced and internally-generated components in projected future East Asian climate trends. We then examine the impact of the internal climate variability upon future climate changes for East Asia, and assess the internal variability-induced uncertainties on multi-decadal timescales.The Community Climate System Model Version 3 (CCSM3) is a comprehensive coupled atmosphere-ocean-sea ice-land general circulation model, which consists of Community Atmospheric Model Version 3 at 2.8° horizontal resolution  and 26 levels in the vertical, Parallel Ocean Program at 1° horizontal resolution with increased resolution to 0.32° at the equator and 40 levels in the vertical, Community Sea Ice Model Version 5 with plastic-elastic-viscous dynamics, and Community Land Model described in Collins, Bitz , to whicThe 40 member ensembles of climate change simulations using the CCSM3 for the period 2000–2060 are conducted. Each of the 40 ensemble members is driven by an identical time-varying radiative forcing: the SRES A1B scenario-based greenhouse gas and stratospheric ozone as well as sulfate aerosol and black-carbon changes . In the http://www.ncl.ucar.edu/Document/Functions/Built-in/kmeans_as136.shtml [https://www.earthsystemgrid.org/dataset/ucar.cgd.ccsm.output.html.Here, we focus on the period of 2005–2055 for each member over East Asia. We consider four important climate parameters: precipitation, near-surface air temperature (SAT), sea level pressure (SLP) and 500hPa geopotential height (Z500) in both boreal winter (December-February) and summer (June-August), and estimate their linear least-square trends over this 51 years period. Given that much of precipitation changes are affected by internal variability , are displayed in In contrast, statistically robust widespread warming across East Asia appears in both winter and summer seasons . For exaTo assess the relative impacts of internal variability and external forcing on future climate trends, we calculate the signal-to-noise ratio (SNR), which is defined as the absolute values of the ensemble mean precipitation  trends divided by the standard deviation of the differences between the individual member precipitation  trends and the 40 member ensemble mean  at each model grid point. If the signal-to-noise ratio value is less than 1, the impact of internal climate variability exceeds that of the external forcing on the projected climate trends .The SNR maps of the projected precipitation, SAT, SLP and Z500 are shown in The much lower SNRs of the projected precipitation trends compared to those of the SAT trends in both seasons imply that the projected changes in precipitation are more impacted by internal climate variability. This is probably because precipitation, compared to SAT, is more affected by complicated atmospheric internal processes . The trends in SLP and Z500 in both seasons show generally increasing SNRs from the north to the south , which sOur results shown above suggest that precipitation trends in the both seasons suffer from large uncertainties owing to internal climate variability that appear to be related to uncertainties in the lower-level atmosphere circulations. We thus apply k-means cluster analysis to depict the occurrence of highly generalized internally-induced precipitation trend patterns across the 40 members. To facilitate the generalized precipitation trends classification, we employ four nodes over the East Asian domain that best represent the internally-induced patterns. We then group the internally-induced SAT, SLP, and Z500 trend patterns by composing each ensemble member that belongs to the corresponding k-means cluster. We evaluate the possible influences of the internally-induced changes in atmospheric circulation patterns on the uncertainties of the projected precipitation and SAT trends.The occurrences of wintertime internally-generated precipitation, SAT and atmospheric circulation trends are categorized in Those internal atmospheric circulation trend patterns over East Asia are linked with broad northern hemisphere wintertime circulation trend patterns. The most frequent pattern  characterizes quasi-barotropic anti-cyclonic circulation trends across northern East Asia and cyclonic circulation trends over southern China . This NoOur results suggest that the magnitudes and spatial patterns of the projected summer precipitation and SAT trends, like those in winter, are also impacted by the internally-generated large-scale atmospheric circulation trends. These internally-generated atmospheric circulation trends in East Asia are also linked with broad structures in the northern hemisphere. For instance, the most frequent pattern (45%) is related to a weak annular pattern in the mid-high latitudes, reminiscent of a positive phase of the AO-like trend pattern  but initiated from a suite of different atmospheric initial states. We present compelling evidence to show that the internally-induced uncertainty in the projected SAT trends in winter is generally larger than that in summer. Moreover, compared to SAT, projection of precipitation trends in both seasons is subject to much larger uncertainty owing to internal climate variability. This suggests that there is generally less confidence in projected changes in precipitation than those in SAT, as was illustrated by . Thus, mS1 File(PDF)Click here for additional data file.


Though depressive symptoms are common in patients with schizophrenia, they are often left untreated and are associated with a high relapse rate, suicidal ideation, increased mortality, reduced social adjustment, and poor quality of life. The present study aims to elucidate the association between depressive symptoms and fronto-temporal activities during a cognitive task in patients with schizophrenia. The fronto-temporal activities of 41 Japanese patients with schizophrenia was evaluated during a verbal fluency task using 52-channel near-infrared spectroscopy (NIRS). Depressive symptoms were assessed using the depression/anxiety component of the Positive and Negative Syndrome Scale (PANSS) five-factor model. The depression/anxiety component of the PANSS five-factor model was negatively correlated with activities of the ventrolateral prefrontal cortex (PFC), right dorsolateral PFC, and left temporal regions. Our findings suggest that reduced fronto-temporal activities on NIRS during a verbal fluency task is related to depressive symptom severity in patients with schizophrenia. The Positive and Negative Syndrome Scale (PANSS) is widely used in the clinical assessment of schizophrenia. Originally, it was divided into three scales: positive, negative, and general psychopathology24478Numerous studies have mostly used functional magnetic resonance imaging (fMRI) and electroencephalography methods to reveal the neural correlates of positive101216171819et al.2223et al.25According to a review by DrevetsThe participants’ demographic data are presented in p < 0.05) in participants with schizophrenia.The mean oxy-Hb levels during the task period were significantly higher than the pre-task baseline for 37 channels  in participants with schizophrenia.The mean deoxy-Hb levels during the task period were significantly higher than those for the pre-task baseline for 28 channels .No significant correlation was found between changes in mean oxy-Hb in any channel and the total, positive, negative, and general psychopathology scores of the PANSS , right dorsolateral PFC , and left temporal  regions (p > 0.05).Mean oxy-Hb changes were significantly negatively correlated with depression/anxiety component scores in the ventrolateral PFC .No significant correlation was observed in any channel between changes in mean deoxy-Hb and component five-factor PANSS scores in participants with schizophrenia  and NIRS data. The present finding is consistent with those of previous studies on patients with schizophrenia indicating that the hemodynamic response in the PFC during a cognitive task was related to the five-factor model of the PANSS, but not to the severity of symptoms as evaluated using the conventional PANSS subscales46p > 0.05). Although antipsychotic dosage levels exhibited significant negative correlations with oxy-Hb changes in two channels , those channels  in which we observed significant correlation between oxy-Hb changes and depression/anxiety component scores were not among these two channels. Therefore, we suspect that the effect of medication on the results may be minimal.The results of the present study must be interpreted with caution due to certain limitations. First, multi-channel NIRS has limited spatial resolution, unlike fMRI and positron emission tomography. Second, because the analysis was based on cross-sectional data, causality could not be determined. Longitudinal studies are required to assess the cause-and-effect relationships. Third, our sample size was not large and is thus subject to type II errors. Further studies involving larger numbers of patients with schizophrenia are required. Further limitations include lack of reliable information on the proportion of patients who met the DSM criteria for current/past MDD and possible confounding effects of different types and dosages of medications. Future longitudinal investigations should be conducted while monitoring diagnoses of depression and medications. Indeed, medication usage may have influenced the results of the present study. Though only three patients in the study were taking antidepressants, all patients were taking antipsychotics at the time of evaluation, the effects of which should not be ignored. However, we found no significant relationship between chlorpromazine equivalent dosage of daily antipsychotic drugs and depression/anxiety component scores Forty-one Japanese patients with schizophrenia who were outpatients at the Tottori University Hospital were included in the study . All patAll participants provided written informed consent and were recruited from January 2008 to December 2012 on the basis of consecutive referrals. All participants were right-handed according to the Edinburgh Handedness Inventoryet al.On the day of the NIRS experiment, psychiatric symptoms and global functioning were evaluated by the same psychiatrist  using the PANSSet al.The task procedure was similar to that of Takizawa http://www.jichi.ac.jp/brainlab/virtual_reg.html)A 52-channel NIRS  machine was used to measure changes in Hb concentration. The NIRS probe comprised 3 × 11 arrays with 17 emitters and 16 detectors. The NIRS probe machine was placed on the fronto-temporal region of each participant, with the midcolumn of the probe located over the Fpz and the lowest probes located along the T3-Fp1-Fpz-Fp2-T4 line, in accordance with the international 10–20 System used in electroencephalography. The distance between pairs of source-detector probes was set at 3 cm, and each measurement area between the pairs of probes was defined as a “channel” (ch). The machine measures activities along the light path at a depth of 2–3 cm below the scalp, which corresponds well with the surface of the cerebral cortex539NIRS measures change in both oxy-Hb and deoxy-Hb at two wavelengths (695 and 830 nm) of infrared light, based on the modified Beer–Lambert lawet al.9The sampling frequency was 10 Hz. To examine VFT-related activities, data for Hb changes were analysed according to the “integral mode”: the pre-task baseline was determined as the mean across 10-s just before the task period, while the post-task baseline was determined as the mean across 5-s, 50-s after the task period. Assuming that task-related activities returns to a pre-task baseline level at a constant interval, linear fitting was performed using the Hb data obtained between the pre-task and post-task baselines. The same procedure has been often used in NIRS studies917The mean changes in oxy-Hb (as opposed to those in deoxy-Hb) were measured during the VFT as an index of cortical activity because oxy-Hb better reflects this activity and shows stronger correlations with blood oxygenation level-dependent signals as measured by fMRI1559Statistical analyses were performed using SPSS 19.0 software .t-tests for each channel to confirm the activities area during the VFT.The mean Hb changes of the pre-task baseline period and that of the task period were compared using paired two-tailed p < 0.05, and multiple comparisons among the 52 channels were corrected using the false discovery rate (FDR) methodBecause the Shapiro–Wilk test revealed that the original three-factor  and five-factor components of the PANSS are not normally distributed, Spearman’s correlation coefficients (r) were calculated to explore the relationships between mean Hb changes during the VFT and the original three-factor and five-factor components of the PANSS. In addition, to elucidate the independent contributions of the depressive symptom components of the PANSS to mean Hb changes in those channels that showed significant correlations, we performed stepwise multiple regression analyses. In these analyses, the mean Hb changes were the dependent variables, and other potential confounding variables were controlled, such as age, gender , premorbid IQ, task performance on the VFT, Global Assessment of Functioning (GAF) score, duration of illness, and daily dosage of antipsychotic drugs used in the analyses of schizophrenia, with a probability of F for a conservative entry and removal criteria of 0.05 and 0.2, respectively. For significant findings, effect sizes were indicated using the standardized regression coefficient (β). Statistical significance was set at How to cite this article: Pu, S. et al. Associations between depressive symptoms and fronto-temporal activities during a verbal fluency task in patients with schizophrenia. Sci. Rep.6, 30685; doi: 10.1038/srep30685 (2016).


Interleukin-13 (IL-13) is a potent pleiotropic cytokine that is produced by activated CD4 T cells. This study was undertaken to determine the relationship between two IL-13 gene single nucleotide polymorphisms (SNP rs1800925 and SNP rs20541) and the incidence of hepatitis B virus-related (HBV) hepatocellular carcinoma (HCC).Three hundred and ninety-eight HBV-positive individuals (192 HCC and 206 patients with chronic hepatitis) and one hundred and ninety-two healthy participants from the First Affiliated Hospital of Guangxi Medical University were enrolled in this study.The results showed no significant differences between the genotype and allele frequencies of the IL-13 gene rs1800925 and rs20541 polymorphisms and chronic hepatitis B risk after adjusting for age, sex, tobacco use, and alcohol intake using binary logistic regression analyses. Regarding the rs20541 SNP, the GA genotype was significantly related to a decreased risk of HCC after adjusting for age, sex, tobacco use, and alcohol intake using binary logistic regression analyses (The odds ratio (OR) = 0.54, 95% confidence intervals (CI) 0.34–0.87). The adjusted OR for the GA and AA genotypes combined was 0.68 (95% CI 0.39–0.90).This study indicates that the functional IL-13 rs20541 polymorphism may contribute to the risk of HCC and that the rs20541 polymorphism is a protective factor for HCC. Hepatocellular carcinoma (HCC) is one of the most common malignancies in humans. Epidemiologic evidence indicates that the main risk factor for HCC is hepatitis B virus (HBV). More than 80% of HCC cases are HBV-related ; howeverInterleukin-13 (IL-13) is a potent pleiotropic cytokine that is produced by activated CD4 T cells . The benSeveral genetic factors like IL-10, IL-18, and IL-12 have been confirmed to be involved in the development of antibodies to the HBV surface antigen in the case of hepatitis B vaccination or natural HBV transmission , 16. It 398 HBV-positive individuals (192 HCC and 206 patients with chronic hepatitis) from the First Affiliated Hospital of Guangxi Medical University were enrolled in this study. The clinical criteria for HBV infection were confirmed by the criteria listed in our previous study . The cliFresh blood samples were collected in EDTA-containing tubes. Genomic DNA was extracted using a QIAamp DNA blood mini kit  as instructed by the manufacturer. The analysis of IL-13 polymorphisms rs1800925 and rs20541 was performed by the classic polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) method. The accuracy of the genotyping results was assessed using the ABI PRISM 3730 to examine the representative PCR-amplified DNA samples  method was used to evaluate the differences in demographic and clinical data among the groups. To test for deviations from the Hardy—Weinberg equilibrium, a χDetailed patient demographics for all groups including gender, age, tobacco use, education, and alcohol intake are listed in The genotype and allele frequencies of IL-13 gene polymorphisms among the chronic hepatitis B patients and the healthy controls are shown in The genotype and allele frequencies of the IL-13 gene polymorphisms among the HBV-related HCC patients and healthy controls are shown in We next investigated whether the differences of genotype and allele frequencies were related to gender. Significant differences in the distributions of the IL-13 gene polymorphisms between HBV-related HCC patients and control groups were observed . Men whohttp://www.ncbi.nlm.nih.gov/snp/). The frequencies of genotype and allele of these two SNPs in our control group were compared with those from the Haplotype Map (HapMap) Project  GA genotype were associated with a decreased risk of HCC compared with patients carrying the GG genotype. It is universally acknowledged that the IL-13 genetic background may be different from other individuals, we compared the genotype and allele frequencies of the two SNPs in our control group with those in different ethnicities from the HapMap. The results showed that the distribution of the two SNPs in the present study was similar to that in the CHB controls. However, when compared with YRI or European populations, it was significantly different, thus suggesting that the distribution of IL-13 gene frequencies might vary among different ethnic groups. Haplotype analyses showed a protective association between the haplotype tagged by the ‘no-risk’ alleles of the rs1800925 and rs20541 SNPs (TG haplotype). Meanwhile, the same ‘no-risk’ alleles of the rs1800925 and rs20541 SNPs (CA haplotype) were significantly associated with increased risk of HCC.IL-13 is well known as a Th2 anti-inflammatory cytokine that is involved in mediating B cell and mast cell proliferation and correlates with IgE synthesis, which is a major regulator in Th2-mediated disease . Based oThe body’s immune response to hepatitis B and C infections, viral clearance, and inflammation have been implicated in cancer diagnosis, prognosis, and therapy , 26. IL-In conclusion, our study provides evidence that the functional IL-13 rs20541 polymorphism may contribute to the risk of HCC. However, the results of this paper were obtained with a relatively limited sample size and single ethnic population. Therefore, larger studies of other ethnic populations, especially studies of the combined effects of the gene-gene and gene—environment are needed to confirm the current results.


Patients showed a reduced volume of the substantia nigra and contrast-to-noise-ratio and both positively correlated with the corresponding striatal DAT density. The apparent transverse relaxation rate and magnetic susceptibility values of the substantia nigra did not differ between patients and healthy controls. The best predictor of DAT reduction was the volume of the substantia nigra. Clinical and imaging correlations were also investigated for the locus coeruleus. Our results suggest that neuromelanin-MRI can be used for quantifying substantia nigra pathology in PD where it closely correlates with dopaminergic striatal innervation loss. Longitudinal studies should further explore the role of Neuromelanin-MRI as an imaging biomarker of PD, especially for subjects at risk of developing the disease.Parkinson's disease (PD) is a progressive neurodegenerative disorder in which the major pathologic substrate is a loss of dopaminergic neurons from the substantia nigra. Our main objective was to determine the correspondence between changes in the substantia nigra, evident in neuromelanin and iron sensitive magnetic resonance imaging (MRI), and dopaminergic striatal innervation loss in patients with PD. Eighteen patients and 18 healthy control subjects were included in the study. Using neuromelanin-MRI, we measured the volume of the substantia nigra and the contrast-to-noise-ratio between substantia nigra and a background region. The apparent transverse relaxation rate and magnetic susceptibility of the substantia nigra were calculated from dual-echo MRI. Striatal dopaminergic innervation was measured as density of dopamine transporter (DAT) by means of single-photon emission computed tomography and [ Parkinson's disease (PD) is characterized by a loss of neuromelanin (NM) containing dopaminergic neurons in the substantia nigra (SN), with a consequent reduction of dopamine concentration in the putamen and caudate nucleus  with [I-123] N-ω-fluoropropyl-2b-carbomethoxy-3b-(4-iodophenyl) tropane (FP-CIT), represent a robust biomarkers for early PD detection and assessing disease progression  that has been widely exploited to characterize iron deposition in the SN  of the SN and LC of PD patients and the corresponding nigro-striatal dopaminergic innervation loss as measured by SPECT with FP-CIT.This study aimed to investigate the correlations between MRI markers  and 18 age-matched healthy controls . The diagnosis of PD was made according to the UK Parkinson Disease Brain Bank criteria. The disease stage was determined using the Hoehn and Yahr scale and the disease severity was evaluated using the Unified Parkinson Disease Rating Scale (UPDRS). UPDRS akinetic-rigid score (UPDRSAK) was derived from the sum of UPDRS items 22 [head item excluded]-23-24-25-26. We calculated this UPDRS sub-score as putaminal FP-CIT binding was shown to exclusively correlate with the extent of rigidity and hypokinesia , as described in Isaias et al. . A histocontra  refers to the side opposite to the clinically most impaired hemibody, and ipsi  refers to the most impaired hemibody. For HC we adopted the conventional of referring to the right side as ipsilateral.where the subscript All subjects underwent MRI on a clinical 3T scanner  with a 32-channel head coil. Conventional MRI scans were obtained to exclude coexisting central nervous system disorders. NM-MRI and multi-echo gradient echo scans were obtained for the comparisons of NM and iron with the SPECT data.2, acquisition/reconstruction resolution 0.5 × 0.6 × 3.0 mm3/0.5 × 0.5 × 3.0 mm3, 12 slices, five averages), with an acquisition time of approximately 7:40 min  with an acquisition time approximately 10 min. Magnitude and phase images were reconstructed from the gradient echo data for each echo time. Again, the sections were in the oblique axial plane perpendicular to the floor of the fourth ventricle.Iron-sensitive imaging made use of a dual-echo, gradient echo sequence . In brief, similar to previous studies  between the SN and the cerebral crus was calculated for each slice and side of the SN as:MNSN and MNCC correspond to the mean signal intensity of the SN and cerebral crus, respectively, and SDCC corresponds to the standard deviation of the cerebral crus. The average CNR of the SN across slices was then calculated for each side. The asymmetry indices (AISN-Volume and AISN-CNR) were calculated for the SN volume and the CNRSN in a way analogous to that for SPECT measurements. Because the results of the above procedure may depend on the placement of the cerebral crus ROIs, the process of ROI drawing and calculation of the volume, CNR and AI values for the SN was repeated four times, and the average and standard deviation across measurements was calculated.where MAX) .Lastly, to allow direct comparisons between * maps were calculated from the magnitude images at different echo times using the scanner manufacturer-supplied software, and inverted to obtain R2* values. Phase and magnitude images for the different echo times were processed offline to calculate quantitative maps of magnetic susceptibility via the morphology-enabled dipole inversion method  , and then applied to the R2* and susceptibility maps. The left and right SN were defined as the hypointense bands between the red nucleus and cerebral peduncle across six slices . A predictive analysis (least squares) between MRI and SPECT measures were then performed. Receiver operating characteristics analysis was performed to assess the diagnostic accuracy of NM-MRI results. The 95% confidence intervals for sensitivity and specificity were calculated according to the Clopper-Pearson method. The intraclass correlation coefficient was used to assess the intra-rater reliability.Statistical analysis were performed with the JMP statistical package, . Gender difference between PD and HC groups was analyzed using Pearson's chi-squared test. Other differences between the PD and HC groups were analyzed by means of Mann-Whitney U test. The Spearman correlation coefficients (ρ) were calculated to investigate statistical dependencies amongst MRI and SPECT measures, demographic and clinical variables. The threshold level of statistical significance was set at AK score was 6 (range: 1–8) and 2 (range: 0–4) for the more and less affected side respectively. The average L-Dopa daily dose was 363 ± 152 mg and the average L-dopa equivalent daily dose was 502 ± 183 mg.There were no significant gender or age differences between the PD patients and the HC group recruited for the present study. There was however, a significant difference in gender between the PD group and the pre-existing HC group (used here as controls for SPECT measures). Median age of the PD patients at motor symptoms onset was 56 years (38–70 years). Median disease duration was 6 years (3–15 years). Eight of the PD patients had disease durations greater than the median value of the PD group ; the remaining 10 patients had referred motor signs for 6 years or less . All patients were at Hoehn and Yahr stage 2. The median UPDRS-III (motor part) score was 14 (range: 5–25) in a “meds-off” phase . The median hemi-body UPDRSAIPutamen and AICaudate). As expected, DAT binding values of the Putamencontra negatively correlated with disease duration , UPDRS-III and UPDRSAK score . It is worth noting that striatal DAT binding did not differ between male and female in our HC cohort.When compared to a pre-existing group of HC, all PD patients had significantly reduced striatal DAT binding values in both putamen and caudate nucleus Table . For botCNRSN, and the CNRLC, respectively, which is an acceptable level of reproducibility. For all NM-MRI measures, there were no significant differences between sides (contra and ipsi) of the SN. In patients, the mean SN volume-contra was lower than SN volume-ipsi, but the difference did not reach statistical significance (p = 0.08). In comparison to the HC group, the NM measures derived from NM-MRI were significantly reduced in PD patients (Table -contra (but not SN volume-ipsi) was negatively correlated with UPDRS-III  and UPDRSAK .Areas of hyperintensity were reliably detected in the NM-MRI images in positions corresponding to the locations of the SN and LC Figure , consistts Table . SN voluCNRSN-contra was positively correlated with CNRLC-contra , and all but four PD patients showed greater %CNRLC-contra values than %CNRSN-contra. AISN-vol and AICNRSN- were not significantly different between PD and HC groups, and, for PD patients, they did not correlate with the corresponding AIPutamen or AICaudate measurements. In fact, in seven PD patients the CNRSN values were lower ipsilateral to the worst affected hemibody and opposite to the putamen with lowest DAT binding values. Despite this, significant positive correlation was also found between both SN volume-contraand-ipsi) , and 66.67% specificity  in discriminating PD from HC ; (2) SN volume-contra = 256 mm3 showing 83% diagnostic accuracy, 66.67% sensitivity  and 100% specificity  ; (3) SN volume-contra = 292 mm3, with the highest diagnostic accuracy and a good trade-off between sensitivity and specificity , and specificity = 83% ).The receiver operating characteristics analysis showed an area under the curve of 0.94, and provided three relevant cut-off values: (1) SN volume* and susceptibility showed a large contrast between the SN and the surrounding brain tissue , Susceptibilityipsi , and Susceptibilitycontra , and with disease duration for p < 0.05), Susceptibilityipsi  and Susceptibilitycontra  , disease duration proved, at a Stepwise regression analysis, to independently correlate with both parameters , whereas the correlation with age was not statistically significant. Lastly, no significant correlation was found when comparing R2* and susceptibility values with NM-MRI or SPECT findings  Figure . NeitherPET and SPECT have been extensively employed to elucidate the functional changes associated with PD and other neurodegenerative disorders  correlated significantly with dopaminergic striatal innervation loss as measured by SPECT with FP-CIT  and specificity (83%).In this study we investigated the correlations between MRI markers of the SN and LC of PD patients and the corresponding nigro-striatal dopaminergic innervation loss as measured by SPECT with FP-CIT. Both of the examined NM-MRI based measures of the SN  was unexpected. In particular, the asymmetry of DAT binding measurement at a striatal level in PD patients was not mirrored by an asymmetry of NM-MRI measurements. In light of the close correlation between post-mortem NM-MRI and the quantity of NM-containing neurons  from NM-MRI has recently been observed to be specific to PD patients with concomitant rapid eye movement sleep behavior disorder  PD-related comorbidities . This may limit our scope for establishing clinical correlations, but was suited to the study design, which was to directly compare MRI metrics of SN pathology and its consequent striatal dopaminergic innervation loss. Second, we obtained cut-off values for SN volume showing high sensitivity and specificity in identifying PD patients, but further studies testing these values in other groups of PD patients and HC are required to validate their usability to differentiate PD patients from HC. The estimates of SN volume, and thus the cut-off points are dependent on the segmentation method and imaging sequence characteristics. In this study, we used a semiautomatic segmentation method based on the approach previously described  of the University Hospital Wuerzburg.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Clinical features and risk factors for atazanavir (ATV)-associated urolithiasis have not been fully investigated.We reviewed all cases of ATV-containing urolithiasis identified by infrared spectrophotometry among HIV-infected patients over a 5-year period to describe their clinical features and outcome. A case-control study was performed to identify risk factors associated with ATV-associated urolithiasis using univariate and multivariate logistic regression analyses.30 cases of ATV-associated urolithiasis were analyzed. Patients were mostly men (87%), median age: 45.5 years, median CD4 cell count: 443 cells/µL and 97% had plasma HIV RNA level <50 cp/mL. Median time between the initiation of ATV-containing regimen and the diagnosis of urolithiasis was 3.1 years. Patients presented with flank pain in 90% and macroscopic hematuria in 82.6%, 34% had renal dysfunction and 44.8% needed ureteroscopic treatment. In univariate analysis, chronic hepatitis C, a history of urolithiasis, prior use of indinavir, ATV duration, undetectable plasma HIV RNA, use of ritonavir as a booster and serum free bilirubin level were associated with ATV-urolithiasis. Multivariate models retained serum free bilirubin level  and either ATV duration  or a history of urolithiasis  when adjusting on serum free bilirubin level as risk factors associated with urolithiasis.ATV-containing urolithiasis are associated with frank clinical symptoms and may require surgical intervention. A high serum bilirubin level, a long exposure to ATV and a history of urolithiasis are risk factors for this rare adverse event. Atazanavir (ATV) is a widely used HIV protease inhibitor (PI) both in treatment-experienced and treatment-naive patients due to its potent antiviral activity and good safety profile Patients with ATV-containing urolithiasis were identified from the computerized databases of three biochemistry laboratories, located in Paris from January 2008 to December 2012. In France HIV infected patients followed in hospitals are all registered in the French Hospital Database on HIV - FHDH and give written informed consent for their clinical records to be used in any epidemiological study.In this study, patient records/information was anonymized and de-identified prior to analysis.In all patients, the presence of ATV in the calculi was confirmed by infrared spectrometry Distributions in cases and controls were compared with the use of Mann-Whitney test for quantitative variables and Fisher's exact test for qualitative variables. Statistical tests were two-sided, and P values of less than 0.05 were considered to indicate statistical significance. Tests for paired samples were not used, as no other factor than the date of occurrence of ATV-associated urolithiasis was used to match cases and controls.A risk factor analysis was conducted to identify factors associated with the occurrence of ATV-containing urolithiasis. We used logistic regression modeling in an univariate analysis among all covariates. Baseline characteristics available at ICU admission and associated with p values lower than 0.1 by univariate analysis or deemed clinically relevant were included in a multivariate logistic regression selection process. Given the number of cases, a maximum of three covariates was allowed in the tested models. Multiple imputation by chained equation was used to handle missing values. A stepwise Bayesian information criterion (BIC)-based selection was repeated in 30 imputed datasets to identify factors independently associated with urolithiasis. Models' calibrations were tested by the le Cessie–van Houwelingen goodness-of-fit test. Statistical analyses were performed with R version 3.0.2 .Sixty-two ATV-containing urolithiasis cases were retrieved from the 3 biochemistry laboratories during the study period but only 30 cases could be analyzed. Patients not enrolled in our study were those who did not live in the Paris area rendering data collection uneasy. Characteristics of cases are depicted in Clinical presentation, treatment and outcome of patients with ATV-associated urolithiasis are described in In univariate analysis, risk factors for ATV-urolithiasis were chronic hepatitis C, a history of urolithiasis, the prior use of indinavir, ATV duration, undetectable HIV plasma RNA, the use of ritonavir as a booster and the serum free bilirubin level . MultivaWe report here a large series of 30 cases of ATV-containing urolithiasis that allowed us to better described the clinical features and outcomes of this rare adverse event in HIV-infected patients receiving an ATV-based antiretroviral regimen.Due to our strict definition of cases based on the identification of ATV in the calculi using infrared spectrometry we could ascertain that the stones obtained in our patients were indeed related to ATV use.2, and a low prevalence of diabetes (3.3%). They were receiving an ATV-based regimen for a median of 3.1 years, a long duration of exposure which is consistent with previous reports Patients with ATV-containing urolithiasis in our study were mostly men, with a median age of 45.5 years, a body mass index of 22.8 kg/mIn a previous report, we showed that whereas lopinavir is not excreted in urine, pretty high concentration of ATV and darunavir can be achieved in urine, with the detection of ATV and darunavir crystals in a minority of patients Since ATV is a widely used PI, it is important to identify risk factors for urolithiasis. Our case-control study was able to identify the level of serum free bilirubin as an independent factor associated with a 2.31-fold increased risk of urolithiasis per 2-fold increase in serum level . This fiIn our study, a history of urolithiasis was also associated with an increased risk of ATV-containing urolithiasis. This finding can be explained by predisposing factors for the development of urolithiasis. Indeed, a number of patients already experienced indinavir-associated urolithiasis in our study, another PI that is prone to induce renal calculi and is also an inhibitor of the UGT1A1 pathway Finally, the duration of ATV use was also associated with an increased risk of ATV-associated urolithiasis in our study. This finding could be explained by the time needed to form ATV-crystals and stones, and is consistent with previous reports underlining that ATV-associated urolithiasis were only reported after a couple of years of ATV-based therapy. This finding is however a matter of concern since it would imply that patients under ATV-based regimen with high serum free bilirubin levels carry a potential risk of developing urolithiasis over time.In summary, our study has shown that ATV-containing urolithiasis can be associated with severe clinical symptoms and are associated with high and prolonged ATV. In those patients, most of them receiving a ritonavir-boosted ATV-based regimen, especially if they have a history of urolithiasis, a switch to another antiretroviral regimen should be considered to avoid the risk of urolithiasis.